WO2013070950A1 - Identification of a dna methylation marker for blood-based detection of ovarian cancer - Google Patents

Abstract

The present invention teaches methods and systems for diagnosing ovarian cancer in a subject, or the recurrence thereof, using the novel marker IFFO1-M. The invention further teaches that this marker can be used in conjunction with additional protein-based markers, such as CA-125, in order to more accurately and effectively monitor disease status.

Description

IDENTIFICATION OF A DNA METHYLATION MARKER FOR BLOOD-BASED

DETECTION OF OVARIAN CANCER

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from US Provisional Patent Application Number

61/557,283, filed on November 8, 2011, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of Grant No. R01-CA096958 awarded by the National Institutes of Health and National Cancer Institute. FIELD OF THE INVENTION

The present invention relates generally to the field of cancer detection, and more specifically to blood-based ovarian cancer detection.

SUMMARY OF THE INVENTION

In some embodiments, the invention teaches a method for diagnosing cancer in a subject, including: obtaining a sample of blood from the subject; and detecting the presence or absence of IFFOl-M in the subject's blood, wherein the subject is diagnosed with cancer if IFFOl-M is present in the subject's blood. In some embodiments, the presence or absence of IFFOl-M is determined by a method selected from the group consisting of: (1) methylation- specific PCR (MSP), (2) whole genome bisulfite sequencing, (3) a HELP assay, (4) ChIP-on- chip assays, (5) restriction landmark genomic scanning, (6) methylated DNA immunoprecipitation (MeDIP), (7) pyrosequencing of bisulfite treated DNA, (8) molecular break light assay for DNA adenine methyltransferase activity, (9) methyl sensitive southern blotting, and (10) combinations thereof.

In some embodiments, the method further includes detecting a level of a protein-based cancer marker in the subject's blood, wherein the subject is diagnosed with cancer if the level of the protein-based cancer marker is elevated, compared to an individual without cancer. In some embodiments, the protein-based cancer marker is CA-125. In some embodiments, the cancer is ovarian cancer. In certain embodiments, the invention teaches a method for diagnosing a recurrence of cancer in a subject, including: obtaining serial blood samples from the subject in whom cancer was previously detected; and measuring a concentration of IFFOl-M in each sample of the subject's blood, wherein recurrence of cancer is diagnosed if the IFFOl-M concentrations measured increase significantly over time and/or are above a threshold level. In some embodiments, the concentration of IFFOl-M is determined by a method selected from the group consisting of: (1) methylation-specific PCR (MSP), (2) whole genome bisulfite sequencing, (3) a HELP assay, (4) ChlP-on-chip assays, (5) restriction landmark genomic scanning, (6) methylated DNA immunoprecipitation (MeDIP), (7) pyrosequencing of bisulfite treated DNA, (8) molecular break light assay for DNA adenine methyltransferase activity, (9) methyl sensitive southern blotting, and (10) combinations thereof. In some embodiments, the cancer is ovarian cancer. In some embodiments, the method further includes measuring a level of a protein-based cancer marker in the subject's blood, wherein the subject is diagnosed with a recurrence of cancer if the level of the protein-based cancer marker is elevated, compared to an individual without cancer. In some embodiments, the protein-based cancer marker is CA-125.

In some embodiments, the invention teaches, in combination, (1) an isolated sample obtained from a subject, including IFF01 and (2) a reagent capable of converting an unmethylated cytosine of a CpG dinucleotide to uracil. In some embodiments, the IFF01 is methylated. In some embodiments, the reagent includes bisulfite.

In some embodiments, the invention teaches a system, including (1) an isolated sample obtained from a subject, including IFF "01 and (2) a reagent capable of converting an unmethylated cytosine of CpG dinucleotide to uracil. In some embodiments, the IFF01 is methylated. In some embodiments, the reagent includes bisulfite.

In various embodiments, the invention teaches a method for treating ovarian cancer in a subject, including: (1) providing a composition comprising a drug for treating ovarian cancer; and (2) administering a therapeutically effective amount of the drug to the subject, so as to treat the ovarian cancer, wherein the subject's blood has been determined to comprise IFFOl-M. In some embodiments, the subject's blood has been serially tested and it has been determined that the concentration of IFFOl-M in the subject's blood is increasing over time. In some embodiments, the subject's blood has been determined to include an elevated level of a protein-based cancer marker, compared to an individual without cancer. In some embodiments, the protein-based cancer marker is CA-125. 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.

Ovarian cancer is the leading cause of gynecological cancer deaths and the fifth leading cause of all cancer-related deaths in women. It has been estimated that one woman in 72 will develop ovarian cancer in her lifetime in the USA, and that one woman in 96 will die of this disease. The five-year overall survival is strongly stage-dependent with rates of 94% for stage I disease and 28% for stage IV disease.

Since early stage disease is often asymptomatic, and there is no effective screening strategy, most patients (62%>) present with advanced-stage (III and IV) disease, in which the cancer has spread throughout the peritoneal cavity or other organs. More than 85% of patients with advanced disease relapse after cessation of primary therapy, despite an initial good response. It is anticipated that effective methods for detection of asymptomatic ovarian cancer before invasion and metastasis has occurred would substantially reduce the mortality rate for this disease. Sensitive detection methods could also be applied to monitoring disease recurrence after tumor resection with or without adjuvant chemotherapy.

Currently, there is no good biomarker or imaging approach with sufficient sensitivity and specificity for the detection of preclinical ovarian cancer. Two protein-based biomarkers, CA-125 and HE4, have been clinically approved to measure disease burden and to evaluate ovarian cancer treatment. However, these markers are not elevated in all ovarian tumors and do not have sufficient positive predictive value for population-based risk assessment or early detection. Given the limitations of current approaches, there is an urgent need to develop more effective strategies for the detection of preclinical ovarian cancer early enough for treatment to be successful.

Epigenetic biomarkers have recently emerged as alternatives to protein biomarkers for the early detection of cancer, including ovarian cancers. Aberrant DNA hypermethylation is frequently observed in cancer cells. Cancer patients have elevated levels of free DNA circulating in the bloodstream. Cancer-associated aberrant DNA methylation, originated at least in part in tumor cells, can be detected in serum or plasma DNA of cancer patients. Methylated DNA is chemically and biologically stable, readily detectable in many types of bodily fluids and therefore well suited for blood-based cancer detection. However, the limited number of DNA methylation markers currently available apply to only a small fraction of ovarian cancers and are non-specific, while the detection technologies lack sensitivity, are largely gel-based, and are non-quantitative. Thus, there is clearly a need in the art for DNA methylation markers for the blood-based detection of ovarian cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are illustrated in the referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

Figure 1 depicts, in accordance with an embodiment of the invention, a schematic representation of the ovarian cancer marker discovery and verification pipeline. The Infinium platform was used to screen 27,578 probes representing 14,489 individual gene loci. The inventors used a systematic stepwise approach to eliminate probes that failed in any of the samples, probes that contained SNPs or repeat sequences, or probes with a beta value higher than 0.2 in any of the PBL samples. The remaining probes were ranked based on their difference between tumors and blood (see Examples section), and the probes with higher DNA methylation in PBL than in any of the tumor samples were eliminated. The top 15 from the remaining 517 markers were transitioned to the MethyLight platform for further verification. All 15 markers passed an independent verification test performed on a publically available TCGA ovarian cancer dataset and additional PBL samples. Three markers failed due to incompatibility issues related to the MethyLight platform, while another ten failed because they were methylated in normal PBL DNA (3 markers) or normal plasma (7 markers). Only one marker, IFFOl, was selected for further verification on patient samples using Digital MethyLight. (The asterisk indicates probes that failed in any of the samples, as well as those that included SNPs and repeat sequences).

