WO2010028313A2 - Phosphoprotein analysis of carcinomas for assessment of drug sensitivity - Google Patents

Abstract

This invention relates generally to a treatment of cancer and more particularly to the identification of carcinomas that are likely to respond to an inhibitor of the ERBB family pathway in the carcinoma cells and to the treatment of patients having such carcinomas with such inhibitors. The invention includes, but is not limited to: 1) a method of determining if a carcinoma in a mammal is likely to respond to an ERBB family pathway inhibitor; 2) a method of treating the mammal with an ERBB family pathway inhibitor; 3) a method for identifying such inhibitors; and 4) a kit for use in such assay.

Description

PHOSPHOPROTEIN ANALYSIS OF CARCINOMAS FOR ASSESSMENT OF DRUG SENSITIVITY

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 61/094,282, filed September 4, 2008, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates generally to the treatment of cancer. More particularly, it relates to the identification of carcinomas that are likely to respond to an inhibitor of the Epidermal Growth Factor (EGF) family of receptors (the "ERBB family"), which contains at least four members, ERB1-ERB4, and their pathways (the "ERBB family pathway") in the carcinoma cells and to the treatment of patients having such carcinomas with such inhibitors.

BACKGROUND OF THE INVENTION

Epidermal growth factor receptor (EGFR) is a member of the ERBB family of receptor tyrosine kinases that regulates cellular growth, survival, and proliferation. EGFR has been extensively characterized regarding its kinase activity (9), amino acid sequence (10), receptor abundance (11), autophosphorylation properties (12), substrates (13-15), and mutation sites (16). Over-expression of EGFR in various malignancies (17), together with the identification of specific EGFR mutations that enhance therapeutic response to small molecule inhibitors, notably in lung adenocarcinoma patients, along with the observation that patients without detectable EGFR kinase domain mutations respond to tyrosine kinase inhibitor therapy, qualifies EGFR as a promising molecular endpoint for individualized therapy (18-21). Mutations in the ERBB family of protein receptors are associated with a significant proportion of carcinomas of all types. Mutations in the receptor are thought to be one way in which the ERBB family pathway becomes hyperactive and drives the growth or metastasis of the cancer cells. It would be desirable to have a method for stratifying patients, especially for distinguishing responders from non-responders, to identify a class of subjects who will benefit from therapy targeted to the ERBB family pathway. There is a need to rapidly identify patients who harbor the mutation(s) and are likely to respond, as well as those patients who do not have the mutation(s), but are sensitive to the therapy because they have an active ERBB family pathway driven by a non-mutation mechanism.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows unsupervised hierarchical clustering of LCM-procured non-small cell lung carcinoma cells analyzed by reverse phase protein microarray. Each row (horizontal axis) represents a patient sample listed by known EGFR tyrosine kinase domain mutation status. Each column (vertical axis) represents a cell signaling protein endpoint. The data cluster into two major clusters: Six of eight EGFR mutant samples in one cluster, and 17 wild type samples in the second cluster.

Figure 2 shows supervised hierarchical clustering of analyte subset determined by Wilcoxon Rank Sum analysis. Each row (horizontal axis) represents a patient sample listed by known EGFR mutation status, each column (vertical axis) represents a protein endpoint found to be significantly different (p<0.04, Table 1) between the wild type EGFR and mutated groups. Figure 3 shows discrimination of wild type or mutated EGFR from microdissected NSCLC samples. Human non-small cell lung cancer samples of known EGFR mutation status were microdissected and key EGFR tyrosine phosphorylation residues were quantitated by reverse phase protein microarray. Specific EGFR related protein ratios were evaluated to distinguish NSCLC cells harboring known EGFR mutations from wild type EGFR. Example ratios that were highly significantly different (p<0.001) were Y1045:Y1068 (left panel), and Y1045: the sum of Y1068 and Yl 148 (right panel).

DESCRIPTION OF THE INVENTION

This invention relates to: 1) a method of determining if a carcinoma in a mammal is likely to respond to an ERBB family pathway inhibitor; 2) a method of treating the mammal with an ERBB family pathway inhibitor; 3) an assay for identifying such inhibitors; and 4) a kit for use in the assay. The invention is not limited to these aspects; other aspects are disclosed and claimed herein.

The singular forms "a," "an," and "the" refer to one or more, unless the context clearly indicates otherwise. In one aspect, the invention provides a method of determining if a carcinoma in a mammal is likely to respond to an ERBB family pathway inhibitor. As used herein, the term "ERBB family pathway inhibitor" means an exogenous agent (including a chemical substance, compound, molecule, or ion, such as an antibody, a drug, or a nucleic acid) which suppresses the signaling activity of the ERBB family receptors. As used herein, the term "respond" means clinical stabilization or inhibition of growth of the carcinoma.

The method comprises the steps of: a) analyzing a sample of the carcinoma to determine the ratio of the relative amounts of phosphorylation of any two or more different amino acid residues in the same protein, or in different proteins, in the ERBB family pathway, and b) comparing the ratio of step (a) to a reference ratio range for the same amino acid residues in carcinoma cells from a reference population of patients with the same carcinoma as the carcinoma in the mammal, wherein the reference ratio range is at least 80% specific for detecting carcinoma cells having a mutation in an ERBB family pathway protein and/or having an activated ERBB family pathway, wherein the carcinoma is likely to respond to the inhibitor, if the ratio of step (a) is within the reference ratio range. Samples are obtained by standard techniques, usually by biopsy.

The mammal may be any mammal, including but not limited to, humans and other primates, pets, such as dogs and cats, farm animals, and laboratory animals, such monkeys, rats, mice, rabbits, and guinea pigs.

The carcinoma may be any carcinoma (any malignant neoplasm that arises from an epithelial cell). These include, but are not limited to, glioblastomas, a melanomas, or carcinomas of the lung, breast, ovary, stomach, pancreas, bladder, head, neck, colon, rectum, or kidney. In one aspect of the invention, the carcinomas are those with a high frequency of ERBB abnormalities, such as lung, breast, melanoma, glioblastoma, bladder, head and neck. In a particular aspect, the carcinoma is a lung carcinoma, such as small cell lung carcinoma or non-small cell lung carcinoma (NSCLS). In another particular aspect, the carcinoma is a breast carcinoma.

The carcinoma sample may be analyzed by various techniques known to those skilled in the art. One such technique comprises a) lysing cancerous epithelial cells isolated from the sample; and b) analyzing the lysate to determine the ratio of the relative amounts of phosphorylation of the amino acid residues. For example, the cells may be isolated by microdissection. Various techniques known to those skilled in the art may be used.

One is laser capture microdissection (LCM), as described in U.S. Patent Nos. 6,251,516 and 6,251,467, as well as in U.S. Appl. No. 10/798,799, each of which is hereby incorporated by reference in its entirety. Briefly, LCM allows for isolation of pure populations or subpopulations of the desired cell type, such as a diseased cell population or a normal cell population, or both even from the same tissue sample. The cells of interest can be identified, e.g., morphology, in situ immunohistochemistry, or fluorescent microscopy. By combining microscopy-based cell identification techniques with laser activation of the polymeric substrate to which the tissue sample is applied, very precise extraction of the desired cells is possible. These cells can then be further characterized, such as for protein biomarkers, or lysed for use in the present invention. Such precision allows for extremely accurate characterization of the desired cells. The lysate is analyzed by various assay techniques in order to measure the amount of phosphorylation of the particular amino acid residues. The techniques include immunoassays, enzyme-linked immunosorbent assay (ELISA), colorimetric assays, assays based on fluorescent readouts, histochemical assays, mass spectrometry, and Western blot. Other techniques include mixing the lysate with beads that bind to the proteins or to nanoparticles that trap the proteins and then detecting the proteins with antibodies. These technologies include the Luminex spectral addressable beads/cell sorter system (Austin, TX), Invitrogen's Dyna Beads (Carlsbad, CA), Ceres Nanosciences' Nanotraps (Manassas, VA), and Ambion's MagnaBeads with MALDI-TOF (Austin, TX).

In one aspect of the invention, the lysate is analyzed by reverse phase protein microarray analysis. A protein microarray is an assay format that utilizes a substrate for simultaneously testing multiple samples as well as for testing multiple target proteins in the same assay. The microarray format is not limited to particular embodiments but can comprise any arrangement and substrate that serves to provide a plurality of individual samples for testing. For example, in some embodiments, the microarray comprises a flat substrate with rows and columns of individual spots, each spot comprising a sample, while in other embodiments, the microarray comprises a flat substrate with a plurality of depressions, for example, a 96-well plate, in which each depression contains one sample. Examples of typical microarray substrates include nitrocellulose, derivatized glass slides, and 3- dimensional substrates, such as hydrogels. Examples of nitrocellulose-coated glass slides include FAST slides (Grace BioLabs, Bend, OR or GE Healthcare), which have protein binding capacities of 75-150 ug/cm2 in a volume of 0.3-2.0 nl/spot. Nitrocellulose-coated glass slides are particularly useful, as a variety of detection methods can be used with this substrate, including chromogenic, fluorometric, and luminescent detection methods.

The number of samples that can be deposited onto a microarray substrate can vary. The size of the substrate can often determine how many samples are located on the substrate. In some embodiments, the protein microarray comprises around 100 spots; in other embodiments, the protein microarray may comprise around 1,000 spots or around 10,000 spots. In yet other embodiments, the microarray comprises from about 1 to about 10,000 spots, about 50 to about 10,000 spots, or about 500 to about 10,000 spots. In some embodiments, the microarray comprises less than about 100,000 spots.

The sample volume which is deposited on each spot and used to form each spot on the microarray can also vary. The volume can depend on diameter of the pin (contact printing), the inherent qualities of the pin hydrophobicity, and the method of supplying the sample. In some embodiments, the amount of sample deposited/printed can range from less than about 1 pico liter to about 100 nano liters.

Samples are placed or loaded onto the substrate using any one of a number of mechanisms known in the art (see Schena, "Microarray biochip technology" Eaton Pub., Natick MA, 2000, incorporated herein by reference in its entirety). For example, in some embodiments, the samples are printed onto the microarray using a printer. The printing technique can be contact or non-contact printing, and can be automated.

Protein microarray formats can fall into two major classes, the Forward Phase Array (FPA) and the Reverse Phase Array (RPMA), depending on whether the analyte is capture from solution phase or bound to solid substrate. Forward Phase Arrays immobilize a bait molecule, such as an antibody designed to capture a specific analyte within a mixture of test sample proteins. In FPAs, the capture molecule specific for the analyte is immobilized on a substrate. The capture molecule is then exposed to the sample, binding the analyte in the sample and immobilizing the analyte onto the substrate. The bound analyte can then be detected using a detectable label. The label can bind to the analyte directly, or can be attached to a secondary "sandwich" antibody that is specific for the analyte. The capture molecule can be any molecule that has specificity for an analyte and includes, but is not limited to, peptides, proteins, antibodies or fragments thereof, oligomers, DNA, RNA, and PNA. In some embodiments, the capture molecule is an antibody or fragment thereof specific for the analyte.

Reverse Phase Arrays (RPMAs) immobilize the test sample analytes on a solid substrate. In RPMAs, the sample is placed directly on the substrate, allowing analyte in the sample to bind directly to the substrate. A detection molecule specific for the analyte is then exposed to the substrate, allowing an analyte-detection molecule complex to form. The detection molecule can comprise a detectable label to indicate the presence of the analyte. Alternatively, a secondary molecule specific for the detection molecule and comprising a detectable label can be provided, allowing for an analyte-detection molecule-labeled secondary molecule complex to form.

RPMAs are highly sensitive and do not require a large amount of sample. The high sensitivity exhibited by RPMAs is due in part to the detection molecule, which can be conjugated to a detectable label, and is also due in part to the fact that the signal from the label can be amplified independently from the immobilized analyte. For example, RPMAs can use tryamide amplification which generates high number of florescent signal on each spot, or florescent signals that are near-IR wavelength, which is outside the emission spectra for nitrocellulose. Amplification chemistries that are available take advantage of methods developed for highly sensitive commercial clinical immunoassays (see, for example, King et al., J. Pathol. 183: 237-241 (1997)). Using commercially available automated equipment, RPMAs can also exhibit excellent "within run" and "between run" analytical precision. RPMAs do not require direct labeling of the sample analyte and do not utilize a two-site antibody sandwich. Therefore, there is no experimental variability introduced due to labeling yield, efficiency, or epitope masking.

