WO2015107430A2

WO2015107430A2 - Methods and biomarkers for detection and prognosis of cervical cancer

Info

Publication number: WO2015107430A2
Application number: PCT/IB2015/000567
Authority: WO
Inventors: Heidi Lyng; Cathinka Halle JULIN; Malin Lando
Original assignee: Oslo Universitetssykehus Hf
Priority date: 2014-01-16
Filing date: 2015-01-16
Publication date: 2015-07-23
Also published as: WO2015107430A3

Abstract

The present invention relates to methods and biomarkers for detection of cervical cancer in biological samples, and in particular to methylation status markers associated with aggressiveness of cervical cancer.

Description

METHODS AND BIOMARKERS FOR DETECTION AND PROGNOSIS OF

CERVICAL CANCER

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Patients with an aggressive cervical cancer are in need of a more comprehensive treatment than what is conventionally given. A large extent of the patients experience recurrence of the disease after treatment, but there are currently no efficient means for identifying these patients. Half of these patients worldwide die of this disease. Cervical cancer is one of the most common malignancies in females worldwide, and is the world's second highest cause of female cancer mortality. Improvement in the handling of this disease will thus affect a large number of women.

A promising and needed strategy for treatment improvement is a prognostic test to identify patients at high risk of failure. This allows for a reduced radiation dose to the low- risk patients and a higher cure probability of the high-risk patients, and be of benefit with respect to increased survival, reduction in side effects, increased life quality, and be a considerable saving for the community due to reduced need for rehabilitation after therapy.

SUMMARY OF THE INVENTION

In some embodiments, the present invention provides methods for predicting a predisposition to cervical cancer in a subject, diagnosing a cervical cancer in a subject, predicting the likelihood of recurrence of cervical cancer in a subject, providing a prognosis for a subject with cervical cancer, or selecting a subject with cervical cancer for treatment with a particular therapy, comprising: a) obtaining DNA from a biological sample of the subject; and b) contacting the DNA with one or more methylation specific detection reagents to determine the level, presence, or frequency of methylation of a nucleic acid polymer corresponding to one or more genes selected from, for example, AK2, AK3L1, ALDOA, B3GNT4, CLK3, C14orf2, C4orfl, C20orf20, DDIT3, FGF11, GAPDH, ISG15, KCTD11, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARB1, SH3GL3, STC2, TRAPPC1, or UPK1A. In some embodiments, the one or more genes is two or more, three or more, four or more, five or more, or all of the genes. In some embodiments, the level or frequency of methylation of a nucleic acid polymer is compared to a reference level or frequency of methylation. In some embodiments, the method further comprises comparing the level, presence, or frequency of methylation of the nucleic acid polymer with a reference level, presence, or frequency of methylation, wherein an altered level, presence, or frequency of methylation for the patient relative to the reference provides an indication of, for example, predicting a predisposition to cervical cancer in a subject, diagnosing a cervical cancer in a subject, predicting the likelihood of recurrence of cervical cancer in a subject, providing a prognosis for a subject with cervical cancer, or selecting a subject with cervical cancer for treatment with a particular therapy. In some embodiments, the method further comprises the step of determining a treatment course of action based on the level, presence, or frequency of methylation. In some embodiments, the treatment is chemotherapy or radiation. In some embodiments, the method further comprises the step of administering the treatment. In some embodiments, the nucleic acid comprises a CpG island, a CpG island shore, or a CpG island shelf. In some embodiments, the CpG island, shore, or shelf is present in a coding region or a regulatory region such as, for example, a promoter. In some embodiments, the determining of the level of altered methylation of a nucleic acid polymer comprises determining the methylation frequency of the CpG island, shore, or shelf. In some embodiments, the determining of the level of a nucleic acid polymer with altered methylation is achieved by a technique selected from, for example, methylation-specific PCR, quantitative methylation- specific PCR, methylation-sensitive DNA restriction enzyme analysis, methylation - insensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, or bisulfite genomic sequencing PCR. In some embodiments, the methylation specific detection reagent is, for example, a pair of amplification primers that specifically hybridizes to said gene, an amplification primer that specifically hybridizes to said gene, a restriction enzyme, or sodium bisulfite, although the present invention is not limited to a particular methylation specific detection reagent. In some embodiments, the reagent (e.g., probe) is one or more (e.g., two) of SEQ ID NOs: 1-106. In some embodiments, the biological sample is a tissue sample, a cell sample, a blood sample, a urine sample, or other biological fluid.

In some embodiments, the present invention provides methods for predicting a predisposition to cervical cancer in a subject, diagnosing a cervical cancer in a subject, predicting the likelihood of recurrence of cervical cancer in a subject, providing a prognosis for a subject with cervical cancer, or selecting a subject with cervical cancer for treatment with a particular therapy, comprising: a) obtaining DNA from a biological sample of the subject; and b) contacting the DNA with one or more methylation specific detection reagents to determine the level, presence, or frequency of methylation of a nucleic acid polymer corresponding to one or more genes (e.g., two or more, three or more, four or more, five or more, or all of the genes) selected from, for example AK2, AK3L1, ALDOA, B3GNT4, C4orf2, FGFll, ISG15, KCTDll, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARB1, SH3GL3, STC2,ox TRAPPC1.

Further embodiments provide the use of a methylation specific nucleic acid detection sequence corresponding to one or more genes selected from, for example, AK2, AK3L1, ALDOA, B3GNT4, CLK3, C14orf2, C4orfl, C20orf20, DDIT3, FGFll, GAPDH, ISG15, KCTDll, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARB1, SH3GL3, STC2, TRAPPCl, or UPK1A for detecting or characterizing cervical cancer in a subject. In some embodiments, an altered level, presence, or frequency of methylation for a patient relative to a reference provides an indication selected from, for example, predicting a predisposition to cervical cancer in a subject, diagnosing a cervical cancer in a subject, predicting the likelihood of recurrence of cervical cancer in a subject, providing a prognosis for a subject with cervical cancer, or selecting a subject with cervical cancer for treatment with a particular therapy.

Yet other embodiments provide the use of a methylation specific nucleic acid detection sequence corresponding to one or more genes selected from, for example, AK2, AK3L1, ALDOA, B3GNT4, C4orf2, FGFll, ISG15, KCTDll, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARB1, SH3GL3, STC2,or TRAPPCl for detecting or characterizing cervical cancer in a subject.

Additional embodiments provide a kit for detecting the presence of a cervical cancer in a mammal, the kit comprising methylation specific detection reagents useful, sufficient, or necessary for detecting and/or characterizing level, presence, or frequency of methylation of one or more genes selected from, for example, AK2, AK3L1, ALDOA, B3GNT4, CLK3, C14orf2, C4orfi, C20orf20, DDIT3, FGFll, GAPDH, ISG15, KCTDll, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARB1, SH3GL3, STC2, TRAPPCl, or UPK1A.

Some embodiments provide a kit for detecting the presence of a cervical cancer in a mammal, the kit comprising methylation specific detection reagents useful, sufficient, or necessary for detecting and/or characterizing level, presence, or frequency of methylation of one or more genes selected from, for example, AK2, AK3L1, ALDOA, B3GNT4, C4orf2, FGFll, ISG15, KCTDll, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARBl, SH3GL3, STC2,ox TRAPPCI.

Yet other embodiments provide a system comprising a computer readable medium comprising instructions for utilizing information on the level, presence, or frequency of methylation of one or more genes selected from, for example, AK2, AK3L1, ALDOA, B3GNT4, CLK3, C14orf2, C4orfl, C20orf20, DDIT3, FGFll, GAPDH, ISG15, KCTDll, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARBl, SH3GL3, STC2, TRAPPCI, or UPK1A to provide an indication selected from, for example, an indication of predicting a predisposition to cervical cancer in a subject, diagnosing a cervical cancer in a subject, predicting the likelihood of recurrence of cervical cancer in a subject, providing a prognosis for a subject with cervical cancer, or selecting a subject with cervical cancer for treatment with a particular therapy.

Other embodiments provide a system comprising a computer readable medium comprising instructions for utilizing information on the level, presence, or frequency of methylation of one or more genes selected from, for example, AK2, AK3L1, ALDOA, B3GNT4, C4orf2, FGFll, ISG15, KCTDll, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARBl, SH3GL3, STC2,or TRAPPCI to provide an indication selected from, for example, an indication of predicting a predisposition to cervical cancer in a subject, diagnosing a cervical cancer in a subject, predicting the likelihood of recurrence of cervical cancer in a subject, providing a prognosis for a subject with cervical cancer, or selecting a subject with cervical cancer for treatment with a particular therapy.

Still further embodiments provide a complex comprising one or more genes selected from the group consisting of AK2, AK3L1, ALDOA, B3GNT4, CLK3, C14orf2, C4orfi, C20orf20, DDIT3, FGFll, GAPDH, ISG15, KCTDll, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARBl, SH3GL3, STC2, TRAPPCI, or UPK1A , wherein each gene is complexed to a methylation status informative reagent.

Further embodiments provide a complex comprising one or more genes selected from the group consisting of AK2, AK3L1, ALDOA, B3GNT4, C4orf2, FGFll, ISG15, KCTDll, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARBl, SH3GL3, STC2,or TRAPPCI , wherein each gene is complexed to a methylation status informative reagent.

Additional embodiments will be apparent to persons skilled in the relevant art based on the teachings contained herein. DESCRIPTION OF THE DRAWINGS

Fig. 1 shows an overview of the 151 methylation probes which significantly

(adj.p<0.1) correlate with the expression of their annotated gene.

Fig. 2 shows Kaplan Meier curves for progression-free survival of patients with low (below median) and high (above median) levels of STC2 (A), PVR (B), and RPL36A (C) methylation, respectively.

Fig. 3 shows Kaplan Meier curves for progression-free survival of patients with low (below median) and high (above median) levels of the methylation score based on 22 probes in 158 cervical cancer patients. P-value from log-rank test is indicated.

Fig. 4 shows an overview of 84 methylation probes.

DEFINITIONS

To facilitate an understanding of the present invention, a number of terms and phrases are defined below:

As used herein, the term "sensitivity" is defined as a statistical measure of

performance of an assay (e.g., method, test), calculated by dividing the number of true positives by the sum of the true positives and the false negatives.

As used herein, the term "specificity" is defined as a statistical measure of

performance of an assay (e.g., method, test), calculated by dividing the number of true negatives by the sum of true negatives and false positives.

As used herein, the term "informative" or "informativeness" refers to a quality of a marker or panel of markers, and specifically to the likelihood of finding a marker (or panel of markers) in a positive sample.

The term "neoplasm" as used herein refers to any new and abnormal growth of tissue. Thus, a neoplasm can be a premalignant neoplasm or a malignant neoplasm. The term "neoplasm-specific marker" refers to any biological material that can be used to indicate the presence of a neoplasm. Examples of biological materials include, without limitation, nucleic acids, polypeptides, carbohydrates, fatty acids, cellular components (e.g., cell membranes and mitochondria), and whole cells.

The term "cervical neoplasm-specific marker" refers to any biological material that can be used to indicate the presence of a cervical neoplasm (e.g., a premalignant cervical neoplasm; a malignant cervical neoplasm). Examples of cervical neoplasm-specific markers include, but are not limited to, AK2, AK3L1, ALDOA, B3GNT4, CLK3, C14orf2, C4orfi, C20orf20, DDIT3, FGF11, GAPDH, ISG15, KCTD11, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARB1, SH3GL3, STC2, TRAPPC1, and UPK1A.

As used herein, the term "methylation status informative reagent" or "methylation specific detection reagent" refers to a reagent or reagents that are informative for

identification of methylation status of a gene. In some embodiments, reagents are primers, probes or antibodies for detection of gene expression products (e.g., RNA transcripts or proteins) of the following genes: AK2, AK3L1, ALDOA, B3GNT4, C14orf2, C4orfl, CLK3, C20orf20, DDIT3, FGF11, GAPDH, ISG15, KCTD11, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARB1, SH3GL3, STC2, TRAPPC1, and UPK1A.

As used herein, the term "amplicon" refers to a nucleic acid generated using primer pairs. The amplicon may be, for example, double or single-stranded DNA (e.g., the result of asymmetric amplification), and either RNA or dsDNA.

The term "amplifying" or "amplification" in the context of nucleic acids refers to the production of multiple copies of a polynucleotide, or a portion of the polynucleotide, typically starting from a small amount of the polynucleotide {e.g. , a single polynucleotide molecule), where the amplification products or amplicons are generally detectable.

Amplification of polynucleotides encompasses a variety of chemical and enzymatic processes. The generation of multiple DNA copies from one or a few copies of a target or template DNA molecule during a polymerase chain reaction (PCR) or a ligase chain reaction (LCR; see, e.g., U.S. Patent No. 5,494,810; herein incorporated by reference in its entirety) are forms of amplification. Additional types of amplification include, but are not limited to, allele-specific PCR (see, e.g., U.S. Patent No. 5,639,611; herein incorporated by reference in its entirety), assembly PCR (see, e.g., U.S. Patent No. 5,965,408; herein incorporated by reference in its entirety), helicase-dependent amplification (see, e.g., U.S. Patent No.

