Cervical Cancer Detection

Abstract

Provided herein is a technique for cervical cancer screening.In particular, provided herein is a method, composition and related use for detecting the presence or absence of cervical cancer, cervical precancer (e.g., cervical associated intraepithelial adenocarcinoma, cervical intraepithelial neoplasia) and cervical cancer subtype (e.g., cervical adenocarcinoma, cervical squamous cell carcinoma) from biological sample (e.g., tissue sample (e.g., cervical tissue), blood sample, plasma sample, serum sample, whole blood sample, secretion sample (e.g., cervical secretion, vaginal secretion), organ secretion sample, CSF sample, saliva sample, urine sample or stool sample), or for distinguishing cervical cancer from other types of gynecological cancer (e.g., endometrial and ovarian cancer).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 157,437, filed March 5, 2021, which is incorporated by reference in its entirety.

[0002] Provided herein is a technique for cervical cancer screening.In particular, provided herein is a method, composition and related use for detecting the presence or absence of cervical cancer, cervical precancer (e.g., cervical associated intraepithelial adenocarcinoma, cervical intraepithelial neoplasia) and cervical cancer subtype (e.g., cervical adenocarcinoma, cervical squamous cell carcinoma) from biological sample (e.g., tissue sample (e.g., cervical tissue), blood sample, plasma sample, serum sample, whole blood sample, secretion sample (e.g., cervical secretion, vaginal secretion), organ secretion sample, CSF sample, saliva sample, urine sample or stool sample), or for distinguishing cervical cancer from other types of gynecological cancer (e.g., endometrial and ovarian cancer). [Background technology]

[0003] Cervical cancer (CC) screening methods continue to evolve and are highly sensitive to the presence of cervical dysplasia and CC. However, the specificity of current molecular-based tests (i.e., high-risk HPV only) is limited given the prevalence of high-risk HPV infections that disappear without neoplastic transformation. Therefore, there is an urgent need to provide improved diagnostic tools for detecting cervical cancer from a single biological sample.

[0004] The present invention meets this need. Summary of the Invention

[0005] Provided herein are technologies for cervical cancer screening, in particular, but not limited to, methods, compositions, and related uses for detecting the presence or absence of cervical cancer, cervical precancer (e.g., cervical associated adenocarcinoma in situ, cervical intraepithelial neoplasia), and subtypes of cervical cancer (e.g., cervical adenocarcinoma, cervical squamous cell carcinoma), as well as for distinguishing cervical cancer from other types of gynecological cancer (e.g., endometrial cancer, and ovarian cancer).

[0006] Indeed, as described in Examples I and II, experiments conducted in the course of identifying embodiments of the present invention identified a novel set of methylation variable regions (DMRs) for discriminating DNA from cervical cancer and precancer from non-neoplastic control DNA, and from other types of gynecological cancers (e.g., endometrial and ovarian cancers).

[0007] These studies list and describe 423 novel DNA methylation markers for distinguishing cervical cancer (subtypes of cervical cancer) and precancer from benign cervical samples (see Tables I-IV, VI-VIII, Example I), and for distinguishing cervical cancer from other types of gynecological cancers (endometrial cancer, ovarian cancer, etc.) (see Tables XI and XII, Example II).

[0008] From these 423 novel DNA methylation markers, further experiments identified the following markers and / or panels of markers that can distinguish cervical cancer from benign cervical samples: any of the markers listed in Table I (see Example I); - any of the markers listed in Table III (see Example I); ●MAX.chr6.58147682-58147771, C1ORF114, ASCL1, ARHGAP12, ZNF773, TTYH1, NEUROG3, ZNF781, N XPH1, MAX.chr9.36739811-36739868, NID2, TMEM200C, CRHR2, ABCB1, ZNF69, ATP10A, MAX.chr18.7 3167725-73167817, MAX.chr2.127783183-127783403, CACNA1C, ZNF382, BARHL1, MAX.chr4.8859853-8859939, ST8SIA1, MAX.chr1.98510958-98511049, C2ORF40, SLC9A3, PRDM12, HOPX_C, and KCNQ5 (Example I); C1orf114, MAX.chr6.58147682-58147771, ZNF773, NEUROG3, ASCL1, NID2, ZNF781, CRHR2, and MAX.chr9.36739811-36739868 (see Table VI and Example I); and - ABCB1, c1orf95, CACNA1C, CACNG8, CHST2, ELMO1, EMID2, FBN1_B, FLT3_A, FLT3_B, GLIS1, GPC6, GREM2, JAM2, KCNK12_A, LOC100129620, MAX.chr15.78112404-78112692, MAX.chr19.4584907-4585088, MAX.chr3.69591689-69591784, NCAM1, NT5C1A, ST8SIA3, ZNF382, ZNF419, ZNF69, and ZSCAN18 (Table VIII, Example I).

[0009] From these 423 novel DNA methylation markers, further experiments identified the following markers and / or panels of markers that can distinguish cervical adenocarcinoma from benign cervical samples: ●ABCB1, ARHGAP12, ASCL1, BARHL1, C1orf114, C2orf40, CACNA1C, CRHR2, HOPX_C, KCNQ5, MAX.chr 1.98510968-98511049, MAX.chr18.73167751-73167791, MAX.chr2.127783183-127783403, MAX .chr4.8859853-8859939, MAX.chr6.58147682-58147771, MAX.chr9.36739811-36739868, NEUROG3, NID2, NXPH1, PRDM12, SLC9A3, TMEM200C, TTYH1, ZNF382, ZNF773, and ZNF781 (see Table VII, Example I).

[0010] From these 423 novel DNA methylation markers, further experiments identified the following markers and / or panels of markers that can distinguish cervical squamous cell carcinoma from benign cervical samples: ●ABCB1, ARHGAP12, ASCL1, ATP10A, BARHL1, C1orf114, CACNA1C, CRHR2, MAX.chr1.985109 68-98511049, MAX.chr18.73167751-73167791, MAX.chr2.127783183-127783403, MAX.ch r4.8859853-8859939, MAX.chr6.58147682-58147771, MAX.chr9.36739811-36739868, NEUROG3, NID2, NXPH1, TMEM200C, TTYH1, ZNF382, ZNF69, ZNF773, and ZNF781 (see Table VII, Example I).

[0011] From these 423 novel DNA methylation markers, further experiments identified the following markers and / or panels of markers that can distinguish cervical-associated adenocarcinoma in situ (AIS) from benign cervical samples: MAX.chr6.58147682-58147771, C1ORF114, ASCL1, ZNF773, TTYH1, NEUROG3, ZNF781, MAX.chr9.36739811-36739868, CRHR2, and NID2 (see Table VI and Example I); and ●ABCB1, ARHGAP12, ASCL1, BARHL1, C1orf114, C2orf40, CACNA1C, CRHR2, HOPX_C, KCNQ5, MAX.chr 1.98510968-98511049, MAX.chr18.73167751-73167791, MAX.chr2.127783183-127783403, MAX .chr4.8859853-8859939, MAX.chr6.58147682-58147771, MAX.chr9.36739811-36739868, NEUROG3, NID2, NXPH1, PRDM12, SLC9A3, TMEM200C, TTYH1, ZNF382, ZNF773, and ZNF781 (see Table VII, Example I).

[0012] From these 423 novel DNA methylation markers, further experiments identified the following markers and / or panels of markers that can distinguish cervical intraepithelial neoplasia (CIN) from benign cervical samples: MAX.chr6.58147682-58147771, C1ORF114, ASCL1, ZNF773, TTYH1, NEUROG3, ZNF781, MAX.chr9.36739811-36739868, CRHR2, and NID2 (see Table VI and Example I); and ●ABCB1, ARHGAP12, ASCL1, ATP10A, BARHL1, C1orf114, CACNA1C, CRHR2, MAX.chr1.985109 68-98511049, MAX.chr18.73167751-73167791, MAX.chr2.127783183-127783403, MAX.ch r4.8859853-8859939, MAX.chr6.58147682-58147771, MAX.chr9.36739811-36739868, NEUROG3, NID2, NXPH1, TMEM200C, TTYH1, ZNF382, ZNF69, ZNF773, and ZNF781 (see Table VII, Example I).

[0013] From these 423 novel DNA methylation markers, further experiments identified the following markers and / or panels of markers that can distinguish cervical cancer from endometrial and / or ovarian cancer: • ABCB1, c1orf95, CACNA1C, CACNG8, CHST2, ELMO1, EMID2, FBN1_B, FLT3_A, FLT3_B, GLIS1, GPC6, GREM2, JAM2, KCNK12_A, LOC100129620, MAX.chr15.78112404-78112692, MAX.chr19.4584907-4585088, MAX.chr3.69591689-69591784, NCAM1, NT5C1A, ST8SIA3, ZNF382, ZNF419, ZNF69, and ZSCAN18 (Table VIII, Example I); any of the markers listed in Table X (see Example I); and - AK5, RABC3, ZNF491, ZNF610, ZNF91, ZNF480, TRPC3_B, and ELMOD1 (see Tables X and XI, Example II).

[0014] As described herein, the present technology provides a large number of methylated DNA markers (MDMs) and their subsets (e.g., a set of 2, 3, 4, 5, 6, 7, 8, 20, 50, 100, 150, 200, 300, 400, 423 markers) that have a high discrimination rate for cervical cancer, various types of cervical cancer (e.g., cervical adenocarcinoma, cervical squamous cell carcinoma), various types of cervical precancer (e.g., cervical associated intraepithelial adenocarcinoma, cervical intraepithelial neoplasia), and to discriminate cervical cancer from other types of gynecological cancer (e.g., endometrial cancer and ovarian cancer). Cervical intraepithelial neoplasia (CIN) can be characterized as CIN1, which refers to abnormal cells affecting about 1 / 3 of the thickness of the cervical epithelium, CIN2, which refers to abnormal cells affecting about 2 / 3 to 2 / 3 of the thickness of the cervical epithelium, and CIN3, which refers to abnormal cells affecting more than 2 / 3 of the cervical epithelium.

[0015] In certain embodiments, the disclosure provides a method for characterizing a biological sample, comprising measuring a methylation level of one or more methylation markers selected from Tables I, III, and X in the biological sample, wherein measuring the methylation level of the one or more methylation markers comprises treating DNA from the biological sample with a reagent that modifies DNA in a methylation-specific manner.

[0016] In some embodiments, the biological sample is from a human. In some embodiments, the human subject has or is suspected of having cervical cancer, cervical cancer subtypes, and / or cervical pre-cancer.

[0017] In some embodiments, the biological sample is selected from a tissue sample, a blood sample, a plasma sample, a serum sample, a whole blood sample, a secretion sample (e.g., cervical secretion, vaginal secretion), an organ secretion sample, a cerebrospinal fluid (CSF) sample, a saliva sample, a urine sample, and a stool sample. In some embodiments, the tissue sample is a cervical tissue sample. In some embodiments, the cervical tissue sample further comprises one or more of vaginal tissue, vaginal cells, endometrial tissue, endometrial cells, ovarian tissue, and ovarian cells. In some embodiments, the secretion sample is a cervical secretion sample. In some embodiments, the cervical secretion sample further comprises one or more of vaginal secretion, endometrial secretion, and ovarian secretion. In some embodiments, the biological sample is collected with a collection device having an absorbent member capable of collecting the biological sample when contacted with a body area. In some embodiments, the absorbent member is a sponge having a shape and size suitable for insertion into a body orifice. In some embodiments, the collection device is a tampon (e.g., a standard tampon), an irrigator that releases fluid into the vagina and recollects the fluid (e.g., a Pantarhei screener), a cervical brush (e.g., a brush that is inserted into the vagina and rotated to collect cells), a Fournier cervical self-sampling device (a tampon-shaped plastic stick), or a cotton swab.

[0018] In some embodiments, the measured methylation level of the one or more methylation markers is compared to the methylation level of the corresponding one or more methylation markers in a control sample that is free of cervical cancer.

[0019] In some embodiments, the method further comprises determining that the individual has cervical cancer if the methylation level measured at the one or more methylation markers is higher than the methylation level measured in the respective control sample. In some embodiments, the method further comprises determining that the individual has cervical cancer, the one or more methylation markers are selected from one of the following groups: • a methylation marker listed in Table I and / or Table III; ●MAX.chr6.58147682-58147771, C1ORF114, ASCL1, ARHGAP12, ZNF773, TTYH1, NEUROG3, ZNF781, N XPH1, MAX.chr9.36739811-36739868, NID2, TMEM200C, CRHR2, ABCB1, ZNF69, ATP10A, MAX.chr18.7 3167725-73167817, MAX.chr2.127783183-127783403, CACNA1C, ZNF382, BARHL1, MAX.chr4.8859853-8859939, ST8SIA1, MAX.chr1.98510958-98511049, C2ORF40, SLC9A3, PRDM12, HOPX_C, and KCNQ5 (Example I); C1orf114, MAX.chr6.58147682-58147771, ZNF773, NEUROG3, ASCL1, NID2, ZNF781, CRHR2, and MAX.chr9.36739811-36739868 (see Table VI and Example I); and - ABCB1, c1orf95, CACNA1C, CACNG8, CHST2, ELMO1, EMID2, FBN1_B, FLT3_A, FLT3_B, GLIS1, GPC6, GREM2, JAM2, KCNK12_A, LOC100129620, MAX.chr15.78112404-78112692, MAX.chr19.4584907-4585088, MAX.chr3.69591689-69591784, NCAM1, NT5C1A, ST8SIA3, ZNF382, ZNF419, ZNF69, and ZSCAN18 (Table VIII, Example I).

[0020] In some embodiments, the method further comprises determining that the individual has a subtype of cervical cancer. In some embodiments, the subtype of cervical cancer is selected from cervical adenocarcinoma and cervical squamous cell carcinoma. In some embodiments, the method further comprises determining that the individual has a subtype of cervical cancer, the one or more methylation markers are selected from one of the following groups: ●ABCB1, ARHGAP12, ASCL1, BARHL1, C1orf114, C2orf40, CACNA1C, CRHR2, HOPX_C, KCNQ5, MAX.chr 1.98510968-98511049, MAX.chr18.73167751-73167791, MAX.chr2.127783183-127783403, MAX .chr4.8859853-8859939, MAX.chr6.58147682-58147771, MAX.chr9.36739811-36739868, NEUROG3, NID2, NXPH1, PRDM12, SLC9A3, TMEM200C, TTYH1, ZNF382, ZNF773, and ZNF781 (see Table VII, Example I).

[0021] In some embodiments, the method further comprises determining that the individual has a cervical cancer precancer. In some embodiments, the cervical cancer precancer is selected from cervical associated intraepithelial adenocarcinoma and cervical intraepithelial neoplasia. In some embodiments, the method further comprises determining that the individual has a cervical cancer precancer, the one or more methylation markers are selected from one of the following groups: MAX.chr6.58147682-58147771, C1ORF114, ASCL1, ZNF773, TTYH1, NEUROG3, ZNF781, MAX.chr9.36739811-36739868, CRHR2, and NID2 (see Table VI and Example I); and ●ABCB1, ARHGAP12, ASCL1, BARHL1, C1orf114, C2orf40, CACNA1C, CRHR2, HOPX_C, KCNQ5, MAX.chr 1.98510968-98511049, MAX.chr18.73167751-73167791, MAX.chr2.127783183-127783403, MAX .chr4.8859853-8859939, MAX.chr6.58147682-58147771, MAX.chr9.36739811-36739868, NEUROG3, NID2, NXPH1, PRDM12, SLC9A3, TMEM200C, TTYH1, ZNF382, ZNF773, and ZNF781 (see Table VII, Example I).

[0022] In some embodiments, the measured methylation levels of the one or more methylation markers are compared to the methylation levels of corresponding one or more methylation markers in the endometrial cancer sample and the ovarian cancer sample. In some embodiments, the method further comprises distinguishing cervical cancer from endometrial cancer and / or ovarian cancer. In some embodiments, the method further comprises distinguishing cervical cancer from endometrial cancer and / or ovarian cancer, the one or more methylation markers are selected from one of the following groups: • Markers listed in Table X; - ABCB1, c1orf95, CACNA1C, CACNG8, CHST2, ELMO1, EMID2, FBN1_B, FLT3_A, FLT3_B, GLIS1, GPC6, GREM2, JAM2, KCNK12_A, LOC100129620, MAX.chr15.78112404-78112692, MAX.chr19.4584907-4585088, MAX.chr3.69591689-69591784, NCAM1, NT5C1A, ST8SIA3, ZNF382, ZNF419, ZNF69, and ZSCAN18 (Table VIII, Example I); and - AK5, RABC3, ZNF491, ZNF610, ZNF91, ZNF480, TRPC3_B, and ELMOD1 (see Tables X and XI, Example II).

[0023] In some embodiments, the reagent that modifies DNA in a methylation-specific manner is a borane reducing agent. In some embodiments, the borane reducing agent is 2-picoline borane. In some embodiments, the reagent that modifies DNA in a methylation-specific manner comprises one or more of a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent. In some embodiments, the reagent that modifies DNA in a methylation-specific manner is a bisulfite reagent, and said treating produces bisulfite-treated DNA.

[0024] In some embodiments, the treated DNA is amplified using a set of primers specific for one or more markers. In some embodiments, the set of primers specific for one or more methylation markers is selected from the group described in Tables V and XII. In some embodiments, the set of primers specific for one or more methylation markers can bind to an amplicon bound by a primer sequence for a particular methylation marker gene described in Tables V and XII, and the amplicon bound by a primer sequence for a methylation marker gene described in Tables V and XII is at least a portion of a gene region of a methylation marker described in Tables I, III, and X. In some embodiments, the set of primers specific for one or more methylation markers is a set of primers that specifically bind to at least a portion of a gene region that includes the chromosomal coordinates of a methylation marker described in Tables I, III, and X.

[0025] In some embodiments, measuring the methylation level of one or more methylation markers comprises multiplex amplification. In some embodiments, measuring the methylation level of one or more methylation markers comprises using one or more methods selected from the group consisting of methylation specific PCR, quantitative methylation specific PCR, methylation specific DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, flap endonuclease assay, PCR-flap assay, and bisulfite genomic sequencing PCR. In some embodiments, measuring the methylation level of one or more methylation markers comprises measuring the methylation of CpG sites of one or more methylation markers. In some embodiments, the CpG sites are in coding or regulatory regions. In some embodiments, the one or more methylation markers are described by the genomic coordinates shown in Tables I, III, and X.

[0026] In certain embodiments, the present disclosure provides a method for preparing a deoxyribonucleic acid (DNA) fraction from a biological sample useful for analyzing one or more genetic loci involved in one or more chromosomal abnormalities, comprising: (a) extracting genomic DNA from a biological sample; (b) (i) treating the extracted genomic DNA with a reagent that modifies DNA in a methylation-specific manner; (ii) amplifying the treated genomic DNA using separate primers specific for one or more of the methylation markers listed in Tables I, III, and X; generating a fraction of the extracted genomic DNA by (c) analyzing one or more loci in the generated fraction of the extracted genomic DNA by measuring a methylation level for each of the one or more methylation markers.

[0027] In some embodiments, the reagent that modifies DNA in a methylation-specific manner is a borane reducing agent. In some embodiments, the borane reducing agent is 2-picoline borane. In some embodiments, the reagent that modifies DNA in a methylation-specific manner comprises one or more of a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent. In some embodiments, the reagent that modifies DNA in a methylation-specific manner is a bisulfite reagent, and said treating produces bisulfite-treated DNA.

[0028] In some embodiments, the set of primers specific for one or more methylation markers is selected from the group set forth in Tables V and XII. In some embodiments, the set of primers specific for one or more methylation markers can bind to an amplicon bound by primer sequences for a particular methylation marker gene set forth in Tables V and XII, and the amplicon bound by primer sequences for a methylation marker gene set forth in Tables V and XII is at least a portion of a gene region of a methylation marker set forth in Tables I, III, and X. In some embodiments, the set of primers specific for one or more methylation markers is a set of primers that specifically bind to at least a portion of a gene region that includes the chromosomal coordinates of a methylation marker set forth in Tables I, III, and X.

[0029] In some embodiments, measuring the methylation level of one or more methylation markers comprises multiplex amplification. In some embodiments, measuring the methylation level of one or more methylation markers comprises using one or more methods selected from the group consisting of methylation specific PCR, quantitative methylation specific PCR, methylation specific DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, flap endonuclease assay, PCR-flap assay, and bisulfite genomic sequencing PCR. In some embodiments, measuring the methylation level of one or more methylation markers comprises measuring the methylation of CpG sites of one or more methylation markers. In some embodiments, the CpG sites are in coding or regulatory regions. In some embodiments, the one or more methylation markers are described by the genomic coordinates shown in Tables I, III, and X.

[0030] In some embodiments, the biological sample is selected from a tissue sample, a blood sample, a plasma sample, a serum sample, a whole blood sample, a secretion sample (e.g., cervical secretions, vaginal secretions), an organ secretion sample, a cerebrospinal fluid (CSF) sample, a saliva sample, a urine sample, and a stool sample. In some embodiments, the tissue sample is a cervical tissue sample. In some embodiments, the cervical tissue sample further comprises one or more of vaginal tissue, vaginal cells, endometrial tissue, endometrial cells, ovarian tissue, and ovarian cells. In some embodiments, the secretion sample is a cervical secretion sample. In some embodiments, the cervical secretion sample further comprises one or more of vaginal secretions, endometrial secretions, and ovarian secretions. In some embodiments, the biological sample is collected with a collection device having an absorbent member capable of collecting tissue and / or cells when contacted with a body area. In some embodiments, the absorbent member is a sponge having a shape and size suitable for insertion into a body orifice. In some embodiments, the collection device is a tampon (e.g., a standard tampon), an irrigator that releases fluid into the vagina and recollects the fluid (e.g., a Pantarhei screener), a cervical brush (e.g., a brush that a woman inserts into her vagina and rotates to collect cells), a Fournier cervical self-sampling device (a tampon-shaped plastic stick), or a cotton swab.

[0031] In some embodiments, the biological sample is from a human. In some embodiments, the human subject has or is suspected of having cervical cancer, cervical cancer subtypes, and / or cervical pre-cancer.

[0032] In some embodiments, the one or more methylation markers are selected from one of the following groups: ●MAX.chr6.58147682-58147771, C1ORF114, ASCL1, ARHGAP12, ZNF773, TTYH1, NEUROG3, ZNF781, NXP H1, MAX.chr9.36739811-36739868, NID2, TMEM200C, CRHR2, ABCB1, ZNF69, ATP10A, MAX.chr18.73167 725-73167817, MAX.chr2.127783183-127783403, CACNA1C, ZNF382, BARHL1, MAX.chr4.8859853-8859939, ST8SIA1, MAX.chr1.98510958-98511049, C2ORF40, SLC9A3, PRDM12, HOPX_C, and KCNQ5 (Example I); • C1orf114, MAX.chr6.58147682-58147771, ZNF773, NEUROG3, ASCL1, NID2, ZNF781, CRHR2, and MAX.chr9.36739811-36739868 (see Table VI and Example I); ●ABCB1, ARHGAP12, ASCL1, BARHL1, C1orf114, C2orf40, CACNA1C, CRHR2, HOPX_C, KCNQ5, MAX.chr 1.98510968-98511049, MAX.chr18.73167751-73167791, MAX.chr2.127783183-127783403, MAX .chr4.8859853-8859939, MAX.chr6.58147682-58147771, MAX.chr9.36739811-36739868, NEUROG3, NID2, NXPH1, PRDM12, SLC9A3, TMEM200C, TTYH1, ZNF382, ZNF773, and ZNF781 (see Table VII, Example I); - ABCB1, c1orf95, CACNA1C, CACNG8, CHST2, ELMO1, EMID2, FBN1_B, FLT3_A, FLT3_B, GLIS1, GPC6, GREM2, JAM2, KCNK12_A, LOC100129620, MAX.chr15.78112404-78112692, MAX.chr19.4584907-4585088, MAX.chr3.69591689-69591784, NCAM1, NT5C1A, ST8SIA3, ZNF382, ZNF419, ZNF69, and ZSCAN18 (Table VIII, Example I); and - AK5, RABC3, ZNF491, ZNF610, ZNF91, ZNF480, TRPC3_B, and ELMOD1 (see Tables X and XI, Example II).

[0033] In certain embodiments, the technology relates to assessing the presence and methylation status of one or more of the MDMs described herein in a biological sample (e.g., a tissue sample (e.g., a cervical sample), a blood sample, a plasma sample, a serum sample, a whole blood sample, a secretion sample (e.g., a cervical secretion, a vaginal secretion), an organ secretion sample, a CSF sample, a saliva sample, a urine sample, or a stool sample). These MDMs include one or more of the methylation variable regions (DMRs) discussed herein, e.g., as shown in Tables I, III, and X. The methylation status is assessed in embodiments of the technology. Thus, the technology provided herein is not limited in the method of measuring the methylation status of a gene, and thus the methylation status of a gene can be measured by any method known in the art.

