JP2017201255A - Cancer detecting method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method by which whether or not a subject person is afflicted by cancer.SOLUTION: A cancer detecting method includes a step of obtaining a sample from a subject person and detecting at least one level selected from the following and out of markers listed in Table 1. The subject person may be afflicted with cancer if he or she is found more variable than the marker levels of other subject persons free from cancer. The sample is ideally taken from eyes (e.g. an isolated tear sample or liquid used for washing eyes), but may as well be taken from saliva or some other body fluid. As eye samples, tear samples containing secretions deriving from other tissues connected to lacrimal gland and lymphatic system are shown.SELECTED DRAWING: None

Description

(Detailed description of the invention)
(background)
This application encompasses proteins and peptide fragments of these proteins, which are produced by proteolytic digestion of the proteins, both of which diagnose cancer or the presence of cancer in an individual. Useful for monitoring.

  Screening mammograms typically have a sensitivity of 75% and a specificity of about 98%, resulting in a false positive rate of approximately 5% per mammogram (Brown, Hound, Sickles, & Kessler, 1995; Kolb, Lichy, & Newhouse, 2002; Luftner & Possinger, 2002). Breast tissue type (more specifically, density) also has a significant impact on mannographic performance. The degree of breast density is classified using the American College of Radiology Breast Imaging Reporting and Data System (BI-RADS). This system consists of four classifications, 1-4; where category 1 is mostly fat (<25% dense); category 2 is scattered fibrand glands density (25 Category 3 is heterogeneously dense (51-75% dense), and Category 4 is very dense (> 75% dense) (Bigenwald, 2008; Klifa, 2010; Scheel, 2014).

  For women with fatty breast tissue, mammography can be an effective screening tool when patients are amenable to annual screening (Tabar, 2001; Pisano, 2005). However, as breast density increases, the effectiveness of mammography decreases, resulting in increased follow-up imaging and, more importantly, failure to diagnose cancer. Mammographic sensitivity has been shown to be as low as 31% for category 1, 27% for category 2, 20% for category 3, and 12.5% for category 4 for high-risk patients ( Bigenwald, 2008). About 50% of the female population is in categories 3 and 4 which are considered to be dense breast tissue (Vachon, 2007). Currently, the best screening option for these women is MRI, which can be up to 10 times more expensive than mammography (Beignwald, 2008). The lack of good screening options is a serious problem. This is because women with 75% or more dense tissue are 4-6 times more likely to develop breast cancer than women with less dense tissue (Boyd, 2007).

  Follow-up imaging to assess false positives costs $ 4 billion in the US, with an additional $ 1.6 billion spent on biopsy alone. In 2010, when 1.6 million biopsies were performed, about 16% (only 261,000) were found to have cancer (Grady, 2012). Solutions to increasing diagnostic parameters of imaging can be found before and after imaging that focuses on genetic and proteomic information, more specifically biomarkers (Armstrong, Handorf, Chen) , & Bristol Demeter, 2013; Li, Zhang, Rosenzweig, Wang, & Chan, 2002).

  Tissue and serum are generally the most logical places to begin biomarker studies, but the large dynamic range of both media makes discovery extremely difficult (Schiess, Worldscheid, & Abersold, 2009). The solution can be in less complex biological fluids such as saliva and tears. The use of tears as a diagnostic medium is not a new application. Because the tear proteome has previously been thoroughly investigated (Boehm et al., 2012; 2011; Lebrecht, Boehm, Schmidt, Koelbl, & Gros, 2009a; Lebrecht et al., U &b; 2007). In this application, a quantitative assay for the detection of a panel of tear-based biomarkers in response to cancer is disclosed. From this quantitative information, the framework of the Certified Laboratory Improvement Amendments (CLIA) protocol is defined.

Brown, M. L., Houn, F., Sickles, E. A., & Kessler, L. G. (1995). Screening Mammography in Community Practice: Positive Predictive. American Journal of Radiology, 165, 1373-1377.

(Summary)
Provided herein are methods for determining whether a subject has cancer. The method comprises obtaining a sample from the subject and detecting the level of at least one of the markers selected from the following and listed in Table 1: Igλ chain V-IV region Hil (LV403), Ig heavy chain V-III BRO (HV305), Ig heavy chain V-III VH26 (HV303), β-2-microglobulin (B2MG), lipocalin-1 (LCN1), zinc-α-2-sugar Protein (ZA2G), cystatin B (CYTB), antileucoproteinase (SLP1), galectin-3 (LEG3), histidine triad nucleotide binding protein 1 (D6RD60), S100A9 (S10A9), S100A8 (S10A8), galectin-3- Binding protein (LG3BP), Igα-1 chain C region cluster Raster (IGHA1), Igκ chain V-III region HAH cluster (KV312), VEGF co-regulated chemokine 1 (VCC1), L-lactate dehydrogenase A chain (LDHA), aldo-ketoreductase family 1 member C (AKR1C1), root Retin (B1AKD8), L-lactate dehydrogenase B chain (LDHB), retinal dehydrogenase 1 (AL1A1), protein of unknown nature (B4E1Z4), α-1-antichymotrypsin (AACT), superoxide dismutase [Cu-Zn] (SODC) , SPARC-like protein 1 (SPRL1), Ig heavy chain V-III region TIL (HV304), keratin (K1C9), cystatin-SN (CYTN), α-actinin-4 (ACTN4), Igλ-3 chain C region (F (IGLC3), immunoglobulin λ-like polypeptide 5 (IGLL5), alcohol dehydrogenase 1C (ADHIG), malate dehydrogenase, mitochondria (MDHM), calmodulin-like protein 5 (CALL5), α-1-antitrypsin (A1AT) , Α-1B-glycoprotein (A1BG), leucine-rich α-2-glycoprotein (A2GL), low molecular weight ubiquitin-like modifier 3 (A8MU27), progradin protein (Aientor gradient protein) 2 homolog (AGR2), profilin -1 (PROF1), Igλ chain V-III region LOI cluster (LV302), prothrombin (E9PIT3), hemopexin (HEMO), Igγ-2 chain C region (IGHG2), ubiquitin- 0S ribosomal protein S27a (RPS27A), afamin (AFAM), apolipoprotein AI (APOA1), apolipoprotein A-IV (APOA4), flavin reductase (NADPH) (BLVRB), prosaposin (PSAP), lacritin (PAP) LACRT), 60S acidic ribosomal protein P1 (RLA1), inter-α-trypsin inhibitor heavy chain H2 (ITIH2), mucin-like protein 1 (MUCL1), S100 A6 (S100A6), Na (+) / H (+) exchange regulation Cofactor NHE-RF1 (NHRF1), thioredoxin domain-containing protein 17 (I3L0K2), lymphocyte-specific protein (LSP1), haptoglobin cluster (H3BS21), myosin regulation Light chain 12A (J2QRS3), ribonuclease inhibitor (RINI), α-enolase (ENOA), cluster of Igκ chain VI region EU (KV106), alcohol dehydrogenase class 4μ / σ chain (ADH7), protein AMBP (AMBP), Angiotensinogen (ANGT), antithrombin-III (ANT3), apolipoprotein A-II (APOA2), calpastatin (B7Z574), brain acid soluble protein 1 (BASP1), α-2-HS-glycoprotein (C9JV77) ), Calreticulin (CALR), calpain-1 catalytic subunit (CAN1), cell division control protein 42 homolog (CDC42), complement C3 (CO3), coronin-1A (COR1A), programmed cell death 6 interaction T Protein (DCD), Defensin 1 (DEF1), F-box only protein 50 (FBX50), γ-glutamylcyclotransferase (GGCT), glutathione reductase, mitochondria (GSHR), keratin, Type II cytoskeleton 1 (K2C1), UMP-CMP kinase (KCY), mesothelin (MSLN), N-acetylmuramoyl-L-alanine amidase (PGRP2), nicotinic acid phosphoribosyltransferase (PNCB), inter-α-trypsin Inhibitor heavy chain H1 (ITIH1), ribonuclease T2 (RNASET2), superoxide dismutase [Mn], mitochondria (SODM), small proline-rich protein 3 (SPRR3), Src substrate cortactin (SRC8), tubulin β-4B chain cluster (TBB4B), tropomyosin α-3 chain (TPM3), serotransferrin (TRFE), glutathione S-transferase P (THIO), vitronectin (VTNC) ), Vitamin D binding protein (Q6LDC6), inter-α-trypsin inhibitor heavy chain H4 (ITIH4), metalloprotease inhibitor (TIMP1), heat shock protein 90 (HSP90), cathepsin B (CATB), ceruloplasmin (CERU), Calprotectin, 14-3-3σ (1433S), α-2-hs-glycoprotein (FETUA), α-2-macroglobulin (A2MG), transthyretin (TTHY). The subject may have cancer if the level of these markers changes compared to the level of the marker in a control sample derived from a subject without cancer. The sample is optimally an eye sample (eg, an isolated tear sample or eye wash), but can also be derived from saliva or other body fluids. An eye sample represents a tear sample containing secretions from the lacrimal gland and other tissues that connect with the lymphatic system.

  Also provided are kits for performing the methods described herein. The kit can include a sample collection platform, a tube for collection and extraction, which can include a protease inhibitor or other protein stabilizer, sample extraction reagents and test equipment.

  For example, the present invention provides the following items.

(Method)
1. A method of determining whether a subject has cancer, the method comprising: taking a sample from the subject; in the sample, of the markers from the list of markers provided in Table 1 Detecting at least one level; and if the level of the marker changes relative to the level of the marker in a cancer-free control sample, the subject has cancer or has cancer A method comprising determining that it may have
2. Item 2. The method according to Item 1, wherein the cancer is breast cancer.
3. Item 3. The method according to any one of Items 1 to 2, wherein the sample is an eye sample.
4). The method of any one of the preceding items, wherein the subject is a human.
5. The method according to any one of the preceding items, wherein the marker is at least 1.5 times, 2 times, 4 times or more lower than the level of the marker in the control sample.
6). The method according to any one of the preceding items, wherein the marker is increased at least 1.5 fold, 2 fold, 4 fold or more compared to the level of the marker in the control sample.
7). The method of any one of the preceding items, wherein the marker combination is used to determine the likelihood of cancer in the subject.
8). The method according to any one of the preceding items, wherein the level of the marker is detected by liquid chromatography-mass spectrometry (LC-MS).
9. The method of any one of the preceding items, wherein the level of the marker is detected by an antibody-based detection method.
10. The method according to any one of the preceding items, wherein the level of the marker is detected by a multiplex protein detection method.
11. The method according to any one of the preceding items, wherein the level of the marker is detected by an mRNA detection method.
12 The method of any one of the preceding items, wherein the subject is suspected of having cancer.
13. The method according to any one of the preceding items, wherein the subject has an increased risk of developing cancer.
14 3. The method of item 2, wherein the subject has a palpable lump suspected of being cancerous.
15. The method of any one of the preceding items, wherein the subject's breast density is Category 1, Category 2, Category 3, or Category 4.
16. The method according to any one of the preceding items, wherein the cancer is detected as a stage of breast cancer.
17. The method according to any one of the preceding items, wherein at least 10 markers are used in combination.
18. The method according to any one of the preceding items, wherein at least 5 markers are used in combination.
19. The method according to any one of the preceding items, wherein at least three markers are used in combination.
20. A kit for performing the method according to any one of the preceding items comprising a collection tube and a protease inhibitor or other protein stabilizer.
21. Item 21. The kit according to Item 20, further comprising an antibody capable of binding to at least one of the markers.
22. The kit according to item 20 or 21, further comprising a primer pair capable of amplifying at least one of the markers.