7,662,594; herein incorporated by reference in its entirety), hot-start PCR (see, e.g., U.S. Patent Nos. 5,773,258 and 5,338,671; each herein incorporated by reference in their entireties), intersequence-specific PCR, inverse PCR (see, e.g., Triglia, et al. (1988) Nucleic Acids Res., 16:8186; herein incorporated by reference in its entirety), ligation-mediated PCR (see, e.g., Guilfoyle, R. et al, Nucleic Acids Research, 25: 1854-1858 (1997); U.S. Patent No. 5,508,169; each of which are herein incorporated by reference in their entireties),

methylation-specific PCR (see, e.g., Herman, et al, (1996) PNAS 93(13) 9821-9826; herein incorporated by reference in its entirety), miniprimer PCR, multiplex ligation-dependent probe amplification (see, e.g., Schouten, et al, (2002) Nucleic Acids Research 30(12): e57; herein incorporated by reference in its entirety), multiplex PCR (see, e.g., Chamberlain, et al., (1988) Nucleic Acids Research 16(23) 11141-11156; Ballabio, et al, (1990) Human Genetics 84(6) 571-573; Hayden, et al, (2008) BMC Genetics 9:80; each of which are herein incorporated by reference in their entireties), nested PCR, overlap-extension PCR (see, e.g., Higuchi, et al, (1988) Nucleic Acids Research 16(15) 7351-7367; herein incorporated by reference in its entirety), real time PCR (see, e.g., Higuchi, etl al, (1992) Biotechnology 10:413-417; Higuchi, et al, (1993) Biotechnology 11 : 1026-1030; each of which are herein incorporated by reference in their entireties), reverse transcription PCR (see, e.g., Bustin, S.A. (2000) J. Molecular Endocrinology 25: 169-193; herein incorporated by reference in its entirety), solid phase PCR, thermal asymmetric interlaced PCR, and Touchdown PCR (see, e.g., Don, et al, Nucleic Acids Research (1991) 19(14) 4008; Roux, K. (1994) Biotechniques 16(5) 812-814; Hecker, et al, (1996) Biotechniques 20(3) 478-485; each of which are herein incorporated by reference in their entireties). Polynucleotide amplification also can be accomplished using digital PCR (see, e.g., Kalinina, et al, Nucleic Acids Research. 25; 1999- 2004, (1997); Vogelstein and Kinzler, Proc Natl Acad Sci USA. 96; 9236-41, (1999);

International Patent Publication No. WO05023091 A2; US Patent Application Publication No. 20070202525; each of which are incorporated herein by reference in their entireties).

As used herein, the terms "complementary" or "complementarity" are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence "5'-A-G-T-3',^M is complementary to the sequence "3'-T-C-A-5\" Complementarity may be "partial," in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon binding between nucleic acids.

As used herein, the term "primer" refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, that is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product that is complementary to a nucleic acid strand is induced (e.g., in the presence of nucleotides and an inducing agent such as a biocatalyst (e.g. , a DNA polymerase or the like) and at a suitable temperature and pH). The primer is typically single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is generally first treated to separate its strands before being used to prepare extension products. In some embodiments, the primer is an

oligodeoxyribonucleotide. The primer is sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method. In certain embodiments, the primer is a capture primer.

As used herein, the term "nucleic acid molecule" refers to any nucleic acid containing molecule, including but not limited to, DNA or RNA. The term encompasses sequences that include any of the known base analogs of DNA and RNA including, but not limited to, 4 acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5- (carboxyhydroxyl-methyl) uracil, 5-fluorouracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil,

dihydrouracil, inosine, N6-isopentenyladenine, 1 -methyladenine, 1-methylpseudo-uracil, 1- methylguanine, 1 -methylinosine, 2,2-dimethyl-guanine, 2-methyladenine, 2-methylguanine, 3-methyl-cytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5- methylaminomethyluracil, 5 -methoxy-amino-methyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N- isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N- uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2- thiocytosine, and 2,6-diaminopurine.

As used herein, the term "nucleobase" is synonymous with other terms in use in the art including "nucleotide," "deoxynucleotide," "nucleotide residue," "deoxynucleotide residue," "nucleotide triphosphate (NTP)," or deoxynucleotide triphosphate (dNTP).

An "oligonucleotide" refers to a nucleic acid that includes at least two nucleic acid monomer units (e.g., nucleotides), typically more than three monomer units, and more typically greater than ten monomer units. The exact size of an oligonucleotide generally depends on various factors, including the ultimate function or use of the oligonucleotide. To further illustrate, oligonucleotides are typically less than 200 residues long (e.g., between 15 and 100), however, as used herein, the term is also intended to encompass longer

polynucleotide chains. Oligonucleotides are often referred to by their length. For example a 24 residue oligonucleotide is referred to as a "24-mer". Typically, the nucleoside monomers are linked by phosphodiester bonds or analogs thereof, including phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like, including associated counterions, e.g., H⁺, NH₄ ⁺, Na⁺, and the like, if such counterions are present. Further, oligonucleotides are typically single-stranded. Oligonucleotides are optionally prepared by any suitable method, including, but not limited to, isolation of an existing or natural sequence, DNA replication or amplification, reverse transcription, cloning and restriction digestion of appropriate sequences, or direct chemical synthesis by a method such as the phosphotriester method of Narang et al. (1979) Meth Enzymol. 68: 90-99; the phosphodiester method of Brown et al. (1979) Meth Enzymol. 68: 109-151 ; the diethylphosphoramidite method of Beaucage et al. (1981) Tetrahedron Lett. 22: 1859-1862; the triester method of Matteucci et al. (1981) J Am Chem Soc. 103 :3185-3191 ; automated synthesis methods; or the solid support method of U.S. Pat. No. 4,458,066, entitled "PROCESS FOR PREPARING POLYNUCLEOTIDES," issued Jul. 3, 1984 to Caruthers et al., or other methods known to those skilled in the art. All of these references are incorporated by reference.

A "sequence" of a biopolymer refers to the order and identity of monomer units (e.g., nucleotides, etc.) in the biopolymer. The sequence (e.g., base sequence) of a nucleic acid is typically read in the 5' to 3' direction.

As used herein, the term "subject" refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

As used herein, the term "non-human animals" refers to all non-human animals including, but are not limited to, vertebrates such as rodents, non-human primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, aves, etc.

The term "gene" refers to a nucleic acid (e.g., DNA) sequence that comprises coding sequences necessary for the production of a polypeptide, RNA (e.g., including but not limited to, mRNA, tRNA and rRNA) or precursor. The polypeptide, RNA, or precursor can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties (e.g. , enzymatic activity, ligand binding, signal transduction, etc.) of the full-length or fragment are retained. The term also encompasses the coding region of a structural gene and the including sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the full-length mRNA. The sequences that are located 5' of the coding region and which are present on the mRNA are referred to as 5' untranslated sequences. The sequences that are located 3' or downstream of the coding region and that are present on the mRNA are referred to as 3' untranslated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns" or "intervening regions" or "intervening sequences". Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or "spliced out" from the nuclear or primary transcript;

introns therefore are absent in the messenger RNA (mRNA) processed transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

The term "locus" as used herein refers to a nucleic acid sequence on a chromosome or on a linkage map and includes the coding sequence as well as 5 ' and 3 ' sequences involved in regulation of the gene.

DETAILED DESCRIPTION OF THE INVENTION

There is an increasing interest in using DNA methylation status of genes as biomarkers in cancer. Methylation of DNA is a stable and heritable covalent modification which mostly occurs in cytosines in CpG dinucleo tides. One of the advantages of using DNA methylation as a biomarker is that it is more specific and sensitive than commonly used protein biomarkers (How et al, 2012). Furthermore, DNA methylation is a stable biomarker which may be analyzed on patient samples without stringent handling requirements, since it is far more stable than mRNA which is the most common material analyzed in gene expression biomarker tests.

A gene signature predictive of poor survival in cervical cancer patients is described in (Halle et al, 2012; WO 2013/124738 A2). This gene signature was developed on the basis of mRNA expression profiles of patients. In light of the advantages of DNA methylation, it was investigated whether any of these 31 genes are regulated by methylation, and the prognostic value for cervical cancer patients.

Until recently, much of the work on DNA methylation has been concerning the methylation level of the promoter region of genes. However, recent studies using whole genome methylation assays have highlighted that methylation throughout the whole gene may have an impact on the level of gene expression. Furthermore, while promoter methylation has been shown to have negative correlation with gene expression, methylation in the body of genes has been shown to be positively associated with the level of expression. The role of gene body methylation remains largely unresolved. However, evidence has been presented indicating that it might be linked with the regulation of processes such as differential splicing, and suppressing the expression of transposable elements (De Angelis et al, 1999).

In this study, the methylation profile of 31 genes of interest was evaluated using the Illumina Infinium 45 OK assay, with a view to identifying which methylation patterns, if any, are associated with disease aggressiveness and survival outcome, and thus may be useful as prognostic markers. Out of the 31 hypoxia-associated genes investigated, 22 were found to have potential as components of a prognostic methylated gene panel.

Accordingly, in some embodiments, the present invention provides methods for predicting a predisposition to cervical cancer in a subject, diagnosing a cervical cancer in a subject, predicting the likelihood of recurrence of cervical cancer in a subject, providing a prognosis for a subject with cervical cancer, or selecting a subject with cervical cancer for treatment with a particular therapy. In some embodiments, the methods comprise

determining the methylation status of one or more (e.g., 2 or more, three or more, four or more, 5 or more, 10 or more, or all of) the genes described herein. In some preferred embodiments, altered methylation status relative to the methylation status in a reference sample (e.g., patients not diagnosed with cervical cancer or patients diagnosed with non- aggressive cervical cancer) is indicative of a predisposition of the subject to cervical cancer, an indication that the subject has cervical cancer, an indication of the likelihood of recurrence of the cervical cancer in the subject, an indication of survival of the subject, and indication of the aggressiveness of the cervical cancer, an indication of the likely outcome of treatment of the cervical cancer or an indication that the subject is a candidate for treatment with a particular therapy.

In some embodiments, detection utilizes methylation specific detection reagents specific for the detection of one or more gene products (e.g., RNA or proteins) or the methylation status of the gene products resulting from the expression of one or more of the following genes: AK2, AK3L1, ALDOA, B3GNT4, CLK3, C14orf2, C4orfl, C20orf20, DDIT3, FGFll, GAPDH, ISG15, KCTDll, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARB1, SH3GL3, STC2, TRAPPC1, or UPK1A. In some embodiments, the genes are selected from AK2, AK3L1, ALDOA, B3GNT4, C4orf2, FGFll, ISG15, KCTDll, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARB1, SH3GL3, STC2,or TRAPPC1.

In some embodiments, compositions, systems, and methods utilize reagents for detection of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or all of the described genes).

Exemplary reagents are described herein and include, but are not limited to, nucleic acid primers, probes, primer pairs, methylation specific restriction enzymes, bisulfite, etc. In some embodiments, the reagent (e.g., probe) is one or more (e.g., two) of SEQ ID NOs: 1- 106. In some embodiments, the reagent is one or more of at least 8 (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or all nucleotides) of SEQ ID NOs: 1-106. In some embodiments, the reagent is one or more of at least 8 (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or all nucleotides of the complement of SEQ ID NOs: l- 106. In some embodiments, detection methods comprise a single probe as described above (e.g., probes indicated as Type II in Table 7). In some embodiments, detection methods comprise a pair of probes (e.g., as indicated as Type I in Table 7; e.g., one or more of SEQ ID NOs: 4:84; 8:85; 13:86; 14:87; 25:88; 26:89; 27:90; 46:91; 37:92; 38:93; 39:94; 44:95; 45:96; 48:97; 49:98; 60:99; 69: 100; 70: 101; 71 : 102; 72:103; 73: 104; 75: 105; or 79: 106).

While the present invention exemplifies several markers specific for detecting cervical cancer, any marker that is correlated with the presence, prognosis, or absence of cervical cancer may be used in conjunction with the identified markers. A marker, as used herein, includes, for example, nucleic acid(s) whose methylation status is characteristic of a cervical neoplasm or the aggressiveness or prognosis of a cervical neoplasm. Depending on the particular set of markers employed in a given analysis, the statistical analysis will vary. For example, where a particular combination of markers is highly specific for cervical cancer, the statistical significance of a positive result will be high. It may be, however, that such specificity is achieved at the cost of sensitivity (e.g., a negative result may occur even in the presence of cervical cancer). By the same token, a different combination may be very sensitive (e.g., few false negatives, but has a lower specificity).

Particular combinations of markers may be used that show optimal function with different ethnic groups or sex, different geographic distributions, different stages of disease, different degrees of specificity or different degrees of sensitivity. Particular combinations may also be developed which are particularly sensitive to the effect of therapeutic regimens on disease progression. In some embodiments, the compositions and methods of the present disclosure find use in determining a treatment course of action of administering, for example, hypoxia-targeting and methylation modulating drugs. Subjects may be monitored after a therapy and/or course of action to determine the effectiveness of that specific therapy and/or course of action.

The methods of the present invention are not limited to particular indicators of cervical neoplasm.

As described above, embodiments of the present invention provide diagnostic and screening methods that utilize the detection of methylation status of gene products resulting from the expression of one or more of the following genes: AK2, AK3L1, ALDOA, B3GNT4, CLK3, C14orf2, C4orfl, C20orf20, DDIT3, FGF11, GAPDH, ISG15, KCTD11, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARBI, SH3GL3, STC2, TRAPPCl, and UPK1A. Exemplary, non- limiting methods are described below.

Any patient sample may be tested according to methods of embodiments of the present invention. By way of non-limiting examples, the sample may be a tissue sample (e.g., a cervical tumor biopsy sample or pelvic lymph node biopsy).

In some embodiments, the patient sample is subjected to preliminary processing designed to isolate or enrich the sample for the gene products or cells that contain the gene products. A variety of techniques known to those of ordinary skill in the art may be used for this purpose, including but not limited to: centrifugation; immunocapture; cell lysis; and, nucleic acid target capture (See, e.g., EP Pat. No. 1 409 727, herein incorporated by reference in its entirety).