The reference ratio range is determined by techniques known to those skilled in the art. In one method, samples of carcinoma cells are obtained from a collection or group of patients, each of whom has the same type of carcinoma. Each sample is analyzed to determine if the ERBB family pathway in the cells is activated and if ERBB proteins in the cells have one or more mutations that affect phosphorylation of one or more amino acid residues in the proteins. In those cells determined to have an activated ERBB family pathway and/or to have ERBB proteins that have one or more mutations that affect phosphorylation of one or more amino acid residues in the proteins, the carcinoma cells are analyzed to determine the ratio of the relative amounts of phosphorylation of any two or more different amino acid residues in the same protein, or in different proteins, in the ERBB family pathway. The relative amounts of phosphorylation can be determined by the reverse phase protein microarray techniques disclosed herein. The previous steps are repeated a sufficient number of times with a sufficient number of samples from different patients to create a range of reference ratios for the amino acid residues, wherein the range is at least 80% specific for detecting carcinoma cells having one or more mutations in one or more ERBB family pathway proteins and/or having an activated ERBB family pathway. As used herein, the term "specific" means providing a true positive, not a false positive. The method of determining the reference ratio range is repeated for different amino acid residues and for different types of carcinoma, such as NSCLC and breast carcinoma. As mentioned herein, the phosphorylation of one or more different amino acid residues is measured. The relative amounts of phosphorylation of two different amino acid residues, i.e., a pair, is determined, which provides a ratio. In one aspect of the invention, only one pair of residues is used. However, the determination of relative phosphorylation need not be limited to one pair of amino acid residues on any of the proteins within the ERBB family pathway. Additional pairs may be measured to provide additional ratios. Also, a ratio can be determined by adding the amounts of phosphorylation of two different amino acid residues and comparing the sum to the amount of phosphorylation of a third residue.

In one aspect of the invention, the method is directed to determining if the carcinoma will respond to an EGFR pathway inhibitor. As used herein, the term "EGFR family pathway inhibitor" means an exogenous agent (including a chemical substance, compound, molecule, or ion, such as an antibody, a drug, or a nucleic acid) which suppresses the signaling activity of the EGFR pathway driven by one or more of the ERBB family receptors. The method comprises the steps of: a) analyzing a sample of the carcinoma to determine the ratio of the relative amounts of phosphorylation of any two or more different amino acid residues in EGFR, or one amino acid residue in EGFR and another amino acid residue in different a different protein in the EGFR pathway, such as Her2, and b) comparing the ratio of step (a) to a range of reference ratios for the same amino acid residues in carcinoma cells from a reference population of patients with the same type of carcinoma, wherein the range of reference ratios is at least 80% specific for detecting carcinoma cells having a mutation in EGFR and/or having an activated EGFR pathway. If the ratio of step (a) is within the range of reference ratios, the carcinoma is likely to respond to the inhibitor. In one aspect of the invention, only two amino acid residues are measured, to provide only one ratio. The amino acid residues may be any sites on an ERBB family protein that can be phosphorylated. These include, but are not limited, to Y 1248 in Her2 and S 1026, S 1070, S1071, Sl 190, S695, S991, T678, T693, Y1016, Y1045, Y1068, Y1092, Y1110, Yl 172, Yl 192, Yl 197, Y869, and Y998 in EGFR. In one aspect of the invention, the sites evaluated are the following sites in EGFR: S1026, S1070, S1071, Sl 190, S695, S991, T678, T693, Y1016, Y1045, Y1068, Y1092, Y1110, Yl 172, Yl 192, Yl 197, Y869, and Y998. If the evaluation is limited to selected epitope sites, then the amino acid residues can be selected from the following: Y845, Y1045, Y1068, Yl 148, Yl 173, and L858R, and especially the following pairs: Y1045 and Y1068; Y1045 and Yl 148; Y1045 and Y845; Y1045 and Yl 173; and Yl 045 and L858R. In one particular aspect, the pair is Y 1045 and Y 1068.

In another aspect of the invention, the ratio is calculated by comparing to the relative phosphorylation of one residue to the sum of the relative phosphorylations of two other residues. The following residues are useful: Y1045:(Y1068+Yl 148), L858R:(Y1148+Y1068), and Her2 Y1248:(Y1148+Y1068). These ratio combinations were selected based on statistical significance within the reference population. The individual epitopes were chosen because of their functional role in the coupling of the receptor to downstream signaling pathways or their influence on the kinase activity of the receptor.

An "ERBB family pathway inhibitor" is an exogenous agent (including a chemical substance, compound, molecule, or ion, such as an antibody, a drug, or a nucleic acid) that suppresses the signaling activity of the ERBB family receptors. This includes small molecules that inhibit phosphorylation of one or more amino acid residues in a protein in the ERBB family pathway, large molecules, such as antibodies that bind to a receptor in the ERBB family pathway, and nucleic acid-based inhibitors.

Small molecule inhibitors include kinase inhibitors, especially tyrosine kinase inhibitors. Examples include Erlotinib (Tarceva®), Gefitinib (Iressa®), Lapatinib (Tykerb®), Vandetanib (Zactima™), Cl- 1033 (Pfizer), EKB-569 (Wyeth), GW2016 (GlaxoSmithKline), GW572016 (Glaxo SmithKline), and PHI166 (Novartis). In one aspect of the invention, the inhibitor is Erlotinib (Tarceva®).

Large molecules include proteins, such antibodies. Antibodies include polyclonal and monoclonal antibodies. Monoclonal antibodies include Cetuximab (Erbitux®),

(Panitumumab (Vectibix®), Trastuzumab (Herceptin®), Pertuzumab (Omnitarg®), EMD72000 (Merck), and MDX447 (Medarex/Merck). In one aspect of the invention, the monoclonal antibody is Trastuzumab (Herceptin®). Nucleic acid-based inhibitors include antisense RNA, ribozymes, and interfering RNA (RNAi) molecules, including small interfering RNA (siRNA) molecules. These inhibitors block or degrade mRNA that encodes a protein in the ERBB family pathway or a microRNA (miRNA) molecule that controls expression of one or more of these proteins. In one aspect, the invention provides a method of determining if a person with a non- small cell lung carcinoma will respond to a tyrosine kinase inhibitor. The method comprises a first step of analyzing a sample of the carcinoma to determine the ratio of the relative amounts of phosphorylation of two or more different tyrosine residues in an EGFR protein in the sample, wherein the two tyrosine residues are selected from the group consisting of: Y845, Y1045, Y1068, Yl 148, Yl 173, and L858R, for example the pair Y1045 and Y1068. Cancerous epithelial cells are isolated from a sample of the carcinoma by laser capture microdissection, and the cells are lysed by standard techniques. The lysate is analyzed by placing it onto a reverse phase protein microarray, contacting it with phospho-antibodies to the two tyrosine residues, and determining the ratio of the relative amounts of phosphorylation by measuring the amount of phosphorylation of each of the two residues. In the second step, the ratio determined by the first step is compared to a reference ratio range for the same tyrosine residues in non-small cell lung carcinoma cells from a reference population of persons with non- small cell lung carcinoma. The reference ratio range is at least 80% specific for detecting carcinoma cells having a mutation in EGFR and/or having an activated EGFR pathway. If the ratio from the first step is within the reference ratio range, the carcinoma is likely to respond to the inhibitor. Useful tyrosine kinase inhibitors include Erlotinib (Tarceva®), Gefitinib (Iressa®), and Lapatinib (Tykerb®).

The invention also includes a method of treating a mammal with a carcinoma. The diagnostic method disclosed herein is used to determine if the carcinoma is likely to respond to an ERBB family pathway inhibitor. If it is likely to respond, then a pharmaceutically effective amount of an ERBB family pathway inhibitor is administered to the mammal.

In one aspect, the method comprises the step of administering a pharmaceutically effective amount of an ERBB family pathway inhibitor to the mammal if, in a sample of the mammal's carcinoma, the ratio of the relative amounts of phosphorylation of any two or more different amino acid residues in the same protein, or in different proteins, in the ERBB family pathway is within a reference ratio range for the same amino acid residues in carcinoma cells from a reference population of patients with the same carcinoma as the carcinoma in the mammal. The reference ratio range is at least 80% specific ( 80% probability of a true positive correct determination that a mutation or activated receptor is present) for detecting carcinoma cells having a mutation in an ERBB family pathway protein and/or having an activated ERBB family pathway.

As mentioned above, the carcinoma may be any carcinoma, including but not limited to, glioblastomas, melanomas, or carcinomas of the lung, breast, ovary, stomach, pancreas, bladder, head, neck, colon, rectum, or kidney, or any malignant neoplasm considered for treatment with any of the aforementioned inhibitors. In one aspect of the invention, the carcinoma is a lung carcinoma, such as small cell lung carcinoma and non-small cell lung carcinoma (NSCLS). In another aspect of the invention, the carcinoma is a breast carcinoma. As also mentioned above, the mammal may be any mammal, including but not limited to, humans and other primates, dogs, cats, rats, mice, rabbits, guinea pigs, and farm animals. The principal use of the invention is expected to be to select therapeutic regimens for humans with carcinomas and then treat them with the selected regimen. However, the invention can be used to treat pets and farm animals, and it can be used on laboratory animals to test inhibitors and to find and develop new ones.

As mentioned above, the inhibitors used for treatment include small molecules that inhibit phosphorylation of one or more amino acid residues in a protein in the ERBB family pathway, antibodies that bind to a receptor in the ERBB family pathway, and interfering RNA (RNAi) molecules, including small interfering RNA (siRNA) molecules that block or degrade mRNA that encodes a protein in the ERBB family pathway. Examples of phosphorylation inhibitors include Erlotinib (Tarceva®), Gefitinib (Iressa®), Lapatinib (Tykerb®), Vandetanib (Zactima™), Cl- 1033 (Pfizer), EKB-569 (Wyeth), GW2016 (GlaxoSmithKline), GW572016 (Glaxo SmithKline), and PHI166 (Novartis). Examples of monoclonal antibody inhibitors include Cetuximab (Erbitux®), (Panitumumab (Vectibix®), Trastuzumab (Herceptin®), EMD72000 (Merck), and MDX447 (Medarex/Merck). More than one of these compounds can be administered at the same time or at different times.

Persons skilled in the art will understand that the inhibitors included within the scope of the claimed invention are not limited to the specific chemical compounds and drug products disclosed herein. Any compound, molecule, or other agent that inhibits the ERBB family pathway is included.

In one aspect, the invention provides a method for treating a person with a non-small cell lung carcinoma. The method comprises the step of administering a pharmaceutically effective amount of an EGFR tyrosine kinase inhibitor to the person if, in a sample of the person's carcinoma, the ratio of the relative amounts of phosphorylation of any one or more pairs of two different tyrosine residues in the EGRF protein is within a reference ratio range for the same pairs of amino acid residues in carcinoma cells from a reference population of patients with non-small cell lung carcinoma. The reference ratio range is at least 80% specific for detecting carcinoma cells having a mutation in EGFR and/or having an activated EGFR pathway. The tyrosine residues are selected from the group consisting of: Y845, Y1045, Y1068, Yl 148, Yl 173, and L858R. The residues Y1045 and Y1068 are especially useful. The tyrosine kinase inhibitor is selected from the group consisting of Erlotinib (Tarceva®), Gefϊtinib (Iressa®), and Lapatinib (Tykerb®). The treatment methods of the invention may further include the step of administering to the mammal a pharmaceutically effective amount of an inhibitor that inhibits phosphorylation of a protein that is downstream of EGFR in the EGFR signal transduction pathway. This is a second inhibitor and is administered in addition to the first. It may be administered at approximately the same time as the first or before or after the first. Such inhibitors include tyrosine kinase inhibitors, serine kinase inhibitors, threonine kinase inhibitors, monoclonal antibodies that bind to EGF or EGFR, and nucleic acid molecules that block or degrade mRNA that encodes a protein in the ERBB family pathway or an miRNA molecule that controls expression of one or more of these proteins. The following compounds are examples of inhibitors that may be administered: Erlotinib (Tarceva®), Gefitinib (Iressa®), Lapatinib (Tykerb®), Vandetanib (Zactima™),Cl-1033 (Pfizer), EKB- 569 (Wyeth), GW2016 (Glaxo SmithKline), GW572016 (GlaxoSmithKline), and PHI166 (Novartis), Cetuximab (Erbitux®), (Panitumumab (Vectibix®), Trastuzumab (Herceptin®), EMD72000 (Merck), and MDX447 (Medarex/Merck). More than one of these downstream inhibitors can be administered at the same time. The dosages, routes of administration, and frequency of administration will be known to, or readily determinable by, persons skilled in the art. For FDA approved drugs, such information will be found in the package insert. More than one inhibitor can be administered at the same time or at different times.

