Compositions and methods for detecting oropharyngeal cancer - Patents.com

Abstract

The present disclosure relates to detecting one or more types of oropharyngeal cancer in a biological sample from a subject. In particular, the present disclosure relates to detecting one or more types of oropharyngeal cancer (e.g., HPV) in a biological sample from a subject having or suspected of having oropharyngeal cancer. + The present invention provides compositions and methods for detecting the presence or absence of oropharyngeal squamous cell carcinoma.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 276,058, filed November 5, 2021, the entirety of which is incorporated by reference herein for all purposes.

[0002] Sequence Listing The text of the computer readable sequence listing entitled "40013_601_SequenceListing", submitted herewith, which was created on November 4, 2022 and has a file size of 154,000 bytes, is hereby incorporated by reference in its entirety.

[0003] Technical Field The present disclosure relates to detecting one or more types of oropharyngeal cancer in a biological sample from a subject. In particular, the present disclosure relates to detecting one or more types of oropharyngeal cancer (e.g., HPV) in a biological sample from a subject having or suspected of having oropharyngeal cancer. + The present invention provides compositions and methods for detecting the presence or absence of oropharyngeal squamous cell carcinoma. [Background technology]

[0004] Oropharyngeal (or head and neck) cancer accounts for 3% of cancers diagnosed annually in the United States and is projected to cause approximately 11,000 deaths in 2021. While the incidence of HPV- cancers has remained relatively constant, the incidence of HPV+ cancer subtypes has increased. There is an urgent need for non-invasive molecular tests to screen patients at high risk for oropharyngeal cancer, as they will likely reduce the burden of this disease and save lives. Various embodiments disclosed herein address this need. Summary of the Invention

[0005] Embodiments of the present disclosure provide methods, compositions, and systems for screening one or more types of oropharyngeal cancer from a biological sample. According to these embodiments, the present disclosure includes, but is not limited to, methods and compositions for detecting the presence of one or more types or subtypes of oropharyngeal cancer from a biological sample. In some embodiments, the biological sample is a tissue sample, a blood sample, a plasma sample, a serum sample, a whole blood sample, a buffy coat sample, a secretion sample, an organ secretion sample, a cerebrospinal fluid (CSF) sample, a saliva sample, a urine sample, and / or a stool sample. In some embodiments, the tissue sample is an oropharyngeal tissue sample, including one or more of soft palate cells or tissue, throat cells or tissue, tongue cells or tissue, and tonsil cells or tissue. In some embodiments, the tissue sample is an HPV(+) tissue sample. In some embodiments, the subject is a human.

[0006] As further described herein, embodiments of the present disclosure each individually include a method for treating oropharyngeal cancer (e.g., HPV + Oropharyngeal squamous cell carcinoma (HPV +The present invention relates to a method for treating OPSCC (Osteoarthritis Pelvic Floor Cancer) comprising: (a) detecting a pulmonary fibrosis (OPSCC) in a patient with pulmonary fibrosis (OPSCC) from a control sample (e.g., benign tissue, including, but not limited to, oropharyngeal tissue, cervical tissue, tonsil tissue, buffy coat samples, and saliva samples); and (benign tissue, including, but not limited to, oropharyngeal tissue, cervical tissue, tonsil tissue, buffy coat samples, and saliva samples). According to these embodiments, the novel DMR(s) include ABCB1, ARHGAP12, ASCL1, C1orf114, EMX1, GRIN2D, LOC645323, MAX.chr6.58147682-58147771, MAX.chr9.36739811-36739868, NEUROG3, NID2, TBX15, TMEM200C, TSPYL5, TTYH1, VWC2, ZNF610, ZNF69, ZNF773, ZNF781, ALX4, ATP10A, C1Q L3, CA8, CACNA1A, CACNG8, CALCA, CCNA1, CLIC6, CLSTN2, CR1, CTNND2, DAB1, DGKG, DOK1, DOK6, DPP4, DUXA, ELMO1, EMBP1, EPDR1, FGF1 2, FLJ43390, FMN2, FOXB2, FOXD4, FREM3, GALR1, GDF6, GFRA1, GRIK3, HOXB3, HOXB4, HPSE2, LDLRAD2, LHX2, LOC100131366, LOC345643, LOC386758, LOC648809, LOC728392, MAML3, MAPRE2, MAX.chr1.226288154-226288189, MAX.chr1.2375078-2375126, MAX.chr1.2415 87339-241587784, MAX.chr1.50798781-50799423, MAX.chr10.22765150-22765477, MAX.chr10.23462342-23462436, MAX.chr11.1 4926602-14927044, MAX.chr11.58903531-58903592, MAX.chr13.28527984-28528214, MAX.chr13.29106641-29107037, MAX.chr14 .100784488-100784782, MAX.chr16.3221176-3221223, MAX.chr16.3222040-3222098, MAX.chr16.71460171-71460282, MAX.chr19.11805263-11805639、MAX.chr19.16394457-16394646、MAX.chr19.21657626-21657769、MAX.chr19.22034646-22034887、MAX.chr19.23299989-23300156、MAX.chr19.30713427-30713588、MAX.chr19.30716926-30717074、MAX.chr19.30718373-30719719、MAX.chr2.118981724-118982174、MAX.chr2.127783107-127783403、MAX.chr2.173099712-173099791、MAX.chr2.66808635-66808731、MAX.chr22.50064113-50064259、MAX.chr3.137489884-137490061、MAX.chr5.138923141-138923219、MAX.chr5.42995180-42995535、MAX.chr6.38683091-38683226、MAX.chr7.121952014-121952084、MAX.chr7.155166980-155167310、MAX.chr8.99986792-99986864、MAX.chr9.79627078-79627116、MAX.chr9.79638034-79638077、MAX.chr9.98789824-98789847、MDFI、MECOM、MED12L、MIR129-2、MIR196A1、NELL1、NPY、ONECUT2、OPCML、PARP15、PDGFD、PEX5L、PRR15、SEMA6A、SFMBT2、SGIP1、SIM2、SLC35F3、SLCO4C1、SORCS3、ST6GALNAC5、ST8SIA5、SV2C、TACC2、TFAP2E、TLX2、TLX3、TRH、TRIM58、VAV3、VSTM2B、WDR17、ZNF254、ZNF43、ZNF486、ZNF491、ZNF518B、ZNF542、ZNF625、ZNF665、ZNF671、ZNF763、ZNF844、AGRN、ANKRD35、ARHGAP27、ARHGAP30、BCL2L11、BIN2、C10orf114、C4orf31、C6orf132、C6orf186、CCDC88B、CRHBP、DAPK1、DNMT3A、DPP10、FAM19A2、FLJ45983、FOSL1、FOXB1、GREM1、HMHA1、HOXA9、IFFO1、INPP4B、ITGB2、ITGB4、ITPKB、KCNIP2、KLHDC7B、LAT、LHX6、LIMK1、LOC100128239、LOC100192379、LOC646278、MAP2K2、MAX.chr1.210426156-210426257、MAX.chr1.84326495-84326656、MAX.chr10.119312785-119312882、MAX.chr15.67326025-67326060、MAX.chr16.54316401-54316453、MAX.chr16.85482306-85482494、MAX.chr17.74994454-74994572、MAX.chr17.76339840-76339972、MAX.chr2.7571082-7571136、MAX.chr21.45577347-45577679、MAX.chr3.14852538-14852568、MAX.chr3.187676564-187676668、MAX.chr4.174430662-174430793、MAX.chr5.177411809-177411836、MAX.chr6.45631561-45631625、MAX.chr7.25892382-25892451, MAX.chr7.402563-402641, MAX.chr7.64349554-64349606, MAX.chr8.142046239-142046398, MAX.chr8.145900842-145901246, MAX.chr9.126101804-126101848, MAX.chr9.126978999-126979182, MAX.chr9.36458633-36458725, MAX.chr9.87905315-87905326, MBP, MFNG, MT1A, The gene is derived from a gene selected from MT1IP, NCOR2, NFATC1, NKX3-2, NRN1, OLIG1, PALLD, PAPLN, PDLIM2, PKN1, PRDM14, PRKG1, PRMT7, PTGER2, PTK2B, RAD52, RBM38, RHOF, RNF220, RTN4RL1, RXRA, SDCCAG8, SHROOM1, SKI, SLC12A8, SLC25A47, SPEG, SUCLG2, TBC1D10C, TMEM132E, VIPR2, WDR66, WNT6, ZDHHC18, ZNF382, and ZNF626.

[0007] Embodiments of the present disclosure also include novel variably methylated regions (DMRs), each of which can individually distinguish oropharyngeal cancer (e.g., oropharyngeal squamous cell carcinoma (HPV(+)OPSCC)) and / or cervical squamous cell carcinoma (HPV(+)CSCC) from control tissue samples (e.g., normal oropharyngeal tissue or normal cervical tissue). According to these embodiments, the novel DMR(s) are derived from a gene selected from ABCB1, ARHGAP12, ASCL1, C1orf114, EMX1, GRIN2D, LOC645323, MAX.chr6.58147682-58147771, MAX.chr9.36739811-36739868, NEUROG3, NID2, TBX15, TMEM200C, TSPYL5, TTYH1, VWC2, ZNF610, ZNF69, ZNF773, and ZNF781.

[0008] Each of the embodiments of the present disclosure is directed to a method for treating oropharyngeal cancer (e.g., HPV + Oropharyngeal squamous cell carcinoma (HPV+The present invention also includes novel variably methylated regions (DMRs) that can distinguish OPSCC) from control or benign tissue (e.g., tonsil tissue controls). According to these embodiments, the novel DMR(s) include ALX4, ATP10A, C1orf114, C1QL3, CA8, CACNA1A, CACNG8, CALCA, CCNA1, CLIC6, CLSTN2, CR1, CTNND2, DAB1, DGKG, DOK1, DOK6, DPP4, DUXA, ELMO1, EMBP1, EPDR1, FGF12, FLJ43390, FMN2, FOXB2, FOXD4, FREM3, GALR1, GDF6, GFRA1, GRIK3, HOXB3, HOXB4, HPSE2, LDLRAD2, LHX2, LOC100131366, LOC345643, LOC386758, LOC645323, LOC648809, LOC728392, MAML3, MAPRE2, MAX.chr1.226288154-2 26288189, MAX.chr1.2375078-2375126, MAX.chr1.241587339-241587784, MAX.chr1.50798781-50799423, MAX.chr10.22765150-227654 77, MAX.chr10.23462342-23462436, MAX.chr11.14926602-14927044, MAX.chr11.58903531-58903592, MAX.chr13.28527984-28528214 , MAX.chr13.29106641-29107037, MAX.chr14.100784488-100784782, MAX.chr16.3221176-3221223, MAX.chr16.3222040-3222098, MAX. chr16.71460171-71460282, MAX.chr19.11805263-11805639, MAX.chr19.16394457-16394646, MAX.chr19.21657626-21657769, MAX.chr 19.22034646-22034887, MAX.chr19.23299989-23300156, MAX.chr19.30713427-30713588, MAX.chr19.30716926-30717074, MAX.chr19.30718373-30719719, MAX.chr2.118981724-118982174, MAX.chr2.127783107-127783403, MAX.chr2.1730997 12-173099791, MAX.chr2.66808635-66808731, MAX.chr22.50064113-50064259, MAX.chr3.137489884-137490 061, MAX.chr5.138923141-138923219, MAX.chr5.42995180-42995535, MAX.chr6.38683091-38683226, MAX.c hr7.121952014-121952084, MAX.chr7.155166980-155167310, MAX.chr8.99986792-99986864, MAX.chr9.7962 7078-79627116, MAX.chr9.79638034-79638077, MAX.chr9.98789824-98789847, MDFI, MECOM, MED12L, MIR129 -2, MIR196A1, NELL1, NPY, ONECUT2, OPCML, PARP15, PDGFD, PEX5L, PRR15, SEMA6A, SFMBT2, SGIP1, SIM2, SLC35F3 , SLCO4C1, SORCS3, ST6GALNAC5, ST8SIA5, SV2C, TACC2, TFAP2E, TLX2, TLX3, TRH, TRIM58, VAV3, VSTM2B, WDR17, ZNF254, ZNF43, ZNF486, ZNF491, ZNF518B, ZNF542, ZNF625, ZNF665, ZNF671, ZNF763, and ZNF844.

[0009] Each of the embodiments of the present disclosure is directed to a method for treating oropharyngeal cancer (e.g., HPV + Oropharyngeal squamous cell carcinoma (HPV +The present invention also includes novel variably methylated regions (DMRs) that can distinguish OPSCC) from control or benign tissue (e.g., normal buffy coat control). According to these embodiments, the novel DMR(s) include AGRN, ANKRD35, ARHGAP27, ARHGAP30, BCL2L11, BIN2, C10orf114, C4orf31, C6orf132, C6orf186, CCDC88B, CRHBP, DAPK1, DNMT3A, DPP10, ELMO1, EPDR1, FAM19A2, FLJ45983, FOSL1, FOXB1, GREM1, HMHA1, HOXA9, IFFO1, INPP4B, ITGB2, ITGB4, ITPKB, KC NIP2, KLHDC7B, LAT, LHX6, LIMK1, LOC100128239, LOC100192379, LOC646278, MAP2K2, MAX.chr1.210426156-210426257, MAX.chr1.843264 95-84326656, MAX.chr10.119312785-119312882, MAX.chr15.67326025-67326060, MAX.chr16.54316401-54316453, MAX.chr16.85482306 -85482494, MAX.chr17.74994454-74994572, MAX.chr17.76339840-76339972, MAX.chr2.7571082-7571136, MAX.chr21.45577347-45577 679, MAX.chr3.14852538-14852568, MAX.chr3.187676564-187676668, MAX.chr4.174430662-174430793, MAX.chr5.177411809-17741183 6, MAX.chr6.45631561-45631625, MAX.chr7.25892382-25892451, MAX.chr7.402563-402641, MAX.chr7.64349554-64349606, MAX.chr8. 142046239-142046398, MAX.chr8.145900842-145901246, MAX.chr9.126101804-126101848, MAX.chr9.126978999-126979182, MAX.chr9.36458633-36458725, MAX.chr9.87905315-87905326, MBP, MFNG, MT1A, MT1IP, NCOR2, NFATC1, NKX3-2, NRN1, OLIG1, PALLD, PAPLN, PDLIM2, PKN1, PRDM14, PRKG1, PRMT7, PTGER2, PTK2B, RAD52, RBM38, RHOF, RNF220, RTN4RL1, RXRA, SDCCAG8, SHROOM1, SKI, SLC12A8, SLC25A47, SPEG, SUCLG2, TBC1D10C, TMEM132E, VIPR2, WDR66, WNT6, ZDHHC18, ZNF382, and ZNF626.

[0010] Each of the embodiments of the present disclosure is directed to a method for treating oropharyngeal cancer (e.g., HPV + Oropharyngeal squamous cell carcinoma (HPV +The present invention also includes novel variably methylated regions (DMRs) that can distinguish OPSCC from control or benign tissue (e.g., normal tissue controls). According to these embodiments, the novel DMR(s) are selected from the group consisting of ALX4, C1orf114, CA8, CCNA1, CLSTN2, CR1, DAB1, DOK1, EMBP1, EPDR1, FLJ43390, FMN2, GDF6, GFRA1, HOXB3, LDLRAD2, LOC648809, MAPRE2, MAX.chr1.241587339-241587784, MAX.chr1.50798781-50799423, MAX.chr13.28527984-28528214, MAX.chr16.3221176-3221223, MAX.chr19.11805263-11805639, MAX.chr19.2203 4646-22034887, MAX.chr19.30718373-30719719, MAX.chr2.173099712-173099791, MAX.chr2.66808635-66808731, MAX.chr6.38683091-38683226, MAX.chr9.79638034-79638077, MECOM, ONECUT2, PARP15, SGIP1, SIM2, SORCS3, ST6GALNAC5, ST8SIA5, TFAP2E, TLX2, TLX3, VSTM2B, WDR17, ZNF254, ZNF43, ZNF491, ZNF763, and ZNF844.

[0011] Each of the embodiments of the present disclosure is directed to a method for treating oropharyngeal cancer (e.g., HPV + Oropharyngeal squamous cell carcinoma (HPV +The present invention also includes novel variably methylated regions (DMRs) that can distinguish OPSCC) from control or benign tissue (e.g., normal buffy coat controls). According to these embodiments, the novel DMR(s) are derived from a gene selected from FAM19A2, IFFO1, ITGB4, LOC100192379, MAX.chr1.84326495-84326656, MAX.chr16.85482306-85482494, MAX.chr6.45631561-45631625, MAX.chr7.25892382-25892451, MT1IP, NCOR2, OLIG1, RAD52, SHROOM1, SLC12A8, and TBC1D10C.

[0012] Each of the embodiments of the present disclosure is directed to a method for treating oropharyngeal cancer (e.g., HPV + Oropharyngeal squamous cell carcinoma (HPV + The present invention also includes novel variably methylated regions (DMRs) that can distinguish OPSCC) from control or benign tissue (e.g., normal tissue or normal buffy coat control). According to these embodiments, the novel DMR(s) are derived from a gene selected from MAX.chr19.30718373-30719719, ITGB4, MAX.chr7.25892382-25892451, RAD52, SHROOM1, SLC12A8, and TBC1D10C.

[0013] Each of the embodiments of the present disclosure is directed to a method for treating oropharyngeal cancer (e.g., HPV + Oropharyngeal squamous cell carcinoma (HPV +The present invention also includes novel variably methylated regions (DMRs) that can distinguish OPSCC) from control or benign tissue (e.g., normal tissue or normal buffy coat control). According to these embodiments, the novel DMR(s) include ALX4, C1orf114, CA8, CCNA1, CLSTN2, CR1, DAB1, DOK1, EMBP1, EPDR1, FAM19A2, FLJ43390, FMN2, GDF6, GFRA1, HOXB3, IFFO1, ITGB4, LDLRAD2, LOC100192379, LOC648809, MAPRE2, MAX.chr1.241587339-24 1587784, MAX.chr1.50798781-50799423, MAX.chr1.84326495-84326656, MAX.chr13.28527984-28528214, MAX.ch r16.3221176-3221223, MAX.chr16.85482306-85482494, MAX.chr19.11805263-11805639, MAX.chr19.22034646-22 034887, MAX.chr19.30718373-30719719, MAX.chr2.173099712-173099791, MAX.chr2.66808635-66808731, MAX.c hr6.38683091-38683226, MAX.chr6.45631561-45631625, MAX.chr7.25892382-25892451, MAX.chr9.79638034-796 38077, MECOM, MT1IP, NCOR2, OLIG1, ONECUT2, PARP15, RAD52, SGIP1, SHROOM1, SIM2, SLC12A8, SORCS3, ST6GALNAC5, ST8SIA5, TBC1D10C, TFAP2E, TLX2, TLX3, VSTM2B, WDR17, ZNF254, ZNF43, ZNF491, ZNF763, and ZNF844.

[0014] Each of the embodiments of the present disclosure is directed to a method for treating oropharyngeal cancer (e.g., HPV + Oropharyngeal squamous cell carcinoma (HPV +The present invention also includes novel variably methylated regions (DMRs) that can distinguish OPSCC from control or benign tissue (e.g., normal tissue or normal buffy coat control). According to these embodiments, the novel DMR(s) are derived from a gene selected from CA8, EMBP1, HOXB3, IFFO1, ITGB4, LOC100192379, LOC648809, MAX.chr1.84326495-84326656, MAX.chr16.3221176-3221223, MAX.chr16.85482306-85482494, MAX.chr19.30718373-30719719, MAX.chr9.79638034-79638077, MT1IP, ONECUT2, SHROOM1, SIM2, SLC12A8, TLX3, and ZNF763.

[0015] Each of the embodiments of the present disclosure is directed to a method for treating oropharyngeal cancer (e.g., HPV + Oropharyngeal squamous cell carcinoma (HPV +The present invention also includes novel variably methylated regions (DMRs) that can distinguish OPSCC) from control or benign tissue (e.g., normal tissue or normal buffy coat control). According to these embodiments, the novel DMR(s) include C1orf114, CA8, CCNA1, EMBP1, EPDR1, FAM19A2, FMN2, HOXB3, IFFO1, ITGB4, LDLRAD2, LOC100192379, LOC648809, MAPR2, MAX.chr1.50798781-50799423, MAX.chr1.84326495-84326656, MAX.chr16.3221176-3221223, MAX.chr19.11805263-11805639, MAX .chr2.66808635-66808731, MAX.chr6.38683091-38683226, MAX.chr6.45631561-45631625, MAX.chr9.79638034-79638077, MECOM, MT1IP, ONECUT2, PARP15, SHROOM1, SIM2, SLC12A8, SORCS3, ST6GALNAC5, ST8SIA5, TBC1D10C, TLX3, ZNF254, ZNF491, ZNF763, and ZNF844.

[0016] Each of the embodiments of the present disclosure is directed to a method for treating oropharyngeal cancer (e.g., HPV + Oropharyngeal squamous cell carcinoma (HPV + The present invention also includes novel variably methylated regions (DMRs) that can distinguish OPSCC) from control or benign tissue (e.g., normal tissue or normal buffy coat control). According to these embodiments, the novel DMR(s) are derived from a gene selected from CA8, EMBP1, HOXB3, IFFO1, ITGB4, LOC100192379, LOC648809, MAX.chr1.84326495-84326656, MAX.chr16.3221176-3221223, MAX.chr9.79638034-79638077, MT1IP, ONECUT2, SHROOM1, SIM2, SLC12A8, TLX3, and ZNF763.

[0017] The embodiments of the present disclosure each individually detect oropharyngeal cancer (e.g., HPV) in a saliva sample from a subject. + Oropharyngeal squamous cell carcinoma (HPV + The present invention also includes novel variably methylated regions (DMRs) that can distinguish OPSCC from control or benign tissue (e.g., saliva control samples). According to these embodiments, the novel DMR(s) are derived from a gene selected from TLX3, MAX.chr16.3221176-3221223, TBC1D10C, and SHROOM1.

[0018] In some embodiments, the novel DMR(s) capable of distinguishing oropharyngeal cancer from control samples were validated based on at least one of the area under the ROC curve (AUC), fold change in methylation, percentage of methylation, and / or percentage of hypermethylation between test and control samples using at least one 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.

