WO2008097229A1

WO2008097229A1 - Method for spectroscopic quantitation of her-2 in biological samples

Info

Publication number: WO2008097229A1
Application number: PCT/US2007/003478
Authority: WO
Inventors: Thomas P. Conrads; Timothy D. Veenstra; Brian L. Hood; David B. Krizman; Marlene M. Darfler
Original assignee: The Government Of The Usa As Represented By The Secretary Of The Dept. Of Health And Human Services
Priority date: 2007-02-09
Filing date: 2007-02-09
Publication date: 2008-08-14

Abstract

Disclosed are methods for the detection of HER-2 in a biological sample using mass spectrometry. Specifically, these methods detect a cysteine-free HER-2 fragment peptide in the biological sample. The presence of HER-2 in a biological sample can be used to determine if a subject has a HER-2 associated cancer. The presence of HER-2 associated cancer can be used to determine if a subject would benefit from a HER-2 targeted therapy, such as an anti-HER-2 antibody. In one aspect, these methods can be used to quantify HER-2 in a biological sample via mass spectrometry by using an optionally isotopically labeled peptide standard.

Description

METHOD FOR SPECTROSCOPIC QUANTITATION OF HER-2 IN BIOLOGICAL SAMPLES

FIELD This disclosure relates, to the field of diagnostic testing. More specifically, this disclosure relates to methods of detecting and determining certain characteristics of cancer using mass spectrometric detection of HER-2 peptides. This disclosure also relates to peptide standards and their use in quantitative mass spectrometric analyses.

BACKGROUND

Cancer is the second leading cause of death in the United States, only exceeded by heart disease. Cancer is characterized as an abnormal state in which uncontrolled proliferation of one or more cell populations interferes with normal biological functioning. The transformation of a normal cell into a cancer cell can result from amplification in the number of copies of a proto-oncogene, which in turn can result in overproduction of the protein and its concomitant effects. Amplified proto-oncogenes have been found in subjects with cancer.

The proto-oncogene encoding the human epidermal growth factor receptor-2 (HER- 2 or ErbB2) has been found to be amplified in a range of tumor types including colon, prostate, stomach, thyroid, ovarian, bladder, salivary gland, endometrial, pancreatic, renal, and non-small-cell lung cancer (NSCLC). The presence of overexpressed HER-2 has also been implicated in disease initiation and progression, and is associated with poor prognosis.

HER-2 is a transmembrane protein of approximately 185 kilodaltons and is a member of the ErbB family of receptor tyrosine kinases. This family of receptor tyrosine kinases are important mediators of cell growth, differentiation, and survival. It is believed that HER-2 overexpression leads to tumor growth via ligand-independent activation of the HER-2 intracellular kinase domain.

Several anti-HER-2 monoclonal antibodies (MAbs) have been produced to specifically antagonize the function of the HER-2 receptor in HER-2-positive tumors. The most notable of these anti-HER-2 antibodies is the recombinant humanized version of the murine anti-HER-2 antibody 4D5 (trastuzumab, huMAb4D5-8, rhuMAb HER-2, and the subject of U.S. Patent. Nos. 5,821,337 and 5,720,954, the specifications of which are incorporated herein by reference in their entirety) marketed under the trade name HERCEPTIN™. Trastuzumab has been shown to be clinically active in patients with HER- 2 overexpressing metastatic breast cancers that have received extensive prior anti-cancer therapy. Preclinical data suggest that anti-HER-2 MAbs, such as trastuzumab, suppress the proliferation of ovarian, gastric and NSCLC cell lines that overexpress the HER-2 receptor. The prevalence of HER-2 overexpression and/or gene amplification in various tumor types raises the possibility of using anti-HER-2 MAbs to antagonize the abnormal function of overexpressed HER-2 receptors in HER-2-positive tumors other than breast. Trastuzumab offers tremendous therapeutic benefits to cancer subjects diagnosed with HER-2 positive tumors. Various assays for HER-2 positive cancer have been developed and include fluorescent in situ hybridization (FISH), which can reveal gene amplification, immunohistochemistry (IHC), which detects the amount of expressed HER-2 protein, and tests for serum levels of HER-2, which detect the amount of circulating HER-2 extracellular domain. However, the diagnosis of HER-2 positive cancers with these methods is non-ideal. FISH is a good method for detecting HER-2, but is technically difficult to implement. IHC is relatively easy to perform, but is inherently difficult to standardize. Finally, serum HER-2 tests measure circulating levels of the shed extracellular domain of HER-2, not gene amplification or overexpression on the surface of tumor cells, and therefore may not be a true measure of the amount of cellular HER-2. Due to the difficulties in the current methods used to diagnosis HER-2 positive cancers, there is a need for improved methods of detecting HER-2 in biological samples, especially histopathologically processed tumor tissue.

SUMMARY

Disclosed herein are methods for detecting HER-2 in a biological sample. The disclosed methods are useful in determining if a subject has HER-2 associated cancer. In one embodiment, the disclosed methods include obtaining a biological sample from a subject, digesting the biological sample with a protein cleavage agent (such as a serine protease, for example trypsin), optionally chromatographing the sample, and detecting a cysteine-free HER-2 fragment peptide in the protein digest. The presence of a cysteine-free HER-2 fragment peptide in the protein digest indicates the presence of HER-2 associated cancer. In some embodiments, the cysteine-free HER-2 fragment peptide is detected by mass spectrometry. In some embodiments, the cysteine-free HER-2 fragment peptide is detected by the detection of fragment ions of the cysteine-free HER-2 fragment peptide, for example using tandem mass spectrometry. Cysteine residues are relatively chemically reactive and can undergo oxidation prior to detection by mass spectrometry leading to peptides of varying masses. For example, cysteine residues readily undergo side reactions such as disulfide formation that results in products having differing masses. Thus, cysteine- free HER-2 fragment peptides are particularly useful for determining the presence of a HER-2 in a sample by mass spectrometry.

Also disclosed are methods of quantitating the amount of the cysteine-free HER-2 fragment peptide in a biological sample. In one aspect, such methods include comparing an amount of the cysteine-free HER-2 fragment peptide to an optionally isotopically labeled cysteine-free peptide standard of known amount. Peptide standards for use in quantitating HER-2 in a biological sample are also disclosed. Such peptide standards contain a cysteine- free peptide corresponding to about 8 to about 45 amino acid residues of HER-2 and are optionally isotopically labeled. In one embodiment, the disclosed methods can be used to determine the progression of the cancer, for example by correlating the detected amount of the cysteine-free HER-2 fragment peptide to progression of the cancer. In addition, the disclosed methods can be used to select a treatment based the presence or absence of the cysteine-free HER-2 fragment peptide in the protein digest. By way of example, the presence of a cysteine-free HER-2 fragment peptide in the protein digest indicates that the subject can be treated with anti-HER-2 therapy, such as an anti-HER-2 antibody therapy.

The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a set of four representative mass spectra obtained from a tryptic digest of HER-2.

Fig. 2 is a representative tandem mass spectrum showing the fragment ions of the HER-2 fragment peptide of interest (SGGDLTLGLEPS EEEAPR, SEQ ID NO: 14) at m/z 914.4, 1043.5, and 1213.6.

Fig. 3 is a set of four representative mass spectra obtained from a tryptic digest of HER-2 showing that the HER-2 fragment peptide SGGDLTLGLEPSEEEAPR (SEQ ID NO: 14) can be accurately measured without ambiguity using a combination of parent and/or fragment ions.

Fig. 4 is a set of three representative mass spectra of a tryptic digest of a sample obtained from a subject's tumor biopsy after LC/MS/MS analysts.

Fig. 5 is a representative tandem mass spectrum of a tryptic digest of a sample obtained from a subject's tumor biopsy showing the fragment ions from HER-2 fragment peptide SGGDLTLGLEPSEEEAPR (SEQ ID NO: 14). Fig 6 is a set of three tandem mass spectra showing the intact HER-2 fragment peptide SGGDLTLGLEPSEEEAPR (SEQ ID NO: 14) and fragment ions of the HER-2 fragment peptide SGGDLTLGLEPSEEEAPR (SEQ ID NO.14).

SEQUENCE LISTING

The amino acid sequences listed in the accompanying sequence listing are shown using standard three letter abbreviations for amino acids, as defined in 37 C.F.R. § 1.822.

SEQ ID NOs: 1—18 are exemplary amino acid sequences corresponding to portions of the amino acid sequence of HER-2. SEQ ID NOs: 19-20 are exemplary amino acid sequences of human HER-2.

DETAILED DESCRIPTION I. Abbreviations

AMU: atomic mass unit CAD/CAM: computer-aided design/computer-aided machining

CCD: charge-coupled device

CE: capillary electrophoresis

CID: collision induced dissociation

EI: electron-impact ionization ESI: electrospray ionization

FFPE: formalin-fixed paraffin-embedded

FISH: fluorescent in situ hybridization

FT-ICR: Fourier-transform ion cyclotron resonance

HER-2 or ErbB2: Human epidermal growth factor receptor 2 HPLC: high performance liquid chromatography

IHC: immunohistochemistry

IMPS: ion mobility spectrometer

IR: infrared

LC: liquid chromatography LIT: linear ion trap

LT: LIQUID TISSUE™

MAbs: monoclonal antibodies

MALDI: matrix-assisted laser desorption-ionization

MARS: multiple affinity removal system MS/MS: tandem mass spectrometry nano-RPLC: nano-reversed-phase liquid chromatography NSCLC: non-small-cell lung cancer PAGE: poly acrylamide gel electrophoresis Q: quadrupole mass analyzer

RP-HPLC: reverse phase high performance liquid chromatography SELDI: surface-enhanced laser desorption-ionization TOF: time-of-flight

SILAC: stable isotope labeling by amino acids in cell culture SRM: selected reaction monitoring

II. Explanation of Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al.

(eds.), The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471 186341); and George P. Redei, Encyclopedic Dictionary of Genetics, Genomics, and Proteomics, 2nd Edition, 2003 (ISBN: 0-471 -26821 -6).

As used herein, the singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Also, as used herein, the term "comprises" means "includes." Hence "comprising A or B" means including A, B, or A and B. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described below. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

To facilitate review of the various embodiments of the invention, the following explanations of specific terms are provided: Administering: Administering refers to the introduction of a composition into a subject by a chosen route. For example, if the chosen route is intravenous, the composition is administered by introducing the composition into a vein of the subject.