In another aspect, the invention includes a method to identify a novel ERBB family pathway inhibitor. The method comprises the steps of: (a) contacting a chemical compound with carcinoma cells, wherein the ratio of the relative amounts of phosphorylation of any two or more different amino acid residues in the same protein, or in different proteins, in the ERBB family pathway in the cells is within a reference ratio range for the same amino acid residues in carcinoma cells from a reference population of patients with the same carcinoma, wherein the reference ratio range is at least 80% specific for detecting carcinoma cells having a mutation in an ERBB family pathway protein and/or having an activated ERBB family pathway ; and (b) determining if a significant number of cancerous cells in the carcinoma have been killed or have had their growth suppressed. In one example, the amino acid residues are in EGFR and the reference ratio range is at least 80% specific for detecting carcinoma cells having an EGFR mutation and/or having an activated EGFR pathway. In an aspect of this example, the inhibitor is a tyrosine kinase inhibitor.

In a further aspect, the invention includes a kit for performing the assay for identifying novel ERBB family pathway inhibitors. The kit comprises the carcinoma cells described herein and a chemical compound to be evaluated. The cells are ones wherein the ratio of the relative amounts of phosphorylation of any two or more different amino acid residues in the same protein, or in different proteins, in the ERBB family pathway in the cells is within a reference ratio range for the same amino acid residues in carcinoma cells from a reference population of patients with the same carcinoma, wherein the reference ratio range is at least 80% specific for detecting carcinoma cells having a mutation in an ERBB family pathway protein and/or having an activated ERBB family pathway. The compound is contacted with the carcinoma cells to determine if a significant number of cancerous cells in the carcinoma are killed by it. The diagnostic and treatment methods of the invention are especially useful in personalized medicine; i.e., the design and implementation of therapeutic regimens for an individual patient. A further application of this approach involves the use of the diagnostic method to determine which of several inhibitors, which could provide a therapeutic benefit, would actually provide the greatest benefit. A sample of the carcinoma is analyzed as described herein to determine the ratio of the relative amounts of phosphorylation of any two or more different amino acid residues in the same protein, or in different proteins, in the ERBB family pathway. The ratio is compared to a reference ratio range for those amino acid residues as described herein. If the ratio is within the reference ratio range, then two or more chemical compounds to be tested are contacted with the carcinoma cells, and the compound that kills or suppresses the growth of the largest number of cancerous cells in the carcinoma is determined. In one example of this aspect, the amino acid residues are in EGFR and the reference ratio range is at least 80% specific for detecting carcinoma cells having an EGFR mutation and/or having an activated EGFR pathway; the ratio of residues is selected from the group consisting of: Y1045 and Y1068; Y1045 and Yl 148; Y1045 and Y845; Y1045 and Yl 173; and Y 1045 and L858R; and the carcinoma is non-small cell lung carcinoma.

The following examples illustrate certain aspects of the invention and should not be construed as limiting the scope thereof.

EXAMPLES

EGFR signaling involves five distinct steps: 1) ligand binding, 2) conformational change in the receptor, 3) homodimerization and/or heterodimerization with other ERBB family receptors, 4) autophosphorylation of tyrosine residues, and 5) transphosphorylation of downstream kinases. Somatic mutations within the kinase domain may affect each of these steps independently or result in a cascade of altered downstream signaling events dependent on the specific receptor mutation.

Very little is known about the state of lung carcinoma EGF kinase pathway signaling within the context of the human lung tissue microenvironment. Each individual patient's carcinoma specimen contains a variable, heterogeneous proportion of stroma, lung parenchyma, bronchial epithelium, inflammatory cells, and endothelial cells. All of these non-carcinoma subpopulations may contain EGF receptors, participate in EGFR signaling, or may contribute EGFR related ligands. Furthermore, it is unknown what proportion of the carcinoma population at any point in time is undergoing active signaling for a specific pathway. Consequently, the state of the EGFR kinase signaling network within the lung carcinoma cells in a tumor specimen cannot be adequately studied using heterogeneous, ground-up tumor tissue, or cultured cell lines (22-24). Laser capture microdissection (LCM) addresses the problem of sample cellular heterogeneity by providing a means to separate tumor cells from the large number of non-tumor cells within the complex microenvironment (22-26).

We quantitatively profiled the phosphorylation and abundance of signal pathway proteins relevant to the EGF receptor within Laser Capture Microdissected (LCM) untreated, human non-small cell carcinomas (NSCLC) (n=25) of known EGFR tyrosine kinase domain mutation status. We used laser capture microdissection to procure pure tumor cell populations from human lung biopsy specimens. Reverse phase protein array quantitation of non-small lung cancer cells was performed to evaluate whether EGFR mutation status in vivo was associated with the coordinated phosphorylation of specific multiple phosphorylation sites on the EGFR, ERBB family receptors, and downstream proteins. Tissue samples were sequenced for exons 18-21 of the EGF receptor (EGFR tyrosine kinase domain) to identify known mutations. To assess the global activity of the EGF receptor, we measured the activation state of six EGFR phosphorylation sites: Y845, Y992, Y1045, Y1068, Yl 148, and Yl 173, as well as total EGFR and Her2 Y 1248. Methods

Tissue samples and Laser Capture Microdissection. Fresh frozen lung adenocarcinoma specimens, stage I/II/III (n=25), and relevant clinical data were obtained the National Institutes of Health, National Cancer Institute, Laboratory of Human Carcinogenesis (38, 39). All patient samples were collected with informed consent, as approved by their respective institutional review boards. An independent board-certified pathologist (LAL) verified the presence of adenocarcinoma tissue prior to laser capture microdissection. Eight μm cryostat sections were sectioned on silanized glass microscope slides (ThermoFisher, Atlanta, GA) or PEN membrane slides (Molecular Devices, Sunnyvale, CA). The frozen sections were stored at -80⁰C prior to staining and microdissection. The frozen section slides were fixed briefly in 70% ethanol, rinsed in water, stained with Mayer's Hematoxylin (Sigma Aldrich, St. Louis, MO), developed in Scott's Tap Water (ThermoFisher) and dehydrated in an ethanol gradient (70%, 95%, 100%) with a final rinse in xylene (Sigma). The sections were allowed to air dry briefly prior to laser capture microdissection. Pure tumor cell populations were microdissected with a PixCell He or Veritas LCM instrument (Molecular Devices). Microdissected cells were stored at -80⁰C prior to microarray construction.

Reverse Phase Protein Microarray construction. The microdissected cells were subjected to lysis with a 2.5% solution of 2-mercaptoethanol (Sigma, St. Louis, MO) in T- PER™ (Pierce, Rockford, IL)/2X SDS Tris-glycine 2X SDS buffer (Invitrogen). Reverse phase protein microarrays were printed in duplicate with whole cell protein lysates as described by Petricoin et al (40). Briefly, the lysates were printed on glass backed nitrocellulose array slides (FAST Slides Whatman, Florham Park, NJ) using a GMS 417 arrayer (Affymetrix, Santa Clara, CA) equipped with 500 μm pins or an Aushon 2470 arrayer equipped with 350 μm pins (Aushon Biosystems, Billerica, MA). Each lysate was printed in a dilution curve representing neat, 1 :2, 1 :4, 1 :8, 1 :16 and negative control dilutions. The slides were stored with desiccant (Drierite, W.A. Hammond, Xenia, OH) at -20⁰C prior to immunostaining. Reverse phase protein microarray immunostaining. Immunostaining was performed on an automated slide stainer per manufacturer's instructions (Autostainer CSA kit, Dako, Carpinteria, CA). Each slide was incubated with a single primary antibody at room temperature for 30 minutes. Each array was probed with a single polyclonal or monoclonal primary antibody. The negative control slide was incubated with antibody diluent. Secondary antibody was goat anti-rabbit IgG H+L (1 :5000) (Vector Labs, Burlingame, CA) or rabbit anti-mouse IgG (1 :10) (Dako). Subsequent protein detection was amplified via horseradish peroxidase mediated biotinyl tyramide with chromogenic detection (Diaminobenzidine) per manufacturer's instructions (Dako). Total protein per microarray spot was determined with Sypro Ruby blot stain (Invitrogen) per manufacturer's directions. Imaging was performed with an Alpha Innotech FluorChem imager (San Leandro, CA).

Antibody validation and phosphoprotein specificity. Primary antibodies were validated prior to use by immunoblotting with complex cellular lysates such as commercial cell lysates or human tissue lysates . Criteria for antibody validation were a) a single band at the correct molecular weight, or b) if two bands were present, 80% of the signal must have been at the correct molecular weight. Specificity of the phosphoprotein- specific antibodies was verified by peptide competition on an immunoblot when the corresponding peptide/antibody pair was available. Specificity criteria were a reduction in signal intensity in the presence of the corresponding peptide compared to the antibody alone. Further specificity for phosphospecific antibodies was verified by peptide/antibody reactivity on a

RPPA. A series of peptides and peptide mixtures were immobilized on a nitrocellulose coated slide. The slide was probed with a single antibody. An antibody was considered specific if the spot signal intensity was not greater than 2 S. D. above background for any peptide other than its corresponding peptide or a mixture of peptides containing the cognate peptide. The antibody was specific if it bound to its cognate peptide or a mixture containing its corresponding peptide.

Statistical analysis. The Ward method for two-way hierarchical clustering was performed using JMP v5.0 (SAS Institute, Cary NC). Spearman's Rho non-parametric analysis was used to compute the likelihood of correlations between endpoints. When data was normally distributed, two-sample t-test was used (SAS ver9.1.3). Wilcoxon rank sum test was used to compare values between two groups if data was not normally distributed (R ver2.6.1, http://www,R-project.org). /? values less than 0.05 were considered significant. Results PCR and sequencing methods for genomic DNA. Tissue samples were sequenced as described previously (38,39) for exons 18-21 (EGFR tyrosine kinase domain). For verification of known EGFR and KRAS mutations in H 1975 and A549 cell lines, total RNA was extracted from both cell lines using RNeasy mini kit (Qiagen) according to manufacturer's instructions. RNA was DNAse treated (DNA- free™, Ambion) and RNA quality was measured by denaturing agarose gel analysis. Reverse transcription with BioRad's iScript cDNA Synthesis Kit was subsequently performed. EGFR exons 17-22 were amplified with forward primer (CCTAAGATCCCGTCCATCG) and reverse primer (AGGCGTTCTCCTTTCTCCAG). Forward primer (AGGCCTGCTGAAAATGACTG) and reverse primer (TCCTGAGCCTGTTTTGTGTCT) were used to amplify exons 1 -4 for K- RAS sequencing. PCR was executed with Platinum® PCR Supermix (Invitrogen) according to manufacturer's recommendations with an annealing temperature of 55°C. Amplicons were purified using QiaQuick PCR purification kit (Qiagen). Sequencing was completed by Northwoods DNA, Inc. using forward primer (CCAACCAAGCTCTCTTGAGG) to determine presence or absence of T790M and EGFR L858R mutations and reverse primer (TGACCTGCTGTGTCGAGAAT) to determine presence or absence of K-RAS mutation.