Figure 2 depicts, in accordance with an embodiment of the invention, a heat map representation of the marker selection process. A) The 12,194 markers remaining after the elimination of the probes that failed in any of the samples, and of the probes containing SNPs or repetitive elements. Markers are ranked in an ascending order based on the mean DNA methylation β value of the two PBL samples. B) The 8,701 markers remaining after eliminating probes with DNA methylation β values > 0.2 in any of the two PBL samples. Probes were ranked in a descending order based on the difference in DNA methylation between the tumor with the lowest β value (T_L) and the PBL sample with the highest β value (PBL_R). C) The 517 markers with higher DNA methyl ation values in any of the tumor than in any of the PBL samples. The markers are ranked in a descending order based on the difference between the tumors and the PBL DNA methylation values. D) The top-ranked 15 markers that were transitioned to the MethyLight platform for further verification.

Figures 3A and 3B depict, in accordance with an embodiment of the invention, dot plot displays of the top 15 -ranked marker distribution in two independent data sets of ovarian cancer samples and ten normal PBL samples. The Infinium-derived β values (Y-axis) for the top 15-ranked markers were compared in the present study (PS) data set (41 ovarian cancers of mixed subtypes), the TCGA data set (284 serous ovarian cancers) and ten normal PBL samples. The horizontal lines represent the median values for each group.

Figure 4 depicts, in accordance with an embodiment of the invention, a representation of the verification phase on the MethyLight platform of the top-ranked 15 DNA methylation markers. Technical controls for the MethyLight (ML) platform led to the elimination of four markers (crossed gray boxes) due to design incompatibility, and failure to amplify the in vitro methylated DNA positive control for MethyLight reactions (M.SssI test). Eleven markers were tested in normal PBL samples using an excess of PBL DNA (50ng). Markers with a cycle threshold (Ct) higher than 35 (dotted boxes) in the two normal PBL samples were retained and markers with a Ct less than 35 (light gray boxes) were eliminated. MethyLight assays with Ct values <35 indicate appreciably detectable amounts of methylated DNA at these loci. Further testing in normal control plasma samples (ΙΟΟμΙ) resulted in the elimination of seven of the eight remaining markers. One remaining marker, IFFOl, was tested in 15 ovarian cancers of different histological subtypes. The MethyLight results for the normal plasma control and the ovarian cancer samples are expressed as Percent of Methylated Reference (PMR). Dotted boxes represent PMR values less than 10, light gray boxes indicate PMR values between 10 and 50, whereas white boxes signify PMR values higher than 50. The types of ovarian tumors used in the analysis are as follows: clear cell carcinomas (CC), mixed clear cell and endometrioid (CC/E), endometrioid (E), mucinous (M), and serous (S).

Figure 5 depicts, in accordance with an embodiment of the invention, the performance of the IFFOl -M marker in the baseline serum samples of ovarian cancer patients and disease- free control women. A) IFFOl -M levels (expressed as the number of IFFOl -M methylated molecules detected in 1ml sera) in the baseline samples of 16 patients and eight normal controls were determined by Digital MethyLight. The number of molecules in patient #1 is an approximation, since counts higher than 15 hits/96-well plate/1 ΟΟμΙ tested could reflect the presence of more than one molecule/well. The histological subtype of the tumors is indicated in parenthesis as follows: serous (S), mucinous (M), and endometrioid (E). The asterisks indicate the patient from whom samples were used in the subsequent longitudinal analysis. B) Receiver operating characteristic curve for IFFOl-M. AUC = area under the curve.

Figure 6 demonstrates, in accordance with an embodiment of the invention, a comparison between the CA-125 and IFFOl-M performance in serially collected serum samples of nine ovarian cancer patients. Blood collected from ovarian cancer patients at the time of surgery (baseline samples) and at subsequent follow-up visits was used to measure CA-125 and IFFOl-M levels. The CA-125 levels (gray bars) are expressed in units/ml of blood, and the IFFOl-M methylation levels (black bars) are expresses as number of detected molecules/ml of sera on the Y-axis. The methylation analysis was performed using Digital MethyLight in DNA extracted from ΙΟΟμΙ of serum. The number of weeks since the baseline sample was collected is represented on the X-axis. The horizontal dashed line set at 35 u/ml represents the normal cut off value for CA-125. All patients except patients #5 and #18 had elevated levels of CA-125 in the baseline samples (> 35 u/ml). The arrow labeled S indicates the time of surgery and the arrow labeled R indicates the time of tumor relapse as determined by CA-125 and/or imaging techniques. Due to the large range of CA-125 values, the inventors restricted the Y-axis to a scale of 400 for both of the markers, and indicated the measurements that exceeded this scale by an asterisk. The values for these determinations are listed in the Table 4.

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. For purposes of the present invention, certain terms are defined below.

As used herein, the acronym "MEIA" means micro-particle enzyme-immunoassay.

As used herein, the acronym "SNPs" means single nucleotide polymorphisms.

"Mammal" as used herein refers to a member of the class Mammalia, including, without limitation, humans as well as nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like. The term does not denote a particular age. Thus, newborn subjects and infant subjects, as well as fetuses, are intended to be included within the scope of this term.

As used herein, the terms "detect," "detecting," or "detection" may describe either the general act of discovering or discerning or the specific observation of a detectably labeled substance, or a substance that doesn't require labeling for detection.

In some embodiments, the numbers expressing quantities of ingredients, properties such reaction conditions, and so forth, used to describe and claim certain embodiments of the application 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 application are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.

In some embodiments, the samples that could be used in connection with the invention include, but are in no way limited to, blood, serum, plasma, cerebrospinal fluid, pus, amniotic fluid, saliva, lung aspirate, vaginal discharge, urethral discharge, tumors and the like. In some embodiments, the sample is obtained from an animal. In some embodiments, the sample is obtained from a mammal. In some embodiments, the sample is obtained from a human. As disclosed herein, the inventors used the Illumina Infinium platform to analyze the DNA methylation status of 27,578 CpG sites in 41 ovarian tumors. A marker selection strategy was employed that emphasized sensitivity by requiring consistency of methylation across tumors, while achieving specificity by excluding markers with methylation in control leukocyte or serum DNA. The verification strategy disclosed herein involved testing the ability of identified markers to monitor disease burden in serially collected serum samples from ovarian cancer patients who had undergone surgical tumor resection compared to CA- 125 levels.

The inventors identified one marker in particular, IFFOl promoter methylation (IFFOl -M), that is frequently methylated in ovarian tumors and that is rarely detected in the blood of normal controls. When tested in 127 serially collected sera from ovarian cancer patients, IFFOl -M showed post-resection kinetics significantly correlated with serum CA- 125 measurements in six out of 16 patients.

As disclosed herein, the invention teaches an effective marker screening and verification strategy that resulted in the identification of IFFOl -M as a blood-based marker for sensitive detection of ovarian cancer. Serum levels of IFFOl -M displayed post-resection kinetics consistent with a reflection of disease burden. Importantly, IFFOl -M can provide effective disease detection capability, either when used alone, or as a complement to one or more previously existing biomarkers. The present invention is based, at least in part, on these findings as well as others further described herein.