[0034] Also provided herein are compositions and kits for carrying out these methods. For example, in some embodiments, one or more MDM-specific reagents (e.g., primers, probes) are provided, either alone or in sets (e.g., a set of primer pairs for amplifying multiple markers). Additional reagents for carrying out detection assays may also be provided (e.g., QuARTS, PCR, enzymes for sequencing, buffers, positive and negative controls, bisulfite, ten-eleven translocation (TET) enzymes (e.g., human TET1, human TET2, human TET3, mouse TET1, mouse TET2, mouse TET3, Naegleria TET (NgTET), Coprinopsis cinerea (CcTET)), or variants thereof), organoboranes, or other assays). In some embodiments, the kits include reagents capable of modifying DNA in a methylation-specific manner (e.g., methylation-sensitive restriction enzymes, methylation-dependent restriction enzymes, and bisulfite reagents) (e.g., methylation-sensitive restriction enzymes, methylation-dependent restriction enzymes, ten-eleven translocation (TET) enzymes (e.g., human TET1, human TET2, human TET3, mouse TET1, mouse TET2, mouse TET3, Naegleria TET (NgTET), Coprinopsis cinerea (CcTET)), or variants thereof), organoborane). In some embodiments, kits are provided that include one or more reagents necessary, sufficient, or useful for carrying out the methods. Reaction mixtures including these reagents are also provided. Master mix reagent sets including multiple reagents that can be added to each other and / or to a test sample to complete a reaction mixture are further provided. In some embodiments, the kit includes a control nucleic acid that includes one or more sequences of DMR1-423 (from Tables I, III, and X) and has a methylation status associated with a subject having cervical cancer, a cervical cancer subtype (e.g., cervical adenocarcinoma, cervical squamous cell carcinoma), and / or a cervical precancer (e.g., adenocarcinoma in situ, cervical intraepithelial neoplasia).In some embodiments, the kit comprises a sample collector for obtaining a sample from a subject (e.g., a tissue sample (e.g., a cervical sample), a blood sample, a plasma sample, a serum sample, a whole blood sample, a secretion sample (e.g., cervical secretions, vaginal secretions), an organ secretion sample, a CSF sample, a saliva sample, a urine sample, and a stool sample). In some embodiments, the kit comprises an oligonucleotide as described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] definition To facilitate understanding of the present technology, several terms and phrases are defined below. Further definitions are set forth throughout the detailed description.

[0036] Throughout this specification and claims, the following terms have the meanings expressly associated therewith, unless the context clearly indicates otherwise. The phrase "in one embodiment" as used herein may refer to the same embodiment, but does not necessarily refer to the same embodiment. Additionally, the phrase "in another embodiment" as used herein does not necessarily refer to different embodiments, but may. Thus, as described below, various embodiments of the invention can be readily combined without departing from the scope or spirit of the invention.

[0037] Additionally, as used herein, unless the context clearly indicates otherwise, the term "or" is an inclusive "or" operator and is equivalent to the term "and / or." The term "based on" is not exclusive and allows for based on additional unlisted factors, unless the context clearly dictates otherwise. Additionally, throughout this specification, the meanings of "a," "an," and "the" include plural referents. The meaning of "in" includes "in" and "on."

[0038] The transitional phrase "consisting essentially of," when used in the claims of this application, limits the scope of the claim to certain substances or steps "and which do not materially affect the basic and novel characteristic(s)" of the claimed invention, as stated in In re Herz, 537 F.2d 549,551-52,190 USPQ 461,463 (CCPA 1976). For example, a composition "consisting essentially of" recited elements may contain unrecited contaminants at levels that are present but do not alter the function of the recited composition when compared to a pure composition, i.e., a composition "consisting of" the recited ingredients.

[0039] The term "one or more" as used herein refers to a number greater than 1. For example, the term "one or more" includes any of the following: 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or more than 12, 13 or more, 14 or more, 15 or more, 20 or more, 50 or more, 100 or more, or more.

[0040] "1 or more but less than a larger number," "2 or more but less than a larger number," "3 or more but less than a larger number," "4 or more but less than a larger number," "5 or more but less than a larger number," "6 or more but less than a larger number," "7 or more but less than a larger number," "8 or more but less than a larger number," "9 or more but less than a larger number," "10 or more but less than a larger number," "11 or more but less than a larger number," "12 or more but less than a larger number," "13 or more but less than a larger number," "14 or more but less than a larger number," or "15 or more but less than a larger number" is not limited to a certain larger number. For example, the larger number can be 10,000, 1,000, 100, 50, etc. For example, the larger number may be about 64 (e.g., 64, 63, 62, 61, 60, 59, 58, 57, 56, 55), 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 32, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2). For example, the larger number may be about 423.

[0041] The terms "one or more methylation markers" or "one or more DMRs" or "one or more genes" or "one or more markers" or "multiple methylation markers" or "multiple markers" or "multiple genes" or "multiple DMRs" are likewise not limited to a specific number combination. Indeed, any number of combinations of methylation markers are contemplated (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, 1-19, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-26, 1-27, 1-28, 1-29, 1-30, 1-31, 1-32, 1-33, 1-34, 1-35, 1-36, 1-37, 1-38, 1-39, 1 ~40, 1~41, 1~42, 1~43, 1~44, 1~45, 1~46, 1~47, 1~48, 1~49, 1~50, 1~51, 1~52, 1~53, 1~54, 1~55, 1~56, 1~57, 1~58, 1~59, 1~60, 1~61, 1~62, 1~63, 1~64 methylation markers) (e.g., 2~3, 2~4, 2~5, 2~6, 2~7, 2~8, 2~9, 2~10, 2~11, 2~12, 2~13, 2~14, 2~15, 2~16, 2~17, 2~18, 2~19, 2-20, 2-21, 2-22, 2-23, 2-24, 2-25, 2-26, 2-27, 2-28, 2-29, 2-30, 2-31, 2-32, 2-33, 2-34, 2-35, 2-36, 2-37, 2-38, 2-39, 2-40, 2-41, 2-42, 2-43, 2-44, 2-45, 2-46, 2-47, 2-48, 2-49, 2-50, 2-51, 2-52, 2-53, 2-54, 2-55, 2-56, 2-57, 2-58, 2-59, 2-60, 2-61, 2-62, 2- 63, 2-64) (e.g., 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-11, 3-12, 3-13, 3-14, 3-15, 3-16, 3-17, 3-18, 3-19, 3-20, 3-21, 3-22, 3-23, 3-24, 3-25, 3-26, 3-27, 3-28, 3-29, 3-30, 3-31, 3-32, 3-33, 3-34, 3-35, 3-36, 3-37, 3-38, 3-39, 3-40, 3-41, 3-42, 3-43, 3-44, 3-45,3-46, 3-47, 3-48, 3-49, 3-50, 3-51, 3-52, 3-53, 3-54, 3-55, 3-56, 3-57, 3-58, 3-59, 3-60, 3-61, 3-62, 3-63, 3-64) (e.g., 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4~11, 4~12, 4~13, 4~14, 4~15, 4~16, 4~17, 4~18, 4~19, 4~20, 4~21, 4~22, 4~23, 4~24, 4~25, 4~26, 4~27, 4~28, 4~29, 4~30, 4~31, 4~32, 4~33, 4~34, 4~35, 4~36, 4~37, 4~38, 4~39, 4~40, 4~41, 4~42, 4~43, 4~44, 4~45, 4~46, 4~47, 4~48, 4~49, 4~50, 4~51, 4~52, 4~53, 4~54, 4~55, 4~56, 4~57, 4~58, 4~59, 4~60, 4-61, 4-62, 4-63, 4-64) (e.g., 5-6, 5-7, 5-8, 5-9, 5-10, 5-11, 5-12, 5-13, 5-14, 5-15, 5-16, 5-17, 5-18, 5-19, 5-20, 5-21, 5-22, 5-23, 5-24, 5-25, 5-26 , 5~27, 5~28, 5~29, 5~30, 5~31, 5~32, 5~33, 5~34, 5~35, 5~36, 5~37, 5~38, 5~39, 5~40, 5~41, 5~42, 5~43, 5~44, 5~45, 5~46, 5~47, 5~48, 5~49, 5~50, 5~51 , 5-52, 5-53, 5-54, 5-55, 5-56, 5-57, 5-58, 5-59, 5-60, 5-61, 5-62, 5-63, 5-64) (e.g., 6-7, 6-8, 6-9, 6-10, 6-11, 6-12, 6-13, 6-14, 6-15, 6-16, 6-17, 6- 18, 6~19, 6~20, 6~21, 6~22, 6~23, 6~24, 6~25, 6~26, 6~27, 6~28, 6~29, 6~30, 6~31, 6~32, 6~33, 6~34, 6~35, 6~36, 6~37, 6~38, 6~39, 6~40, 6~41, 6~42, 6~ 43, 6-44, 6-45, 6-46, 6-47, 6-48, 6-49, 6-50, 6-51, 6-52, 6-53, 6-54, 6-55, 6-56, 6-57, 6-58, 6-59, 6-60, 6-61, 6-62, 6-63, 6-64) (e.g., 7-8, 7-9, 7-10,7~11, 7~12, 7~13, 7~14, 7~15, 7~16, 7~17, 7~18, 7~19, 7~20, 7~21, 7~22, 7~23, 7~24, 7~25, 7~26, 7~27, 7~28, 7~29, 7~30, 7~31, 7~32, 7~33, 7~34, 7~35, 7~36, 7~37, 7~38, 7~39, 7~40, 7~41, 7~42, 7~43, 7~44, 7~45, 7~46, 7~47, 7~48, 7~49, 7~50, 7~51, 7~52, 7~53, 7~54, 7~55, 7~56, 7~57, 7~58, 7~59, 7~60, 7-61, 7-62, 7-63, 7-64) (e.g., 8-9, 8-10, 8-11, 8-12, 8-13, 8-14, 8-15, 8-16, 8-17, 8-18, 8-19, 8-20, 8-21, 8-22, 8-23, 8-24, 8-25, 8-26, 8-27, 8-28, 8 ~29, 8~30, 8~31, 8~32, 8~33, 8~34, 8~35, 8~36, 8~37, 8~38, 8~39, 8~40, 8~41, 8~42, 8~43, 8~44, 8~45, 8~46, 8~47, 8~48, 8~49, 8~50, 8~51, 8~52, 8~53, 8 ~54, 8~55, 8~56, 8~57, 8~58, 8~59, 8~60, 8~61, 8~62, 8~63, 8~64) (e.g., 9~10, 9~11, 9~12, 9~13, 9~14, 9~15, 9~16, 9~17, 9~18, 9~19, 9~20, 9~21, 9~22, 9 ~23, 9~24, 9~25, 9~26, 9~27, 9~28, 9~29, 9~30, 9~31, 9~32, 9~33, 9~34, 9~35, 9~36, 9~37, 9~38, 9~39, 9~40, 9~41, 9~42, 9~43, 9~44, 9~45, 9~46, 9~47, 9 ~48, 9~49, 9~50, 9~51, 9~52, 9~53, 9~54, 9~55, 9~56, 9~57, 9~58, 9~59, 9~60, 9~61, 9~62, 9~63, 9~64) (e.g., 10~11, 10~12, 10~13, 10~14, 10~15, 10~16, 10~17, 10~18, 10~19, 10~20, 10~21, 10~22, 10~23, 10~24, 10~25, 10~26, 10~27, 10~28, 10~29, 10~30, 10~31, 10~32, 10~33, 10~34, 10~35, 10~36, 10~37,10~38, 10~39, 10~40, 10~41, 10~42, 10~43, 10~44, 10~45, 10~46, 10~47, 10~48, 10~49, 10~50, 10~51, 10~52, 10~53, 10~54, 10~55, 10~56, 10~57, 10~5 8, 10-59, 10-60, 10-61, 10-62, 10-63, 10-64) (e.g., 11-12, 11-13, 11-14, 11-15, 11-16, 11-17, 11-18, 11-19, 11-20, 11-21, 11-22, 11-23, 11-24, 11-25, 11-26, 11-27, 11-28, 11-29, 11-30, 11-31, 11-32, 11-33, 11-34, 11-35, 11-36, 11-37, 11-38, 11-39, 11-40, 5, 11~26, 11~27, 11~28, 11~29, 11~30, 11~31, 11~32, 11~33, 11~34, 11~35, 11~36, 11~37, 11~38, 11~39, 11~40, 11~41, 11~42, 11~43, 11~44, 11~45, 11~ 46, 11-47, 11-48, 11-49, 11-50, 11-51, 11-52, 11-53, 11-54, 11-55, 11-56, 11-57, 11-58, 11-59, 11-60, 11-61, 11-62, 11-63, 11-64) (e.g., 12-13, 12- 14, 12~15, 12~16, 12~17, 12~18, 12~19, 12~20, 12~21, 12~22, 12~23, 12~24, 12~25, 12~26, 12~27, 12~28, 12~29, 12~30, 12~31, 12~32, 12~33, 12~34, 12 ~35, 12~36, 12~37, 12~38, 12~39, 12~40, 12~41, 12~42, 12~43, 12~44, 12~45, 12~46, 12~47, 12~48, 12~49, 12~50, 12~51, 12~52, 12~53, 12~54, 12~55, 1 2-56, 12-57, 12-58, 12-59, 12-60, 12-61, 12-62, 12-63, 12-64) (e.g., 13-14, 13-15, 13-16, 13-17, 13-18, 13-19, 13-20, 13-21, 13-22, 13-23, 13-24, 1 3~25, 13~26, 13~27, 13~28, 13~29, 13~30, 13~31, 13~32, 13~33, 13~34, 13~35, 13~36, 13~37, 13~38, 13~39, 13~40, 13~41, 13~42, 13~43, 13~44, 13~45,13~46, 13~47, 13~48, 13~49, 13~50, 13~51, 13~52, 13~53, 13~54, 13~55, 13~56, 13~57, 13~58, 13~59, 13~60, 13~61, 13~62, 13~63, 13~64) (e.g., 14~15 , 14-16, 14-17, 14-18, 14-19, 14-20, 14-21, 14-22, 14-23, 14-24, 14-25, 14-26, 14-27, 14-28, 14-29, 14-30, 14-31, 14-32, 14-33, 14-34, 14-35, 14-36, 14-37, 14-39, 14-40, 14-41, 14-42, 14-43, 14-44, 14-45, 14-46, 14-47, 14-48, 14-49, 14-50, 14-51, 14-52, 6, 14~37, 14~38, 14~39, 14~40, 14~41, 14~42, 14~43, 14~44, 14~45, 14~46, 14~47, 14~48, 14~49, 14~50, 14~51, 14~52, 14~53, 14~54, 14~55, 14~56, 14~ 57, 14-58, 14-59, 14-60, 14-61, 14-62, 14-63, 14-64) (e.g., 15-16, 15-17, 15-18, 15-19, 15-20, 15-21, 15-22, 15-23, 15-24, 15-25, 15-26, 15-27, 15- 28, 15-29, 15-30, 15-31, 15-32, 15-33, 15-34, 15-35, 15-36, 15-37, 15-38, 15-39, 15-40, 15-41, 15-42, 15-43, 15-44, 15-45, 15-46, 15-47, 15-48, 15 ~49, 15~50, 15~51, 15~52, 15~53, 15~54, 15~55, 15~56, 15~57, 15~58, 15~59, 15~60, 15~61, 15~62, 15~63, 15~64) (e.g., 16~17, 16~18, 16~19, 16~20, 16 ~21, 16~22, 16~23, 16~24, 16~25, 16~26, 16~27, 16~28, 16~29, 16~30, 16~31, 16~32, 16~33, 16~34, 16~35, 16~36, 16~37, 16~38, 16~39, 16~40, 16~41, 1 6~42, 16~43, 16~44, 16~45, 16~46, 16~47, 16~48, 16~49, 16~50, 16~51, 16~52, 16~53, 16~54, 16~55, 16~56, 16~57, 16~58, 16~59, 16~60, 16~61, 16~62,16-63, 16-64) (e.g., 17-18, 17-19, 17-20, 17-21, 17-22, 17-23, 17-24, 17-25, 17-26, 17-27, 17-28, 17-29, 17-30, 17-31, 17-32, 17-33, 17-34, 17-35, 5, 17-36, 17-37, 17-38, 17-39, 17-40, 17-41, 17-42, 17-43, 17-44, 17-45, 17-46, 17-47, 17-48, 17-49, 17-50, 17-51, 17-52, 17-53, 17-54, 17-55, 17 ~56, 17~57, 17~58, 17~59, 17~60, 17~61, 17~62, 17~63, 17~64) (e.g., 18~19, 18~20, 18~21, 18~22, 18~23, 18~24, 18~25, 18~26, 18~27, 18~28, 18~29, 1 8~30, 18~31, 18~32, 18~33, 18~34, 18~35, 18~36, 18~37, 18~38, 18~39, 18~40, 18~41, 18~42, 18~43, 18~44, 18~45, 18~46, 18~47, 18~48, 18~49, 18~50 , 18-51, 18-52, 18-53, 18-54, 18-55, 18-56, 18-57, 18-58, 18-59, 18-60, 18-61, 18-62, 18-63, 18-64) (e.g., 19-20, 19-21, 19-22, 19-23, 19-24, 19-25 , 19~26, 19~27, 19~28, 19~29, 19~30, 19~31, 19~32, 19~33, 19~34, 19~35, 19~36, 19~37, 19~38, 19~39, 19~40, 19~41, 19~42, 19~43, 19~44, 19~45, 19~ 46, 19-47, 19-48, 19-49, 19-50, 19-51, 19-52, 19-53, 19-54, 19-55, 19-56, 19-57, 19-58, 19-59, 19-60, 19-61, 19-62, 19-63, 19-64) (e.g., 20-21, 20- 22, 20-23, 20-24, 20-25, 20-26, 20-27, 20-28, 20-29, 20-30, 20-31, 20-32, 20-33, 20-34, 20-35, 20-36, 20-37, 20-38, 20-39, 20-40, 20-41, 20-42, 2 0~43, 20~44, 20~45, 20~46, 20~47, 20~48, 20~49, 20~50, 20~51, 20~52, 20~53, 20~54, 20~55, 20~56, 20~57, 20~58, 20~59, 20~60, 20~61, 20~62, 20~63,20-64) (e.g., 21-22, 21-23, 21-24, 21-25, 21-26, 21-27, 21-28, 21-29, 21-30, 21-31, 21-32, 21-33, 21-34, 21-35, 21-36, 21-37, 21-38, 21-39, 21-40 , 21-41, 21-42, 21-43, 21-44, 21-45, 21-46, 21-47, 21-48, 21-49, 21-50, 21-51, 21-52, 21-53, 21-54, 21-55, 21-56, 21-57, 21-58, 21-59, 21-60, 21-61, 1, 21-62, 21-63, 21-64) (e.g., 22-23, 22-24, 22-25, 22-26, 22-27, 22-28, 22-29, 22-30, 22-31, 22-32, 22-33, 22-34, 22-35, 22-36, 22-37, 22-38, 22-39, 22-40, 22-41, 22-42, 22-43, 22-44, 22-45, 22-46, 22-47, 22-48, 22-49, 22-50, 22-51, 22-52, 22-53, 22-54, 9, 22~40, 22~41, 22~42, 22~43, 22~44, 22~45, 22~46, 22~47, 22~48, 22~49, 22~50, 22~51, 22~52, 22~53, 22~54, 22~55, 22~56, 22~57, 22~58, 22~59, 22~ 60, 22-61, 22-62, 22-63, 22-64) (e.g., 23-24, 23-25, 23-26, 23-27, 23-28, 23-29, 23-30, 23-31, 23-32, 23-33, 23-34, 23-35, 23-36, 23-37, 23-38, 23 ~39, 23~40, 23~41, 23~42, 23~43, 23~44, 23~45, 23~46, 23~47, 23~48, 23~49, 23~50, 23~51, 23~52, 23~53, 23~54, 23~55, 23~56, 23~57, 23~58, 23~59, 2 3-60, 23-61, 23-62, 23-63, 23-64) (e.g., 24-25, 24-26, 24-27, 24-28, 24-29, 24-30, 24-31, 24-32, 24-33, 24-34, 24-35, 24-36, 24-37, 24-38, 24-39, 2 4~40, 24~41, 24~42, 24~43, 24~44, 24~45, 24~46, 24~47, 24~48, 24~49, 24~50, 24~51, 24~52, 24~53, 24~54, 24~55, 24~56, 24~57, 24~58, 24~59, 24~60,24~61, 24~62, 24~63, 24~64) (e.g., 25~26, 25~27, 25~28, 25~29, 25~30, 25~31, 25~32, 25~33, 25~34, 25~35, 25~36, 25~37, 25~38, 25~39, 25~40, 25~41 , 25~42, 25~43, 25~44, 25~45, 25~46, 25~47, 25~48, 25~49, 25~50, 25~51, 25~52, 25~53, 25~54, 25~55, 25~56, 25~57, 25~58, 25~59, 25~60, 25~61, 25~6 2, 25-63, 25-64) (e.g., 26-27, 26-28, 26-29, 26-30, 26-31, 26-32, 26-33, 26-34, 26-35, 26-36, 26-37, 26-38, 26-39, 26-40, 26-41, 26-42, 26-43, 26- 44, 26~45, 26~46, 26~47, 26~48, 26~49, 26~50, 26~51, 26~52, 26~53, 26~54, 26~55, 26~56, 26~57, 26~58, 26~59, 26~60, 26~61, 26~62, 26~63, 26~64) (e.g. For example, 27~28, 27~29, 27~30, 27~31, 27~32, 27~33, 27~34, 27~35, 27~36, 27~37, 27~38, 27~39, 27~40, 27~41, 27~42, 27~43, 27~44, 27~45, 27~46, 27~47, 2 7-48, 27-49, 27-50, 27-51, 27-52, 27-53, 27-54, 27-55, 27-56, 27-57, 27-58, 27-59, 27-60, 27-61, 27-62, 27-63, 27-64) (e.g., 28-29, 28-30, 28-31, 2 8~32, 28~33, 28~34, 28~35, 28~36, 28~37, 28~38, 28~39, 28~40, 28~41, 28~42, 28~43, 28~44, 28~45, 28~46, 28~47, 28~48, 28~49, 28~50, 28~51, 28~52, 28-53, 28-54, 28-55, 28-56, 28-57, 28-58, 28-59, 28-60, 28-61, 28-62, 28-63, 28-64) (e.g., 29-30, 29-31, 29-32, 29-33, 29-34, 29-35, 29-36, 29-37,29~38, 29~39, 29~40, 29~41, 29~42, 29~43, 29~44, 29~45, 29~46, 29~47, 29~48, 29~49, 29~50, 29~51, 29~52, 29~53, 29~54, 29~55, 29~56, 29~57, 29~5 8, 29-59, 29-60, 29-61, 29-62, 29-63, 29-64) (e.g., 30-31, 30-32, 30-33, 30-34, 30-35, 30-36, 30-37, 30-38, 30-39, 30-40, 30-41, 30-42, 30-43, 30-44, 30-45, 30-46, 30-47, 30-48, 30-49, 30-50, 30-51, 30-52, 30-53, 30-54, 30-55, 30-56, 30-57, 30-58, 30-59, 4, 30~45, 30~46, 30~47, 30~48, 30~49, 30~50, 30~51, 30~52, 30~53, 30~54, 30~55, 30~56, 30~57, 30~58, 30~59, 30~60, 30~61, 30~62, 30~63, 30~64) (e.g. For example, 31-32, 31-33, 31-34, 31-35, 31-36, 31-37, 31-38, 31-39, 31-40, 31-41, 31-42, 31-43, 31-44, 31-45, 31-46, 31-47, 31-48, 31-49, 31-50, 31-51, 31 ~52, 31~53, 31~54, 31~55, 31~56, 31~57, 31~58, 31~59, 31~60, 31~61, 31~62, 31~63, 31~64) (e.g., 32~33, 32~34, 32~35, 32~36, 32~37, 32~38, 32~39, 3 2~40, 32~41, 32~42, 32~43, 32~44, 32~45, 32~46, 32~47, 32~48, 32~49, 32~50, 32~51, 32~52, 32~53, 32~54, 32~55, 32~56, 32~57, 32~58, 32~59, 32~60, 32-61, 32-62, 32-63, 32-64) (e.g., 33-34, 33-35, 33-36, 33-37, 33-38, 33-39, 33-40, 33-41, 33-42, 33-43, 33-44, 33-45, 33-46, 33-47, 33-48, 33-49, 33-50, 33-51, 33-52, 33-53, 33-54, 33-55, 33-56, 33-57, 33-58, 33-59, 33-60, 33-61, 33-62, 33-63, 33-64) (e.g., 34-35, 34-36, 34-37, 34-38, 34-39,34~40, 34~41, 34~42, 34~43, 34~44, 34~45, 34~46, 34~47, 34~48, 34~49, 34~50, 34~51, 34~52, 34~53, 34~54, 34~55, 34~56, 34~57, 34~58, 34~59, 34~6 0, 34-61, 34-62, 34-63, 34-64) (e.g., 35-36, 35-37, 35-38, 35-39, 35-40, 35-41, 35-42, 35-43, 35-44, 35-45, 35-46, 35-47, 35-48, 35-49, 35-50, 35-5 1, 35-52, 35-53, 35-54, 35-55, 35-56, 35-57, 35-58, 35-59, 35-60, 35-61, 35-62, 35-63, 35-64) (e.g., 36-37, 36-38, 36-39, 36-40, 36-41, 36-42, 36-43, 36-44) 3, 36~44, 36~45, 36~46, 36~47, 36~48, 36~49, 36~50, 36~51, 36~52, 36~53, 36~54, 36~55, 36~56, 36~57, 36~58, 36~59, 36~60, 36~61, 36~62, 36~63, 36~ 64) (e.g., 37-38, 37-39, 37-40, 37-41, 37-42, 37-43, 37-44, 37-45, 37-46, 37-47, 37-48, 37-49, 37-50, 37-51, 37-52, 37-53, 37-54, 37-55, 37-56, 37-57, 37-58, 37-59, 37-60, 37-61, 37-62, 37-63, 37-64) (e.g., 38-39, 38-40, 38-41, 38-42, 38-43, 38-44, 38-45, 38-46, 38-47, 38-48, 38-49, 38-50, 38 ~51, 38~52, 38~53, 38~54, 38~55, 38~56, 38~57, 38~58, 38~59, 38~60, 38~61, 38~62, 38~63, 38~64), (e.g., 39~40, 39~41, 39~42, 39~43, 39~44, 39~45, 3 9-46, 39-47, 39-48, 39-49, 39-50, 39-51, 39-52, 39-53, 39-54, 39-55, 39-56, 39-57, 39-58, 39-59, 39-60, 39-61, 39-62, 39-63, 39-64), (e.g., 40-41,40~42、40~43、40~44、40~45、40~46、40~47、40~48、40~49、40~50、40~51、40~52、40~53、40~54、40~55、40~56、40~57、40~58、40~59、40~60、40~61、40~62、40~63、40~6、 4), (e.g., 41-42, 41-43, 41-44, 41-45, 41-46, 41-47, 41-48, 41-49, 41-50, 41-51, 41-52, 41-53, 41-54, 41-55, 41-56, 41-57, 41-58, 41-59, 41-60, 41-61, 41-62, 41-63, 41-64), (e.g., 42-43, 42-44, 42-45, 42-46, 42-47, 42-48, 42-49, 42-50, 42-51, 42-52, 42-53, 42-54, 42-55, 42-56, 42-57, 42-58, 4 2-59, 42-60, 42-61, 42-62, 42-63, 42-64) (e.g., 43-44, 43-45, 43-46, 43-47, 43-48, 43-49, 43-50, 43-51, 43-52, 43-53, 43-54, 43-55, 43-56, 43-57, 43-58, 43-59, 43-60, 43-61, 43-62, 43-63, 43-64) (e.g., 44-45, 44-46, 44-47, 44-48, 44-49, 44-50, 44-51, 44-52, 44-53, 44-54, 44-55, 44-56, 44-57, 44-58, 44-59, 44-60, 44-61, 44-62, 44-63, 44-64) (e.g., 45-46, 45-47, 45-48, 45-49, 45-50, 45-51, 45-52, 45-53, 45-54, 45-55, 45-56, 45-57, 45-58 , 45-59, 45-60, 45-61, 45-62, 45-63, 45-64) (e.g., 46-47, 46-48, 46-49, 46-50, 46-51, 46-52, 46-53, 46-54, 46-55, 46-56, 46-57, 46-58, 46-59, 46-60 , 46-61, 46-62, 46-63, 46-64) (e.g., 47-48, 47-49, 47-50, 47-51, 47-52, 47-53, 47-54, 47-55, 47-56, 47-57, 47-58, 47-59, 47-60, 47-61, 47-62, 47-63, 47-64) (e.g., 48-49, 48-50, 48-51, 48-52, 48-53, 48-54, 48-55, 48-56, 48-57, 48-58, 48-59, 48-60, 48-61, 48-62, 48-63, 48-64) (e.g., 49-50, 49-51,49-52, 49-53, 49-54, 49-55, 49-56, 49-57, 49-58, 49-59, 49-60, 49-61, 49-62, 49-63, 49-64) (e.g. 50-51, 50-52, 50-53, 50-54, 50-55, 50-56, 50-57 , 50-58, 50-59, 50-60, 50-61, 50-62, 50-63, 50-64) (e.g., 51-52, 51-53, 51-54, 51-55, 51-56, 51-57, 51-58, 51-59, 51-60, 51-61, 51-62, 51-63, 51-64 ) (e.g., 52-53, 52-54, 52-55, 52-56, 52-57, 52-58, 52-59, 52-60, 52-61, 52-62, 52-63, 52-64) (e.g., 53-54, 53-55, 53-56, 53-57, 53-58, 53-59, 53-60, 53-61, 53-62, 53-63, 53-64) (e.g., 54-55, 54-56, 54-57, 54-58, 54-59, 54-60, 54-61, 54-62, 54-63, 54-64) (e.g., 55-56, 55-57, 55-58, 55-59, 55-60, 55-61, 55-62, 55-63, 55-64) (e.g., 56-57, 56-58, 56-59, 56-60, 56-61, 56-62, 56-63, 56-64) (e.g., 57-58, 57-59, 57-60, 57-61, 57-62, 57-63, 57-64) (e.g., 58-59, 58-60, 58-61, 58-62, 58-63, 58-64) (e.g., 59-60, 59-61, 59-62, 59-63, 59-64) (e.g., 60-61, 60-62, 60-63, 60-64) (e.g., 61-62, 61-63, 61- 64) (e.g., 62-63, 62-64) (e.g., 63-64) (e.g., 1-423, 10-423, 20-423, 41-423, 50-423, 62-423, 64-423, 100-423, 150-423, 200-423, 300-423, 320-423, 361-423, 400-423, 410-423, 415-423, 420-423, 421-423, 422-423) (e.g., 423 or less, 400 or less, 350 or less, 361 or less, 320 or less, 300 or less, 250 or less, 200 or less, 150 or less, 100 or less, 75 or less,70 or less, 65 or less, 64 or less, 62 or less; 61 or less; 60 or less; 59 or less; 58 or less; 57 or less; 56 or less; 55 or less; 54 or less; 53 or less; 52 or less; 51 or less; 50 or less Lower; 49 or less; 48 or less; 47 or less; 46 or less; 45 or less; 44 or less; 43 or less; 42 or less; 41 or less; 40 or less; 39 or less; 38 or less; 37 or less; 36 or less; 35 or less; 34 or less;33 or less;32 or less;31 or less;30 or less;29 or less;28 or less;27 or less;26 or less;25 or less;24 or less;23 or less;22 or less;21 or less;20 or less;19 or less;18 or less;17 or less;16 or less;15 or less;14 or less;13 or less;12 or less;11 or less;10 or less;9 or less;8 or less;7 or less;6 or less;5 or less;4 or less;3 or less;2 or 1).

