WO2014062218A1

WO2014062218A1 - Colorectal cancer dna methylation markers

Info

Publication number: WO2014062218A1
Application number: PCT/US2013/029715
Authority: WO
Inventors: Peter W. Laird; Mihaela Campan; Christopher P. E. LANGE; Daniel J. Weisenberger; Toshinori HINOUE
Original assignee: University Of Southern California
Priority date: 2012-10-16
Filing date: 2013-03-07
Publication date: 2014-04-24

Abstract

Biomarkers for early detection of colorectal cancer are disclosed. A genome-scale marker discovery method or enhanced nucleic acid detection techniques such as digital MethyLight PCR may be used to identify and verify candidate DNAmethylation biomarkers for blood -based detection of colorectal cancer.

Description

COLORECTAL CANCER DNA METHYLATION MARKERS

FIELD OF THE INVENTION

The present invention relates to blood-based DNA methylation biomarkers for colorectal cancer (CRC) using a genome-scale DNA methylation approach for marker discovery. Using enhanced nucleic acid detection techniques, such as digital MethyLight PCR, markers suitable for prognostic and diagnostic applications were identified from preoperative clinical blood specimens obtained from patients undergoing curative surgery for

CRC.

BACKGROUND

All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

Colorectal cancer (CRC) is a common disease with an estimated 143,460 new cases in the USA in 2012. CRC is the third most frequently diagnosed cancer in males and females in the Western world and a significant percentage of patients who present with CRC will have distant metastases (stage IV) at diagnosis, which is often incurable. It is clear that localized cancer (stage I/II) detected early is more amenable to curative therapy, offering superior prognosis. Accordingly, diagnostic methods that result in early detection of malignant or even premalignant disease could have considerable clinical benefits, reducing mortality and morbidity of patients with colorectal cancer. Available potential screening techniques for CRC include fecal occult blood test, double contrast barium enema, endoscopy, with preference for pancolonoscopy, and virtual colonoscopy. The measurement of serum carcinoembryonic antigen (CEA) has also been suggested as a possible screening modality but it lacks sufficient sensitivity to detect CRC at an early stage, and its level is also elevated in non-malignant diseases (e.g. diverticulitis, gastritis, diabetes).

An optimal screening test is expected to be highly sensitive and specific, pose no risk to the patients, and have high patient acceptance. It should also be cost effective and easy to perform. As current screening procedures lack sufficient positive predictive value, require unpleasant preparation or cause discomfort, there is a need to develop new non-invasive tests for the detection of CRC at a stage early enough for treatment to be successful. DNA methylation markers are promising tools that could be useful for early cancer detection. In the past decade it has become clear that cancer cells have aberrant patterns of DNA methylation and that these abnormalities can be detected in tumor-derived DNA found in the plasma or serum of cancer patients. Several studies have documented the presence of free DNA derived from solid tumors in the bloodstream of cancer patients, which raises the possibility of detecting these cancer-specific molecules in subjects with existing disease.

A number of studies have already reported the use of DNA methylation markers for blood-based detection of CRC with varying results. However, most of these studies have relied on testing a limited number of pre-selected genes and on the use of non-quantitative detection methods, such as gel-based methylation-specific PCR.

Described herein is a systematic genome-wide marker discovery approach and verification study for blood-based DNA methylation markers. Combining enhanced nucleic acid detection approaches, such as genome-wide methylation screening, digital PCR, and MethyLight detection, the inventors have identified at least two novel colorectal cancer biomarkers, THBD-M and C9orf50-M, which demonstrate excellent ability to distinguish between CRC tumors and matched normal colon tissue in clinical samples.

SUMMARY OF THE INVENTION

Described herein is a method of determining a diagnosis of cancer in an individual suspected of having cancer, including obtaining a sample from an individual suspected of having cancer, determining the presence or absence of a high level of expression in the individual relative to a normal baseline standard for a single diagnostic panel including the following biomarkers: TFPI2, THBD, C9ORF50, ADHFE1, FGF12, PTPRT, ZNF568, KIAA1026, SFMBT2, and/or AUTS2, and diagnosing a case of cancer if the individual demonstrates the presence of a high level of expression relative to a normal baseline standard of at least one biomarker. In another embodiment, the at least one biomarker includes THBD and C9ORF50. In another embodiment, the at least one biomarker includes THBD. In another embodiment, the cancer is colorectcal cancer. In another embodiment, obtaining a sample from an individual includes drawing blood, serum, or plasma from the individual. In another embodiment, the diagnosis provides a molecular subtype classification for the diagnosed case of cancer in the individual. In another embodiment, the diagnosis provides a therapeutic selection for the diagnosed individual, selected from the group consisting of: chemotherapy, radiotherapy, surgery, and combinations thereof. In another embodiment, determining the presence or absence includes use of digital PCR. In another embodiment, determining the presence or absence is capable of detecting methylation.

In another aspect, further described herein is a method of determining a prognosis of cancer in an individual, including: determining the presence or absence of a high level of expression in the individual relative to a normal baseline standard for a single prognostic panel including the following biomarkers: TFPI2, THBD, C9ORF50, ADHFE1, FGF12, PTPRT, ZNF568, KIAA1026, SFMBT2, and/or AUTS2, and prognosing a case of cancer if the individual demonstrates the presence of a high level of expression relative to a normal baseline standard of at least one biomarker. In another embodiment, the at least one biomarker includes THBD and C9ORF50. In another embodiment, the at least one biomarker includes THBD. In another embodiment, the cancer is colorectal cancer. In another embodiment, determining the presence or aabsence of a high level of expression in the individual includes drawing a blood, serum or plasma sample from the individual. In another embodiment, the prognosis provides a therapeutic selection for the prognosed individual, selected from the group consisting of: chemotherapy, radiotherapy, surgery, and combinations thereof. In another embodiment, determining the presence or absence includes use of digital PCR. In another embodiment, determining the presence or absence is capable of detecting methylation.

In another aspect, described herein is a kit for detecting colorectal cancer biomarkers including: a nucleic acid capable of detecting at least one colorectal cancer biomarker selected from the group consisting of: TFPI2, THBD, C9ORF50, ADHFE1,FGF12, PTPRT, ZNF568, KIAA1026, SFMBT2, and AUTS2, at least one reagent adapted for detection of the at least one colorectal cancer biomarker, and instructions for using the kit to detect the at least one colorectal cancer biomarker. In another embodiment, the at least one colorectal cancer biomarker is THBD and C9ORF50. In another embodiment, the at least one colorectal cancer biomarker is THBD. In another embodiment, the at least one reagent is adapted for detecting methylation levels in the at least one colorectal cancer biomarker. In another embodiment, detecting at least one colorectal cancer biomarker includes use of digital PCR. In another embodiment, detecting at least one colorectal cancer biomarker includes detecting methylation. In another aspect, also described herein is a method of determining the subtype of cancer in a subject, including: obtaining a test sample from a subject, determining the expression level of at least one biomarker in the test sample, comparing the expression level of the at least one biomarker in the test sample with the expression level the at least one biomarker in a reference sample from a healthy individual, and determining that the subject has a particular subtype of cancer based on the level of expression of the at least one biomarker in the test sample compared to the level of expression of the at least one biomarker in the reference sample from the healthy individual. In another embodiment, the test sample includes a blood, serum or plasma sample from the subject. In another embodiment, the at least one biomarker is TFPI2, THBD, C9ORF50, ADHFE1, FGF12, PTPRT, ZNF568, KIAA1026, SFMBT2, and/or AUTS2. In another embodiment, determining the expression level of the at least one biomarker includes analyzing the transcription level of the at least one biomarker or analyzing the protein level of the at least one biomarker. In another embodiment, the cancer is colorectal cancer. In another embodiment, the cancer is a subset of colorectal cancer. In another embodiment, determining the expression level includes use of digital PCR. In another embodiment, determining the expression level includes detecting methylation.

Also described herein is a method of determining an increased susceptibility of a subject to cancer, including: obtaining a test sample from the subject, determining the expression level of at least one biomarker in the test sample, comparing the expression level of the at least one biomarker in the test sample with the expression level of the at least one biomarker in a reference sample from a healthy individual, and determining that the subject has an increased susceptibility to cancer based on the level of expression of the at least one biomarker in the test sample compared to the level of expression of the at least one biomarker in the reference sample from the healthy individual. In another embodiment, the sample includes a blood, serum or plasma sample from the subject. In another embodiment, the at least one biomarker is TFPI2, THBD, C9ORF50, ADHFE1, FGF12, PTPRT, ZNF568, KIAA1026, SFMBT2, and/or AUTS2. In another embodiment, determining the expression level of the at least one biomarker includes analyzing the transcription level of the at least one biomarker or analyzing the protein level of the at least one biomarker. In another embodiment, the cancer is colorectal cancer. In another embodiment, the the cancer is a subset of colorectal cancer. In another embodiment, determining the expression level includes use of digital PCR. In another embodiment, determining the expression level includes detecting methylation.