[0019] According to the above, the control sample includes a sample from a subject without cancer, a sample from a subject without oropharyngeal cancer, a sample from a subject with a type of cancer that is not oropharyngeal cancer, or a sample from a subject with HPV(+) cancer that is not oropharyngeal cancer. In some embodiments, the control sample is derived from a tissue sample, a blood sample, a plasma sample, a serum sample, a whole blood sample, a buffy coat sample, a secretion sample, an organ secretion sample, a cerebrospinal fluid (CSF) sample, a saliva sample, a urine sample, or a stool sample. In some embodiments, the control sample is derived from an oropharyngeal tissue sample, including one or more of soft palate cells or tissue, throat cells or tissue, tongue cells or tissue, and tonsil cells or tissue. In some embodiments, the tissue sample is an HPV(+) tissue sample.

[0020] In some embodiments, the novel DMR(s) capable of distinguishing oropharyngeal cancer from control samples are associated with an area under the ROC curve (AUC) of 0.5 or greater, where the ROC curve distinguishes between subjects with or suspected of having OPSCC and control DNA samples. In some embodiments, the novel DMR(s) capable of distinguishing oropharyngeal cancer from control samples are associated with an area under the ROC curve (AUC) of 0.6 or greater, where the ROC curve distinguishes between subjects with or suspected of having OPSCC and control DNA samples. In some embodiments, the novel DMR(s) capable of distinguishing oropharyngeal cancer from control samples are associated with an area under the ROC curve (AUC) of 0.7 or greater, where the ROC curve distinguishes between subjects with or suspected of having OPSCC and control DNA samples. In some embodiments, the novel DMR(s) capable of distinguishing oropharyngeal cancer from control samples are associated with an area under the ROC curve (AUC) of 0.8 or greater, where the ROC curve distinguishes between subjects having or suspected of having OPSCC and control DNA samples. In some embodiments, the novel DMR(s) capable of distinguishing oropharyngeal cancer from control samples are associated with an area under the ROC curve (AUC) of 0.9 or greater, where the ROC curve distinguishes between subjects having or suspected of having OPSCC and control DNA samples.

[0021] In some embodiments, the novel DMR(s) capable of distinguishing oropharyngeal cancer from a control sample comprises an increased rate of hypermethylation compared to a control DNA sample. In some embodiments, the novel DMR(s) capable of distinguishing oropharyngeal cancer from a control sample comprises an increased rate of hypermethylation compared to a control DNA sample.

[0022] In some embodiments, a biological sample is obtained from the subject, and the method further comprises extracting a DNA sample from the biological sample. In some embodiments, the biological sample is collected with a collection device having an adsorption element capable of collecting the biological sample upon contact. In some embodiments, the biological sample is collected with a collection device having an extraction element capable of extracting the biological sample. In some embodiments, the adsorption or extraction element is configured for insertion into an orifice (mouth, nose, or throat).

[0023] In some embodiments, the reagent that modifies DNA in a methylation-specific manner is a borane reducing agent. 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.

[0024] In some embodiments, determining the methylation profile of the at least one DMR comprises amplifying at least a portion of the DMR using a set of primers (e.g., Table 3 and Table 12). In some embodiments, determining the methylation profile of the at least one DMR comprises performing at least one 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, determining the methylation profile of the at least one DMR comprises determining the presence or absence of methylation at CpG sites. In some embodiments, the one or more CpG sites are present in a coding region, a non-coding region, and / or a regulatory region of a gene (e.g., any one of the genes disclosed herein).

[0025]

[0023] Embodiments of the present disclosure also include methods for identifying oropharyngeal cancer. According to these embodiments, the methods include determining a methylation profile in at least one variably methylated region (DMR) of a DNA sample obtained from a subject having or suspected of having oropharyngeal cancer by treating the sample with a reagent that modifies DNA in a methylation-specific manner. In some embodiments, the methylation profile is determined based on whether the subject has oropharyngeal cancer (e.g., HPV + Oropharyngeal squamous cell carcinoma (HPV + In some embodiments, the method further comprises treating the subject with an anti-cancer therapy. [Brief description of the drawings]

[0026] [Figure 1] Representative boxplots showing the ability of GRIND2-derived DMRs to distinguish oropharyngeal cancer (HPV(+) oropharyngeal squamous cell carcinoma (OSPCC)) from controls (normal oropharyngeal tissue (NOP) and normal cervical tissue (NCS)) and HPV(+) cervical squamous cell carcinoma (CSCC). [Diagram 2] Representative boxplots showing the ability of EMX1-derived DMRs to distinguish oropharyngeal cancer (HPV(+) oropharyngeal squamous cell carcinoma (OSPCC)) from controls (normal oropharyngeal tissue (NOP) and normal cervical tissue (NCS)) and HPV(+) cervical squamous cell carcinoma (CSCC). [Diagram 3] Representative boxplots showing the ability of VWC2-derived DMRs to distinguish oropharyngeal cancer (HPV(+) oropharyngeal squamous cell carcinoma (OSPCC)) from controls (normal oropharyngeal tissue (NOP) and normal cervical tissue (NCS)) and HPV(+) cervical squamous cell carcinoma (CSCC). [Figure 4] Representative boxplots showing the ability of ZNF610-derived DMRs to distinguish oropharyngeal cancer (HPV(+) oropharyngeal squamous cell carcinoma (OSPCC)) from controls (normal oropharyngeal tissue (NOP) and normal cervical tissue (NCS)) and HPV(+) cervical squamous cell carcinoma (CSCC). [Diagram 5]Representative boxplots showing the ability of ZNF781.A-derived DMRs to distinguish oropharyngeal cancer (HPV(+) oropharyngeal squamous cell carcinoma (OSPCC)) from controls (normal oropharyngeal tissue (NOP) and normal cervical tissue (NCS)) and HPV(+) cervical squamous cell carcinoma (CSCC). [Figure 6] Representative boxplots showing the ability of TBX15-derived DMRs to distinguish oropharyngeal cancer (HPV(+) oropharyngeal squamous cell carcinoma (OSPCC)) from controls (normal oropharyngeal tissue (NOP) and normal cervical tissue (NCS)) and HPV(+) cervical squamous cell carcinoma (CSCC). [Figure 7] Representative boxplots showing the ability of TSPYL5-derived DMRs to distinguish oropharyngeal cancer (HPV(+) oropharyngeal squamous cell carcinoma (OSPCC)) from controls (normal oropharyngeal tissue (NOP) and normal cervical tissue (NCS)) and HPV(+) cervical squamous cell carcinoma (CSCC). [Figure 8] Representative boxplots showing the ability of LOC645323-derived DMRs to distinguish oropharyngeal cancer (HPV(+) oropharyngeal squamous cell carcinoma (OSPCC)) from controls (normal oropharyngeal tissue (NOP) and normal cervical tissue (NCS)) and HPV(+) cervical squamous cell carcinoma (CSCC). [Figure 9] Representative boxplots showing the ability of ASCL1-derived DMRs to distinguish oropharyngeal cancer (HPV(+) oropharyngeal squamous cell carcinoma (OSPCC)) from controls (normal oropharyngeal tissue (NOP) and normal cervical tissue (NCS)) and HPV(+) cervical squamous cell carcinoma (CSCC). [Figure 10] Representative boxplots showing the ability of ABCB1-derived DMRs to distinguish oropharyngeal cancer (HPV(+) oropharyngeal squamous cell carcinoma (OSPCC)) from controls (normal oropharyngeal tissue (NOP) and normal cervical tissue (NCS)) and HPV(+) cervical squamous cell carcinoma (CSCC). [Figure 11]Representative boxplots showing the ability of ZNF69-derived DMRs to distinguish oropharyngeal cancer (HPV(+) oropharyngeal squamous cell carcinoma (OSPCC)) from controls (normal oropharyngeal tissue (NOP) and normal cervical tissue (NCS)) and HPV(+) cervical squamous cell carcinoma (CSCC). [Figure 12] Representative boxplots showing the ability of DMRs derived from MAX.chr9.36739811-36739868 to distinguish oropharyngeal cancer (HPV(+) oropharyngeal squamous cell carcinoma (OSPCC)) from controls (normal oropharyngeal tissue (NOP) and normal cervical tissue (NCS)) and HPV(+) cervical squamous cell carcinoma (CSCC). [Figure 13] Representative boxplots showing the ability of ARHGAP12-derived DMRs to distinguish oropharyngeal cancer (HPV(+) oropharyngeal squamous cell carcinoma (OSPCC)) from controls (normal oropharyngeal tissue (NOP) and normal cervical tissue (NCS)) and HPV(+) cervical squamous cell carcinoma (CSCC). [Figure 14] Representative boxplots showing the ability of C1orf114-derived DMRs to distinguish oropharyngeal cancer (HPV(+) oropharyngeal squamous cell carcinoma (OSPCC)) from controls (normal oropharyngeal tissue (NOP) and normal cervical tissue (NCS)) and HPV(+) cervical squamous cell carcinoma (CSCC). [Figure 15] Representative boxplot showing the ability of DMRs derived from MAX.chr6.58147682-58147771 to distinguish oropharyngeal cancer (HPV(+) oropharyngeal squamous cell carcinoma (OSPCC)) from controls (normal oropharyngeal tissue (NOP) and normal cervical tissue (NCS)) and HPV(+) cervical squamous cell carcinoma (CSCC). [Figure 16] Representative boxplots showing the ability of NEUROG3-derived DMRs to distinguish oropharyngeal cancer (HPV(+) oropharyngeal squamous cell carcinoma (OSPCC)) from controls (normal oropharyngeal tissue (NOP) and normal cervical tissue (NCS)) and HPV(+) cervical squamous cell carcinoma (CSCC). [Figure 17]Representative boxplots showing the ability of NID2-derived DMRs to distinguish oropharyngeal cancer (HPV(+) oropharyngeal squamous cell carcinoma (OSPCC)) from controls (normal oropharyngeal tissue (NOP) and normal cervical tissue (NCS)) and HPV(+) cervical squamous cell carcinoma (CSCC). [Figure 18] Representative boxplots showing the ability of TMEM200C-derived DMRs to distinguish oropharyngeal cancer (HPV(+) oropharyngeal squamous cell carcinoma (OSPCC)) from controls (normal oropharyngeal tissue (NOP) and normal cervical tissue (NCS)) and HPV(+) cervical squamous cell carcinoma (CSCC). [Figure 19] Representative boxplots showing the ability of TTYH1-derived DMRs to distinguish oropharyngeal cancer (HPV(+) oropharyngeal squamous cell carcinoma (OSPCC)) from controls (normal oropharyngeal tissue (NOP) and normal cervical tissue (NCS)) and HPV(+) cervical squamous cell carcinoma (CSCC). [Figure 20] Representative boxplots showing the ability of ZNF773-derived DMRs to distinguish oropharyngeal cancer (HPV(+) oropharyngeal squamous cell carcinoma (OSPCC)) from controls (normal oropharyngeal tissue (NOP) and normal cervical tissue (NCS)) and HPV(+) cervical squamous cell carcinoma (CSCC). [Figure 21] Representative boxplots showing the ability of ZNF781.B-derived DMRs to distinguish oropharyngeal cancer (HPV(+) oropharyngeal squamous cell carcinoma (OSPCC)) from controls (normal oropharyngeal tissue (NOP) and normal cervical tissue (NCS)) and HPV(+) cervical squamous cell carcinoma (CSCC). [Figure 22] Representative box plots of β-actin as a control for Figures 1-21. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Oropharyngeal (or head and neck) cancer accounts for 3% of cancers diagnosed annually in the United States and is projected to cause approximately 11,000 deaths in 2021. While the incidence of HPV- cancers has remained relatively constant, the incidence of HPV+ cancer subtypes has been increasing. Noninvasive molecular tests to screen patients at high risk for oropharyngeal cancer are urgently needed as they will likely reduce the burden of this disease and save lives. At present, there are no accurate, easy-to-use, and widely accessible screening tools for the ideal clinical management of oropharyngeal tumors. To address these clinical gaps, we conducted experiments to develop a new approach focused on marker detection within tissues, across multiple tissue compartments, and in body fluids to target novel, highly discriminatory methylated DNA markers with the ability to predict primary tumor characteristics using an exquisitely sensitive analytical platform.

[0028] Therefore, various experiments described herein were performed to discover novel methylated DNA markers in tissues of malignant oropharyngeal tumors by unbiased whole methylome sequencing (RRBS), validate the top candidates in independent tissues, evaluate the accuracy of oropharyngeal cancer detection by assaying the top methylated DNA markers in plasma, identify the accuracy of oropharyngeal cancer detection by assaying the top methylated DNA markers in body fluids, and identify methylated DNA markers with potential oropharyngeal site specificity by in silico comparing the discovered candidates against a whole methylome database created for tumors across multiple organs.

[0029] New molecular technologies offer an opportunity to rethink how cancer screening is performed. With well-identifiable markers and sensitive assay tools, detection of multiple cancers at their earliest stages by blood or other remote media (e.g., urine or saliva) for oropharyngeal tumors may be possible. Thus, the traditional approach of single-organ screening may be replaced by a new paradigm of multi-organ cancer screening using a single non-invasive test. The potential gains in cost-efficiency and benefits in reducing cancer deaths could be staggering. Blood testing represents the most attractive approach to universal cancer screening. However, long-standing blood testing methods have largely failed to detect early cancer with sufficient sensitivity or specificity to achieve clinical utility or efficacy. Historically, blood tests for cancer have focused on transparent compartments (i.e., plasma or serum) and targeted various proteins or acquired genetic alterations associated with cancer. Such markers often lack site specificity, which creates diagnostic ambiguity and confounds downstream clinical evaluation.

[0030] As described further herein, various embodiments of the present disclosure provide solutions to these technical and biological barriers. Analytical sensitivity is improved by orders of magnitude compared to past methods, falling within the zone required for detection of low abundance markers with early stage disease. In addition, the data provided herein demonstrates that marker assays provide complementary value to plasma testing alone for detection of the earliest stage lesions, since alternative compartments may address other mechanisms of marker entry into the blood.

[0031] The section headings used in this section and throughout this disclosure are for organizational purposes only and are not intended to be limiting.

[0032] 1.Definition Throughout this specification and claims, the following terms have the meanings expressly associated therewith, unless the context clearly dictates otherwise. The phrase "in one embodiment" as used herein may, but does not necessarily, refer to the same embodiment. Additionally, the phrase "in another embodiment" as used herein may, but does not necessarily refer to different embodiments. Thus, as described below, various embodiments of the invention can be readily combined without departing from the scope or spirit of the invention.

[0033] Additionally, as used herein, the term "or" is an inclusive "or" operator and is equivalent to the term "and / or" unless the context clearly dictates otherwise. 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."

[0034] The transitional phrase "consisting essentially of," when used in the claims of this application, limits the scope of the claim to the particular materials or steps of the claimed invention and "which do not materially affect the basic and novel characteristic(s)," as discussed in In re Herz, 537 F.2d 549,551-52,190 USPQ 461,463 (CCPA 1976). For example, a composition "consisting essentially of" may contain unrecited contaminants, although the recited elements are present, at levels that do not alter the function of the recited composition compared to the pure composition, i.e., a composition "consisting of" the recited components.

[0035] The term "one or more" as used herein refers to a number greater than 1. For example, the term "one or more" encompasses any of the following: two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, twenty or more, fifty or more, a hundred or more, or even greater numbers.

[0036] The terms "one or more but less than a larger number," "two or more but less than a larger number," "three or more but less than a larger number," "four or more but less than a larger number," "five or more but less than a larger number," "six or more but less than a larger number," "seven or more but less than a larger number," "eight or more but less than a larger number," "nine or more but less than a larger number," "ten or more but less than a larger number," "eleven or more but less than a larger number," "twelve or more but less than a larger number," "thirteen or more but less than a larger number," "fourteen or more but less than a larger number," or "fifteen or more but less than a larger number" are not limited to a larger number. For example, the larger number can be 10,000, 1,000, 100, 50, etc. For example, some larger number can be about 50 (e.g., 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).

[0037] The terms "one or more methylation markers", or "one or more DMRs", or "one or more genes", "one or more markers", or "multiple methylation markers", or "multiple markers", or "multiple genes", or "multiple DMRs" are similarly not limited to specific numerical combinations. In fact, any numerical combination of methylation markers (e.g., methylation markers 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) ( For example, 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) (for example, 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) (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) (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) (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) (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) (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) (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) (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, 1 0~24, 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) (e.g. 11~12, 11~13, 11~14, 11~15, 11~16, 1 1~17, 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) (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) (e.g., 13-14, 13-15, 13-16, 13-17, 13-18, 13-19, 13-20, 13-21, 13-22, 13-23, 13-24, 13-25, 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) (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, 14-38) (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) (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 )(For example, 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, 17~36, 17~37 , 17-38) (e.g., 18-19, 18-20, 18-21, 18-22, 18-23, 18-24, 18-25, 18-26, 18-27, 18-28, 18-29, 18-30, 18-31, 18-32, 18-33, 18-34, 18-35, 18-36, 18-37,18-38) (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) (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) (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) (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) (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) (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) (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) (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) (e.g., 27-28, 27-29, 27-30, 27-31, 27-32, 27-33, 27-34, 27-35, 27-36, 27-37, 27-38) (e.g., 28-29, 28-30, 28-31, 28-32, 28-33, 28-34, 28-35, 28-36, 28-37, 28-38) (e.g., 29-30, 29-31, 29-32, 29-33, 29-34, 29-35, 29-36, 29-37, 29-38) (e.g., 30-31,30-32, 30-33, 30-34, 30-35, 30-36, 30-37, 30-38) (e.g., 31-32, 31-33, 31-34, 31-35, 31-36, 31-37, 31-38) (e.g., 32-33, 32-34, 32-35, 32-36, 32-37, 32-38) (e.g., 33-34, 33-35, 33-36, 33-37, 33-38) (e.g., 34-35, 34-36, 34-37, 34-38) (e.g., 35-36, 35-37, 35-38) ( For example, 36-37, 36-38) (e.g., 37-38) (e.g., 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) are contemplated.

[0038] The term "one or more protein markers" is similarly not limited to a particular combination of values. Indeed, any combination of values ​​of protein markers (e.g., 1-2 protein markers, 1-3, 1-4, 1-5) (e.g., 2-3, 2-4, 2-5) (e.g., 3-4, 3-5) (e.g., 4-5) (e.g., 5 or less, 4 or less, 3 or less, 2, or 1) is contemplated.

[0039] The terms "multiple types of cancer", or "one or more types of cancer", or "one or more subtypes of cancer", or "multiple different types or subtypes of cancer" are similarly not limited to any particular combination of values. + Any numerical combination of oropharyngeal cancer types or subtypes may be specified, including but not limited to oropharyngeal squamous cell carcinoma.

[0040] 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 a backbone modified for stability or for other reasons is a "nucleic acid". As used herein, the term "nucleic acid" encompasses such chemically, enzymatically, or metabolically modified forms of nucleic acid, as well as the chemical forms of DNA characteristic of viruses and cells (including, for example, simple and complex cells).

[0041] The term "oligonucleotide", or "polynucleotide", or "nucleotide", or "nucleic acid" refers to a molecule having two or more, preferably three or more, and usually ten or more deoxyribonucleotides or ribonucleotides. The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. Oligonucleotides can be produced in any manner, 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.

[0042] 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.

[0043] 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.

[0044] 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 a 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 fragments of that gene. These fragments can range in size from a few nucleotides to the entire gene sequence minus one nucleotide. Thus, "nucleotides comprising at least a portion of a gene" can include fragments of a gene or the entire gene.

[0045] The term "gene" also includes the coding region of a structural gene and includes sequences (e.g., including coding, regulatory, structural and other sequences) located adjacent to the coding region at both the 5' and 3' ends, at a distance of about 1 kb on either end, such that the gene corresponds to the length of the full-length mRNA. Sequences that are 5' of the coding region and present on the mRNA are referred to as 5' non-translated or untranslated sequences. Sequences that are 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 some 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.

[0046] 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.

[0047] When used in reference 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 used in reference 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 used in reference to a protein, the term "wild type" refers to a protein that has the characteristics of a naturally occurring protein. When used in reference to an object, the term "naturally occurring" refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a natural source and has not been intentionally modified by the hand of man in a laboratory 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 used in reference to a gene or gene product, the term "modified" or "mutant" refers to a gene or gene product, respectively, that exhibits modifications in sequence and / or functional properties (i.e., altered characteristics) when compared to the 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.

[0048] The term "allele" refers to genetic variations, including, but not limited to, variants and mutations, polymorphic loci, and single nucleotide polymorphic loci, frameshifts, and splice variants. Alleles may occur naturally in a population or may arise during the lifetime of any particular individual in a population.

[0049] Thus, the terms "variant" and "mutant" when used in reference to a nucleotide sequence refer to a nucleic acid sequence that differs by one or more nucleotides from another, usually related nucleotide sequence. A "variation" is the difference between two different nucleotide sequences, typically one sequence being a reference sequence.

[0050] The term "primer" refers to an oligonucleotide that can serve as a point of initiation of synthesis when placed under conditions that induce synthesis of a primer extension product that is complementary to a nucleic acid template strand, whether naturally occurring, for example, as a nucleic acid fragment from a restriction digest, or synthetically produced (e.g., in the presence of nucleotides and an inducing agent, such as DNA polymerase, at a suitable 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 sufficiently long to prime the synthesis of the extension product in the presence of the inducing agent. The exact length of the primer will depend on many factors, including temperature, source of primer, and use of the method. In some embodiments, the primer pairs are specific for a specific variably methylated region (e.g., a DMR in Tables 1, 2, 6, and 7) and specifically bind to at least a portion of a genetic region that includes the DMR (e.g., the chromosomal coordinates in Tables 1, 2, 6, and 7).

[0051] The term "probe" refers to an oligonucleotide (e.g., a sequence of nucleotides) that can hybridize to another oligonucleotide of interest, whether naturally occurring, such as a purified restriction digest, synthetically produced, recombinantly produced, or produced by PCR amplification. 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 embodiments of the present disclosure may, in some embodiments, be labeled with any "reporter molecule," which is detectable by any detection system, including, but not limited to, enzymes (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the various embodiments of the present disclosure be limited to any particular detection system or label.