In certain embodiments, methods, kits, and systems of the present invention involve determination of methylation state of a locus of interest (e.g., in human DNA) (e.g., in human DNA extracted from a blood sample, from a serum sample, from a plasma sample, from a cell sample, etc). Any appropriate method can be used to determine whether a particular DNA is hypermethylated or hypomethylated. Standard PCR techniques, for example, can be used to determine which residues are methylated, since unmethylated cytosines converted to uracil are replaced by thymidine residues during PCR. PCR reactions can contain, for example, 10 μΐ_^ οΐ captured DNA that either has or has not been treated with sodium bisulfite, IX PCR buffer, 0.2 mM dNTPs, 0.5 μΜ sequence specific primers (e.g., primers flanking a CpG island or CpG shore within the captured DNA), and 5 units DNA polymerase (e.g., Amplitaq DNA polymerase from PE Applied Biosystems, Norwalk, CT) in a total volume of 50 μΐ. A typical PCR protocol can include, for example, an initial denaturation step at 94°C for 5 min, 40 amplification cycles consisting of 1 minute at 94°C, 1 minute at 60°C, and 1 minute at 72°C, and a final extension step at 72°C for 5 minutes.

To analyze which residues within a captured DNA are methylated, the sequences of PCR products corresponding to samples treated with and without sodium bisulfite can be compared. The sequence from the untreated DNA will reveal the positions of all cytosine residues within the PCR product. Cytosines that were unmethylated will be converted to thymidine residues in the sequence of the bisulfite-treated DNA, while residues that were methylated will be unaffected by bisulfite treatment.

In some embodiments, nucleic acid sequencing methods are utilized for detection. In some embodiments, the sequencing is Second Generation (a.k.a. Next Generation or Next- Gen), Third Generation (a.k.a. Next-Next-Gen), or Fourth Generation (a.k.a. N3-Gen) sequencing technology including, but not limited to, pyrosequencing, sequencing-by-ligation, single molecule sequencing, sequence-by-synthesis (SBS), semiconductor sequencing, massive parallel clonal, massive parallel single molecule SBS, massive parallel single molecule real-time, massive parallel single molecule real-time nanopore technology, etc. Morozova and Marra provide a review of some such technologies in Genomics, 92: 255 (2008), herein incorporated by reference in its entirety. Those of ordinary skill in the art will recognize that because RNA is less stable in the cell and more prone to nuclease attack experimentally RNA is usually reverse transcribed to DNA before sequencing.

DNA sequencing techniques include fluorescence-based sequencing methodologies (See, e.g., Birren et al, Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor, N.Y.; herein incorporated by reference in its entirety). In some embodiments, the sequencing is automated sequencing. In some embodiments, the sequenceing is parallel sequencing of partitioned amplicons (PCT Publication No: WO2006084132 to Kevin McKernan et al., herein incorporated by reference in its entirety). In some embodiments, the sequencing is DNA sequencing by parallel oligonucleotide extension (See, e.g., U.S. Pat. No. 5,750,341 to Macevicz et al., and U.S. Pat. No. 6,306,597 to Macevicz et al., both of which are herein incorporated by reference in their entireties). Additional examples of sequencing techniques include the Church polony technology (Mitra et al., 2003, Analytical Biochemistry 320, 55- 65; Shendure et al, 2005 Science 309, 1728-1732; U.S. Pat. No. 6,432,360, U.S. Pat. No. 6,485,944, U.S. Pat. No. 6,511 ,803; herein incorporated by reference in their entireties), the 454 picotiter pyrosequencing technology (Margulies et al, 2005 Nature 437, 376-380; US 20050130173; herein incorporated by reference in their entireties), the Solexa single base addition technology (Bennett et al, 2005, Pharmacogenomics, 6, 373-382; U.S. Pat. No. 6,787,308; U.S. Pat. No. 6,833,246; herein incorporated by reference in their entireties), the Lynx massively parallel signature sequencing technology (Brenner et al. (2000). Nat.

Biotechnol. 18:630-634; U.S. Pat. No. 5,695,934; U.S. Pat. No. 5,714,330; herein

incorporated by reference in their entireties), and the Adessi PCR colony technology (Adessi et al. (2000). Nucleic Acid Res. 28, E87; WO 00018957; herein incorporated by reference in its entirety).

Next-generation sequencing (NGS) methods share the common feature of massively parallel, high-throughput strategies, with the goal of lower costs in comparison to older sequencing methods (see, e.g., Voelkerding et al, Clinical Chem., 55: 641-658, 2009;

MacLean et al., Nature Rev. Microbiol., 7: 287-296; each herein incorporated by reference in their entirety). NGS methods can be broadly divided into those that typically use template amplification and those that do not. Amplification-requiring methods include pyrosequencing commercialized by Roche as the 454 technology platforms (e.g., GS 20 and GS FLX), Life Technologies/Ion Torrent, the Solexa platform commercialized by Illumina, GnuBio, and the Supported Oligonucleotide Ligation and Detection (SOLiD) platform commercialized by Applied Biosystems. Non-amplification approaches, also known as single-molecule sequencing, are exemplified by the HeliScope platform commercialized by Helicos

Biosciences, and emerging platforms commercialized by VisiGen, Oxford Nanopore

Technologies Ltd., and Pacific Biosciences, respectively.

In pyrosequencing (Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLean et al, Nature Rev. Microbiol, 7: 287-296; U.S. Pat. No. 6,210,891; U.S. Pat. No. 6,258,568; each herein incorporated by reference in its entirety), template DNA is fragmented, end- repaired, ligated to adaptors, and clonally amplified in-situ by capturing single template molecules with beads bearing oligonucleotides complementary to the adaptors. Each bead bearing a single template type is compartmentalized into a water-in-oil microvesicle, and the template is clonally amplified using a technique referred to as emulsion PCR. The emulsion is disrupted after amplification and beads are deposited into individual wells of a picotitre plate functioning as a flow cell during the sequencing reactions. Ordered, iterative

introduction of each of the four dNTP reagents occurs in the flow cell in the presence of sequencing enzymes and luminescent reporter such as luciferase. In the event that an appropriate dNTP is added to the 3' end of the sequencing primer, the resulting production of ATP causes a burst of luminescence within the well, which is recorded using a CCD camera. It is possible to achieve read lengths greater than or equal to 400 bases, and 10⁶ sequence reads can be achieved, resulting in up to 500 million base pairs (Mb) of sequence.

In the Solexa/Illumina platform (Voelkerding et al, Clinical Chem., 55: 641-658, 2009; MacLean et al, Nature Rev. Microbiol, 7: 287-296; U.S. Pat. No. 6,833,246; U.S. Pat. No. 7,115,400; U.S. Pat. No. 6,969,488; each herein incorporated by reference in its entirety), sequencing data are produced in the form of shorter-length reads. In this method, single- stranded fragmented DNA is end-repaired to generate 5'-phosphorylated blunt ends, followed by Klenow-mediated addition of a single A base to the 3' end of the fragments. A-addition facilitates addition of T-overhang adaptor oligonucleotides, which are subsequently used to capture the template-adaptor molecules on the surface of a flow cell that is studded with oligonucleotide anchors. The anchor is used as a PCR primer, but because of the length of the template and its proximity to other nearby anchor oligonucleotides, extension by PCR results in the "arching over" of the molecule to hybridize with an adjacent anchor oligonucleotide to form a bridge structure on the surface of the flow cell. These loops of DNA are denatured and cleaved. Forward strands are then sequenced with reversible dye terminators. The sequence of incorporated nucleotides is determined by detection of post-incorporation fluorescence, with each fluor and block removed prior to the next cycle of dNTP addition. Sequence read length ranges from 36 nucleotides to over 250 nucleotides, with overall output exceeding 1 billion nucleotide pairs per analytical run.

Sequencing nucleic acid molecules using SOLiD technology (Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; U.S. Pat. No. 5,912,148; U.S. Pat. No. 6,130,073; each herein incorporated by reference in their entirety) also involves fragmentation of the template, ligation to oligonucleotide adaptors, attachment to beads, and clonal amplification by emulsion PCR. Following this, beads bearing template are immobilized on a derivatized surface of a glass flow-cell, and a primer complementary to the adaptor oligonucleotide is annealed. However, rather than utilizing this primer for 3' extension, it is instead used to provide a 5' phosphate group for ligation to interrogation probes containing two probe-specific bases followed by 6 degenerate bases and one of four fluorescent labels. In the SOLiD system, interrogation probes have 16 possible combinations of the two bases at the 3' end of each probe, and one of four fluors at the 5' end. Fluor color, and thus identity of each probe, corresponds to specified color-space coding schemes. Multiple rounds (usually 7) of probe annealing, ligation, and fluor detection are followed by denaturation, and then a second round of sequencing using a primer that is offset by one base relative to the initial primer. In this manner, the template sequence can be computationally re-constructed, and template bases are interrogated twice, resulting in increased accuracy. Sequence read length averages 35 nucleotides, and overall output exceeds 4 billion bases per sequencing run.

In certain embodiments, sequencing is nanopore sequencing (see, e.g., Astier et al, J. Am. Chem. Soc. 2006 Feb 8; 128(5): 1705-10, herein incorporated by reference). The theory behind nanopore sequencing has to do with what occurs when a nanopore is immersed in a conducting fluid and a potential (voltage) is applied across it. Under these conditions a slight electric current due to conduction of ions through the nanopore can be observed, and the amount of current is exceedingly sensitive to the size of the nanopore. As each base of a nucleic acid passes through the nanopore, this causes a change in the magnitude of the current through the nanopore that is distinct for each of the four bases, thereby allowing the sequence of the DNA molecule to be determined.

In certain embodiments, sequencing is HeliScope by Helicos Biosciences

(Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev.

Microbiol., 7: 287-296; U.S. Pat. No. 7,169,560; U.S. Pat. No. 7,282,337; U.S. Pat. No. 7,482,120; U.S. Pat. No. 7,501,245; U.S. Pat. No. 6,818,395; U.S. Pat. No. 6,911,345; U.S. Pat. No. 7,501,245; each herein incorporated by reference in their entirety). Template DNA is fragmented and polyadenylated at the 3' end, with the final adenosine bearing a fluorescent label. Denatured polyadenylated template fragments are ligated to poly(dT) oligonucleotides on the surface of a flow cell. Initial physical locations of captured template molecules are recorded by a CCD camera, and then label is cleaved and washed away. Sequencing is achieved by addition of polymerase and serial addition of fluorescently-labeled dNTP reagents. Incorporation events result in fluor signal corresponding to the dNTP, and signal is captured by a CCD camera before each round of dNTP addition. Sequence read length ranges from 25-50 nucleotides, with overall output exceeding 1 billion nucleotide pairs per analytical run.

The Ion Torrent technology is a method of DNA sequencing based on the detection of hydrogen ions that are released during the polymerization of DNA (see, e.g., Science

327(5970): 1190 (2010); U.S. Pat. Appl. Pub. Nos. 20090026082, 20090127589,

20100301398, 20100197507, 20100188073, and 20100137143, incorporated by reference in their entireties for all purposes). A microwell contains a template DNA strand to be sequenced. Beneath the layer of microwells is a hypersensitive ISFET ion sensor. All layers are contained within a CMOS semiconductor chip, similar to that used in the electronics industry. When a dNTP is incorporated into the growing complementary strand a hydrogen ion is released, which triggers a hypersensitive ion sensor. If homopolymer repeats are present in the template sequence, multiple dNTP molecules will be incorporated in a single cycle. This leads to a corresponding number of released hydrogens and a proportionally higher electronic signal. This technology differs from other sequencing technologies in that no modified nucleotides or optics are used. The per-base accuracy of the Ion Torrent sequencer is -99.6% for 50 base reads, with -100 Mb to 100Gb generated per run. The read- length is 100-300 base pairs. The accuracy for homopolymer repeats of 5 repeats in length is -98%. The benefits of ion semiconductor sequencing are rapid sequencing speed and low upfront and operating costs.

In some embodiments, sequencing is the technique developed by Stratos Genomics, Inc. and involves the use of Xpandomers. This sequencing process typically includes providing a daughter strand produced by a template-directed synthesis. The daughter strand generally includes a plurality of subunits coupled in a sequence corresponding to a contiguous nucleotide sequence of all or a portion of a target nucleic acid in which the individual subunits comprise a tether, at least one probe or nucleobase residue, and at least one selectively cleavable bond. The selectively cleavable bond(s) is/are cleaved to yield an Xpandomer of a length longer than the plurality of the subunits of the daughter strand. The Xpandomer typically includes the tethers and reporter elements for parsing genetic information in a sequence corresponding to the contiguous nucleotide sequence of all or a portion of the target nucleic acid. Reporter elements of the Xpandomer are then detected. Additional details relating to Xpandomer-based approaches are described in, for example, U.S. Pat. Pub No. 20090035777, entitled "High Throughput Nucleic Acid Sequencing by Expansion," filed June 19, 2008, which is incorporated herein in its entirety.

Other emerging single molecule sequencing methods include real-time sequencing by synthesis using a VisiGen platform (Voelkerding et al., Clinical Chem., 55: 641-58, 2009; U.S. Pat. No. 7,329,492; U.S. Pat. App. Ser. No. 11/671956; U.S. Pat. App. Ser. No.

11/781166; each herein incorporated by reference in their entirety) in which immobilized, primed DNA template is subjected to strand extension using a fluorescently-modified polymerase and florescent acceptor molecules, resulting in detectible fluorescence resonance energy transfer (FRET) upon nucleotide addition.

Similarly, in some embodiments, methods of the present invention involve the determination (e.g., assessment, ascertaining, quantitation) of methylation level of an indicator of cervical neoplasm (e.g., the methylation level of a CpG island or CpG shore in the coding or regulatory region of a gene locus) in a sample (e.g., a DNA sample extracted from stool, bile or blood). A skilled artisan understands that an increased, decreased, informative, or otherwise distinguishably different methylation level is articulated with respect to a reference (e.g., a reference level, a control level, a threshold level, or the like). For example, the term "elevated methylation" as used herein with respect to the methylation status (e.g., CpG DNA methylation) of a gene locus (e.g.,) is any methylation level that is above a median methylation level in a sample from a random population of mammals (e.g., a random population of 10, 20, 30, 40, 50, 100, or 500 mammals) that do not have a cervical neoplasm (e.g., cervical cancer). Elevated levels of methylation can be any level provided that the level is greater than a corresponding reference level. For example, an elevated methylation level of a locus of interest (e.g.,) methylation can be 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fold greater than the reference level methylation observed in a normal sample. It is noted that a reference level can be any amount. The term "elevated methylation score" as used herein with respect to detected methylation events in a matrix panel of particular nucleic acid markers is any methylation score that is above a median methylation score in a sample from a random population of mammals (e.g., a random population of 10, 20, 30, 40, 50, 100, or 500 mammals) that do not have a cervical neoplasm. An elevated methylation score in a matrix panel of particular nucleic acid markers can be any score provided that the score is greater than a corresponding reference score. For example, an elevated score of methylation in a locus of interest (e.g.,) can be 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fold greater than the reference methylation score observed in a normal sample. It is noted that a reference score can be any amount.

