WO2006084018A2

WO2006084018A2 - Methods for determining responsiveness to cancer therapy

Info

Publication number: WO2006084018A2
Application number: PCT/US2006/003660
Authority: WO
Inventors: Mengxiang Tang; Liching Cao; Rajiv Dua; Ali Mukherjee; Herjit Pannu; Jagrup Pannu; Yining Shi; Yuping Tan; Sharat Singh; Colombe Chappey
Original assignee: Monogram Biosciences, Inc.
Priority date: 2005-02-02
Filing date: 2006-02-01
Publication date: 2006-08-10
Also published as: WO2006084018A3

Abstract

In certain aspects, the present invention provides methods and compositions for determining whether a cancer cells is likely to respond to treatment with a Her 1 -acting agent. In one aspect, the invention provides a method for determining whether a cancer cell is likely to respond to treatment with a Her 1 -acting agent, comprising detecting on the cancer cell at least about 600 Her 1 -Her 1 dimers, wherein the presence of the at least about 600 Her 1 -Her 1 dimers indicates that the cancer is likely to respond to treatment with the Her 1 -acting agent. In another aspect, the invention provides a method for determining whether a cancer cell is likely to respond to treatment with a Her 1 -acting agent, comprising detecting on a cell of the cancer at least about 600 Her 1 -Her 1 dimers, at least about 1000 Herl-Her2 dimers, and fewer than about 1000 Her2-Her3 dimers, wherein the presence of the at least about 600 Her 1 -Her 1 dimers, the at least about 1000 Herl-Her2 dimers, and the fewer than about 1000 Her2-Her3 dimers indicates that the cancer cell is likely to respond to treatment with the Her 1 -acting agent. Preferably, methods of the invention are implemented by using sets of binding compounds having releasable molecular tags that are specific for multiple components of one or more types of receptor dimers. After binding, molecular tags are released and separated from the assay mixture for analysis.

Description

Methods for Determining Responsiveness to Cancer Therapy

1. Field of the Invention

[0001] The present invention relates generally to biomarkers, and more particularly, to the use of ErbB cell surface receptor complexes, such as dimers and oligomers, as biomarkers for determining responsiveness of a cancer to anticancer therapy, particularly Gefitinib therapy.

2. Background of the Invention

[0002] A biomarker is generally a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. See Atkinson et ah, 2001, Clin. Pharmacol. Th er. 69:89-95. Biomarkers vary widely in nature, ease of measurement, and correlation with physiological states of interest. See, e.g., Frank et ah, 2003, Nature Reviews Drug Discovery 2:566-580. It is widely believed that the development of new validated biomarkers will lead both to significant reductions in healthcare and drag development costs and to significant improvements in treatment for a wide variety of diseases and conditions. Thus, a great deal of effort has been directed to using new technologies to find new classes of biomarkers. See, e.g., Petricoin et ah, 2002, Nature Reviews Drug Discovery, 1:683-695; and Sidransky, 2002, Nature Reviews Cancer 2:210-219.

[0003] The interactions of cell surface membrane components play crucial roles in transmitting extracellular signals to a cell in normal physiology, and in disease conditions, In particular, many types of cell surface receptors undergo dimerization, oligomerization, or clustering in connection with the transduction of an extracellular event or signal such as, for example, ligand-receptor binding, into a cellular response, such as, e.g., proliferation, increased or decreased gene expression, or the like. See, e.g., George et ah, 2002, Nature Reviews Drug Discovery 1:808-820; Mellado et al, 2001, Ann. Rev. Immunol. 19:397-421; Schlessinger, 2000, Cell 103:211-225; and Yarden, 2001, Eur. J. Cancer 37:S3-S8. The role of such signal transduction events in diseases, such as cancer, has been the object of intense research and has led to the development of several new drags and drag candidates. See, e.g., Herbst and Shin, 2002, Cancer 94:1593-1611; Yarden and Sliwkowski, 2001, Nature Reviews Molecular Cell Biology 2:127-137; McCormick, 1999, Trends in Cell Biology 9:53-56 (1999); and Blume- Jensen and Hunter, 2001, Nature 411:355-365.

[0004] Expression of individual cell surface receptors has been used successfully as biomarkers. For example, U.S. Patent 4,968,603 describes Her2 expression as a cancer biomarker. However, mere detection of individual receptor expression alone is not always a reliable indicator of a disease status or condition. See Chow et al., 2001, Clin. Cancer Res., 7:1957-1962. Further, no biomarkers or assays are available to determine that indicate a cancer is likely to respond to treatment with a Her 1 -acting agent, such as, for example, Gefitinib. Thus, there remains a need for assays and biomarkers that can indicate whether a cancer is likely to respond to treatment with such agents, including Gefitinib. Such assays could be used to identify patients likely to respond to Gefitinib treatment, thereby avoiding administration of costly and potentially toxic compounds to patients who are unlikely to respond. These and other unmet needs are provided by the present invention.

3. SUMMARY OF THE INVENTION

[0005] In one aspect, the invention provides a method for determining whether a cancer cell is likely to respond to treatment with a Her 1 -acting agent, In certain embodiments, the methods comprise detecting on the cancer cell at least about 750 Herl-Herl dimers, wherein the presence of the at least about 750 Herl-Herl dimers indicates that the cancer is likely to respond to treatment with the Her 1 -acting agent. In a preferred embodiment, the Herl-acting agent is Gefitinib.

[0006] In another aspect, the invention provides a method for determining whether a cancer or cancer cell is likely to respond to treatment with a Herl-acting agent, comprising determining a Diagnostic Index for the cancer or cancer cell according to a formula of the invention as described hereinafter, wherein the Diagnostic Index indicates the probability that the cancer or cancer cell is likely to respond to treatment with a Herl- acting agent. In certain preferred embodiments, the Herl-acting agent is Gefitinib.

[0007] In another aspect, the invention provides a method for determining whether a cancer or cancer cell is likely to respond to treatment with a Herl-acting agent, comprising determining a balanced dimer score for the cancer cell determined according to a formula of the invention as described hereinafter, wherein the balanced dimer score indicates that the subject is likely to respond to treatment with a Her 1 -acting agent. In certain preferred embodiments, the Her 1 -acting agent is Gefitinib.

[0008] In another aspect, the invention provides a method for determining whether a subject with cancer is likely to respond to treatment with a Her 1 -acting agent, comprising determining a Diagnostic Index for a cell in a biological sample from the subject's cancer according to a formula of the invention as described hereinafter, wherein the Diagnostic Index indicates the probability that the subject is likely to respond to treatment with a Her 1 -acting agent. In certain preferred embodiments, the Her 1 -acting agent is Gefitinib.

[0009] In another aspect, the invention provides a method for determining whether a subject with cancer is likely to respond to treatment with a Herl-acting agent, comprising determining a balanced dimer score for a cell in a biological sample from the subject's cancer determined according to a formula of the invention as described hereinafter, wherein a balanced dimer score indicates that the subject is likely to respond to treatment with a Herl-acting agent. In certain preferred embodiments, the Herl-acting agent is Gefitinib.

[0010] In another aspect, the invention provides methods of treating a subject with cancer. In one aspect, the methods comprise determining that the subject is afflicted with a cancer comprising a cancer cell that is likely to respond to treatment with a Herl- acting agent according to a method of the invention, and administering an effective amount of a Herl-acting agent to the subject. In another aspect, the methods comprise determining that a subject is afflicted with a cancer comprising a cancer cell that is likely to respond to treatment with a Herl-acting agent according to a method of the invention, then advising a medical professional of the treatment option of administering to the subject an effective amount of a Herl-acting agent. In certain embodiments, the Herl- acting agent is Gefitinib. In certain embodiments, the cancer is lung cancer, e.g., non small cell lung cancer.

4. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figures 1 A-IF provide diagrams illustrating the use of releasable molecular tags to measure receptor dimer populations. [0012] Figures IG- IH provide diagrams illustrating the use of releasable molecular tags to measure cell surface receptor complexes in fixed tissue specimens.

[0013] Figures 2A-2E provide diagrams illustrating an embodiment of the method of the invention for profiling relative amounts of dimers of a plurality of receptor types.

[0014] Figures 3 A-3D provide diagrams illustrating methods for attaching molecular tags to antibodies.

[0015] Figures 4A-4B present representative electropherograms showing expression of total Her2 and Herl-Her2 dimers in patient samples.

[0016] Figures 5 presents a representative electropherogram showing expression of total Her2 and Her2-Her3 dimers in patient samples.

[0017] Figures 6A-6B present representative electropherograms showing expression of total Herl, Herl-Herl dimers, and phosphorylated Herl in patient samples.

[0018] Figure 7 presents a representative electropherogram showing expression of Herl - Her3 dimers in patient samples.

[0019] Figure 8 presents representative data showing amounts of total Herl expression (HIT), Herl-Herl dimer expression (HIl dimers per cell), phosphorylated Herl, internal control expression, and % Tumor cells observed in the samples by immunohistochemical analysis.

[0020] Figures 9A-9G present graphs showing amounts of Herl total, phosphorylated Herl, Her2 total, phosphorylated Her2, Her3 total, Herl -HErI dimer, Herl-Her2 dimer, Her2-Her3 dimer, and Herl-Her3 dimer expression observed on representative patient samples.

[0021] Figures 10A-10B present graphs in arithmetic and logarithmic scale, respectively, plotting responsiveness versus amount of Her receptor and dimer expression.

[0022] Figure 11 shows a computer system in accordance with the present invention. [0023] Figure 12 presents three Classification Trees showing segregation of subjects responsive to treatment with a Her 1 -acting agent from subjects unresponsive to treatment with the Herl -acting agent by levels of Herl-Herl dimer expression, total Her2 expression, total Herl expression, Herl-Her2 dimer expression, and/or Herl-Her3 dimer expression.

[0024] Figure 13 presents diagrammatic representations showing univariate analyses (Wilcoxson Rank Sum Test) of dimer levels observed in responders and non-responders performed for each of HER 1:1 (HIlD), 1:2(H12D), 1:3(H13D), and 2:3(H23D) dimers. Squares = progressive disease (PD); circles = CR/PR/SD (see text).

[0025] Figure 14 presents a diagrammatic representation of HIlD levels in clinical responders and non-responders demonstrating that all but 5 non-responders had Hl ID levels below approximately 1600, while roughly 2/3 of the responders had HIlD levels > 1600. Squares = PD; diamonds = CR/PR/SD (see text).

[0026] Figure 15 presents a diagrammatic representation of Hl ID levels in clinical responders and non-responders using recursive partitioning.

[0027] Figure 16 presents a representative plot on logarithmic scale of Herl-Herl dimers versus Her2-Her3 dimers for responders versus non-responders.

5. Detailed Description of the Invention 5.1 Definitions

[0028] The term "about," as used herein, unless otherwise indicated, refers to a value that is no more than 10% above or below the value being modified by the term. For example, the term "about 5 μ.g/kg" means a range of from 4.5 μg/kg to 5.5 μg/kg. As another example, "about 1 hour" means a range of from 48 minutes to 72 minutes.

[0029] "Antibody" means an immunoglobulin that specifically binds to, and is thereby defined as complementary with, a particular spatial and polar organization of another molecule. The antibody can be monoclonal, polyclonal, or recombinant and can be prepared by techniques that are well known in the art such as immunization of a host and collection of sera (polyclonal) or by preparing continuous hybrid cell lines and collecting the secreted protein (monoclonal), or by cloning and expressing nucleotide sequences or mutagenized versions thereof coding at least for the amino acid sequences required for specific binding of natural antibodies. Antibodies may include a complete immunoglobulin or fragment thereof, which immunoglobulins include the various classes and isotypes, such as IgA, IgD, IgE, IgGl, IgG2a, IgG2b and IgG3, IgM, etc. Fragments thereof may include Fab, Fv and F(ab')2, Fab', and the like. Antibodies may also be single-chain antibodies, or an antigen-binding fragment thereof, chimeric antibodies, humanized antibodies, or any other antibody derivative known to one of skill in the art that retains binding activity that is specific for a particular binding site. In addition, aggregates, polymers, and conjugates of immunoglobulins or their fragments can be used where appropriate so long as binding affinity for a particular binding site is maintained. Guidance in the production and selection of antibodies and antibody derivatives for use in immunoassays, including such assays employing releasable molecular tag (as described below) can be found in readily available texts and manuals, e.g., Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York; Howard and Bethell, 2001, Basic Methods in Antibody Production and Characterization, CRC Press; Wild, ed., 1994, The Immunoassay Handbook, Stockton Press, New York.

[0030] "Antibody binding composition" means a molecule or a complex of molecules that comprises one or more antibodies, or antigen-binding fragment, and derives its binding specificity from such antibody or antibody fragment. Antibody binding compositions include, but are not limited to, (i) antibody pairs in which a first antibody binds specifically to a target molecule and a second antibody binds specifically to a constant region of the first antibody; a biotinylated antibody that binds specifically to a target molecule and a streptavidin protein, which protein is derivatized with moieties such as molecular tags or photosensitizers, or the like, via a biotin moiety; (ii) antibodies specific for a target molecule and conjugated to a polymer, such as dextran, which, in turn, is derivatized with moieties such as molecular tags or photosensitizers, either directly by covalent bonds or indirectly via streptavidin-biotin linkages; (iii) antibodies specific for a target molecule and conjugated to a bead, or microbead, or other solid phase support, which, in turn, is derivatized either directly or indirectly with moieties such as molecular tags or photosensitizers, or polymers containing the latter.

[0031] As used herein, the term "fragment" in the phrase "antigen-binding antibody fragment" refers to a peptide or polypeptide comprising an amino acid sequence of at least about 5 contiguous amino acid residues, at least about 10 contiguous amino acid residues, at least about 15 contiguous amino acid residues, at least about 20 contiguous amino acid residues, at least about 25 contiguous amino acid residues, at least about 40 contiguous amino acid residues, at least about 50 contiguous amino acid residues, at least about 60 contiguous amino residues, at least about 70 contiguous amino acid residues, at least about 80 contiguous amino acid residues, at least about 90 contiguous amino acid residues, at least about 100 contiguous amino acid residues, at least about 110 contiguous amino acid residues, or at least about 120 contiguous amino acid residues, of the amino acid sequence of another polypeptide, e.g., an antibody that preferentially binds an ErbB receptor.

[0032] "Antigenic determinant," or "epitope" means a site on the surface of a molecule, usually a protein, to which a single antibody molecule binds. Generally, a protein has several or many different antigenic determinants and reacts with antibodies of many different specificities. A preferred antigenic determinant is a phosphorylation site of a protein.

[0033] "Binding moiety" means any molecule to which molecular tags can be directly or indirectly attached that is capable of specifically binding to an analyte. Binding moieties include, but are not limited to, antibodies, antibody binding compositions, peptides, proteins, nucleic acids, and organic molecules having a molecular weight of up to about 1000 daltons and containing atoms selected from the group consisting of hydrogen, carbon, oxygen, nitrogen, sulfur, and phosphorus. Preferably, binding moieties are antibodies or antibody binding compositions.

[0034] "Cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, lung cancer, e.g., small-cell lung cancer or non-small cell lung cancer; gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer. [0035] "Complex" as used herein means an assemblage or aggregate of molecules in direct or indirect contact with one another. In one aspect, "contact," or more particularly, "direct contact" in reference to a complex of molecules, or in reference to specificity or specific binding, means two or more molecules are close enough so that attractive noncovalent interactions, such as Van der Waal forces, hydrogen bonding, ionic and hydrophobic interactions, and the like, dominate the interaction of the molecules, In such an aspect, a complex of molecules is stable in that under assay conditions the complex is thermodynamically more favorable than a non-aggregated, or non- complexed, state of its component molecules. As used herein, "complex" usually refers to a stable aggregate of two or more proteins, and is equivalently referred to as a "protein-protein complex." Most typically, a "complex" refers to a stable aggregate of two proteins.

[0036] "Dimer" in reference to cell surface membrane receptors means a complex of two or more membrane-bound receptor proteins that may be the same or different. Dimers of identical receptors are referred to as "homodimers" and dimers of different receptors are referred to as "heterodimers." Dimers usually consist of two receptors in contact with one another. Dimers may be created in a cell surface membrane by passive processes, such as Van der Waal interactions, and the like, as described above in the definition of "complex," or dimers may be created by active processes, such as by ligand-induced dimerization, covalent linkages, interaction with intracellular components, or the like. See, e.g., Schlessinger, 2000, Cell 103:211-225. As used herein, the term "dimer" is understood to refer to "cell surface membrane receptor dimer," unless understood otherwise from the context.

[0037] "Disease status" includes, but is not limited to, the following features: likelihood of contracting a disease, presence or absence of a disease, prognosis of disease severity, and likelihood that a patient, or cancer or cancer cell obtained from a patient or subject, will respond to treatment by a particular therapeutic agent that acts through a receptor complex, In regard to cancer, "disease status" further includes detection of precancerous or cancerous cells or tissues, the selection of patients that are likely to respond to treatment by a therapeutic agent that acts through one or more receptor complexes, such as one or more receptor dimers, and the ameliorative effects of treatment with such therapeutic agents, In one aspect, disease status in reference to Her receptor complexes means likelihood that a cancer patient will respond to treatment with a Her, or ErbB, dimer-acting drag. In another aspect, disease status in reference to Her receptor complexes refers to the likelihood that a cancer patient will respond to treatment with a Her 1 -acting drag. Preferably, such cancer patient is a lung cancer patient and such Herl- acting drags include Gefitinib (IRESSA^®).

[0038] "Diagnostic Index" as used herein refers to a number that reflects the likelihood that a cancer or cancer cell will respond to treatment with a Her 1 -acting agent, In general, a threshold Diagnostic Index is determined for each appropriate formula of the invention that distinguishes cancers or cancer cells that are likely to respond to treatment with a Her 1 -acting agent from cancers or cancer cells that are not likely to respond to treatment. Preferably, the Diagnostic Index is determined for each appropriate formula of the invention such that cancers or cancer cells likely to respond to treatment with a Her 1 -acting agent are significantly more likely to respond to treatment with a Herl- acting agent than cancers or cancer cells not likely to respond to treatment with a Herl- acting agent. In certain, non-limiting, preferred embodiments, the Diagnostic Index is a number between 0 and 1. In certain embodiments, cancers or cancer cells that have a Diagnostic Index above the threshold Diagnostic Index are likely to respond to treatment with a Her 1 -acting agent, while cancers or cancer cells that have a Diagnostic Index below the threshold Diagnostic Index are likely to respond to treatment with a Herl- acting agent. Preferably, in such embodiments, cancers or cancer cells that have a Diagnostic Index above the threshold Diagnostic Index are significantly more likely to respond to treatment with a Her 1 -acting agent than cancers or cancer cells that have a Diagnostic Index below the threshold Diagnostic Index.

[0039] "ErbB receptor" or "Her receptor" is a receptor protein tyrosine kinase which belongs to the ErbB receptor family and includes EGFR ("Herl"), ErbB2 ("Her2"), ErbB3 ("Her3") andErbB4 ("Her4") receptors. The ErbB receptor generally comprises an extracellular domain, which may bind an ErbB ligand; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; and a carboxyl-terminal signaling domain harboring several tyrosine residues which can be phosphorylated. The ErbB receptor may comprise a native ErbB receptor sequence or an amino acid sequence variant thereof. Preferably the ErbB receptor comprises a native human ErbB receptor sequence, In one aspect, ErbB receptor includes truncated versions of Her receptors, including but not limited to, EGFRvIII and p95Her2 as discussed in Chu et al, 1997, Biochem. J. 324:855-861 and Xia et al, 2004, Oncogene 23:646-653.

[0040] The terms "ErbBl," "epidermal growth factor receptor," "EGFR," and "Herl" are used interchangeably herein and refer to native EGFR, and allelic variants thereof, as disclosed, for example, in Carpenter et al, 1987, Ann. Rev. Biochem. 56:881-914, including such variants as, for example, a deletion mutant EGFR as in Humphrey et al, 1987, P.N.A.S. USA 87:4207-4211. Unless indicated otherwise, the terms " ErbBl" " EGFR " and "Herl" when used herein refer to the human protein. The gene encoding Herl is referred to herein as "erbBl." Examples of antibodies which bind to Herl include MAb 579 (ATCC CRL RB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) and variants thereof, such as chimerized 225 (C225) and reshaped human 225 (H225) as disclosed in, for example, U.S. Patent No. 4,943,533, and International Patent Publication WO 96/40210.

[0041] "Her2", "ErbB2" "c-Erb-B2" are used interchangeably herein and refer to native Her2, and allelic variants thereof, as described, for example, in Semba et al, 1985, P.N.A.S. USA 82:6497-650 and Yamamoto et al, 1986, Nature 319:230-234 and Genebank accession number X03363. Unless indicated otherwise, the terms "ErbB2" "c-Erb-B2" and "Her2" when used herein refer to the human protein. The gene encoding Her2 is referred to herein as "erbB2." Examples of antibodies that specifically bind to Her2 are disclosed in, for example, U.S. patents 5,677,171; 5,772,997; and Fendly et al, 1990, Cancer Res. 50: 1550-1558.

[0042] "ErbB3" and "Her3" are used interchangeably herein and refer to native Her3, and allelic variants thereof, as described, for example, in U.S. Pat. Nos. 5,183,884 and 5,480,968 as well as Kraus et al, 1989, P.N.A.S. (USA) 86:9193-9197. Unless indicated otherwise, the terms "ErbB3" and "Her3" when used herein refer to the human protein. The gene encoding Her3 is referred to herein as "erbB3." Examples of antibodies which bind Her3 include, for example, the 8B8 antibody (ATCC HB 12070) as described in, for example, U.S. Pat. No. 5,968,511.

[0043] The terms "ErbB4" and "Her4" are used interchangeably herein and refer to native Her4, and allelic variants thereof, as described, for example, in E.P. Pat. App. No. 599,274; Plowman et al, 1993, P.N.A.S. (USA) 90:1746-1750; and Plowman et al, 1993, Nature 366:473-475, including such variants as, e.g., the Her4 isoforms disclosed in International Patent Publication No. WO 99/19488. Unless indicated otherwise, the terms "ErbB4" and "Her4" when used herein refer to the human protein. The gene encoding Her4 is referred to herein as "erbB4."

[0044] A "Herl -acting agent," as used herein, refers to a compound that can inhibit a biological activity of Herl . Such biological activities include, but are not limited to, dimerization, autophosphorylation, phosphorylation of another receptor, signal transduction, and the like. Exemplary Herl -acting agents include, but are not limited to, Gefitinib, tarceva, and erbitux.

[0045] "Insulin-like growth factor-1 receptor" or "IGF-IR," as used herein, refers to a human receptor tyrosine kinase, or homolog or variant thereof, such as those disclosed in Ullrich et al., 1986, EMBO J, 5:2503-2512 and Steele-Perkins et al, 1988, J. Biol. Chem. 263:11486-11492.

[0046] "Isolated" in reference to a polypeptide or protein means substantially separated from the components of its natural environment. Preferably, an isolated polypeptide or protein is a composition that consists of at least eighty percent of the polypeptide or protein identified by sequence on a weight basis as compared to components of its natural environment; more preferably, such composition consists of at least ninety-five percent of the polypeptide or protein identified by sequence on a weight basis as compared to components of its natural environment; and still more preferably, such composition consists of at least ninety-nine percent of the polypeptide or protein identified by sequence on a weight basis as compared to components of its natural environment. Most preferably, an isolated polypeptide or protein is a homogeneous composition that can be resolved as a single spot after conventional separation by two- dimensional gel electrophoresis based on molecular weight and isoelectric point. Protocols for such analysis by conventional two-dimensional gel electrophoresis are well known to one of ordinary skill in the art, such as, e.g., the procedures described by Hames and Rickwood, eds., 1981, Gel Electrophoresis of Proteins: A Practical Approach, IRL Press, Oxford; Scopes, 1982, Protein Purification, Springer- Verlag, New York; and Rabilloud, ed., 2000, Proteome Research: Two-Dimensional Gel Electrophoresis and Identification Methods, Springer- Verlag, Berlin. [0047] A "kit" refers to any delivery system for delivering materials or reagents for carrying out a method of the invention. In the context of reaction assays, such delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g., probes, enzymes, etc. in the appropriate containers) and/or supporting materials (e.g., buffers, written instructions for performing the assay etc.) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and/or supporting materials. Such contents may be delivered to the intended recipient together or separately. For example, a first container may contain an enzyme for use in an assay, while a second container may contain probes.

[0048] "Likely to," as used herein, refers to an increased probability that an item, object, or thing exhibiting certain assayed characteristics will also exhibit non-assayed or assayable characteristics relative to a reference that does not exhibit the assayed characteristics. Thus, in one example, a cancer cell that is likely to respond to treatment with Gefitinib as determined according to a method of the invention has an increased probability of responding to treatment with Gefitinib relative to a reference cancer cell, for example, a cancer cell with a probability of responding to treatment that is the average cancer cell's probability of responding to treatment with Gefitinib. In certain embodiments, the average cancer cell's probability of responding to treatment with Gefitinib is the average response observed for a statistically significant number of cancer cells treated with Gefitinib.

[0049] As used herein, the terms "manage," "managing" and "management" refer to the beneficial effects that a subject derives when the methods provided herein are practiced on the subject, which does not result in a cure of the disease, In certain embodiments, a subject is administered a therapy as described herein to "manage" a disorder so as to prevent or slow the progression or worsening of the disorder, In certain embodiments, a subject is administered a therapy as described herein to "manage" a disorder so as to lengthen of the life of the subject over his or her theoretical life expectancy without being administered therapy for the disorder.

[0050] As used herein, the term "monoclonal antibody" refers to an antibody that is derived from a single cellular clone, including any eukaryotic, prokaryotic, or phage clone, and is not dependent upon the method by which it is produced. Therefore, a "monoclonal antibody" can refer to a composition comprising a population of antibodies that each bind to a single epitope wherein said composition lacks antibodies that bind a different epitope than the single epitope to which the population of antibodies bind. It is noted, of course, that in certain instances, a single epitope is present in a polypeptide at multiple positions, In such instances, although the monoclonal antibody may bind to multiple positions, it is, nonetheless, still considered to be binding to a single epitope.

[0051] "Percent identical," "percent identity," or similar terms, used in respect of the comparison of a reference sequence and another sequence (i.e. a "candidate" sequence) means that in an optimal alignment between the two sequences, the candidate sequence is identical to the reference sequence in a number of subunit positions equivalent to the indicated percentage, the subunits being nucleotides for polynucleotide comparisons or amino acids for polypeptide comparisons. As used herein, an "optimal alignment" of sequences being compared is one that maximizes matches between subunits and minimizes the number of gaps employed in constructing an alignment. Percent identities may be determined with commercially available implementations of algorithms described by Needleman and Wunsch, 1970, J. MoI. Biol. 48:443-453 ("GAP" program of Wisconsin Sequence Analysis Package, Genetics Computer Group, Madison, WI). Other software packages in the art for constructing alignments and calculating percentage identity or other measures of similarity include the "BestFit" program, based on the algorithm of Smith and Waterman, 1981, Advances in Applied Mathematics 2:482-489 (Wisconsin Sequence Analysis Package, Genetics Computer Group, Madison, WI). In other words, for example, to obtain a polypeptide having an amino acid sequence at least 95 percent identical to a reference amino acid sequence, up to five percent of the amino acid residues in the reference sequence many be deleted or substituted with another amino acid, or a number of amino acids up to five percent of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence many occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence of in one or more contiguous groups with in the references sequence. It is understood that in making comparisons with reference sequences of the invention that candidate sequence may be a component or segment of a larger polypeptide or polynucleotide and that such comparisons for the purpose computing percentage identity is to be carried out with respect to the relevant component or segment.

[0052] As used herein, the terms "prevent", "preventing" and "prevention" refer to the impedition of the recurrence or onset of a disorder or one or more symptoms of a disorder in a subject.

[0053] "Phosphatidylinositol-3 kinase protein," or equivalently a "PDK protein," refers to a human intracellular protein of the set of human proteins described under NCBI accession numbers NP_852664, NP_852556, and NP_852665, and homologs or variants thereof, and proteins having amino acid sequences substantially identical thereto.

[0054] "Platelet-derived growth factor receptor" or "PDGFR" means a human receptor tyrosine kinase protein that is substantially identical to PDGFRα or PDGFRβ, or homologs or variants thereof, as described in Heldin et ah, 1999, Physiological Reviews 79:1283-1316. In one aspect, the invention includes determining the status of cancers, pre-cancerous conditions, fibrotic or sclerotic conditions by measuring one or more dimers of the following group: PDGFRα homodimers, PDGFRβ homodimers, and PDGFRα-PDGFRβ heterodimers. In particular, fibrotic conditions include lung or kidney fibrosis, and sclerotic conditions include atherosclerosis. Cancers include, but are not limited to, breast cancer, colorectal carcinoma, glioblastoma, and ovarian carcinoma. Reference to "PDGFR" alone is understood to mean "PDGFRα" or "PDGFRβ."

[0055] A "polypeptide" refers to a class of compounds composed of amino acid residues chemically bonded together by amide linkages with elimination of water between the carboxy group of one amino acid and the amino group of another amino acid. A polypeptide is a polymer of amino acid residues, which may contain a large number of such residues. Peptides are similar to polypeptides, except that, generally, they are comprised of a lesser number of amino acids. Peptides are sometimes referred to as oligopeptides. There is no clear-cut distinction between polypeptides and peptides. For convenience, in this disclosure and claims, the term "polypeptide" will be used to refer generally to peptides and polypeptides. The amino acid residues may be natural, i.e., one of the twenty amino acids ordinarily found in human proteins, or non-natural. Further, a polypeptide may be expressed by an organism or synthesized synthetically.

[0056] "Protein" refers to a polypeptide, usually synthesized by a biological cell, folded into a defined three-dimensional structure. Proteins are generally from about 5,000 to about 5,000,000 or more in molecular weight, more usually from about 5,000 to about 1,000,000 molecular weight, and may include posttranslational modifications, such acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, farnesylation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, phosphorylation, prenylation, racemization, selenoylation, sulfation, and ubiquitination. See, e.g., Wold, 1983, "Post-translational Protein Modifications: Perspectives and Prospects," Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, pp.1-12. Proteins include, by way of illustration and not limitation, cytokines or interleukins, enzymes such as, e.g., kinases, proteases, galactosidases and so forth, protamines, histones, albumins, immunoglobulins, scleroproteins, phosphoproteins, mucoproteins, chromoproteins, lipoproteins, nucleoproteins, glycoproteins, T-cell receptors, other receptors, proteoglycans, and the like.

[0057] "Reference sample" means one or more cell, xenograft, or tissue samples that are representative of a normal or non-diseased state to which measurements on patient samples are compared to determine whether a receptor complex is present in excess or in reduced amount in the patient sample. The nature of the reference sample is a matter of design choice for a particular assay and may be derived or determined from normal tissue of the patient him- or herself, or from tissues from a healthy individual or a population of healthy individuals. Preferably, values relating to amounts of receptor complexes in reference samples are obtained under essentially identical experimental conditions as corresponding values for patient samples being tested. Reference samples may be from the same kind of tissue as that the patient sample, or it may be from different tissue types, and the population from which reference samples are obtained may be selected for characteristics that match those of the patient, such as age, sex, race, and the like. Typically, in assays of the invention, amounts of receptor complexes on patient samples are compared to corresponding values of reference samples that have been previously tabulated and are provided as average ranges, average values with standard deviations, or like representations.

[0058] "Receptor complex" means a complex that comprises at least one cell surface membrane receptor. Receptor complexes may include a dimer of cell surface membrane receptors, or one or more intracellular proteins, such as adaptor proteins, that form links in the various signaling pathways. Exemplary intracellular proteins that may be part of a receptor complex includes, but is not limit to, PDK proteins, Grb2 proteins, Grb7 proteins, She proteins, and Sos proteins, Src proteins, CbI proteins, PLCγ proteins, Shp2 proteins, GAP proteins, Nek proteins, Vav proteins, and Crk proteins. In one aspect, receptor complexes include PDK or She proteins.

[0059] "Receptor tyrosine kinase," or "RTK," refers to a human receptor protein having intracellular kinase activity and being selected from the RTK family of proteins, such as those described in Schlessinger, 2000, Cell 103: 211-225 and Blume- Jensen and Hunter, supra. "Receptor tyrosine kinase dimer" refers to a complex in a cell surface membrane comprising two receptor tyrosine kinase proteins. In some aspects, a receptor tyrosine kinase dimer may comprise two covalently linked receptor tyrosine kinase proteins. Exemplary RTK dimers are listed in Table 1. RTK dimers of particular interest are Her receptor dimers and VEGFR dimers.

[0060] "Responsiveness," to "respond" to treatment, and other forms of this verb, as used herein, refer to the reaction of a cancer cell to treatment with a Her 1 -acting agent. In one example, a cancer cell responds to treatment with a Her 1 -acting agent if growth of a tumor comprising the cancer cell is retarded about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more, In another example, a cancer cell responds to treatment with a Herl-acting agent if a tumor comprising the cancer cell shrinks by about 5%, 10%, 20%, 30%, 40%, 50% or more as determined by any appropriate measure, e.g., by mass or volume, In still another example, a cancer cell responds to treatment with a Herl-acting agent if a patient with a tumor comprising the cancer cell experiences a life expectancy extended by about 5%, 10%, 20%, 30%, 40%, 50% or more beyond the life expectancy predicted if no treatment is administered.

[0061] "Sample" or "tissue sample" or "patient sample" or "patient cell or tissue sample" or "specimen" each refers to a collection of similar cells obtained from a tissue of a subject or patient. The source of the tissue sample may be solid tissue as from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate; blood or any blood constituents; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; or cells from any time in gestation or development of the subject. The tissue sample may contain compounds which are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like. In one aspect of the invention, tissue samples or patient samples are fixed, particularly conventional formalin-fixed paraffin-embedded samples. Such samples are typically used in an assay for receptor complexes in the form of thin sections, e.g. 3-10 μm thick, of fixed tissue mounted on a microscope slide, or equivalent surface. Such samples also typically undergo a conventional re-hydration procedure, and optionally, an antigen retrieval procedure as a part of, or preliminary to, assay measurements.

[0062] "Separation profile" in reference to the separation of molecular tags means a chart, graph, curve, bar graph, or other representation of signal intensity data versus a parameter related to the molecular tags, such as retention time, mass, or the like, that provides a readout, or measure, of the number of molecular tags of each type produced in an assay. A separation profile may be an electropherogram, a chromatogram, an electrochromatogram, a mass spectrogram, or like graphical representation of data depending on the separation technique employed. A "peak" or a "band" or a "zone" in reference to a separation profile means a region where a separated compound is concentrated. There may be multiple separation profiles for a single assay if, for example, different molecular tags have different fluorescent labels having distinct emission spectra and data is collected and recorded at multiple wavelengths. In one aspect, released molecular tags are separated by differences in electrophoretic mobility to form an electropherogram wherein different molecular tags correspond to distinct peaks on the electropherogram. A measure of the distinctness, or lack of overlap, of adjacent peaks in an electropherogram is "electrophoretic resolution," which may be taken as the distance between adjacent peak niaximums divided by four times the larger of the two standard deviations of the peaks. Preferably, adjacent peaks have a resolution of at least 1.0, and more preferably, at least 1.5, and most preferably, at least 2.0. In a given separation and detection system, the desired resolution may be obtained by selecting a plurality of molecular tags whose members have electrophoretic mobilities that differ by at least a peak-resolving amount, such quantity depending on several factors well known to those of ordinary skill, including signal detection system, nature of the fluorescent moieties, the diffusion coefficients of the tags, the presence or absence of sieving matrices, nature of the electrophoretic apparatus, e.g. presence or absence of channels, length of separation channels, and the like. Electropherograms may be analyzed to associate features in the data with the presence, absence, or quantities of molecular tags using analysis programs, such as disclosed in U.S. Patent Application Publication 2003/0170734 Al.

[0063] "SHC" (standing for "Src homology 2/α-collagen-related") means any one of a family of adaptor proteins (66, 52, and 46 kDalton) in RTK signaling pathways substantially identical to those described in Pelicci et al., 1992, Cell 70: 93-104, or variants or homologs thereof. In one aspect, SHC means the human versions of such adaptor proteins.

[0064] "Signaling pathway" or "signal transduction pathway" refers to a series of molecular events usually beginning with the interaction of a cell surface receptor with an extracellular ligand or with the binding of an intracellular molecule to a phosphorylated site of a cell surface receptor that triggers a series of molecular interactions, wherein the series of molecular interactions results in a regulation of gene expression in the nucleus of a cell. "Ras-MAPK pathway" means a signaling pathway that includes the phosphorylation of a MAPK protein subsequent to the formation of a Ras-GTP complex. "PI3K-Akt pathway" means a signaling pathway that includes the phosphorylation of an Akt protein by a PI3K protein.

[0065] "Specific" or "specificity" in reference to the binding of one molecule to another molecule, such as a binding compound, or probe, for a target analyte or complex, means the recognition, contact, and formation of a stable complex between the probe and target, together with substantially less recognition, contact, or complex formation of the probe with other molecules. In one aspect, "specific" in reference to the binding of a first molecule to a second molecule means that to the extent the first molecule recognizes and forms a complex with another molecules in a reaction or sample, it forms the largest number of the complexes with the second molecule. In certain embodiments, this largest number is at least fifty percent of all such complexes form by the first molecule. Generally, molecules involved in a specific binding event have areas on their surfaces or in cavities giving rise to specific recognition between the molecules binding to each other. Examples of specific binding include antibody-antigen interactions, enzyme- substrate interactions, formation of duplexes or triplexes among polynucleotides and/or oligonucleotides, receptor-ligand interactions, and the like.

[0066] "Spectrally resolvable" in reference to a plurality of fluorescent labels means that the fluorescent emission bands of the labels are sufficiently distinct, i.e. sufficiently non- overlapping, that molecular tags to which the respective labels are attached can be distinguished on the basis of the fluorescent signal generated by the respective labels by standard photodetection systems, e.g. employing a system of band pass filters and photomultiplier tubes, or the like, as exemplified by the systems described in U.S. Pat. Nos. 4,230,558; 4,811,218, or the like, or in Wheeless et al, 1985, Flow Cytometry: Instrumentation and Data Analysis, Academic Press, New York, pp. 21-76.

[0067] "Substantially identical" in reference to proteins or amino acid sequences of proteins in a family of related proteins that are being compared means either that one protein has an amino acid sequence that is at least fifty percent identical to the other protein or that one protein is an isoform or splice variant of the same gene as the other protein. In certain embodiments, substantially identical means one protein, or amino acid sequence thereof, is at least eighty percent identical to the other protein, or amino acid sequence thereof.

[0068] As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, the terms "subject" and "subjects" refer to an animal, preferably a mammal including a non-primate (e.g., a cow, pig, horse, donkey, goat, camel, cat, dog, guinea pig, rat, mouse, sheep) and a primate (e.g. , a monkey, such as a cynomolgous monkey, gorilla, chimpanzee, and a human), preferably a human. In one embodiment, the subject is a subject with cancer, for example, ovarian cancer. [0069] "Treat," "treatment," and other forms of this word, as used herein, refers to the administration of a Her 1 -acting agent to impede growth of a cancer, to cause a cancer to shrink by weight or volume, to extend the expected survival time of the subject, and the like.

5.2 Methods for Determining Responsiveness to Therapy [0070] In certain aspects, the invention provides methods for determining whether a cancer is likely to respond to treatment with a Her 1 -acting agent. In certain embodiments, the methods comprise determining a probability that the cancer will respond to treatment with the Her 1 -acting agent based on application of a formula of the invention to one or more biomarkers associated with responsiveness and/or non- responsiveness to treatment with the Her 1 -acting agent as described herein. In certain embodiments, the biomarkers associated with responsiveness to treatment with the Herl- acting agent comprise the presence and/or amount of expression of one or more of Her 1- Herl dimers, Herl-Her2 dimers, Herl-Her3 dimers, and/or Her2-Her3 dimers and/or presence and/or amount of Her 1 phosphorylation and/or Her2 phosphorylation, or any combination thereof. In certain embodiments, the presence and/or amount of the biomarker(s) positively correlate with responsiveness to treatment of a cancer or cancer cell with a Herl -acting agent. In certain embodiments, the likelihood that the cancer will respond to treatment with the Herl -acting agent is presented as a Diagnostic Index.

[0071] In a number of embodiments presented below, the methods comprise detecting the number of one or more types of Her- 1 containing dimers (Herl -Herl, Herl-Her2, Herl-Her3) per cell. Generally, the embodiments presented below comprise detecting whether the number or numbers of Herl -containing dimers per cell is greater than a particular value, whereby, if greater, this indicates that the cell or cancer is likely to respond to a Herl -acting agent. It is to be understood that the invention also encompasses methods whereby if the number or numbers of such Her 1 -containing dimers per cell is determined to be less than the particular value presented in such embodiments, then this indicates that the cell or cancer is not likely to respond to the Herl -acting agent. For example, in an embodiment presented below that comprises detecting the number of Herl-Herl dimers per cell whereby if the number is greater than about 1000 this indicates that the cell is likely to respond to a Herl -acting agent, it should be understood that the present invention also encompasses a method that comprises detecting the number of Herl-Herl dimers per cell such that if the number is less than about 1000 this indicates that the cell is not likely to respond to a Her 1 -acting agent.

[0072] Thus, in certain embodiments, the invention provides a method for determining whether a subject with cancer is likely to respond to treatment with a Her 1 -acting agent that comprises determining a Diagnostic Index for the cancer based upon a formula of the invention, wherein application of the formula to one or more biomarkers on the cells of the cancer indicates that the cancer is likely to respond to treatment with a Her 1 -acting agent.

[0073] In certain embodiments, formulae of the invention for computing a Diagnostic Index for a cancer or cancer cell are according to General Formula A:

Log(p/(l+p)) = a₀ + ai*log(bi*Hl ID + b₂*H13D) + a₂*log(H12D) + a₃*log(H23D) + a₄*log(H2P)

General Formula A

where Hl ID is the number of Herl-Herl dimers detected per cancer cell, Hl 3D is the number of Herl-Her3 dimers detected per cancer cell, H12D is the number of Herl-Her2 dimers detected per cancer cell, H23D is the number of Her2-Her3 dimers detected per cancer cell, H2P is the number of phosphorylated Her2 receptors detected per cel\,p is the Diagnostic Index used to predict the cancer's probability of responding to treatment with the drug, and a₀, a_l5 a₂, a₃, a₄, b_1; and b₂ are each coefficients selected to conform the formula to responsiveness of cancer or cancer cells to treatment with Her 1 -acting agents observed in clinical studies. In certain embodiments, the Diagnostic Index/), determined according to General Formula A, can be a number between 0 and 1, where a larger value for p indicates a greater probability that the cancer will respond to treatment with a Her 1 -acting agent, In certain embodiments, the Diagnostic Index p multiplied by 100% indicates the probability by percentage that the cancer will respond to treatment with a Herl -acting agent.

[0074] In certain embodiments, the Diagnostic Index p can be compared with a threshold Diagnostic Index, In certain embodiments, where the Diagnostic Index p is 1reater than the threshold Diagnostic Index, the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent. In certain embodiments, where the Diagnostic Index p is less than the threshold Diagnostic Index, the cancer or cancer cell is not likely to respond to treatment with a Herl -acting agent.

[0075] The coefficients of the formulae of the invention can be derived using any statistical analysis method known by one of skill in the art without limitation. In certain embodiments, the coefficients are derived using linear regression analysis. In certain embodiments, the coefficients are derived using logistic regression analysis. Preferably, the coefficients are selected to minimize the whole model fitting p-value as determined by likelihood ratio. Thus, in certain embodiments, the whole model fitting p-value as determined by likelihood ratio is less than about 0.1, preferably less than about 0.05, more preferably less than about 0.01, more preferably less than about 0.005, and still more preferably less than about 0.001. In certain embodiments, the p-value for one or more coefficients as determined by row exclusion is less than about 0.1, preferably less than about 0.05, more preferably less than about 0.01, more preferably less than about 0.005, and still more preferably less than about 0.001. Preferably, the p-value for each coefficient as determined by row exclusion is less than about 0.1, more preferably less than about 0.05, more preferably less than about 0.01, more preferably less than about 0.005, and still more preferably less than about 0.001. In some embodiments, the p- value for each coefficient as determined can show that the individual coefficient is not significant, so long as the combination of coefficients selected for the formula renders the formula as a whole significant as assessed according to any technique known by one of skill in the art without limitation.

[0076] As one of skill in the art will recognize, not all formulae of the invention need take into account every variable appearing in General Formula A. Accordingly, in certain embodiments, a₀, a₁, a₂, a₃, a₄, b₁, and b₂ can each independently be 0, though not all of a₀, a₁, a₂, a₃, a₄, b₁, and b₂ can simultaneously be 0. Further, in certain embodiments, a₀, a₁, a₂, a₃, a₄, b₁, and b₂ can each independently be 1.

[0077] In certain embodiments, the values of each of a₀, a₁, a₂, a₃, a₄, bi, or b₂ can be selected independently fro6 the values selected for any other of a_o, a₁, a₂, a₃, a_4, b₁, or b₂. In certain embodiments, the values selected for one of a a₁, a₂, a₃, a₄,

or b₂ can depend on the value selected for any other of a_o, a₁, a₂, a₃, a₄, b₁, or b₂.

[0078] In certain embodiments, a₀ is a positive number. In certain embodiments, a₀ is a negative number, In certain embodiments, a₀ is between about -40 and about 40. In certain embodiments, a₀ is between about -10 and about 10. In certain embodiments, a₀ is between about -8 and about 8. In certain embodiments, a₀ is between about -5 and about 5. In certain embodiments, a₀ is between about -4 and about 4. In certain embodiments, a₀ is between about -3 and about 3. In certain embodiments, a₀ is between about -2 and about 2. In certain embodiments, a₀ is between about -1 and about 1. In certain embodiments, a₀ is between about -10 and about -0.001. In certain embodiments, a₀ is between about -7 and about -0.001. In certain embodiments, a₀ is between about -5 and about -0.001. In certain embodiments, a₀ is between about -4 and about -0.001. In certain embodiments, a₀ is between about -3 and about -0.001. In certain embodiments, a₀ is between about -2 and about -0.001. In certain embodiments, a₀ is between about -1 and about -0.001. In certain embodiments, a₀ is between about -3.5 and about -1. In certain embodiments, a₀ is between about -3.1 and about -1. In certain embodiments, a₀ is between about -3.1 and about -1.5. In certain embodiments, a₀ is between about -3.1 and about -2. In certain embodiments, a₀ is between about -3.1 and about -2.5. In certain embodiments, a₀ is between about -2.1 and about -1. In certain embodiments, a₀ is between about -2.1 and about -1.5. In certain embodiments, a₀ is about -4. In certain embodiments, a₀ is about -3. In certain embodiments, a₀ is about -2. In certain embodiments, a₀ is about -1.5. In certain embodiments, a₀ is about -1. In certain embodiments, a₀ is about -2.09. In certain embodiments, a₀ is about -3.061. In certain embodiments, a₀ is about -3.014. In certain embodiments, a₀ is about -∏ . In certain embodiments, a₀ is about -1.947. In certain embodiments, a₀ is about -1.098. In certain embodiments, a₀ is about -1.632. In certain embodiments, a₀ is about -3.162.

[0079] In certain embodiments, a₁ is a positive number, In certain embodiments, a₁ is a negative number, In certain embodiments, a₁ is between about -10 and about 10. In certain embodiments, a₁ is between about -8 and about 8. In certain embodiments, a₁ is between about -5 and about 5. In certain embodiments, a₁ is between about -4 and about 4. In certain embodiments, ai is between about -3 and about 3. In certain embodiments, a.\ is between about -2 and about 2. In certain embodiments, a₁ is between about -1 and about 1. In certain embodiments, ai is between about 0 and about 15. In certain embodiments, ai is between about 0 and about 10. In certain embodiments, a₁ is between about 0 and about 2. In certain embodiments, a₁ is between about 0.001 and about 10. In certain embodiments, a₁ is between about 0.001 and about 7. In certain embodiments, a₁ is between about 0.001 and about 5. In certain embodiments, a₁ is between about 0.001 and about 4. In certain embodiments, a₁ is between about 0.001 and about 3. In certain embodiments, ai is between about 0.001 and about 2. In certain embodiments, a\ is between about 0.001 and about 1. In certain embodiments, a₁ is between about 0.3 and about 1.5. In certain embodiments, ai is between about 0.5 and about 1.5. In certain embodiments, ai is between about 0.3 and about 1.2. In certain embodiments, a₁ is between about 0.5 and about 1.2. In certain embodiments, a₁ is between about 0.7 and about 1.5. In certain embodiments, a₁ is between about 0.7 and about 1.2. In certain embodiments, a₁ is between about 0.7 and about 0.9. In certain embodiments, a₁ is between about 0.9 and about 1.5. In certain embodiments, a₁ is between about 0.9 and about 1.2. In certain embodiments, a₁ is about 4. In certain embodiments, ai is about 3. In certain embodiments, a₁ is about 2. In certain embodiments, a₁ is about 1. In certain embodiments, ai is about 0.992. In certain embodiments, a₁ is about 1.123. In certain embodiments, ai is about 1.096. In certain embodiments, a₁ is about 0.904. In certain embodiments, ai is about 0.58. In certain embodiments, a₁ is about 0.732. In certain embodiments, ai is about 0.912.

[0080] In certain embodiments, a₂ is a positive number. In certain embodiments, a₂ is a negative number. In certain embodiments, a₂ is between about -15 and about 15. In certain embodiments, a₂ is between about -10 and about 10. In certain embodiments, a₂ is between about -8 and about 8. In certain embodiments, a₂ is between about -5 and about 5. In certain embodiments, a₂ is between about -4 and about 4. In certain embodiments, a₂ is between about -3 and about 3. In certain embodiments, a₂ is between about -3 and about 2. In certain embodiments, a₂ is between about -2 and about 2. In certain embodiments, a₂ is between about -1 and about 1. In certain embodiments, a₂ is between about -10 and about -0.001. In certain embodiments, a₂is between about -7 and about -0.001. In certain embodiments, a₂ is between about -5 and about -0.001. In certain embodiments, a₂ is between about -4 and about -0.001. In certain embodiments, a₂ is between about -3 and about -0.001. In certain embodiments, a₂ is between about -2 and about -0.001. In certain embodiments, a₂ is between about -1 and about -0.001. In certain embodiments, a₂ is between about -0.5 and about -0.001. In certain embodiments, a₂ is between about -0.8 and about -0.2. In certain embodiments, a₂ is between about -0.6 and about -0.2. In certain embodiments, a₂ is between about -0.5 and about -0.2. In certain embodiments, a₂ is between about -0.8 and about -0.4. In certain embodiments, a₂ is between about -0.6 and about -0.4. In certain embodiments, a₂ is between about -0.5 and about -0.4. In certain embodiments, a₂ is about -4. In certain embodiments, a₂ is about -3. In certain embodiments, a₂ is about -2. In certain embodiments, a₂ is about -1.5. In certain embodiments, a₂ is about -1. In certain embodiments, a₂ is about -0.433. In certain embodiments, a₂ is about -0.448. In certain embodiments, a₂ is about -0.476. In certain embodiments, a₂ is 0.

