Detection and prognosis of cervical cancer

Abstract

The present invention relates to methods and kits for identifying, diagnosing, prognosing, and monitoring cervical cancer. These methods include determining the methylation status or the expression levels of particular genes, or a combination thereof.

1 . A method for identifying, in a test sample, cervical cancer or its precursor, or predisposition to cervical cancer, said method comprising: providing a test sample comprising cervical cells or nucleic acids from cervical cells; detecting in said test sample epigenetic modification of at least one gene selected from the group consisting of JAM3, LMX1A, CDO1, NID2, ALX3, ALX4, AR, ARID4A, ATM, AURKA, B4GALT1, BMP2, BMP6, BNIP3, C13orf18, C16orf48, C9orf19, CALCA, CAMK4, CCNA1, CCND2, CDH1, CDH4, CDK6, CDKN1B, CDKN2B, CLSTN2, CLU, COL1A1, CPT1C, CTDSPL, CYCLIND2, DAPK1, DBC1, DDX19B, DKK2, EGFR, EGR4, EPB41L3, FOS, FOXE1, GADD45A, GATA4, GDAP1L1, GNB4, GPNMB, GREM1, Gst-Pi, HHIP, HIN1, HOOK2, HOXA1, HOXA11, HOXA7, HOXD1, IGSF4, ISYNA1, JPH3, KNDC1, KRAS, LAMA1, LOC285016, LOX, LTB4R, MAL, MTAP, MYO18B, NDRG2, NOL4, NPTX1, NPTX2, OGFOD2, PAK3, PAX1, PDCD4, PHACTR3, POMC, PRKCE, RAD23B, RALY, RARA, RASSF1A, RBP4, RECK, RPRM, SALL4, SEMA3F, SLC5A8, SLIT1, SLIT2, SLIT3, SMPD1, SOCS1, SOX1, SOX17, SPARC, SPN, SST, TAC1, TERT, TFPI-2, TLL1, TNFAIP1, TRMT1, TWIST1, UGT1A1, WIF1, WIT1, WT1, XRCC3, and ZGPAT; wherein epigenetic modification in said test sample indicates the presence of cells that are neoplastic, precursor to neoplastic, or predisposed to neoplasia, or the presence of nucleic acids from cells that are neoplastic, precursor to neoplastic, or predisposed to neoplasia. 
     
     
         2 . A method for identifying, in a test sample, cervical cancer or its precursor, or predisposition to cervical cancer, said method comprising:
 providing a test sample comprising cervical cells or nucleic acids from cervical cells;   detecting in said test sample epigenetic modification of at least one gene selected from   the group of JAM3, LMX1A, CDO1, NID2, CCNA1, HOXA11, GREM1 and TAC1; wherein epigenetic modification in said test sample indicates the presence of cells that are neoplastic, precursor to neoplastic, or predisposed to neoplasia, or the presence of nucleic acids from cells that are neoplastic, precursor to neoplastic, or predisposed to neoplasia.   
     
     
         3 . Method for cervical cancer detection or screening comprising the steps of:
 a) providing a test sample comprising cervical cells or nucleic acids from cervical cells;   b) assaying the test sample of step a) for high-risk human papillomavirus (hr-HPV);   c) if b) is positive for the presence of hr-HPV, assaying the methylation status of at least one gene selected from the group consisting of JAM3, LMX1A, CDO1, NID2, ALX3, ALX4, AR, ARID4A, ATM, AURKA, B4GALT1, BMP2, BMP6, BNIP3, C13orf18, C16orf48, C9orf19, CALCA, CAMK4, CCNA1, CCND2, CDH1, CDH4, CDK6, CDKN1B, CDKN2B, CLSTN2, CLU, COL1A1, CPT1C, CTDSPL, CYCLIND2, DAPK1, DBC1, DDX19B, DKK2, EGFR, EGR4, EPB41L3, FOS, FOXE1, GADD45A, GATA4, GDAP1L1, GNB4, GPNMB, GREM1, Gst-Pi, HHIP, HIN1, HOOK2, HOXA1, HOXA11, HOXA7, HOXD1, IGSF4, ISYNA1, JPH3, KNDC1, KRAS, LAMA1, LOC285016, LOX, LTB4R, MAL, MTAP, MYO18B, NDRG2, NOL4, NPTX1, NPTX2, OGFOD2, PAK3, PAX1, PDCD4, PHACTR3, POMC, PRKCE, RAD23B, RALY, RARA, RASSF1A, RBP4, RECK, RPRM, SALL4, SEMA3F, SLC5A8, SLIT1, SLIT2, SLIT3, SMPD1, SOCS1, SOX1, SOX17, SPARC, SPN, SST, TAC1, TERT, TFPI-2, TLL1, TNFAIP1, TRMT1, TWIST1, UGT1A1, WIF1, WIT1, WT1, XRCC3, and ZGPAT;   d) if the at least one gene of c) is methylated, refer the woman for colposcopy;   e) if the at least one gene of c) is unmethylated, refer the woman to a more regular screening for the presence of hr-HPV.   
     