Biological sample: Any solid or fluid sample obtained from, excreted by or secreted by any living organism, including without limitation, multicellular organisms

(animals, including samples from a healthy or apparently healthy human subject or a human patient affected by a condition or disease to be diagnosed or investigated, such as cancer). For example, a biological sample can be a biological fluid obtained from, for example, blood, plasma, serum, urine, bile, ascites, saliva, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion, a transudate, an exudate (for example, fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (for example, a normal joint or a joint affected by disease such as a rheumatoid arthritis, osteoarthritis, gout or septic arthritis). A biological sample can also be a sample obtained from any organ or tissue (including a biopsy or autopsy specimen, such as a tumor biopsy) or can comprise a cell (whether a primary cell or cultured cell) or medium conditioned by any cell, tissue or organ. A biological sample can also be a sample which has been chemically treated, for example a sample which is, fixed (such as fixed in formalin or any other chemical fixative known in the art) and/or paraffin embedded.

Chromatographing: The process of separating a mixture. It involves passing a mixture through a stationary phase, which separates molecules of interest from other molecules in the mixture and allows it to be isolated. Examples of methods of chromatographic separation include capillary-action chromatography such as paper chromatography, thin layer chromatography (TLC), column chromatography, fast protein liquid chromatography (FPLC), nano-reversed phase liquid chromatography, ion exchange chromatography, gel chromatography such as gel filtration chromatography, size exclusion chromatography, affinity chromatography, high performance liquid chromatography (HPLC), and reverse phase high performance liquid chromatography (RP-HPLC) amongst others.

Corresponding: The term "corresponding" is a relative term indicating similarity in position, purpose or structure. In one embodiment, a peptide standard "corresponding" to a HER-2 amino acid sequence has an amino acid sequence identical to a portion of HER-2 (regardless of whether or not they have the same mass). Such a "peptide standard" can be used to quantitate the amount of a fragment peptide of identical sequence, such as a HER-2 fragment peptide. In other embodiments, mass spectral signals in a mass spectrum that are due to corresponding peptides of identical structure but differing masses are "corresponding" mass spectral signals. A mass spectral signal due to a particular peptide is also referred to as a signal corresponding to the peptide.

DNA (deoxyribonucleic acid): DNA is a long chain polymer which comprises the genetic material of most living organisms (some viruses have genes comprising ribonucleic acid (RNA)). The repeating units in DNA polymers are four different nucleotides, each of which comprises one of the four bases, adenine, guanine, cytosine and thymine bound to a deoxyribose sugar to which a phosphate group is attached. Triplets of nucleotides (referred to as codons) code for each amino acid in a polypeptide, or for a stop signal. The term codon is also used for the corresponding (and complementary) sequences of three nucleotides in the mRNA into which the DNA sequence is transcribed.

Unless otherwise specified, any reference to a DNA molecule is intended to include the reverse complement of that DNA molecule. DNA molecules, though written to depict only a single strand, encompass both strands of a double-stranded DNA molecule. Expression: The process whereby the genetic information contained in a nucleotide sequence is converted into other cellular components, such as mRNA and protein. Generally, expression of a nucleotide sequence takes place within a cell, but can also take place in a cell-free system.

Fragment peptide: A peptide generated by proteolytic cleavage of a protein or polypeptide with a protein cleavage agent, for example in a protein digest. Such proteolytic peptides include peptides produced by treatment of a protein with one or more endoproteases such as trypsin, chymotrypsin, endoprotease ArgC, endoprotease aspN, endoprotease gluC, and endoprotease lysC, as well as peptides produced by cleavage using chemical agents, such as cyanogen bromide, formic acid, and thiotrifluoroacetic acid. One or more cleavage peptides from a particular protein can be mass identifiers for the protein. In several examples, a fragment peptide is a HER-2 fragment peptide.

HER-2: HER-2 (also known as neu and ErbB2) is a member of the epidermal growth factor receptor (EGFR) family and is implicated in the pathogenesis of cancer. It is a cell membrane surface-bound tyrosine kinase and is involved in the signal transduction pathways leading to cell growth and differentiation. Exemplary amino acid sequences of HER-2 can be found at GENBANK® accession numbers NP_004439 and NM_001005862 as available November 18, 2006. In the context of the HER-2 protein as set forth as SEQ ID NO: 19, HER-2 is composed of an extracellular portion from about residue number 23 to about residue number 653 of SEQ ID NO: 19, a transmembrane portion from about residue number 653 to about residue number 675 of SEQ ID NO: 19, and a cytoplasmic portion from about residue number 676 to about residue number 1255 of SEQ ID NO: 19. The kinase domain is contained within the cytoplasmic portion of HER-2 and includes from about residue number 720 to about residue number 987 of SEQ ID NO: 19. The amino acid sequence given as GENBANK® accession number NP_004439 is set forth below as SEQ ID NO: 19.

melaalcrwg lllallppga astqvctgtd mklrlpaspe thldmlrhly qgcqwqgnl eltylptnas lsfIqdiqev qgyvliahnq vrqvplqrlr ivrgtqlfed nyalavldng dplnnttpvt gaspgglrel qlrslteilk ggvliqrnpq lcyqdhilwk difhknnqla ltlidtnrsr achpcspmck gsrcwgesse dcqsltrtvc aggcarckgp lptdccheqc aagctgpkhs dclaclhfnh sgicelhcpa lvtyntdtfe smpnpegryt fgascvtacp ynylstdvgs ctlvcplhnq evtaedgtqr cekcskpcar vcyglgmehl revravtsan iqefagckki fgslaflpes fdgdpasnta plqpeqlqyf etleeitgyl yisawpdslp dlsvfqnlqv irgrilhnga ysltlqglgi swlglrslre lgsglalihh nthlcfvhtv pwdqlfrnph qa11htanrp edecvgegla chqlcarghc wgpgptqcvn csqflrgqec veecrvlggl preyvnarhc lpchpecqpq ngsvtcfgpe adqcvacahy kdppfcvarc psgvkpdlsy mpiwkfpdee gacqpcpinc thscvdlddk gcpaeqrasp ltsiisawg illvwlgw fgilikrrqq kirkytmrrl lqetelvepl tpsgampnqa qmrilketel rkvkvlgsga fgtvykgiwi pdgenvkipv aikvlrents pkankeilde ayvmagvgsp yvsrllgicl tstvqlvtql mpygclldhv renrgrlgsq dllnwctnqia kgmsyledvr lvhrdlaarn vlvkspnhvk itdfglarll dideteyhad ggkvpikwma lesilrrrft hqsdvwsygv tvwelmtfga kpydgipare ipdllekger lpqppictid vymimvkcwm idsecrprfr elvsefsrma rdpqrfwiq nedlgpaspl dstfyrslle dddmgdlvda eeylvpqqgf .fcpdpapgag gmvhhrhrss strsgggdlt lglepseeea prsplapseg agsdvfdgdl gmgaakglqs lpthdpsplq rysedptvpl psetdgyvap ltcspqpeyv nqpdvrpqpp spregplpaa rpagatlerp ktlspgkngv vkdvfafgga venpeyltpq ggaapqphpp pafspafdnl yywdqdpper gappstfkgt ptaenpeylg ldvpv(SEQ ID NO: 19)

The amino acid sequence given as GENBANK® accession number NM OO 1005862 is set forth below as SEQ ID NO:20.

mklrlpaspe thldmlrhly qgcqwqgnl eltylptnas lsfIqdiqev qgyvliahnq vrqvplqrlr ivrgtqlfed nyalavldng dplnnttpvt gaspgglrel qlrslteilk ggvliqrnpq lcyqdtilwk difhknnqla ltlidtnrsr achpcspmck gsrcwgesse dcqsltrtvc aggcarckgp lptdccheqc aagctgpkhs dclaclhfnh sgicelhcpa lvtyntdtfe smpnpegryt fgascvtacp ynylstdvgs ctlvcplhnq evtaedgtqr cekcskpcar vcyglgmehl revravtsan iqefagckki fgslaflpes fdgdpasnta plqpeqlqyf etleeitgyl yisawpdslp dlsvfqnlqv irgri1hnga ysltlqglgi swlglrslre lgsglalihh nthlcfvhtv pwdqlfrnph qallhtanrp edecvgegla chqlcarghc wgpgptqcvn csqflrgqec veecrvlqgl preyvnarhc lpchpecqpq ngsvtcfgpe adqcvacahy kdppfcvarc psgvkpdlsy mpiwkfpdee gacqpcpinc thscvdlddk gcpaeqrasp ltsiisawg illvwlgw fgilikrrqq kirkytmrrl lqetelvepl tpsgampnqa qmrilketel rkvkvlgsga fgtvykgiwi pdgenvkipv aikvlrents pkankeilde ayvmagvgsp yvsrllgicl tstvqlvtql mpygclldhv renrgrlgsq dllnwcmqia kgmsyledvr lvhrdlaarn vlvkspnhvk itdfglarll dideteyhad ggkvpikwma lesilrrrft hqsdvwsygv tvwelmtfga kpydgipare ipdllekger lpqppictid vymimvkcwm idsecrprfr elvsefsrma rdpqrfwiq nedlgpaspl dstfyrslle dddmgdlvda eeylvpqqgf fcpdpapgag gmvhhrhrss strsgggdlt lglepseeea prsplapseg agsdvfdgdl gmgaakglqs lpthdpsplq rysedptvpl psetdgyvap ltcspqpeyv nqpdvrpqpp spregplpaa rpagatlerp ktlspgkngv vkdvfafgga venpeyltpq ggaapqphpp pafspafdnl yywdqdpper gappstfkgt ptaenpeylg ldvpv (SEQ ID NO: 20) HER-2 fragment peptide: A peptide that is derived from the full length HER-2 protein by proteolytic cleavage. In several examples, a HER-2 fragment peptide includes an amino acid sequence as set forth as SEQ ID NOs: 1-29. HER-2 fragment peptides can be used as mass identifiers for HER-2 to identify the presence of HER-2 in a sample, such as a sample obtained from a subject.

Host cell: A host cell is a cell that is used to express a nucleic acid sequence coding for a peptide standard. Examples of host cells include microorganisms such as bacteria, protozoans, yeast, viruses and algae, and cultured cells such as cultured human, porcine and murine cell lines. Isolated: An "isolated" biological component (such as a nucleic acid, peptide or protein) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs or is transgenically expressed, that is, other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides and proteins which have been "isolated" thus include nucleic acids and proteins purified by standard or non-standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized peptides and nucleic acids.

Isotopic analog: "Isotopic analog" refers to a molecule that differs from another molecule in the relative isotopic abundance of an atom it contains. For example, peptide sequences containing identical sequences of amino acids, but differing in the isotopic abundance of an atom, are isotopic analogs of each other. The term "isotopic analog" is a relative term that does necessarily not imply that the isotopic analog necessarily contains an isotope that is present in less or greater abundance in nature. For example, a mass identifier containing a natural abundance of ¹²C and ¹³C is an isotopic analog of a corresponding mass identifier having non-natural abundances of these isotopes, and vice versa.