[0081] In certain embodiments, a₃ is a positive number. In certain embodiments, a₃ is a negative number. In certain embodiments, a₃ is between about -30 and about 15. In certain embodiments, a₃ is between about -10 and about 7. In certain embodiments, a₃ is between about -10 and about 10. In certain embodiments, a₃ is between about -8 and about 8. In certain embodiments, a₃ is between about -5 and about 5. In certain embodiments, a₃ is between about -4 and about 4. In certain embodiments, a₃ is between about -3 and about 3. In certain embodiments, a₃ is between about -3 and about 2. In certain embodiments, a₃ is between about -2 and about 2. In certain embodiments, a₃ is between about -1 and about 1. In certain embodiments, a₃ is between about -10 and about -0.001. In certain embodiments, a₃ is between about -7 and about -0.001. In certain embodiments, a₃ is between about -5 and about -0.001. In certain embodiments, a₃ is between about -4 and about -0.001. In certain embodiments, a₃ is between about -3 and about -0.001. In certain embodiments, a₃ is between about -2 and about -0.001. In certain embodiments, a₃ is between about -1 and about -0.001. In certain embodiments, a₃ is between about -0.5 and about -0.001. In certain embodiments, a₃ is between about 0.001 and about 10. In certain embodiments, a₃ is between about 0.001 and about 7. In certain embodiments, a₃ is between about 0.001 and about 5. In certain embodiments, a₃ is between about 0.001 and about 4. In certain embodiments, a₃ is between about 0.001 and about 3. In certain embodiments, a₃ is between about 0.001 and about 2. In certain embodiments, a₃ is between about 0.001 and about 1. In certain embodiments, a₃ is between about 0.001 and about 0.5. In certain embodiments, a₃ is between about -1 and about 1. In certain embodiments, a₃ is between about -0.5 and about 0.5. In certain embodiments, a₃ is between about -0.2 and about 0.5. In certain embodiments, a₃ is between about -0.1 and about 0.5. In certain embodiments, a₃ is between about -0.5 and about 0.2. In certain embodiments, a₃ is between about -0.5 and about 0.1. In certain embodiments, a₃ is between about -0.2 and about 0.2. In certain embodiments, a₃ is between about -0.1 and about 0.1. In certain embodiments, a₃ is about -4. In certain embodiments, a₃ is about -3. In certain embodiments, a₃ is about -2. In certain embodiments, a₃ is about -1. In certain embodiments, a₃ is about -0.5. In certain embodiments, a₃ is about -0.1. In certain embodiments, a₃ is about 4. In certain embodiments, a₃ is about 3. In certain embodiments, a₃ is about 2. In certain embodiments, a₃ is about 1. In certain embodiments, a₃ is about 0.5. In certain embodiments, a₃ is about 0.1. In certain embodiments, a₃ is about -0.187. In certain embodiments, a₃ is about -0.089. In certain embodiments, a₃ is about -0.141. In certain embodiments, a₃ is about 0.041. In certain embodiments, a₃ is about -0.192.

[0082] In certain embodiments, a₄ is a positive number. In certain embodiments, a₄ is a negative number. In certain embodiments, a₄ is between about -10 and about 10. In certain embodiments, 0₄ is between about -8 and about 8. In certain embodiments, a₄ is between about -5 and about 5. In certain embodiments, a₄ is between about -4 and about 4. In certain embodiments, a₄ is between about -3 and about 3. In certain embodiments, a₀ is between about -2 and about 2. In certain embodiments, a₄ is between about -1 and about 1. In certain embodiments, a₄ is between about -10 and about -0.001. In certain embodiments, a₄ is between about -7 and about -0.001. In certain embodiments, a₄ is between about -5 and about -0.001. In certain embodiments, a₄ is between about -4 and about -0.001. In certain embodiments, a₄ is between about -3 and about -0.001. In certain embodiments, a₄ is between about -2 and about -0.001. In certain embodiments, a₄ is between about -1 and about -0.001. In certain embodiments, a₄ is between about -0.8 and about -0.1. In certain embodiments, a₄ is between about -0.6 and about -0.1. In certain embodiments, a₄ is between about -0.5 and about -0.1. In certain embodiments, a₄ is between about -0.8 and about -0.3. In certain embodiments, a₄ is between about -0.6 and about -0.3. In certain embodiments, a₄ is between about -0.5 and about -0.3. In certain embodiments, a₄ is about -4. In certain embodiments, a₄ is about -3. In certain embodiments, a₄ is about -2. In certain embodiments, a₄ is about -1.5. In certain embodiments, a₄ is about -1. In certain embodiments, a₄ is about -0.4. In certain embodiments, a₄ is about -0.393. In certain embodiments, a₄ is about -0.397. In certain embodiments, a₄ is about -0.39.

[0083] In certain embodiments, bi is between about 0 and about 10. In certain embodiments, bi is between about 0 and about 5. In certain embodiments, bi is between about 0 and about 2. In certain embodiments, b₁ is 0. In certain embodiments, bi is 1.

[0084] In certain embodiments, b₂ is between about -10 and about 10. In certain embodiments, b₂ is between about -5 and about 5. In certain embodiments, b₂ is between about -2 and about 2. In certain embodiments, b₂ is 0. In certain embodiments, b₂ is 1.

[0085] In certain embodiments, a₀ is between about -40 and about 40, ai is between about -10 and about 10, a₂ is between about -15 and about 15, a₃ is between about -30 and about 15, a₄ is between about -10 and about 10, b₁ is between about 0 and about 10, and b₂ is between about -10 and about 10. In certain embodiments, a₀ is between about -40 and about 40, a₁ is between about -10 and about 10, a₂ is between about -15 and about 15, a₃ is between about -30 and about 15, a₄ is between about -10 and about 10, b₁ is 1, and b₂ is 1. In certain embodiments, a₀ is between about -40 and about 40, a₁ is between about -10 and about 10, a₂ is between about -15 and about 15, a₃ is between about -30 and about 15, a₄ is between about -10 and about 10, b₁ is 0, and b₂ is 1. In certain embodiments, a₀ is between about -40 and about 40, a₁ is between about -10 and about 10, a₂ is between about -15 and about 15, a₃ is between about -30 and about 15, a₄ is between about -10 and about 10, b₁ is 0, and b₂ 1. In certain embodiments, a₀ is between about -40 and about 40, a₁ is between about -10 and about 10, a₂ is between about -15 and about 15, a₃ is between about -30 and about 15, a₄ is between about -10 and about 10, b₁ is 0, and b₂ is 0.

[0086] In certain embodiments, a₀ is between about -5 and about 5, a₁ is between about -1 and about 5, a₂ is between about -15 and about 15, a₃ is between about -2 and about 2, a₄ is between about -2 and about 2, b₁ is between about 0 and about 1, and b₂ is between about -1 and about 1. In certain embodiments, a₀ is between about -5 and about 5, a₁ is between about -1 and about 5, a₂ is between about -15 and about 15, a₃ is between about - 2 and about 2, a₄ is between about -2 and about 2, b₁ is 1, and b₂ is 1. In certain embodiments, a₀ is between about -5 and about 5, a₁ is between about -1 and about 5, a₂ is between about -15 and about 15, a₃ is between about -2 and about 2, a₄ is between about -2 and about 2, h_\ is 0, and b₂ is 1. In certain embodiments, a₀ is between about -5 and about 5, a₁ is between about -1 and about 5, a₂ is between about -15 and about 15, a₃ is between about -2 and about 2, a₄ is between about -2 and about 2,

is 0, and b₂ 1. In certain embodiments, a₀ is between about -5 and about 5, a₁ is between about -1 and about 5, a₂ is between about -15 and about 15, a₃ is between about -2 and about 2, a₄ is between about -2 and about 2, b₁ is 0, and b₂ is 0.

[0087] In certain embodiments, a₀ is between about -3.5 and about -1.5, ai is between about 0.5 and about 1.5, a₂ is between about -1 and about 2, a₃ is between about -1 and about 0, a₄ is between about -1 and about 0, b₁ is between about 0 and about 1, and b₂ is between about -1 and about 1. In certain embodiments, a₀ is between about -3.5 and about -1.5, a₁ is between about 0.5 and about 1.5, a₂is between about -1 and about 2, a₃ is between about -1 and about 0, a₄ is between about -1 and about 0, b₁ is 1, and b₂ is 1. In certain embodiments, a₀ is between about -3.5 and about -1.5, ai is between about 0.5 and about 1.5, a₂ is between about -1 and about 2, a₃ is between about -1 and about 0, a4 is between about -1 and about 0, bi is 0, and b₂ is 1. In certain embodiments, a₀ is between about -3.5 and about -1.5, ai is between about 0.5 and about 1.5, a₂ is between about -1 and about 2, a₃ is between about -1 and about 0, a₄ is between about -1 and about 0, b] is 0, and b₂ 1. In certain embodiments, a₀ is between about -3.5 and about - 1.5, ai is between about 0.5 and about 1.5, a₂ is between about -1 and about 2, a₃ is between about -1 and about 0, a₄ is between about -1 and about 0, bi is 0, and b₂ is 0.

[0088] In certain embodiments, a₀ is between about -3.5 and about -1.5, ai is between about 0.5 and about 1.5, a₂ is between about -1 and about 2, a₃ is between about -1 and about 0, a₄ is 0, bi is between about 0 and about 1, and b₂ is between about -1 and about 1. In certain embodiments, a₀ is between about -3.5 and about -1.5, ai is between about 0.5 and about 1.5, a₂ is between about -1 and about 2, a₃ is between about -1 and about 0, a₄ is 0, bi is 1, and b₂ is 1. In certain embodiments, a₀ is between about -3.5 and about - 1.5, ai is between about 0.5 and about 1.5, a₂ is between about -1 and about 2, a₃ is between about -1 and about 0, a₄ is 0, bi is 0, and b₂ is 1. In certain embodiments, a₀ is between about -3.5 and about -1.5, ai is between about 0.5 and about 1.5, a₂ is between about -1 and about 2, a₃ is between about -1 and about 0, a₄ is 0, bi is 0, and b₂ 1. In certain embodiments, a₀ is between about -3.5 and about -1.5, ai is between about 0.5 and about 1.5, a₂ is between about -1 and about 2, a₃ is between about -1 and about 0, a₄ is 0, bi is 0, and b₂ is 0.

[0089] In certain embodiments, a₀ is between about -3.5 and about -1.5, a₁ is between about 0.5 and about 1.5, a₂ is between about -1 and about 2, a₃ is 0, a₄ is between about - 1 and about 0, bi is between about 0 and about 1, and b₂ is between about -1 and about 1. In certain embodiments, a₀ is between about -3.5 and about -1.5, ai is between about 0.5 and about 1.5, a₂ is between about -1 and about 2, a₃ is 0, a₄ is between about -1 and about 0, bi is 1, and b₂ is 1. In certain embodiments, a₀ is between about -3.5 and about - 1.5, ai is between about 0.5 and about 1.5, a₂ is between about -1 and about 2, a₃ is 0, a₄ is between about -1 and about 0, bi is 0, and b₂ is 1. In certain embodiments, a₀ is between about -3.5 and about -1.5, a₁ is between about 0.5 and about 1.5, a₂ is between about -1 and about 2, a₃ is 0, a₄ is between about -1 and about 0, b₁ is 0, and b₂ 1. In certain embodiments, a₀ is between about -3.5 and about -1.5, a₁ is between about 0.5 and about 1.5, a₂ is between about -1 and about 2, a₃ is 0, a₄ is between about -1 and about 0, b₁ is 0, and b₂ is 0.

[0090] In certain embodiments, a₀ is between about -3.5 and about -1.5, a₁ is between about 0.5 and about 1.5, a₂ is 0, a₃ is between about -1 and about 0, a₄ is between about - 1 and about 0, bi is between about 0 and about 1, and b₂ is between about -1 and about 1. In certain embodiments, a₀ is between about -3.5 and about -1.5, a₁ is between about 0.5 and about 1.5, a₂ is 0, a₃ is between about -1 and about 0, a₄ is between about -1 and about 0, b₁ is 1, and b₂ is 1. In certain embodiments, a₀ is between about -3.5 and about - 1.5, ai is between about 0.5 and about 1.5, a₂ is 0, a₃ is between about -1 and about 0, a₄ is between about -1 and about 0, b₁ is 0, and b₂ is 1. In certain embodiments, a₀ is between about -3.5 and about -1.5, a₁ is between about 0.5 and about 1.5, a₂ is 0, a₃ is between about -1 and about 0, a₄ is between about -1 and about 0, b₁ is 0, and b₂ 1. In certain embodiments, a₀ is between about -3.5 and about -1.5, a₁ is between about 0.5 and about 1.5, a₂ is 0, a₃ is between about -1 and about 0, a₄ is between about -1 and about 0, b₁ is 0, and b₂ is 0.

[0091] In certain embodiments, the Diagnostic Index p is determined to be about 0.01. In certain embodiments, the Diagnostic Index p is determined to be about 0.02. In certain embodiments, the Diagnostic Index p is determined to be about 0.03. In certain embodiments, the Diagnostic Index /? is determined to be about 0.04. In certain embodiments, the Diagnostic Index p is determined to be about 0.05. In certain embodiments, the Diagnostic Index /? is determined to be about 0.06. In certain embodiments, the Diagnostic Index p is determined to be about 0.07. In certain embodiments, the Diagnostic Index p is determined to be about 0.08. In certain embodiments, the Diagnostic Index p is determined to be about 0.09. In certain embodiments, the Diagnostic Index p is determined to be about 0.10. In certain embodiments, the Diagnostic Index p is determined to be about 0.11. In certain embodiments, the Diagnostic Index p is determined to be about 0.12. In certain embodiments, the Diagnostic Index p is determined to be about 0.13. In certain embodiments, the Diagnostic Index p is determined to be about 0.14. In certain embodiments, the Diagnostic Index p is determined to be about 0.15. In certain embodiments, the Diagnostic Index p is determined to be about 0.16. In certain embodiments, the Diagnostic Index p is determined to be about 0.17. In certain embodiments, the Diagnostic Index p is determined to be about 0.18. In certain embodiments, the Diagnostic Index p is determined to be about 0.19. In certain embodiments, the Diagnostic Index p is determined to be about 0.20. In certain embodiments, the Diagnostic Index p is determined to be about 0.21. In certain embodiments, the Diagnostic Index p is determined to be about 0.22. In certain embodiments, the Diagnostic Index p is determined to be about 0.23. In certain embodiments, the Diagnostic Index p is determined to be about 0.24. In certain embodiments, the Diagnostic Index p is determined to be about 0.25. In certain embodiments, the Diagnostic Index p is determined to be about 0.26. In certain embodiments, the Diagnostic Index p is determined to be about 0.27. In certain embodiments, the Diagnostic Index p is determined to be about 0.28. In certain embodiments, the Diagnostic Index p is determined to be about 0.29. In certain embodiments, the Diagnostic Index p is determined to be about 0.30. In certain embodiments, the Diagnostic Index p is determined to be about 0.31. In certain embodiments, the Diagnostic Index p is determined to be about 0.32. In certain embodiments, the Diagnostic Index p is determined to be about 0.33. In certain embodiments, the Diagnostic Index p is determined to be about 0.34. In certain embodiments, the Diagnostic Index p is determined to be about 0.35. In certain embodiments, the Diagnostic Index p is determined to be about 0.36. In certain embodiments, the Diagnostic Index p is determined to be about 0.37. In certain embodiments, the Diagnostic Index p is determined to be about 0.38. In certain embodiments, the Diagnostic Index p is determined to be about 0.39. In certain embodiments, the Diagnostic Index p is determined to be about 0.40. In certain embodiments, the Diagnostic Index p is determined to be about 0.41. In certain embodiments, the Diagnostic Index p is determined to be about 0.42. In certain embodiments, the Diagnostic Index p is determined to be about 0.43. In certain embodiments, the Diagnostic Index p is determined to be about 0.44. In certain embodiments, the Diagnostic Index p is determined to be about 0.45. In certain embodiments, the Diagnostic Index p is determined to be about 0.46. In certain embodiments, the Diagnostic Index p is determined to be about 0.47. In certain embodiments, the Diagnostic Index p is determined to be about 0.48. In certain embodiments, the Diagnostic Index p is determined to be about 0.49. In certain embodiments, the Diagnostic Index p is determined to be about 0.50. In certain embodiments, the Diagnostic Index p is determined to be about 0.51. In certain embodiments, the Diagnostic Index p is determined to be about 0.52. In certain embodiments, the Diagnostic Index p is determined to be about 0.53. In certain embodiments, the Diagnostic Index p is determined to be about 0.54. In certain embodiments, the Diagnostic Index p is determined to be about 0.55. In certain embodiments, the Diagnostic Index p is determined to be about 0.56. In certain embodiments, the Diagnostic Index p is determined to be about 0.57. In certain embodiments, the Diagnostic Index p is determined to be about 0.58. In certain embodiments, the Diagnostic Index p is determined to be about 0.59. In certain embodiments, the Diagnostic Index p is determined to be about 0.60. In certain embodiments, the Diagnostic Index p is determined to be about 0.61. In certain embodiments, the Diagnostic Index p is determined to be about 0.62. In certain embodiments, the Diagnostic Index p is determined to be about 0.63. In certain embodiments, the Diagnostic Index p is determined to be about 0.64. In certain embodiments, the Diagnostic Index p is determined to be about 0.65. In certain embodiments, the Diagnostic Index p is determined to be about 0.66. In certain embodiments, the Diagnostic Index p is determined to be about 0.67. In certain embodiments, the Diagnostic Index p is determined to be about 0.68. In certain embodiments, the Diagnostic Index p is determined to be about 0.69. In certain embodiments, the Diagnostic Index p is determined to be about 0.70. In certain embodiments, the Diagnostic Index p is determined to be about 0.71. In certain embodiments, the Diagnostic Index p is determined to be about 0.72. In certain embodiments, the Diagnostic Index p is determined to be about 0.73. In certain embodiments, the Diagnostic Index p is determined to be about 0.74. In certain embodiments, the Diagnostic Index p is determined to be about 0.75. In certain embodiments, the Diagnostic Index p is determined to be about 0.76. In certain embodiments, the Diagnostic Index p is determined to be about 0.77. In certain embodiments, the Diagnostic Index p is determined to be about 0.78. In certain embodiments, the Diagnostic Index p is determined to be about 0.79. In certain embodiments, the Diagnostic Index p is determined to be about 0.80. In certain embodiments, the Diagnostic Index p is determined to be about 0.81. In certain embodiments, the Diagnostic Index p is determined to be about 0.82. In certain embodiments, the Diagnostic Index p is determined to be about 0.83. In certain embodiments, the Diagnostic Index p is determined to be about 0.84. In certain embodiments, the Diagnostic Index p is determined to be about 0.85. In certain embodiments, the Diagnostic Index p is determined to be about 0.86. In certain embodiments, the Diagnostic Index p is determined to be about 0.87. In certain embodiments, the Diagnostic Index p is determined to be about 0.88. In certain embodiments, the Diagnostic Index p is determined to be about 0.89. In certain embodiments, the Diagnostic Index p is determined to be about 0.90. In certain embodiments, the Diagnostic Index p is determined to be about 0.91. In certain embodiments, the Diagnostic^' Index p is determined to be about 0.92. In certain embodiments, the Diagnostic Index p is detennined to be about 0.93. In certain embodiments, the Diagnostic Index p is determined to be about 0.94. In certain embodiments, the Diagnostic Index p is determined to be about 0.95. In certain embodiments, the Diagnostic Index p is determined to be about 0.96. In certain embodiments, the Diagnostic Index p is detennined to be about 0.97. In certain embodiments, the Diagnostic Index p is determined to be about 0.98. In certain embodiments, the Diagnostic Index p is determined to be about 0.99.

[0092] In certain embodiments, the threshold Diagnostic Index is about 0.01. In certain embodiments, the threshold Diagnostic Index is about 0.02. In certain embodiments, the threshold Diagnostic Index is about 0.03. In certain embodiments, the threshold Diagnostic Index is about 0.04. In certain embodiments, the threshold Diagnostic Index is about 0.05. In certain embodiments, the threshold Diagnostic Index is about 0.06. In certain embodiments, the threshold Diagnostic Index is about 0.07. In certain embodiments, the threshold Diagnostic Index is about 0.08. In certain embodiments, the threshold Diagnostic Index is about 0.09. In certain embodiments, the threshold Diagnostic Index is about 0.10. In certain embodiments, the threshold Diagnostic Index is about 0.11. In certain embodiments, the threshold Diagnostic Index is about 0.12. In certain embodiments, the threshold Diagnostic Index is about 0.13. In certain embodiments, the threshold Diagnostic Index is about 0.14. In certain embodiments, the threshold Diagnostic Index is about 0.15. In certain embodiments, the threshold Diagnostic Index is about 0.16. In certain embodiments, the threshold Diagnostic Index is about 0.17. In certain embodiments, the threshold Diagnostic Index is about 0.18. In certain embodiments, the threshold Diagnostic Index is about 0.19. In certain embodiments, the threshold Diagnostic Index is about 0.20. In certain embodiments, the threshold Diagnostic Index is about 0.21. In certain embodiments, the threshold Diagnostic Index is about 0.22. In certain embodiments, the threshold Diagnostic Index is about 0.23. In certain embodiments, the threshold Diagnostic Index is about 0.24. In certain embodiments, the threshold Diagnostic Index is about 0.25. In certain embodiments, the threshold Diagnostic Index is about 0.26. In certain embodiments, the threshold Diagnostic Index is about 0.27. In certain embodiments, the threshold Diagnostic Index is about 0.28. In certain embodiments, the threshold Diagnostic Index is about 0.29. In certain embodiments, the threshold Diagnostic Index is about 0.30. In certain embodiments, the threshold Diagnostic Index is about 0.31. In certain embodiments, the threshold Diagnostic Index is about 0.32. In certain embodiments, the threshold Diagnostic Index is about 0.33. In certain embodiments, the threshold Diagnostic Index is about 0.34. In certain embodiments, the threshold Diagnostic Index is about 0.35. In certain embodiments, the threshold Diagnostic Index is about 0.36. In certain embodiments, the threshold Diagnostic Index is about 0.37. In certain embodiments, the threshold Diagnostic Index is about 0.38. In certain embodiments, the threshold Diagnostic Index is about 0.39. In certain embodiments, the threshold Diagnostic Index is about 0.40. In certain embodiments, the threshold Diagnostic Index is about 0.41. In certain embodiments, the threshold Diagnostic Index is about 0.42. In certain embodiments, the threshold Diagnostic Index is about 0.43. In certain embodiments, the threshold Diagnostic Index is about 0.44. In certain embodiments, the threshold Diagnostic Index is about 0.45. In certain embodiments, the threshold Diagnostic Index is about 0.46. In certain embodiments, the threshold Diagnostic Index is about 0.47. In certain embodiments, the threshold Diagnostic Index is about 0.48. In certain embodiments, the threshold Diagnostic Index is about 0.49. In certain embodiments, the threshold Diagnostic Index is about 0.50. In certain embodiments, the threshold Diagnostic Index is about 0.51. In certain embodiments, the threshold Diagnostic Index is about 0.52. In certain embodiments, the threshold Diagnostic Index is about 0.53. In certain embodiments, the threshold Diagnostic Index is about 0.54. In certain embodiments, the threshold Diagnostic Index is about 0.55. In certain embodiments, the threshold Diagnostic Index is about 0.56. In certain embodiments, the threshold Diagnostic Index is about 0.57. In certain embodiments, the threshold Diagnostic Index is about 0.58. In certain embodiments, the threshold Diagnostic Index is about 0.59. In certain embodiments, the threshold Diagnostic Index is about 0.60. In certain embodiments, the threshold Diagnostic Index is about 0.61. In certain embodiments, the threshold Diagnostic Index is about 0.62. In certain embodiments, the threshold Diagnostic Index is about 0.63. In certain embodiments, the threshold Diagnostic Index is about 0.64. In certain embodiments, the threshold Diagnostic Index is about 0.65. In certain embodiments, the threshold Diagnostic Index is about 0.66. In certain embodiments, the threshold Diagnostic Index is about 0.67. In certain embodiments, the threshold Diagnostic Index is about 0.68. In certain embodiments, the threshold Diagnostic Index is about 0.69. In certain embodiments, the threshold Diagnostic Index is about 0.70. In certain embodiments, the threshold Diagnostic Index is about 0.71. In certain embodiments, the threshold Diagnostic Index is about 0.72. In certain embodiments, the threshold Diagnostic Index is about 0.73. In certain embodiments, the threshold Diagnostic Index is about 0.74. In certain embodiments, the threshold Diagnostic Index is about 0.75. In certain embodiments, the threshold Diagnostic Index is about 0.76. In certain embodiments, the threshold Diagnostic Index is about 0.77. In certain embodiments, the threshold Diagnostic Index is about 0.78. In certain embodiments, the threshold Diagnostic Index is about 0.79. In certain embodiments, the threshold Diagnostic Index is about 0.80. In certain embodiments, the threshold Diagnostic Index is about 0.81. In certain embodiments, the threshold Diagnostic Index is about 0.82. In certain embodiments, the threshold Diagnostic Index is about 0.83. In certain embodiments, the threshold Diagnostic Index is about 0.84. In certain embodiments, the threshold Diagnostic Index is about 0.85. In certain embodiments, the threshold Diagnostic Index is about 0.86. In certain embodiments, the threshold Diagnostic Index is about 0.87. In certain embodiments, the threshold Diagnostic Index is about 0.88. In certain embodiments, the threshold Diagnostic Index is about 0.89. In certain embodiments, the threshold Diagnostic Index is about 0.90. In certain embodiments, the threshold Diagnostic Index is about 0.91. In certain embodiments, the threshold Diagnostic Index is about 0.92. In certain embodiments, the threshold Diagnostic Index is about 0.93. In certain embodiments, the threshold Diagnostic Index is about 0.94. In certain embodiments, the threshold Diagnostic Index is about 0.95. In certain embodiments, the threshold Diagnostic Index is about 0.96. In certain embodiments, the threshold Diagnostic Index is about 0.97. In certain embodiments, the threshold Diagnostic Index is about 0.98. In certain embodiments, the threshold Diagnostic Index is about 0.99.

[0093] In certain embodiments, the threshold Diagnostic Index can be selected prior to selection of the coefficients used in the formula of the invention, then the coefficients selected to render the threshold Diagnostic Index indicative of the likelihood of the cancer or cancer cell to respond to Herl -acting agent, e.g., Gefitinib, therapy.

[0094] In certain embodiments, the formula of the invention is according to Formula I:

log(p/(l+p)) = -2.09 + 0.992*log(HllD+H13D) -0.39*log(H2P) -0.187*log(H23D)

Formula I

where Hl ID is the number of Herl-Herl dimers detected per cancer cell, H13D is the number of Herl-Her3 dimers detected per cancer cell, H23D is the number of Her2-Her3 dimers detected per cancer cell, H2P is the number of phosphorylated Her2 receptors detected per cancer cell, and/? is the Diagnostic Index used to predict the cancer's probability of responding to treatment with the drug. The Diagnostic Index p, determined according to Formula I, is a number between 0 and 1, where a larger value for/? indicates a greater probability that the cancer will respond to treatment with a Herl acting agent. In certain embodiments, p multiplied by 100% indicates the probability by percentage that the cancer will respond to treatment with a Her-1 acting agent, e.g., Gefltinib.

[0095] In certain embodiments, the formula of the invention is according to Formula II:

log(p/(l+p))=-3.207+1.098*log(HHD+H13D)-0.142* log(H23D)-0.307*log(H12D)

Formula II

where H11D is the number of Herl-Herl dimers detected per cancer cell, H13D is the number of Herl-Her3 dimers detected per cancer cell, H12D is the number of Herl-Her2 dimers detected per cancer cell, H23D is the number of Her2-Her3 dimers detected per cancer cell, and p is the Diagnostic Index used to predict the cancer's probability of responding to treatment with the drug. The Diagnostic Indexp, determined according to Formula II, is a number between 0 and 1, where a larger value for p indicates a greater probability that the cancer will respond to treatment with a Herl acting agent. In certain embodiments, p multiplied by 100% indicates the probability by percentage that the cancer will respond to treatment with a Her-1 acting agent, e.g., Gefitinib.

[0096] In certain embodiments, the formula of the invention is according to Formula III:

log(p/(1+p))=-3.126+1.047*log(H11D+H13D)-0.322*log(H12D),

Formula III

where H11D is the number of Herl-Herl dimers detected per cancer cell, H13D is the number of Herl-Her3 dimers detected per cancer cell, H12D is the number of Herl-Her2 dimers detected per cancer cell, and p is the Diagnostic Index used to predict the cancer's probability of responding to treatment with the drug. The Diagnostic Index p, determined according to Formula III, is a number between 0 and 1, where a larger value for p indicates a greater probability that the cancer will respond to treatment with a Herl acting agent. In certain embodiments, p multiplied by 100% indicates the probability by percentage that the cancer will respond to treatment with a Her-1 acting agent, e.g., Gefitinib.

[0097] In certain embodiments, the formula of the invention is according to Formula IV: log(p/(1+p)) = -1.947 + 0.904*log(H11D+H13D) -0.393*log(H2P)

Formula IV

where H11D is the number of Herl-Herl dimers detected per cancer cell, H13D is the number of Herl-Her3 dimers detected per cancer cell, H2P is the number of phosphorylated Her2 receptors detected per cancer cell, and p is the Diagnostic Index used to predict the cancer's probability of responding to treatment with the drug. The Diagnostic Index p, determined according to Formula IV, is a number between 0 and 1, where a larger value for p indicates a greater probability that the cancer will respond to treatment with a Herl acting agent. In certain embodiments, p multiplied by 100% indicates the probability by percentage that the cancer will respond to treatment with a Her-1 acting agent, e.g., Gefitinib.

[0098] In certain embodiments, the formula of the invention is according to Formula V:

log(p/(1+p)) = -1.098 + 0.58*log(H11D) - 0.141* log(H23D) + 0.322*log(H13D) -

0.397*log(H2P) Formula V

where H11D is the number of Herl-Herl dimers detected per cancer cell, H23D is the number of Her2-Her3 dimers detected per cancer cell, H13D is the number of Her1-Her3 dimers detected per cancer cell, H2P is the number of phosphorylated Her2 receptors detected per cancer cell, and p is the Diagnostic Index used to predict the cancer's probability of responding to treatment with the drug. The Diagnostic Index p, determined according to Formula V, is a number between 0 and 1, where a larger value for p indicates a greater probability that the cancer will respond to treatment with a Her1 acting agent. In certain embodiments, p multiplied by 100% indicates the probability by percentage that the cancer will respond to treatment with a Her-1 acting agent, e.g., Gefitinib.

[0099] In certain embodiments, the formula of the invention is according to Formula VI:

log(p/(1+p))=-1.828+0.747*log(H11D)+0.009* log(H23D)+0.158*log(H13D)-

0.388*log(H12D) Formula VI where Hl ID is the number of Herl-Herl dimers detected per cancer cell, H23D is the number of Her2-Her3 dimers detected per cancer cell, Hl 3D is the number of Herl-Her3 dimers detected per cancer cell, H2P is the number of phosphorylated Her2 receptors detected per cancer cell, and p is the Diagnostic Index used to predict the cancer's probability of responding to treatment with the drag. The Diagnostic Index p, determined according to Formula VI, is a number between 0 and 1, where a larger value for p indicates a greater probability that the cancer will respond to treatment with a Herl acting agent. In certain embodiments, p multiplied by 100% indicates the probability by percentage that the cancer will respond to treatment with a Her-1 acting agent, e.g., Gefitinib.

[0100] In certain embodiments, the formula of the invention is according to

Formula VII:

log(p/(l+p)) = -3.162+0.912*log(Hl lD+H13D)-0.192*log(H23D),

Formula VII

where Hl ID is the number of Herl-Herl dimers detected per cancer cell, H23D is the number of Her2-Her3 dimers detected per cancer cell, Hl 3D is the number of Herl-Her3 dimers detected per cancer cell, and is the Diagnostic Index used to predict the cancer's probability of responding to treatment with the drag. The Diagnostic Index p, determined according to Formula VII, is a number between 0 and 1, where a larger value for p indicates a greater probability that the cancer will respond to treatment with a Herl acting agent. In certain embodiments, p multiplied by 100% indicates the probability by percentage that the cancer will respond to treatment with a Her-1 acting agent, e.g., Gefitinib. In certain embodiments, the threshold Diagnostic Index for Formula VII is about 0.27. In certain embodiments, the threshold Diagnostic Index for Formula VII is about 0.4.

[0101] In certain embodiments, the Diagnostic Index p determined according to

Formula VII indicates that the cancer or cancer cell is likely to partially respond to treatment. In certain embodiments, the Diagnostic Index p determined according to Formula VII indicates that the cancer or cancer cell is likely to respond to treatment with a Her-1 acting agent. In certain embodiments, the Diagnostic Index p determined according to Formula VII is less than about 0.27, thereby indicating that the cancer or cancer cell is not likely to respond to treatment with a Her-1 acting agent. In certain embodiments, the Diagnostic Index p determined according to Formula VII is between about 0.27 and about 0.40, thereby indicating that the cancer or cancer cell is likely to partially respond to treatment with a Her-1 acting agent. In certain embodiments, the Diagnostic Index p determined according to Formula VII is more than about 0.40, thereby indicating that the cancer or cancer cell is likely to respond to treatment with a Her-1 acting agent.

[0102] In certain embodiments, the formula of the invention is according to

Formula VIII:

log(p/(l+p)) = -1.807+0.273*log(Hl lD+H13D)-0.241*log(H12D),

Formula VIII

where Hl ID is the number of Herl-Herl dimers detected per cancer cell, H13D is the number of Herl-Her3 dimers detected per cancer cell, H12D is the number of Herl-Her2 dimers detected per cancer cell, and p is the Diagnostic Index used to predict the cancer's probability of responding to treatment with the drug. The Diagnostic Index p, determined according to Formula VIII, is a number between 0 and 1, where a larger value fox p indicates a greater probability that the cancer will respond to treatment with a Herl acting agent. In certain embodiments, p multiplied by 100% indicates the probability by percentage that the cancer will respond to treatment with a Her-1 acting agent, e.g., Gefitinib. In certain embodiments, the threshold Diagnostic Index for Formula VII is about 0.28.

[0103] In certain embodiments, the Diagnostic Index/* determined according to

Formula VII indicates that the cancer or cancer cell is likely to partially respond to treatment, In certain embodiments, the Diagnostic Index/) determined according to Formula VII indicates that the cancer or cancer cell is likely to respond to treatment with a Her-1 acting agent, In certain embodiments, the Diagnostic Index p determined according to Formula VII is less than about 0.28, thereby indicating that the cancer or cancer cell is not likely to respond to treatment with a Her-1 acting agent, In certain embodiments, the Diagnostic Index p determined according to Formula VII is more than about 0.28, thereby indicating that the cancer or cancer cell is likely to respond to treatment with a Her-1 acting agent.

[0104] In certain embodiments, the probability that the cancer will respond to treatment with a Her-1 acting agent, e.g., Gefitinib, is about 1%. In certain embodiments, the probability that the cancer will respond to treatment with a Her-1 acting agent, e.g., Gefitinib, is about 5%. In certain embodiments, the probability that the cancer will respond to treatment with a Her-1 acting agent, e.g., Gefitinib, is about 10%. In certain embodiments, the probability that the cancer will respond to treatment with a Her-1 acting agent, e.g., Gefitinib, is about 15%. In certain embodiments, the probability that the cancer will respond to treatment with a Her-1 acting agent, e.g., Gefitinib, is about 20%. In certain embodiments, the probability that the cancer will respond to treatment with a Her-1 acting agent, e.g., Gefitinib, is about 25%. In certain embodiments, the probability that the cancer will respond to treatment with a Her-1 acting agent, e.g., Gefitinib, is about 30%. In certain embodiments, the probability that the cancer will respond to treatment with a Her-1 acting agent, e.g., Gefitinib, is about 35%. In certain embodiments, the probability that the cancer will respond to treatment with a Her-1 acting agent, e.g., Gefitinib, is about 40%. In certain embodiments, the probability that the cancer will respond to treatment with a Her-1 acting agent, e.g., Gefitinib, is about 45%. In certain embodiments, the probability that the cancer will respond to treatment with a Her-1 acting agent, e.g., Gefitinib, is about 50%. In certain embodiments, the probability that the cancer will respond to treatment with a Her-1 acting agent, e.g., Gefitinib, is about 55%. m certain embodiments, the probability that the cancer will respond to treatment with a Her-1 acting agent, e.g., Gefitinib, is about 60%. In certain embodiments, the probability that the cancer will respond to treatment with a Her-1 acting agent, e.g., Gefitinib, is about 65%. In certain embodiments, the probability that the cancer will respond to treatment with a Her-1 acting agent, e.g., Gefitinib, is about 70%. In certain embodiments, the probability that the cancer will respond to treatment with a Her-1 acting agent, e.g., Gefitinib, is about 75%. In certain embodiments, the probability that the cancer will respond to treatment with a Her-1 acting agent, e.g., Gefitinib, is about 80%. In certain embodiments, the probability that the cancer will respond to treatment with a Her-1 acting agent, e.g., Gefitinib, is about 85%. In certain embodiments, the probability that the cancer will respond to treatment with a Her-1 acting agent, e.g., Gefitinib, is about 90%. In certain embodiments, the probability that the cancer will respond to treatment with a Her-1 acting agent, e.g., Gefitinib, is about 95%. In certain embodiments, the probability that the cancer will respond to treatment with a Her-1 acting agent, e.g., Gefitinib is about 99%.

[0105] In certain embodiments, the responsiveness of the cancer to Her-1 acting agent, e.g., Gefitinib, therapy manifests as a reduction in cancer tumor size. In certain embodiments, the responsiveness of the cancer to Her-1 acting agent, e.g., Gefitinib, therapy manifests as a reduction in growth rate of the cancer. In certain embodiments, a subject predicted not to respond to treatment with a Her 1 -acting agent will nonetheless experience a reduction in the growth rate of the cancer. In certain embodiments, the responsiveness of the cancer to Her-1 acting agent, e.g., Gefitinib, therapy manifests as an extension of survival time of the patient with the cancer. In certain embodiments, the responsiveness of the cancer to Her-1 acting agent, e.g., Gefitinib, therapy manifests as amelioration of one or more symptoms associated with the cancer.

[0106] In another aspect, the present invention provides methods for determining whether a cancer cell is likely to respond to treatment with a Her 1 -acting agent. In certain embodiments, the methods comprise detecting on the cancer cell the presence and/or amount of ErbB dimers correlated with responsiveness to treatment with a Herl- acting agent, thereby determining whether the cancer cell is likely to respond to treatment with a Her 1 -acting agent. In certain embodiments, the activation state of the receptors within the ErbB dimers can be detected. For example, in certain embodiments, the phosphorylation state of one of the Her receptors in the dimers can be assessed as a measure of expression and activation of the dimers. As is well known in the art, phosphorylation of a Her receptor indicates that the receptor has been activated and is the mechanism for transducing the downstream signal.

[0107] In certain embodiments, the methods comprise determining a balanced dimer score for the cancer or cancer cell, where the balanced dimer score indicates whether the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent. A balanced dimer score according to the present invention can be calculated according to General Formula B:

Balanced Dimer Score = a₀*(b₀Herl/l + biHerl/2 + b₂Herl/3) - a_!*Her2/3 General Formula B

where Herl/1 is the number of Herl-Herl dimers detected per cancer cell, Hl/3 is the number of Herl-Her3 dimers detected per cancer cell, Hl/2 is the number of Herl-Her2 dimers detected per cancer cell, and H2/3D is the number of Her2-Her3 dimers detected per cancer cell, and a₀, a₁, b_o, b₁, and b₂ are each coefficients selected with statistical methods to conform the formula to responsiveness to treatment with Her 1 -acting agents observed in clinical studies. A cut-off thresho Id value can then be determined for the balanced dimer score calculated according to General Formula B by testing such thresholds until one that best predicts responsiveness to treatment with a Herl -acting agent is identified. For example, a two-by-two contingency table can be constructed as shown in the examples below to assess the predictive value of any particular cut-off for the Balanced Dimer Score determined with defined coefficients. The predictive value for the Balanced Dimer Score for predicting whether a cancer or cancer cell will respond to treatment with a Herl-acting agent can be at least about 55%, more preferably at least about 60%, more preferably at least about 65%, more preferably at least about 70%, more preferably at least about 75%, more preferably at least about 80%. In general, the cut-off for the Balanced Dimer Score is preferably selected in order to minimize false negatives, e.g., to minimize the identification of patients who would benefit from treatment with a Herl-acting agent as patients who would not benefit from such treatment.

[0108] As one of skill in the art will recognize, not all formulae of the invention need take into account every variable appearing in General Formula B. Accordingly, in certain embodiments, a₀, a₁, b_o, b₁, or b₂ can each independently be 0, though not all of a₀, ai, bi, and b₂ can simultaneously be 0. Further, in certain embodiments, a₀, a₁, b_o, b₁, or b₂ can each independently be 1.

[0109] In certain embodiments, the values of each of a₀, a₁, b_o, b₁, or b₂ can be selected independently from the values selected for any other of a₀, a₁, b_o, b₁, or b₂. In certain embodiments, the values selected for one of a₀, a₁, b_o, b₁, or b₂ can depend on the value selected for any other of a_o, a₁, b_o, b₁, or b₂.

[0110] In certain embodiments, a_o is a positive number. In certain embodiments, ao is between about 10 and about 1. In certain embodiments, a_o is between about 10 and about 3. In certain embodiments, a₀ is between about 10 and about 5. In certain embodiments, a₀ is between about 10 and about 7. In certain embodiments, a₀ is between about 10 and about 8. In certain embodiments, a₀ is between about 8 and about 1. In certain embodiments, a₀ is between about 6 and about 1. In certain embodiments, a₀ is between about 4 and about 1. In certain embodiments, a₀ is between about 8 and about 2. In certain embodiments, a₀ is between about 6 and about 2. In certain embodiments, a₀ is between about 4 and about 2. In certain embodiments, a₀ is about 1. In certain embodiments, a₀ is about 2. In certain embodiments, a₀ is about 3. In certain embodiments, a₀ is about 4. In certain embodiments, a₀ is about 5. In certain embodiments, a₀ is about 7. In certain embodiments, a₀ is about 10. In a preferred embodiment, a₀ is about 3.2.

[0111] In certain embodiments, a₁ is a positive number. In certain embodiments, a₁ is between about 50 and about 1. In certain embodiments, a₁ is between about 50 and about 5. In certain embodiments, a1 is between about 50 and about 10. In certain embodiments, a₁ is between about 30 and about 1. In certain embodiments, a₁ is between about 30 and about 5. In certain embodiments, a₁ is between about 30 and about 10. In certain embodiments, ai is between about 20 and about 1. In certain embodiments, a₁ is between about 20 and about 5. In certain embodiments, a₁ is between about 20 and about 10. In certain embodiments, a₁ is between about 15 and about 5. In certain embodiments, a₁ is between about 12 and about 8. In certain embodiments, a₁ is about 10. In certain embodiments, a₁ is about 12. In certain embodiments, a₁ is about 13. In certain embodiments, a₁ is about 8. In certain embodiments, a₁ is about 9. In certain embodiments, a₁ is about 7. In certain embodiments, a₁ is about 15. In a preferred embodiment, a₁ is about 10.5.

[0112] In certain embodiments, the methods comprise determining a balanced dimer score for the cancer or cancer cell, where the balanced dimer score is calculated according to Formula IX :

Balanced Dimer Score = 3.2 * (Her 1/1 + Herl/2 + Herl/3) - 10.5 * Her2/3

Formula IX [0113] In certain embodiments, the methods comprise detecting more than about

1000 Herl-Herl dimers per cell or determining a balanced dimer score according to Formula IX for the cancer or cancer cell. In certain embodiments, the methods comprise detecting more than about 1050 Herl-Herl dimers per cell or determining a balanced dimer score according to Formula IX for the cancer or cancer cell. In certain embodiments, the methods comprise detecting more than about 1100 Herl-Herl dimers per cell or determining a balanced dimer score according to Formula IX for the cancer or cancer cell. In certain embodiments, the methods comprise detecting more than about 1150 Herl-Herl dimers per cell or determining a balanced dimer score according to Formula IX for the cancer or cancer cell. In certain embodiments, the methods comprise detecting more than about 1175 Herl-Herl dimers per cell or determining a balanced dimer score according to Formula IX for the cancer or cancer cell. In certain embodiments, the methods comprise detecting more than about 1185 Herl-Herl dimers per cell or determining a balanced dimer score according to Formula IX for the cancer or cancer cell. In certain embodiments, the methods comprise detecting more than about 1200 Herl-Herl dimers per cell or determining a balanced dimer score according to Formula IX for the cancer or cancer cell. In certain embodiments, the methods comprise detecting more than about 1250 Herl-Herl dimers per cell or determining a balanced dimer score according to Formula IX for the cancer or cancer cell. In certain embodiments, the methods comprise detecting more than about 1300 Herl-Herl dimers per cell or determining a balanced dimer score according to Formula IX for the cancer or cancer cell. In certain embodiments, the methods comprise detecting more than about 1350 Herl-Herl dimers per cell or determining a balanced dimer score according to Formula IX for the cancer or cancer cell. In certain embodiments, the methods comprise detecting more than about 1400 Herl-Herl dimers per cell or determining a balanced dimer score according to Formula IX for the cancer or cancer cell. In certain embodiments, the methods comprise detecting more than about 1500 Herl-Herl dimers per cell or determining a balanced dimer score according to Formula IX for the cancer or cancer cell. In certain embodiments, the methods comprise detecting more than about 1600 Herl-Herl dimers per cell or determining a balanced dimer score according to Formula IX for the cancer or cancer cell. [0114] In certain embodiments, the methods comprise detecting more than about

1000 Her 1 -Her 1 dimers per cell and determining a balanced dimer score according to Formula IX for the cancer or cancer cell. In certain embodiments, the methods comprise detecting more than about 1050 Her 1 -Her 1 dimers per cell and determining a balanced dimer score according to Formula IX for the cancer or cancer cell. In certain embodiments, the methods comprise detecting more than about 1100 Her 1 -Her 1 dimers per cell and determining a balanced dimer score according to Formula IX for the cancer or cancer cell. In certain embodiments, the methods comprise detecting more than about 1150 Herl-Herl dimers per cell and determining a balanced dimer score according to Formula IX for the cancer or cancer cell. In certain embodiments, the methods comprise detecting more than about 1175 Herl-Herl dimers per cell and determining a balanced dimer score according to Formula IX for the cancer or cancer cell. In certain embodiments, the methods comprise detecting more than about 1185 Herl-Herl dimers per cell and determining a balanced dimer score according to Formula IX for the cancer or cancer cell. In certain embodiments, the methods comprise detecting more than about 1200 Herl-Herl dimers per cell and determining a balanced dimer score according to Formula IX for the cancer or cancer cell. In certain embodiments, the methods comprise detecting more than about 1250 Herl-Herl dimers per cell and determining a balanced dimer score according to Formula IX for the cancer or cancer cell. In certain embodiments, the methods comprise detecting more than about 1300 Herl-Herl dimers per cell and determining a balanced dimer score according to Formula IX for the cancer or cancer cell. In certain embodiments, the methods comprise detecting more than about 1350 Herl-Herl dimers per cell and determining a balanced dimer score according to Formula IX for the cancer or cancer cell. In certain embodiments, the methods comprise detecting more than about 1400 Herl-Herl dimers per cell and determining a balanced dimer score according to Formula IX for the cancer or cancer cell, In certain embodiments, the methods comprise detecting more than about 1500 Herl-Herl dimers per cell and determining a balanced dimer score according to Formula IX for the cancer or cancer cell. In certain embodiments, the methods comprise detecting more than about 1600 Herl-Herl dimers per cell and determining a balanced dimer score according to Formula IX for the cancer or cancer cell. [0115] In certain embodiments, the balanced dimer score determined according to

Formula IX is at least about 5,000. In certain embodiments, the balanced dimer score determined according to Formula IX is at least about 7,500. In certain embodiments, the balanced dimer score is determined to be at least about 10,000. In certain embodiments, the balanced dimer score determined according to Formula IX is at least about 10,250. In certain embodiments, the balanced dimer score determined according to Formula IX is at least about 10,500. In certain embodiments, the balanced dimer score determined according to Formula IX is at least about 10,750. In certain embodiments, the balanced dimer score determined according to Formula IX is at least about 10,800. In certain embodiments, the balanced dimer score determined according to Formula IX is at least about 10,850. In certain embodiments, the balanced dimer score determined according to Formula IX is at least about 10,900. In certain embodiments, the balanced dimer score determined according to Formula IX is at least about 10,950. In certain embodiments, the balanced dimer score determined according to Formula IX is at least about 11,000. In certain embodiments, the balanced dimer score determined according to Formula IX is at least about 12,000. In certain embodiments, the balanced dimer score determined according to Formula IX is at least about 13,000. In certain embodiments, the balanced dimer score determined according to Formula IX is at least about 14,000. In a preferred embodiment, the balanced dimer score determined according to Formula IX is at least about 15,000. In certain embodiments, the balanced dimer score determined according to Formula IX is at least about 17,500. In certain embodiments, the balanced dimer score determined according to Formula IX is at least about 20,000. In certain embodiments, the balanced dimer score determined according to Formula IX is at least about 22,500. In certain embodiments, the balanced dimer score determined according to Formula IX is at least about 25,000. In certain embodiments, the balanced dimer score determined according to Formula IX is at least about 27,500. In certain embodiments, the balanced dimer score determined according to Formula IX is at least about 30,000. In certain embodiments, the balanced dimer score determined according to Formula IX is at least about 32,500. In certain embodiments, the balanced dimer score determined according to Formula IX is at least about 35,000. [0116] In certain embodiments, a balanced dimer score determined according to

Formula IX that is at least about 5,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Herl -acting agent. In certain embodiments, a balanced dimer score determined according to Formula IX that is at least about 7,500 indicates that the cancer or cancer cell is likely to respond to treatment with a Herl -acting agent. In certain embodiments, a balanced dimer score determined according to Formula IX that is at least about 10,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Herl -acting agent. In certain embodiments, a balanced dimer score determined according to Formula IX that is at least about 11,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Herl -acting agent. In certain embodiments, a balanced dimer score determined according to Formula IX that is at least about 12,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Herl -acting agent. In certain embodiments, a balanced dimer score determined according to Formula IX that is at least about 13,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Herl -acting agent. In certain embodiments, a balanced dimer score determined according to Formula IX that is at least about 14,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Herl- acting agent. In a preferred embodiment, a balanced dimer score determined according to Formula IX that is at least about 15,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Herl -acting agent. In certain embodiments, a balanced dimer score determined according to Formula IX that is at least about 17,500 indicates that the cancer or cancer cell is likely to respond to treatment with a Herl- acting agent. In certain embodiments, a balanced dimer score determined according to Formula IX that is at least about 20,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Herl -acting agent. In certain embodiments, a balanced dimer score determined according to Formula IX that is at least about 22,500 indicates that the cancer or cancer cell is likely to respond to treatment with a Herl -acting agent. In certain embodiments, a balanced dimer score determined according to Formula IX that is at least about 25,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Herl -acting agent. In certain embodiments, a balanced dimer score determined according to Formula IX that is at least about 27,500 indicates that the cancer or cancer cell is likely to respond to treatment with a Herl -acting agent, In certain embodiments, a balanced dimer score determined according to Formula IX that is at least about 30,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent. In certain embodiments, a balanced dimer score determined according to Formula IX that is at least about 32,500 indicates that the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent, In certain embodiments, a balanced dimer score determined according to Formula IX that is at least about 35,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Herl- acting agent. In certain embodiments, the Her 1 -acting agent is Gefitinib, Tarceva, or erbitux. In a preferred embodiment, the Herl -acting agent is Gefitinib.