One of skill in the art would readily appreciate that there are many well-known methods that can be used to assess the DNA methylation described herein. Merely by way of example, DNA methylation can be detected by any of the following well-known assays. (1) Methylation-Specific PCR (MSP), which is based on a chemical reaction of sodium bisulfite with DNA that converts unmethylated cytosines of CpG dinucleotides to uracil or UpG, followed by traditional PCR. Importantly, methylated cytosines will not be converted in this process, and primers are designed to overlap the CpG site of interest, which allows one to determine methylation status as methylated or unmethylated. (2) Whole genome bisulfite sequencing, also known as BS-Seq, which is a high-throughput genome-wide analysis of DNA methylation. It is based on aforementioned sodium bisulfite conversion of genomic DNA, which is then sequenced on a next-generation sequencing platform. The sequences obtained are then re-aligned to the reference genome to determine methylation states of CpG dinucleotides based on mismatches resulting from the conversion of unmethylated cytosines into uracil. (3) The HELP assay, which is based on restriction enzymes' differential ability to recognize and cleave methylated and unmethylated CpG DNA sites. (4) ChlP-on-chip assays, which are based on the ability of commercially prepared antibodies to bind to DNA methylation-associated proteins like MeCP2. (5) Restriction landmark genomic scanning, an assay based upon restriction enzymes' differential recognition of methylated and unmethylated CpG sites; the assay is similar in concept to the HELP assay. (6) Methylated DNA immunoprecipitation (MeDIP), analogous to chromatin immunoprecipitation, immunoprecipitation is used to isolate methylated DNA fragments for input into DNA detection methods such as DNA microarrays (MeDIP-chip) or DNA sequencing (MeDIP- seq). (7) Pyro sequencing of bisulfite treated DNA, which is sequencing of an amplicon made by a normal forward primer but a biotenylated reverse primer to PCR the gene of choice. The Pyrosequencer then analyzes the sample by denaturing the DNA and adding one nucleotide at a time to the mix according to a sequence given by the user. If there is a mis-match, it is recorded and the percentage of DNA for which the mis-match is present is noted. This gives the user a percentage methylation per CpG island. (8) Molecular break light assay for DNA adenine methyltransferase activity - an assay that relies on the specificity of the restriction enzyme Dpnl for fully methylated (adenine methylation) GATC sites in an oligonucleotide labeled with a fluorophore and quencher. The adenine methyltransferase methylates the oligonucleotide making it a substrate for Dpnl. Cutting of the oligonucleotide by Dpnl gives rise to a fluorescence increase. (9) Methyl sensitive Southern blotting is similar to the HELP assay, although uses Southern blotting techniques to probe gene-specific differences in methylation using restriction digests. This technique is used to evaluate local methylation near the binding site for the probe. One of skill in the art would also readily appreciate additional techniques and reagents, capable of achieving similar results to those described above, could also be used in conjunction with the inventive method.

In various embodiments, the invention teaches a method for diagnosing cancer in a subject. In some embodiments, the method includes: obtaining a sample of a bodily fluid and/or a tissue from a subject; and detecting the presence or absence of IFF01-M in the subject's bodily fluid and/or tissue, wherein the subject is diagnosed with cancer if IFF01-M is present in the subject's bodily fluid and/or tissue. In some embodiments, the bodily fluid described herein is blood. In other embodiments, the bodily fluid and/or tissue can include, but are in no way limited to those described above as potential sample sources. In various embodiments, the presence or absence of IFF01-M is determined by any of the following methods, described in greater detail above, and well-known to one of skill in the art: (1) methylation-specific PCR (MSP), (2) whole genome bisulfite sequencing, (3) the HELP assay, (4) ChlP-on-chip assays, (5) restriction landmark genomic scanning, (6) methylated DNA immunoprecipitation (MeDIP), (7) pyrosequencing of bisulfite treated DNA, (8) molecular break light assay for DNA adenine methyltransferase activity, (9) methyl sensitive southern blotting, and (10) combinations thereof. In some embodiments, the method also includes detecting a level of a protein-based cancer marker in the subject's blood, wherein the subject is diagnosed with cancer if the level of the protein-based cancer marker is elevated, compared to an individual without cancer. In some embodiments, the protein-based cancer marker is CA-125. In some embodiments, the cancer diagnosed is ovarian cancer. In some embodiments, the subject is a human.

In various embodiments, the invention teaches a method for diagnosing a recurrence of cancer in a subject, including: obtaining serial bodily fluid and/or tissue samples from a subject in whom cancer was previously detected; and measuring a concentration of IFF01-M in each sample of the subject's bodily fluid and/or tissue, wherein recurrence of cancer is diagnosed if the IFF01-M levels measured increase significantly over time. In some embodiments, the concentration of IFF01-M is determined by a method selected from the group consisting of: (1) methylation-specific PCR (MSP), (2) whole genome bisulfite sequencing, (3) the HELP assay, (4) ChlP-on-chip assays, (5) restriction landmark genomic scanning, (6) methylated DNA immunoprecipitation (MeDIP), (7) pyrosequencing of bisulfite treated DNA, (8) molecular break light assay for DNA adenine methyltransferase activity, (9) methyl sensitive southern blotting, and combinations thereof. In some embodiments, the cancer is ovarian cancer. In some embodiments, the method further includes determining a level of one or more known protein-based cancer markers in the subject's blood, wherein the subject is diagnosed with a recurrence of cancer if the level of the protein-based cancer marker is elevated, compared to a subject without cancer. In some embodiments, the protein-based cancer marker is CA-125.

In some embodiments, the invention teaches, in combination (1) an isolated sample obtained from a subject including IFF01 and (2) a reagent capable of converting an unmethylated cytosine of a CpG dinucleotide to uracil. In some embodiments, the IFF01 is methylated. In some embodiments, the IFF01 is isolated prior to interaction with the reagent. In some embodiments, the reagent includes bisulfite.

In some embodiments, the invention teaches a system including (1) an isolated sample obtained from a subject, wherein the sample includes IFF01, and (2) a reagent capable of converting an unmethylated cytosine of CpG dinucleotide to uracil. In some embodiments, the isolated sample is derived from the subject's bodily fluid or tissue, as described herein. In some embodiments, the IFF01 is methylated. In some embodiments, the reagent includes bisulfite.

In various embodiments, the invention teaches a method for treating ovarian cancer in a subject, including providing a composition comprising a drug for treating ovarian cancer; and administering a therapeutically effective amount of the drug to the subject so as to treat the ovarian cancer, wherein one or more of the subject's bodily fluids and/or tissues have been determined to contain IFF01-M. In some embodiments, the bodily fluid containing IFF01-M is blood. In certain embodiments, the subject's bodily fluids and/or tissues have been serially tested, and it has been determined that the concentration of IFF01-M in the bodily fluids and/or tissues is increasing over time. In certain embodiments the concentration of IFF01-M has increased in the subject's blood over time. In some embodiments, the subject's blood has been determined to contain an elevated level of a protein-based cancer marker, compared to an individual without cancer. In some embodiments, the protein-based cancer marker is CA-125.

