JP2014502729A - Protein biomarkers for recurrent breast cancer - Google Patents

Abstract

  The patent application was found to be differentially expressed between primary tumor breast cancer cells that did not have recurrent breast cancer disease after initial treatment and primary breast cancer cells that had recurrent breast cancer disease after initial treatment. The disclosed proteins are disclosed and described. These proteins can be used individually or in specific combinations against biological samples from breast cancer patients in diagnostic and prognostic protein assays, and the breast cancer patient's cancer may recur after initial diagnosis and treatment Can be shown. Determining the differential expression of these proteins can also be useful to indicate additional therapies that combat the potential for recurrent breast cancer. Intact full-length proteins can be assayed, or peptides derived from these proteins can be assayed as reporters for these proteins. These proteins can also be identified as “companion diagnostic” proteins, where differentially expressed proteins used as diagnostic and prognostic indicators are also used as targets for therapeutic intervention in breast cancer be able to. Isotope-labeled versions of protein-derived peptides are also disclosed and described herein.

Description

  No. 61 / 428,145, filed Dec. 29, 2010, entitled “Protein Biomarkers of Recurrent Breast Cancer”, the contents of which are hereby incorporated by reference in their entirety. Insist on the benefits.

  In the United States, an estimated 180,000 new cases of invasive breast cancer are diagnosed among women on an annual basis, and approximately 40,000 people are predicted to die of breast cancer each year. Only lung cancer is responsible for more cancer deaths in women. Based on the latest data, the relative survival rate of women diagnosed with breast cancer is 89% surviving 5 years after diagnosis, 81% after 10 years, and 73% after 15 years. However, the 5-year relative survival rate is lower among women in more advanced stages (more invasive) at the time of diagnosis, in which case the 5-year relative survival rate is 98% for localized disease (stage 0 and 1) 84% for local disease (stage 2) and 27% for distant disease (stages 3 and 4). Thus, providing the ability to initially identify patients at greater risk of having late cancers (stages 3 and 4) that may appear to be early cancers (stage 0-2) can improve survival. Most important to increase. This is because, at an earlier time, based on identifying those patients with more invasive disease, an enhanced, more informed decision can be made, which Life is rescued.

  When breast cancer is diagnosed in a patient, a typical initial treatment is to remove the tumor by surgery, followed by death of all remaining cancer cells that have not been removed by surgery. Designed. Breast cancer stage recognition is important for patient treatment because different stages / stages of breast cancer respond differently to different treatment strategies. Determination of breast cancer stage, stage, and / or grade is best determined by analyzing actual breast tumor tissue after removal from the patient. Tumor cells within the breast tumor tissue are the best therapeutics to use to determine the stage, stage, and / or extent of breast cancer and for all remaining tumor cells in the patient. To identify, it can be analyzed histologically and molecularly. The most widely and conveniently available form of cancer patient tissue is formalin-fixed paraffin-embedded tissue.

  Formaldehyde / formalin fixation of tissues removed by surgery is by far the most common method of preserving cancer tissue worldwide and is an accepted practice in standard pathology practice. An aqueous solution of formaldehyde is called formalin. Formaldehyde / formalin fixation typically uses an aqueous formaldehyde solution called formalin. “100%” formalin consists of a saturated solution of water containing formaldehyde (about 40% by volume formaldehyde or 37% by weight) and has a small amount of stabilizer, usually methanol, to limit the degree of oxidation and polymerization. The most common method of tissue preservation is to immerse the whole tissue in an aqueous formaldehyde solution, generally called 10% neutral buffered formalin, for a long period of time (8-48 hours), followed by the fixation of the whole tissue. It is embedded in paraffin wax and stored at room temperature for a long time. Thus, molecular analysis methods for analyzing formalin-fixed cancer tissue are the most accepted and frequently used methods for the analysis of cancer patient tissues.

  An important issue for determining breast cancer treatment initially appears to have non-invasive localized disease (stage 0-2) but may actually have more invasive disease (stage 3-4) The identification of patients who have sex and who will relapse with high probability regardless of surgery and initial chemotherapy treatment. If the patient is better identified as having a high probability of recurrence because the patient is actually a more invasive form of breast cancer than would be thought from histopathology or other means Those patients undergoing more aggressive surgery (eg, typical mastectomy as opposed to fistula resection, also known as “mastectomy”), initial chemotherapy, or additional secondary therapy Can be implemented.

  There are molecular tests designed to identify patients whose breast cancer is more invasive than others by analyzing patient-derived formalin-fixed tissue, such as the Oncotype Dx test from GenomicHealth and the Mammaprint test from Agendia It is. However, these tests increase the number of patients in an intermediate category that cannot indicate the possibility of disease recurrence or non-recurrence. Furthermore, existing tests analyze nucleic acids and do not analyze actual functional entities, proteins that are differentially present in breast cancer tissues / cells. Tests that use proteins as an invasive indicator of breast cancer are more advantageous because they are primarily proteins that perform the functions of the cells, not the nucleic acids, and cause the cells to become cancerous This is because it is an abnormally expressed protein. In addition, abnormally expressed proteins can be targeted to drugs that selectively or specifically attack cancer cells. Thus, diagnostic tests that analyze proteins and proteomic techniques that perform analysis are advantageous.

  The field of proteomics strives to establish the identity, quantity, structure, and biochemistry and cellular function of all proteins in an organism. The application of proteomics has historically progressed mainly on the basis of one protein at a time. The human proteome contains hundreds of thousands of proteins, and using recently developed proteomics technology, the changes in proteins over-expressed in cells in solid tissues and proteins shed in body fluids during disease exacerbations Now it can be inspected. Specific proteins and protein patterns that have been found to be differentially expressed in diseased versus normal cells reflect certain disease states and can be diagnostic.

  In recent years, advanced technologies and methods have been developed that provide an interface between clinical medicine / pathology and proteomics. High throughput exhaustive proteomic analysis techniques such as liquid chromatography-tandem mass spectrometry (LC-MS / MS) can be used to create a proteomic profile from disease specific biological samples. Such an exhaustive profile can be performed on all types of biological samples, such as frozen tissue, formalin-fixed tissue, and body fluids.

  Without convenient and reliable target screening / diagnostic tests for cancer, the lack of molecular diagnostic assays plagues the health care system and continues to complicate efforts to detect and treat malignant tumors early in them. Breast cancer protein biomarkers that are differentially expressed in early, invasive versus early, non-invasive breast cancer tissues can significantly ameliorate breast cancer diagnosis, diagnosis and prognostic potential, thereby afflicting women with breast cancer. It will form the basis of a “personalized medicine” approach to reduce and provide a target for the development of drugs that can treat breast cancer more effectively. In addition, the presence of these biomarkers in bodily fluids due to localized excretion into the breast tissue lumen and ultimately into the blood can be sampled for proteomics-based screening and early detection. Will provide a bodily fluid that can. The development of proteomics-based diagnostic / screening tests and treatment strategies for early breast cancer would represent an important medical advancement to a “personalized medicine” approach for breast cancer diagnosis, prognosis, and therapy.