In another aspect, further described herein is a method of selecting a treatment for a cancer patient, including: assaying a biological sample from the patient by detecting the expression level of at least one biomarker in the test sample, comparing the expression level of the at least one biomarker in the test sample with the expression level the at least one biomarker in a reference sample from a healthy individual, and determining that the subject has a particular subtype of cancer based on the level of expression of the at least one biomarker in the test sample compared to the level of expression of the at least one biomarker in the reference sample from the healthy individual, and based on that determination, selecting a treatment for the patient. In another embodiment, the at least one biomarker is TFPI2, THBD, C9ORF50, ADHFE1, FGF12, PTPRT, ZNF568, KIAA1026, SFMBT2, and/or AUTS2. In another embodiment, determining the expression level of the at least one biomarker includes analyzing the transcription level of the at least one biomarker or analyzing the protein level of the at least one biomarker. In another embodiment, the cancer is colorectal cancer. In another embodiment, the cancer is a subset of colorectal cancer. In another embodiment, detecting the expression level includes use of digital PCR. In another embodiment, determining detecting the expression level includes detecting methylation.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Schematic representation of colorectal cancer marker discovery and verification pipeline. The inventors used DNA methylation data from the Infinium HumanMethylation27 Beadchip (HM27) and HumanMethylation450 Beadchip (HM450) Infmium platforms to screen 27,578 (HM27) and 482,421 (HM450) CpG loci for their methylation status in CRC samples, PBL samples from healthy subjects, paired normal colorectal tissue samples (NC) and 15 other types of cancer (OC). The inventors used a stepwise approach eliminating probes that failed in any of the samples, probes that contained SNPs or repeat sequences, probes with a highest PBL β-value (β-PBLn) or a mean normal colon tissue β-value (P-NC_M) higher than the associated 10th percentile of CRC tumor β- values (β-CRCio) or higher than 0.2 in any of the PBL or NC samples (Infinium panel). The remaining probes were ranked based on the difference between β-CRCio and β-PBLn and the top 25 were selected from both datasets (HM27 and HM450) for filtering against OC samples. Probes with a mean OC β-value higher than the associated mean CRC β-value (β- CRCM) were eliminated. A total of 15 MethyLight reactions (markers) were designed for 10 probes and tested in a sequence of verification steps (MethyLight panel). Markers were eliminated if their performance was suboptimal in controls such as in vitro methylated Sssl DNA, PBL and plasma samples from healthy controls and CRC tumor tissues. Markers were also eliminated if they failed to detect CRC methylated DNA in pooled plasma and serum from CRC patients. Two markers met all the selection criteria and were advanced in the pipeline for further verification on individual patient samples. (*Probes that failed in any of the samples, as well as those that included SNPs and repeat sequences; **Other cancer types used in this study are summarized in Table 1 , ***M &sI treated DNA).

Figure 2. Scatterplot representation of marker discovery process and receiver operating characteristic (ROC) curves. (A) (top figure: HM27, bottom figure: HM450), scatterplots of the highest PBL β-value (β-PBLn) of 10 (HM27) and 2 (HM450) healthy control samples (X-axis) against the associated 10th percentile of CRC tumor β-values (β- CRCio) on the Y-axis. The blue dots represent the eliminated probes (HM27: n=23,049; HM450: n=367,833) and the red dots (HM27: n=695; HM450: n=30,207) represent the retained probes with a β-CRCio > β-PBLn or a β-PBLn <0.2. (B) scatterplots of the mean normal colon tissue β-value (β-NC_M) for the retained probes from Panel A (X-axis) against the associated β-CRCio (Y-axis). The red dots (HM27: n=512; HM450: n=28,428) represent the eliminated probes, the green dots represent the retained probes (HM27: n=183; HM450: n=1779) with a P-CRCio > β-NCM or a β-NCM <0.2. (C) scatterplots of the retained probes from Panel B (green) displayed by the difference between β-CRCio and β-PBLn (X-axis) against the associated β-CRCio (Y-axis). The dots within the yellow square are the probes selected for additional filtering against other types of cancer. The white arrows point out the probes of the two candidate markers. (D) ROC curves for the probes used in the multiplex reaction based on methylation β-values of 335 independent colorectal cancer samples and 23 independent matched normal colorectal tissue samples (the DNA methylation data of these samples were not used in the marker discovery pipeline). The dark grey color is the area under the curve.

Figure 3. DNA methylation β-values of THBD and C9orf50 in various types of samples. Jitter plots representing Infinium-based DNA methylation β-values of (A) THBD and (B) C9orf50 in 335 independent CRC tumors, matched normal colon tissues, a variety of other cancer types and PBL from healthy individuals. The specific number of samples for each tissue type is described in Table 1. Figure 4. Detection of THBD-M and C9orf50-M in plasma and serum from CRC patients and controls. Digital MethyLight was performed in 1 ml plasma (A) and serum (D) to detect THBD-M and C9orf50-M in CRC and control samples. The absolute number of molecules detected by the multiplex (sum of the two markers) reaction is recorded on the y- axis. The CRC samples are arranged by stage. Asterisks (*) indicate samples with more than 25 molecules detected (up to 153 molecules in plasma and 157 molecules in serum). Receiver operating characteristic (ROC) curves and area under curves (AUCs) (95% confidence intervals) of the different CRC stages in plasma (B) and serum (E) based on the number of detected molecules. ROC analysis and AUCs (95% confidence intervals) for THBD-M, C9orf50-M as individual reactions and as a multiplex reaction in plasma (C) and serum (F).

Figure 5 Preoperative carcinoembryonic antigen (CEA) serum levels of all patients with the associated number of detected THBD-M and C9orf50-M molecules per 1 ml of (A) plasma and (B) serum.

DESCRIPTION OF THE INVENTION

All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3^rd ed., J. Wiley & Sons (New York, NY 2001); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 5^th ed., J. Wiley & Sons (New York, NY 2001); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2001), provide one skilled in the art with a general guide to many of the terms used in the present application.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.

As used herein,the meaning of "a," "an," and "the" includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of "in" includes "in" and "on" unless the context clearly dictates otherwise.

As used herein, the term "CRC" means colorectal cancer. As used herein, "diagnose" or "diagnosis" refers to determining the nature or the identity of a condition or disease. A diagnosis may be accompanied by a determination as to the severity of the disease.

As used herein, the term "dysfunctional signaling" refers to signaling mechanisms that are considered to be abnormal and not ordinarily found in a healthy subject or typically found in a population examined as a whole with an average amount of incidence.

As used herein, the designations of "normal," "low," and "high" gene or marker expression levels are determined relative to a normal baseline standard of gene or marker expression level. There are various methods known to one of skill in the art in determining the "normal baseline standard" of expression level. For example, as described herein, biomarker detection capability can be described in terms of a comparing measurements from tumor tissue, colon tissue suspected of harboring disease and/or matched normal colon tissue. These comparison can be expressed in the form of confidence intervals, such as receiver operating characteristic (ROC) curves or areas under the curve (AUCs), demonstrating the capability of a biomarker assay to distinguish among measurements levels. Levels of gene or marker expression can be determined using various methods known to one of skill in the art. For example, as described herein, the inventors used enhanced nucleic acid detection techniques of high sensitivity, with quantitative measurement and detection of DNA state. For example, this includes digital PCR using a sample distribution over multiple polymerase chain reaction (PCR) chambers, methylation-specific approaches such as MethyLight, or other methods that amplify sequences based on DNA methylation status. Similarly, as readily apparent to one of skill in the art, detection and quantification of various compositions that might include polynucleotides, polypeptides as well as various reporter molecules may be used to aid in determining levels of gene or marker expression.

As used herein, "prognostic" or "prognosis" refers to predicting the outcome or prognosis of a disease.

As used herein, "treatment" or "treating" should be understood to include any indicia of success in the treatment, alleviation or amelioration of an injury, pathology or condition. This may include parameters such as abatement, remission, diminishing of symptoms, slowing in the rate of degeneration or decline, making the final point of degeneration less debilitating; improving a patient's physical or mental well-being; or, in some situations, preventing the onset of disease. Colorectal cancer (CRC) is a common disease that, when detected early, can be amenable to curative therapy, and offering superior prognosis. However, diagnostic methods that result in early detection of malignant or even premalignant disease are lacking. Currently, no blood-based markers have yet been approved by the FDA for the use of early detection of CRC. Serum CEA is a blood-based biomarker that is in use for CRC detection, but it lacks the sensitivity for primary CRC detection. Serum CEA measurement is used mainly as a follow-up tool after initial treatment, and yields a sensitivity of approximately 72% for the detection of liver metastasis and 60% for local recurrence with specificities of 91% and 86% respectively. Therefore, there is a need in the art for new approaches for early stage detection of colorectal cancer.