[0052] The term "target" as used herein refers to a nucleic acid that is sought 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, however, in some embodiments of assays in which the target DNA is not amplified, for example, invasive cleavage assays, the target includes the site where the probe and invasive oligonucleotide (e.g., INVADER oligonucleotide) bind to form an invasive cleavage structure such that the presence of the target nucleic acid can be detected. A "segment" is defined as a region of nucleic acid within the target sequence.

[0053] Thus, as used herein, "non-target", when used to describe a nucleic acid such as, for example, 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, a non-target nucleic acid may refer to, for example, a nucleic acid present in a sample that does not contain a target sequence, but in some embodiments, a non-target may refer to a nucleic acid that is not derived from a sample that contains or is suspected of containing an exogenous nucleic acid (i.e., a target nucleic acid) and is added to a reaction, for example, to normalize the activity of an enzyme (e.g., a polymerase) in the reaction to reduce variability in the performance of the enzyme in the reaction.

[0054] 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.

[0055] As used herein, the term "amplification reagents" refers to those reagents necessary for amplification, excluding primers, nucleic acid template, and amplification enzymes (deoxyribonucleoside triphosphates, buffers, etc.) Typically, amplification reagents are placed with other reaction components and placed in a reaction vessel.

[0056] As used herein, the term "control" when used in reference to nucleic acid detection or analysis refers to a nucleic acid with known characteristics (e.g., known sequence, known copy number per cell) for use in comparison with an experimental target (e.g., a nucleic acid of unknown concentration). A control can be an endogenous, preferably invariant, gene to which a test or target nucleic acid in an assay can be normalized. Such normalization controls for sample-to-sample variations that may occur, for example, in sample processing, assay efficiency, etc., allow accurate sample-to-sample data comparison. Genes useful for normalizing nucleic acid detection assays for human samples include, for example, b-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 located on Chr16 (16q22.1) of human DNA and encoding a protein characterized as zinc finger DHHC type-containing 1, belonging to the DHHC palmitoyltransferase family.

[0057] A control can also be an external control. For example, in quantitative assays such as qPCR, QuARTS, etc., a "calibrator" or "calibration control" is a nucleic acid of known sequence, e.g., having the same sequence as a portion of an experimental target nucleic acid, and having a known concentration or series of concentrations (e.g., a control target serially diluted for the generation of a calibration curve in quantitative PCR). Typically, the calibration control is 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 are easily provided in equimolar amounts. In some 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, incorporated herein by reference.

[0058] As further described herein, a "control" or "control sample" may include, but is not limited to, a tissue sample, a blood sample, a plasma sample, a serum sample, a whole blood sample, a buffy coat sample, a secretion sample, an organ secretion sample, a cerebrospinal fluid (CSF) sample, a saliva sample, a urine sample, or a stool sample. In some embodiments, the control sample is derived from an oropharyngeal tissue sample, including one or more of soft palate cells or tissue, throat cells or tissue, tongue cells or tissue, and tonsil cells or tissue. In some embodiments, the control sample is a sample from a subject without cancer, a sample from a subject without oropharyngeal cancer, a sample from a subject with a type of cancer that is not oropharyngeal cancer, or a sample from a subject with HPV(+) cancer that is not oropharyngeal cancer. In some embodiments, the tissue sample is an HPV(+) tissue sample.

[0059] 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.

[0060] As used herein, a "methylated nucleic acid molecule" refers to a nucleic acid molecule that contains one or more methylated nucleotides.

[0061] 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.

[0062] As used herein, the term "methylation level", when applied to a methylation marker, refers to the amount of methylation within a particular methylation marker. Methylation level may also refer to the amount of methylation within 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 fraction of cells in a sample that have or do not have a methylation group on such cytosine. Alternatively, methylation level may also describe whether a single CpG dinucleotide is methylated.

[0063] 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 about the position within the sequence where methylation occurs.

[0064] 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).

[0065] The methylation status may optionally be represented or indicated by a "methylation value" (e.g., representing a methylation frequency, fraction, ratio, percentage, etc.). Methylation values ​​may 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.

[0066] As used herein, "methylation frequency" or "percent (%) methylation" refers to the number of instances where a molecule or locus is methylated compared to the number of instances where the molecule or locus is unmethylated.

[0067] The term "methylation score" as used herein is a score indicating the methylation events detected in a marker or panel of markers compared to the median methylation events for the marker or panel of markers from a random population of mammals without tumors of a particular interest (e.g., a random population of 10, 20, 30, 40, 50, 100, or 500 individual mammals). The increase in the methylation score of a marker or panel of markers can be any score, as long as the score is greater than the corresponding reference score. For example, the increase in the methylation score of 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.

[0068] Thus, methylation state describes the state of methylation of a nucleic acid (e.g., a genomic sequence). In addition, methylation state refers to the characteristics of a nucleic acid segment at a particular genomic locus that are 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 (e.g., due to different allelic origins). 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 referred to as "hypermethylated" or "increased methylation", whereas if a cytosine (C) residue(s) in a DNA sequence is unmethylated, it can be referred to as "hypomethylated" or "decreased 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 hypomethylated or have reduced 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 have hypomethylated or reduced 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 percentage, but can have different methylation patterns when the number of methylated and unmethylated nucleotides is the same or similar throughout this 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 the 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 may also refer to the difference in the level or pattern between patients who have cancer recurrence after surgery and those who do not. Variable methylation, and specific levels or patterns of DNA methylation, are prognostic and predictive biomarkers, for example, when precise cutoffs or predictive characteristics are defined.

[0069] 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 in 50% of cases and unmethylated in 50% of cases. 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, if 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 will be 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.

[0070] 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). Most 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).

[0071] As used herein, "CpG island" (or "cytosine-phosphate-guanine island") refers to a G:C-rich region of genomic DNA that contains an increased number of CpG dinucleotides relative to the total genomic DNA. A CpG island can be at least 100, 200, or more base pairs long, 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. It will be appreciated that other sequences in the human genome are prone to DNA methylation, such as CpA and CpT (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).

[0072] 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 can change 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 can comprise contacting the nucleic acid molecule with the reagent, and can be optionally combined with additional steps that achieve the desired change in the nucleotide sequence. Such methods can 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.

[0073] 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.

[0074] 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.

[0075] The term "MS AP-PCR" (methylation-sensitive arbitrarily primed polymerase chain reaction) refers to an art-recognized technique that uses CG-rich primers to allow global scanning of the genome, focusing on regions most likely to contain CpG dinucleotides, as described in Gonzalgo et al. (1997) Cancer Research 57:594-599.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] The term "Ms-SNuPE" (Methylation-Sensitive Single Nucleotide Primer Extension) refers to the art-recognized assay described by Gonzalgo & Jones (1997) Nucleic Acids Res. 25:2529-2531.

[0080] 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 US Pat. No. 5,786,146.

[0081] The term "COBRA" (Combined Bisulfite Restriction Analysis) refers to the art-recognized methylation assay described by Xiong & Laird (1997) Nucleic Acids Res. 25:2532-2534.

[0082] The term "MCA" (Methylated CpG Island Amplification) refers to the methylation assay described by Toyota et al. (1999) Cancer Res. 59:2307-12 and WO00 / 26401A1.

[0083] 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 a 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.

[0084] 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. In the case of a restriction enzyme that specifically cleaves when the recognition site is unmethylated or hemimethylated (methylation-sensitive enzyme), cleavage does not occur (or is significantly less efficient) when the recognition site is methylated on one or both strands. In the case of a restriction enzyme that specifically cleaves only when the recognition site is methylated (methylation-dependent enzyme), cleavage does not occur (or is significantly less efficient) when the recognition site is unmethylated. Preferably, it is a methylation-specific restriction enzyme, the recognition sequence of which contains a CG dinucleotide (e.g., a 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.

[0085] As used herein, the "sensitivity" of a given marker (or set of markers used together) refers to the proportion 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 tumor 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 tumor 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 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.

[0086] As used herein, the "specificity" of a given marker (or set of markers used together) refers to the proportion of non-neoplastic samples that report DNA methylation values ​​below a threshold that distinguishes between neoplastic and non-neoplastic samples. In some embodiments, a negative is defined as a histologically confirmed non-neoplastic sample that reports a DNA methylation value below a threshold (e.g., a non-disease associated range), and a false negative is defined as a histologically confirmed non-neoplastic sample that reports a DNA methylation value above a threshold (e.g., a 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 within 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.

[0087] 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).

[0088] 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.

[0089] As used herein, the term "tumor-specific marker" refers to any biomaterial or element that can be used to indicate the presence of a tumor. Examples of biomaterials 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, an intragenic region, a specific locus, etc.). A region of a nucleic acid that is a marker can be referred to, for example, as a "marker gene," "marker region," "marker sequence," "marker locus," etc.

[0090] 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.

[0091] The terms "precancerous" or "preneoplastic" and their equivalents refer to any cell proliferative disorder that is undergoing malignant transformation.

[0092] A "site" of a tumor, adenoma, cancer, etc., is a tissue, organ, cell type, anatomical region, body part, etc. within a subject's body in which the tumor, adenoma, cancer, etc. is located.

[0093] 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 the likelihood of progression or regression) of a subject with a disease or condition, and determining the effect of a treatment on 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 tumor, or the likelihood that such a subject will respond favorably to a compound (e.g., a pharmaceutical, e.g., a drug) or other treatment.

[0094] The term "isolated", when used in reference to a nucleic acid, such as "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 context 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 naturally occur. Examples of non-isolated nucleic acids include a given DNA sequence (e.g., a gene) that is found on a host cell chromosome in close proximity to adjacent genes. An RNA sequence, such as a particular mRNA sequence that encodes a particular protein, 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, by way of 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 different nucleic acid sequence than that found in nature. An isolated nucleic acid or oligonucleotide can be present in single-stranded or double-stranded form. When an isolated nucleic acid or oligonucleotide is utilized to express a protein, the oligonucleotide will contain at least 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 being isolated from its natural or typical environment. For example, the isolated nucleic acid may be present in a host cell in which it is placed, for example, for heterologous expression.

[0095] 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.

[0096] 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.).

[0097] 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 sample types applicable to various embodiments of the present disclosure.

[0098] As used herein, a "remote sample", when used in some contexts, refers to a sample that is indirectly collected from a site that is not the source of the cells, tissues, or organs of the sample. For example, when sample material derived from the pancreas is evaluated in a stool sample, the sample is a remote sample.

[0099] As used herein, the term "patient" or "subject" refers to an organism that is the subject of the various tests described herein. 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. Further with respect to the diagnostic methods, the preferred subject is a vertebrate subject. The preferred vertebrates are warm-blooded animals, and the preferred warm-blooded vertebrates are mammals. The preferred mammal is most preferably a human. As used herein, the term "subject" includes both human and animal subjects. Thus, veterinary therapeutic uses are provided herein. Thus, the present disclosure provides for the diagnosis of mammals, such as humans, as well as mammals of importance due to being endangered species, such as Siberian tigers, mammals of economic importance, such as animals raised on farms for human consumption, and / or animals of social importance 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, ruminants and / or ungulates such as swine (including pigs, hogs, and wild boars), cows, bulls, sheep, giraffes, deer, goats, bison, and camels, pinnipeds, and horses. Thus, diagnosis and treatment of livestock (including, but not limited to, domesticated pigs, ruminants, ungulates, horses (including race horses), and the like) are also provided. Embodiments of the present disclosure further include a system for diagnosing one or more types or subtypes of oropharyngeal cancer in a subject. The system may be provided as a commercially available kit and may be used, for example, to screen for risk of one or more types or subtypes of oropharyngeal cancer or to diagnose one or more types or subtypes of oropharyngeal cancer in a subject from whom a biological sample was taken. An exemplary system provided according to various embodiments of the present disclosure includes assessing the methylation state or profile of a marker as described herein.

[0100] 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 enclosures (e.g., boxes) that contain the relevant reaction reagents and / or supporting equipment. As used herein, the term "fragmented kit" refers to a delivery system that includes two or more separate containers, each of which contains a portion of the total kit components. The containers can be delivered to the intended recipient together or separately. For example, a first container can contain an enzyme for use in the assay, while a second container contains an oligonucleotide. The term "separated kit" is intended to encompass, but is not limited to, a kit that contains an analyte-specific reagent (ASR) regulated under Section 520(e) of the Federal Food, Drug, and Cosmetic Act. Indeed, any delivery system that comprises two or more separate containers, each containing a portion of the overall kit components, is included in the term "split kit." In contrast, a "combined kit" refers to a delivery system that contains all of the components of a reaction assay in a single container (e.g., a single box housing each of the desired components). The term "kit" includes both fragmented and combined kits.

[0101] 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 related to 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 allele identities, statistical correlations between the presence of an allele and characteristics of a subject (e.g., a human subject), the presence or absence of an allele in an individual or population, the percentage of the likelihood that an allele is present in an individual with one or more particular characteristics, and the like.

[0102] 2. Methylation DNA markers and biomarker panels Embodiments of the present disclosure provide methods, compositions, and systems for screening one or more types of oropharyngeal cancer from a biological sample. According to these embodiments, the present disclosure includes, but is not limited to, methods and compositions for detecting the presence of one or more types or subtypes of oropharyngeal cancer from a biological sample. In some embodiments, the biological sample is a tissue sample, a blood sample, a plasma sample, a serum sample, a whole blood sample, a buffy coat sample, a secretion sample, an organ secretion sample, a cerebrospinal fluid (CSF) sample, a saliva sample, a urine sample, and / or a stool sample. In some embodiments, the tissue sample is an oropharyngeal tissue sample, including one or more of soft palate cells or tissue, throat cells or tissue, tongue cells or tissue, and tonsil cells or tissue. In some embodiments, the tissue sample is an HPV(+) tissue sample. In some embodiments, the subject is a human.

[0103] As further described herein, embodiments of the present disclosure each individually include a method for treating oropharyngeal cancer (e.g., HPV + Oropharyngeal squamous cell carcinoma (HPV +The present invention relates to a method for treating OPSCC (Osteoarthritis Pelvic Floor Cancer) comprising: (a) detecting a pulmonary fibrosis (OPSCC) in a patient with pulmonary fibrosis (OPSCC) from a control sample (e.g., benign tissue, including, but not limited to, oropharyngeal tissue, cervical tissue, tonsil tissue, buffy coat samples, and saliva samples); and (benign tissue, including, but not limited to, oropharyngeal tissue, cervical tissue, tonsil tissue, buffy coat samples, and saliva samples). According to these embodiments, the novel DMR(s) include ABCB1, ARHGAP12, ASCL1, C1orf114, EMX1, GRIN2D, LOC645323, MAX.chr6.58147682-58147771, MAX.chr9.36739811-36739868, NEUROG3, NID2, TBX15, TMEM200C, TSPYL5, TTYH1, VWC2, ZNF610, ZNF69, ZNF773, ZNF781, ALX4, ATP10A, C1Q L3, CA8, CACNA1A, CACNG8, CALCA, CCNA1, CLIC6, CLSTN2, CR1, CTNND2, DAB1, DGKG, DOK1, DOK6, DPP4, DUXA, ELMO1, EMBP1, EPDR1, FGF1 2, FLJ43390, FMN2, FOXB2, FOXD4, FREM3, GALR1, GDF6, GFRA1, GRIK3, HOXB3, HOXB4, HPSE2, LDLRAD2, LHX2, LOC100131366, LOC345643, LOC386758, LOC648809, LOC728392, MAML3, MAPRE2, MAX.chr1.226288154-226288189, MAX.chr1.2375078-2375126, MAX.chr1.2415 87339-241587784, MAX.chr1.50798781-50799423, MAX.chr10.22765150-22765477, MAX.chr10.23462342-23462436, MAX.chr11.1 4926602-14927044, MAX.chr11.58903531-58903592, MAX.chr13.28527984-28528214, MAX.chr13.29106641-29107037, MAX.chr14 .100784488-100784782, MAX.chr16.3221176-3221223, MAX.chr16.3222040-3222098, MAX.chr16.71460171-71460282, MAX.chr19.11805263-11805639、MAX.chr19.16394457-16394646、MAX.chr19.21657626-21657769、MAX.chr19.22034646-22034887、MAX.chr19.23299989-23300156、MAX.chr19.30713427-30713588、MAX.chr19.30716926-30717074、MAX.chr19.30718373-30719719、MAX.chr2.118981724-118982174、MAX.chr2.127783107-127783403、MAX.chr2.173099712-173099791、MAX.chr2.66808635-66808731、MAX.chr22.50064113-50064259、MAX.chr3.137489884-137490061、MAX.chr5.138923141-138923219、MAX.chr5.42995180-42995535、MAX.chr6.38683091-38683226、MAX.chr7.121952014-121952084、MAX.chr7.155166980-155167310、MAX.chr8.99986792-99986864、MAX.chr9.79627078-79627116、MAX.chr9.79638034-79638077、MAX.chr9.98789824-98789847、MDFI、MECOM、MED12L、MIR129-2、MIR196A1、NELL1、NPY、ONECUT2、OPCML、PARP15、PDGFD、PEX5L、PRR15、SEMA6A、SFMBT2、SGIP1、SIM2、SLC35F3、SLCO4C1、SORCS3、ST6GALNAC5、ST8SIA5、SV2C、TACC2、TFAP2E、TLX2、TLX3、TRH、TRIM58、VAV3、VSTM2B、WDR17、ZNF254、ZNF43、ZNF486、ZNF491、ZNF518B、ZNF542、ZNF625、ZNF665、ZNF671、ZNF763、ZNF844、AGRN、ANKRD35、ARHGAP27、ARHGAP30、BCL2L11、BIN2、C10orf114、C4orf31、C6orf132、C6orf186、CCDC88B、CRHBP、DAPK1、DNMT3A、DPP10、FAM19A2、FLJ45983、FOSL1、FOXB1、GREM1、HMHA1、HOXA9、IFFO1、INPP4B、ITGB2、ITGB4、ITPKB、KCNIP2、KLHDC7B、LAT、LHX6、LIMK1、LOC100128239、LOC100192379、LOC646278、MAP2K2、MAX.chr1.210426156-210426257、MAX.chr1.84326495-84326656、MAX.chr10.119312785-119312882、MAX.chr15.67326025-67326060、MAX.chr16.54316401-54316453、MAX.chr16.85482306-85482494、MAX.chr17.74994454-74994572、MAX.chr17.76339840-76339972、MAX.chr2.7571082-7571136、MAX.chr21.45577347-45577679、MAX.chr3.14852538-14852568、MAX.chr3.187676564-187676668、MAX.chr4.174430662-174430793、MAX.chr5.177411809-177411836、MAX.chr6.45631561-45631625、MAX.chr7.25892382-25892451, MAX.chr7.402563-402641, MAX.chr7.64349554-64349606, MAX.chr8.142046239-142046398, MAX.chr8.145900842-145901246, M AX.chr9.126101804-126101848, MAX.chr9.126978999-126979182, MAX.chr9.36458633-36458725, MAX.chr9.87905315-87905326, MBP, MFNG, MT1A, MT 1IP, NCOR2, NFATC1, NKX3-2, NRN1, OLIG1, PALLD, PAPLN, PDLIM2, PKN1, PRDM14, PRKG1, PRMT7, PTGER2, PTK2B, RAD52, RBM38, RHOF, RNF220, RTN4RL1, RXRA, SDCCAG8, SHROOM1, SKI, SLC12A8, SLC25A47, SPEG, SUCLG2, TBC1D10C, TMEM132E, VIPR2, WDR66, WNT6, ZDHHC18, ZNF382, and ZNF626 (Table 1).

[0104] Embodiments of the present disclosure also include novel variably methylated regions (DMRs), each of which can individually distinguish oropharyngeal cancer (e.g., oropharyngeal squamous cell carcinoma (HPV(+)OPSCC)) and / or cervical squamous cell carcinoma (HPV(+)CSCC) from control tissue samples (e.g., normal oropharyngeal tissue or normal cervical tissue). According to these embodiments, the novel DMR(s) are derived from a gene selected from ABCB1, ARHGAP12, ASCL1, C1orf114, EMX1, GRIN2D, LOC645323, MAX.chr6.58147682-58147771, MAX.chr9.36739811-36739868, NEUROG3, NID2, TBX15, TMEM200C, TSPYL5, TTYH1, VWC2, ZNF610, ZNF69, ZNF773, and ZNF781 (Table 2).

[0105] Each of the embodiments of the present disclosure is directed to a method for treating oropharyngeal cancer (e.g., HPV +Oropharyngeal squamous cell carcinoma (HPV +The present invention also includes novel variably methylated regions (DMRs) that can distinguish OPSCC) from control or benign tissue (e.g., tonsil tissue controls). According to these embodiments, the novel DMR(s) include ALX4, ATP10A, C1orf114, C1QL3, CA8, CACNA1A, CACNG8, CALCA, CCNA1, CLIC6, CLSTN2, CR1, CTNND2, DAB1, DGKG, DOK1, DOK6, DPP4, DUXA, ELMO1, EMBP1, EPDR1, FGF12, FLJ43390, FMN2, FOXB2, FOXD4, FREM3, GALR1, GDF6, GFRA1, GRIK3, HOXB3, HOXB4, HPSE2, LDLRAD2, LHX2, LOC100131366, LOC345643, LOC386758, LOC645323, LOC648809, LOC728392, MAML3, MAPRE2, MAX.chr1.226288154-2 26288189, MAX.chr1.2375078-2375126, MAX.chr1.241587339-241587784, MAX.chr1.50798781-50799423, MAX.chr10.22765150-227654 77, MAX.chr10.23462342-23462436, MAX.chr11.14926602-14927044, MAX.chr11.58903531-58903592, MAX.chr13.28527984-28528214 , MAX.chr13.29106641-29107037, MAX.chr14.100784488-100784782, MAX.chr16.3221176-3221223, MAX.chr16.3222040-3222098, MAX. chr16.71460171-71460282, MAX.chr19.11805263-11805639, MAX.chr19.16394457-16394646, MAX.chr19.21657626-21657769, MAX.chr 19.22034646-22034887, MAX.chr19.23299989-23300156, MAX.chr19.30713427-30713588, MAX.chr19.30716926-30717074, MAX.chr19.30718373-30719719, MAX.chr2.118981724-118982174, MAX.chr2.127783107-127783403, MAX.chr2.17309971 2-173099791, MAX.chr2.66808635-66808731, MAX.chr22.50064113-50064259, MAX.chr3.137489884-1374900 61, MAX.chr5.138923141-138923219, MAX.chr5.42995180-42995535, MAX.chr6.38683091-38683226, MAX.chr 7.121952014-121952084, MAX.chr7.155166980-155167310, MAX.chr8.99986792-99986864, MAX.chr9.796270 78-79627116, MAX.chr9.79638034-79638077, MAX.chr9.98789824-98789847, MDFI, MECOM, MED12L, MIR129-2, MIR196A1, NELL1, NPY, ONECUT2, OPCML, PARP15, PDGFD, PEX5L, PRR15, SEMA6A, SFMBT2, SGIP1, SIM2, SLC35F3, SL Originating from genes selected from CO4C1, SORCS3, ST6GALNAC5, ST8SIA5, SV2C, TACC2, TFAP2E, TLX2, TLX3, TRH, TRIM58, VAV3, VSTM2B, WDR17, ZNF254, ZNF43, ZNF486, ZNF491, ZNF518B, ZNF542, ZNF625, ZNF665, ZNF671, ZNF763, and ZNF844 (Table 6).