  The present disclosure provides, among other things, a method of diagnosing the presence of recurrent breast cancer disease that is masked by a histological appearance as a less invasive, non-recurrent form of the disease. A sample is obtained from the patient. The sample is a bodily fluid such as serum or aspirate that may contain breast cancer tissue, breast cancer cells, or cells / proteins derived from the patient's cancer tissue. The presence and level of expression of at least one, two, three, four, five, six, seven, eight or more proteins listed in Table 1 are determined in the sample. The level of protein expression detected in (early, recurrent breast cancer tissue) is compared to the expression level of the same protein in early non-recurrent breast cancer. Differential expression of at least one protein or a combination of proteins indicates the presence of breast cancer disease that tends to recur in patients who are unrelated to either current or past treatment. In this way, prognosis can be determined, which predicts whether breast cancer (eg, early breast cancer) tends to recur after initial treatment. In one embodiment, the protein, or peptide fragment thereof, is detected by mass spectrometry, and the expression level of at least one or more proteins is determined by spectral count quantization mass spectrometry or selective reaction monitoring (SRM) mass spectrometry; Is also referred to as multiple reaction monitoring (MRM) mass spectrometry and is hereinafter alternatively referred to as SRM / MRM assay (s). In another embodiment, proteins are detected and their expression levels are determined by protein microarray or immunoassay.

  This disclosure also provides a method for identifying protein targets for therapeutic intervention in breast cancer. The presence and expression levels of one, two, three, four, five, six, seven, eight or more proteins listed in Table 1 are detected in the sample. The expression level of the protein detected in early breast cancer tissue is compared to the expression level of the same protein in early, non-recurrent) breast cancer. Differential expression of one, two, three, four, five, six, seven, eight or more proteins indicates the choice of therapy and defines a specific target for therapeutic intervention in breast cancer can do.

  Sample selection for assessing protein expression includes solid tissue (normal or diseased) and bodily fluids obtained from the patient by surgical means such as biopsy and aspiration. Protein expression is most conveniently detected and measured in cells or tissue samples from solid tumor tissue. This is because they are the actual cells that proliferate and cause disease. However, sometimes it is less invasive and more comfortable for the patient to collect bodily fluids, such as blood, lymph and / or ascites surrounding the tumor itself. These fluid sources can include many of the proteins listed in Table 1, because they can be secreted by the tumor cells into the surrounding fluid, or the tumor cells themselves are from the solid tumor. Because they can be found and now found in fluids, often they are easier samples to collect from breast cancer patients. The proteins listed in Table 1 can be detected in either solid tissues or body fluids from breast cancer patients and levels can be measured.

In one embodiment, the collection of biomarkers provides a prognosis that early primary breast cancer can recur in patients after initial treatment,
(A) measuring the expression level of one, two, three, four, five, six, seven, eight or more proteins listed in Table 1 in a sample from a human patient; Wherein the sample comprises breast cancer tissue, breast cancer cells, or a body fluid containing protein from the patient's breast cancer sample, such as blood or ascites; and (b) Table 1 in non-recurrent breast cancer The one, two, three, four, five, six, seven, eight or more listed in Table 1 in recurrent breast cancer when compared to the expression levels of those proteins listed Determining an increase and / or decrease in the expression of the protein;
Here, the identification of one, two, three, four, five, six, seven, eight or more of those proteins is more prone to recurrence of primary breast cancer in the patient Show the potential. In another embodiment of the prognostic method, increasing one, two, three, four, five, six, seven, eight or more proteins listed in Table 1 are examined. In yet another embodiment of the prognostic method, one, two, three, four, five, six, seven, eight or more proteins listed in decreasing Table 1 are examined. In yet another embodiment of the prognostic method, increasing 1, 2, 3, 4, 4, 5, 6, 7, 8 or more proteins listed in Table 1 are decreased. Are tested in combination with one, two, three, four, five, six, seven, eight or more proteins listed in. The greater the number of biomarker proteins in Table 1 found to increase (overexpression) and / or decrease (underexpression) in a sample obtained from a patient, the more likely that primary breast cancer will recur in that patient. Becomes higher.