(index)
A1. A method wherein the level of at least one of the markers from the list of markers provided in Table 1 is used as an indicator for determining whether a subject has cancer, said method comprising: Detecting in a sample obtained from the body the level in said sample of at least one of the markers from the list of markers provided in Table 1, wherein said level in a control sample without cancer A method optionally comprising a step wherein a change in the level of the marker relative to the level of the marker indicates that the subject has or may have cancer.
A2. The method of item A1, wherein the cancer is breast cancer.
A3. The method according to any of the preceding items, wherein the sample is an eye sample.
A4. The method of any one of the preceding items, wherein the subject is a human.
A5. The method according to any one of the preceding items, wherein the marker is at least 1.5 times, 2 times, 4 times or more lower than the level of the marker in the control sample.
A6. The method according to any one of the preceding items, wherein the marker is increased at least 1.5 fold, 2 fold, 4 fold or more compared to the level of the marker in the control sample.
A7. The method of any one of the preceding items, wherein the marker combination is used to determine the likelihood of cancer in the subject.
A8. The method according to any one of the preceding items, wherein the level of the marker is detected by liquid chromatography-mass spectrometry (LC-MS).
A9. The method of any one of the preceding items, wherein the level of the marker is detected by an antibody-based detection method.
A10. The method according to any one of the preceding items, wherein the level of the marker is detected by a multiplex protein detection method.
A11. The method according to any one of the preceding items, wherein the level of the marker is detected by an mRNA detection method.
A12. The method of any one of the preceding items, wherein the subject is suspected of having cancer.
A13. The method according to any one of the preceding items, wherein the subject has an increased risk of developing cancer.
A14. The method of item A2, wherein the subject has a palpable lump suspected of being cancerous.
A15. The method of any one of the preceding items, wherein the subject's breast density is Category 1, Category 2, Category 3, or Category 4.
A16. The method according to any one of the preceding items, wherein the cancer is detected as a stage of breast cancer.
A17. The method according to any one of the preceding items, wherein at least 10 markers are used in combination.
A18. The method according to any one of the preceding items, wherein at least 5 markers are used in combination.
A19. The method according to any one of the preceding items, wherein at least three markers are used in combination.
A20. A kit for performing the method according to any one of the preceding items comprising a collection tube and a protease inhibitor or other protein stabilizer.
A21. The kit of item A20, further comprising an antibody capable of binding to at least one of the markers.
A22. The kit according to item A20 or A21, further comprising a primer pair capable of amplifying at least one of the markers.

(marker)
B1. A marker for determining whether a subject has a cancer comprising at least one of the markers from the list of markers provided in Table 1, wherein said marker in a control sample without cancer A marker, wherein a change in the level of the marker in a sample derived from the subject when compared to the level of indicates that the subject has or may have cancer.
B2. The marker according to item B1, wherein the cancer is breast cancer.
B3. The marker according to any of the preceding items, wherein the sample is an eye sample.
B4. The marker according to any one of the preceding items, wherein the subject is a human.
B5. The marker according to any one of the preceding items, wherein the marker is at least 1.5 times, 2 times, 4 times or more lower than the level of the marker in the control sample.
B6. The marker according to any one of the preceding items, wherein the marker is increased by at least 1.5 fold, 2 fold, 4 fold or more compared to the level of the marker in the control sample.
B7. The marker combination according to any one of the preceding items, wherein the marker is used to determine the likelihood of cancer in the subject.
B8. The marker or combination according to any one of the preceding items, wherein the level of the marker is detected by liquid chromatography-mass spectrometry (LC-MS).
B9. The marker or combination according to any one of the preceding items, wherein the level of the marker is detected by an antibody-based detection method.
B10. The marker or combination according to any one of the preceding items, wherein the level of the marker is detected by a multiplex protein detection method.
B11. The marker or combination according to any one of the preceding items, wherein the level of the marker is detected by an mRNA detection method.
B12. The marker or combination according to any one of the preceding items, wherein the subject is suspected of having cancer.
B13. The marker or combination according to any one of the preceding items, wherein the subject has an increased risk of developing cancer.
B14. The marker or combination of item B2, wherein the subject has a palpable lump suspected of being cancerous.
B15. The marker or combination according to any one of the preceding items, wherein the subject's breast density is Category 1, Category 2, Category 3, or Category 4.
B16. The marker or combination according to any one of the preceding items, wherein the cancer is detected as a stage of breast cancer.
B17. The marker or combination according to any one of the preceding items, wherein at least 10 markers are used in combination.
B18. The marker or combination according to any one of the preceding items, wherein at least 5 markers are used in combination.
B19. The marker or combination according to any one of the preceding items, wherein at least three markers are used in combination.
B20. A kit for determining whether a subject has cancer using a marker or combination according to any one of the preceding items, comprising a collection tube and a protease inhibitor, or other protein stabilizer. .
B21. The kit of item B20, further comprising an antibody capable of binding to at least one of the markers.
B22. The kit according to item B20 or B21, further comprising a primer pair capable of amplifying at least one of the markers.

(Detection agent)
C1. A detection agent for detecting whether a subject has cancer, wherein the detection agent is at least one of the markers in the list of markers provided in Table 1 in the sample of the subject. An agent or means for detecting a level; wherein the change in the level of the marker as compared to the level of the marker in a cancer free control sample is whether the subject has cancer or cancer A detection agent which indicates that it may have.
C2. The detection agent according to item C1, wherein the cancer is breast cancer.
C3. The detection agent according to any one of the preceding items, wherein the sample is an eye sample.
C4. The detection agent according to any one of the preceding items, wherein the subject is a human.
C5. The detection agent according to any one of the preceding items, wherein the marker is at least 1.5 times, 2 times, 4 times or more lower than the level of the marker in the control sample. .
C6. The detection agent according to any one of the preceding items, wherein the marker is increased by at least 1.5-fold, 2-fold, 4-fold or more compared to the level of the marker in the control sample. .
C7. The detection agent according to any one of the preceding items, wherein the combination of markers is used to determine the likelihood of cancer in the subject.
C8. The detection agent according to any one of the preceding items, wherein the level of the marker is detected by liquid chromatography-mass spectrometry (LC-MS).
C9. The detection agent according to any one of the preceding items, wherein the level of the marker is detected by an antibody-based detection method.
C10. The detection agent according to any one of the preceding items, wherein the level of the marker is detected by a multiplex protein detection method.
C11. The detection agent according to any one of the preceding items, wherein the level of the marker is detected by an mRNA detection method.
C12. The detection agent according to any one of the preceding items, wherein the subject is suspected of having cancer.
C13. The detection agent according to any one of the preceding items, wherein the subject has an increased risk of developing cancer.
C14. The detection agent according to item C2, wherein the subject has a palpable lump suspected of being cancerous.
C15. The detection agent according to any one of the preceding items, wherein the breast density of the subject is Category 1, Category 2, Category 3, or Category 4.
C16. The detection agent according to any one of the preceding items, wherein the cancer is detected as a stage of breast cancer.
C17. The detection agent according to any one of the preceding items, wherein at least 10 types of markers are used in combination.
C18. The detection agent according to any one of the preceding items, wherein at least 5 types of markers are used in combination.
C19. The detection agent according to any one of the preceding items, wherein at least three types of markers are used in combination.
C20. A kit for determining whether a subject has cancer, comprising the detection agent, collection tube and protease inhibitor, or other protein stabilizer of any one of the preceding items.
C21. The kit of item C20, further comprising an antibody capable of binding to at least one of the markers.
C22. The kit according to item C20 or C21, further comprising a primer pair capable of amplifying at least one of the markers.

(Diagnostic agent)
D1. A diagnostic agent for determining whether a subject has cancer, wherein the diagnostic agent is at least one of the markers in the list of markers provided in Table 1 in the sample of the subject. An agent or means for detecting a level; wherein the change in the level of the marker as compared to the level of the marker in a cancer free control sample is whether the subject has cancer or cancer A diagnostic agent which indicates that it may have.
D2. The diagnostic agent according to Item D1, wherein the cancer is breast cancer.
D3. The diagnostic agent according to any one of the preceding items, wherein the sample is an eye sample.
D4. The diagnostic agent according to any one of the preceding items, wherein the subject is a human.
D5. The diagnostic agent according to any one of the preceding items, wherein the marker is at least 1.5 times, 2 times, 4 times or more lower than the level of the marker in the control sample. .
D6. The diagnostic agent according to any one of the preceding items, wherein the marker is increased by at least 1.5 fold, 2 fold, 4 fold or more compared to the level of the marker in the control sample. .
D7. The diagnostic agent according to any one of the preceding items, wherein the combination of markers is used to determine the likelihood of cancer in the subject.
D8. The diagnostic agent according to any one of the preceding items, wherein the level of the marker is detected by liquid chromatography-mass spectrometry (LC-MS).
D9. The diagnostic agent according to any one of the preceding items, wherein the level of the marker is detected by an antibody-based detection method.
D10. The diagnostic agent according to any one of the preceding items, wherein the level of the marker is detected by a multiplex protein detection method.
D11. The diagnostic agent according to any one of the preceding items, wherein the level of the marker is detected by an mRNA detection method.
D12. The diagnostic agent according to any one of the preceding items, wherein the subject is suspected of having cancer.
D13. The diagnostic agent according to any one of the preceding items, wherein the subject has an increased risk of developing cancer.
D14. The diagnostic agent according to Item D2, wherein the subject has a palpable lump suspected of being cancerous.
D15. The diagnostic agent according to any one of the preceding items, wherein the subject has a breast density of Category 1, Category 2, Category 3, or Category 4.
D16. The diagnostic agent according to any one of the preceding items, wherein the cancer is detected as a stage of breast cancer.
D17. The diagnostic agent according to any one of the preceding items, wherein at least 10 types of markers are used in combination.
D18. The diagnostic agent according to any one of the preceding items, wherein at least 5 types of markers are used in combination.
D19. The diagnostic agent according to any one of the preceding items, wherein at least three types of markers are used in combination.
D20. A kit for determining whether a subject has cancer, comprising the diagnostic agent, collection tube and protease inhibitor, or other protein stabilizer of any one of the preceding items.
D21. The kit of D20, further comprising an antibody capable of binding to at least one of the markers.
D22. The kit according to item D20 or D21, further comprising a primer pair capable of amplifying at least one of the markers.

(system)
E1. A system for determining whether a subject has cancer, the system comprising: an agent for obtaining a sample from the subject or a marker provided in Table 1 in the sample An agent or means for performing the step of detecting the level of at least one of the markers in the list; and when the level of the marker changes relative to the level of the marker in a cancer-free control sample, A system comprising an agent or means for determining that a subject has or is likely to have cancer.
E2. The system of item E1, wherein the cancer is breast cancer.
E3. The system according to any of the preceding items, wherein the sample is an eye sample.
E4. The system of any one of the preceding items, wherein the subject is a human.
E5. The system according to any one of the preceding items, wherein the marker is at least 1.5 times, 2 times, 4 times or more lower than the level of the marker in the control sample.
E6. The system according to any one of the preceding items, wherein the marker is increased at least 1.5 fold, 2 fold, 4 fold or more compared to the level of the marker in the control sample.
E7. The system of any one of the preceding items, wherein a combination of markers is used to determine the likelihood of cancer in the subject.
E8. The system of any preceding item, wherein the level of the marker is detected by liquid chromatography-mass spectrometry (LC-MS).
E9. The system of any preceding item, wherein the level of the marker is detected by an antibody-based detection method.
E10. The system according to any one of the preceding items, wherein the level of the marker is detected by a multiplex protein detection method.
E11. The system according to any one of the preceding items, wherein the level of the marker is detected by an mRNA detection method.
E12. The system according to any one of the preceding items, wherein the subject is suspected of having cancer.
E13. The system according to any one of the preceding items, wherein the subject has an increased risk of developing cancer.
E14. The system of item E2, wherein the subject has a palpable lump suspected of being cancerous.
E15. The system of any one of the preceding items, wherein the subject's breast density is Category 1, Category 2, Category 3, or Category 4.
E16. The system according to any one of the preceding items, wherein the cancer is detected as a stage of breast cancer.
E17. The system according to any one of the preceding items, wherein at least 10 markers are used in combination.
E18. The system according to any one of the preceding items, wherein at least 5 markers are used in combination.
E19. The system according to any one of the preceding items, wherein at least three markers are used in combination.
E20. The system according to any one of the preceding items, further comprising a collection tube and a protease inhibitor, or other protein stabilization system.
E21. The system of item E20, further comprising an antibody capable of binding to at least one of the markers.
E22. The system of item E20 or E21, further comprising a primer pair capable of amplifying at least one of the markers.

(Summary of this disclosure)
Methods and kits for detecting cancer, and particularly breast cancer, in a subject by measuring the level of at least one of a series of biomarkers when compared to a control sample without cancer. The expression of the biomarker is either increased or decreased in a sample derived from a subject with cancer when compared to the expression level in a subject without cancer. The sample is optimally an eye sample (eg, an isolated tear sample or eye wash), but can be derived from saliva or other body fluids as well. The kit can include a collection tube and a protease inhibitor or protein stabilizer.