[0106] Each of the embodiments of the present disclosure is directed to a method for treating oropharyngeal cancer (e.g., HPV + Oropharyngeal squamous cell carcinoma (HPV +The present invention also includes novel variably methylated regions (DMRs) that can distinguish OPSCC) from control or benign tissue (e.g., normal buffy coat control). According to these embodiments, the novel DMR(s) include AGRN, ANKRD35, ARHGAP27, ARHGAP30, BCL2L11, BIN2, C10orf114, C4orf31, C6orf132, C6orf186, CCDC88B, CRHBP, DAPK1, DNMT3A, DPP10, ELMO1, EPDR1, FAM19A2, FLJ45983, FOSL1, FOXB1, GREM1, HMHA1, HOXA9, IFFO1, INPP4B, ITGB2, ITGB4, ITPKB, KC NIP2, KLHDC7B, LAT, LHX6, LIMK1, LOC100128239, LOC100192379, LOC646278, MAP2K2, MAX.chr1.210426156-210426257, MAX.chr1.843264 95-84326656, MAX.chr10.119312785-119312882, MAX.chr15.67326025-67326060, MAX.chr16.54316401-54316453, MAX.chr16.85482306 -85482494, MAX.chr17.74994454-74994572, MAX.chr17.76339840-76339972, MAX.chr2.7571082-7571136, MAX.chr21.45577347-45577 679, MAX.chr3.14852538-14852568, MAX.chr3.187676564-187676668, MAX.chr4.174430662-174430793, MAX.chr5.177411809-17741183 6, MAX.chr6.45631561-45631625, MAX.chr7.25892382-25892451, MAX.chr7.402563-402641, MAX.chr7.64349554-64349606, MAX.chr8. 142046239-142046398, MAX.chr8.145900842-145901246, MAX.chr9.126101804-126101848, MAX.chr9.126978999-126979182, MAX.chr9.36458633-36458725, MAX.chr9.87905315-87905326, MBP, MFNG, MT1A, MT1IP, NCOR2, NFATC1, NKX3-2, NRN1, OLIG1, PALLD, PAPLN, PDLIM2, PKN1, PRDM14, PRKG1, PRMT7, PTGER2, PTK2B, RAD52, RBM38, RHOF, RNF220, RTN4RL1, RXRA, SDCCAG8, SHROOM1, SKI, SLC12A8, SLC25A47, SPEG, SUCLG2, TBC1D10C, TMEM132E, VIPR2, WDR66, WNT6, ZDHHC18, ZNF382, and ZNF626 (Table 7).

[0107] Each of the embodiments of the present disclosure is directed to a method for treating oropharyngeal cancer (e.g., HPV + Oropharyngeal squamous cell carcinoma (HPV +The present invention also includes novel variably methylated regions (DMRs) that can distinguish OPSCC from control or benign tissue (e.g., normal tissue controls). According to these embodiments, the novel DMR(s) are selected from the group consisting of ALX4, C1orf114, CA8, CCNA1, CLSTN2, CR1, DAB1, DOK1, EMBP1, EPDR1, FLJ43390, FMN2, GDF6, GFRA1, HOXB3, LDLRAD2, LOC648809, MAPRE2, MAX.chr1.241587339-241587784, MAX.chr1.50798781-50799423, MAX.chr13.28527984-28528214, MAX.chr16.3221176-3221223, MAX.chr19.11805263-11805639, MAX.chr19.220346 46-22034887, MAX.chr19.30718373-30719719, MAX.chr2.173099712-173099791, MAX.chr2.66808635-66808731, MAX.chr6.38683091-38683226, MAX.chr9.79638034-79638077, MECOM, ONECUT2, PARP15, SGIP1, SIM2, SORCS3, ST6GALNAC5, ST8SIA5, TFAP2E, TLX2, TLX3, VSTM2B, WDR17, ZNF254, ZNF43, ZNF491, ZNF763, and ZNF844 (Table 8).

[0108] Each of the embodiments of the present disclosure is directed to a method for treating oropharyngeal cancer (e.g., HPV + Oropharyngeal squamous cell carcinoma (HPV +The present invention also includes novel variably methylated regions (DMRs) that can distinguish OPSCC) from control or benign tissue (e.g., normal buffy coat control). According to these embodiments, the novel DMR(s) are derived from genes selected from FAM19A2, IFFO1, ITGB4, LOC100192379, MAX.chr1.84326495-84326656, MAX.chr16.85482306-85482494, MAX.chr6.45631561-45631625, MAX.chr7.25892382-25892451, MT1IP, NCOR2, OLIG1, RAD52, SHROOM1, SLC12A8, and TBC1D10C (Table 8).

[0109] Each of the embodiments of the present disclosure is directed to a method for treating oropharyngeal cancer (e.g., HPV + Oropharyngeal squamous cell carcinoma (HPV + The present invention also includes novel variably methylated regions (DMRs) that can distinguish OPSCC) from control or benign tissue (e.g., normal tissue or normal buffy coat control). According to these embodiments, the novel DMR(s) are derived from genes selected from MAX.chr19.30718373-30719719, ITGB4, MAX.chr7.25892382-25892451, RAD52, SHROOM1, SLC12A8, and TBC1D10C (Table 8).

[0110] Each of the embodiments of the present disclosure is directed to a method for treating oropharyngeal cancer (e.g., HPV + Oropharyngeal squamous cell carcinoma (HPV +The present invention also includes novel variably methylated regions (DMRs) that can distinguish OPSCC) from control or benign tissue (e.g., normal tissue or normal buffy coat control). According to these embodiments, the novel DMR(s) include ALX4, C1orf114, CA8, CCNA1, CLSTN2, CR1, DAB1, DOK1, EMBP1, EPDR1, FAM19A2, FLJ43390, FMN2, GDF6, GFRA1, HOXB3, IFFO1, ITGB4, LDLRAD2, LOC100192379, LOC648809, MAPRE2, MAX.chr1.241587339-241 587784, MAX.chr1.50798781-50799423, MAX.chr1.84326495-84326656, MAX.chr13.28527984-28528214, MAX.chr1 6.3221176-3221223, MAX.chr16.85482306-85482494, MAX.chr19.11805263-11805639, MAX.chr19.22034646-2203 4887, MAX.chr19.30718373-30719719, MAX.chr2.173099712-173099791, MAX.chr2.66808635-66808731, MAX.chr6 .38683091-38683226, MAX.chr6.45631561-45631625, MAX.chr7.25892382-25892451, MAX.chr9.79638034-796380 77, MECOM, MT1IP, NCOR2, OLIG1, ONECUT2, PARP15, RAD52, SGIP1, SHROOM1, SIM2, SLC12A8, SORCS3, ST6GALNAC5, ST8SIA5, TBC1D10C, TFAP2E, TLX2, TLX3, VSTM2B, WDR17, ZNF254, ZNF43, ZNF491, ZNF763, and ZNF844 (Table 9).

[0111] Each of the embodiments of the present disclosure is directed to a method for treating oropharyngeal cancer (e.g., HPV + Oropharyngeal squamous cell carcinoma (HPV +The present invention also includes novel variably methylated regions (DMRs) that can distinguish OPSCC from control or benign tissue (e.g., normal tissue or normal buffy coat control). According to these embodiments, the novel DMR(s) are derived from a gene selected from CA8, EMBP1, HOXB3, IFFO1, ITGB4, LOC100192379, LOC648809, MAX.chr1.84326495-84326656, MAX.chr16.3221176-3221223, MAX.chr16.85482306-85482494, MAX.chr19.30718373-30719719, MAX.chr9.79638034-79638077, MT1IP, ONECUT2, SHROOM1, SIM2, SLC12A8, TLX3, and ZNF763 (Table 9).

[0112] Each of the embodiments of the present disclosure is directed to a method for treating oropharyngeal cancer (e.g., HPV + Oropharyngeal squamous cell carcinoma (HPV +The present invention also includes novel variably methylated regions (DMRs) that can distinguish OPSCC) from control or benign tissue (e.g., normal tissue or normal buffy coat control). According to these embodiments, the novel DMR(s) include C1orf114, CA8, CCNA1, EMBP1, EPDR1, FAM19A2, FMN2, HOXB3, IFFO1, ITGB4, LDLRAD2, LOC100192379, LOC648809, MAPR2, MAX.chr1.50798781-50799423, MAX.chr1.84326495-84326656, MAX.chr16.3221176-3221223, MAX.chr19.11805263-11805639, MAX.chr17.2221176-2221223, MAX.chr19.11805263-11805639, MAX.chr18.2221176-2221223, MAX.chr19.11805263-11805639 ... r2.66808635-66808731, MAX.chr6.38683091-38683226, MAX.chr6.45631561-45631625, MAX.chr9.79638034-79638077, MECOM, MT1IP, ONECUT2, PARP15, SHROOM1, SIM2, SLC12A8, SORCS3, ST6GALNAC5, ST8SIA5, TBC1D10C, TLX3, ZNF254, ZNF491, ZNF763, and ZNF844 (Table 10).

[0113] Each of the embodiments of the present disclosure is directed to a method for treating oropharyngeal cancer (e.g., HPV + Oropharyngeal squamous cell carcinoma (HPV + The present invention also includes novel variably methylated regions (DMRs) that can distinguish OPSCC) from control or benign tissue (e.g., normal tissue or normal buffy coat control). According to these embodiments, the novel DMR(s) are derived from genes selected from CA8, EMBP1, HOXB3, IFFO1, ITGB4, LOC100192379, LOC648809, MAX.chr1.84326495-84326656, MAX.chr16.3221176-3221223, MAX.chr9.79638034-79638077, MT1IP, ONECUT2, SHROOM1, SIM2, SLC12A8, TLX3, and ZNF763 (Table 10).

[0114] The embodiments of the present disclosure each individually detect oropharyngeal cancer (e.g., HPV) in a saliva sample from a subject. + Oropharyngeal squamous cell carcinoma (HPV + The present invention also includes novel variably methylated regions (DMRs) that can distinguish OPSCC from control or benign tissue (e.g., saliva control samples). According to these embodiments, the novel DMR(s) are derived from genes selected from TLX3, MAX.chr16.3221176-3221223, TBC1D10C, and SHROOM1 (Table 11).

[0115] As described in the preceding Examples, experiments were conducted to identify DMRs (also referred to herein as methylated DNA markers (MDMs)) that can distinguish types and subtypes of oropharyngeal cancer from controls (e.g., healthy samples, benign samples, etc.). These experiments included validation studies of the utility and performance of a panel of methylated DNA markers for detecting one or more types or subtypes of oropharyngeal cancer by testing an independent set of case / control samples with a refined panel of markers. Such experiments included the identification of DMRs (also referred to herein as methylated DNA markers (MDMs)) that can distinguish types and subtypes of oropharyngeal cancer from controls (e.g., healthy samples, benign samples, etc.). + Oropharyngeal squamous cell carcinoma (HPV + The present invention has led to the identification of MDMs useful for simultaneously detecting the presence of one or more types of OPSCC. The control sample can be a sample from a subject without cancer, a sample from a subject without oropharyngeal cancer, a sample from a subject with a type of cancer that is not oropharyngeal cancer, or a sample from a subject with HPV(+) cancer that is not oropharyngeal cancer. In some embodiments, the control sample is derived from a tissue sample, a blood sample, a plasma sample, a serum sample, a whole blood sample, a buffy coat sample, a secretion sample, an organ secretion sample, a cerebrospinal fluid (CSF) sample, a saliva sample, a urine sample, and a stool sample. In some embodiments, the control sample is derived from an oropharyngeal tissue sample, including one or more of soft palate cells or tissue, throat cells or tissue, tongue cells or tissue, and tonsil cells or tissue. In some embodiments, the tissue sample is an HPV(+) tissue sample.

[0116] In some embodiments, the present disclosure provides compositions and methods for identifying, determining, and / or classifying one or more types of oropharyngeal cancer from a biological sample (e.g., a tissue sample, a blood sample, a plasma sample, a serum sample, a whole blood sample, a buffy coat sample, a secretion sample, an organ secretion sample, a cerebrospinal fluid (CSF) sample, a saliva sample, a urine sample, and / or a stool sample). The methods generally include determining a methylation profile of at least one methylation marker in a biological sample isolated from a subject. In some embodiments, a change in the methylation status or profile of the marker indicates the presence of a particular type, class, or site of oropharyngeal cancer. In general, such methods are useful for detecting the presence or absence of a particular type or subtype of oropharyngeal cancer. In some embodiments, types and subtypes of oropharyngeal cancer include HPV markers, HPV markers, and / or HPV markers. + Oropharyngeal squamous cell carcinoma (HPV + OPSCC) are included, but are not limited to:

[0117] In some embodiments, methods are provided that include contacting nucleic acid (e.g., genomic DNA) in a biological sample obtained from a subject with at least one reagent or set of reagents that distinguish between methylated and unmethylated nucleotides (e.g., CpG dinucleotides) within at least one methylation marker, and detecting the presence or absence of one or more types or subtypes of oropharyngeal cancer (e.g., providing a sensitivity of 80% or greater and a specificity of 80% or greater).

[0118] In some embodiments, a method is provided that includes measuring the methylation level of one or more genes or methylated DNA markers in a biological sample from a human individual by treating genomic DNA in the biological sample with a reagent that modifies the DNA in a methylation-specific manner, amplifying the treated genomic DNA using a set of primers for the selected one or more genes or methylation markers, and determining the methylation level of the one or more genes or methylation markers.

[0119] In some embodiments, a method is provided that includes measuring the amount of one or more methylated DNA markers or genes in DNA from a biological sample, measuring the amount of at least one reference marker in the DNA, and calculating the amount of the at least one methylation marker gene measured in the DNA as a percentage of the amount of the reference marker gene measured in the DNA, wherein the value indicates the amount of the at least one methylation marker DNA measured in the biological sample.

[0120] In some embodiments, a method is provided that includes measuring the methylation level of CpG sites for one or more genes in a biological sample of a human individual by treating genomic DNA in the biological sample with bisulfite, a reagent capable of modifying DNA in a methylation-specific manner, amplifying the modified genomic DNA using a set of primers for the selected one or more genes, and determining the methylation level of CpG sites for the selected one or more genes.

[0121] In some embodiments, the disclosure provides a method of characterizing a biological sample, comprising measuring one or both of the methylation levels of CpG sites for one or more genes in a biological sample of a human individual by treating genomic DNA in the biological sample with bisulfite, amplifying the bisulfite-treated genomic DNA using a set of primers for the selected one or more genes, and determining the methylation levels of the CpG sites. In some embodiments, the method comprises comparing one or both of the methylation levels of the methylation markers with the methylation levels of the corresponding set of genes in a control sample that does not have the particular type of cancer, and / or determining that the subject has one or more types or subtypes of oropharyngeal cancer if one or both of the methylation levels measured for the one or more genes are higher than the methylation levels measured in the respective control sample.

[0122] In some embodiments, the present disclosure provides methods for measuring the methylation level of one or more genes or markers in a biological sample by treating genomic DNA in the biological sample with bisulfite, amplifying the bisulfite-treated genomic DNA using a set of primers for one or more selected genes, and determining the methylation level of one or more genes or markers.

[0123] In some embodiments, the present disclosure provides a method for screening for one or more types or subtypes of oropharyngeal cancer in a sample obtained from a subject. According to these embodiments, the method includes assaying the methylation status or profile of one or more methylated DNA markers, and identifying the subject as having one or more types or subtypes of oropharyngeal cancer if the methylation status or profile of the markers differs from the methylation status or profile of the markers assayed in a subject that does not have one or more types of cancer.

[0124] In some embodiments, the disclosure provides a method comprising measuring the methylation level of one or more genes or markers in a biological sample of a human individual by treating genomic DNA in the biological sample with a reagent that modifies the DNA in a methylation-specific manner, amplifying the treated genomic DNA using a set of primers for the selected one or more genes or markers, and determining the methylation level of the one or more genes or markers.

[0125] In some embodiments, the disclosure provides methods of characterizing a biological sample comprising: measuring the amount of at least one methylated DNA marker in DNA extracted from the biological sample; treating genomic DNA in the biological sample with bisulfite; amplifying the bisulfite-treated genomic DNA using primers specific for a CpG site for each marker, wherein the primers specific for each marker are capable of binding to an amplicon bounded by a primer sequence for the markers listed in Tables 3 and 12, and wherein the amplicon bounded by the primer sequence for the markers listed in Tables 3 and 12 is at least a portion of a gene region for a methylated marker listed in Tables 1, 2, 6, or 7; and determining the methylation level of the CpG site for one or more genes.

[0126] In some embodiments, the disclosure provides a method comprising: extracting genomic DNA from a biological sample of a human individual having or suspected of having one or more types or subtypes of oropharyngeal cancer, measuring a methylation level of one or more methylated DNA markers in the DNA extracted from the biological sample; treating the extracted genomic DNA with bisulfite; amplifying the bisulfite-treated genomic DNA with primers specific for one or more markers, wherein the primers specific for the one or more markers are capable of binding to at least a portion of the bisulfite-treated genomic DNA for a chromosomal region of a marker listed in Tables 1, 2, 6, or 7; and measuring a methylation level of the one or more methylation markers.

[0127] In some embodiments, the disclosure provides a method comprising: extracting genomic DNA from a biological sample of a human individual having or suspected of having one or more types or subtypes of oropharyngeal cancer, measuring a methylation level of one or more methylated DNA markers in the DNA extracted from the biological sample; treating the extracted genomic DNA with bisulfite; amplifying the bisulfite-treated genomic DNA with primers specific for one or more markers, wherein the primers specific for the one or more markers are capable of binding to at least a portion of the bisulfite-treated genomic DNA for chromosomal regions of the markers listed in Table 1; and measuring a methylation level of the one or more methylation markers.

[0128] In some embodiments, the disclosure provides a method comprising: extracting genomic DNA from a biological sample of a human individual having or suspected of having one or more types or subtypes of oropharyngeal cancer, measuring a methylation level of one or more methylated DNA markers in the DNA extracted from the biological sample; treating the extracted genomic DNA with bisulfite; amplifying the bisulfite-treated genomic DNA with primers specific for one or more markers, wherein the primers specific for the one or more markers are capable of binding to at least a portion of the bisulfite-treated genomic DNA for chromosomal regions of the markers listed in Table 2; and measuring a methylation level of the one or more methylation markers.

[0129] In some embodiments, the disclosure provides a method comprising: extracting genomic DNA from a biological sample of a human individual having or suspected of having one or more types or subtypes of oropharyngeal cancer, measuring a methylation level of one or more methylated DNA markers in the DNA extracted from the biological sample; treating the extracted genomic DNA with bisulfite; amplifying the bisulfite-treated genomic DNA with primers specific for one or more markers, wherein the primers specific for the one or more markers are capable of binding to at least a portion of the bisulfite-treated genomic DNA for chromosomal regions of the markers listed in Table 6; and measuring a methylation level of the one or more methylation markers.

[0130] In some embodiments, the disclosure provides a method comprising: extracting genomic DNA from a biological sample of a human individual having or suspected of having one or more types or subtypes of oropharyngeal cancer, measuring a methylation level of one or more methylated DNA markers in the DNA extracted from the biological sample; treating the extracted genomic DNA with bisulfite; amplifying the bisulfite-treated genomic DNA with primers specific for one or more markers, wherein the primers specific for the one or more markers are capable of binding to at least a portion of the bisulfite-treated genomic DNA for chromosomal regions of the markers listed in Table 7; and measuring a methylation level of the one or more methylation markers.

[0131] In some embodiments, the disclosure provides a method comprising extracting genomic DNA from a biological sample of a human individual having or suspected of having cancer, treating the extracted genomic DNA with bisulfite, amplifying the bisulfite-treated genomic DNA using separate primers specific for CpG sites for one or more methylated DNA markers, and measuring the methylation level of the CpG sites for each of the one or more markers.

[0132] In some embodiments, the present disclosure provides a method for preparing a DNA fraction from a biological sample of a human individual, which is useful for analyzing one or more loci involved in one or more chromosomal abnormalities.According to these embodiments, the method includes: extracting genomic DNA from a biological sample of a human individual; treating the extracted genomic DNA with a reagent that modifies DNA in a methylation-specific manner to generate a fraction of extracted genomic DNA; amplifying the bisulfite-treated genomic DNA using separate primers specific for one or more methylated DNA markers; and analyzing one or more loci in the generated fraction of extracted genomic DNA by measuring the methylation level of CpG sites for each of the one or more markers.