Certain embodiments of the invention are described below.
1. A method of prognosing that early primary breast cancer can recur in a patient after initial treatment, comprising the following steps:
a) measuring the expression level of at least one, at least two, at least three, or more and combinations and combinations of the proteins listed in Table 1 in a sample from a human patient, the sample comprising: A body fluid containing a protein from said sample of breast cancer tissue, breast cancer cells, or breast cancer of said patient, such as blood or ascites; and b) said at least one of the proteins listed in Table 1 in non-recurrent breast cancer Said at least one, at least two, at least two of the proteins listed in Table 1 in recurrent breast cancer when compared to the expression level of one or more, at least two, at least three, or more and combinations and combinations Determining an increase and / or decrease in expression of three or more, or multiple and combinations of primary milk Showing the likelihood that cancer is more likely to recur in said patient.
2. The method of embodiment 1, wherein the breast cancer sample consists essentially of breast epithelial cells.
3. The method of embodiment 1, wherein the bodily fluid includes, but is not limited to, fractionated or unfractionated blood, serum, plasma, lymph, or fluid collected by pleural effusion.
4). The method of embodiment 1, wherein the tissue is harvested by biopsy or surgical procedure.
5. The method of embodiment 4, wherein the tissue is chemically fixed and stored.
6). The method of embodiment 5, wherein the chemical fixation and storage comprises formalin fixation and paraffin embedding.
7). The method of embodiment 4 or 5, wherein the tissue is frozen.
8). The method of embodiment 1, wherein the protein is measured as an intact, full-length protein, or measured by measuring multiple or individual peptides obtained by fragmentation of an intact, full-length protein.
9. The method of any of embodiments 1-8, wherein the protein is detected by mass spectrometry and the measured expression level of the protein is determined by spectral count quantification after the mass analysis.
10. The method of any of embodiments 1-8, wherein the protein is detected by mass spectrometry and the measured expression level of the protein is determined by a selective reaction monitoring (SRM) assay.
11. The protein is detected by mass spectrometry, and the measured expression level of the protein is determined by a multiplex SRM assay called a multiple reaction monitoring (MRM) assay, where more than one protein is detected in a single mass analysis The method of any of embodiments 1-8, wherein the method is quantified.
12 The mass spectrometry is performed using LC-ESI-MS / MS, MALDI-MS, tandem MS, TOF / TOF, TOF-MS, TOF-MS / MS, triple quadrupole MS, and triple quadrupole MS. The method of any of embodiments 8-11, selected from the group consisting of / MS.
13. Embodiment 13. The method of embodiment 12, wherein the mass spectrometry comprises liquid chromatography-tandem mass spectrometry.
14 The method of any of embodiments 1-8, wherein said proteins are detected and their expression levels are determined by protein microarray or immunoassay.
15. The method of embodiment 14, wherein the immunoassay is selected from the group consisting of immunohistochemistry, Western blot, dot blot, and ELISA.
16. A method showing the selection of the primary breast cancer therapy most likely to recur after initial treatment, including the following steps:
a) detecting the presence of at least one, at least two, at least three, or more and combinations of the proteins listed in Table 1 in a sample derived from a human patient and measuring the expression level thereof. Wherein said sample comprises breast cancer tissue, breast cancer cells, or a bodily fluid containing protein from said patient's breast cancer said sample, eg, blood or ascites; and b) listed in Table 1 in non-recurrent breast cancer The at least one or more of the proteins listed in Table 1 in recurrent breast cancer when compared to the expression level of said at least one, at least two, at least three, or more and combinations of said protein, Increase and / or decrease expression of at least 2 or more, at least 3 or more, or multiple and combinations Determining that the primary breast cancer is more likely to recur in the patient.
17. Quantifying the amount of one or more, two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more proteins or their peptide fragments of Table 1. Including.
18. One or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or ten or more proteins, their peptides, or antibodies to them in Table 1. A composition comprising.
19. One or more, two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more peptides of the proteins of Table 1 each derived from a different protein The composition of Embodiment 18.
20. The composition of Embodiment 19 wherein each of the peptides is labeled with one or more isotopes independently selected from the group consisting of: 18O, 17O, 34S, 15N, 13C, 2H or combinations thereof.
1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 of the proteins in Table 1 increased in tissues derived from primary tumors that recurred within 21.2 years The composition of any of embodiments 19-20, comprising the above, or eight or more peptides.
22. One or more, two or more, three or more, four or more, five or more, six or more, seven of the proteins in Table 1 decrease in tissue from a primary tumor that has recurred within 22.2 years The composition of any of embodiments 19-21, comprising the above or eight or more peptides.
23. The composition of any of embodiments 18-22, wherein said composition is substantially pure or does not include other cellular components selected from any combination of other proteins, membrane lipids and / or nucleic acids.
24. Embodiments further comprising evaluating and / or determining the level (amount) or sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleic acids in the protein digest. The method of any one of 1-17.
25. 25. The method of embodiment 24, wherein the nucleic acid has a length independently selected from greater than about 15, 20, 25, 30, 35, 40, 50, 60, 75, or 100 nucleotide lengths.
26. The nucleic acid is about 150, 200, 250, 300, 350, 400, 500, 600, 750, 1,000, 2,000, 4,000, 5,000, 7,500, 10,000, 15,000. Or the method of embodiment 25, having a length independently selected from nucleotide lengths less than 20,000.
27. Evaluating and / or determining the level (amount) or sequence comprises determining either the sequence of nucleotides in the nucleic acid and / or the characteristics of the nucleic acid according to any one or more of the following: Any of 24-26 methods: performing nucleic acid sequencing, performing restriction fragment polymorphism analysis, performing hybridization with another nucleic acid, identifying one or more deletions and / or insertions, and / or mutations, For example, without limitation, determination of the presence of single base pair polymorphisms, transitions and / or transversions.
28. The method of any of embodiments 24-27, wherein the two or more nucleic acids encode the proteins of Table 1.
29. The method of any of embodiments 24-28, wherein the nucleic acid encodes a protein of the following SEQ ID NO: 1-20, 21-41, 1-10, 11-20, 21-30, 31-41 of Table 1, or Fragments of them.

Methods at the biomarker clinical medicine / pathology and proteomics interface were used to identify differentially expressed proteins between early, non-recurrent breast cancer epithelial cells and early, recurrent breast cancer epithelial cells. The protein list of the proteins provided in Table 1 provides comprehensive LC-MS / MS proteomic profiling of cells obtained from early, non-recurrent breast cancer tissue and early, recurrent breast cancer tissue; and early, non-recurrent breast cancer cells Were consistently overexpressed or underexpressed by comparing to early, recurrent breast cancer cells. It should be noted that many or all of these proteins can be readily assayed in body fluids derived from breast cancer cells, such as fluids derived from ascites or blood, such as plasma and serum. It is either breast-derived tissue, breast epithelial cells, or body fluid that is assayed for diagnostic assessment of breast cancer by assaying for specific protein expression from the list described herein . One or more of the same proteins also form the basis for a targeted therapeutic approach where drugs are directed towards these proteins. Identification of these proteins is most likely to recur at time points after initial treatment in various biological samples taken from subjects, such as fixed and frozen tissues, and body fluid samples derived from both blood and ascites Provides the ability to detect early breast cancer. Diagnostic and prognostic endpoints for disease analysis include both single analyte and proteomic patterns. Proteomic patterns can be composed of many individual proteins, each of which does not individually identify breast cancers that tend to recur, but collectively can identify breast cancers that have an increased probability of recurrence . Individual proteins, protein patterns, and / or collections of multiple proteins used for diagnosis, prognosis, and therapy of recurrent breast cancer are also provided.

  The methods provided herein enable assessment of the likelihood of recurrence of primary breast cancer and therapeutic strategies for subjects (patients) with breast cancer. The method is more invasive when breast cancer that appears to be early non-invasive by visual histological methods is potentially relapsed after a first assessment of the presence, absence, nature and / or extent of breast cancer Useful to determine if there is a possibility of developing advanced breast cancer. Breast cancer can be diagnosed in a subject by measuring one, two, three, four, five, six, seven, eight or more proteins from the list of proteins in Table 1. The prognosis of the subject can be determined and a drug specific for the subject's disease can be selected. Samples of tissue, such as those obtained surgically from a subject or biopsied, frozen, or chemically fixed, or bodily fluids such as blood, lymph, serum, plasma, and / or ascites Examine and protein expression is assessed and measured.

  The observed difference in the protein from the list of proteins in Table 1 found in a biological sample from a subject with breast cancer that is non-recurrent versus a biological sample from a subject with breast cancer that is recurrent is determined by the disease protein profile. Representing the presence, absence, nature or extent of cancer pathology in the patient.

  In one embodiment, the difference between the recurrent breast cancer protein profile and the reference non-recurrent breast cancer protein profile is one, two, three, four, five, six, seven from the list in Table 1. Including differences in the amount of eight or more biomarker proteins. A method for assessing breast cancer pathology in a subject comprises distinguishing between different disease states or between a disease state and a normal state. Such profiles are also used to determine prognosis, which aims to monitor the extent and prediction of breast cancer progression or regression in a subject. To this end, a recurrent breast cancer protein profile can be derived from a biological sample previously obtained from a subject, such as a biological sample obtained before treatment or as part of a general health screen.

  The method is also adapted to assess the effectiveness of therapeutic decisions, such as drugs or surgery. In the case of drug therapy selection, one or more proteins within the breast cancer protein profile may serve as targets for drug therapy. In one embodiment, the drug specifically interacts with individual, specific proteins from the list of proteins in Table 1. In another embodiment, the drug interacts with a binding partner of a protein from the list of Table 1, thereby altering the ability of the protein of Table 1 to interact with that binding partner or perform a biological function. . In yet another embodiment, the expression profile of one, two, three, four, five, six, seven, eight or more proteins selects drug therapy and / or period / regimen Can be used for.