The methods are not limited to a particular type of mammal. In some embodiments, the mammal is a human. In some embodiments, the cancer is premalignant. In some embodiments, the cancer is malignant. In some embodiments, the cancer is an aggressive cervical cancer. In some embodiments, the compositions and methods described herein differentiate between aggressive and non-aggressive cervical cancers.

The present invention also provides methods and materials to assist medical or research professionals in determining whether or not a mammal has a cervical cancer or to determine the aggressiveness of the cervical cancer. Medical professionals can be, for example, doctors, nurses, medical laboratory technologists, and pharmacists. Research professionals can be, for example, principle investigators, research technicians, postdoctoral trainees, and graduate students. A professional can be assisted by (1) determining the ratio of particular markers in a sample, and (2) communicating information about the ratio to that professional, for example.

After the level (score, frequency) of particular markers in a blood, serum, or plasma sample is reported, a medical professional can take one or more actions that can affect patient care. For example, a medical professional can record the results in a patient's medical record. In some cases, a medical professional can record a diagnosis of a cervical cancer, or otherwise transform the patient's medical record, to reflect the patient's medical condition. In some cases, a medical professional can review and evaluate a patient's entire medical record, and assess multiple treatment strategies, for clinical intervention of a patient's condition. In some cases, a medical professional can record a prediction of tumor occurrence with the reported indicators. In some cases, a medical professional can review and evaluate a patient's entire medical record and assess multiple treatment strategies, for clinical intervention of a patient's condition.

A medical professional can initiate or modify treatment of a cervical cancer after receiving information regarding the level (score, frequency) associated with markers in a patient's stool, blood, serum, bile or plasma sample. In some cases, a medical professional can compare previous reports and the recently communicated level (score, frequency) of markers, and recommend a change in therapy. In some cases, a medical professional can enroll a patient in a clinical trial for novel therapeutic intervention of cervical cancer neoplasm. In some cases, a medical professional can elect waiting to begin therapy until the patient's symptoms require clinical intervention.

For example, in some embodiments, patients identified as having aggressive cervical cancer are given adjuvant radiation or chemotherapy following surgical treatment while patient identified as having non-aggressive cervical cancer are not given adjuvant therapy. In some embodiments, the assay is repeated after adjuvant treatment. In some embodiments, the assay to determine methylation status of the described genes is performed one or more times before, during, or after, primary or adjuvant therapy.

A medical professional can communicate the assay results to a patient or a patient's family. In some cases, a medical professional can provide a patient and/or a patient's family with information regarding cervical neoplasia, including treatment options, prognosis, and referrals to specialists, e.g., oncologists and/or radiologists. In some cases, a medical professional can provide a copy of a patient's medical records to communicate assay results to a specialist. A research professional can apply information regarding a subject's assay results to advance cervical neoplasm research. For example, a researcher can compile data on the assay results, with information regarding the efficacy of a drug for treatment of a cervical cancer to identify an effective treatment. In some cases, a research professional can obtain assay results to evaluate a subject's enrollment, or continued participation in a research study or clinical trial. In some cases, a research professional can classify the severity of a subject's condition, based on assay results. In some cases, a research professional can communicate a subject's assay results to a medical professional. In some cases, a research professional can refer a subject to a medical professional for clinical assessment of cervical neoplasia, and treatment thereof. Any appropriate method can be used to communicate information to another person (e.g., a professional). For example, information can be given directly or indirectly to a professional. For example, a laboratory technician can input the assay results into a computer-based record. In some cases, information is communicated by making a physical alteration to medical or research records. For example, a medical professional can make a permanent notation or flag a medical record for communicating a diagnosis to other medical professionals reviewing the record. In addition, any type of communication can be used to communicate the information. For example, mail, e-mail, telephone, and face-to-face interactions can be used. The information also can be communicated to a professional by making that information electronically available to the professional. For example, the information can be communicated to a professional by placing the information on a computer database such that the professional can access the information. In addition, the information can be communicated to a hospital, clinic, or research facility serving as an agent for the professional.

It is noted that a single sample can be analyzed for one cervical cancer-specific marker or for multiple cervical neoplasm-specific markers. In preferred embodiments, a single sample is analyzed for multiple cervical neoplasm-specific markers, for example, using multi-marker assays. In addition, multiple samples can be collected for a single mammal and analyzed as described herein. In some embodiments, a sample is split into first and second portions, where the first portion undergoes cytological analysis and the second portion undergoes further purification or processing (e.g., sequence-specific capture step(s) (e.g., for isolation of specific markers for analysis of methylation levels). In some embodiments, the sample undergoes one or more preprocessing steps before being split into portions. In some embodiments, the sample is treated, handled, or preserved in a manner that promotes DNA integrity and/or inhibits DNA degradation (e.g., through use of storage buffers with stabilizing agents (e.g., chelating agents, DNase inhibitors) or handling or processing techniques that promote DNA integrity (e.g., immediate processing or storage at low temperature (e.g., -80 degrees C)).

Some embodiments of the invention provides a diagnostic kit for the diagnosis or screening of cancer comprising one or reagents for detection of methylation status of the genes selected from, for example one or more those described herein. For example, in some embodiments, the reagents comprise nucleic acids (e.g., oligonucleotides, primers, probes, etc.). In some embodiments, kits provide reagents useful, necessary or sufficient for detecting methylation status and/or providing a diagnosis or prognosis. Compositions for use in the diagnostic methods described herein include, but are not limited to, kits comprising one or more methylation status informative reagents as described above. In some embodiments, the kits comprise one or more methylation status informative reagents for detecting altered gene expression in a sample from a subject having or suspected of having cervical cancer, wherein the reagents are specific detection of one or more gene products from the following genes: AK3L1, ALDOA, B3GNT4, CLK3, C14orfi, C4orfl, C20orf20, DDIT3, FGF11, GAPDH, ISG15, KCTD11, P4HA2, PFKFB4, PVR, PYGL, RHOC, RPL36A, S100A2, SCARB1, SH3GL3, STC2, TRAPPC1, and UPK1A.

In some embodiments, the present disclosure provides complexes of one or more of the above-described genes and a methylation status informative reagent.

The diagnostic kits may further comprise any reagent or media necessary, sufficient or useful to perform analyses, such as PCR analyses, such as methylation specific polymerase chain reaction (MSP) sequence analyses, bisulphite treatment, bisulphite sequencing, electrophoresis, pyrosequencing, mass spectrometry and sequence analyses by restriction digestion, next generation sequencing, quantitative and/or qualitative methylation, pyrosequencing, Southern blotting, restriction landmark genome scanning (RLGS), single nucleotide primer extension, CpG island microarray, SNUPE, COBRA, mass spectrometry, by use of methylation specific restriction enzymes or by measuring the expression level of said genes. In particular, the kit may further comprise one or more methylation specific detection components selected from, for example, deoxyribonucleoside triphosphates, buffers, stabilizers, thermostable DNA polymerases, restriction endonucleases (including methylation specific endonucleases), and labels (including fiuorescent, chemiluminescent and radioactive labels). The diagnostic assay according to the invention may further comprise one or more reagents required for isolation of DNA.

In some embodiments, the kits of the present invention include a means for containing the reagents in close confinement for commercial sale such as, e.g., injection or blow-molded plastic containers into which the desired reagent are retained. Other containers suitable for conducting certain steps of the disclosed methods also may be provided.

In some embodiments, the compositions and methods disclosed herein are useful in monitoring the treatment of cervical cancers. For example, in some embodiments, the methods may be performed immediately before, during and/or after a treatment to monitor treatment success or determine a treatment course of action. In some embodiments, the methods are performed at intervals on disease free patients to ensure treatment success. In some embodiments, the present disclosure provides compositions and method for determining a treatment course of action and administering the treatment. In some embodiments, the methods are repeated and the results are used to determine a treatment course of action (e.g., to start, stop, or modify a treatment).

The present invention also provides a variety of computer-related embodiments. Specifically, in some embodiments the invention provides computer programming for analyzing and comparing a pattern of cervical cancer-specific marker detection results in a sample obtained from a subject to, for example, a library of such marker patterns known to be indicative of the presence or absence of a cervical cancer, or a particular stage or prognosis of a cervical cancer.

In some embodiments, the present invention provides computer programming for analyzing and comparing a first and a second pattern of cervical cancer-specific marker detection results from a sample taken at least two different time points. In some

embodiments, the first pattern may be indicative of a pre-cancerous condition and/or low risk condition for a cervical cancer and/or progression from a pre-cancerous condition to a cancerous condition. In such embodiments, the comparing provides for monitoring of the progression of the condition from the first time point to the second time point.

In yet another embodiment, the invention provides computer programming for analyzing and comparing a pattern of cervical cancer-specific marker detection results from a sample to a library of cervical cancer-specific marker patterns known to be indicative of the presence or absence of a cervical cancer, wherein the comparing provides, for example, a differential diagnosis between an aggressively malignant cervical cancer and a less aggressive cervical cancer (e.g., the marker pattern provides for staging and/or grading of the cancerous condition).

The methods and systems described herein can be implemented in numerous ways. In one embodiment, the methods involve use of a communications infrastructure, for example the internet. Several embodiments of the invention are discussed below. It is also to be understood that the present invention may be implemented in various forms of hardware, software, firmware, processors, distributed servers (e.g., as used in cloud computing) or a combination thereof. The methods and systems described herein can be implemented as a combination of hardware and software. The software can be implemented as an application program tangibly embodied on a program storage device, or different portions of the software implemented in the user's computing environment (e.g., as an applet) and on the reviewer's computing environment, where the reviewer may be located at a remote site (e.g., at a service provider's facility).

For example, during or after data input by the user, portions of the data processing can be performed in the user-side computing environment. For example, the user-side computing environment can be programmed to provide for defined test codes to denote platform, carrier/diagnostic test, or both; processing of data using defined flags, and/or generation of flag configurations, where the responses are transmitted as processed or partially processed responses to the reviewer's computing environment in the form of test code and flag configurations for subsequent execution of one or more algorithms to provide a results and/or generate a report in the reviewer's computing environment.

The application program for executing the algorithms described herein may be uploaded to, and executed by, a machine comprising any suitable architecture. In general, the machine involves a computer platform having hardware such as one or more central processing units (CPU), a random access memory (RAM), and input/output (I/O) interface(s). The computer platform also includes an operating system and microinstruction code. The various processes and functions described herein may either be part of the microinstruction code or part of the application program (or a combination thereof) which is executed via the operating system. In addition, various other peripheral devices may be connected to the computer platform such as an additional data storage device and a printing device.

As a computer system, the system generally includes a processor unit. The processor unit operates to receive information, which generally includes test data (e.g., specific gene products assayed), and test result data (e.g., the pattern of cervical neoplasm-specific marker detection results from a sample). This information received can be stored at least temporarily in a database, and data analyzed in comparison to a library of marker patterns known to be indicative of the presence or absence of a pre-cancerous condition, or known to be indicative of a stage and/or grade of cervical cancer.

Part or all of the input and output data can also be sent electronically; certain output data (e.g., reports) can be sent electronically or telephonically (e.g., by facsimile, e.g., using devices such as fax back). Exemplary output receiving devices can include a display element, a printer, a facsimile device and the like. Electronic forms of transmission and/or display can include email, interactive television, and the like. In some embodiments, all or a portion of the input data and/or all or a portion of the output data (e.g., usually at least the library of the pattern of cervical neoplasm-specific marker detection results known to be indicative of the presence or absence of a pre-cancerous condition) are maintained on a server for access, e.g., confidential access. The results may be accessed or sent to professionals as desired.

A system for use in the methods described herein generally includes at least one computer processor (e.g., where the method is carried out in its entirety at a single site) or at least two networked computer processors (e.g., where detected marker data for a sample obtained from a subject is to be input by a user (e.g., a technician or someone performing the assays)) and transmitted to a remote site to a second computer processor for analysis (e.g., where the pattern of cervical cancer-specific marker) detection results is compared to a library of patterns known to be indicative of the presence or absence of a pre-cancerous condition), where the first and second computer processors are connected by a network, e.g., via an intranet or internet). The system can also include a user component(s) for input; and a reviewer component(s) for review of data, and generation of reports, including detection of a pre-cancerous condition, staging and/or grading of a cervical cancer, or monitoring the progression of a pre-cancerous condition or a cervical cancer. Additional components of the system can include a server component(s); and a database(s) for storing data (e.g., as in a database of report elements, e.g., a library of marker patterns known to be indicative of the presence or absence of a pre-cancerous condition and/or known to be indicative of a grade and/or a stage of a cervical cancer, or a relational database (RDB) which can include data input by the user and data output. The computer processors can be processors that are typically found in personal desktop computers (e.g., IBM, Dell, Macintosh), portable computers, mainframes, minicomputers, tablet computer, smart phone, or other computing devices.

The input components can be complete, stand-alone personal computers offering a full range of power and features to run applications. The user component usually operates under any desired operating system and includes a communication element (e.g., a modem or other hardware for connecting to a network using a cellular phone network, Wi-Fi, Bluetooth, Ethernet, etc.), one or more input devices (e.g., a keyboard, mouse, keypad, or other device used to transfer information or commands), a storage element (e.g., a hard drive or other computer-readable, computer-writable storage medium), and a display element (e.g., a monitor, television, LCD, LED, or other display device that conveys information to the user). The user enters input commands into the computer processor through an input device.