However, a common shortcoming in traditional approaches for identification of new

CRC biomarkers, including published CRC studies, is reliance on a candidate gene approach for marker discovery. These approaches are often based on a nonsystematic selection of candidate marker genes, which are tested in healthy and cancerous tissues and then validated in a patient population. Although some of these studies have resulted in promising biomarkers for early CRC detection, the lack of a thorough biomarker discovery strategy raises the question whether superior markers may have been overlooked. With the more advanced technologies currently available, it is possible to obtain genome-scale DNA methylation data that can be useful for biomarker discovery.

As described herein, the inventors have relied upon a genome-scale multistep marker discovery to identify CRC biomarkers using the HM27 and HM450 BeadChip platform; the latter evaluates the DNA methylation status of over 482,000 CpG loci and covers 96% of all UCSC CpG islands. A similar marker-pipeline strategy for ovarian cancer and identified the new sensitive recurrence biomarker IFFOl-M. Campan et al. (2011). The described discovery strategy uses DNA methylation data from 4,201 cancer samples of different origins to optimize CRC specificity. The results described herein show that this discovery strategy works successfully for CRC, resulting in at least two new biomarkers: THBD-M and C9orf50-M. With AUCs of 0.97 and 1.0 respectively on the Infinium assay, these two markers have an excellent ability to distinguish between CRC tumors and matched normal colon tissue.

The present invention includes a method of determining a diagnosis of cancer in an individual suspected of having cancer, including determining the presence or absence of a high level of expression in the individual relative to a normal baseline standard for a prognostic panel including one or more biomarkers, and diagnosing a case of cancer if the individual demonstrates the presence of a high level of expression relative to a normal baseline standard of at least one of the biomarkers in the diagnostic panel. In other embodiments, the individual demonstrates the presence of a high level of expression relative to a normal baseline standard of two, three, four, five, six, seven, eight, nine, ten, or at least ten of the biomarkers in the diagnostic panel. In certain embodiments, the diagnostic panel includes tissue factor pathway inhibitor (TFPI2), thrombomodulin (THBD), chromosome 9 open reading frame 50 (C9ORF50), alcohol dehydrogenase, iron containing, 1 (ADHFE1), fibroblast growth factor (FGF12), tyrosine-protein phosphatase T (PTPRT), zinc finger protein 568 (ZNF568), kazrin (KIAA1026), Scm-like with four mbt domains 2 (SFMBT2), and/or autism susceptibility candidate 2 (AUTS2). In other embodiments, the diagnostic panel includes THBD and C9ORF50. In other embodiments, the diagnostic panel includes THBD. In other embodiments, the marker is methylated, for example, THBD-M or C9ORF50-M. In other embodiments, the methylation is partial or full methylation. In another embodiment, the cancer is colorectcal cancer. In certain embodiments, the colorectcal cancer is non-invasive colorectal cancer. In another embodiment, the diagnosis provides a therapeutic selection for the diagnosed individual, selected from the group consisting of: chemotherapy, radiotherapy, surgery, and/or combinations thereof. In various embodiments, the diagnosis provides a molecular subtype classification of the cancer type. In various embodiments, the diagnosis distinguishes between stage I, II, III, and IV colorectal cancers. In another embodiment, determining the presence or absence of a high level of expression in the individual relative to a normal baseline standard includes obtaining a sample from the individual. In another embodiment, obtaining a sample includes obtaining blood, serum, and/or plasma from the individual. In different embodiments, probes are capable of detecting circulating free cancer DNA in the bloodstream of the individual.

In in various embodiments, tissue factor pathway inhibitor 2 (TFPI2) includes variant 1 encoded by SEQ ID NO: 44, Genbank accession no. NM 006528.3, or TFPI2 variant 2 encoded by SEQ ID NO: 45, accession no. NM 001271003.1, TFPI2 variant 2 encoded by a nucleic acid sequence SEQ ID NO: 46, accession no. NM 001271004.1. In another embodiment, thombomodulin (THBD) is encoded by nucleic acid sequence SEQ ID NO: 47, accession no. NM 000361. In another embodiment, chromosome 9 open reading frame 50 (C9ORF50) is encoded by SEQ ID NO: 48, accession no. NM 199350.3. In another embodiment, alcohol dehydrogenase, iron containing, 1 (ADHFE1) is encoded by SEQ ID NO: 49, accession no. NM 144650. In another embodiment, fibroblast growth factor -12 (FGF-12) includes variant 1 encoded by nucleic acid sequence SEQ ID NO: 50, accession no. NM 021032, or FGF-12 variant 2 encoded by SEQ ID NO: 51, accession no. NM 0041 13.5. In another embodiment, protein tyrosine phosphatase, receptor type, T (PTPRT) variant 1 encoded by SEQ ID NO: 52, accession no. NM 133170, or PTPRT variant 2 encoded by SEQ ID NO: 53, accession no. NM 007050. In another embodiment, zinc finger protein 568 (ZNF568) includes variant 1 encoded by SEQ ID NO: 54, accession no. NM_198539, or ZNF568 variant 2 encoded by SEQ ID NO: 55, accession no. NM_001204835, ZNF568 variant 3 encoded by SEQ ID NO: 56, accession no. NM 001204836, or ZNF568 variant 4 encoded by SEQ ID NO: 57, encoded by accession no. NM 001204837, or ZNF568 encoded by [SEQ ID NO: 58, accession no. NM 001204838, ZNF568 variant 6 encoded by SEQ ID NO: 59, accession no. NM 001204839. In another embodiment, kazrin (KIAA1026) is encoded by SEQ ID NO: 60, accession no. BC035501. In another embodiment, Scm-like with four MBT domains protein 2 (SFMBT2) is encoded by SEQ ID NO: 61, accession no. NM 001018039. In another embodiment, autism susceptibility candidate 2 (AUTS2) includes variant 1 encoded by SEQ ID NO: 62, accession no. NM 015570, AUTS2 variant 2 encoded by SEQ ID NO: 63, accession no. NM_001127232, AUTS2 variant 3 encoded by SEQ ID NO: 64, accession no. NM 001127231. As described herein, the present invention includes a method of determining a prognosis of cancer in an individual including determining the presence or absence of a high level of expression in the individual relative to a normal baseline standard for a prognostic panel including one or more biomarkers, and prognosing a case of cancer if the individual demonstrates the presence of a high level of expression relative to a normal baseline standard of at least one of the biomarkers in the prognostic panel. In other embodiments, the individual demonstrates the presence of a high level of expression relative to a normal baseline standard of two, three, four, five, six, seven, eight, nine, ten, or at least ten of the biomarkers in the prognostic panel. In certain embodiments, the prognostic panel includes TFPI2, THBD, C9ORF50, ADHFE1, FGF12, PTPRT, ZNF568, KIAA1026, SFMBT2, and/or AUTS2. In other embodiments, the prognostic panel includes THBD and C9ORF50. In other embodiments, the prognostic panel includes THBD. In other embodiments, the marker is methylated, for example, THBD-M or C9ORF50-M. In other embodiments, the methylation is partial or full methylation. In another embodiment, the cancer is colorectcal cancer. In certain embodiments, the colorectcal cancer is non-invasive colorectal cancer. In another embodiment, the prognosis provides a therapeutic selection for the prognosed individual, selected from the group consisting of: chemotherapy, radiotherapy, surgery, and/or combinations thereof. In various embodiments, the prognosis provides a molecular subtype classification of the cancer type. In various embodiments, the molecular subtype classification is tied to clinical features such as chemoresistance, recurrence, malignancy, and metastases. In another embodiment, determining the presence or absence of a high level of expression in the individual relative to a normal baseline standard includes obtaining a sample from the individual. In another embodiment, obtaining a sample from includes obtaining blood, serum, and/or plasma from the individual. In different embodiments, probes are capable of detecting circulating free cancer DNA in the bloodstream of the individual.

The present invention is also directed to a kit to diagnose, prognose and/or treat cancer, such as for practicing the method of analyzing the expression of a diagnostic panel including one or more markersfor the diagnosis of colorectcal cancer. In another embodiment, the cancer is colorectcal cancer, the colorectcal cancer is non-invasive colorectal cancer. For example, the kit may contain compositions for analyzing a diagnostic panel with any of the following biomarkers: TFPI2, THBD, C9ORF50, ADHFEl, FGF12, PTPRT, ZNF568, KIAA1026, SFMBT2, and/or AUTS2. In some embodiments the kit contains compositions detecting and amplifying expression of TFPI2, THBD, C9ORF50, ADHFEl, FGF12, PTPRT, ZNF568, KIAA1026, SFMBT2, and/or AUTS2, as described above.