EGFR mutations are associated with site-specific phosphorylation of EGF receptor in NSCL lung carcinoma cells procured by LCM. To elucidate cell signaling pathways relevant to human lung adenocarcinomas, we used laser capture microdissection to procure pure tumor cell populations from human lung biopsy specimens. An advantage of this study was the collection of snap frozen tumor specimens at the time of primary surgical diagnosis. Therefore, we were not evaluating the effects of treatment on the cellular signaling pathways but rather the state of the tumor at time of procurement in treatment naϊve patients. A total of 31 cases were available for this study. We applied a set of quality assessment criteria to the samples to judge their adequacy for microdissection (22). Criteria were: a) presence of tumor cells, b) sufficient size and quantity of tissue, and c) absence of dehydration or freeze-thaw artifacts. 1 case was judged inadequate due to freezer dehydration artifacts. 4 cases consisted of connective tissue with minimal tumor cells. One case was not analyzed due to insufficient total protein on the microarray. 83% of the cases were judged to be adequate for microdissection and array analysis. 10-15 cryosections were prepared for each case, with 1-2 sections/slide. The average number of laser pulses (shots) per slide was 1149, with an average microdissection efficiency of 95%, resulting in approximately 15,000 - 30,000 cells/sample, collected from multiple cryosections. This was sufficient material for adequate sensitivity and specificity on the reverse phase protein microarray (RPPA) (40, 42-44). Human Endothelial (HE) cell lysates treated with pervanadate were used as a model of phosphorylated VEGF receptor sensitivity and precision. Human endothelial cells are known to express approximately 100,000 VEGF receptors/cell. Sensitivity of the arrays probed with anti- VEGFR Y951 was found to be approximately 3,000 - 4,000 receptor molecules. To determine inter-slide precision, HE cells treated with pervanadate were printed in duplicate on 8 slides and probed with anti-VEGFR Y951. Excellent dose response curves were observed between arrays (CV%: 5.0% to 17.8%, n=8). Within run variation (n=12) was found to be within 2.0% to 18.1% for the HE+pervandate cell lysate with good linearity (R²=0.9693).

Post-translational modifications of EGFR and associated downstream proteins were quantitatively measured using RPPA technology. To assess the global activity of the EGF receptor, we measured the activation state of six EGFR phosphorylation sites: Y845, Y992, Y1045, Y1068, Yl 148, and Yl 173, as well as total EGFR. We used the L858R mutation specific antibody to validate our mutation sequencing results. This antibody recognizes

EGFR with the L858R point mutation ,which is one member of the group of mutations found to correlate with sensitivity to gefitinib therapy. This antibody only stained the H 1975 mutant cell line and the known L858R tissue samples, providing an independent means of cross-checking the array data and mutation analysis (Figures 1-2). Unsupervised hierarchical clustering analysis revealed the presence of two major groups with 6/8 mutants clustered distinctly from the 17 wild type samples (Figure 2). All the mutation cases were associated with higher levels of EGF receptor phosphorylation on residues Yl 148 and Y1068 and lower levels of phosphorylation for Smad/IRS-1/Her2 endpoints. This was confirmed by t-test and Wilcoxon rank sum analysis of the individual analytes (Table 1).

Five of six patient samples possessing either a L858R or deletion exon 19 EGFR mutation had a concomitant unique double phosphorylation of EGFR residues Y 1068 and Yl 148, as well as a reduction in Her2 Y1248, IRS-I S612 and Smad2 S465/467 compared to the wild type. Moreover, the mutant samples exhibited reduced levels of EGFR Y 1045, Y845, and Yl 173 compared to the wild type (Figure 1). Thus, the mutant EGFR as a class appeared different from the wild type with regard to elevated phosphorylation of specific EGFR sites, while the wild type as a class had elevation in Her2 Y1248, IRS-I S612 and Smad2 S465/467. EGFR mutant tissue carcinoma cell populations exhibit distinct pairwise correlations compared to wild type. In order to further understand these correlations, Spearman's rho non-parametric analysis was conducted to examine the strength of the linear relationship between pairs of endpoints among all the endpoints analyzed. Spearman's rho correlation coefficient is computed on the ranks of the data using the formula for the Pearson's correlation. The Pearson product-moment correlation coefficient measures the strength of the linear relationship between two variables. The results are shown in Table 2, which displays those correlations which have a significant p value (p<0.01) and correlation coefficient >0.80. As a class, the mutant carcinomas were found to have a correlation between pairs of phosphorylation sites on EGFR and AKT (Table 2). In contrast the wild type samples lacked correlations of these same two proteins and phosphorylation sites. The wild type samples were scored as having strong correlations between mTOR S2481 and IRS-I S612; Cox2 and IRS-I S612; and EGFR Y992 and 14-3-3 ζγη (zeta/gamma/eta). These correlations were not observed in the mutant. Instead, the mutant samples showed a significant correlation between 11 protein pairs including Src Y527 and She Y317, as well as EGFR Y 1045 and 14- 3-3 ζγη. The point mutation specific antibody, anti-EGFR L858R, did not show any statistically significant differences in our study set of 8 EGFR mutated samples and 17 wild type samples, even though there was clustering of L858R mutant samples by unsupervised hierarchical clustering (Figure 1). This is most likely due to the inclusion of multiple classes of mutations in the mutated group and the specificity of the antibody for the L858R mutation. Although we do not know the extent to which other gene mutations such as p53, K-ras or Rb may influence AKT signaling, the observed signaling profiles in this study set may provide clues indicating the organization of the EGFR related protein network linkages regarding differences between the wild type and mutant adenocarcinoma samples. Comparisons of specific EGFR related protein ratios. Specific EGFR related protein ratios were evaluated to distinguish NSCLC cells harboring known EGFR mutations from wild type EGFR. Monotonic variables or ratios of phosphorylated ERBB family proteins, or total EGFR, were found to be statistically different in the EGFR mutated cohort compared to the wild type EGFR group. Ratios were calculated by: a) determining the mean of each group (mutant versus wild type) for specific protein endpoints (i.e. Y1045), or b) calculating a ratio of the monotonic variable to the sum of specific endpoints (i.e. Y1068+Y1148). See Table 3. Table 1. Differentially activated proteins in microdissected NSCLC samples as a comparison of EGFR tyrosine kinase domain mutation status (wild type versus mutant).

Mean of Mean of

EGFR Wild

SubMutation Type cellular P value cohort cohort location Protein Cellular Function (p<0.05) (n=8) (n=17)

Membrane EGFR Prosurvival 0.231 0.479 0.384

EGFR L858R Prosurvival 0.75 0.141 0.033

EGFR Y845 0.021 0.125 0.222

Prosurvival

EGFR Y992 Prosurvival 0.210 0.219 0.199

EGR Y1045 0.002 0.176 0.625

Prosurvival

EGFR Y1068 Prosurvival 0.026 0.299 0.093

EGFR Y1148 Prosurvival 0.044 0.686 0.512

EGFR Y1173 Prosurvival 0.030 0.525 0.681

HER2 Y1248 Prosurvival 0.015 0.199 0.596

Cytoplasm 14-3-3 ζvη Signal transduction 0.341 0.465 0.563

AKT S473 Prosurvival 0.083 0.364 0.562

AKT T308 Prosurvival 0.071 0.502 0.663

APC2 Cell Cycle 0.646 0.757 0.728

BUB3 Mitotic checkpoint 0.157 0.667 0.756

BCL-2 S70 Anti-apoptotic 0.239 0.434 0.556

ERK T202/Y204 Growth/Differentiation 0.919 0.561 0.549

COX2 Inflammation 0.136 0.657 0.503

Cyclin D1 Cell Cycle 0.382 0.528 0.446

Cyclin E Cell Cycle 0.193 0.609 0.501

FOX01 (FKHR) Cell survival/Cell 0.693 0.633 0.597

T24 Cycle

IRS-1 S612 Insulin <0.001 0.170 0.546 signaling/Growth

MTOR S2481 Growth/Protein 0.068 0.674 0.493 translation

SHC Y317 Scaffold protein for 0.138 0.358 0.151 receptor tyrosine kinases

SRC Y416 Growth/Differentiation 0.345 0.355 0.498

(upregulation)

SRC Y527 Growth/Differentiation 0.075 0.610 0.450

(down regulation)

Nucleus STAT3 S727 Transcription control 0.324 0.633 0.545

SMAD2 Transcription 0.011 0.409 0.634

S465/467 control Table 2. Spearman's rho non-parametric correlations for microdissected human NSCLC samples.

EGFR Mutant Phenotype EGFR Wild Type Phenotype

Protein Protein Spearman Profc»Rho Protein Protein Spearman Profc»Rho endpoint endpoint Rho (p value) endpoint endpoint Rho (p value)

AKT AKT

0.9762 MTOR IRS-1

0.00003 0.8676 0.000006 T308 S473 S2481 S612

EGFR

COX2 0.9762 IRS-1 0.00003

Y1148 COX2 0.8578 S612 0.00001

EGFR

COX2 0.9322 0.0007 14-3-3 EGFR

0.8162 0.00006

Y845 ζyη Y992

EGFR EGFR Y992 0.8838 0.004

Y1045

EGFR

EGFR 0.8809 0.004

Y1148

MTOR

APC2 0.8809 0.004 S2481 SRC SHC 0.8809 0.004 Y527 Y317 EGFR EGFR 0.8684 0.005 Y845 Y1148 14-3-3 EGFR 0.8625 0.006 ζyη Y1045

MTOR

EGFR 0.8571 0.006

S2481

EGFR C0X2 0.8095 0.015

Table 3 : EGFR Mutation vs Wild Type

Mean of EGFR Mean of

Mutation Wild Type Statistical

Protein Ratio p Value (N=8) (N=17) Result

EGFR:Y1068+Y1148* 0.1272 0.4030 0.1964 Not Different

Statistically

Y1045:Y1068 3.9691e-05 0.3496 29.9051 Different

Statistically

Y1045:Y1148 0.0002 0.2675 1.2418 Different

Statistically

Y1045:Y1068+Y1148 8.1546e-05 0.1789 1.0360 Different

Statistically

Y1045:Y845 0.0005 1.0338 5.4119 Different

Statistically

Y1045:Y1173 1.7044e-05 0.2590 0.9242 Different

Y1148:EGFR 0.2621 1.5142 1.4209 Not Different

Maybe

Y1068:EGFR 0.0784 0.6125 0.2669 Different

Statistically

Y1045:EGFR 0.0004 0.3895 1.7058 Different

Y992:EGFR 0.4397 0.4734 0.5722 Not Different

Statistically

Y845:EGFR 0.0181 0.2216 0.6208 Different

Maybe

L858R:Y1148+Y1068 0.0883 0.1524 0.0573 Different

Statistically

Her2Y1248:Yl 148+Yl 068 0.0077 0.3197 0.9823 Different

Statistically

Y1173:EGFR 0.0008 1.1542 1.8777 Different

Statistically

Y1045:L858R 2.50174e-05 2.6975 19.7959 Different

*The ratio is indicated by a colon :

The sum of the relative intensity of two endpoints is indicated by a plus sign

Conclusions

Reverse phase protein array quantitation of NSCLC revealed simultaneous increased phosphorylation of EGFR residues Yl 148(p<0.044) and Y1068(p<0.026), and decreased phosphorylation of EGFR Y1045(p<0.002), Her2 Y1248(p<0.015), IRS-I S612(p<0.001), and Smad S465/467(p<0.011), across all classes of mutated EGFR patient samples compared to wild type. These data strongly support the concepts that: a) EGF receptor mutation status, known to correlate with sensitivity to EGF pathway inhibitors, may be identified via quantitative comparison of at least two phosphorylated epitopes on the EGF receptor, or comparison of phosphorylated epitopes to total EGFR, b) specific ratios of protein endpoints can distinguish cells with EGFR mutation from wild type, and c) patients exhibiting greater phosphorylation of total EGFR, Y1068, Yl 173, and/or Yl 148 compared to Y1045 may benefit from tyrosine kinase inhibitor therapy regardless of EGFR mutation status.