  The method further includes from the protein list of Table 1 between a test protein profile of a biological sample from a subject suspected of having recurrent breast cancer and a reference protein profile from a biological sample from a subject not having recurrent breast cancer. A classification model or algorithm based on one or more protein differences.

  In some embodiments, recurrent or non-recurrent breast cancer protein profiles or both are generated using mass spectrometry. In such embodiments, the mass spectrometry method used may conveniently use an ion trap instrument or a triple quadrupole instrument. In general, for analysis by mass spectrometry, intact full-length proteins are reduced to individual peptides by treatment of protein samples with proteolytic enzymes such as trypsin, papain, chymotrypsin, etc. It is considered as a composite lysate. Such peptide lysates are a preferred form of a sample for the analysis of proteins from biological samples by mass spectrometry, where the quantitative presence of a specific individual peptide is the intact full-length protein from which the peptide is derived. The quantitative presence of In one embodiment, analysis of all peptides simultaneously in an exhaustive manner can be conveniently performed on an ion trap mass spectrometer. In one embodiment, analysis of individual specific peptides and thus target peptides that specifically concentrate the assay on the protein from which they are derived is performed on a triple quadrupole mass spectrometer. Implementation of targeted quantitative protein analysis by triple quadrupole mass spectrometry can be accomplished using SRM / MRM methods. The method can be used to create a protein profile to investigate the likelihood of recurrent breast cancer in a subject from which a biological sample was obtained.

  Prior to analysis by mass spectrometry, the peptides in the lysate may be subjected to various techniques that facilitate their analysis and measurement by mass spectrometry. In one embodiment, the peptide is suitable by affinity techniques such as immunological purification (eg immunoaffinity chromatography), chromatography on ion selective media, or if the peptide is modified. Separation can be achieved by separation using a simple medium such as a lectin for separation of carbohydrate modified peptides. In one embodiment, a SISCAPA method that employs immunological separation of peptides prior to mass spectrometry is used. SISCAPA technology is described, for example, in US Pat. No. 7,632,686. In other embodiments, lectin affinity methods (eg, affinity purification and / or chromatography can be used to separate peptides from lysates prior to analysis by mass spectrometry. Methods for separation of groups of peptides, For example, lectin-based methods are described, for example, in Geng et al., J. Chromatography B, 752: 293-306 (2001) Immunoaffinity chromatography techniques, lectin affinity techniques, and other forms of affinity. Separation and / or chromatography (eg, reverse phase, size-based separation, ion exchange) can be used in any suitable combination to facilitate analysis of the peptide by mass spectrometry.

  Another assay method is to immobilize proteins and / or protein-derived peptides on a microarray (eg, using immobilized antibodies) prior to detecting the protein using antibody-based methods including sandwich-type assays. Including) Other assay methods include immunohistochemical analysis using antibody-based protein detection methods on thin tissue sections, where the protein remains intact in the tissue section (not subject to proteolysis). ). Still other assay methods include antibody-based Western blots and ELISA protein detection methods, in which the protein preparation examined is an intact full-length protein and / or a derived peptide. All of these described protein detection methods can be used to detect individual polypeptides derived from the entire intact protein, and thus these methods do not necessarily require detection of the entire intact protein. Although not, it can include detection of peptides derived from the whole intact protein. These methods can be used alone or in any combination, for example in combination with a method based on mass spectrometry. Any suitable reporting / detection system known in the art can be used with such assays, including but not limited to fluorescence, UV / Vis chromatophore expression, plasmon resonance, metal staining, etc. Is mentioned.

  Accordingly, methods useful for detecting proteins from the protein list of Table 1 and polypeptides derived from these proteins are provided. The presence, absence, nature or extent of breast cancer pathology indicative of recurrent breast cancer disease in a patient is expressed by one or more expressed biomarker proteins from the list and / or expression of one or more derived peptides from the same protein Can be evaluated.

  In yet another embodiment, a method is provided for screening a patient or population of patients for breast cancer by assaying for the presence of one or more proteins found in Table 1, or their derived peptides. . The assay (s) used can be mass spectrometry assays, immunoassays such as Western blots, enzyme-linked immunosorbent assays (ELISAs), or immunohistochemical methods on intact tissue sections, or any combination thereof May be included. As described above, multiple (eg, one, two, three, four, five, six, seven, eight or more) proteins that increase or decrease with increasing likelihood of breast cancer recurrence or Derived peptides can be analyzed, thus increasing the predictive power of screening assays. In one embodiment, one, two, three, four, five, six, seven, eight, eight or more proteins listed in Table 1 that increase in one embodiment are listed in Table 1 that decreases. In combination with one, two, three, four, five, six, seven, eight or more proteins.

Biomarker Identification Protein biomarkers (eg, the proteins in Table 1) are breast cancer epithelial cells obtained from primary tumors that caused recurrent breast cancer after surgery and recurrent after surgery that is unrelated to current or conventional treatment. Selection was based on the differential pattern of their expression observed in breast cancer epithelial cells obtained from primary tumors that were breast cancers that did not cause breast cancer. The levels of some proteins were increased in cancer cells obtained from recurrent breast cancer tissue and the levels of other proteins were decreased in recurrent cancer tissue cells.

  The data in Table 1 were collected by mass spectrometry analysis of protein lysates from tissue and cells of patients suffering from recurrence of breast cancer and non-breast cancer. Protein lysates obtained from the cells of the two patient populations contain all the necessary information about differential protein expression. Protein lysates from cells of these patient populations were prepared using the Liquid Tissue ™ protocol and reagents. In the preparation method, cells (tissue sample) are collected into a tube by a tissue microdissection, and then the cells (tissue sample) are maintained at a high temperature in a buffer solution for a long time (for example, about 80 ° C. to about 100 Reversing or releasing protein cross-linking at a temperature of about 10 minutes to about 4 hours). The buffer used is a neutral buffer (eg, a Tris-based buffer or a buffer containing a surfactant), conveniently a buffer that does not interfere with mass spectrometry. As soon as the formalin-induced crosslinking is negatively affected, the cells are then fully digested in a predictable manner using a protease (eg, trypsin). The result of heating and proteolysis is a liquid, soluble, dilutable biomolecule lysate.

  The prepared lysate is then analyzed by exhaustive proteomic mass spectrometry and the data is initially presented as an identification of the total number of peptides in each protein lysate. As soon as as many peptides as possible were identified in a single MS analysis of a single lysate, that list of peptides was then compared to the list of peptides identified across all lysates in the study set. . Thus, the starting point for determining differential protein expression by mass spectrometry is a list of peptides found to be expressed in one sample and / or a group of similar samples, a second sample and / or Or compared to a list of peptides found to be expressed in groups of similar samples. The first group of 4 (4) Liquid Tissue ™ samples is derived from primary breast cancer tissue from patients who did not recur at least 2 years after initial treatment, and 5 (5) Liquid Tissue ™. The second group of samples was derived from early primary breast cancer tissue from patients who relapsed within 2 years after initial treatment. Comparison of those proteins differentially expressed in recurrent early breast cancer versus non-recurrent early breast cancer between these two patient groups formed the initial study set of proteins described in Table 1.