[0133] In some embodiments, the present disclosure provides a method for preparing a DNA fraction from a biological sample of a human individual, which is useful for analyzing one or more DNA fragments involved in one or more chromosomal abnormalities.According to these embodiments, the method includes: extracting genomic DNA from a biological sample of a human individual; treating the extracted genomic DNA with a reagent that modifies DNA in a methylation-specific manner to generate a fraction of extracted genomic DNA; amplifying the bisulfite-treated genomic DNA using separate primers specific for one or more methylated DNA markers; and analyzing one or more DNA fragments in the generated fraction of extracted genomic DNA by measuring the methylation level of CpG sites for each of the one or more markers.

[0134] As will be understood by those skilled in the art based on the present disclosure, the various methods described herein are not limited to the use of any one specific methylated DNA marker, methylation marker gene, methylation gene, and / or DMR. That is, one or more of the methylated DNA markers, methylation marker genes, methylation genes, and / or DMRs of the present disclosure (including any combination thereof) can be used to distinguish and / or identify one or more types or subtypes of oropharyngeal cancer. In addition, the methylated DNA markers, methylation marker genes, methylation genes, and / or DMRs of the present disclosure can include any region or subregion (e.g., genes on chromosomes, single nucleotides, CpG islands, etc.) of the markers listed in Tables 1, 2, 6, and 7.

[0135] In some embodiments, the DMRs are ABCB1, ARHGAP12, ASCL1, C1orf114, EMX1, GRIN2D, LOC645323, MAX.chr6.58147682-58147771, MAX.chr9.36739811-36739868, NEUROG3, NID2, TBX15, TMEM200C, TSPYL5, TTYH1, VWC2, ZNF610, ZNF69, ZNF773, ZNF781, ALX4, ATP10A, C1QL3, CA8, CACNA1A, CACNG8, CALCA, CCNA1, CACNG1, CACNG2, CACNG3, CACNG4, CACNG5, CACNG6, CACNG7, CACNG8, CACNG9, CACNG1, CACNG1, CACNG1, CACNG1, CACNG1, CACNG1, CACNG1, CACNG2 ... LIC6, CLSTN2, CR1, CTNND2, DAB1, DGKG, DOK1, DOK6, DPP4, DUXA, ELMO1, EMBP1, EPDR1, FGF12, FLJ43390, FMN2, FOXB2, FOXD4, FREM3, GALR1, GDF6, GFR A1, GRIK3, HOXB3, HOXB4, HPSE2, LDLRAD2, LHX2, LOC100131366, LOC345643, LOC386758, LOC648809, LOC728392, MAML3, MAPRE2, MAX.chr1.22628815 4-226288189, MAX.chr1.2375078-2375126, MAX.chr1.241587339-241587784, MAX.chr1.50798781-50799423, MAX.chr10.22765150-22765477, MA X.chr10.23462342-23462436, MAX.chr11.14926602-14927044, MAX.chr11.58903531-58903592, MAX.chr13.28527984-28528214, MAX.chr13.291 06641-29107037, MAX.chr14.100784488-100784782, MAX.chr16.3221176-3221223, MAX.chr16.3222040-3222098, MAX.chr16.71460171-7146028 2, MAX.chr19.11805263-11805639, MAX.chr19.16394457-16394646, MAX.chr19.21657626-21657769, MAX.chr19.22034646-22034887, MAX.chr19.23299989-23300156、MAX.chr19.30713427-30713588、MAX.chr19.30716926-30717074、MAX.chr19.30718373-30719719、MAX.chr2.118981724-118982174、MAX.chr2.127783107-127783403、MAX.chr2.173099712-173099791、MAX.chr2.66808635-66808731、MAX.chr22.50064113-50064259、MAX.chr3.137489884-137490061、MAX.chr5.138923141-138923219、MAX.chr5.42995180-42995535、MAX.chr6.38683091-38683226、MAX.chr7.121952014-121952084、MAX.chr7.155166980-155167310、MAX.chr8.99986792-99986864、MAX.chr9.79627078-79627116、MAX.chr9.79638034-79638077、MAX.chr9.98789824-98789847、MDFI、MECOM、MED12L、MIR129-2、MIR196A1、NELL1、NPY、ONECUT2、OPCML、PARP15、PDGFD、PEX5L、PRR15、SEMA6A、SFMBT2、SGIP1、SIM2、SLC35F3、SLCO4C1、SORCS3、ST6GALNAC5、ST8SIA5、SV2C、TACC2、TFAP2E、TLX2、TLX3、TRH、TRIM58、VAV3、VSTM2B、WDR17、ZNF254、ZNF43、ZNF486、ZNF491、ZNF518B、ZNF542、ZNF625、ZNF665、ZNF671、ZNF763、ZNF844、AGRN、ANKRD35、ARHGAP27、ARHGAP30、BCL2L11、BIN2、C10orf114、C4orf31、C6orf132、C6orf186、CCDC88B、CRHBP、DAPK1、DNMT3A、DPP10、FAM19A2、FLJ45983、FOSL1、FOXB1、GREM1、HMHA1、HOXA9、IFFO1、INPP4B、ITGB2、ITGB4、ITPKB、KCNIP2、KLHDC7B、LAT、LHX6、LIMK1、LOC100128239、LOC100192379、LOC646278、MAP2K2、MAX.chr1.210426156-210426257、MAX.chr1.84326495-84326656、MAX.chr10.119312785-119312882、MAX.chr15.67326025-67326060、MAX.chr16.54316401-54316453、MAX.chr16.85482306-85482494、MAX.chr17.74994454-74994572、MAX.chr17.76339840-76339972、MAX.chr2.7571082-7571136、MAX.chr21.45577347-45577679、MAX.chr3.14852538-14852568、MAX.chr3.187676564-187676668、MAX.chr4.174430662-174430793、MAX.chr5.177411809-177411836、MAX.chr6.45631561-45631625、MAX.chr7.25892382-25892451, MAX.chr7.402563-402641, MAX.chr7.64349554-64349606, MA X.chr8.142046239-142046398, MAX.chr8.145900842-145901246, MAX.chr9.12610 1804-126101848, MAX.chr9.126978999-126979182, MAX.chr9.36458633-36458725 , MAX.chr9.87905315-87905326, MBP, MFNG, MT1A, MT1IP, NCOR2, NFATC1, NKX3-2, NRN 1, OLIG1, PALLD, PAPLN, PDLIM2, PKN1, PRDM14, PRKG1, PRMT7, PTGER2, PTK2B, RAD52, RBM38, RHOF, RNF220, RTN4RL1, RXRA, SDCCAG8, SHROOM1, SKI, SLC12A8, SLC25A47, SPEG, SUCLG2, TBC1D10C, TMEM132E, VIPR2, WDR66, WNT6, ZDHHC18, ZNF382, and ZNF626 (Table 1), and the subject has or is suspected of having oropharyngeal cancer (e.g., oropharyngeal squamous cell carcinoma (HPV(+)OPSCC)). In some embodiments, determining the methylation profile of the DMR includes comparing the methylation profile to a corresponding region from a control DNA sample (e.g., a control oropharyngeal tissue or a control buffy coat sample).

[0136] In some embodiments, the DMR is from a gene selected from ABCB1, ARHGAP12, ASCL1, C1orf114, EMX1, GRIN2D, LOC645323, MAX.chr6.58147682-58147771, MAX.chr9.36739811-36739868, NEUROG3, NID2, TBX15, TMEM200C, TSPYL5, TTYH1, VWC2, ZNF610, ZNF69, ZNF773, and ZNF781 (Table 2), and the subject has or is suspected of having oropharyngeal cancer (e.g., oropharyngeal squamous cell carcinoma (HPV(+)OPSCC). In some embodiments, determining the methylation profile of the DMR comprises comparing the methylation profile to a corresponding region from a control DNA sample (e.g., a control oropharyngeal tissue or a control buffy coat sample).

[0137] In some embodiments, the DMR is ALX4, ATP10A, C1orf114, C1QL3, CA8, CACNA1A, CACNG8, CALCA, CCNA1, CLIC6, CLSTN2, CR1, CTNND2, DAB1, DGKG, DOK1, DOK6, DPP4, DUXA, ELMO1, EMBP1, EPDR1, FGF12, FLJ43390, FMN2, FOXB2, FOXD4, FREM3, GALR1, GDF6, GFRA1, GRIK3, HOXB3, HOXB4, HPSE2, LDLRAD2, LHX2, LOC100131 366, LOC345643, LOC386758, LOC645323, LOC648809, LOC728392, MAML3, MAPRE2, MAX.chr1.226288154-226288189, MAX.chr1.2375078-2375126, MAX .chr1.241587339-241587784, MAX.chr1.50798781-50799423, MAX.chr10.22765150-22765477, MAX.chr10.23462342-23462436, MAX.chr11.149266 02-14927044, MAX.chr11.58903531-58903592, MAX.chr13.28527984-28528214, MAX.chr13.29106641-29107037, MAX.chr14.100784488-10078478 2, MAX.chr16.3221176-3221223, MAX.chr16.3222040-3222098, MAX.chr16.71460171-71460282, MAX.chr19.11805263-11805639, MAX.chr19.1639 4457-16394646, MAX.chr19.21657626-21657769, MAX.chr19.22034646-22034887, MAX.chr19.23299989-23300156, MAX.chr19.30713427-3071358 8, MAX.chr19.30716926-30717074, MAX.chr19.30718373-30719719, MAX.chr2.118981724-118982174, MAX.chr2.127783107-127783403, MAX.chr2.173099712-173099791, MAX.chr2.66808635-66808731, MAX.chr22.50064113-50064259, MAX.chr3.137489884-1374900 61, MAX.chr5.138923141-138923219, MAX.chr5.42995180-42995535, MAX.chr6.38683091-38683226, MAX.chr7.121952 014-121952084, MAX.chr7.155166980-155167310, MAX.chr8.99986792-99986864, MAX.chr9.79627078-79627116, MAX. chr9.79638034-79638077, MAX.chr9.98789824-98789847, MDFI, MECOM, MED12L, MIR129-2, MIR196A1, NELL1, NPY, ONECUT 2, OPCML, PARP15, PDGFD, PEX5L, PRR15, SEMA6A, SFMBT2, SGIP1, SIM2, SLC35F3, SLCO4C1, SORCS3, ST6GALNAC5, ST8SIA5, S V2C, TACC2, TFAP2E, TLX2, TLX3, TRH, TRIM58, VAV3, VSTM2B, WDR17, ZNF254, ZNF43, ZNF486, ZNF491, ZNF518B, ZNF542, ZNF6 25, ZNF665, ZNF671, ZNF763, and ZNF844 (Table 6), and the subject has or is suspected of having oropharyngeal cancer (e.g., oropharyngeal squamous cell carcinoma (HPV(+)OPSCC). In some embodiments, determining the methylation profile of the DMR includes comparing the methylation profile to a corresponding region from a control DNA sample (e.g., a control oropharyngeal tissue or a control buffy coat sample).

[0138] In some embodiments, the DMR is AGRN, ANKRD35, ARHGAP27, ARHGAP30, BCL2L11, BIN2, C10orf114, C4orf31, C6orf132, C6orf186, CCDC88B, CRHBP, DAPK1, DNMT3A, DPP10, ELMO1, EPDR1, FAM19A2, FLJ45983, FOSL1, FOXB1, GREM1, HMHA1, HOXA9, IFFO1, INPP4B, ITGB2, ITGB4, ITPKB, KCNIP2, KLHDC7B, LAT, LHX6, LIMK1, LOC100128239, LOC100192379, LOC646278, MAP2K2, MAX.chr1.210426156-210426257, MAX.chr1.84326495-84326656, MAX. chr10.119312785-119312882, MAX.chr15.67326025-67326060, MAX.chr16.54316401-54316453, MAX.chr16.85482306-85482494, MAX.chr1 7.74994454-74994572, MAX.chr17.76339840-76339972, MAX.chr2.7571082-7571136, MAX.chr21.45577347-45577679, MAX.chr3.1485253 8-14852568, MAX.chr3.187676564-187676668, MAX.chr4.174430662-174430793, MAX.chr5.177411809-177411836, MAX.chr6.45631561-45 631625, MAX.chr7.25892382-25892451, MAX.chr7.402563-402641, MAX.chr7.64349554-64349606, MAX.chr8.142046239-142046398, MAX.c hr8.145900842-145901246, MAX.chr9.126101804-126101848, MAX.chr9.126978999-126979182, MAX.chr9.36458633-36458725, MAX.chr9.87905315-87905326, MBP, MFNG, MT1A, MT1IP, NCOR2, NFATC1, NKX3-2, NRN1, OLIG1, PALLD, PAPLN, PDLIM2, PKN1, PRDM14, PRKG1, PRMT 7, PTGER2, PTK2B, RAD52, RBM38, RHOF, RNF220, RTN4RL1, RXRA, SDCCAG8, SHROOM1, SKI, SLC12A8, SLC25A47, SPEG, SUCLG2, TBC1D10C, The DMR is derived from a gene selected from TMEM132E, VIPR2, WDR66, WNT6, ZDHHC18, ZNF382, and ZNF626 (Table 7), and the subject has or is suspected of having oropharyngeal cancer (e.g., oropharyngeal squamous cell carcinoma (HPV(+)OPSCC). In some embodiments, determining the methylation profile of the DMR includes comparing the methylation profile to a corresponding region from a control DNA sample (e.g., a control oropharyngeal tissue or a control buffy coat sample).

[0139] In some embodiments, the DMRs are ALX4, C1orf114, CA8, CCNA1, CLSTN2, CR1, DAB1, DOK1, EMBP1, EPDR1, FLJ43390, FMN2, GDF6, GFRA1, HOXB3, LDLRAD2, LOC648809, MAPR2, MAX.chr1.241587339-241587784, MAX.chr1.50798781-5079 9423, MAX.chr13.28527984-28528214, MAX.chr16.3221176-3221223, MAX.chr19.11805263-11805639, MAX. chr19.22034646-22034887, MAX.chr19.30718373-30719719, MAX.chr2.173099712-173099791, MAX.chr2.6 6808635-66808731, MAX.chr6.38683091-38683226, MAX.chr9.79638034-79638077, MECOM, ONECUT2, PARP15, SGIP1, SIM2, SORCS3, ST6GALNAC5, ST8SIA5, TFAP2E, TLX2, TLX3, VSTM2B, WDR17, ZNF254, ZNF43, ZNF491, ZNF763, and ZNF844 (Table 8), and the subject has or is suspected of having oropharyngeal cancer (e.g., oropharyngeal squamous cell carcinoma (HPV(+)OPSCC). In some embodiments, determining the methylation profile of the DMR comprises comparing the methylation profile to a corresponding region from a control DNA sample (e.g., a control oropharyngeal tissue or a control buffy coat sample).

[0140] In some embodiments, the DMRs are FAM19A2, IFFO1, ITGB4, LOC100192379, MAX.chr1.84326495-84326656, MAX.chr16.85482306-85482494, MAX.chr6.45631561-45631625, MAX.chr7.25892382-25892451, MT1IP, NCOR2, OLIG1, RAD52, SHROOM 1, SLC12A8, and TBC1D10C (Table 8), and the subject has or is suspected of having oropharyngeal cancer (e.g., oropharyngeal squamous cell carcinoma (HPV(+)OPSCC). In some embodiments, determining the methylation profile of the DMR comprises comparing the methylation profile to a corresponding region from a control DNA sample (e.g., a control oropharyngeal tissue or a control buffy coat sample).

[0141] In some embodiments, the DMR is from a gene selected from MAX.chr19.30718373-30719719, ITGB4, MAX.chr7.25892382-25892451, RAD52, SHROOM1, SLC12A8, and TBC1D10C (Table 8), and the subject has or is suspected of having oropharyngeal cancer (e.g., oropharyngeal squamous cell carcinoma (HPV(+)OPSCC). In some embodiments, determining the methylation profile of the DMR comprises comparing the methylation profile to a corresponding region from a control DNA sample (e.g., a control oropharyngeal tissue or a control buffy coat sample).

[0142] In some embodiments, the DMRs are ALX4, C1orf114, CA8, CCNA1, CLSTN2, CR1, DAB1, DOK1, EMBP1, EPDR1, FAM19A2, FLJ43390, FMN2, GDF6, GFRA1, HOXB3, IFFO1, ITGB4, LDLRAD2, LOC100192379, LOC648809, MAPRE2, MAX.chr1.241587339-241587784, MAX.chr1.50798781-50799423, M AX.chr1.84326495-84326656, MAX.chr13.28527984-28528214, MAX.chr16.3221176-3221223, MAX.chr16.85482306-85482494, MAX.chr1 9.11805263-11805639, MAX.chr19.22034646-22034887, MAX.chr19.30718373-30719719, MAX.chr2.173099712-173099791, MAX.chr2.668 08635-66808731, MAX.chr6.38683091-38683226, MAX.chr6.45631561-45631625, MAX.chr7.25892382-25892451, MAX.chr9.79638034-79 638077, MECOM, MT1IP, NCOR2, OLIG1, ONECUT2, PARP15, RAD52, SGIP1, SHROOM1, SIM2, SLC12A8, SORCS3, ST6GALNAC5, ST8SIA5, TBC1D10C, TF The DMR is derived from a gene selected from AP2E, TLX2, TLX3, VSTM2B, WDR17, ZNF254, ZNF43, ZNF491, ZNF763, and ZNF844 (Table 9), and the subject has or is suspected of having oropharyngeal cancer (e.g., oropharyngeal squamous cell carcinoma (HPV(+)OPSCC). In some embodiments, determining the methylation profile of the DMR comprises comparing the methylation profile to a corresponding region from a control DNA sample (e.g., a control oropharyngeal tissue or a control buffy coat sample).

[0143] In some embodiments, the DMRs are CA8, EMBP1, HOXB3, IFFO1, ITGB4, LOC100192379, LOC648809, MAX.chr1.84326495-84326656, MAX.chr16.3221176-3221223, MAX.chr16.85482306-85482494, MAX.chr19.30718373-30719719, MAX.chr9.79638034-79638077, MT1IP, O The DMR is derived from a gene selected from NECUT2, SHROOM1, SIM2, SLC12A8, TLX3, and ZNF763 (Table 9), and the subject has or is suspected of having oropharyngeal cancer (e.g., oropharyngeal squamous cell carcinoma (HPV(+)OPSCC). In some embodiments, determining the methylation profile of the DMR comprises comparing the methylation profile to a corresponding region from a control DNA sample (e.g., a control oropharyngeal tissue or a control buffy coat sample).

[0144] In some embodiments, the DMRs are Clorf114, CA8, CCNA1, EMBP1, EPDR1, FAM19A2, FMN2, HOXB3, IFFO1, ITGB4, LDLRAD2, LOC100192379, LOC648809, MAPR2, MAX.chr1.50798781-50799423, MAX.chr1.84 326495-84326656, MAX.chr16.3221176-3221223, MAX.chr19.11805263-11805639, MAX.chr 2.66808635-66808731, MAX.chr6.38683091-38683226, MAX.chr6.45631561-45631625, MAX. chr9.79638034-79638077, MECOM, MT1IP, ONECUT2, PARP15, SHROOM1, SIM2, SLC12A8, SORCS3, ST6GALNAC5, ST8SIA5, TBC1D10C, TLX3, ZNF254, ZNF491, ZNF763, and ZNF844 (Table 10), and the subject has or is suspected of having oropharyngeal cancer (e.g., oropharyngeal squamous cell carcinoma (HPV(+)OPSCC). In some embodiments, determining the methylation profile of the DMR comprises comparing the methylation profile to a corresponding region from a control DNA sample (e.g., a control oropharyngeal tissue or a control buffy coat sample).

[0145] In some embodiments, the DMR is from a gene selected from CA8, EMBP1, HOXB3, IFFO1, ITGB4, LOC100192379, LOC648809, MAX.chr1.84326495-84326656, MAX.chr16.3221176-3221223, MAX.chr9.79638034-79638077, MT1IP, ONECUT2, SHROOM1, SIM2, SLC12A8, TLX3, and ZNF763 (Table 10), and the subject has or is suspected of having oropharyngeal cancer (e.g., oropharyngeal squamous cell carcinoma (HPV(+)OPSCC). In some embodiments, determining the methylation profile of the DMR comprises comparing the methylation profile to a corresponding region from a control DNA sample (e.g., a control oropharyngeal tissue or a control buffy coat sample).

[0146] In some embodiments, the DMR is from a gene selected from TLX3, MAX.chr16.3221176-3221223, TBC1D10C, and SHROOM1 (Table 11), and the subject has or is suspected of having oropharyngeal cancer (e.g., oropharyngeal squamous cell carcinoma (HPV(+) OPSCC). In some embodiments, determining a methylation profile of the DMR comprises comparing the methylation profile to a corresponding region from a control DNA sample (e.g., a saliva sample).

[0147] In some embodiments, the DMR(s) that can distinguish oropharyngeal cancer from a control sample are associated with an area under the ROC curve (AUC) of 0.5 or greater, and the ROC curve distinguishes between subjects with or suspected of having OPSCC and control DNA samples. In some embodiments, the DMR(s) that can distinguish oropharyngeal cancer from a control sample are associated with an area under the ROC curve (AUC) of 0.6 or greater, and the ROC curve distinguishes between subjects with or suspected of having OPSCC and control DNA samples. In some embodiments, the DMR(s) that can distinguish oropharyngeal cancer from a control sample are associated with an area under the ROC curve (AUC) of 0.7 or greater, and the ROC curve distinguishes between subjects with or suspected of having OPSCC and control DNA samples. In some embodiments, the DMR(s) that can distinguish oropharyngeal cancer from a control sample are associated with an area under the ROC curve (AUC) of 0.8 or greater, where the ROC curve distinguishes between subjects having or suspected of having OPSCC and control DNA samples. In some embodiments, the DMR(s) that can distinguish oropharyngeal cancer from a control sample are associated with an area under the ROC curve (AUC) of 0.9 or greater, where the ROC curve distinguishes between subjects having or suspected of having OPSCC and control DNA samples.

[0148] In some embodiments, the DMR(s) that can distinguish oropharyngeal cancer from a control sample comprises an increased rate of hypermethylation compared to a control DNA sample. In some embodiments, the DMR(s) that can distinguish oropharyngeal cancer from a control sample comprises an increased rate of hypermethylation compared to a control DNA sample.