[0117] In certain embodiments, more than about 1000, 1050, 1100, 1150, 1175,

1185, 1200, or 1250 Herl-Herl dimers per cell and a balanced dimer score determined according to Formula IX that is at least about 9,500, 10,000, 10,250, 10,500, 10,750, 10,800, 10,850, 10,875, 10,900, or 11,000 indicate that the cancer or cancer cell is likely to respond to treatment with a Herl -acting agent, In certain embodiments, more than about 1100 Herl-Herl dimers per cell and a balanced dimer score determined according to Formula IX that is at least about 10,000 indicate that the cancer or cancer cell is likely to respond to treatment with a Herl-acting agent. In certain embodiments, more than about 1100 Herl-Herl dimers per cell and a balanced dimer score determined according to Formula IX that is at least about 10,500 indicate that the cancer or cancer cell is likely to respond to treatment with a Herl-acting agent, In certain embodiments, more than about 1100 Herl-Herl dimers per cell and a balanced dimer score determined according to Formula IX that is at least about 11,000 indicate that the cancer or cancer cell is likely to respond to treatment with a Herl-acting agent, In certain embodiments, more than about 1150 Herl-Herl dimers per cell and a balanced dimer score determined according to Formula IX that is at least about 10,000 indicate that the cancer or cancer cell is likely to respond to treatment with a Herl-acting agent. In certain embodiments, more than about 1150 Herl-Herl dimers per cell and a balanced dimer score determined according to Formula IX that is at least about 10,500 indicate that the cancer or cancer cell is likely to respond to treatment with a Herl-acting agent. In certain embodiments, more than about 1150 Herl-Herl dimers per cell and a balanced dimer score determined according to Formula IX that is at least about 11,000 indicate that the cancer or cancer cell is likely to respond to treatment with a Herl-acting agent, In another embodiment, more than about 1185 Herl-Herl dimers per cell and a balanced dimer score determined according to Formula IX that is at least about 10,875 indicate that the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent. In certain embodiments, the Herl- acting agent is Gefitinib, Tarceva, or erbitux. In a preferred embodiment, the Herl- acting agent is Gefitinib.

[0118] In certain embodiments, the methods comprise determining a balanced dimer score for the cancer or cancer cell, wherein the balanced dimer score is determined according to Formula X:

Balanced Dimer Score = 3.2 * Herl/3 - 10.5 * Her2/3 Formula X

In certain embodiments, the methods comprise detecting more than 1000 Herl-Herl dimers per cell or determining a balanced dimer score according to Formula X for the cancer or cancer cell. In certain embodiments, the methods comprise detecting more than 1000 Herl-Herl dimers per cell and determining a balanced dimer score according to Formula X for the cancer or cancer cell. In certain embodiments, the methods comprise detecting more than 1000 Herl-Herl dimers per cell, deteraiining a balanced dimer score according to Formula IX for the cancer or cancer cell, or determining a balanced dimer score according to Formula X for the cancer or cancer cell. In certain embodiments, the methods comprise detecting more than 1000 Herl-Herl dimers per cell, determining a balanced dimer score according to Formula IX for the cancer or cancer cell, and determining a balanced dimer score according to Formula X for the cancer or cancer cell.

[0119] In certain embodiments, the balanced dimer score determined according to

Formula X is at least about 500. In certain embodiments, the balanced dimer score determined according to Formula X is at least about 1,000. In certain embodiments, the balanced dimer score determined according to Formula X is at least about 1,500. In certain embodiments, the balanced dimer score determined according to Formula X is at least about 2,000. In certain embodiments, the balanced dimer score determined according to Formula X is at least about 2,500. In certain embodiments, the balanced dimer score determined according to Formula X is at least about 3,000. In certain embodiments, the balanced dimer score determined according to Formula X is at least about 3,500. In a preferred embodiment, the balanced dimer score determined according to Formula X is at least about 4,000. In certain embodiments, the balanced dimer score determined according to Formula X is at least about 4,500. In certain embodiments, the balanced dimer score determined according to Formula X is at least about 5,000. In certain embodiments, the balanced dimer score determined according to Formula X is at least about 7,500. In certain embodiments, the balanced dimer score determined according to Formula X is at least about 10,000. In certain embodiments, the balanced dimer score determined according to Formula X is at least about 15,000.

[0120] In certain embodiments, a balanced dimer score determined according to

Formula X that is at least about 500 indicates that the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent. In certain embodiments, a balanced dimer score determined according to Formula X that is at least about 1,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent. In certain embodiments, a balanced dimer score determined according to Formula X that is at least about 1,500 indicates that the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent. In certain embodiments, a balanced dimer score determined according to Formula X that is at least about 2,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Herl -acting agent. In certain embodiments, a balanced dimer score determined according to Formula X that is at least about 2,500 indicates that the cancer or cancer cell is likely to respond to treatment with a Herl -acting agent. In certain embodiments, a balanced dimer score determined according to Formula X that is at least about 3,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Herl -acting agent. In certain embodiments, a balanced dimer score determined according to Formula X that is at least about 3,500 indicates that the cancer or cancer cell is likely to respond to treatment with a Herl- acting agent. In a preferred embodiment, a balanced dimer score determined according to Formula X that is at least about 4,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Herl -acting agent. In certain embodiments, a balanced dimer score determined according to Formula X that is at least about 4,500 indicates that the cancer or cancer cell is likely to respond to treatment with a Herl -acting agent. In certain embodiments, a balanced dimer score determined according to Formula X that is at least about 5,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent. In certain embodiments, a balanced dimer score determined according to Formula X that is at least about 7,500 indicates that the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent. In certain embodiments, a balanced dimer score determined according to Formula X that is at least about 10,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Herl -acting agent, In certain embodiments, the Her 1 -acting agent is Gefitinib, Tarceva, or erbitux. In a preferred embodiment, the Herl -acting agent is Gefitinib.

[0121] In certain embodiments, the methods comprise determining a balanced dimer score for the cancer or cancer cell, wherein the balanced dimer score is determined according to Formula XI:

Balanced Dimer Score = 3.2 * Herl/1 + 5.0*Herl/2 + 3.1*Herl/3 - 10.5 * Her2/3

Formula XI

In certain embodiments, the methods comprise detecting more than 1000 Herl -Herl dimers per cell or determining a balanced dimer score according to Formula XI for the cancer or cancer cell. In certain embodiments, the methods comprise detecting more than 1000 Herl -Herl dimers per cell and determining a balanced dimer score according to Formula XI for the cancer or cancer cell, In certain embodiments, the methods comprise detecting more than 1000 Herl -Herl dimers per cell, determining a balanced dimer score according to Formula X for the cancer or cancer cell, or determining a balanced dimer score according to Formula XI for the cancer or cancer cell, In certain embodiments, the methods comprise detecting more than 1000 Herl -Herl dimers per cell, determining a balanced dimer score according to Formula X for the cancer or cancer cell, and determining a balanced dimer score according to Formula XI for the cancer or cancer cell.

[0122] In certain embodiments, the-balanced dimer score determined according to

Formula XI is at least about 2,000. In certain embodiments, the balanced dimer score determined according to Formula XI is at least about 4,000. In certain embodiments, the balanced dimer score determined according to Formula XI is at least about 6,000. In certain embodiments, the balanced dimer score determined according to Formula XI is at least about 8,000. In certain embodiments, the balanced dimer score determined according to Formula XI is at least about 10,000. In certain embodiments, the balanced dimer score determined according to Formula XI is at least about 12,000. In certain embodiments, the balanced dimer score determined according to Formula XI is at least about 14,000. In a preferred embodiment, the balanced dimer score determined according to Formula XI is at least about 15,000. In certain embodiments, the balanced dimer score determined according to Formula XI is at least about 17,500. In certain embodiments, the balanced dimer score determined according to Formula XI is at least about 20,000. In certain embodiments, the balanced dimer score determined according to Formula XI is at least about 25,500. In certain embodiments, the balanced dimer score determined according to Formula XI is at least about 30,000. In certain embodiments, the balanced dimer score determined according to Formula XI is at least about 15,000.

[0123] In certain embodiments, a balanced dimer score determined according to

Formula XI that is at least about 1,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent. In certain embodiments, a balanced dimer score determined according to Formula XI that is at least about 2,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent. In certain embodiments, a balanced dimer score determined according to Formula XI that is at least about 4,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent. In certain embodiments, a balanced dimer score determined according to Formula XI that is at least about 6,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent. In certain embodiments, a balanced dimer score determined according to Formula XI that is at least about 8,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent. In certain embodiments, a balanced dimer score determined according to Formula XI that is at least about 10,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent. In certain embodiments, a balanced dimer score determined according to Formula XI that is at least about 12,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Herl- acting agent. In a preferred embodiment, a balanced dimer score determined according to Formula XI that is at least about 14,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent. In certain embodiments, a balanced dimer score determined according to Formula XI that is at least about 15,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Herl- acting agent, In certain embodiments, a balanced dimer score determined according to Formula XI that is at least about 17,500 indicates that the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent, In certain embodiments, a balanced dimer score determined according to Formula XI that is at least about 20,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent, In certain embodiments, a balanced dimer score determined according to Formula XI that is at least about 10,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent. In certain embodiments, the Herl -acting agent is Gefitinib, Tarceva, or erbitux. In a preferred embodiment, the Herl -acting agent is Gefitinib.

[0124] ^' In certain embodiments, the methods comprise determining a balanced dimer score for the cancer or cancer cell, wherein the balanced dimer score is determined according to Formula XII:

Balanced Dimer Score = 3.2 * Herl/1 + 5.0*Herl/2 + 3.1*Herl/3 - 6.5 * Her2/3

Formula XII In certain embodiments, the methods comprise detecting more than 1000 Herl -Herl dimers per cell or determining a balanced dimer score according to Formula XII for the cancer or cancer cell. In certain embodiments, the methods comprise detecting more than 1000 Herl -Herl dimers per cell and determining a balanced dimer score according to Formula XII for the cancer or cancer cell. In certain embodiments, the methods comprise determining if the cancer or cancer cell expresses more than 1000 Herl -Herl dimers per cell, and if more than 1000 Herl -Herl dimers per cell are not expressed, then determining a balanced dimer score according to Formula XII for the cancer or cancer cell. In certain embodiments, the methods comprise detecting more than 1000 Herl- Herl dimers per cell, determining a balanced dimer score according to Formula X for the cancer or cancer cell, or determining a balanced dimer score according to Formula XII for the cancer or cancer cell, In certain embodiments, the methods comprise determining if the cancer or cancer cell expresses more than 1000 Herl -Herl dimers per cell, and if more than 1000 Herl -Herl dimers are not determined, then determining a balanced dimer score according to Formula X for the cancer or cancer cell, and if the balanced dimer score determined according to Formula X is not at least about 15,000, then determining a balanced dimer score according to Formula XII for the cancer or cancer cell. In certain embodiments, the methods comprise detecting more than 1000 Herl- Herl dimers per cell, determining a balanced dimer score according to Formula X for the cancer or cancer cell, and determining a balanced dimer score according to Formula XII for the cancer or cancer cell.

[0125] In certain embodiments, the balanced dimer score determined according to

Formula XII is at least about 2,000. In certain embodiments, the balanced dimer score determined according to Formula XII is at least about 4,000. In certain embodiments, the balanced dimer score determined according to Formula XII is at least about 6,000. In certain embodiments, the balanced dimer score determined according to Formula XII is at least about 8,000. In certain embodiments, the balanced dimer score determined according to Formula XII is at least about 10,000. In certain embodiments, the balanced dimer score determined according to Formula XII is at least about 12,000. In certain embodiments, the balanced dimer score determined according to Formula XII is at least about 14,000. In a preferred embodiment, the balanced dimer score determined according to Formula XII is at least about 15,000. In certain embodiments, the balanced dimer score determined according to Formula XII is at least about 17,500. In certain embodiments, the balanced dimer score determined according to Formula XII is at least about 20,000. In certain embodiments, the balanced dimer score determined according to Formula XII is at least about 25,500. In certain embodiments, the balanced dimer score determined according to Formula XII is at least about 30,000. In certain embodiments, the balanced dimer score determined according to Formula XII is at least about 15,000.

[0126] In certain embodiments, a balanced dimer score determined according to

Formula XII that is at least about 1,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent. In certain embodiments, a balanced dimer score determined according to Formula XII that is at least about 2,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent. In certain embodiments, a balanced dimer score determined according to Formula XII that is at least about 4,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent, In certain embodiments, a balanced dimer score determined according to Formula XII that is at least about 6,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent. In certain embodiments, a balanced dimer score determined according to Formula XII that is at least about 8,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent. In certain embodiments, a balanced dimer score determined according to Formula XII that is at least about 10,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent. In certain embodiments, a balanced dimer score deteπnined according to Formula XII that is at least about 12,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Herl- acting agent. In a preferred embodiment, a balanced dimer score determined according to Formula XII that is at least about 14,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent. In certain embodiments, a balanced dimer score determined according to Formula XII that is at least about 15,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Herl- acting agent. In certain embodiments, a balanced dimer score determined according to Formula XII that is at least about' 17,500 indicates that the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent. In certain embodiments, a balanced dimer score determined according to Formula XII that is at least about 20,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent. In certain embodiments, a balanced dimer score determined according to Formula XII that is at least about 10,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent. In certain embodiments, the Her 1 -acting agent is Gefitinib, Tarceva, or erbitux. In a preferred embodiment, the Herl -acting agent is Gefitinib.

[0127] In certain embodiments, the methods comprise detecting on the cancer cell at least about 600 Herl -Herl dimers, wherein the presence of the at least about 600 Herl -Herl dimers indicates that the cancer is likely to respond to treatment with the Herl -acting agent, In certain embodiments, the Herl -acting agent is Gefitinib, Tarceva, or erbitux. In a preferred embodiment, the Herl -acting agent is Gefitinib.

[0128] In certain embodiments, a cancer cell that is likely to respond to treatment with a Herl -acting agent has a probability of treatment that is increased about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90, 100%, or more over a reference cancer cell, In certain embodiments, the reference cancer cell is a cancer cell that does not respond to treatment with a Her 1 -acting agent. In certain embodiments, the reference cancer cell is a cancer cell wherein the responsiveness of the cancer cell to treatment with a Her 1 -acting agent has not been determined, but is rather the average responsiveness of a cancer cell to treatment with the Her 1 -acting agent, In certain embodiments, the average responsiveness to treatment with the Her 1 -acting agent is about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 18%, 20%, or 25%. In certain embodiments, a cancer cell that has been determined to be likely to respond to treatment with a Herl- acting agent is more likely than not to respond to treatment with the Her 1 -acting agent. In still another aspect, a cancer cell that is likely to respond to treatment with a Herl- acting agent has a about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more probability of responding to treatment with the Her 1 -acting agent.

[0129] In certain embodiments, at least about 750 Herl-Herl dimers are detected.

In certain embodiments, at least about 800 Herl-Herl dimers are detected, In certain embodiments, at least about 900 Herl-Herl dimers are detected. In certain embodiments, at least about 1000 Herl-Herl dimers are detected, In certain embodiments, at least about 1100 Herl-Herl dimers are detected. In certain embodiments, at least about 1200 Herl-Herl dimers are detected, In certain embodiments, at least about 1300 Herl-Herl dimers are detected, In certain embodiments, at least about 1325 Herl-Herl dimers are detected, In certain embodiments, at least about 1400 Herl-Herl dimers are detected, In certain embodiments, at least about 1500 Herl-Herl dimers are detected, In certain embodiments, at least about 1600 Herl-Herl dimers are detected, In certain embodiments, at least about 1800 Herl-Herl dimers are detected. In certain embodiments, at least about 1900 Herl-Herl dimers are detected, In certain embodiments, at least about 2000 Herl-Herl dimers are detected, In certain embodiments, at least about 2100 Herl-Herl dimers are detected, In certain embodiments, at least about 2200 Herl-Herl dimers are detected. In certain embodiments, at least about 2300 Herl-Herl dimers are detected. In certain embodiments, at least about 2400 Herl-Herl dimers are detected, In certain embodiments, at least about 2500 Herl-Herl dimers are detected, In certain embodiments, at least about 2600 Herl-Herl dimers are detected, In certain embodiments, at least about 2700 Her 1 -Her 1 dimers are detected. In certain embodiments, at least about 2781 Herl-Herl dimers are detected. In certain embodiments, at least about 2800 Herl-Herl dimers are detected. In certain embodiments, at least about 2900 Herl-Herl dimers are detected. In certain embodiments, at least about 3000 Herl-Herl dimers are detected. In certain embodiments, at least about 3500 Herl-Herl dimers are detected. In certain embodiments, between about 600 and about 100,000 Herl-Herl dimers are detected. In certain embodiments, between about 600 and about 10,000 Herl-Herl dimers are detected. In certain embodiments, between about 600 and about 30,000 Herl-Herl dimers are detected. In certain embodiments, between about 600 and about 50,000 Herl- Herl dimers are detected. In certain embodiments, between about 600 and about 70,000 Herl-Herl dimers are detected. In certain embodiments, between about 600 and about 90,000 Herl-Herl dimers are detected. In certain embodiments, between about 1100 and about 100,000 Herl-Herl dimers are detected. In certain embodiments, between about 1100 and about 10,000 Herl-Herl dimers are detected. In certain embodiments, between about 1100 and about 30,000 Herl-Herl dimers are detected. In certain embodiments, between about 1100 and about 50,000 Herl-Herl dimers are detected. In certain embodiments, between about 1100 and about 70,000 Herl-Herl dimers are detected. In certain embodiments, between about 1100 and about 90,000 Herl-Herl dimers are detected.

[0130] In certain embodiments, detecting the Herl-Herl dimers is accomplished by contacting the cell with a binding compound having a molecular tag attached thereto by a cleavable linkage, and a cleaving probe having a cleavage inducing-moiety; activating the cleaving agent such that, if the binding compound is within an effective proximity of the cleavage-inducing moiety of the cleaving probe, the cleavage-inducing moiety cleaves the cleavable linker so that the molecular tag is released; and quantifying the amount of released molecular tag, thereby detecting the Herl-Herl dimers. In certain embodiments, the binding compound and the cleaving probe each specifically bind Herl . In certain embodiments, binding of a binding compound or a cleaving probe to a Herl monomer precludes binding of another binding compound or cleaving probe to the same Herl monomer. In certain embodiments, the binding compound and the cleaving probe each specifically binds a Herl epitope. In certain embodiments, the binding compound and the cleaving probe each specifically binds the same Herl epitope. In certain embodiments, the binding compound and the cleaving probe each comprises a monoclonal antibody or an antigen-binding fragment. In certain embodiments, the binding compound and the cleaving probe each comprises the same monoclonal antibody or antigen-binding fragment. In certain embodiments, the binding compound and the cleaving probe each specifically binds a Herl ligand binding site, In certain embodiments, the binding compound and the cleaving probe each comprises a Herl ligand.

[0131] In certain embodiments, the cancer cell is a breast cancer cell, lung cancer cell, colorectal cancer cell, prostate cancer cell, or ovarian cancer cell. In a preferred embodiment, the cancer cell is a lung cancer cell. In certain embodiments, the cancer cell is a carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid cancer cell. In certain embodiments, the cancer cell is a squamous cancer cell (e.g. epithelial squamous cell cancer), lung cancer cell (e.g. small-cell lung cancer cell, non-small cell lung cancer cell, lung adenocarcinoma cancer cell, lung squamous cancer cell, epidermoid carcinoma cells, adenocarcinoma cells, large cell carcinoma cells, carcinoid tumor cells, bronchial gland tumor cells, mesothelioma cells, sarcoma cells or cells from mixed lung tumors), cancer cell from the peritoneum, hepatocellular cancer cell, gastric or stomach cancer cell (e.g., gastrointestinal cancer cell, pancreatic cancer cell, or glioblastoma cancer cell), cervical cancer cell, ovarian cancer cell, liver cancer cell, bladder cancer cell, urinary tract cancer cell, hepatoma cell, breast cancer cell, colon cancer cell, rectal cancer cell, colorectal cancer cell, endometrial or uterine carcinoma cancer cell, salivary gland carcinoma cancer cell, kidney or renal cancer cell, prostate cancer cell, vulval cancer cell, thyroid cancer cell, hepatic carcinoma cancer cell, anal carcinoma cancer cell, penile carcinoma cancer cell, melanoma cell, multiple myeloma and/or B-cell lymphoma cell, brain cancer cell, head and/or neck cancer cell, and cells from associated metastases thereof.

[0132] In certain embodiments, the Herl -Herl dimers on the cancer cell are detected directly on a patient sample. In certain embodiments, the patient sample is a fixed tissue sample, a frozen tissue sample, or a sample purified from circulating epithelial cells, In certain embodiments, the patient sample is a lung tissue sample, a breast tissue sample, a colorectal tissue sample, a prostate tissue sample, or an ovarian issue sample. In a preferred embodiment, the patient sample is a lung tissue sample. In certain preferred embodiments, the cancer cell is obtained from a biological sample of a subject having or suspected of having a cancer.

[0133] In certain embodiments, the methods comprise detecting on the cancer cell at least about 1750 Herl-Her3 dimers, wherein the presence of the at least about 1750 Herl-Her3 dimers indicates that the cancer is likely to respond to treatment with the Herl -acting agent, In certain embodiments, the Her 1 -acting agent is Gefitinib, Tarceva, or erbitux. In a preferred embodiment, the Herl -acting agent is Gefitinib.

[0134] In certain embodiments, a cancer cell that is likely to respond to treatment with a Herl-acting agent has a probability of treatment that is increased about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90, 100%, or more over a reference cancer cell. In certain embodiments, the reference cancer cell is a cancer cell that does not respond to treatment with a Herl-acting agent. In certain embodiments, the reference cancer cell is a cancer cell wherein the responsiveness of the cancer cell to treatment with a Herl-acting agent has not been determined, but is rather the average responsiveness of a cancer cell to treatment with the Herl-acting agent. In certain embodiments, the average responsiveness to treatment with the Herl-acting agent is about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 18%, 20%, or 25%. In certain embodiments, a cancer cell that has been determined to be likely to respond to treatment with a Herl- acting agent is more likely than not to respond to treatment with the Herl-acting agent. In still another aspect, a cancer cell that is likely to respond to treatment with a Herl- acting agent has a about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more probability of responding to treatment with the Herl-acting agent.

[0135] In certain embodiments, at least about 750 Herl-Her3 dimers are detected.

In certain embodiments, at least about 800 Herl-Her3 dimers are detected, In certain embodiments, at least about 900 Herl-Her3 dimers are detected, In certain embodiments, at least about 1000 Herl-Her3 dimers are detected. In certain embodiments, at least about 1100 Herl-Her3 dimers are detected, In certain embodiments, at least about 1200 Herl-Her3 dimers are detected, In certain embodiments, at least about 1300 Herl-Her3 dimers are detected, In certain embodiments, at least about 1325 Herl-Her3 dimers are detected, In certain embodiments, at least about 1400 Herl-Her3 dimers are detected. In certain embodiments, at least about 1500 Herl-Her3 dimers are detected. In certain embodiments, at least about 1600 Herl-Her3 dimers are detected. In certain embodiments, at least about 1700 Herl-Her3 dimers are detected. In certain embodiments, at least about 1800 Herl-Her3 dimers are detected. In certain embodiments, at least about 1900 Herl-Her3 dimers are detected. In certain embodiments, at least about 2000 Herl-Her3 dimers are detected. In certain embodiments, at least about 2100 Herl-Her3 dimers are detected. In certain embodiments, at least about 2200 Herl-Her3 dimers are detected. In certain embodiments, at least about 2300 Herl-Her3 dimers are detected. In certain embodiments, at least about 2400 Herl-Her3 dimers are detected. In certain embodiments, at least about 2500 Herl-Her3 dimers are detected. In certain embodiments, at least about 2600 Herl-Her3 dimers are detected. In certain embodiments, at least about 2700 Herl-Her3 dimers are detected. In certain embodiments, at least about 2800 Herl-Her3 dimers are detected. In certain embodiments, at least about 2900 Herl-Her3 dimers are detected. In certain embodiments, at least about 3000 Herl-Her3 dimers are detected. In certain embodiments, at least about 3500 Herl-Her3 dimers are detected. In certain embodiments, between about 600 and about 100,000 Herl-Her3 dimers are detected. In certain embodiments, between about 600 and about 10,000 Herl-Her3 dimers are detected. In certain embodiments, between about 600 and about 30,000 Herl-Her3 dimers are detected. In certain embodiments, between about 600 and about 50,000 Herl- Her3 dimers are detected. In certain embodiments, between about 600 and about 70,000 Herl-Her3 dimers are detected. In certain embodiments, between about 600 and about 90,000 Herl-Her3 dimers are detected. In certain embodiments, between about 1100 and about 100,000 Herl-Her3 dimers are detected. In certain embodiments, between about 1100 and about 10,000 Herl-Her3 dimers are detected. In certain embodiments, between about 1100 and about 30,000 Herl-Her3 dimers are detected. In certain embodiments, between about 1100 and about 50,000 Herl-Her3 dimers are detected. In certain embodiments, between about 1100 and about 70,000 Herl-Her3 dimers are detected. In certain embodiments, between about 1100 and about 90,000 Herl-Her3 dimers are detected. [0136] In certain embodiments, the methods of the invention further comprise detecting on a cancer cell at least about 1000 Herl-Her3 dimers, wherein the presence of at least about 600 Her 1 -Her 1 dimers and at least about 1000 Herl-Her3 dimers indicates that the cancer is likely to respond to treatment with the Herl -acting agent, In certain embodiments, at least about 800 Herl -Herl dimers are detected. In certain embodiments, at least about 900 Herl -Herl dimers are detected. In certain embodiments, at least about 1000 Herl -Herl dimers are detected. In certain embodiments, at least about 1100 Herl- Herl dimers are detected, In certain embodiments, at least about 1200 Herl-Her3 dimers are detected. In certain embodiments, at least about 1400 Herl-Her3 dimers are detected. In certain embodiments, at least about 1600 Herl-Her3 dimers are detected, In certain embodiments, at least about 1700 Herl-Her3 dimers are detected. In certain embodiments, at least about 1800 Herl-Her3 dimers are detected, In certain embodiments, at least about 1100 Herl -Herl dimers and at least about 1800 Herl-Her3 dimers are detected, In certain embodiments, between about 1000 and about 100,000 Herl-Her3 dimers are detected. In certain embodiments, between about 1000 and about 10,000 Herl-Her3 dimers are detected, In certain embodiments, between about 1000 and about 20,000 Herl-Her3 dimers are detected, In certain embodiments, between about 1000 and about 40,000 Herl-Her3 dimers are detected, In certain embodiments, between about 1000 and about 60,000 Herl-Her3 dimers are detected, In certain embodiments, between about 1000 and about 80,000 Herl-Her3 dimers are detected. In certain embodiments, between about 1800 and about 100,000 Herl-Her3 dimers are detected. In certain embodiments, between about 1800 and about 10,000 Herl-Her3 dimers are detected, In certain embodiments, between about 1800 and about 20,000 Herl-Her3 dimers are detected. In certain embodiments, between about 1800 and about 40,000 Herl-Her3 dimers are detected, In certain embodiments, between about 1800 and about 60,000 Herl-Her3 dimers are detected. In certain embodiments, between about 1800 and about 80,000 Herl-Her3 dimers are detected.

[0137] In certain embodiments, between about 600 and about 100,000 Herl-Herl dimers and between about 1000 and about 100,000 Herl-Her3 dimers are detected, In certain embodiments, between about 600 and about 10,000 Herl-Herl dimers and between about 1000 and about 10,000 Herl-Her3 dimers are detected, In certain embodiments, between about 600 and about 20,000 Herl-Herl dimers and between about 1000 and about 20,000 Herl-Her3 dimers are detected. In certain embodiments, between about 600 and about 40,000 Her 1 -Her 1 dimers and between about 1000 and about 40,000 Herl-Her3 dimers are detected. In certain embodiments, between about 600 and about 60,000 Herl-Herl dimers and between about 1000 and about 60,000 Herl-Her3 dimers are detected. In certain embodiments, between about 600 and about 80,000 Herl-Herl dimers and between about 1000 and about 80,000 Herl-Her3 dimers are detected. In certain embodiments, between about 1000 and about 100,000 Herl-Herl dimers and between about 1800 and about 100,000 Herl-Her3 dimers are detected. In certain embodiments, between about 1000 and about 10,000 Herl-Herl dimers and between about 1800 and about 10,000 Herl-Her3 dimers are detected. In certain embodiments, between about 1000 and about 20,000 Herl-Herl dimers and between about 1800 and about 20,000 Herl-Her3 dimers are detected. In certain embodiments, between about 1000 and about 40,000 Herl-Herl dimers and between about 1800 and about 40,000 Herl-Her3 dimers are detected. In certain embodiments, between about 1000 and about 60,000 Herl-Herl dimers and between about 1800 and about 60,000 Herl-Her3 dimers are detected. In certain embodiments, between about 1000 and about 80,000 Herl-Herl dimers and between about 1800 and about 80,000 Herl-Her3 dimers are detected.

[0138] In certain embodiments, detecting the Herl-Her3 dimers is accomplished by contacting the cell with a binding compound having a molecular tag attached thereto by a cleavable linkage, and a cleaving probe having a cleavage inducing-moiety; activating the cleaving agent such that, if the binding compound is within an effective proximity of the cleavage-inducing moiety of the cleaving probe, the cleavage-inducing moiety cleaves the cleavable linker so that the molecular tag is released; and quantifying the amount of released molecular tag, thereby detecting the Herl-Her3 dimers. In certain embodiments, the binding compound and the cleaving probe each specifically binds either Herl or Her3. In certain embodiments, the cleaving probe and the binding probe do not both bind the same receptor. In certain embodiments, the binding compound specifically binds a Herl epitope. In certain embodiments, the binding compound comprises a monoclonal antibody or antigen-binding fragment. In certain embodiments, the binding compound specifically binds a Herl ligand binding site. In certain embodiments, the binding compound comprises a Herl ligand. In certain embodiments, the binding compound specifically binds a Her3 epitope. In certain embodiments, the binding compound specifically binds a Her3 ligand binding site. In certain embodiments, the binding compound comprises a Her3 ligand. In certain embodiments, the cleaving probe specifically binds a Herl epitope. In certain embodiments, the cleaving probe comprises a monoclonal antibody or antigen-binding fragment. In certain embodiments, the cleaving probe specifically binds a Herl ligand binding site, In certain embodiments, the cleaving probe comprises a Herl ligand. In certain embodiments, the cleaving probe specifically binds a Her3 epitope, In certain embodiments, the cleaving probe specifically binds a Her3 ligand binding site. In certain embodiments, the cleaving probe comprises a Her3 ligand.

[0139] In certain embodiments, at least about 750 Herl-Her3 dimers are detected.

In certain embodiments, at least about 800 Herl-Her3 dimers are detected, In certain embodiments, at least about 900 Herl-Her3 dimers are detected. In certain embodiments, at least about 1000 Herl-Her3 dimers are detected, In certain embodiments, at least about 1100 Herl-Her3 dimers are detected, In certain embodiments, at least about 1200 Herl-Her3 dimers are detected. In certain embodiments, at least about 1300 Herl-Her3 dimers are detected, In certain embodiments, at least about 1325 Herl-Her3 dimers are detected, In certain embodiments, at least about 1400 Herl-Her3 dimers are detected. In certain embodiments, at least about 1500 Herl-Her3 dimers are detected, In certain embodiments, at least about 1600 Herl-Her3 dimers are detected, In certain embodiments, at least about 1700 Herl-Her3 dimers are detected, In certain embodiments, at least about 1800 Herl-Her3 dimers are detected. In certain embodiments, at least about 1900 Herl-Her3 dimers are detected. In certain embodiments, at least about 2000 Herl-Her3 dimers are detected, In certain embodiments, at least about 2100 Herl-Her3 dimers are detected. In certain embodiments, at least about 2200 Herl-Her3 dimers are detected. In certain embodiments, at least about 2300 Herl-Her3 dimers are detected, In certain embodiments, at least about 2400 Herl-Her3 dimers are detected. In certain embodiments, at least about 2500 Herl-Her3 dimers are detected, In certain embodiments, at least about 2600 Herl-Her3 dimers are detected, In certain embodiments, at least about 2700 Herl-Her3 dimers are detected, In certain embodiments, at least about 2800 Herl-Her3 dimers are detected. In certain embodiments, at least about 2900 Herl-Her3 dimers are detected. In certain embodiments, at least about 3000 Herl-Her3 dimers are detected. In certain embodiments, at least about 3500 Herl-Her3 dimers are detected. In certain embodiments, between about 600 and about 100,000 Herl-Her3 dimers are detected. In certain embodiments, between about 600 and about 10,000 Herl-Her3 dimers are detected. In certain embodiments, between about 600 and about 30,000 Herl-Her3 dimers are detected. In certain embodiments, between about 600 and about 50,000 Herl- Her3 dimers are detected. In certain embodiments, between about 600 and about 70,000 Herl-Her3 dimers are detected. In certain embodiments, between about 600 and about 90,000 Herl-Her3 dimers are detected. In certain embodiments, between about 1100 and about 100,000 Herl-Her3 dimers are detected. In certain embodiments, between about 1100 and about 10,000 Herl-Her3 dimers are detected. In certain embodiments, between about 1100 and about 30,000 Herl-Her3 dimers are detected. In certain embodiments, between about 1100 and about 50,000 Herl-Her3 dimers are detected. In certain embodiments, between about 1100 and about 70,000 Herl-Her3 dimers are detected. In certain embodiments, between about 1100 and about 90,000 Herl-Her3 dimers are detected.

[0140] In certain embodiments, the methods of the invention comprise detecting on a cancer cell more than about 1000 Her2-Her3 dimers, wherein the presence of more than about 1000 Her2-Her3 dimers indicates that the cancer cell is not likely to respond to treatment with the Her 1 -acting agent, In certain embodiments, more than about 900 Her2-Her3 dimers are detected. In certain embodiments, more than about 800 Her2- Her3 dimers are detected, In certain embodiments, more than about 700 Her2-Her3 dimers are detected, In certain embodiments, more than about 600 Her2-Her3 dimers are detected. In certain embodiments, fewer than about 550 Her2-Her3 dimers are detected, In certain embodiments, more than about 500 Her2-Her3 dimers are detected, In certain embodiments, more than about 475 Her2-Her3 dimers are detected, In certain embodiments, more than about 450 Her2-Her3 dimers are detected. In certain embodiments, more than about 425 Her2-Her3 dimers are detected, In certain embodiments, more than about 400 Her2-Her3 dimers are detected, In certain embodiments, more than about 375 Her2-Her3 dimers are detected, In certain embodiments, more than about 350 Her2-Her3 dimers are detected. In certain embodiments, more than about 300 Her2-Her3 dimers are detected. In certain embodiments, between about 1 and about 1000 Her2-Her3 dimers are detected. In certain embodiments, between about 1 and about 900 Her2-Her3 dimers are detected. In certain embodiments, between about 1 and about 800 Her2-Her3 dimers are detected. In certain embodiments, between about 1 and about 700 Her2-Her3 dimers are detected. In certain embodiments, between about 1 and about 600 Her2-Her3 dimers are detected. In certain embodiments, between about 1 and about 500 Her2-Her3 dimers are detected. In certain embodiments, between about 1 and about 400 Her2-Her3 dimers are detected. In certain embodiments, between about 1 and about 300 Her2-Her3 dimers are detected. In certain embodiments, between about 1 and about 200 Her2-Her3 dimers are detected. In certain embodiments, between about 1 and about 100 Her2-Her3 dimers are detected. In certain embodiments, between about 1 and about 50 Her2-Her3 dimers are detected. In certain embodiments, between about 1 and about 2000 Her2-Her3 dimers are detected.

[0141] In certain aspects, the methods comprise determining the number of Herl-

Herl dimers per cell and the number of Her2-Her3 dimers per cell. In other aspects, the methods comprise determining the number of Her 1 -Her 1 dimers per cell, the number of Her2-Her3 dimers per cell, and determining a balanced dimer score according to one of the formulas presented herein for the cancer or the cancer cell. In one embodiment, the formula is Formula IX.

[0142] In certain embodiments, the methods of the invention comprise detecting

Her2-Her3 dimers and Her 1 -Her 1 dimers on a cancer cell, wherein the presence of at least about 1000, 1050, 1100, 1150, 1175, 1185, 1200, 1250, 1300, 1350, 1400, 1500, or 1600 Herl-Herl dimers and fewer than about 1100, 1000, 900, 800, 700, 600, 550, 500, 475, 450, 425, 400, 375, 350, or 300 Her2-Her3 dimers indicates that the cancer cell is likely to respond to treatment with the Her 1 -acting agent. In certain embodiments, the presence of at least about 1150, 1175 or 1185 Herl-Herl dimers and fewer than about 400, 425 or 450 indicates that the cancer cell is likely to respond to treatment with the Her 1 -acting agent. In certain embodiments, a partial response is observed as a reduction in cancer growth rate. [0143] In another aspect, the present invention provides methods for determining whether a cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent, wherein said method comprises determining the ratio of Herl-Her3 dimers per cell to Her2-Her3 dimers per cell. In one embodiment, a determination of a ratio of Herl-Her3 dimers per cell to Her2-Her3 dimers per cell of greater than about 0.50 to greater than about 0.60 indicates that the cell is likely to respond to treatment with a Her 1 -acting agent. In another embodiment, a determination of a ratio of Herl-Her3 dimers per cell to Her2-Her3 dimers per cell of greater than about 0.55 to greater than about 0.57 indicates that the cell is likely to respond to treatment with a Her 1 -acting agent. In yet another embodiment, a determination of a ratio of Herl-Her3 dimers per cell to Her2-Her3 dimers per cell of greater than about 0.25, 0.30, 0.35, 0.40 or 0.45 indicates that the cell is likely to respond to treatment with a Her 1 -acting agent, In yet another embodiment, a determination of a ratio of Herl-Her3 dimers per cell to Her2-Her3 dimers per cell of greater than about 0.65, 0.70, or 0.75 indicates that the cell is likely to respond to treatment with a Her 1 -acting agent. In certain embodiments, the Herl -acting agent is Gefitinib, tarceva, or erbitux. In a preferred embodiment, the Herl -acting agent is Gefϊtinib.

[0144] In another embodiment, a determination of a ratio of Herl-Her3 dimers per cell to Her2-Her3 dimers per cell of less than about 0.50 to less than about 0.60 indicates that the cell is not likely to respond to treatment with a Herl -acting agent. In another embodiment, a determination of a ratio of Herl-Her3 dimers per cell to Her2-Her3 dimers per cell of less than about 0.55 to less than about 0.57 indicates that the cell is not likely to respond to treatment with a Herl -acting agent, In yet another embodiment, a determination of a ratio of Herl-Her3 dimers per cell to Her2-Her3 dimers per cell of less than about 0.25, 0.30, 0.35, 0.40 or 0.45 indicates that the cell is not likely to respond to treatment with a Herl -acting agent. In yet another embodiment, a determination of a ratio of Herl-Her3 dimers per cell to Her2-Her3 dimers per cell of less than about 0.65, 0.70, or 0.75 indicates that the cell is not likely to respond to treatment with a Herl -acting agent, In certain embodiments, the Herl -acting agent is Gefitinib, tarceva, or erbitux. In a preferred embodiment, the Herl -acting agent is Gefitinib. [0145] In another aspect, the present invention provides methods for determining whether a cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent, wherein said method comprises determining the ratio of the sum of Her 1 -Her 1 dimers per cell and Herl-Her2 dimers per cell to Her2-Her3 dimers per cell; that is: (Herl-Herl dimers per cell + Herl-Her2 dimers per cell)/Her2-Her3 dimers per cell. In one embodiment, a determination of this ratio as greater than about 5.0 to greater than about 5.2 indicates that the cell is likely to respond to treatment with a Her 1 -acting agent. In another embodiment, a determination of this ratio as greater than about 5.1, 5.13, 5.15, or 5.17 indicates that the cell is likely to respond to treatment with a Her 1 -acting agent. In yet another embodiment, a determination of this ratio as greater than about 2, 2.5, 3.0, 3.5, 4.0, 4.5, or 4.75 indicates that the cell is likely to respond to treatment with a Herl- acting agent. In yet another embodiment, a determination of this ratio as greater than about 5.3, 5.4, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 indicates that the cell is likely to respond to treatment with a Herl -acting agent. In certain embodiments, the Herl -acting agent is Gefitinib, tarceva, or erbitux. In a preferred embodiment, the Herl- acting agent is Gefitinib.

[0146] In another embodiment, a determination of this ratio as less than about 5.0 to less than about 5.2 indicates that the cell is not likely to respond to treatment with a Herl -acting agent. In another embodiment, a determination of this ratio as less than about 5.1, 5.13, 5.15, or 5.17 indicates that the cell is not likely to respond to treatment with a Herl -acting agent. In yet another embodiment, a determination of this ratio as less than about 2, 2.5, 3.0, 3.5, 4.0, 4.5, or 4.75 indicates that the cell is not likely to respond to treatment with a Herl -acting agent. In yet another embodiment, a determination of this ratio as less than about 5.3, 5.4, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 indicates that the cell is not likely to respond to treatment with a Herl -acting agent. In certain embodiments, the Herl -acting agent is Gefitinib, tarceva, or erbitux. In a preferred embodiment, the Herl -acting agent is Gefitinib.

[0147] In certain aspects, the present invention provides methods for determining whether a cancer or cancer cell is likely to respond to treatment with a Herl -acting agent, wherein the method comprises determining the ratio of Herl-Herl dimers per cell and Herl-Her2 dimers per cell to Her2-Her3 dimers per cell as above, and further comprises determining the number of Her2-Her3 dimers per cell. In another embodiment, if the number of Her2-Her3 dimers per cell is greater than about 400, 410, 420, 425, 430, 435, 440, 445, or 450, the cell is unlikely to respond to treatment with a Her 1 -acting agent. In another embodiment, if the number of Her2-Her3 dimers per cell is greater than about 425, the cell is unlikely to respond to treatment with a Her 1 -acting agent. In yet another embodiment, if the number of Her2-Her3 dimers per cell is greater than about 300, 325, 350, 375 or 390, the cell is unlikely to respond to treatment with a Herl-acting agent. In another embodiment, if the number of Her2-Her3 dimers per cell is greater than about 460, 475, 500, 550, 600, 650, 675, 700, 750, 800, 850, 900, 950, or 1000, the cell is unlikely to respond to treatment with a Herl-acting agent. In certain embodiments, the Herl-acting agent is Gefitinib, tarceva, or erbitux. In a preferred embodiment, the Herl- acting agent is Gefitinib.

[0148] In certain embodiments, detecting the Her2-Her3 dimers is accomplished by contacting the cell with a binding compound having a molecular tag attached thereto by a cleavable linkage, and a cleaving probe having a cleavage inducing-moiety; activating the cleaving agent such that, if the binding compound is within an effective proximity of the cleavage-inducing moiety of the cleaving probe, the cleavage-inducing moiety cleaves the cleavable linker so that the molecular tag is released; and quantifying the amount of released molecular tag, thereby detecting the Her2-Her3 dimers. In certain embodiments, the binding compound and the cleaving probe each specifically binds either Her2 or Her3. In certain embodiments, the cleaving probe and the binding probe do not both bind the same receptor. In certain embodiments, the binding compound specifically binds a Her2 epitope. In certain embodiments, the binding compound comprises a monoclonal antibody or antigen-binding fragment. In certain embodiments, wherein the binding compound specifically binds a Her2 ligand binding site, In certain embodiments, the binding compound comprises a Her2 ligand. In certain embodiments, the binding compound specifically binds a Her3 epitope, In certain embodiments, the binding compound specifically binds a Her3 ligand binding site, In certain embodiments, the binding compound comprises a Her3 ligand. In certain embodiments, the cleaving probe specifically binds a Her2 epitope, In certain embodiments, the cleaving probe comprises a monoclonal antibody or antigen-binding fragment, In certain embodiments, the cleaving probe specifically binds a Her2 ligand binding site, In certain embodiments, the cleaving probe comprises a Her2 ligand. In certain embodiments, the cleaving probe specifically binds a Her3 epitope. In certain embodiments, the cleaving probe specifically binds a Her3 ligand binding site. In certain embodiments, the cleaving probe comprises a Her3 ligand.

[0149] In another aspect, the invention provides a method for determining whether a cancer cell is likely to respond to treatment with a Her 1 -acting agent, comprising detecting on a cell of the cancer at least about 600 Her 1 -Her 1 dimers, at least about 1000 Herl-Her2 dimers, and fewer than about 1000 Her2-Her3 dimers, wherein the presence of the at least about 600 Herl-Herl dimers, the at least about 1000 Herl-Her2 dimers, and the fewer than about 1000 Her2-Her3 dimers indicates that the cancer cell is likely to respond to treatment with the Her 1 -acting agent. In certain preferred embodiments, the Her 1 -acting agent is Gefϊtinib. In certain embodiments, at least about 800 Herl-Herl dimers are detected. In certain embodiments, at least about 900 Herl-Herl dimers are detected. In certain embodiments, at least about 1000 Herl-Herl dimers are detected. In certain embodiments, at least about 1100 Herl-Herl dimers are detected. In certain embodiments, between about 600 and about 100,000 Herl-Herl dimers are detected.

[0150] In certain embodiments, at least about 800 Herl-Herl dimers are detected.

In certain embodiments, at least about 900 Herl-Herl dimers are detected. In certain embodiments, at least about 1000 Herl-Herl dimers are detected, In certain embodiments, at least about 1100 Herl-Herl dimers are detected, In certain embodiments, at least about 1200 Herl-Her3 dimers are detected, In certain embodiments, at least about 1400 Herl-Her3 dimers are detected, In certain embodiments, at least about 1600 Herl-Her3 dimers are detected, In certain embodiments, at least about 1700 Herl-Her3 dimers are detected, In certain embodiments, at least about 1800 Herl-Her3 dimers are detected. In certain embodiments, between about 1000 and about 1000,000 Herl-Her3 dimers are detected.

[0151] In certain embodiments, at least about 1100 Herl-Herl dimers and at least about 1800 Herl-Her3 dimers are detected, In certain embodiments, fewer than about 900 Her2-Her3 dimers are detected. In certain embodiments, fewer than about 800 Her2-Her3 dimers are detected, In certain embodiments, fewer than about 700 Her2- Her3 dimers are detected, In certain embodiments, fewer than about 600 Her2-Her3 dimers are detected. In certain embodiments, wherein between about 1 and about 1000 Her2-Her3 dimers are detected. In certain embodiments, at least about 1100 Herl-Herl dimers and fewer than about 600 Her2-Her3 dimers are detected. In certain embodiments, at least about 1100 Herl-Herl dimers, at least about 1800 Her2-Her3 dimers, and fewer than about 600 Her2-Her3 dimers are detected. In certain embodiments, between about 600 and about 100,000 Herl-Herl dimers and between about 1 and about 1000 Her2-Her3 dimers are detected. In certain embodiments, between about 600 and about 100,000 Herl-Herl dimers and between about 1000 and about 100,000 Herl-Her3 dimers are detected. In certain embodiments, between about 600 and about 100,000 Herl-Herl dimers, between about 1000 and about 100,000 Herl- Her3 dimers, and between about 1 and about 1000 Her2-Her3 dimers are detected.

[0152] In certain embodiments, detecting the Herl-Herl dimers is accomplished by contacting the cell with a binding compound having a molecular tag attached thereto by a cleavable linkage, and a cleaving probe having a cleavage inducing-moiety; activating the cleaving agent such that, if the binding compound is within an effective proximity of the cleavage-inducing moiety of the cleaving probe, the cleavage-inducing moiety cleaves the cleavable linker so that the molecular tag is released; and quantifying the amount of released molecular tag, thereby detecting the Herl-Herl dimers. In certain embodiments, wherein the binding compound and the cleaving probe each specifically bind Herl. In certain embodiments, binding of a binding compound or a cleaving probe to a Herl monomer precludes binding of another binding compound or cleaving probe to the same Herl monomer.