Figure 1: The following proteins identified in Experiment 1: (A) Ig heavy chain V-IV region HiL, (B) Ig heavy chain region V-III region BRO, (C) Ig heavy chain V-III region VH26, (D) Antileucoproteinase, (E) β2 microglobulin, (F) Calmodulin-like protein 5, (G) Lipocalin 1, (H) Cystatin B, (I) Galectin 3, (J) Zinc-α2 glycoprotein spectrum ANOVA of strength. FIG. 2: Ig heavy chain V-IV region HiL, Ig heavy chain region V-III region BRO, Ig heavy chain V-III region VH26, antileucoproteinase, β2 microglobulin, calmodulin-like protein 5, lipocalin 1, cystatin B, Cancer, benign and control ROC curves from Experiment 1 using Galectin 3, zinc-α2 glycoprotein. FIG. 3: Mosaic plot of breast density distribution for 66 of the 75 samples tested in Experiment 1. Same as above FIG. 4: ANOVA of the spectral intensity of the protein identified in experiment 2. The proteins shown are: (A) 14-3-3 protein σ, (B) 6-phosphogluconate dehydrogenase, decarboxylation, (C) α-actinin-4, (D) retinal dehydrogenase, (E ) Argininosuccinate synthase, (F) β-2-microglobulin, (G) calmodulin, (H) ceruloplasmin, (I) cofilin, (J) cystatin-SN, (K) enolase 1, (L) gelsolin, M) heat shock protein β-1, (N) Igα-1 chain C region, (O) lacritin, (P) lysozyme, (Q) basement membrane specific heparin sulfate proteoglycan core protein, (R) polymer immunoglobulin receptor, (S) Profilin 1, (T) S100A8, (U) S100A9, (V) Secretoglobin Family 1D (W) VEGF co-regulatory chemokine 1, (X) histidine triad nucleotide binding protein 1, (Y) defensin 1, (Z) L-lactate dehydrogenase A chain, (AA) SPARC-like protein 1, (BB) ) Annexin 5, (CC) Ig heavy chain V-III region TIL, (DD) inter-α-trypsin inhibitor heavy chain 1, (EE) α-1B-glycoprotein, (FF) α-1-antitrypsin. Same as above Same as above Same as above Same as above Same as above Same as above Same as above Same as above Same as above Same as above Same as above Same as above Same as above Same as above Same as above FIG. 5: ANOVA comparing the expression level of S100A9 in breast cancer samples and control samples as determined by ELISA. FIG. 6: ANOVA comparing LG3BP expression levels in breast cancer samples and control samples as determined by ELISA. FIG. 7: ROC curve of S100A9. Figure 8: LG3BP ROC curve Figure 9: ROC curve combining S100A9 and LG3BP Figure 10: ANOVA with S100A9 expression based on breast tissue category. FIG. 11: ANOVA of LG3BP expression based on breast tissue category.

(Description of Embodiment)
(Detailed explanation)
Provided herein are proteins derived from eye samples and peptide fragments obtained by trypsin digestion. The polypeptide produced is selected from the following and listed in Table 1: Igλ chain V-IV region Hil (LV403), Ig heavy chain V-III BRO (HV305), Ig heavy chain V- III VH26 (HV303), β-2-microglobulin (B2MG), lipocalin-1 (LCN1), zinc-α-2-glycoprotein (ZA2G), cystatin B (CYTB), antileucoproteinase (SLP1), galectin- 3 (LEG3), histidine triad nucleotide binding protein 1 (D6RD60), S100A9 (S10A9), S100A8 (S10A8), galectin-3-binding protein (LG3BP), Igα-1 chain C region cluster (IGHA1), Igκ chain Cluster of V-III region HAH (KV312 , VEGF co-regulatory chemokine 1 (VCC1), L-lactate dehydrogenase A chain (LDHA), aldo-ketoreductase family 1 member C (AKR1C1), root retin (B1AKD8), L-lactate dehydrogenase B chain (LDHB), retinal Dehydrogenase 1 (AL1A1), protein of unknown nature (B4E1Z4), α-1-antichymotrypsin (AACT), superoxide dismutase [Cu—Zn] (SODC), SPARC-like protein 1 (SPRL1), Ig heavy chain V-III region TIL (HV304), keratin (K1C9), cystatin-SN (CYTN), α-actinin-4 (ACTN4), Igλ-3 chain C region (fragment) (IGLC3), immunoglobulin λ-like polypeptide 5 (IGLL5), Alcohol dehydrogenase 1C (ADHIG), malate dehydrogenase, mitochondria (MDHM), calmodulin-like protein 5 (CALL5), α-1-antitrypsin (A1AT), α-1B-glycoprotein (A1BG), leucine-rich α-2- Glycoprotein (A2GL), small ubiquitin-like modifier 3 (A8MU27), pro-gradient protein 2 homolog (AGR2), profilin-1 (PROF1), Igλ chain V-III region LOI cluster (LV302), prothrombin (E9PIT3 ), Hemopexin (HEMO), Igγ-2 chain C region (IGHG2), ubiquitin-40S ribosomal protein S27a (RPS27A), afamin (AFAM), apolipoprotein AI (APOA1), apolipop Rotin A-IV (APOA4), flavin reductase (NADPH) (BLVRB), prosaposin (PSAP), lacritin (LACRT), 60S acidic ribosomal protein P1 (RLA1), inter-α-trypsin inhibitor heavy chain H2 (ITIH2), mucin Like protein 1 (MUCL1), S100 A6 (S100A6), Na (+) / H (+) exchange regulatory cofactor NHE-RF1 (NHRF1), thioredoxin domain-containing protein 17 (I3L0K2), lymphocyte specific protein (LSP1) ), Haptoglobin cluster (H3BS21), myosin regulatory light chain 12A (J2QRS3), ribonuclease inhibitor (RINI), α-enolase (ENOA), Igκ chain VI region EU cluster (KV1) 06), alcohol dehydrogenase class 4μ / σ chain (ADH7), protein AMBP (AMBP), angiotensinogen (ANGT), antithrombin-III (ANT3), apolipoprotein A-II (APOA2), calpastatin (B7Z574) Brain acid soluble protein 1 (BASP1), α-2-HS-glycoprotein (C9JV77), calreticulin (CALR), calpain-1 catalytic subunit (CAN1), cell division control protein 42 homolog (CDC42), Complement C3 (CO3), coronin-1A (COR1A), programmed cell death 6-interacting protein (DCD), defensin 1 (DEF1), F-box only protein 50 (FBX50), γ-glutamyl cyclotransferase ( GCT), glutathione reductase, mitochondria (GSHR), keratin, type II cytoskeleton 1 (K2C1), UMP-CMP kinase (KCY), mesothelin (MSLN), N-acetylmuramoyl-L-alanine amidase (PGRP2), nicotine Acid phosphoribosyltransferase (PNCB), inter-α-trypsin inhibitor heavy chain H1 (ITIH1), ribonuclease T2 (RNASET2), superoxide dismutase [Mn], mitochondria (SODM), small proline-rich protein 3 (SPRR3), Src Substrate cortactin (SRC8), tubulin β-4B chain cluster (TBB4B), tropomyosin α-3 chain (TPM3), serotransferrin (TRFE), glutathione S -Transferase P (THIO), vitronectin (VTNC), vitamin D binding protein (Q6LDC6), inter-α-trypsin inhibitor heavy chain H4 (ITIH4), metalloprotease inhibitor (TIMP1), heat shock protein 90 (HSP90), cathepsin B (CATB), ceruloplasmin (CERU), calprotectin, 14-3-3σ (1433S), α-2-hs-glycoprotein (FETUA), α-2-macroglobulin (A2MG), transthyretin (TTHY) ). The trypsin sequence and the full-length amino acid sequence of the protein identified as down-regulated in the cancer sample are provided in Appendix I.

  It is shown in the examples that the proteins and peptides are either increased or decreased in the biological sample depending on the presence of breast cancer compared to the control. These proteins and peptides are biomarkers and are used to determine the disease state of a patient or other subject.

  Subjects include humans, domesticated animals such as cats, dogs, cows, pigs, or other animals that are susceptible to cancer. “Patient” refers to a subject who has been diagnosed with a disease, has been diagnosed with cancer, or is under examination for having cancer. Thus, the terms subject and patient can be used interchangeably herein. The subject can be suspected of having cancer. The subject is cancer (breast cancer, acoustic nerve tumor, acute lymphoblastic leukemia, acute myeloid leukemia, adrenal tumor, AIDS-related cancer, basal cell cancer, benign hematologic disorder, bladder cancer, bone cancer, brain tumor. (Metastatic and primary), breast cancer, cancer of unknown primary origin, cervical cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancer, esophageal cancer, gallbladder cancer and bile duct cancer, Gastrointestinal neuroendocrine tumor, GERD, Barrett's esophagus and achalasia, gestational choriocarcinoma, head and neck cancer, Kaposi's sarcoma, kidney cancer, leukemia, liver cancer, liver metastasis, low-grade glioma, lung cancer, Lymphoma, male breast cancer, melanoma, Merkel cell carcinoma, mesothelioma, multiple myeloma, myelodysplastic syndrome, ovarian cancer, pancreatic cancer, pancreatic cyst, pituitary tumor, prostate cancer, lung neuroendocrine tumor Rare blood disorders, skin cancer, Tissue sarcoma, bone marrow tumor, squamous cell carcinoma, stomach (gastric) cancer, testicular cancer (germline tumor), thymoma and other thymic tumors, tracheal disease, uterus (endometrium) ) Cancer, uterine sarcoma) may be suspected. The subject may have an increased risk of developing breast cancer. For example, the subject may be at risk of cancer due to a positive test for a gene (eg, BRCA) known to increase cancer risk by detecting a mass in the breast due to a positive mammographic result. It may have increased or may be suspected of having cancer, or has already undergone resection, biopsy, or other procedure to remove the cancer. The subject may be in the process of being treated for cancer, or may have been previously treated, and the methods and kits herein may be used to monitor the progress of treatment or alternatively It is used to monitor the recurrence or spread of the cancer.

  Also provided herein are methods and kits for collecting ocular samples for use in determining the expression level of an identified protein or polypeptide in lacrimal gland secretions. The use of collection strips and tubes containing protease inhibitors or protein stabilizers is disclosed. The kit further includes a buffer or reagent for eluting the breast cancer biomarker from a collection strip that has been in contact with the eye to collect lacrimal secretions. Also disclosed is the design of a device for collecting proteins from the eye cavity and packaging of the device with a container for containing the collection device and elution buffer.