[0149] In some embodiments, determining the methylation profile of at least one DMR comprises amplifying at least a portion of the DMR using a set of primers (e.g., Tables 3 and 12). In some embodiments, determining the methylation profile of at least one DMR comprises performing at least one 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, determining the methylation profile of at least one DMR comprises determining the presence or absence of methylation at CpG sites. In some embodiments, the one or more CpG sites are present in a coding region, a non-coding region, and / or a regulatory region of a gene (e.g., any one of the genes disclosed herein). In some embodiments, the DMR(s) capable of distinguishing oropharyngeal cancer from control samples can be validated using at least one of methylation-specific PCR, quantitative methylation-specific PCR, methylation-specific DNA restriction enzyme analysis, bisulfite pyrosequencing, flap endonuclease assay, PCR flap assay, and bisulfite genomic sequencing PCR. In some embodiments, the DMR(s) capable of distinguishing oropharyngeal cancer from control samples can be evaluated based on at least one of the area under the ROC curve (AUC), fold change in methylation, percentage of methylation, and / or percentage of hypermethylation between the test sample and the control sample.

[0150] As one of skill in the art would understand based on the present disclosure, one or more types or subtypes of oropharyngeal cancer may be predicted by various combinations of markers (e.g., identified by statistical techniques related to the specificity and sensitivity of the prediction). Embodiments of the present disclosure provide methods for identifying predictive combinations and validated predictive combinations of one or more types or subtypes of oropharyngeal cancer.

[0151] Such methods are not limited to a subject type. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. Such methods are not limited to a particular manner or technique for measuring protein expression and / or activity. Techniques for measuring protein expression and / or activity levels are known in the art. Indeed, any known technique for measuring protein expression and / or activity levels is contemplated and incorporated herein.

[0152] Such methods are not limited to a particular format or technique for 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.

[0153] In some embodiments, measuring the methylation state or profile of a methylated DNA marker in a sample comprises determining the methylation state of one nucleotide base. In some embodiments, measuring the methylation state of a methylated DNA marker in a sample comprises determining the degree of methylation at a plurality of nucleotide bases. Further, in some embodiments, the methylation state or profile of a methylated DNA marker comprises increased methylation of the marker compared to a normal methylation state or profile of the marker. In some embodiments, the methylation state or profile of a marker comprises decreased methylation of the marker compared to a normal methylation state of the marker. In some embodiments, the methylation state or profile of a marker comprises a different pattern of methylation of the marker compared to a normal methylation state or profile of the marker.

[0154] Further, in some embodiments, the marker is a region of 100 or less nucleotide bases. In some embodiments, the marker is a region of 500 or less nucleotide bases. In some embodiments, the marker is a region of 1000 or less nucleotide bases. In some embodiments, the marker is a region of 5000 or less nucleotide bases. In some embodiments, the marker is one nucleotide base. In some embodiments, the marker is in a high CpG density promoter region.

[0155] In certain embodiments, methods of analyzing nucleic acids for the presence of 5-methylcytosine include treating DNA with a reagent that modifies the 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.

[0156] A frequently used method for analyzing nucleic acids for the presence of 5-methylcytosine is based on the bisulfite method for detection of 5-methylcytosine in DNA described by Frommer et al. (Frommer et al. (1992) Proc. Natl. Acad. Sci. USA 89:1827-31, expressly incorporated herein by reference in its entirety for all purposes), or a variation thereof. The bisulfite method for mapping 5-methylcytosine is based on the finding that cytosine, but not 5-methylcytosine, reacts with the ion of bisulfite (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 using bisulfite genomic sequencing (Grigg G, & Clark S, Bioessays (1994) 16:431-36; Grigg G, DNA Seq. (1996) 6:189-98), methylation specific PCR (MSP) (e.g., as disclosed in U.S. Pat. No. 5,786,146), or assays involving sequence specific probe cleavage (e.g., 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), as well as U.S. Pat. Nos. 8,361,720, 8,715,937, 8,916,344, and 9,212,392).

[0157] In some embodiments, conventional techniques include methods that involve encapsulating the DNA to be analyzed in an agarose matrix, thereby preventing diffusion and renaturation of the DNA (bisulfite only reacts with single-stranded DNA), and replacing 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 methylation status, demonstrating the utility 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.

[0158] Bisulfite techniques typically involve amplifying short specific fragments of known nucleic acids after bisulfite treatment, then assaying the products by sequencing (Olek & Walter (1997) Nat. Genet. 17:275-6) or analyzing the positions of individual cytosines using primer extension reactions (Gonzalgo & Jones (1997) Nucleic Acids Res. 25:2529-31, WO 95 / 00669, U.S. Patent No. 6,251,594). 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., WO 99 / 28498). In addition, the use of bisulfite technology 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; WO 9746705; WO 9515373).

[0159] Various methylation assay procedures can be used in conjunction with bisulfite treatment according to embodiments of the present disclosure. 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 others, sequencing of the bisulfite-treated nucleic acid, PCR (for sequence-specific amplification), Southern blot analysis, and the use of methylation-specific restriction enzymes, e.g., methylation-sensitive or methylation-dependent enzymes.

[0160] For example, the use of bisulfite treatment has simplified genome sequencing for the analysis of methylation patterns and 5-methylcytosine distribution (Frommer et al. (1992) Proc. Natl. Acad. Sci. USA 89:1827-1831). In addition, restriction enzyme digestion of PCR products amplified from bisulfite converted DNA is useful for assessment of methylation status, for example, as described in Sadri & Hornsby (1997) Nucl. Acids Res. 24:5058-5059, or as embodied in the method known as COBRA (Combined Bisulfite Restriction Analysis) (Xiong & Laird (1997) Nucleic Acids Res. 25:2532-2534).

[0161] 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 detection using specific labeled hybridization probes. Methylation levels in the original DNA sample are represented by the relative amounts of digested and undigested PCR products in a linear quantification approach across a wide range of DNA methylation levels. In addition, this technique can be reliably applied to DNA obtained from microdissected paraffin-embedded tissue samples.

[0162] Typical reagents for COBRA™ analysis (e.g., as found in a typical COBRA™-based kit) may include, but are not limited to, the following: PCR primers for specific loci (e.g., specific genes, markers, DMRs, regions of genes, regions of markers, bisulfite-treated DNA sequences, CpG islands, etc.), restriction enzymes and appropriate buffers, gene hybridization oligonucleotides, control hybridization oligonucleotides, kinase labeling kits of oligonucleotide probes, and labeled nucleotides. In addition, bisulfite conversion reagents may include DNA denaturation buffers, sulfonation buffers, DNA recovery reagents or kits (e.g., precipitation, ultrafiltration, affinity columns), desulfonation buffers, and DNA recovery components.

[0163] Assays such as "MethyLight™" (a 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.

[0164] The "HeavyMethyl™" assay, technology, 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 nucleic acid samples.

[0165] 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.

[0166] Typical reagents for HeavyMethyl™ analysis (e.g., as may be found in a typical MethyLight™-based kit) may include, but are not limited to, the following: PCR primers for specific loci (e.g., specific genes, markers, regions of genes, regions of markers, bisulfite-treated DNA sequences, CpG islands, or bisulfite-treated DNA sequences or CpG islands, etc.), blocking oligonucleotides, optimized PCR buffer and deoxynucleotides, and Taq polymerase.

[0167] 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., those that may be found in a typical MSP-based kit) may include, but are not limited to, methylated and unmethylated PCR primers, optimized PCR buffers and deoxynucleotides, and specific probes for specific loci (e.g., specific genes, markers, regions of genes, regions of markers, bisulfite-treated DNA sequences, CpG islands, etc.).

[0168] The MethyLight™ assay is a high-throughput quantitative methylation assay that utilizes fluorescence-based real-time PCR (e.g., TaqMan®) that 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.

[0169] The MethyLight™ assay is used as a quantitative test for methylation patterns in nucleic acids, 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 input DNA amount is provided by a reaction in which neither the primers nor the probe cover 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.

[0170] The MethyLight™ process is used with any suitable probe (e.g., “TaqMan®” probe, Lightcycler® probe, etc.). 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 a TaqMan® probe, e.g., with MSP primers and / or HeavyMethyl blocker oligonucleotides, 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 as it enzymatically synthesizes new strands 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.

[0171] Typical reagents for MethyLight™ analysis (e.g., as may be found in a typical MethyLight™-based kit) may include, but are not limited to, the following: PCR primers for specific loci (e.g., specific genes, markers, regions of genes, regions of markers, bisulfite-treated DNA sequences, CpG islands, etc.), TaqMan® or Lightcycler® probes, optimized PCR buffer and deoxynucleotides, and Taq polymerase.

[0172] 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 input DNA amount is provided by a reaction in which neither the primers nor the probe cover 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.

[0173] 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 as it enzymatically synthesizes new strands 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) can include, but are not limited to: PCR primers for specific loci (e.g., specific genes, markers, regions of genes, regions of markers, bisulfite-treated DNA sequences, CpG islands, etc.), TaqMan® or Lightcycler® probes, optimized PCR buffer and deoxynucleotides, and Taq polymerase.

[0174] Ms-SNuPE™ technology is a quantitative method for evaluating methylation differences at specific CpG sites based on bisulfite treatment of DNA followed by single nucleotide primer extension (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. Then, amplification of the desired target sequence is performed using PCR primers specific for bisulfite-converted DNA, and the resulting products are isolated and used as templates for methylation analysis at CpG sites of interest. It is possible to analyze small amounts of DNA (e.g., microdissected pathology sections), avoiding the use of restriction enzymes to determine methylation status at CpG sites.

[0175] Typical reagents for Ms-SNuPE™ analysis (e.g., as may be found in a typical Ms-SNuPE™-based kit) may include, but are not limited to, the following: PCR primers for specific loci (e.g., specific genes, markers, regions of genes, regions of markers, 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 the Ms-SNuPE reaction), and labeled nucleotides. In addition, bisulfite conversion reagents may include DNA denaturation buffers, sulfonation buffers, DNA recovery reagents or kits (e.g., precipitation, ultrafiltration, affinity columns), desulfonation buffers, and DNA recovery components.

[0176] 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, including frequent restriction enzyme cleavage sites in these regions.

[0177] 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, ligating adapters, bisulfite conversion, and PCR. See, for example, Meissner et al. (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.

[0178] In some embodiments, a quantitative allele-specific real-time target and signal amplification (QuARTS) assay is used to assess the 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 (e.g., 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, thus liberating multiple fluorophores per flap and providing exponential signal amplification. QuARTS can detect multiple targets in a single reaction well by using FRET cassettes with different dyes. See, for example, Zou et al. (2010) "Sensitive quantification of methylated markers with a novel methylation specific technology" Clin Chem 56:A199), as well as U.S. Patent Nos. 8,361,720, 8,715,937, 8,916,344, and 9,212,392 (each of which is incorporated herein by reference for all purposes).

[0179] 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 such 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, such as, for example, 6-hydroxy-2,5,7,8,-tetramethylchroman 2-carboxylic acid or trihydroxybenzone, and a derivative thereof, such as, for example, gallic acid (see PCT / EP2004 / 011715, incorporated by reference in its entirety). In certain preferred embodiments, the bisulfite reaction includes treatment with ammonium bisulfite, for example, as described in WO2013 / 116375.

[0180] In some embodiments, fragments of treated DNA are amplified using a set of primer oligonucleotides (see, e.g., Tables 3 and 12) and an amplification enzyme according to the methods and compositions described herein. Amplification of several DNA segments can be carried out simultaneously in one and the same reaction vessel. Typically, amplification is carried out using the polymerase chain reaction (PCR). Amplicons are usually 100-2000 base pairs in length.

[0181] In some embodiments of the method, the methylation status or profile of CpG positions in or near variably methylated regions (e.g., Tables 1, 2, 6, and 7) can be detected by 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 for the discrimination 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 includes at least one CpG dinucleotide. MSP primers specific for unmethylated DNA contain a "T" at the C position in the CpG.

[0182] 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 methylated DNA markers. In some embodiments, the primers or primer pairs are listed in Tables 3 and 12 (SEQ ID NOs: 1-176). In some embodiments, the primers or primer pairs specific for each methylation marker gene can bind to an amplicon bound by the primer sequence of the marker gene listed in Tables 3 and 12, where the amplicon bound by the primer sequence of the marker gene listed in Tables 3 and 12 is at least a portion of a gene region of a methylation marker gene listed in Tables 1, 2, 6, or 7. In some embodiments, the primers or primer pairs of the methylation markers are a set of primers that specifically bind to at least a portion of a gene region that includes chromosomal coordinates for a particular methylation marker listed in Tables 1, 2, 6, or 7.

[0183] In another embodiment, the present disclosure 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 Publication No. 202000370114). The method involves reacting oxidized 5mC residues selected from 5-formylcytosine (5fC), 5-carboxymethylcytosine (5caC), and combinations thereof, with a borane reducing agent. The oxidized 5mC residue may be naturally occurring or, more typically, may be the result of previous oxidation of a 5mC or 5hmC residue, e.g., oxidation of 5mC or 5hmC by a TET family enzyme (e.g., TET1, TET2, or TET3), or chemical oxidation of 5mC or 5hmC, e.g., by potassium perruthenate (KRuO4) or inorganic peroxo compounds, or compositions such as peroxotungstate (see, e.g., Okamoto et al. (2011) Chem. Commun. 47:11231-33) and combinations of copper(II) perchlorate / 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) (see, e.g., Matsushita et al. (2017) Chem. Commun. 53:5756-59).

[0184] The borane reducing agent may be characterized as a complex of borane and a nitrogen-containing compound selected from a nitrogen heterocycle and a tertiary amine. The nitrogen heterocycle may be monocyclic, bicyclic, or polycyclic, but typically is 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 may 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.

[0185] The reaction of borane reducing agents with oxidized 5mC residues in cell-free DNA is advantageous insofar as non-toxic reagents and mild reaction conditions can be used, and does not require any hydrogen sulfate or any other potentially DNA-degrading reagents. Furthermore, the conversion of oxidized 5mC residues to dihydrouracils using borane reducing agents can be carried out in a "one-pot" or "one-tube" reaction without the need for isolation of any intermediates. This is crucial because the conversion involves multiple steps, namely, (1) reduction of the alkene bond linking C-4 and C-5 in the oxidized 5mC, (2) deamination, and (3) either decarboxylation if the oxidized 5mC is 5caC, or deformylation if the oxidized 5mC is 5fC.

[0186] In addition to the method for converting oxidized 5-methylcytosine residues in cell-free DNA to dihydrouracil residues, the present disclosure also provides a reaction mixture related to the aforementioned 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 either decarboxylate or deformylate the at least one oxidized 5-methylcytosine residue. The borane reducing agent is a complex of borane and a nitrogen-containing compound selected from nitrogen heterocycles and tertiary amines, as described above. In a preferred embodiment, the reaction mixture is substantially free of bisulfite, which means that it is substantially free of bisulfite ions and bisulfite salts. Ideally, the reaction mixture is free of bisulfite.

[0187] In a related aspect of the disclosure, a kit for converting 5mC residues in cell-free DNA to dihydrouracil residues is provided, 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 either reduce, deaminate, and decarboxylate or deformylate the oxidized 5mC residue. The kit may also include instructions for using the components to practice the above method.

[0188] In another embodiment, a method utilizing the above oxidation reaction is provided, 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 thereon, which allows for the 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 containing unmodified 5mC residues; and (c) oxidizing the unmodified 5mC residues to produce 5caC, 5fC, 5hm ... 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 dihydrosyl residue in place of the oxidized 5mC residue; (e) amplifying and sequencing the DNA containing the dihydrosyl residue; and (f) determining a 5-methylation pattern from the sequencing results of (e).

[0189] In some embodiments, the disclosure provides a method for identifying 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC) in a target nucleic acid. In some embodiments, the method includes modifying the target nucleic acid by 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 such that one or more 5caC or 5fC residues are generated, converting 5caC and / or 5fC to dihydrouracil (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, and detecting a sequence of the modified target nucleic acid, wherein a cytosine (C) to thymine (T) transition or a cytosine (C) to DHU transition in the sequence of the modified target nucleic acid compared to the target nucleic acid provides the position of either 5mC or 5hmC in the target nucleic acid. In some embodiments, the borane reducing agent is 2-picoline borane.

[0190] 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. 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). In some embodiments, the method further comprises blocking one or more modified cytosines. In some embodiments, the blocking step comprises adding sugars to 5hmC. In some embodiments, the method further comprises amplifying the copy number of one or more nucleic acid sequences. In some embodiments, the oxidizing agent is potassium perruthenate or Cu(II) / TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl).

[0191] Cell-free DNA is typically extracted from a biological sample from a subject, which may be whole blood, plasma, urine, saliva, mucosal excretion, organ secretions, sputum, feces, or tears. In some embodiments, the cell-free DNA is derived from a tumor (e.g., an oropharyngeal tumor). In other embodiments, the cell-free DNA is derived from a patient with a disease or other pathogenic condition. The cell-free DNA may or may not be derived from a tumor. In some embodiments, the cell-free DNA in which the 5hmC residues are to be modified is in purified, fragmented, and adaptor-ligated form. DNA purification in this context can be performed using any suitable method known to those of skill in the art and / or described in the relevant literature, and although the cell-free DNA itself may be highly fragmented, occasionally 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) have been 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 blunted fragments are also provided with 3' overhangs containing a single 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, which is ligated to both ends of the cell-free DNA fragments and contains at least one molecular barcode. The use of adaptors also allows for selective PCR enrichment of the DNA fragments to which the adaptors are ligated.

[0192] In some embodiments, the "purified and fragmented cell-free DNA" comprises adaptor-ligated DNA fragments. Modification of 5hmC residues in these cell-free DNA fragments with affinity tags is performed to allow for subsequent removal of the modified 5hmC-containing DNA from the cell-free DNA. 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 the biotin moiety as an affinity tag allows for easy removal by streptavidin (e.g., streptavidin beads, magnetic streptavidin beads, etc.).

[0193] Tagging of 5hmC residues with biotin moieties or other affinity tags can be achieved by covalently attaching a chemoselective group to the 5hmC residues of DNA fragments, which can undergo reaction 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 spontaneous 1,3-cycloaddition reaction with an alkyne-functionalized biotin moiety, as described in Robertson et al. (2011) Biochem. Biophys. Res. Comm. 411(1):40-3, U.S. Patent No. 8,741,567, and WO2017 / 176630. Thus, the addition of an alkyne-functionalized biotin moiety results in the covalent attachment of a biotin moiety to each 5hmC residue.

[0194] The affinity tagged DNA fragments can then be pulled down using streptavidin, in one embodiment in the form of streptavidin beads, magnetic streptavidin beads, etc., and set aside for subsequent analysis, if desired. The supernatant remaining after removal of the affinity tagged fragments contains DNA with unmodified 5mC residues and no 5hmC residues.

[0195] In some embodiments, unmodified 5mC residues are oxidized to provide 5caC and / or 5fC residues using any suitable means. The oxidizing agent is selected to oxidize 5mC residues beyond hydroxymethylation, i.e., to provide 5caC and / or 5fC residues. The oxidation can be performed enzymatically using a catalytically active TET family enzyme. "TET family enzyme" or "TET enzyme" as these terms are 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 (see 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 anion 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), as well as inorganic peroxo compounds and compositions such as peroxotungstate or copper(II) perchlorate / TEMPO combinations. At this point, it is not necessary to separate the 5fC-containing fragments from the 5caC-containing fragments, as long as both the 5fC and 5caC residues are converted to dihydrouracil (DHU) in the next step of the process.

[0196] In some embodiments, 5-hydroxymethylcytosine residues are blocked with β-glucosyltransferase (β3GT), while 5-methylcytosine residues are oxidized with a TET enzyme effective to provide 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 obtain dihydrouracil. In a variation of this embodiment, the 5hmC-containing fragments are not removed. Rather, in "TET-assisted picoline borane sequencing (TAPS)", the 5mC-containing fragments and the 5hmC-containing fragments are enzymatically oxidized together to provide 5fC-containing fragments and 5caC-containing fragments. The reaction with 2-picoline borane results in DHU residues if 5mC and 5hmC residues were originally present. "Chemically assisted picoline borane sequencing (CAPS)" involves selective oxidation of 5hmC-containing fragments with potassium perruthenate, leaving the 5mC residues unchanged.

[0197] In a related embodiment, the method further comprises identifying hydroxymethylation patterns in the 5hmC-containing DNA removed from the cell-free DNA. This can be performed using the techniques described in detail in WO2017 / 176630. This process can be performed without intermediate removal or isolation in a one-tube method. For example, first, cell-free DNA fragments, preferably adapter-ligated DNA fragments, are functionalized with βGT-catalyzed uridine diphosphoglucose 6-azide, followed by biotinylation via a chemoselective azide group. This procedure results in covalently attached biotin at each 5hmC site. In the next step, the biotinylated strand and the unmodified (native) 5mC-containing strand are simultaneously pulled down for further processing. The native 5mC-containing strand is pulled down using an anti-5mC antibody or a methyl-CpG binding domain (MBD) protein, as known in the art. With the 5hmC residues blocked, unmodified 5mC residues are then selectively oxidized using any suitable technique to convert 5mC to 5fC and / or 5caC, as described elsewhere herein.

[0198] The fragments obtained by amplification can carry 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 the label is a mass label, 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).

[0199] Methods for isolating DNA suitable for these assay techniques are known in the art. In particular, some embodiments involve the isolation of nucleic acids as described in U.S. Patent Application Serial No. 13 / 470,251 ("Isolation of Nucleic Acids"), which is incorporated herein by reference in its entirety.

[0200] In some embodiments, the markers described herein are useful in a QUARTS assay performed on a fecal 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 free of substances that substantially and / or effectively inhibit assays (e.g., PCR, INVADER, QuARTS assays, etc.) used to test the DNA sample. Such DNA samples are useful 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 certain mutant alleles or certain methylation states, and thus detecting and / or quantifying such mutant alleles or methylation states has predictive value in cancer diagnosis and treatment.

[0201] Many valuable genetic markers are present in very low amounts in samples, and many of the events that generate such markers are rare.As a result, even highly sensitive detection methods such as PCR require large amounts of DNA to provide a sufficient amount of low-abundance targets to meet or exceed the detection threshold of the assay.In addition, even the presence of small amounts of inhibitors can impair the accuracy and precision of these assays that are aimed at detecting such low-abundance targets.Therefore, provided herein is a method for generating such DNA samples that provides the necessary volume and concentration control.