These findings have relevance to the individualized therapy of lung cancer with drugs that inhibit the EGFR pathway. An elevated level of phosphorylation of one or more of these residues in proteins of associated downstream pathways, compared to a baseline value, may indicate that the patient is likely to be responsive to a) EGFR therapy (e.g. to treatment with an agent that inhibits the kinase activity of EGFR), b) a kinase inhibitor directed towards an associated downstream pathway, or c) a combination of an EGFR inhibitor and an inhibitor of a member of one of the identified downstream pathways. This is because activity of the downstream proteins indicates that the entire EGF associated pathway is active and in use in the cancer cells. Since the entire pathway is active, this may indicate that the cancer cell is more likely being driven by this pathway, and thus would be effectively treated by blocking multiple nodes along this pathway.

Moreover, the measured ratio(s) of EGFR phosphoproteins, as well as individual or pairs of phosphoprotein data, may be used to design individualized therapy regimens for single or combination agents. This therapy may be a) EGFR therapy (e.g. to treatment with an agent that inhibits the kinase activity of EGFR), b) a kinase inhibitor directed towards an associated downstream pathway, or c) a combination of an EGFR inhibitor and an inhibitor of a member of one of the identified downstream pathways. In addition this information may be used to predict potential response to tyrosine kinase inhibitor therapy.

REFERENCES

1. Jain, M., Arvanitis, C, Chu, K., Dewey, W., Leonhardt, E., Trinh, M., Sundberg, C. D., Bishop, J. M., Felsher, D. W. (2002) Sustained loss of a neoplastic phenotype by brief inactivation of MYC. Science. 297, 102-4 2. Weinstein, I. B. (2000) Disorders in cell circuitry during multistage carcinogenesis: the role of homeostasis. Carcinogenesis. 21, 857-64 3. Boockvar, J. A., Kapitonov, D., Kapoor, G., Schouten, J., Counelis, G. J., Bogler, O., Snyder, E. Y., Mclntosh, T. K., O'Rourke, D. M. (2003) Constitutive EGFR signaling confers a motile phenotype to neural stem cells. MoI Cell Neurosci. 24, 1116-30

4. Ayuso-Sacido, A., Graham, C, Greenfield, J. P., Boockvar, J. A. (2006) The duality of epidermal growth factor receptor (EGFR) signaling and neural stem cell phenotype: cell enhancer or cell transformer? Curr Stem Cell Res Ther. 1, 387-94

5. Haugh, J. M., Huang, A. C, Wiley, H. S., Wells, A., Lauffenburger, D. A. (1999) Internalized epidermal growth factor receptors participate in the activation of p21(ras) in fibroblasts. J Biol Chem. 21 A, 34350-60 6. Jaramillo, M. L., Banville, M., Collins, C, Paul-Roc, B., Bourget, L., O'Connor McCourt, M. (2008) Differential Sensitivity of A549 Non Small Lung Carcinoma Cell Responses to Epidermal Growth Factor Receptor Pathway Inhibitors. Cancer Biol Ther. 7, 557-68

7. Mattoon, D. R., Lamothe, B., Lax, L, Schlessinger, J. (2004) The docking protein Gabl is the primary mediator of EGF-stimulated activation of the PI-3K/Akt cell survival pathway. BMC Biol. 2, 24

8. Riedel, R. F., Febbo, P. G. (2005) Epidermal growth factor receptor mutations predict sensitivity to gefϊtinib in patients with non-small-cell lung cancer. Future Oncol. 1, 461-6

9. Cohen, S. (1983) Purification of the receptor for epidermal growth factor from A-431 cells: its function as a tyrosyl kinase. Methods Enzymol. 99, 379-87

10. Ullrich, A., Coussens, L., Hayflick, J. S., Dull, T. J., Gray, A., Tarn, A. W., Lee, J., Yarden, Y., Libermann, T. A., Schlessinger, J., et al. (1984) Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. Nature. 309, 418-25 11. Dittadi, R., Gion, M., Brazzale, A., Bruscagnin, G. (1990) Radioligand binding assay of epidermal growth factor receptor: causes of variability and standardization of the assay. Clin Chem. 36, 849-54

12. Weber, W., Bertics, P. J., Gill, G. N. (1984) Immunoaffmity purification of the epidermal growth factor receptor. Stoichiometry of binding and kinetics of self- phosphorylation. J Biol Chem. 259, 14631-6

13. Haigler, H. T., Schlaepfer, D. D., Burgess, W. H. (1987) Characterization of lipocortin I and an immunologically unrelated 33-kDa protein as epidermal growth factor receptor/kinase substrates and phospholipase A2 inhibitors. J Biol Chem. 262, 6921-30 14. Citri, A., Yarden, Y. (2006) EGF-ERBB signalling: towards the systems level. Nat Rev MoI Cell Biol 7, 505-16

15. Burgess, A. W., Cho, H. S., Eigenbrot, C, Ferguson, K. M., Garrett, T. P., Leahy, D. J., Lemmon, M. A., Sliwkowski, M. X., Ward, C. W., Yokoyama, S. (2003) An open-and- shut case? Recent insights into the activation of EGF/ErbB receptors. MoI Cell. 12, 541-52

16. Sharma, S. V., Bell, D. W., Settleman, J., Haber, D. A. (2007) Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer. 7, 169-81

17. Hwang, D. L., Tay, Y. C, Lin, S. S., Lev-Ran, A. (1986) Expression of epidermal growth factor receptors in human lung tumors. Cancer. 58, 2260-3 18. Lynch, T. J., Bell, D. W., Sordella, R., Gurubhagavatula, S., Okimoto, R. A.,

Brannigan, B. W., Harris, P. L., Haserlat, S. M., Supko, J. G., Haluska, F. G., Louis, D. N., Christiani, D. C, Settleman, J., Haber, D. A. (2004) Activating mutations in the epidermal growth factor receptor underlying responsiveness of non- small-cell lung cancer to gefitinib. N EnglJMed. 350, 2129-39 19. Paez, J. G., Janne, P. A., Lee, J. C, Tracy, S., Greulich, H., Gabriel, S., Herman, P., Kaye, F. J., Lindeman, N., Boggon, T. J., Naoki, K., Sasaki, H., Fujii, Y., Eck, M. J., Sellers, W. R., Johnson, B. E., Meyerson, M. (2004) EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science. 304, 1497-500

20. Sordella, R., Bell, D. W., Haber, D. A., Settleman, J. (2004) Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways. Science. 305, 1163-7

21. Takano, T., Ohe, Y., Sakamoto, H., Tsuta, K., Matsuno, Y., Tateishi, U., Yamamoto, S., Nokihara, H., Yamamoto, N., Sekine, L, Kunitoh, H., Shibata, T., Sakiyama, T., Yoshida, T., Tamura, T. (2005) Epidermal growth factor receptor gene mutations and increased copy numbers predict gefitinib sensitivity in patients with recurrent non-small-cell lung cancer. J Clin Oncol. 23, 6829-37

22. Espina, V., Wulfkuhle, J. D., Calvert, V. S., VanMeter, A., Zhou, W., Coukos, G., Geho, D. H., Petricoin, E. F., 3rd, Liotta, L. A. (2006) Laser-capture microdissection. Nat Protoc. 1, 586-603

23. Espina, V., Heiby, M., Pierobon, M., Liotta, L. A. (2007) Laser capture microdissection technology. Expert Rev MoI Diagn. 7, 647-57

24. Wulfkuhle, J. D., Speer, R., Pierobon, M., Laird, J., Espina, V., Deng, J., Mammano, E., Yang, S. X., Swain, S. M., Nitti, D., Esserman, L. J., Belluco, C, Liotta, L. A., Petricoin, E. F., 3rd (2008) Multiplexed Cell Signaling Analysis of Human Breast Cancer Applications for Personalized Therapy. J Pro teome Res. 7, 1508-1517

25. Emmert-Buck, M. R., Bonner, R. F., Smith, P. D., Chuaqui, R. F., Zhuang, Z., Goldstein, S. R., Weiss, R. A., Liotta, L. A. (1996) Laser capture microdissection. Science. 274, 998-1001

26. Bonner, R. F., Emmert-Buck, M., Cole, K., Pohida, T., Chuaqui, R., Goldstein, S., Liotta, L. A. (1997) Laser capture microdissection: molecular analysis of tissue. Science. 278, 1481,1483

27. Morgillo, F., Woo, J. K., Kim, E. S., Hong, W. K., Lee, H. Y. (2006) Heterodimerization of insulin-like growth factor receptor/epidermal growth factor receptor and induction of survivin expression counteract the antitumor action of erlotinib. Cancer Res. 66, 10100-11

28. Ciardiello, F., Troiani, T., Bianco, R., Orditura, M., Morgillo, F., Martinelli, E., Morelli, M. P., Cascone, T., Tortora, G. (2006) Interaction between the epidermal growth factor receptor (EGFR) and the vascular endothelial growth factor (VEGF) pathways: a rational approach for multi-target anticancer therapy. Ann Oncol. 17 Suppl 7, viilO9-14

29. Kobayashi, S., Shimamura, T., Monti, S., Steidl, U., Hetherington, C. J., Lowell, A. M., Golub, T., Meyerson, M., Tenen, D. G., Shapiro, G. L, Halmos, B. (2006) Transcriptional profiling identifies cyclin Dl as a critical downstream effector of mutant epidermal growth factor receptor signaling. Cancer Res. 66, 11389-98

30. Liu, X., Carlisle, D. L., Swick, M. C, Gaither-Davis, A., Grandis, J. R., Siegfried, J. M. (2007) Gastrin-releasing peptide activates Akt through the epidermal growth factor receptor pathway and abrogates the effect of gefitinib. Exp Cell Res. 313, 1361-72.

31. Pawson, T., Gish, G. D., Nash, P., (2001) SH2 domains, interaction modules and cellular wiring. Trends Cell Biol. 11 , 504-11

32. Schlessinger, J. (2004) Common and distinct elements in cellular signaling via EGF and FGF receptors. Science. 306, 1506-7

33. Gilmer, T. M., Cable, L., Alligood, K., Rusnak, D., Spehar, G., Gallagher, K. T., Woldu, E., Carter, H. L., Truesdale, A. T., Shewchuk, L., Wood, E. R. (2008) Impact of common epidermal growth factor receptor and HER2 variants on receptor activity and inhibition by lapatinib. Cancer Res. 68, 571-9

34. Janne, P. A., Borras, A. M., Kuang, Y., Rogers, A. M., Joshi, V. A., Liyanage, H., Lindeman, N., Lee, J. C, Halmos, B., Maher, E. A., Distel, R. J., Meyerson, M., Johnson, B. E. (2006) A rapid and sensitive enzymatic method for epidermal growth factor receptor mutation screening. Clin Cancer Res. Yl, 751-8

35. Jackman, D. M., Yeap, B. Y., Sequist, L. V., Lindeman, N., Holmes, A. J., Joshi, V. A., Bell, D. W., Huberman, M. S., Halmos, B., Rabin, M. S., Haber, D. A., Lynch, T. J., Meyerson, M., Johnson, B. E., Janne, P. A. (2006) Exon 19 deletion mutations of epidermal growth factor receptor are associated with prolonged survival in non-small cell lung cancer patients treated with gefitinib or erlotinib. Clin Cancer Res. 12, 3908-14

36. Uchida, A., Hirano, S., Kitao, H., Ogino, A., Rai, K., Toyooka, S., Takigawa, N., Tabata, M., Takata, M., Kiura, K., Tanimoto, M. (2007) Activation of downstream epidermal growth factor receptor (EGFR) signaling provides gefϊtinib-resistance in cells carrying EGFR mutation. Cancer Sci. 98, 357-63

37. Okabe, T., Okamoto, L, Tamura, K., Terashima, M., Yoshida, T., Satoh, T., Takada, M., Fukuoka, M., Nakagawa, K. (2007) Differential constitutive activation of the epidermal growth factor receptor in non-small cell lung cancer cells bearing EGFR gene mutation and amplification. Cancer Res. 67, 2046-53