Isotopically-labeled or labeled: "Isotopically-labeled" or "labeled" refer to a molecule that includes one or more stable heavy isotopes in a greater-than-natural abundance. Heavy stable isotopes include, for example ²H, ¹³C, ¹⁵N, ³⁴S, ¹⁷O, and ¹⁸O. Mass spectrometry: Mass spectrometry is a method wherein, a sample is analyzed by generating gas phase ions from the sample, which are then separated according to their mass-to-charge ratio (m/z) and detected. Methods of generating gas phase ions from a sample include electrospray ionization (ESI), matrix-assisted laser desorption-ionization (MALDI), surface-enhanced laser desorption-ionization (SELDI), chemical ionization, and electron-impact ionization (EI). Separation of ions according to their m/z ratio can be accomplished with any type of mass analyzer, including quadrupole mass analyzers (Q), time-of-flight (TOF) mass analyzers, magnetic sector mass analyzers, 3D and linear ion traps (IT), Fourier-transform ion cyclotron resonance (FT-ICR) analyzers, and combinations thereof (for example, a quadrupole-time-of-flight analyzer, or Q-TOF analyzer). Prior to separation, the sample may be subjected to one or more dimensions of chromatographic separation, for example, one or more dimensions of liquid or size exclusion chromatography.

Nucleotide: A base, such as a pyrimidine, purine, or synthetic analogs thereof, linked to a sugar, plus a phosphate, which forms one monomer in a polynucleotide. A nucleotide sequence refers to the sequence of bases in a polynucleotide.

Oligonucleotide or "oligo": Multiple nucleotides (that is, molecules comprising a sugar (for example, ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (Py) (for example, cytosine (C), thymine (T) or uracil (U)) or a substituted purine (Pu) (for example, adenine (A) or guanine (G)). The term "oligonucleotide" as used herein refers to both oligoribonucleotides and oligodeoxyribonucleotides. Oligonucleotides can be obtained from existing nucleic acid sources (for example, genomic or cDNA), but are preferably synthetic (that is, produced by oligonucleotide synthesis).

Peptide/Proteiπ/Polypeptide: All of these terms refer to a polymer of amino acids and/or amino acid analogs that are joined by peptide bonds or peptide bond mimetics. The twenty naturally-occurring amino acids and their single-letter and three-letter designations are as follows:

Predictable mass difference: A predictable mass difference is a difference in the molecular mass of two molecules or ions (such as two peptides, peptide ions) that can be calculated from the molecular formulas and isotopic contents of the two molecules or ions. Although predictable mass differences exist between molecules or ions of differing molecular formulas, they also can exist between two molecules or ions that have the same molecular formula but include different isotopes of their constituent atoms. A predictable mass difference is present between two molecules or ions of the same formula when a known number of atoms of one or more type in one molecule or ion are replaced by lighter or heavier isotopes of those atoms in the other molecule or ion. For example, replacement of a ¹²C atom in a molecule with a ¹³C atom (or vice versa) provides a predictable mass difference of about 1 atomic mass unit (amu), replacement of a ¹⁴N atom with a ¹⁵N atom (or vice versa) provides a predictable mass difference of about 1 amu, and replacement of a ¹H atom with a ²H (or vice versa) provides a predictable mass difference of about 1 amu. Such differences between the masses of particular atoms in two different molecules or ions are summed over all of the atoms in the two molecules or ions to provide a predictable mass difference between the two molecules or ions. Thus, for example, if two molecules have the formula CeHe, where one molecule includes 6 '³C atoms and the other includes 6 ¹²C atoms, the predictable mass difference between the two molecules is about 6 amu (1 amu difference/carbon atom).

Prognosis: The probable course or outcome of a disease process. In several examples, the prognosis of a subject with cancer can indicate the likelihood of survival and/or the likelihood of metastasis. The prognosis of a subject with cancer can indicate the likelihood that the subject will survive for a period of time, such as about one, about two, about three, about four, about five or about ten years. The prognosis of a subject with cancer can also indicate the likelihood of a cure, of the likelihood that the subject will remain disease-free following treatment for a period of time, such as about one, about two, about three, about four, about five or about ten years.

Protease or proteolytic enzymes: Examples of proteolytic enzymes include endoproteases such as trypsin, chymotrypsin, eπdoprotease ArgC, endoprotease aspN, endoprotease gluC, and endoprotease lysC. Examples of chemical protein cleavage agents include cyanogen bromide, formic acid, and thiotrifluoroacetic acid. The specific bonds cleaved by an endoprotease or a chemical protein cleavage agents may be more specifically referred to as "endoprotease cleavage sites" and "chemical protein cleavage agent sites," respectively. Proteins typically contain one or more intrinsic protein cleavage agent sites that are recognized by one or more protein cleavage agents by virtue of the amino acid sequence of the protein. Standard: A standard is a substance or solution of a substance of known amount, purity or concentration. A standard can be compared (such as by spectrometric, chromatographic, or spectrophotometric analysis) to an unknown sample (of the same or similar substance) to determine the presence of the substance in the sample and/or determine the amount, purity or concentration of the unknown sample. In one embodiment a standard is a peptide standard. An internal standard is a compound that is added in a known amount to a sample prior to sample preparation and/or analysis and serves as a reference for calculating the concentrations of the components of the sample. Isotopically-labeled peptides are particularly useful as internal standards for peptide analysis since the chemical properties of the labeled peptide standards are almost identical to their non-labeled counterparts. Thus, during chemical sample preparation steps (such as chromatography, for example, HPLC) any loss of the non-labeled peptides is reflected in a similar loss of the labeled peptides.

Subject: Living multi-cellular vertebrate organisms, a category that includes both human and veterinary subjects, including human and non-human mammals. Therapeutically effective amount: A quantity of a specific substance sufficient to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to inhibit or suppress growth of a tumor. In one embodiment, a therapeutically effective amount is the amount necessary to eliminate a tumor. When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations (for example, in tumors) that has been shown to achieve a desired in vitro effect.

Treating: Inhibiting the full development of a disease or condition, for example, in a subject who is at risk for a disease such as cancer. "Treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. The term "ameliorating," with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to a particular cancer. A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.

Tumor: The product of neoplasia is a neoplasm (a tumor), which is an abnormal growth of tissue that results from excessive cell division. A tumor that does not metastasize is referred to as "benign." A tumor that invades the surrounding tissue and/or can metastasize is referred to as "malignant." A tumor can be a primary tumor meaning it has originated in the same organ in which it is present, and has not metastasized to it. A tumor can be a secondary tumor, meaning that it has migrated away from the original organ (site of a primary tumor). Secondary tumors are also referred to as metastatic tumors. A malignant tumor is generally classified as cancer.

Examples of hematological cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblasts, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, and myelodysplasia.

Examples of solid cancers, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, bladder carcinoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma).

III. Description of Several Embodiments

Human epidermal growth factor receptor 2 (HER-2) is an important proto- oncoprotein whose expression level has been shown to be directly indicative of cancer, such as aggressive breast cancer. The connection between HER-2 and cancer appears to be especially important in breast cancer. HER-2 positive breast cancer is a more aggressive disease with a greater likelihood of recurrence, a poorer prognosis, and a decreased chance of survival. It has been estimated that HER-2 positive breast cancer cases account for approximately 25—30% of patients with primary or metastatic breast cancer and has been shown to be predictive of breast cancer outcome. In addition, many other cancers are known to be associated with HER-2 amplification and/or overexpression. Examples of cancers associated with HER-2 include some cancers of the bladder, breast, colon, endometrial, kidney, lung, ovarian, pancreas, prostate, salivary gland, stomach, and thyroid. Targeted therapies have been developed to combat HER-2 associated cancers. However, to determine if a subject will benefit from such therapies the HER-2 status of the subjects should be known. This disclosure relates to methods of detecting HER-2 in a biological sample, for example in a sample obtained from a subject. Among the methods described herein are those that allow the direct detection and/or quantification of HER-2 from biological samples, such as tissue samples. Accordingly, disclosed embodiments include methods for quantitating HER-2 in subjects with cancer. For example, to determine if a subject would benefit from HER-2 targeted therapy, such as antibody-based anti-tumor therapy with anti-HER-2 antibodies.

A. Detection of HER-2 Peptides in Biological Samples Methods are provided herein to determine if a subject has cancer associated with

HER-2 expression. The methods include obtaining a biological sample from a subject and screening the biological sample for the presence of HER-2. The presence of HER-2 in a biological sample is determined by obtaining a protein digest from the biological sample and detecting the presence of a cysteine-free HER-2 fragment peptide in the protein digest. The presence of a cysteine-free HER-2 fragment peptide in the protein digest indicates that the subject has HER-2 associated cancer. Examples of cancers known to be associated with HER-2 amplification and/or overexpression include certain cancers of the bladder, the breast, the colon, the endometrium, the kidney, the lung, the ovaries, the pancreas, the prostate, the salivary gland, the stomach, and the thyroid. One particular advantageous aspect of the disclosed method is that it can be used to determine the presence of HER-2 in different types of biological samples, for example a solid biological sample obtained from a subject, such as a tissue sample, or a fluid sample obtained from a subject. In particular embodiments, a biological sample obtained from the subject is a blood sample, a urine sample, a serum sample, an ascites sample, a saliva sample, a cell, or portion of tissue, although any biological sample of interest can be used. Tissue samples, such as a portion of a tissue, can be obtained by a variety of invasive, minimally invasive, and/or non-invasive methods. It will appreciated that any method of obtaining tissue from a subject can be utilized,; and that the selection of the method used will depend upon various factors such as the type of tissue, age of the subject, or procedures available to the practitioner. Examples of tissue samples that can be used include, but are not limited to, bladder, breast, colon, endometrium, kidney, lung, ovarian, pancreas, prostate, salivary gland, stomach, or thyroid tissue samples. The tissue sample can be obtained by a variety of procedures including, but not limited to, surgical excision, aspiration, or biopsy. In some embodiments, the tissue sample is obtained from a tumor from a subject. In some examples, the tumor is a primary tumor. In other examples, the tumor is a secondary tumor, for example when the cancer is a metastatic cancer. In particular examples, the cancer is cancer of the bladder, breast, colon, endometrium, kidney, lung, ovarian, pancreas, prostate, salivary gland, stomach, or thyroid.

Fragment peptides, such as HER-2 fragment peptides, can be obtained by proteolytic cleavage of the biological sample with a protein or polypeptide with a protein cleavage agent, such as in a protein digest. The HER-2 fragment peptide can be excised from an intracellular or extracellular portion of HER-2. Such fragment peptides are excised from the full length protein. By way of example, a HER-2 fragment peptide is removed from the full length protein such that it does not include the full length HER-2 protein. Proteolytic peptides (such as HER-2 fragment peptides) include peptides produced by treatment of a protein with one or more endoproteases such as trypsin, chymotrypsin, endoprotease ArgC, endoprotease aspN, endoprotease gluC, and endoprotease lysC, as well as peptides produced by chemical cleavage reactions, such as those that employ cyanogen bromide, formic acid, and thiotrtfluoroacetic acid as is well known to those of skill in the art. In some embodiments, the proteolytic cleavage agent is serine protease. In one embodiment, the proteolytic cleavage agent is trypsin, and the resulting digest is a trypsin digest.