[0042] As used herein, "nucleic acid" or "nucleic acid molecule" generally refers to any ribonucleic acid or deoxyribonucleic acid, which may be unmodified or modified DNA or RNA. "Nucleic acid" includes, but is not limited to, single-stranded and double-stranded nucleic acids. As used herein, the term "nucleic acid" also includes DNA as described above that contains one or more modified bases. Thus, DNA with backbone modifications for stability or other reasons is a "nucleic acid". As used herein, the term "nucleic acid" encompasses chemically, enzymatically, or metabolically modified forms of nucleic acid, as well as chemical forms of DNA characteristic of viruses and cells, including, for example, single cells and complex cells.

[0043] The term "oligonucleotide" or "polynucleotide" or "nucleotide" or "nucleic acid" refers to a molecule containing two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and usually more than ten. The exact size depends on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. Oligonucleotides can be produced by any method, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof. Typical deoxyribonucleotides of DNA are thymine, adenine, cytosine, and guanine. Typical ribonucleotides of RNA are uracil, adenine, cytosine, and guanine.

[0044] As used herein, the term "locus" or "region" of a nucleic acid refers to a small region of a nucleic acid, e.g., a gene on a chromosome, a single nucleotide, a CpG island, and the like.

[0045] The terms "complementary" and "complementarity" refer to nucleotides (e.g., a single nucleotide) or polynucleotides (e.g., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence 5'-AGT-3' is complementary to the sequence 3'-TCA-5'. Complementarity can be "partial," in which only a portion of the nucleic acid bases match according to the base-pairing rules. Alternatively, there can be "complete" or "total" complementarity between nucleic acids. The degree of complementarity between strands of nucleic acids affects the efficiency and strength of hybridization between strands of nucleic acids. This is particularly important in amplification reactions and in detection methods that depend on binding between nucleic acids.

[0046] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of an RNA or a polypeptide or its precursor. A functional polypeptide 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 property of the polypeptide (e.g., enzymatic activity, ligand binding, signal transduction, etc.) is retained. When used in reference to a gene, the term "portion" refers to a fragment of that gene. These fragments can range in size from a few nucleotides to the entire gene sequence minus one nucleotide. Thus, "a nucleotide sequence comprising at least a portion of a gene" can include a fragment of a gene or the entire gene.

[0047] The term "gene" also includes the coding region of a structural gene and includes sequences located adjacent to the coding region at both the 5' and 3' ends, for example, at a distance of about 1 kb on either end, such that the gene corresponds to the length of the full-length mRNA (e.g., including coding, regulatory, structural and other sequences). Sequences located 5' of the coding region and present on the mRNA are referred to as 5' non-translated or untranslated sequences. Sequences located 3' or downstream of the coding region and present on the mRNA are referred to as 3' non-translated or untranslated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. In certain organisms (e.g., eukaryotes), genomic forms or clones of a gene contain the coding region interrupted by non-coding sequences called "introns" or "intervening regions" or "intervening sequences". Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA) and introns may contain regulatory elements such as enhancers. Introns are removed or "spliced ​​out" from the nuclear or primary transcript and are therefore absent in the messenger RNA (mRNA) transcript, which functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

[0048] In addition to containing introns, genomic forms of a gene may also include sequences located both 5' and 3' to the sequences present in the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the untranslated sequences present in the mRNA transcript). The 5' flanking region may contain regulatory sequences such as promoters and enhancers that control or influence the transcription of the gene. The 3' flanking region may contain sequences that direct the termination of transcription, post-transcriptional cleavage, and polyadenylation.

[0049] When referring to a gene, the term "wild type" refers to a gene that has the characteristics of a gene isolated from a naturally occurring source. When referring to a gene product, the term "wild type" refers to a gene product that has the characteristics of a gene product isolated from a naturally occurring source. When referring to a gene, the term "wild type" refers to a gene that has the characteristics of a gene isolated from a naturally occurring source. The term "naturally occurring" when applied to an object refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that can be isolated from a natural source and has not been intentionally modified by the hand of man in a laboratory or present in an organism (including viruses) is naturally occurring. A wild type gene is often that gene or allele that is most frequently observed in a population and is therefore arbitrarily referred to as the "normal" or "wild type" form of the gene. In contrast, when referring to a gene or gene product, the term "modified" or "mutated" refers to a gene or gene product, respectively, that exhibits modifications in sequence and / or functional properties (i.e., altered characteristics) when compared to a wild type gene or gene product. It is noted that naturally occurring mutants can be isolated, which are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.

[0050] The term "allele" refers to variants of a gene, including, but not limited to, variants and mutants, polymorphic loci, and single nucleotide polymorphic loci, frameshifts, and splice variants. An allele can occur naturally in a population, or it can arise during the lifetime of any particular individual in this population.

[0051] Thus, when used in reference to a nucleotide sequence, the terms "variant" and "mutant" refer to a nucleic acid sequence that differs from another, normally related, nucleotide sequence by one or more nucleotides. A "mutation" is a difference between two different nucleotide sequences, typically one sequence being a reference sequence.

[0052] The term "primer" refers to an oligonucleotide, whether naturally occurring as a nucleic acid fragment from a purified restriction digest or synthesized, that can act as an initiation point for synthesis when placed under conditions to induce synthesis of a primer extension product that is complementary to a template strand of nucleic acid (e.g., in the presence of nucleotides and an inducing agent such as DNA polymerase, at an appropriate temperature and pH). The primer is preferably single-stranded to maximize the efficiency of amplification, but may alternatively be double-stranded. If double-stranded, the primer is first treated to separate its strands and then used to prepare the extension product. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be long enough to prime the synthesis of the extension product in the presence of the inducing agent. The exact length of the primer depends on many factors, including temperature, primer source, and use of the method. In some embodiments, the primer pair is specific for a particular MDM (e.g., MDMs of Tables I, III, and X) and specifically binds to at least a portion of the genetic region that contains the MDM (e.g., the chromosomal coordinates of Tables I, III, and / or X).

[0053] The term "probe" refers to an oligonucleotide (e.g., a sequence of nucleotides) that is either naturally occurring, as in a purified restriction digest, or produced synthetically, recombinantly, or by PCR amplification, which can hybridize to another oligonucleotide of interest. Probes can be single-stranded or double-stranded. Probes are useful for the detection, identification, and isolation of specific gene sequences (e.g., "capture probes"). It is contemplated that any probe used in the present invention can, in some embodiments, be labeled with any "reporter molecule" and thus detectable in any detection system, including, but not limited to, enzymatic (e.g., ELISA, and enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.

[0054] The term "target" as used herein refers to a nucleic acid that is to be sorted out from other nucleic acids, for example by probe binding, amplification, isolation, capture, etc. For example, when used in reference to the polymerase chain reaction, "target" refers to the region of nucleic acid bound by the primers used for the polymerase chain reaction, whereas when used in assays in which the target DNA is not amplified, for example, in some embodiments of an invasion cleavage assay, the target includes the site where the probe and the invading oligonucleotide (e.g., INVADER oligonucleotide) bind to form an invasion cleavage structure, which allows the presence of the target nucleic acid to be detected. A "segment" is defined as a region of nucleic acid within the target sequence.

[0055] Thus, as used herein, "non-target", for example when used to describe a nucleic acid such as DNA, refers to a nucleic acid that may be present in a reaction but is not the subject of detection or characterization by the reaction. In some embodiments, non-target nucleic acid may refer to a nucleic acid present in a sample that does not contain a target sequence, for example, but in some embodiments, non-target may also refer to an exogenous nucleic acid, i.e., a nucleic acid that is not derived from a sample that contains or is suspected to contain a target nucleic acid, and is added to a reaction, for example, to reduce variability in the performance of an enzyme (e.g., a polymerase) in the reaction, to normalize the activity of the enzyme.

[0056] As used herein, "methylation" refers to cytosine methylation at the C5 or N4 position of cytosine, the N6 position of adenine, or other types of nucleic acid methylation. In vitro amplified DNA is usually not methylated, since typical in vitro DNA amplification methods do not preserve the methylation pattern of the amplified template. However, "unmethylated DNA" or "methylated DNA" can also refer to amplified DNA whose original template was not methylated or was methylated, respectively.

[0057] As used herein, the term "amplification reagents" refers to the reagents necessary for amplification, excluding primers, nucleic acid template, and amplification enzymes (deoxyribonucleoside triphosphates, buffers, etc.) Typically, amplification reagents are placed and contained within a reaction vessel along with other reaction components.

[0058] As used herein, the term "control" when used in relation to nucleic acid detection or analysis refers to a nucleic acid with known characteristics (e.g., known sequence, known copy number per cell) that is used to compare with an experimental target (e.g., a nucleic acid of unknown concentration, etc.). A control can be an endogenous, preferably invariant, gene to which the test or target nucleic acid in the assay can be normalized. Such normalization reduces sample-to-sample variability that may occur, for example, in sample processing, assay efficiency, etc., allowing accurate sample-to-sample data comparison. Genes used to normalize nucleic acid detection assays in human samples include, for example, β-actin, ZDHHC1, and B3GALT6 (see, for example, U.S. Patent Application Nos. 14 / 966,617 and 62 / 364,082, each of which is incorporated herein by reference). As used herein, "ZDHHC1" refers to a gene that encodes a protein belonging to the DHHC palmitoyltransferase family, characterized as zinc finger DHHC-type containing 1, located on human DNA chromosome 16 (16q22.1).

[0059] Controls can also be external. For example, in quantitative assays such as qPCR, QuARTS, etc., a "calibrator" or "calibration control" is a nucleic acid of known sequence, e.g., the same sequence as a portion of an experimental target nucleic acid, and a known concentration or series of concentrations (e.g., serially diluted control target for generating a standard curve for quantitative PCR). Typically, calibration controls are analyzed using the same reagents and reaction conditions as those used for the experimental DNA. In certain embodiments, the measurement of the calibrator is performed simultaneously with the experimental assay, e.g., in the same thermal cycler. In preferred embodiments, multiple calibrators can be included in a single plasmid, such that different calibrator sequences can be easily provided in equimolar amounts. In particularly preferred embodiments, the plasmid calibrator is digested, e.g., with one or more restriction enzymes, to release the calibrator portion from the plasmid vector. See, e.g., WO2015 / 066695, which is incorporated herein by reference.

[0060] As used herein, "methylated nucleotide" or "methylated nucleotide base" refers to the presence of a methyl moiety on a nucleotide base, which is not present in recognized typical nucleotide bases. For example, cytosine does not contain a methyl moiety on its pyrimidine ring, but 5-methylcytosine contains a methyl moiety at the 5th position of its pyrimidine ring. Thus, cytosine is not a methylated nucleotide, but 5-methylcytosine is a methylated nucleotide. In another example, thymine contains a methyl moiety at the 5th position of its pyrimidine ring, but since thymine is a typical nucleotide base of DNA, for the purposes of this specification, thymine is not considered to be a methylated nucleotide when present in DNA.

[0061] As used herein, a "methylated nucleic acid molecule" refers to a nucleic acid molecule that contains one or more methylated nucleotides.

[0062] As used herein, the "methylation state," "methylation profile," and "methylation status" of a nucleic acid molecule refer to the presence or absence of one or more methylated nucleotide bases in a nucleic acid molecule. For example, a nucleic acid molecule that contains a methylated cytosine is considered to be methylated (e.g., the methylation state of the nucleic acid molecule is methylated). A nucleic acid molecule that does not contain any methylated nucleotides is considered to be unmethylated.

[0063] As used herein, the term "methylation level" applied to a methylation marker refers to the amount of methylation in a particular methylation marker. Methylation level may also refer to the amount of methylation in a particular methylation marker compared to an established standard or control. Methylation level may also refer to whether one or more cytosine residues present in a CpG context have a methylation group. Methylation level may also refer to the proportion of cells in a sample that have or do not have a methylation group at such cytosine. Methylation level may also represent whether a single CpG dinucleotide is methylated.

[0064] The methylation state of a particular nucleic acid sequence (e.g., a genetic marker, or a DNA region, as described herein) can indicate the methylation state of all bases in the sequence, or it can indicate the methylation state of a subset of these bases (e.g., one or more cytosines) within the sequence, or it can indicate information about the methylation density of a region within the sequence, with or without providing precise information of the position within the sequence where methylation occurs.

[0065] The methylation state of a nucleotide locus in a nucleic acid molecule refers to the presence or absence of a methylated nucleotide at a particular locus in a nucleic acid molecule.For example, the methylation state of a cytosine at the 7th nucleotide in a nucleic acid molecule is methylated if the nucleotide present at the 7th nucleotide in the nucleic acid molecule is 5-methylcytosine.Similarly, the methylation state of a cytosine at the 7th nucleotide in a nucleic acid molecule is unmethylated if the nucleotide present at the 7th nucleotide in the nucleic acid molecule is cytosine (and is not 5-methylcytosine).

[0066] The methylation status can optionally be represented or indicated by a "methylation value" (e.g., representing a methylation frequency, fraction, proportion, percent, etc.). Methylation values ​​can be generated, for example, by quantifying the amount of intact nucleic acid present after restriction digestion with a methylation-dependent restriction enzyme, or by comparing amplification profiles after a bisulfite reaction, or by comparing sequences of bisulfite-treated and untreated nucleic acids, or by comparing TET-treated and untreated nucleic acids. Thus, a value, e.g., a methylation value, represents the methylation status and can thereby be used as a quantitative indicator of the methylation status across multiple copies of a locus. This is of particular use when it is desirable to compare the methylation status of sequences in a sample to a threshold or reference value.

[0067] As used herein, "methylation frequency" or "percent (%) methylation" refers to the number of cases where a molecule or locus is methylated compared to the number of cases where the molecule or locus is unmethylated.

[0068] The term "methylation score" as used herein refers to a score that indicates the methylation events detected in a marker or panel of markers compared to the median value of the methylation events in a marker or panel of markers from a random population of mammals that do not have a particular tumor of interest (e.g., a random population of 10, 20, 30, 40, 50, 100, or 500 mammals). A high methylation score in a marker or panel of markers can be any score, as long as it is greater than the corresponding reference score. For example, a high methylation score in a marker or panel of markers can be 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more, greater than the reference methylation score.

[0069] Thus, a methylation state represents the state of methylation of a nucleic acid (e.g., a genomic sequence). In addition, a methylation state refers to a characteristic of a nucleic acid segment at a particular genomic locus that is related to methylation. Such characteristics include, but are not limited to, whether any of the cytosine (C) residues in this DNA sequence are methylated, the location of the methylated C residue(s), the frequency or percentage of methylated C throughout any particular region of the nucleic acid, and allelic differences in methylation due to, for example, differences in allelic origin. The terms "methylation state", "methylation profile", and "methylation state" also refer to the relative concentration, absolute concentration, or pattern of methylated or unmethylated C throughout any particular region of a nucleic acid in a biological sample. For example, if a cytosine (C) residue(s) in a nucleic acid sequence is methylated, it can be said to have "hypermethylated" or "high methylation", whereas if a cytosine (C) residue(s) in the DNA is unmethylated, it can be said to have "hypomethylated" or "low methylation". Similarly, if a cytosine (C) residue(s) in a nucleic acid sequence is methylated compared to another nucleic acid sequence (e.g., from a different region or from a different individual), the sequence is considered to be hypermethylated or have high methylation compared to the other nucleic acid sequence. Alternatively, if a cytosine (C) residue(s) in a DNA sequence is unmethylated compared to another nucleic acid sequence (e.g., from a different region or from a different individual), the sequence is considered to be hypomethylated or have low methylation compared to the other nucleic acid sequence. In addition, as used herein, the term "methylation pattern" refers to the collection of sites of methylated and unmethylated nucleotides across a nucleic acid region. Two nucleic acids can have the same or similar methylation frequency or methylation pattern, but have different methylation patterns when the number of methylated and unmethylated nucleotides is the same or similar throughout the region, but the positions of the methylated and unmethylated nucleotides are different.Sequences are referred to as having "variable methylation" or "differences in methylation" or "different methylation states" when they differ in the degree (e.g., one has increased or decreased methylation compared to the other), frequency, or pattern of methylation. The term "variable methylation" refers to a difference in the level or pattern of nucleic acid methylation in a cancer-positive sample compared to the level or pattern of nucleic acid methylation in a cancer-negative sample. It can also refer to a difference in the level or pattern between patients who have recurrence of cancer after surgery and those who do not. Variable methylation, and specific levels or patterns of DNA methylation, are prognostic and predictive biomarkers, for example, if precise cutoffs or predictive characteristics are defined.

[0070] Methylation state frequencies can be used to describe a population of individuals or a sample from a single individual. For example, a nucleotide locus with a methylation state frequency of 50% is methylated 50% of the time and unmethylated 50% of the time. Such frequencies can be used to describe, for example, the degree to which a nucleotide locus or nucleic acid region is methylated in a population of individuals or a collection of nucleic acids. Thus, when the methylation in a first population or pool of nucleic acid molecules is different from the methylation in a second population or pool of nucleic acid molecules, the frequency of the methylation state of the first population or pool is different from the frequency of the methylation state of the second population or pool. Such frequencies can also be used to describe, for example, the degree to which a nucleotide locus or nucleic acid region is methylated in a single individual. For example, such frequencies can be used to describe the degree to which a group of cells from a tissue sample is methylated or unmethylated at a nucleotide locus or nucleic acid region.

[0071] Typically, methylation of human DNA occurs on dinucleotide sequences containing adjacent guanines and cytosines, where the cytosine is located 5' to the guanine (also referred to as CpG dinucleotide sequences). Many of the cytosines within CpG dinucleotides are methylated in the human genome, but some remain unmethylated in specific CpG dinucleotide-rich genomic regions known as CpG islands (see, e.g., Antequera et al. (1990) Cell 62:503-514).