  The classification of differential protein expression from the list of peptides found in patients with relapses and patients without recurrent breast cancers first determined which proteins were represented by the given peptide list; This was then achieved by counting the total number of peptides identified for each protein. The data collation method is known as a spectral count method (SC). Thus, the spectral count for a protein is based on the total number of peptides identified for that protein in a single lysate, which is a relative indicator for the abundance of that protein in the lysate analyzed by MS. Spectral counting is a mathematical method that provides the ability to compare the relative protein abundance for a protein from one sample and / or group of similar samples with the next sample and / or group of similar samples. This approach can also be used to distinguish protein abundance among individual proteins within a sample.

  Spectral counts between thousands of individual proteins derived from breast cancer epithelial cells derived from tumors from multiple primary patients that caused recurrent breast cancer and from multiple primary patients that did not cause recurrent breast cancer In samples obtained from breast cancer epithelial cells obtained from different tumors.

  Thus, protein abundance was obtained by mass spectrometry of protein lysates from multiple breast cancer tissues using peptide spectral counting (SC). In addition, peptides whose sequences were located in multiple protein isoforms were grouped according to the principle of saving. To determine statistically significant changes in protein abundance across patient samples by disease stage subgroup, identified from non-relapsed stage II (NRII) versus stage with recurrent disease (stage IIR) patient samples A hierarchical supervised cluster analysis of peptides was performed. In this case, the Mann-Whitney rank corresponding to the filter criteria requires that the variance of the total spectral count peptide identified identifies that 60% of the samples in the supervised group have a minimum peptide count of 2 or more for a protein. Determined using sum test (significance level p ≦ 0.05, Fisher exact test).

  The selection of proteins in Table 1 is significant in stage II breast cancer tissue from patients with recurrent disease (stage IIR) versus breast cancer tissue from patients who did not have recurrent disease (stage NRII) within 2 years ( Significance level p ≦ 0.05, Fisher exact probability test) Limited to those proteins that showed high or low spectral count abundance.

Table 1 shows the names of 41 proteins, 31 being increased in abundance and 11 being decreased, which significantly differentiate between stage NRII versus stage IIR patients. In one embodiment, the prognostic method uses at least one or more proteins having an increased level, in another embodiment, a decreased level, and in yet another embodiment, increased and decreased. Use a combination of levels. In addition, prognostic methods can include specific combinations of decreased and / or increased expression across multiple proteins in a single assay, indicating a pattern of protein expression changes indicative of and prognostic of early recurrent breast cancer Is given. The information shown from left to right along the top of Table 1 is: 1) Uniprot accession number, 2) Log ₂ ratio spectral count change between recurrent breast cancer and non-recurrent breast cancer, 3) Protein Abbreviations and 4) names of proteins. All the proteins in this list meet the criteria for a P value of less than 0.05 indicating their significance, so that each of these proteins is most likely to relapse and diagnose invasive breast cancer, Identified as a candidate biomarker for breast cancer that can be used for prognosis or therapeutic targets.

  The methods include those using related proteins as well as diagnostic, prognostic, therapeutic treatment methods and compositions using the proteins listed in Table 1. In one embodiment, the related protein comprises a protein / polypeptide that shares at least some amino acid sequence with the protein of Table 1, which is another transcript (or from a gene encoding the protein of Table 1 (or Produced by translation of a separately processed transcript). In another embodiment, related proteins include proteins / polypeptides that share at least some amino acid sequence with the proteins of Table 1 produced by translation or alteration at the post-translational level (eg, post-translational modification). In any embodiment, the related protein may comprise more than 5, 6, 7, 8, 10, 12, 15, 18, or 20 contiguous amino acids that are identical to the sequence found in the protein of Table 1.

Embodiments provided herein include one or more, two or more, three or more, four or more, five or more, six or more, eight or more, or ten or more proteins of Table 1, or those A composition comprising a polypeptide fragment of In some embodiments, the composition comprises two or more, three or more, four or more, five or more, six or more, or seven or more proteins found in Table 1 or peptide fragments of those proteins. An antibody that specifically binds to. A composition comprising a peptide may comprise one or more, two or more, three or more, four or more, five or more, six or more, eight or more, or ten or more peptides that are isotopically labeled. Each of the peptides can be labeled with one or more isotopes independently selected from the group consisting of: ¹⁸ O, ¹⁷ O, ³⁴ S, ¹⁵ N, ¹³ C, ² H or combinations thereof. A composition comprising a peptide derived from any of the proteins in Table 1 regardless of whether it is isotopically labeled, should contain all of the peptides derived from any given protein (eg, a complete set of tryptic peptides). There is no. In some embodiments, the composition is one for two, three, four, five, six, seven, eight, nine, ten or more proteins appearing in Table 1 or Table 2. Contains only three, four, five, six, or seven peptides. The composition comprising the peptide can take the form of a dried or lyophilized material, a liquid (eg, aqueous) solution or suspension, an array, or a blot.

Use of Biomarkers The protein biomarkers described herein can be advantageously used to improve the treatment of patients with breast cancer. Using the ability to assay overexpression and / or underexpression of one or more proteins in recurrent breast cancer versus non-recurrent breast cancer and this overexpression and / or underexpression in biological samples, It can be determined whether or not it has a type of cancer that tends to be. In the case of a protein profile that suggests that the patient has a form of breast cancer that tends to recur, the results may also be different from those used for patients with non-recurrent breast cancer Can indicate selection. Furthermore, decisions based on altered expression of multiple proteins are likely to be more effective as indicators of recurrent breast cancer than individual assessments of one or two proteins. The method includes and provides for simultaneous assessment and correlation of multiple proteins in a single biological sample from an individual suspected of suffering from a recurrent form of breast cancer.

  The best course of disease treatment using the overexpression and / or underexpression of one or more proteins in recurrent breast cancer versus non-recurrent breast cancer and the ability to assay this overexpression and / or underexpression in a biological sample Can help determine which therapeutics are selected to achieve this. One or more of the proteins shown herein can be directly targeted by a drug, so that breast cancer cells are preferred over normal cells in tissues that do not express one or more of these proteins. Can be killed.

  Types of biological samples that are assayed using one or more of these proteins as biomarkers for recurrent breast cancer include tissue that has been biopsied or surgically removed. The tissue can be fresh, frozen, and / or chemically fixed, such as stored in formalin and other similar chemical fixatives. Another form of biological sample that can be taken is a fractionated or non-fractionated biological fluid sample, such as serum, plasma, whole blood, and ascites. All of these forms of patient-derived biological samples can be assayed for expression of one or more proteins in Table 1.