[0153] In certain embodiments, detecting the Herl-Her3 dimers is accomplished by contacting the cell with a binding compound having a molecular tag attached thereto by a cleavable linkage, and a cleaving probe having a cleavage inducing-moiety; activating the cleaving agent such that, if the binding compound is within an effective proximity of the cleavage-inducing moiety of the cleaving probe, the cleavage-inducing moiety cleaves the cleavable linker so that the molecular tag is released; and quantifying the amount of released molecular tag, thereby detecting the Herl-Her3 dimers. In certain embodiments, the binding compound and the cleaving probe each specifically binds either Herl or Her3. In certain embodiments, the cleaving probe and the binding probe do not both bind the same receptor. [0154] In certain embodiments, detecting the Herl-Her2 dimers is accomplished by contacting the cell with a binding compound having a molecular tag attached thereto by a cleavable linkage, and a cleaving probe having a cleavage inducing-moiety, wherein the binding compound and the cleaving probe each specifically binds either Herl or Her2, and the cleaving probe and the binding probe do not both bind the same receptor, and wherein if the binding compound is within an effective proximity of the cleavage- inducing moiety of the cleaving probe, the cleavage-inducing moiety cleaves the cleavable linker so that the molecular tag is released; and quantifying the amount of released molecular tag, thereby detecting the Herl-Her2 dimers. In certain embodiments, activating the cleaving probe cleaves the cleavable linker. In certain embodiments, the binding compound and the cleaving probe each specifically binds either Herl or Her2. In certain embodiments, the cleaving probe and the binding probe do not both bind the same receptor.

[0155] In certain embodiment, detecting the Her2-Her3 dimers is accomplished by contacting the cell with a binding compound having a molecular tag attached thereto by a cleavable linkage, and a cleaving probe having a cleavage inducing-moiety, wherein the binding compound and the cleaving probe each specifically binds either Her2 or Her3, and the cleaving probe and the binding probe do not both bind the same receptor, and wherein if the binding compound is within an effective proximity of the cleavage- inducing moiety of the cleaving probe, the cleavage-inducing moiety cleaves the cleavable linker so that the molecular tag is released; and quantifying the amount of released molecular tag, thereby detecting the Her2-Her3 dimers. In certain embodiments, activating the cleaving probe cleaves the cleavable linker. In certain embodiments, the binding compound and the cleaving probe each specifically binds either Her2 or Her3. In certain embodiments, the cleaving probe and the binding probe do not both bind the same receptor.

[0156] In certain embodiments, the Herl-Herl dimers, Herl-Her3 dimers, Herl-

Her2 dimers, Her2-Her3 dimers, total Herl, total Her2, and/or total Her3 are detected in a single assay, In certain embodiments, the cancer cell is a breast cancer cell, a lung cancer cell, a colorectal cancer cell, a prostate cancer cell, or an ovarian cancer cell. In a preferred embodiment, the cancer cell is a lung cancer cell. [0157] In certain embodiments, the Her 1 -Her 1 dimers on the cancer cell are detected directly on a patient sample. In certain embodiments, the Herl-Her2 dimers on the cancer cell are detected directly on a patient sample. In certain embodiments, the Herl-Her3 dimers on the cancer cell are detected directly on a patient sample, In certain embodiments, the Her2-Her3 dimers on the cancer cell are detected directly on a patient sample, In certain embodiments, the total Herl expressed on the cancer cell is detected directly on a patient sample. In certain embodiments, the total Her2 expressed on the cancer cell is detected directly on a patient sample, In certain embodiments, the total Her3 expressed on the cancer cell is detected directly on a patient sample. In certain embodiments, the patient sample is a fixed tissue sample, a frozen tissue sample, or a sample purified from circulating epithelial cells.

[0158] In another aspect, the invention provides a method for determining whether a subject with cancer is likely to respond to treatment with a Herl -acting agent, comprising detecting in a biological sample from the subject's cancer at least about 600 Herl -Herl dimers per cancer cell. In certain embodiments, the presence of the at least about 600 Herl -Herl dimers per cancer cell indicates that the cancer is likely to respond to treatment with the Herl -acting agent. In certain embodiments, at least about 1100 Herl -Herl dimers per cancer cell are detected.

[0159] In certain embodiments, the methods further comprise detecting in the biological sample at least about 1000 Herl-Her3 dimers per cancer cell, In certain embodiments, the presence of the at least about 600 Herl -Herl dimers per cancer cell and the at least about 1000 Herl-Her3 dimers indicate that the cancer is likely to respond to treatment with the Herl -acting agent. In certain embodiments, at least about 1800 Herl-Her3 dimers are detected.

[0160] In certain embodiments, the methods further comprise detecting in the biological sample fewer than about 1000 Her2-Her3 dimers per cancer cell, In certain embodiments, the presence of the at least about 600 Herl -Herl dimers per cancer cell and the fewer than about 1000 Herl-Her3 dimers indicate that the cancer is likely to respond to treatment with the Herl -acting agent. In certain embodiments, fewer than about 600 Her2-Her3 dimers are detected. [0161] In certain embodiments, the methods comprise detecting on the cancer cell fewer than about 130 Herl-Her2 dimers, wherein the presence of the fewer than about 130 Herl-Her2 dimers indicates that the cancer is likely to respond to treatment with the Her 1 -acting agent. In certain embodiments, the Her 1 -acting agent is Gefitinib, Tarceva, or erbitux. In a preferred embodiment, the Herl -acting agent is Gefitinib.

[0162] In certain embodiments, a cancer cell that is likely to respond to treatment with a Herl -acting agent has a probability of treatment that is increased about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90, 100%, or more over a reference cancer cell. In certain embodiments, the reference cancer cell is a cancer cell that does not respond to treatment with a Herl -acting agent, In certain embodiments, the reference cancer cell is a cancer cell wherein the responsiveness of the cancer cell to treatment with a Herl -acting agent has not been determined, but is rather the average responsiveness of a cancer cell to treatment with the Herl -acting agent, In certain embodiments, the average responsiveness to treatment with the Herl -acting agent is about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 18%, 20%, or 25%. In certain embodiments, a cancer cell that has been determined to be likely to respond to treatment with a Herl- acting agent is more likely than not to respond to treatment with the Herl -acting agent. In still another aspect, a cancer cell that is likely to respond to treatment with a Herl- acting agent has a about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more probability of responding to treatment with the Herl -acting agent.

[0163] In certain embodiments, fewer than about 130 Herl-Her2 dimers are detected. In certain embodiments, fewer than about 50 Herl-Her2 dimers are detected, In certain embodiments, fewer than about 100 Herl-Her2 dimers are detected. In certain embodiments, fewer than about 200 Herl-Her2 dimers are detected, In certain embodiments, fewer than 300 Herl-Her2 dimers are detected, In certain embodiments, fewer than 400 Herl-Her2 dimers are detected. In certain embodiments, fewer than 500 Herl-Her2 dimers are detected, In certain embodiments, fewer than 600 Herl-Her2 dimers are detected, In certain embodiments, fewer than 800 Herl-Her2 dimers are detected. In certain embodiments, fewer than 900 Herl-Her2 dimers are detected, In certain embodiments, fewer than 1000 Herl-Her2 dimers are detected, In certain embodiments, fewer than 1100 Herl-Her2 dimers are detected, In certain embodiments, fewer than 1200 Herl-Her2 dimers are detected, In certain embodiments, fewer than 1300 Herl-Her2 dimers are detected. In certain embodiments, fewer than 1400 Herl- Her2 dimers are detected. In certain embodiments, fewer than 1500 Herl-Her2 dimers are detected. In certain embodiments, fewer than 1600 Herl-Her2 dimers are detected. In certain embodiments, fewer than 1700 Herl-Her2 dimers are detected. In certain embodiments, fewer than 1800 Herl-Her2 dimers are detected. In certain embodiments, fewer than 2000 Herl-Her2 dimers are detected. In certain embodiments, fewer than 2400 Herl-Her2 dimers are detected. In certain embodiments, fewer than 2700 Herl- Her2 dimers are detected. In certain embodiments, fewer than 3000 Herl-Her2 dimers are detected. In certain embodiments, fewer than 3500 Herl-Her2 dimers are detected. In certain embodiments, fewer than 4000 Herl-Her2 dimers are detected. In certain embodiments, fewer than 5000 Herl-Her2 dimers are detected. In certain embodiments, between about 600 and about 100,000 Herl-Her2 dimers are detected. In certain embodiments, between about 600 and about 10,000 Herl-Her2 dimers are detected. In certain embodiments, between about 600 and about 30,000 Herl-Her2 dimers are detected. In certain embodiments, between about 600 and about 50,000 Herl-Her2 dimers are detected. In certain embodiments, between about 600 and about 70,000 Herl- Her2 dimers are detected. In certain embodiments, between about 600 and about 90,000 Herl-Her2 dimers are detected. In certain embodiments, between about 1100 and about 100,000 Herl-Her2 dimers are detected. In certain embodiments, between about 1100 and about 10,000 Herl-Her2 dimers are detected. In certain embodiments, between about 1100 and about 30,000 Herl-Her2 dimers are detected. In certain embodiments, between about 1100 and about 50,000 Herl-Her2 dimers are detected. In certain embodiments, between about 1100 and about 70,000 Herl-Her2 dimers are detected. In certain embodiments, between about 1100 and about 90,000 Herl-Her2 dimers are detected.

[0164] In certain embodiments, the methods comprise detecting on the cancer cell an amount of Her 1 receptors that results in fewer than about 875 relative fluorescent units determined according to the method of Example 1, wherein the presence of the amount of Her 1 receptors indicates that the cancer is likely to respond to treatment with the Her 1 -acting agent. In certain embodiments, the Her 1 -acting agent is Gefitinib, Tarceva, or erbitux. In a preferred embodiment, the Herl -acting agent is Gefitinib. [0165] One of skill in the art will recognize that the method of determining the amount of Herl receptors described in Example 1 can readily be modified; such modifications are also within the scope of the present invention. For example, the number of fluorescent units can be normalized against any common protein present in the appropriate cell line, including, but not limited to, actin, myosin, cytokeratin, and the like. Further, the signal need not necessarily be a fluorescent signal, but rather any convenient signal for detecting the amount of a receptor that can be normalized against an appropriate protein can be used. For example, the receptor can also be detected with a radiolabeled antibody and normalized against a fluorescent signal, or vice versa. Any such system known by one of skill in the art without limitation can be used to determine the relative amount of Herl, Her2, or Her3 receptor or Her receptor phosphorylation.

[0166] In certain embodiments, an amount of Herl receptors that results in fewer than about 300 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Herl receptors that results in fewer than about 400 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Herl receptors that results in fewer than about 500 relative fluorescent units determined according to the method of Example 1 is detected, In certain embodiments, an amount of Herl receptors that results in fewer than about 600 relative fluorescent units determined according to the method of Example 1 is detected, In certain embodiments, an amount of Herl receptors that results in fewer than about 700 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Herl receptors that results in fewer than about 800 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Herl receptors that results in fewer than about 900 relative fluorescent units determined according to the method of Example 1 is detected, In certain embodiments, an amount of Herl receptors that results in fewer than about 1000 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Herl receptors that results in fewer than about 1100 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Herl receptors that results in fewer than about 1200 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Herl receptors that results in fewer than about 1300 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Her 1 receptors that results in fewer than about 1400 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Herl receptors that results in fewer than about 1500 relative fluorescent units determined according to the method of Example 1 is detected, In certain embodiments, an amount of Herl receptors that results in fewer than about 1600 relative fluorescent units determined according to the method of Example 1 is detected, In certain embodiments, an amount of Herl receptors that results in fewer than about 1700 relative fluorescent units determined according to the method of Example 1 is detected, In certain embodiments, an amount of Herl receptors that results in fewer than about 1800 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Herl receptors that results in fewer than about 1900 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Herl receptors that results in fewer than about 2000 relative fluorescent units determined according to the method of Example 1 is detected, In certain embodiments, an amount of Herl receptors that results in fewer than about 2500 relative fluorescent units determined according to the method of Example 1 is detected, In certain embodiments, an amount of Herl receptors that results in fewer than about 3000 relative fluorescent units determined according to the method of Example 1 is detected, In certain embodiments, an amount of Herl receptors that results in fewer than about 3500 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Herl receptors that results in fewer than about 4000 relative fluorescent units determined according to the method of Example 1 is detected, In certain embodiments, an amount of Herl receptors that results in fewer than about 4500 relative fluorescent units determined according to the method of Example 1 is detected, In certain embodiments, an amount of Herl receptors that results in fewer than about 5000 relative fluorescent units determined according to the method of Example 1 is detected, In certain embodiments, an amount of Herl receptors that results in between about 300 and about 100,000 relative fluorescence units is detected. In certain embodiments, an amount of Herl receptors that results in between about 300 and about 10,000 relative fluorescence units is detected, In certain embodiments, an amount of Herl receptors that results in between about 300 and about 30,000 relative fluorescence units is detected, In certain embodiments, an amount of Herl receptors that results in between about 300 and about 50,000 relative fluorescence units is detected. In certain embodiments, an amount of Herl receptors that results in between about 300 and about 70,000 relative fluorescence units is detected, In certain embodiments, an amount of Herl receptors that results in between about 300 and about 90,000 relative fluorescence units is detected, In certain embodiments, an amount of Herl receptors that results in between about 800 and about 100,000 relative fluorescence units is detected. In certain embodiments, an amount of Herl receptors that results in between about 800 and about 10,000 relative fluorescence units is detected, In certain embodiments, an amount of Herl receptors that results in between about 800 and about 30,000 relative fluorescence units is detected. In certain embodiments, an amount of Herl receptors that results in between about 800 and about 50,000 relative fluorescence units is detected. In certain embodiments, an amount of Herl receptors that results in between about 800 and about 70,000 relative fluorescence units is detected. In certain embodiments, an amount of Herl receptors that results in between about 800 and about 90,000 relative fluorescence units is detected.

[0167] In certain embodiments, detecting the amount of Herl receptors dimers is accomplished by contacting the cell with a binding compound having a molecular tag attached thereto by a cleavable linkage, and a cleaving probe having a cleavage inducing- moiety; activating the cleaving agent such that, if the binding compound is within an effective proximity of the cleavage-inducing moiety of the cleaving probe, the cleavage- inducing moiety cleaves the cleavable linker so that the molecular tag is released; and quantifying the amount of released molecular tag, thereby detecting the amount of Herl receptors. In certain embodiments, the binding compound and the cleaving probe each specifically binds Herl . In certain embodiments, the binding compound and the cleaving probe bind different epitopes of Herl.

[0168] In certain embodiments, the methods comprise detecting on the cancer cell an amount of Her2 receptors that results in fewer than about 4000 relative fluorescent units determined according to the method of Example 1, wherein the presence of the amount of Her2 receptors indicates that the cancer is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the Herl -acting agent is Gefitinib, Tarceva, or erbitux. In a preferred embodiment, the Herl -acting agent is Gefitinib. [0169] In certain embodiments, an amount of Her2 receptors that results in fewer than about 500 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Her2 receptors that results in fewer than about 800 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Her2 receptors that results in fewer than about 1000 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Her2 receptors that results in fewer than about 1250 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Her2 receptors that results in fewer than about 1500 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Her2 receptors that results in fewer than about 1750 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Her2 receptors that results in fewer than about 2000 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Her2 receptors that results in fewer than about 2500 relative fluorescent units determined according to the method of Example 1 is detected, In certain embodiments, an amount of Her2 receptors that results in fewer than about 3000 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Her2 receptors that results in fewer than about 3250 relative fluorescent units determined according to the method of Example 1 is detected, In certain embodiments, an amount of Her2 receptors that results in fewer than about 3500 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Her2 receptors that results in fewer than about 3600 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Her2 receptors that results in fewer than about 3700 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Her2 receptors that results in fewer than about 3800 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Her2 receptors that results in fewer than about 3900 relative fluorescent units determined according to the method of Example 1 is detected, In certain embodiments, an amount of Her2 receptors that results in fewer than about 4000 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Her2 receptors that results in fewer than about 4100 relative fluorescent units deteπnined according to the method of Example 1 is detected. In certain embodiments, an amount of Her2 receptors that results in fewer than about 4200 relative fluorescent units deteπnined according to the method of Example 1 is detected. In certain embodiments, an amount of Her2 receptors that results in fewer than about 4300 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Her2 receptors that results in fewer than about 4400 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Her2 receptors that results in fewer than about 4500 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Her2 receptors that results in fewer than about 4750 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Her2 receptors that results in fewer than about 5000 relative fluorescent units determined according to the method of Example 1 is detected, In certain embodiments, an amount of Her2 receptors that results in fewer than about 7500 relative fluorescent units determined according to the method of Example 1 is detected, In certain embodiments, an amount of Her2 receptors that results in fewer than about 10000 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Her2 receptors that results in fewer than about 15000 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Her2 receptors that results in fewer than about 20000 relative fluorescent units determined according to the method of Example 1 is detected. In certain embodiments, an amount of Her2 receptors that results in between about 300 and about 100,000 relative fluorescence units is detected. In certain embodiments, an amount of Her2 receptors that results in between about 300 and about 10,000 relative fluorescence units is detected. In certain embodiments, an amount of Her2 receptors that results in between about 300 and about 30,000 relative fluorescence units is detected. In certain embodiments, an amount of Her2 receptors that results in between about 300 and about 50,000 relative fluorescence units is detected. In certain embodiments, an amount of Her2 receptors that results in between about 300 and about 70,000 relative fluorescence units is detected. In certain embodiments, an amount of Her2 receptors that results in between about 300 and about 90,000 relative fluorescence units is detected. In certain embodiments, an amount of Her2 receptors that results in between about 800 and about 100,000 relative fluorescence units is detected. In certain embodiments, an amount of Her2 receptors that results in between about 800 and about 10,000 relative fluorescence units is detected. In certain embodiments, an amount of Her2 receptors that results in between about 800 and about 30,000 relative fluorescence units is detected. In certain embodiments, an amount of Her2 receptors that results in between about 800 and about 50,000 relative fluorescence units is detected. In certain embodiments, an amount of Her2 receptors that results in between about 800 and about 70,000 relative fluorescence units is detected. In certain embodiments, an amount of Her2 receptors that results in between about 800 and about 90,000 relative fluorescence units is detected.

[0170] In certain embodiments, detecting the amount of Her2 receptors dimers is accomplished by contacting the cell with a binding compound having a molecular tag attached thereto by a cleavable linkage, and a cleaving probe having a cleavage inducing- moiety; activating the cleaving agent such that, if the binding compound is within an effective proximity of the cleavage-inducing moiety of the cleaving probe, the cleavage- inducing moiety cleaves the cleavable linker so that the molecular tag is released; and quantifying the amount of released molecular tag, thereby detecting the amount of Her2 receptors. In certain embodiments, the binding compound and the cleaving probe each specifically binds Her2. In certain embodiments, the binding compound and the cleaving probe bind different epitopes of Her2.

[0171] In certain embodiments, the invention provides a method for determining whether a cancer cell is likely to respond to treatment with a Her 1 -acting agent, comprising detecting on the cancer cell at least about 1600 Her 1 -Her 1 dimers or at least about 850 Herl-Her3 dimers, and fewer than about 600 Her2-Her3 dimers, wherein the presence of the 1600 Herl-Herl dimers or at least about 850 Herl-Her3 dimers and fewer than about 600 Her2-Her3 dimers indicates that the cancer is likely to respond to treatment with the Her 1 -acting agent. In certain embodiments, the Her 1 -acting agent is Gefitinib, tarceva, or erbitux. In certain embodiments, the Her 1 -acting agent is Gefitinib.

[0172] In certain embodiments, at least about 750 Herl-Herl dimers are detected.

In certain embodiments, at least about 800 Herl-Herl dimers are detected, In certain embodiments, at least about 900 Herl-Herl dimers are detected, In certain embodiments, at least about 1000 Herl-Herl dimers are detected, In certain embodiments, at least about 1100 Herl-Herl dimers are detected. In certain embodiments, at least about 1200 Herl-Herl dimers are detected. In certain embodiments, at least about 1300 Herl-Herl dimers are detected. In certain embodiments, at least about 1325 Herl-Herl dimers are detected. In certain embodiments, between about 600 and about 100,000 Herl-Herl dimers are detected.

[0173] In certain embodiments, detecting the Herl-Herl dimers is accomplished by contacting the cell with a binding compound having a molecular tag attached thereto by a cleavable linkage, and a cleaving probe having a cleavage inducing-moiety, wherein the binding compound and the cleaving probe each specifically bind Herl, and wherein binding of a binding compound or a cleaving probe to a Herl monomer precludes binding of another binding compound or cleaving probe to the same Herl monomer, and wherein if the binding compound is within an effective proximity of the cleavage- inducing moiety of the cleaving probe, the cleavage-inducing moiety cleaves the cleavable linker so that the molecular tag is released; and quantifying the amount of released molecular tag, thereby detecting the Herl-Herl dimers.

[0174] In certain embodiments, activating the cleavage-inducing moiety cleaves the cleavable linker. In certain embodiments, the binding compound and the cleaving probe each specifically binds a Herl epitope. In certain embodiments, the binding compound and the cleaving probe each specifically binds an identical Herl epitope, In certain embodiments, the binding compound and the cleaving probe each comprises an antibody or antigen-binding fragment, In certain embodiments, the binding compound and the cleaving probe each comprises an antibody or antigen-binding fragment. In certain embodiments, the binding compound and the cleaving probe each specifically binds a Herl ligand binding site, In certain embodiments, the binding compound and the cleaving probe each comprises a Herl ligand.

[0175] In certain embodiments, the cancer cell is a breast cancer cell, lung cancer cell, colorectal cancer cell, prostate cancer cell, or ovarian cancer cell. In certain embodiments, the cancer cell is a lung cancer cell, In certain embodiments, the Herl- Herl dimers on the cancer cell are detected directly on a patient sample, the patient sample is a fixed tissue sample, a frozen tissue sample, or a sample purified from circulating epithelial cells, In certain embodiments, the patient sample is a lung tissue sample, a breast tissue sample, a colorectal tissue sample, a prostate tissue sample, or an ovarian tissue sample. In certain embodiments, the patient sample is a lung tissue sample. In certain embodiments, the cancer cell is obtained from a biological sample of a subject having or suspected of having a cancer.

[0176] In certain embodiments, detecting the Herl-Her3 dimers is accomplished by contacting the cell with a binding compound having a molecular tag attached thereto by a cleavable linkage, and a cleaving probe having a cleavage inducing-moiety, wherein the binding compound and the cleaving probe each specifically binds either Herl or Her3, and the cleaving probe and the binding probe do not both bind the same receptor, and wherein if the binding compound is within an effective proximity of the cleavage- inducing moiety of the cleaving probe, the cleavage-inducing moiety cleaves the cleavable linker so that the molecular tag is released; and quantifying the amount of released molecular tag, thereby detecting the Herl-Her3 dimers. In certain embodiments, activating the cleavage-inducing moiety cleaves the cleavable linker.

[0177] In certain embodiments, the binding compound specifically binds a Herl epitope, In certain embodiments, the binding compound comprises an antibody or antigen-binding fragment. In certain embodiments, the binding compound specifically binds a Herl ligand binding site. In certain embodiments, the binding compound comprises a Herl ligand. In certain embodiments, the binding compound specifically binds a Her3 epitope, In certain embodiments, the binding compound specifically binds a Her3 ligand binding site. In certain embodiments, the binding compound comprises a Her3 ligand. In certain embodiments, the cleaving probe specifically binds a Herl epitope. In certain embodiments, the cleaving probe comprises an antibody or antigen- binding fragment, In certain embodiments, the cleaving probe specifically binds a Herl ligand binding site. In certain embodiments, the cleaving probe comprises a Herl ligand. In certain embodiments, the cleaving probe specifically binds a Her3 epitope. In certain embodiments, the cleaving probe specifically binds a Her3 ligand binding site. In certain embodiments, the cleaving probe comprises a Her3 ligand.

[0178] In certain embodiments, detecting the Her2-Her3 dimers is accomplished by contacting the cell with a binding compound having a molecular tag attached thereto by a cleavable linkage, and a cleaving probe having a cleavage inducing-moiety, wherein the binding compound and the cleaving probe each specifically binds either Her2 or Her3, and the cleaving probe and the binding probe do not both bind the same receptor, and wherein if the binding compound is within an effective proximity of the cleavage-inducing moiety of the cleaving probe, the cleavage-inducing moiety cleaves the cleavable linker so that the molecular tag is released; and quantifying the amount of released molecular tag, thereby detecting the Her2-Her3 dimers.

[0179] In certain embodiments, activating the cleavage-inducing moiety cleaves the cleavable linker. In certain embodiments, the binding compound specifically binds a Her2 epitope. In certain embodiments, the binding compound comprises an antibody or antigen-binding fragment. In certain embodiments, the binding compound specifically binds a Her2 ligand binding site. In certain embodiments, the binding compound comprises a Her2 ligand. In certain embodiments, the binding compound specifically binds a Her3 epitope. In certain embodiments, the binding compound specifically binds a Her3 ligand binding site. In certain embodiments, the binding compound comprises a Her3 ligand. In certain embodiments, the cleaving probe specifically binds a Her2 epitope. In certain embodiments, the cleaving probe comprises an antibody or antigen- binding fragment, In certain embodiments, the cleaving probe specifically binds a Her2 ligand binding site. In certain embodiments, the cleaving probe comprises a Her2 ligand. In certain embodiments, the cleaving probe specifically binds a Her3 epitope, In certain embodiments, the cleaving probe specifically binds a Her3 ligand binding site, In certain embodiments, the cleaving probe comprises a Her3 ligand.

[0180] In certain embodiments, at least about 1700 Herl-Herl dimers are detected.

In certain embodiments, at least about 1800 Herl-Herl dimers are detected, In certain embodiments, at least about 1900 Herl-Herl dimers are detected. In certain embodiments, at least about 2000 Herl-Herl dimers are detected. In certain embodiments, at least about 2100 Herl-Herl dimers are detected, In certain embodiments, at least about 2200 Herl-Herl dimers are detected, In certain embodiments, at least about 2300 Herl-Herl dimers are detected. In certain embodiments, at least about 2400 Herl-Herl dimers are detected, In certain embodiments, at least about 2500 Herl-Herl dimers are detected, In certain embodiments, at least about 2600 Herl-Herl dimers are detected, In certain embodiments, at least about 2700 Herl-Herl dimers are detected. Li certain embodiments, at least about 2800 Herl-Herl dimers are detected. In certain embodiments, at least about 2900 Herl-Herl dimers are detected. In certain embodiments, at least about 3000 Herl-Herl dimers are detected.

[0181] In certain embodiments, at least about 900 Herl-Her3 dimers are detected.

In certain embodiments, at least about 1000 Herl-Her3 dimers are detected. In certain embodiments, at least about 1100 Herl-Her3 dimers are detected. In certain embodiments, at least about 1200 Herl-Her3 dimers are detected. In certain embodiments, at least about 1300 Herl-Her3 dimers are detected. In certain embodiments, at least about 1400 Herl-Her3 dimers are detected. In certain embodiments, at least about 1500 Herl-Her3 dimers are detected. In certain embodiments, at least about 1600 Herl-Her3 dimers are detected. In certain embodiments, at least about 1700 Herl-Her3 dimers are detected. In certain embodiments, at least about 1800 Herl-Her3 dimers are detected. In certain embodiments, at least about 1900 Herl-Her3 dimers are detected. In certain embodiments, at least about 2000 Herl-Her3 dimers are detected.

[0182] In certain embodiments, fewer than about 550 Her2-Her3 dimers are detected. In certain embodiments, fewer than about 500 Her2-Her3 dimers are detected. In certain embodiments, fewer than about 450 Her2-Her3 dimers are detected. In certain embodiments, fewer than about 400 Her2-Her3 dimers are detected. In certain embodiments, fewer than about 350 Her2-Her3 dimers are detected. In certain embodiments, fewer than about 300 Her2-Her3 dimers are detected. In certain embodiments, fewer than about 250 Her2-Her3 dimers are detected. In certain embodiments, fewer than about 200 Her2-Her3 dimers are detected. In certain embodiments, fewer than about 150 Her2-Her3 dimers are detected. In certain embodiments, fewer than about 100 Her2-Her3 dimers are detected. In certain embodiments, fewer than about 50 Her2-Her3 dimers are detected.

[0183] In another aspect, the invention provides a method for determining whether a cancer cell is likely to respond to treatment with a Herl -acting agent, comprising detecting on the cancer cell an amount of one or more ErbB dimers described herein as indicating that the cancer is likely to respond to treatment with the Herl -acting agent and detecting a mutation in a gene that is associated with responsiveness to treatment with a Herl-acting agent. For example, certain mutations in EGFR (Herl) are correlated with responsiveness to treatment with an exemplary Herl-acting agent, Gefitinib.

[0184] In certain embodiments, an amount of ErbB dimers that indicates that the cancer cell is not likely to respond to treatment is detected and a mutation associated with responsiveness is detected, and the cancer is determined to be likely to be respond to treatment. In certain embodiments, an amount of ErbB dimers that indicates that the cancer cell is not likely to respond to treatment is detected and a mutation associated with responsiveness is detected, and the cancer is determined to be not likely to be respond to treatment. In certain embodiments, an amount of ErbB dimers that indicates that the cancer cell is likely to respond to treatment is detected and a mutation associated with responsiveness is not detected, and the cancer is determined to be likely to be respond to treatment. In certain embodiments, an amount of ErbB dimers that indicates that the cancer cell is likely to respond to treatment is detected and a mutation associated with responsiveness not is detected, and the cancer is determined to be not likely to be respond to treatment.

[0185] In another aspect, the invention provides a method for determining whether a cancer cell is likely to respond to treatment with a Herl-acting agent, comprising detecting on the cancer cell an amount of one or more ErbB dimers described herein as indicating that the cancer is likely to respond to treatment with the Herl-acting agent and detecting a mutation in a gene that is associated with non-responsiveness to treatment with a Herl-acting agent. For example, certain mutations in KRAS are correlated with non-responsiveness to treatment with an exemplary Herl-acting agent, Gefitinib. (Lynch, T. et al, 2004, N. Engl. J. Med. 350: 1-11; Pao, W. et al, 2005, PIoS. 2:57-61; Gumerlock, P.H. et al, ASCO 2005, Abst. 7008)

[0186] In certain embodiments, an amount of ErbB dimers that indicates that the cancer cell is not likely to respond to treatment is detected and a mutation associated with non-responsiveness is detected, and the cancer is determined to be likely to be respond to treatment. In certain embodiments, an amount of ErbB dimers that indicates that the cancer cell is not likely to respond to treatment is detected and a mutation associated with non-responsiveness is detected, and the cancer is determined to be not likely to be respond to treatment. In certain embodiments, an amount of ErbB dimers that indicates that the cancer cell is likely to respond to treatment is detected and a mutation associated with non-responsiveness is not detected, and the cancer is determined to be likely to be respond to treatment. In certain embodiments, an amount of ErbB dimers that indicates that the cancer cell is likely to respond to treatment is detected and a mutation associated with non-responsiveness not is detected, and the cancer is determined to be not likely to be respond to treatment.

[0187] In another aspect, the invention provides a method for determining whether a cancer cell is likely to respond to treatment with a Her 1 -acting agent, comprising detecting on the cancer cell (i) fewer than about 230 Her2-Her3 dimers, and (ii) at least about 500 Herl-Herl dimers and fewer than about 220 Herl-Her2 dimers or at least about 1600 Herl-Herl dimers and fewer than about 150 Herl-Her3 dimers, wherein satisfaction of conditions (i) and (ii) indicates that the cancer cell is likely to respond to treatment with the Her 1 -acting agent. In certain embodiments, the Her 1 -acting agent is gefitinib, tarceva, or erbitux. In certain embodiments, the Her 1 -acting agent is Gefitinib.

[0188] In certain embodiments, fewer than about 200 Her2-Her3 dimers are detected. In certain embodiments, fewer than about 150 Her2-Her3 dimers are detected. In certain embodiments, fewer than about 100 Her2-Her3 dimers are detected, In certain embodiments, fewer than about 50 Her2-Her3 dimers are detected. In certain embodiments, no Her2-Her3 dimers are detected.

[0189] In certain embodiments, at least about 500 Herl-Herl dimers are detected.

In certain embodiments, at least about 600 Herl-Herl dimers are detected, In certain embodiments, at least about 700 Herl-Herl dimers are detected, In certain embodiments, at least about 750 Herl-Herl dimers are detected. In certain embodiments, at least about 900 Herl-Herl dimers are detected. In certain embodiments, at least about 1000 Herl-Herl dimers are detected. In certain embodiments, at least about 1100 Herl-Herl dimers are detected, In certain embodiments, at least about 1200 Herl-Herl dimers are detected. Li certain embodiments, at least about 1300 Herl-Herl dimers are detected. In certain embodiments, at least about 1400 Herl-Herl dimers are detected, In certain embodiments, at least about 1500 Herl-Herl dimers are detected, In certain embodiments, at least about 1600 Herl-Herl dimers are detected, In certain embodiments, at least about 1700 Her 1 -Her 1 dimers are detected. In certain embodiments, at least about 1800 Herl-Herl dimers are detected. In certain embodiments, at least about 1900 Herl-Herl dimers are detected. In certain embodiments, at least about 2000 Herl-Herl dimers are detected. In certain embodiments, at least about 2500 Herl-Herl dimers are detected. In certain embodiments, at least about 3000 Herl-Herl dimers are detected.

[0190] In certain embodiments, fewer than about 200 Herl-Her2 dimers are detected. In certain embodiments, fewer than about 150 Herl-Her2 dimers are detected. In certain embodiments, fewer than about 100 Herl-Her2 dimers are detected. In certain embodiments, fewer than about 50 Herl-Her2 dimers are detected. In certain embodiments, no Herl-Her2 dimers are detected.

[0191] In certain embodiments, fewer than about 100 Herl-Her3 dimers are detected. In certain embodiments, fewer than about 50 Herl-Her3 dimers are detected. In certain embodiments, no Herl-Her3 dimers are detected.

[0192] In certain embodiments, detecting the Herl-Herl dimers is accomplished by contacting the cell with (i) a binding compound having a molecular tag attached thereto by a cleavable linkage, and (ii) a cleaving probe having a cleavage inducing- moiety, wherein the binding compound and the cleaving probe each specifically bind Herl, and wherein binding of a binding compound or a cleaving probe to a Herl monomer precludes binding of another binding compound or cleaving probe to the same Herl monomer, and wherein if the binding compound is within an effective proximity of the cleavage-inducing moiety of the cleaving probe, the cleavage-inducing moiety cleaves the cleavable linker so that the molecular tag is released; and quantifying the amount of released molecular tag, thereby detecting the Herl-Herl dimers.

[0193] In certain embodiments, activating the cleavage-inducing moiety cleaves the cleavable linker. In certain embodiments, the binding compound and the cleaving probe each specifically binds a Herl epitope. In certain embodiments, the binding compound and the cleaving probe each specifically binds an identical Herl epitope, In certain embodiments, the binding compound and the cleaving probe each comprises an antibody or antigen-binding fragment. In certain embodiments, the binding compound and the cleaving probe each comprises an antibody or antigen-binding fragment. In certain embodiments, the binding compound and the cleaving probe each specifically binds a Herl ligand binding site. In certain embodiments, the binding compound and the cleaving probe each comprises a Herl ligand.

[0194] In certain embodiments, the cancer cell is a breast cancer cell, lung cancer cell, colorectal cancer cell, prostate cancer cell, or ovarian cancer cell. In certain embodiments, the cancer cell is a lung cancer cell, In certain embodiments, the Herl- Herl dimers, Herl-Her2 dimers, Herl-Her3 dimers, and/or Her2-Her3 dimers on the cancer cell are detected directly on a patient sample.

[0195] In certain embodiments, the patient sample is a fixed tissue sample, a frozen tissue sample, or a sample purified from circulating epithelial cells. In certain embodiments, the patient sample is a lung tissue sample, a breast tissue sample, a colorectal tissue sample, a prostate tissue sample, or an ovarian tissue sample. In certain embodiments, the patient sample is a lung tissue sample. In certain embodiments, the cancer cell is obtained from a biological sample of a subject having or suspected of having a cancer.

[0196] In certain embodiments, detecting the Her2-Her3 dimers is accomplished by contacting the cell with (i) a binding compound having a molecular tag attached thereto by a cleavable linkage, and. (H) a cleaving probe having a cleavage inducing- moiety, wherein the binding compound and the cleaving probe each specifically binds either Her2 or Her3, and the cleaving probe and the binding probe do not both bind the same receptor, and wherein if the binding compound is within an effective proximity of the cleavage-inducing moiety of the cleaving probe, the cleavage-inducing moiety cleaves the cleavable linker so that the molecular tag is released; and quantifying the amount of released molecular tag, thereby detecting the Her2-Her3 dimers.

[0197] In certain embodiments, activating the cleavage-inducing moiety cleaves the cleavable linker, In certain embodiments, the binding compound specifically binds a Her2 epitope. In certain embodiments, the binding compound comprises an antibody or antigen-binding fragment, In certain embodiments, the binding compound specifically binds a Her2 ligand binding site, In certain embodiments, the binding compound comprises a Her2 ligand. In certain embodiments, the binding compound specifically binds a Her3 epitope. In certain embodiments, the binding compound specifically binds a Her3 ligand binding site. In certain embodiments, the binding compound comprises a Her3 ligand.

[0198] In certain embodiments, the cleaving probe specifically binds a Her2 epitope. In certain embodiments, the cleaving probe comprises an antibody or antigen- binding fragment. In certain embodiments, the cleaving probe specifically binds a Her2 ligand binding site. In certain embodiments, the cleaving probe comprises a Her2 ligand. In certain embodiments, the cleaving probe specifically binds a Her3 epitope. In certain embodiments, the cleaving probe specifically binds a Her3 ligand binding site. In certain embodiments, the cleaving probe comprises a Her3 ligand.

[0199] In certain embodiments, detecting the Herl-Her3 dimers is accomplished by contacting the cell with (i) a binding compound having a molecular tag attached thereto by a cleavable linkage, and (ii) a cleaving probe having a cleavage inducing- moiety, wherein the binding compound and the cleaving probe each specifically binds either Herl or Her3, and the cleaving probe and the binding probe do not both bind the same receptor, and wherein if the binding compound is within an effective proximity of the cleavage-inducing moiety of the cleaving probe, the cleavage-inducing moiety cleaves the cleavable linker so that the molecular tag is released; and quantifying the amount of released molecular tag, thereby detecting the Herl-Her3 dimers.

[0200] In certain embodiments, activating the cleavage-inducing moiety cleaves the cleavable linker. In certain embodiments, the binding compound specifically binds a Herl epitope. In certain embodiments, the binding compound comprises an antibody or antigen-binding fragment, In certain embodiments, the binding compound specifically binds a Herl ligand binding site. In certain embodiments, the binding compound comprises a Herl ligand. In certain embodiments, the binding compound specifically binds a Her3 epitope. In certain embodiments, the binding compound specifically binds a Her3 ligand binding site. In certain embodiments, the binding compound comprises a Her3 ligand.

[0201] In certain embodiments, the cleaving probe specifically binds a Herl epitope. In certain embodiments, the cleaving probe comprises an antibody or antigen- binding fragment, hi certain embodiments, the cleaving probe specifically binds a Herl ligand binding site. In certain embodiments, the cleaving probe comprises a Herl ligand. In certain embodiments, the cleaving probe specifically binds a Her3 epitope. In certain embodiments, the cleaving probe specifically binds a Her3 ligand binding site. In certain embodiments, the cleaving probe comprises a Her3 ligand.

[0202] In certain embodiments, detecting the Herl-Her2 dimers is accomplished by contacting the cell with (i) a binding compound having a molecular tag attached thereto by a cleavable linkage, and (ii) a cleaving probe having a cleavage inducing- moiety, wherein the binding compound and the cleaving probe each specifically binds either Herl or Her2, and the cleaving probe and the binding probe do not both bind the same receptor, and wherein if the binding compound is within an effective proximity of the cleavage-inducing moiety of the cleaving probe, the cleavage-inducing moiety cleaves the cleavable linker so that the molecular tag is released; and quantifying the amount of released molecular tag, thereby detecting the Herl-Her2 dimers. In certain embodiments, activating the cleavage-inducing moiety cleaves the cleavable linker.

[0203] In certain embodiments, the binding compound specifically binds a Herl epitope. In certain embodiments, the binding compound comprises an antibody or antigen-binding fragment. In certain embodiments, the binding compound specifically binds a Herl ligand binding site. In certain embodiments, the binding compound comprises a Herl ligand. In certain embodiments, the binding compound specifically binds a Her2 epitope. In certain embodiments, the binding compound specifically binds a Her2 ligand binding site. In certain embodiments, the binding compound comprises a Her2 ligand.

[0204] In certain embodiments, the cleaving probe specifically binds a Herl epitope. In certain embodiments, the cleaving probe comprises an antibody or antigen- binding fragment. In certain embodiments, the cleaving probe specifically binds a Herl ligand binding site, m certain embodiments, the cleaving probe comprises a Herl ligand. In certain embodiments, the cleaving probe specifically binds a Her2 epitope. In certain embodiments, the cleaving probe specifically binds a Her2 ligand binding site. In certain embodiments, the cleaving probe comprises a Her2 ligand.

[0205] In certain embodiments, the Herl -Herl dimers, Herl-Her3 dimers, and

Her2-Her3 dimers are detected in a single assay. [0206] In another aspect, the invention provides a method for determining whether a cancer cell is likely to respond to treatment with a Her 1 -acting agent, comprising detecting on the cancer cell (i) fewer than about 1000 Her2-Her3 dimers, wherein the presence of fewer than about 230 Her2-Her3 dimers indicates that the cancer cell is likely to respond to treatment with the Her 1 -acting agent. In certain embodiments, the Her 1 -acting agent is gefitinib, tarceva, or erbitux. In certain embodiments, the Herl- acting agent is Gefitinib.

[0207] In certain embodiments, fewer than about 1000 Her2-Her3 dimers are detected. In certain embodiments, fewer than about 900 Her2-Her3 dimers are detected. In certain embodiments, fewer than about 800 Her2-Her3 dimers are detected. In certain embodiments, fewer than about 700 Her2-Her3 dimers are detected. In certain embodiments, fewer than about 600 Her2-Her3 dimers are detected. In certain embodiments, fewer than about 500 Her2-Her3 dimers are detected. In certain embodiments, fewer than about 400 Her2-Her3 dimers are detected. In certain embodiments, fewer than about 300 Her2-Her3 dimers are detected. In certain embodiments, fewer than about 200 Her2-Her3 dimers are detected. In certain embodiments, fewer than about 100 Her2-Her3 dimers are detected. In certain embodiments, fewer than about 50 Her2-Her3 dimers are detected. In certain embodiments, no Her2-Her3 dimers are detected.

[0208] In another aspect, the invention provides a method for determining whether a cancer cell is likely to respond to treatment with a Herl -acting agent, comprising detecting on a cell of the cancer an amount of Herl -Herl dimers that indicates that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl - Her2 dimers that indicates that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl-Her3 dimers that indicates that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Her2-Her3 dimers that indicates that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl receptors that indicates that the cancer cell is likely to respond to treatment with the Her 1 -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Her2 receptors that indicates that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Her3 receptors that indicates that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl - Herl dimers and Herl-Her2 dimers that together indicate that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl -Herl dimers and Herl- Her3 dimers that together indicate that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl-Her2 dimers and Herl-Her3 dimers that together indicate that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl -Herl dimers, Herl-Her2 dimers, and Herl-Her3 dimers that together indicate that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl -Herl dimers and an amount of Herl receptors that together indicate that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl- Herl dimers and an amount of Her2 receptors that together indicate that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl-Herl dimers and an amount of Her3 receptors that together indicate that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl-Her2 dimers and an amount of Herl receptors that together indicate that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl-Her2 dimers and an amount of Her2 receptors that together indicate that the cancer cell is likely to respond to treatment with the Herl- acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl-Her2 dimers and an amount of Her3 receptors that together indicate that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl-Her3 dimers and an amount of Her 1 receptors that together indicate that the cancer cell is likely to respond to treatment with the Her 1 -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Her 1- Her3 dimers and an amount of Her2 receptors that together indicate that the cancer cell is likely to respond to treatment with the Her 1 -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl-Her3 dimers and an amount of Her3 receptors that together indicate that the cancer cell is likely to respond to treatment with the Her 1 -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Her 1 -Her 1 dimers and Herl-Her2 dimers and an amount of Herl receptors that together indicate that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl -Herl dimers and Herl- Her2 dimers and an amount of Her2 receptors that together indicate that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl -Herl dimers and Herl-Her2 dimers and an amount of Her3 receptors that together indicate that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl-Herl dimers and Herl-Her3 dimers and an amount of Herl receptors that together indicate that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl- Herl dimers and Herl-Her3 dimers and an amount of Her2 receptors that together indicate that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl-Herl dimers and Herl-Her3 dimers and an amount of Her3 receptors that together indicate that the cancer cell is likely to respond to treatment with the Herl- acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl-Her2 dimers and Herl-Her3 dimers and an amount of Herl receptors that together indicate that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl-Her2 dimers and Herl-Her3 dimers and an amount of Her2 receptors that together indicate that the cancer cell is likely to respond to treatment with the Her 1 -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl-Her2 dimers and Herl-Her3 dimers and an amount of Her3 receptors that together indicate that the cancer cell is likely to respond to treatment with the Her 1 -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl-Herl dimers, Herl-Her2 dimers, and Herl-Her3 dimers and an amount of Herl receptors that together indicate that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl-Herl dimers, Herl-Her2 dimers, and Herl-Her3 dimers and an amount of Her2 receptors that together indicate that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl-Herl dimers, Herl-Her2 dimers, and Herl-Her3 dimers and an amount of Her3 receptors that together indicate that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl-Herl dimers, Herl-Her2 dimers, and Herl-Her3 dimers and an amount of Herl receptors and Her2 receptors that together indicate that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl-Herl dimers, Herl-Her2 dimers, and Herl-Her3 dimers and an amount of Herl receptors and Her3 receptors that together indicate that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl-Herl dimers, Herl-Her2 dimers, and Herl-Her3 dimers and an amount of Her2 receptors and Her3 receptors that together indicate that the cancer cell is likely to respond to treatment with the Herl- acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl-Herl dimers and an amount of Herl receptors and Her2 receptors that together indicate that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl-Herl dimers and an amount of Herl receptors and Her3 receptors that together indicate that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl-Herl dimers and an amount of Her2 receptors and Her3 receptors that together indicate that the cancer cell is likely to respond to treatment with the Her 1 -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl-Her2 dimers and an amount of Herl receptors and Her2 receptors that together indicate that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl-Her2 dimers and an amount of Herl receptors and Her3 receptors that together indicate that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl-Her2 dimers and an amount of Her2 receptors and Her3 receptors that together indicate that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl-Her3 dimers and an amount of Herl receptors and Her2 receptors that together indicate that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl-Her3 dimers and an amount of Herl receptors and Her3 receptors that together indicate that the cancer cell is likely to respond to treatment with the Herl -acting agent. In certain embodiments, the methods comprise detecting on a cell of the cancer an amount of Herl-Her3 dimers and an amount of Her2 receptors and Her3 receptors that together indicate that the cancer cell is likely to respond to treatment with the Herl -acting agent.

[0209] In certain embodiments, the methods comprise detecting the amount of

Herl -Herl dimers on a cancer cell present on a cancer cell, then detecting the amount of Herl-Her2 dimers on the cancer cell, In certain embodiments, the methods comprise detecting the amount of Herl -Herl dimers on a cancer cell present on a cancer cell, then detecting the amount of Herl-Her3 dimers on the cancer cell. In certain embodiments, the methods comprise detecting the amount of Herl-Her2 dimers on a cancer cell present on a cancer cell, then detecting the amount of Herl-Her3 dimers on the cancer cell, In certain embodiments, the methods comprise detecting the amount of Herl-Her3 dimers on a cancer cell present on a cancer cell, then detecting the amount of Herl -Herl dimers on the cancer cell, In certain embodiments, the methods comprise detecting the amount of Herl -Her 3 dimers on a cancer cell present on a cancer cell, then detecting the amount of Herl-Her2 dimers on the cancer cell, In certain embodiments, the methods comprise detecting the amount of Herl-Her2 dimers on a cancer cell present on a cancer cell, then detecting the amount of Her 1 -Her 1 dimers on the cancer cell. In certain embodiments, the methods comprise detecting the amount of Herl-Her2 dimers on a cancer cell present on a cancer cell, then detecting the amount of Herl-Her3 dimers on the cancer cell, In certain embodiments, the methods comprise detecting the amount of Her 1 -Her 1 dimers on a cancer cell present on a cancer cell, then detecting the amount of Her2-Her3 dimers on the cancer cell. In certain embodiments, the methods comprise detecting the amount of Herl-Her3 dimers on a cancer cell present on a cancer cell, then detecting the amount of Her2-Her3 dimers on the cancer cell. In certain embodiments, the methods comprise detecting the amount of Herl-Her2 dimers on a cancer cell present on a cancer cell, then detecting the amount of Her2-Her3 dimers on the cancer cell. In certain embodiments, the methods further comprise detecting the total amount of Her 1, Her2, and/or Her3, or any combination thereof, subsequent to detecting the amount of Herl-Herl dimers, Herl-Her2 dimers, and/or Herl-Her3 dimers, or any combination thereof.

[0210] In another aspect, the invention provides a method of using ErbB cell surface receptor complexes as biomarkers for the status of a disease or other physiological conditions in a biological organism, particularly a cancer status in a human, In one aspect, ErbB receptor complexes are measured directly from patient samples; that is, measurements are made without culturing, formation of xenografts, or the use of like techniques, that require extra labor and expense and that may introduce artifacts and/or noise into the measurement process, In certain embodiments, measurements of one or more receptor complexes are made directly on tissue lysates of frozen patient samples or on sections of fixed patient samples. In a preferred embodiment, one or more ErbB receptor complexes are measured in sections of formalin-fixed paraffin-embedded (FFPE) samples.