[0072] As used herein, "CpG island" or "cytosine-phosphate-guanine-island" refers to a G:C-rich region of genomic DNA that contains more CpG dinucleotides compared to the total genomic DNA. A CpG island can be at least 100, 200 base pairs long, or longer, where the G:C content of the region is at least 50% and the ratio of observed CpG frequency to expected frequency is 0.6, and in some cases, a CpG island can be at least 500 base pairs long, where the G:C content of the region is at least 55% and the ratio of observed CpG frequency to expected frequency is 0.65. The observed CpG frequency to expected frequency can be calculated according to the method provided in Gardiner-Garden et al (1987) J.Mol.Biol.196:261-281. For example, the observed CpG frequency to the expected frequency can be calculated according to the formula R=(A×B) / (C×D), where R is the ratio of the observed CpG frequency to the expected frequency, A is the number of CpG dinucleotides in the analyzed sequence, B is the total number of nucleotides in the analyzed sequence, C is the total number of C nucleotides in the analyzed sequence, and D is the total number of G nucleotides in the analyzed sequence. Methylation status is usually determined in CpG islands, e.g., in promoter regions. However, it will be appreciated that other sequences in the human genome, such as CpA and CpT, are also subject to DNA methylation (see Ramsahoye (2000) Proc. Natl. Acad. Sci. USA 97:5237-5242; Salmon and Kaye (1970) Biochim. Biophys. Acta. 204:340-351; Grafstrom (1985) Nucleic Acids Res. 13:2827-2842; Nyce (1986) Nucleic Acids Res. 14:4353-4367; Woodcock (1987) Biochem. Biophys. Res. Commun. 145: 888-894).

[0073] As used herein, a "methylation specific reagent" refers to a reagent that modifies the nucleotides of a nucleic acid molecule as a function of the methylation state of the nucleic acid molecule, or a methylation specific reagent refers to a compound or composition, or other agent, that is capable of altering the nucleotide sequence of a nucleic acid molecule in a manner that reflects the methylation state of the nucleic acid molecule. Methods of treating a nucleic acid molecule with such reagents may include contacting the nucleic acid molecule with the reagent, optionally in combination with additional steps, to obtain a desired change in the nucleotide sequence. Such methods may be applied in a manner in which unmethylated nucleotides (e.g., each unmethylated cytosine) are modified into a different nucleotide. For example, in some embodiments, such reagents can deaminate unmethylated cytosine nucleotides to generate deoxyuracil residues. Examples of such reagents include, but are not limited to, methylation-sensitive restriction enzymes, methylation-dependent restriction enzymes, bisulfite reagents, TET enzymes, and borane reducing agents.

[0074] Alteration of a nucleic acid nucleotide sequence with a methylation specific reagent can also result in a nucleic acid molecule in which each methylated nucleotide is modified to a different nucleotide.

[0075] The term "methylation assay" refers to any assay for determining the methylation status of one or more CpG dinucleotide sequences within a nucleic acid sequence.

[0076] The term "MS AP-PCR" (methylation-sensitive arbitrarily primed polymerase chain reaction) refers to an art-recognized technique that uses CG-rich primers to globally scan the genome and focus on regions most likely to contain CpG dinucleotides, and is described by Gonzalgo et al. (1997) Cancer Research 57:594-599.

[0077] The term "MethyLight™" refers to the art-recognized fluorescence-based real-time PCR technology described by Eads et al. (1999) Cancer Res. 59:2302-2306.

[0078] The term "HeavyMethyl™" refers to an assay in which methylation-specific inhibitory probes (also referred to herein as inhibitors) that cover the CpG positions between or covered by the amplification primers enable methylation-specific selective amplification of a nucleic acid sample.

[0079] The term "HeavyMethyl™ MethyLight™" assay refers to the HeavyMethyl™ MethyLight™ assay, which is a variation of the MethyLight™ assay in which the MethyLight™ assay is combined with a methylation-specific blocking probe that covers the CpG positions between the amplification primers.

[0080] The term "Ms-SNuPE" (Methylation-Sensitive Single-Base Primer Extension) refers to the art-recognized assay described in Gonzalgo & Jones (1997) Nucleic Acids Res. 25: 2529-2531.

[0081] The term "MSP" (methylation specific PCR) refers to the art-recognized methylation assay described by Herman et al. (1996) Proc. Natl. Acad. Sci. USA 93:9821-9826 and in U.S. Pat. No. 5,786,146.

[0082] The term "COBRA" (Combined Bisulfite Restriction Analysis) refers to an art-recognized methylation assay described in Xiong & Laird (1997) Nucleic Acids Res. 25:2532-2534.

[0083] The term "MCA" (Methylated CpG Island Amplification) refers to the methylation assay described in Toyota et al. (1999) Cancer Res. 59: 2307-12 and WO00 / 26401A1.

[0084] As used herein, a "selected nucleotide" refers to one of the four nucleotides typically occurring in a nucleic acid molecule (C, G, T, and A for DNA and C, G, U, and A for RNA) and can include methylated derivatives of a typically occurring nucleotide (e.g., when C is a selected nucleotide, both methylated and unmethylated C are included in the meaning of the selected nucleotide), but a methylated selected nucleotide specifically refers to a typically occurring nucleotide that is methylated and an unmethylated selected nucleotide specifically refers to a typically occurring nucleotide that is unmethylated.

[0085] The term "methylation-specific restriction enzyme" refers to a restriction enzyme that selectively digests nucleic acids depending on the methylation state of its recognition site. For restriction enzymes that specifically cleave when the recognition site is unmethylated or hemimethylated (methylation-sensitive enzymes), cleavage does not occur (or occurs much less efficiently) when the recognition site is methylated on one or both strands. For restriction enzymes that specifically cleave only when the recognition site is methylated (methylation-dependent enzymes), cleavage does not occur (or occurs much less efficiently) when the recognition site is unmethylated. Preferably, it is a methylation-specific restriction enzyme, whose recognition sequence includes a CG dinucleotide (e.g., recognition sequence such as CGCG or CCCGGG). More preferred for some embodiments are restriction enzymes that do not cleave when the cytosine in this dinucleotide is methylated at the carbon atom C5.

[0086] As used herein, the "sensitivity" of a particular marker (or set of markers used in combination) refers to the percentage of samples reporting DNA methylation values ​​above a threshold that distinguishes between neoplastic and non-neoplastic samples. In some embodiments, a positive is defined as a histologically confirmed neoplasm reporting a DNA methylation value above a threshold (e.g., a range associated with disease) and a false negative is defined as a histologically confirmed neoplasm reporting a DNA methylation value below a threshold (e.g., a range associated with non-disease). Thus, the sensitivity value reflects the probability that a DNA methylation measurement value of a given marker obtained from a known diseased sample will be within the range of disease-associated measurements. As defined herein, the clinical relevance of a calculated sensitivity value represents an estimate of the probability that a given marker will detect the presence of a clinical condition when applied to a subject with that clinical condition.

[0087] As used herein, the "specificity" of a particular marker (or set of markers used in combination) refers to the percentage of non-tumor samples that report DNA methylation values ​​below a threshold that distinguishes between tumor and non-tumor samples. In some embodiments, a negative is defined as a histologically confirmed non-tumor sample that reports a DNA methylation value below the threshold (e.g., in the non-disease associated range), and a false positive is defined as a histologically confirmed non-tumor sample that reports a DNA methylation value above the threshold (e.g., in the disease associated range). Thus, the specificity value reflects the probability that a DNA methylation measurement of a given marker obtained from a known non-neoplastic sample will be in the range of non-disease associated measurements. As defined herein, the clinical relevance of a calculated specificity value represents an estimate of the probability that a given marker will detect the absence of a clinical condition when applied to patients who do not have that clinical condition.

[0088] The term "AUC" used herein is an abbreviation for "area under the curve". In particular, AUC refers to the area under the receiver operating characteristic (ROC) curve. The ROC curve is a plot of the true positive rate against the false positive rate for different possible cut points of a diagnostic test. The ROC curve shows the trade-off between sensitivity and specificity depending on the cut point selected (any increase in sensitivity will be accompanied by a decrease in specificity). The area under the ROC curve (AUC) is a measure of the accuracy of a diagnostic test (the larger the area, the better, with 1 being optimal, and a random test has a ROC curve that is located on the diagonal, with an area of ​​0.5. See: J.P.Egan. (1975) Signal Detection Theory and ROC Analysis, Academic Press, New York).

[0089] As used herein, the term "tumor" refers to any new abnormal growth of tissue. Thus, a tumor can be a pre-malignant tumor or a malignant tumor.

[0090] The term "tumor-specific marker" as used herein refers to any biological material or element that can be used to indicate the presence of a tumor. Examples of biological materials include, but are not limited to, nucleic acids, polypeptides, carbohydrates, fatty acids, cellular components (e.g., cell membranes and mitochondria), and whole cells. In some cases, the marker is a specific nucleic acid region (e.g., a gene, a region within a gene, a specific locus, etc.). A region of a nucleic acid that is a marker may be referred to, for example, as a "marker gene," "marker region," "marker sequence," "marker locus," etc.

[0091] As used herein, the term "adenoma" refers to a benign tumor of glandular origin. These growths are benign, although over time they can progress to become malignant.

[0092] The terms "precancerous" or "preneoplastic" and their equivalents refer to any cell proliferative disorder that has undergone malignant transformation.

[0093] The "site" of a tumor, adenoma, cancer, etc., is the tissue, organ, cell type, anatomical region, body part, etc. in a subject in which the tumor, adenoma, cancer, etc. is located.

[0094] As used herein, the application of a "diagnostic" test includes the detection or identification of a disease state or condition in a subject, including determining the likelihood that a subject suffers from a given disease or condition, determining the likelihood that a subject with a disease or condition will respond to a treatment, determining the prognosis (or likelihood of progression or regression) of a subject with a disease or condition, and determining the efficacy of a treatment for a subject with a disease or condition. For example, diagnostics can be used to detect the presence or likelihood of a subject suffering from a neoplasm, or the likelihood that such a subject will respond successfully to a compound (e.g., a pharmaceutical, e.g., a drug) or other treatment.

[0095] The term "isolated", when used in reference to a nucleic acid, as in "isolated oligonucleotide", refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid that is normally associated with its natural source. An isolated nucleic acid is present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids, such as DNA and RNA, are found in the state in which they occur in nature. Examples of non-isolated nucleic acids include a given DNA sequence (e.g., a gene) found on a host cell chromosome adjacent to adjacent genes, an RNA sequence, such as a particular mRNA sequence that encodes a particular protein, that is found in a cell as a mixture with many other mRNAs that encode many proteins. However, an isolated nucleic acid that encodes a particular protein includes as an example, a nucleic acid in a cell that normally expresses the protein, where the nucleic acid is in a different chromosomal location than that of the natural cell, or is otherwise adjacent to a nucleic acid sequence that is different from that found in nature. An isolated nucleic acid or oligonucleotide may be single-stranded or double-stranded. When an isolated nucleic acid or oligonucleotide is used to express a protein, the oligonucleotide will minimally contain a sense or coding strand (i.e., the oligonucleotide may be single-stranded), but may also contain both a sense and an antisense strand (i.e., the oligonucleotide may be double-stranded). The isolated nucleic acid may be combined with other nucleic acids or molecules after isolation from its natural or normal environment. For example, the isolated nucleic acid may be present in a host cell, for example, in which it is placed for heterologous expression.

[0096] The term "purified" refers to a molecule, either a nucleic acid or an amino acid sequence, that has been removed, isolated, or separated from its natural environment. Thus, an "isolated nucleic acid sequence" can be a purified nucleic acid sequence. "Substantially purified" molecules are at least 60% free, preferably at least 75% free, and more preferably at least 90% free from other components with which they are naturally associated. As used herein, the term "purified" or "purify" also refers to the removal of contaminants from a sample. The removal of contaminating proteins results in an increase in the percentage of the polypeptide or nucleic acid of interest in a sample. In another example, recombinant polypeptides are expressed in plant, bacterial, yeast, or mammalian host cells, and these polypeptides are purified by the removal of host cell proteins, thereby increasing the percentage of recombinant polypeptides in a sample.

[0097] The term "composition comprising" a given polynucleotide sequence or polypeptide refers broadly to any composition that contains the given polynucleotide sequence or polypeptide. Compositions can include aqueous solutions containing salts (e.g., NaCl), detergents (e.g., SDS), and other components (e.g., Denhardt's solution, milk powder, salmon sperm DNA, etc.).

[0098] The term "sample" is used in its broadest sense. In one sense, it can refer to animal cells or tissues. In another sense, it refers to specimens or cultures obtained from any source, as well as biological and environmental samples. Biological samples can be obtained from plants or animals (including humans) and include fluids, solids, tissues, and gases. Environmental samples include environmental materials such as surface materials, soil, water, and industrial samples. These examples should not be construed as limiting the types of samples applicable to the present invention.

[0099] As used herein, a "remote sample" as used in some contexts refers to a sample that is indirectly collected from a site that is not the sample source of the cell, tissue, or organ. For example, when evaluating sample material derived from the cervix in a fecal sample, the sample is a remote sample.

[0100] As used herein, the term "patient" or "subject" refers to an organism that is the subject of the various tests provided by the technology. The term "subject" includes animals, preferably mammals, including humans. In a preferred embodiment, the subject is a primate. In an even more preferred embodiment, the subject is a human. Furthermore, with respect to diagnostic methods, the preferred subject is a vertebrate subject. The preferred vertebrates are warm-blooded, and the preferred warm-blooded vertebrates are mammals. The preferred mammals are most preferably humans. As used herein, the term "subject" includes both human and animal subjects. Thus, veterinary therapeutic uses are provided herein. As such, the technology of the present invention is provided for the diagnosis of mammals, such as humans, and those mammals that are economically important, such as animals raised on farms for human consumption, important because they are endangered, such as the Amur tiger, and / or animals that are socially important to humans, such as animals kept as pets or in zoos. Examples of such animals include, but are not limited to, carnivores such as cats and dogs; swine, including pigs, hogs, and boars; ruminants and / or ungulates, such as cows, oxen, sheep, giraffes, deer, goats, bison, and camels; pinnipeds; and horses. Thus, also provided are diagnostics and treatments for livestock, including, but not limited to, domesticated pigs, ruminants, ungulates, horses (including racehorses), and the like. The subject matter disclosed herein further includes a system for diagnosing cervical cancer, subtypes of cervical cancer (e.g., cervical adenocarcinoma, cervical squamous cell carcinoma), and / or cervical precancer (e.g., cervical associated intraepithelial adenocarcinoma, cervical intraepithelial neoplasia) in a subject. The system may be provided as a commercially available kit that can be used to screen for risk of cervical cancer, cervical cancer subtypes (e.g., cervical adenocarcinoma, cervical squamous cell carcinoma), and / or cervical precancer (e.g., cervical associated adenocarcinoma in situ, cervical intraepithelial neoplasia) in a subject from whom a biological sample was taken, or to diagnose cervical cancer, cervical cancer subtypes, and / or cervical precancer.Exemplary systems provided in accordance with the present technology include assessing the methylation status of the markers described herein.

[0101] As used herein, the term "kit" refers to any delivery system for delivering materials. In the context of a reaction assay, such delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g., oligonucleotides in appropriate containers, enzymes, etc.) and / or supporting materials (e.g., buffers, written instructions for performing the assay, etc.) from one location to another. For example, a kit includes one or more enclosed members (e.g., boxes) that contain the relevant reaction reagents and / or supporting materials. As used herein, the term "fragmentation kit" refers to a delivery system that includes two or more separate containers, each of which contains a sub-portion of the overall kit components. The containers can be delivered to the intended recipient together or separately. For example, a first container may contain an enzyme for use in the assay, while a second container contains an oligonucleotide. The term "fragmentation kit" is intended to encompass, but is not limited to, a kit that includes an analyte specific reagent (ASR) as defined by section 520(e) of the Federal Food, Drug, and Cosmetic Act. Indeed, any delivery system that includes two or more separate containers, each housing a portion of the overall kit's components, is included in the term "fragmented kit." In contrast, a "combined kit" refers to a delivery system that contains all components of a reaction assay in a single container (e.g., in a single box housing each of the desired components). The term "kit" includes both fragmented and combined kits.

[0102] As used herein, the term "information" refers to any collection of facts or data. With respect to information stored or processed using a computer system(s), including but not limited to the Internet, the term refers to any data stored in any format (e.g., analog, digital, optical, etc.). As used herein, the term "information about a subject" refers to facts or data about a subject (e.g., a human, a plant, or an animal). The term "genomic information" refers to information about a genome, including but not limited to nucleic acid sequences, genes, methylation rates, allele frequencies, RNA expression levels, protein expression, phenotypes correlated to genotypes, and the like. "Allele frequency information" refers to facts or data about allele frequencies, including but not limited to the identity of the allele, statistical correlation between the presence of the allele and characteristics of the subject (e.g., a human subject), the presence or absence of the allele in an individual or population, the percentage of the likelihood of the allele being present in an individual with one or more particular characteristics, and the like.

[0103] Provided herein are techniques for screening for cervical cancer, in particular but not limited to methods, compositions, and / or related uses for detecting the presence of cervical cancer and / or specific forms of cervical cancer (e.g., cervical adenocarcinoma, cervical squamous cell carcinoma, cervical-associated intraepithelial adenocarcinoma, cervical intraepithelial neoplasia) from biological samples (e.g., tissue samples (e.g., cervical samples), blood samples, plasma samples, serum samples, whole blood samples, secretion samples (e.g., cervical secretions, vaginal secretions), organ secretion samples, CSF samples, saliva samples, urine samples, and stool samples) or for distinguishing cervical cancer from other types of gynecological cancers (e.g., endometrial cancer, and ovarian cancer).

[0104] Indeed, as described in Examples I and II, experiments conducted in the course of identifying embodiments of the present invention have identified a set of novel methylation variable regions (DMRs) for distinguishing cervical cancer-derived DNA from non-neoplastic control DNA and for distinguishing cervical cancer tissue from endometrial and ovarian cancer. These experiments list and describe 423 novel DNA methylation markers for distinguishing cervical cancer, cervical cancer subtypes, and cervical pre-cancerous tissue from benign cervical tissue (see Tables I-IV, VI-VIII, Example I), as well as for distinguishing cervical cancer from other types of gynecological cancers (e.g., endometrial cancer, ovarian cancer), and cervical cancer tissue from endometrial and ovarian cancer tissue (see Tables XI and XII, Example II).

[0105] In certain aspects, the technology provides compositions and methods for identifying, determining, and / or classifying cancers, such as cervical cancer, subtypes of cervical cancer (e.g., cervical adenocarcinoma, cervical squamous cell carcinoma), and / or cervical precancer (e.g., cervical associated adenocarcinoma in situ, cervical intraepithelial neoplasia). The methods include determining the methylation status of at least one methylation marker in a biological sample isolated from a subject (e.g., a tissue sample (e.g., cervical tissue), a blood sample, a plasma sample, a serum sample, a whole blood sample, a secretion sample (e.g., cervical secretion, vaginal secretion), an organ secretion sample, a CSF sample, a saliva sample, a urine sample, or a stool sample), wherein a change in the methylation status of the marker indicates the presence, class, or site of cervical cancer, subtypes of cervical cancer (e.g., cervical adenocarcinoma, cervical squamous cell carcinoma), and / or cervical precancer (e.g., cervical associated adenocarcinoma in situ, cervical intraepithelial neoplasia). Certain embodiments relate to markers comprising methylation variable regions (DMRs, e.g., DMRs 1-423, see Tables I, III, and X) that are used to diagnose (e.g., screen for) cervical cancer, subtypes of cervical cancer, and / or cervical pre-cancer, or to distinguish cervical cancer from other types of gynecological cancers (e.g., endometrial and ovarian cancer).

[0106] In a particular embodiment of the present technology, the following steps are performed: 1) contacting nucleic acid (e.g., genomic DNA) in a biological sample obtained from a subject (e.g., a tissue sample (e.g., a cervical sample), a blood sample, a plasma sample, a serum sample, a whole blood sample, a secretion sample (e.g., a cervical secretion, a vaginal secretion), an organ secretion sample, a CSF sample, a saliva sample, a urine sample, or a stool sample) with at least one reagent or set of reagents that distinguish between methylated dinucleotides (e.g., CpG dinucleotides) and unmethylated dinucleotides within one or more methylation markers; 2) detecting cervical cancer, subtypes of cervical cancer (e.g., cervical adenocarcinoma, cervical squamous cell carcinoma), cervical precancer (e.g., cervical associated adenocarcinoma in situ, cervical intraepithelial neoplasia), or distinguishing cervical cancer from other types of gynecological cancer (e.g., endometrial and ovarian cancer) (e.g., providing a sensitivity of 80% or greater and a specificity of 80% or greater).

[0107] In a particular embodiment of the present technology, the following steps are performed: 1) measuring the methylation level of one or more genes or methylation markers in a biological sample from a human individual (e.g., a tissue sample (e.g., a cervical sample), a blood sample, a plasma sample, a serum sample, a whole blood sample, a secretion sample (e.g., a cervical secretion, a vaginal secretion), an organ secretion sample, a CSF sample, a saliva sample, a urine sample, or a stool sample) by treating genomic DNA in the biological sample with a reagent that modifies DNA in a methylation-specific manner; 2) amplifying the treated genomic DNA using a set of primers for the selected one or more genes or methylated markers; 3) determining the methylation level of said one or more genes or methylated markers.

[0108] In a particular embodiment of the present technology, the following steps are performed: 1) measuring the amount of one or more methylated marker genes in DNA from a biological sample (e.g., a tissue sample (e.g., a cervical sample), a blood sample, a plasma sample, a serum sample, a whole blood sample, a secretion sample (e.g., cervical secretions, vaginal secretions), an organ secretion sample, a CSF sample, a saliva sample, a urine sample, or a stool sample); 2) measuring the amount of at least one reference marker in the DNA; 3) calculating a value of the amount of the at least one methylated marker gene measured in the DNA as a percentage of the amount of the reference marker gene measured in the DNA, said value being indicative of the amount of the at least one methylated marker DNA measured in the biological sample.

[0109] In a particular embodiment of the present technology, the following steps are performed: 1) measuring the methylation level of CpG sites of one or more genes in a biological sample from a human individual (e.g., a tissue sample (e.g., a cervical sample), a blood sample, a plasma sample, a serum sample, a whole blood sample, a secretion sample (e.g., a cervical secretion, a vaginal secretion), an organ secretion sample, a CSF sample, a saliva sample, a urine sample, or a stool sample) by treating genomic DNA in the biological sample with bisulfite, a reagent capable of modifying DNA in a methylation-specific manner; 2) amplifying the modified genomic DNA using a set of primers for the selected one or more genes; 3) determining the methylation level of said CpG sites of said selected one or more genes.

[0110] In certain embodiments, the present technology provides a method for characterizing a biological sample, comprising: (a) measuring the methylation level of CpG sites of one or more genes in a biological sample from a human individual (e.g., a tissue sample (e.g., a cervical sample), a blood sample, a plasma sample, a serum sample, a whole blood sample, a secretion sample (e.g., a cervical secretion, a vaginal secretion), an organ secretion sample, a CSF sample, a saliva sample, a urine sample, or a stool sample) by treating genomic DNA in the biological sample with bisulfite and determining the methylation level of the CpG sites; (b) comparing the methylation level of the one or more genes to the methylation level of a corresponding set of genes in a control sample free of cervical cancer; (c) determining that the individual has cervical cancer, a subtype of cervical cancer (e.g., cervical adenocarcinoma, cervical squamous cell carcinoma), and / or cervical precancer (e.g., cervical associated intraepithelial adenocarcinoma, cervical intraepithelial neoplasia) if the methylation level measured for the one or more genes is higher than the methylation level measured in the respective control sample.

[0111] In certain embodiments, the present technology provides a method for distinguishing cervical cancer from other types of gynecological cancer (e.g., endometrial and ovarian cancer) in a biological sample, comprising: (a) measuring the methylation level of CpG sites of one or more genes in a biological sample from a human individual (e.g., a tissue sample (e.g., a cervical sample), a blood sample, a plasma sample, a serum sample, a whole blood sample, a secretion sample (e.g., a cervical secretion, a vaginal secretion), an organ secretion sample, a CSF sample, a saliva sample, a urine sample, or a stool sample) by treating genomic DNA in the biological sample with bisulfite and determining the methylation level of the CpG sites; (b) comparing said methylation level of said one or more genes to the methylation level of a corresponding set of genes in an endometrial cancer sample and / or an ovarian cancer sample; (c) determining that the individual has cervical cancer if the methylation level measured in the one or more genes is higher than the methylation level measured in the respective endometrial and / or ovarian cancer samples.