     
         4 . Method for cervical cancer detection or screening comprising the steps of:
 a) providing a test sample comprising cervical cells or nucleic acids from cervical cells;   b) assaying the test sample of step a) for hr-HPV;   c) if b) is positive for the presence of hr-HPV, assaying the methylation status of at least one gene selected from the group consisting of JAM3, LMX1A, CDO1, NID2, ALX3, ALX4, AR, ARID4A, ATM, AURKA, B4GALT1, BMP2, BMP6, BNIP3, C13orf18, C16orf48, C9orf19, CALCA, CAMK4, CCNA1, CCND2, CDH1, CDH4, CDK6, CDKN1B, CDKN2B, CLSTN2, CLU, COL1A1, CPT1C, CTDSPL, CYCLIND2, DAPK1, DBC1, DDX19B, DKK2, EGFR, EGR4, EPB41L3, FOS, FOXE1, GADD45A, GATA4, GDAP1L1, GNB4, GPNMB, GREM1, Gst-Pi, HHIP, HIN1, HOOK2, HOXA1, HOXA11, HOXA7, HOXD1, IGSF4, ISYNA1, JPH3, KNDC1, KRAS, LAMA1, LOC285016, LOX, LTB4R, MAL, MTAP, MYO18B, NDRG2, NOL4, NPTX1, NPTX2, OGFOD2, PAK3, PAX1, PDCD4, PHACTR3, POMC, PRKCE, RAD23B, RALY, RARA, RASSF1A, RBP4, RECK, RPRM, SALL4, SEMA3F, SLC5A8, SLIT1, SLIT2, SLIT3, SMPD1, SOCS1, SOX1, SOX17, SPARC, SPN, SST, TAC1, TERT, TFPI-2, TLL1, TNFAIP1, TRMT1, TWIST1, UGT1A1, WIF1, WIT1, WT1, XRCC3, and ZGPAT, and/or typing the hr-HPV as HPV16 and/or HPV18;   d) if the at least one gene of c) is methylated, and/or the sample is typed HPV16 and/or HPV 18 positive, refer the woman for colposcopy;   e) if the at least one gene of c) is unmethylated, refer the woman to a more regular screening for the presence of hr-HPV.   
     
     
         5 . The method according to  claim 1  wherein the test sample comprises squamous cells, nucleic acids from squamous cells, adenocarcinoma cells, nucleic acids from adenocarcinoma cells, adenosquamous cell carcinoma cells, nucleic acids from adenosquamous carcinoma cells, or any combination thereof. 
     
     
         6 . The method according to  claim 1  wherein the test sample is from a specimen selected from the group consisting of a tissue specimen, a biopsy specimen, a surgical specimen, a cytological specimen, cervical scrapings, cervical smear, cervical washing, vaginal excretions, and blood. 
     
     
         7 . The method of  claim 6  wherein the test sample is from a biopsy specimen and surgical removal of neoplastic tissue is recommended to the patient. 
     
     
         8 . The method of  claim 6  wherein the specimen is a surgical specimen and adjuvant chemotherapy or adjuvant radiation therapy is recommended to the patient. 
     
     
         9 . The method according to  claim 1  wherein epigenetic modification of at least two genes is detected. 
     