38. Yang, S. H., Mechanic, L. E., Yang, P., Landi, M. T., Bowman, E. D., Wampfler, J., Meerzaman, D., Hong, K. M., Mann, F., Dracheva, T., Fukuoka, J., Travis, W., Caporaso, N. E., Harris, C. C, Jen, J. (2005) Mutations in the tyrosine kinase domain of the epidermal growth factor receptor in non-small cell lung cancer. Clin Cancer Res. 11, 2106-10 39. Yang, S. H., Mechanic, L. E., Yang, P., Landi, M. T., Bowman, E. D., Wampfler, J., Meerzaman, D., Hong, K. M., Mann, F., Dracheva, T., Fukuoka, J., Travis, W., Caporaso, N. E., Harris, C. C, Jen, J. (2005) Mutations in the tyrosine kinase domain of the epidermal growht factor receptor in non-small cell lung cancer. Clin Cancer Res. 11, 7960-7961

40. Petricoin, E. F., 3rd, Espina, V., Araujo, R. P., Midura, B., Yeung, C, Wan, X., Eichler, G. S., Johann, D. J., Jr., Qualman, S., Tsokos, M., Krishnan, K., Helman, L. J.,

Liotta, L. A. (2007) Phosphoprotein pathway mapping: Akt/mammalian target of rapamycin activation is negatively associated with childhood rhabdomyosarcoma survival. Cancer Res. 67, 3431-40

41. Shigematsu, H., Lin, L., Takahashi, T., Nomura, M., Suzuki, M., Wistuba, II, Fong, K. M., Lee, H., Toyooka, S., Shimizu, N., Fujisawa, T., Feng, Z., Roth, J. A., Herz, J., Minna,

J. D., Gazdar, A. F. (2005) Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J Natl Cancer Inst. 97, 339-46 42. Gulmann, C, Espina, V., Petricoin, E., 3rd, Longo, D. L., Santi, M., Knutsen, T., Raffeld, M., Jaffe, E. S., Liotta, L. A., Feldman, A. L. (2005) Proteomic analysis of apoptotic pathways reveals prognostic factors in follicular lymphoma. Clin Cancer Res. 11, 5847-55

43. Paweletz, C. P., Charboneau, L., Bichsel, V. E., Simone, N. L., Chen, T., Gillespie, J. W., Emmert-Buck, M. R., Roth, M. J., Petricoin, I. E., Liotta, L. A. (2001) Reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front. Oncogene. 20, 1981-9

44. Rapkiewicz, A., Espina, V., Zujewski, J. A., Lebowitz, P. F., Filie, A., Wulfkuhle, J., Camphausen, K., Petricoin, E. F., 3rd, Liotta, L. A., Abati, A. (2007) The needle in the haystack: application of breast fine -needle aspirate samples to quantitative protein microarray technology. Cancer. I l l, 173-84

45. Brognard, J., Clark, A. S., Ni, Y., Dennis, P. A. (2001) Akt/protein kinase B is constitutively active in non-small cell lung cancer cells and promotes cellular survival and resistance to chemotherapy and radiation. Cancer Res. 61, 3986-97 46. Wells, A. (1999) EGF receptor. IntJBiochem Cell Biol. 31, 637-43

47. Sawyers, C. L. (2008) The cancer biomarker problem. Nature. 452, 548-52

48. Zhang, J., Kalyankrishna, S., Wislez, M., Thilaganathan, N., Saigal, B., Wei, W., Ma, L., Wistuba, II, Johnson, F. M., Kurie, J. M. (2007) SRC-family kinases are activated in non- small cell lung cancer and promote the survival of epidermal growth factor receptor- dependent cell lines. Am J Pathol. 170, 366-76

49. Fu, H., Xia, K., Pallas, D. C, Cui, C, Conroy, K., Narsimhan, R. P., Mamon, H., Collier, R. J., Roberts, T. M. (1994) Interaction of the protein kinase Raf-1 with 14-3-3 proteins. Science. 266, 126-9

50. Engelman, J. A., Zejnullahu, K., Mitsudomi, T., Song, Y., Hyland, C, Park, J. O., Lindeman, N., Gale, C. M., Zhao, X., Christensen, J., Kosaka, T., Holmes, A. J., Rogers, A.

M., Cappuzzo, F., Mok, T., Lee, C, Johnson, B. E., Cantley, L. C, Janne, P. A. (2007) MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science. 316, 1039-43

51. Uramoto, H., Mitsudomi, T. (2007) Which biomarker predicts benefit from EGFR- TKI treatment for patients with lung cancer? Br J Cancer. 96, 857-63

52. Apgar, J. F., Toettcher, J. E., Endy, D., White, F. M., Tidor, B. (2008) Stimulus design for model selection and validation in cell signaling. PLoS Comput Biol. 4, e30 53. Araujo, R. P., Liotta, L. A. (2006) A control theoretic paradigm for cell signaling networks: a simple complexity for a sensitive robustness. Curr Opin Chem Biol. 10, 81-7

54. Wu, S. L., Kim, J., Bandle, R. W., Liotta, L., Petricoin, E., Karger, B. L. (2006) Dynamic profiling of the post-translational modifications and interaction partners of epidermal growth factor receptor signaling after stimulation by epidermal growth factor using Extended Range Proteomic Analysis (ERPA). MoI Cell Proteomics. 5, 1610-27

55. Oksvold, M. P., Thien, C. B., Widerberg, J., Chantry, A., Huitfeldt, H. S., Langdon, W. Y. (2003) Serine mutations that abrogate ligand-induced ubiquitination and internalization of the EGF receptor do not affect c-Cbl association with the receptor. Oncogene. 22, 8509-18 56. Hynes, N. E., Lane, H. A. (2005) ERBB receptors and cancer: the complexity of targeted inhibitors. Nat Rev Cancer. 5, 341-54

57. Pao, W., Miller, V., Zakowski, M., Doherty, J., Politi, K., Sarkaria, L, Singh, B., Heelan, R., Rusch, V., Fulton, L., Mardis, E., Kupfer, D., Wilson, R., Kris, M., Varmus, H. (2004) EGF receptor gene mutations are common in lung cancers from "never smokers" and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci USA. 101, 13306-11

58. Yarden, Y., Sliwkowski, M. X. (2001) Untangling the ErbB signalling network. Nat Rev MoI Cell Biol. 2, 127-37

59. Pawson, T. (2004) Specificity in signal transduction: from phosphotyrosine-SH2 domain interactions to complex cellular systems. Cell. 116, 191-203

60. Pawson, T., Nash, P. (2003) Assembly of cell regulatory systems through protein interaction domains. Science. 300, 445-52

61. Olayioye, M. A., Neve, R. M., Lane, H. A., Hynes, N. E. (2000) The ErbB signaling network: receptor heterodimerization in development and cancer. Embo J. 19, 3159-67 62. Rikova, K., Guo, A., Zeng, Q., Possemato, A., Yu, J., Haack, H., Nardone, J., Lee, K., Reeves, C, Li, Y., Hu, Y., Tan, Z., Stokes, M., Sullivan, L., Mitchell, J., Wetzel, R., Macneill, J., Ren, J. M., Yuan, J., Bakalarski, C. E., Villen, J., Kornhauser, J. M., Smith, B., Li, D., Zhou, X., Gygi, S. P., Gu, T. L., Polakiewicz, R. D., Rush, J., Comb, M. J. (2007) Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell. 131, 1190-203

63. Han, W., Zhang, T., Yu, H., Foulke, J. G., Tang, C. K. (2006) Hypophosphorylation of residue Y 1045 leads to defective downregulation of EGFRvIII. Cancer Biol Ther. 5, 1361- 8 64. Huang, F., Goh, L. K., Sorkin, A. (2007) EGF receptor ubiquitination is not necessary for its internalization. Proc Natl Acad Sd USA. 104, 16904-9

65. Padron, D., Sato, M., Shay, J. W., Gazdar, A. F., Minna, J. D., Roth, M. G. (2007) Epidermal growth factor receptors with tyrosine kinase domain mutations exhibit reduced CbI association, poor ubiquitylation, and down-regulation but are efficiently internalized. Cancer Res. 67, 7695-702

66. Oksvold, M. P., Huitfeldt, H. S., Langdon, W. Y. (2004) Identification of 14-3-3zeta as an EGF receptor interacting protein. FEBS Lett. 569, 207-10

67. Pallas, D. C, Fu, H., Haehnel, L. C, Weller, W., Collier, R. J., Roberts, T. M. (1994) Association of polyoma virus middle tumor antigen with 14-3-3 proteins. Science. 265, 535-7

68. Rittinger, K., Budman, J., Xu, J., Volinia, S., Cantley, L. C, Smerdon, S. J., Gamblin, S. J., Yaffe, M. B. (1999) Structural analysis of 14-3-3 phosphopeptide complexes identifies a dual role for the nuclear export signal of 14-3-3 in ligand binding. MoI Cell. 4, 153-66

69. Yaffe, M. B., Rittinger, K., Volinia, S., Caron, P. R., Aitken, A., Leffers, H., Gamblin, S. J., Smerdon, S. J., Cantley, L. C. (1997) The structural basis for 14-3-

3 :phosphopeptide binding specificity. Cell. 91, 961-71

70. Theroux, S. J., Taglienti-Sian, C, Nair, N., Countaway, J. L., Robinson, H. L., Davis, R. J. (1992) Increased oncogenic potential of ErbB is associated with the loss of a COOH- terminal domain serine phosphorylation site. J Biol Chem. 267, 7967-70 71. Countaway, J. L., McQuilkin, P., Girones, N., Davis, R. J. (1990) Multisite phosphorylation of the epidermal growth factor receptor. Use of site-directed mutagenesis to examine the role of serine/threonine phosphorylation. J Biol Chem. 265, 3407-16 72. Akhurst, R. J. (2002) TGF-beta antagonists: why suppress a tumor suppressor? J Clin Invest. 109, 1533-6 73. Derynck, R., Akhurst, R. J., Balmain, A. (2001) TGF-beta signaling in tumor suppression and cancer progression. Nat Genet. 29, 117-29

74. Wakefield, L. M., Roberts, A. B. (2002) TGF-beta signaling: positive and negative effects on tumorigenesis. Curr Opin Genet Dev. 12, 22-9

75. Hida, T., Yatabe, Y., Achiwa, H., Muramatsu, H., Kozaki, K., Nakamura, S., Ogawa, M., Mitsudomi, T., Sugiura, T., Takahashi, T. (1998) Increased expression of cyclooxygenase

2 occurs frequently in human lung cancers, specifically in adenocarcinomas. Cancer Res. 58, 3761-4 76. Hida, T., Kozaki, K., Muramatsu, H., Masuda, A., Shimizu, S., Mitsudomi, T., Sugiura, T., Ogawa, M., Takahashi, T. (2000) Cyclooxygenase-2 inhibitor induces apoptosis and enhances cytotoxicity of various anticancer agents in non- small cell lung cancer cell lines. Clin Cancer Res. 6, 2006-11 77. Huang, M., Stolina, M., Sharma, S., Mao, J. T., Zhu, L., Miller, P. W., Wollman, J., Herschman, H., Dubinett, S. M. (1998) Non-small cell lung cancer cyclooxygenase-2- dependent regulation of cytokine balance in lymphocytes and macrophages: up-regulation of interleukin 10 and down-regulation of interleukin 12 production. Cancer Res. 58, 1208-16

78. Winters, M. E., Mehta, A. L, Petricoin, E. F., 3rd, Kohn, E. C, Liotta, L. A. (2005) Supra-additive growth inhibition by a celecoxib analogue and carboxyamido-triazole is primarily mediated through apoptosis. Cancer Res. 65, 3853-60

79. Gual, P., Le Marchand-Brustel, Y., Tanti, J. F. (2005) Positive and negative regulation of insulin signaling through IRS-I phosphorylation. Biochimie. 87, 99-109

80. Cosaceanu, D., Carapancea, M., Alexandru, O., Budiu, R., Martinsson, H. S., Starborg, M., Vrabete, M., Kanter, L., Lewensohn, R., Dricu, A. (2007) Comparison of three approaches for inhibiting insulin-like growth factor I receptor and their effects on NSCLC cell lines in vitro. Growth Factors. 25, 1-8

81. Han, C. H., Cho, J. Y., Moon, J. T., Kim, H. J., Kim, S. K., Shin, D. H., Chang, J., Ahn, C. M., Chang, Y. S. (2006) Clinical significance of insulin receptor substrate-I down- regulation in non-small cell lung cancer. Oncol Rep. 16, 1205-10

82. Tremblay, F., Marette, A. (2001) Amino acid and insulin signaling via the mTOR/p70 S6 kinase pathway. A negative feedback mechanism leading to insulin resistance in skeletal muscle cells. J Biol Chem. 276, 38052-60

83. Wan, X., Harkavy, B., Shen, N., Grohar, P., Helman, L. J. (2007) Rapamycin induces feedback activation of Akt signaling through an IGF-lR-dependent mechanism. Oncogene.