In various embodiments, TFPI1 can be detected using, for example, the primer combination described by SEQ ID NO: 1 and 2, in further combination with a probe, such as SEQ ID NO. 31. In another embodiment, THBD can be detected using, for example, the primer combination described by SEQ ID NO: 3 and 4, in further combination with a probe, such as SEQ ID NO. 32. In another embodiment, can be detected using, for example, the primer combination described by SEQ ID NO: 5 and 6, SEQ ID NO: 7 and 8, SEQ ID NO: 9 and 10, or SEQ ID NO: 29 and 30 in further combination with a probe, such as SEQ ID NO. 33, 34, or 43. In another embodiment, ADHFEl can be detected using, for example, the primer combination described by SEQ ID NO: 11 and 12, or SEQ ID NO: 13 and 14, in further combination with a probe, such as SEQ ID NO. 35. In another embodiment, FGF-12 can be detected using, for example, the primer combination described by SEQ ID NO: 15 and 16, in further combination with a probe, such as SEQ ID NO. 36. In another embodiment, PTPRT can be detected using, for example, the primer combination described by SEQ ID NO: 17 and 18, in further combination with a probe, such as SEQ ID NO. 37. In another embodiment, ZNF568 can be detected using, for example, the primer combination described by SEQ ID NO: 19 and 20, in further combination with a probe, such as SEQ ID NO. 38. In another embodiment, KIAA1026 can be detected using, for example, the primer combination described by SEQ ID NO: 21 and 22, in further combination with a probe, such as SEQ ID NO. 39. In another embodiment, SFMBT2 can be detected using, for example, the primer combination described by SEQ ID NO: 23 and 24, or SEQ ID NO: 23 and 24 in further combination with a probe, such as SEQ ID NO. 41 or 42. In another embodiment, AUTS2 can be detected using, for example, the primer combination described by SEQ ID NO: 27 and 28 in further combination with a probe, such as SEQ ID NO. 42.

In some embodiments the kit contains a composition including biomarkers for detecting and amplifying expression of THBD and C9ORF50. In some embodiments the kit contains compositions for detecting and amplifying expression of THBD. In other embodiments, the marker is methylated, for example, THBD-M or C9ORF50-M. In other embodiments, the methylation is partial or full methylation. In some embodiments the kit contains a composition for detection a biomarker from a sample of blood, serum, and/or plasma. In various embodiments, the kit is an assemblage of materials or components, including at least one of the inventive compositions described herein. In various embodiments, at least one of the inventive compositions is a nucleic acid sequence described in Table 3.

Instructions for use may be included in the kit. "Instructions for use" typically include a tangible expression describing the technique to be employed in using the components of the kit to effect a desired outcome, such as to prognose or predict progression of bladder cancer. Optionally, the kit also contains other useful components, such as, diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, bandaging materials or other useful paraphernalia as will be readily recognized by those of skill in the art.

The materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility. For example the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated or frozen temperatures. The components are typically contained in suitable packaging material(s). As employed herein, the phrase "packaging material" refers to one or more physical structures used to house the contents of the kit, such as inventive compositions and the like. The packaging material is constructed by well known methods, preferably to provide a sterile, contaminant-free environment. As used herein, the term "package" refers to a suitable solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding the individual kit components. The packaging material generally has an external label which indicates the contents and/or purpose of the kit and/or its components. The present invention is also directed to a method of detecting a biomarker using digital polymerase chain reaction (PCR). In various embodiment, digital PCR is used in combination with methylation-specific PCR, such as MethyLight In different embodiments, the digital PCR used in combination with methylation-specific PCR includes probes allowing for multiplex detection of at least two, three, four, five, or six biomarkers in a sample. In different embodiments, the probes are specific for TFPI2, THBD, C9ORF50, ADHFE1, FGF12, PTPRT, ZNF568, KIAA1026, SFMBT2, and/or AUTS2. In different embodiments, probes for multiplex detection are specific for THBD and C9ORF50. In other embodiments, the probes are capable of specifically detecting methylated forms of a marker, for example, THBD-M or C9ORF50-M. In other embodiments, the methylation is partial or full methylation. In other embodiments, the probes are selective for unmethylated, partially, or fully methylated forms of a marker. In different embodiments, probes are capable of detecting circulating free cancer DNA. In another embodiment, the sample includes nucleic acid isolated from obtaining blood, serum, and/or plasma from an individual. In different embodiments, probes are capable of detecting circulating free cancer DNA in the bloodstream of an individual. In other embodiments, the method of detecting a biomarker includes quantifying amount of unmethylated, partially or fully methylated biomarkers in a sample.

A variety of methods can be used to determine the presence or absence of gene expression. As an example, enzymatic amplification of nucleic acid from an individual may be used to obtain nucleic acid for subsequent analysis. The presence or absence of gene expression may also be determined directly from the individual's nucleic acid without enzymatic amplification.

Analysis of the nucleic acid from an individual, whether amplified or not, may be performed using any of various techniques. Useful techniques include, without limitation, polymerase chain reaction based analysis, sequence analysis and electrophoretic analysis. As used herein, the term "nucleic acid" means a polynucleotide such as a single or double- stranded DNA or RNA molecule including, for example, genomic DNA, cDNA and mRNA. The term nucleic acid encompasses nucleic acid molecules of both natural and synthetic origin as well as molecules of linear, circular or branched configuration representing either the sense or antisense strand, or both, of a native nucleic acid molecule.

The presence or absence of gene expression may involve amplification of an individual's nucleic acid by the polymerase chain reaction. Use of the polymerase chain reaction for the amplification of nucleic acids is well known in the art (see, for example, Mullis et al. (Eds.), The Polymerase Chain Reaction, Birkhauser, Boston, (1994)). PCR can be modified in a limited dilution assay to allow for detection of a specific product in only a minority of reactions, according to the technique known as digital PCR.

Other molecular methods useful for determining the presence or absence of gene expression are known in the art and useful in the methods of the invention. Other well-known approaches for determining the presence or absence of gene expression include automated sequencing and RNAase mismatch techniques (Winter et al, Proc. Natl. Acad. Sci. 82:7575- 7579 (1985)). Furthermore, one skilled in the art understands that, where the presence or absence of expression of multiple genes is to be determined, individual gene expression can be detected by any combination of molecular methods. See, in general, Birren et al. (Eds.) Genome Analysis: A Laboratory Manual Volume 1 (Analyzing DNA) New York, Cold Spring Harbor Laboratory Press (1997). In addition, one skilled in the art understands that expression of multiple genes can be detected in individual reactions or in a single reaction (a "multiplex" assay). As described herein, a variety of methylation-specific detection approaches can be applied, including the use of methylation-specific PCR, such as MethyLight. Some methylation-specific PCR techniques, require bisulfite conversion of DNA template. In view of the above, one skilled in the art realizes that the methods of the present invention for prognosing an individual may be practiced using one or any combination of the well known assays described above or another art-recognized genetic assay. There are also many techniques readily available in the field for detecting the presence or absence of polypeptides or other biomarkers, including protein microarrays. For example, some of the detection paradigms that can be employed to this end include optical methods, electrochemical methods (voltametry and amperometry techniques), atomic force microscopy, and radio frequency methods, e.g., multipolar resonance spectroscopy. Illustrative of optical methods, in addition to microscopy, both confocal and non-confocal, are detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a grating coupler waveguide method or inter ferometry).

Prior to detection, biomarkers may also be fractionated to isolate them from other components in a solution or of blood that may interfere with detection. Fractionation may include platelet isolation from other blood components, sub-cellular fractionation of platelet components and/or fractionation of the desired biomarkers from other biomolecules found in platelets using techniques such as chromatography, affinity purification, ID and 2D mapping, and other methodologies for purification known to those of skill in the art. In one embodiment, a sample is analyzed by means of a biochip. Biochips generally include solid substrates and have a generally planar surface, to which a capture reagent (also called an adsorbent or affinity reagent) is attached. Frequently, the surface of a biochip includes a plurality of addressable locations, each of which has the capture reagent bound there.

EXAMPLES

The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

Example 1

Study Design The inventors used DNA methylation data from 711 colorectal tumors, 53 matched adjacent-normal colonic tissue samples, 286 healthy blood samples and 4,201 tumor samples of 15 different cancer types. DNA methylation data were generated by the Illumina Infmium HumanMethylation27 and the HumanMethylation450 platforms, which determine the methylation status of 27,578 and 482,421 CpG sites respectively. The inventors first performed a multistep marker selection to identify candidate markers with high methylation across all colorectal tumors while harboring low methylation in healthy samples and other cancer types. The inventors then used pre-therapeutic plasma and serum samples from 107 colorectal cancer patients and 98 controls without colorectal cancer, confirmed by colonoscopy, to verify candidate markers. The inventors selected two markers for further evaluation: methylated THBD (THBD-M) and methylated C9orf50 (C9orf50-M). When tested on clinical plasma and serum samples these markers outperformed carcinoembryonic antigen (CEA) serum measurement and resulted in a high sensitive and specific test performance for early colorectal cancer detection.