     
         10 . The method according to  claim 1  wherein epigenetic modification is detected by detecting methylation of a CpG dinucleotide motif in the gene, or a promoter region thereof. 
     
     
         11 . The method according to  claim 1  wherein epigenetic modification is detected by detecting expression of mRNA of the gene. 
     
     
         12 . The method of  claim 10  wherein methylation is detected by: contacting at least a portion of the gene or promoter region thereof of the test sample with a chemical reagent that selectively modifies a non-methylated cytosine residue relative to a methylated cytosine residue, or selectively modifies a methylated cytosine residue relative to a non-methylated cytosine residue; and detecting a product generated due to said contacting. 
     
     
         13 . The method of  claim 12  wherein the step of detecting a product employs amplification with at least one primer that hybridizes to a sequence comprising a modified non-methylated CpG dinucleotide motif but not to a sequence comprising an unmodified methylated CpG dinucleotide motif thereby forming amplification products. 
     
     
         14 . The method of  claim 12  wherein the step of detecting a product comprises amplification with at least one primer that hybridizes to a sequence comprising an unmodified methylated CpG dinucleotide motif but not to a sequence comprising a modified non-methylated CpG dinucleotide motif thereby forming amplification products. 
     
     
         15 . The method of  claim 12  wherein the product is detected by a method selected from the group consisting of electrophoresis, hybridization, amplification, sequencing, ligase chain reaction, chromatography, mass spectrometry, and combinations thereof. 
     
     
         16 . The method of  claim 12  wherein the chemical reagent comprises bisulfite ions. 
     
     
         17 . The method of  claim 16 , further comprising treating the bisulfite ion-contacted, at least a portion of the gene with alkali. 
     
     
         18 . The method according to  claim 1  wherein the step of detecting employs amplification of at least a portion of the at least one gene using an oligonucleotide primer that specifically hybridizes under amplification conditions to a region of a gene selected from the group consisting of JAM3, LMX1A, CDO1, NID2, ALX3, ALX4, AR, ARID4A, ATM, AURKA, B4GALT1, BMP2, BMP6, BNIP3, C13orf18, C16orf48, C9orf19, CALCA, CAMK4, CCNA1, CCND2, CDH1, CDH4, CDK6, CDKN1B, CDKN2B, CLSTN2, CLU, COL1A1, CPT1C, CTDSPL, CYCLIND2, DAPK1, DBC1, DDX19B, DKK2, EGFR, EGR4, EPB41L3, FOS, FOXE1, GADD45A, GATA4, GDAP1L1, GNB4, GPNMB, GREM1, Gst-Pi, HHIP, HIN1, HOOK2, HOXA1, HOXA11, HOXA7, HOXD1, IGSF4, ISYNA1, JPH3, KNDC1, KRAS, LAMA1, LOC285016, LOX, LTB4R, MAL, MTAP, MYO18B, NDRG2, NOL4, NPTX1, NPTX2, OGFOD2, PAK3, PAX1, PDCD4, PHACTR3, POMC, PRKCE, RAD23B, RALY, RARA, RASSF1A, RBP4, RECK, RPRM, SALL4, SEMA3F, SLC5A8, SLIT1, SLIT2, SLIT3, SMPD1, SOCS1, SOX1, SOX17, SPARC, SPN, SST, TACT, TERT, TFPI-2, TLL1, TNFAIP1, TRMT1, TWIST1, GT1A1, WIF1, WIT1, WT1, XRCC3, and ZGPAT; wherein the region is within about 10 kb of said gene's transcription start site. 
     