[0112] In certain embodiments, the subject technology provides a method for screening for cervical cancer, subtypes of cervical cancer (e.g., cervical adenocarcinoma, cervical squamous cell carcinoma), and / or cervical pre-cancer (e.g., cervical associated intraepithelial adenocarcinoma, cervical intraepithelial neoplasia) in a biological sample obtained from a subject (e.g., a tissue sample (e.g., cervical sample), a blood sample, a plasma sample, a serum sample, a whole blood sample, a secretion sample (e.g., cervical secretions, vaginal secretions), an organ secretion sample, a CSF sample, a saliva sample, a urine sample, or a stool sample), comprising: 1) assaying the methylation status of one or more DNA methylation markers; and 2) identifying the subject as having cervical cancer, a subtype of cervical cancer (e.g., cervical adenocarcinoma, cervical squamous cell carcinoma), and / or cervical precancer (e.g., cervical associated intraepithelial adenocarcinoma, cervical intraepithelial neoplasia) if the methylation status of the marker differs from the methylation status of the marker assayed in subjects without cervical cancer.

[0113] In certain embodiments, the technology provides a method for characterizing a biological sample (e.g., a tissue sample (e.g., a cervical sample), a blood sample, a plasma sample, a serum sample, a whole blood sample, a secretion sample (e.g., a cervical secretion, a vaginal secretion), an organ secretion sample, a CSF sample, a saliva sample, a urine sample, or a stool sample) comprising measuring the amount of one or more methylated markers in DNA extracted from the biological sample, treating genomic DNA in the biological sample with bisulfite, and isoforms of the bisulfite-treated genomic DNA using primers specific for CpG sites of each marker gene. and amplifying the CpG sites of one or more genes, wherein the primers specific for each marker gene are capable of binding to an amplicon bound by a primer sequence for the marker gene (e.g., a primer described in Table V and / or XII), the amplicon bound by the primer sequence for the marker gene being at least a portion of a gene region of the methylated marker gene described in Tables I, III, and / or X.

[0114] In certain embodiments, the technology provides a method comprising: measuring the methylation level of one or more methylated marker genes in DNA extracted from a biological sample (e.g., a tissue sample (e.g., a cervical sample), a blood sample, a plasma sample, a serum sample, a whole blood sample, a secretion sample (e.g., a cervical secretion, a vaginal secretion), an organ secretion sample, a CSF sample, a saliva sample, a urine sample, or a stool sample) by extracting genomic DNA from the biological sample of a human individual suspected of having or having cancer; treating the extracted genomic DNA with bisulfite; amplifying the bisulfite-treated genomic DNA with primers specific for the one or more genes, wherein the primers specific for the one or more genes are capable of binding to at least a portion of the bisulfite-treated genomic DNA in the chromosomal regions of the markers listed in Tables I, III, and X; and measuring the methylation level of one or more methylated marker genes.

[0115] In certain embodiments, the technology provides a method for preparing a DNA fraction useful for analyzing one or more genetic loci involved in one or more chromosomal abnormalities from a biological sample of a human individual (e.g., a tissue sample (e.g., a cervical sample), a blood sample, a plasma sample, a serum sample, a whole blood sample, a secretion sample (e.g., cervical secretions, vaginal secretions), an organ secretion sample, a CSF sample, a saliva sample, a urine sample, or a stool sample), comprising: (a) extracting genomic DNA from a biological sample from a human individual; (b) (i) treating the extracted genomic DNA with a reagent that modifies DNA in a methylation-specific manner; (ii) amplifying the bisulfite-treated genomic DNA using separate primers specific for one or more methylation markers; generating a fraction of the extracted genomic DNA by (c) analyzing one or more loci in the generated fraction of the extracted genomic DNA by measuring the methylation level of the CpG sites of each of the one or more methylation markers.

[0116] In certain embodiments, the technology provides a method for preparing a DNA fraction useful for analyzing one or more DNA fragments involved in one or more chromosomal abnormalities from a biological sample of a human individual (e.g., a tissue sample (e.g., a cervical sample), a blood sample, a plasma sample, a serum sample, a whole blood sample, a secretion sample (e.g., cervical secretions, vaginal secretions), an organ secretion sample, a CSF sample, a saliva sample, a urine sample, or a stool sample), comprising: (a) extracting genomic DNA from a biological sample from a human individual; (b) (i) treating the extracted genomic DNA with a reagent that modifies DNA in a methylation-specific manner; (ii) amplifying the bisulfite-treated genomic DNA using separate primers specific for one or more methylation markers; generating a fraction of the extracted genomic DNA by (c) analyzing one or more DNA fragments in the generated fraction of the extracted genomic DNA by measuring the methylation level of the CpG site of each of the one or more methylation markers.

[0117] Preferably, the sensitivity of such a method is about 70% to about 100%, or about 80% to about 90%, or about 80% to about 85%. Preferably, the specificity is about 70% to about 100%, or about 80% to about 90%, or about 80% to about 85%.

[0118] Such methods are not limited to a particular methylation marker, methylation marker gene, gene, DMR, and / or DNA methylation marker. In some embodiments, the one or more methylation markers, methylation marker genes, genes, DMRs, and / or DNA methylation markers comprise bases in a DMR selected from the group consisting of DMRs 1-423 shown in Tables I, III, and X.

[0119] In some embodiments, the one or more methylation markers, methylation marker genes, genes, DMRs, and / or DNA methylation markers are selected from Tables I and / or III.

[0120] In some embodiments, the one or more methylation markers, methylation marker genes, genes, DMRs, and / or DNA methylation markers are selected from the group consisting of MAX.chr6.58147682-58147771, C1ORF114, ASCL1, ARHGAP12, ZNF773, TTYH1, NEUROG3, ZNF781, NXPH1, MAX.chr9.36739811-36739868, NID2, TMEM200C, CRHR2, ABCB1 , ZNF69, ATP10A, MAX.chr18.73167725-73167817, MAX.chr2.127783183-127783403, CACNA1C, ZNF382, BARHL1, MAX.chr4.8859853-8859939, ST8SIA1, MAX.chr1.98510958-98511049, C2ORF40, SLC9A3, PRDM12, HOPX_C, and KCNQ5 (Example I).

[0121] In some embodiments, the one or more methylation markers, methylation marker genes, genes, DMRs, and / or DNA methylation markers are selected from C1orf114, MAX.chr6.58147682-58147771, ZNF773, NEUROG3, ASCL1, NID2, ZNF781, CRHR2, and MAX.chr9.36739811-36739868 (see Table VI and Example I).

[0122] In some embodiments, the one or more methylation markers, methylation marker genes, genes, DMRs, and / or DNA methylation markers are selected from the group consisting of ABCB1, ARHGAP12, ASCL1, BARHL1, C1orf114, C2orf40, CACNA1C, CRHR2, HOPX_C, KCNQ5, MAX.chr1.98510968-98511049, MAX.chr18.73167751-73167791, MA X.chr2.127783183-127783403, MAX.chr4.8859853-8859939, MAX.chr6.58147682-58147771, MAX.chr9.36739811-36739868, NEUROG3, NID2, NXPH1, PRDM12, SLC9A3, TMEM200C, TTYH1, ZNF382, ZNF773, and ZNF781 (Table VII, Example I).

[0123] In some embodiments, the one or more methylation markers, methylation marker genes, genes, DMRs, and / or DNA methylation markers are selected from ABCB1, c1orf95, CACNA1C, CACNG8, CHST2, ELMO1, EMID2, FBN1_B, FLT3_A, FLT3_B, GLIS1, GPC6, GREM2, JAM2, KCNK12_A, LOC100129620, MAX.chr15.78112404-78112692, MAX.chr19.4584907-4585088, MAX.chr3.69591689-69591784, NCAM1, NT5C1A, ST8SIA3, ZNF382, ZNF419, ZNF69, and ZSCAN18 (see Table VIII, Example I).

[0124] In some embodiments, the one or more methylation markers, methylation marker genes, genes, DMRs, and / or DNA methylation markers are selected from Table X.

[0125] In some embodiments, the one or more methylation markers, methylation marker genes, genes, DMRs, and / or DNA methylation markers are selected from AK5, RABC3, ZNF491, ZNF610, ZNF91, ZNF480, TRPC3_B, and ELMOD1 (Tables X and XI, Example II).

[0126] Such method is not limited to a specific sample or type of biological sample.For example, in some embodiments, biological sample is tissue sample (e.g., cervical tissue), blood sample, plasma sample, serum sample, whole blood sample, secretion sample (e.g., cervical secretion, vaginal secretion), organ secretion sample, CSF sample, saliva sample, urine sample, or stool sample.

[0127] Various cancers (e.g., cervical cancer and subtypes of cervical cancer) and precancers (e.g., cervical precancer) are predicted by various combinations of markers, as identified, for example, by statistical methods for specificity and sensitivity of prediction. The technology further provides methods for identifying predictive combinations and validated predictive combinations for several cancers.

[0128] Such methods are not limited to the type of subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0129] Such methods are not limited to a particular method or technique for determining, characterizing, measuring, or assaying the methylation of one or more methylation markers, methylation marker genes, genes, DMRs, and / or DNA methylation markers. In some embodiments, such techniques are based on analysis of the methylation state (e.g., CpG methylation state) of at least one marker, region of a marker, or base of a marker that comprises a DMR.

[0130] In some embodiments, measuring the methylation state of the methyl marker in the sample comprises determining the methylation state of a single base. In some embodiments, measuring the methylation state of the marker in the sample comprises determining the degree of methylation at a plurality of bases. Further, in some embodiments, the methylation state of the marker comprises an increase in methylation of the marker relative to a normal methylation state of the marker. In some embodiments, the methylation state of the marker comprises a decrease in methylation of the marker relative to a normal methylation state of the marker. In some embodiments, the methylation state of the marker comprises a different pattern of methylation of the marker compared to a normal methylation state of the marker.

[0131] Further, in some embodiments, the marker is a region of 100 or less bases, the marker is a region of 500 or less bases, the marker is a region of 1000 or less bases, the marker is a region of 5000 or less bases, or in some embodiments, the marker is a single base. In some embodiments, the marker is in a promoter with high CpG density.

[0132] In certain embodiments, methods of analyzing nucleic acids for the presence of 5-methylcytosine include treating DNA with reagents that modify DNA in a methylation-specific manner, examples of such reagents include, but are not limited to, methylation-sensitive restriction enzymes, methylation-dependent restriction enzymes, bisulfite reagents, TET enzymes, and borane reducing agents.

[0133] A frequently used method for analyzing nucleic acids for the presence of 5-methylcytosine is based on the bisulfite method described by Frommer, et al. for the detection of 5-methylcytosine in DNA (Frommer et al. (1992) Proc. Natl. Acad. Sci. USA 89:1827-31, expressly incorporated herein by reference in its entirety for all purposes) or their variations. The bisulfite method for mapping 5-methylcytosine is based on the finding that cytosine, but not 5-methylcytosine, reacts with hydrogen sulfite ions (also known as bisulfite). This reaction is usually carried out according to the following steps: First, cytosine reacts with bisulfite to form sulfonated cytosine. Next, spontaneous deamination of the sulfonated reaction intermediate results in sulfonated uracil. Finally, the sulfonated uracil is desulfonated under alkaline conditions to form uracil. Detection is possible because uracil base pairs with adenine (and thus behaves like thymine), whereas 5-methylcytosine base pairs with guanine (and thus behaves like cytosine). This allows for distinguishing methylated from unmethylated cytosines, for example, by bisulfite genomic sequencing (Grigg G, & Clark S, Bioessays (1994) 16:431-36; Grigg G, DNA Seq. (1996) 6:189-98), methylation specific PCR (MSP) as disclosed in U.S. Pat. No. 5,786,146, or using assays involving sequence specific cleavage, such as the QuARTS flap endonuclease assay (see, e.g., Zou et al. (2010) "Sensitive quantification of methylated markers with a novel methylation specific technology" Clin Chem 56:A199; and U.S. Pat. Nos. 8,361,720, 8,715,937, 8,916,344, and 9,212,392).

[0134] Some conventional techniques involve encapsulating the DNA to be analyzed in an agarose matrix, thereby preventing diffusion and renaturation of the DNA (bisulfite reacts only with single-stranded DNA), and replacing the precipitation and purification steps with high-speed dialysis (Olek A, et al. (1996) "A modified and improved method for bisulfite based cytosine methylation analysis" Nucleic Acids Res. 24:5064-6). It is therefore possible to analyze individual cells for their methylation status, demonstrating the usefulness and sensitivity of the method. An overview of conventional methods for detecting 5-methylcytosine is provided by Rein, T., et al. (1998) Nucleic Acids Res. 26:2255.

[0135] Bisulfite methods generally involve bisulfite treatment followed by amplification of short, specific fragments of known nucleic acids and then assaying the products by sequencing (Olek & Walter (1997) Nat Genet. 17:275-6) or primer extension reactions (Gonzalgo & Jones (1997) Nucleic Acids Res. 25: 2529-31; WO 95 / 00669; U.S. Patent No. 6,251,594) to analyze individual cytosine positions. Some methods use enzymatic digestion (Xiong & Laird (1997) Nucleic Acids Res. 25:2532-4). Detection by hybridization has also been described in the art (Olek et al., WO99 / 28498). Additionally, the use of bisulfite techniques for methylation detection on individual genes has been described (Grigg & Clark (1994) Bioessays 16:431-6; Zeschnigk et al. (1997) Hum Mol Genet. 6:387-95; Feil et al. (1994) Nucleic Acids Res. 22: 695; Martin et al. (1995) Gene 157:261-4; WO9746705; WO9515373).

[0136] Various methylation assay techniques can be used in conjunction with bisulfite treatment according to the technology of the present invention. These assays allow for the determination of the methylation state of one or more CpG dinucleotides (e.g., CpG islands) within a nucleic acid sequence. Such assays involve, among other techniques, sequencing of bisulfite-treated nucleic acids, PCR (for sequence-specific amplification), Southern blot analysis, and the use of methylation-specific enzymes, e.g., methylation-sensitive or methylation-dependent enzymes.

[0137] For example, genome sequencing has been simplified for the analysis of methylation patterns and 5-methylcytosine distribution by using bisulfite treatment (Frommer et al. (1992) Proc. Natl. Acad. Sci. USA 89:1827-1831). Furthermore, restriction enzyme digestion of PCR products amplified from bisulfite converted DNA can be used to assess methylation status, for example, as described in Sadri & Hornsby (1997) Nucl. Acids Res. 24:5058-5059, or as achieved in the method known as COBRA (Combined Bisulfite Restriction Analysis) (Xiong & Laird (1997) Nucleic Acids Res. 25:2532-2534).

[0138] The COBRA™ analysis is a quantitative methylation assay useful for determining DNA methylation levels at specific loci in small amounts of genomic DNA (Xiong & Laird, Nucleic Acids Res. 25:2532-2534, 1997). Briefly, restriction enzyme digestion is used to reveal methylation-dependent sequence differences in PCR products of sodium bisulfite-treated DNA. Methylation-dependent sequence differences are first introduced into genomic DNA by standard bisulfite treatment according to the procedure described by Frommer et al. (Proc. Natl. Acad. Sci. USA 89:1827-1831, 1992). PCR amplification of the bisulfite-converted DNA is then performed using primers specific for the CpG island of interest, followed by restriction endonuclease digestion, gel electrophoresis, and a specifically labeled hybridization probe. Methylation levels in the original DNA samples are represented by the relative amounts of digested and undigested PCR products, providing a linear method for quantification over a wide range of DNA methylation levels. In addition, the method can be reliably applied to DNA obtained from microdissected paraffin-embedded tissue samples.

[0139] Typical reagents for COBRA™ analysis (e.g., as found in a typical COBRA™-based kit) include, but are not limited to, PCR primers for specific loci (e.g., specific genes, markers, DMRs, gene regions, marker regions, bisulfite-treated DNA sequences, CpG islands, etc.); restriction enzymes and appropriate buffers; gene hybridization oligonucleotides; control hybridization oligonucleotides; kinase labeling kits for oligonucleotide probes; and labeled nucleotides. Additionally, bisulfite conversion reagents can include DNA denaturation buffers; sulfonation buffers; DNA recovery reagents or kits (e.g., precipitation, ultrafiltration, affinity columns); desulfonation buffers; and DNA recovery components.

[0140] Assays such as "MethyLight™" (fluorescence-based real-time PCR technology) (Eads et al., Cancer Res. 59:2302-2306, 1999), Ms-SNuPE™ (methylation-sensitive single nucleotide primer extension) reactions (Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531, 1997), methylation-specific PCR ("MSP"; Herman et al., Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996; U.S. Patent No. 5,786,146) and methylated CpG island amplification ("MCA"; Toyota et al., Cancer Res. 59:2307-12, 1999) are used alone or in combination with one or more of these methods.

[0141] The "HeavyMethyl™" assay, a technique, is a quantitative method to assess methylation differences based on methylation-specific amplification of bisulfite-treated DNA. Methylation-specific inhibitor probes ("inhibitors") covering the CpG positions between or covered by the amplification primers allow methylation-specific selective amplification of the diffuse sample.

[0142] The term "HeavyMethyl™ MethyLight™" assay refers to the HeavyMethyl™ MethyLight™ assay, which is a variation of the MethyLight™ assay in which the MethyLight™ assay is combined with a methylation-specific blocking probe that covers the CpG positions between the amplification primers. The HeavyMethyl™ assay can also be used in combination with methylation-specific amplification primers.

[0143] Typical reagents for HeavyMethyl™ analysis (e.g., as found in a typical MethyLight™-based kit) include, but are not limited to, PCR primers for specific loci (e.g., specific genes, markers, gene regions, marker regions, bisulfite-treated DNA sequences, CpG islands, or bisulfite-treated DNA sequences or CpG islands, etc.); blocking oligonucleotides; optimized PCR buffers and deoxynucleotides, and Taq polymerase.

[0144] MSP (methylation specific PCR) allows the assessment of the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of methylation sensitive restriction enzymes (Herman et al. Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996, U.S. Patent No. 5,786,146). Briefly, DNA is modified with sodium bisulfite, which converts unmethylated but not methylated cytosines to uracil, and these products are then amplified with primers specific for methylated versus unmethylated DNA. MSP requires only small amounts of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. Typical reagents for MSP analysis (e.g., as found in a typical MSP-based kit) include, but are not limited to, methylated and unmethylated PCR primers for specific loci (e.g., specific genes, markers, gene regions, marker regions, bisulfite-treated DNA sequences, CpG islands, etc.); optimized PCR buffers and deoxynucleotides, and specific probes.

[0145] The MethyLight™ assay is a high-throughput quantitative methylation assay that utilizes fluorescence-based real-time PCR (e.g., TaqMan®) and does not require further manipulation after the PCR step (Eads et al., Cancer Res. 59:2302-2306, 1999). Briefly, the MethyLight™ process begins with a mixed sample of genomic DNA that is converted into a mixed pool of methylation-dependent sequence differences according to standard procedures in a sodium bisulfite reaction (the bisulfite process converts unmethylated cytosine residues to uracil). Fluorescence-based PCR is then performed in a "biased" reaction, for example, with PCR primers that overlap known CpG dinucleotides. Sequence discrimination occurs both at the level of the amplification process and at the level of the fluorescence detection process.

[0146] The MethyLight™ assay is used as a quantitative test for methylation patterns in nucleic acid, e.g., genomic DNA samples, in which sequence discrimination occurs at the level of probe hybridization. In the quantitative version, the PCR reaction results in methylation-specific amplification in the presence of fluorescent probes that overlap specific putative methylation sites. An unbiased control for the input DNA amount is provided by a reaction in which neither the primers nor the probe overlap any CpG dinucleotides. Alternatively, a quantitative test for genomic methylation is achieved by probing a biased PCR pool with either control oligonucleotides that do not cover known methylation sites (e.g., fluorescent-based versions of HeavyMethyl™ and MSP techniques) or oligonucleotides that cover potential methylation sites.

[0147] The MethyLight™ process is used with any suitable probe (e.g., “TaqMan®” probe, Lightcycler® probe). For example, in some applications, double-stranded genomic DNA is treated with sodium bisulfite and subjected to one of two sets of PCR reactions, using, for example, a TaqMan® probe with MSP primers and / or a HeavyMethyl inhibitor oligonucleotide and a TaqMan® probe. The TaqMan® probe is dual-labeled with fluorescent “reporter” and “quencher” molecules and is designed to be specific for relatively GC-rich regions, so that it melts during the PCR cycles at a higher temperature, about 10° C., than the forward or reverse primers. This allows the TaqMan® probe to remain fully hybridized during the PCR annealing / extension step. The Taq polymerase will eventually reach the annealed TaqMan® probe to enzymatically synthesize a new strand during PCR. Taq polymerase 5' to 3' endonuclease activity then displaces the TaqMan® probe by digesting it, releasing a fluorescent reporter molecule for quantitative detection of its unquenched signal using a real-time fluorescence detection system.

[0148] Typical reagents for MethyLight™ analysis (e.g., as found in a typical MethyLight™-based kit) include, but are not limited to, PCR primers for specific loci (e.g., specific genes, markers, gene regions, marker regions, bisulfite-treated DNA sequences, CpG islands, etc.); TaqMan® or Lightcycler® probes; optimized PCR buffers and deoxynucleotides, and Taq polymerase.

[0149] The QM™ (Quantitative Methylation) Assay is an alternative quantitative test for methylation patterns in genomic DNA samples, in which sequence discrimination occurs at the level of probe hybridization. In this quantitative version, the PCR reaction results in unbiased amplification in the presence of fluorescent probes that overlap specific putative methylation sites. An unbiased control for the amount of input DNA is provided by a reaction in which neither the primers nor the probe overlap any CpG dinucleotides. Alternatively, a quantitative test for genomic methylation is achieved by probing a biased PCR pool with either control oligonucleotides that do not cover known methylation sites (fluorescence-based versions of HeavyMethyl™ and MSP techniques) or oligonucleotides that cover potential methylation sites.

[0150] The QM™ process can be used with any suitable probe, such as a “TaqMan®” probe, a Lightcycler® probe, etc., in the amplification process. For example, double-stranded genomic DNA is treated with sodium bisulfite and receives unbiased primers and a TaqMan® probe. The TaqMan® probe is dual-labeled with fluorescent “reporter” and “quencher” molecules and is designed to be specific for relatively GC-rich regions, so that it melts during the PCR cycle at a higher temperature, about 10° C., than the forward or reverse primers. This allows the TaqMan® probe to remain fully hybridized during the PCR annealing / extension step. The Taq polymerase will eventually reach the annealed TaqMan® probe to enzymatically synthesize a new strand during PCR. The Taq polymerase 5' to 3' endonuclease activity then displaces the TaqMan® probe by digesting it, releasing a fluorescent reporter molecule for quantitative detection of its unquenched signal using a real-time fluorescence detection system. Typical reagents for QM™ analysis (e.g., as found in a typical QM™-based kit) include, but are not limited to, PCR primers for specific loci (e.g., specific genes, markers, gene regions, marker regions, bisulfite-treated DNA sequences, CpG islands, etc.); TaqMan® or Lightcycler® probes; optimized PCR buffers and deoxynucleotides, and Taq polymerase.

[0151] The Ms-SNuPE™ technique is a quantitative method that involves single-base primer extension followed by evaluation of methylation differences at specific CpG sites based on bisulfite treatment of DNA (Gonzalgo & Jones, Nucleic Acids Res 25:2529-2531, 1997). Briefly, genomic DNA is reacted with sodium bisulfite, converting unmethylated cytosines to uracil, while leaving 5-methylcytosines unchanged. Amplification of the desired target sequence is then performed using PCR primers specific to the bisulfite-converted DNA, and the resulting products are isolated and used as templates for methylation analysis at CpG sites of interest. Small amounts of DNA can be analyzed (e.g., microdissected pathology sections), which avoids the use of restriction enzymes to determine the methylation status at CpG sites.

[0152] Typical reagents for Ms-SNuPE™ analysis (e.g., as found in a typical Ms-SNuPE™-based kit) include, but are not limited to, PCR primers for specific loci (e.g., specific genes, markers, gene regions, marker regions, bisulfite-treated DNA sequences, CpG islands, etc.); optimized PCR buffers and deoxynucleotides; gel extraction kits; positive control primers; Ms-SNuPE™ primers for specific loci; reaction buffers (for Ms-SNuPE reactions); and labeled nucleotides. Additionally, bisulfite conversion reagents can include DNA denaturation buffers; sulfonation buffers; DNA recovery reagents or kits (e.g., precipitation, ultrafiltration, affinity columns); desulfonation buffers; and DNA recovery components.