Generally, the user interface is a graphical user interface (GUI) written for web browser applications. The server component(s) can be a personal computer, a minicomputer, or a mainframe, or distributed across multiple servers (e.g., as in cloud computing applications) and offers data management, information sharing between clients, network administration and security. The application and any databases used can be on the same or different servers. Other computing arrangements for the user and server(s), including processing on a single machine such as a mainframe, a collection of machines, or other suitable configuration are contemplated. In general, the user and server machines work together to accomplish the processing of the present invention.

Where used, the database(s) is usually connected to the database server component and can be any device which will hold data. For example, the database can be any magnetic or optical storing device for a computer (e.g., CDROM, internal hard drive, tape drive). The database can be located remote to the server component (with access via a network, modem, etc.) or locally to the server component.

Where used in the system and methods, the database can be a relational database that is organized and accessed according to relationships between data items. The relational database is generally composed of a plurality of tables (entities). The rows of a table represent records (collections of information about separate items) and the columns represent fields (particular attributes of a record). In its simplest conception, the relational database is a collection of data entries that "relate" to each other through at least one common field.

Additional workstations equipped with computers and printers may be used at point of service to enter data and, in some embodiments, generate appropriate reports, if desired. The computer(s) can have a shortcut (e.g., on the desktop) to launch the application to facilitate initiation of data entry, transmission, analysis, report receipt, etc. as desired.

In certain embodiments, the present invention provides methods for obtaining a subject's risk profile for developing cervical cancer. In some embodiments, such methods involve obtaining a blood or blood product sample from a subject (e.g., a human at risk for developing cervical cancer; a human undergoing a routine physical examination), detecting the presence, absence, or level (e.g., methylation frequency or score) of one or more markers specific for a cervical cancer in or associated with the blood or blood product sample (e.g., specific for a cervical cancer) in the sample, and generating a risk profile for developing cervical cancer based upon the detected level (score, frequency) or presence or absence of the indicators of cervical cancer. For example, in some embodiments, a generated risk profile will change depending upon specific markers and detected as present or absent or at defined threshold levels. The present invention is not limited to a particular manner of generating the risk profile. In some embodiments, a processor (e.g., computer) is used to generate such a risk profile. In some embodiments, the processor uses an algorithm (e.g., software) specific for interpreting the presence and absence of specific markers as determined with the methods of the present invention. In some embodiments, the presence and absence of specific markers as determined with the methods of the present invention are imputed into such an algorithm, and the risk profile is reported based upon a comparison of such input with established norms (e.g., established norm for pre-cancerous condition, established norm for various risk levels for developing cervical cancer, established norm for subjects diagnosed with various stages of cervical cancer). In some embodiments, the risk profile indicates a subject's risk for developing cervical cancer or a subject's risk for re-developing cervical cancer. In some embodiments, the risk profile indicates a subject to be, for example, a very low, a low, a moderate, a high, and a very high chance of developing or re-developing cervical cancer or having a poor prognosis (e.g., likelihood of long term survival) from cervical cancer. In some embodiments, a health care provider (e.g., an oncologist) will use such a risk profile in determining a course of treatment or intervention (e.g., biopsy, wait and see, referral to an oncologist, referral to a surgeon, etc.).

EXAMPLES

The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

Example 1

METHODS

Patients and tumor specimens

A total of 158 patients with squamous cell cervical carcinoma, prospectively recruited to a chemoradiotherapy protocol at the Norwegian Radium Hospital from 2001 to 2006, were included. All patients were treated with external radiation of 50 Gy to the tumor, parametria, and adjacent pelvic wall, and with 45 Gy to the remaining part of the pelvic region. This was followed by brachytherapy of 21 Gy to point A. Adjuvant cisplatin (40 mg/m ) was given weekly during the course of external radiation. Follow up consisted of clinical examinations, and when symptoms of relapse were seen, MR imaging of pelvis and retroperitoneum as well as X-ray of thorax were carried out. Relapse (progressive disease) was classified as locoregional (regression within the irradiated field), distant, or both. One to 4 tumor biopsies, approximately 5x5x5mm in size, were taken before the start of therapy, immediately snap frozen, stored at 80°C until further analyses. The study was approved by the regional committee of medical research ethics in southern Norway, and written informed consent was achieved from all patients.

Array-based DNA Methylation Analysis with Infinium Methylation 450K

For 158 patients, genomic DNA was isolated from the cervical cancer biopsies according to a standard protocol, including proteinase K, phenol, chloroform, and isoamylalcohol (Jones et al, 2012 Nat. Rev. Genet. 75:484-492). DNA (1 μg) was digested overnight, using Dpnll endonuclease (New England Biolabs, Beverly, MA, USA), and purified using the QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA). Purified DNA quality and concentration were assessed with Quant-iT™ PicoGreen® dsDNA Assay Kit (Life Technologies, Paisley, UK) prior to bisulfite conversion. One μg of purified genomic DNA was bisulfite converted using the EZ DNA Methylation Kit (Zymo Research, Orange, CA, USA) following the manufacturer's protocol. Following the Illumina 450K array protocol, 4 ul of bisulfite converted sample was whole-genome amplified,

enzymatically digested, hybridized to the array and then single nucleotide extension was performed. After scanning, the raw image data of each bead chip were processed in

GenomeStudioV2011.1 Methylation Module VI .9. The percentage of methylation of a given CpG locus was reported as a β-value, which is a continuous variable between 0 and 1. This corresponds to the ratio of intensities between methylated and unmethylated alleles.

To pre-process the data, the pipeline developed by Touleimat & Tost (2012

Epigenomics 4:325-341) was implemented using R (version 2.15.1). In brief, for quality control, probes with less than three functional beads on the array were considered to be nonfunctional, and were assigned a detection p-value equal to 1. "Good quality" samples were defined as having >80% high quality (detection p-value <0.01) probes. All patient samples respected this criterion, and were thus kept for the further analyses. Further, all allosomal probes located on the Y chromosome were removed, since the samples were from female patients.

A color balance adjustment between the two color channels was performed, followed by a separate color background adjustment based on the negative control probes provided by Illumina. Finally, a subset quantile normalization was performed in order to correct for the Infl/Infll shift and normalize between samples. For probe category construction, CpG- categories were built using the more robust "relationToCpG" annotation from the Illumina file. As an additional step, poorly performing probes, defined as having a detection p-value of >0.05 in more than 20 % of the patient samples, were excluded.

Gene expression analysis

Gene expression profiling of 147 patients was performed using the Illumina bead arrays human WG-6 v3 (Illumina Inc.) with 48803 transcripts, as previously described (Lando et al, 2009). In brief, total R A was isolated from the frozen tumor specimens using Trizol reagent (Life Technologies) and from the cell lines using RNeasy MiniKit (Quiagen). cR A was synthesized, labeled, and hybridized to the arrays. Signal extraction and quantile normalization were carried out by the software provided by the manufacturer (Illumina Inc.).

Statistics

In the survival analyses, the endpoint was progression- free survival, where the time from diagnosis to cancer-related death or to the first event of relapse was used. Cox proportional hazard analysis was used to evaluate the prognostic value of various parameters with respect to progression-free survival. Kaplan-Meier curves were compared using log-rank test. Spearman rank correlation analyses were utilized to assess toe correlation between methylation and gene expression values. P-values <0.05 or adjusted p-values <0.1 were considered significant.

RESULTS AND DISCUSSION

There were 548 methylation probes annotated to one of the 31 hypoxia-associated genes, respectively. As illustrated in Figure 1, there were several methylation probes in both the promoter and gene body of the genes which showed a correlation with expression of the relevant/related gene. More detailed information about the methylation probes is listed in Table 1. Most of the promoter methylation and gene body methylation are negatively and positively correlated, respectively, with the gene expression.

A Spearman rank correlation was performed to assess the degree of which these individual methylation sites contributed to the regulation of expression of their annotated gene. Only methylation probes which showed a good correlation (adjusted p-value <0.1) to the expression of their annotated gene were included in further analyses (Table 2), leading to the exclusion of 393 probes and 5 genes (C19orf53, CLK3, EROIL, HMOX1, and SNTA1). Furthermore, the 4 genes (AK2, C14orf2, ECE2, and SPAG7) which only were left with one remaining probe after this filtering step were also excluded. After these selection steps, 151 probes remained, representing 22 of the 31 genes in the signature (Table 2). Seven of the genes had at least one methylation probe which significantly correlated with poor prognosis of the patients (Cox regression analysis, p<0.1). This is exemplified by Stanniocalcin 2 (STC2), Poliovirus receptor (PVR), and ribosomal protein L36a (RPL36A) in Figure 2, and is described in more detail in Table 3.

Even if the probes for the remaining 15 genes did not significantly correlate with survival individually, they may contribute to predicting survival if assessed in combination with the other probes in a "methylation score". Thus, one representative probe for each of the 22 genes was included in the score, and the resulting combined score for the 22 genes was assessed for prognostic significance by Cox regression analysis. A methylation score was calculated to obtain a metric which took into account the methylation level of all these 22 genes. The methylation score was calculated from the methylation probes as described below.

One representative probe per gene was selected. The individual beta values of each probe for each patient were median-centered across all 158 patients for the respective probe. For each patient, the values of the probes showing a positive correlation with survival (positive Cox regression correlation coefficient, Table 3) were averaged, and similarly for those showing a negative correlation with survival. Furthermore, the difference between the resulting average values for each patient was calculated, resulting in a "methylation score". As seen in Figure 3, the level of this methylation score could significantly predict survival of the cervical cancer patients. For patients having a high methylation score, the probability of progression free survival decreased from 80 % to 46 % (p=0.001) (Figure 3). This 22-gene score also appeared to be a strong prognostic factor independent on other clinical variables, like tumor and FIGO stage, in a multivariate Cox regression analysis (Table 4).

In summary, a majority of the genes in the 31 gene signature were regulated by methylation. Furthermore, the methylation level of these genes could predict survival of cervical cancer patients in a highly significant manner, independent of other clinical variables. Thus, a test based on the methylation status of all or a subset of these genes could be valuable for identification of cervical cancer patients of high risk of recurrence who may benefit from more aggressive treatment.

One gene in the study with gene body methylation variation is stanniocalcin 2 (STC2). A previous study has investigated methylation of STC2 in various cancer cell lines, but none of cervical cancer origin, and furthermore, only looked at methylation of the promoter region, showing that hypomethylation of this region was associated with

overexpression (Law et al, 2008 Exp. Cell Res. 574: 1823-1830). Another recent study has also demonstrated changes in methylation status of STC2 in renal cell cancers (Girgis et al, 2012 Cancer Res. 72(20):5273-84). None of the above studies has disclosed a prognostic value of STC2 in cervical cancer. The study supports the negative correlation between promoter methylation and gene expression, but importantly, it was also shown that hypermethylation of large number of methylation sites in the gene body of STC2 are associated with increased levels of STC2 gene expression, and that this methylation has prognostic significance. Furthermore, this gene body methylation appears to be significantly correlated with survival of the cervical cancer patients (Figure 2), while promoter

methylation is not prognostic (data not shown). This demonstrates the importance of also investigating the body region of a gene with regard to methylation status, and highlights the novelty of the findings.

Additional genes in the prognostic methylation signature have previously been described as methylated in one or more other cancer types, but without any disclosure of association with cervical cancer prognosis. These include S100A2, whose hypermethylation has been reported to in association with prostate cancer (Rehman et al, 2006 Prostate

65(4):322-30), breast cancer (Wicki et al, 1997 Cell Calcium 22(4):243-54), lung cancer (Feng et al, 2001 Cancer Res. 61(21):7999-8004), head and neck cancer (Zhang et al, 2007 Head Neck 29(3):236-43) and malignant medulloblastoma (Lindsey et al, 2007 Br J Cancer 97(2):267-74). None of the above disclosed prognostic significance of hypermethylation. Similarly, the signature gene PVR has been associated with leukemia (Wang et al, 2011 J Immunother. 34(4):353-61), KCTD11 was shown to be hypermethylated in multiple cancer types not including cervical cancer (Mancarelli et al, 2011 Mol Cancer 9 : 172), and P4HA2 was reported to be transcriptionally silenced by methylation in lymphoma (Hatzimichael et al, 2012 Br J Cancer 2012 Oct 9;107(8)). The promoter oiSH3GL3 was aberrantly methylated in colorectal cancer patients (Fang et al, 2012 Asian Pac J Cancer Prev.

13(5): 1917-21), while the UPK1A promoter was reported to be hypermethylated in a manner negatively correlated with expression in primary squamous cell carcinoma (Kong et al, 2010 Cancer Res. 70(21):8832-41). None of the above disclosures relate a prognostic significance of the observed hypermethylation.

The expression level of DDIT3 has also been reported to have prognostic value in malignant mesothelioma (Dalton et al, 2013 Br J Cancer 108: 1340-1347), melanoma (Korabiowska et al, 2002 Histol Histopathol 17:805-811) and non-small cell lung cancer (Lee et al, 2012 Oncol Lett. 4:408-412), but without any association with methylation in the relevant malignancies. In another study of an unrelated malignancy, DDIT3 was found to be methylated in chronic myeloid leukemia, but without reference to prognosis (Wang et al, 2010 J Exp Clin Cancer Res. 29:54).

Table 1: Information about the 151 probes which are significantly correlated (adj.p<0.1) with their annotated gene.