HER-2 fragment peptides derived from a full length HER-2 protein can be uniquely associated with the full length HER-2 protein sequence. Thus, these peptides can be used to determine the presence of HER-2 in a biological sample, such as a biological sample obtained from a subject. Identification of the peptide sequence that is uniquely associated with the larger peptide sequence in a sample identifies the larger peptide sequence in the sample. In other words, a HER-2 fragment peptide that is uniquely associated with a full length HER-2 protein is a mass identifier that contains enough sequence information to discriminate between the HER-2 protein and other proteins in the sample. Mass identifiers are peptides (or a set of peptides) having a particular sequence(s) that is (are) uniquely generated from a protein of interest (such as HER-2) by treatment with a particular protein cleavage agent. Detection of a mass identifier for a protein of interest in a sample unambiguously identifies the presence of the protein of interest in a sample treated with the protein cleavage agent, and determination of the concentration or amount of the mass identifier in a sample also determines the concentration or amount of the protein of interest in the sample either directly or after multiplying the concentration of the mass identifier by the number of such mass identifier generated per protein of interest. Mass identifiers can be identified by treating proteins with a protein cleavage agent in vivo, in vitro or in silico. Various methods and algorithms for determining a mass identifier for a protein of interest are known, but all have in common that peptide sequences obtained by digestion (actual or theoretical) of a protein of interest with a protein cleavage agent (such as an endoprotease or a model of an endoprotease' s cleavage specificity) are compared to peptide sequences obtained by digestion of other known proteins with the same cleavage agent to determine one or more peptide sequences that are uniquely produced from the protein of interest (such as HER-2).

During proteolytic cleavage and sample processing the sulfur atom present in cysteine residues can become oxidized and form cystine or even cysteic acid. The multiple oxidation states of cysteine produce peptides with unpredictable masses, which can present problems for the identification and/or quantification of peptides containing cysteines. To overcome the problems associated with the various oxidation states of cysteine, HER-2 fragment peptides useful in the disclosed examples do not contain cysteine residues. Similar to cysteine, the sulfur atom present in methionine residues can become oxidized, thereby complicating the determination of the HER-2 presence in a sample by detecting methionine containing peptide fragments of HER-2. Thus, in certain embodiments, the cysteine-free

HER-2 fragment peptide also is a methionine-free peptide. Tryptophan is an another amino acid that is oxidation sensitive, thus hampering the determination of HER-2 presence in a sample by detecting tryptophan containing peptide fragments of HER-2. In certain embodiments, the cysteine-free HER-2 fragment peptide also is a tryptophan-free peptide. In certain embodiments, the cysteine-free HER-2 fragment peptide also is a methionine- and tryptophan-free peptide. Shi et al. (Shi et al, J. Proteome Res. 4: 1427-1433, 2005) describes the detection of HER-2 by covalently labeling HER-2 fragment peptides with mass tags. The Shi et al. method requires that HER-2 fragment peptides contain cysteine residues that are covalently attached to a tri-alanine peptide mass tag via iodoacetate chemistry. The methods disclosed herein eliminate the need to covalently attach a mass tag, as described by Shi et al.

Cysteine-free HER-2 fragment peptides useful in the disclosed examples are from about 8 to about 45 amino acid residues in length, such as about 9 amino acids, about 10 amino acids, about 1 1 amino acids, about 12 amino acids, about 13 amino acids, about 14 amino acids, about 15 amino acids, about 16 amino acids, about 17 amino acids, about 18 amino acids, about 19 amino acids, about 20 amino acids, about 21 amino acids, about 22 amino acids, about 25 amino acids, about 30 amino acids, about 35 amino acids, about 40 amino acids, or about 45 amino acids in length. In some embodiments, the cysteine-free HER-2 fragment peptide corresponds to an amino acid sequence in the extracellular portion of HER-2. In some embodiments, the cysteine-free HER-2 fragment peptide corresponds to an amino acid sequence in the intracellular portion of HER-2. In particular embodiments, the cysteine-free HER-2 fragment peptide includes an amino acid sequence as set forth as SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO: 17, or SEQ ID NO: 18. In particularly useful embodiments, the cysteine-free HER-2 fragment peptide includes the amino acid sequence as set forth as SEQ ID NO: 14, which is both methionine- and tryptophan-free. In additional embodiments, the cysteine-free HER-2 fragment peptide consist of an amino acid sequence as set forth as one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:1 1,

SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 17, or SEQ ID NO. l 8. In a particularly useful embodiment, the cysteine-free HER-2 fragment peptide consists of an amino acid sequence as set forth as SEQ ID NO: 14.

The cysteine-free HER-2 fragment peptides can be detected by any method that allows for the detection and identification of peptides. Methods particularly suited to the detection and identification of peptides, such as HER-2 fragment peptides, are mass spectrometric methods. In certain embodiments, the cysteine-free HER-2 fragment peptides are detected with mass spectrometry. In certain embodiments, the cysteine-free HER-2 fragment peptides are detected with tandem mass spectrometry. It some embodiments, the cysteine-free HER-2 fragment peptides are detected by detection of ion fragments generated from the cysteine-free HER-2 fragment peptides (for example by collision using tandem mass spectrometry). Exemplary mass spectrometric methods that can be used in the disclosed methods are found in section C below, although it is contemplated that any mass spectrometric technique that identifies peptides could be used. Enzymatic digestion of complex mixtures of proteins followed by mass spectrometric based analysis of the digest is well known in the art (see for example, U.S. Patent No. 6,940,065 and J. Protein Chem., 16: 495-497, 1997). Prior to mass spectrometric analysis, it can be advantageous to fractionate the protein digest, for example by chromatographing the protein digest. Methods of fractionation of a protein sample are well known in the art, and include without limitation paper chromatography, thin layer chromatography (TLC), liquid chromatography, column chromatography, fast protein liquid chromatography (FPLC), ion exchange chromatography, size exclusion chromatography, affinity chromatography, high performance liquid chromatography (HPLC), poly acrylamide gel electrophoresis (PAGE), capillary electrophoresis (CE) and reverse phase high performance liquid chromatography (RP-HPLC) amongst others.

Aspects of the disclosed methods relate to quantitating the amount of cysteine-free HER-2 fragment peptide present in the biological sample. The quantity of cysteine-free HER-2 fragment peptide present in the biological sample is proportional to the amount of HER-2 present in the sample prior to digestion, thus the disclosed method allows for the quantitation of HER-2. Protein expression levels can be quantified by mass spectrometry if peptide standards of known concentration are available. Methods for quantitating a cysteine-free HER-2 fragment peptide include comparing an amount of the cysteine-free HER-2 fragment peptide to a cysteine-free peptide standard of known amount. Typically, the peptide standards are isotopically labeled peptides, and these are added in known amounts to a non-labeled protein digest. However, non-isotopically labeled peptide standards also can be used. By way of example, the change in relative peak intensity before and after the addition of a peptide standard can be used to calculate the amount of a cysteine-free HER-2 fragment peptide present in a biological sample, thus providing quantification of HER-2 in the sample. When a non-isotopically labeled peptide standard is used, a mass spectrum of the protein digest is obtained without addition of the non- isotopically labeled peptide standard and mass spectrum of the protein digest is obtained with the addition of the non-isotopically labeled peptide standard. The ratio of the intensity of the signals with and without the addition of the non-isotopically labeled peptide standard reflects the relative amounts (or concentrations) of the cysteine-free HER-2 fragment peptide present in a biological sample, and thus the amount of HER-2 present in the sample. It is understood that the spectra with and without the peptide standard can be obtained in any order.

When an isotopically labeled peptide standard is used, typically the combined sample (peptide standard plus protein digest) is analyzed by mass spectrometry, and the ratios of the mass spectral signal intensities for the labeled peptide standard and the sample peptides are measured. Typically, the peptide standard is added to the biological sample prior to the protein digest, however in some circumstances it may be advantageous to add the peptide standard after proteolytic digest. A mass spectrum of a sample containing both sample peptides and the added peptide standard typically includes one or more pairs of separated signals that are due to a sample peptide and its corresponding peptide standard. The ratio of the intensity of the signals in each pair reflects the relative amounts (or concentrations) of each peptide present in the sample. Since the amount (or concentration) of the peptide standard is known, the amount (or concentration) of the sample peptide can be calculated by multiplying the ratio of the intensity of the signal for the sample peptide to the intensity of the signal for the peptide standard by the known amount (or concentration) of the peptide standard. Furthermore, since the sample peptides are present in amounts (or concentrations) that are the same as (or related by a known ratio to) the amounts (or concentrations) of the proteins originally in the sample, a determination of the amounts (or concentrations) of the sample peptides also permits a determination of the amounts (or concentrations) of the proteins in the sample. Since the concentrations of the peptide standards are known, the concentration of the sample peptides (and the proteins they are derived from, such as full length HER-2) can be calculated using the ratios.