Example 2

Sample Isolation

Informed consent was obtained from participating patients and controls under the approval of local and regional institutional review boards. Pre-therapeutic plasma and serum samples were obtained from CRC patients in the outpatient clinic via phlebotomy of the median cubital vein from April 2008 to December 2011. Plasma and serum were isolated within 30 minutes of venapuncture. Each plasma or serum sample was further divided into two separate tubes and stored at -80°C until processing. The serum CEA was measured at each venapuncture in CRC patients.

Controls were defined as subjects without CRC or any malignancy in the past five years and were included in this study at the endoscopy department. Individuals undergoing colonoscopy, who showed no sign of a colorectal malignancy, were eligible to participate. Indications for colonoscopy for these patients were surveillance colonoscopies because of inflammatory bowel disease (IBD; Crohn's disease or Ulcerative Colitis), positive family history of CRC, gastro -intestinal complaints or rectal blood loss. An experienced gastroenterologist performed all colonoscopies. Patients with mild, controlled IBD were included as long as it was possible to reliably inspect the colonic mucosa at colonoscopy and if the surveillance biopsies that are routinely taken along the whole colorectal tract were pathologically normal (showing no signs of dysplasia). Plasma and serum samples were isolated from these individuals using the same protocol as for the CRC patients.

CRC tissue was obtained during the surgical resection of the tumor and immediately sent to the pathologist. Dissection of a representative part of the tumor was followed by storage of the fresh-frozen sample at -80°C within one hour after surgical resection. In addition, a pathologically normal colon sample was taken at least 10 cm away from the edge of the tumor and stored in the same way.

Example 3

Marker Discovery: Technologies and Datasets

In the marker discovery phase of this study the inventors used DNA methylation data generated by Illumina Infinium HumanMethylation27 BeadChip® (HM27) and the HumanMethylation450 BeadChip® (HM450) platforms. The Infinium assay quantifies DNA methylation levels at specific cytosine residues adjacent to guanine residues (CpG loci), by calculating the ratio (β-value) of intensities between locus-specific methylated and unmethylated bead-bound probes. The β-value is a continuous variable, ranging from 0 (unmethylated) to 1 (fully methylated). Bibikova M, Lin Z, Zhou L, Chudin E, Garcia EW, et al. (2006) High-throughput DNA methylation profiling using universal bead arrays. Genome research 16: 383-393. The HM27 BeadChip® assesses the DNA methylation level of 27,578 CpG sites located at the promoter regions of 14,495 protein-coding genes. The HM450 BeadChip® evaluates DNA methylation status of 482,421 CpG loci and covers 99% of RefSeq genes and 96% of UCSC CpG islands (www.ncbi.nlm.nih.gov/RefSeq, www. illumina . com) .

Infinium HM27 and HM450 data from 711 colorectal tumors, 53 matched adjacent- normal colonic tissue samples and 10 peripheral blood lymphocyte (PBL) samples of healthy individuals was used to to identify and verify candidate DNA methylation tumor markers. In addition, the inventors used Infinium data from publicly available data sets. The first data set is Gene Expression Omnibus (GEO) database at the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/geo/, accession number GSE 19711). The second data set is The Cancer Genome Atlas (TCGA) Data Portal (http://tcga- data.nci.nih.gov/tcga/tcgaHome2.jsp). These two data sets represent 274 healthy PBL samples and 4,201 malignant tissue specimens from 15 different cancer types to maximize CRC specificity (see Table 1). The β-values of 611 CRC tumors and 24 matched adjacent- normal colonic tissue samples were retrieved from the DNA methylation dataset for CRCs posted on The Cancer Genome Atlas (TCGA). Data of the other 100 CRC tumors and 29 matched adjacent-normal colonic tissue samples were generated at the USC Epigenome Center in a previous study. Hinoue T, Weisenberger DJ, Lange CP, Shen H, Byun HM, et al. (2012) Genome-scale analysis of aberrant DNA methylation in colorectal cancer. Genome research 22: 271-282. Infinium data of 274 PBL samples were downloaded from the Gene Expression Omnibus (GEO) database. The data for the 10 remaining PBL samples were generated using the HM27 platform for a previous marker discovery study at the USC Epigenome Center. Campan M, Moffitt M, Houshdaran S, Shen H, Widschwendter M, et al. (2011) Genome-scale screen for DNA methylation-based detection markers for ovarian cancer. PloS one 6: e28141.

Table 1. Overview of samples and data sets used for biomarker discovery.

*normal samples were obtained from surgical specimens of CRC patients, at least 10cm from the tumor margins.

** these samples were among the samples run on the HM27 platform.

DNA methylation status was assessed using the HM27 BeadChip for 336 CRC samples, 29 normal colon samples and 274 PBL samples (Table 1). For the remaining 375 CRC tumors and 24 normal colon samples, the DNA methylation status was evaluated with the HM450 BeadChip (Table 1). The inventors also generated HM450 data on two PBL samples from the collection of 10 studied on the HM27 platform. Of the 375 CRC, data from 40 samples were used to perform the marker discovery, and the DNA methylation data of the remaining 335 samples was used to verify the final markers selected. Example 4

Marker Discovery: Filter Criteria

The inventors employed a multistep filtering process in the discovery phase of this study. DNA methylation data generated by the two different BeadChips (HM27 and HM450) were analyzed separately, but using the same filtering steps (Figure 1 and 2). Initial filtering included exclusion of all Infinium probes that failed in any of the samples. HM27 probes that were not uniquely aligned to the human genome (hgl9, GRCh37), or that were associated with single nucleotide polymorphisms (SNPs) within 10 basepairs of the target CpG (identified using the NCBI dbSNP builds 126 and 128), or probes that covered repetitive elements (identified by RepeatMasker) were also excluded. Following initial filtering, the inventors determined the highest β-value for each probe in 10 healthy PBL samples (β-PBLn) and the 10^th percentile of the CRC tumor β-values (β-CRCio) (Figure 2A). Further filtering excluded all probes with a β-PBLn higher than 0.2 or higher than the associated β-CRCio. Additional filtering excluded the remaining probes against normal colon tissue and 15 other cancer types. Specifically, the mean β-value of each probe was determined in normal colon tissue (P-NCM) and eliminated all probes that had a β-NCM value higher than 0.2 or higher than the associated β-CRCio value (Figure 2B). The inventors then selected the top 25 markers in both datasets after ranking the probes based on the difference between β-CRCio and β-PBL_fj (Figure 2C). For the filtering against other cancer types, the inventors determined the mean β-value (β-OC_M) for the remaining probes in each cancer type and eliminated all probes that had a β-OCM higher than the mean CRC β-value (β-CRCM)· The remaining probes were selected for MethyLight reaction design and further evaluation.

Example 5

DNA Extraction and Bisulfite Modification

DNA from two healthy PBL samples and 25 CRC tumor samples were extracted. DNA from plasma and serum samples was extracted using the QIAamp® Circulating Nucleic Acid Kit (Qiagen), specially designed to recover a maximum amount of circulating cell-free DNA from serum or blood. The Zymo® EZ DNA methylation kit (Zymo Research) was used to bisulfite convert the extracted DNA. All extractions and conversions were performed according to the manufacture's instructions. The quality and quantity of the bisulfite- con erted DNA, as well as the completeness of the bisulfite conversion, were assessed using a panel of quality control reactions as previously described.

Example 6

MethyLight Analysis

The MethyLight assay was performed as previously described. Trinh BN, Long TI, Laird PW (2001) DNA methylation analysis by MethyLight technology. Methods 25: 456- 462. The sequence of the MethyLight primers and probes used in these analyses are described in Table 3.

The MethyLight reactions were evaluated in four steps. First, M.&sl (New England Biolabs) treated PBL DNA (Promega) was used to determine if the reaction amplified in vitro methylated control DNA. Reactions with a cycle threshold [C(t)] higher than 35 were excluded. Secondly, the reactions were screened against 50ng PBL DNA from two healthy individuals. Reactions with C(t) values lower than 40 were excluded. The remaining reactions were tested on 25 CRC DNA samples, using an ALU-based MethyLight reaction and an M.&sl DNA standard curve to calculate the Percentage of Methylated Reference (PMR). Reactions with a PMR< 10 in more than one CRC tumors were eliminated. Finally, the reactions were tested in 10 plasma samples from healthy donors (equivalent of ΙΟΟμΙ plasma) and ranked according to their C(t) values. Reactions with C(t) values less than 50 in one or more of these samples were eliminated. Example 7

Digital MethyLight Analysis: Pooled clinical samples and Individual Clinical Samples

Digital MethyLight is a quantitative PCR technique in which bisulfite-converted DNA is analyzed using the MethyLight PCR assay in a distributive fashion over 96 reaction chambers for each sample. This technique is an efficient and effective method of obtaining DNA methylation information for samples with small amounts of DNA. The inventors prepared four separate pools of plasma and four pools of serum DNA to test candidate markers (Pool 1 = 16 controls (without IBD), Pool 2 = 16 patients with mild IBD, Pool 3 = 16 patients with stages I/II CRC and Pool 4 = 16 patients with stages III/IV CRC). Each of these pools contained DNA from 190μ1 of plasma or serum from each of the 16 individuals in the pool. For each reaction, the inventors first tested DNA from 50μ1 plasma or serum of each pool. For the reactions that did not result in any PCR amplifications (hits) with 50μ1, the volume was increased to a 150μ1 equivalent. Finally, reactions that did not result in any hits in the CRC pools or gave hits in the controls with or without IBD were excluded.