[0211] In certain embodiments, one or more ErbB receptor complexes are measured on a single biopsy obtained from a subject. More preferably, one or more ErbB receptor complexes are measured on a plurality of biopsies obtained from a subject. In certain embodiments, one or more ErbB receptor complexes are measured on two, three, four, five, six, seven, eight, nine, ten or more biopsies obtained from a subject. [0212] In another aspect, the invention provides an indirect measurement of ErbB receptor phosphorylation through the measurement of complexes that depend on such posttraiislational modifications for their formation. In yet another aspect, the invention provides an indirect measurement of ErbB receptor dimerization and/or activation through measurement of phosphorylation of one or more members of an ErbB dimer.

[0213] In certain embodiments, a plurality of ErbB receptor complexes, such as receptor dimers, are simultaneously measured in the same assay reaction mixture. Preferably, such complexes are measured using binding compounds having one or more molecular tags releasably attached, such that after binding to a protein in a complex, the molecular tags may be released and separated from the reaction, or assay, mixture for detection and/or quantification.

[0214] In another aspect, the invention provides a method for determining a disease status of a patient comprising: measuring an amount of each of one or more ErbB receptor dimers in a patient sample; comparing each such amount to its corresponding amount from a reference sample; and correlating differences in the amounts from the patient sample and the respective corresponding amounts from the reference sample to the presence or severity of a disease condition in the patient. In one preferred embodiment, the step of measuring comprising the steps of: (i) providing one or more binding compounds specific for a protein of each of the one or more receptor dimers, such that each binding compound has one or more molecular tags each attached thereto by a cleavable linkage, and such that the one or more molecular tags attached to different binding compounds have different separation characteristics so that upon separation molecular tags from different binding compounds form distinct peaks in a separation profile; (ii) mixing the binding compounds and the one or more complexes such that binding compounds specifically bind to their respective receptor dimers to form detectable complexes; (iii) cleaving the cleavable linkage of each binding compound forming detectable complexes, and (iv) separating and identifying the released molecular tags to determine the presence or absence or the amount of the one or more receptor dimers.

[0215] In another aspect, the step of measuring the amounts of one or more types of ErbB receptor dimer comprising the following steps: (i) providing for each of the one or more types of receptor dimer a cleaving probe specific for a first receptor in each of the one or more receptor dimers, each cleaving probe having a cleavage-inducing moiety with an effective proximity; (ii) providing one or more binding compounds specific for a second receptor of each of the one or more receptor dimers, such that each binding compound has one or more molecular tags each attached thereto by a cleavable linkage, and such that the one or more molecular tags attached to different binding compounds have different separation characteristics so that upon separation molecular tags from different binding compounds form distinct peaks in a separation profile; (iii) mixing the cleaving probes, the binding compounds, and the one or more types of receptor dimers such that cleaving probes specifically bind to first receptors of the receptor dimers and binding compounds specifically bind to the second receptors of the receptor dimers and such that cleavable linkages of the binding compounds are within the effective proximity of cleavage-inducing moieties of the cleaving probes so that molecular tags are released; and (iv) separating and identifying the released molecular tags to determine the presence or absence or the amount of the one or more types of receptor dimers. Preferably, receptor dimers and first and second receptors are selected from the receptor dimers listed in Table 1.

[0216] In another aspect of the invention, a biological sample, which comprises a mixed cell population suspected of containing the rare cell of interest is obtained from a patient. A sample is then prepared by mixing the biological specimen with magnetic particles which are coupled to a biospecific ligand specifically reactive with an antigen on the rare cell that is different from or not found on blood cells (referred to herein as a "capture antigen"), so that other sample components may be substantially removed. The sample is subjected to a magnetic field which is effective to separate cells labeled with the magnetic particles, including the rare cells of interest, if any are present in the specimen. The cell population so isolated is then analyzed using molecular tags conjugated to binding moieties specific for biomarkers to determine the presence and/or number of rare cells. In a preferred embodiment the magnetic particles used in this method are colloidal magnetic nanoparticles. Preferably, such rare cell populations are circulating epithelial cells, which may be isolated from patient's blood using epithelial- specific capture antigens such as, for example, those disclosed in Hayes et ah, 2002, International J. Oncol. 21:1111-1117; Soria et α/., 1999, Clin. Can. Res. 5:971-975; Ady et ah, 2004, British J. Cancer 90:443-448; each of which are hereby incorporated by reference in its entirety. In particular, monoclonal antibody BerEP4 (Dynal A.S., Oslo, Norway) may be used to capture human epithelial cells with magnetic particles.

[0217] In another aspect, the invention provides a method for determining a cancer status of a patient comprising the following steps: (i) immunomagnetically isolating a patient sample comprising circulating epithelial cells by contacting a sample of patient blood with one or more antibody compositions, each antibody composition being specific for a capture antigen and being attached to a magnetic particle; (ii) measuring an amount of each of one or more ErbB receptor complexes in the patient sample; comparing each such amount to its corresponding amount from a reference sample; and correlating differences in the amounts from the patient sample and the respective corresponding amounts from the reference sample to the presence or severity of a cancer condition in the patient. In one preferred embodiment, the step of measuring comprises the steps of: (i) providing one or more binding compounds specific for a protein of each of the one or more ErbB receptor complexes, such that each binding compound has one or more molecular tags each attached thereto by a cleavable linkage, and such that the one or more molecular tags attached to different binding compounds have different separation characteristics so that upon separation molecular tags from different binding compounds form distinct peaks in a separation profile; (ii) mixing the binding compounds and the one or more ErbB receptor complexes such that binding compounds specifically bind to their respective proteins of the one or more ErbB receptor complexes to form detectable complexes; (iii) cleaving the cleavable linkage of each binding compound forming detectable complexes, and (iv) separating and identifying the released molecular tags to determine the presence or absence or the amount of the one or more ErbB receptor complexes.

[0218] In certain embodiments, the step of measuring the amounts of one or more

ErbB receptor complexes comprising the following steps: (i) providing for each of the one or more ErbB receptor complexes a cleaving probe specific for a first protein in each of the one or more ErbB receptor complexes, each cleaving probe having a cleavage- inducing moiety with an effective proximity; (ii) providing one or more binding compounds specific for a second protein of each of the one or more ErbB receptor complexes, such that each binding compound has one or more molecular tags each attached thereto by a cleavable linkage, and such that the one or more molecular tags attached to different binding compounds have different separation characteristics so that upon separation molecular tags from different binding compounds form distinct peaks in a separation profile; (iii) mixing the cleaving probes, the binding compounds, and the one or more complexes such that cleaving probes specifically bind to first proteins of the ErbB receptor complexes and binding compounds specifically bind to the second proteins of the ErbB receptor complexes and such that cleavable linkages of the binding compounds are within the effective proximity of cleavage-inducing moieties of the cleaving probes so that molecular tags are released; and (iv) separating and identifying the released molecular tags to determine the presence or absence or the amount of the one or more ErbB receptor complexes.

5.2.1 Exemplary Receptor Dimer

Biomarkers and Dimer-Acting Drugs

[0219] Biomarkers of the invention include dimers and oligomers of the following receptors.

Table 1. Exemplary Receptor Complexes of Cell Surface Membranes

[0220] Without intending to be bound to any particular theory or mechanism of action, it is believed that many drugs that are in use or are under development function by inhibiting one or more functions of ErbB receptor dimers, such as the association of component receptors into a dimer structure, or a function, such as an enzymatic activity, e.g., kinase activity, or autophosphorylation, that depends on dimerization. Any such structural or functional activity of the dimers can be monitored as a proxy for an activity known by one of skill in the art to be related thereto. For example, phosphorylation of certain ErbB receptors can be monitored as a proxy for dimerization or ligand binding. Such drugs are referred to herein as "dimer-acting" drugs, or "ErbB dimer-acting" drugs. The number, type, formation, and/or dissociation of receptor dimers in the cells of a patient being treated, or whose treatment is contemplated, have a bearing on the effectiveness or suitability of using a particular ErbB dimer-acting drug. The following ErbB receptor dimers are biomarkers related to the indicated drags. In one aspect, the invention provides biomarkers for monitoring the effect on disease status of an ErbB dimer-acting drug, comprising detecting the presence and/or amount of one or more biomarker associated with the ErbB dimer-acting drag.

Table 2.

Drags Positively or Negatively Associated with Dimers of Cell Surface Membranes Dimer Drag(s)

[0221] The dimer-acting drags listed in Table 2 are described and characterized by, for example, Traxler, 2002, Expert Opin. Ther. Targets 7: 215-234; Baselga, ed., 2002, Oncology Biotherapeutics 2:1-36; Nam et al., 2003, Current Drug Targets 4:159- 179; and Seymour, 2001, Current Drug Targets 2:117-133; each of which is hereby incorporated by reference in its entirety.

[0222] In another aspect, the invention relates to Herl -acting agents, as defined above. The Herl -acting agent can be any such agent known to one of skill in the art, without limitation, In certain embodiments the Herl -acting agent is selected from the group consisting of Gefitinib, tarceva, and erbitux. In a preferred embodiment, the Herl- acting agent is Gefitinib.

[0223] Gefitinib is an anilinoquinazoline with the chemical name 4-

Quinazolinamine, N-(3-chloro-4fluorophenyl)-7-methoxy-6-[3-4-morpholin) propoxy] and the following structural formula:

[0224] It has the molecular formula C₂₂H₂₄ClFN₄O₃, a relative molecular mass of

446.9 and is a white-colored powder. Gefitinib is a free base. The molecule has pKas of 5.4 and 7.2 and therefore ionizes progressively in solution as the pH falls. Gefitinib can be defined as sparingly soluble at pH 1, but is practically insoluble above pH 7, with the, solubility dropping sharply between pH 4 and pH 6. In non-aqueous solvents, Gefitinib is freely soluble in glacial acetic acid and dimethylsulphoxide, soluble in pyridine, sparingly soluble in tetrahydrofuran, and slightly soluble in methanol, ethanol (99.5%), ethyl acetate, propan-2-ol and acetonitrile.

[0225] IRESSA^® (Gefitinib tablets) contain 250 mg of Gefitinib and are available as brown film-coated tablets for daily oral administration. The inactive ingredients of IRESSA^® tablets are as follows: Tablet core: Lactose monohydrate, microcrystalline cellulose, croscarmellose sodium, povidone, sodium lauryl sulfate and magnesium stearate. Coating: Hydroxypropyl methylcellulose, polyethylene glycol 300, titanium dioxide, red ferric oxide and yellow ferric oxide.

5.2.2 Preparation of Samples

[0226] Samples containing molecular complexes suitable for use as biomarkers may come from a wide variety of sources for use with the present invention to relate receptor complexes populations to disease status or health status, including cell cultures, animal or plant tissues, patient biopsies, or the like. Preferably, samples are human patient samples. Samples are prepared for assays of the invention using conventional techniques, which may depend on the source from which a sample is taken.

5.2.2.1 Solid Tissue Samples

[0227] For biopsies and medical specimens, guidance is provided in the following references: Bancroft JD & Stevens A, eds. 1977, Theory and Practice of Histological Techniques, Churchill Livingstone, Edinburgh,; Pearse, 1980, Histochemistry. Theory and applied. 4^th ed., Churchill Livingstone, Edinburgh.

[0228] In the area of cancerous disease status, examples of patient tissue samples that may be used include, but are not limited to, breast, prostate, ovary, colon, lung, endometrium, stomach, salivary gland or pancreas. The tissue sample can be obtained by a variety of procedures including, but not limited to surgical excision, aspiration or biopsy. The tissue may be fresh or frozen. In one embodiment, assays of the invention are carried out on tissue samples that have been fixed and embedded in paraffin or the like; therefore, in such embodiments a step of deparaffmation can be carried out. A tissue sample may be fixed (i.e., preserved) by conventional methodology. See, e.g., Lee G. Luna, HT (ASCP) Ed., 1960, Manual of Histological Staining Method of the Armed Forces Institute of Pathology 3^rd edition, The Blakston Division McGraw-Hill Book Company, New York; Ulreka V. Mikel, Ed., 1994, The Armed Forces Institute of Pathology Advanced Laboratory Methods in Histology and Pathology, Armed Forces Institute of Pathology, American Registry of Pathology, Washington, D. C. One of skill in the art will appreciate that the choice of a fixative is determined by the purpose for which the tissue is to be histologically stained or otherwise analyzed. One of skill in the art will also appreciate that the length of fixation depends upon the size of the tissue sample and the fixative used. By way of example, neutral buffered formalin, Bouin's or paraformaldehyde, may be used to fix a tissue sample.

[0229] Generally, a tissue sample is first fixed and is then dehydrated through an ascending series of alcohols, infiltrated and embedded with paraffin or other sectioning media so that the tissue sample may be sectioned. Alternatively, one may section the tissue and fix the sections obtained. By way of example, the tissue sample may be embedded and processed in paraffin by conventional methodology according to conventional techniques described by the references provided above. Examples of paraffin that may be used include, but are not limited to, Paraplast, Broloid, and Tissuemay. Once the tissue sample is embedded, the sample may be sectioned by a microtome or the like according to conventional techniques such as those described by the references provided above. By way of example for this procedure, sections may have a thickness in a range from about three microns to about twelve microns, and preferably, a thickness in a range of from about 5 microns to about 10 microns, In one aspect, a section may have an area of from about 10 mm to about 1 cm . Once cut, the sections may be attached to slides by several standard methods. Examples of slide adhesives include, but are not limited to, silane, gelatin, poly-L-lysine and the like. By way of example, the paraffin embedded sections may be attached to positively charged slides and/or slides coated with poly-L-lysine.

[0230] If paraffin has been used as the embedding material, the tissue sections are generally deparaffmized and rehydrated to water prior to detection of biomarkers. The tissue sections may be deparaffmized by several conventional standard methodologies. For example, xylenes and a gradually descending series of alcohols may be used according to conventional techniques described by the references provided above. Alternatively, commercially available deparaffmizing non-organic agents such as Hemo- De® (CMS, Houston, Tex.) may be used.

[0231] For mammalian tissue culture cells, fresh tissues, or like sources, samples may be prepared by conventional cell lysis techniques (e.g., 0.14 M NaCl, 1.5 mM MgCl₂, 10 mM Tris-Cl (pH 8.6), 0.5% Nonidet P-40, and protease and/or phosphatase inhibitors as required). For fresh mammalian tissues, sample preparation may also include a tissue disaggregation step, such as, for example, crushing, mincing, grinding, sonication, or the like.

5.2.2.2 Magnetic Isolation of Cells

[0232] In some applications, such as measuring dimers on rare metastatic cells from a patient's blood, an enrichment step may be carried out prior to conducting an assay for surface receptor dimer populations. Immunomagnetic isolation or enrichment may be carried out using a variety of techniques and materials known in the art, as disclosed in the following representative references that are incorporated by reference: U.S. Patent Nos. 6,365,362; 5,646,001; 5,998,224; 5,665,582; 6,048,515; 5,508,164; 5,691,208; 4,452,773; and 4,375,407; Radbruch et al, 1994, Methods in Cell Biology, Vol. 42, Ch. 23, Academic Press, New York,; Uhlen et al, 1994, Advances in Biomagnetic Separation, Eaton Publishing, Natick; Safarik et al, 1999, J Chromatography B 722:33-53; Miltenyi et al, 1990, Cytometry 11 :231-238; Nakamura et al, 2001, Biotechnol. Prog. 17:1145-1155; Moreno et al, 2001, Urology 58:386-392; Racila et al, 1998, Proc. Natl. Acad. ScL USA 95:4589-4594; Zigeuner et al, 2003, J. Urology 169:701-705; Ghossein et al., 2001, Seminars in Surgical Oncology 20:304- 311.

[0233] The preferred magnetic particles for use in carrying out this invention are particles that behave as colloids. Such particles are characterized by their sub-micron particle size, which is generally less than about 200 nanometers (nm) (0.20 microns), and their stability to gravitational separation from solution for extended periods of time, In addition to the many other advantages, this size range makes them essentially invisible to analytical techniques commonly applied to cell analysis. Particles within the range of 90- 150 nm and having between 70-90% magnetic mass are also contemplated for use in the present invention. Suitable magnetic particles are composed of a crystalline core of superparamagnetic material surrounded by molecules which are bonded, e.g., physically absorbed or covalently attached, to the magnetic core and which confer stabilizing colloidal properties. The coating material should preferably be applied in an amount effective to prevent non specific interactions between biological macromolecules found in the sample and the magnetic cores. Such biological macromolecules may include sialic acid residues on the surface of non-target cells, lectins, glyproteins and other membrane components. In addition, the material should contain as much magnetic mass/nanoparticle as possible. The size of the magnetic crystals comprising the core is sufficiently small that they do not contain a complete magnetic domain. The size of the nanoparticles is sufficiently small such that their Brownian energy exceeds their magnetic moment. As a consequence, North Pole, South Pole alignment and subsequent mutual attraction/repulsion of these colloidal magnetic particles does not appear to occur even in moderately strong magnetic fields, contributing to their solution stability. Finally, the magnetic particles should be separable in high magnetic gradient external field separators. That characteristic facilitates sample handling and provides economic advantages over the more complicated internal gradient columns loaded with ferromagnetic beads or steel wool. Magnetic particles having the above-described properties can be prepared by modification of base materials described in U.S. Pat. Nos. 4,795,698, 5,597,531 and 5,698,271, which patents are incorporated by reference in their entireties.

5.3 Assays Using Releasable Molecular Tags [0234] Many advantages are provided by measuring dimer populations using releasable molecular tags, including (1) separation of released molecular tags from an assay mixture provides greatly reduced background and a significant gain in sensitivity; and (2) the use of molecular tags that are specially designed for ease of separation and detection provides a convenient multiplexing capability so that multiple receptor complex components may be readily measured simultaneously in the same assay. Assays employing such tags can have a variety of forms and are disclosed in the following references: U.S. Patent No. 6,627,400; published U.S. Patent Application Nos. 2002/0013126, 2003/0170915, and 2002/0146726; and International Patent Publication No. WO 2004/011900, each of which is incorporated herein by reference in its entirety. For example, a wide variety of separation techniques may be employed that can distinguish molecules based on one or more physical, chemical, or optical differences among molecules being separated including but not limited to electrophoretic mobility, molecular weight, shape, solubility, pKa, hydrophobicity, charge, charge/mass ratio, polarity, or the like. In one aspect, molecular tags in a plurality or set differ in electrophoretic mobility and optical detection characteristics and are separated by electrophoresis, In another aspect, molecular tags in a plurality or set may differ in molecular weight, shape, solubility, pKa, hydrophobicity, charge, polarity, and are separated by normal phase or reverse phase HPLC, ion exchange HPLC, capillary electrochromatography, mass spectroscopy, gas phase chromatography, or like technique.

[0235] Sets of molecular tags are provided that can be separated into distinct bands or peaks by a separation technique after they are released from binding compounds. Identification and quantification of such peaks provides a measure or profile of the presence and/or amounts of receptor dimers. Molecular tags within a set may be chemically diverse; however, for convenience, sets of molecular tags are usually chemically related. For example, they may all be peptides, or they may consist of different combinations of the same basic building blocks or monomers, or they may be synthesized using the same basic scaffold with different substituent groups for imparting different separation characteristics, as described more fully below. The number of molecular tags in a plurality may vary depending on several factors including the mode of separation employed, the labels used on the molecular tags for detection, the sensitivity of the binding moieties, the efficiency with which the cleavable linkages are cleaved, and the like. In one aspect, the number of molecular tags in a plurality for measuring populations of receptor dimers is in the range of from 2 to 20. In other aspects, the size of the plurality may be in the range of from 2 to 18, 2 to 16, 2 to 14, 2 o 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, or 2 to 3.

[0236] Receptor dimers may be detected in assays having homogeneous formats or a non-homogeneous, e.g., heterogeneous, formats. In a homogeneous format, no step is required to separate binding compounds specifically bound to target complexes from unbound binding compounds. In a preferred embodiment, homogeneous formats employ reagent pairs comprising (i) one or more binding compounds with releasable molecular tags and (ii) at least one cleaving probe that is capable of generating an active species that reacts with and releases molecular tags within an effective proximity of the cleaving probe.

[0237] Receptor dimers may also be detected by assays employing a heterogeneous format. Heterogeneous techniques normally involve a separation step, where intracellular complexes having binding compounds specifically bound are separated from unbound binding compounds, and optionally, other sample components, such as proteins, membrane fragments, and the like. Separation can be achieved in a variety of ways, such as employing a reagent bound to a solid support that distinguishes between complex-bound and unbound binding compounds. The solid support may be a vessel wall, e.g., microliter well plate well, capillary, plate, slide, beads, including magnetic beads, liposomes, or the like. The primary characteristics of the solid support are that it (1) permits segregation of the bound and unbound binding compounds and (2) does not interfere with the formation of the binding complex, or the other operations in the determination of receptor dimers. Usually, in fixed samples, unbound binding compounds are removed simply by washing.

[0238] With detection using molecular tags in a heterogeneous format, after washing, a sample may be combined with a solvent into which the molecular tags are to be released. Depending on the nature of the cleavable bond and the method of cleavage, the solvent may include any additional reagents for the cleavage. Where reagents for cleavage are not required, the solvent conveniently may be a separation buffer, e.g. an electrophoretic separation medium. For example, where the cleavable linkage is photolabile or cleavable via an active species generated by a photosensitizer, the medium may be irradiated with light of appropriate wavelength to release the molecular tags into the buffer.

[0239] In either format, if the assay reaction conditions interfere with the separation technique employed, it may be necessary to remove, or exchange, the assay reaction buffer prior to cleavage and separation of the molecular tags. For example, in some embodiments, assay conditions include salt concentrations (e.g., required for specific binding) that degrade separation performance when molecular tags are separated on the basis of electrophoretic mobility. In such embodiments, an assay buffer is replaced by a separation buffer, or medium, prior to release and separation of the molecular tags.

[0240] Assays employing releasable molecular tags and cleaving probes can be made in many different formats and configurations depending on the complexes that are detected or measured. One of skill in the art can, guided by the present disclosure, routinely select the numbers and specificities of particular binding compounds and cleaving probes for use in the methods of the invention.

[0241] In one aspect of the invention, the use of releasable molecular tags to measure dimers of cell surface membranes is shown diagrammatically in Figs. IA and IB. Binding compounds (100) having molecular tags "InT₁" and "mT₂" and cleaving probe (102) having photosensitizer "PS" are combined with biological cells (104). Binding compounds having molecular tag "mTi" are specific for cell surface receptors Ri (106) and binding compounds having molecular tag "111T₂" are specific for cell surface receptors R₂ (108). Cell surface receptors R₁ and R₂ are present as monomers, e.g. (106) and (108), and as dimers (110) in cell surface membrane (112). After these assay components are incubated in a suitable binding buffer to permit the formation (114) of stable complexes between binding compounds and their respective receptor targets and between the cleaving probe and its receptor target. As illustrated, preferably binding compounds and cleaving probes each comprise an antibody binding composition, which permits the molecular tags and cleavage-inducing moiety to be specifically targeted to membrane components, In one aspect, such antibody binding compositions are monoclonal antibodies. In such embodiments, binding buffers may comprise buffers used in conventional ELISA techniques, or the like. After binding compounds and cleaving probes for stable complexes (116), the assay mixture is illuminated (118) to induce photosensitizers (120) to generate singlet oxygen. Singlet oxygen rapidly reacts with components of the assay mixture so that its effective proximity (122) for cleaving cleavable linkages of molecular tags is spatially limited so that only molecular tags that happen to be within the effective proximity are released (124). As illustrated, the only molecular tags released are those on binding compounds that form stable complexes with R₁-R₂ dimers and a cleaving probe. Released molecular tags (126) are removed from the assay mixture and separated (128) in accordance with a separation characteristic so that a distinct peak (130) is formed in a separation profile (132). In accordance with the invention, such removal and separation may be the same step. Optionally, prior to illumination the binding buffer may be removed and replaced with a buffer more suitable for separation, i.e. a separation buffer. For example, binding buffers typically have salt concentrations that may degrade the performance of some separation techniques, such as capillary electrophoresis, for separating molecular tags into distinct peaks. In one embodiment, such exchange of buffers maybe accomplished by membrane filtration.

[0242] An embodiment that illustrates ratiometric measurement of heterodimers is illustrated in Fig. 1C, in which an additional binding compound is employed to give a measure of the total amount of protein (1104) in a sample. Reagents (1122) of the invention comprise (i) cleaving probes (1108), first binding compound (1106), and second binding compound (1107), wherein first binding compound (1106) is specific for protein (1102) and second binding compound (1107) is specific for protein (1104) at a different antigenic determinant than that cleaving probe (1108) is specific for. After binding of the reagents, cleaving probe (1108) is activated to produce active species that cleave the cleavable linkages of the molecular tags within the effective proximity of the photosensitizer. In this embodiment, molecular tags are released from monomers of protein (1104) that have both reagents (1107) and (1108) attached and from heterodimers that have reagent (1108) attached and either or both of reagents (1106) and (1107) attached. Released molecular tags (1123) are separated, and peaks (1118 and 1124) in a separation profile (1126) are correlated to the amounts of the released molecular tags, In this embodiment, relative peak heights, or areas, may reflect (i) the differences in affinity of the first and second binding compounds for their respective antigenic determinants, and/or (ii) the presence or absence of the antigenic determinant that the binding compound is specific for. The later situation is important whenever a binding compound is used to monitor the post-translational state of a protein, such as, for example, a phosphorylation state.

[0243] Homodimers may be measured as illustrated in Fig. ID. As above, an assay may comprise three reagents (1128): cleaving probes (1134), first binding compound (1130), and second binding compound (1132). First binding compound (1130) and cleaving probe (1134) are constructed to be specific for the same antigenic determinant (1135) on protein (1138) that exists (1140) in a sample as either a homodimer (1136) or a monomer (1138). After reagents (1128) are combined with a sample under conditions that promote the formation of stable complexes between the reagents and their respective targets, multiple complexes (1142 through 1150) form in the assay mixture. Because cleaving probe (1134) and binding compound (1130) are specific for the same antigenic determinant (1135), four different combinations (1144 through 1150) of reagents may form complexes with homodimers. Of the complexes in the assay mixture, only those (1143) with both a cleaving probe (1134) and at least one binding compound will contribute released molecular tags (1151) for separation and detection (1154). In this embodiment, the size of peak (1153) is proportional to the amount of homodimer in the assay mixture, while the size of peak (1152) is proportional to the total amount of protein (1138) in the assay mixture, both in monomelic form (1142) or in homodimeric form (1146 and 1148). Fig. IE illustrates the analogous measurements for cell surface receptors that form heterodimers in cell surface membrane (1161). One skilled in the art would understand that dimers may be measured in either lysates of cells or tissues, or in fixed samples whose membranes have been permeabilized or removed by the fixing process. In such cases, binding compounds may be specific for either extracellular or intracellular domains of cell surface membrane receptors.

[0244] As illustrated in Figs. IE and IF, releasable molecular tags may also be used for the simultaneous detection or measurement of multiple dimers and intracellular complexes in a cellular sample. Cells (160), which may be from a sample from in vitro cultures or from a specimen of patient tissue, are lysed (172) to render accessible molecular complexes associated with the cell membrane, and/or post-translational modification sites, such as phosphorylation sites, within the cytoplasmic domains of the membrane molecules. After lysing, the resulting lysate (174) is combined with assay reagents (176) that include multiple cleaving probes (175) and multiple binding compounds (177). Assay conditions are selected (178) that allow reagents (176) to specifically bind to their respective targets, so that upon activation cleavable linkages within the effective proximity (180) of the cleavage-inducing moieties are cleaved and molecular tags are released (182). As above, after cleavage, the released molecular tags are separated (184) and identified in a separation profile (186), such as an electropherogram, and based on the number and quantities of molecular tags measured, a profile is obtained of the selected molecular complexes in the cells of the sample.

[0245] Figs. IG and IH illustrate an embodiment of the invention for measuring receptor complexes in fixed or frozen tissue samples. Fixed tissue sample (1000), e.g. a formalin- fixed paraffin-embedded sample, is sliced to provide a section (1004) using a microtome, or like instrument, which after placing on surface (1006), which may be a microscope slide, it is de-waxed and re-hydrated for application of assay reagents. Enlargement (1007) shows portion (1008) of section (1004) on portion (1014) of microscope slide (1006). Receptor dimer molecules (1018) are illustrated as embedded in the remnants of membrane structure (1016) of the fixed sample. In accordance with this aspect of the invention, cleaving probe and binding compounds are incubated with the fixed sample so that they bind to their target molecules. For example, cleaving probes (1012) (illustrated in the figure as an antibody having a photosensitizer ("PS") attached) and first binding compound (1010)(illustrated as an antibody having molecular tag "HiT₁" attached) specifically bind to receptor (1011) common to all of the dimers shown, second binding compound (1017)(with "mT₂") specifically binds to receptor (1015), and third binding compound (1019)(with "mT₃") specifically binds to receptor (1013). After washing to remove binding compounds and cleaving probe that are not specifically bound to their respective target molecules, buffer (1024) (referred to as "illumination buffer" in the figure) is added. For convenience, buffer (1024) may be contained on section (1004), or a portion thereof, by creating a hydrophobic barrier on slide (1006), e.g. with a wax pen. After illumination of photosensitizers and release of molecular tags (1026), buffer (1024) now containing release molecular tags (1025) is transferred to a separation device, such as a capillary electrophoresis instrument, for separation (1028) and identification of the released molecular tags in, for example, electropherogram (1030).

[0246] Measurements made directly on tissue samples, particularly as illustrated in

Figs. IG and IH, may be normalized by including measurements on cellular or tissue targets that are representative of the total cell number in the sample and/or the numbers of particular subtypes of cells in the sample. Such tissue targets are referred to herein as "tissue indicators." The additional measurement may be preferred, or even necessary, because of the cellular and tissue heterogeneity in patient samples, particularly tumor samples, which may comprise substantial fractions of normal cells. For example, in Fig. IH, values for the total amount of receptor (1011) may be given as a ratio of the following two measurements: area of peak (1030) of molecular tag CmT₁") and the area of a peak corresponding to a molecular tag correlated with a cellular or tissue component common to all the cells in the sample, e.g., tubulin, or the like. In embodiments where all the cells in the sample are epithelial cells, cytokeratin or similar markers may be used. Accordingly, detection methods based on releasable molecular tags may include an additional step of providing a binding compound (with a distinct molecular tag) specific for a normalization protein, such as, e.g., tubulin.

[0247] Figures 2A-2E illustrate another embodiment of the invention for profiling dimerization among a plurality of receptor types. Figure 2A outlines the basic steps of such an assay. Cell membranes (200) that are to be tested for dimers of cell surface receptors are combined with sets of binding compounds (202) and (204) and cleaving probe (206). Membrane fractions (200) contain three different types of monomer receptor molecules ("1," "2," and "3") in its cell membrane which associate to form three different heterodimers: 1-2, 1-3, and 2-3. This arrangement reflects some of the dimers that can form between, for example, Herl, Her2, and Her3. Three antibody reagents (202) and (204) are combined with membrane fraction (200), each of the antibody reagents having binding specificity for one of the three receptor molecules, where antibody (206) is specific for receptor molecule 1, antibody (204) is specific for receptor molecule 2, and antibody (202) is specific for receptor molecule 3. The antibody for the first receptor molecule is covalently coupled to a photosensitizer molecule, labeled PS. The antibodies for the second and third receptor molecules are linked to two different tags, labeled T₂ and T₃, respectively, through a linkage that is cleavable by an active species generated by the photosensitizer moiety.

[0248] After mixing, the antibodies are allowed to bind (208) to molecules on the surface of the membranes. The photosensitizer is activated (210), cleaving the linkage between tags and antibodies that are within an actionable distance from a sensitizer molecule, thereby releasing tags into the assay medium. Material from the reaction is then separated (212), e.g., by capillary electrophoresis, as illustrated. As shown at the bottom of Figure 2 A, the tags T₂ and T₃ are released, and separation by electrophoresis will reveal two bands corresponding to these tags. Because the tags are designed to have a known electrophoretic mobility, each of the bands can be uniquely assigned to one of the tags used in the assay.

[0249] As shown in Fig. 2 A, only two of the three different heterodimers that are present in the cell membrane will bind both a photosensitizer-containing antibody and a tag-containing antibody, and thus only these two species should give rise to released tags. However, multiple experiments are required to measure the relative amounts of the different dimers. Fig. 2B provides a table listing five different assay combinations. In Fig. 2C are the illustrative results for each assay composition. Assay I represents the results from the complete assay, as described in Figure 2A. In Assay II, the antibody specific for receptor molecule 1, which is linked to the photosensitizer, is omitted. This assay yields no signal, indicating that the T₂ and T₃ signals obtained in Assay I require the photosensitizer reagent. Similarly, Assay V shows that the tag signals require the presence of the membranes. Assays III and IV show that each tagged reagent does not require the presence of the other to be cleaved. These results, when considered together, allow one to draw conclusions regarding the presence and composition of receptor heterodimers present in the membrane, as given in Figure 2C, i.e., that both the 1-2 and the 1-3 heterodimer are present. Furthermore, the relative signal intensities from each tag allow one to estimate the relative abundance of each of the heterodimers.

[0250] A conclusion regarding existence of the 2-3 heterodimer cannot be made with the combination of reagents used in this assay, however. No signal representing this complex will be obtained, whether or not the complex is present, because it will not have a photosensitizer reagent bound to it. In order to draw conclusions regarding every possible dimeric combination of the three monomers, either a fourth reagent must be used that can be localized to every possible oligomer comprising monomers 1, 2, and/or 3, or the three binding agents used in this experiment must be coupled in different combinations to tags and sensitizer molecules. The later strategy is illustrated in Figures 2D and 2E. Three possible combinations of photosensitizer and tag distribution among the three antibody reagents are listed in the table on the left of Figure 2D. The first combination comprises a photosensitizer coupled to the antibody specific for monomer number 1, and is the same combination used in the illustration of Figure 2A-2C, and has the same dimer population as in Figure 2C. The second combination comprises a photosensitizer coupled to the antibody specific for monomer number 2, and the population profile yields the same number for heterodimer 1-2, plus a value for heterodimer 2-3. The third combination comprises a photosensitizer coupled to the antibody specific for monomer number 3, and the population profile yields the same number for heterodimer 1-3 and 2-3 as obtained from the first two combinations. These results can be combined to yield the overall heterodimer population profile given in Figure 2E.

[0251] A preferred embodiment for measuring relative amounts of receptor dimers containing a common component receptor is described below. In this assay design, two different receptor dimers ("1-2" and "2-3") each having a common component, "2," may be measured ratiometrically with respect to the common component. An assay design is shown for measuring receptor heterodimer comprising receptor "1" and receptor "2" and receptor heterodimer comprising receptor "2" and receptor "3". A key feature of this embodiment is that cleaving probe be made specific for the common receptor of the pair of heterodimers. Binding compound specific for receptor "2" provides a signal related to the total amount of receptor "2" in the assay, whereas binding compound specific for receptor "1" and binding compound specific for receptor "3" provide signals related only to the amount of receptor "1" and receptor "3" present as heterodimers with receptor "2," respectively. This design may be generalized to more than two receptor complexes that contain a common component by simply adding binding compounds specific for the components of the additional complexes.

5.3.1 Binding Compounds [0252] As mentioned above, mixtures containing pluralities of different binding compounds may be provided, wherein each different binding compound has one or more molecular tags attached through cleavable linkages. The nature of the binding compound, cleavable linkage and molecular tag may vary widely. A binding compound may comprise an antibody binding composition, an antibody, a peptide, a peptide or non- peptide ligand for a cell surface receptor, a protein, an oligonucleotide, an oligonucleotide analog, such as a peptide nucleic acid, a lectin, or any other molecular entity that is capable of specifically binding to a target protein or molecule or stable complex formation with an analyte of interest, such as a complex of proteins, In one aspect, a binding compound, which can be represented by the formula below, comprises one or more molecular tags attached to a binding moiety.

B-(L-E)_k

wherein B is binding moiety; L is a cleavable linkage; and E is a molecular tag. In homogeneous assays, cleavable linkage, L, may be an oxidation-labile linkage, and more preferably, it is a linkage that may be cleaved by singlet oxygen. The moiety "-(L-E)_k" indicates that a single binding compound may have multiple molecular tags attached via cleavable linkages. In one aspect, k is an integer greater than or equal to one, but in other embodiments, k may be greater than several hundred, e.g. 100 to 500, or k is greater than several hundred to as many as several thousand, e.g. 500 to 5000. Usually each of the plurality of different types of binding compound has a different molecular tag, E. Cleavable linkages, e.g. oxidation-labile linkages, and molecular tags, E, are attached to B by way of conventional chemistries.

[0253] Preferably, B is an antibody binding composition that specifically binds to a target, such as a predetermined antigenic determinant of a target protein, such as a cell surface receptor. Such compositions are readily formed from a wide variety of commercially available antibodies, either monoclonal and polyclonal, specific for proteins of interest, In particular, antibodies specific for epidermal growth factor receptors are disclosed in U.S. Patent Nos. 5,677,171; 5,772,997; 5,968,511; 5,480,968; 5,811,098, each of which are incorporated by reference in its entirety. U.S. Patent No. 6,488,390, hereby incorporated by reference in its entirety, discloses antibodies specific for a G-protein coupled receptor, CCR4. U.S. Patent 5,599,681, hereby incorporated by reference in its entirety, discloses antibodies specific for phosphorylation sites of proteins. Commercial vendors, such as Cell Signaling Technology (Beverly, MA), Biosource International (Camarillo, CA), and Upstate (Charlottesville, VA), also provide monoclonal and polyclonal antibodies specific for many receptors.

[0254] Cleavable linkage, L, can be virtually any chemical linking group that may be cleaved under conditions that do not degrade the structure or affect detection characteristics of the released molecular tag, E. Whenever a cleaving probe is used in a homogeneous assay format, cleavable linkage, L, is cleaved by a cleavage agent generated by the cleaving probe that acts over a short distance so that only cleavable linkages in the immediate proximity of the cleaving probe are cleaved. Typically, such an agent must be activated by making a physical or chemical change to the reaction mixture so that the agent produces a short lived active species that diffuses to a cleavable linkage to effect cleavage. In a homogeneous format, the cleavage agent is preferably attached to a binding moiety, such as an antibody, that targets prior to activation the cleavage agent to a particular site in the proximity of a binding compound with releasable molecular tags. In such embodiments, a cleavage agent is referred to herein as a "cleavage-inducing moiety," which is discussed more fully below.

[0255] In a non-homogeneous format, because specifically bound binding compounds are separated from unbound binding compounds, a wider selection of cleavable linkages and cleavage agents are available for use. Cleavable linkages may not only include linkages that are labile to reaction with a locally acting reactive species, such as hydrogen peroxide, singlet oxygen, or the like, but also linkages that are labile to agents that operate throughout a reaction mixture, such as base-labile linkages, photocleavable linkages, linkages cleavable by reduction, linkages cleaved by oxidation, acid-labile linkages, peptide linkages cleavable by specific proteases, and the like. References describing many such linkages include, for example, Greene and Wuts, 1991, Protective Groups in Organic Synthesis, Second Edition, John Wiley & Sons, New York; Hermanson,1996, Bioconjugate Techniques, Academic Press, New York; and U.S. Patent No. 5,565,324.

[0256] In one aspect, commercially available cleavable reagent systems may be employed with the invention. For example, a disulfide linkage may be introduced between an antibody binding composition and a molecular tag using a heterofunctional agent such as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), succinimidyloxycarbonyl-o;-methyl-Q;-(2-pyridyldithio)toluene (SMPT), or the like, available from vendors such as Pierce Chemical Company (Rockford, IL). Disulfide bonds introduced by such linkages can be broken by treatment with a reducing agent, such as dithiothreitol (DTT), dithioerythritol (DTE), 2-mercaptoethanol, sodium borohydride, or the like. Typical concentrations of reducing agents to effect cleavage of disulfide bonds are in the range of from 10 to 100 mM. An oxidatively labile linkage may be introduced between an antibody binding composition and a molecular tag using the homobifunctional NHS ester cross-linking reagent, disuccinimidyl tartarate (DST)(available from Pierce) that contains central cis-diols that are susceptible to cleavage with sodium periodate (e.g., 15 mM periodate at physiological pH for 4 hours). Linkages that contain esterified spacer components may be cleaved with strong nucleophilic agents, such as hydroxylamine, e.g., 0.1 N hydroxylamine, pH 8.5, for 3-6 hours at 37⁰C. Such spacers can be introduced by a homobifunctional cross-linking agent such as ethylene glycol bis(succinimidylsuccinate)(EGS) available from Pierce (Rockford, IL). A base labile linkage can be introduced with a sulfone group. Homobifunctional cross-linking agents that can be used to introduce sulfone groups in a cleavable linkage include bis[2-(succinimidyloxycarbonyloxy)ethyl]sulfone (BSOCOES), and 4,4-difluoro-3,3-dinitrophenylsulfone (DFDNPS). Exemplary basic conditions for cleavage include 0.1 M sodium phosphate, adjusted to pH 11.6 by addition of Tris base, containing 6 M urea, 0.1% SDS, and 2 mM DTT, with incubation at 37 ⁰C for 2 hours. Photocleavable linkages also include those disclosed in U.S. Patent No. 5,986,076.

[0257] When L is oxidation labile, L may be a thioether or its selenium analog; or an olefin, which contains carbon-carbon double bonds, wherein cleavage of a double bond to an oxo group, releases the molecular tag, E. Illustrative oxidation labile linkages are disclosed in U.S. Patent Nos. 6,627,400 and 5,622,929 and in published U.S. Patent Application Nos. 2002/0013126 and 2003/0170915; each of which is hereby incorporated herein by reference in its entirety.

[0258] Molecular tag, E, in the present invention may comprise an electrophone tag as described in the following references when separation of pluralities of molecular tags are carried out by gas chromatography or mass spectrometry: See, e.g., Zhang et al, 2002, Bioconjugate Chem. 13:1002-1012; Giese, 1983, Anal. Chem. 2:165-168; and U.S. Patent Nos. 4,650,750; 5,360,819; 5,516,931; and 5,602,273, each of which is hereby incorporated by reference in its entirety.

[0259] Molecular tag, E, is preferably a water-soluble organic compound that is stable with respect to the active species, especially singlet oxygen, and that includes a detection or reporter group. Otherwise, E may vary widely in size and structure. In one aspect, E has a molecular weight in the range of from about 50 to about 2500 daltons, more preferably, from about 50 to about 1500 daltons. Preferred structures of E are described more fully below. E may comprise a detection group for generating an electrochemical, fluorescent, or chromogenic signal. In embodiments employing detection by mass, E may not have a separate moiety for detection purposes. Preferably, the detection group generates a fluorescent signal.

[0260] Molecular tags within a plurality are selected so that each has a unique separation characteristic and/or a unique optical property with respect to the other members of the same plurality. In one aspect, the chromatographic or electrophoretic separation characteristic is retention time under set of standard separation conditions conventional in the art, e.g., voltage, column pressure, column type, mobile phase, electrophoretic separation medium, or the like. In another aspect, the optical property is a fluorescence property, such as emission spectrum, fluorescence lifetime, fluorescence intensity at a given wavelength or band of wavelengths, or the like. Preferably, the fluorescence property is fluorescence intensity. For example, each molecular tag of a plurality may have the same fluorescent emission properties, but each will differ from one another by virtue of a unique retention time. On the other hand, or two or more of the molecular tags of a plurality may have identical migration, or retention, times, but they will have unique fluorescent properties, e.g. spectrally resolvable emission spectra, so that all the members of the plurality are distinguishable by the combination of molecular separation and fluorescence measurement.

[0261] Preferably, released molecular tags are detected by electrophoretic separation and the fluorescence of a detection group, In such embodiments, molecular tags having substantially identical fluorescence properties have different electrophoretic mobilities so that distinct peaks in an electropherogram are formed under separation conditions. Preferably, pluralities of molecular tags of the invention are separated by conventional capillary electrophoresis apparatus, either in the presence or absence of a conventional sieving matrix. Exemplary capillary electrophoresis apparatus include Applied Biosystems (Foster City, CA) models 310, 3100 and 3700; Beckman (Fullerton, CA) model P/ACE MDQ; Amersham Biosciences (Sunnyvale, CA) MegaBACE 1000 or 4000; SpectruMedix genetic analysis system; and the like. Electrophoretic mobility is proportional to q/M^2/3, where q is the charge on the molecule and M is the mass of the molecule. Desirably, the difference in mobility under the conditions of the determination between the closest electrophoretic labels will be at least about 0.001, usually 0.002, more usually at least about 0.01, and may be 0.02 or more. Preferably, in such conventional apparatus, the electrophoretic mobilities of molecular tags of a plurality differ by at least one percent, and more preferably, by at least a percentage in the range of from 1 to 10 percent. Molecular tags are identified and quantified by analysis of a separation profile, or more specifically, an electropherogram, and such values are correlated with the amounts and kinds of receptor dimers present in a sample. For example, during or after electrophoretic separation, the molecular tags are detected or identified by recording fluorescence signals and migration times (or migration distances) of the separated compounds, or by constructing a chart of relative fluorescent and order of migration of the molecular tags (e.g., as an electropherogram). Preferably, the presence, absence, and/or amounts of molecular tags are measured by using one or more standards as disclosed by published U.S. Patent Application No. 2003/0170734A1, which is hereby incorporated by reference in its entirety.

[0262] Pluralities of molecular tags may also be designed for separation by chromatography based on one or more physical characteristics that include but are not limited to molecular weight, shape, solubility, pKa, hydrophobicity, charge, polarity, or the like, e.g. as disclosed in published U.S. Patent Application No. 2003/0235832, which hereby is incorporated by reference in its entirety. A chromatographic separation technique is selected based on parameters such as column type, solid phase, mobile phase, and the like, followed by selection of a plurality of molecular tags that may be separated to form distinct peaks or bands in a single operation. Several factors determine which HPLC technique is selected for use in the invention, including the number of molecular tags to be detected (i.e., the size of the plurality), the estimated quantities of each molecular tag that will be generated in the assays, the availability and ease of synthesizing molecular tags that are candidates for a set to be used in multiplexed assays, the detection modality employed, and the availability, robustness, cost, and ease of operation of HPLC instrumentation, columns, and solvents. Generally, columns and techniques are favored that are suitable for analyzing limited amounts of sample and that provide the highest resolution separations. Guidance for making such selections can be found in the literature, such as, for example, Snyder et ah, 1988, Practical HPLC Method Development, John Wiley & Sons, New York; Millner, 1999, High Resolution Chromatography: A Practical Approach, Oxford University Press, New York; Chi-San Wu, 1999, Column Handbook for Size Exclusion Chromatography, Academic Press, San Diego; and Oliver, 1989, HPLC of Macromolecules: A Practical Approach, Oxford University Press, Oxford, England. In particular, procedures are available for systematic development and optimization of chromatographic separations given conditions, such as column type, solid phase, and the like. See, e.g., Haber et ah, 2000, J. Chromatogr. Sd. 38:386-392; Outinen et ah, 1998, Eur. J. Pharm. ScI 6:197-205; Lewis et ah, 1992, J. Chromatogr. 592:183-195 and 197- 208; and the like. An exemplary HPLC instrumentation system suitable for use with the present invention is the Agilent 1100 Series HPLC system (Agilent Technologies, Palo Alto, CA).

[0263] In one aspect, molecular tag, E, is (M, D), where M is a mobility- modifying moiety and D is a detection moiety. The notation "(M, D)" is used to indicate that the ordering of the M and D moieties may be such that either moiety can be adjacent to the cleavable linkage, L. That is, "B-L-(M, D)" designates binding compound of either of two forms: "B-L-M-D" or "B-L-D-M."

[0264] Detection moiety, D, may be a fluorescent label or dye, a chromogenic label or dye, an electrochemical label, or the like. Preferably, D is a fluorescent dye. Exemplary fluorescent dyes for use with the invention include water-soluble rhodamine dyes, fluoresceins, 4,7-dichlorofluoresceins, benzoxanthene dyes, and energy transfer dyes, as disclosed in the following references: Anonymous, 2002, Handbook of Molecular Probes and Research Reagents, 8^th ed., Molecular Probes, Eugene, OR; U.S. Patent Nos. 6,191,278, 6,372,907, 6,096,723, 5,945,526, 4,997,928, and 4,318,846; and Lee et al., 1997, Nucleic Acids Research 25:2816-2822. Preferably, D is a fluorescein or a fluorescein derivative.

[0265] In an embodiment illustrated in Fig. 3A, binding compounds comprise a biotinylated antibody (300) as a binding moiety. Molecular tags are attached to binding moiety (300) by way of avidin or streptavidin bridge (306). Preferably, in operation, binding moiety (300) is first reacted with a target complex, after which avidin or streptavidin is added (304) to form antibody-biotin-avidin complex (305). To such complexes (305) are added (308) biotinylated molecular tags (310) to form binding compound (312).

[0266] In still another embodiment illustrated in Fig. 3B, binding compounds comprise an antibody (314) derivatized with a multi-functional moiety (316) that contains multiple functional groups (318) that are reacted (320) molecular tag precursors to give a final binding compound having multiple molecular tags (322) attached. Exemplary multi-functional moieties include aminodextran, and like materials.

[0267] Once each of the binding compounds is separately derivatized by a different molecular tag, it is pooled with other binding compounds to form a plurality of binding compounds. Usually, each different kind of binding compound is present in a composition in the same proportion; however, proportions may be varied as a design choice so that one or a subset of particular binding compounds are present in greater or lower proportion depending on the desirability or requirements for a particular embodiment or assay. Factors that may affect such design choices include, but are not limited to, antibody affinity and avidity for a particular target, relative prevalence of a target, fluorescent characteristics of a detection moiety of a molecular tag, and the like.

5.3.2 Cleavage-Inducing Moiety Producing Active Species

[0268] A cleavage-inducing moiety, or cleaving agent, is a group that produces an active species that is capable of cleaving a cleavable linkage, preferably by oxidation. Preferably, the active species is a chemical species that exhibits short-lived activity so that its cleavage-inducing effects are only in the proximity of the site of its generation. Either the active species is inherently short lived, so that it will not create significant background because beyond the proximity of its creation, or a scavenger is employed that efficiently scavenges the active species, so that it is not available to react with cleavable linkages beyond a short distance from the site of its generation. Illustrative active species include singlet oxygen, hydrogen peroxide, NADH, and hydroxyl radicals, phenoxy radical, superoxide, and the like. Illustrative quenchers for active species that cause oxidation include polyenes, carotenoids, vitamin E, vitamin C, amino acid-pyrrole N-conjugates of tyrosine, histidine, and glutathione, and the like. See, e.g. Beutner et al., 2000, Meth. Enzymol. 319:226-241.