26, 1932-40

84. Oliveira, J. C, Souza, K. K., Dias, M. M., Faria, M. C, Ropelle, E. R., Flores, M. B., Ueno, M., Velloso, L. A., Saad, S. T., Saad, M. J., Carvalheira, J. B. (2008) Antineoplastic effect of rapamycin is potentiated by inhibition of IRS-I signaling in prostate cancer cells xenografts. J Cancer Res Clin Oncol

85. Perez-Torres, M., Guix, M., Gonzalez, A., Arteaga, C. L. (2006) Epidermal growth factor receptor (EGFR) antibody down-regulates mutant receptors and inhibits tumors expressing EGFR mutations. J Biol Chem. 281, 40183-92 86. Blons, H., Cote, J. F., Le Corre, D., Riquet, M., Fabre-Guilevin, E., Laurent-Puig, P., Danel, C. (2006) Epidermal growth factor receptor mutation in lung cancer are linked to bronchioloalveolar differentiation. Am J Surg Pathol. 30, 1309-15

87. Pugh et al., Correlations of EGFR mutations and increases in EGFR and HER2 copy number to gefϊtinib response in a retrospective analysis of lung cancer patients. BMC

Cancer. 7, 128 (2007)

88. Bjorling, E., and Uhlen, M. (2008) Antibodypedia - a portal for sharing antibody and antigen validation data. MoI Cell Proteomics. July 29, (paper in press)

All publications, including issued patents and published applications, and all database entries identified by url addresses or accession numbers are incorporated herein by reference in their entirety.

Although this invention has been described in relation to certain embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.

Claims

WHAT IS CLAIMED IS:

1. A method of determining if a carcinoma in a mammal is likely to respond to an ERBB family pathway inhibitor comprising the steps of: a) analyzing a sample of the carcinoma to determine the ratio of the relative amounts of phosphorylation of any two or more different amino acid residues in the same protein, or in different proteins, in the ERBB family pathway, and b) comparing the ratio of step (a) to a reference ratio range for the same amino acid residues in carcinoma cells from a reference population of patients with the same carcinoma as the carcinoma in the mammal, wherein the reference ratio range is at least 80% specific for detecting carcinoma cells having a mutation in an ERBB family pathway protein and/or having an activated ERBB family pathway, wherein the carcinoma is likely to respond to the inhibitor if the ratio of step (a) is within the reference ratio range.

2. The method of claim 1 wherein the mammal is a mouse or a rat.

3. The method of claim 1 wherein the mammal is a human.

4. The method of claim 3 wherein the inhibitor is selected from the group consisting of a small molecule that inhibits phosphorylation of one or more amino acid residues in a protein in the ERBB family pathway, an antibody that binds to a receptor in the ERBB family pathway, and a nucleic acid that blocks or degrades mRNA that encodes a protein in the ERBB family pathway or a microRNA (miRNA) molecule that controls expression of one or more of the proteins.

5. The method of claim 4 wherein the inhibitor is an EGFR pathway inhibitor and the reference ratio range is at least 80% specific for detecting carcinoma cells having an EGFR mutation and/or having an activated EGFR pathway.

6. The method of claim 5 wherein only two different amino acid residues are analyzed.

7. The method of claim 5 wherein the inhibitor is a small molecule.

8. The method of claim 7 wherein the small molecule inhibitor is a kinase inhibitor.

9. The method of claim 8 wherein the kinase inhibitor is a tyrosine kinase inhibitor.

10. The method of claim 5 wherein the inhibitor is an antibody.

11. The method of claim 10 wherein the antibody is a monoclonal antibody.

12. The method of claim 5 wherein the inhibitor is a nucleic acid.

13. The method of claim 12 wherein the nucleic acid is an siRNA molecule.

14. The method of claim 8 wherein the kinase inhibitor is selected from the group consisting of Erlotinib (Tarceva®), Gefitinib (Iressa®), Lapatinib (Tykerb®), Vandetanib (Zactima™), Cl- 1033 (Pfizer), EKB-569 (Wyeth), GW2016 (Glaxo SmithKline), GW572016 (GlaxoSmithKline), and PHI 166 (Novartis).

15. The method of claim 14 wherein the kinase inhibitor is Erlotinib (Tarceva®).

16. The method of claim 10 wherein the antibody inhibitor is selected from the group consisting of Cetuximab (Erbitux®), (Panitumumab (Vectibix®), Trastuzumab (Herceptin®), Pertuzumab (Omnitarg®), EMD72000 (Merck), and MDX447 (Medarex/Merck).

17. The method of claim 16 wherein the antibody is Trastuzumab (Herceptin®).

18. The method of claim 5 wherein the amino acid residues are selected from the group consisting of S1026, S1070, S1071, Sl 190, S695, S991, T678, T693, Y1016, Y1045, Y1068, Y1092, Yl 110, Yl 172, Yl 192, Yl 197, Y869, and Y998 in EGFR and Y1248 in Her2.

19. The method of claim 18 wherein the amino acid residues are selected from the group consisting of S1026, S1070, S1071, Sl 190, S695, S991, T678, T693, Y1016, Y1045, Y1068, Y1092, Yl 110, Yl 172, Yl 192, Yl 197, Y869, and Y998 in EGFR.

20. The method of claim 18 wherein the amino acid residues are selected from the group consisting of: Y845, Y1045, Y1068, Yl 148, Yl 173, L858R, in EGFR and Y1248 in Her2.

21. The method of claim 20 wherein the amino acid residues are selected from the group consisting of the following: Y1045 and Y1068; Y1045 and Yl 148; Y1045 and Y845; Y1045 and Yl 173; Y 1045 and L858R; Y 1045 and (Y1068+Y1148); L858R and (Yl 148 + Y 1068); and Her2Y1248 and (Yl 148 + Y1068).

22. The method of claim 21 wherein the ratios of the relative amounts of phosphorylation of one of the following amino acid residue pairs are determined: Y1045 and Y1068; Y1045 and Yl 148; Y1045 and Y845; Y1045 and Yl 173; and Y1045 and L858R.

23. The method of claim 22 wherein the pair of amino acid residues is Y 1045 and Y 1068.

24. The method of any one of claims 1-23 wherein the carcinoma is a glioblastoma, a melanoma, or a carcinoma of the lung, breast, ovary, stomach, pancreas, bladder, head, neck, colon, rectum, or kidney.

25. The method of claim 24 wherein the carcinoma has a high frequency of ERBB abnormalities.

26. The method of claim 25 wherein the carcinoma is a glioblastoma, a melanoma, or a carcinoma of the lung, breast, bladder, head, or neck.

27. The method of claim 24 wherein the carcinoma is a lung carcinoma.

28. The method of claim 27 wherein the lung carcinoma is non-small cell lung carcinoma.

29. The method of claim 24 wherein the carcinoma is a breast carcinoma.

30. The method of claim 24 wherein the step of analyzing a sample comprises the steps of: a) lysing cancerous epithelial cells isolated from the sample; and b) analyzing the lysate to determine the ratio of the relative amounts of phosphorylation of any two or more different amino acid residues in the same protein, or in different proteins, in the ERBB family pathway in the lysate.

31. The method of claim 30, wherein the cancerous epithelial cells are isolated by microdissection.

32. The method of claim 31 , wherein the microdissection comprises laser capture microdissection.

33. The method of claim 30, wherein the step of analyzing the lysate comprises using an assay selected from the group consisting of immunoassays, enzyme-linked immunosorbent assay (ELISA), colorimetric assays, assays based on fluorescent readouts, histochemical assays, mass spectrometry, and Western blot.

34. The method of claim 33, wherein the lysate is placed onto a reverse phase protein microarray and then analyzed by an immunoassay.

35. The method of claim 34, wherein the immunoassay comprises contacting the lysate on the microarray with phospho-antibodies to at least two different tyrosine residues in an EGFR protein.

36. The method of claim 24 wherein the reference ratio range is determined by: a) obtaining samples of carcinoma cells from a collection of patients, each of whom has the same type of carcinoma; b) for each sample, determining if the ERBB family pathway in the cells is activated and/or if ERBB proteins in the cells of each sample have one or more mutations that affect phosphorylation of one or more amino acid residues in the proteins; c) in those cells determined by step (b) to have an activated ERBB family pathway and/or to have ERBB proteins that have one or more mutations that affect phosphorylation of one or more amino acid residues in the proteins, analyzing the carcinoma cells to determine the ratio of the relative amounts of phosphorylation of any two or more different amino acid residues in the same protein, or in different proteins, in the ERBB family pathway; and d) repeating steps (b) and (c) a sufficient number of times with a sufficient number of samples from different patients to create a range of reference ratios for the amino acid residues, wherein the range is at least 80% specific for detecting carcinoma cells having one or more mutations in one or more ERBB family pathway proteins and/or having an activated ERBB family pathway.

37. The method of claim 36 wherein only two different amino acid residues are analyzed.

38. The method of claim 37 comprising repeating steps (a) - (d) for a different pair of amino acid residues.

39. The method of claim 38 comprising repeating steps (a) - (d) for a different type of carcinoma.

40. The method of claim 39 wherein the inhibitor is an EGFR pathway inhibitor, the amino acid residues are in EGFR, and the reference ratio range is at least 80% specific for detecting carcinoma cells having an EGFR mutation and/or having an activated EGFR pathway.

41. The method of claim 40 wherein the carcinoma is a non-small cell lung carcinoma.

42. The method of claim 40 wherein the carcinoma is a breast carcinoma.

43. A method of determining if a person with a non-small cell lung carcinoma will likely respond to a tyrosine kinase inhibitor comprising the steps of: a) analyzing a sample of the carcinoma to determine the ratio of the relative amounts of phosphorylation of two different tyrosine residues in an EGFR protein in the sample, wherein the two tyrosine residues are selected from the group consisting of: Y845, Y 1045, Y1068, Yl 148, Yl 173, and L858R; and b) comparing the ratio of step (a) to a reference ratio range for the same two tyrosine residues in non-small cell lung carcinoma cells from a reference population of persons with non-small cell lung carcinoma, wherein the reference ratio range is at least 80% specific for detecting carcinoma cells having a mutation in EGFR and/or having an activated EGFR pathway, wherein the carcinoma is likely to respond to the inhibitor if the ratio of step (a) is within the reference ratio range.

44. The method of claim 43 wherein the step of analyzing comprises lysing cancerous epithelial cells isolated from the sample by laser capture microdissection and analyzing the lysate to determine the ratio of the relative amounts of phosphorylation of the two tyrosine residues.

45. The method of claim 44 wherein the lysate is analyzed by placing the lysate onto a reverse phase protein microarray, contacting it with phospho-antibodies to the residues, and determining the ratio of the relative amounts of phosphorylation.

46. The method of claim 45 wherein the two different tyrosine residues are Y 1045 and Y1068.

47. The method of any one of claims 43-46 wherein the tyrosine kinase inhibitor is selected from the group consisting of Erlotinib (Tarceva®), Gefitinib (Iressa®), and Lapatinib (Tykerb®).