The two candidate markers that survived this elimination process were labeled with different fluorophores. This enabled reaction specific colored PCR outcomes that allowed distinguishing hits from each of these markers when they were run together (multiplex). All probes and primers were synthesized by Biosearch Technology, Inc, Novota, California, USA. The two-marker multiplex was tested on plasma and serum samples from 75 independent CRC patients and 70 controls with a test volume of lml. Tables 2A and 2B give an overview of the clinical characteristics of the CRC patients and control subjects used for clinical marker testing in this study.

Table 2A. Clinical characteristics of controls used for plasma and serum analysis.

Table 2B. Clinical characteristics of CRC patients used for plasma and serum analysis.

Example 8

Statistical Analysis

The computation of confidence intervals of areas under the curve (AUCs) and the statistical tests were conducted in R (version 2.14.0), with the R package pROC. Robin X, Turck N, Hainard A, Tiberti N, Lisacek F, et al. (2011) pROC: an open-source package for R and S+ to analyze and compare ROC curves. BMC bioinformatics 12: 77. The method as proposed by DeLong and colleagues was used for the test. DeLong ER, DeLong DM, Clarke-Pearson DL (1988) Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach. Biometrics 44: 837-845.

Example 9

Marker Discovery in Genome-Scale DNA Methylation Data Sets The inventors performed a stepwise marker discovery analysis using available DNA methylation data sets from a large number of CRC tumors, 15 different other cancer types, and control samples from plasma, PBL and matched adjacent-normal colonic tissues (Figures 1 and 2, Table 1) to identify CRC DNA methylation markers. The inventors generated data using two different Illumina Infinium HumanMethylation BeadChip platforms, HM27 and HM450, as described. After removing potentially problematic probes, probe sequences that overlapped SNPs or repetitive elements, and probes that failed to perform in all samples, there were 23,049 HM27 probes and 367,254 HM450 probes.

Of these probes, 695 remained in the HM27 group and 29,640 in the HM450 group, after eliminating those that had higher DNA methylation levels in the healthy PBL samples than in CRC tumors. In addition, the inventors excluded all probes with higher DNA methylation in normal colon tissue than in CRC and ranked the remaining probes based on the difference between healthy PBL and CRC tumor DNA methylation. The DNA methylation status of the combined top 50 probes (ranked by the greatest difference between healthy PBL and CRC DNA methylation) were compared between CRC and 15 other types of cancer. Probes with a higher mean DNA methylation level in any other type of cancer than in CRC were excluded. Ten CRC-specific candidate probes (associated with ten unique gene promoters) remained, which showed higher mean DNA methylation values in CRC than the mean corresponding DNA methylation value of all the other cancers.

Example 10

Candidate Marker MethyLight Assay Development and Verification

The inventors designed and tested a total of 15 real time PCR-based MethyLight assays (markers) for the ten remaining probes. MethyLight-based techniques are highly sensitive methods for detection of methylated DNA molecules. The primer and probe sequences for these reactions are described in Table 3. The sequence of verification tests performed on these markers is illustrated in the right panel of Figure 1.

Three MethyLight reactions did not amplify in vitro methylated (M &sl-treated) control DNA and were therefore eliminated. Five markers that were positive in healthy PBL samples and three markers that had a PMR<10 in more than one of 25 CRC tumors tested by MethyLight were also eliminated. The four remaining markers were tested in plasma samples from ten healthy donors and none of them were detected in these samples. All four markers were next analyzed by Digital MethyLight in pooled serum and plasma samples from controls with or without IBD and CRC patients. Two markers that could not be detected in the pooled CRC samples were eliminated. The final two candidate DNA methylation biomarkers that met all of the stringent selection criteria: THBD (THBD-M) and C9orf50 (C9orf50-M).

Example 11

Preliminary performance evaluation of THBD and C9orf50 The inventors evaluated the performance of THBD (Infinium probe number cg24562819) and C9orf50 (Infinium probe number cgl4015706) in discriminating CRC tissue and adjacent-normal colorectal tissue in an independent data set of 335 CRC tumors (Table 1). THBD and C9orf50 had β-values >0.4 in 95% and 100% of all analyzed CRC tumors respectively. Figure 2 shows the receiver operating characteristic (ROC) curves for THBD and C9orf50 in the discrimination of CRC tumor samples versus normal colonic tissue. The AUCs for THBD and C9orf50 were 0.97, and 1.0, respectively.

Importantly, both markers revealed lower DNA methylation levels in all other cancer types including breast, lung, prostate, thyroid, uterine, kidney, ovarian, gastric, pancreatic and bladder cancers, as well as melanoma, acute myeloid leukemia, glioblastoma multiforme and head and neck squamous cell carcinoma (Figure 3).

Example 12

Testing the performance of THBD-M and C9orf50-M in individual clinical samples

The inventors developed a multiplex reaction for the two markers using different reporter dyes for each of the reactions. The THBD-M probe was labeled with a QUASAR fluorophore that results in a red fluorescent signal and the C9orf50-M probe was labeled with the blue FAM fluorophore. The primers and probes of the two markers were tested for interference by combining them in one solution at various concentrations using M.&sl treated control DNA for MethyLight and Digital MethyLight assays (data not shown). Since the multiplex reaction of the two markers performed as well as the individual reactions the inventors used the former for further clinical testing.

A total of 107 CRC patients and 98 controls without CRC, verified by colonoscopy, were included in this prospective study. Paired serum and plasma samples were available from all controls and 104 CRC patients, while only plasma was obtained from three CRC patients. Although stage IV CRC was an exclusion criterion in this study, aspecific abnormalities were seen on pre-operative imaging diagnostics for three patients (e.g. small pulmonary nodules on CT-thorax) which later, but before surgery, turned out to be distant metastasis. These patients were subsequently upstaged to stage IV CRC. Thirty-two plasma and serum samples from controls and CRC patients were previously used in the pooled sample analysis as mentioned above.

The inventors tested the multiplexed Digital MethyLight assays for THBD-M and C9orf50-M markers on individual plasma samples from 75 CRC and 66 controls and on individual serum samples from 72 CRC and 66 controls. Figure 4 shows the number of molecules (sum of the two markers) detected in 1 ml of plasma (Figure 4A) and serum (Figure 4D) for different stages of CRC compared to controls. The ROC curves illustrate the test performance for the multiplex reaction per disease stage (Figure 4B) and for both markers separately in plasma (Figure 4C) and serum (Figure 4E and 4F). The AUCs per disease stage are described in Figure 4. For all stages, there was borderline significantly improved test performance for serum compared to plasma as the test medium (p=0.06 for all stages of CRC). THBD-M performed significantly better compared to C9orf50-M in both plasma and serum (p<0.001). The addition of C9orf50-M to THBD-M for the detection of CRC did not improve test performance. The AUCs per stage, for each marker separately and the multiplex reaction, are summarized in Table 4.

The inventors determined CEA levels in preoperative serum samples from 107 CRC patients. An elevated serum CEA (>5.0ng/ml) was observed in 35/107 (33%) patients. For stage I CRC serum CEA was elevated in 14%, for stage II in 33%, for stage III in 39% and for stage IV in 67%. Figures 5A and 5B summarizes preoperative CEA serum levels of all patients with the associated number of detected THBD-M and C9orf50-M molecules per 1 ml of plasma and serum. Example 13

Improved Marker Discovery Platform

As described, a critical shortcoming in existing biomarker discovery approaches, including published CRC biomarker studies, is the reliance reliance on a candidate gene approach for marker discovery. As described, advanced technologies currently available, such as genome wide detection of methylation status, quantitative approaches to measuring DNA states, such as the described digital MethyLight approach described, is shown to be effective in a genome-scale DNA methylation-focused approach that can be useful for biomarker discovery. The described discovery strategy uses DNA methylation data from 4,201 cancer samples of different origins to optimize CRC specificity. The results described herein show that this discovery strategy works successfully for CRC, resulting in two new biomarkers: THBD-M and C9orf50-M. With AUCs of 0.97 and 1.0 respectively on the Infinium assay, these two markers have an excellent ability to distinguish between CRC tumors and matched normal colon tissue.