Merely by way of non- limiting example, the therapeutic composition can include one or more of the following (1) a platinum agent, including but in no way limited to Parap latin (carboplatin and cispiatin); (2) a taxane, including but in no way limited to Taxol (paclitaxel) and Taxotere (docetaxei), (3) an anthrac cline, including but in no way limited to Adriamycm (doxorubicin) and Doxil (liposomal doxorubicin), (4) gemcitabine, and (5) topotecan.

In certain embodiments, the invention teaches a method of screening for and verifying DNA methylation markers for blood-based screening for ovarian cancer in a subject, including screening and verifying potential markers according to the methodology disclosed in Figure 1.

One of skill in the art would readily appreciate that by appropriately modifying the methodology described in Figure 1 , and elsewhere herein, other candidate blood-based DNA methylation markers could be determined for a variety of different cancer types.

EXAMPLES

Example 1

Introduction

The inventors conducted a large-scale systematic marker discovery for DNA methylation markers of ovarian cancer that are not present in the blood of women without ovarian cancer. DNA methylation markers have been found to have moderate clinical sensitivity in many prior reports. In considering how to improve the sensitivity of DNA methylation markers, the inventors recognized that the methylation status of normal ovary is irrelevant, as long as normal ovary DNA does not normally leak into the bloodstream and the markers are negative in healthy controls. Therefore, the inventors modified their discovery strategy to focus on a direct comparison of tumor vs. blood, as opposed to tumor vs. normal tissue. In the selection process, the inventors emphasized marker sensitivity by requiring consistency of tumor methylation, and marker specificity by excluding markers with methylation in control leukocyte or serum DNA. The inventors identified a DNA methylation marker, IFFOl-M, which was tested as a blood-based biomarker in case and control sera. To provide evidence that the candidate IFFOl-M marker measures disease burden in the blood, the inventors analyzed the temporal patterns of IFFOl-M levels in serial blood samples drawn before and after resection of the primary tumor, and compared these to a validated marker for disease burden, CA-125. This within- subject comparison allows each patient to serve as her own control, with no variation in genetic background between the serial blood samples. The inventors' studies disclose the quantitative digital analysis of IFFOl-M in serial samples from nine patients, for a total of 127 blood samples.

Example 2

Patients and controls specimen collection and processing

The 41 ovarian tumor samples used in the Infinium-based marker discovery phase of the study were obtained from patients that underwent surgery at two institutions, Duke University Medical Center (30 samples) and University of Southern California Medical Center (1 1 samples). All tumor samples were obtained from patients who provided written informed consent, which was approved by the Institutional Review Boards of the respective institutions. Among the tumor samples collected, there was one mixed (clear cell and endometrioid), three clear cell, four mucinous, four endometrioid, and 32 serous epithelial ovarian carcinoma samples (Table 1). Tumor tissues were flash- frozen in liquid nitrogen and stored at -80°C until processed. Peripheral blood leukocyte (PBL) and plasma samples used in the discovery and verification stages were obtained from 10 healthy post-menopausal women whose bloods were commercially purchased (HemaCare Corporation). Plasma was isolated from blood collected in tubes containing EDTA. The tubes were spun for 10 min at 300g at 4°C. Without removing the plasma from the tube after the first centrifugation, the inventors spun the tubes for an additional 10 minutes at l,600g at 4°C. The separated plasma was transferred to microcentrifuge tubes and spun again at 16,000g for 10 min at 4°C. The supernatant was collected and stored at -80°C until ready to use. The thin peripheral blood leukocytes layer that sedimented above the red blood cells was collected and stored -80°C until ready to use for DNA extraction.

DNA was extracted from tissues using standard protocols. DNA was extracted from the PBL samples using the QIAamp® DNA Blood kit (Qiagen), while free DNA from plasma and sera was extracted using the QIAamp® UltraSens Virus Kit (Qiagen). In both experiments, DNA was extracted following the manufacturer's instructions.

The blood samples used in the longitudinal analyses were collected from 16 patients treated for ovarian cancer between 1992-2000 at the Department of Obstetrics and Gynecology, Innsbruck University Hospital (Innsbruck, Austria) in compliance with and approved by the Innsbruck University Institutional Review Board. The clinical and pathological characteristics of these patients are listed in Table 3. The first blood samples drawn, referred to as baseline samples, were obtained before the surgery for eleven of the patients and several days after the surgery for five patients (see complete information in Table 4). Additional blood was collected from all patients at each follow-up visit for periods of times ranging from 37 to 246 weeks (Table 4). For serum isolation, blood was allowed to coagulate for 1-4 hours at room temperature (RT) and centrifuged for 10 minutes at 2000g at RT. Serum was isolated from the clot, aliquoted into microcentrifuge tubes, and stored at - 80°C until analysis. Control sera from eight healthy women were commercially purchased (Innovative Research). Free circulating DNA was isolated from the patients and controls sera using the QIAamp® UltraSens Virus Kit (Qiagen) following the manufacturer's instructions. Levels of CA-125 were determined by a micro-particle enzyme -immunoassay (MEIA) using the IMX analyzer (Abbott Laboratories).

Example 3

DNA methylation analysis

All DNA specimens were subjected to bisulfite modification using the EZ DNA Methylation Kit (Zymo Research) according to the manufacturer's instructions. For the Infmium-based analysis, 1 μg genomic DNA from each sample was bisulfite converted in 96 well plate format using the EZ96 DNA methylation kit (Zymo Research). The quality and quantity of the bisulfite-converted DNA, as well as the completeness of the bisulfite conversion, were assessed using a panel of quality control reactions as previously described in Campan et al. MethyLight. Methods Mol Biol 507: 325-337, incorporated herein by reference in its entirety as though fully set forth. Following the conversion, the modified DNA was eluted in 18μ1 elution buffer supplied with the kit, and 5μ1 of each sample was used in the Illumina Infinium DNA methylation assay as specified by the manufacturer.

For MethyLight analysis, ^g genomic DNA from each tumor and PBL sample was treated with bisulfite using the Zymo EZ DNA methylation kit (Zymo Research). Similarly, the Zymo EZ DNA methylation kit was used to bisulfite convert the DNA extracted from plasma or sera samples. In general 1ml of plasma or sera was processed in one column of the Zymo kit. After purification, all bisulfite modified DNA samples were eluted in ΙΟμΙ of elution buffer and further diluted as follows: the PBL-DNA was diluted to a final concentration of 0.5¾/μ1. The tumor DNA was diluted based on the cycle threshold (Ct) of an ALU-based MethyLight reaction. Only DNAs with a Ct less than 21 for this ALU-based reaction were used in any of the subsequent analyses. The plasma DNA was diluted such that every Ιμΐ of modified DNA represented ΙΟμΙ of the initial volume of plasma or sera used. For each MethyLight reaction ΙΟμΙ of the diluted tumor, PBL or plasma bisulfite converted DNA were used. For Digital MethyLight analysis, the entire amount of DNA extracted from lml of serum was bisulfite converted, and the samples were diluted such that every Ιμΐ of each bisulfite-converted DNA sample represented Ιμΐ of the initial serum volume used. In each Digital MethyLight analysis, ΙΟΟμΙ of the diluted DNA was used.