  The methods disclosed herein encompass the use (single or multiple) of these cancer biomarkers in a CLIA-based protocol that utilizes a triple quadrupole LC-MS / MS platform, which includes Conducted in a centralized laboratory testing facility. Eye samples collected from individuals can be transported to the testing facility in this embodiment. The identified proteins and their subsequent proteolytic fragments (as shown in Appendix I) are used for quantitative analysis of diagnostic peptides produced in triple quadrupoles. Polypeptides selected from the following and listed in Table 1: Igλ chain V-IV region Hil (LV403), Ig heavy chain V-III BRO (HV305), Ig heavy chain V-III VH26 (HV303), β -2-microglobulin (B2MG), lipocalin-1 (LCN1), zinc-α-2-glycoprotein (ZA2G), cystatin B (CYTB), antileucoproteinase (SLP1), galectin-3 (LEG3), histidine tri Adnucleotide binding protein 1 (D6RD60), S100A9 (S10A9), S100A8 (S10A8), galectin-3-binding protein (LG3BP), cluster of Igα-1 chain C region (IGHA1), cluster of Igκ chain V-III region HAH (KV312), VEGF co-regulatory Mokaine 1 (VCC1), L-lactate dehydrogenase A chain (LDHA), aldo-ketoreductase family 1 member C (AKR1C1), Root retin (B1AKD8), L-lactate dehydrogenase B chain (LDHB), retinal dehydrogenase 1 (AL1A1) , Protein of unknown nature (B4E1Z4), α-1-antichymotrypsin (AACT), superoxide dismutase [Cu—Zn] (SODC), SPARC-like protein 1 (SPRL1), Ig heavy chain V-III region TIL (HV304), Keratin (K1C9), cystatin-SN (CYTN), α-actinin-4 (ACTN4), Igλ-3 chain C region (fragment) (IGLC3), immunoglobulin λ-like polypeptide 5 (IGLL5), alcohol dehydrogenation Ase 1C (ADHIG), malate dehydrogenase, mitochondria (MDHM), calmodulin-like protein 5 (CALL5), α-1-antitrypsin (A1AT), α-1B-glycoprotein (A1BG), leucine-rich α-2- Glycoprotein (A2GL), small ubiquitin-like modifier 3 (A8MU27), pro-gradient protein 2 homolog (AGR2), profilin-1 (PROF1), Igλ chain V-III region LOI cluster (LV302), prothrombin (E9PIT3 ), Hemopexin (HEMO), Igγ-2 chain C region (IGHG2), ubiquitin-40S ribosomal protein S27a (RPS27A), afamin (AFAM), apolipoprotein AI (APOA1), apolipoprotein A-IV (A OA4), flavin reductase (NADPH) (BLVRB), prosaposin (PSAP), lacritin (LACRT), 60S acidic ribosomal protein P1 (RLA1), inter-α-trypsin inhibitor heavy chain H2 (ITIH2), mucin-like protein 1 (MUCL1) ), S100 A6 (S100A6), Na (+) / H (+) exchange regulatory cofactor NHE-RF1 (NHRF1), thioredoxin domain-containing protein 17 (I3L0K2), lymphocyte-specific protein (LSP1), haptoglobin cluster (H3BS21), myosin regulatory light chain 12A (J2QRS3), ribonuclease inhibitor (RINI), α-enolase (ENOA), cluster of Igκ chain VI region EU (KV106), alcohol Hydrogenase class 4μ / σ chain (ADH7), protein AMBP (AMBP), angiotensinogen (ANGT), antithrombin-III (ANT3), apolipoprotein A-II (APOA2), calpastatin (B7Z574), brain acid soluble Protein 1 (BASP1), α-2-HS-glycoprotein (C9JV77), calreticulin (CALR), calpain-1 catalytic subunit (CAN1), cell division control protein 42 homolog (CDC42), complement C3 ( CO3), coronin-1A (COR1A), programmed cell death 6-interacting protein (DCD), defensin 1 (DEF1), F-box only protein 50 (FBX50), gamma glutamyl cyclotransferase (GGCT), glutathione Reductase, mitochondria (GSHR), keratin, type II cytoskeleton 1 (K2C1), UMP-CMP kinase (KCY), mesothelin (MSLN), N-acetylmuramoyl-L-alanine amidase (PGRP2), phosphoribosyl nicotinate Transferase (PNCB), inter-α-trypsin inhibitor heavy chain H1 (ITIH1), ribonuclease T2 (RNASET2), superoxide dismutase [Mn], mitochondria (SODM), small molecule proline rich protein 3 (SPRR3), Src substrate cortactin ( SRC8), cluster of tubulin β-4B chain (TBB4B), tropomyosin α-3 chain (TPM3), cellotransferrin (TRFE), glutathione S-transferase P (THIO), vitronectin (VTNC), vitamin D binding protein (Q6LDC6), inter-α-trypsin inhibitor heavy chain H4 (ITIH4), metalloprotease inhibitor (TIMP1), heat shock protein 90 (HSP90), cathepsin B (CATB) ), Ceruloplasmin (CERU), calprotectin, 14-3-3σ (1433S), α-2-hs-glycoprotein (FETUA), α-2-macroglobulin (A2MG), transthyretin (TTHY) A threshold or relative or actual value for the directly related polypeptide concentration can be defined, or the sample can be compared directly to a non-cancerous control. Quantitative information in report form can be provided to physicians to help make decisions regarding patient care pathways. A physician can make treatment decisions based on these results, and the final step can include the administration of an appropriate anti-cancer therapy to the subject.

  In an alternative embodiment, a polypeptide as selected from and listed in Table 1: Igλ chain V-IV region Hil (LV403), Ig heavy chain V-III BRO (HV305), Ig heavy chain V-III VH26 (HV303), β-2-microglobulin (B2MG), lipocalin-1 (LCN1), zinc-α-2-glycoprotein (ZA2G), cystatin B (CYTB), antileucoproteinase (SLP1), galectin-3 (LEG3), histidine triad nucleotide binding protein 1 (D6RD60), S100A9 (S10A9), S100A8 (S10A8), galectin-3-binding protein (LG3BP), Igα-1 chain C region cluster (IGHA1), Igκ chain V -III region HAH cluster (KV3 2), VEGF co-regulating chemokine 1 (VCC1), L-lactate dehydrogenase A chain (LDHA), aldo-ketoreductase family 1 member C (AKR1C1), root retin (B1AKD8), L-lactate dehydrogenase B chain (LDHB) , Retinal dehydrogenase 1 (AL1A1), protein with unknown properties (B4E1Z4), α-1-antichymotrypsin (AACT), superoxide dismutase [Cu-Zn] (SODC), SPARC-like protein 1 (SPRL1), Ig heavy chain V- III region TIL (HV304), keratin (K1C9), cystatin-SN (CYTN), α-actinin-4 (ACTN4), Igλ-3 chain C region (fragment) (IGLC3), immunoglobulin λ-like polypeptide 5 (IGLL5 Alcohol dehydrogenase 1C (ADHIG), malate dehydrogenase, mitochondria (MDHM), calmodulin-like protein 5 (CALL5), α-1-antitrypsin (A1AT), α-1B-glycoprotein (A1BG), leucine-rich α-2 -Glycoprotein (A2GL), small molecule ubiquitin-like modifier 3 (A8MU27), pro-gradient protein 2 homolog (AGR2), profilin-1, (PROF1), cluster of Igλ chain V-III region LOI (LV302), prothrombin (E9PIT3), hemopexin (HEMO), Igγ-2 chain C region (IGHG2), ubiquitin-40S ribosomal protein S27a (RPS27A), afamin (AFAM), apolipoprotein AI (APOA1), apo Poprotein A-IV (APOA4), flavin reductase (NADPH) (BLVRB), prosaposin (PSAP), lacritin (LACRT), 60S acidic ribosomal protein P1 (RLA1), inter-α-trypsin inhibitor heavy chain H2 (ITIH2), Mucin-like protein 1 (MUCL1), S100 A6 (S100A6), Na (+) / H (+) exchange regulatory cofactor NHE-RF1 (NHRF1), thioredoxin domain-containing protein 17 (I3L0K2), lymphocyte-specific protein ( LSP1), haptoglobin cluster (H3BS21), myosin regulatory light chain 12A (J2QRS3), ribonuclease inhibitor (RINI), α-enolase (ENOA), Igκ chain VI region EU cluster ( KV106), alcohol dehydrogenase class 4μ / σ chain (ADH7), protein AMBP (AMBP), angiotensinogen (ANGT), antithrombin-III (ANT3), apolipoprotein A-II (APOA2), calpastatin (B7Z574) Brain acid soluble protein 1 (BASP1), α-2-HS-glycoprotein (C9JV77), calreticulin (CALR), calpain-1 catalytic subunit (CAN1), cell division control protein 42 homolog (CDC42), Complement C3 (CO3), coronin-1A (COR1A), programmed cell death 6-interacting protein (DCD), defensin 1 (DEF1), F-box only protein 50 (FBX50), gamma glutamyl cyclotransferase GGCT, glutathione reductase, mitochondria (GSHR), keratin, type II cytoskeleton 1 (K2C1), UMP-CMP kinase (KCY), mesothelin (MSLN), N-acetylmuramoyl-L-alanine amidase (PGRP2) Nicotinate phosphoribosyltransferase (PNCB), inter-α-trypsin inhibitor heavy chain H1 (ITIH1), ribonuclease T2 (RNASET2), superoxide dismutase [Mn], mitochondria (SODM), small molecule proline rich protein 3 (SPRR3) , Src substrate cortactin (SRC8), tubulin β-4B chain cluster (TBB4B), tropomyosin α-3 chain (TPM3), serotransferrin (TRFE), glutati ON S-transferase P (THIO), vitronectin (VTNC), vitamin D binding protein (Q6LDC6), inter-α-trypsin inhibitor heavy chain H4 (ITIH4), metalloprotease inhibitor (TIMP1), heat shock protein 90 (HSP90), Cathepsin B (CATB), ceruloplasmin (CERU), calprotectin, 14-3-3σ (1433S), α-2-hs-glycoprotein (FETUA), α-2-macroglobulin (A2MG), transthyretin (TTHY) implements a binding agent, eg, an antibody, peptoid, or coated surface, and a reagent that provides specific binding interactions for these proteins, and a detectable signal (eg, fluorescence) , Color change, or Based on the generation of the V absorption) it can be detected by causing a reaction which can be quantified. It is also provided to perform these components in the cartridge with a partner reading device (eg, a point-of-care device that can be used at the point of care). These protein and polypeptide binders can also be used for detection in lateral flow devices. Thus, methods of detecting the level of protein expression in the sample using a binding partner (eg, an antibody) can be used in immunoassays to detect the markers provided herein.

  The immunoassay typically comprises a step of contacting a test sample with an antibody or antigen that specifically binds or otherwise recognizes a biomarker, and a complex of antibody or antigen bound to the biomarker in the sample. Detecting the presence of a body. The immunoassay procedures include a wide variety of immunoassay procedures known in the art with recognition of antibody / antigen complexes, including enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), and Western blots, and multiplex Can be selected from the use of assays (including the use of antibody arrays), where some desired antibodies can be placed on a support (eg, glass beads or plates) and allowed to react with the test sample Or else it can be contacted. Such assays are well known to those skilled in the art.