[0269] One consideration in designing assays employing a cleavage-inducing moiety and a cleavable linkage is that they not be so far removed from one another when bound to a receptor complex that the active species generated by the cleavage-inducing moiety cannot efficiently cleave the cleavable linkage. In one aspect, cleavable linkages preferably are within about 1000 nm, and preferably within about 20-200 nm, of a bound cleavage-inducing moiety. More preferably, for photosensitizer cleavage-inducing moieties generating singlet oxygen, cleavable linkages are within about 20-100 nm of a photosensitizer in a receptor complex. The range within which a cleavage-inducing moiety can effectively cleave a cleavable linkage (that is, cleave enough molecular tag to generate a detectable signal) is referred to herein as its "effective proximity." One of ordinary skill in the art will recognize that the effective proximity of a particular sensitizer may depend on the details of a particular assay design and may be determined or modified by routine experimentation.

[0270] A sensitizer is a compound that can be induced to generate a reactive intermediate, or species, usually singlet oxygen. Preferably, a sensitizer used in accordance with the invention is a photosensitizer. Other sensitizers included within the scope of the invention are compounds that on excitation by heat, light, ionizing radiation, or chemical activation will release a molecule of singlet oxygen. The best known members of this class of compounds include the endoperoxides such as 1,4- biscarboxyethyl- 1 ,4-naphthalene endoperoxide, 9, 10-diphenylanthracene-9, 10- endoperoxide and 5,6,11,12-tetraphenyl naphthalene 5,12-endoperoxide. Heating or direct absorption of light by these compounds releases singlet oxygen. Further sensitizers are disclosed by Di Mascio et al, 1994, FEBS Lett. 355:287 (peroxidases and oxygenases); and Kanofsky, 1983, J.Biol. Chem. 258:5991-5993 (lactoperoxidase); Pierlot et al., 2000, Meth. Enzymol. 319:3-20 (thermal lysis of endoperoxides). Attachment of a binding agent to the cleavage-inducing moiety may be direct or indirect, covalent or non-covalent, and can be accomplished by well-known techniques commonly available in the literature. See, e.g., Ichiro Chibata, 1978, Immobilized Enzymes, Halsted Press, New York; and Cuatrecasas, 1970, J. Biol. Chem. 245:3059.

[0271] As mentioned above, the preferred cleavage-inducing moiety in accordance with the present invention is a photosensitizer that produces singlet oxygen. As used herein, "photosensitizer" refers to a light-adsorbing molecule that when activated by light converts molecular oxygen into singlet oxygen. Photosensitizers may be attached directly or indirectly, via covalent or non-covalent linkages, to the binding agent of a class-specific reagent. Guidance for constructing such compositions, particularly for antibodies as binding agents, available in the literature, e.g. in the fields of photodynamic therapy, immunodiagnostics, and the like. Exemplary guidance may be found in Ullman et al, 1994, Proc. Natl. Acad. Sci. USA 91, 5426-5430; Strong et al, 1994, Ann. New York Acad. Sci. 745: 297-320; Yarmush et al, 1993, Crit. Rev. Therapeutic Drug Carrier Syst. 10: 197-252; and U.S. Patent Nos. 5,709,994, 5,340,716, 6,251,581, and 5,516,636.

[0272] A large variety of light sources are available to photo-activate photosensitizers to generate singlet oxygen. Both polychromatic and monochromatic sources may be used as long as the source is sufficiently intense to produce enough singlet oxygen in a practical time duration. The length of the irradiation depends on the nature of the photosensitizer, the nature of the cleavable linkage, the power of the source of irradiation, and its distance from the sample, and so forth. In general, the period for irradiation may be less than about a microsecond to as long as about 10 minutes, usually in the range of about one millisecond to about 60 seconds. The intensity and length of irradiation should be sufficient to excite at least about 0.1% of the photosensitizer molecules, usually at least about 30% of the photosensitizer molecules and preferably, substantially all of the photosensitizer molecules. Exemplary light sources include, by way of illustration and not limitation, lasers such as, e.g., helium-neon lasers, argon lasers, YAG lasers, He/Cd lasers, and ruby lasers; photodiodes; mercury, sodium and xenon vapor lamps; incandescent lamps such as, e.g., tungsten and tungsten/halogen; flashlamps; and the like. An exemplary photoactivation device suitable for use in the methods of the invention is disclosed International Patent Publication No. WO 03/051669. In such embodiments, the photoactivation device is an array of light emitting diodes (LEDs) mounted in housing that permits the simultaneous illumination of all the wells in a 96-well plate. A suitable LED for use in the present invention is a high power GaAIAs IR emitter, such as model OD-880W manufactured by OPTO DIODE CORP. (Newbury Park, CA).

[0273] Examples of photosensitizers that may be utilized in the present invention are those that have the above properties and those disclosed by U.S. Patent Nos. 5,536,834, 5,763,602, 5,565,552, 5,709,994, 5,340,716, 5,516,636, 6,251,581, and 6,001,673; published European Patent Application No. 0484027; Martin et al, 1990, Methods Enzymol. 186:635-645; and Yarmush et al, 1993, Crit. Rev. Therapeutic Drug Carrier Syst. 10:197-252.

[0274] As with sensitizers, in certain embodiments, a photosensitizer may be associated with a solid phase support by being covalently or non-covalently attached to the surface of the support or incorporated into the body of the support. In general, the photosensitizer is associated with the support in an amount necessary to achieve the necessary amount of singlet oxygen. Generally, the amount of photosensitizer is determined empirically according to routine methods.

[0275] In one embodiment, a photosensitizer is incorporated into a latex particle to form photosensitizer beads, e.g. as disclosed by U.S. Patent Nos. 5,709,994 and 6,346,384; and International Patent Publication No. WO 01/84157. Alternatively, photosensitizer beads may be prepared by covalently attaching a photosensitizer, such as rose bengal, to 0.5 micron latex beads by means of chloromethyl groups on the latex to provide an ester linking group, as described in J Amer. Chem. Soc, 91:31 Al (1975). Use of such photosensitizer beads is illustrated in Fig. 3C and 3D. As described in Fig. 3 C for heteroduplex detection, complexes (230) are formed after combining reagents (1122) with a sample. This reaction may be carried out, for example, in a conventional 96-well or 384-well microtiter plate, or the like, having a filter membrane that forms one wall, e.g. the bottom, of the wells that allows reagents to be removed by the application of a vacuum. This allows the convenient exchange of buffers, if the buffer required for specific binding of binding compounds is different that the buffer required for either singlet oxygen generation or separation. For example, in the case of antibody-based binding compounds, a high salt buffer is required. If electrophoretic separation of the released tags is employed, then better performance is achieved by exchanging the buffer for one that has a lower salt concentration suitable for electrophoresis. In this embodiment, instead of attaching a photosensitizer directly to a binding compound, such as an antibody, a cleaving probe comprises two components: antibody (232) derivitized with a capture moiety, such as biotin (indicated in Fig. 3C as "bio") and photosensitizer bead (338) whose surface is derivatized with an agent (234) that specifically binds with the capture moiety, such as avidin or streptavidin. Complexes (230) are then captured (236) by photosensitizer beads by way of the capture moiety, such as biotin (bio). Conveniently, if the pore diameter of the filter membrane is selected so that photosensitizer beads (338) cannot pass, then a buffer exchange also serves to remove unbound binding compounds, which leads to an improved signal. After an appropriate buffer for separation has been added, if necessary, photosensitizer beads (338) are illuminated (240) so that singlet oxygen is generated (242) and molecular tags are released (244). See Figure 3D. Such released molecular tags (346) are then separated to form separation profile (352) and dimers are quantified ratiometrically from peaks (348) and (350). See Figure 3D. Photosensitizer beads may be used in either homogeneous or heterogeneous assay formats.

[0276] Preferably, when analytes, such as cell surface receptors, are being detected or antigen in a fixed sample, a cleaving probe may comprise a primary haptenated antibody and a secondary anti-hapten binding protein derivatized with multiple photosensitizer molecules. A preferred primary haptenated antibody is a biotinylated antibody, and preferred secondary anti-hapten binding proteins may be either an anti- biotin antibody or streptavidin. Other combinations of such primary and secondary reagents are well known in the art. Exemplary combinations of such reagents are taught by Haugland, 2002, Handbook of Fluorescent Probes and Research Reagents, Ninth Edition, Molecular Probes, Eugene, OR. An exemplary combination of such reagents is described below. There binding compounds having releasable tags CmT₁" and "mT₂"), and primary antibody derivatized with biotin are specifically bound to different epitopes of receptor dimer in membrane. Biotin-specific binding protein, e.g. streptavidin, is attached to biotin bringing multiple photosensitizers into effective proximity of binding compounds. Biotin-specific binding protein may also be an anti-biotin antibody, and photosensitizers may be attached via free amine group on the protein by conventional coupling chemistries, e.g. , Hermanson (supra). An exemplary photosensitizer for such use is an NHS ester of methylene blue prepared as disclosed in published European Patent Application 0510688.

5.3.3 Conditions for Assays Employing Releasable Molecular Tags

[0277] The following general discussion of methods and specific conditions and materials are by way of illustration and not limitation. One of skill in the art will understand how the methods described herein can be adapted to other applications, particularly with using different samples, cell types and target complexes.

[0278] In conducting the methods of the invention, a combination of the assay components is made, including the sample being tested, the binding compounds, and optionally the cleaving probe. Generally, assay components may be combined in any order. In certain applications, however, the order of addition may be relevant. For example, one may wish to monitor competitive binding, such as in a quantitative assay. Or one may wish to monitor the stability of an assembled complex. In such applications, reactions may be assembled in stages, and may require incubations before the complete mixture has been assembled, or before the cleaving reaction is initiated.

[0279] The amounts of each reagent can generally be determined empirically. The amount of sample used in an assay will be determined by the predicted number of target complexes present and the means of separation and detection used to monitor the signal of the assay. In general, the amounts of the binding compounds and the cleaving probe can be provided in molar excess relative to the expected amount of the target molecules in the sample, generally at a molar excess of at least about 1.5, more desirably about 10- fold excess, or more. In specific applications, the concentration used may be higher or lower, depending on the affinity of the binding agents and the expected number of target molecules present on a single cell. Where one is determining the effect of a chemical compound on formation of oligomeric cell surface complexes, the compound may be added to the cells prior to, simultaneously with, or after addition of the probes, depending on the effect being monitored.

[0280] The assay mixture can be combined and incubated under conditions that provide for binding of the probes to the cell surface molecules, usually in an aqueous medium, generally at a physiological pH (comparable to the pH at which the cells are cultures), maintained by a buffer at a concentration in the range of about 10 to 200 mM. Conventional buffers may be used, as well as other conventional additives as necessary, such as salts, growth medium, stabilizers, etc. Physiological and constant temperatures are normally employed. Incubation temperatures normally range from about 4° to 70°C, usually from about 15° to 45°C, more usually about 25° to 37⁰C.

[0281] After assembly of the assay mixture and incubation to allow the probes to bind to cell surface molecules, the mixture can be treated to activate the cleaving agent to cleave the tags from the binding compounds that are within the effective proximity of the cleaving agent, releasing the corresponding tag from the cell surface into solution. The nature of this treatment will depend on the mechanism of action of the cleaving agent. For example, where a photosensitizer is employed as the cleaving agent, activation of cleavage can comprise irradiation of the mixture at the wavelength of light appropriate to the particular sensitizer used.

[0282] Following cleavage, the sample can then be analyzed to determine the identity of tags that have been released. Where an assay employing a plurality of binding compounds is employed, separation of the released tags will generally precede their detection. The methods for both separation and detection are determined in the process of designing the tags for the assay. A preferred mode of separation employs electrophoresis, in which the various tags are separated based on known differences in their electrophoretic mobilities.

[0283] As mentioned above, in some embodiments, if the assay reaction conditions may interfere with the separation technique employed, it may be necessary to remove, or exchange, the assay reaction buffer prior to cleavage and separation of the molecular tags. For example, assay conditions may include salt concentrations (e.g. required for specific binding) that degrade separation performance when molecular tags are separated on the basis of electrophoretic mobility. Thus, such high salt buffers may be removed, e.g., prior to cleavage of molecular tags, and replaced with another buffer suitable for electrophoretic separation through filtration, aspiration, dilution, or other means.

5.4 Methods of Treatment [0284] In yet another aspect, the invention further provides methods of treating a subject with cancer, In one aspect, the methods comprise detenrήning that the subject has a cancer comprising a cancer cell that is likely to respond to treatment with a Herl- acting according to a method of the invention, and administering an effective amount of a Her 1 -acting agent to the subject. In certain embodiments, the Her 1 -acting agent is Gefitinib.

[0285] In another aspect, the methods comprise determining that a subject has a cancer comprising a cancer cell that is likely to respond to treatment with a Her 1 -acting agent according to a method of the invention, then advising a medical professional of the treatment option of administering to the subject an effective amount of a Her 1 -acting agent. In certain embodiments, the Herl -acting agent is Gefitinib.

[0286] In another aspect, the methods comprise determining that a subject is has cancer comprising a cancer cell that is likely to respond to treatment with a Herl -acting agent according to a method of the invention, then advising a medical professional to treat the subject with an effective amount of a Herl -acting agent. In certain embodiments, the Herl -acting agent is Gefitinib.

[0287] In still another aspect, the methods comprise determining that a subject has a pre-cancerous condition that is likely to respond to treatment with a Herl -acting according to a method of the invention, and administering an effective amount of a Herl - acting agent to the subject, In certain embodiments, the Herl -acting agent is Gefitinib.

[0288] In still another aspect, the methods comprise determining that a subject has a pre-cancerous condition that is likely to respond to treatment with a Herl -acting according to a method of the invention, then advising a medical professional of the treatment option of administering to the subject an effective amount of a Herl -acting agent. In certain embodiments, the Herl -acting agent is Gefitinib.

[0289] In yet another aspect, the methods comprise determining that a subject has a pre-cancerous condition that is likely to respond to treatment with a Herl -acting agent according to a method of the invention, then advising a medical professional to treat the subject with an effective amount of a Herl -acting agent, In certain embodiments, the Herl -acting agent is Gefitinib. [0290] In still another aspect, the methods comprise deteraiining that a subject has a cancer or pre-cancerous condition that is likely to respond to treatment with a Herl- acting agent according to a method of the invention at a first time, then determining that the subject remains with a cancer or pre-cancerous condition that is likely to respond to treatment with a Her 1 -acting agent according to a method of the invention at a later second time. In other embodiments, the methods comprise determining that a subject has a cancer or pre-cancerous condition that is likely to respond to treatment with a Herl- acting agent according to a method of the invention at a first time, then determining that the subject remains with a cancer or pre-cancerous condition that is not likely to respond to treatment with a Her 1 -acting agent according to a method of the invention at a later second time.

[0291] In certain embodiments, the subject has locally advanced or metastatic non- small cell lung cancer. In certain embodiments, the cancer has failed to respond to platinum-based chemotherapy. In certain embodiments, the cancer has failed to respond to docetaxel. m certain embodiments, about 250 mg Gefitinib is administered. In certain embodiments, about 500 mg Gefitinib is administered. In certain embodiments, between about 10 mg and about 500 mg Gefitinib is administered.

5.5 Other Embodiments of the Invention

[0292] In still other aspects, the invention provides several additional embodiments of the invention. In one such aspect, the invention provides a computer- implemented method for determining whether a cancer cell is likely to respond to treatment with a Her 1 -acting agent. Such methods generally comprise performing a method of the invention with a computer system adapted to perform the method of the invention. Such adaptation is well within the skill of those in the art.

[0293] In certain embodiments, the methods comprise calculating a Diagnostic

Index of a cancer or a probability that the cancer will respond to treatment with an Herl- acting agent using a formula of the invention with a computer. In certain embodiments, the method further comprises the step of displaying the Diagnostic Index of the cancer or the probability of responding to treatment with a Her 1 -acting agent on a computer display. In other embodiments, the method further comprises the step of printing the Diagnostic Index of the cancer or the probability of responding to treatment with a Herl- acting agent onto a tangible medium, such as, for example, paper.

[0294] In another aspect, the invention provides a display that indicates that a cancer or cancer cell is likely to respond to treatment with a Herl -acting agent. In certain embodiments, the display is a computer display. Preferably, the likelihood of the cancer or cancer cell to respond to such treatment is determined according to a method or formula of the invention.

[0295] In still another aspect, the invention provides a paper document that indicates that a cancer or cancer cell is likely to respond to treatment with a Herl -acting agent. In certain embodiments, the paper document is a printed document. In certain embodiments, the printed document is a computer print-out. Preferably, the likelihood of the cancer or cancer cell to respond to such treatment is determined according to a method or formula of the invention.

[0296] In still another aspect, the invention provides a computer-readable memory that comprises data indicating that a cancer or cancer cell is likely to respond to treatment with a Herl-acting agent. In certain embodiments, the computer-readable memory is a random-access memory, In certain embodiments, the computer-readable memory is a fixed disk. In certain embodiments, the computer-readable memory is a floppy disk. In certain embodiments, the computer-readable memory is a portable memory device, such as, e.g., a USB key or an iPod™. Preferably, the likelihood of the cancer or cancer cell to respond to such treatment is determined according to a method of the invention.

[0297] In still another aspect, the invention provides a computer-readable memory that comprises data comprising a number of biomarkers on a cancer or cancer cell that are associated with responsiveness to Gefitinib therapy as described herein and computer-readable instructions for determining the Diagnostic Index of the cancer or cancer cell or probability that the cancer or cancer cell will respond to Gefitinib therapy, In certain embodiments, the computer-readable memory is a random-access memory. In certain embodiments, the computer-readable memory is a fixed disk, In certain embodiments, the computer-readable memory is a floppy disk. In certain embodiments, the computer-readable memory is a portable memory device, such as, e.g., a USB key or an iPod™. Preferably, the likelihood of the cancer or cancer cell to respond to such treatment is determined according to a method or formula of the invention.

[0298] In yet another aspect, the invention also provides kits that are useful in determining whether a cancer or cancer cell is likely to respond to treatment with a Herl- acting agent, In certain embodiments, the kits of the present invention comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195 or 200 or more binding compounds that can be used to detect and/or quantify one or more biomarkers correlated with responsiveness to treatment with a Her 1 -acting agent. In other embodiments, the kits of the present invention comprise at least 2, but as many as several hundred or more such binding compounds. The kit may also comprise one or more cleaving probes for use in a method of the invention. The kit may also comprise at least one internal standard to be used in generating the biomarker profiles of the present invention. Likewise, the internal standard or standards can be any of the classes of compounds described above.

[0299] The kits of the present invention may also include reagents such as buffers, or other reagents that can be used in detecting the biomarker(s) associated with responsiveness to treatment with a Her 1 -acting agent. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like.

[0300] Some kits of the invention may further comprise a computer program product for use in conjunction with a computer system, wherein the computer program product comprises a computer readable storage medium and a computer program mechanism embedded therein, In such kits, the computer program mechanism comprises instructions for evaluating whether a plurality of features in a biomarker profile of a cancer or cancer cell satisfies a first value set. Satisfying the first value set predicts that the cancer or cancer cell is likely to respond to treatment with a Herl acting agent, hi one embodiment, the plurality of features corresponds to the presence and/or amount of expression of Herl -Herl dimers, Herl-Her2 dimers, Herl-Her3 dimers, Her2-Her3 dimers, Herl phosphorylation, Her2 phosphorylation, Her3 phosphorylation, or any other biomarker described herein as correlated with responsiveness or non-responsiveness to treatment with a Her 1 -acting agent. In some kits, the computer program product further comprises instructions for evaluating whether the plurality of features in the biomarker profile of the test subject satisfies a second value set. Satisfying the second value set predicts that the cancer or cancer cell is not likely to respond to treatment with a Herl acting agent.

[0301] Some kits of the present invention comprise a computer having a central processing unit and a memory coupled to the central processing unit. The memory stores instructions for evaluating whether a plurality of features in a biomarker profile of a cancer or cancer cell satisfies a first value set. Satisfying the first value set predicts that the cancer or cancer cell is likely to respond to treatment with a Herl acting agent, In one embodiment, the plurality of features corresponds the presence and/or amount of expression of Herl -Herl dimers, Herl-Her2 dimers, Herl-Her3 dimers, Her2-Her3 dimers, Herl phosphorylation, Her2 phosphorylation, Her3 phosphorylation, or any other biomarker described herein as correlated with responsiveness or non-responsiveness to treatment with a Herl -acting agent.

[0302] Fig. 11 details an exemplary system that supports the functionality described above. The system is preferably a computer system 10 having:

a central processing unit 22; a main non- volatile storage unit 14, for example, a hard disk drive, for storing software and data, the storage unit 14 controlled by storage controller 12; a system memory 36, preferably high speed random-access memory (RAM), for storing system control programs, data, and application programs, comprising programs and data loaded from non-volatile storage unit 14; system memory 36 may also include read-only memory (ROM); a user interface 32, comprising one or more input devices (e.g., keyboard 28) and a display 26 or other output device; a network interface card 20 for connecting to any wired or wireless communication network 34 (e.g., a wide area network such as the Internet); an internal bus 30 for interconnecting the aforementioned elements of the system; and a power source 24 to power the aforementioned elements.

[0303] Operation of computer 10 is controlled primarily by operating system 40, which is executed by central processing unit 22. Operating system 40 can be stored in system memory 36. In addition to operating system 40, in a typical implementation system memory 36 includes:

file system 42 for controlling access to the various files and data structures used by the present invention; a training data set 44 for use in construction one or more decision rules in accordance with the present invention; a data analysis algorithm module 54 for processing training data and constructing decision rules; one or more decision rules 56; a biomarker profile evaluation module 60 for determining whether a plurality of features in a biomarker profile of a cancer or cancer cell satisfies a first value set; a test subject biomarker profile 62 comprising biomarkers 64 and, for each such biomarkers, features 66; and a database 68 of select biomarkers of the present invention as described herein.

[0304] Training data set 46 comprises data for a plurality of subjects 46. For each subject 46 there is a subject identifier 48 and a plurality of biomarkers 50. For each biomarker 50, there is at least one feature 52. Although not shown in Figure 35, for each feature 52, there is a feature value. For each decision rule 56 constructed using data analysis algorithms, there is at least one decision rule value set 58.

[0305] As illustrated in Figure 11, computer 10 comprises software program modules and data structures. The data structures stored in computer 10 include training data set 44, decision rules 56, test subject biomarker profile 62, and biomarker database 68. Each of these data structures can comprise any form of data storage system including, but not limited to, a flat ASCII or binary file, an Excel spreadsheet, a relational database (SQL), or an on-line analytical processing (OLAP) database (MDX and/or variants thereof). In some specific embodiments, such data structures are each in the form of one or more databases that include hierarchical structure (e.g., a star schema). In some embodiments, such data structures are each in the form of databases that do not have explicit hierarchy {e.g., dimension tables that are not hierarchically arranged).

[0306] In some embodiments, each of the data structures stored or accessible to system 10 are single data structures. In other embodiments, such data structures in fact comprise a plurality of data structures (e.g., databases, files, archives) that may or may not all be hosted by the same computer 10. For example, in some embodiments, training data set 44 comprises a plurality of Excel spreadsheets that are stored either on computer 10 and/or on computers that are addressable by computer 10 across wide area network 34. In another example, training data set 44 comprises a database that is either stored on computer 10 or is distributed across one or more computers that are addressable by computer 10 across wide area network 34.

[0307] It will be appreciated that many of the modules and data structures illustrated in Figure 11 can be located on one or more remote computers. For example, some embodiments of the present application are web service-type implementations, In such embodiments, biomarker profile evaluation module 60 and/or other modules can reside on a client computer that is in communication with computer 10 via network 34. In some embodiments, for example, biomarker profile evaluation module 60 can be an interactive web page.

[0308] In some embodiments, training data set 44, decision rules 56, and/or biomarker database 68 illustrated in Figure 11 are on a single computer (computer 10) and in other embodiments one or more of such data structures and module are hosted by one or more remote computers (not shown). Any arrangement of the data structures and software modules illustrated in Figure 11 on one or more computers is within the scope of the present invention so long as these data structures and software modules are addressable with respect to each other across network 34 or by other electronic means. Thus, the present invention fully encompasses a broad array of computer systems.

6. EXAMPLES

[0309] The following examples provide methods and exemplary results showing detection of ErbB monomers and dimers on patient samples and statistical correlations between expression levels of ErbB dimers and responsiveness to an exemplary Herl- acting agent, Gefitinib.

6.1 Sources of Materials Used in Examples

[0310] Antibodies that specifically bind Her receptors, adaptor molecules, and normalization standards are obtained from commercial vendors, including Labvision, Cell Signaling Technology, and BD Biosciences. All cell lines were purchased from ATCC (Manassas, VA).

[0311] The molecular tag-antibody conjugates used below are formed by reacting

NHS esters of the molecular tag with a free amine on the indicated antibody using conventional procedures. Molecular tags are disclosed in U.S. Published Application Nos. 2003/017915 and 2002/0013126, which are each incorporated by reference. Briefly, binding compounds below are molecular tag-monoclonal antibody conjugates formed by reacting an NHS ester of a molecular tag with free amines of the antibodies in a conventional reaction.

6.2 Example 1: Detection of Herl Dimers, Herl-Her2 Dimers, and Her2-Her3 Dimers and Receptor Phosphorylation

[0312] This example describes detection of Herl -Herl homodimers and Herl-

Her2 and Her2-Her3 heterodimers and receptor phosphorylation.

Sample Preparation

[0313] Samples were prepared according to the following protocol. For double- coated formalin-fixed paraffin embedded (FFPE slides) tissue samples, the FFPE samples were incubated in solvent baths filled with xylene at 55°C for 20 minutes. The slides were then transferred to fresh xylene jars and incubated for 10 min at 55°C. The slides were then treated as regular FFPE slides, as follows.

[0314] First, any excess paraffin particles along the edges of all slides was scrapped off with razor blade, as needed. Next, the FFPE slides were incubated back to back in xylene at 55⁰C for 5 min. Slides were agitated up and down twice during the deparaffmization procedure. Next, the slides were transferred to fresh xylene jars and soaked for additional 5 min at 55⁰C. Slides were checked for any residual paraffin particles and, if any was found, the slides were soaked for additional 5 min at 55⁰C. Next, the slides were removed from the xylene solution, excess xylene was blotted off by tapping the bottom of the slides on paper towel briefly, and the slides were transferred to a 100% ethanol bath and soaked for 5 min. The slides were agitated up and down in the bath several times during soaking. The 100% ethanol wash was then repeated once.

[0315] Next, the slides were placed in a 70% ethanol bath and soaked for 5 min.

The slides were agitated up and down in the bath for several times during soaking. The 70% ethanol wash was then repeated once. After removing the slides from the 70% ethanol wash, the slides were soaked for 5 min in a deionized water bath. The slides were agitated up and down in the bath several times during soaking. The deionized water wash was then repeated once. Next, the slides were soaked for additional 5 min in IxPBS. Finally, the slides were placed in fresh IxPBS, whereupon the slides were stored in the IxPBS at 4⁰C for future experiments.

Tissue Pre-digestion

[0316] First, the slide was removed from IxPBS bath and placed slides in Ventana

Discovery System (Ventana model Discovery® XT; Ventana Medical Systems, Tucson, AZ) and digested with protease digestion on Ventana with Protease 2 (Ventana cat # 760-2019) using protocol 29. Protocol 29 contains no deparaffmization procedure and uses protease 2 for 8 min for digestion. The slides were then removed from the apparatus and cleaned with protease wash buffer, followed by a DI water rinse, both using squirt bottle.

Heat-induced epitope retrieval

[0317] First, slides were placed in a staining dish containing 250 ml citrate buffer, then heated in a microwave oven and on power level 10 for 4 min followed by power level 3 for 10 min, ensuring that the buffer boils during heating. Next, the slides are cooled to room temperature for 1 h. Next, the slides were removed from the citrate buffer, placed in a DI water bath and soaked for 5 min; ensuring that all slides are immersed under the water level. The slides were agitated up and down in the bath several times during soaking. The DI water was then replaced with IxPBS and soaked for an additional 5 min.

eTas Assay for Dirtier Detection [0318] Slides were removed from the IxPBS bath and briefly blotted to remove excess liquid on edges of the slides with Kimwipes or vacuum, while ensuring the sample sections were hydrated at all times. Hydrophobic circles were drawn around the sections, whenever possible with a gap distance of at least 3-5 mm. Slides were placed in black humidified chamber box (Scientific Device Lab cat # 197BL; Scientific Device Lab, Des Plaines, IL) and 50 μl of blocking buffer was added to each section, then blocked for 1 hour at room temperature. Next, blocking buffer (see Table 3, below) was removed via vacuum and 30 μl of antibody mix (see Tables 4-8, below) was added to each section and incubated overnight at 4 ⁰C. Next, the antibody mix was removed via vacuum. Using a disposable transfer pipet, ~50 μl of eTag™ wash buffer was dropped onto each section. Wash buffer was removed via vacuum and then repeated twice.

Table 4: HER 1/1 Ab mix in blocking buffer

Table 6: HER 1/3 Ab mix in blocking buffer

Table 7: HER 2/2 Ab mix in blocking buffer

[0319] Working scissors (cleavage probe) solution was then prepared in the dark room. The blocking buffer was removed via vacuum and a variable amount of scissors solution (prepared according to Table 9, below) was applied to each section and incubated at room temperature for 1 hour. The volume of scissors solution added depended on the diameter of the section: for 2 mm diameter sections and smaller, 10 μ\ scissors was added; for 2-9 mm diameter sections, 30 ju.1 scissors was added; and for 10 mm diameter sections and larger, 80 μl scissors was added.

Table 9: Scissors solution in eTa ™ wash buffer

[0320] Next, scissors solution was removed via vacuum and the slides were transferred to a DI water bath with a 24-slide rack. The slide rack was agitated up and down, then rinsed again two more times. The slides were then removed from the water bath, then excess water on and around the sections was removed briefly with vacuum. Then, 25 μl of illumination buffer (prepared according to Table 10, below) was added to each section, then illuminated at 680 nm for 2 hours under cold condition using a 20- slot illuminator device.

Table 10: Illumination buffer

[0321] Next, the samples were transferred to sample plates with defined sample layout, and the buffer containing the sample on the sections was mixed briefly prior to transferring to the sample plates. Each section was blocked with 30 μ.1 of blocking buffer a for second incubation or slides stored in IxPBS at 4 ⁰C for future use.

[0322] eTag™ reporters were then detected from each sample using an ABI 3100

(Applied Biosystems Inc, Foster City, CA) with for 1080s on a 36cm capillary according to the conditions presented in Table 11. Representative electropherograms showing amounts of Her 1, Her2, and Her3 monomers and Herl-Herl, Herl-Her2, and Her2-Her3 dimers are presented in Figures 4-7.

Table 11

Data Analysis

[0323] Analysis of CE separations of eTag™ reporters was carried out using eTag™ Informer™ software (Aclara, Mountain View, CA; catalog # PN1500-0010). All eTag™ reporter peaks in the electropherogram were identified and labeled with the appropriate target protein name, quantitative results based on peak area and calibration curves were determined. The illumination buffer contained the CE Standard (CES: Aclara cat # ACLA-P-M-I) reagent with three CE internal standards (markers): MF, M3 and ML. Peak areas were normalized against an M3, which minimizes well-to-well variation. The normalized peak area was used to calculate the protein and dimer concentrations of the samples. Figures 8-10 resent exemplary data and bar graphs generated by this process.

6.3 Example 2: Correlations Between ErbB Dimer

Expression Levels and Responsiveness to Gefitinib Therapy

[0324] This example provides methods and results for experiments to correlate expression levels of particular ErbB receptor dimers with responsiveness to therapy with an exemplary Herl -acting agent, Gefitinib. ha particular, this example describes determinations of correlations between amounts of Herl -Her dimer expression, Herl- Her3 dimer expression, and Her2-Her3 dimer expression and responsiveness to Gefitinib therapy.

[0325] The samples of forty-eight lung cancer patients treated with Gefitinib

(IRESSA^®) were analyzed. eTag™ assays were conducted on the 48 samples according to the methods described in Example 1, above, and the amounts of the following receptors were measured for each sample: Herl total; Herl -Herl dimer; phosphorylated Herl; extracellular Her2 total; extracellular Her 1-Her2 dimer; intracellular Her2 total; intracellular Herl-Her2 dimer; phosphorylated Her2; Her3 total; Her2-Her3 dimer; and Herl-Her3 dimer.

[0326] All measurements of dimer amounts calculated in units of dimer per cell, while all other measurements were calculated in relative fluorescence units (RFU). The tumor percentage of each sample was also measured using the immunohistochemistry (IHC) or hematoxylin and eosin (H&E) image method and used to calculate the number of cancer cells present in each sample and the ratio of cancer cells to normal cells in the sample. Briefly, samples were scored for tumor cell percentage relative to normal cell based on cell morphology, differentiation patterns, and grade and invasiveness of the cancer based on standard histological techniques. During measurements of dimer expression, molecular tags that recognize cytokeratin and/or tubulin were used to estimate the total number of cells, both normal and cancerous, present in the sample. The total number of cells was then multiplied by the proportion of cancer cells in the sample to identify the number of cancer cells in the sample, which were then used to normalize the numbers of dimers expressed per cell.

[0327] In the 48 patients, 9 patients exhibited a partial response to treatment with

Gefitinib (PR), 7 patients exhibited stable disease (SD), and 32 patients exhibited progression of disease (PD). The nine patients exhibiting PR were #1, #2, #6, #8, #28, #31, #34, #40, and #44. The seven patients exhibiting SD were #7, #18, #20, #23, #36, #39, and #43. All the remaining patients were patients with PD.

Data Screening

[0328] Since the eTag assays measure the protein expression, protein interaction, and protein activation in tumor cells, patient #39 (patient with SD) and patient #41 (patient with PD) were dropped from the sample pool due to zero percent tumor cell observed from IHC image. Further, the samples for patient #22 and patient #49 were actually separate samples obtained from the same patient; the sample from patient #22 was preserved on a FFPE slide while the sample from patient #49 was preserved as a lysate. The results were very consistent between the two samples. Therefore, to avoid duplicated data, only the FFPE results of patient #22 were used in the statistical analysis.

[0329] In total, 46 samples were used in the statistical analysis. The patients were divided to two groups, responsive group and non-responsive group. The responsive group included all patients with PR and SD, and the non-responsive group included all patients with PD. There were 15 patients in responsive group and 31 patients in non- responsive group in our sample pool used for the statistical analysis.

[0330] First, expression of the tested receptors was plotted versus response to make an initial estimate of factors correlating with responsiveness. Expression of all of the measured receptors was plotted against their responsiveness to Gefitinib as shown in Figure 1OA and 1OB. Figures 1OA and 1OB present the same data; Figure 1OA is drawn in arithmetic scale, while Figure 1OB is drawn in log scale to better show middle to high expression levels.

[0331] Figures 1OA and 1OB show that the expression levels of Herl-Herl dimer separates responsive versus non-responsive patients fairly well. Most patients in non- responsive group had smaller Herl/1 dimer than that of patients in responsive group. In addition, for some receptors, the low level data spread over patients in both groups. There were some extremely high values only appearing in patients in non-responsive group, such as, for example, extra Herl/2 dimer, Her2 phospho, H2/3 dimer, and Her3 total, as shown in Table 12, below.

Table 12: Overexpressed Rece tors in Non-Res onders

Two sample analysis

[0332] To confirm the observation discussed above, two-sample tests were used to assess the equality of the means of the two groups for each measurement. The most popular two-sample test is t-test, which assumes normality of the two groups. However, Figures 1OA and 1OB indicates that most of the measurements of receptor expression do not exhibit normal distribution.

[0333] Therefore, the Shapiro- WiIk test was used to test the normality of the two groups for each measurement. Only Her2 phospho measurement exhibited normal distribution in both groups, while all other ten measurements did not show significant agreement with normality at all (data not shown).

[0334] In view of this result, the two-sample Wilcoxon test was used instead of the t-test, since the two-sample Wilcoxon test only assumes a common continuous distribution. The Wilcoxon test showed that the Herl/1 dimer measurement in the two groups was not equal with statistical significance (p-value = 0.0097). The other measurements did not show any significant difference between the two groups with the test (data not shown) in isolation. However, Her2 phospho measurement had statistically significant difference between the two groups by t-test (p-value is 0.044).

Classification By Measurements

[0335] In view of the above basic analysis, Herl/1 dimer measurement was chosen as the first variable to classify patients to two groups. The cut-off value of 1100 dimers/cell was selected to maximize the sensitivity of the method without adversely affecting specificity. Therefore, a patient with more than 1100 Herl/1 dimers per tumor cell was termed responsive group, and a patient with less than 1100 Herl/1 dimers per tumor cell was put in non-responsive group. A two by two contingency table was constructed, and sensitivity, specificity, positive predictive value, and negative predictive value were calculated directly from the table. The two-tailed p-value was calculated by Fisher's exact test, and the 95% confidence intervals for sensitivity, specificity, positive predictive value, and negative predictive value were obtained from Yates-Corrected-Chi- Square test and shown in Table 13. Table 14 shows the same data as Table 13, but the responsive patients from Table 13 were split into PR and SD as shown in Table 14.

Table 13

Table 14

[0336] The observation that a patient did not respond to Gefltinib when expression of some receptors are extremely high could be explained by considering that there may be negative factors that influence a patient's responsiveness to Gefltinib, since Gefltinib is designed to block Herl phosphorylation. Therefore, Her2 phospho and Her2-Her3 dimer were tested as determinant variables to classify patients further, and the results are shown in Tables 15 and 16. Table 16 shows the same data as Table 15, but the responsive patients from Table 15 were split into PR and SD as shown in Table 16.

Table 15

Table 16

[0337] Next, Herl-Her3 tested as an additional variable to classify the patients, since Herl-Her3 could possibly act as a positive factor in predicting a patient's responsiveness to Gefitinib. Three patients that responded to Gefitinib were assigned to non-responsive group by the above two classification methods, and some of these patients had reasonably high Herl-Her3 dimer expression. Two different Herl/3 cut-off values were studied to improve the sensitivity with or without hurting the specificity, and the results are shown in Tables 17 and 19. Tables 18 and 20 show the same data as Tables 17 and 19, respectively, but the responsive patients from Tables 17 and 19 were split into PR and SD as shown in Tables 18 and 20.

Table 17

Table 18

Table 19

Table 20

[0338] Next, the same test was performed without taking into account expression levels of Her2 phospho. Tables 21, 22, and 23, below, show the predictive power of expression levels Her 1 -Her 1 and Her2-Her3 dimers with two cut-off values for Herl- Her3 dimer expression. The data show that Her2 phospho expression levels do not add additional specificity or sensitivity to the prediction of responsiveness when Herl-Her3 expression levels are considered.

Table 21

Table 22

Table 23

[0339] Without intending to be bound by any particular theory or mechanism of action, it is believed that Her 1 -Her 1 homodimer expression levels best predict responsiveness to Her 1 -acting agents such as Gefitinib because such agents act by inhibiting a biological function of Her 1. Thus, if a cancer cell expresses Herl-Herl dimers in sufficient amounts, the cancer cell would be expected to respond to treatment with the Her 1 -acting agent. However, expression levels of Herl-Herl dimers cannot identify all cancers that would be expected to respond to treatment with a Herl -acting agent because of the propensity of Herl to dimerize with other ErbB receptors, such as, for example, Her3. Further, a cancer cell that expresses ErbB receptor dimers that do not comprise Herl would not be expected to respond to therapy with a Herl -acting agent, since the Herl -acting agent would not be expected to effectively inhibit any biological activity of a non-Herl receptor. Further explanation of the mechanism believed to underlie this phenomenon may be found, for example, In Pinkas-Kramarski et ah, 1996, EMBO J. 15:2452-2467, which is hereby incorporated by reference in its entirety.

[0340] Patient #34 was the only patient in this study who responded to Gefitinib, but was predicted to be non-responsive based on expression patterns of Herl-Herl, Herl-Her3, and Her2-Her3. This sample had low levels (below the cut-off values) of Herl/1 dimer and Herl/3 dimer. This patient's gene encoding EGFR comprised a deletion, and therefore it was hypothesized that the mutation could be slowing down receptor internalization and the mutant receptors were more sensitive than wild type receptor to inhibition by Gefitinib. Accordingly, the genes encoding EGFR from the remaining patients were sequenced to perform a mutational analysis.

[0341] Based on sequencing information of EGFR of the 46 patients, patient #22,

#28, #31, and #34 exhibited mutant EGFR. Out of the four patients with mutant EGFR, patient #25, #31, and #34 responded to Gefitinib, while patient #22 did not respond. Interestingly, the mutational analysis did not identify 6 PRs and 7 SDs in the sample pool, suggesting that mutational analysis alone cannot identify a substantial fraction of patients responsive to Gefitinib therapy.

6.4 Example 3: Logistic Regression Analysis Showing Correlations Between ErbB Dimer Expression Levels and Responsiveness to Gefitinib Therapy

[0342] This example describes a statistical analysis of ErbB dimer expression and phosphorylation on a second dataset. In the example, Herl-Herl dimer expression, Herl-Her2 dimer expression, Herl-Her3 dimer expression, Her2-Her3 dimer expression, and Her2 phosphorylation were analyzed on fifty samples from non-small cell lung cancer (NSCLC) patients treated with Gefitinib (IRESSA^®) selected from three clinical studies. The samples were selected for analysis based on the tumor percentage in the assay slides; samples with more 10% tumor cells of total cells on the slides were included in the data analysis. All measurements of receptor expression and phosphorylation were performed using the eTag™ assay as described above in Example 2. The results of the analysis are provided in Table 24, below.

Table 24

Legend

[0343] Sample: Sample Identification

Resp.: Responsiveness to gefitinb therapy; P = disease progression, S = stable disease, and R = disease response; responsiveness was scored by measuring amount of tumor shrinkage following Gefitinib therapy

HIT: Total number of Herl receptors detected per cell

Hl-Hl: Number of Herl-Herl dimers detected per cell

HlP: Number of phosphorylated Herl receptors detected per cell

H2T: Total number of Her2 receptors detected per cell

H1-H2: Number of Herl-Her2 dimers detected per cell

H2P: Number of phosphorylated Her2 receptors detected per cell

H3T: Total number of Herl receptors detected per cell

H2-H3: Number of Her2-Her3 dimers detected per cell

H1-H3: Number of Herl -Her 3 dimers detected per cell

% Tumor: percentage of tumor cells to total cells in assayed slide

[0344] Based on logistic regression analysis, described below, certain combinations of Herl-Herl dimer expression levels, Herl-Her2 dimer expression levels, Herl-Her3 dimer expression levels, Her2-Her3 dimer expression levels, and Her2 phosphorylation were identified as biomarkers for predicting NSCLC patients' responsiveness to Gefitinib therapy. Statview 5.0.1 (SAS Research Institute, North Carolina) was used to perform the logistic regression analysis. Multiple models, based on combinations of these biomarkers, were found to have strong predictive power as shown below.

6.4.1 Model I

[0345] This example describes a logistic regression model based on Herl-Herl dimer (Hl ID) expression levels, Herl-Her3 dimer (H 13D) expression levels, Her2-Her3 dimer (H23D) expression levels, and Her2 phosphorylation (H2P). The model has the formula:

log(p/(l+p)) = -2.09 + 0.992*log(HllD+H13D) -0.39*log(H2P) -0.187*log(H23D), where/) is the Diagnostic Index used to predict a patient's odds to respond to the drug, and p is between 0 and 1. This formula is referred to above as Formula I.

[0346] The whole model fitting p-value is 0.036 by likelihood ratio, and the coefficients table is displayed below as Table 25:

Table 25

Coef Std. Error Coef/SE Chi-Square P-Value Exp(Coef) constant -2.090 1.482 -1.410 1.989 .1584 .124

H11 D+H13D .992 .431 2.303 5.304 .0213 2.696

H2P -.390 .280 -1.395 1.945 .1631 .677

H23D -.187 .274 -.684 .467 .4942 .829

[0347] The predicted Diagnostic Index based on the model is presented in Table

26 below and is sorted by Diagnostic Index. For the formula used in model 1, a Diagnostic Index threshold of 0.4 distinguished cancers likely to respond to treatment with a Herl -acting agent, e.g., Gefitinib, from cancers unlikely to respond to such treatment, e.g., a cancer with a Diagnostic Index less than 0.4 was determined to be unlikely to respond to Herl -acting agent, e.g., Gefitinib, treatment, while a cancer with a Diagnostic Index greater than 0.4 was determined to be likely to respond to Herl -acting agent, e.g., Gefitinib, treatment. Application of this threshold Diagnostic Index to the Diagnostic Indexes determined according to Formula I identified 31 subjects not likely to respond to treatment with a Herl -acting agent, of which 4 were false negatives, and 31 subjects likely to respond to treatment with a Herl-acting agent, of which 11 were false positives.

Table 26

6.4.2 Model II

[0348] This example describes a logistic regression model based on Herl -Herl dimer (HIlD) expression levels, Herl-Her3 dimer (H13D) expression levels, Herl-Her2 dimer (H12D) expression levels, and Her2-Her3 dimer (H23D) expression levels. The model has the formula:

log(p/(1+p))=-3.207+1.098*log(HllD+H13D)-0.142*log(H23D)-0.307*log(H12D) where p is the Diagnostic Index used to predict a patient's odds to respond to the drug, and p is between 0 and 1. This formula is referred to above as Formula II.

[0349] The whole model fitting p-value is 0.0203 by likelihood ratio, and the coefficients table is displayed below in Table 27:

Table 27

[0350] The predicted Diagnostic Index based on model 2 is listed below in Table

28 and was sorted by Diagnostic Index. For the formula used in model 2, a Diagnostic Index threshold of 0.4 distinguished cancers likely to respond to treatment with a Herl- acting agent, e.g., Gefitinib, from cancers unlikely to respond to such treatment, e.g., a cancer with a Diagnostic Index less than 0.4 was determined to be unlikely to respond to Herl-acting agent, e.g., Gefitinib, treatment, while a cancer with a Diagnostic Index greater than 0.4 was determined to be likely to respond to Herl-acting agent, e.g., Gefitinib, treatment. Application of this threshold Diagnostic Index to the Diagnostic Indexes determined according to Formula II identified 29 subjects not likely to respond to treatment with a Herl-acting agent, of which 4 were false negatives, and 33 subjects likely to respond to treatment with a Herl-acting agent, of which 13 were false positives. Table 28

6.4.3 Model III

[0351] This example describes a logistic regression model based on Herl-Herl dimer (H11D) expression levels, Herl-Her3 dimer (H13D) expression levels, and Herl- Her2 dimer expression levels. The model has the formula:

log(p/(l+p))=-3.126+1.047*log(H11D+H13D)-0.322*log(H12D), where p is the Diagnostic Index used to predict a patient's odds to respond to the drug, and p is between 0 and 1. This formula is referred to above as Formula III.

[0352] The whole model fitting p-value is 0.0078 by likelihood ratio, and the coefficients table is displayed below in Table 29:

Table 29

Coef Std. Error Coef/SE Chi-Square P-Value Exp(Coef) constant -3.014 1.444 -2.087 4.357 .0369 .049

H11D+H13D 1.096 .453 2.417 5.843 .0156 2.991 lnH12D -.448 .238 -1.882 3.542 .0598 .639

[0353] The predicted Diagnostic Index based on model 3 is listed below in Table

30 and was sorted by Diagnostic Index. For the formula used in model 3, a Diagnostic Index threshold of 0.35 distinguished cancers likely to respond to treatment with a Herl- acting agent, e.g., Gefitinib, from cancers unlikely to respond to such treatment, e.g., a cancer with a Diagnostic Index less than 0.35 was determined to be unlikely to respond to Her 1 -acting agent, e.g., Gefitinib, treatment, while a cancer with a Diagnostic Index greater than 0.35 was determined to be likely to respond to Herl -acting agent, e.g., Gefitinib, treatment. Application of this threshold Diagnostic Index to the Diagnostic Indexes determined according to Formula III identified 23 subjects not likely to respond to treatment with a Herl-acting agent, of which 3 were false negatives, and 39 subjects likely to respond to treatment with a Herl-acting agent, of which 19 were false positives.

Table 30

6.4.4 Model IV

[0354] This example describes a logistic regression model based on Herl-Herl dimer (HIlD) expression levels, Herl-Her3 dimer (H13D) expression levels, and Her2 phosphorylation (H2P) expression levels. The model has the formula:

log(p/(l+p)) = -1.947 + 0.904*log(Hl lD+H13D) -0.393*log(H2P), where p is the Diagnostic Index used to predict a patient's odds to respond to the drug, and p is between 0 and 1. This formula is referred to above as Formula IV.

[0355] The whole model fitting p-value is 0.0177 by likelihood ratio, and the coefficients table is displayed below in Table 31 : Table 31

Coef Std. Error Coef/SE Chi-Square P-Value Exp(Coef) constant -1.947 1.420 -1.372 1.881 .1702 .143

H11 D+H13D .904 .394 2.296 5.273 .0217 2.469

H2P -.393 .278 -1.415 2.001 .1572 .675

[0356] The predicted Diagnostic Index based on model 4 is listed below in Table

32 and was sorted by Diagnostic Index. For the formula used in model 4, a Diagnostic Index threshold of 0.36 distinguished cancers likely to respond to treatment with a Herl- acting agent, e.g., Gefitinib, from cancers unlikely to respond to such treatment, e.g., a cancer with a Diagnostic Index less than 0.36 was determined to be unlikely to respond to Her 1 -acting agent, e.g., Gefitinib, treatment, while a cancer with a Diagnostic Index greater than 0.36 was determined to be likely to respond to Herl-acting agent, e.g., Gefitinib, treatment. Application of this threshold Diagnostic Index to the Diagnostic Indexes determined according to Formula IV identified 26 subjects not likely to respond to treatment with a Herl-acting agent, of which 3 were false negatives, and 36 subjects likely to respond to treatment with a Herl-acting agent, of which 15 were false positives.