Peptide standards useful in the disclosed method correspond to an amino acid sequence of about 8 to about 45 amino acid residues of HER-2, such as about 9 amino acids, about 10 amino acids, about 1 1 amino acids, about 12 amino acids, about 13 amino acids, about 14 amino acids, about 15 amino acids, about 16 amino acids, about 17 amino acids, about 18 amino acids, about 19 amino acids, about 20 amino acids, about 21 amino acids, about 22 amino acids, about 25 amino acids, about 30 amino acids, about 35 amino acids, about 40 amino acids, or about 45 amino acids of HER-2. In some embodiments, the peptide standard corresponds to an amino acid sequence in the extracellular portion of HER- 2. In some embodiments, the peptide standard corresponds to an amino acid sequence in the intracellular portion of HER-2. In certain embodiments, the peptide standard also is a methionine-free peptide. In certain embodiments, the peptide standard also is atryptophan- free peptide. In some embodiments, the peptide standard includes an amino acid sequence as set forth as SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO: 1 1, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 17, or SEQ ID NO: 18. In particular embodiments, the peptide standard includes an amino acid sequence set forth as SEQ ID NO: 14. In some embodiments, the peptide standard consists of an amino acid sequence as set forth as one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 1 1, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 17, or SEQ ID NO: 18. In particular embodiments, the peptide standard consists of an amino acid sequence set forth as SEQ ED NO: 14. In some embodiments, the peptide standard is labeled with an isotope, such as a heavy stable isotope. Examples of particularly useful heavy stable isotopes are ¹⁸0, ¹⁷0, ³⁴S, ¹⁵N, ¹³C, and ²H. Peptide standards can be labeled with one ore more isotopes, for example a labeled peptide can contain ¹⁸O₇ ¹⁷O, ¹⁵N, ³⁴S, ¹³C, and ²H or any combination thereof. Methods of labeling peptides with heavy isotopes are well known in the art and exemplary methods are given below in section D. One advantage of the present technique is that it can be used to detect HER-2 in fixed and paraffin embedded tissues. Because most pathology laboratories use formalin fixation and paraffin embedding to store tissues, this approach is particularly useful for tracking HER-2 tumor status over time. Tissue samples can be fresh, frozen, or fixed (i.e., preserved), for example in formalin, such as buffered formalin. The disclosed methods accommodate the use of paraffin embedded tissue samples, such as archived paraffin embedded biopsy samples. The tissue sample can be fixed by conventional methodology (see for example, Manual of Histological Staining Method of the Armed Forces Institute of Pathology, 3^rd Edition Lee G. Luna, H.T. (ASCP) Editor, The Blakston Division McGraw- Hill Book Company: New York; (1960); The Armed Forces Institute of Pathology Advanced Laboratory Methods in Histology and Pathology ( 1994) Ulreka V. Mikel, Editor, Armed Forces Institute of Pathology, American Registry of Pathology, Washington, D.C.). The choice of a fixative is determined by the purpose for which the tissue is to be histologically stained or otherwise analyzed. The length of fixation depends upon the size of the tissue sample and the fixative used. By way of example, tissue samples can be fixed with neutral buffered formalin, Bouin's fluid, paraformaldehyde, and the like. In one embodiment, the tissue sample is fixed and optionally embedded in paraffin or the like.

Generally, a tissue sample is first fixed and is then dehydrated through an ascending series of alcohols, infiltrated, and embedded with paraffin or other sectioning media so that the tissue sample can be sectioned. Alternatively, one can section the tissue and fix the sections obtained. Paraffin is readily obtained from commercial sources such as

PARAPLAST® (MCCORMICK™ Scientific, St. Louis, MO) and POLYFIN™ available from (Polysciences, Inc, Warrington, PA). Tissue samples can be embedded and processed in paraffin by conventional methodology. Once the tissue sample is embedded, the sample can be sectioned by a microtome or the like. By way of example, sections typically range from about three micrometers to about five micrometers in thickness, although the disclosed methods can accommodate larger sections, such as sections about ten micrometers are greater in thickness. Paraffin processing can be performed via a routine overnight or accelerated cycle in an automated tissue processor. Once embedded, thin sections typically are mounted onto slides (such as glass or quartz slides). If paraffin has been used as the embedding material, the tissue sections are generally deparaffϊnized and rehydrated in water. The tissue sections can be deparaffϊnized by several conventional standard methodologies. For example, xylenes and a gradually descending series of alcohols can be used. Alternatively, commercially available deparaffinizing agents such as HEMO-DE™ (Scientific Safety Solvents, Keller, TX) or HISTOCLEAR™ (East Sussex, England) can be used. The sections can be stained by standard techniques such as hematoxylin and eosin, methylene green nuclear stain, fluorescent in situ hybridization, or immunohistochemistry for identification of tissue morphology and cell populations of interest.

^■In some circumstances, the tissue sample is subjected to laser-mediated dissection (such as laser capture dissection or laser microdissection and pressure catapulting) prior to protein digestion. For example, laser-mediated dissection can be used when the tissue sample contains both cancerous and non-cancerous cells. In this circumstance, laser mediated dissection is used to select only cancer cells for protein digestion. Methods of laser-mediated dissection are well known in the art and exemplary methods are given below in section E.

B. Methods of Diagnosis and Treatment

The disclosed methods are particularly suited for monitoring disease progression in a subject. Such methods involve detecting an amount of HER-2 in a biological sample from a subject at a first time point, detecting an amount of HER-2 in a biological sample from a subject at a second time point, and comparing the amount of HER-2 at the two time points. It has been found that the expression level of HER-2 in a tumor correlates with the severity of the cancer. Thus, a decrease in the amount of HER-2 present in a biological sample, for example as measured by the presence of a cysteine-free HER-2 fragment peptide, would correlate with regression of the cancer. Conversely, an increase in the amount of cysteine- free HER-2 fragment peptide (indicating an increase in HER-2) could correlate to a progression of the cancer, for example progression to a metastatic form of cancer.

The disclosed methods are particularly useful for selecting a treatment for a subject having a cancer in which HER-2 expression is correlated with tumor development or severity. Such methods involve detecting the presence of an amount of cysteine-free HER-2 fragment peptide in a biological sample from a subject, such as in protein digest obtained from the sample. The presence of the cysteine-free HER-2 fragment peptide indicates that a treatment can be selected that specifically targets HER-2 positive cancer. In one embodiment, an antibody treatment specific for HER-2 positive cancers is selected, such as treatment with trastuzumab. In some embodiments, when the cysteine-free HER-2 fragment peptide is present, the subject is treated with a therapeutically effective amount of an anti- HER-2 antibody, such as trastuzumab. In another embodiment, a HER-2 kinase inhibitor is selected. In some examples, the biological sample is HER-2 negative, in such a case a therapy specific for HER-2 positive cancer would not be selected. In this case a treatment such as the use of chemotherapeutic agents, immunotherapeutic agents, radiotherapy, or surgical intervention can be selected.

The disclosed methods are also useful for determining if a subject, such as a subject with cancer, will benefit from treatment with a HER-2 targeted therapy, such as an anti- HER-2 antibody therapy, for example trastuzumab therapy. Such methods include selecting a subject for evaluation of their HER-2 status, such as the HER-2 status of their tumor(s). The presence of an amount of cysteine-free HER-2 fragment peptide in a biological sample obtained from the subject, such as in a protein digest obtained from the sample, is determined. The presence of the cysteine-free HER-2 fragment, and thus a HER-2 associated cancer, indicates that the subject will benefit from treatment with the anti-HER-2 therapy, such as an anti-HER-2 antibody therapy, for example trastuzumab therapy. One of ordinary skill in the art can select suitable anti-HER-2 antibodies for administration to a subject with HER-2 associated cancer. Examples of suitable anti-HER-2 antibodies that can be administered to a subject with HER-2 associated cancer can be found in U.S. Patent Nos. 5,821,337, 5,720,954, 5,783,186, and 6,627,196; U.S. Patent Publication No. 2006/0018899; and International Patent Publication Nos. WO 94/00136, WO 89/06692, and amongst others. Additional anti-HER2 antibodies that can be used with the disclosed methods have been described in Tagliabue et al. Int. J. Cancer 47:933— 937 (1991); McKenzie et al. Oncogene 4:543-548 (1989); Maier et al Cancer Res. 51 :5361-5369 (1991); Bacus ef α/. Molecular Carcinogenesis 3:350-362 (1990); Stancovski et al. PNAS (USA) 88:8691-8695 (1991); Bacus et al. Cancer Research

52:2580-2589 (1992); Xu et al. Int. J. Cancer 53:401-408 (1993); Kasprzyk et al. Cancer Research 52:2771-2776 (1992); Hancock et al. Cancer Res. 51 :4575-4580 (1991); Shawver et al. Cancer Res. 54: 1367-1373 (1994); Arteaga et al. Cancer Res. 54:3758-3765 (1994); Haπverth et al. J. Biol. Chem. 267:15160-15167 (1992); and Klapper et al. Oncogene 14:2099-2109 (1997). C. Mass Spectrometric Methods

Mass spectrometry is particularly suited to the identification of peptides from biological samples, such as peptides excised from HER-2. Mass spectrometry also is particularly useful in the quantitation of peptides in a biological sample, for example using isotopically labeled peptide standards. The application of mass spectrometric techniques to identify proteins in biological samples is known in the art and is described for example in Akhilesh et al, Nature, 405:837-846, 2000; Dutt et al, Curr. Opin. Biotechnol., 1 1 -.176- 179, 2000; Gygi et al, Curr. Opin. Chem. Biol, 4 (5): 489-94, 2000; Gygi et al, Anal. Chem., 72 (6): 1 1 12-8, 2000; and Anderson et al, Curr. Opin. Biotechnol., 1 1 :408-Φ 12, 2000.

Typically, mass spectrometers generate gas phase ions from a sample (such as a sample containing HER-2 fragment peptides and/or peptide standards). The gas phase ions are then separated according to their mass-to-charge ratio (m/z) and detected. Suitable techniques for producing vapor phase ions for use in the disclosed methods include without limitation electrospray ionization (ESI), matrix-assisted laser desorption-ionization (MALDI), surface-enhanced laser desorption-ionization (SELDI), chemical ionization, and electron-impact ionization (EI).

Separation of ions according to their m/z ratio can be accomplished with any type of mass analyzer, including quadrupole mass analyzers (Q), time-of-flight (TOF) mass analyzers (for example linear or reflecting) analyzers, magnetic sector mass analyzers, 3D and linear ion traps (IT), Fourier-transform ion cyclotron resonance (FT-ICR) analyzers, and combinations thereof (for example, a quadrupole-time-of-flight analyzer, or Q-TOF analyzer). In some embodiments, the mass spectrometric technique is tandem mass spectrometry (MS/MS) and the presence of a HER-2 fragment peptide is detected. Typically, in tandem mass spectrometry a HER-2 fragment peptide entering the tandem mass spectrometer is selected and subjected to collision induced dissociation (CID). The spectra of the resulting fragment ion is recorded in the second stage of the mass spectrometry, as a so-called CID spectrum. Because the CID process usually causes fragmentation at peptide bonds and different amino acids for the most part yield peaks of different masses, a CID spectrum alone often provides enough information to determine the presence of a peptide such as a HER-2 fragment peptide. Suitable mass spectrometer systems for MS/MS include an ion fragmentor and one, two, or more mass spectrometers, such as those described above. Examples of suitable ion fragmentors include, but are not limited to, collision cells (in which ions are fragmented by causing them to collide with neutral gas molecules), photo dissociation cells (in which ions are fragmented by irradiating them with a beam of photons), and surface dissociation fragmentor (in which ions are fragmented by colliding them with a solid or a liquid surface). Suitable mass spectrometer systems can also include ion reflectors.