  Detection of the biomarkers described herein in a sample can be performed in a variety of ways. In one embodiment, the method provides reverse transcription of complementary DNA from mRNA obtained from the sample. The fluorescent dye-labeled complementary RNA can be transcribed from the complementary DNA, which is then hybridized to an array of oligonucleotide probes. The color of the fluorescence generated by hybridization can be read by a machine (eg, SureScan microarray scanner (Agilent Technologies)), data can be obtained, and software (eg, Agilent Feature Extraction Software (9.1)) can be obtained. Can be processed using. Such array-based methods include microarray analysis to generate gene expression profiles. As used herein, the term “gene expression profile” refers to the expression level of mRNA or protein of a panel of genes in the subject. As used herein, the term “diagnostic panel” refers to a panel of genes, peptides or proteins whose expression levels can be relied upon to diagnose or infer a disease state. Included in this panel are genes, peptides and proteins selected from the following and listed in Table 1: Igλ chain V-IV region Hil (LV403), Ig heavy chain V-III BRO (HV305) Ig heavy chain V-III VH26 (HV303), β-2-microglobulin (B2MG), lipocalin-1 (LCN1), zinc-α-2-glycoprotein (ZA2G), cystatin B (CYTB), antileucoproteinase (SLP1), galectin-3 (LEG3), histidine triad nucleotide binding protein 1 (D6RD60), S100A9 (S10A9), S100A8 (S10A8), galectin-3-binding protein (LG3BP), cluster of Igα-1 chain C region (IGHA1), Igκ chain V-III region HAH cluster (KV312), VEGF co-regulatory chemokine 1 (VCC1), L-lactate dehydrogenase A chain (LDHA), aldo-ketoreductase family 1 member C (AKR1C1), root retin (B1AKD8), L-lactate dehydrogenase B Chain (LDHB), retinal dehydrogenase 1 (AL1A1), protein of unknown nature (B4E1Z4), α-1-antichymotrypsin (AACT), superoxide dismutase [Cu-Zn] (SODC), SPARC-like protein 1 (SPRL1), Ig Heavy chain V-III region TIL (HV304), keratin (K1C9), cystatin-SN (CYTN), α-actinin-4 (ACTN4), Igλ-3 chain C region (fragment) (IGLC3), immunoglobulin λ Polypeptide 5 (IGLL5), alcohol dehydrogenase 1C (ADHIG), malate dehydrogenase, mitochondria (MDHM), calmodulin-like protein 5 (CALL5), α-1-antitrypsin (A1AT), α-1B-glycoprotein (A1BG) , Leucine-rich α-2-glycoprotein (A2GL), small ubiquitin-like modifier 3 (A8MU27), pro-gradient protein 2 homolog (AGR2), profilin-1 (PROF1), Igλ chain V-III region LOI cluster (LV302), prothrombin (E9PIT3), hemopexin (HEMO), Igγ-2 chain C region (IGHG2), ubiquitin-40S ribosomal protein S27a (RPS27A), afamin (AFAM), apolipoprotein -I (APOA1), apolipoprotein A-IV (APOA4), flavin reductase (NADPH) (BLVRB), prosaposin (PSAP), lacritin (LACRT), 60S acidic ribosomal protein P1 (RLA1), inter-α-trypsin inhibitor heavy Chain H2 (ITIH2), mucin-like protein 1 (MUCL1), S100 A6 (S100A6), Na (+) / H (+) exchange regulatory cofactor NHE-RF1 (NHRF1), thioredoxin domain-containing protein 17 (I3L0K2), Lymphocyte-specific protein (LSP1), haptoglobin cluster (H3BS21), myosin regulatory light chain 12A (J2QRS3), ribonuclease inhibitor (RINI), α-enolase (ENOA), Igκ chain V -I region EU cluster (KV106), alcohol dehydrogenase class 4μ / σ chain (ADH7), protein AMBP (AMBP), angiotensinogen (ANGT), antithrombin-III (ANT3), apolipoprotein A-II (APOA2 ), Calpastatin (B7Z574), brain acid soluble protein 1 (BASP1), α-2-HS-glycoprotein (C9JV77), calreticulin (CALR), calpain-1 catalytic subunit (CAN1), cell division control Protein 42 homolog (CDC42), complement C3 (CO3), coronin-1A (COR1A), programmed cell death 6-interacting protein (DCD), defensin 1 (DEF1), F-box only protein 50 (FBX50), γ Guru Milcyclotransferase (GGCT), glutathione reductase, mitochondria (GSHR), keratin, type II cytoskeleton 1 (K2C1), UMP-CMP kinase (KCY), mesothelin (MSLN), N-acetylmuramoyl-L-alanine amidase ( PGRP2), nicotinic acid phosphoribosyltransferase (PNCB), inter-α-trypsin inhibitor heavy chain H1 (ITIH1), ribonuclease T2 (RNASET2), superoxide dismutase [Mn], mitochondria (SODM), small molecule proline rich protein 3 ( SPRR3), Src substrate cortactin (SRC8), cluster of tubulin β-4B chain (TBB4B), tropomyosin α-3 chain (TPM3), cellotransfer Phosphorus (TRFE), glutathione S-transferase P (THIO), vitronectin (VTNC), vitamin D binding protein (Q6LDC6), inter-α-trypsin inhibitor heavy chain H4 (ITIH4), metalloprotease inhibitor (TIMP1), heat shock protein 90 (HSP90), cathepsin B (CATB), ceruloplasmin (CERU), calprotectin, 14-3-3σ (1433S), α-2-hs-glycoprotein (FETUA), α-2-macroglobulin (A2MG) ), Transthyretin (TTHY), and any combination thereof (as provided herein).

  In other embodiments, the complementary DNA is reverse transcribed from the mRNA obtained from the sample, amplified, and real-time PCR (which is normalized to (absolute copy number or DNA input or further normalizing gene). As a relative quantity, where possible), allowing simultaneous detection and quantification of the specific gene product in the complementary DNA sample and the original mRNA sample.

  The version of the method of the present invention includes detecting at least one biomarker. However, any number of biomarkers can be detected. It is preferred that at least two biomarkers are detected in the analysis. However, it is realized that three, four, or more (including all) of the biomarkers described herein can be utilized in the analysis. Thus, not only one or more markers can be detected, but any number or combination of markers can also be used in the detection. Furthermore, other biomarkers not described herein can be used in combination with any of the biomarkers of the present disclosure to assist in the diagnosis of cancer. Furthermore, any combination of the above biomarkers can be detected according to the version of the present invention.

  Markers selected from the following and listed in Table 1: Igλ chain V-IV region Hil (LV403), Ig heavy chain V-III BRO (HV305), Ig heavy chain V-III VH26 (HV303), β- 2-microglobulin (B2MG), lipocalin-1 (LCN1), zinc-α-2-glycoprotein (ZA2G), cystatin B (CYTB), antileucoproteinase (SLP1), galectin-3 (LEG3), histidine tri Adnucleotide binding protein 1 (D6RD60), S100A9 (S10A9), S100A8 (S10A8), galectin-3-binding protein (LG3BP), cluster of Igα-1 chain C region (IGHA1), cluster of Igκ chain V-III region HAH (KV312), VEGF co-regulatory ke Cain 1 (VCC1), L-lactate dehydrogenase A chain (LDHA), aldo-ketoreductase family 1 member C (AKR1C1), root retin (B1AKD8), L-lactate dehydrogenase B chain (LDHB), retinal dehydrogenase 1 (AL1A1) , Protein of unknown nature (B4E1Z4), α-1-antichymotrypsin (AACT), superoxide dismutase [Cu—Zn] (SODC), SPARC-like protein 1 (SPRL1), Ig heavy chain V-III region TIL (HV304), Keratin (K1C9), cystatin-SN (CYTN), α-actinin-4 (ACTN4), Igλ-3 chain C region (fragment) (IGLC3), immunoglobulin λ-like polypeptide 5 (IGLL5), alcohol dehydrogena ZE1C (ADHIG), malate dehydrogenase, mitochondria (MDHM), calmodulin-like protein 5 (CALL5), α-1-antitrypsin (A1AT), α-1B-glycoprotein (A1BG), leucine-rich α-2-saccharide Protein (A2GL), small molecule ubiquitin-like modifier 3 (A8MU27), pro-gradient protein 2 homolog (AGR2), profilin-1 (PROF1), cluster of Igλ chain V-III region LOI (LV302), prothrombin (E9PIT3) , Hemopexin (HEMO), Igγ-2 chain C region (IGHG2), ubiquitin-40S ribosomal protein S27a (RPS27A), afamin (AFAM), apolipoprotein AI (APOA1), apolipoprotein A-IV (AP A4), flavin reductase (NADPH) (BLVRB), prosaposin (PSAP), lacritin (LACRT), 60S acidic ribosomal protein P1 (RLA1), inter-α-trypsin inhibitor heavy chain H2 (ITIH2), mucin-like protein 1 (MUCL1) ), S100 A6 (S100A6), Na (+) / H (+) exchange regulatory cofactor NHE-RF1 (NHRF1), thioredoxin domain-containing protein 17 (I3L0K2), lymphocyte-specific protein (LSP1), haptoglobin cluster (H3BS21), myosin-regulated light chain 12A (J2QRS3), ribonuclease inhibitor (RINI), α-enolase (ENOA), Igκ chain VI region EU cluster (KV106), alcohol Drogenase class 4μ / σ chain (ADH7), protein AMBP (AMBP), angiotensinogen (ANGT), antithrombin-III (ANT3), apolipoprotein A-II (APOA2), calpastatin (B7Z574), brain Acid soluble protein 1 (BASP1), α-2-HS-glycoprotein (C9JV77), calreticulin (CALR), calpain-1 catalytic subunit (CAN1), cell division control protein 42 homolog (CDC42), complement C3 (CO3), coronin-1A (COR1A), programmed cell death 6-interacting protein (DCD), defensin 1 (DEF1), F-box only protein 50 (FBX50), γ-glutamyl cyclotransferase (GGCT), glutathio Reductase, mitochondria (GSHR), keratin, type II cytoskeleton 1 (K2C1), UMP-CMP kinase (KCY), mesothelin (MSLN), N-acetylmuramoyl-L-alanine amidase (PGRP2), nicotinic acid phosphoribosyltransferase (PNCB), inter-α-trypsin inhibitor heavy chain H1 (ITIH1), ribonuclease T2 (RNASET2), superoxide dismutase [Mn], mitochondria (SODM), small molecule proline rich protein 3 (SPRR3), Src substrate cortactin (SRC8) ), Tubulin β-4B chain cluster (TBB4B), tropomyosin α-3 chain (TPM3), serotransferrin (TRFE), glutathione S-transferase P (THIO), vitronectin (VTNC), vitamin D binding protein (Q6LDC6), inter-α-trypsin inhibitor heavy chain H4 (ITIH4), metalloprotease inhibitor (TIMP1), heat shock protein 90 (HSP90), cathepsin B (CATB) , Ceruloplasmin (CERU), calprotectin, 14-3-3σ (1433S), α-2-hs-glycoprotein (FETUA), α-2-macroglobulin (A2MG), transthyretin (TTHY), May be increased or decreased by at least 1.5 times, 2 times, 4 times, 5 times, 8 times, 10 times or more compared to the level of the marker in the control sample . The control sample can be a sample from a subject without cancer, a pooled sample from a subject without cancer, or an average expression level of a subject without cancer Can be known control or baseline expression levels.

  Several terms are used throughout this disclosure and are intended to be defined as commonly used in the art or as specifically provided herein. As provided herein, mass spectrometry or MS is an analysis that generates an electric or magnetic field and determines the mass-to-charge ratio of peptides and compounds to identify or determine peptide sequences and chemical structures. A technique. LC-MS / MS spectroscopy is an analytical technique that combines the resolution of high performance liquid chromatography (HPLC) with the mass spectrometry of mass spectrometry. Triple quadrupole mass spectrometry refers to a tandem mass spectrometer that has three ionization chambers (Q1, Q2, and Q3). This technique allows target detection of the molecule of interest. An ion pair is a parent peptide that is detected at Q1 in its double or triple charged form, and the resulting y ion produced by Q2 when detected at Q3 of a triple quadrupole mass spectrometer or b ion. A SIS internal peptide refers to a synthetic isotopically labeled peptide having the same sequence as the peptide monitored in Q1 and used as an internal reference standard for quantifying the peptide of interest. -Y ion means the ion produced | generated from the C terminal of a peptide fragment. -B ion means the ion produced | generated from the N terminal of a peptide fragment. Quantitative ions refer to the selected highest intensity y ion or b ion used to determine the amount of its parent protein in a biological sample. Qualitative ion refers to the qualitative ion vs. the selected protein of interest and the labeled peptide vs. the ion (s) selected to ensure the integrity of the selected standard.

  CLIA is a clinical laboratory improvement amendment, which is a federal regulatory standard that applies to all laboratory tests conducted on humans in the United States, except for clinical trials and basic research. Yes (CLIA-related Federal Gazette and Federal Regulations published). A CLIA-approved laboratory conducts laboratory testing of human specimens for the diagnosis, prevention, or treatment of a disease or disorder, and is approved and monitored by a regulatory agency approved by the US Food and Drug Administration (FDA). Clinical laboratory (CLIA laws and regulations, 2013). Tests that CLIA refrains from apply refer to clinical tests that meet specific criteria for risk, error, and complexity as defined by the FDA.

  A point-of-care device includes a binding agent for a biomarker or series of biomarkers of interest, and can generate information regarding its presence, absence, and in some cases, the concentration of a detected biomarker, and A device or cartridge that can be used at a doctor's place of care. Analyte refers to any measurable biomarker, which can be a protein, peptide, macromolecule, metabolite, small molecule, or autoantibody. Biological fluid as used herein refers to tears, whole blood, serum, urine and saliva. A biomarker is any substance (eg, protein, peptide, metabolite, polynucleotide sequence) whose concentration level changes, eg, increases or decreases, in the body as a result of a disease or condition. Markers and biomarkers can be used interchangeably herein.

  A lateral flow test refers to a device that uses a porous paper of sintered polymer to determine the presence of an analyte in a biological fluid. ELISA refers to an enzyme-linked immunosorbent assay that uses antibodies or antigens to detect the presence and concentration of the analyte of interest. A diagnostic panel refers to a group of molecules, such as proteins or peptides, the combined concentration of which is used to diagnose a disease state, such as cancer. A breast cancer marker is a molecule: eg, protein, peptide, metabolite, polynucleotide sequence whose concentration level changes as a result of the presence or absence of cancer in the body: eg, increases or decreases Say things.