[0202] In some embodiments, the biological sample is a tissue sample, a blood sample, a plasma sample, a serum sample, a whole blood sample, a buffy coat sample, a secretion sample, an organ secretion sample, a cerebrospinal fluid (CSF) sample, a saliva sample, a urine sample, and / or a stool sample. In some embodiments, the tissue sample is an oropharyngeal tissue sample, including one or more of soft palate cells or tissue, throat cells or tissue, tongue cells or tissue, and tonsil cells or tissue. In some embodiments, the tissue sample is an HPV(+) tissue sample. In some embodiments, the subject is a human. Such samples can be obtained by any number of means known in the art, such as those that will be apparent to those of skill in the art. Acellular or substantially acellular samples can be obtained by subjecting the sample to a variety of techniques known to those of skill in the art, including, but not limited to, centrifugation and filtration. In general, it is preferred that no invasive techniques are used to obtain the sample, although it may still be preferred to obtain samples such as tissue homogenates, tissue sections, and biopsy specimens. The present technology is not limited to the methods used to prepare the sample and provide nucleic acids for testing. For example, in some embodiments, DNA is isolated from a sample (e.g., a tissue sample, a blood sample, a plasma sample, a serum sample, a whole blood sample, a buffy coat sample, a secretion sample, an organ secretion sample, a cerebrospinal fluid (CSF) sample, a saliva sample, a urine sample, and / or a stool sample) using direct gene capture or related methods, e.g., as detailed in U.S. Pat. Nos. 8,808,990 and 9,169,511, and WO 2012 / 155072.

[0203] 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 efficiently process multiple samples and potentially provide higher diagnostic and / or prognostic accuracy. In addition, 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 can allow 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.

[0204] 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.

[0205] 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 clinical sample types, including neoplastic or pre-neoplastic material, are suitable for use with the method, for example, cell lines, histological slides, biopsies, paraffin-embedded tissues, body fluids, feces, tissues, colonic effluent, urine, plasma, serum, whole blood, isolated blood cells, cells isolated from blood, and combinations thereof.

[0206] 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 fecal samples, blood samples, or plasma samples by using direct gene capture or related methods, for example, as described in US Patent Application No. 61 / 485386.

[0207] The genomic DNA sample is then treated with at least one reagent, or a set of reagents, that distinguishes between methylated and unmethylated CpG dinucleotides within at least one marker that comprises a DMR (e.g., a DMR in Tables 1, 2, 6, or 7).

[0208] 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, however, in some embodiments, the reagent may be a methylation-sensitive restriction enzyme.

[0209] In some embodiments, the genomic DNA sample is treated in such a manner 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, disulfite) followed by alkaline hydrolysis.

[0210] The processed 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 at least one DMR, e.g., a DMR selected from the DMRs of Tables 1, 2, 6, or 7). Methods of analysis can be selected from those listed herein, e.g., those known in the art, including QuARTS and MSP, as described herein.

[0211] Such samples can be obtained by any number of means known in the art, such as those that will be apparent to those of skill in the art. For example, urine and fecal samples are easily 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 known to those of skill in the art, including, but not limited to, centrifugation and filtration. In general, it is preferred that no invasive techniques are used to obtain the sample, although it may still be preferred to obtain samples such as tissue homogenates, tissue sections, and biopsy specimens.

[0212] The embodiments of the present disclosure further provide compositions. In some embodiments, the present disclosure provides compositions comprising a nucleic acid comprising a DMR and a bisulfite reagent. In some embodiments, compositions are provided comprising a nucleic acid comprising a DMR and one or more oligonucleotides as set forth in SEQ ID NOs: 1-176. In certain embodiments, compositions are provided comprising a nucleic acid comprising a DMR and a methylation-sensitive restriction enzyme. In certain embodiments, compositions are provided comprising a nucleic acid comprising a DMR and a polymerase.

[0213] 3.Treatment method In some embodiments, the disclosure provides methods for treating a subject (e.g., a patient having or suspected of having one or more types or subtypes of oropharyngeal cancer). According to these embodiments, the methods include determining the methylation status or profile of one or more methylated DNA markers 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. In some embodiments, treating a subject includes 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.

[0214] In some embodiments, a method for diagnosing a particular type of cancer in a subject is provided. 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 whether a subject 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, for example, one or more biomarkers (e.g., one or more methylation markers, methylation marker genes, genes, DMRs, and / or DNA methylation markers disclosed herein), whose methylation status indicates the presence, severity, or absence of the condition.

[0215] In addition to diagnosis, clinical cancer prognosis is concerned with determining the aggressiveness of cancer and the likelihood of tumor recurrence, and planning the most effective therapy. If it is possible to perform a more accurate prognosis, or even to assess the potential risk of suffering from cancer, it is possible to select an appropriate, and in some cases, less harsh, treatment for the patient. The evaluation of cancer biomarkers (e.g., determining methylation status) is useful for distinguishing subjects with good prognosis and / or low risk of developing cancer, who do not require therapy or who require limited therapy, from subjects who are more likely to develop cancer or suffer from cancer recurrence, who can benefit from more intensive treatment.

[0216] As such, "making a diagnosis" or "diagnosing" as used herein further includes determining the risk of developing cancer or determining a prognosis, which may be provided to predict clinical outcomes (with or without medical treatment), select appropriate treatment (or whether treatment is effective), or monitor current treatment to potentially modify treatment, based on measurements of diagnostic biomarkers (e.g., DMRs) disclosed herein. Furthermore, in some embodiments of the subject matter disclosed herein, multiple determinations of biomarkers over time may be made to facilitate diagnosis and / or prognosis. Changes in biomarkers over time may be used to predict clinical outcomes, monitor progression of the cancer or cancer subtype, and / or monitor the effectiveness of appropriate therapies against the cancer. In such embodiments, for example, one may expect to see changes in the methylation status of one or more biomarkers (e.g., DMRs) disclosed herein in biological samples over time (and potentially one or more additional biomarker(s) if monitored) over the course of an effective therapy.

[0217] The subject matter of the present disclosure further provides a method for determining whether to initiate or continue cancer prevention or treatment in a subject. In some embodiments, the method includes providing a series of biological samples from a subject over a period of time, analyzing the series of biological samples to determine the methylation state or profile of at least one marker disclosed herein in each of the biological samples, and comparing any measurable changes in the methylation state of one or more biomarkers in each of the biological samples. Any changes over a period of time can be used to predict the risk of developing cancer, predict clinical outcomes, determine whether to initiate or continue cancer prevention or therapy, and whether the current therapy 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 state 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 state of the biomarker levels from the different samples can be correlated with a particular cancer risk, prognosis, determining treatment efficacy, and / or cancer progression in the subject. In some embodiments, the disclosed methods and compositions are for the treatment or diagnosis of disease at an early stage, e.g., before symptoms of the disease appear, hi some embodiments, the disclosed methods and compositions are for the treatment or diagnosis of disease at a clinical stage.

[0218] In some embodiments, multiple determinations of one or more diagnostic or prognostic biomarkers can be made, and the temporal changes in the markers 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 such embodiments, an increase in a marker from the first time to the second time can be diagnostic of a particular type or severity of 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, comparative measurements of the same biomarkers can be made at multiple time points, and a given biomarker can be measured at a first time point and a second biomarker at a second time point, and the comparison of these markers can provide diagnostic information.

[0219] 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 that a given course or outcome is more or less likely to occur predictably based on the methylation status of a biomarker (e.g., DMR and / or protein marker). Instead, a person skilled in the art will understand that the term "prognosis" refers to an 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, in individuals that do not exhibit the condition (e.g., have normal methylation status of one or more DMRs), the probability of a given outcome (e.g., suffer from a certain type of cancer) may be very low.

[0220] In some embodiments, the statistical analysis correlates the prognostic indicator with a predisposition to adverse outcomes. For example, in some embodiments, a methylation status that differs from that in a normal control sample obtained from a patient without 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. In addition, 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 and determining a confidence interval and / or p-value. See, e.g., 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.

[0221] In other embodiments, a threshold change in the methylation status of a prognostic or diagnostic biomarker disclosed herein (e.g., DMR; protein marker) can be established, and the change in the methylation status of the biomarker in a biological sample is simply compared to the threshold 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. Because reference is made to individual sample measurements rather than population averages, those skilled in the art are familiar with using such nomograms to relate the two values, with the understanding that the uncertainty of the measurements is the same as the uncertainty of the marker concentration.

[0222] In some embodiments, a control sample is analyzed simultaneously with the biological sample so that results obtained from the biological sample can be compared to results obtained from the control sample. In addition, it is contemplated that a standard curve can be provided and the assay results of the biological sample can be compared to the standard curve. Such a standard curve presents the methylation state of the biomarker according to the assay unit, e.g., fluorescent signal intensity if a fluorescent label is used. Samples taken from multiple donors can be used to provide a standard curve for the control methylation state of one or more biomarkers in normal tissues, as well as the "risk" level of one or more biomarkers in plasma taken from donors with a particular type of cancer. In certain embodiments of the method, a subject is identified as having cancer by identifying an abnormal methylation state of one or more DMRs provided herein in a biological sample obtained from the subject. In other embodiments of the method, detection of an abnormal methylation state of one or more of these biomarkers in a biological sample obtained from the subject results in the subject being identified as having cancer.

[0223] 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 efficiently process multiple samples and potentially provide higher diagnostic and / or prognostic accuracy. In addition, 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 can allow 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.

[0224] 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.

[0225] In some embodiments, a subject is diagnosed as having a certain type of cancer if there is a measurable difference in the methylation status of at least one biomarker in the sample compared to the control methylation status. Conversely, if no change in methylation status is identified in the biological sample, the subject may be identified as not having, not at risk of, or at low risk of a certain type of cancer. In this regard, subjects with cancer or at risk may be differentiated from subjects with cancer or at low risk to subjects with substantially no cancer. Subjects with a risk of developing a certain type of cancer may be placed on a more intensive and / or regular screening schedule. Meanwhile, those subjects with low risk or substantially no risk may avoid undergoing additional testing (e.g., invasive procedures) for cancer risk until future screening, e.g., screening performed according to various embodiments of the present disclosure, indicates that the subjects have developed a risk of cancer risk.

[0226] As mentioned above, depending on the embodiment of the method of the present disclosure, detecting a change in the methylation state of one or more biomarkers can be a qualitative or quantitative determination. Thus, diagnosing a subject as having or at risk of developing a particular type of cancer indicates that a certain threshold measurement is made, for example, the methylation state of one or more biomarkers in a biological sample changes from a predetermined control methylation state. In some embodiments of the method, the control methylation state is any detectable methylation state of the biomarker. In other embodiments of the method in which 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 method, the predetermined methylation state is based on and / or identified by a standard curve. In other embodiments of the method, the predetermined methylation state is a specific state or range of states. Thus, the predetermined methylation state can be selected in part based on the embodiment of the method to be performed and the desired specificity, etc., within acceptable limits that will be apparent to one of skill in the art.

[0227] Further with respect to the diagnostic method, the preferred subject is a vertebrate subject. The preferred vertebrate is a warm-blooded animal, and the preferred warm-blooded vertebrate is a mammal. The preferred mammal is most preferably a human. As used herein, the term "subject" includes both human and animal subjects. Thus, veterinary therapeutic uses are provided herein. Thus, embodiments of the present disclosure provide for the diagnosis of mammals such as humans, as well as mammals of importance due to being endangered species, such as Siberian tigers, mammals of economic importance, such as animals raised on farms for human consumption, and / or animals of social importance to humans, such as animals raised as pets or in zoos. Examples of such animals include, but are not limited to, carnivores, such as cats and dogs, ruminants and / or ungulates, such as swine (including pigs, hogs, and wild boars), cows, bulls, sheep, giraffes, deer, goats, bison, and camels, and horses. Thus, diagnosis and treatment of livestock, including but not limited to domesticated pigs, ruminants, ungulates, horses (including racehorses), and the like, is also provided.

[0228] 4. Samples, Kits, and Controls The embodiments of the present disclosure provide techniques for screening one or more types of oropharyngeal cancer from a biological sample. According to these embodiments, the present disclosure includes, but is not limited to, methods and compositions for detecting the presence of one or more types and / or subtypes of oropharyngeal cancer from a biological sample. In some embodiments, the biological sample is a tissue sample, a blood sample, a plasma sample, a serum sample, a whole blood sample, a buffy coat sample, a secretion sample, an organ secretion sample, a cerebrospinal fluid (CSF) sample, a saliva sample, a urine sample, and / or a stool sample. In some embodiments, the tissue sample is an oropharyngeal tissue sample, including one or more of soft palate cells or tissue, throat cells or tissue, tongue cells or tissue, and tonsil cells or tissue. In some embodiments, the tissue sample is an HPV(+) tissue sample. In some embodiments, the subject is a human.

[0229] In other embodiments, "sample", "test sample", and "biological sample" refer to a fluid sample that contains or is suspected of containing the methylated DNA markers of the present disclosure. The sample may be from any suitable source. In some cases, the sample may include a liquid, a flowable particulate solid, or a fluid suspension of solid particles. In some cases, the sample may be processed prior to the analysis described herein. For example, the sample may be separated or purified from its source prior to analysis. In certain examples, the source is a mammalian (e.g., human) bodily substance (e.g., bodily fluid, blood (whole blood, serum, plasma, etc.), urine, saliva, sweat, sputum, semen, mucus, tears, lymph, amniotic fluid, interstitial fluid, cerebrospinal fluid, feces, tissue, organ, one or more dried blood spots). Tissues include, but are not limited to, oropharyngeal tissue samples, including one or more of soft palate cells or tissue, throat cells or tissue, tongue cells or tissue, and tonsil cells or tissue. The sample may be a liquid sample or a liquid extract of a solid sample. In some embodiments, the source of the sample may be an organ or tissue, such as a biopsy sample and / or a secretory sample (e.g., oropharyngeal secretions), which may be solubilized by tissue disruption / cell lysis. In addition, the sample may be a nasopharyngeal or oropharyngeal sample obtained using one or more swabs, which are placed into a sterile tube containing a viral transport medium (VTM) or universal transport medium (UTM) for testing.

[0230] A wide range of fluid sample volumes can be analyzed. In some exemplary embodiments, the sample volume can be about 0.5 nL, about 1 nL, about 3 nL, about 0.01 μL, about 0.1 μL, about 1 μL, about 5 μL, about 10 μL, about 100 μL, about 1 mL, about 5 mL, about 10 mL, etc. In some cases, the volume of the fluid sample is about 0.01 μL to about 10 mL, about 0.01 μL to about 1 mL, about 0.01 μL to about 100 μL, or about 0.1 μL to about 10 μL.

[0231] In some cases, the fluid sample may be diluted before use in the assay. For example, in embodiments where the source containing the methylated DNA marker is a human body fluid (e.g., blood, serum, secretions), the fluid may be diluted with a suitable solvent (e.g., a buffer such as PBS buffer). The fluid sample may be diluted about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 10-fold, about 100-fold, or more before use. In other cases, the fluid sample is not diluted before use in the assay.

[0232] In some cases, the sample may undergo pre-analysis treatment. Pre-analysis treatment may provide additional functions such as non-specific protein removal and / or effective yet inexpensively implemented mixing functions. Common methods of pre-analysis treatment may include the use of electrokinetic trapping, AC electrokinetics, surface acoustic waves, isotachophoresis, dielectrophoresis, electrophoresis, or other pre-concentration techniques known in the art. In some cases, the fluid sample may be concentrated before use in the assay. For example, in embodiments where the source containing the methylated DNA marker is a human body fluid (e.g., blood, serum, secretions), the fluid may be concentrated by precipitation, evaporation, filtration, centrifugation, or a combination thereof. The fluid sample may be concentrated about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 10-fold, about 100-fold, or more, before use.

[0233] It may be desirable to include a control. The control may be analyzed simultaneously with the sample from the subject, as described above. The results obtained from the subject sample may be compared with the results obtained from the control sample. A standard curve may be provided, and the assay results of the biological sample may be compared. Such a standard curve shows the level of one or more methylated DNA markers as a function of assay units. Samples taken from multiple donors may be used to provide standard curves for reference levels of methylated DNA markers in normal healthy tissues, as well as "risk" levels of methylated DNA markers in tissues taken from donors that may have one or more characteristics of oropharyngeal cancer.

[0234] The embodiments of the present disclosure also include kits for carrying out the methods described herein. The kits include embodiments of the compositions, devices, apparatus, etc. described herein, and instructions for using the kits. Such instructions describe appropriate methods for preparing an analyte from a sample, e.g., methods for collecting a sample and preparing nucleic acid from the sample. 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., box(es)) for convenient storage, transportation, and / or use by a user of the kit. It is understood that liquid components (e.g., buffers) may be provided in lyophilized form to be reconstituted by the user. The kits 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.

[0235] In some embodiments, the disclosure provides a composition (e.g., a reaction mixture). In some embodiments, the disclosure provides a composition comprising a nucleic acid comprising a DMR and a reagent capable of modifying 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 a composition comprising a nucleic acid comprising a DMR and an oligonucleotide described herein. Some embodiments provide a composition comprising a nucleic acid comprising a DMR and a methylation-sensitive restriction enzyme. Some embodiments provide a composition comprising a nucleic acid comprising a DMR and a polymerase.

[0236] In some embodiments, the science and technology described herein is associated with a programmable machine designed to perform an array of arithmetic or logical operations, such as those provided by the methods described herein. For example, some embodiments of the science and technology are associated with (e.g., implemented in) computer software and / or computer hardware. In one aspect, the technology relates 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 performing a sequence of instructions to read, manipulate, and store data (e.g., the methods provided herein). In some embodiments, the microprocessor is part of a system for determining methylation status (e.g., of one or more DMRs in Tables 1, 2, 6, or 7), comparing methylation status, generating a standard curve, 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, analyzing sequences, or any of the above 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 in Tables 1, 2, 6, or 7), 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, analyzing sequences, or any of the above described herein or known in the art.

[0237] In some embodiments, the software or hardware component receives results of the multiple assays and determines to report to a user a single value result indicative of risk of cancer based on the results of the multiple assays (e.g., determining the methylation status of one or more DMRs in Tables 1, 2, 6, or 7). Related embodiments calculate a risk factor based on a mathematical combination (e.g., weighted combination, linear combination) of results from the multiple assays (e.g., determining the methylation status of one or more DMRs in Tables 1, 2, 6, or 7). In some embodiments, the methylation status of the DMRs defines a dimension and may have values ​​in a multidimensional space, and the coordinates defined by the methylation status of the multiple DMRs are a result (e.g., for reporting to a user or related to cancer risk).

[0238] In some embodiments, various embodiments of the present disclosure are associated with multiple programmable devices that work in concert to perform the methods 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 computer architecture that relies on complete computers (on-board CPU, storage, power, network interfaces, etc.) being connected to a network (private, public, or Internet) by traditional network interfaces such as Ethernet, optical fiber, etc., or by wireless networking technology.

[0239] 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. Such processors 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, for example, stores instructions that, when executed by the processor, cause the processor to perform the steps described herein.

[0240] 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. All such components, computers, and systems described herein as related to the technology may be logical or virtual.

[0241] In some embodiments, the present disclosure provides a system for screening one or more types or subtypes of oropharyngeal cancer in a sample obtained from a subject. Exemplary embodiments of the system include, for example, a system for screening multiple types or subtypes of oropharyngeal cancer in a sample obtained from a subject (e.g., a tissue sample, a blood sample, a plasma sample, a serum sample, a whole blood sample, a buffy coat sample, a secretion sample, an organ secretion sample, a cerebrospinal fluid (CSF) sample, a saliva sample, a urine sample, and / or a stool sample). In some embodiments, the system includes an analysis component configured for one or both of determining a methylation status of one or more methylation markers in the sample, a software component configured to compare the methylation status of one or more methylation markers in the 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.

[0242] In some embodiments, the alert is determined by a software component that receives results from multiple assays (e.g., determines the methylation status of one or more methylation markers), calculates a value or result, and reports based on the multiple results.

[0243] 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 alert to report 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 combination of one or more results from the multiple assays that is indicative of risk of cancer in a subject. Such methods are not limited to a particular methylation marker. In such methods and systems, the one or more methylation markers include bases in a DMR selected from the DMRs of Tables 1, 2, 6, and 7.

[0244] In this detailed description of the 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 the 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 may be altered and still be within the spirit and scope of the various embodiments disclosed herein.

[0245] The various components of the kit are optionally provided in suitable containers, as needed. The kit may further include a container for holding or storing the sample (e.g., a container or cartridge for urine, whole blood, plasma, serum sample, tissue, or bodily secretion sample). If applicable, the kit may also optionally contain reaction vessels, mixing vessels, and other components that facilitate preparation of reagents or test samples. The kit may also include one or more instruments to aid in obtaining the test sample, such as a syringe, pipette, forceps, measuring spoon, etc. In some embodiments, the instrument is a collection device. In some embodiments, a biological sample is obtained from a subject, and the method further includes extracting a DNA sample from the biological sample using an extraction element. In some embodiments, the biological sample is collected with a collection device having an adsorbent element that can collect the biological sample upon contact. In some embodiments, the adsorbent element is a sponge configured to be inserted into an orifice (e.g., mouth, throat, or nose). EXAMPLES

[0246] 5. Working Example It will be appreciated by those skilled in the art that other suitable modifications and adaptations of the disclosed methods described herein are readily applicable and recognizable, and may be made using suitable equivalents without departing from the scope of the disclosure or the aspects and embodiments disclosed herein.Though the disclosure has been described in detail above, it will be more clearly understood by reference to the following examples.These examples are intended merely to illustrate some aspects and embodiments of the disclosure, and should not be considered as limiting the scope of the disclosure.The disclosures of all journal references, US patents, and publications referenced herein are incorporated herein by reference in their entirety.

[0247] The present disclosure has multiple aspects, which are illustrated by the following non-limiting examples.