Table 32

6.4.5 Model V

[0357] This example describes a logistic regression model based on Herl-Herl dimer (HIlD) expression levels, Herl-Her3 dimer (Hl 3D) expression levels, Her2-Her3 dimer (H23D) expression levels, and Her2 phosphorylation (H2P). The model has the formula: log(p/(l+/?)) = -1.098 + 0.58*log(HllD) - 0.141*log(H23D) + 0.322*log(H13D) -

0.397*log(H2P), where p is the Diagnostic Index used to predict a patient's odds to respond to the drug, and p is between 0 and 1. This formula is referred to above as Formula V.

[0358] The whole model fitting p-value is 0.0292 by likelihood ratio, and the coefficients table is displayed below in Table 33:

Table 33

constant H11 D H2P H23D H13D

[0359] The predicted Diagnostic Index based on model 5 is listed below in Table

34 and was sorted by Diagnostic Index. For the formula used in model 5, a Diagnostic Index threshold of 0.35 distinguished cancers likely to respond to treatment with a Herl- acting agent, e.g., Gefitinib, from cancers unlikely to respond to such treatment, e.g., a cancer with a Diagnostic Index less than 0.35 was determined to be unlikely to respond to Her 1 -acting agent, e.g., Gefitinib, treatment, while a cancer with a Diagnostic Index greater than 0.35 was determined to be likely to respond to Herl-acting agent, e.g., Gefitinib, treatment. Application of this threshold Diagnostic Index to the Diagnostic Indexes determined according to Formula V identified 25 subjects not likely to respond to treatment with a Herl-acting agent, of which 4 were false negatives, and 37 subjects likely to respond to treatment with a Herl-acting agent, of which 17 were false positives.

Table 34

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Z0 fZ

Z6

61-

68

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6.4.6 Model VI

[0360] This example describes a logistic regression model based on Herl -Herl dimer (H11D) expression levels, Herl-Her3 dimer (H13D) expression levels, Herl-Her2 dimer expression levels, and Her2-Her3 dimer (H23D) expression levels. The model has the formula:

log(p/(1+p))=-1.828+0.747*log(HHD)+0.009*log(H23D)+0.158*log(H13D)-

0.388*log(H12D) where p is the Diagnostic Index used to predict a subject's likelihood of responding to the drug, and p is between 0 and 1. This formula is referred to above as Formula VI.

[0361] The whole model fitting p-value is 0.0123 by likelihood ratio, and the coefficients table is displayed below in Table 35:

Table 35

constant H11 D H23D H13D lnH12D

[0362] The predicted Diagnostic Index based on the model is listed below in Table

36 and was sorted by Diagnostic Index. For the formula used in model 6, a Diagnostic Index threshold of 0.45 distinguished cancers likely to respond to treatment with a Herl- acting agent, e.g., Gefitinib, from cancers unlikely to respond to such treatment, e.g., a cancer with a Diagnostic Index less than 0.45 was determined to be unlikely to respond to Herl -acting agent, e.g., Gefitinib, treatment, while a cancer with a Diagnostic Index greater than 0.45 was determined to be likely to respond to Herl -acting agent, e.g., Gefitinib, treatment. Application of this threshold Diagnostic Index to the Diagnostic Indexes determined according to Formula VI identified 33 subjects not likely to respond to treatment with a Her 1 -acting agent, of which 6 were false negatives, and 29 subjects likely to respond to treatment with a Her 1 -acting agent, of which 11 were false positives.

Table 36

6.4.7 Model VII

[0363] This example describes a logistic regression model based on Herl-Herl dimer (HIlD) expression levels, Herl-Her3 dimer (H13D) expression levels, and Her2- Her3 dimer (H23D) expression levels. The model has the formula:

log(p/(1+p)) = -3.162+0.912*log(H11D+H13D)-0.192*log(H23D),

Formula VII

where p is the Diagnostic Index used to predict a patient's odds to respond to the drug, and p is between 0 and 1.

[0364] The whole model fitting p-value is 0.0405 by likelihood ratio, and the coefficients table is displayed below in Table 37:

Table 37

Coef Std. Error Coef/SE Chi-Square P-Value Exp(Coef) constant -3.162 1.457 -2.169 4.706 .0301 .042

H11 D+H13D .912 .443 2.058 4.236 .0396 2.490

H23D -.192 .265 -.726 .527 .4678 .825

[0365] The predicted Diagnostic Index based on model VII is listed below in

Table 38 and was sorted by Diagnostic Index. For the formula used in model VII, a Diagnostic Index threshold of 0.4 distinguished cancers likely to respond to treatment with a Herl -acting agent, e.g., Gefitinib, from cancers unlikely to respond to such treatment, e.g., a cancer with a Diagnostic Index less than 0.4 was determined to be unlikely to respond to Herl -acting agent, e.g., Gefitinib, treatment, while a cancer with a Diagnostic Index greater than 0.4 was detennined to be likely to respond to Herl -acting agent, e.g., Gefitinib, treatment. Application of this threshold Diagnostic Index to the Diagnostic Indexes determined according to Formula VII identified 28 subjects not likely to respond to treatment with a Herl -acting agent, of which 4 were false negatives, and 34 subjects likely to respond to treatment with a Herl-acting agent, of which 14 were false positives.

Table 38

6.4.8 Model VIII

[0366] This example describes a logistic regression model based on Her 1 -Herl dimer (Hl ID) expression levels, Herl-Her2 dimer (H12D) expression levels, and Herl- Her3 dimer (Hl 3D) expression levels. In this analysis, subjects with Stable Disease were scored as nonresponsive to treatment with a Herl -acting agent. Further, this analysis was performed on a slightly larger dataset than the examples above. For convenience, the complete data table is presented as Table 40, below. It should be noted that certain of the subjects are repeated from Table 24, above. The model has the formula:

log(p/(l+p)) = -1.807+0.273*log(HllD+H13D)-0.241*log(H12D), Formula VIII

where p is the Diagnostic Index used to predict a patient's odds to respond to the drug, andjo is between 0 and 1.

[0367] The whole model fitting p-value is 0.0035 by likelihood ratio. The coefficients table showing predictive value, sensitivity, and specificity of the algorithm is presented below in Table 39:

[0368] The predicted Diagnostic Index based on model 7 is listed below in Table

40. For the formula used in model 7, a Diagnostic Index threshold of 0.28 distinguished cancers likely to respond to treatment with a Her 1 -acting agent, e.g., Gefitinib, from cancers unlikely to respond to such treatment, e.g. , a cancer with a Diagnostic Index less than 0.28 was determined to be unlikely to respond to Herl -acting agent, e.g., Gefitinib, treatment, while a cancer with a Diagnostic Index greater than 0.28 was determined to be likely to respond to Herl -acting agent, e.g., Gefitinib, treatment. Application of this threshold Diagnostic Index to the Diagnostic Indexes determined according to Formula VII identified 60 subjects not likely to respond to treatment with a Herl -acting agent, of which 19 were false negatives, and 21 subjects likely to respond to treatment with a Herl -acting agent, of which 6 were false positives.

6.5 Example 4: Correlations Between ErbB Dimer

Expression Levels and Responsiveness to Gefitinib Therapy

[0369] This example provides methods and results for experiments to correlate levels of particular ErbB receptor dimers with responsiveness to therapy with an exemplary Herl -acting agent, Gefitinib. In particular, this example describes determinations of correlations between amounts of Herl -Her dimer expression, Herl- Her3 dimer expression, and Her2-Her3 dimer expression and responsiveness to Gefitinib therapy. [0370] The samples of ninety-two lung cancer patients treated with Gefitinib

(IRESSA^®) were analyzed; the 92 samples analyzed include the 46 samples discussed in Example 2, above. eTag™ assays were conducted on the 92 samples according to the methods described in Example 1, above, and the amounts of the following receptors were measured for each sample: Herl total; Herl-Herl dimer; phosphorylated Herl; extracellular Her2 total; extracellular Herl-Her2 dimer; intracellular Her2 total; intracellular Herl -Her2 dimer; phosphorylated Her2; Her3 total; Her2-Her3 dimer; and Herl-Her3 dimer.

[0371] AU measurements of dimer amounts calculated in units of dimer per cell, while all other measurements were calculated in relative fluorescence units (RFU). The tumor percentage of each sample was also measured using the immunohistochemistry (EHC) or hematoxylin and eosin (H&E) image method and used to calculate the number of cancer cells present in each sample and the ratio of cancer cells to normal cells in the sample. Briefly, samples were scored for tumor cell percentage relative to normal cell based on cell morphology, differentiation patterns, and grade and invasiveness of the cancer based on standard histological techniques. During measurements of dimer expression, molecular tags that recognize cytokeratin and/or tubulin were used to estimate the total number of cells, both normal and cancerous, present in the sample. The total number of cells was then multiplied by the proportion of cancer cells in the sample to identify the number of cancer cells in the sample, which were then used to normalize the numbers of dimers expressed per cell. The samples with small tumor percentage (less than 5% tumor cells relative to total cells) and small sections were excluded from the analysis.

[0372] Table 41, below, summarizes the patient samples used in this example. In

Table 41, CR indicates the number of patients with complete response, PR indicates the number of patients with a partial response, SD indicates the number of patients with stable disease, and PD indicates the number of patients with disease progression. In this study, responsiveness is scored by assessing lung tumor size according to the RECIST standard. See Therasse et ah, 2000, J Natl Cancer Inst 92:205-16. Table 41

[0373] Three different predictive algorithms were tested using two-by-two contingency tables to assess the predictive power of the algorithms for responsiveness to Gefitinib therapy. The algorithms and contingency tables are set forth below.

6.5.1 Predictive Formula IX

[0374] This example assesses the predictive power of algorithms that include

Formula IX, in which a cancer is predicted to respond to Her 1 -acting agent, e.g., Gefitinib, therapy if

• The cancer expresses more than 1000 Her 1 -Her 1 dimers per cell, or

• The cancer's balanced dimer score is above 15,000, where the balanced dimer score is calculated by Formula IX:

3.2 * (Herl/1 + Herl/2 + Herl/3) - 10.5 * Her2/3 Formula IX

[0375] In Formula IX, Herl/1 = number of Her 1 -Her 1 dimers per cancer cell;

Herl/2 = number of Herl-Her2 dimers per cancer cell; Herl/3 = number of Herl-Her3 dimers per cancer cell; and Her2/3 = number of Her2-Her3 dimers per cancer cell.

[0376] A two by two contingency table was constructed for the combined data from studies 1, 2, 3, and 4 to assess the predictive power of algorithms including Formula IX, and p-values were calculated by Fisher's exact test using GraphPad (GraphPad Software Inc., San Diego, CA). Sensitivities (Sn) and specificities (Sp) were also calculated. The classification based on the combined data together was statistically significant (p-value=0.0001). The contingency table is shown in Table 42, below. Table 42

[0377] Of seven false negatives (subjects predicted not to respond to treatment who in fact did respond to treatment), four were scored as Partial Response and three were scored as Stable Disease, according to the RECIST standard.

6.5.2 Predictive Formula X

[0378] This example assess the predictive power of algorithms that include

Formula X. In this example, a cancer is predicted to respond to Her 1 -acting agent, e.g., Gefitinib, therapy if

• The cancer expresses more than 1000 Her 1 -Her 1 dimers per cell, or

• The cancer's first balanced dimer score is above 15,000, or

• The patients' second balanced dimer score is above 4000, where

The first balanced dimer score is calculated by Formula IX

3.2 * (Herl/1 + Herl/2 + Herl/3) - 10.5 * Her2/3; and

Formula IX

The second balanced dimer score is calculated by Formula X

3.2 * Herl/3 - 10.5 * Her2/3 Formula X

[0379] In Formula IX, Herl/1 = number of Herl-Herl dimers per cancer cell;

[0380] Two by two contingency tables were constructed for the combined data from studies 1, 2, 3, and 4 to assess the predictive power of algorithms including Formula X, and p-values were calculated by Fisher's exact test using GraphPad (GraphPad Software Inc., San Diego, CA). Sensitivities (Sn) and specificities (Sp) were also calculated. The classification based on the combined data together was statistically significant (p-value=0.004). The contingency table is shown in Table 43, below.

Table 43

[0381] Of six false negatives (subjects predicted not to respond to treatment who in fact did respond to treatment), three were scored as Partial Response and three were scored as Stable Disease, according to the RECIST standard.

6.5.3 Predictive Formula XIII

[0382] This example assess the predictive power of algorithms including Formula

XIII, in which a cancer is predicted respond to Gefitinib therapy if

• The cancer expresses more than 750 Herl-Herl dimers per cell.

Formula XIII

[0383] Two by two contingency tables were constructed for the combined data from studies 1, 2, 3, and 4 to assess the predictive power of algorithms including Formula XIII, and p-values were calculated by Fisher's exact test using GraphPad (GraphPad Software Inc., San Diego, CA). Sensitivities (Sn) and specificities (Sp) were also calculated. The classification based on the combined data together was statistically significant (p-value=0.01). The contingency table is shown in Table 44, below.

Table 44

[0384] Of thirteen false negatives (subjects predicted not to respond to treatment who in fact did respond to treatment), five were scored as Partial Response and seven were scored as Stable Disease.

6.6 Example 5: Correlations between ErbB Dimer Expression and

Responsiveness to Gefitinib Therapy: Classification Tree Analysis

[0385] This example describes a statistical analysis of the dataset presented in

Table 24, above, to identify rules that predict whether a subject is likely to respond to treatment with a Her 1 -acting agent, e.g., Gefitinib. In this example, Classification and Regression Tree (CART 5.0; Salford Systems, San Diego, CA) analysis was used to segregate subjects responsive to treatment with a Herl -acting agent from patients unresponsive to such treatment. In this example, subjects with Stable Disease according to Table 24 were scored as unresponsive to treatment with a Herl -acting agent.

[0386] Three Classification Trees were generated in this analysis, shown in Figure

12. In all three trees, the first rule used to segregate subjects responsive to treatment with a Herl -acting agent from unresponsive subjects was the amount of Herl -Herl dimers and total Her2 expressed by the subject's cancer. Of subjects with cancers expressing fewer than 1325 Herl-Herl dimers per cell and expressing an amount of Her2 receptors resulting in fewer than 4000 relative fluorescence units (hereinafter Group A), 8 were responsive to treatment with a Herl -acting agent, while 2 were not responsive to such treatment. Of subjects with cancers expressing fewer than 1325 Herl-Herl dimers per cell or expressing an amount of Her2 receptors resulting in more than 4000 relative fluorescence units (hereinafter Group B), 34 were not responsive to treatment with a Herl -acting agent, while 6 were responsive to such treatment.

[0387] In Trees 1 and 2, the next rule used to segregate responders from non- responders was the amount of Herl monomer expressed by the subject's cancer. The 40 subjects from Group B were segregated according to whether the cancer cells express an amount of Herl receptors resulting in more than or fewer than 872 relative fluorescent units per cell. Of 16 subjects with cancers expressing an amount of Herl resulting in more than 872 relative fluorescent units, none were responsive to treatment with a Herl- acting agent. Of 24 subjects with cancers expressing an amount of Herl resulting in fewer than 872 relative fluorescent units (hereinafter Group C), 6 were responsive to treatment with a Her 1 -acting agent and 18 were not responsive to such treatment. This rule was the last rule applied in Tree 1.

[0388] For Tree 2, the next rule used to segregate responders from non-responders was the amount of Her 1 -Her 1 dimers expressed per cancercell. The 24 subjects of Group C were segregated according to whether the subject's cancer expresses more or fewer than 2781 Herl-Herl dimers per cell. Of 3 subjects with cancers expressing more than 2781 Herl-Herl dimers per cell, all were responsive to treatment with a Herl -acting agent. Of 21 subjects with cancers expressing fewer than 2781 Herl-Herl dimers per cell, 18 were not responsive to treatment with a Herl -acting agent, and 3 were responsive to such treatment.

[0389] To generate Tree 3, a different rule from the second step of Trees 1 and 2 was applied to Group B. Specifically, the 40 subjects of Group B were segregated according to the amount of expression of Herl-Her2 dimers and Herl-Her3 dimers. Of 37 subjects with cancers expressing more than 130 Herl-Her2 dimers or fewer than 1750 Herl-Her3 dimers, 33 were not responsive to treatment with a Herl-acting agent and 4 were responsive to such treatment. Of 3 subjects with cancers expressing fewer than 130 Herl-Her2 dimers and more than 1750 Herl-Her3 dimers, 2 were responsive to treatment with a Herl-acting agent and 1 was not responsive to such treatment.

6.7 Example 6: Correlations between ErbB Dimer Expression and Responsiveness to Gefitinib Therapy: Nearest Neighbor Analysis

[0390] This example describes a statistical analysis of 48 samples, described below, to identify rules that predict whether a subject is likely to respond to treatment with a Herl-acting agent, e.g., Gefitinib. In this example, nearest neighbor analysis was used to segregate subjects responsive to treatment with a Herl-acting agent from patients unresponsive to such treatment.

[0391] Forty-eight FFPE samples derived from patients with stage IIIB or IV Non-

Small-Cell Lung Cancer (NSCLC) who had failed at least 2 prior chemotherapy regimens and had been enrolled either in IDEAL-2 (N=31) or the Gefitinib expanded access program (EAP, N=I 7) were retrospectively analyzed with the eTag assay to examine whether there was a correlation between HER-family dimer profiles and clinical response as assessed by RECIST criteria. The mean age of the population was 64 years with a range of 33 to 86 years. There were 28 females (58%) and 20 males (42%). Two samples (#39 and #41) had zero percent tumor by H&E stain and thus were not tested. Two samples (#34 and #22) were taken from the same patient, one (#34) prior to treatment with Gefitinib and the other (#22) following relapse on Gefitinib therapy. Fifty- four percent of the specimens were metastatic lesions (N=26) while forty-six percent were primary lung lesions (N=22)(see Table 45). AU samples were blinded during testing. The 46 samples tested had dimer profiles determined by the eTag assay and the data were analyzed for patterns that correlated with clinical response to Gefitinib (see Table 46). Correlations between dimer levels and clinical outcomes were examined and clinical breakpoints were sought. Algorithms for predicting response to Gefitinib were developed. The data indicate an interpretation of EGFR pathway activation status as assessed by HER- family dimerization patterns.

Table 45

Table 46

Legend to Table 46 Clinical and pathological characteristics of the 48 patients analyzed from IDEAL-2 and the Gefitinib EAP with their eTag dimer measurements, EGFR and KRAS mutations, predicted responses given by the formula ^N* [(Hl ID > 1600 or H13D > 850) and H23D < 600) predicts response], and clinical responses as assessed by RECIST ciiteria eTag values aie in dimers/cell.

Abbreviations Hl ID = HERl.1 dimers, H13D = HERl 3 dimers, H23D = HER2.3 dimers, Non-Small-Cell Lung Cancer - NSCLC, Adenocarcinoma = AC, Bronchiolo- Alveolar Carcinoma = BAC, Squamous Cell Carcinoma = SCC, Large Cell Carcinoma = LCC, Large Cell Neuroendocrine Caicmoma = LCNC, Mixed Neuroendocrine/ Adenocarcinoma = NE/ AC, Mixed Adenocaicrnoma/Bronchiolo- Alveolar Carcinoma = AC/BAC, SD = stable disease, PR = partial response, PD = progressive disease, R = response, NR = no iesponse [0392] Several approaches were used to test whether dimer measurements a) correlated with and b) were predictive of clinical responses as determined by RECIST criteria. In the first, two-thirds of the specimens (31/46) were randomly selected to serve as a "training" dataset, and the remaining 1/3 (15/46) were used as a "test" dataset. K- nearest neighbor (K-NN) analyses were performed to see whether the correct clinical outcome could be predicted by comparing dimer measurements for each of the test specimens to the dimer patterns observed for the training specimens matched with their corresponding clinical outcomes. Throughout this example, those patients who were judged by RECIST criteria to have achieved a complete response (CR), a partial response (PR), or stable disease (SD) were considered "responders" and those who were assessed as having progressive disease (PD) were considered "non-responders." The results of the K-NN analysis are shown below in Table 47. In Tables 47, 48 and 49, R = clinical response (CR/PR/SD by RECIST criteria); NR = non-response (PD by RECIST criteria); 1 = predicted response when compared to training dataset; 0 = predicted non-response when compared to training dataset; Sn = sensitivity; Sp = specificity; PPV = positive predictive value; NPV = negative predictive value.

Table 47

[0393] As shown in Table 47, the training/test dataset approach yielded a sensitivity for response of 80%, a specificity of 70%, a positive predictive value (PPV) of 57% and a negative predictive value of 87%. Although these are reasonably good predictive measures, the p-value trended toward significance but did not reach statistical significance.

[0394] In the next approach, the data were analyzed using a "leave one out" cross validation method whereby 45 of the 46 sample measurements were used as the comparison dataset for the remaining test sample in a K-NN analysis. This was repeated 46 times, once for each sample in the dataset. The results are shown in Table 48, below. The cross validation exercise demonstrates a sensitivity for response of 66%, a specificity of 87%, a PPV of 71%, and a NPV of 84%.

Table 48

[0395] Next, analysis was performed to determine which dimer measurements were best correlated with response to Gefitinib in this dataset as well as begin to understand the relationship between the absolute measurements of HER- family dimers present on the tumor cells and clinical responsiveness as assessed by RECIST criteria. These experiments were designed to identify potential "clinical breakpoints" for predicting response based on the relative amounts of HER 1:1 (HIlD), 1:2(H12D), 1:3(H13D), and 2:3(H23D) dimers present on the tumor cells to develop a predictive algorithm that can be applied to independent datasets.

[0396] Briefly, univariate analyses (Wilcoxson Rank Sum Test) indicated that the presence of Hl ID was positively correlated with Gefitinib responsiveness, while the presence of H12D and Hl 3D were negatively correlated with response. H23D did not appear to be correlated with Gefitinib responsiveness (Figure 13). Close examination of HIlD levels in clinical responders and non-responders demonstrated that all but 5 non- responders had Hl ID levels below approximately 1600, while 11/15 of the responders had Hl ID levels >1600. See Figure 14. Recursive partitioning analyses (Figure 15) yielded a similar breakpoint for Hl ID at 1618 dimers/cell. Additional analyses of the correlates of Gefitinib responsiveness yielded the algorithm [(Hl ID > 1600 OR H13D > 850) AND H23D < 600] as the one that best correlates with response.

Table 49

[0397] When this algorithm is applied to the dataset, it resulted in 87% accuracy

(40/46 predictions agreed with the actual clinical responses by RECIST), a sensitivity for response of 80%, specificity of 90%, corresponding to a PPV of 80% and a NPV of 90% (p = 0.0001), as shown in Table 49. The identification of the clinical breakpoints was useful in this regard. For example, despite the fact that the univariate analyses suggest no relationship between H23D and responsiveness to Gefitinib (see Figure 13), close examination revealed that the four patients with the highest levels of H23D (> 600, see Table 2) did not respond to Gefitinib, in some cases despite extremely high lvels of HI lD.

[0398] The distribution of dimer measurements for Hl ID, H12D, and H13D as shown in Figure 13 demonstrates that higher levels of Hl ID correlate with response whereas higher levels of H12D and Hl 3D correlate better with non-response. The logistic regression model shown in Tables 50 and 51 confirmed that Hl ID levels best correlate with response to Gefitinib in this cohort. As shown in Table 50, the Sn, Sp, PPV, and NPV change only slightly as the breakpoints are adjusted empirically to arrive at the algorithm that best correlates with the observed outcomes. Inclusion of Hl 3D as a term in the predictive algorithm results in the identification of one additional responder and slightly elevates the sensitivity and PPV of the algorithm. Likewise, elimination of the Hl 3D term from the algorithm has only a minor effect on the correlation with outcome, and, as such, in one embodiment can be omitted from the algorithm.

Table 51

!ogistf(formula = Resp - log(H11 D) + log(H12D) + log(H13D))

Model fitted by Penalized ML

Confidence intervals and p-values by Profile Likelihood coef se(coef) lower 0.95 Upper 0.95 Chisq p-value

(Intercept) -1.3321096 1.1044919 -3.9650718 0.561107343 1.798511 0.179892629 log(H11D) 0.4047708 0.1706777 0.1173783 0.817100764 8.651616 0.003267715 log{H12D) -0.3184645 0.1471257 -0.6348410 -0.055134757 5.713575 0.016834228 log(H13D) -0.2548601 0.1356871 -0.5394585 -0.009709435 4.161984 0.041340960

Likelihood ratio test=17.17168 on 3 df, p=0.0006515445, n=46

Thus, in one embodiment, tumor cells exhibiting Hl ID, Hl 2D and/or Hl 3D, as measured by the eTag techniques described herein, are predicted to respond to Gefitnib. Likewise, individuals with tumors exhibiting such dimers are predicted to respond to Gefitnib.

In another embodiment, tumor cells exhibiting Hl ID, as measured by the eTag techniques described herein, are predicted to respond to Gefitnib. Likewise, individuals with tumors exhibiting Hl ID dimers are predicted to respond to Gefitnib. In another embodiment, tumor cells exhibiting H12D and/or H13D, as measured by the eTag techniques described herein, are predicted to be unlikely to respond to standard dosages of Gefitnib, said dosages being well known to those of skill in the art. Likewise, in such an embodiment, individuals with tumors exhibiting H12D and/or Hl 3D are predicted to be unlikely to respond to standard dosages of Gefitnib.

Moreover, in one embodiment, tumor cells exhibiting H23D, even in the presence of Hl ID, H12D, and/or H13D dimers, as measured by the eTag techniques described herein, are predicted to be unlikely to respond to Gefitnib. Likewise, individuals with tumors exhibiting H23D dimers, even in the presence of Her 1- containing dimers, are predicted to be unlikely to respond to Gefitnib.

6.8 Example 7: Effects of KRAS or EGFR

Mutations on Responsiveness to Gefitinib Therapy

[0399] In addition to the analysis described in Example 6, the samples comprising the dataset of Example 6 were examined to assess the genotypes of KRAS and EGFR to assess whether the tumors comprised mutations associated with responsiveness or non- responsiveness to Gefitinib therapy. Six specimens in the dataset exhibited mutations either in EGFR or KRAS as shown in Table 52.

Table 52

[0400] Six specimens exhibited mutations either in EGFR or K-RAS (Table 46).

The presence of particular mutations in exons 18-21 of the EGFR-TK has been shown to increase the affinity of binding with Gefitinib and has been associated with improved responses to the drug. Conversely, K-RAS mutations, typically found in exon 2, codons 12 and 13, have been associated with failure to respond to Gefitinib (Lynch, T. et al., 2004, N. Engl. J. Med. 350: 1-11; Pao, W. et al., 2005, PIoS. 2:57-61; Gumerlock, P.H. et al., ASCO 2005, Abst. 7008). While there were fifteen patients in the cohort who experienced an objective response, only three possessed EGFR-TK mutations. Of the six patients who had mutations, five had clinical outcomes that were consistent with the predictions made by the eTag algorithm. Patient #34 was predicted to be a non- responder by the eTag algorithm, but did respond initially to therapy and did have an EGFR mutation (L858R). The same patient (#22) subsequently failed treatment with Gefitinib, was re-biopsied, and in addition to the L858R mutation in exon 21 of EGFR- TK, was found to have acquired an additional EGFR exon 20 mutation (T86M) as well as mutation in exon 1 of K-RAS (Q25R). Of note, one patient with a K-RAS mutation (#7) was assessed as having stable disease, consistent with the prediction made by the eTag assay. 6.9 Example 9: Correlations between ErbB Dimer Expression and Responsiveness to Gefitinib Therapy: Manual Projection Pursuit

[0401] This example describes the use of manual projection pursuit to construct a model for predicting response to therapy with a Herl-acting agent, e.g., gefitinib. The model was generated from measured Her dimer content in the 46 patient samples presented in Table 46, above.

[0402] Manual projection pursuit was used to stratify patients into responder or non-responder groups. Briefly, this entailed generating two-dimensional plots of each of the 6 possible pair-wise groupings of parameters, specifically, HIlD vs. H12D, HI lD vs. H13D, HIlD vs. H23D, H12D vs. H13D, H12D vs. H23D and H13D vs. H23D. This provided visual clues as to the most sensitive and specific stratification rules. For example, a plot of Hl ID vs. H23D (Figure 16) revealed that high H23D correlates negatively with response. This result suggested that H23D<230 was required for patients in this data set to respond to gefitinib therapy. The data were then replotted, removing omiting patients with H23D>230, and the process was repeated iteratively using the remaining parameters. The distinguishing criteria were ultimately selected to minimize the number of stratification rules while maximizing accuracy of prediction.

[0403] This analysis generated Formula XIV, below, which predicts that patients are likely to respond to gefitinib therapy if:

[(Hl 1D>5OO and H12D<220) or (Hl 1D>16OO and H13D<150) ] and H23D<230

Formula XIV

[0404] In Formula XIV, Hl ID is the number of Herl -Her 1 dimers per cancer cell,

H12D is the number of Herl-Her2 dimers per cancer cell, and H23D is the number of Her2-Her3 dimers per cancer cell.

[0405] Formula XIV correctly predicted the outcome of 43 of the 46 patients.

This algorithm gave a sensitivity (% responders correctly predicted) of 87%, a specificity (% non-responders correctly predicted) of 97%, positive predictive value (% predicted responders who actually responded) of 93% and a negative predictive value (% predicted non-responders who actually failed to respond) of 94%, as shown below in the two-by- two contingency table presented as Table 53.

Table 53

[0406] The foregoing description of the present invention is provided by way of illustration, and are not intended to limit the scope of the invention in any manner. Further, various equivalents and adaptations will be readily apparent to those of skill in the art. Such equivalents and adaptations are also intended to be within the scope of the invention claimed herein. Further, all documents, references, patents, patent publications, and other printed materials referred to herein are hereby incorporated by reference in their entireties, whether or not such documents are explicitly incorporated by reference at the time the document is named.

Claims

WHAT IS CLAIMED IS:

1. A method for determining whether a cancer cell is likely to respond to treatment with a Her 1 -acting agent, comprising detecting on the cancer cell at least about 600 Herl-Herl dimers, wherein the presence of the at least about 600 Herl-Herl dimers indicates that the cancer is likely to respond to treatment with the Herl- acting agent.

2. The method of claim 1, wherein the Herl -acting agent is Gefitinib, tarceva, or erbitux.

3. The method of claim 1, wherein the Herl-acting agent is Gefitinib.

4. The method of claim 1, wherein at least about 750 Herl-Herl dimers are detected.

5. The method of claim 1, wherein at least about 800 Herl-Herl dimers are detected.

6. The method of claim 1, wherein at least about 900 Herl-Herl dimers are detected.

7. The method of claim 1, wherein at least about 1000 Herl-Herl dimers are detected.

8. The method of claim 1, wherein at least about 1100 Herl-Herl dimers are detected.

9. The method of claim 1, wherein at least about 1200 Herl-Herl dimers are detected.

10. The method of claim 1, wherein at least about 1300 Herl-Herl dimers are detected.

11. The method of claim 1, wherein at least about 1325 Herl-Herl dimers are detected.

12. The method of claim 1, wherein between about 600 and about 100,000 Herl- Herl dimers are detected.

13. The method of claim 1 , wherein detecting the Herl-Herl dimers is accomplished by:

(a) contacting the cell with:

(i) a binding compound having a molecular tag attached thereto by a cleavable linkage, and

(ii) a cleaving probe having a cleavage inducing-moiety, wherein the binding compound and the cleaving probe each specifically bind Herl, and wherein binding of a binding compound or a cleaving probe to a Herl monomer precludes binding of another binding compound or cleaving probe to the same Herl monomer, and wherein if the binding compound is within an effective proximity of the cleavage- inducing moiety of the cleaving probe, the cleavage-inducing moiety cleaves the cleavable linker so that the molecular tag is released; and

(b) quantifying the amount of released molecular tag, thereby detecting the Herl-Herl dimers.

14. The method of claim 13, wherein activating the cleavage-inducing moiety cleaves the cleavable linker.

15. The method of claim 13, wherein the binding compound and the cleaving probe each specifically binds a Herl epitope.

16. The method of claim 15, wherein the binding compound and the cleaving probe each specifically binds an identical Herl epitope.

17. The method of claim 13, wherein the binding compound and the cleaving probe each comprises an antibody or antigen-binding fragment.

18. The method of claim 15, wherein the binding compound and the cleaving probe each comprises an antibody or antigen-binding fragment.

19. The method of claim 13, wherein the binding compound and the cleaving probe each specifically binds a Herl ligand binding site.

20. The method of claim 13, wherein the binding compound and the cleaving probe each comprises a Herl ligand.

21. The method of claim 1, wherein the cancer cell is a breast cancer cell, lung cancer cell, colorectal cancer cell, prostate cancer cell, or ovarian cancer cell.

22. The method of claim 21, wherein the cancer cell is a lung cancer cell.

23. The method of claim 1, wherein the Herl -Herl dimers on the cancer cell are detected directly on a patient sample.

24. The method of claim 23, wherein the patient sample is a fixed tissue sample, a frozen tissue sample, or a sample purified from circulating epithelial cells.

25. The method of claim 23, wherein the patient sample is a lung tissue sample, a breast tissue sample, a colorectal tissue sample, a prostate tissue sample, or an ovarian tissue sample.

26. The method of claim 25, wherein the patient sample is a lung tissue sample.

27. The method of claim 1, wherein the cancer cell is obtained from a biological sample of a subject having or suspected of having a cancer.

28. The method of claim 1, further comprising detecting on a cancer cell at least about 1000 Herl-Her3 dimers, wherein the presence of the at least about 600 Herl -Herl dimers and the at least about 1000 Herl-Her3 dimers indicates that the cancer is likely to respond to treatment with the Herl -acting agent.

29. The method of claim 28, wherein at least about 800 Herl-Herl dimers are detected.

30. The method of claim 28, wherein at least about 900 Herl-Herl dimers are detected.

31. The method of claim 28, wherein at least about 1000 Herl-Herl dimers are detected.

32. The method of claim 28, wherein at least about 1100 Herl-Herl dimers are detected.

33. The method of claim 28, wherein at least about 1200 Herl-Her3 dimers are detected.

34. The method of claim 28, wherein at least about 1400 Herl-Her3 dimers are detected.

35. The method of claim 28, wherein at least about 1600 Herl-Her3 dimers are detected.

36. The method of claim 28, wherein at least about 1700 Herl-Her3 dimers are detected.

37. The method of claim 28, wherein at least about 1800 Herl-Her3 dimers are detected.

38. The method of claim 28, wherein at least about 1100 Herl -Herl dimers and at least about 1800 Herl-Her3 dimers are detected.

39. The method of claim 28, wherein between about 1000 and about 100,000 Herl- Her3 dimers are detected.

40. The method of claim 28, wherein between about 600 and about 100,000 Her 1 - Herl dimers and between about 1000 and about 100,000 Herl-Her3 dimers are detected.

41. The method of claim 28, wherein detecting the Herl-Her3 dimers is accomplished by:

(a) contacting the cell with:

(ii) a cleaving probe having a cleavage inducing-moiety, wherein the binding compound and the cleaving probe each specifically binds either Herl or Her3, and the cleaving probe and the binding probe do not both bind the same receptor, and wherein if the binding compound is within an effective proximity of the cleavage-inducing moiety of the cleaving probe, the cleavage-inducing moiety cleaves the cleavable linker so that the molecular tag is released; and

(b) quantifying the amount of released molecular tag, thereby detecting the Herl-Her3 dimers.

42. The method of claim 41, wherein activating the cleavage-inducing moiety cleaves the cleavable linker.

43. The method of claim 41, wherein the binding compound specifically binds a Herl epitope.

44. The method of claim 41, wherein the binding compound comprises an antibody or antigen-binding fragment.

45. The method of claim 41, wherein the binding compound specifically binds a Herl ligand binding site.

46. The method of claim 41, wherein the binding compound comprises a Herl ligand.

47. The method of claim 41, wherein the binding compound specifically binds a Her3 epitope.

48. The method of claim 41, wherein the binding compound specifically binds a Her3 ligand binding site.

49. The method of claim 41 , wherein the binding compound comprises a Her3 ligand.

50. The method of claim 41, wherein the cleaving probe specifically binds a Herl epitope.

51. The method of claim 41 , wherein the cleaving probe comprises an antibody or antigen-binding fragment.

52. The method of claim 41, wherein the cleaving probe specifically binds a Herl ligand binding site.

53. The method of claim 41, wherein the cleaving probe comprises a Herl ligand.

54. The method of claim 41, wherein the cleaving probe specifically binds a Her3 epitope.

55. The method of claim 41, wherein the cleaving probe specifically binds a Her3 ligand binding site.

56. The method of claim 41, wherein the cleaving probe comprises a Her3 ligand.

57. The method of claim 1, further comprising detecting on a cancer cell fewer than about 1000 Her2-Her3 dimers, wherein the presence of the at least about 600 Herl -Herl dimers and the fewer than about 1000 Her2-Her3 dimers indicates that the cancer cell is likely to respond to treatment with the Herl -acting agent.

58. The method of claim 57, wherein at least about 800 Her 1 -Her 1 dimers are detected.

59. The method of claim 57, wherein at least about 900 Herl-Herl dimers are detected.

60. The method of claim 57, wherein at least about 1000 Herl-Herl dimers are detected.

61. The method of claim 57, wherein at least about 1100 Herl-Herl dimers are detected.

62. The method of claim 57, wherein fewer than about 900 Her2-Her3 dimers are detected.

63. The method of claim 57, wherein fewer than about 800 Her2-Her3 dimers are detected.

64. The method of claim 57, wherein fewer than about 700 Her2-Her3 dimers are detected.

65. The method of claim 57, wherein fewer than about 600 Her2-Her3 dimers are detected.

66. The method of claim 57, wherein between about 1 and about 1000 Her2-Her3 dimers are detected.

67. The method of claim 57, wherein at least about 1100 Herl-Herl dimers and fewer than about 600 Her2-Her3 dimers are detected.

68. The method of claim 57, wherein detecting the Her2-Her3 dimers is accomplished by:

(a) contacting the cell with: (i) a binding compound having a molecular tag attached thereto by a cleavable linkage, and (ii) a cleaving probe having a cleavage inducing-moiety,

(b) wherein the binding compound and the cleaving probe each specifically binds either Her2 or Her3, and the cleaving probe and the binding probe do not both bind the same receptor, and wherein if the binding compound is within an effective proximity of the cleavage-inducing moiety of the cleaving probe, the cleavage-inducing moiety cleaves the cleavable linker so that the molecular tag is released; and

(c) quantifying the amount of released molecular tag, thereby detecting the Her2-Her3 dimers.

69. The method of claim 68, wherein activating the cleavage-inducing moiety cleaves the cleavable linker.

70. The method of claim 68, wherein the binding compound specifically binds a Her2 epitope.

71. The method of claim 68, wherein the binding compound comprises an antibody or antigen-binding fragment.

72. The method of claim 68, wherein the binding compound specifically binds a Her2 ligand binding site.

73. The method of claim 68, wherein the binding compound comprises a Her2 ligand.

74. The method of claim 68, wherein the binding compound specifically binds a Her3 epitope.

75. The method of claim 68, wherein the binding compound specifically binds a Her3 ligand binding site.

76. The method of claim 68, wherein the binding compound comprises a Her3 ligand.

77. The method of claim 68, wherein the cleaving probe specifically binds a Her2 epitope.

78. The method of claim 68, wherein the cleaving probe comprises an antibody or antigen-binding fragment.

79. The method of claim 68, wherein the cleaving probe specifically binds a Her2 ligand binding site.

80. The method of claim 68, wherein the cleaving probe comprises a Her2 ligand.

81. The method of claim 68, wherein the cleaving probe specifically binds a Her3 epitope.

82. The method of claim 68, wherein the cleaving probe specifically binds a Her3 ligand binding site.

83. The method of claim 68, wherein the cleaving probe comprises a Her3 ligand.

84. The method of claim 1, further comprising detecting an amount of Her2 receptors expressed on the cancer cell that results in fewer than about 4000 relative fluorescent units, wherein this amount of Her2 receptors indicates that the cancer cell is likely to respond to treatment with a Her 1 -acting agent and wherein the amount of relative fluorescent units is determined by:

(a) contacting the cell with:

(i) a binding compound having a molecular tag attached thereto by a cleavable linkage, and (ii) a cleaving probe having a cleavage inducing-moiety,

(b) wherein the binding compound and the cleaving probe each specifically binds an epitope of Her2, and the cleaving probe and the binding probe do not both bind the same epitope, and wherein if the binding compound is within an effective proximity of the cleavage-inducing moiety of the cleaving probe, the cleavage-inducing moiety cleaves the cleavable linker so that the molecular tag is released; and

(c) quantifying the amount of released molecular tag, determining the amount of relative fluorescent units.

85. The method of claim 84, wherein an amount of Her2 receptors expressed on the cancer cell that results in fewer than about 5000 relative fluorescent units is detected.

86. The method of claim 84, wherein an amount of Her2 receptors expressed on the cancer cell that results in fewer than about 7500 relative fluorescent units is detected.

87. The method of claim 84, wherein an amount of Her2 receptors expressed on the cancer cell that results in fewer than about 10000 relative fluorescent units is detected.

88. The method of claim 84, wherein an amount of Her2 receptors expressed on the cancer cell that results in fewer than about 3500 relative fluorescent units is detected.

89. The method of claim 84, wherein an amount of Her2 receptors expressed on the cancer cell that results in fewer than about 3000 relative fluorescent units is detected.

90. The method of claim 84, wherein an amount of Her2 receptors expressed on the cancer cell that results in fewer than about 2500 relative fluorescent units is detected.

91. The method of claim 84, wherein an amount of Her2 receptors expressed on the cancer cell that results in fewer than about 2000 relative fluorescent units is detected.

92. The method of claim 84, wherein an amount of Her2 receptors expressed on the cancer cell that results in fewer than about 1500 relative fluorescent units is detected.

93. The method of claim 84, wherein an amount of Her2 receptors expressed on the cancer cell that results in fewer than about 1000 relative fluorescent units is detected.

94. The method of claim 1 or 84, further comprising detecting on the cell fewer than about 130 Herl-Her2 dimers, wherein the presence of the fewer than about 130 Herl-Her2 dimers indicates that the cancer is likely to respond to treatment with a Her 1 -acting agent.

95. The method of claim 94, wherein fewer than about 100 Herl-Her2 dimers are detected.

96. The method of claim 94, wherein fewer than about 50 Herl-Her2 dimers are detected.

97. The method of claim 94, wherein fewer than about 200 Herl-Her2 dimers are detected.

98. The method of claim 94, wherein fewer than about 300 Herl-Her2 dimers are detected.

99. The method of claim 94, wherein fewer than about 400 Herl-Her2 dimers are detected.

100. The method of claim 94, wherein fewer than about 500 Herl-Her2 dimers are detected.

101. The method of claim 94, wherein fewer than about 750 Herl-Her2 dimers are detected.

102. The method of claim 94, wherein fewer than about 1000 Herl -Her2 dimers are detected.

103. The method of claim 94, wherein fewer than about 1500 Herl -Her2 dimers are detected.

104. The method of claim 94, wherein fewer than about 2000 Herl -Her2 dimers are detected.

105. The method of claim 1 or 84, further comprising detecting on the cell more than about 1750 Herl-Her3 dimers, wherein the presence of the more than about 1750 Herl-Her3 dimers indicates that the cancer is likely to respond to treatment with a Herl -acting agent.

106. The method of claim 105, wherein more than about 1800 Herl-Her3 dimers are detected.

107. The method of claim 105, wherein more than about 1500 Herl-Her3 dimers are detected.

108. The method of claim 105, wherein more than about 2000 Herl-Her3 dimers are detected.

109. The method of claim 105, wherein more than about 2200 Herl-Her3 dimers are detected.

110. The method of claim 105, wherein more than about 2400 Herl-Her3 dimers are detected.

111. The method of claim 105, wherein more than about 3000 Herl-Her3 dimers are detected.

112. The method of claim 105, wherein more than about 3500 Herl-Her3 dimers are detected.

113. The method of claim 105, wherein more than about 4000 Her 1 -Her3 dimers are detected.

114. The method of claim 105, wherein more than about 4500 Herl-Her3 dimers are detected.

115. The method of claim 105, wherein more than about 5000 Herl-Her3 dimers are detected.

116. The method of claim 1 or 84, further comprising detecting on the cell more than about 1750 Herl-Her3 dimers and fewer than about 130 Herl-Her2 dimers, wherein the presence of the more than about 1750 Herl-Her3 dimers and the fewer than about 130 Herl-Her2 dimers indicates that the cancer is likely to respond to treatment with a Her 1 -acting agent.

117. The method of claim 116, wherein more than about 1800 Herl -Her3 dimers are detected.

118. The method of claim 116, wherein more than about 1500 Herl -Her3 dimers are detected.

119. The method of claim 116, wherein more than about 2000 Herl-Her3 dimers are detected.

120. The method of claim 116, wherein more than about 2200 Herl-Her3 dimers are detected.

121. The method of claim 116, wherein more than about 2400 Herl-Her3 dimers are detected.

122. The method of claim 116, wherein more than about 3000 Herl-Her3 dimers are detected.

123. The method of claim 116, wherein more than about 3500 Her 1 -Her3 dimers are detected.

124. The method of claim 116, wherein more than about 4000 Herl-Her3 dimers are detected.

125. The method of claim 116, wherein more than about 4500 Herl-Her3 dimers are detected.

126. The method of claim 116, wherein more than about 5000 Herl-Her3 dimers are detected.

127. The method of claim 116, wherein fewer than about 100 Herl-Her2 dimers are detected.

128. The method of claim 116, wherein fewer than about 50 Herl-Her2 dimers are detected.

129. The method of claim 116, wherein fewer than about 200 Herl-Her2 dimers are detected.

130. The method of claim 116, wherein fewer than about 300 Herl-Her2 dimers are detected.

131. The method of claim 116, wherein fewer than about 400 Herl-Her2 dimers are detected.

132. The method of claim 116, wherein fewer than about 500 Herl-Her2 dimers are detected.

133. The method of claim 116, wherein fewer than about 750 Herl-Her2 dimers are detected.

134. The method of claim 116, wherein fewer than about 1000 Her 1 -Her2 dimers are detected.

135. The method of claim 116, wherein fewer than about 1500 Herl-Her2 dimers are detected.

136. The method of claim 116, wherein fewer than about 2000 Herl-Her2 dimers are detected.

137. A method for determining whether a cancer cell is likely to respond to treatment with a Her 1 -acting agent, comprising detecting on a cell of the cancer at least about 600 Herl-Herl dimers, at least about 1000 Herl-Her3 dimers, and fewer than about 1000 Her2-Her3 dimers, wherein the presence of the at least about 600 Herl-Herl dimers, the at least about 1000 Herl-Her3 dimers, and the fewer than about 1000 Her2-Her3 dimers indicates that the cancer cell is likely to respond to treatment with the Her 1 -acting agent.

138. The method of claim 86, wherein the Herl-acting agent is Gefitinib.

139. The method of claim 86, wherein at least about 800 Herl-Herl dimers are detected.

140. The method of claim 86, wherein at least about 900 Herl-Herl dimers are detected.

141. The method of claim 86, wherein at least about 1000 Herl-Herl dimers are detected.

142. The method of claim 86, wherein at least about 1100 Herl-Herl dimers are detected.

143. The method of claim 86, wherein between about 600 and about 100,000 Herl- Herl dimers are detected.

144. The method of claim 86, wherein at least about 800 Herl-Herl dimers are detected.

145. The method of claim 86, wherein at least about 900 Herl-Herl dimers are detected.

146. The method of claim 86, wherein at least about 1000 Herl-Herl dimers are detected.

147. The method of claim 86, wherein at least about 1100 Herl-Herl dimers are detected.

148. The method of claim 86, wherein at least about 1200 Herl-Her3 dimers are detected.

149. The method of claim 86, wherein at least about 1400 Herl-Her3 dimers are detected.

150. The method of claim 86, wherein at least about 1600 Herl-Her3 dimers are detected.

151. The method of claim 86, wherein at least about 1700 Her 1 -Her3 dimers are detected.

152. The method of claim 86, wherein at least about 1800 Herl-Her3 dimers are detected.

153. The method of claim 86, wherein between about 1000 and about 100,000 Herl- Her3 dimers are detected.

154. The method of claim 86, wherein at least about 1100 Herl-Herl dimers and at least about 1800 Herl-Her3 dimers are detected.

155. The method of claim 86, wherein fewer than about 900 Her2-Her3 dimers are detected.

156. The method of claim 86, wherein fewer than about 800 Her2-Her3 dimers are detected.

157. The method of claim 86, wherein fewer than about 700 Her2-Her3 dimers are detected.

158. The method of claim 86, wherein fewer than about 600 Her2-Her3 dimers are detected.

159. The method of claim 86, wherein between about 1 and about 1000 Her2-Her3 dimers are detected.

160. The method of claim 86, wherein at least about 1100 Her 1 -Her 1 dimers and fewer than about 600 Her2-Her3 dimers are detected.

161. The method of claim 86, wherein at least about 1100 Herl-Herl dimers, at least about 1800 Her2-Her3 dimers, and fewer than about 600 Her2-Her3 dimers are detected.

162. The method of claim 86, wherein between about 600 and about 100,000 Herl- Herl dimers and between about 1 and about 1000 Her2-Her3 dimers are detected.

163. The method of claim 86, wherein between about 600 and about 100,000 Herl- Herl dimers and between about 1000 and about 100,000 Herl-Her3 dimers are detected.

164. The method of claim 86, wherein between about 600 and about 100,000 Herl- Herl dimers, between about 1000 and about 100,000 Herl-Her3 dimers, and between about 1 and about 1000 Her2-Her3 dimers are detected.

165. The method of claim 86, wherein detecting the Herl -Herl dimers is accomplished by:

(a) contacting the cell with:

(ii) a cleaving probe having a cleavage inducing-moiety, wherein the binding compound and the cleaving probe each specifically bind Herl , and wherein binding of a binding compound or a cleaving probe to a Herl monomer precludes binding of another binding compound or cleaving probe to the same Herl monomer, and wherein if the binding compound is within an effective proximity of the cleavage- inducing moiety of the cleaving probe, the cleavage-inducing moiety cleaves the cleavable linker so that the molecular tag is released; and

(b) quantifying the amount of released molecular tag, thereby detecting the Herl -Herl dimers.