[0153] Reduced representation bisulfite sequencing (RRBS) begins with bisulfite treatment of nucleic acids to convert all unmethylated cytosines to uracils, followed by restriction enzyme digestion (e.g., with an enzyme that recognizes sites containing CG sequences, such as Mspl), and completing the sequencing of the fragments after coupling to an adaptor ligand. The choice of restriction enzyme enriches for fragments in CpG-dense regions, reducing the number of redundant sequences that may map to multiple gene locations during the analysis. As such, RRBS reduces the complexity of the nucleic acid sample by selecting a subset of restriction fragments for sequencing (e.g., by size selection using preparative gel electrophoresis). In contrast to whole genome bisulfite sequencing, all fragments generated by restriction enzyme digestion contain DNA methylation information for at least one CpG dinucleotide. As such, RRBS provides an assay to assess the methylation status of one or more genomic loci, since it enriches the sample for promoters, CpG islands, and other genomic features with high frequency of restriction enzyme cleavage sites in these regions.

[0154] A typical protocol for RRBS includes the steps of digesting a nucleic acid sample with a restriction enzyme such as MspI, filling in overhangs and A-tailing, adaptor ligation, bisulfite conversion, and PCR. See, e.g., (2005) "Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution" Nat Methods 7:133-6; Meissner et al. (2005) "Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis" Nucleic Acids Res. 33: 5868-77.

[0155] In some embodiments, a quantitative allele-specific real-time target and signal amplification (QuARTS) assay is used to assess methylation status. In each QuARTS assay, three reactions occur in succession, the primary reaction includes amplification (reaction 1) and target probe cleavage (reaction 2), and the secondary reaction includes FRET cleavage and fluorescent signal generation (reaction 3). When the target nucleic acid is amplified with a specific primer, a specific detection probe with a flap sequence is loosely bound to the amplicon. The presence of a specific invasive oligonucleotide at the target binding site causes a 5' nuclease, such as FEN-1 endonuclease, to cleave between the detection probe and the flap sequence, thereby releasing the flap sequence. The flap sequence is complementary to the non-hairpin portion of the corresponding FRET cassette. Thus, the flap sequence functions as an invasive oligonucleotide on the FRET cassette, resulting in cleavage between the FRET cassette fluorophore and the quencher, generating a fluorescent signal. The cleavage reaction can cleave multiple probes per target, thereby releasing multiple fluorophores per flap, resulting in exponential signal amplification. QuARTS is capable of detecting multiple targets in a single reaction well by using FRET cassettes containing different dyes (see, e.g., Zou et al. (2010) "Sensitive quantification of methylated markers with a novel methylation specific technology" Clin Chem 56:A199), and U.S. Patent Nos. 8,361,720, 8,715,937, 8,916,344, and 9,212,392, each of which is incorporated by reference herein for all purposes.

[0156] The term "bisulfite reagent" refers to a reagent comprising bisulfite, disulfite, hydrogen sulfite, or a combination thereof, useful for distinguishing between methylated and unmethylated CpG dinucleotide sequences, as disclosed herein. Methods of said treatment are known in the art (e.g., PCT / EP2004 / 011715 and WO2013 / 116375, each of which is incorporated by reference in its entirety). In some embodiments, the bisulfite treatment is carried out in the presence of a denaturing solvent, such as, but not limited to, n-alkylene glycol or diethylene glycol dimethyl ether (DME), or in the presence of dioxane or a dioxane derivative. In some embodiments, the denaturing solvent is used at a concentration of 1% to 35% (v / v). In some embodiments, the bisulfite reaction is carried out in the presence of a scavenger, such as, but not limited to, a chroman derivative, e.g., 6-hydroxy-2,5,7,8,-tetramethylchroman 2-carboxylic acid or trihydroxybenzoic acid, and derivatives thereof, e.g., gallic acid (see PCT / EP2004 / 011715, which is incorporated herein by reference in its entirety). In certain preferred embodiments, the bisulfite reaction includes treatment with ammonium bisulfite, e.g., as described in WO2013 / 116375.

[0157] In some embodiments, the treated DNA fragments are amplified using a set of primer oligonucleotides according to the invention (see, for example, Tables V and VII) and an amplification enzyme. It is possible to carry out the amplification of several DNA segments simultaneously in one reaction vessel and in the same reaction vessel. Usually, the amplification is carried out using the polymerase chain reaction (PCR). The amplicons are usually 100-2000 base pairs long.

[0158] In another embodiment of the method, the methylation status of CpG positions in or near markers containing DMRs (e.g., DMRs 1-423, Tables I, III and X) can be detected using methylation-specific primer oligonucleotides. This technique (MSP) is described in U.S. Patent No. 6,265,171 to Herman. The use of methylation status-specific primers for the amplification of bisulfite-treated DNA allows differentiation between methylated and unmethylated nucleic acids. MSP primer pairs contain at least one primer that hybridizes to bisulfite-treated CpG dinucleotides. Thus, the sequence of the primer contains at least one CpG dinucleotide. MSP primers specific for unmethylated DNA contain a "T" at the C position in the CpG.

[0159] Such methods are not limited to a particular type or kind of primer or primer pair associated with one or more methylation markers, methylation marker genes, genes, DMRs, and / or DNA methylation markers. In some embodiments, the primers or primer pairs are set forth in Table V (SEQ ID NOs: 1-76). In some embodiments, the primers or primer pairs specific for each methylation marker gene can bind to an amplicon bound by a primer sequence for a marker gene set forth in Tables V and XII, where the amplicon bound by a primer sequence for a marker gene set forth in Tables V and XII is at least a portion of a gene region of a methylation marker set forth in Tables I, III, and X. In some embodiments, the primers or primer pairs for the methylation markers are a set of primers that specifically bind to at least a portion of a gene region that includes the chromosomal coordinates of a particular methylation marker set forth in Tables I, III, and X.

[0160] In another embodiment, the invention provides a method for converting oxidized 5-methylcytosine residues in cell-free DNA to dihydrouracil residues (see Liu et al., 2019, Nat Biotechnol. 37, pp. 424-429; U.S. Patent Application Publication No. 202000370114). The method comprises reacting an oxidized 5mC residue selected from 5-formylcytosine (5fC), 5-carboxymethylcytosine (5caC), and combinations thereof, with a borane reducing agent. Oxidized 5mC residues may be naturally occurring or, more typically, may result from prior oxidation of a 5mC or 5hmC residue, e.g., oxidation of 5mC or 5hmC by an enzyme of the TET family (e.g., TET1, TET2, or TET3), or chemical oxidation of 5mC or 5hmC (see, e.g., Okamato et al. (2011) Chem. Commun. 47:11231-33), e.g., using an inorganic peroxo compound or composition such as potassium perruthenate (KRuO4) or peroxotungstate and a combination of copper(II) perchlorate / 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) (see, e.g., Matsushita et al. (2017) Chem. Commun. 53:5756-59).

[0161] The borane reducing agent can be characterized as a complex of borane and a nitrogen-containing compound selected from a nitrogen heterocycle and a tertiary amine. The nitrogen heterocycle can be monocyclic, bicyclic, or polycyclic, but is typically monocyclic in the form of a 5- or 6-membered ring containing a nitrogen heteroatom and optionally one or more additional heteroatoms selected from N, O, and S. The nitrogen heterocycle can be aromatic or alicyclic. Preferred nitrogen heterocycles herein include 2-pyrroline, 2H-pyrrole, 1H-pyrrole, pyrazolidine, imidazolidine, 2-pyrazoline, 2-imidazoline, pyrazole, imidazole, 1,2,4-triazole, 1,2,4-triazole, pyridazine, pyrimidine, pyrazine, 1,2,4-triazine, and 1,3,5-triazine, any of which may be unsubstituted or substituted with one or more non-hydrogen substituents. Typical non-hydrogen substituents are alkyl groups, particularly lower alkyl groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, etc. Exemplary compounds include pyridine borane, 2-methylpyridine borane (also called 2-picoline borane), and 5-ethyl-2-pyridine.

[0162] The reaction of borane reducing agents with oxidized 5mC residues of cell-free DNA is advantageous insofar as non-toxic reagents and mild reaction conditions can be used, without the need for hydrogen sulfate or other reagents that may degrade DNA. Furthermore, the conversion of oxidized 5mC residues to dihydrolauracils with borane reducing agents can be carried out in a "one-pot" or "one-tube" reaction without the need for isolation of intermediates. This is crucial since the conversion involves multiple steps, namely (1) reduction of the alkene bond connecting C-4 and C-5 of oxidized 5mC, (2) deamination, and (3) either decarboxylation if oxidized 5mC is 5caC or deformylation if oxidized 5mC is 5fC.

[0163] In addition to the method of converting oxidized 5-methylcytosine residues in cell-free DNA to dihydrouracil residues, the present invention also provides a reaction mixture related to the above method. The reaction mixture includes a sample of cell-free DNA containing at least one oxidized 5-methylcytosine residue selected from 5caC, 5fC, and combinations thereof, and a borane reducing agent effective to reduce, deaminate, and decarboxylate or deformylate at least one oxidized 5-methylcytosine residue. As explained above, the borane reducing agent is a complex of borane and a nitrogen-containing compound selected from nitrogen heterocycles and tertiary amines. In a preferred embodiment, the reaction mixture is substantially free of bisulfite, meaning that it is substantially free of bisulfite ions and bisulfite salts. Ideally, the reaction mixture is free of bisulfite salts.

[0164] In a related aspect of the invention, a kit is provided for converting 5mC residues in cell-free DNA to dihydrouracil residues, the kit comprising a reagent for blocking 5hmC residues, a reagent for oxidizing 5mC residues beyond hydroxymethylation to provide oxidized 5mC residues, and a borane reducing agent effective to reduce, deaminate, and decarboxylate or deformylate the oxidized 5mC residues. The kit may also include instructions for using the components to practice the above method.

[0165] In another embodiment, a method is provided that utilizes the above oxidation reaction, which allows for the detection of the presence and location of 5-methylcytosine residues in cell-free DNA, and includes the steps of: (a) modifying 5hmC residues in the fragmented, adaptor-ligated cell-free DNA to provide an affinity tag to the residue, which affinity tag allows for removal of the modified 5hmC-containing DNA from the cell-free DNA; (b) removing the modified 5hmC-containing DNA from the cell-free DNA, leaving DNA that contains unmodified 5mC residues; (c) oxidizing unmodified 5mC residues to obtain DNA comprising oxidized 5mC residues selected from 5caC, 5fC, and combinations thereof; (d) contacting the DNA containing the oxidized 5mC residue with a borane reducing agent effective to reduce, deaminate, and decarboxylate or deformylate the oxidized 5mC residue, thereby providing DNA containing a dihydrouracil residue in place of the oxidized 5mC residue; (e) amplifying and sequencing DNA containing dihydrouracil residues; (f) determining the 5-methylation pattern from the sequencing results of (e).

[0166] In another embodiment, a method for identifying 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC) in a target nucleic acid is provided, comprising the steps of: Providing a biological sample containing a target nucleic acid; Modifying a target nucleic acid, comprising: converting 5mC and 5hmC in the nucleic acid sample to 5-carboxylcytosine (5caC) and / or 5-formylcytosine (5fC) by contacting the nucleic acid sample with a TET enzyme to generate one or more 5caC or 5fC residues; modifying the target nucleic acid, comprising converting 5caC and / or 5fC to dihydrolauracil (DHU) by treating the target nucleic acid with a borane reducing agent to provide a modified nucleic acid sample comprising the modified target nucleic acid; A step of detecting the sequence of the modified target nucleic acid, wherein a change from cytosine (C) to thymine (T) or a change from cytosine (C) to DHU in the sequence of the modified target nucleic acid compared to the target nucleic acid provides a position of 5mC or 5hmC in the target nucleic acid.

[0167] In some embodiments, the borane reducing agent is 2-picoline borane.

[0168] In some embodiments, detecting the sequence of the modified target nucleic acid comprises one or more of chain termination sequencing, microarray, high throughput sequencing, and restriction enzyme analysis.

[0169] In some embodiments, the TET enzyme is selected from the group consisting of human TET1, TET2, and TET3; mouse Tet1, Tet2, and Tet3; Naegleria TET (NgTET); and Coprinopsis cinerea (CcTET).

[0170] In some embodiments, the method further comprises blocking one or more modified cytosines, hi some embodiments, the blocking comprises adding a sugar to the 5hmC.

[0171] In some embodiments, the method further comprises amplifying the copy number of one or more nucleic acid sequences.

[0172] In some embodiments, the oxidizing agent is potassium perruthenate or Cu(II) / TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl).

[0173] Cell-free DNA is extracted from a subject's body sample, which is usually whole blood, plasma, or serum, most commonly plasma, but may also be tissue (e.g., cervical tissue), secretions (e.g., cervical secretions, vaginal secretions), organ secretions, CSF, urine, saliva, mucosal excretions, sputum, stool, or tears. In some embodiments, the cell-free DNA is derived from a tumor. In other embodiments, the cell-free DNA is derived from a patient with a disease or other pathological condition. The cell-free DNA may or may not be derived from a tumor. It is noted that in step (a), the cell-free DNA in which the 5hmC residues are modified is in purified, fragmented form, and adapter ligated. DNA purification in this context can be performed using any suitable method known to those skilled in the art and / or described in the relevant literature, and the cell-free DNA itself may be highly fragmented, although in some cases further fragmentation may be desirable, for example as described in US Patent Publication No. 2017 / 0253924. Cell-free DNA fragments generally range in size from about 20 nucleotides to about 500 nucleotides, more typically from about 20 nucleotides to about 250 nucleotides. The purified cell-free DNA fragments modified in step (a) are end-repaired using conventional means (e.g., restriction enzymes) so that the fragments have blunt ends at each 3' and 5' end. In a preferred method, the blunt-ended fragments are given a 3' overhang containing one adenine residue using a polymerase such as Taq polymerase, as described in WO2017 / 176630. This facilitates the subsequent ligation of a selected universal adaptor, i.e., an adaptor such as a Y adaptor or a hairpin adaptor containing at least one molecular barcode, which is ligated to both ends of the cell-free DNA fragments. The use of adaptors also allows for selective PCR enrichment of adaptor-ligated DNA fragments.

[0174] Thus, in step (a), the "purified fragmented cell-free DNA" comprises adaptor-ligated DNA fragments. The modification of 5hmC residues of these cell-free DNA fragments with affinity tags is performed to allow for the subsequent removal of modified 5hmC-containing DNA from the cell-free DNA, as described in step (a). In one embodiment, the affinity tag comprises a biotin moiety, such as biotin, desthiobiotin, oxybiotin, 2-iminobiotin, diaminobiotin, biotin sulfoxide, biocytin, etc. The use of a biotin moiety as an affinity tag facilitates removal by streptavidin, e.g., streptavidin beads, magnetic streptavidin beads, etc.

[0175] Tagging of 5hmC residues with biotin moieties or other affinity tags is achieved by covalently attaching a chemoselective group to the 5hmC residues in the DNA fragment, which can react with a functionalized affinity tag to attach the affinity tag to the 5hmC residue. In one embodiment, the chemoselective group is UDP glucose-6-azide, which undergoes a 1,3-cycloaddition reaction with an alkyne-functionalized biotin moiety (described in Robertson et al. (2011) Biochem. Biophys. Res. Comm. 411(1):40-3, U.S. Patent No. 8,741,567, and WO 2017 / 176630). Thus, the addition of an alkyne-functionalized biotin moiety results in the covalent attachment of the biotin moiety to each 5hmC residue.

[0176] The affinity-tagged DNA fragments can then be removed in step (b) using streptavidin, in one embodiment in the form of streptavidin beads, magnetic streptavidin beads, etc., and set aside for later analysis if desired. The supernatant remaining after removal of the affinity-tagged fragments contains DNA with unmodified 5mC residues and no 5hmC residues.

[0177] In step (c), the unmodified 5mC residue is oxidized to give 5caC and / or 5fC residues using any suitable means. The oxidizing agent is selected to oxidize the 5mC residue beyond hydroxymethylation, i.e. to give 5caC and / or 5fC residues. The oxidation can be performed enzymatically using a catalytically active TET family enzyme. The term "TET family enzyme" or "TET enzyme" as used herein refers to a catalytically active "TET family protein" or "TET catalytically active fragment" as defined in U.S. Pat. No. 9,115,386, the disclosure of which is incorporated herein by reference. A preferred TET enzyme in this context is TET2 (Ito et al. (2011) Science 333(6047):1300-1303). The oxidation can also be performed chemically using a chemical oxidizing agent, as described in the previous section. Examples of suitable oxidizing agents include, but are not limited to, perruthenate anions in the form of inorganic or organic perruthenates, including metal perruthenates such as potassium perruthenate (KRuO4), tetraalkylammonium perruthenates such as tetrapropylammonium perruthenate (TPAP) and tetrabutylammonium perruthenate (TBAP), and polymer-supported perruthenate (PSP); and inorganic peroxo compounds and compositions, such as peroxotungstate or a combination of copper(II) perchlorate / TEMPO. At this point, it is not necessary to separate the 5fC-containing fragment from the 5caC-containing fragment, as long as both the 5fC and 5caC residues are converted to dihydrouracil (DHU) in the next step of the process, step (e).

[0178] In some embodiments, 5-hydroxymethylcytosine residues are blocked with β-glucosyltransferase (β3GT), while 5-methylcytosine residues are oxidized with TET enzymes, which are effective in giving a mixture of 5-formylcytosine and 5-carboxymethylcytosine. The mixture containing both of these oxidized species can be reacted with 2-picoline borane or another borane reducing agent to give dihydrouracil. In a variation of this embodiment, the 5hmC-containing fragments are not removed in step (b). Instead, 5mC-containing fragments and 5hmC-containing fragments are enzymatically oxidized together by "TET-assisted picoline borane sequencing (TAPS)" to give 5fC-containing fragments and 5caC-containing fragments. The reaction with 2-picoline borane generates DHU residues where the 5mC and 5hmC residues were originally located. In "chemically assisted picoline borane sequencing (CAPS)", 5hmC-containing fragments are selectively oxidized with potassium perruthenate, leaving the 5mC residues unchanged.

[0179] The method of this embodiment has many advantages: no bisulfite is required, non-toxic reagents and reactants are used, and the process proceeds under mild conditions. Furthermore, the entire process can be carried out in a single test tube, without the need for isolation of intermediates.

[0180] In a related embodiment, the method includes as a further step (g) determining the hydroxymethylation pattern of the 5hmC-containing DNA removed from the cell-free DNA in step (b). This can be done using the techniques described in detail in WO2017 / 176630. This process can be done in a one-tube method without intermediate removal or isolation. For example, cell-free DNA fragments, preferably adapter-ligated DNA fragments, are first subjected to functionalization with βGT-catalyzed uridine diphosphoglucose 6-azide and then biotinylated via a chemoselective azide group. This procedure results in covalent attachment of biotin to each 5hmC site. In the next step, the biotinylated strands and the strands containing unmodified (natural) 5mC are simultaneously removed for further processing. The natural 5mC-containing strands are removed using anti-5mC antibodies or methyl-CpG binding domain (MBD) proteins, as is well known to those skilled in the art. With the 5hmC residues blocked, unmodified 5mC residues are then selectively oxidized using any suitable technique for converting 5mC to 5fC and / or 5caC, as described elsewhere herein.

[0181] These fragments obtained by amplification can have directly or indirectly detectable labels.In some embodiments, these labels are fluorescent labels, radionuclides, or detachable molecular fragments with typical masses that can be detected in mass spectrometers.When these labels are mass labels, some embodiments provide that the labeled amplicons have a single positive or negative effective charge, allowing for better detectability in mass spectrometers.This detection can be performed and visualized, for example, by matrix-assisted laser desorption / ionization mass spectrometry (MALDI) or using electron spray mass spectrometry (ESI).

[0182] Methods for isolating DNA suitable for these assay techniques are well known in the art. In particular, some embodiments involve isolating nucleic acids as described in U.S. Patent Application Serial No. 13 / 470,251 ("Isolation of Nucleic Acids"), which is incorporated by reference in its entirety.

[0183] In some embodiments, the markers described herein can be used in a QUARTS assay performed on a biological sample (e.g., a tissue sample (e.g., a cervical sample), a blood sample, a plasma sample, a serum sample, a whole blood sample, a secretion sample (e.g., cervical secretions, vaginal secretions), an organ secretion sample, a CSF sample, a saliva sample, a urine sample, or a stool sample). In some embodiments, methods are provided for generating DNA samples, particularly DNA samples that contain highly purified low abundance nucleic acids in small volumes (e.g., less than 100 microliters, less than 60 microliters) and that are substantially and / or effectively free of substances that inhibit assays used to test the DNA sample (e.g., PCR, INVADER, QuARTS assays, etc.). Such DNA samples can be used in diagnostic assays that qualitatively detect the presence or quantitatively measure the activity, expression or amount of genes, genetic variants (e.g., alleles), or genetic modifications (e.g., methylation) present in a sample taken from a patient. For example, some cancers are correlated with the presence of particular mutant alleles or particular methylation states, and therefore, detection and / or quantification of such mutant alleles or methylation states has predictive value in cancer diagnosis and treatment.

[0184] Many useful genetic markers are present in very small amounts in samples, and many of the events that generate such markers are rare.Thus, even highly sensitive detection methods such as PCR require large amounts of DNA to provide low-abundance targets that are sufficient to meet or exceed the detection threshold of the assay.Furthermore, the presence of even small amounts of inhibitors impairs the accuracy and precision of these assays aimed at detecting such low-abundance targets.Therefore, methods are provided herein that provide the necessary volume and concentration control for generating such DNA samples.

[0185] In some embodiments, the sample comprises a tissue sample (e.g., a cervical sample), blood, plasma, serum, whole blood, secretions (e.g., cervical secretions, vaginal secretions), organ secretions, CSF, saliva, urine, or stool. In some embodiments, the subject is a human. Samples such as these can be obtained by any number of means known in the art, as would be apparent to one of skill in the art. For example, urine and fecal samples are readily achievable, while blood, ascites, serum, or pancreatic juice samples can be obtained parenterally, for example, by using a needle and syringe. Acellular or substantially acellular samples can be obtained by subjecting the sample to a variety of techniques, including, but not limited to, centrifugation and filtration. Although it is generally preferred to obtain samples using non-invasive techniques, it may be preferable to obtain samples such as tissue homogenates, tissue sections, and biopsy samples.

[0186] In some embodiments, the sample is obtained using any type or kind of collection device capable of obtaining the desired sample type, for example, the collection device may be a device capable of obtaining a cervical tissue sample. In certain embodiments, the collection device is a device capable of obtaining tissue or cells from or near the cervix. In some embodiments, cervical tissue samples include, for example, samples containing any cervical tissue or cervical cells, which may include tissue or cells from areas anatomically near the cervix (e.g., vaginal tissue, vaginal cells, endometrial tissue, endometrial cells, ovarian tissue, ovarian cells, etc.) in addition to cervical tissue or cells. In another embodiment, cervical secretion samples include, for example, samples containing any cervical secretion(s), including secretions from areas anatomically near the cervix (e.g., vaginal secretions, endometrial secretions, ovarian secretions, etc.). In some embodiments, the collection device has an absorbent member capable of collecting a sample (e.g., tissue, secretion, and / or cells) when contacted with a body area (e.g., cervix, vaginal canal). In some embodiments, the absorbent member is a sponge having a shape and size suitable for insertion into a body orifice (e.g., cervix, vaginal canal), e.g., having a cylindrical shape. In some embodiments, the collection device is a tampon (e.g., a standard tampon), an irrigator that releases liquid into the vagina and recollects the liquid (e.g., a Pantarhei screener), a cervical brush (e.g., a brush that is inserted into the vagina and rotated to collect cells), a Fournier cervical self-collection device (a tampon-like plastic stick), or a cotton swab. In some embodiments, the absorbent member is made of a material that allows for the collection of the desired sample. In some embodiments, the absorbent member is a sponge material such as rayon and / or cotton.

[0187] Acellular or substantially acellular samples can be obtained by subjecting the sample to various techniques, including, but not limited to, centrifugation and filtration. Although it is generally preferred to obtain samples using non-invasive techniques, it may be preferred to obtain samples such as tissue homogenates, tissue sections, and biopsy samples. The present technology is not limited to the method used to prepare the sample and provide nucleic acid for testing. For example, in some embodiments, DNA is isolated from a sample (e.g., a tissue sample (e.g., cervical tissue), a blood sample, a plasma sample, a serum sample, a whole blood sample, a secretion sample (e.g., cervical secretion, vaginal secretion), an organ secretion sample, a CSF sample, a saliva sample, a urine sample, or a stool sample) using direct gene capture, as described in detail in U.S. Pat. Nos. 8,808,990 and 9,169,511, and WO2012 / 155072, or related methods.