The Infinium analysis was performed in the USC Epigenome center using the HumanMethylation27 BeadArray (Illumina). The results of the Infinium assay were compiled for each locus using Illumina BeadStudio software (Illumina) and are reported as beta (β) values which are DNA methylation scores ranging from 0 to 1 that reflect the fractional DNA methylation level of a single CpG site. The MethyLight assay and data analysis were performed as previously described in Eads et al. MethyLight: a high- throughput assay to measure DNA methylation. Nucleic Acids Res 28: E32; and Weisenberger et al. CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer. Nat Genet 38: 787-793, both of which are incorporated herein by reference as though fully set forth.

The primers and probes for these analyses are listed in the Table 2. The Digital MethyLight assay and data analysis were performed as previously described in Weisenberger et al. DNA methylation analysis by digital bisulfite genomic sequencing and digital MethyLight. Nucleic Acids Res 36: 4689-4698, which is incorporated herein by reference in its entirety as though fully set forth. One significant difference is that the PCR reactions were performed in a volume of ΙΟμΙ instead of 30μ1.

Example 4

Marker selection

Infinium probes that failed in any of the samples were excluded from the analysis. Probes associated with single nucleotide polymorphisms (SNPs), identified using the NCBI dbSNP builds 126 and 128, or repetitive elements identified by RepeatMasker were also excluded. The two PBL samples used in the analysis were run in duplicate on the Infinium DNA methylation platform and the averaged β values for each sample were used for marker filtering. The inventors eliminated any probes with a β value higher or equal to 0.2 in any of the two averaged PBL samples. The inventors identified the tumor sample with the lowest DNA methylation value (T_L) and the PBL sample with the highest DNA methylation value (PBL_R) for each probe and calculated the difference between the β values of these two samples (T_L- PBL_H). The probes were ranked based on this difference and the probes for which the difference was less or equal to 0 were eliminated.

Example 5

Verification of the 15 top-ranked markers using The Cancer Genome Atlas (TCGA) data

The β values of the 15 top-ranked markers were retrieved from the publically available DNA methylation dataset for serous ovarian cancers posted on the TCGA Data Portal. The inventors compared the distribution of Infinium-generated β values of these 15 markers in all the 41 ovarian cancer samples or just the 29 serous ovarian cancers of this study to those obtained from 284 serous ovarian cancer samples in the TCGA study and a total of 10 normal PBL samples using dot-diagrams from the GraphPad Prism Software (GraphPad Software Inc.). Example 6

Statistical analysis

The ability of IFFOl-M to discriminate between case and control samples was assessed by plotting the receiver operating characteristics curve, which associates the true positive rate (sensitivity) to the false positive rate (1 -specificity) and by computing area under the curve (AUC). The 95% Confidence Interval (CI) of the AUC was computed with 2000 bootstrap samples using the pROC R package. Pearson product-moment correlation coefficient (Pearson's r) was calculated to measure the degree of correlation between the IFFOl-M DNA methylation levels and the CA-125 levels in each of the nine patients. Statistical test against the null hypothesis that r = 0 was performed and a p value cutoff of 0.05 was used to declare significance. All statistical analyses were performed using the R 2.13 software.

Example 7

DNA methylation-based marker discovery pipeline The inventors devised a comprehensive and systematic strategy to identify and evaluate blood-based DNA methylation markers for ovarian cancer that are only present in the blood from individuals afflicted with the disease. Figure 1 illustrates the steps undertaken to achieve this goal. Each of these steps is described in more detail in the subsequent sections. Example 8

Screening for and selection of DNA methylation markers for ovarian cancer

The inventors first conducted a large-scale DNA methylation analysis of 41 ovarian cancer samples (Table 1) and two PBL samples from disease-free postmenopausal women. The inventors used the Infmium DNA methylation BeadArray that simultaneously interrogates the DNA methylation status of 27,578 probes spanning 14,489 unique genetic loci. The inventors began the marker selection process by filtering out all probes that failed (detection p-value>0.05) in any of the samples, as well as probes containing single-nucleotide polymorphisms (SNPs), and repeat sequences (Fig 1 and 2A). The inventors next eliminated all the probes with high DNA methylation levels in PBL (β > 0.2 in any one of the PBL sample) (Fig. 1). The remaining 13,628 probes (8701 unique genes) were ranked in a descending order based on an algorithm that calculates the difference between the least methylated ovarian tumor sample and the most methylated blood sample for each probe (Fig. 1 and 2B). The 554 probes (517 unique genes) with higher DNA methylation in any of the ovarian tumors compared to the two normal blood samples were retained for future evaluation (Fig 1 and 2C). The inventors next performed confirmatory analyses with the top 15-ranked markers (Fig. 1 and 2D). The choice of testing only this limited number of markers was motivated by cost and patient sample availability constrains. Example 9

Independent assessment of reproducibility of the top 15 -ranked makers

Since the number of ovarian cancer samples used in the screening step was relative small, the inventors took advantage of the publicly available TCGA DNA methylation data on serous ovarian cancers to confirm the performance of the 15 top-ranked markers in an independent larger dataset. This analysis was facilitated by the fact that the TCGA data were generated using the same technology as in the inventors' study. The distribution of the DNA methylation beta values for all 15 markers in the two tumor datasets (present study (PS) and TCGA) in comparison to a set of ten healthy control PBL samples is shown in Figure 3. The range of DNA methylation values in the inventors' experimental set of 41 serous, mucinous, clear-cell, and endometrioid ovarian cancers was similar to that of the 284 serous ovarian cancer samples from TCGA, with both showing much higher DNA methylation levels than in the ten healthy control PBL samples. The results did not differ when the analysis was restricted to the 29 of the 41 ovarian cancer patients with serous histology (data not shown).

Example 10

MethyLight assays development and verification in control samples for the 15 top-ranked markers

The inventors next transitioned the 15 markers to the more sensitive PCR-based DNA methylation detection platforms, MethyLight and Digital MethyLight. Unlike Illumina Infinium DNA methylation probes, which assay the DNA methylation status of a single cytosine, MethyLight-based primers and probe interrogate concordant DNA methylation of several methylated cytosines simultaneously over a short genomic region. Consequently, MethyLight results for a specific genetic locus may differ from those registered by an Illumina Infinium probe at the same location due to the presence of neighboring cytosines and variations in the primers/probe positioning. The inventors were successful in developing MethyLight reactions for 12 of the 15 candidate markers (Table 2). The DNA sequences adjacent to the Infinium targeted cytosines for three of the 15 markers were not suitable for MethyLight design (Fig 4). An additional marker was eliminated from the pipeline because it failed to amplify in vitro methylated (M.SssI -treated) control DNA (Fig. 4). The inventors subjected the remaining 11 markers to a stringent counter screen against excess amounts of PBL DNA (50ng) from two disease-free postmenopausal women, to exclude DNA methylation markers that would present background problems for the sensitive detection of ovarian DNA methylation markers in blood. This MethyLight-based screen yielded eight markers with very low levels of DNA methylation in PBL (MethyLight cycle thresholds (Ct) higher than 35 in both samples).

These eight markers were subsequently evaluated on concentrated free plasma DNA samples from ten healthy postmenopausal women, yielding only one marker, IFFOl, with almost undetectable DNA methylation in all of these control plasma samples (PMRs < 5) (Fig. 4). The inventors next tested IFFOl on 15 ovarian tumors of various histological subtypes, 14 of which have also been used in the initial screening step (Table 1), and found significant levels of DNA methylation (PMR > 20) in all the tumors analyzed (Fig 4). This is in agreement with the Infinium DNA methylation analysis from the same tumor samples (Fig 2). This marker was therefore selected for subsequent clinical verification in a set of serum samples collected longitudinally from a separate set of ovarian cancer patients.