[0248] Example 1 Oropharyngeal cancer (e.g., HPV + Experiments were conducted to evaluate the feasibility of a panel of variably methylated regions (DMRs) for the detection of oropharyngeal squamous cell carcinoma. These regions are listed in Table 1 below. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14] [Table 1-15]

[0249] Example 2 The rate of human papillomavirus-associated oropharyngeal squamous cell carcinoma (HPV(+)OPSCC) continues to increase worldwide. Although studies on circulating tumor HPV DNA (ctHPVDNA) and pan-cancer assays are promising, data distinguishing anogenital from HPV(+)OPSCC are lacking. Therefore, experiments were performed to evaluate the feasibility of a targeted assay of a panel of methylated DNA markers (MDM) for the detection of OPSCC. A panel of methylated markers for human papillomavirus-associated cervical squamous cell carcinoma (HPV(+)CSCC) was analyzed and validated for use as markers for HPV(+)OPSCC.

[0250] Patients who met the inclusion criteria had a primary (non-recurrent) tumor, no history of pelvic or head and neck cancer or tumors, no exposure to chemotherapy agents within the past year, no previous radiation therapy to the target area, no transplants, adequate clinical history, adequate target tissue availability (>5 mm), and were ≥18 years old. Methylated DNA markers (MDMs) identified from sequenced variably methylated regions were selected from a panel previously validated for HPV(+) OPSCC and evaluated against DNA from independent formalin-fixed paraffin-embedded HPV(+) OPSCC, HPV(+) CSCC, normal oropharyngeal tissue, and normal cervical tissue using methylation-specific polymerase chain reaction. White blood cells (WBCs) were used as background controls.

[0251] Thirty-four patients with HPV(+) OPSCC, 36 with HPV(+) CSCC, 26 with normal oropharyngeal tonsillar tissue, and 24 with normal cervical tissue met the inclusion criteria. Compared with all others, HPV(+) OPSCC patients were slightly older (57 vs. 44, p=0.027), more frequently exposed to alcohol (85% vs. 64%, p=0.02), but had similar ACE-27 comorbidity scores (p=0.078) and smoking (p=0.066). Approximately 88% of patients with HPV(+) OPSCC and 58% of patients with normal tonsillar tissue were male. 0% of HPV(+) CSCC and 0% of patients with normal cervical tissue had a previous abnormal PAP smear. Tumor stage was stage I (83% vs. 47%), stage II (11% vs. 44%), and stage III (6% vs. 9%) for HPV(+)CSCC and HPV(+)OPSCC, respectively. Twenty-one MDMs were evaluated, and the area under the receiver operating characteristic curve (AUC) was reported for HPV(+)OPSCC and HPV(+)CSCC. Table 2 shows the 21 markers, the source of each marker, and the respective chromosomal information. Table 3 shows the relevant primer sequence information for the markers listed in Table 2. [Table 2]

[0252] [Table 3-1] [Table 3-2]

[0253] As shown in Table 4, within HPV(+)CSCC, 18 / 21 (86%) MDMs achieved AUC ≥ 0.9, and all MDMs showed better classification than chance compared to control cervical tissue (all p < 0.0001). For the HPV(+)OPSCC cohort, the majority of MDMs had a lower AUC compared to that of HPV(+)CSCC. However, 5 / 21 (24%) achieved AUC ≥ 0.90, 15 / 21 (71%) achieved AUC ≥ 0.8, and 19 / 21 (90%) showed better classification than chance compared to control tonsillar tissue (all p < 0.001). [Table 4-1] [Table 4-2]

[0254] Table 5 shows the patient characteristics of the above experimental sample. [Table 5]

[0255] In accordance with the above data, the following materials and methods were used.

[0256] Samples. Up to 10 (10um) / 2 (2mm) FFPE tissue cores of the area of ​​interest were obtained from each tissue block. Smaller size tissue cores were obtained from multiple tissue blocks depending on the tissue size of the block. At least two 2mm tissue cores or 10 slides of 10um were required to obtain sufficient quality DNA because the quality of DNA from FFPE tissue is usually low and the DNA is fragmented. Using a core punch preserves more tissue on the block because only a small area of ​​interest is taken instead of many sections of the entire block. In addition, slides are stained with P16 for HPV testing. All tissue samples were reviewed by a Mayo Clinic pathologist to confirm histology. Quantitative methylation specific PCR assays (qMSP) have already been developed for top cancer tissue specific marker candidates from previous studies. These markers were validated in target tissues from the patient groups identified above. Tissues were grossly dissected and histologically reviewed by an expert gastrointestinal pathologist. Samples were age-sex matched, randomized, and blinded. DNA from was purified using the QIAamp DNA FFPE Tissue Kit (FFPE tissue) and QIAamp DNA Blood Mini Kit (buffy coat samples) (Qiagen, Valencia CA). DNA was repurified with AMPure XP beads (Beckman-Coulter, Brea CA) and quantified with PicoGreen (Thermo-Fisher, Waltham MA). DNA integrity was assessed using qPCR.

[0257] Biomarker Selection. Previously identified and validated CSCC biomarkers were selected to test the OPSCC sample cohort. Thirteen methylated DNA markers (MDMs) from an independent sample set were able to distinguish cancer from normal cervical tissue with individual "area under the ROC curve" (AUC) performances above 0.90 and at least a 5-fold difference in methylation. In addition, we included eight MDMs identified from a previous pan-GI discovery study that showed high levels of hypermethylation in esophageal squamous cell carcinoma. These eight were later tested in a small cohort of head and neck cancers and compared to normal esophageal epithelium and shown to be highly methylated in these cancers as well (see, e.g., Table 2).

[0258] Biomarker testing. Up to 300ng of sample DNA was treated with sodium bisulfite and repurified using the Zymo EZ DNA methylation method (Zymo Research, Irvine CA). Quantitative methylation-specific PCR (qMSP) assays with oligos specific for differentially methylated CpGs were performed on the converted DNA. Approximately 10ng of converted DNA (per marker) was amplified using SYBR Green detection on Roche 480 LightCyclers (Roche, Basel Switzerland). Serially diluted universal 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.

[0259] Statistics. Sample data were extensively analyzed using descriptive statistics. These experiments were performed to evaluate the similarity of methylation markers between CSCC and OPSCC patients. An initial analysis of variance between the two groups was performed with Student's t-test.

[0260] Example 3 In this example, experiments were performed to identify additional DMRs that can distinguish oropharyngeal cancer (eg, OPSCC) from control samples (eg, tissue and buffy coat controls).

[0261] Utilizing a proprietary methodology of sample preparation, sequencing, analysis pipeline, and filters, we identified variably methylated regions (DMRs) to identify and refine these oropharyngeal cancers to excel in a clinical trial setting. From tissue-to-tissue analysis, 129 hypermethylated OPSCC DMRs were identified (Table 1 above, Table 6 below). These included OPSCC-specific regions as well as regions frequently methylated in several or more epithelial cancer types. From OPSCC tissue, buffy coat analysis yielded 105 hypermethylated tissue DMRs with an AUC of >0.95 and less than 1% noise in leukocytes (Table 1 above, Table 7 below). [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7] [Table 6-8]

[0262] [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6]

[0263] For validation in OPSCC, 62 candidates were selected (Table 8). These were the top-ranked MDMs in terms of AUC, fold change, delta methylation, and p-value, among others. Methylation-specific PCR assays were developed to test discovery tissue samples. Short amplicon primers (<150 bp) were designed to target the most discriminatory CpGs in the DMRs, and the assays were checked against controls to ensure that fully methylated fragments were robustly and linearly amplified, while unmethylated and / or unconverted fragments were not amplified. [Table 8-1] [Table 8-2] [Table 8-3]

[0264] Results were logistically analyzed to determine AUC and fold change. Analyses of tissue and buffy coat controls were performed separately. Table 9 provides the results of the qMSP assay. One DMR, ZNF763, was 100% discriminative in separating cancer from benign tissue, and 18 MDMs completely discriminated cancer from buffy coat samples (an important feature for liquid biopsy applications). [Table 9-1] [Table 9-2] [Table 9-3]

[0265] In addition, 39 of the 62 DMRs were utilized for further validation studies (Table 10). These DMRs had a between-tissue AUC greater than 0.80 and / or a between-tissue buffy coat AUC greater than 0.90. [Table 10-1] [Table 10-2]

[0266] DNA from 10 normal salivary cell pellets was tested at the seven DMRs to confirm that the assay as currently configured is suitable for this sample type (Table 11). Note that three of the seven DMRs are essentially silent, while the others show different degrees of hypermethylation. These results are consistent with those predicted from the RRBS data. [Table 11]

[0267] In summary, the DMRs developed for the detection of oropharyngeal cancer have demonstrated excellent performance through validation on both normal tissue and normal WBC (buffy coat) control samples. The DMR markers disclosed in this disclosure for oropharyngeal cancer, as well as the assays developed to evaluate them, are uniquely suitable for detecting these cancers in a non-invasive clinical setting.

[0268] According to the above data, the following materials and methods were used. Table 12 lists the primer sequences used in the above assay. Please note that for five DMRs, two different versions of primer pairs were used due to the number of distinguishable CpGs. These DMRs include EMBP1, FLJ43390, MAX_chr1_241587339_241587784, MAX_chr19_30718373_30719719, and SORCS3. [Table 12-1] [Table 12-2] [Table 12-3] [Table 12-4] [Table 12-5]

[0269] Samples. FFPE tissues were obtained from 18 HPV+ oropharyngeal squamous cell carcinoma (OPSCC) patients (9 stage I, 7 stage II, and 2 stage III) and 18 non-cancer patients (tonsils). Samples were age- and sex-matched. All tissues were provided by the Mayo Clinic Tissue Registry. Eighteen normal buffy coat samples from the NOMAD collection were also included in the study. Genomic DNA was purified using QIAamp FFPE Mini Kit (FFPE) and QIAamp DNA Blood Mini Kit (Buffy Coat) (Qiagen, Valencia CA). DNA was repurified with AMPure XP beads (Beckman-Coulter, Brea CA) and quantified with PicoGreen (Thermo-Fisher, Waltham MA). DNA integrity was assessed using qPCR.

[0270] Sequencing. RRBS sequencing libraries were prepared using the Ovation RRBS Methyl-Seq Library Preparation Kit (Tecan Genomics, Redwood City CA) with modifications. Briefly, samples were digested with Msp1, ligated to indexing flow cell adapters, bisulfite converted (twice), amplified, assembled in a 4-plex format, and sequenced by the Mayo Genomics facility on an Illumina HiSeq 4000 instrument (Illumina, San Diego CA). 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 coverage ≥10x and base quality score ≥20, methylation rates were determined by calculating C / (C+T) or, conversely, G / (G+A) in the case of reads mapping to the opposite strand.

[0271] Biomarker selection. Variable methylation regions (DMRs) were derived using a proprietary identification pipeline and regression package. Differences in mean percent methylation were compared between cases, tissue controls, and buffy coat controls. Tiling reading frames within 100 base pairs of each mapped CpG was used to identify DMRs with <5% methylation in controls, although this cutoff varied depending on the stringency required. DMRs were only analyzed if the total depth of coverage was, on average, 10 reads per subject and the variance across subgroups was >0.

[0272] Following regression, DMRs were ranked by p-value, area under the receiver operating characteristic curve (AUC), and fold change difference between cases and controls. No adjustment for false positives was performed at this stage, as independent validation was planned a priori.

[0273] Specifically, individual CpGs within DMRs were ranked by their hypermethylation ratio, i.e., the number of methylated cytosines at a given locus relative to the total number of cytosines at that site. For cases, the ratio had to be ≥ 0.20 (20%); for tissue controls, ≤ 0.05 (5%); and for buffy coat controls, ≤ 0.01 (1%). DMRs ranged from 60 to 200 bp and contained a minimum cutoff of 5 CpGs per region. DMRs with excessively high CpG density (> 30%) were excluded to avoid GC-related amplification issues in the validation phase. For each candidate region, a 2-D methylation intensity heat map was created and individual CpGs within the region plotted against samples from the case-control group. Methylation CpG patterns of OPSCC were analyzed against their respective benign controls and / or non-cancer buffy coats. The final selection required coordinated and consecutive hypermethylation (in some cases) of individual CpGs across the entire DMR sequence, per sample level. Conversely, control samples had to have at least 10-fold less methylation than the cases and the CpG patterns had to be empirically discordant.

[0274] Biomarker validation. A subset of DMRs was selected for further development. Criteria were primarily logistic-derived area under the ROC 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 tissue-to-tissue comparisons, and 0.95 for tissue-to-buffy coat comparisons. In addition, methylation fold change (mean cancer hypermethylation rate / mean control hypermethylation rate) was calculated, using a lower limit of 10 for tissue-to-tissue comparisons and 20 for tissue-to-buffy coat comparisons. P values ​​had to be <0.01. DMRs had to be concordantly methylated in cancer and discordant (or unmethylated) in controls. Case-control comparisons included: OPSCC vs. tonsil tissue controls, and OPSCC vs. normal buffy coat.

[0275] Quantitative methylation specific PCR (qMSP) primers were designed for the candidate genomic hg19 region using MethPrimer (Li LC and Dahiya R. MethPrimer: designing primers for methylation PCRs. Bioinformatics 2002 Nov;18(11):1427-31 PMID:12424112) and QC checked with 20ng (6250 equivalents) of positive and negative genomic methylation controls. Multiple annealing temperatures were evaluated for optimal discrimination. Validation was performed by qMSP on sequenced DNA samples. This was done to verify that the DMRs contained truly discriminatory CpGs by testing on an independent non-NGS targeted PCR platform.

[0276] DNA purification was performed as previously described. The EZ-96 DNA Methylation Kit (Zymo Research, Irvine CA) was used for the bisulfite conversion step. 10 ng of converted DNA (per marker) was amplified using SYBR Green detection on Roche 480 LightCyclers (Roche, Basel Switzerland). Serially diluted universal methylated genomic DNA (Zymo Research) was used as a quantification standard. The 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.

[0277] Statistics. Results were logistically analyzed for the performance of individual MDMs (methylated DNA markers).

[0278] All publications and patents mentioned in the above specification are incorporated herein 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 technology as described. Although the technology has been described in connection with specific exemplary embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are 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.

[0279] The present disclosure has multiple aspects, which are illustrated by the following non-limiting examples.

Claims

1. A method for characterizing a sample, said method comprising: The method comprises determining a methylation profile in at least one variably methylated region (DMR) of a DNA sample obtained from a subject having or suspected of having oropharyngeal cancer by treating the sample with a reagent that modifies DNA in a methylation-specific manner.

2. The methylation profile at the at least one DMR is determined to be + Oropharyngeal squamous cell carcinoma (HPV + 10. The method of claim 1, wherein the patient is diagnosed with or suspected of having OPSCC.

3. The at least one DMR is selected from the group consisting of EMBP1, ABCB1, ARHGAP12, ASCL1, C1orf114, EMX1, GRIN2D, LOC645323, MAX. chr6.58147682-58147771, MAX. chr9.36739811-36739868, NEUROG3, NID2, TBX15, TMEM200C, TSPYL5, TTYH1, VWC2, ZNF610, ZNF69, ZNF773, ZNF7 81, ALX4, ATP10A, C1QL3, CA8, CACNA1A, CACNG8, CALCA, CCNA1, CLIC6, CLSTN2, CR1, CTNND2, DAB1, DGKG, DOK1, DO K6, DPP4, DUXA, ELMO1, EPDR1, FGF12, FLJ43390, FMN2, FOXB2, FOXD4, FREM3, GALR1, GDF6, GFRA1, GRIK3, HOXB3, H OXB4, HPSE2, LDLRAD2, LHX2, LOC100131366, LOC345643, LOC386758, LOC648809, LOC728392, MAML3, MAPRE2, MAX. chr1.226288154-226288189, MAX. chr1.2375078-2375126, MAX. chr1.241587339-241587784, MAX. chr1.50798781-50799423, MAX. chr10.22765150-22765477, MAX. chr10.23462342-23462436, MAX. chr11.14926602-14927044, MAX. chr11.58903531-58903592, MAX. chr13.28527984-28528214, MAX. chr13.29106641-29107037, MAX. chr14.100784488-100784782, MAX. chr16.3221176-3221223, MAX. chr16.3222040-3222098, MAX. chr16.71460171-71460282, MAX. chr19.11805263-11805639, MAX. chr19.16394457-16394646, MAX. chr19.21657626-21657769, MAX. chr19.22034646-22034887, MAX. chr19.23299989-23300156,MAX.chr19.30713427-30713588、MAX.chr19.30716926-30717074、MAX.chr19.30718373-30719719、MAX.chr2.118981724-118982174、MAX.chr2.127783107-127783403、MAX.chr2.173099712-173099791、MAX.chr2.66808635-66808731、MAX.chr22.50064113-50064259、MAX.chr3.137489884-137490061、MAX.chr5.138923141-138923219、MAX.chr5.42995180-42995535、MAX.chr6.38683091-38683226、MAX.chr7.121952014-121952084、MAX.chr7.155166980-155167310、MAX.chr8.99986792-99986864、MAX.chr9.79627078-79627116、MAX.chr9.79638034-79638077、MAX.chr9.98789824-98789847、MDFI、MECOM、MED12L、MIR129-2、MIR196A1、NELL1、NPY、ONECUT2、OPCML、PARP15、PDGFD、PEX5L、PRR15、SEMA6A、SFMBT2、SGIP1、SIM2、SLC35F3、SLCO4C1、SORCS3、ST6GALNAC5、ST8SIA5、SV2C、TACC2、TFAP2E、TLX2、TLX3、TRH、TRIM58、VAV3、VSTM2B、WDR17、ZNF254、ZNF43、ZNF486、ZNF491、ZNF518B、ZNF542、ZNF625、ZNF665、ZNF671、ZNF763、ZNF844、AGRN、ANKRD35、ARHGAP27、ARHGAP30、BCL2L11、BIN2、C10orf114、C4orf31、C6orf132、C6orf186、CCDC88B、CRHBP、DAPK1、DNMT3A、DPP10、FAM19A2、FLJ45983、FOSL1、FOXB1、GREM1、HMHA1、HOXA9、IFFO1、INPP4B、ITGB2、ITGB4、ITPKB、KCNIP2、KLHDC7B、LAT、LHX6, LIMK1, LOC100128239, LOC100192379, LOC646278, MAP2K2, MAX. chr1.210426156-210426257, MAX. chr1.84326495-84326656, MAX. chr10.119312785-119312882, MAX. chr15.67326025-67326060, MAX. chr16.54316401-54316453, MAX. chr16.85482306-85482494, MAX. chr17.74994454-74994572, MAX. chr17.76339840-76339972, MAX. chr2.7571082-7571136, MAX. chr21.45577347-45577679, MAX. chr3.14852538-14852568, MAX. chr3.187676564-187676668, MAX. chr4.174430662-174430793, MAX. chr5.177411809-177411836, MAX. chr6.45631561-45631625, MAX. chr7.25892382-25892451, MAX. chr7.402563-402641, MAX. chr7.64349554-64349606, MAX. chr8.142046239-142046398, MAX. chr8.145900842-145901246, MAX. chr9.126101804-126101848, MAX. chr9.126978999-126979182, MAX. chr9.36458633-36458725, MAX. chr9.87905315-87905326, MBP, MFNG, MT1A, MT1IP, NCOR2, NFATC1, NKX3-2, NRN1, OLIG1 , PALLD, PAPLN, PDLIM2, PKN1, PRDM14, PRKG1, PRMT7, PTGER2, PTK2B, RAD52, RBM38, RHOF , RNF220, RTN4RL1, RXRA, SDCCAG8, SHROOM1, SKI, SLC12A8, SLC25A47, SPEG, SUCLG2, TBC1D10C, TMEM132E, VIPR2, WDR66, WNT6, ZDHHC18, ZNF382, and / or ZNF626;The method of claim 1.

4. 10. The method of claim 1, wherein the at least one DMR is associated with an area under the ROC curve (AUC) of 0.5 or greater, and the ROC curve distinguishes between DNA samples from subjects having or suspected of having oropharyngeal cancer and control DNA samples.

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

6. The method of claim 5, wherein the tissue sample is selected from an oropharyngeal tissue sample, a soft palate tissue sample, a throat tissue sample, a tongue tissue sample, and / or a tonsil tissue sample.

7. 10. The method of claim 1, wherein the at least one DMR comprises an increased percentage of methylation compared to a control DNA sample.

8. 10. The method of claim 1, wherein the at least one DMR comprises an increased hypermethylation rate compared to a control DNA sample.

9. The method described in claim 1, wherein the at least one DMR distinguishes between a subject having or suspected of having oropharyngeal cancer and a control DNA sample, and the control sample is derived from a subject not having oropharyngeal cancer.

10. 10. The method of claim 9, wherein the control DNA sample is selected from a tissue sample, a blood sample, a plasma sample, a serum sample, a whole blood sample, a buffy coat sample, a secretion sample, an organ secretion sample, a cerebrospinal fluid (CSF) sample, a saliva sample, a urine sample, and / or a stool sample.

11. 11. The method of claim 10, wherein the tissue sample is selected from an oropharyngeal tissue sample, a soft palate tissue sample, a throat tissue sample, a tongue tissue sample, and / or a tonsil tissue sample.

12. The method of claim 1 , wherein the subject is a human.

13. 10. The method of claim 1, wherein the sample is obtained from the subject, and the method further comprises extracting the DNA sample from the sample.

14. 10. The method of claim 1, wherein the sample is collected with a collection device.

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

16. 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.

17. 10. The method of claim 1, wherein determining the methylation profile of at least one DMR comprises amplifying at least a portion of the at least one DMR using a set of primers.

18. 2. The method of claim 1, wherein determining the methylation profile of at least one DMR comprises performing at least one 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.

19. 10. The method of claim 1, wherein determining the methylation profile of at least one DMR comprises determining the presence or absence of methylation at one or more CpG sites.

20. 20. The method of claim 19, wherein the one or more CpG sites are present in a coding region, a non-coding region, and / or a regulatory region of a gene.

21. 10. The method of claim 1, wherein determining the methylation profile of at least one DMR comprises determining a methylation frequency.

22. 10. The method of claim 1, wherein determining the methylation profile of at least one DMR comprises determining a methylation pattern.

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