Although DNA methylation of these genes has not yet been reported in association with CRC early detection, a recent study showed that aberrant THBD DNA methylation was linked to gastric cancer carcinogenesis. Shin CM, Kim N, Jung Y, Park JH, Kang GH, et al. (2010) Role of Helicobacter pylori infection in aberrant DNA methylation along multistep gastric carcinogenesis. Cancer science 101 : 1337-1346. This is consistent with the slightly higher DNA methylation levels observed for this marker in gastric cancer samples compared to other types of cancer (Figure 3). Nevertheless, the inventors found significantly higher levels of THBD DNA methylation in CRC than in gastric cancer (pO.001). Moreover, the application of Digital PCR to multiplexed MethyLight assays allowed for efficient use of valuable samples by simultaneously analyzing more than one marker without loss of sensitivity. This technology allows for the detection of single methylated DNA molecules against a large background of unmethylated molecules, and provides a quantitative PCR test result.

Circulating free cancer DNA (cfDNA) has the potential to be tumor-specific and has a relatively short half-life making it suitable as biomarker. To EW, Chan KC, Leung SF, Chan LY, To KF, et al. (2003) Rapid clearance of plasma Epstein-Barr virus DNA after surgical treatment of nasopharyngeal carcinoma. Clinical cancer research : an official journal of the American Association for Cancer Research 9: 3254-3259. Fleischhacker M, Schmidt B (2007) Circulating nucleic acids (CNAs) and cancer—a survey. Biochimica et biophysica acta 1775: 181-232.

Example 14

Technical Factors Impacting Sensitivity

Although our two markers were consistently methylated in almost all CRC tumors, and tested positive in 25 clinical CRC samples using MethyLight, the described techniques appear presently unable to detect DNA methylated at these loci in 1ml samples of peripheral serum or plasma for some CRC cases. It is conceivable that the use of larger analyte volumes would increase sensitivity, but some tumors may not shed substantial amounts of tumor- derived DNA into the bloodstream. It is thought that tumor DNA enters the bloodstream by secretion or as a result of apoptosis or necrosis of cancer cells from the primary tumor or metastatic deposits. While cancer patients tend to have higher cfDNA levels than healthy subjects, concentrations of cfDNA in peripheral blood may vary significantly between individuals.

One of the technical factors that could influence diagnostic performance of a biomarker is test volume. For example, the SEPT9 assay utilizes 4-5 ml of plasma. A few studies, however, have demonstrated that SEPT9 is also methylated in other types of cancer such as in lung adenocarcinoma, breast cancer and head and neck squamous cell carcinoma. The inventors' results described herein show that THBD and C9orf50 harbor low levels of DNA methylation in 15 types of cancer other than CRC, including most high-incidence cancers. Further assay optimization should produce substantially improved marker performance for both THBD-M and C9orf50-M. While in this study, the use of serum resulted in a slightly higher test performance of

THBD-M and the multiplex compared to plasma, this difference was of borderline significance. Although it has been reported that serum contains more cfDNA than plasma, no large-scale studies have been published comparing serum and plasma as test medium for blood-based detection of malignant diseases. Hence, it remains unclear whether serum or plasma is the optimal test specimen.

THBD-M outperformed C9orf50-M, and combining the two markers in a multiplexed assay did not increase test sensitivity. With a detection threshold of zero molecules per 1ml plasma, THBD-M was able to detect 71% of all CRCs at a specificity of 80%. Interestingly, for stage I/II the detection rate in CRC was 74% with this marker. THBD-M had a higher sensitivity for the detection of colon cancers (77% for all stages) than rectal tumors (53% for all stages) in plasma. This difference was marginally significant (p=0.07). Early stage colon cancers were also detected by this marker at a relatively high percentage, 75% for stage I, and 77%) for stage II. It is known that a subset of right- sided colon tumors exhibits high frequency of DNA hypermethylation at multiple promoter CpG islands, which is designated as CIMP. In addition it has been described that CIMP-high frequency increases gradually from the rectum to the right-sided colon. There did not appear to be significant differences in DNA methylation of THBD-M or C9orf50-M between CIMP and non-CIMP colorectal tumors. Both markers showed high levels of DNA methylation in all colorectal cancers, regardless of their CIMP status.

The fact that this diagnostic test detected a considerable fraction of mostly curable

CRCs, with 5-year survival rates of 72%>-93%>, seems promising. With an AUC of 0.80 in plasma and 0.82 in serum, THBD-M compares favorably to or outperforms other published blood-based DNA methylation biomarkers. In the present study, serum CEA detected only 33% of the primary CRCs.

In conclusion, using an improved marker discovery platform, the inventors have identified two novel blood-based DNA methylation markers for early detection of CRC though a systematic genome-scale marker discovery and verification study. Of these two markers, THBD-M had a promising performance in clinical samples justifying its further optimization and clinical testing. The various methods and techniques described above provide a number of ways to carry out the invention. Of course, it is to be understood that not necessarily all objectives or advantages described may be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as may be taught or suggested herein. A variety of advantageous and disadvantageous alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several advantageous features, while others specifically exclude one, another, or several disadvantageous features, while still others specifically mitigate a present disadvantageous feature by inclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be mixed and matched by one of ordinary skill in this art to perform methods in accordance with principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments.

Although the invention has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the invention extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof.

Many variations and alternative elements have been disclosed in embodiments of the present invention. Still further variations and alternate elements will be apparent to one of skill in the art. Among these variations, without limitation, are methods of identifying, or detecting colorectal markers, methods of using colorectal markers in various prognostic, diagnostic, and therapeutic methods, or kits therein. Various embodiments of the invention can specifically include or exclude any of these variations or elements.

In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term "about." Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

In some embodiments, the terms "a" and "an" and "the" and similar references used in the context of describing a particular embodiment of the invention (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the invention can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this invention include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above cited references and printed publications are herein individually incorporated by reference in their entirety.

In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that can be employed can be within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present invention are not limited to that precisely as shown and described.

Table 3. MethylLight Primers and Probes Sequences

;: -;:

::^:

:. ί ί J ¾ a

Table 4. AUCs of biomarkers separately and combined tested in 1 ml of plamsa (A) and serum (B)

Table 4. A JCs of oma rkers separatey aid m Mn ed tested ¾ Ml ^ M I _. & serum (8)

Case Oss&ase

T Stage ΤϋΒΟ-ϋ CSwSS-M

Ssf R -11

te. per

ml ^* an! ml

1 t 3 i 0 0

2 7 i 0 07

WA mi mk 1,4

4 WA mm. WA 15

5 8 i 2 1J8

7 1 1 i 0 1€

8 1 ^■0 i δ 1J8

it A WA 2.2

12 » Wh ^'mk mA 2.2

IS 1 1 0 G 2.4

14 3 ^■8 8 0 2.e

15 MA mi 2*

ie WA mm. 2J8

1:7 2? 15 « 27

IS 1 1 i Q 27

1§ 1 3 i 1 27

WA ^β s 2M

21 WA fflh E.2

22 m £ 2 1 3.1

23 § 0: G 4.2

24 urn A ^'mk 4.2

.25 ? 0: 1 G 7.4

26 2 1 0 G 9.-1

27 N£& 10.3

2B § 2 1 28.8

2§ 1 11 30 IS 05

30 1 MA 1 0 10

31 1 & 15 ^■8 17 1.

32 A WA 1.0

S3 1 t k MA 1

34 1 MA wh WA 11

5 1 w 11 2 2 17

3§ 11 2 2 i 0 17

11 m 7 18 22 2.3

38 1 i 2 8 1 2.3

38 1 8 10 ^■5 4 25

40 1 1 e i 2 32

41 11 2 2 i 0 34

42 11 1 ^■0 i 0 E,:6

43 WA »¼. WA 3€

5 II i 2 :0 4.4

47 11 § :0 1 4.5

4S 11 urn A ^'mk 5.8

11 2 :0 G -m CEft cssgesper Mote* 2utes er

si m

58 2 & i Qi S.2

51 M 12 4 3- B.i

52 §5 m 2 S-* 12.7

53 M 7 -S 7 13.1

54 2 1 Qi 145

55 M 13 m S- 2 22.1]

Qi 24.1

57 M 3 1 † 3 -3

5S 3 7B.