  In addition to being useful for diagnosing cancer, and particularly breast cancer, in a subject, the kits and methods provided herein treat cancer in an individual previously diagnosed with cancer. Or it can be used to monitor recurrence. Thus, markers selected from the following and listed in Table 1: Igλ chain V-IV region Hil (LV403), Ig heavy chain V-III BRO (HV305), Ig heavy chain V-III VH26 (HV303), β-2-microglobulin (B2MG), lipocalin-1 (LCN1), zinc-α-2-glycoprotein (ZA2G), cystatin B (CYTB), antileucoproteinase (SLP1), galectin-3 (LEG3), histidine Triad nucleotide binding protein 1 (D6RD60), S100A9 (S10A9), S100A8 (S10A8), galectin-3-binding protein (LG3BP), cluster of Igα-1 chain C region (IGHA1), Igκ chain V-III region HAH Cluster (KV312), VEGF co-ordination Sexual chemokine 1 (VCC1), L-lactate dehydrogenase A chain (LDHA), aldo-ketoreductase family 1 member C (AKR1C1), root retin (B1AKD8), L-lactate dehydrogenase B chain (LDHB), retinal dehydrogenase 1 (AL1A1) ), Protein of unknown properties (B4E1Z4), α-1-antichymotrypsin (AACT), superoxide dismutase [Cu—Zn] (SODC), SPARC-like protein 1 (SPRL1), Ig heavy chain V-III region TIL (HV304) , Keratin (K1C9), cystatin-SN (CYTN), α-actinin-4 (ACTN4), Igλ-3 chain C region (fragment) (IGLC3), immunoglobulin λ-like polypeptide 5 (IGLL5), alcohol dehydrate Genase 1C (ADHIG), malate dehydrogenase, mitochondria (MDHM), calmodulin-like protein 5 (CALL5), α-1-antitrypsin (A1AT), α-1B-glycoprotein (A1BG), leucine-rich α-2-sugar Protein (A2GL), small molecule ubiquitin-like modifier 3 (A8MU27), pro-gradient protein 2 homolog (AGR2), profilin-1 (PROF1), cluster of Igλ chain V-III region LOI (LV302), prothrombin (E9PIT3) , Hemopexin (HEMO), Igγ-2 chain C region (IGHG2), ubiquitin-40S ribosomal protein S27a (RPS27A), afamin (AFAM), apolipoprotein AI (APOA1), apolipoprotein A-IV APOA4), flavin reductase (NADPH) (BLVRB), prosaposin (PSAP), lacritin (LACRT), 60S acidic ribosomal protein P1 (RLA1), inter-α-trypsin inhibitor heavy chain H2 (ITIH2), mucin-like protein 1 (MUCL1) ), S100 A6 (S100A6), Na (+) / H (+) exchange regulatory cofactor NHE-RF1 (NHRF1), thioredoxin domain-containing protein 17 (I3L0K2), lymphocyte-specific protein (LSP1), haptoglobin cluster (H3BS21), myosin regulatory light chain 12A (J2QRS3), ribonuclease inhibitor (RINI), α-enolase (ENOA), Igκ chain VI region EU cluster (KV106), alcohol Rudehydrogenase class 4μ / σ chain (ADH7), protein AMBP (AMBP), angiotensinogen (ANGT), antithrombin-III (ANT3), apolipoprotein A-II (APOA2), calpastatin (B7Z574), brain acid Soluble protein 1 (BASP1), α-2-HS-glycoprotein (C9JV77), calreticulin (CALR), calpain-1 catalytic subunit (CAN1), cell division control protein 42 homolog (CDC42), complement C3 (CO3), coronin-1A (COR1A), programmed cell death 6-interacting protein (DCD), defensin 1 (DEF1), F-box only protein 50 (FBX50), gamma glutamyl cyclotransferase (GGCT), glu Thion reductase, mitochondria (GSHR), keratin, type II cytoskeleton 1 (K2C1), UMP-CMP kinase (KCY), mesothelin (MSLN), N-acetylmuramoyl-L-alanine amidase (PGRP2), phosphoribosyl nicotinate Transferase (PNCB), inter-α-trypsin inhibitor heavy chain H1 (ITIH1), ribonuclease T2 (RNASET2), superoxide dismutase [Mn], mitochondria (SODM), small molecule proline rich protein 3 (SPRR3), Src substrate cortactin ( SRC8), tubulin β-4B chain cluster (TBB4B), tropomyosin α-3 chain (TPM3), serotransferrin (TRFE), glutathione S-transfer Hase P (THIO), vitronectin (VTNC), vitamin D binding protein (Q6LDC6), inter-α-trypsin inhibitor heavy chain H4 (ITIH4), metalloprotease inhibitor (TIMP1), heat shock protein 90 (HSP90), cathepsin B (CATB), ceruloplasmin (CERU), calprotectin, 14-3-3σ (1433S), α-2-hs-glycoprotein (FETUA), α-2-macroglobulin (A2MG), transthyretin (TTHY) ) Levels increase or decrease over time in the same subject after treatment, additional chemotherapeutic agents targeting the cancer can be administered.

  The methods and kits can also be used to monitor the effectiveness of chemotherapeutic treatment. In this alternative embodiment, biomarkers as selected from and listed in Table 1: Igλ chain V-IV region Hil (LV403), Ig heavy chain V-III BRO (HV305), Ig heavy chain V- III VH26 (HV303), β-2-microglobulin (B2MG), lipocalin-1 (LCN1), zinc-α-2-glycoprotein (ZA2G), cystatin B (CYTB), antileucoproteinase (SLP1), galectin- 3 (LEG3), histidine triad nucleotide binding protein 1 (D6RD60), S100A9 (S10A9), S100A8 (S10A8), galectin-3-binding protein (LG3BP), Igα-1 chain C region cluster (IGHA1), Igκ Cluster of chain V-III region HAH ( V312), VEGF co-regulatory chemokine 1 (VCC1), L-lactate dehydrogenase A chain (LDHA), aldo-ketoreductase family 1 member C (AKR1C1), root retin (B1AKD8), L-lactate dehydrogenase B chain (LDHB) , Retinal dehydrogenase 1 (AL1A1), protein with unknown properties (B4E1Z4), α-1-antichymotrypsin (AACT), superoxide dismutase [Cu-Zn] (SODC), SPARC-like protein 1 (SPRL1), Ig heavy chain V- III region TIL (HV304), keratin (K1C9), cystatin-SN (CYTN), α-actinin-4 (ACTN4), Igλ-3 chain C region (fragment) (IGLC3), immunoglobulin λ-like polypeptide 5 (IG L5), alcohol dehydrogenase 1C (ADHIG), malate dehydrogenase, mitochondria (MDHM), calmodulin-like protein 5 (CALL5), α-1-antitrypsin (A1AT), α-1B-glycoprotein (A1BG), leucine-rich α 2-glycoprotein (A2GL), small molecule ubiquitin-like modifier 3 (A8MU27), pro-gradient protein 2 homolog (AGR2), profilin-1 (PROF1), cluster of Igλ chain V-III region LOI (LV302), Prothrombin (E9PIT3), hemopexin (HEMO), Igγ-2 chain C region (IGHG2), ubiquitin-40S ribosomal protein S27a (RPS27A), afamin (AFAM), apolipoprotein AI (APOA1), Polypoprotein A-IV (APOA4), flavin reductase (NADPH) (BLVRB), prosaposin (PSAP), lacritin (LACRT), 60S acidic ribosomal protein P1 (RLA1), inter-α-trypsin inhibitor heavy chain H2 (ITIH2) , Mucin-like protein 1 (MUCL1), S100 A6 (S100A6), Na (+) / H (+) exchange regulatory cofactor NHE-RF1 (NHRF1), thioredoxin domain-containing protein 17 (I3L0K2), lymphocyte-specific protein (LSP1), haptoglobin cluster (H3BS21), myosin regulatory light chain 12A (J2QRS3), ribonuclease inhibitor (RINI), α-enolase (ENOA), Igκ chain V-I region EU cluster -(KV106), alcohol dehydrogenase class 4 μ / σ chain (ADH7), protein AMBP (AMBP), angiotensinogen (ANGT), antithrombin-III (ANT3), apolipoprotein A-II (APOA2), calpastatin ( B7Z574), brain acid soluble protein 1 (BASP1), α-2-HS-glycoprotein (C9JV77), calreticulin (CALR), calpain-1 catalytic subunit (CAN1), cell division control protein 42 homolog (CDC42) ), Complement C3 (CO3), coronin-1A (COR1A), programmed cell death 6-interacting protein (DCD), defensin 1 (DEF1), F-box only protein 50 (FBX50), γ-glutamylcyclotransferase Rase (GGCT), glutathione reductase, mitochondria (GSHR), keratin, type II cytoskeleton 1 (K2C1), UMP-CMP kinase (KCY), mesothelin (MSLN), N-acetylmuramoyl-L-alanine amidase (PGRP2) Nicotinate phosphoribosyltransferase (PNCB), inter-α-trypsin inhibitor heavy chain H1 (ITIH1), ribonuclease T2 (RNASET2), superoxide dismutase [Mn], mitochondria (SODM), small molecule proline rich protein 3 (SPRR3) , Src substrate cortactin (SRC8), tubulin β-4B chain cluster (TBB4B), tropomyosin α-3 chain (TPM3), cellotransferrin (TRFE), glu Tathione S-transferase P (THIO), vitronectin (VTNC), vitamin D binding protein (Q6LDC6), inter-α-trypsin inhibitor heavy chain H4 (ITIH4), metalloprotease inhibitor (TIMP1), heat shock protein 90 (HSP90), Cathepsin B (CATB), ceruloplasmin (CERU), calprotectin, 14-3-3σ (1433S), α-2-hs-glycoprotein (FETUA), α-2-macroglobulin (A2MG), transthyretin The level of (TTHY) may increase or decrease over time if the treatment regimen is effective, and will not change or change if the treatment regimen is not effective in a single subject. Raw being examined in the body Depending on the marker (s) is either may increase over time or reduced.

  Cancer treatment includes reducing the number of cancer cells or the size of a tumor or mass, reducing the progression of cancer to a less aggressive form in the above subject, and proliferating cancer cells. Or reduce the rate of tumor growth, kill cancer cells, reduce cancer cell metastasis in the subject, or reduce the likelihood of cancer recurrence. However, it is not limited to these. Treatment of a subject, as used herein, benefits a subject suffering from or at risk of developing the disease (in the condition of the subject, eg, in one or more symptoms). Improvement, delay of progression of the disease, delay of onset of symptoms, or delay of progression of symptoms, etc.).

  The present disclosure is not limited to the specific details of the assembly, component arrangement, or method steps set forth herein. The compositions and methods disclosed herein can be made, implemented, used, performed and / or formed in a variety of ways apparent to those skilled in the art in light of the disclosure that follows. . The terminology and terminology used herein is for the purpose of description only and is not to be considered limited to the scope of the claims. Usual indicators, for example, first, second, and third, are used in the specification and claims to refer to various structures or method steps, and any particular It is not intended to be construed as indicating any structure or step in the formula, or any specific order or configuration for such structures or steps. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. Use of any and all examples or exemplary phrases provided herein, eg, “such as” (eg,), facilitates the disclosure unless otherwise claimed. And is not intended to imply any limitation on the scope of the disclosure. The language shown in the description and in the drawings are not intended to indicate that any unclaimed element is essential to the practice of the disclosed subject matter. The terms “including”, “comprising” or “having” and their use herein are the elements listed thereafter. And its equivalents, as well as additional elements. Embodiments in which certain elements are described as “including”, “comprising”, or “having” are from those elements “from” Also contemplated as “consisting essentially of” and “consisting of”.

  The description of a range of values herein is intended only to serve as a convenient way to individually reference each distinct value that falls within the above range, unless otherwise indicated herein. Each distinct value is incorporated herein as if it were individually described herein. For example, if the concentration range is stated as 1% to 50%, values such as 2% to 40%, 10% to 30%, or 1% to 3% are explicitly listed herein. Is intended. These are merely examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest and highest values listed are explicitly stated in this disclosure. Should be considered. The use of the word “about” to describe a particular stated quantity or range of quantities is considered due to manufacturing tolerances, instrumental errors and human error in forming measurements, etc. Values that are close to the quantities described above, such as values that are naturally taken into account, are included in the quantities. All percentages referring to amounts are by weight unless otherwise indicated.