Gene Symbol Probe ID Chromosome Mapinfo* Position**

AK3L1 cel0482024 1 65613042 Promoter

AK3L1 ce00009916 1 65613819 Promoter

AK3L1 ce24049468 1 65615281 Promoter

AK3L1 CS23983726 1 65642217 Bodv

AK3L1 cel0716862 1 65674876 Bodv

ALDOA ce24780263 16 30064201 Promoter

ALDOA ce00300822 16 30075511 Bodv

ALDOA ceOl 165575 16 30075904 Bodv

ALDOA ce02891495 16 30075969 Bodv

ALDOA cgl0103580 16 30076325 Bodv

ALDOA ce05048002 16 30077837 Bodv

B3GNT4 ce27049761 12 122688708 Promoter

B3GNT4 cel8552482 12 122690827 Bodv

B3GNT4 ce06994572 12 122691993 Bodv

C20ORF20 cel4529610 20 61426508 Promoter

C20ORF20 cel4582546 20 61427684 Promoter

C20ORF20 ce04788221 20 61427686 Promoter

C20ORF20 ce03211173 20 61427693 Promoter

C20ORF20 ce24999924 20 61428215 Promoter

DDIT3 ce20727555 12 57910480 Bodv

DDIT3 cel9510318 12 57913710 Promoter

DDIT3 cel3148824 12 57914715 Promoter

FGF11 ce03613649 17 7341191 Promoter

FGF11 cel3492227 17 7341436 Promoter

FGF11 cel8105842 17 7341440 Promoter

FGF11 cgl9056772 17 7341616 Promoter

FGF11 ce25563256 17 7341641 Promoter

FGF11 eel 3194425 17 7341936 Promoter

FGF11 ce06462874 17 7342585 Promoter

FGF11 ce01557754 17 7342661 Promoter

FGF11 cel5068733 17 7342846 Promoter

FGF11 ce27579532 17 7342894 Promoter

FGF11 ce00795277 17 7343105 Promoter

FGF11 ce05513300 17 7345301 Bodv

FGF11 cel7788227 17 7347145 Promoter

GAPDH cel5350627 12 6642201 Promoter

GAPDH ce00252813 12 6642229 Promoter

GAPDH ce00241355 12 6643629 Promoter

ISG15 ce20062691 1 949392 Promoter

ISG15 ce03811829 1 949449 Promoter

ISG15 eel 1211792 1 949634 Promoter

ISG15 ce04788999 1 949850 Promoter

ISG15 cel6526047 1 949893 Promoter

KCTD11 ce07148458 17 7254301 Bodv KCTD11 ce03666441 17 7254671 Bodv

KCTD11 ce07639694 17 7254909 Promoter

KCTD11 ce22084563 17 7255046 Promoter

KCTD11 ce05164926 17 7255624 Promoter

KCTD11 ce02550691 17 7255736 Promoter

KCTD11 cg26144202 17 7256361 Promoter

KCTD11 cel4696468 17 7256577 Promoter

KCTD11 ce21507719 17 7256673 Promoter

KCTD11 ce05779246 17 7258070 Bodv

LOC401152 cel3155599 4 120221765 Promoter

LOC401152 ce05238812 4 120221776 Promoter

P4HA2 ce09830083 5 131561058 Bodv

P4HA2 cel4527110 5 131561292 Bodv

P4HA2 cel7861653 5 131561363 Bodv

P4HA2 ce24117468 5 131562848 Promoter

P4HA2 cel8318560 5 131563015 Promoter

PFKFB4 ce21141812 3 48556323 Bodv

PFKFB4 ce04498104 3 48573748 Bodv

PFKFB4 cel0319474 3 48573819 Bodv

PFKFB4 ce20732160 3 48590040 Bodv

PFKFB4 ce08568670 3 48593395 Promoter

PVR ce02415834 19 45146246 Promoter

PVR ce04566018 19 45146967 Promoter

PVR cel4538146 19 45146976 Promoter

PVR cel0777702 19 45147005 Promoter

PVR ce07917289 19 45147056 Promoter

PVR ce05012825 19 45147078 Promoter

PVR ce05878558 19 45147316 Promoter

PVR ce25328384 19 45165815 Bodv

PYGL cgl9839026 14 51410501 Promoter

PYGL ce07035145 14 51410689 Promoter

PYGL ce21805221 14 51411140 Promoter

PYGL cel3008339 14 51411481 Promoter

PYGL cel2117827 14 51411647 Promoter

RHOC ce04859477 1 113249989 Promoter

RHOC ceOOO 16968 1 113250448 Promoter

RHOC cel9507217 1 113251308 Promoter

RPL36A ce01410230 X 100645860 Promoter

RPL36A ce09738386 X 100645872 Promoter

RPL36A ce07825508 X 100645891 Promoter

RPL36A cel2440269 X 100645895 Promoter

RPL36A ce04025582 X 100645906 Promoter

RPL36A ce22682567 X 100645938 Promoter

RPL36A cel2920408 X 100645993 Promoter

RPL36A cel8507125 X 100646106 Promoter

RPL36A eel 1390763 X 100648809 Bodv

S100A2 ce23656322 1 153533922 Bodv

S100A2 ce07353685 1 153536278 Bodv

S100A2 ce00647881 1 153536528 Promoter

S100A2 ce27310485 1 153536563 Promoter

S100A2 ce23023513 1 153537369 Promoter

S100A2 ce22700686 1 153538764 Promoter

S100A2 ce21196487 1 153538964 Promoter

S100A2 ce08105396 1 153539058 Promoter S100A2 cel9907725 1 153539397 Promoter

S100A2 ce02332117 1 153539404 Promoter

SCARB1 cel0911287 12 125271306 Bodv

SCARB1 ce25043237 12 125297360 Bodv

SCARB1 cel9950933 12 125299563 Bodv

SCARB1 ce23476025 12 125317455 Bodv

SCARB1 cel2079885 12 125328475 Bodv

SCARB1 cel8036143 12 125329150 Bodv

SCARB1 ce22775642 12 125332183 Bodv

SCARB1 cel3075279 12 125333267 Bodv

SCARB1 cel4846380 12 125333322 Bodv

SCARB1 ce23460943 12 125333358 Bodv

SCARB1 ce06719671 12 125347797 Promoter

SCARB1 ce27180443 12 125348871 Promoter

SH3GL3 ce23940614 15 84115479 Promoter

SH3GL3 ce04779631 15 84115895 Promoter

SH3GL3 ce26215967 15 84115897 Promoter

SH3GL3 ce27021357 15 84115969 Promoter

SH3GL3 cel6100530 15 84146864 Bodv

SH3GL3 ce03752741 15 84168459 Bodv

SH3GL3 ce02750133 15 84181244 Bodv

SH3GL3 ce02030270 15 84215725 Bodv

SH3GL3 cel5564896 15 84287328 Bodv

STC2 ce03846076 5 172743837 Bodv

STC2 ce08839053 5 172744670 Bodv

STC2 ce03983713 5 172744704 Bodv

STC2 cel9878194 5 172744882 Bodv

STC2 ce23428905 5 172744941 Bodv

STC2 ce20977312 5 172748917 Bodv

STC2 cel0625081 5 172750450 Bodv

STC2 cel3329346 5 172750611 Bodv

STC2 cel9481287 5 172750923 Bodv

STC2 cel5420720 5 172751061 Bodv

STC2 ce26605683 5 172751269 Bodv

STC2 ceO 1984743 5 172751331 Bodv

STC2 cel9773855 5 172754417 Bodv

STC2 ce02138264 5 172754465 Bodv

STC2 ce06439005 5 172754848 Promoter

STC2 cel8680788 5 172754896 Promoter

STC2 cel l457817 5 172755103 Promoter

STC2 ce02135395 5 172755183 Promoter

STC2 cel8335607 5 172755413 Promoter

STC2 cel9673350 5 172755421 Promoter

STC2 cel8281743 5 172755856 Promoter

STC2 ce22834998 5 172755995 Promoter

STC2 ce08711858 5 172756550 Promoter

STC2 cel6443866 5 172756558 Promoter

STC2 cel7802144 5 172756776 Promoter

STC2 cel9586881 5 172756999 Promoter

TRAPPC1 cel3697368 17 7833663 Promoter

TRAPPC1 ce02146374 17 7835269 Promoter

UPK1A ce22802439 19 36157825 Promoter

UPK1A ce09968702 19 36160759 Bodv *Mapinfo; The genomic position of C in the CG dinucleotide which the probe is designed to detect

** "Promoter" is defined as +/- 2000 bp from the transcriptional start site of the gene, while "body" designates all other regions.

Table 2: Selection of methylation probes for further analyses

Number of

Total

probes

Gene number of

correlated with

probes

gene expression

AK2 13 1

AK3L1 26 5

ALDOA 27 6

B3GNT4 12 3

C4orf3 13 2

C14orf2 12 1

C19orf53 10 0

C20orf20 16 5

CLK3 24 0

DDIT3 16 3

ECE2 21 1

EROIL 16 0

FGF11 19 13

GAPDH 16 3

HMOX1 9 0

ISG15 10

3021 5

KCTD11 15 10

P4HA2 23 5

PFKFB4 17 5

PVR 18 8

PYGL 14 5

RHOC 13 3 RPL36A 14 9

S100A2 11 10

SCARB1 47 12

SH3GL3 19 9

SNTA1 14 0

SPAG7 19 1

STC2 34 26

TRAPPC1 17 2

UPK1A 13 2

Table 3: The 22 genes for which gene expression was regulated by methylation levels.

Gene expression Cox regression correlation analysis

Position of Adjusted Correlation Hazard

Gene name p-value

probe^a p-value coefficient Ratio

STC2 Body 1.3E-08 0.49 0.001 2.65

PVR Body 1.0E-04 0.36 0.014 6.50

KCTD11 Promoter 1.6E-05 -0.39 0.032 -4.32

RPL36A Promoter 0.01 -0.26 0.036 -2.72

S100A2 Promoter 0.09 -0.18 0.037 -3.11

PYGL Promoter 0.01 -0.25 0.042 -5.15

FGF11 Promoter 1.3E-09 -0.52 0.085 -1.87

P4HA2 Promoter 1.9E-04 -0.35 0.123 -3.09

TRAPPC1 Promoter 0.01 -0.25 0.159 5.13

SH3GL3 Body 4.5E-07 0.44 0.200 1.84

AK3L1 Body 1.4E-05 -0.39 0.282 1.15

SCARB1 Body 0.05 0.21 0.367 2.26

C4orf3 Promoter 0.02 0.23 0.400 -19.6

C20ORF20 Promoter 0.06 -0.20 0.467 -1.50

ISG15 Promoter 9.2E-04 -0.31 0.511 -0.54

GAPDH Promoter 0.02 -0.23 0.554 -4.51

RHOC Promoter 0.02 -0.24 0.574 0.72 DDIT3 Body 0.09 -0.18 0.586 2.37

UPK1A Promoter 0.08 0.19 0.620 0.56

PFKFB4 Body 8.2E-04 -0.32 0.648 -0.58

B3GNT4 Body 3.3E-03 0.28 0.719 0.49

ALDOA Promoter 0.05 -0.21 0.767 -0.48 a The methylation probe was defined as being located in the promoter of a gene if positioned <2000bp from its transcriptional start site (TSS). If positioned >2000bp from the TSS, the probe was defined as being located in the body of the gene.

Table 4: Cox regression analysis of the 22-gene methylation score and clinical variables

Abbreviations: CI, Confidence Interval; FIGO, Federation International de Gynecologie et d'Obstetrique. Tumor volume and methylation score were divided into two groups based on median values of all patients, respectively. FIGO stage was divided into two groups; lb-2b and 3a-4a.

Example 2

In Example 1, gene expression data and clinical data from 46 patients was used to develop a prognostic hypoxia gene signature, while 109 independent patients were used for validation (Halle et al., Cancer Res., 72: 5285-5295, 2012). In Example 1, the combined samples (designated cohort 1 for the purposes of this Example) were used to investigate correlation of methylation levels of the originally identified 31 hypoxia-associated genes with expression and clinical outcome. In this Example, data for the 109 validation patients was used to investigate the relevance of methylation of the signature genes, to be able to have an independent analysis of the gene methylation without interference from the previous analysis. Methylation data were available for 107 of these patients, which thus constitutes sub-cohort 1 in the current analysis.

The initial total number of methylation probes annotated to the 31 genes was 587. Of these probes, 39 were annotated to a different isoform than the one included in the hypoxia score in a previous study (Halle et al, supra), and were therefore excluded. The 32 probes that were located within 10 bp of a known SNP were not included in further analyses.

Methylation probes that showed little variation across patients, as defined by a standard deviation of <0.06 were removed. After these selection steps were performed, 168

methylation probes were selected for further analyses.

Eighty four of these methylation probes had a significant correlation (p<0.05) with its annotated gene expression probe. Thus, for 21 of the 31 genes, at least one methylation probe significantly correlated with gene expression of the annotated gene (Table 5). Importantly, none of the 84 probes were cross-reactive against other parts of the genome, according to the investigation of Infinium 450K probes performed by Chen et al. (Epigenetics., 8: 203-209, 2013).

The methylation probes in the study that showed a correlation with expression of their annotated gene were located both in the promoter and in the body of the genes (Table 5). Moreover, most of the promoter methylation and gene body methylation were positively and negatively correlated with gene expression, respectively.

To validate the associations between methylation and gene expression levels of the hypoxia signature genes, the Infinium 450K DNA methylation assay was performed on an independent cohort of 117 cervical cancer patients, and gene expression profiling was assessed by the Illumina bead arrays human HT12-v4. A Spearman rank correlation analysis was performed to evaluate the correlation between methylation and gene expression for all the genes in the hypoxia score. A correlation between methylation and gene expression was validated for all but three of the 21 genes from the test cohort (sub-cohort 1). Thus, 70 of the methylation probes, representing 18 genes, were significantly correlated with gene expression. The correlations between gene expression and correlation for both cohorts are visualized in Figure 4, while the correlation data are listed in table 5. In Figure 4, correlations between methylation and gene expression for all candidate methylation probes for each gene are indicated with colors for cohort 1 (CI) and cohort 2 (C2). Colors indicate significant (p<0.05) negative and positive correlations, respectively, while white represents nonsignificant and thus not validated probes. Promoter was defined as +/- 2000 bp from the transcriptional start site, while gene body was defined as the remaining part of the transcript.