  Since both nucleic acids and proteins can be analyzed from the same biomolecular lysate preparation used herein (eg, US Pat. No. 7,473,532), from the same sample, disease diagnosis and drug treatment decisions Additional information about can be created. For example, additional information regarding cellular status and their uncontrolled growth, potential drug resistance, and potential for cancer development can be obtained by analyzing nucleic acids from their lysate preparations. By using lysate preparations for both protein / peptide analysis and nucleic acid analysis, any 1, 2, 3, 4, 5 or more genes and / or nucleic acids and / or the state of the protein they encode Information (eg, mRNA molecules and their expression levels or splice variants) can be obtained from the same biomolecule lysate preparation. For example, information about any one, two, three, four, five or more peptides in Table 1 and / or proteins from which they are derived or nucleic acids encoding those proteins can be evaluated. Nucleic acids can be examined, for example, by: one or more sequencing methods, performing restriction fragment polymorphism analysis, performing hybridization with another nucleic acid, deletion, insertion identification and / or mutation For example, but not limited to, the determination of the presence of single base pair polymorphisms, transitions and / or transversions. Such testing may be performed in any suitable format, such as, but not limited to, an array, microarray, blot, or in solution (eg, by polymerase chain reaction “PCR” or ligase chain reaction “LCR”). Can do.

  If hybridization with another nucleic acid is used, the assay or test can be performed in any suitable format (eg, array / microarray, blot, etc.) with the appropriate stringency of the nucleic acid to obtain specific binding. It can be carried out by contacting under conditions. The required “stringency” of a hybridization reaction can be determined by one skilled in the art and generally includes empirical calculations that depend on probe length, wash temperature, and salt concentration. In general, the longer the probe, the higher the temperature required for proper annealing, and the shorter the probe, the lower the required temperature. Hybridization generally depends on the ability of denatured DNA to anneal with complementary strands if they are present in an environment below their melting temperature. The higher the desired degree of homology between the probe and hybridizable sequence, the higher the relative temperature that can be used, the higher the relative temperature, the more likely the hybridization reaction will be stringent, The reverse is also true. See, for example, Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995). Hybridization reactions typically use stringent conditions or moderately stringent conditions.

  “Stringent conditions” are typically low ionic strength, with or without denaturing agents (eg, formamide), and high temperatures for washing, eg, 0.015 M sodium chloride / 0.0015 M citric acid at 50 ° C. Sodium acid / 0.1% sodium dodecyl sulfate is used.

  “Moderately stringent conditions” can be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, as described above. Use of less stringent wash solutions and hybridization conditions (eg, temperature, ionic strength and% SDS). An example of moderately stringent conditions is overnight incubation at 37 ° C. in a solution containing: 20% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (PH 7.6) 5X Denhardt's solution, 10% dextran sulfate, and 20 mg / ml denatured sheared salmon sperm DNA, followed by washing in 1XSSC at about 37-50 ° C. One skilled in the art will recognize how to adjust temperature, ionic strength, etc. as needed to accommodate factors such as probe length.

  In one embodiment, the sample is one, two, three, four, five, six, seven, eight, nine or more peptides generated from the proteins of Table 1 and / or One or more of those peptides or they are analyzed against a nucleic acid encoding a protein obtained by proteolysis. In one embodiment, the sample is 2, 3, 4, 5, 6, 7, 8, 9 or more peptides generated from the proteins of Table 1 and / or from Table 1. Analyzed against 2, 3, 4, 5, 6, 7, 8, 9 or more nucleic acids encoding the protein, where the protein from Table 1 is any range of proteins represented by the following SEQ ID NOs: Selected from: 1-20, 21-41, 1-10, 11-20, 21-30, 31-41 of Table 1.

EXAMPLE 5 (5) formalin-fixed breast cancer tissue samples obtained from patients who relapsed breast cancer within 2 years and 4 (4) breast cancer tissue samples obtained from patients who did not relapse breast cancer within 2 years Were examined for differential protein expression correlated with cancer. These proteins can be used to improve breast cancer diagnosis, prognosis, and therapy.

  Tissue sections were prepared from each tissue for histological analysis and epithelial cancer cell acquisition was performed by tissue microdissection. Soluble protein lysate was prepared from a microdissected breast cancer tissue sample using Liquid Tissue ™ MS Protein Prep Kit (Expression Pathology, Inc.). Each lysate consisted of the total protein content of microdissected cells digested into peptide fragments predictable by the protease trypsin. In this form, each and every protein lysate can be evaluated by mass spectrometric techniques for the identification and quantification of the proteins present in each lysate. In addition, using total mass spectrometry data across all samples, differential protein expression between individual samples and between primary tumors from patients with non-recurrent breast cancer and patients with recurrent breast cancer decide.

  Mass analysis of each trypsin digested protein lysate was performed as follows. Liquid chromatography (LC) was performed using a Dionex Ultimate 3000 system coupled to a ThermoFisher linear ion trap mass spectrometer (MS) online. Sample separation was performed using a 75 μm ID × 360 μm OD × 10 cm long fused silica capillary column 5 μm, 300 Å pore size Jupiter C-18 stationary phase. After injection of 5 μl of resuspended protein lysate, the column is washed with 98% mobile phase A (water with 0.1% formic acid) for 30 minutes, and the peptide is washed with 2% mobile phase B (acetonitrile with 0.1% formic acid). ) Elution with a linear gradient of -42% mobile phase B at 140 minutes followed by 98% B for an additional 20 minutes (all at a constant flow rate of 250 nL / min). A linear ion trap mass spectrometer (LIT-MS) is operated in a data-dependent MS / MS mode, where 7 MS / MS scans after each full MS scan (precursor ion selective scan range of m / z 350-1800) , Where the seven most abundant peptide molecular ions were selected for tandem MS using 35% relative collision induced dissociation (CID) energy. Dynamic exclusion was used to minimize redundant selection of peptides for CID.

Peptide identification was performed on a SEQUEST (BioWorks) on a 72-node Beowulf cluster using the following parameters against the UniProt derived human proteome database (version 10/08, 56,301 protein entries) obtained from the European Bioinformatics Institute (EBI). , V3.2, Thermo Scientific) obtained by examining LC-MS / MS data: trypsin (KR); total enzymatic cleavage; two missing cleavage sites; 1.5 Da peptide mass resistant peptide resistance, Various modifications for 0.5 Da fragment ion resistance and methionine oxidation (m / z 15.99492). The resulting peptide identification was filtered according to specific SEQUEST scoring criteria: δ correlation (ΔC _n ) ≧ 0.08 and charge state dependent cross correlation (Xcorr) ≧ 1.9 ([M + H] ¹⁺ ), ≧ 2. 2 ([M + 2H] ²⁺ ), and ≧ 3.5 ([M + 3H] ³⁺ ) (Supplementary Table 1). By these criteria, 5.84% false discovery rate (FDR) for all peptides identified as determined by examining the entire data set against a decoy human database with reversed protein sequences It became. Protein abundance was derived by spectral counting (SC), and peptides whose sequences were located in multiple protein isoforms were grouped according to the principle of saving. Stage II patient samples with non-recurrent disease stage II (IINR) versus recurrent disease (stage IIR) to determine statistically significant changes in protein abundance across patient samples by disease stage subgroup A hierarchical control cluster analysis of the identified peptides was performed, where the variance of the total spectral count peptides identified was 60% of the samples in the supervised group had a minimum peptide count of 2 or more for a protein. Mann-Whitney rank sum test (significance level p ≦ 0.05, Fisher's exact test) corresponding to the filter criteria required to have.