58 Ml n 0 1.0

88 Ml 8 Q- Qi IS

S Ml 7 3. 0 1.1

82 Ml 2 & Qi 1.1

S3 ffi 1 1; 0 1.2

64 Ml 4 Q- 3 Qi 1.2 ss Ml mm MA mA 1.3

SS Ml 2 4 1 Qi 1.«

§7 Ml 0 0 £ 1.3

S§ Ml 2 0 Q 1.S

§§ Ml mm iA 1.§

Ml m W 5- 7 2.S

71 Ml i 0 0 2.S

72 Ml WA Wk A 2.S

73 Ml 4 0 0 2.1

74 Ml 3 I 0 Q 2.2

75 Ml 2 0 I 2.3

7® Ml WA Wk mA. 23

77 Ml mm MA 2.5

7§ Ml WA 2 mA. Q 2.«

7S Ml 4 iA 1 ?A 2.5

SB Ml Q- 0 Q 3.1

84 Ml WA Mk WA. ?ΦΑ 3¾

SS Ml 21 te4 2 4.

S7 Ml mt net mk HiA

SS Ml β § 0 Qi S.-1 ss Ml mk HiA

ss Ml s 1 Qi 7.4

§ Ml mt net mk HiA

§2 Ml 2 2 0

SS Ml 2 I 0 Qi i.s

§ Ml 0 3- 11.§

Ss Ml 4 4 2 S.3

§g Ml i 3. ^•s J

S7 Ml iMiA Mk WA. 2S7

§€ Ml mt mk mk HiA 2

8δ Ml WA HiA WA Wk 22V1

IDS Ml MA k mk. HiA 239 Table 4c, A tJCs of b mm arfe rs sgp rately t¾d M M tested M 1 ff>Ia« ¾ ( Al Μ s roro (B) w ta mm cs m CEA

Plasm Serusi fss Plasma Se?¾m

Mcesufes per Mesukss ps¾' siscsigs ;per Mo¾c-ygs per

mm. WA 32.4

102 so 14 35.7

10:3 Nfit S4..

184 m §2

105 !! t WA

106 if 21 0 Ί

107 i

0s WA mk

Claims

THE CLAIMS

1. A method of determining a diagnosis of cancer in an individual suspected of having cancer, comprising:

obtaining a sample from an individual suspected of having cancer;

determining the presence or absence of a high level of expression in the individual relative to a normal baseline standard for a single diagnostic panel comprising the following biomarkers:

TFPI2, THBD, C9ORF50, ADHFEl, FGF12, PTPRT, ZNF568, KIAA1026, SFMBT2, and/or AUTS2; and

diagnosing a case of cancer if the individual demonstrates the presence of a high level of expression relative to a normal baseline standard of at least one biomarker.

2. The method of claim 1, wherein the at least one biomarker comprises THBD.

3. The method of claim 1, wherein the at least one biomarker comprises THBD and C9ORF50.

4. The method of claim 1, wherein the cancer is colorectcal cancer.

5. The method of claim 1, wherein obtaining a sample from an individual includes drawing blood, serum, or plasma from the individual.

6. The method of claim 1, wherein the diagnosis provides a molecular subtype classification for the diagnosed case of cancer in the individual.

7. The method of claim 1, wherein the diagnosis provides a therapeutic selection for the diagnosed individual, selected from the group consisting of: chemotherapy, radiotherapy, surgery, and combinations thereof.

8. The method of claim 1, wherein determining the presence or absence comprises use of digital PCR.

9. The method of claim 1 , wherein determining the presence or absence is capable of detecting methylation.

10. A method of determining a prognosis of cancer in an individual, comprising:

determining the presence or absence of a high level of expression in the individual relative to a normal baseline standard for a single prognostic panel comprising the following biomarkers:

TFPI2, THBD, C9ORF50, ADHFE1, FGF12, PTPRT, ZNF568, KIAA1026, SFMBT2, and/or AUTS2; and

prognosing a case of cancer if the individual demonstrates the presence of a high level of expression relative to a normal baseline standard of at least one biomarker.

11. The method of claim 10 wherein the at least one biomarker comprises THBD.

12. The method of claim 10, wherein the at least one biomarker comprises THBD and C9ORF50.

13. The method of claim 10, wherein the cancer is colorectal cancer.

14. The method of claim 10, wherein determining the presence or absence of a high level of expression in the individual comprises drawing a blood, serum or plasma sample from the individual.

15. The method of claim 10, wherein the prognosis provides a therapeutic selection for the prognosed individual, selected from the group consisting of: chemotherapy, radiotherapy, surgery, and combinations thereof.

16. The method of claim 10, wherein determining the presence or absence comprises use of digital PCR.

17. The method of claim 10, wherein determining the presence or absence is capable of detecting methylation.

18. A kit for detecting colorectal cancer biomarkers comprising:

a nucleic acid capable of detecting at least one colorectal cancer

biomarker selected from the group consisting of: TFPI2, THBD, C9ORF50, ADHFE1, FGF12, PTPRT, ZNF568, KIAA1026, SFMBT2, and AUTS2;

at least one reagent adapted for detection of the at least one colorectal cancer biomarker; and

instructions for using the kit to detect the at least one colorectal cancer biomarker.

19. The kit of claim 18, wherein the at least one colorectal cancer biomarker is THBD.

20. The kit of claim 18, wherein the at least one colorectal cancer biomarker is THBD and C9ORF50.

21. The kid of claim 18, whrein the at least one reagent is adapted for detecting methylation levels in the at least one colorectal cancer biomarker.

22. The method of claim 18, wherein detecting at least one colorectal cancer biomarker comprises use of digital PCR.

23. The method of claim 18, wherein detecting at least one colorectal cancer biomarker comprises detecting methylation.

24. A method of determining the subtype of cancer in a subject, comprising:

obtaining a test sample from a subject;

determining the expression level of at least one biomarker in the test sample; comparing the expression level of the at least one biomarker in the test sample with the expression level the at least one biomarker in a reference sample from a healthy individual; and

determining that the subject has a particular subtype of cancer based on the level of expression of the at least one biomarker in the test sample compared to the level of expression of the at least one biomarker in the reference sample from the healthy individual.

25. The method of claim 24, wherein the test sample comprises a blood, serum or plasma sample from the subject.

26. The method of claim 24, wherein the at least one biomarker is TFPI2, THBD, C9ORF50, ADHFE1, FGF12, PTPRT, ZNF568, KIAA1026, SFMBT2, and/or AUTS2.

27. The method of claim 24, wherein determining the expression level of the at least one biomarker comprises analyzing the transcription level of the at least one biomarker or analyzing the protein level of the at least one biomarker.

28. The method of claim 24, wherein the cancer is colorectal cancer.

29. The method of claim 24, wherein the cancer is a subset of colorectal cancer.

30. The method of claim 24, wherein determining the expression level comprises use of digital PCR.

31. The method of claim 24, wherein determining the expression level comprises detecting methylation.

32. A method of determining an increased susceptibility of a subject to cancer, comprising:

obtaining a test sample from the subject;

determining the expression level of at least one biomarker in the test sample; comparing the expression level of the at least one biomarker in the test sample with the expression level of the at least one biomarker in a reference sample from a healthy individual; and

determining that the subject has an increased susceptibility to cancer based on the level of expression of the at least one biomarker in the test sample compared to the level of expression of the at least one biomarker in the reference sample from the healthy individual.

33. The method of claim 32, wherein the sample comprises a blood, serum or plasma sample from the subject.

34. The method of claim 32, wherein the at least one biomarker is TFPI2, THBD, C9ORF50, ADHFE1, FGF12, PTPRT, ZNF568, KIAA1026, SFMBT2, and/or AUTS2.

35. The method of claim 32, wherein determining the expression level of the at least one biomarker comprises analyzing the transcription level of the at least one biomarker or analyzing the protein level of the at least one biomarker.

36. The method of claim 32, wherein the cancer is colorectal cancer.

37. The method of claim 32, wherein the cancer is a subset of colorectal cancer.

38. The method of claim 32, wherein determining the expression level comprises use of digital PCR.

39. The method of claim 32, wherein determining the expression level comprises detecting methylation.

40. A method of selecting a treatment for a cancer patient, comprising:

assaying a biological sample from the patient by

detecting the expression level of at least one biomarker in the test sample; comparing the expression level of the at least one biomarker in the test sample with the expression level the at least one biomarker in a reference sample from a healthy individual; and

determining that the subject has a particular subtype of cancer based on the level of expression of the at least one biomarker in the test sample compared to the level of expression of the at least one biomarker in the reference sample from the healthy individual; and

based on that determination, selecting a treatment for the patient.

41. The method of claim 40, wherein the at least one biomarker is TFPI2, THBD, C9ORF50, ADHFE1, FGF12, PTPRT, ZNF568, KIAA1026, SFMBT2, and/or AUTS2.

42. The method of claim 40, wherein detecting the expression level of the at least one biomarker comprises analyzing the transcription level of the at least one biomarker or analyzing the protein level of the at least one biomarker.

43. The method of claim 40, wherein the cancer is colorectal cancer.

44. The method of claim 40, wherein the cancer is a subset of colorectal cancer.

45. The method of claim 40, wherein detecting the expression level comprises use of digital PCR.

46. The method of claim 40, wherein determining detecting the expression level comprises detecting methylation.