In Example 1 , cohort 1 was used to assess the correlation between gene expression probes and methylation probes for the 31 genes. In the current analysis (Example 2), this correlation was re-assessed in an optimized sub-group of cohort 1 , called sub-cohort 1 , and further validated in cohort 2. Table 6 summarizes which genes had correlating gene expression and methylation probes in the various cohorts. Twenty genes had correlating probes in independent cohorts (at least one from cohort 1 and sub-cohort 1, plus cohort 2). These genes constitute are highlighted in table 6.

Table 5: Validation of the correlating methylation probes

Gene expression correlation

Sub-Cohort 1 Cohort 2

Methylation Correlati< m p- Corre ition

Symbol Position* p-value Validated probe coef. value coef.

cgl 1254053 AK2 Body -0.26 0.006 -0.27 0.003

cg24049468 AK3L1 Promoter -0.35 2E-04 -0.45 4E-07 V cg26036018 AK3L1 Body 0.24 0.015 -0.05 0.596

cg23983726 AK3L1 Body -0.3" 0.001 -0.40 6E-06 V cg00300822 ALDOA Body -()."- 0.005 -0.02 0.850

cgl8552482 B3GNT4 Body 0.32 0.001 0.29 0.002 V cg26332630 C14orf2 Body -0. 0.024 -0.27 0.004

cg03211173 C20orf20 Promoter -0." l 0.027 -0.14 0.129

cg26223996 CLK3 Body -0.20 0.043 -0.12 0.205

cg03613649 FGF11 Promoter -0.32 0.00 1 -0.46 1E-07

cgl 3492227 FGF11 Promoter -0.54 3E-09 -0.61 3E-13 V cgl8105842 FGF11 Promoter -0.55 7E-10 -0.64 5E-15 V cgl9056772 FGF11 Promoter -0.5^' 2E-10 -0.57 lE-11 V cg25563256 FGF11 Promoter -0.55 9E-10 -0.56 6E-11 V cgl 3194425 FGF11 Promoter -0.65 4E-14 -0.65 2E-15 V cg06462874 FGF11 Promoter -0.5 1 2E-08 -0.55 lE-10 V cg01557754 FGF11 Promoter -0.60 7E-12 -0.6S 4E-17 V

cgl 8507125 RPL36A Promoter ii -0.3 1 0.001 -0.08 0.380 cgl 1390763 RPL36A Body -0.2S 0.004 -0.16 0.082 cgl 9907725 S100A2 Promoter ii -0.30 0.002 -0.13 0.157 cg23023513 S100A2 Promoter ii -0.4 1 2E-05 -0.39 1E-05 V cg27310485 S100A2 Promoter ii -0.47 4E-07 -0.44 7E-07 V cg07353685 S100A2 Body -0.5S 4E-09 -0.5 1 4E-09 V cg23656322 S100A2 Body -0.4- 5E-07 -0.4^" 1E-07 V cg27180443 SCARB1 Promoter i -0.23 0.015 -0.32 4E-04 V cg23460943 SCARB1 Body 0.37 9E-05 0.3 1 8E-04 V cg22775642 SCARB1 Body 0.24 0.013 0 03 0.782 cgl2079885 SCARB1 Body 0.50 5E-08 0.40 8E-06 V cg03423228 SCARB1 Body . 0 0.002 0.30 0.001 V cgl9950933 SCARB1 Body 0.35 2E-04 0.30 0.001 V cg25043237 SCARB1 Body 0.34 4E-04 0.33 3E-04 V cgl091 1287 SCARB1 Body 0.5 1 2E-08 0.40 7E-06 V cg23940614 SH3GL3 Promoter ii -0.23 0.015 -0.35 1E-04 V cgl6100530 SH3GL3 Body 0.46 5E-07 0.20 0.028 V cg03752741 SH3GL3 Body 0.35 2E-04 0.40 9E-06 V cgl5564896 SH3GL3 Body 0.39 4E-05 0.29 0.002 V cgl6443866 STC2 Promoter ii -0.33 5E-04 -0.27 0.004 V cg0871 1858 STC2 Promoter ii -0.36 1E-04 -0.25 0.007 V cgl 8680788 STC2 Promoter ii -0.45 1E-06 -0.42 2E-06 V cgl9773855 STC2 Body -0.44 2E-06 -0.40 1E-05 V cgO 1984743 STC2 Body 0.45 1E-06 0.33 3E-04 V cg26605683 STC2 Body 0.46 6E-07 0.25 0.006 V cgl 5420720 STC2 Body 0.44 2E-06 0.29 0.002 V cgl9481287 STC2 Body 0.39 4E-05 0.20 0.029 V cgl3329346 STC2 Body o 1 1 ^; 04 () ΛΛ IV 07 V cgl0625081 STC2 Body 0.34 3E-04 0.30 0.001 V cg20977312 STC2 Body 0.26 0.007 0 1 1 0.253 cg23428905 STC2 Body 0.5 1 2E-08 0.49 3E-08 V cgl9878194 STC2 Body n ΛΛ 111 } | I· ΠΛ II u V cg08839053 STC2 Body 0.52 1E-08 <1E-12 V cg03846076 STC2 Body 0.55 8E-10 0. ¾2 4E-04 V cg01837574 TRAPPC1 Promoter -0.2 1 0.031 -0 . 0.037 V ^a The 84 probes which were significantly correlated (p<0.05) with their annotated gene in cohort 1.

b Promoter was defined as +/- 2000 bp from the transcriptional start site, while gene body was defined as the remaining part of the transcript.

c Significant negative and positive correlations are emphasized with green and red colors, respectively, while white indicates non-significant correlations.

Table 6: Summary of genes displaying significant correlation between gene expression and methylation from the various analyses and cohorts

Gene* Cohort 1 Sub cohort 1 C

AK2

AK3L1 V V V

ALDOA V V

B3GNT4 V V V

C4orf3 V

C14orf2 V V

C19orf53

C20orf20 V V

CLK3 V

DDIT3 V

ECE2

EROIL

FGF11 V V V

GAPDH V

HMOX1

ISG15 V V V

KCTD11 V V V

P4HA2 V V V

PFKFB4 V V V

PVR V V V PYGL V V V

RHOC V V V

RPL36A V V V

S100A2 V V V

SCARB1 V V V

SH3GL3 V V V

SNTA1

SPAG7

STC2 V V V

TRAPPC1 V V V

UPK1A V

*The highlighted genes (in bold) represent those with correlating gene expression and methylation probes in multiple independent cohorts, representing the focus list of prognostically significantly methylated genes.

Table 7 shows probe exemplary probe sequences. Two Infinium design types were used. Type I design involves two probes per methylation locus (e.g., allele A and allele B), while type II design has one probe per locus.

Table 7

AAATATACCCC II AAAATACAATA AAAAAATACCT AAAAATATTTA

cg06462874 FGF11 AACCAC 16

ATATACTCCTC II

AATAAATCCCA

CCCTCRAATTTT

ACCTCCTATTA

cg01557754 FGF11 AAAAC 17

TATCTAACTATT II

AACTTTAACCT

AAACCRAAACC

AATCTCAAATA

cgl7788227 FGF11 ACCAC 18

AAACCTATATA II AC CCTACAAA ACTAAAACCTA ACRATAAAAAT

cg20062691 ISG15 ACTAAC 19

RAATTCCAAAT II ATCCCTAAACA ACTCCATATCR ATATCAAAACT

cg03811829 ISG15 AAAAAC 20

AAACAAATACR II

ACRAACCTCTA

AACATCCTAAT

AAAAAATAACA

cgl 1211792 ISG15 AAAACC 21

TATTTCCRACCC II

TTAATCCTACTC

RAATACTAATA

AAAACCCTTAA

cg04788999 ISG15 CTCC 22

AAACACAAACA II

CAAAAACRACC

ATTTCTCTTTAC

AACAACCTTTA

cgl6526047 ISG15 TTTCC 23

AAACCTAAAAA II

ATAAAATAAAT

ATATTCTTTACC

TACCTACCCCT

cg07148458 KCTD11 AAACC 24

CTCAAATCACA CTCGAATCAC I ACATATAAAAA AACGTATAAA TCATAACTACA AATCATAACT ACAACTCAACT ACGACGACTC

cg03666441 KCTD11 TCTCCA 25 AACTTCTCCG 88

AAATAAAAA I

AAATAAAAACC CCGAACTAAC AAACTAACAAA GAAAACGAA AACAAATAACA TAACGAAACC AAACCCCCTTA CCCTTATATC

cg05164926 KCTD11 TATCCA 26 CG 89

CCACAAAACAA CCACGAAACA I ATCCAAACAAC AATCCAAACG CCAACCTCAAA ACCCAACCTC

cg26144202 KCTD11 AAATTAAAAAT 27 AAAAAATTAA 90 ATACCA AAATATACCG

CTTCCRAACCA II

ACCTTTTCTACA

CCRACTCTAAA

TATCTAAATAC

cg21507719 KCTD11 TTTAC 28

AAACAACCATA II

AACCTATTACT

AAAAAATAACT

AAAAAAAACCC

cg05779246 KCTD11 AAAACC 29

TAATTCRT AA II CTAATTCAACA AACRAAACCCT TCT AT ATACCA

cg24117468 P4HA2 AAAACC 30

TCCCCTAACAC II ATAACCAACTA CCAATATAAAA CAACAATCTCA

cgl4527110 P4HA2 AAATAC 31

CTTAAAAAAAT II CTAAAACTAAA AACAAAACCTT AACTAATATAT

cg09830083 P4HA2 AACCAC 32

ATAAAATTATT II

AAAAAACTCAC

CTCTCCTAAAA

TCCCAAATATT

cg08568670 PFKFB4 TAAACC 33

AAACCRATAAA II

CAACTAAAACC

C CATTATACA

TTATTTACATA

cg20732160 PFKFB4 ATTCCC 34

TATAAACCAAA II ACTACRTAATA AACCRTATAAC TAACCACATCC

eg 10319474 PFKFB4 AAAACC 35

GAAAAAACA I

AAAAAAACAAT ATCCTAAATC

CCTAAATCCCC CCCGCCAATC

ACCAATCCAAC CGACCCTTAA

CCTTAAAATTA AATTAAACTC

cg04498104 PFKFB4 AACTCA 36 G 91

AAATTACCTTC AAATTACCTT I

CTAATACACAC CCTAATACAC

ACCAAAATACT ACACCGAAAT

CAAAAAAAAAA ACTCGAAAAA

cg21141812 PFKFB4 CTACCA 37 AAAACTACCG 92

ATAAAAAAA I

ATAAAAAAACA CGAAAACAC

AAAACACACAA ACGAAAAAA

AAAAATTCAAC TTCAACAACT

AACTTACTAAC TACTAACGAC

cgl4538146 PVR AACTCA 38 TCG 93 AATATAAACAA AATATAAACG I CCTCACACAAA ACCTCACACA AAACCACCTCT AAAAACCGCC TCTAATAAACA TCTTCTAATA