  Reliable peptide data was used to combine peptide lists for samples, eliminate redundant peptide identification, and generate a unique list of peptides. Each peptide in the list is already associated with a protein, so the list was easily converted to a list of proteins, and specifically a unique protein list was created for each patient sample. Based on these data, a quantitative analysis to determine differential protein expression between recurrent breast cancer and non-recurrent breast cancer was performed using a spectral count quantification method. Spectral count quantification is the process of counting the number of unique peptides associated with each protein. A value of 4 beside the protein name reflects the presence of four unique peptides associated with that particular protein. There could be many repeated identifications for any of the individual peptides, but the count was based on the unique peptide and not the total peptide. This count directly correlates with the relative abundance of each particular protein, so the more unique peptides identified for a protein, the greater the relative expression of that protein in any particular sample.

  The goal of this data analysis was to identify those proteins whose quantitative expression levels derived from them showed significant differences in expression between recurrent breast cancer and non-recurrent breast cancer samples. These proteins, identified by a much larger number of unique peptides in recurrent breast cancer cells than non-recurrent breast cancer cells, are the most likely candidates for novel biomarkers of invasive recurrent breast cancer Established standards. Cluster analysis is a statistical method to determine which items are significantly different between two separate groups, identifying 41 proteins differentially expressed between recurrent breast cancer cells and non-recurrent breast cancer These are listed in Table 1.

Selective Response Monitoring Assay for Analysis of Patient Tissues The SRM / MRM assay described herein is a relative or absolute quantitative analysis of one or more specific peptides derived from one or more proteins listed in Table 1. Level can be measured. Using this method, the amount of a peptide, peptides, proteins, or proteins can be transferred to a biological sample of a patient, eg, a peptide / protein preparation or formalin-fixed paraffin-embedded tissue obtained from a body fluid. A means of measuring by mass spectrometry on the derived Liquid Tissue ™ lysate is provided. The SRM / MRM assay can measure peptides directly in complex protein lysates prepared from patient tissue samples such as formalin-fixed cancer patient tissue.

  Methods for preparing protein samples from formalin-fixed tissue are described in US Pat. No. 7,473,532, the contents of which are hereby incorporated by reference in their entirety. The method described in that patent is described in Expression Pathology, Inc. (Liquid Tissue ™ reagent available from (Rockville, MD).

  Using the results from the SRM / MRM assay, correlate an accurate, accurate quantitative level of a peptide, multiple peptides, proteins, or multiple proteins with the specific cancer of the patient from whom the biological sample was taken Can be made. This not only provides diagnostic information about the cancer, but also allows a doctor or other health care professional to determine the appropriate therapy for the patient. Such assays provide diagnostically and therapeutically important information about protein expression levels in diseased tissue or other patient samples, such as body fluids, and are referred to as companion diagnostic assays. For example, such an assay can be designed to diagnose the stage or extent of cancer and to determine the therapeutic agent or course of therapy that is most likely to respond to the patient and yields positive results. . An SRM / MRM assay can measure the relative or absolute level of a protein, or a specific unmodified peptide derived from a protein, and can also determine the absolute or relative level of a specific modified peptide derived from a protein. Examples of modifications include phosphorylated amino acid residues and glycosylated amino acid residues that are present on the peptide.

  A relative quantitative level of a peptide, a plurality of peptides, a protein, or a plurality of proteins is determined by the SRM / MRM method, where individual proteins from one protein or a plurality of proteins in a biological sample are determined. The signature peak area (or peak height if the peaks are well separated) derived from mass spectrometry of the peptide, or multiple peptides, using the same method in one or more additional and different biological samples , Compared to the signature peak area determined for the same single protein, or the same identical peptide from multiple proteins, or multiple peptides. Thus, the amount of a certain protein, or a specific peptide from a plurality of proteins, or a plurality of peptides is equal to the same one protein, or the same one peptide from a plurality of proteins, or a plurality of peptides. In contrast, it is determined under the same experimental conditions across two or more biological samples. In addition, relative quantification can be performed on a single peptide or a plurality of peptides derived from a single protein within a single sample by using the SRM / MRM method to calculate the signature peak area for that peptide against that protein. Can be determined by comparing the signature peak areas for different proteins in the same protein preparation, or for different, different peptides, or peptides from multiple proteins. Thus, the amount of a particular peptide from a protein, and thus the amount of that protein, is determined relative to each other within the same sample. These approaches generate quantification between samples and within a sample for the amount of an individual peptide or peptides from one protein to another peptide or peptides. In some cases, the amounts determined by the signature peak area are relative to each other, regardless of the absolute weight-by-volume or weight-by-weight amount of the peptide in the protein preparation from the biological sample. Relative quantitative data for individual signature peak areas between different samples is normalized to the amount of protein analyzed per sample. Relative quantification can be performed across many peptides simultaneously, in a single sample, and / or across many samples, providing relative protein content, insights about one peptide / protein relative to other peptides / proteins.

  The absolute quantitative level of a protein or proteins is determined by the SRM / MRM method, where the SRM / MRM signature peak areas of individual peptides from a protein within a biological sample are known The amount of “added” internal standard SRM / MRM signature peak area is compared. In one embodiment, the internal standard is a synthetic version of the same exact peptide that includes one or more amino acid residues that are labeled with one or more heavy isotopes. Such isotopically labeled internal standards are synthesized so that mass spectrometry produces distinct, predictable and consistent SRM / MRM signature peaks that are distinct from the native peptide signature peaks Can be used as a vessel peak. Thus, when an internal standard is added in a known amount over a protein or peptide preparation from a biological sample and analyzed by mass spectrometry, the signature peak area of the native peptide is compared to the signature peak area of the internal standard peptide This numerical comparison indicates either the absolute molar concentration and / or the absolute weight of the natural peptide present in the original protein preparation from the biological sample. Absolute quantitative data for fragmented peptides is displayed according to the amount of protein analyzed per sample. Absolute quantification can be performed across many peptides, and therefore proteins, in a single sample and / or across many samples simultaneously, providing insights into the amount of absolute protein in individual biological samples and the entire cohort of individual samples. can get.