48. The method of claim 47 wherein the tyrosine kinase inhibitor is Erlotinib (Tarceva®).

49. A method of treating a mammal with a carcinoma comprising the step of: administering a pharmaceutically effective amount of an ERBB family pathway inhibitor to the mammal if, in a sample of the mammal's carcinoma, the ratio of the relative amounts of phosphorylation of any two or more different amino acid residues in the same protein, or in different proteins, in the ERBB family pathway is within a reference ratio range for the same amino acid residues in carcinoma cells from a reference population of patients with the same carcinoma as the carcinoma in the mammal, wherein the reference ratio range is at least 80% specific for detecting carcinoma cells having a mutation in an ERBB family pathway protein and/or having an activated ERBB family pathway.

50. The method of claim 49 wherein the mammal is a human.

51. The method of claim 50 wherein the inhibitor is an EGFR pathway inhibitor, the amino acid residues are in EGFR, and the reference ratio range is at least 80% specific for detecting carcinoma cells having an EGFR mutation and/or having an activated EGFR pathway.

52. The method of claim 51 wherein the inhibitor is a kinase inhibitor.

53. The method of claim 51 wherein the inhibitor is a monoclonal antibody.

54. The method of claim 52 wherein the kinase inhibitor is selected from the group consisting of Erlotinib (Tarceva®), Gefitinib (Iressa®), Lapatinib (Tykerb®), Vandetanib (Zactima™), Cl- 1033 (Pfizer), EKB-569 (Wyeth), GW2016 (Glaxo SmithKline), GW572016 (GlaxoSmithKline), and PHI 166 (Novartis).

55. The method of claim 54 wherein the kinase inhibitor is Erlotinib (Tarceva®).

56. The method of claim 51 wherein the inhibitor is an antibody selected from the group consisting of Cetuximab (Erbitux®), (Panitumumab (Vectibix®), Trastuzumab (Herceptin®), Pertuzumab (Omnitarg®), EMD72000 (Merck), and MDX447 (Medarex/Merck).

57. The method of claim 56 wherein the antibody is Trastuzumab (Herceptin®).

58. The method of claim 51 wherein the amino acid residues are selected from the group consisting of S1026, S1070, S1071, Sl 190, S695, S991, T678, T693, Y1016, Y1045, Y1068, Y1092, Yl 110, Yl 172, Yl 192, Yl 197, Y869, Y998 in EGFR.

59. The method of claim 58 wherein the amino acid residues are selected from the group consisting of: Y845, Y1045, Y1068, Yl 148, Yl 173, and L858R.

60. The method of claim 59 wherein the amino acid residues are selected from the group consisting of: Y1045 and Y1068; Y1045 and Yl 148; Y1045 and Y845; Y1045 and Yl 173; and Y1045 and L858R; Y1045 and (Y1068+Y1148); and L858R and (Yl 148 + Y1068).

61. The method of claim 60 wherein the ratios of the relative amounts of phosphorylation of one of the following amino acid residue pairs are determined: Y1045 and Y1068; Y1045 and Yl 148; Y1045 and Y845; Y1045 and Yl 173; and Y1045 and L858R.

62. The method of claim 61 wherein the pair of amino acid residues is Y1045 and Y1068.

63. The method of any one of claims 49-62 wherein the carcinoma is a non-small cell lung cancer.

64. The method of any one of claims 49-62 wherein the carcinoma is a breast carcinoma.

65. The method of any one of claims 49-62 wherein the step of analyzing a sample comprises: a) lysing cancerous epithelial cells isolated from the sample; and b) analyzing the lysate to determine the ratio of the relative amounts of phosphorylation of any two different amino acid residues in the same protein, or in different proteins, in the ERBB family pathway in the lysate.

66. The method of claim 65, wherein the cancerous epithelial cells are isolated by laser capture microdissection.

67. The method of claim 66, wherein the lysate is placed onto a reverse phase microarray and then analyzed by an immunoassay.

68. The method of claim 67, wherein the immunoassay comprises contacting the lysate on the microarray with phospho-antibodies to at least two different tyrosine residues in an EGFR protein.

69. The method of any one of claims 49-62 wherein the reference ratio range is determined by the steps of: a) obtaining samples of carcinoma cells from a collection of patients, each of whom has the same type of carcinoma; b) for each sample, determining if the ERBB family pathway in the cells is activated and if ERBB proteins in the cells of each sample have one or more mutations that affect phosphorylation of one or more amino acid residues in the proteins; c) in those cells determined by step (b) to have an activated ERBB family pathway and/or to have ERBB proteins that have one or more mutations that affect phosphorylation of one or more amino acid residues in the proteins, analyzing the carcinoma cells to determine the ratio of the relative amounts of phosphorylation of any two or more different amino acid residues in the same protein, or in different proteins, in the ERBB family pathway; and d) repeating steps (b) and (c) a sufficient number of times with a sufficient number of samples from different patients to create a range of reference ratios for the amino acid residues, wherein the range is at least 80% specific for detecting carcinoma cells having one or more mutations in one or more ERBB family pathway proteins and/or having an activated ERBB family pathway.

70. The method of claim 69 wherein only two different amino acid residues are analyzed.

71. The method of claim 70 comprising repeating steps (a) - (d) for a different pair of amino acid residues.

72. The method of claim 71 comprising repeating steps (a) - (d) for a different type of carcinoma.

73. The method of claim 72 wherein the inhibitor is an EGFR pathway inhibitor, the amino acid residues are in EGFR, and the reference ratio range is at least 80% specific for detecting carcinoma cells having an EGFR mutation and/or having an activated EGFR pathway.

74. The method of claim 73 wherein the carcinoma is a non- small cell lung carcinoma.

75. The method of claim 73 wherein the carcinoma is a breast carcinoma.

76. The method of claim 49 further comprising the step of: administering to the patient a pharmaceutically effective amount of a compound that inhibits phosphorylation of a protein that is downstream of EGFR in the EGFR signal transduction pathway.

77. The method of claim 76 wherein the compound that inhibits phosphorylation of a protein that is downstream of EGFR in the EGFR signal transduction pathway is selected from the group consisting of tyrosine kinase inhibitors, serine kinase inhibitors, threonine kinase inhibitors and monoclonal antibodies.

78. The method of claim 76 or 77 wherein the compound is selected from the group consisting of Erlotinib (Tarceva®), Gefitinib (Iressa®), Lapatinib (Tykerb®), Vandetanib (Zactima™), Cl- 1033 (Pfizer), EKB-569 (Wyeth), GW2016 (Glaxo SmithKline), GW572016 (Glaxo SmithKline), and PHI 166 (Novartis), Cetuximab (Erbitux®), (Panitumumab (Vectibix®), Trastuzumab (Herceptin®), Pertuzumab (Omnitarg®), EMD72000 (Merck), and MDX447 (Medarex/Merck).

79. The method of claim 78 wherein the compound is Erlotinib (Tarceva®) or Trastuzumab (Herceptin®).

80. A method of treating a person with a non-small cell lung carcinoma comprising the step of : administering a pharmaceutically effective amount of an EGFR tyrosine kinase inhibitor to the person if, in a sample of the person's carcinoma, the ratio of the relative amounts of phosphorylation of any two different amino acid residues in the EGRF protein is within reference ratio range for the same two amino acid residues in carcinoma cells from a reference population of patients with non- small cell lung carcinoma, wherein the reference ratio range is at least 80% specific for detecting carcinoma cells having a mutation in EGFR and/or having an activated EGFR pathway.

81. The method of claim 80 wherein the two tyrosine residues are selected from the group consisting of: Y845, Y1045, Y1068, Yl 148, Yl 173, and L858R.

82. The method of claim 80 wherein the two different tyrosine residues are Y1045 and

Y1068.

83. The method of any one of claims 80, 81 , or 82 wherein the tyrosine kinase inhibitor is selected from the group consisting of Erlotinib (Tarceva®), Gefitinib (Iressa®), and

Lapatinib (Tykerb®).

84. The method of claim 83 wherein the tyrosine kinase inhibitor is Erlotinib (Tarceva®).

85. A method to identify an ERBB family pathway inhibitor comprising the steps of: (a) contacting a chemical compound with carcinoma cells, wherein the ratio of the relative amounts of phosphorylation of any two or more different amino acid residues in the same protein, or in different proteins, in the ERBB family pathway in the cells is within a reference ratio range for the same amino acid residues in carcinoma cells from a reference population of patients with the same carcinoma, wherein the reference ratio range is at least

80% specific for detecting carcinoma cells having a mutation in an ERBB family pathway protein and/or having an activated ERBB family pathway; and (b) determining if a significant number of cancerous cells in the carcinoma have been suppressed in their growth rate or die.

86. The method of claim 85 wherein the amino acid residues are in EGFR and the reference ratio range is at least 80% specific for detecting carcinoma cells having an EGFR mutation and/or having an activated EGFR pathway.

87. The method of claim 86 wherein the inhibitor is a tyrosine kinase inhibitor.

88. The method of claim 86 wherein the ratio of residues is selected from the group consisting of: Y1045, Y1068; Y1045, Yl 148; Y1045, Y845; Y1045, Yl 173; and Y1045, L858R.

89. The method of claim 88 wherein the residues are Y1045 and Y1068.

90. The method of any one of claims 85-89 wherein the carcinoma is a non-small cell lung carcinoma.

91. The method of any one of claims 85-89 wherein the carcinoma is a breast carcinoma.

92. A kit comprising: carcinoma cells, wherein the ratio of the relative amounts of phosphorylation of any two or more different amino acid residues in the same protein, or in different proteins, in the ERBB family pathway in the cells is within a reference ratio range for the same amino acid residues in carcinoma cells from a reference population of patients with the same carcinoma, wherein the reference ratio range is at least 80% specific for detecting carcinoma cells having a mutation in an ERBB family pathway protein and/or having an activated ERBB family pathway ; and a chemical compound to be contacted with the carcinoma cells to determine if a significant number of cancerous cells in the carcinoma are suppressed in their growth rate or are killed by the compound.

93. The kit of claim 92 wherein wherein the amino acid residues are in EGFR and the reference ratio range is at least 80% specific for detecting carcinoma cells having an EGFR mutation and/or having an activated EGFR pathway.

94. The kit of claim 93 wherein the inhibitor is a tyrosine kinase inhibitor.

95. The kit of claim 93 wherein the ratio of residues is selected from the group consisting of: Y1045, Y1068; Y1045, Yl 148; Y1045, Y845; Y1045, Yl 173; and Y1045, L858R.

96. The kit of claim 93 wherein the pair of residues is Y1045 and Y1068.

97. A method to identify an ERBB family pathway inhibitor, from among several such inhibitors, that will best treat a patient's carcinoma comprising the steps of: a) analyzing a sample of the carcinoma to determine the ratio of the relative amounts of phosphorylation of any two or more different amino acid residues in the same protein, or in different proteins, in the ERBB family pathway; b) comparing the ratio of step (a) to a reference ratio range for the same amino acid residues in carcinoma cells from a reference population of patients with the same carcinoma as the carcinoma in the mammal, wherein the reference ratio range is at least 80% specific for detecting carcinoma cells having a mutation in an ERBB family pathway protein and/or having an activated ERBB family pathway; c) if the ratio of step (a) is within the reference ratio range, contacting two or more inhibitors to be tested with the carcinoma cells; and d) determining which inhibitor kills or inhibits the growth rate of the largest number of cancerous cells in the carcinoma.

98. The method of claim 97 wherein the amino acid residues are in EGFR and the reference ratio range is at least 80% specific for detecting carcinoma cells having an EGFR mutation and/or having an activated EGFR pathway.

99. The method of claim 98 wherein the inhibitor is a tyrosine kinase inhibitor.

100. The method of claim 98 wherein the ratio of residues is selected from the group consisting of: Y1045, Y1068; Y1045, Yl 148; Y1045, Y845; Y1045, Yl 173; and Y1045, L858R.

101. The method of claim 98 wherein the pair of residues is Y 1045 and Y 1068.

102. The method of any one of claims 97-101 wherein the carcinoma is a non-small cell lung carcinoma.

103. The method of any one of claims 97- 101 wherein the carcinoma is a breast carcinoma.