     
         19 . The method according to  claim 1  wherein the step of detecting employs amplification of at least a portion of the at least one gene using at least one pair of oligonucleotide primers that specifically hybridizes under amplification conditions to a region of a gene selected from the group consisting of genes according to JAM3, LMX1A, CDO1, NID2, ALX3, ALX4, AR, ARID4A, ATM, AURKA, B4GALT1, BMP2, BMP6, BNIP3, C13orf18, C16orf48, C9orf19, CALCA, CAMK4, CCNA1, CCND2, CDH1, CDH4, CDK6, CDKN1B, CDKN2B, CLSTN2, CLU, COL1A1, CPT1C, CTDSPL, CYCLIND2, DAPK1, DBC1, DDX19B, DKK2, EGFR, EGR4, EPB41L3, FOS, FOXE1, GADD45A, GATA4, GDAP1L1, GNB4, GPNMB, GREM1, Gst-Pi, HHIP, HIN1, HOOK2, HOXA1, HOXA11, HOXA7, HOXD1, IGSF4, ISYNA1, JPH3, KNDC1, KRAS, LAMA1, LOC285016, LOX, LTB4R, MAL, MTAP, MYO18B, NDRG2, NOL4, NPTX1, NPTX2, OGFOD2, PAK3, PAX1, PDCD4, PHACTR3, POMC, PRKCE, RAD23B, RALY, RARA, RASSF1A, RBP4, RECK, RPRM, SALL4, SEMA3F, SLC5A8, SLIT1, SLIT2, SLIT3, SMPD1, SOCS1, SOX1, SOX17, SPARC, SPN, SST, TAC1, TERT, TFPI-2, TLL1, TNFAIP1, TRMT1, TWIST1, UGT1A1, WIF1, WIT1, WT1, XRCC3, and ZGPAT; wherein the region is within about 10 kb of said gene's transcription start site. 
     
     
         20 . A kit for assessing cervical cancer or its precursor, or predisposition to cervical cancer in a test sample containing cervical cells or nucleic acids from cervical cells, said kit comprising in a package:
 a reagent that (a) modifies methylated cytosine residues but not non-methylated cytosine residues, or that (b) modifies non-methylated cytosine residues but not methylated cytosine residues; and   at least one pair of oligonucleotide primers that specifically hybridizes under amplification conditions to a region of a gene selected from the group consisting of JAM3, LMX1A, CDO1, NID2, ALX3, ALX4, AR, ARID4A, ATM, AURKA, B4GALT1, BMP2, BMP6, BNIP3, C13orf18, C16orf48, C9orf19, CALCA, CAMK4, CCNA1, CCND2, CDH1, CDH4, CDK6, CDKN1B, CDKN2B, CLSTN2, CLU, COL1A1, CPT1C, CTDSPL, CYCLIND2, DAPK1, DBC1, DDX19B, DKK2, EGFR, EGR4, EPB41L3, FOS, FOXE1, GADD45A, GATA4, GDAP1L1, GNB4, GPNMB, GREM1, Gst-Pi, HHIP, HIN1, HOOK2, HOXA1, HOXA11, HOXA7, HOXD1, IGSF4, ISYNA1, JPH3, KNDC1, KRAS, LAMA1, LOC285016, LOX, LTB4R, MAL, MTAP, MYO18B, NDRG2, NOL4, NPTX1, NPTX2, OGFOD2, PAK3, PAX1, PDCD4, PHACTR3, POMC, PRKCE, RAD23B, RALY, RARA, RASSF1A, RBP4, RECK, RPRM, SALL4, SEMA3F, SLC5A8, SLIT1, SLIT2, SLIT3, SMPD1, SOCS1, SOX1, SOX17, SPARC, SPN, SST, TAC1, TERT, TFPI-2, TLL1, TNFAIP1, TRMT1, TWIST1, UGT1A1, WIF1, WIT1, WT1, XRCC3, and ZGPAT; wherein the region is within about 10 kb of said gene's transcription start site.   
     
     
         21 . The kit of  claim 20  wherein the at least one pair of primers is selected from primers according to SEQ ID NO: 1-264 and/or SEQ ID NO: 426-437. 
     
     
         22 . The kit of  claim 20  wherein the at least one pair of oligonucleotide primers amplifies an amplicon selected from SEQ ID NO: 265-396 and/or SEQ ID NO: 438-443. 
     
     
         23 . The kit of  claim 20  further comprising at least one oligonucleotide probe which hybridizes to an amplicon selected from SEQ ID NO: 265-396 and/or SEQ ID NO: 438-443. 
     
     
         24 . The kit of  claim 23  wherein the oligonucleotide probe is selected from the group consisting of SEQ ID NO: 397-425. 
     
     
         25 . The kit of  claim 20  further comprising a DNA polymerase for amplifying DNA. 
     
     
         26 . An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-449.