Example 11

IFFOl gene promoter DNA methylation (IFFOl -M) marker performance in serum samples

The inventors first evaluated the performance of IFFOl -M marker in serum samples obtained from eight healthy older women controls and 16 ovarian cancer patients with advanced (stage III and IV) disease using the highly sensitive and quantitative Digital MethyLight assay [23]. The clinicopatho logical characteristics of the ovarian cancer patients included in this analysis are summarized in Table 3. The output for Digital MethyLight is measured by counting the individually methylated DNA molecules. The inventors detected the IFFOl -M marker in all patient samples. In contrast, IFFOl -M was detected at very low levels in only two of the eight control samples (Fig. 5A). Ten of the patient sera had more IFFOl -M DNA methylation than any of the positive control samples. The Receiver Operating Characteristics (ROC) analysis with an estimated area under the curve of 0.95 [95% CI, 0.8359 to 1] indicated a good discriminatory potential for the IFFOl -M marker (Fig. 5B). Since these results might have been influenced by differences in the way the control and case samples were collected and processed the inventors continued the testing of IFFOl -M in serially collected serum samples from ovarian cancer patients.

Example 12

Verification of the IFFOl -M marker in a longitudinal screen for recurring ovarian cancer

The inventors tested whether IFFOl -M marker could measure disease burden in serially collected serum samples of ovarian cancer patients and compared its performance to that of CA-125. This within- subject comparison eliminates the need of control samples since each patient serves as her own control. Serum samples were collected around the time of surgery (baseline samples) and during subsequent follow-up examinations for 16 ovarian cancer patients. The baseline samples from each of these 16 patients were used in the previous analysis (Table 3). Eleven of the baseline samples were collected before the surgery (mean = 11 +/- 5 days), while five of them were collected after the surgery (mean = 13 +/- 10 days) (Table 4). On average, 15 serum samples (ranging from 8 to 23) were collected at follow-up visits from each of the patients, with an average follow up time of 92 weeks (ranging from 37 to 246 weeks). All but three patients had abnormal CA-125 levels (> 35 U/ml) in the baseline samples.

The inventors used nine of the 16 patients with high levels of IFFOl-M in the baseline samples (Fig. 5 A) to longitudinally compare the performance of the IFFOl-M marker to that of CA-125 in the serum samples collected during follow-up (Fig. 6). Of these, eight patients had baseline IFFOl-M serum levels well above background levels (Fig. 5 A and Table 4). The inventors included one additional patient (patient #18) with borderline baseline IFFOl-M levels, but with negative CA-125 measurements.

Two of the nine patients had normal CA-125 levels in the baseline samples (patients #5 and #18). Clinical recurrence occurred in all but one of the patients (patient #17). In the weeks immediately following surgery, both CA-125 levels and IFFOl-M DNA methylation measurements dropped in all patient samples relative to baseline. The decrease in both these markers paralleled the reduction in the tumor burden following the surgery. No increase of either CA-125 or IFFOl-M occurred after the initial drop in the patient #17 who never had a clinical relapse.

CA-125 levels eventually rose and exceeded normal levels (35 U/ml) in the follow-up samples of six of the eight patients with recurrent disease (patients #1, #2, #8, #14, #15, and #21). The IFFOl-M marker increased in four of the eight patients with recurrent disease. In three of these patients (patients #1, #8, and #15) the increase in IFFOl-M paralleled CA-125, whereas in one (patient #18) the IFFOl-M increase was not accompanied by an increase in the CA-125 levels. The increase of the IFFO-M occurred in three patients with serous ovarian cancer and in one with mucinous ovarian cancer. In combination, CA-125 and IFFOl-M DNA methylation markers tracked the disease status in eight of the nine analyzed patients. The IFFOl-M DNA methylation and CA-125 levels were correlated with each other in six out of nine patients (p<0.05, Pearson product-moment correlation test). The correlation coefficients were, 0.97, 0.81, 0.70, 0.97, 0.95, and 0.74 for patients #1, #2, #5, #14, #15 and #17 respectively. While not wishing to be bound by any one particular theory, these data strongly suggest that the IFFOl-M marker correlates with disease status and that it may complement CA-125 in detecting disease recurrence in some cases.

Example 13

Discussion

The inventors used a new strategy to identify blood-based candidate DNA methylation markers of ovarian cancer and to verify their potential to detect recurrent disease. The inventors sought to circumvent some of the limitations associated with the biomarker development process.

While not wishing to be bound by any one particular theory, one problem with biomarkers identified by high throughput technologies is their lack of sufficient specificity. The use of a genome-scale screening approach presented the inventors with the challenge of defining a clear marker selection strategy that would emphasize both marker sensitivity and specificity and help prioritize among the hundreds of potential biomarkers. In most studies of epigenetic biomarkers for blood-based detection of cancer, marker specificity is initially inferred from normal vs. tumor tissue comparisons. The inventors emphasized specificity of blood-based detection by directly comparing tumors from ovarian cancer patients to blood DNA from women without ovarian cancer, and eliminating markers found to be methylated in blood from age-matched healthy controls. The inventors included ovarian cancer samples from four different ovarian cancer subtypes in the analysis (Table 1) to maximize marker sensitivity for detection for all of these types of ovarian cancer. During the verification process the inventors counter-screened their markers with large quantities of PBL DNA and then with both serum- and plasma-derived DNA to exclude markers with low specificity.

DNA methylation markers generally suffer from poor clinical sensitivity. In order to emphasize sensitivity in their marker selection strategy the inventors used very stringent criteria that required consistently higher DNA methylation in all tumors than in any of the normal blood samples. The inventors anticipated that this approach would enrich for markers with a high prevalence of DNA methylation in ovarian cancers, which in turn, would translate into a higher sensitivity for detection of ovarian cancer in patient blood than for markers with a lower frequency of tumor DNA methylation.

Promising biomarkers emerging from large-scale discovery efforts have often performed poorly when tested on independent validation samples due in part to lack of randomization of case and control blood samples at baseline in observational diagnostic studies. Diagnostic biomarker studies usually rely on subject selection on the basis of diagnosis, which can result in baseline differences between cases and controls. This can lead to false-positive identification of disease-associated markers. Population-based cohort studies with prediagnostic blood samples can also be an excellent source for nested case- control comparisons (also referred to as a prospective-specimen collection, retrospective- blinded-evaluation (PRoBE) design. However, the number of incident cases in cohorts is usually limiting and the high demand for these precious samples generally precludes their use for early-stage candidate DNA methylation biomarker evaluation. Also, since ovarian cancer is a disease with low incidence, the use of a prospective population-based study for early- phase ovarian cancer markers validation is not very practical.

In this study the inventors tested an alternative verification scheme that evaluates the marker's correlation with a validated marker, CA-125, known to be associated with disease status in post-resection serially collected blood samples. This within-subject approach circumvents some of the drawbacks of traditional case-control designs, since each case serves as its own genetically matched control. The information regarding the ability of the inventors' top marker, IFFOl-M, to measure disease status was extracted from the temporal pattern across many serial samples for each subject (8-21 samples per patient). The comparison to the validated biomarker CA-125 lent further support to the conclusion that IFFOl-M is measuring disease status.