[0188] The analysis of markers can be performed separately or simultaneously with additional markers in one test sample. For example, it is possible to combine several markers in one test to allow efficient processing of multiple samples and potentially provide higher diagnostic and / or prognostic accuracy. Furthermore, those skilled in the art will recognize the value of testing multiple samples from the same subject (e.g., at successive time points). Such testing of successive samples allows the identification of changes in the methylation status of markers over time. Changes in methylation status, and the absence of changes in methylation status, can provide useful information about disease status, including, but not limited to, revealing the approximate time from the occurrence of this event, the presence and amount of salvageable tissue, suitability of drug therapy, efficacy of various therapies, and identification of the subject's outcome, including risk of future events.

[0189] Biomarker analysis can be performed in a variety of physical formats, for example using microtiter plates or automated applications to facilitate the processing of multiple test samples, or single sample formats can be developed to facilitate immediate treatment and diagnosis in a timely manner, for example in an outpatient or emergency room setting.

[0190] Genomic DNA can be isolated by any means, including the use of commercially available kits. Briefly, if the DNA of interest is encapsulated by a cell membrane, the biological sample must be disrupted and dissolved by enzymatic, chemical or mechanical means. Proteins and other contaminants can then be removed from the DNA solution, for example, by digestion with proteinase K. The genomic DNA is then recovered from the solution. This can be done by a variety of methods, including salting out, organic extraction, or binding of DNA to a solid support. The choice of method is influenced by several factors, including time, cost, and the amount of DNA required. All types of clinical samples, including neoplastic or pre-neoplastic material, are suitable for use in the method, such as tissues (e.g., cervical tissue), cell lines, histological slides, biopsies, paraffin-embedded tissues, secretions (e.g., cervical secretions, vaginal secretions), body fluids, feces, tissues, colonic discharge, urine, plasma, serum, whole blood, isolated blood cells, cells isolated from blood, and combinations thereof.

[0191] The present technology is not limited to the method used to prepare samples and provide nucleic acid for testing.For example, in some embodiments, DNA is isolated from biological samples (e.g., tissue samples (e.g., cervical tissue), blood samples, plasma samples, serum samples, whole blood samples, secretion samples (e.g., cervical secretion, vaginal secretion), organ secretion samples, CSF samples, saliva samples, urine samples, or stool samples) using direct gene capture, as described in detail in US Patent Application Publication No. 61 / 485386, or related methods.

[0192] The genomic DNA sample is then treated with at least one reagent, or a series of reagents, that distinguishes between methylated and unmethylated CpG dinucleotides within at least one marker that comprises a DMR (e.g., DMRs 1-423, e.g., as shown in Tables I, III, and X).

[0193] In some embodiments, the reagent converts cytosine bases that are not methylated at the 5' position to uracil, thymine, or another base that differs from cytosine in terms of hybridization behavior, although in some embodiments the reagent can be a methylation-sensitive restriction enzyme.

[0194] In some embodiments, the genomic DNA sample is treated in such a way that cytosine bases that are not methylated at the 5' position are converted to uracil, thymine, or another base that differs from cytosine in terms of hybridization behavior. In some embodiments, this treatment is carried out with bisulfite (hydrogen sulfite), followed by alkaline hydrolysis.

[0195] The treated nucleic acid is then analyzed to determine the methylation status of the target gene sequence (at least one gene, genomic sequence, or nucleotide from a marker that includes a DMR, e.g., at least one DMR selected from DMRs 1-423, e.g., as set forth in Tables I, III, and X). Analytical methods can be selected from those known in the art, including analytical methods listed herein, e.g., QuARTS and MSP as described herein.

[0196] Aberrant methylation, and more specifically, hypermethylation of markers that include DMRs (e.g., DMRs 1-423, e.g., as shown in Tables I, III, and X), is associated with cervical cancer, subtypes of cervical cancer, and cervical precancer.

[0197] In some embodiments, the technology relates to a method of treating a patient (e.g., a patient with cervical cancer and / or a subtype of cervical cancer), comprising: 1) determining one or both of the methylation statuses of one or more methylation markers as provided herein, and administering a treatment to the patient based on the results of determining the methylation status. The treatment can be administering a pharmaceutical compound, administering a vaccine, performing a surgery, imaging the patient, performing another test. Preferably, the use is in methods of clinical screening, methods of prognostic evaluation, methods of monitoring the outcome of a therapy, methods of identifying patients most likely to respond to a particular therapeutic treatment, methods of imaging a patient or subject, and methods for drug screening and development.

[0198] In some embodiments of the present technology, a method is provided for diagnosing a particular type of cancer (e.g., cervical cancer or subtypes of cervical cancer) and / or precancer (e.g., cervical precancer) in a subject. The terms "diagnose" and "diagnosis" as used herein refer to a method by which a person skilled in the art can estimate and even determine whether a subject suffers from a given disease or condition, or is likely to develop a given disease or condition in the future. A person skilled in the art often makes a diagnosis based on one or more diagnostic indicators, such as one or more biomarkers (e.g., one or more methylation markers, methylation marker genes, genes, DMRs, and / or DNA methylation markers as disclosed herein), the methylation status of which indicates the presence, severity, or absence of a condition.

[0199] In addition to diagnosis, clinical cancer prognosis is concerned with determining the aggressiveness of cancer and the possibility of tumor recurrence, and planning the most effective therapy. If it is possible to make a more accurate diagnosis, or even if it is possible to evaluate the potential risk of suffering from cancer, it is possible to select appropriate treatment, and in some cases, treatment that is less harsh for the patient. The evaluation of cancer biomarkers (e.g., determining methylation status) is useful for dividing subjects with good prognosis and / or low risk of suffering from cancer (e.g., cervical cancer or subtypes of cervical cancer) that do not require treatment, or subjects with a higher probability of suffering from cancer into subjects with limited treatment, or subjects with cancer recurrence that may benefit from more intensive treatment.

[0200] As such, "making a diagnosis" or "diagnosing" as used herein further includes determining the risk of suffering from cancer (e.g., cervical cancer or a subtype of cervical cancer) or determining a prognosis, which can be provided to predict clinical outcomes (with or without medical treatment), select appropriate treatment (or whether treatment is effective), or monitor current treatment and potentially modify treatment based on measurements of diagnostic biomarkers (e.g., DMR) disclosed herein. Furthermore, in some embodiments of the subject matter disclosed herein, multiple determinations of biomarkers over time can be made to facilitate diagnosis and / or prognosis. Temporal changes in biomarkers can be used to predict clinical outcomes, monitor the progression of cancer or a subtype of cancer (e.g., cervical cancer or a subtype of cervical cancer), and / or monitor the effectiveness of appropriate therapies directed against cancer. In such embodiments, one would expect to see changes in the methylation status of one or more biomarkers (e.g., DMR) disclosed herein (and optionally one or more additional biomarkers, if monitored) in biological samples (e.g., tissue samples (e.g., cervical samples), blood samples, plasma samples, serum samples, whole blood samples, secretion samples (e.g., cervical secretions, vaginal secretions), organ secretion samples, CSF samples, saliva samples, urine samples, and stool samples) over time during the course of an effective therapy.

[0201] The subject matter disclosed herein further provides, in some embodiments, a method for determining whether to initiate or continue prevention or treatment of cancer (e.g., cervical cancer or a subtype of cervical cancer) in a subject. Any changes in the time period can be used to predict the risk of suffering from cancer (e.g., cervical cancer or a subtype of cervical cancer), predict clinical regression, determine whether to initiate or continue prevention or treatment of cancer, and whether the current treatment is effectively treating the cancer. For example, a first time point can be selected before the start of treatment, and a second time point can be selected at a time point after the start of treatment. The methylation status can be measured in each of the samples taken at different time points, and qualitative and / or quantitative differences are recorded. Changes in the methylation status of biomarker levels from different samples can be correlated with the risk, prognosis, treatment efficacy determination, and / or cancer progression of a particular cancer (e.g., cervical cancer or a subtype of cervical cancer) in the subject.

[0202] In preferred embodiments, the methods and compositions of the invention are for the treatment or diagnosis of disease at an early stage, e.g., before symptoms of the disease (e.g., cervical cancer or subtypes of cervical cancer) are present. In some embodiments, the methods and compositions of the invention are for the treatment or diagnosis of disease at a clinical stage.

[0203] As described, in some embodiments, multiple determinations of one or more diagnostic or prognostic biomarkers can be performed, and changes in the markers over time can be used to determine a diagnosis or prognosis. For example, a diagnostic marker can be determined a first time and again a second time. In these and other embodiments, an increase in a marker from the first time to the second time can be a diagnosis of a particular type or severity of cancer (e.g., cervical cancer or a subtype of cervical cancer), or a given prognosis. Similarly, a decrease in a marker from the first time to the second time can indicate a particular type or severity of cancer, or a given prognosis. Furthermore, the degree of change in one or more markers can be related to the severity of cancer, and future adverse events. Those skilled in the art will understand that in certain embodiments, relative measurements of the same biomarkers can be performed at multiple time points, and a particular biomarker can be measured at one time point and a second biomarker at a second time point, and a comparison of these markers can provide diagnostic information.

[0204] As used herein, the phrase "determining prognosis" refers to a method that allows a person skilled in the art to predict the course or outcome of a condition in a subject. The term "prognosis" does not refer to the ability to predict the course or outcome of a condition with 100% accuracy, or even the ability that a given course or outcome is more or less likely to occur based on the methylation status of a biomarker (e.g., DMR). Instead, a person skilled in the art will understand that the term "prognosis" refers to the increased probability that a certain course or outcome will occur, i.e., that a course or outcome is more likely to occur in a subject that exhibits a given condition when compared to those individuals that do not exhibit the condition. For example, an individual that does not exhibit a condition (e.g., has a normal methylation status of one or more DMRs) may have a very low probability of a given outcome (e.g., suffering from a certain type of cancer (e.g., cervical cancer or subtype of cervical cancer)).

[0205] In some embodiments, statistical analysis correlates the prognostic indicator with a predisposition to adverse outcomes. For example, in some embodiments, a methylation status different from that in a normal control sample obtained from a patient without cancer (e.g., cervical cancer or a subtype of cervical cancer) may indicate that the subject is more likely to suffer from cancer than a subject with a level more similar to the methylation status in the control sample, as determined by the level of statistical significance. Furthermore, the change in methylation status from the baseline (e.g., "normal") level may reflect the subject's prognosis, and the degree of change in methylation status may be related to the severity of an adverse event. Statistical significance is often determined by comparing two or more populations to determine a confidence interval and / or p-value. See, for example, Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983, which is incorporated herein by reference in its entirety. Exemplary confidence intervals of the present subject matter are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%, while exemplary p-values ​​are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, and 0.0001.

[0206] In other embodiments, a threshold degree of change in the methylation status of a prognostic or diagnostic biomarker (e.g., DMR) disclosed herein can be established, and the degree of change in the methylation status of the biomarker in a biological sample is simply compared to the threshold degree of change in the methylation status. Preferred threshold changes in the methylation status of the biomarkers provided herein are about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 50%, about 75%, about 100%, and about 150%. In yet other embodiments, a "nomogram" can be established, which directly relates the methylation status of a prognostic or diagnostic indicator (biomarker, or combination of biomarkers) to the associated properties for a given outcome. Those skilled in the art will recognize that the use of such a nomogram reflects individual sample measurements, not population averages, and therefore relates the two values ​​with the understanding that the uncertainty in the measurements is the same as the uncertainty in the marker concentration.

[0207] In some embodiments, a control sample is analyzed simultaneously with a biological sample (e.g., a tissue sample (e.g., a cervical sample), a blood sample, a plasma sample, a serum sample, a whole blood sample, a secretion sample (e.g., cervical secretion, vaginal secretion), an organ secretion sample, a CSF sample, a saliva sample, a urine sample, or a stool sample) so that results obtained from the biological sample can be compared to results obtained from the control sample. It is further contemplated that a standard curve can be provided and assay results of the biological sample compared to the standard curve. Such a standard curve provides a methylation status of the biomarker according to assay units, e.g., fluorescent signal intensity if a fluorescent label is used. Using samples from multiple donors, standard curves can be obtained for control methylation status of one or more biomarkers in normal tissues and for "at risk" levels of one or more biomarkers in plasma from donors with a particular type of cancer (e.g., cervical cancer or subtypes of cervical cancer) or precancer (e.g., cervical precancer). In certain embodiments of this method, a subject is identified as having cancer (e.g., cervical cancer or a subtype of cervical cancer) or precancer (e.g., cervical precancer) if an aberrant methylation state of one or more DMRs provided herein is identified in a biological sample obtained from the subject. In other embodiments of this method, a subject is identified as having cancer (e.g., cervical cancer or a subtype of cervical cancer) or precancer (e.g., cervical precancer) if an aberrant methylation state of one or more of these biomarkers is detected in a biological sample obtained from the subject.

[0208] The analysis of markers can be performed separately or simultaneously with additional markers in one test sample. For example, it is possible to combine several markers in one test to allow efficient processing of multiple samples and potentially provide higher diagnostic and / or prognostic accuracy. Furthermore, those skilled in the art will recognize the value of testing multiple samples from the same subject (e.g., at successive time points). Such testing of successive samples allows the identification of changes in the methylation status of markers over time. Changes in methylation status, and the absence of changes in methylation status, can provide useful information about disease status, including, but not limited to, revealing the approximate time from the occurrence of this event, the presence and amount of salvageable tissue, suitability of drug therapy, efficacy of various therapies, and identification of the subject's outcome, including risk of future events.

[0209] Biomarker analysis can be performed in a variety of physical formats, for example using microtiter plates or automated applications to facilitate the processing of multiple test samples, or single sample formats can be developed to facilitate immediate treatment and diagnosis in a timely manner, for example in an outpatient or emergency room setting.

[0210] In some embodiments, a subject is diagnosed as having cervical cancer, cervical cancer subtypes, and / or cervical precancer if there is a measurable difference in the methylation status of at least one biomarker in the sample compared to a control methylation status. Conversely, if no change in the methylation status in the biological sample is identified, the subject can be identified as not having cervical cancer, cervical cancer subtypes, and / or cervical precancer, not at risk of cancer or precancer, or at low risk of cancer or precancer. In this regard, subjects with cancer or at risk can be distinguished from subjects with cancer or at low risk from subjects with substantially no cancer or precancer. Subjects at risk of developing cervical cancer, cervical cancer subtypes, and / or cervical precancer can be placed on a more intensive and / or regular screening schedule. On the other hand, subjects at low risk to subjects with substantially no risk can be avoided from being subjected to additional testing (e.g., invasive procedures) for risk of cancer until future screening, e.g., screening performed according to the present technology, indicates that the subjects are at risk of cancer.

[0211] As mentioned above, depending on the embodiment of the method of the present technology, detecting a change in the methylation state of one or more biomarkers can be a qualitative determination, or it can be a quantitative determination. Thus, diagnosing a subject as suffering from or at risk of developing cervical cancer or cervical cancer subtypes indicates that a certain threshold measurement is made, for example, the methylation state of one or more biomarkers in a biological sample (e.g., a tissue sample (e.g., a cervical sample), a blood sample, a plasma sample, a serum sample, a whole blood sample, a secretion sample (e.g., a cervical secretion, a vaginal secretion), an organ secretion sample, a CSF sample, a saliva sample, a urine sample, or a stool sample) is changed from a predetermined control methylation state. In some embodiments of the present method, the control methylation state is a detectable methylation state of any of the biomarkers. In other embodiments of the method, where a control sample is tested simultaneously with the biological sample, the predetermined methylation state is the methylation state in the control sample. In other embodiments of the present method, the predetermined methylation state is based on and / or identified by a standard curve. In other embodiments of the method, the predetermined methylation is a specific state or range of states. Thus, the predetermined methylation state can be selected based in part on the embodiment of the method being performed, the specificity desired, etc., within acceptable limits that will be apparent to one of skill in the art.

[0212] In certain embodiments, the technology provides for the treatment of nucleic acids containing DMRs with reagents that can modify nucleic acids in a methylation-specific manner (e.g., methylation-sensitive restriction enzymes, methylation-dependent restriction enzymes, and bisulfite reagents) (e.g., methylation-sensitive restriction enzymes, methylation-dependent restriction enzymes, ten-eleven translocation (TET) enzymes, and bisulfite reagents). (e.g., human TET1, human TET2, human TET3, mouse TET1, mouse TET2, mouse TET3, Naegleria TET (NgTET), Coprinopsis cinerea (CcTET), or a variant thereof) to, for example, produce a methylation-specifically modified nucleic acid; sequencing the methylation-specifically modified nucleic acid to obtain a nucleotide sequence of the methylation-specifically modified nucleic acid; comparing the nucleotide sequence of the methylation-specifically modified nucleic acid to a nucleotide sequence of a nucleic acid comprising a DMR from a subject not having cervical cancer or a subtype of cervical cancer to identify a difference between the two sequences; and, if a difference is present, identifying the subject as having cervical cancer or a subtype of cervical cancer.

[0213] The present technology further provides compositions. In certain embodiments, the present technology provides compositions comprising a nucleic acid comprising a DMR and a bisulfite reagent. In certain embodiments, compositions comprising a nucleic acid comprising a DMR and one or more oligonucleotides set forth in SEQ ID NOs: 1-76 are provided. In certain embodiments, compositions comprising a nucleic acid comprising a DMR and a methylation-sensitive restriction enzyme are provided. In certain embodiments, compositions comprising a nucleic acid comprising a DMR and a polymerase are provided.

[0214] The present technology further provides kits. The kits include embodiments of the compositions, devices, apparatuses, etc. described herein, and instructions for using the kits. Such instructions include suitable methods for preparing specimens from samples, for example, for collecting samples (e.g., tissue samples (e.g., cervical samples), blood samples, plasma samples, serum samples, whole blood samples, secretion samples (e.g., cervical secretions, vaginal secretions), organ secretion samples, CSF samples, saliva samples, urine samples, or stool samples) and preparing nucleic acids from the samples. In some embodiments, the kits include one or more collection devices (e.g., tampons (e.g., standard tampons), irrigators that release liquid into the vagina and recollect the liquid (e.g., Pantarhei screeners), cervical brushes (e.g., brushes that are inserted into the vagina and rotated to collect cells), Fournier cervical self-collection devices (tampon-like plastic sticks), or cotton swabs) that can obtain samples (e.g., tissue, secretions, and / or cells) from the cervix or near the cervix. The individual components of the kit are packaged in suitable containers and packaging (e.g., vials, boxes, blister packs, ampoules, jars, bottles, tubes, etc.), and the components are packaged together in suitable containers (e.g., one or more boxes) for convenient storage, transport, and / or use by the user of the kit. It is understood that the liquid components (e.g., buffers) may be provided in lyophilized form to be reconstituted by the user. The kit may include controls or references to evaluate, verify, and / or ensure the performance of the kit. For example, a kit for assaying the amount of nucleic acid present in a sample may include a control containing a known concentration of the same or another nucleic acid for comparison, and in some embodiments, a detection reagent (e.g., primers) specific for the control nucleic acid. The kit is suitable for use in a clinical setting and, in some embodiments, for use in the user's home. The components of the kit, in some embodiments, provide the functionality of a system for preparing a nucleic acid solution from a sample. In some embodiments, certain components of the system are provided by the user.

[0215] In certain embodiments, the technology relates to compositions (e.g., reaction mixtures). In some embodiments, compositions are provided that include a nucleic acid that includes a DMR and a reagent that can modify DNA in a methylation-specific manner (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent) (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, a ten-eleven translocation (TET) enzyme (e.g., human TET1, human TET2, human TET3, mouse TET1, mouse TET2, mouse TET3, Naegleria TET (NgTET), Coprinopsis cinerea (CcTET)), or variants thereof), a borane reducing agent). Some embodiments provide compositions that include a nucleic acid that includes a DMR and an oligonucleotide described herein. Some embodiments provide compositions that include a nucleic acid that includes a DMR and a methylation-sensitive restriction enzyme. Some embodiments provide compositions that include a nucleic acid that includes a DMR and a polymerase.

[0216] In some embodiments, the techniques described herein are associated with programmable machines designed to perform sequences of arithmetic or logical operations, such as those provided by the methods described herein. For example, some embodiments of the techniques are associated with (e.g., implemented in) computer software and / or computer hardware. In one aspect, the techniques relate to a computer that includes a form of memory, elements for performing arithmetic and logical operations, and a processing element (e.g., a microprocessor) for executing a set of instructions (e.g., the methods provided herein) for reading, manipulating, and storing data. In some embodiments, the microprocessor is part of a system for determining methylation status (e.g., of one or more DMRs, e.g., DMRs 1-423 shown in Tables I, III, and X), comparing methylation status, generating standard curves, determining Ct values, calculating methylation fractions, frequencies, or percentages, identifying CpG islands, determining assay or marker specificity and / or sensitivity, calculating ROC curves and associated AUCs, and performing sequence analysis, any of which are described herein or known in the art. In some embodiments, the microprocessor is part of a system for determining methylation status (e.g., of one or more DMRs, e.g., DMRs 1-423 shown in Tables I, III, and X), comparing methylation statuses, generating standard curves, determining Ct values, calculating methylation rates, frequencies, or percentages, identifying CpG islands, determining assay or marker specificity and / or sensitivity, calculating ROC curves and associated AUCs, and performing sequence analysis, any of which are described herein or known in the art.

[0217] In some embodiments, the software or hardware component receives results of the multiple assays and determines and reports to a user a single value result indicative of cancer risk (e.g., cervical cancer or subtype of cervical cancer) or precancer risk (e.g., cervical precancer) based on the results of the multiple assays (e.g., determining the methylation status of multiple DMRs (e.g., as shown in Tables I, III, and X)). Related embodiments calculate a risk factor based, for example, on a mathematical combination (e.g., weighted combination, linear combination) of results from the multiple assays (e.g., determining the methylation status of multiple DMRs shown in Tables I, III, and X). In some embodiments, the methylation status of the DMRs defines dimensions and can have values ​​in a multidimensional space, and the coordinates defined by the methylation status of the multiple DMRs are a result, e.g., related to cancer risk, for reporting, e.g., to a user.

[0218] In some embodiments, the technology provided herein involves multiple programmable devices that work in concert to perform the methods as described herein. For example, in some embodiments, multiple computers (e.g., connected by a network) can operate in parallel to collect and process data, for example in an implementation of cluster computing or grid computing or some other distributed computing architecture that relies on complete computers (on-board CPU, storage, power, network interfaces, etc.) connected to a network (private, public, or Internet) by traditional network interfaces such as Ethernet, optical fiber, etc., or by wireless networking technology.

[0219] For example, some embodiments provide a computer including a computer-readable medium. The embodiments include a random access memory (RAM) coupled to a processor. The processor executes computer-executable program instructions stored in the memory. Processors such as these may include microprocessors, ASICs, state machines, or other processors, and may be any of a number of computer processors, such as processors from Intel Corporation of Santa Clara, California, Motorola Corporation of Schaumburg, Illinois. Processors such as these may include or be in communication with a medium, such as a computer-readable medium, that stores instructions that, for example, when executed by the processor, cause the processor to perform the steps described herein.

[0220] The computer is connected to a network in some embodiments. The computer may also include multiple external or internal devices, such as a mouse, CD-ROM, DVD, keyboard, display, or other input or output devices. Examples of computers are personal computers, digital assistants, personal digital assistants, cellular phones, mobile phones, smartphones, pagers, digital tablets, laptop computers, Internet appliances, and other processor-based devices. In general, these computers related to the aspects of the technology provided herein can be any type of processor-based platform that runs on any operating system, such as Microsoft Windows, Linux, UNIX, Mac OS X, and can support one or more programs, including the technology provided herein. Some embodiments include personal computers that run other application programs (e.g., applications). Applications can be stored in memory and can include, for example, word processing applications, spreadsheet applications, email applications, instant messenger applications, presentation applications, Internet browser applications, calendar / organizer applications, and any other applications executable by a client device.

[0221] All such components, computers, and systems described herein as relating to the present technology may be logical or virtual.

[0222] In certain embodiments, the technology provides a system for screening for cervical cancer, cervical cancer subtypes, and / or cervical precancer in a sample obtained from a subject. Exemplary embodiments of the system include, for example, a system for screening for cervical cancer or cervical cancer subtypes in a sample obtained from a subject (e.g., a tissue sample (e.g., a cervical sample), a blood sample, a plasma sample, a serum sample, a whole blood sample, a secretion sample (e.g., a cervical secretion, a vaginal secretion), an organ secretion sample, a CSF sample, a saliva sample, a urine sample, or a stool sample), comprising: an analytical component configured to perform one or both of the following: determining the methylation status of one or more methylated markers in the sample; a software component configured to compare the methylation status of one or more methylated markers in a sample with a control or reference sample recorded in a database; and an alert component configured to alert a user of a cancer-related condition.

[0223] In some embodiments, the alert is determined by a software component that receives results from multiple assays (e.g., determining the methylation status of one or more methylated markers) and calculates a reported value or result based on the multiple results.