Prior to mass spectrometry the sample can be subjected to one or more dimensions of chromatographic separation, for example, one or more dimensions of liquid or size exclusion chromatography. Representative examples of chromatographic separation include paper chromatography, thin layer chromatography (TLC), liquid chromatography, column chromatography, fast protein liquid chromatography (FPLC), ion exchange chromatography, size exclusion chromatography, affinity chromatography, high performance liquid chromatography (HPLC), nano-reverse phase liquid chromatography (nano-RPLC), poly acrylamide gel electrophoresis (PAGE), capillary electrophoresis (CE), reverse phase high performance liquid chromatography (RP-HPLC) or other suitable chromatographic techniques. Thus, in some embodiments, the mass spectrometry technique is directly or indirectly coupled with a liquid chromatography technique, such as column chromatography, fast protein liquid chromatography (FPLC), ion exchange chromatography, size exclusion chromatography, affinity chromatography, high performance liquid chromatography (HPLC), nano-reverse phase liquid chromatography (nano-RPLC), poly acrylamide gel electrophoresis (PAGE), capillary electrophoresis (CE) or reverse phase high performance liquid chromatography (RP-HPLC) to further resolve the biological sample prior to mass spectrometric analysis.

The reagents (such as buffers and the like), and particularly LIQUID TISSUE^® reagents, used in accordance with the disclosed methods are preferably chosen such as to not significantly interfere with mass spectral analysis, such as tandem mass spectrometric methods. Preferably, but not necessarily, the reagents are selected so as to impart desirable characteristics to the analysis. Examples of such characteristics include for example decreasing the energy required to volatilize the peptide, facilitating ionization, creating predominantly singly charged ions, reducing the peak width, and increasing the sensitivity and/or selectivity of the desired analysis product.

D. Isotopically-Iabeled Peptides

In some embodiments, the peptide standard is labeled with an isotope, such as a heavy stable isotope. Examples of particularly useful heavy stable isotopes are ¹⁸0, ¹⁷0, ³⁴S, ¹⁵N, ¹³C, and ²H. Useful isotopically labeled peptide standards have a predictable number of sites where the heavy isotope replaces a non-heavy isotope yielding a peptide with a predictable mass difference from a peptide that does not have any heavy isotopes incorporated. Thus, the isotopic- label ing of the peptide standards yields separate distinct mass spectrometric signals from peptides obtained from the biological samples. These isotopically labeled peptide standards can be used to quantify proteins present in biological samples.

Stable isotopes can be incorporated into peptides either biologically {in vivo) or chemically {in vitro). Methods for providing isotopically-labeled peptide standards are to express the peptides in a host cell (such as E. colϊ) grown on an isotopically-altered medium. Alternatively, peptides can be expressed in cell free systems. Isotopically-altered medium is a growth medium that is enriched in one or more stable heavy isotopes of an element or elements relative to their natural isotopic abundances. For example, a growth medium including greater amounts of ²H, ¹³C, ³⁴S, ¹⁵N, ¹⁷O, and/or ¹⁸O than are found in nature is an isotopically-altered medium. Enrichment of the medium with stable heavy isotopes can be partial (where both heavy and light isotopes of a particular element are present in the medium), or uniform (where substantially only heavy isotopes of a particular element are present, such as greater than 90%, 95%, 98% or 99% of the atoms of an element are the heavy isotope). Stable heavy isotopes can be added to the medium in any form. For example, the isotopes can be added in the form of a simple chemical substance such as ¹⁵NH₃, ^uC-glucose, ²H₂O, or can be added in the form of a more complex substance such as an isotopically-altered amino acid (for example, amino acids labeled with ²H, ¹³C, ³⁴S, ¹⁵N, ¹⁷O, and/or ¹⁸O, such as deuterium-enriched leucine, serine, and/or tyrosine). Uniform labeling media refers to a growth medium wherein substantially ali atoms (such as greater than 90%, 95%, 98% or 99% of all atoms) of a particular element present in the medium are present in the form of a particular isotope of the element. A uniformly-labeled growth medium provides a particular type of atom substantially in the form of a single isotope of the atom. For example, a medium that provides nitrogen in the form Of ¹⁵NH₃ as the sole nitrogen source for host cells grown on the medium is a uniformly-labeled media. Thus, a peptide standard expressed in host cells grown in media with ¹⁵NH₃ as the sole nitrogen source will be uniformly isotopically labeled with ¹⁵N.

Biological isotopic labeling schemes for quantitative mass spectrometry include stable isotope labeling by amino acids in cell culture (SILAC) and related techniques (see, for example, Ong et ah, Molecular & Cellular Proteomics, 1.5:376-386, 2002). In the SILAC technique, isotopically-labeled amino acids are added to an amino acid deficient cell culture, and are incorporated into proteins or peptides during cell growth. Alternatively, peptides or proteins can be expressed in host cells grown in media with a stable heavy isotope as the sole source for a particular element.

In some instances peptides expressed in a host cell are unstable and prone to degradation (see, for example, Lindhout et al., Protein Science, 12: 1786—1791, 2003). One solution to the stability problem is to express peptides in a fusion construct with a larger fusion protein partner. The fusion protein approach has been applied to produce a single peptide or a few copies of a single peptide (a homopolymer of peptides) as part of the fusion protein (see, Jones et al., Biochemistry, 39: 1870-1878, 2000; Majerle et al., J. Biomol. NMR, 18: 145-151, 2000; Sharon et al, Protein Expr. Purif., 24: 374-383, 2002; and Lindhout et al., Protein Science, 12: 1786-1791, 2003). The peptide standard is then excised from the fusion construct, for example by proteolytic cleavage with an endopeptidase such as trypsin. Peptide standards can also be produced from HER-2 or a portion thereof that is expressed in an isotopically-altered medium, (for example in a host cell). The resulting HER-2 protein or a portion thereof can be digested with an endoprotease (such as trypsin) to excise HER-2 fragment peptides that can be used as peptide standards. Peptide standards, such as a peptide corresponding to a portion of HER- 2, can be subsequently purified and quantitated for use as a peptide standard of known molecular weight and concentration.

Isotopically-labeled peptide standards of known concentration and molecular weight can be synthesized from isotopically labeled amino acids. Peptide synthesis is well known in the art (see for example, Atherton and Sheppard, Solid Phase Peptide Synthesis: a Practical Approach published by published by Oxford University Press, USA, and Chan and White Fmoc Solid Phase Peptide Synthesis: A Practical Approach, published by Oxford University Press, USA). Peptide standards useful in the disclosed method corresponds to an amino acid sequence of about 8 to about 45 amino acid residues of HER-2, such as about 9 amino acids, about 10 amino acids, about 1 1 amino acids, about 12 amino acids, about 13 amino acids, about 14 amino acids, about 15 amino acids, about 16 amino acids, about 17 amino acids, about 18 amino acids, about 19 amino acids, about 20 amino acids, about 21 amino acids, about 22 amino acids, about 25 amino acids, about 30 amino acids, about 35 amino acids, about 40 amino acids, or about 45 amino acids of HER-2.

In some embodiments, the peptide standard comprises an amino acid sequence as set forth as SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ED NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 17, or SEQ ID NO: 18. In particular embodiments, the peptide standard comprises an amino acid sequence set forth as SEQ ID NO: 14. In some embodiments, the peptide standard consists of an amino acid sequence as set forth as one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO.8, SEQ ID NO: 1 1, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 17, or SEQ ID NO: 18. In particular embodiments, the peptide standard consists of an amino acid sequence set forth as SEQ ID NO: 14.

E. Laser-Mediated Microdissection

Laser mediated microdissection, such as laser capture microdissection, laser microdissection and pressure catapulting (LMPC) as well as other technical approaches, are a methods for procuring pure cells from specific microscopic regions of tissue sections. Typically, tissue samples obtained from a subject, such as tissue biopsies, include heterogeneous cell populations, with different cell types exhibiting strong adhesive interactions with adjacent cells, connective stroma, blood vessels, glandular and muscle components, adipose cells, inflammatory, or immune cells amongst others. In disease pathologies, the diseased cells of interest, such as precancerous cells, cancer cells, or invading groups of cancer cells, are surrounded by these heterogeneous tissue elements. In some situations, the cell types undergoing similar molecular changes, such as cancer cells, may constitute less than 5% of the volume of the tissue biopsy sample. Laser-mediated microdissection can be applied to obtain pure cell populations, such as populations from homogenous tissue samples.

Laser capture microdissection typically uses a laser beam that focally activates a transfer film capable of specifically bonding to cells identified and targeted by microscopy within the tissue sample. The transfer film with the bonded cells is then lifted off the thin tissue section, leaving all unwanted cells behind (which would contaminate the molecular purity of subsequent analysis). The transparent transfer film (such as ethylene vinyl acetate polymer film containing a naphthalocyanine near infrared (IR) absorbing dye) is applied to the surface of the thin tissue section overlying the target. Under the microscope, the operator (such as diagnostic pathologist) views the thin tissue section through the slide (such as a glass or quartz slide) on which it is mounted and chooses microscopic clusters of cells to study. When the cells of choice are in the center of the field of view, the operator activates a laser (such as a near ER. laser diode) integral with the microscope optics. The pulsed laser beam activates a precise spot on the transfer film immediately above the cells of interest in order to heat the polymer to its melting point. At this precise location the film melts and the melted polymer expands downward making contact with the cell surface and flowing into microscopic air spaces in the desiccated tissue section and fuses with the underlying cells of choice. The heat is rapidly conducted away in the slide within a couple of milliseconds of the end of the pulse causing the cooled polymer to rapidly resolidify and bond to the targeted tissue. The strong bond formed allows the bonded cell(s) to be ripped out of the surrounding tissue when the film is subsequently lifted off the tissue. By focusing the laser to a small spot and using a short pulse, the region of polymer melting, expansion, and bonding can be confined to an individual cell. When the film is removed the chosen cell(s) are tightly held within the focally expanded polymer while the rest of the tissue is left behind. This allows multiple homogeneous samples within the tissue section or cytological preparation to be targeted and pooled for extraction of proteins and or peptides for analysis. For a successful laser capture dissection, the strength of the bond between polymer film and the targeted tissue must be stronger than that between the tissue and the underlying slide. Therefore, for most tissue types, techniques which increase the adhesion of the tissue section to the slide should be avoided. In a commercial system, such as the instruments produced by Arcturus (Mountain

View, CA), the film is permanently bonded to the underside of a transparent vial cap. A mechanical arm precisely positions the transfer surface onto the tissue. The microscope focuses the laser beam to discrete sizes (presently either 30 or 60 micrometer diameters), delivering precise pulsed doses to the targeted film. Targeted cells are transferred to the cap surface, and the cap is placed directly onto a vial for molecular processing. The size of the targeting pulses is selected by the operator. Cells adherent to the film retain their morphologic features, and the operator can verify that the correct cells have been procured.