cg05012825 PVR CCAACA 39 AACACCGACG 94

T AAT AAAAAT C II

TTAAACCACAC

RATATTAACTT

AATATATAAAA

cg25328384 PVR TCAACC 40

ACTCTCAACAT II

TTAATCAAAAA

ATAACRAAAAC

CTCTTATCCTAT

cgl9839026 PYGL ATACC 41

TTCTAAATCCC II

AACTATAAATT

CTATTCCCACTC

CTAAATCTAAC

cgOOO 16968 RHOC TTAAC 42

AATCRAAATAA II

ATAAACCACCC

TTTAAACRAAA

TCCTTCCTTAAA

cg01410230 RPL36A CTCCC 43

AATATAAAAC I

AATATAAAACT TCAACGCAAC CAACACAACAC GCCCTCGAAC CCTCAAACAAC GACTAAAAA TAAAAAAACTC AACTCAATTC

cg09738386 RPL36A AATTCA 44 G 95

TCAATAAAAAC TCGATAAAAA I AATCATAAAAT CAATCATAAA ATAAAACTCAA ATATAAAACT CACAACACCCT CAACGCAACG

cg07825508 RPL36A CAAACA 45 CCCTCGAACG 96

AAAAATCRATA II AAAACAATCAT AAAATATAAAA CTCAAC CAAC

eg 12440269 RPL36A RCCCTC 46

CACTTCCTCAT II

AAAAATCRATA

AAAACAATCAT

AAAATATAAAA

cg04025582 RPL36A CTCAAC 47

CAAACGAAA I

CAAACAAAAAT ATATATAAAA

ATATAAAAAAT AATTCCCGAT

TCCCAATCACA CGCACTTCCT

CTTCCTCATAA CATAAAAATC

cg22682567 RPL36A AAATCA 48 G 97

CATAAAAAA I

CATAAAAAAAT ATACGATCGA

ACAATCAAAAA AAACCTCCTA

CCTCCTATATA TATACTTCCG

CTTCCATTTACC TTTACCTCGC

cgl2920408 RPL36A TCACA 49 G 98

ATTAAATTACT II

cgl 8507125 RPL36A CTAATATATCR 50 AATCCACACAA

AAACCTAAACC CATCCC

TTTATTTCCTTA II

TATTTTTCTAAT

ATCCTTTTTCTA

TTCTAAAACCC

cgl 1390763 RPL36A CAC 51

AATAACATAAC II

AAAAAAATCAA

ACTAAAAACAA

ACTAATCCCTT

cgl9907725 S100A2 CCTAAC 52

AACCCTTCTTTA II

AAAATCTAAAC

CTAAAAACAAA

AAATATACATA

cg23023513 S100A2 TAAAC 53

ATACCATCAAA II AAAAACTTAAT AACTCCCAAAC ACAAAAAAAAC

cg27310485 S100A2 TTTAAC 54

TCCTTATACAA II

AAATTCCTTCA

TTTCCCCCTTAC

TCAACTTAAAC

cg07353685 S100A2 TTATC 55

AACACTCATCA II CTATCATATAC AATAACTTCTT CCAAAACTACC

cg23656322 S100A2 CAAACC 56

CCTCCTCTATCR II

AAACCATCATC

ATAATTAATCA

CTATAAATAAA

cg27180443 SCARB1 ACTTC 57

TAAAAAATCAA II

AACRTAATCAT

CACCAAAAAAT

CCCAACTAACT

cg23460943 SCARB1 AAAAAC 58

ATCCRCCATCT II

CTTCCCATATA

ATAACACTAAA

ATAAACAAAAA

cg22775642 SCARB1 CTAACC 59

TACACCACATA TACACCACGT I CCCTCACTAAA ACCCTCACTA ACCCCAAACAT AAACCCCAAA TCTCAAAAACT CATTCTCGAA

cgl2079885 SCARB1 AAATCA 60 AACTAAATCG 99

TAAATCRTTTTC II

RCCCATTACRA

ACAATACTACT

ATACACATTCT

cg03423228 SCARB1 TATAC 61

CTCACCAAAAC II

cgl9950933 SCARB1 CAAAATATTAA 62 ACATAACRATA

TAATCRCTCTCC

RAACC

AATAACTCTAA II TTATTCTTAACT CCACATAAACA CRTAACAAACA

cg25043237 SCARB1 CACCC 63

CAAAACCATAT II

AATCRTATCCT

AACCAATAAAA

AATAAACCTAA

cgl0911287 SCARB1 CCTCAC 64

RCTATCAACRA II CACTAAAAACA ACCATCTCCAA AATACCATAAA

cg23940614 SH3GL3 AAACTC 65

CCTAAAATCTA II

ATCTTCTCAAA

ATAACTAAACC

CTATCCCTTCA

cgl6100530 SH3GL3 ACTTTC 66

ATTAAAACACT II AATAATCATTT ACTCAAATTTA CAATTAACACA

cg03752741 SH3GL3 ATTCTC 67

TTTACTTCCTAT II

AACTAAAAATA

AATCTCACCCA

TTACAATTATA

cgl5564896 SH3GL3 TCAAC 68

AAACACCACTT AAACGCCGCT I

AAAACACCTCT TAAAACGCCT

TCCCCTACTAT CTTCCCCTAC

ATATATACATA TATATATATA

cgl6443866 STC2 TACACA 69 CGTATACACG 100

AAAAAAATTAA II

ACRCCRCTTAA

AACRCCTCTTC

CCCTACTATAT

cg08711858 STC2 ATATAC

AAAACCACGC I

AAAACCACACA AAACTCTTAA AACTCTTAAAA AATAACACGA TAACACAAAAC AACGTACAAA ATACAAAAAAA AAAAAACGC

cgl 8680788 STC2 AACACA 70 G 101

CGCCATCCCC I

CACCATCCCCA AACTCCTAAC ACTCCTAACCA CAAATCAACA AATCAACAAAC AACCAAAAA CAAAAAATAAT ATAATAACAC

cgl9773855 STC2 AACACA 71 G 102

TCTCCAACAAC TCTCCGACGA I

AACTAATTTCT CAACTAATTT

ATAATAATATA CTATAATAAT

AAATACAAAAA ATAAAATACG

cg01984743 STC2 TTCCCA 72 AAAATTCCCG 103 ACAATACCTC I

ACAATACCTCC CGAACTACGC

AAACTACACAA GAAATTAATA

AATTAATAAAA AAAAAACAA

AAACAACACCA CACCGAACTC

cg26605683 STC2 AACTCA 73 G 104

CAAATACTACC II AAACAAAATAC CAAACCAACCT TTTAAAAACAA

eg 15420720 STC2 TAATCC 74

ATCCAAATCTC ATCCAAATCT I

ACACATTTCCTT CACACGTTTC

TTTCCCTTAATT CTTTTTCCCTT

CCAAAAACAAC AATTCCAAAA

cgl9481287 STC2 ACCA 75 ACGACACCG 105

AATAAACATCC II CTTATTACCCA ATACTAAAAAT ATCATATCACT

cgl3329346 STC2 CTAAAC 76

ACATTTCAAAA II

CRTCTTTAATA

AATAACTTACC

CTAAAAACACA

cgl0625081 STC2 CAAACC 77

ACTCTTACCAA II

AAACAAAACAA

TATTACATTTTC

CTCRAATTAAA

cg20977312 STC2 CTAAC 78

AATACCAAAA I

AATACCAAAAA ATAAACGAA TAAACAAAATA ATAACAAAA ACAAAAACCAC ACCACCCAAA CCAAACACCCA CGCCCATACC

cg23428905 STC2 TACCCA 79 CG 106

TAACCAAACCT II TTCATTTCACCT CCRAATATCAA AATACTCAAAC

cgl9878194 STC2 TATTC 80

CCTTAAATAC II TATAAATACAA AATTCACCAAA CACCCCTAAAA

cg08839053 STC2 CCCCAC 81

AAAAAATACAC II

ACCTAAAAAAT

ACATTTAACAA

ATTTCCCTTAA

cg03846076 STC2 AATTTC 82

TTCAAATTCTTT II

TCCAATCCRAC

TAACAAACTTC

TRAPPC RACTCAATTCTT

cg01837574 1 AAAC 83 All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the medical sciences are intended to be within the scope of the following claims.

Claims

1. A method for predicting a predisposition to cervical cancer in a subject, diagnosing a cervical cancer in a subject, predicting the likelihood of recurrence of cervical cancer in a subject, providing a prognosis for a subject with cervical cancer, or selecting a subject with cervical cancer for treatment with a particular therapy, comprising:

a) obtaining DNA from a biological sample of said subject; and

b) contacting said DNA with one or more methylation specific detection reagents to determine the level, presence, or frequency of methylation of a nucleic acid polymer corresponding to one or more genes selected from the group consisting of STC2, PVR, KCTDll, RPL36A, S100A2, PYGL, FGFll, P4HA2, AK2, AK3L1, ALDOA, B3GNT4, C14orf2, C4orfi, C20orf20, CLK3, DDIT3, GAPDH, ISG15, PFKFB4, RHOC, SCARBI, SH3GL3, TRAPPC1, and UPK1A.

2. The method of claim 1, wherein said one or more genes is two or more genes.

3. The method of claim 1, wherein said one or more genes is three or more genes.

4. The method of claim 1, wherein said one or more genes is five or more genes.

5. The method of claim 1, wherein said one or more genes is all of said more genes.

6. The method of claim 1, wherein said genes are two or more of STC2, PVR, KCTDll, RPL36A, S100A2, PYGL, FGFll, P4HA2, AK2, AK3L1, ALDOA, B3GNT4, C4orf2, ISG15, PFKFB4, RHOC, SCARBI, SH3GL3, and TRAPPC1.

7. The method of claim 1 , wherein said genes are STC2, PVR, KCTDll, RPL36A, S100A2, PYGL, FGFll, P4HA2, AK2, AK3L1, ALDOA, B3GNT4, C4orf2, ISG15 , PFKFB4, RHOC, SCARBI, SH3GL3, and TRAPPC1.

8. The method of any of Claims 1 to 7, wherein the level or frequency of methylation of a nucleic acid polymer is compared to a reference level or frequency of methylation.

9. The method of any of Claims 1 to 8, further comprising comparing the level, presence, or frequency of methylation of said nucleic acid polymer with a reference level, presence, or frequency of methylation, wherein an altered level, presence, or frequency of methylation for said patient relative to said reference provides an indication selected from the group consisting of predicting the likelihood of recurrence of cervical cancer in a subject, an indication of a predisposition of the subject to a cervical cancer, an indication that the subject has a cervical cancer, an indication of prognosis, and the response of a subject to treatment with a particular therapy.

10. The method of claim 9, further comprising the step of determining a treatment course of action based on said level, presence, or frequency of methylation.

11. The method of claim 10, wherein said treatment is chemotherapy or radiation.

12. The method of claim 11, further comprising the step of administering said treatment.

13. The method of any of Claims 1 to 12, wherein said nucleic acid comprises a region selected from the group consisting of a CpG island, a CpG island shelf, and a CpG island shore.

14. The method of claim 13, wherein said CpG island, CpG island shelf, or shore is present in a coding region or a regulatory region.

15. The method of claim 14, wherein said regulatory region is a promoter.

16. The method of claim 14, wherein said determining of the level of altered methylation of a nucleic acid polymer comprises determining the methylation frequency of said CpG island, island shelf, or island shore.

17. The method of any of Claims 1 to 16, wherein said determining of the level of a nucleic acid polymer with altered methylation is achieved by a technique selected from the group consisting of methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, methylation - insensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, and bisulfite genomic sequencing PCR.

18. The method of claim 1, wherein said methylation specific detection reagent is selected from the group consisting of a pair of amplification primers that specifically hybridizes to said gene, an amplification primer that specifically hybridizes to said gene, a restriction enzyme, and sodium bisulfite.

19. The method of claim 18, wherein said reagent is one or more of SEQ ID NOs: 1 - 106.

20. The method of any of Claims 1 to 18, wherein said biological sample is selected from the group consisting of a tissue sample, a cell sample, and a blood sample.

21. Use of a methylation specific nucleic acid detection sequence corresponding to one or more genes selected from the group consisting of STC2, PVR, KCTDll, RPL36A, S100A2, PYGL, FGFll, P4HA2, AK2, AK3L1, ALDOA, B3GNT4, C14orf2, C4orfl, C20orf20, CLK3, DDIT3, GAPDH, ISG15, PFKFB4, RHOC, RPL36A, SCARB1, SH3GL3, TRAPPC1, and UPK1A for detecting a cervical cancer in a subject.

22. The use of claim 21, wherein an altered level, presence, or frequency of methylation for a patient relative to a reference provides an indication selected from the group consisting of an indication of predicting the likelihood of recurrence of cervical cancer in a subject, an indication of a predisposition of the subject to a cervical cancer, an indication that the subject has a cervical cancer, an indication of prognosis, and the response of a subject to treatment with a particular therapy.

23. The use of claim 21, wherein said one or more genes are selected from the group consisting of STC2, PVR, KCTDll, RPL36A, S100A2, PYGL, FGFll, P4HA2, AK2, AK3L1, ALDOA, B3GNT4, C4orf2, ISG15, PFKFB4, RHOC, RPL36A, SCARB1, SH3GL3, and TRAP PCI.

24. The use of claim 21, wherein said reagent is one or more of SEQ ID NOs: 1-106.

25. A kit for detecting the presence of a cervical cancer in a mammal, said kit comprising methylation specific detection reagents useful, sufficient, or necessary for detecting and/or characterizing level, presence, or frequency of methylation of one or more genes selected from the group consisting of STC2, PVR, KCTDll, RPL36A, S100A2, PYGL, FGFll, P4HA2, AK2, AK3L1, ALDOA, B3GNT4, C14orf2, C4orfi, C20orf20, CLK3, DDIT3, GAPDH, ISG15, PFKFB4, , RHOC, RPL36A, SCARB1, SH3GL3, TRAPPC1, and UPK1A.

26. The kit of claim 25, wherein said methylation specific detection reagent is selected from the group consisting of a pair of amplification primers that specifically hybridizes to said gene, an amplification primer that specifically hybridizes to said gene, a restriction enzyme, and sodium bisulfite.

27. The kit of claim 26, wherein said reagent is one or more of SEQ ID NOs: 1-106.

28. The kit of claim 25, wherein said one or more genes are selected from the group consisting of STC2, PVR, KCTDll, RPL36A, S100A2, PYGL, FGFll, P4HA2, AK2, AK3L1, ALDOA, B3GNT4, C4orf2, ISG15, PFKFB4, RHOC, SCARB1, SH3GL3, and TRAPPC1.

29. A system comprising a computer readable medium comprising instructions for utilizing information on the level, presence, or frequency of methylation of one or more genes selected from the group consisting of STC2, PVR, KCTDll, RPL36A, S100A2, PYGL, FGFll, P4HA2, AK2, AK3L1, ALDOA, B3GNT4, C14orf2, C4orfl, C20orf20, CLK3, DDIT3, GAPDH, ISG15, PFKFB4, , RHOC, SCARB1, SH3GL3, TRAPPC1, and UPK1A to provide an indication selected from the group consisting of predicting the likelihood of recurrence of cervical cancer in a subject, an indication of a predisposition of the subject to a cervical cancer, an indication that the subject has a cervical cancer, an indication of prognosis, and the response of a subject to treatment with a particular therapy.

30. The system of claim 29, wherein said one or more genes are selected from the group consisting of STC2, PVR, KCTDll, RPL36A, S100A2, PYGL, FGFll, P4HA2, AK2, AK3L1, ALDOA, B3GNT4, C4orf2, ISG15, PFKFB4, RHOC, SCARB1, SH3GL3, and TRAPPC1.

31. The system of claim 29, wherein said reagent is one or more of SEQ ID NOs: 1-106.

32. A complex comprising one or more genes selected from the group consisting of STC2, PVR, KCTDll, RPL36A, S100A2, PYGL, FGFll, P4HA2, AK2, AK3L1, ALDOA, B3GNT4, C14orf2, C4orfi, C20orf20, CLK3, DDIT3, GAPDH, ISG15, PFKFB4, , RHOC, SCARBl, SH3GL3, TRAPPCl, and UPK1A, wherein each gene is complexed to a methylation status informative reagent.

33. The complex of claim 32, wherein said methylation specific detection reagent is selected from the group consisting of a pair of amplification primers that specifically hybridizes to said gene, an amplification primer that specifically hybridizes to said gene, a restriction enzyme, and sodium bisulfite.

34. The complex of claim 33, wherein said reagent is one or more of SEQ ID NOs: 1-106.

35. The complex of claim 32, wherein said one or more genes are selected from the group consisting of STC2, PVR, KCTDll, RPL36A, S100A2, PYGL, FGFll, P4HA2, AK2, AK3L1, ALDOA, B3GNT4, C4orf2, ISG15, PFKFB4, , RHOC, SCARBl, SH3GL3, and TRAPPCl.