  No admission is made that any reference, including any non-patent or patent literature cited herein, constitutes prior art. In particular, unless otherwise stated, approval that any reference to any document herein forms part of the common general knowledge in the field in the United States or any other country. It is understood that does not constitute. Any discussion of the above references will state what the author claims, and ensure the right of the applicant to challenge the accuracy and appropriateness of any of the documents cited herein. . All references cited herein are fully incorporated by reference unless explicitly indicated otherwise. In the event of any inconsistency between any definitions and / or descriptions found in the cited references, the present disclosure shall control. None of the references mentioned is admitted to be prior art with respect to the present invention.

  The following examples are meant to be illustrative only and are not meant as limitations on the scope of embodiments of the present invention or the scope of the appended claims.

(Example 1: Participant selection)
Study participants were recruited from patients who had been seen at Breast Health and Surgical Centers AR, OK, TN and WA with the approval of the institutional review board. Consent was obtained from all patients and a copy of informed consent was obtained prior to sample collection or before completing the patient information sheet. Samples were collected by clinic personnel (ie, nurses and professional technicians) and / or Ascendant Dx personnel. Inclusion / exclusion criteria used for patient selection were as follows:
Inclusion criteria:
As long as the individual presents a physical examination of an individual between the ages of 18 and 100, or presents an abnormal test or examination evaluation, or presents a palpable mass evaluation, or as long as a portion of the mass remains, Presenting mass before or after biopsy, or recently diagnosed with breast cancer, but has not yet received any kind of treatment.

Exclusion criteria:
It is associated with infection or trauma of an individual eye younger than 18 years and over 100 years of age, or is currently suffering from acute conjunctivitis, or is diagnosed with breast cancer and undergoing treatment.

  Control samples were collected from patients who had been diagnosed for routine screening mammograms and did not respond to a call back for further procedures. Benign samples were collected at the time of biopsy and included in the benign group after pathological results were determined. Cancer samples from patients undergoing MRI prior to surgery and from patients undergoing sentinel node procedures prior to surgery are also collected at the time of biopsy and cancer is detected after pathological results are known. Included in group.

  Data collected from participants included: age, gender, race, birth control or hormone replacement therapy, ophthalmic infections, current or recent chemotherapy treatment, cancer family History, genetic testing (BRAC1 / 2) if available, cancer stage (I, II, III, IV), cancer type (non-invasive ductal carcinoma, invasive ductal carcinoma, invasive lobular carcinoma, Noninvasive lobular carcinoma, and unknown), hormone receptor status (ER +/−, PR +/−, HER2 +/−), mass size, tumor grade (I, II, III), breast density score (categories 1, 2) Density identified as 3, or 4) and previous cancer history.

(Example 2: Schirmer strip sample collection procedure)
Institutional review board approval was obtained for tear collection using Schirmer strips (Gulden Optalmics Elkins Park, PA). For collection, the rounded tip of the Schirmer strip was folded at a 0 mm line forming a lip. The folded part was placed on the participant's lower eyelid and they were asked to close their eyes and hold their eyes closed for 5 minutes. After 5 minutes, the strip was removed and placed in a sterile 1.5 mL pre-labeled snap top tube and placed at -20 ° C or -80 ° C, depending on availability. The collection criteria stipulated that the strip could be removed if the 35 mm mark was reached before a time of 5 minutes. Samples collected at participating clinics were collected by Ascendant Dx personnel on a weekly basis, and placed in dry ice to the Ascendant Dx research facility.

(Example 3: Schirmer strip sample processing)
Protein elution was performed by first dicing the strip with sterile forceps and scissors and placing it in a clean sterile 1.5 mL snap top tube. 200 μL of 1 × phosphate buffered saline (PBS) was added to the diced strip and the sample was incubated for 3 hours at 4 ° C. with gentle shaking. Following elution, the sample was briefly centrifuged in a tabletop centrifuge to collect the strip fragment at the bottom of the tube and transfer the supernatant to a new clean 1.5 mL snap top tube. Total protein content was determined using a standard bicinchoninic acid (BCA) assay and the samples were stored at -80 ° C until further use.

  The total protein content of each pool was determined using a BCA assay kit (Pierce) with a 1:20 (v / v) standard and unknown to working reagent and with an incubation time of 30 minutes at 37 ° C. Were determined. To ensure reliable total protein content calculations, serial dilutions were made for each sample (eg, 1: 2, 1: 4, 1: 6) and all dilutions were plated in triplicate. . Diluted albumin (2 mg / ml, 1.5 mg / ml, 1 mg / ml, 0.75 mg / ml, 0.5 mg / ml, 0.25 mg / ml, 0.125 mg / ml, 0.025 mg / ml and 0 mg / ml ) Was generated and blank subtraction was applied to all standards and unknowns. The protein concentration of each unknown was calculated using the four parameter fit of the standard curve. The concentration was multiplied by the dilution factor and averaged to give an accurate total protein concentration calculation. An assay was considered valid if the coefficient of variation (% CV) was 15% or less.

Example 4: Methods and results for unlabeled quantification by LC MS / MS
Experiment 1 : Trypsin digestion in solution, followed by LC MS / MS on 25 breast cancer samples, 25 benign samples, and 25 control samples of University of Arkansas for Medical Sciences (UAMS) . Solution digestion was performed after reduction in 10 mM Tris [2-carboxyethyl] phosphine (Pierce) and alkylation in 50 mM iodoacetamide (Sigma-Aldrich) followed by addition of 100 ng porcine trypsin (Promega) and 12 at 37 ° C. All 75 samples were performed in 100 mM ammonium bicarbonate (Sigma-Aldrich) with a ~ 16 hour incubation. The peptide product was then acidified in 0.1% formic acid (Fluka). Tryptic peptide was separated by reverse phase Jupiter Proteo resin (Phenomenex) on a 100 × 0.075 mm column using a nanoAcquity UPLC system (Waters). Peptides were eluted using an 80 minute gradient from buffer A: B ratio 97: 3 to 35:65 [buffer A = 0.1% formic acid, 0.05% acetonitrile; buffer B = 0. 1% formic acid, 75% acetonitrile]. The eluted peptide was ionized by electrospray (1.8 kV) followed by MS / MS analysis using collision-induced dissociation on an LTQ Orbitrap Velos mass spectrometer (Thermo). MS data was acquired using an FTMS analyzer in profile mode at a resolution of 60,000 over a range of 375-1500 m / z. MS / MS data was acquired for the top 15 peaks from each MS scan using the ion trap analyzer in centroid mode and normal mass range with normalized collision energy of 35.0. Proteins were identified from MS / MS spectra by database search with Mascot search engine (Matrix Science) or MaxQuant quantitative proteomics software (Max Plan Institute). Mascot search results were edited using Scaffold (Proteome Software).

  The following criteria were set to select a group of proteins that could exhibit altered breast physiology: 1) Protein has a fold change of 1.5 or more (positive or negative for cancer) In any direction). 2) The fold change should be accompanied by a p-value <0.05. 3) Protein is present in 12 out of 25 cancer samples. Using these criteria, the over 500 list was narrowed down to the following proteins: α-1-antitrypsin (A1AT), antileucoproteinase (SLP1), cofilin (COF1), antithrombin-III (ANT3), β-2-microglobulin (B2MG), protein-glutamine-γ-glutamyltransferase (B4DIT7), protein of unknown nature (B8ZZQ6), calmodulin-like protein 5 (CALL5), cystatin-B (CYTB), neutrophil fencing ( Neutrophyldefening-1 (DEF1), Destrin (DEST), Kariocin-1 (E7ER44), Rab GDP dissociation inhibitor β cluster (E7EU23), Elongation factor 1-α ( F1A1), ezrin (EZRI), heme binding protein 2 (HEB2), heat shock cognate 71 (HSP7C), heat shock protein β1 (HSPB1), Ig heavy chain V-III (HV303), Ig heavy chain V-III region BRO cluster (HV305), lipocalin-1 (LCN1), galectin-3 (LEG3), Igλ chain V-IV (LV403), Igμ chain c region isoform 2 (P01871), isoform 2 heat shock protein cluster (P07900-2), 14-3-3 protein ζδ cluster (P63104), perilipin-3 isoform 3 (PLIN3), proteasome activator complex subunit 1 (Q06323), UMP-CMP kinase (Q5T0D2), Protein S 00-A4 (S10A4), S100-A8 (S10A8), protein S100-A9 (S10A9), protein S100-A11 (S10AB), submandibular gland androgen regulatory protein (SMR3B), zinc-α-2-glycoprotein (ZA2G) ), Zymogen granule protein 16 homolog B (ZG16B).

Experiment 2 : In solution, trypsin digestion followed by LC MS / MS was repeated using 50 samples in each group (breast cancer, benign, control). The same method as described for Experiment 1 was used for Experiment 2. The increase in sample size eliminated some of the trends observed in Experiment 1, while the trends appearing in Experiment 1 were more pronounced in Experiment 2. From this data, the list of 500 proteins described in Experiment 1 was further refined to the desired 103 proteins. This list includes: histidine triad nucleotide binding protein 1 (D6RD60), S100A9 (S10A9), S100A8 (S10A8), galectin-3-binding protein (LG3BP), cluster of Igα-1 chain C region (IGHA1) , Igκ chain V-III region HAH cluster (KV312), VEGF co-regulated chemokine 1 (VCC1), L-lactate dehydrogenase A chain (LDHA), aldo-ketoreductase family 1 member C (AKR1C1), root retin (B1AKD8) ), L-lactate dehydrogenase B chain (LDHB), retinal dehydrogenase 1 (AL1A1), protein of unknown nature (B4E1Z4), α-1-antichymotrypsin (AACT), superoxide dismutase [C -Zn] (SODC), SPARC-like protein 1 (SPRL1), Ig heavy chain V-III region TIL (HV304), keratin (K1C9), cystatin-SN (CYTN), α-actinin-4 (ACTN4), Igλ- 3-chain C region (fragment) (IGLC3), immunoglobulin λ-like polypeptide 5 (IGLL5), alcohol dehydrogenase 1C (ADHIG), malate dehydrogenase, mitochondria (MDHM), calmodulin-like protein 5 (CALL5), α-1- Antitrypsin (A1AT), α-1B-glycoprotein (A1BG), leucine-rich α-2-glycoprotein (A2GL), low molecular weight ubiquitin-like modifier 3 (A8MU27), pro-gradient protein 2 homolog (AGR2), profilin -1 (PROF ), Igλ chain V-III region LOI cluster (LV302), prothrombin (E9PIT3), hemopexin (HEMO), Igγ-2 chain C region (IGHG2), ubiquitin-40S ribosomal protein S27a (RPS27A), afamin (AFAM), Apolipoprotein AI (APOA1), apolipoprotein A-IV (APOA4), flavin reductase (NADPH) (BLVRB), prosaposin (PSAP), lacritin (LACRT), 60S acidic ribosomal protein P1 (RLA1), inter-α- Trypsin inhibitor heavy chain H2 (ITIH2), mucin-like protein 1 (MUCL1), S100 A6 (S100A6), Na (+) / H (+) exchange regulatory cofactor NHE-RF1 (NHRF1), Redoxin domain-containing protein 17 (I3L0K2), lymphocyte-specific protein (LSP1), cluster of haptoglobin (H3BS21), myosin regulatory light chain 12A (J2QRS3), ribonuclease inhibitor (RINI), α-enolase (ENOA), Igκ Cluster of chain VI region EU (KV106), alcohol dehydrogenase class 4 μ / σ chain (ADH7), protein AMBP (AMBP), angiotensinogen (ANGT), antithrombin-III (ANT3), apolipoprotein A- II (APOA2), calpastatin (B7Z574), brain acid soluble protein 1 (BASP1), α-2-HS-glycoprotein (C9JV77), calreticulin (CALR), calpain-1 catalytic sub Knit (CAN1), cell division control protein 42 homolog (CDC42), complement C3 (CO3), coronin-1A (COR1A), programmed cell death 6-interacting protein (DCD), defensin 1 (DEF1), F-box Only protein 50 (FBX50), γ-glutamyl cyclotransferase (GGCT), glutathione reductase, mitochondria (GSHR), keratin, type II cytoskeleton 1 (K2C1), UMP-CMP kinase (KCY), mesothelin (MSLN), N-acetyl Muramoyl-L-alanine amidase (PGRP2), nicotinic acid phosphoribosyltransferase (PNCB), inter-α-trypsin inhibitor heavy chain H1 (ITIH1), ribonuclease T2 (RNASE 2), superoxide dismutase [Mn], mitochondria (SODM), low molecular weight proline rich protein 3 (SPRR3), Src substrate cortactin (SRC8), cluster of tubulin β-4B chain (TBB4B), tropomyosin α-3 chain ( TPM3), serotransferrin (TRFE), glutathione S-transferase P (THIO), vitronectin (VTNC), vitamin D binding protein (Q6LDC6), inter-α-trypsin inhibitor heavy chain H4 (ITIH4), metalloprotease inhibitor (TIMP1) , Heat shock protein 90 (HSP90), cathepsin B (CATB), ceruloplasmin (CERU), calprotectin, 14-3-3σ (1433S), α-2-hs-sugar tamper Quality (FETUA), α-2- macroglobulin (A2MG), transthyretin (TTHY) (Appendix 1). A complete list of identified proteins was exported from Scaffold software for analysis using the JMPpro11 statistical software package.