Typically in laser microdissection and pressure catapulting, a pulsed ultra-violet (UV-A) laser of high beam quality is interfaced into the microscope and focused through an objective to a beam spot size of less than 1 micrometer in diameter for to excise tissues of interest (such as cells of interest) from the surrounding tissue. Laser mediated excision is a locally restricted ablative process that occurs without heating of the adjacent tissue and results in a clear cut gap between the tissue of interest and the surrounding tissue. After microdissection, the isolated specimens are ejected out of the object plane and catapulted directly into the cap of a common microfuge tube. This is performed in an entirely non- contact manner with the help of a single defocused laser pulse. Commercial systems such as the Zeiss PALM MicroLaser System, and the Leica LMD6000 amongst others can be used for pressure catapulting laser microdissection. In addition, certain commercially-available tissue slides (such as DIRECTOR™ slides) have been designed specifically for dissection of tissue by laser microdissection and pressure catapulting. In some embodiments, computer software is used to control the tissue microdissection process and to store data records, including digital images of the microdissected cells before and after transfer. These records can be integrated with subsequent molecular analysis results.

The following examples are provided to illustrate particular features of certain embodiments. However, the particular features described below should not be construed as limitations on the scope of the invention, but rather as examples from which equivalents will be recognized by those of ordinary skill in the art.

EXAMPLES

Example 1

Tissue Sample Preparation

This example describes representative methods for the preparation, extraction, and trypsin digests of peptides from formalin fixed paraffin embedded (FFPE) tissues.

FFPE whole-mount tissue samples were histologically analyzed by standard methods. Sections were cut and placed on glass slides and stained with hematoxylin and eosin for identification of histologically distinct tissue regions. Histological analysis was performed on an Olympus BX51 microscope, and images (10 X 20 magnification) were taken with an Olympus Q color camera mounted on the microscope.

Ten micrometer thick sections were cut from FFPE whole-mount tissue blocks, placed on the coated DIRECTOR™ slides (Expression Pathology Inc.), and heated for 60 minutes at 58 ⁰C. Paraffin was removed by treatment in SUBX® organic solvent (SURGIP ATH® Medical Industries, Richmond, IL) twice for 5 minutes. Tissues were rehydrated with multiple graded ethanol solutions and distilled water. Tissues were counterstained with Mayer's hematoxylin, dehydrated through graded ethanol solutions, and air-dried. Tissue was rehydrated prior to microdissection with 50% glycerol in water for 5 minutes. Slides were placed upside down below the 10x objective and visualized to locate . cellular regions with specific histological features, which were mapped using the accompanying stage software. The microdissection was automated, allowing for modulation of the laser and scanning of the receiving substrate via computer-aided design/computer- aided machining (CAD/CAM).

The tissue microdissection instrument is based on an optical setup designed to direct a pulsed laser onto a target slide holding a ten micrometer thick tissue section. An excimer laser (MPB Technologies PSX-100) operating at the following conditions was utilized for microdissection: 248 nm wavelength, 2.5 ns pulse, E_max = 5 mJ, repetition rate = 0.1 to 100 Hz. Target slides are made of optically transparent quartz with an energy transfer coating with the exact dimensions of a standard histology glass slide. The slide stage is a computer controlled, XY translation stage with a maximum translation speed of 200 millimeter/second. The laser is split by a 1/8 beam splitter to an energy meter, and the remaining beam travels to an UV reflective mirror and directed down (-Z) to a 10x microscope objective (LMU-IOx-UVR, OFR), which focuses the laser onto the slide and allows for observation of the dissection process via a confocally aligned CCD camera. Microdissection was performed by software directed laser pulses to strike at a constant velocity and rate throughput over the previously defined and mapped cellular regions to achieve complete transfer of cells within the selected area into a 1.5 milliliter low binding microcentrifuge receiving tube.

Example 2 Extraction of Peptides from Microdissected Tissue -

This example describes illustrative procedures for the extraction and digestion of peptides from microdissected tissue.

Cells collected by microdissection for nano-RPLC-MS/MS analysis were processed using LIQUID TISSUE^®-MS reagents and protocol (Expression Pathology Inc) according to the manufacturer's recommendations. Microdissected tissue was suspended in 20 microliters of LT-MS reaction buffer, incubated at 95 ⁰C for 90 minutes, then cooled on ice for 3 minutes at which time 1 microliter of trypsin (15—18 U) was added followed by incubation at 37 ⁰C overnight. DTT was added to a final concentration of 10 millimolar, and the samples were heated for 5 minutes at 95°C to reduce cysteine residues. Digestates were stored at — 20 ⁰C until analysis.

Example 3 Plasma Immunodepletion

This example describes illustrative methods for the immunodepletion of major plasma proteins from serum prior to trypsin digestion.

Using the multiple affinity removal system (MARS, Agilent technologies ) serum is immunodepletion of the six highest abundance proteins albumin, IgG, antitrypsin, IgA, transferrin and haptoglobin (representing 85-90% of total serum protein mass) according to manufacturer's protocols. Depleted samples are exchanged in to 50 millimolar ammonium bicarbonate using a vivaspinconcentrator (5000 molecular weight cut off, Vivascience). Immunodepleted samples are denatured and reduced by incubation in 5% SDS and 5 millimolar tris-(2-carboxyethyl)phosphine at 60 ⁰C for 15 minutes. Trypsin is then added and the samples digested for 5 hours. Digestates are stored at -20 ⁰C until analysis.

Example 4

Mass Spectrometric Analysis of Peptides Using Nano Reverse Phase Liquid

Chromatography

This example describes illustrative methods for the analysis of peptides using nano reverse phase liquid chromatography followed by mass spectrometric identification of peptides.

Nano reverse phase liquid chromatography (RPLC) was performed using an Agilent 1 100 capillary LC system (Agilent Technologies, Palo Alto, CA) coupled on-line to eithera linear ion trap (LIT) mass spectrometer (LTQ₅ Thermo Electron, San Jose, CA) or a hybrid LIT-FT-ICR MS. Nano- RPLC separations of each sample were performed using 75 micrometer inner diameter X 360 outer diameter X 10 centimeter long fused silica capillary columns (Polymicro Technologies, Phoenix, AZ) that were slurry packed in-house with 3 micrometer, 300 A pore size C-18 silica bonded stationary phase (Vydac, Hysperia, CA). After injecting 1 microliter of sample, the column was washed for 30 minutes with 98% mobile-phase A (0.1% formic acid in water) at a flow rate of 0.5 microliter/minute. Peptides were eluted using a linear gradient of 2% mobile-phase B (0.1% formic acid in ACN) to 40% solventB in 1 10 minutes, then to 98% B in an additional 30 minutes, all ata constant flow rate of 0.25 microliter/minute.

The LIT-MS was operated in a data-dependent MS/MS mode in which each full MS scan is followed by five MS/MS scans where peptide molecular ions are selected for collision-induced dissociation (CID) using a normalized collision energy of 35%. Dynamic exclusion was utilized to minimize redundant selection of peptides previously selected for CID. The heated capillary temperature and electrospray voltage were set at 160 ⁰C and 1.5 kV, respectively. Data were collected over a broad mass to charge (jnlz) precursor ion selection scan range.

Example 5

Selection of HER-2 Peptide Standards

This example describes the methodology used to select exemplary peptide standards for use in the quantitation of HER-2 in biological samples. HER-2 was digested with trypsin and analyzed by nano-RPLC-MS/MS to facilitate selection of a readily detectable peptide with the best signal intensity and chromatographic peak shape. Eighteen identifiable HER-2 fragment peptides were observed from a 100 nanogram injection of a HER-2 tryptic digest (see Table 1). Table 1. HER-2 Fragment Peptides Identified.

# is the number of peptides, xC, cross-correlation value; dCN, delta correlation value.

Of the eighteen initial peptides, six peptides containing cysteine, methionine, or tryptophan were eliminated for further study due to potential complications due to oxidation and/or cystine formation. In addition, eight peptides containing internal, missed tryptic cleavage sites also were eliminated for further study.

Based on these analyses, a tryptic peptide spanning amino acid residues 1054 to 1072 of HER-2 (SGGGDLTLGLEPSEEEAPR, SEQ ID NO: 14) was selected for use as a peptide standard for the quantitation of HER-2 in a biological sample. The HER-2 fragment peptide corresponding to the amino acid sequence SGGGDLTLGLEPSEEEAPR (SEQ ID NO: 14) had an observable mass signal at m/z = 957.5. This corresponds to the doubly charged peptide with two additional hydrogens ([M+2H]²⁺) (see Fig. 1 spectrum A). Fig. 1, spectrum A shows a representative base peak spectrum obtained from a tryptic digest of HER-2 centered on m/z ration 957 to 958, which is roughly the m/z of HER-2 fragment peptide SGGDLTLGLEPSEEEAPR (SEQ ID NO:14). The spectrum is shown as relative abundance versus retention time (minutes). The spectrum was collected with a gradient of 0-40 minutes at 1% mobile phase B/minute, and 250 nanoliters/minute. Individual peaks in the spectrum are indicated by retention time. Three observable fragment ions of this peptide could also be detected using spectral windows of 913.9— 914.9 m/z, 1043-1044 m/z, and 1213-1214 m/z (see Fig. 1, spectra B and C respectively). The selected HER-2 fragment peptide SGGGDLTLGLEPSEEEAPR (SEQ ID NO: 14) fragments under tandem mass spectrometric analysis to give a distinctive triplet of peaks at m/z 914.4, 1043.5, and 1213.6, as shown in Fig. 2. The spectrum shown in Fig. 2 is given as relative abundance versus m/z ratio over the m/z ratio from of 250 to 1930. The positions of the three readily monitored transition ions are indicated. Because the selected peptide provides a distinctive, readily recognized fragmentation pattern rather than a single peak, it can be detected with increased selectivity. Using the mass signal of the selected peptide and/or the fragmentation ions observed the presence and amount of HER-2 can be accurately measured without ambiguity. The fragment ions can be used to quantitate the amount of HER-2 in a biological sample. With reference to Fig. 3, the first spectrum labeled A is a mass spectrum with an expanded scale roughly centered on the doubly charged intact peptide (m/z 957). Fig. 3, spectrum B, C, and D is a tandem mass spectrum with an expanded scale roughly centered on the HER-2 fragment peptide SGGDLTLGLEPSEEEAPR (SEQ ID NO: 14) fragment ion at m/z 914, the fragment ion at m/z 1043, and the fragment ion at m/z 1213, respectively. The set of spectra shown in Fig. 3 demonstrates that the HER-2 fragment peptide SGGDLTLGLEPSEEEAPR (SEQ ID NO: 14) can be accurately measured without ambiguity using a combination of parent and/or fragment ions.