The rapid decrease in the serum levels of IFFOl-M in all nine patients in the weeks immediately following surgery provides compelling evidence that IFFOl-M serum levels reflect tumor burden. In many cases, IFFOl-M closely tracked CA-125 serum levels in the post-resection serum specimens. IFFOl-M rose at the time of disease recurrence in three of the nine patients in a similar manner to CA-125, and even outperformed CA-125 in one additional patient in which CA-125 never increased over its normal values, despite disease relapse (Figure 6, Patient #18).

CA-125 surpassed IFFOl-M performance in three of the patients (#2, #14, and #21). This however, could be a direct consequence of the small volume of serum (100 μΐ) used for the DNA methylation analyses, and better performance of IFFOl-M should be expected in future studies using larger volumes of sera. In combination, CA-125 and IFFOl-M corresponded to relapse in seven of the eight patients with recurrent disease, indicating that IFFOl-M could complement CA-125 in monitoring residual disease.

Despite increased efforts to identify new protein-based ovarian cancer biomarkers, CA-125 still remains the best marker for detecting early disease, up to three years in advance of the clinical diagnosis in some patients, and to monitor disease recurrence. This is the first time that a DNA methylation marker has been shown to have a concordant behavior with a protein marker with recognized clinical use. The analysis of post-resection serially collected samples can provide an effective method to evaluate whether a candidate marker has the potential to detect recurrent disease prior to the onset of symptoms or clinical evidence of disease and to help in the triage process of candidate markers that could be advanced for further analysis in valuable samples from larger population-based studies.

Example 14

Tables

12864 use tumor Mixed Clear 34 4

Cell/Endomet

rioid

12865 use tumor Clear Cell 35 2

12767 use tumor Clear Cell 36 1

12855 use tumor Clear Cell 37 3

12766 use tumor Mucinous 38 9

12852 use tumor Mucinous 39 10

12863 use tumor Mucinous 40 12

12866 use tumor Mucinous 41 11

12868 use tumor Serous 13

10703 HemaCare Control PBL 1

10704 HemaCare Control PBL 2

10707 HemaCare Control PBL 3

10705 HemaCare Control PBL 4

10706 HemaCare Control PBL 5

10708* HemaCare Control PBL 6

10709 HemaCare Control PBL 7

10710* HemaCare Control PBL 8

10711 HemaCare Control PBL 9

10712 HemaCare Control PBL 10

11162 HemaCare Control 1

plasma

11163 HemaCare Control 2

plasma

11164 HemaCare Control 3

plasma

11165 HemaCare Control 4

plasma

11166 HemaCare Control 5

plasma

11167 HemaCare Control 6

plasma

Table 1 . Sample's source, histo ogy, and alternative IDs in the various

utilized assays (continued).

Illumina

Laird Infinium Assay MethyLight Digital

ID. Source Sample type Histology ID ID MethyLight ID

11168 HemaCare Control 7

plasma

11169 HemaCare Control 8

plasma

11170 HemaCare Control 9

plasma

11171 HemaCare Control 10

plasma

11480 Innovative Control sera 1

Res

11481 Innovative Control sera 2

Res

11482 Innovative Control sera 3

Res

11681 Innovative Control sera 4

Res

11682 Innovative Control sera 5

Res

11683 Innovative Control sera 6

Res 11684 Innovative Control sera 7 Res

11973 Innovative Control sera 8

Res

10817 Innsbruck Patient sera Serous 1

10818 Innsbruck Patient sera Serous 2

10820 Innsbruck Patient sera Mucinous 4

10821 Innsbruck Patient sera Serous 5

10822 Innsbruck Patient sera Serous 6

10824 Innsbruck Patient sera Mucinous 8

10825 Innsbruck Patient sera Serous 9

10828 Innsbruck Patient sera Endometrioid 12

10829 Innsbruck Patient sera Serous 13

10830 Innsbruck Patient sera Endometrioid 14

10831 Innsbruck Patient sera Serous 15

10832 Innsbruck Patient sera Serous 16

10833 Innsbruck Patient sera Endometrioid 17

10834 Innsbruck Patient sera Serous 18

10835 Innsbruck Patient sera Serous 19

10837 Innsbruck Patient sera Serous 21

Table 2: Meth lLi ht Primers and Probes Se uences.

Table 2: Meth lLi ht Primers and Probes Se uences Continued .

24

30661875- 6FAM-CGACCACGTAACTACCGATACCCCGTTA-BHQ1

OSM 22 HB-765

30661967

25

36233422- 6FAM-TTCAACATAAAAACCTTCTTCTACCGCCCC-

TMEM149 19 HB-820

36233530 BHQ1

26

202129775- 6FAM-ACAACCGACCCCTAATAACAACGACAACA-

PTPN7 1 HB-807

202129885 BHQ1

27

76127469-

TMC8 17 HB-819 6FAM-CCCCTCTACGCCGACGTCGAA-BHQ 1

76127564

28

6665179- 6 FAM-CCCTACTCCTACACCGATCTACATCTCCCAA-

IFF01 12 HB-757

6665271 BHQ1

29

51718059- 6FAM-CGAAAACTAACCTCGAACGAACGCTACCTA-

BIN2 12 HB-822

51718168 BHQ1

30

29757309- 6FAM-CTTCATCCGAAACCGAACCTCTACGTCTAA-

C160rf54 16 HB-796

29757378 BHQ1

31

TBC1D10 67171693- 6FAM-AACCAAACCCGCTAAACTCTAAATCGAACC-

11 HB-802

C 67171804 BHQ1

32

TNFRSF2 6526161-

1 HB-080 6FAM-CGCCCAAAAACTTCCCGACTCCGTA-BHQ-1

5 6526229

33

113931499- 6FAM-TATAACTATAACGACTAAAACTTCCGACTT-

PSD4 2 HB-751

113931588 MGBNFQ

Sequence ID numbers refer to sequences excluding markers and quenchers

The coordinates for the MethyLight amplicons are given using the February 2009 Assembly from the University of California Santa Cruz (UCSC) Genome Browser. The primers and probes sequences are written in the 5 ' to 3 ' orientation. All probes contain at the 5' end a 6FAM fluorophore and either a Black Hole Quencher (BHQ) or a Minor Groove Binding Non-Fluorescent Quencher (MGBNFQ) and the 3 ' end.

Table 3: Clinical and Pathological Characteristics of the Ovarian Cancer Patients and the Age of the Normal Controls used for testing of the IFF01-M in Serum Samples by Digital MethylLight.

11149 21 Case 59 10/25/99 Serous 3C 2

10875 4 Case 48 3/20/96 Mucinous 3C 3

10910 6 Case 61 4/20/98 Serous 3C 2

10965 9 Case 63 1/29/97 Serous 3C 3

11019 12 Case 58 3/19/98 Endometrioid 3C 3

11030 13 Case 67 6/27/95 Serous 3C 3

11081 16 Case 63 05/6/98 Serous 3C 2

11127 19 Case 58 8/19/97 Serous 3C 2

11480 1 Control 57

11481 2 Control 56

11482 3 Control 57

11681 4 Control 50

11682 5 Control 51

11683 6 Control 52

11684 7 Control 62

11973 8 Control NA

(Continued).