  The SRM / MRM assay method, for example, directly aids in the diagnosis of the stage of cancer in a patient-derived tissue, eg, formalin-fixed tissue, and is the most advantageous therapeutic agent for use in treating the patient, and / or Or it can be used to help determine a treatment strategy. For example, cancer tissue removed from a patient can be analyzed by surgery for therapeutic removal of partial or whole tumors, or by biopsy procedures performed to determine the presence or absence of a suspected disease. It is determined whether the specific protein, or proteins, and which form of protein is present in the patient tissue. In addition, the expression level of one or more proteins can be determined and compared to “normal” or baseline levels found in healthy tissue or tissues exhibiting different stages / stages of cancer. This information can then be used to assign a stage or stage to a particular cancer and can be adapted to strategies for treating patients based on the determined level of specific proteins. Adapting specific information about the level of a protein or proteins as determined by the SRM / MRM assay to a therapeutic strategy based on the level of these proteins in cancer cells derived from a patient Define what is called a personalized medical approach to treatment. The SRM / MRM assay methods described herein form the basis of an individualized medical approach by using analysis of proteins from the patient's own tissue as a source for diagnostic and therapeutic decisions. The SRM / MRM method described herein can be used to specifically assay the proteins in Table 1.

  Although the present invention has been described in connection with certain embodiments thereof, many details have been set forth for purposes of illustration, and the present invention is susceptible to additional embodiments and has been described herein. It will be apparent to those skilled in the art that some of the details described in can be varied significantly without departing from the basic principles of the invention described herein.

Claims (29)

A method of prognosing that early primary breast cancer can recur in patients after initial treatment, comprising the following steps:
a) measuring the expression level of at least one, at least two, at least three, or more and combinations and combinations of the proteins listed in Table 1 in a sample from a human patient, the sample comprising: A body fluid containing a protein from said sample of breast cancer tissue, breast cancer cells, or breast cancer of said patient, such as blood or ascites; and b) said at least one of the proteins listed in Table 1 in non-recurrent breast cancer Said at least one, at least two, at least two of the proteins listed in Table 1 in recurrent breast cancer when compared to the expression level of one or more, at least two, at least three, or more and combinations and combinations Determining an increase and / or decrease in expression of three or more, or multiple and combinations of primary milk Showing the likelihood that cancer is more likely to recur in said patient.   The method of claim 1, wherein the breast cancer sample consists essentially of breast epithelial cells.   2. The method of claim 1, wherein the bodily fluid includes, but is not limited to, fractionated or non-fractionated blood, serum, plasma, lymph, or fluid collected by pleural effusion.   The method of claim 1, wherein the tissue is collected by biopsy or surgical procedure.   The method of claim 4, wherein the tissue is chemically fixed and stored.   6. The method of claim 5, wherein the chemical fixation and storage includes formalin fixation and paraffin embedding.   The method of claim 4, wherein the tissue is frozen.   2. The method of claim 1, wherein the protein is measured by measuring multiple or individual peptides, measured as intact, full-length protein, or obtained by fragmentation of intact, full-length protein.   The method according to claim 1, wherein the protein is detected by mass spectrometry, and the measured expression level of the protein is determined by spectral count quantification after the mass analysis.   9. The method of any one of claims 1 to 8, wherein the protein is detected by mass spectrometry and the measured expression level of the protein is determined by a selective reaction monitoring (SRM) assay.   The protein is detected by mass spectrometry, and the measured expression level of the protein is determined by a multiplex SRM assay called a multiple reaction monitoring (MRM) assay, where more than one protein is detected in a single mass analysis The method according to claim 1, wherein the method is quantified.   The mass spectrometry is performed using LC-ESI-MS / MS, MALDI-MS, tandem MS, TOF / TOF, TOF-MS, TOF-MS / MS, triple quadrupole MS, and triple quadrupole MS. 9. The method of claim 8, wherein the method is selected from the group consisting of / MS.   The method of claim 12, wherein the mass spectrometry comprises liquid chromatography-tandem mass spectrometry.   9. The method according to any one of claims 1 to 8, wherein the proteins are detected and their expression levels are determined by protein microarray or immunoassay.   15. The method of claim 14, wherein the immunoassay is selected from the group consisting of immunohistochemistry, Western blot, dot blot, and ELISA. A method showing the choice of therapy for the primary breast cancer most likely to recur after initial treatment, including the following steps:
a) detecting the presence of at least one, at least two, at least three, or more and combinations of the proteins listed in Table 1 in a sample derived from a human patient and measuring the expression level thereof. Wherein said sample comprises breast cancer tissue, breast cancer cells, or a bodily fluid containing protein from said patient's breast cancer said sample, eg, blood or ascites; and b) listed in Table 1 in non-recurrent breast cancer The at least one or more of the proteins listed in Table 1 in recurrent breast cancer when compared to the expression level of said at least one, at least two, at least three, or more and combinations of said protein, Increase and / or decrease expression of at least 2 or more, at least 3 or more, or multiple and combinations And indicating the likelihood that primary breast cancer is more likely to recur in said patient.   Quantifying the amount of one or more, two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more proteins or their peptide fragments of Table 1. Including.   One or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or ten or more proteins of Table 1, peptides thereof, or antibodies thereto A composition comprising.   One or more, two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more peptides of the proteins of Table 1 each derived from a different protein The composition according to claim 18.   20. The composition of claim 19, wherein each of said peptides is labeled with one or more isotopes independently selected from the group consisting of: 18O, 17O, 34S, 15N, 13C, 2H or theirs combination.   1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more of the proteins of Table 1 that increase in tissue from a primary tumor that has recurred within 2 years, 21. The composition of claim 19 or 20, comprising 8 or more peptides.   1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more of the proteins of Table 1 that decrease in tissue from a primary tumor that has recurred within 2 years, 21. The composition of claim 19 or 20, comprising 8 or more peptides.   1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more of the proteins of Table 1 that decrease in tissue derived from a primary tumor that recurs in 2 years, Or the composition of claim 21 comprising 8 or more peptides.   The method further comprises evaluating and / or determining the level (amount) or sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleic acids in the protein digest. The method as described in any one of 1-8.   25. The method of claim 24, wherein the nucleic acid has a length independently selected from a nucleotide length greater than about 15, 20, 25, 30, 35, 40, 50, 60, 75, or 100.   The nucleic acid is about 150, 200, 250, 300, 350, 400, 500, 600, 750, 1,000, 2,000, 4,000, 5,000, 7,500, 10,000, 15,000. 26. The method of claim 25, having a length independently selected from nucleotide lengths of less than 20,000.   Evaluating and / or determining the level (amount) or sequence comprises determining either the sequence of nucleotides in the nucleic acid and / or the characteristics of the nucleic acid according to any one or more of the following: The method described in 24: nucleic acid sequencing, performing restriction fragment polymorphism analysis, performing hybridization with another nucleic acid, identifying one or more deletions and / or insertions, and / or mutations such as Not determined, but the presence of single base pair polymorphisms, transitions and / or transversions.   25. The method of claim 24, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleic acids encode the proteins of Table 1.   27. The method of claim 26, wherein the nucleic acid encodes a protein of the following SEQ ID NO: 1-20, 21-41, 1-10, 11-20, 21-30, 31-41 of Table 1 or fragments thereof .