A synthetic peptide corresponding to residues 1054 to 1072 of HER-2 (SGGGDLTLGLEPSEEEAPR, SEQ ID NO: 14) was synthesized with ¹³C labeling to serve as an internal standard for quantification of HER-2 in biological samples. The linear range and limits of detection and quantitation were evaluated by analyzing mixtures of the peptide isotopomers by nanoflow reversed-phase liquid chromatography (nano-RPLC) coupled online with a linear ion trap mass spectrometer (MS) operation in tandem MS mode operating in a selected reaction monitoring (SRM) mode. The properties of these peptide candidates were further evaluated in terms of their suitability for conducting SRM measurements from a complex mixture by nano-RPLC- MS/MS analysis of stage III metastatic breast carcinoma cells known to express high levels (2.2 million copies/cell) of HER-2. Example 6 Analysis of HER-2 Peptides in Tumor Biopsies

This example describes the determination of the presence of HER-2 fragment peptides in biological samples obtained from tumor biopsies. Trypsin peptide digests were obtained from formalin fixed breast cancer biopsy samples using the LIQUID TISSUE^® reagents and protocols as described. The peptide digests were subjected to LC/MS/MS analysis to determine if the HER-2 fragment peptide corresponding to amino acid residues 1054 to 1072 of HER-2 (SGGGDLTLGLEPSEEEAPR, SEQ ID NO: 14) was detectable in a complex biological sample, as shown the set of three spectra in Fig. 4. With reference to Fig. 4, spectrum A shows the spectrum obtained with a spectral window of 956.1— 959.1 m/z. This spectral window corresponds to the m/z of the doubly charged HER-2 fragment peptide SGGGDLTLGLEPSEEEAPR (SEQ ID NO: 14). In this spectrum, the HER-2 fragment peptide is observable at a retention time of 85.19 minutes. MS/MS selection and analysis of peptides at m/z = 957.5 reduced the number of additional peaks as shown in Fig. 4, spectrum B. Further refinement yielded a reproducible peak corresponding to the ion fragments of the HER-2 fragment peptide SGGGDLTLGLEPSEEEAPR (SEQ ID NO: 14), (see Fig. 4, spectrum C and Fig 5).

Example 7

Mass Spectrometric Analysis of HER-2 and Breast Cancer Tissue

This example describes exemplary methods for the determination of the amount of HER-2 protein in tissue samples obtained from formalin fixed human breast cancer tissue. Breast carcinoma cells were microdissected from formalin fixed paraffin embedded (FFPE) breast cancer tissue using DIRECTOR™ slides as described. Known concentrations of HER-2 peptide standard (SGGGDLTLGLEPSEEEAPR, SEQ ID NO: 14) were added to the samples obtained via tissue microdissection. The samples were processed using the LIQUID TISSUE^® reagents and protocol according to manufacturer's recommendations (Expression Pathology Inc). Known concentrations of a HER-2 peptide standard (SGGGDLTLGLEPSEEEAPR, SEQ ID NO: 14) were added to the microdissected tissue samples. The digests were analyzed by nano-RPLC-MS/MS. The amount of HER-2 was quantified by calculating relative peak areas generated from reconstructed ion chromatograms of selected transition ions directly detected from the SRM analysis of the selected peptide ion (see Fig. 6, spectra A-C). With reference to Fig. 6, spectrum A is a mass spectrum of an m/z window of 956.01 to 959,01, showing the presence of the HER-2 fragment peptide (SGGGDLTLGLEPSEEEAPR, SEQ ID NO: 14) Fig 6, spectrum B shows a tandem mass spectrum of the intact HER-2 fragment peptide SGGDLTLGLEP S EEEAPR (SEQ ID NO: 14), while Fig 6, spectrum C is a tandem mass spectrum of the fragment ions of the HER-2 fragment peptide SGGDLTLGLEPSEEEAPR (SEQ ID NO: 14).

While this disclosure has been described with an emphasis upon particular embodiments, it will be obvious to those of ordinary skill in the art that variations of the particular embodiments may be used, and it is intended that the disclosure may be practiced otherwise than as specifically described herein. Features, characteristics, compounds, chemical moieties, or examples described in conjunction with a particular aspect, embodiment, or example of the invention are to be understood to be applicable to any other aspect, embodiment, or example of the invention. Accordingly, this disclosure includes all modifications encompassed within the spirit and scope of the disclosure as defined by the following claims.

Claims

We claim:

1. A method for detecting HER-2 in a biological sample, comprising: producing a protein digest from the biological sample; and detecting a cysteine-free HER-2 fragment peptide in the protein digest, thereby determining the presence of HER-2 in the biological sample.

2. The method of claim 1 , wherein the cysteine-free HER-2 fragment is derived from the extracellular domain of HER-2.

3. The method of claim 1 , wherein the cysteine-free HER-2 fragment is derived from the intracellular domain of HER-2.

4. The method of claim 1, wherein the cysteine-free HER-2 fragment peptide is a methionine-free peptide.

5. The method of claim 1, wherein the cysteine-free HER-2 fragment peptide is a tryptophan-free peptide.

6. The method of claim 1, further comprising chromatographing the protein digest.

7. The method of claim 6, wherein chromatographing comprises one or more of gel electrophoresis, liquid chromatography, capillary electrophoresis, nano-reversed phase liquid chromatography, high performance liquid chromatography, or reverse phase high performance liquid chromatography.

8. The method of claim 1, wherein the protein digest comprises a serine protease digest.

9. The method of claim 8, wherein the serine protease digest comprises a trypsin digest.

10. The method of claim 1, wherein detecting comprises mass spectrometry.

11. The method of claim 1, wherein detecting comprises tandem mass spectrometry.

12. The method of claim 1, wherein the cysteine-free HER-2 fragment peptide comprises from about 8 to about 45 amino acid residues of SEQ ID NO: 19.

13. The method of claim 12, wherein the cysteine-free HER-2 fragment peptide comprises an amino acid sequence as set forth as SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:12, SEQ ED NO:14, SEQ ID NO.17, or SEQ ID NO: 18.

14. The method of claim 13, wherein the cysteine-free HER-2 fragment peptide comprises an amino acid sequence set forth as SEQ ID NO: 14.

15. The method of claim 12, wherein the cysteine-free HER-2 fragment peptide consists of the amino acid sequence as set forth as one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO-I l, SEQ IDNO.-12, SEQ ID NO:14, SEQ ED NO: 17, or SEQ ED NO: 18.

16. The method of claim 15, wherein the cysteine-free HER-2 fragment peptide consists of the amino acid sequence set forth as SEQ ID NO: 14.

17. The method of claim 1, wherein the biological sample is a blood sample, a urine sample, a serum sample, an ascites sample, a saliva sample, a cell, or a portion of a tissue.

18. The method of claim 17, wherein the portion of the tissue is obtained by biopsy.

19. The method of claim 17, wherein the portion of the tissue is formalin fixed.

20. The method of claim 17, wherein the portion of the tissue is paraffin embedded.

21. The method of claim 17, wherein the portion of the tissue is obtained by performing laser-mediated dissection.

22. The method of ciaim 17, wherein the portion of the tissue is obtained from a tumor.

23. The method of claim 22, wherein the tumor is a primary tumor.

24. The method of claim 22, wherein the tumor is a secondary tumor.

25. The method of claim I, further comprising quantifying the cysteine-free HER-2 fragment peptide.

26. The method of claim 25, wherein quantifying the cysteine-free HER-2 fragment peptide comprises comparing an amount of the cysteine-free HER-2 fragment peptide to a cysteine-free peptide standard of known amount, wherein the cysteine-free peptide standard corresponds to an amino acid sequence of about 8 to about 45 amino acid residues of HER-2.

27. The method of claim 26, wherein the cysteine-free peptide standard comprises an amino acid sequence as set forth as SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 1 1, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:17, or SEQ ID NO: 18.

28. The method of claim 27 wherein the cysteine-free peptide standard comprises an amino acid sequence set forth as SEQ ID NO: 14.

29. The method of claim 26, wherein the cysteine-free peptide standard consists of an amino acid sequence as set forth as one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ED NO:8, SEQ ID NO: 1 1, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 17, or SEQ ID NO: 18.

30. The method of claim 29, wherein cysteine-free peptide standard consists of an amino acid sequence set forth as SEQ ID NO: 14.

31. The method of claim 26, wherein the cysteine-free peptide standard is an isotopically labeled peptide.

32. The method of claim 31, wherein the isotopically labeled peptide comprises one or more heavy stable isotopes selected from ¹⁸O, ¹⁷O, ³⁴S, ¹⁵N, ¹³C, ²H or combinations thereof.

33. The method of claim 1, further comprising obtaining the biological sample from a subject, wherein detecting the cysteine-free HER-2 fragment peptide in the protein digest indicates the presence of HER-2 associated cancer in the subject.

34. The method of claim 33, wherein the cancer is metastatic cancer.

35. The method of claim 33, further comprising correlating a detected amount of the cysteine-free HER-2 fragment peptide to progression of the cancer.

36. The method of claim 33, wherein the HER-2 associated cancer is bladder, breast, colon, endometrial, kidney, lung, ovarian, pancreas, prostate, salivary gland, stomach, or thyroid cancer.

37. The method of any one of claims 33—36, further comprising selecting a treatment for the subject based the presence or absence of the cysteine-free HER-2 fragment peptide in the protein digest.

38. The method of claim any one of claims 33— 36, further comprising administering a therapeutically effective amount of an anti-HER-2 antibody to the subject.

39. The method of claim 38, wherein the anti-HER-2 antibody is trastuzumab.

40. A method for selecting a subject for anti-HER-2 antibody therapy comprising detecting the presence of a HER-2 associated cancer according to the method of any one of claims 33—36.

41. The method of claim 40, further comprising administering a therapeutically effective amount of an anti-HER-2 antibody to the subject if the presence of HER-2 associated cancer is detected.

42. The method of claim 40, wherein the anti-HER-2 antibody is trastuzumab.

43. A peptide standard for quantifying HER-2 in a biological sample, comprising a cysteine-free peptide corresponding to from about 8 to about 45 amino acid residues of HER-2.

44. The peptide standard of claim 43, wherein the peptide standard comprises an amino acid sequence as set forth as SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 17, or SEQ ID NO: 18.

45. The peptide standard of claim 44, wherein the peptide standard comprises an amino acid sequence set forth as SEQ ID NO: 14.

46. The peptide standard of claim 43, wherein the peptide standard consists of an amino acid sequence as set forth as one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,

SEQ ID NO:5, SEQ ED NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 17, or SEQ ID NO: 18.

47. The peptide standard of claim 46, wherein the peptide standard consists of an amino acid sequence set forth as SEQ ID NO: 14.

48. The peptide standard of claim 43, wherein the peptide standard is labeled with an isotope.

49. The peptide standard of claim 48, wherein the isotope comprises a heavy stable isotope, wherein the heavy stable isotope is ¹⁸O, ¹⁷O₅ ³⁴S₃ ¹⁵N, ¹³C, ²H, or a combination thereof.