166. The method of claim 165, wherein activating the cleavage-inducing moiety cleaves the cleavable linker.

167. The method of claim 86, wherein detecting the Herl-Her3 dimers is accomplished by:

(a) contacting the cell with:

168. The method of claim 167, wherein activating the cleavage-inducing moiety cleaves the cleavable linker.

169. The method of claim 86, wherein detecting the Her2-Her3 dimers is accomplished by:

(a) contacting the cell with:

(ii) a cleaving probe having a cleavage inducing-moiety, wherein the binding compound and the cleaving probe each specifically binds either Her2 or Her3, and the cleaving probe and the binding probe do not both bind the same receptor, and wherein if the binding compound is within an effective proximity of the cleavage-inducing moiety of the cleaving probe, the cleavage-inducing moiety cleaves the cleavable linker so that the molecular tag is released; and

170. The method of claim 169, wherein activating the cleavage-inducing moiety cleaves the cleavable linker.

171. The method of claim 86, wherein the Her 1 -Her 1 dimers, Herl-Her3 dimers, and Her2-Her3 dimers are detected in a single assay.

172. The method of claim 171, wherein a single assay comprises the methods of claims 165, 167, and 169.

173. The method of claim 172, wherein the methods of claims 165, 167, and 169are performed concurrently.

174. The method of claim 86, wherein the cancer cell is a breast cancer cell, lung cancer cell, colorectal cancer cell, prostate cancer cell, or ovarian cancer cell.

175. The method of claim 174, wherein the cancer cell is a lung cancer cell.

176. The method of claim 86, wherein the Herl-Herl dimers on the cancer cell are detected directly on a patient sample.

177. The method of claim 176, wherein the patient sample is a fixed tissue sample, a frozen tissue sample, or a sample purified from circulating epithelial cells.

178. A method for determining whether a subject with cancer is likely to respond to treatment with a Herl -acting agent, comprising detecting in a biological sample from the subject's cancer at least about 600 Herl-Herl dimers per cancer cell, wherein the presence of the at least about 600 Herl-Herl dimers per cancer cell indicates that the cancer is likely to respond to treatment with the Herl -acting agent.

179. The method of claim 178, wherein at least about 1100 Herl-Herl dimers per cancer cell are detected.

180. The method of claim 178, further comprising detecting in the biological sample at least about 1000 Herl-Her3 dimers per cancer cell, wherein the presence of the at least about 600 Herl-Herl dimers per cancer cell and the at least about 1000 Herl-Her3 dimers indicate that the cancer is likely to respond to treatment with the Herl -acting agent.

181. The method of claim 180, wherein at least about 1800 Her 1 -Her3 dimers are detected.

182. The method of claim 178, further comprising detecting in the biological sample fewer than about 1000 Her2-Her3 dimers per cancer cell, wherein the presence of the at least about 600 Herl-Herl dimers per cancer cell and the fewer than about 1000 Herl-Her3 dimers indicate that the cancer is likely to respond to treatment with the Herl -acting agent.

183. The method of claim 182, wherein fewer than about 600 Her2-Her3 dimers are detected.

184. A method for determining whether a subject with cancer is likely to respond to treatment with a Herl-acting agent, comprising determ ining a Diagnostic Index for a cell in a biological sample from the subject's cancer according to

log(p/(1+p )) = -2.09 + 0.992*log(HllD+H13D) -0.39*log(H2P) -0.187*log(H23D)

Formula I,

wherein the Diagnostic Index indicates whether the subject is likely to respond to treatment with a Herl-acting agent, and wherein Hl ID is the number of Herl- Herl dimers detected per cancer cell, H13D is the number of Herl-Her3 dimers detected per cancer cell, H23D is the number of Her2-Her3 dimers detected per cancer cell, H2P is the number of phosphorylated Her2 receptors detected per cancer cell, anάp is the Diagnostic Index used to determine whether the subject is likely to respond to treatment with the Herl-acting agent.

185. A method for determining whether a subject with cancer is likely to respond to treatment with a Herl-acting agent, comprising determining a Diagnostic Index for a cell in a biological sample from the subject's cancer according to

log(p/(l+p))=-3.207+1.098*log(HHD+H13D)-0.142*log(H23D)-0.307*log(H12D)

Formula II

wherein the Diagnostic Index indicates whether the subject is likely to respond to treatment with a Herl-acting agent, and wherein Hl ID is the number of Herl- Herl dimers detected per cancer cell, Hl 3D is the number of Herl-Her3 dimers detected per cancer cell, Hl 2D is the number of Herl-Her2 dimers detected per cancer cell, H23D is the number of Her2-Her3 dimers detected per cancer cell, and p is the Diagnostic Index used to determine whether the subject is likely to respond to treatment with the Herl-acting agent.

186. A method for determining whether a subject with cancer is likely to respond to treatment with a Herl-acting agent, comprising determining a Diagnostic Index for a cell in a biological sample from the subject's cancer according to log(p/(1+p ))=-3.126+1.047*log(H11D+H13D)-0.322*log(H12D),

Formula III

wherein the Diagnostic Index indicates whether the subject is likely to respond to treatment with a Herl-acting agent, and wherein Hl ID is the number of Herl- Herl dimers detected per cancer cell, H13D is the number of Her 1 -Her 3 dimers detected per cancer cell, H12D is the number of Herl-Her2 dimers detected per cancer cell, and p is the Diagnostic Index used to determine whether the subject is likely to respond to treatment with the Herl-acting agent.

187. A method for determining whether a subject with cancer is likely to respond to treatment with a Herl-acting agent, comprising determining a Diagnostic Index for a cell in a biological sample from the subject's cancer according to

log(p/(1+p)) = -1.947 + 0.904*log(HllD+H13D) -0.393*log(H2P)

Formula IV

wherein the Diagnostic Index indicates whether the subject is likely to respond to treatment with a Herl-acting agent, and wherein Hl ID is the number of Herl- Herl dimers detected per cancer cell, Hl 3D is the number of Herl-Her3 dimers detected per cancer cell, H2P is the number of phosphorylated Her2 receptors detected per cancer cell, and p is the Diagnostic Index used to determine whether the subject is likely to respond to treatment with the Herl-acting agent.

188. A method for determining whether a subject with cancer is likely to respond to treatment with a Herl-acting agent, comprising determining a Diagnostic Index for a cell in a biological sample from the subject's cancer according to

log(p/(1+p)) = -1.098 + 0.58*log(H11D) - 0.141*log(H23D) + 0.322*log(H13D) -

0.397*log(H2P) Formula V

wherein the Diagnostic Index indicates whether the subject is likely to respond to treatment with a Herl-acting agent, and wherein Hl ID is the number of Herl- Herl dimers detected per cancer cell, H23D is the number of Her2-Her3 dimers detected per cancer cell, Hl 3D is the number of Herl-Her3 dimers detected per cancer cell, H2P is the number of phosphorylated Her2 receptors detected per cancer cell, and p is the Diagnostic Index used to determine whether the subject is likely to respond to treatment with the Herl -acting agent

189. A method for determining whether a subj ect with cancer is likely to respond to treatment with a Herl -acting agent, comprising determining a Diagnostic Index for a cell in a biological sample from the subject's cancer according to

log(p/(1+p))=-1.828+0.747*log(Hl lD)+0.009*log(H23D)+0.158*log(H13D)-

0.388*log(H12D) Formula VI

wherein the Diagnostic Index indicates whether the subject is likely to respond to treatment with a Herl-acting agent, and wherein Hl ID is the number of Herl- Herl dimers detected per cancer cell, H23D is the number of Her2-Her3 dimers detected per cancer cell, Hl 3D is the number of Herl-Her3 dimers detected per cancer cell, H2P is the number of phosphorylated Her2 receptors detected per cancer cell, and p is the Diagnostic Index used to determine whether the subject is likely to respond to treatment with the Herl-acting agent.

190. A method for determining whether a subj ect with cancer is likely to respond to treatment with a Herl-acting agent, comprising determining a Diagnostic Index for a cell in a biological sample from the subject's cancer according to

log(p/(1+p)) = -3.162+0.912*log(Hl lD+H13D)-0.192*log(H23D),

Formula VII

wherein the Diagnostic Index indicates whether the subject is likely to respond to treatment with a Herl-acting agent, and wherein Hl ID is the number of Herl- Herl dimers detected per cancer cell, H23D is the number of Her2-Her3 dimers detected per cancer cell, H13D is the number of Herl-Her3 dimers detected per cancer cell, and p is the Diagnostic Index used to determine whether the subject is likely to respond to treatment with the Her 1 -acting agent.

191. The method of claim 184, 185, 186, 187, 188, 189, or 190, wherein the Diagnostic Index is about 0.01.

192. The method of claim 184, 185, 186, 187, 188, 189, or 190, wherein the Diagnostic Index is about 0.05.

193. The method of claim 184, 185, 186, 187, 188, 189, or 190, wherein the Diagnostic Index is about 0.1.

194. The method of claim 184, 185, 186, 187, 188, 189, or 190, wherein the Diagnostic Index is about 0.15.

195. The method of claim 184, 185, 186, 187, 188, 189, or 190, wherein the Diagnostic Index is about 0.2.

196. The method of claim 184, 185, 186, 187, 188, 189, or 190, wherein the Diagnostic Index is about 0.25.

197. The method of claim 184, 185, 186, 187, 188, 189, or 190, wherein the Diagnostic Index is about 0.3.

198. The method of claim 184, 185, 186, 187, 188, 189, or 190, wherein the Diagnostic Index is about 0.35.

199. The method of claim 184, 185, 186, 187, 188, 189, or 190, wherein the Diagnostic Index is about 0.4.

200. The method of claim 184, 185, 186, 187, 188, 189, or 190, wherein the Diagnostic Index is about 0.45.

201. The method of claim 184, 185, 186, 187, 188, 189, or 190, wherein the Diagnostic Index is about 0.5.

202. The method of claim 184, 185, 186, 187, 188, 189, or 190, wherein the Diagnostic Index is about 0.55.

203. The method of claim 184, 185, 186, 187, 188, 189, or 190, wherein the Diagnostic Index is about 0.6.

204. The method of claim 184, 185, 186, 187, 188, 189, or 190, wherein the Diagnostic Index is about 0.65.

205. The method of claim 184, 185, 186, 187, 188, 189, or 190, wherein the Diagnostic Index is about 0.7.

206. The method of claim 184, 185, 186, 187, 188, 189, or 190, wherein the Diagnostic Index is about 0.75.

207. The method of claim 184, 185, 186, 187, 188, 189, or 190, wherein the Diagnostic Index is about 0.8.

208. The method of claim 184, 185, 186, 187, 188, 189, or 190, wherein the Diagnostic Index is about 0.85.

209. The method of claim 184, 185, 186, 187, 188, 189, or 190, wherein the Diagnostic Index is about 0.9.

210. The method of claim 184, 185, 186, 187, 188, 189, or 190, wherein the Diagnostic Index is about 0.95.

211. The method of claim 184, 185, 186, 187, 188, 189, or 190, wherein the Diagnostic Index is about 0.99.

212. A method for determining whether a subject with cancer is likely to respond to treatment with a Herl -acting agent, comprising determining a balanced dimer score for a cell in a biological sample from the subject's cancer, wherein the balanced dimer score is calculated according to Formula IX:

3.2 * (Herl/1 + Herl/2 + Herl/3) - 10.5 * Her2/3

Formula IX, wherein a balanced dimer score determined according to Formula IX greater than about 15,000 indicates that the subject is likely to respond to treatment with a Herl-acting agent, and wherein Herl/1 is the number of Herl-Herl dimers expressed per cancer cell, Herl/2 is the number of Herl-Her2 dimers expressed per cancer cell, Herl/3 is the number of Herl-Her3 dimers expressed per cancer cell, and Her2/3 is the number of Her2-Her3 dimers expressed per cancer cell.

213. The method of claim 212, further comprising detecting more than 1000 Herl- Herl dimers per cancer cell, wherein the presence of more than 1000 Herl-Herl dimers per cancer cell or a balanced dimer score determined according to Formula IX greater than about 15,000 indicates that the subject with cancer is likely to respond to treatment with a Herl-acting agent.

214. The method of claim 212, wherein the balanced dimer score determined according to Formula IX is at least about 5,000.

215. The method of claim 212, wherein the balanced dimer score determined according to Formula IX is at least about 7,500.

216. The method of claim 212, wherein the balanced dimer score determined according to Formula IX is at least about 10,000.

217. The method of claim 212, wherein the balanced dimer score determined according to Formula IX is at least about 11,000.

218. The method of claim 212, wherein the balanced dimer score determined according to Formula IX is at least about 12,000.

219. The method of claim 212, wherein the balanced dimer score determined according to Formula IX is at least about 13,000.

220. The method of claim 212, wherein the balanced dimer score determined according to Formula IX is at least about 14,000.

221. The method of claim 212, wherein the balanced dimer score determined according to Formula IX is at least about 15,000.

222. The method of claim 212, wherein the balanced dimer score determined according to Formula IX is at least about 17,500.

223. The method of claim 212, wherein the balanced dimer score determined according to Formula IX is at least about 20,000.

224. A method for determining whether a subject with cancer is likely to respond to treatment with a Herl -acting agent, comprising determining a balanced dimer score for a cell in a biological sample from the subject's cancer, wherein the balanced dimer score is calculated according to Formula X:

3.2 * Herl/3 - 10.5 * Her2/3

Formula X

wherein a balanced dimer score determined according to Formula X greater than about 4,000 indicates that the subject with cancer is likely to respond to treatment with a Herl -acting agent, and wherein Herl/3 is the number of Herl-Her3 dimers expressed per cancer cell and Her2/3 is the number of Her2-Her3 dimers expressed per cancer cell.

225. The method of claim 224, further comprising detecting more than 1000 Herl - Herl dimers per cancer cell, wherein the presence of more than 1000 Herl -Herl dimers per cancer cell or a balanced dimer score determined according to Formula X greater than about 4,000 indicates that the subject with cancer is likely to respond to treatment with a Herl -acting agent.

226. The method of claim 224, further comprising determining a balanced dimer score for a cell in a biological sample from the subject's cancer, wherein the balanced dimer score is calculated according to Formula IX:

3.2 * (Herl/1 + Herl/2 + Herl/3) - 10.5 * Her2/3

Formula IX,

wherein Herl/1 is the number of Herl-Herl dimers expressed per cancer cell, Herl/2 is the number of Herl-Her2 dimers expressed per cancer cell, Herl/3 is the number of Her 1 -Her 3 dimers expressed per cancer cell, and Her2/3 is the number of Her2-Her3 dimers expressed per cancer cell, and wherein a balanced dimer score determined according to Formula IX greater than about 15,000 or a balanced dimer score determined according to Formula X greater than about 4,000 indicates that the subject is likely to respond to treatment with a Herl- acting agent.

227. The method of claim 224, further comprising detecting more than 1000 Herl- Herl dimers per cancer cell and determining a balanced dimer score for a cell in a biological sample from the subject's cancer, wherein the balanced dimer score is calculated according to Formula IX:

3.2 * (Herl/1 + Herl/2 + Herl/3) - 10.5 * Her2/3

Formula IX,

wherein Herl/1 is the number of Herl-Herl dimers expressed per cancer cell, Herl/2 is the number of Herl-Her2 dimers expressed per cancer cell, Herl/3 is the number of Herl-Her3 dimers expressed per cancer cell, and Her2/3 is the number of Her2-Her3 dimers expressed per cancer cell, and wherein a balanced dimer score determined according to Formula IX greater than about 15,000 indicates that the subject is likely to respond to treatment with a Her 1 -acting agent, the presence of more than 1000 Herl-Herl dimers per cancer cell, or a balanced dimer score determined according to Formula X greater than about 4,000 indicates that the subject with cancer is likely to respond to treatment with a Her 1 -acting agent.

228. The method of claim 224, wherein the balanced dimer score determined according to Formula X is at least about 500.

229. The method of claim 224, wherein the balanced dimer score determined according to Formula X is at least about 1,000.

230. The method of claim 224, wherein the balanced dimer score determined according to Formula X is at least about 1,500.

231. The method of claim 224, wherein the balanced dimer score determined according to Formula X is at least about 2,000.

232. The method of claim 224, wherein the balanced dimer score determined according to Formula X is at least about 2,500.

233. The method of claim 224, wherein the balanced dimer score determined according to Formula X is at least about 3,000.

234. The method of claim 224, wherein the balanced dimer score determined according to Formula X is at least about 3,500.

235. The method of claim 224, wherein the balanced dimer score determined according to Formula X is at least about 4,000.

236. The method of claim 224, wherein the balanced dimer score determined according to Formula X is at least about 4,500.

237. The method of claim 224, wherein the balanced dimer score determined according to Formula X is at least about 5,000.

238. The method of claim 224, wherein the balanced dimer score determined according to Formula X is at least about 7,500.

239. The method of claim 224, wherein the balanced dimer score determined according to Formula X is at least about 10,000.

240. The method of claim 224, wherein the balanced dimer score determined according to Formula X is at least about 15,000.

241. A method for determining whether a cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent, comprising determining a Diagnostic Index for the cancer or cancer cell according to

log(p/(l+p)) = -2.09 + 0.992*log(Hl lD+H13D) -0.39*log(H2P) -0.187*log(H23D)

Formula I,

wherein the Diagnostic Index indicates whether the cancer or cancer cell is likely to respond to treatment with a Herl-acting agent, and wherein Hl ID is the number of Herl-Herl dimers detected per cancer cell, H13D is the number of Herl-Her3 dimers detected per cancer cell, H23D is the number of Her2-Her3 dimers detected per cancer cell, H2P is the number of phosphorylated Her2 receptors detected per cancer cell, and p is the Diagnostic Index used to determine whether the cancer or cancer cell is likely to respond to treatment with the Herl-acting agent.

242. A method for determining whether a cancer or cancer cell is likely to respond to treatment with a Herl-acting agent, comprising determining a Diagnostic Index for the cancer or cancer cell according to

log(p/(1+p))=-3.207+1.098* log(Hl lD+H13D)-0.142*log(H23D)-0.307*log(H12D)

Formula II

wherein the Diagnostic Index indicates whether the cancer or cancer cell is likely to respond to treatment with a Herl-acting agent, and wherein Hl ID is the number of Herl-Herl dimers detected per cancer cell, Hl 3D is the number of Herl-Her3 dimers detected per cancer cell, H12D is the number of Herl-Her2 dimers detected per cancer cell, H23D is the number of Her2-Her3 dimers detected per cancer cell, and p is the Diagnostic Index used to determine whether the cancer or cancer cell is likely to respond to treatment with the Her 1 -acting agent.

243. A method for determining whether a cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent, comprising determining a Diagnostic Index for the cancer or cancer cell according to

log(p/(1+p))=-3.126+1.047*log(HHD+H13D)-0.322*log(H12D),

Formula III

wherein the Diagnostic Index indicates whether the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent, and wherein Hl ID is the number of Herl-Herl dimers detected per cancer cell, H13D is the number of Herl-Her3 dimers detected per cancer cell, H12D is the number of Herl-Her2 dimers detected per cancer cell, and p is the Diagnostic Index used to determine whether the cancer or cancer cell is likely to respond to treatment with the Herl- acting agent.

244. A method for determining whether a cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent, comprising determining a Diagnostic Index for the cancer or cancer cell according to

log(p/(1+p)) = -1.947 + 0.904*log(H11D+H13D) -0.393*log(H2P)

Formula IV

wherein the Diagnostic Index indicates whether the cancer or cancer cell is likely to respond to treatment with a Herl-acting agent, and wherein Hl ID is the number of Herl-Herl dimers detected per cancer cell, Hl 3D is the number of Herl-Her3 dimers detected per cancer cell, H2P is the number of phosphorylated Her2 receptors detected per cancer cell, and p is the Diagnostic Index used to determine whether the cancer or cancer cell is likely to respond to treatment with the Herl-acting agent.

245. A method for determining whether a cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent, comprising determining a Diagnostic Index for the cancer or cancer cell according to

log(p/(l+p)) = -1.098 + 0.58*log(Hl lD) - 0.141*log(H23D) + 0.322*log(H13D) -

0.397*log(H2P) Formula V

wherein the Diagnostic Index indicates whether the cancer or cancer cell is likely to respond to treatment with a Herl-acting agent, and wherein Hl ID is the number of Herl-Herl dimers detected per cancer cell, H23D is the number of Her2-Her3 dimers detected per cancer cell, H13D is the number of Herl-Her3 dimers detected per cancer cell, H2P is the number of phosphorylated Her2 receptors detected per cancer cell, and p is the Diagnostic Index used to determine whether the cancer or cancer cell is likely to respond to treatment with the Herl-acting agent.

246. A method for determining whether a cancer or cancer cell is likely to respond to treatment with a Herl-acting agent, comprising determining a Diagnostic Index for the cancer or cancer cell according to

log(p/(l+p))=-1.828+0.747* log(HllD)+0.00* log(H23D)+0.158*log(H13D)-

0.388*log(H12D) Formula VI

247. A method for determining whether a cancer or cancer cell is likely to respond to treatment with a Herl -acting agent, comprising determining a Diagnostic Index for the cancer or cancer cell according to

log(1/(1+p)) = -3.162+0.912*log(HllD+H13D)-0.192*log(H23D),

Formula VII

wherein the Diagnostic Index indicates whether the cancer or cancer cell is likely to respond to treatment with a Herl -acting agent, and wherein Hl ID is the number of Herl -Herl dimers detected per cancer cell, H23D is the number of Her2-Her3 dimers detected per cancer cell, Hl 3D is the number of Herl-Her3 dimers detected per cancer cell, and p is the Diagnostic Index used to determine whether the cancer or cancer cell is likely to respond to treatment with the Herl- acting agent.

248. The method of claim 241, 242, 243, 244, 245, 246, or 247, wherein the Diagnostic Index is about 0.01.

249. The method of claim 241 , 242, 243, 244, 245, 246, or 247, wherein the Diagnostic Index is about 0.05.

250. The method of claim 241 , 242, 243, 244, 245, 246, or 247, wherein the Diagnostic Index is about 0.1.

251. The method of claim 241 , 242, 243, 244, 245, 246, or 247, wherein the Diagnostic Index is about 0.15.

252. The method of claim 241 , 242, 243, 244, 245, 246, or 247, wherein the Diagnostic Index is about 0.2.

253. The method of claim 241 , 242, 243 , 244, 245, 246, or 247, wherein the Diagnostic Index is about 0.25.

254. The method of claim 241 , 242, 243, 244, 245, 246, or 247, wherein the Diagnostic Index is about 0.3.

255. The method of claim 241, 242, 243, 244, 245, 246, or 247, wherein the Diagnostic Index is about 0.35.

256. The method of claim 241, 242, 243, 244, 245, 246, or 247, wherein the Diagnostic Index is about 0.4.

257. The method of claim 241, 242, 243, 244, 245, 246, or 247, wherein the Diagnostic Index is about 0.45.

258. The method of claim 241, 242, 243, 244, 245, 246, or 247, wherein the Diagnostic Index is about 0.5.

259. The method of claim 241 , 242, 243, 244, 245, 246, or 247, wherein the Diagnostic Index is about 0.55.

260. The method of claim 241, 242, 243, 244, 245, 246, or 247, wherein the Diagnostic Index is about 0.6.

261. The method of claim 241, 242, 243, 244, 245, 246, or 247, wherein the Diagnostic Index is about 0.65.

262. The. method of claim 241 , 242, 243, 244, 245, 246, or 247, wherein the Diagnostic Index is about 0.7.

263. The method of claim 241 , 242, 243 , 244, 245 , 246, or 247, wherein the Diagnostic Index is about 0.75.

264. The method of claim 241, 242, 243, 244, 245, 246, or 247, wherein the Diagnostic Index is about 0.8.

265. The method of claim 241, 242, 243, 244, 245, 246, or 247, wherein the Diagnostic Index is about 0.85.

266. The method of claim 241 , 242, 243, 244, 245, 246, or 247, wherein the Diagnostic Index is about 0.9.

267. The method of claim 241 , 242, 243 , 244, 245 , 246, or 247, wherein the Diagnostic Index is about 0.95.

268. The method of claim 241, 242, 243, 244, 245, 246, or 247, wherein the Diagnostic Index is about 0.99.

269. A method for determining whether a cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent, comprising determining a balanced dimer score for the cancer or cancer cell, wherein the balanced dimer score is calculated according to Formula IX:

3.2 * (Herl/1 + Herl/2 + Herl/3) - 10.5 * Her2/3

Formula IX, wherein a balanced dimer score determined according to Formula IX greater than about 15,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Herl-acting agent, and wherein Herl/1 is the number of Herl- Herl dimers expressed per cancer cell, Herl/2 is the number of Herl-Her2 dimers expressed per cancer cell, Herl/3 is the number of Herl-Her3 dimers expressed per cancer cell, and Her2/3 is the number of Her2-Her3 dimers expressed per cancer cell.

270. The method of claim 212, further comprising detecting more than 1000 Herl- Herl dimers per cancer cell, wherein the presence of more than 1000 Herl-Herl dimers per cancer cell or a balanced dimer score determined according to Formula IX greater than about 15,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Herl-acting agent.

271. The method of claim 212, wherein the balanced dimer score determined according to Formula IX is at least about 5,000.

272. The method of claim 212, wherein the balanced dimer score determined according to Formula IX is at least about 7,500.

273. The method of claim 212, wherein the balanced dimer score determined according to Formula IX is at least about 10,000.

274. The method of claim 212, wherein the balanced dimer score determined according to Formula IX is at least about 11,000.

275. The method of claim 212, wherein the balanced dimer score determined according to Formula IX is at least about 12,000.

276. The method of claim 212, wherein the balanced dimer score determined according to Formula IX is at least about 13,000.

277. The method of claim 212, wherein the balanced dimer score determined according to Formula IX is at least about 14,000.

278. The method of claim 212, wherein the balanced dimer score determined according to Formula IX is at least about 15,000.

279. The method of claim 212, wherein the balanced dimer score determined according to Formula IX is at least about 17,500.

280. The method of claim 212, wherein the balanced dimer score determined according to Formula IX is at least about 20,000.

281. A method for determining whether a cancer or cancer cell is likely to respond to treatment with a Herl-acting agent, comprising determining a balanced dimer score for the cancer or cancer cell, wherein the balanced dimer score is calculated according to Formula X: 3.2 * Herl/3 - 10.5 * Her2/3 Formula X

wherein a balanced dimer score determined according to Formula X greater than about 4,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Herl-acting agent, and wherein Herl/3 is the number of Herl- Her3 dimers expressed per cancer cell and Her2/3 is the number of Her2-Her3 dimers expressed per cancer cell.

282. The method of claim 224, further comprising detecting more than 1000 Herl- Herl dimers per cancer cell, wherein the presence of more than 1000 Herl-Herl dimers per cancer cell or a balanced dimer score determined according to Formula X greater than about 4,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Herl-acting agent.

283. The method of claim 224, further comprising determining a balanced dimer score for the cancer or cancer cell, wherein the balanced dimer score is calculated according to Formula IX:

3.2 * (Herl/1 + Herl/2 + Herl/3) - 10.5 * Her2/3

Formula IX,

wherein Herl/1 is the number of Herl-Herl dimers expressed per cancer cell, Herl/2 is the number of Herl-Her2 dimers expressed per cancer cell, Herl/3 is the number of Herl-Her3 dimers expressed per cancer cell, and Her2/3 is the number of Her2-Her3 dimers expressed per cancer cell, and wherein a balanced dimer score determined according to Formula IX greater than about 15,000 or a balanced dimer score determined according to Formula X greater than about 4,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Herl-acting agent.

284. The method of claim 224, further comprising detecting more than 1000 Herl- Herl dimers per cancer cell and determining a balanced dimer score for the cancer or cancer cell, wherein the balanced dimer score is calculated according to Formula IX:

3.2 * (Herl/1 + Herl/2 + Herl/3) - 10.5 * Her2/3

Formula IX,

wherein Herl/1 is the number of Herl-Herl dimers expressed per cancer cell, Herl/2 is the number of Herl-Her2 dimers expressed per cancer cell, Herl/3 is the number of Her 1 -Her 3 dimers expressed per cancer cell, and Her2/3 is the number of Her2-Her3 dimers expressed per cancer cell, and wherein a balanced dimer score determined according to Formula IX greater than about 15,000, the presence of more than 1000 Herl-Herl dimers per cancer cell, or a balanced dimer score determined according to Formula X greater than about 4,000 indicates that the cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent.

285. The method of claim 224, wherein the balanced dimer score determined according to Formula X is at least about 500.

286. The method of claim 224, wherein the balanced dimer score determined according to Formula X is at least about 1,000.

287. The method of claim 224, wherein the balanced dimer score determined according to Formula X is at least about 1,500.

288. The method of claim 224, wherein the balanced dimer score determined according to Formula X is at least about 2,000.

289. The method of claim 224, wherein the balanced dimer score determined according to Formula X is at least about 2,500.

290. The method of claim 224, wherein the balanced dimer score determined according to Formula X is at least about 3,000.

291. The method of claim 224, wherein the balanced dimer score determined according to Formula X is at least about 3,500.

292. The method of claim 224, wherein the balanced dimer score determined according to Formula X is at least about 4,000.

293. The method of claim 224, wherein the balanced dimer score determined according to Formula X is at least about 4,500.

294. The method of claim 224, wherein the balanced dimer score determined according to Formula X is at least about 5,000.

295. The method of claim 224, wherein the balanced dimer score determined according to Formula X is at least about 7,500.

296. The method of claim 224, wherein the balanced dimer score determined according to Formula X is at least about 10,000.

297. The method of claim 224, wherein the balanced dimer score determined according to Formula X is at least about 15,000.

298. The method of claim 224, wherein the balanced dimer score determined according to Formula X is at least about 20,000.

299. A computer program product for use in conjunction with a computer system, wherein the computer program product comprises a computer readable storage medium and a computer program mechanism embedded therein, the computer program mechanism comprising instructions for evaluating whether a plurality of features in a biomarker profile of a test subject cancer or cancer cell satisfies a first value set, wherein satisfying the first value set predicts that the test subject cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent, and wherein the plurality of features comprise one or more of the presence or amount of expression of Herl-Herl dimers, Herl-Her2 dimers, Herl-Her3 dimers, Her2-Her3 dimers, Herl phosphorylation, or Her2 phosphorylation.

300. A computer comprising:

(a) a central processing unit;

(b) a memory coupled to the central processing unit, the memory storing: instructions for evaluating whether a plurality of features in a biomarker profile of a test subject cancer or cancer cell satisfies a first value set, wherein satisfying the first value set predicts that the test subject cancer or cancer cell is likely to respond to treatment with a Her 1 -acting agent, and wherein the plurality of features comprise one or more of the presence or amount of expression of Her 1- Herl dimers, Herl-Her2 dimers, Herl-Her3 dimers, Her2-Her3 dimers, Herl phosphorylation, or Her2 phosphorylation.

301. A method for determining whether a cancer cell is likely to respond to treatment with a Herl -acting agent, comprising detecting on the cancer cell

(a) at least about 1600 Herl-Herl dimers or at least about 850 Herl-Her3 dimers, and

(b) fewer than about 600 Her2-Her3 dimers, wherein the presence of the 1600 Herl-Herl dimers or at least about 850 Herl- Her3 dimers and fewer than about 600 Her2-Her3 dimers indicates that the cancer is likely to respond to treatment with the Herl -acting agent.

302. The method of claim 301, wherein the Herl-acting agent is Geiϊtinib, tarceva, or erbitux.

303. The method of claim 301, wherein the Herl-acting agent is Gefitinib.

304. The method of claim 301, wherein at least about 750 Herl-Herl dimers are detected.

305. The method of claim 301, wherein at least about 800 Herl-Herl dimers are detected.

306. The method of claim 301, wherein at least about 900 Herl-Herl dimers are detected.

307. The method of claim 301, wherein at least about 1000 Herl-Herl dimers are detected.

308. The method of claim 301, wherein at least about 1100 Herl-Herl dimers are detected.

309. The method of claim 301, wherein at least about 1200 Herl-Herl dimers are detected.

310. The method of claim 301, wherein at least about 1300 Herl-Herl dimers are detected.

311. The method of claim 301, wherein at least about 1325 Herl-Herl dimers are detected.

312. The method of claim 301, wherein between about 600 and about 100,000 Herl- Herl dimers are detected.

313. The method of claim 301, wherein detecting the Herl-Herl dimers is accomplished by:

(a) contacting the cell with:

(ii) a cleaving probe having a cleavage inducing-moiety, wherein the binding compound and the cleaving probe each specifically bind Herl, and wherein binding of a binding compound or a cleaving probe to a Herl monomer precludes binding of another binding compound or cleaving probe to the same Herl monomer, and wherein if the binding compound is within an effective proximity of the cleavage- inducing moiety of the cleaving probe, the cleavage-inducing moiety cleaves the cleavable linker so that the molecular tag is released; and (b) quantifying the amount of released molecular tag, thereby detecting the Herl-Herl dimers.

314. The method of claim 313, wherein activating the cleavage-inducing moiety cleaves the cleavable linker.

315. The method of claim 313, wherein the binding compound and the cleaving probe each specifically binds a Herl epitope.

316. The method of claim 315, wherein the binding compound and the cleaving probe each specifically binds an identical Herl epitope.

317. The method of claim 313, wherein the binding compound and the cleaving probe each comprises an antibody or antigen-binding fragment.

318. The method of claim 315, wherein the binding compound and the cleaving probe each comprises an antibody or antigen-binding fragment.

319. The method of claim 313, wherein the binding compound and the cleaving probe each specifically binds a Herl ligand binding site.

320. The method of claim 313, wherein the binding compound and the cleaving probe each comprises a Herl ligand.

321. The method of claim 301 , wherein the cancer cell is a breast cancer cell, lung cancer cell, colorectal cancer cell, prostate cancer cell, or ovarian cancer cell.

322. The method of claim 321 , wherein the cancer cell is a lung cancer cell.

323. The method of claim 301, wherein the Herl-Herl dimers on the cancer cell are detected directly on a patient sample.

324. The method of claim 323, wherein the patient sample is a fixed tissue sample, a frozen tissue sample, or a sample purified from circulating epithelial cells.

325. The method of claim 323, wherein the patient sample is a lung tissue sample, a breast tissue sample, a colorectal tissue sample, a prostate tissue sample, or an ovarian tissue sample.

326. The method of claim 325, wherein the patient sample is a lung tissue sample.

327. The method of claim 301, wherein the cancer cell is obtained from a biological sample of a subject having or suspected of having a cancer.

328. The method of claim 301, wherein detecting the Herl-Her3 dimers is accomplished by:

(a) contacting the cell with:

329. The method of claim 328, wherein activating the cleavage-inducing moiety cleaves the cleavable linker.

330. The method of claim 328, wherein the binding compound specifically binds a Herl epitope.

331. The method of claim 328, wherein the binding compound comprises an antibody or antigen-binding fragment.

332. The method of claim 328, wherein the binding compound specifically binds a Herl ligand binding site.

333. The method of claim 328, wherein the binding compound comprises a Herl ligand.

334. The method of claim 328, wherein the binding compound specifically binds a Her3 epitope.

335. The method of claim 328, wherein the binding compound specifically binds a Her3 ligand binding site.

336. The method of claim 328, wherein the binding compound comprises a Her3 ligand.

337. The method of claim 328, wherein the cleaving probe specifically binds a Herl epitope.

338. The method of claim 328, wherein the cleaving probe comprises an antibody or antigen-binding fragment.

339. The method of claim 328, wherein the cleaving probe specifically binds a Herl ligand binding site.

340. The method of claim 328, wherein the cleaving probe comprises a Herl ligand.

341. The method of claim 328, wherein the cleaving probe specifically binds a Her3 epitope.

342. The method of claim 328, wherein the cleaving probe specifically binds a Her3 ligand binding site.

343. The method of claim 328, wherein the cleaving probe comprises a Her3 ligand.

344. The method of claim 301, wherein detecting the Her2-Her3 dimers is accomplished by:

(a) contacting the cell with:

345. The method of claim 344, wherein activating the cleavage-inducing moiety cleaves the cleavable linker.

346. The method of claim 344, wherein the binding compound specifically binds a Her2 epitope.

347. The method of claim 68, wherein the binding compound comprises an antibody or antigen-binding fragment.

348. The method of claim 344, wherein the binding compound specifically binds a Her2 ligand binding site.

349. The method of claim 344, wherein the binding compound comprises a Her2 ligand.

350. The method of claim 344, wherein the binding compound specifically binds a Her3 epitope.

351. The method of claim 344, wherein the binding compound specifically binds a Her3 ligand binding site.

352. The method of claim 344, wherein the binding compound comprises a Her3 ligand.

353. The method of claim 344, wherein the cleaving probe specifically binds a Her2 epitope.

354. The method of claim 344, wherein the cleaving probe comprises an antibody or antigen-binding fragment.

355. The method of claim 344, wherein the cleaving probe specifically binds a Her2 ligand binding site.

356. The method of claim 344, wherein the cleaving probe comprises a Her2 ligand.

357. The method of claim 344, wherein the cleaving probe specifically binds a Her3 epitope.

358. The method of claim 344, wherein the cleaving probe specifically binds a Her3 ligand binding site.

359. The method of claim 344, wherein the cleaving probe comprises a Her3 ligand.

360. The method of claim 301, wherein at least about 1700 Herl-Herl dimers are detected.

361. The method of claim 301, wherein at least about 1800 Herl-Herl dimers are detected.

362. The method of claim 301, wherein at least about 1900 Herl-Herl dimers are detected.

363. The method of claim 301, wherein at least about 2000 Herl-Herl dimers are detected.

364. The method of claim 301, wherein at least about 2100 Herl-Herl dimers are detected.

365. The method of claim 301, wherein at least about 2200 Herl-Herl dimers are detected.

366. The method of claim 301, wherein at least about 2300 Herl-Herl dimers are detected.

367. The method of claim 301, wherein at least about 2400 Herl-Herl dimers are detected.

368. The method of claim 301, wherein at least about 2500 Herl-Herl dimers are detected.

369. The method of claim 301, wherein at least about 2600 Herl-Herl dimers are detected.

370. The method of claim 301, wherein at least about 2700 Herl-Herl dimers are detected.

371. The method of claim 301, wherein at least about 2800 Herl-Herl dimers are detected.

372. The method of claim 301, wherein at least about 2900 Herl-Herl dimers are detected.

373. The method of claim 301, wherein at least about 3000 Herl-Herl dimers are detected.

374. The method of claim 301, wherein at least about 900 Herl-Her3 dimers are detected.

375. The method of claim 301, wherein at least about 1000 Herl-Her3 dimers are detected.

376. The method of claim 301, wherein at least about 1100 Herl-Her3 dimers are detected.

377. The method of claim 301, wherein at least about 1200 Herl-Her3 dimers are detected.

378. The method of claim 301, wherein at least about 1300 Herl-Her3 dimers are detected.

379. The method of claim 301, wherein at least about 1400 Herl-Her3 dimers are detected.

380. The method of claim 301, wherein at least about 1500 Herl-Her3 dimers are detected.

381. The method of claim 301, wherein at least about 1600 Herl-Her3 dimers are detected.

382. The method of claim 301, wherein at least about 1700 Herl-Her3 dimers are detected.

383. The method of claim 301, wherein at least about 1800 Herl-Her3 dimers are detected.

384. The method of claim 301, wherein at least about 1900 Herl-Her3 dimers are detected.

385. The method of claim 301, wherein at least about 2000 Herl-Her3 dimers are detected.

386. The method of claim 301, wherein fewer than about 550 Her2-Her3 dimers are detected.

387. The method of claim 301, wherein fewer than about 500 Her2-Her3 dimers are detected.

388. The method of claim 301, wherein fewer than about 450 Her2-Her3 dimers are detected.

389. The method of claim 301, wherein fewer than about 400 Her2-Her3 dimers are detected.

390. The method of claim 301, wherein fewer than about 350 Her2-Her3 dimers are detected.

391. The method of claim 301, wherein fewer than about 300 Her2-Her3 dimers are detected.

392. The method of claim 301 , wherein fewer than about 250 Her2-Her3 dimers are detected.

393. The method of claim 301, wherein fewer than about 200 Her2-Her3 dimers are detected.

394. The method of claim 301 , wherein fewer than about 150 Her2-Her3 dimers are detected.

395. The method of claim 301, wherein fewer than about 100 Her2-Her3 dimers are detected.

396. The method of claim 301, wherein fewer than about 50 Her2-Her3 dimers are detected.

397. A method for determining whether a cancer cell is likely to respond to treatment with a Her 1 -acting agent, comprising detecting on the cancer cell

(a) fewer than about 230 Her2-Her3 dimers, and

(b) at least about 500 Her 1 -Her 1 dimers and fewer than about 220 Herl-Her2 dimers or at least about 1600 Her 1 -Her 1 dimers and fewer than about 150 Herl-Her3 dimers, wherein satisfaction of conditions (a) and (b) indicates that the cancer cell is likely to respond to treatment with the Herl -acting agent.

398. The method of claim 397, wherein the Herl-acting agent is Gefitinib, tarceva, or erbitux.

399. The method of claim 397, wherein the Herl -acting agent is Gefitinib.

400. The method of claim 397, wherein fewer than about 200 Her2-Her3 dimers are detected.

401. The method of claim 397, wherein fewer than about 150 Her2-Her3 dimers are detected.

402. The method of claim 397, wherein fewer than about 100 Her2-Her3 dimers are detected.

403. The method of claim 397, wherein fewer than about 50 Her2-Her3 dimers are detected.

404. The method of claim 397, wherein no Her2-Her3 dimers are detected.

405. The method of claim 397, wherein at least about 500 Herl -Herl dimers are detected.

406. The method of claim 397, wherein at least about 600 Herl-Herl dimers are detected.

407. The method of claim 397, wherein at least about 700 Herl-Herl dimers are detected.

408. The method of claim 397, wherein at least about 750 Herl-Herl dimers are detected.

409. The method of claim 397, wherein at least about 900 Herl-Herl dimers are detected.

410. The method of claim 397, wherein at least about 1000 Herl-Herl dimers are detected.

411. The method of claim 397, wherein at least about 1100 Herl-Herl dimers are detected.

412. The method of claim 397, wherein at least about 1200 Herl-Herl dimers are detected.

413. The method of claim 397, wherein at least about 1300 Herl-Herl dimers are detected.

414. The method of claim 397, wherein at least about 1400 Herl-Herl dimers are detected.

415. The method of claim 397, wherein at least about 1500 Herl-Herl dimers are detected.

416. The method of claim 397, wherein at least about 1600 Herl-Herl dimers are detected.

417. The method of claim 397, wherein at least about 1700 Her 1 -Her 1 dimers are detected.

418. The method of claim 397, wherein at least about 1800 Herl-Herl dimers are detected.

419. The method of claim 397, wherein at least about 1900 Herl-Herl dimers are detected.

420. The method of claim 397, wherein at least about 2000 Herl-Herl dimers are detected.

421. The method of claim 397, wherein at least about 2500 Herl-Herl dimers are detected.

422. The method of claim 397, wherein at least about 3000 Herl-Herl dimers are detected.

423. The method of claim 397, wherein fewer than about 200 Herl-Her2 dimers are detected.

424. The method of claim 397, wherein fewer than about 150 Herl-Her2 dimers are detected.

425. The method of claim 397, wherein fewer than about 100 Herl-Her2 dimers are detected.

426. The method of claim 397, wherein fewer than about 50 Herl-Her2 dimers are detected.

427. The method of claim 397, wherein no Herl-Her2 dimers are detected.

428. The method of claim 397, wherein fewer than about 100 Herl-Her3 dimers are detected.

429. The method of claim 397, wherein fewer than about 50 Herl-Her3 dimers are detected.

430. The method of claim 397, wherein no Herl-Her3 dimers are detected.

431. The method of claim 397, wherein detecting the Herl-Herl dimers is accomplished by:

(a) contacting the cell with:

432. The method of claim 431 , wherein activating the cleavage-inducing moiety cleaves the cleavable linker.

433. The method of claim 431 , wherein the binding compound and the cleaving probe each specifically binds a Herl epitope.

434. The method of claim 433, wherein the binding compound and the cleaving probe each specifically binds an identical Herl epitope.

435. The method of claim 431 , wherein the binding compound and the cleaving probe each comprises an antibody or antigen-binding fragment.

436. The method of claim 433, wherein the binding compound and the cleaving probe each comprises an antibody or antigen-binding fragment.

437. The method of claim 431 , wherein the binding compound and the cleaving probe each specifically binds a Herl ligand binding site.

438. The method of claim 431 , wherein the binding compound and the cleaving probe each comprises a Herl ligand.

439. The method of claim 397, wherein the cancer cell is a breast cancer cell, lung cancer cell, colorectal cancer cell, prostate cancer cell, or ovarian cancer cell.

440. The method of claim 439, wherein the cancer cell is a lung cancer cell.

441. The method of claim 397, wherein the Herl -Herl dimers on the cancer cell are detected directly on a patient sample.

442. The method of claim 441, wherein the patient sample is a fixed tissue sample, a frozen tissue sample, or a sample purified from circulating epithelial cells.

443. The method of claim 441, wherein the patient sample is a lung tissue sample, a breast tissue sample, a colorectal tissue sample, a prostate tissue sample, or an ovarian tissue sample.

444. The method of claim 443, wherein the patient sample is a lung tissue sample.

445. The method of claim 397, wherein the cancer cell is obtained from a biological sample of a subject having or suspected of having a cancer.

446. The method of claim 397, wherein detecting the Her2-Her3 dimers is accomplished by:

(a) contacting the cell with:

447. The method of claim 446, wherein activating the cleavage-inducing moiety cleaves the cleavable linker.

448. The method of claim 446, wherein the binding compound specifically binds a Her2 epitope.

449. The method of claim 446, wherein the binding compound comprises an antibody or antigen-binding fragment.

450. The method of claim 446, wherein the binding compound specifically binds a Her2 ligand binding site.

451. The method of claim 446, wherein the binding compound comprises a Her2 ligand.

452. The method of claim 446, wherein the binding compound specifically binds a Her3 epitope.

453. The method of claim 446, wherein the binding compound specifically binds a Her3 ligand binding site.

454. The method of claim 446, wherein the binding compound comprises a Her3 ligand.

455. The method of claim 446, wherein the cleaving probe specifically binds a Her2 epitope.

456. The method of claim 446, wherein the cleaving probe comprises an antibody or antigen-binding fragment.

457. The method of claim 446, wherein the cleaving probe specifically binds a Her2 ligand binding site.

458. The method of claim 446, wherein the cleaving probe comprises a Her2 ligand.

459. The method of claim 446, wherein the cleaving probe specifically binds a Her3 epitope.

460. The method of claim 446, wherein the cleaving probe specifically binds a Her3 ligand binding site.

461. The method of claim 446, wherein the cleaving probe comprises a Her3 ligand.

462. The method of claim 397, wherein detecting the Herl-Her3 dimers is accomplished by:

(a) contacting the cell with:

463. The method of claim 462, wherein activating the cleavage-inducing moiety cleaves the cleavable linker.

464. The method of claim 462, wherein the binding compound specifically binds a Herl epitope.

465. The method of claim 462, wherein the binding compound comprises an antibody or antigen-binding fragment.

466. The method of claim 462, wherein the binding compound specifically binds a Herl ligand binding site.

467. The method of claim 462, wherein the binding compound comprises a Herl ligand.

468. The method of claim 462, wherein the binding compound specifically binds a Her3 epitope.

469. The method of claim 462, wherein the binding compound specifically binds a Her3 ligand binding site.

470. The method of claim 462, wherein the binding compound comprises a Her3 ligand.

471. The method of claim 462, wherein the cleaving probe specifically binds a Herl epitope.

472. The method of claim 462, wherein the cleaving probe comprises an antibody or antigen-binding fragment.

473. The method of claim 328, wherein the cleaving probe specifically binds a Herl ligand binding site.

474. The method of claim 462, wherein the cleaving probe comprises a Herl ligand.

475. The method of claim 462, wherein the cleaving probe specifically binds a Her3 epitope.

476. The method of claim 462, wherein the cleaving probe specifically binds a Her3 ligand binding site.

477. The method of claim 462, wherein the cleaving probe comprises a Her3 ligand.

478. The method of claim 397, wherein detecting the Herl-Her2 dimers is accomplished by:

(a) contacting the cell with:

(ii) a cleaving probe having a cleavage inducing-moiety, wherein the binding compound and the cleaving probe each specifically binds either Herl or Her2, and the cleaving probe and the binding probe do not both bind the same receptor, and wherein if the binding compound is within an effective proximity of the cleavage-inducing moiety of the cleaving probe, the cleavage-inducing moiety cleaves the cleavable linker so that the molecular tag is released; and

(b) quantifying the amount of released molecular tag, thereby detecting the Herl-Her2 dimers.

479. The method of claim 478, wherein activating the cleavage-inducing moiety cleaves the cleavable linker.

480. The method of claim 478, wherein the binding compound specifically binds a Herl epitope.

481. The method of claim 478, wherein the binding compound comprises an antibody or antigen-binding fragment.

482. The method of claim 478, wherein the binding compound specifically binds a Herl ligand binding site.

483. The method of claim 478, wherein the binding compound comprises a Herl ligand.

484. The method of claim 478, wherein the binding compound specifically binds a Her2 epitope.

485. The method of claim 478, wherein the binding compound specifically binds a Her2 ligand binding site.

486. The method of claim 478, wherein the binding compound comprises a Her2 ligand.

487. The method of claim 478, wherein the cleaving probe specifically binds a Herl epitope.

488. The method of claim 478, wherein the cleaving probe comprises an antibody or antigen-binding fragment.

489. The method of claim 478, wherein the cleaving probe specifically binds a Herl ligand binding site.

490. The method of claim 478, wherein the cleaving probe comprises a Herl ligand.

491. The method of claim 478, wherein the cleaving probe specifically binds a Her2 epitope.

492. The method of claim 478, wherein the cleaving probe specifically binds a Her2 ligand binding site.

493. The method of claim 478, wherein the cleaving probe comprises a Her2 ligand.

494. The method of claim 397, wherein the Herl-Herl dimers, Herl-Her3 dimers, and Her2-Her3 dimers are detected in a single assay.

495. The method of claim 494, wherein a single assay comprises the methods of claims 431, 446, 462, and 478.

496. The method of claim 495, wherein the methods of claims 431 , 446, 462, and 478 are performed concurrently.