Example 5: Statistical analysis using JMPpro11 software
Experiment 1 : Data were organized by using Microsoft Excel® spreadsheet software and the spectral intensity of each of the proteins previously identified for all 75 samples. Sample status (cancer, benign, control), cancer type, tumor size grouping (1 = 0.1 mm to 1 cm, 2 = 1.1 to 2.9 cm, 3 = 3 cm or more) and tumor malignancy status Was also included in the spreadsheet. This spread seed was uploaded to the JMPpro11 software package for analysis.

  Binary linear regression was applied between the cancer sample and the control sample. Six proteins were identified as significant based on their sigmoid curves and p-values: Igλ chain V-IV (LV403), Ig heavy chain V-III region BRO (HV305), Ig heavy chain V− III VH26 (HV303), lipocalin-1 (LCN1), β-2-microglobulin (B2MG), and zinc-α-2-glycoprotein (ZA2G). All six proteins appeared to be down-regulated against cancer. To determine whether the down-regulation of these proteins is specific for breast cancer, binary linear regression was repeated between benign and control. This comparison showed that the six previously identified proteins were also down-regulated in benign samples, and four more proteins were identified as significant between benign and control; antileucoproteinase (SLP1), calmodulin-like protein 5 (CALL5), cystatin B (CYTB), and galectin-3 (LEG3).

  Apparent logistic regression was used to determine the degree of differentiation between all groups using all 3 groups and all 10 proteins. The degree of misclassification for the group of 10 proteins was 12%. The receiver operator curves for each category yielded an AUC value of 0.96 for cancer, 0.94 for benign, and 0.98 for control (FIG. 1). A one-way ANOVA analysis was performed to ensure a shifted average for the three categories of samples with each protein (Figure 3).

Experiment 2 : One-way ANOVA analysis was performed using the spectral intensities recorded for all proteins identified from mass spectrometry. Trypsin digested peptide products were analyzed for total protein. Proteins were compared in three ways: 1) control vs. cancer, 2) cancer vs. benign, 3) benign vs. control. An alpha level of 0.05 was used as an indicator of significant expression change between groups. Variations in spectral intensity of trypsin digested fragments were compared. A series of proteins whose expression levels were increased in breast cancer samples relative to controls were identified. Proteins whose expression was reduced in breast cancer samples relative to controls were also identified. To ensure the effectiveness of the experiment, it was established that there were proteins whose expression remained constant across all groups tested (FIG. 4).

  The initial list of 500 proteins was narrowed down to the desired 103 proteins. Within this group of proteins are proteins that have increased expression for breast cancer and proteins that have decreased expression for breast cancer (FIG. 4). The diagnostic capabilities of total protein sequences, trypsin digested peptides, and sequence anomaly, which may be present as a result of disease, were all of interest.

(Example 6: ELISA analysis)
A series of proteins with increased expression levels in cancer samples relative to control samples were observed by mass spectrometry and several candidates were selected for further validation using ELISA. S100A9 was selected as a representative example of increased expression in tears of breast cancer patients compared to controls. LG3BP is shown as a representative of the protein with reduced expression levels in breast cancer samples compared to control samples. A standard ELISA protocol was used to assess expression levels in tears. S100A9 and LG3BP data were obtained using a kit purchased as DuoSets from R & D Systems (Minneapolis, Minn.). The ELISA procedure was performed according to the instructions provided by R & D systems. Briefly, 100 μL / well of capture antibody diluted in 1 × PBS to the recommended working concentration (GAL3BP 1.0 μg / ml; S100A9 0.5 μg / ml) is added to the high binding 96-well plate at room temperature. Incubate overnight (approximately 16 hours). The plate was rinsed 4 times with 1 × PBS containing 0.05% Tween and blotted against clean absorbent paper to remove any traces of liquid after each rinse. A rinse cycle was performed after each step of the assay. Blocking buffer (1 × PBS with 1% BSA) was added to all wells (approximately 200 μL / well) and the plates were incubated at room temperature for 2 hours. Tear samples were diluted using 1 × PBS with 1% BSA at dilutions of 1:10 and 1:50 for S100A9 and 1: 100 and 1: 500 for galectin3-binding protein. Dilutions known to fall within the linear region of the standard curve for each assay were selected based on the results of previous optimization assays. Samples and standards were tested in duplicate using 100 μL / well and incubated for 2 hours at room temperature. The detection antibody was diluted to its recommended working concentration (GAL3BP 2 μg / ml and S100A9 1 μg / ml), 100 μL was added to each well, and the plate was incubated at room temperature for 2 hours. Streptavidin-HRP solution was added to each well (100 μL / well) and incubated for 15 minutes at room temperature. Visualization was achieved by addition to the TMB substrate solution. After 15 minutes, 50 μL of 1 MH ₂ SO ₄ was added to each well and the absorbance at 450 nm was determined. ELISA data was analyzed using Prism version 6.0 available from GraphPad.

  Statistical analysis of ELISA data: The concentration of each protein (as calculated by Prism software) was exported to JMP Pro11 for statistical evaluation. A number of candidates were selected and investigated using ELISA assays against control samples, cancer samples or benign samples. The results of two proteins (S100A9 and galectin-3-binding protein) are shown to provide representative examples of proteins with increased and decreased protein expression in breast cancer tear samples compared to controls (FIG. 5 respectively). And FIG. 6). With respect to 63 breast cancer samples, 79 control samples, and 92 benign samples, S100A9 ANOVA produced a p-value of 0.0005 when all three groups were evaluated. The group average for S100A9 was: Breast cancer = 5673.02 pg / ml; Control = 2130.18 pg / ml; Benign = 6179.10 pg / ml. S100A9 expression is increased 2.6-fold in cancer samples compared to control samples and 2.9-fold in benign samples compared to control samples. ANOVA of galectin-3 binding protein for 66 breast cancer samples, 55 control samples, and 54 benign samples yielded a p-value <0.0001 when all three groups were evaluated. The group mean for LG3BP was as follows: breast cancer = 24448.1 pg / ml; control = 70242.2 pg / ml; benign = 62329.7 pg / ml. LG3BP has a 2.8-fold increase in control samples compared to cancer samples and a 2.5-fold increase in benign samples compared to cancer.

  Apparent logistic regression analysis for breast cancer samples and control samples was performed using two representative proteins. The ROC curve generated for S100A9 has an AUC of 0.78220 (FIG. 7) and LG3BP has an AUC of 0.76088 (FIG. 8). The above analysis was repeated using two proteins, yielding an AUC of 0.84250 (FIG. 9).

  Sample population characteristics: Clinical data (eg, breast density, cancer type, and tumor size) were obtained for as many samples as possible.

  Experiment 1: Of the 75 samples tested, a breast density score was obtained for 41 of the samples. The classification of the sample population by breast density was 14% Category 1, 36.6% Category 2, 43.9% Category 3, 4.7% Category 4 (FIG. 2). 18 of the 25 cancer samples were collected from patients diagnosed with intra ductal carcinoma (IDC). Two patients were diagnosed with non-invasive ductal carcinoma (DCIS), one patient was diagnosed with non-invasive lobular carcinoma (LCIS), and one patient with intralobular carcinoma (ILC). Diagnosed.

A scale was designed for classification. Here, the classification “small” was given to tumors <20 mm, the classification “medium” was assigned to tumors between 21 mm and 99 mm, and the classification “large” was assigned to tumors> 1 cm. Of the samples collected from breast cancer patients, 17 samples were classified as large tumors, 2 samples were classified as moderate, and 6 samples were classified as small. Receptor type pathology information was obtained for 8 of the cancer samples. 1 sample is ER ⁻ / HER 2 ⁻ , 1 sample is ER ⁻ / PR ⁻ , 2 samples are ER ⁺ / HER 2 ⁻ , 3 samples are ER ⁺ / HER ^{2 +} and 1 sample is ER ⁺ / PR ^- it was.

  Experiment 2: The sample pool analyzed in this study consisted of 5.9% category 1 breast tissue; 50% category 2, 37.8% category 3 and 5.4% category 4. ANOVA analysis was performed on several proteins assessed by ELISA to determine whether breast tissue type affects protein expression levels. Representative data is provided for the two proteins studied (FIGS. 10 and 11). Using an alpha level of 0.05, there was no statistically significant difference in the mean value of the 4 types of breast type for each of the proteins evaluated in the ELISA shown in FIG. 10 and FIG.

Samples were collected from various breast cancer types: 17 samples from patients diagnosed with DCIS, 44 samples from patients diagnosed with IDC, 4 samples from patients with both DCIS and IDC, and ILC Results were 5 samples from patients with Tumor sizes were extremely diverse. The classification system described with respect to Experiment 1 is used for samples provided with tumor size, 33 samples from patients with large tumors, 9 samples from patients with moderate tumor sizes, and small tumor sizes Twelve samples from patients with Of the 38 samples provided with the receptor type, 1 sample is ER ⁻ , 2 samples are ER ⁻ / HER 2 ⁻ , 2 samples are triple negative (ER ⁻ / HER 2 ⁻ / PR ⁻ ), and 1 sample is ER ⁻ / HER ^{2 +} , 11 samples were ER ⁺ , ER ⁺ / HER 2 16 and 5 samples were ER ⁺ / HER ^{2 +} . To date, no distinction has been observed for the proteins studied, based on tumor size, cancer type, or receptor status.

References:

Claims (22)

A method for determining whether a subject has cancer comprising:
Obtaining a sample from the subject;
Detecting the level of at least one of the markers from the list of markers provided in Table 1 in the sample; and the subject has a level of the marker in a control sample free of cancer. Determining that it has or is likely to have cancer if it changes compared to the level;
Including the method. The method of claim 1, wherein the cancer is breast cancer. A method according to any preceding claim, wherein the sample is an eye sample. The method according to any one of the preceding claims, wherein the subject is a human. The method according to any one of the preceding claims, wherein the marker is at least 1.5 times, 2 times, 4 times or more lower than the level of the marker in the control sample. . The method according to any one of the preceding claims, wherein the marker is increased by at least 1.5 fold, 2 fold, 4 fold or more compared to the level of the marker in the control sample. The method of any one of the preceding claims, wherein a combination of markers is used to determine the likelihood of cancer in the subject. The method according to any one of the preceding claims, wherein the level of the marker is detected by liquid chromatography-mass spectrometry (LC-MS). The method according to any one of the preceding claims, wherein the level of the marker is detected by an antibody-based detection method. The method according to any one of the preceding claims, wherein the level of the marker is detected by a multiplex protein detection method. The method according to any one of the preceding claims, wherein the level of the marker is detected by an mRNA detection method. The method according to any one of the preceding claims, wherein the subject is suspected of having cancer. The method according to any one of the preceding claims, wherein the subject has an increased risk of developing cancer. The method of claim 2, wherein the subject has a palpable lump suspected of being cancerous. The method according to any one of the preceding claims, wherein the subject's breast density is Category 1, Category 2, Category 3, or Category 4. The method according to claim 1, wherein the cancer is detected as a stage of breast cancer. The method according to any one of the preceding claims, wherein at least 10 markers are used in combination. At least 5 types of markers are used together. A method according to any one of the preceding claims. The method according to any one of the preceding claims, wherein at least three markers are used in combination. A kit for performing the method according to any one of the preceding claims, comprising a collection tube and a protease inhibitor or other protein stabilizer. 21. The kit of claim 20, further comprising an antibody that can bind to at least one of the markers. 21. The kit of claim 20, further comprising a primer pair that can amplify at least one of the markers.