Table 4. CA-125 and IFFOl-M levels in the serially collected serum samples of nine patients with ovarian cancer.

(U/ml) Methylated

Molec/ΙΟΟμΙ

4 17 5/27/1997 63 3

4 18 7/1/1997 68 2

4 19 8/21/1997 75 4

4 20 11/19/1997 88 13

4 21 2/18/1998 101 246

4 22 3/4/1998 103 267

4 23 3/10/1998 104 286

ratient 8

IFFOl-M

CA-125* No. of

Sample Collection Surgery Baseline Weeks of Levels Methylated

ID Date Date Sample Follow-up (U/ml) Molec/ΙΟΟμΙ

8 1 5/9/94 5/26/94 Preoperative 0 561 14

8 2 7/11/94 9 191 3

8 3 8/10/94 13 68 1

8 4 9/9/94 18 32 0

8 5 10/7/94 22 15 2

8 6 11/11/94 27 14 8

8 7 1/6/95 35 12 3

8 8 2/16/95 40 7 1

8 9 3/16/95 44 12 2

8 10 5/3/95 51 8 1

8 11 8/1/95 64 20 6

8 12 8/18/95 67 27 2

8 13 11/29/95 81 111 22

Table 4 (continued)

Table 4 (continued)

Table 4 (continued)

Table 4 (continued)

18 13 2/3/94 101 7 1

18 14 3/24/94 108 6 0

18 15 7/13/94 124 2 1

18 16 4/5/95 162 4 8

18 17 10/4/95 188 3 1

18 18 11/22/95 195 3 2

18 19 12/4/95 197 4 2

18 20 11/16/96 246 8 32

Patient 19

IFFOl-M

CA-125* No. of

Sample Collection Surgery Baseline Weeks of Levels Methylated

ID Date Date Sample Follow-up (U/ml) Molec/ΙΟΟμΙ

19 1 9/18/1997 8/19/97 Postoperative 0 884 3

19 2 10/9/1997 3 75

19 3 10/30/1997 6 26

19 4 11/21/1997 9 13

19 5 12/11/1997 12 9

19 6 1/7/1998 16 6

19 7 2/26/1998 23 6

19 8 11/12/1998 60 42

19 9 12/15/1998 65 46

19 10 2/18/1999 74 51

19 11 6/16/1999 91 89

19 12 7/15/1999 95 116

Table 4 (continued)

NA = not available

* Interpretation of serum CA-125 levels have been based on a normal value of less than 35 U/ml.

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 can 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 taught or suggested herein. A variety of alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several features, while others specifically exclude one, another, or several features, while still others mitigate a particular 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 employed in various combinations by one of ordinary skill in this art to perform methods in accordance with the principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments.

Although the application 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 application extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof.

In some embodiments, the terms "a" and "an" and "the" and similar references used in the context of describing a particular embodiment of the application (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 (for example, "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the application and does not pose a limitation on the scope of the application otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application.

Preferred embodiments of this application are described herein, including the best mode known to the inventors for carrying out the application. 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 application can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this application 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 application unless otherwise indicated herein or otherwise clearly contradicted by context.

All patents, patent applications, publications of patent applications, and other material, such as articles, books, specifications, publications, documents, things, and/or the like, referenced herein are hereby incorporated herein by this reference in their entirety for all purposes, excepting any prosecution file history associated with same, any of same that is inconsistent with or in conflict with the present document, or any of same that may have a limiting affect as to the broadest scope of the claims now or later associated with the present document. By way of example, should there be any inconsistency or conflict between the description, definition, and/or the use of a term associated with any of the incorporated material and that associated with the present document, the description, definition, and/or the use of the term in the present document shall prevail.

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

Claims

CLAIMS What is claimed is:

1. A method for diagnosing cancer in a subject, comprising:

obtaining a sample of blood from the subject; and

detecting the presence or absence of IFFOl-M in the subject's blood, wherein the subject is diagnosed with cancer if IFFOl-M is present in the subject's blood.

2. The method of claim 1, wherein the presence or absence of IFFOl-M is determined by a method selected from the group consisting of: (1) methylation-specific PCR (MSP), (2) whole genome bisulfite sequencing, (3) a HELP assay, (4) ChlP-on-chip assays, (5) restriction landmark genomic scanning, (6) methylated DNA immunoprecipitation (MeDIP), (7) pyrosequencing of bisulfite treated DNA, (8) molecular break light assay for DNA adenine methyltransferase activity, (9) methyl sensitive southern blotting, and (10) combinations thereof.

3. The method of claim 1, further comprising detecting a level of a protein-based cancer marker in the subject's blood, wherein the subject is diagnosed with cancer if the level of the protein-based cancer marker is elevated, compared to an individual without cancer.

4. The method of claim 3, wherein the protein-based cancer marker is CA-125.

5. The method of claim 1, wherein the cancer is ovarian cancer.

6. A method for diagnosing a recurrence of cancer in a subject, comprising:

obtaining serial blood samples from the subject in whom cancer was previously detected; and

measuring a concentration of IFFOl-M in each sample of the subject's blood, wherein recurrence of cancer is diagnosed if the IFFOl-M concentrations measured increase significantly over time and/or are above a threshold level.

7. The method of claim 6, wherein the concentration of IFF01-M is determined by a method selected from the group consisting of: (1) methylation-specific PCR (MSP), (2) whole genome bisulfite sequencing, (3) a HELP assay, (4) ChlP-on-chip assays, (5) restriction landmark genomic scanning, (6) methylated DNA immunoprecipitation (MeDIP), (7) pyrosequencing of bisulfite treated DNA, (8) molecular break light assay for DNA adenine methyltransferase activity, (9) methyl sensitive southern blotting, and (10) combinations thereof.

8. The method of claim 6, wherein the cancer is ovarian cancer.

9. The method of claim 6, further comprising measuring a level of a protein-based cancer marker in the subject's blood, wherein the subject is diagnosed with a recurrence of cancer if the level of the protein-based cancer marker is elevated, compared to an individual without cancer.

10. The method of claim 9, wherein the protein-based cancer marker is CA-125.

11. In combination, (1) an isolated sample obtained from a subject, comprising IFF01 and (2) a reagent capable of converting an unmethylated cytosine of a CpG dinucleotide to uracil.

12. The combination of claim 11, wherein the IFF01 is methylated.

13. The combination of claim 11 or 12, wherein the reagent comprises bisulfite.

14. A system, comprising (1) an isolated sample obtained from a subject, comprising IFF 01 and (2) a reagent capable of converting an unmethylated cytosine of CpG dinucleotide to uracil.

15. The system of claim 14, wherein the IFF01 is methylated.

16. The system of claim 14 or 15, wherein the reagent comprises bisulfite.

17. A method for treating ovarian cancer in a subject, comprising:

(i) providing a composition comprising a drug for treating ovarian cancer; and

(ii) administering a therapeutically effective amount of the drug to the subject, so as to treat the ovarian cancer, wherein the subject's blood has been determined to comprise IFFOl-M.

18. The method of claim 17, wherein the subject's blood has been serially tested and it has been determined that the concentration of IFFOl-M in the subject's blood is increasing over time.

19. The method of claim 17, wherein the subject's blood has been determined to comprise an elevated level of a protein-based cancer marker, compared to an individual without cancer.

20. The method of claim 19, wherein the protein-based cancer marker is CA-125.