[0224] Some embodiments provide a database of weighting parameters associated with each methylation marker provided herein for use in calculating a value or result and / or reporting an alert to a user (e.g., a doctor, nurse, clinician, etc.). In some embodiments, all results from the multiple assays are reported. In some embodiments, one or more results are used to provide a score, value, or result based on a combined result of one or more results from the multiple assays that is indicative of a subject's risk of cancer. Such methods are not limited to a particular methylation marker.

[0225] In such methods and systems, the one or more methylation markers comprise bases in a DMR selected from the group consisting of DMRs 1-423 shown in Tables I, III, and X.

[0226] In this detailed description of various embodiments, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the disclosed embodiments. However, those skilled in the art will appreciate that these various embodiments may be practiced with or without these specific details. In other instances, structures and mechanisms are shown in block diagram form. Moreover, those skilled in the art will readily appreciate that the particular order in which the methods are presented and performed is illustrative, and that the order can be changed and still be contemplated to be within the spirit and scope of the various embodiments disclosed herein. EXAMPLES

[0227] Example I This example describes experiments performed to evaluate the feasibility of a targeted assay of a panel of methylated DNA markers (MDM) for the detection of cervical cancer.

[0228] Using a proprietary methodology of sample preparation, sequencing, analytical pipeline, and filters, methylation variable regions (DMRs) were identified and narrowed down to DMRs that accurately indicate cervical cancer and excel in a clinical trial setting. Analysis by tissue identified 320 hypermethylated cervical cancer (CC) DMRs (Table I). Table II shows the area under the curve (AUC), fold change, and p-values ​​in cervical cancer tissues and benign cervical tissues for the markers listed in Table I. The 320 hypermethylated CC DMRs identified included CC-specific and CC subtype-specific regions.

[0229] [Table 1] JPEG2024509851000002.jpg208169JPEG2024509851000003.jpg203169JPEG2024509851000004.jpg203169JPEG2024509851000005.jpg203169 JPEG2024509851000006.jpg203169JPEG2024509851000007.jpg203169JPEG2024509851000008.jpg203169JPEG2024509851000009.jpg155169

[0230] [Table 2] JPEG2024509851000011.jpg70169JPEG2024509851000012.jpg208169JPEG20245098510 00013.jpg208169JPEG2024509851000014.jpg192169JPEG2024509851000015.jpg200169 JPEG2024509851000016.jpg200169JPEG2024509851000017.jpg210169JPEG20245098510 00018.jpg208169JPEG2024509851000019.jpg208169JPEG2024509851000020.jpg192169

[0231] Analysis of tissue versus white blood cells (buffy coat) yielded DMRs for 41 hypermethylated cervical tissues with less than 1% noise in WBCs (Table III, Table IV).

[0232] [Table 3] JPEG2024509851000022.jpg46169

[0233] [Table 4] JPEG2024509851000024.jpg134169

[0234] From these markers, we selected 29 candidates as initial pilots: MAX.chr6.58147682-58147771, C1ORF114, ASCL1, ARHGAP12, ZNF773, TTYH1, NEUROG3, ZNF781, NXPH1, MAX.chr9.36739811-36739868, NID2, TMEM200C, CRHR2, ABCB1, ZNF69, A TP10A, MAX.chr18.73167725-73167817, MAX.chr2.127783183-127783403, CACNA1C, ZNF382, BARHL1, MAX.chr4.8859853-8859939, ST8SIA1, MAX.chr1.98510958-98511049, C2ORF40, SLC9A3, PRDM12, HOPX_C, and KCNQ5. A quantitative methylation-specific PCR assay was developed and tested on independent samples. Short amplicon primers (<150 bp) were designed to target the most discriminatory CpGs within the DMR and tested on controls to ensure that fully methylated fragments were stably and linearly amplified, whereas unmethylated and / or unconverted fragments were not amplified. The 60 primer sequences are shown in Table V.

[0235] [Table 5] JPEG2024509851000026.jpg219169

[0236] 29 MDMs (MAX.chr6.58147682-58147771, C1ORF114, ASCL1, ARHGAP12, ZNF773, TTYH1, NEUROG3, ZNF781, NXPH1, MAX.chr 9.36739811-36739868, NID2, TMEM200C, CRHR2, ABCB1, ZNF69, ATP10A, MAX.chr18.73167725-73167817, MAX.chr2.1277 Each of the ten MDMs (83183-127783403, CACNA1C, ZNF382, BARHL1, MAX.chr4.8859853-8859939, ST8SIA1, MAX.chr1.98510958-98511049, C2ORF40, SLC9A3, PRDM12, HOPX_C, and KCNQ5) had an area under the curve (AUC) >0.90 and highly discriminated CC from benign cervicovaginal (BCV) tissue (Table VI). CC MDMs also highly discriminated adenocarcinoma in situ (AIS) from BCV, but performed poorly in cervical intraepithelial neoplasia (CIN) 2 / 3 and CIN1 (Table VI).

[0237] [Table 6]

[0238] Previous studies have recognized that the epigenetics of each cancer subtype within a given organ is distinct and the best panel would be derived from a combination of subtype markers. The results are shown in Table VII. The discrimination strength of each marker assay is given as a numerical value. Many assays almost completely discriminated both adenocarcinoma and squamous cell carcinoma from benign tissue. Adenocarcinoma in situ samples were detected with AUCs in the high 80s and CIN2 / 3 in the low 80s by several marker assays. Methylation FC (%) of controls versus cancer ranged from 2 to 340.

[0239] [Table 7]

[0240] Experiments were also performed to investigate the potential of the CC MDM assay to discriminate between gynecological malignancies. The organ site specificity of the assay was evaluated, which will be important in clinical trials with tampons. Table VIII shows the methylation rate (%) of 27 selected MDMs in the different cohorts studied. The 27 MDMs were highly methylated in the cervical cancer cohort, but generally less than 5% in endometrial cancer.

[0241] [Table 8] JPEG2024509851000030.jpg91169

[0242] Whole-methylome sequencing, stringent filtering criteria, and biological validation yielded excellent candidate MDMs for cervical cancer. Some MDMs discriminate both CC histological types from benign cervix with relatively high sensitivity, whereas others show subtype selectivity.

[0243] sample: Tissues and blood were obtained from the Mayo Clinic Biospecimen Repository under institutional IRB oversight. Samples were selected in strict compliance with subject study approval and inclusion / exclusion criteria. Cervical subtypes included 1) adenocarcinoma and 2) squamous cell carcinoma. Controls included benign cervicovaginal (BCV) tissue and whole blood derived leukocytes. Endometrial cancer and controls were also performed. Tissues were macro-dissected and an expert gynecologic pathologist reviewed the tissues. Samples were age- and sex-matched, randomized, and blinded. DNA from 113 frozen tissues (16 grade 1 / 2 endometrioid (G1 / 2E), 16 grade 3 endometrioid (G3E), 11 serous, 11 clear cell EC, 15 uterine carcinosarcomas, 44 benign endometrial (BE) tissues (14 proliferative, 12 atrophic, 18 irregular proliferation), 70 formalin-fixed paraffin-embedded (FFPE) cervical carcinomas (CC) (36 squamous cell, 34 adenocarcinoma), and 18 buffy coats from cancer-free women was purified using the QIAamp DNA Tissue Mini Kit (frozen tissues), QIAamp DNA FFPE Tissue Kit (FFPE tissues), and QIAamp DNA Blood Mini Kit (buffy coat samples) (Qiagen, Valencia CA). DNA was repurified with AMPure XP beads (Beckman-Coulter, Brea CA) and purified using PicoGreen (Thermo-Fisher, Waltham DNA integrity was assessed using qPCR.

[0244] Sequencing: RRBS sequencing libraries were prepared according to the Meissner protocol (Gu et al. Nature Protocols 2011) with modifications. Each sample was pooled in a 4-plex format and sequenced on an Illumina HiSeq 2500 instrument (Illumina, San Diego CA) by the Mayo Genomics Facility. Reads were processed by Illumina pipeline modules for image analysis and base calling. Secondary analysis was performed using SAAP-RRBS, a bioinformatics suite developed by Mayo. Briefly, reads were cleaned using Trim-Galore and aligned to the GRCh37 / hg19 reference genome constructed with BSMAP. For CpGs with ≥10x coverage and base quality score ≥20, methylation rates were determined by calculating C / (C+T) or, conversely, G / (G+A) for reads mapping to the opposite strand.

[0245] Biomarker Selection: Individual CpGs were ranked by their hypermethylation ratio, i.e., the number of methylated cytosines relative to the total number of cytosines at that location, at a given locus. For cases, the ratio had to be ≥ 0.20 (20%); for BCV tissue controls, ≤ 0.05 (5%); for buffy coat controls, ≤ 0.01 (1%). CpGs that did not meet these criteria were discarded. Candidate CpGs were subsequently binned by genomic location into DMRs (variably methylated regions) ranging from approximately 60 to 200 bp with a minimum cutoff of 5 CpGs per region. DMRs with excessively high CpG density (> 30%) were excluded to avoid GC-related amplification issues during the validation phase. For each candidate region, a 2D matrix was created comparing individual CpGs sample by sample for both cases and controls. Overall CC versus all benign endometrium and / or non-cancer buffy coat, as well as subtype comparisons, were analyzed. These CpG matrices were then compared to the reference sequences to assess whether genomically contiguous methylated sites were discarded during the initial filtering. From this subset of regions, final selection required, at the per-sample level, coordinated and contiguous (in cases) hypermethylation of individual CpGs across the DMR sequences. Conversely, control samples were required to have at least one-tenth as many methylated as cases, and the CpG pattern had to be more random and less coordinated. At least 10% of the cancer samples within the subtype cohort were required to have at least 50% hypermethylation rates at all CpG sites within the DMR.

[0246] In a separate analysis, we derived DMRs based on the mean methylation values ​​of CpGs using a proprietary DMR identification pipeline and regression package. Differences in mean methylation percentages were compared between CC cases, tissue controls, and buffy coat controls, and tiled reading frames within 100 base pairs of each mapped CpG were used to identify DMRs with less than 5% methylation in controls. DMRs were analyzed only if the total coverage depth was an average of 10 reads per subject and the variance between subgroups was greater than 0. Assuming a biologically relevant increase in odds ratio of more than 3-fold and a coverage depth of 10 reads, ≥18 samples per group were required to achieve 80% power at a two-sided test with a significance level of 5% and assuming a binomial variance inflation factor of 1.

[0247] After regression, DMRs were ranked by the difference in p-value, area under the receiver operating characteristic curve (AUC), and fold change between cases and all controls. No adjustment for false positives was performed at this stage because independent validation was planned a priori.

[0248] Biomarker Validation: A subset of cervical cancer DMRs was selected for further expansion. Criteria were primarily logistic-derived area under the receiver operating characteristic curve measures, which provide a performance assessment of the discriminatory potential of the region. An AUC of 0.85 was selected as the cutoff for case vs. control tissue comparisons; 0.95 was used as the cutoff for case vs. blood comparisons. Additionally, methylation fold change (mean cancer hypermethylation rate / mean control hypermethylation rate) was calculated, with a lower limit of 20 for tissue vs. tissue comparisons and 50 for tissue vs. buffy coat comparisons. P values ​​had to be less than 0.05 and / or 0.001, respectively. DMRs had to be included in both the average and individual CpG selection processes. Quantitative methylation specific PCR (qMSP) primers were designed for the candidate regions using MethPrimer (see Li LC and Dahiya R. Bioinformatics 2002 Nov;18(11):1427-31) and QC-tested with 20ng (6250 equivalents) of positive and negative genomic methylation controls. Multiple annealing temperatures were tested for optimal discrimination. Validation was performed by qMSP on a comparable set of independent tissue samples. Additional cohorts included adenocarcinoma in situ (AIS) and cervical intraepithelial neoplasia (CIN1-3). Patient demographics are shown in Table IX.

[0249] [Table 9] JPEG2024509851000032.jpg136169

[0250] These tissues were identified as before by expert clinical and pathological review. DNA purification was performed as described previously. For the bisulfite conversion step, the EZ-96 DNA Methylation kit (Zymo Research, Irvine CA) was used. 10 ng of converted DNA (per marker) was amplified using SYBR Green detection on a Roche 480 LightCycler (Roche, Basel Switzerland). Serially diluted universally methylated genomic DNA (Zymo Research) was used as a quantification standard. A CpG-independent ACTB (β-actin) assay was used as input reference and normalization control. Results were expressed as copies methylated (specific marker) / copies of ACTB.

[0251] Statistics: Results were logistically analyzed for the performance of individual MDMs (methylated DNA markers). For marker combinations, random forest regression (rForest) was used to generate 500 individual models fitted to bootstrap samples of the original data (approximately 2 / 3 of the data for training) to estimate the cross-validation error for the entire MDM panel (1 / 3 of the data for testing), which was repeated 500 times to avoid spurious splits that would under- or overestimate the true cross-validation measure. Results were then averaged over the 500 repetitions.

[0252] Example II. This example describes the identification of methylation markers that can distinguish cervical cancer from endometrial and ovarian cancer.

[0253] Aiming to reveal organ-specific hypermethylated regions among three gynecologic cancers (i.e. cervical, endometrial, and ovarian cancers) and their respective subtypes, we performed an experiment to combine or integrate unique previously generated, validated, and published tissue RRBS (reduced representation bisulfite sequencing) datasets of these cancers. Only CpGs common to all three studies were analyzed. Regions were required to contain at least six consecutive CpGs in either the sense or antisense strand. Discovery samples included 34 cervical adenocarcinomas, 36 cervical squamous cell carcinomas, 15 grade 1 or 2 uterine endometrioid carcinomas, 16 grade 3 uterine endometrioid carcinomas, 11 serous and clear cell uterine carcinomas, 15 uterine carcinosarcomas, as well as 18 serous ovarian carcinomas, 15 clear cell ovarian carcinomas, 6 mucinous ovarian carcinomas, and 18 endometrioid ovarian carcinomas. Benign controls included 18 endocervical-vaginal samples, 44 endometrial tissues (14 proliferative, 12 atrophic, 18 irregularly proliferative, and 10 secretory), 20 fallopian tube samples, and 36 noncancerous buffy coat or peripheral blood leukocyte samples.

[0254] For this application, we focused on markers specific for cervical cancer (both adenocarcinoma and squamous cell carcinoma types). As a first step, all regions of variably methylated had to have very low background or noise (<1%) in benign cervicovaginal cells and tissues. DNA from this tissue type represents by far the highest proportion of nucleic acids found on tampons. Secondly, to avoid potential signals from inflammation, we excluded regions with less than 1% methylation in leukocyte DNA. The remaining CpGs were used to comprehensively compare cervical cancer with endometrial and ovarian cancer. Adenocarcinoma and squamous cell carcinoma were analyzed separately. Three indicators were evaluated: 1) the ratio of methylation in cervical cancer to that in endometrial and ovarian cancer, 2) the intensity, frequency, and continuity (read level) of hypermethylation observed in cervical cancer samples, and 3) the absolute methylation rate in cervical cancer. For the first one, we used cutoff values ​​of CC / EC>20 and CC / OC>50. These values ​​were empirically chosen to reduce the number of DMR candidates from thousands to hundreds. For the third, a cutoff value of less than 20% was used, which reduced the number of candidates below 100. For the second indicator, a scoring system was used to identify concordant hypermethylation across the DMR. Regions with probabilistic discordant CpG methylation were excluded. This analysis resulted in the identification of a panel of 64 DMRs, shown in Table X. Table XI shows the ratio of cervical cancer methylation to endometrial cancer, ovarian cancer, and white blood cell (buffy coat) methylation for the markers listed in Table X.

[0255] [Table 10] JPEG2024509851000034.jpg155169

[0256] [Table 11] JPEG2024509851000036.jpg160169

[0257] Among these, due to the limited amount of DNA, the following eight DMRs were selected for conversion to a qMSP (quantitative methylation specific PCR) assay and validation in an independent sample set: AK5, RABC3, ZNF491, ZNF610, ZNF91, ZNF480, TRPC3_B, and ELMOD1. Primer sequences for the eight DMRs (AK5, RABC3, ZNF491, ZNF610, ZNF91, ZNF480, TRPC3_B, and ELMOD1) are shown in Table XII.

[0258] [Table 12]

[0259] Samples included 38 cervical adenocarcinomas, 36 cervical squamous cell carcinomas, 18 grade 1 or 2 endometrioid carcinomas, 24 grade 3 endometrioid carcinomas, 16 serous and 7 clear cell uterine carcinomas, 18 uterine carcinosarcomas, and 36 serous, 21 clear cell, 4 mucinous, and 21 endometrioid ovarian carcinomas. Benign controls included 29 endocervical vaginal samples, 14 endometrial tissues (8 proliferative, 2 atrophic, 3 irregularly proliferative, and 1 secretory), and 29 fallopian tube samples. Overall cervical cancer was compared logistically to overall endometrial and ovarian cancers.

[0260] The performance of individual MDMs (methylated DNA markers) ranged from 30% sensitivity at 98% specificity to 73% sensitivity at 99% specificity. Panels of 2-3 MDMs were complementary; for example, combining AK5 and RABC3 had a sensitivity of 80% at 98% specificity. Thus, the combination of markers detected a specific cervical cancer methylation 4 / 5 times, with a false positive rate of 2%.

[0261] In conclusion, these eight MDMs (AK5, RABC3, ZNF491, ZNF610, ZNF91, ZNF480, TRPC3_B, and ELMOD1) and the remaining 56 DMRs can indicate with high accuracy the presence of cervical cancer, regardless of whether it is an adenocarcinoma or squamous subtype, distinct from the other two gynecological organ cancers.

[0262] All publications and patents cited in the above specification are hereby incorporated by reference in their entirety for all purposes. Various modifications and variations of the compositions, methods, and applications of the described technology will be apparent to those skilled in the art without departing from the scope and spirit of the described technology. Although such technology has been described in connection with specific exemplary embodiments, it should be understood that the invention as claimed should not be unnecessarily limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are apparent to those skilled in the art of pharmacology, biochemistry, medicine, or related fields are intended to be within the scope of the appended claims.

Claims

1. A method for characterizing a biological sample from a subject, comprising: treating DNA from the biological sample with a reagent that modifies DNA in a methylation-specific manner; and The method comprises measuring a methylation level of one or more methylation markers selected from Tables I, III, and / or X in the biological sample, comprising measuring the methylation level of the one or more methylation markers.

2. 2. The method of claim 1, wherein the biological sample is selected from a tissue sample, a blood sample, a plasma sample, a serum sample, a whole blood sample, a secretion sample, an organ secretion sample, a cerebrospinal fluid (CSF) sample, a saliva sample, a urine sample, and / or a stool sample.

3. The method of claim 2 , wherein the tissue sample is a cervical tissue sample.

4. 4. The method of claim 3, wherein the cervical tissue sample further comprises one or more of vaginal tissue, vaginal cells, endometrial tissue, endometrial cells, ovarian tissue, and / or ovarian cells.

5. The method of claim 2 , wherein the secretion sample is a cervical secretion sample.

6. 6. The method of claim 5, wherein the cervical secretion sample further comprises one or more of a vaginal secretion, an endometrial secretion, and / or an ovarian secretion.

7. The method of claim 1, wherein the method comprises comparing the measured methylation levels of the one or more methylation markers with the methylation levels of one or more corresponding methylation markers from a control sample without cervical cancer.

8. 8. The method of claim 7, further comprising determining that the subject has cervical cancer if the methylation levels measured for the one or more methylation markers are higher than the methylation levels of one or more corresponding methylation markers from the respective control samples.

9. 9. The method of claim 8, wherein the one or more methylation markers are selected from one of the following groups: - a methylation marker listed in Table I and / or Table III; ●MAX. chr6.58147682-58147771, C1ORF114, ASCL1, ARHGAP12, ZNF773, TTYH1, NEUROG3, ZNF781, NXPH1, MAX. chr9.36739811-36739868, NID2, TMEM200C, CRHR2, ABCB1, ZNF69, ATP10A, MAX. chr18.73167725-73167817, MAX. chr2.127783183-127783403, CACNA1C, ZNF382, BARHL1, MAX. chr4.8859853-8859939, ST8SIA1, MAX.chr1.98510958-98511049, C2ORF40, SLC9A3, PRDM12, HOPX_C, and / or KCNQ5; C1orf114, MAX. chr6.58147682-58147771, ZNF773, NEUROG3, ASCL1, NID2, ZNF781, CRHR2, and / or MAX. chr9.36739811-36739868; and / or ABCB1, c1orf95, CACNA1C, CACNG8, CHST2, ELMO1, EMID2, FBN1_B, FLT3_A, FLT3_B, GLIS1, GPC6, GREM2, JAM2, KCNK12_A, LOC100129620, MAX.chr15.78112404-78112692, MAX.chr19.4584907-4585088, MAX.chr3.69591689-69591784, NCAM1, NT5C1A, ST8SIA3, ZNF382, ZNF419, ZNF69, and / or ZSCAN18.

10. 10. The method of claim 8, further comprising determining that the subject has a subtype of cervical cancer.

11. 10. The method of claim 8, further comprising determining that the subject has a pre-cervical cancer.

12. The method of claim 1, wherein the method comprises comparing the measured methylation levels of the one or more methylation markers with the methylation levels of one or more corresponding methylation markers from an endometrial cancer sample and an ovarian cancer sample.

13. The method of claim 1 , wherein the reagent that modifies DNA in a methylation-specific manner is a borane reducing agent.

14. 2. The method of claim 1, wherein the reagent that modifies DNA in a methylation-specific manner comprises one or more of a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and / or a bisulfite reagent.

15. 2. The method of claim 1, wherein the reagent that modifies DNA in a methylation-specific manner is a bisulfite reagent and the treating produces bisulfite-treated DNA.

16. 10. The method of claim 1, wherein the treated DNA is amplified using a set of primers specific for the one or more markers.

17. 2. The method of claim 1, wherein measuring the methylation level of the one or more methylation markers comprises using one or more methods selected from the group consisting of methylation specific PCR, quantitative methylation specific PCR, methylation specific DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, flap endonuclease assay, PCR-flap assay, and / or bisulfite genomic sequencing PCR.

18. The method of claim 1 , wherein measuring the methylation level of the one or more methylation markers comprises measuring methylation of CpG sites of the one or more methylation markers.

19. 19. The method of claim 18, wherein the CpG site is in a coding or regulatory region.

20. The method of claim 1 , wherein the biological sample is from a human subject.

21. 21. The method of claim 20, wherein the human subject has and / or is suspected of having cervical cancer, a cervical cancer subtype, or cervical precancer.

22. 3. The method of claim 2, wherein the biological sample is collected with a collection device having an absorbent member capable of collecting the biological sample when contacted with an area of ​​the body.

23. A method for preparing a deoxyribonucleic acid (DNA) fraction from a biological sample useful for analyzing one or more genetic loci involved in one or more chromosomal abnormalities, comprising: (a) extracting genomic DNA from a biological sample; (b) (i) treating the extracted genomic DNA with a reagent that modifies DNA in a methylation-specific manner; (ii) amplifying the treated genomic DNA using separate primers specific for one or more methylation markers listed in Tables I, III, and / or X; generating a fraction of the extracted genomic DNA by (c) analyzing one or more loci in the generated fraction of the extracted genomic DNA by measuring a methylation level for each of the one or more methylation markers.

24. The method of claim 23, wherein the biological sample is selected from a tissue sample, a blood sample, a plasma sample, a serum sample, a whole blood sample, a secretion sample, an organ secretion sample, a cerebrospinal fluid (CSF) sample, a saliva sample, a urine sample, and / or a stool sample.

25. The method of claim 23, wherein the one or more methylation markers are selected from one of the following groups: A methylation marker listed in Table I and / or Table III; MAX. chr6.58147682-58147771, C1ORF114, ASCL1, ARHGAP12, ZNF773, TTYH1, NEUROG3, ZNF781, NXPH1, MAX. chr9.36739811-36739868, NID2, TMEM200C, CRHR2, ABCB1, ZNF69, ATP10A, MAX. chr18.73167725-73167817, MAX. chr2.127783183-127783403, CACNA1C, ZNF382, BARHL1, MAX. chr4.8859853-8859939, ST8SIA1, MAX.chr1.98510958-98511049, C2ORF40, SLC9A3, PRDM12, HOPX_C, and / or KCNQ5; C1orf114, MAX. chr6.58147682-58147771, ZNF773, NEUROG3, ASCL1, NID2, ZNF781, CRHR2, and / or MAX. chr9.36739811-36739868; and / or ABCB1, c1orf95, CACNA1C, CACNG8, CHST2, ELMO1, EMID2, FBN1_B, FLT3_A, FLT3_B, GLIS1, GPC6, GREM2, JAM2, KCNK12_A, LOC100129620, MAX.chr15.78112404-78112692, MAX.chr19.4584907-4585088, MAX.chr3.69591689-69591784, NCAM1, NT5C1A, ST8SIA3, ZNF382, ZNF419, ZNF69, and / or ZSCAN18.