Detection and prognosis of cervical cancer
View Patent ↗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 and performing screening or treating, said method comprising: providing a test sample comprising cervical cells or nucleic acids from cervical cells; detecting in said test sample methylation of at least one gene selected from the group consisting of EPB41L3, JAM3, and TERT 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, the product obtained by contacting at least a portion of the TERT gene or promoter region thereof comprising SEQ ID NO:384; wherein methylation 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, wherein
if the at least one gene is methylated, refer the woman for colposcopy; or
if the at least one gene is unmethylated, refer the woman to screening for the presence of hr-HPV; or
if the at least one gene is methylated, refer the woman for cervical resection.
2. The method of claim 1 , further comprising detecting in said test sample the methylation of C13orf18.
3. 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.
4. 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.
5. The method of claim 4 , wherein the test sample is from a biopsy specimen and surgical removal of neoplastic tissue is recommended to the patient.
6. The method of claim 4 , wherein the specimen is a surgical specimen and adjuvant chemotherapy or adjuvant radiation therapy is recommended to the patient.
7. The method according to claim 1 , wherein epigenetic modification of at least two genes is detected.
8. The method according to claim 1 , wherein said methylation is at a CpG dinucleotide motif in the gene, or a promoter region thereof.
9. The method of claim 1 , 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.
10. The method of claim 1 , 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.
11. The method of claim 1 , 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.
12. The method of claim 1 , wherein the chemical reagent comprises bisulfite ions.
13. The method of claim 12 , further comprising treating the bisulfite ion-contacted, at least a portion of the gene with alkali.
14. 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 EPB41L3, JAM3, and TERT; wherein the region is within about 10 kb of said gene's transcription start site.
15. 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 EPB41L3, JAM3, and TERT; wherein the region is within about 10 kb of said gene's transcription start site.
16. The method of claim 15 , further comprising one pair of oligonucleotide primers that specifically hybridize under amplification conditions to C13orf18.
17. A method for identifying, in a test sample, cervical cancer or its precursor, or predisposition to cervical cancer and performing screening or treating, said method comprising: providing a test sample comprising cervical cells or nucleic acids from cervical cells; detecting in said test sample methylation of at least one gene selected from the group consisting of EPB41L3, JAM3, and TERT 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, the product comprising at least one of SEQ ID NO:309, SEQ ID NO:310, SEQ ID NO:331, and SEQ ID NO:384; wherein methylation 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, wherein
if the at least one gene is methylated, refer the woman for colposcopy; or
if the at least one gene is unmethylated, refer the woman to screening for the presence of hr-HPV; or
if the at least one gene is methylated, refer the woman for cervical resection.
18. The method of claim 17 , further comprising detecting in said test sample the methylation of C13orf18.
19. The method of claim 17 , wherein the test sample is from a biopsy specimen and surgical removal of neoplastic tissue is recommended to the patient.
20. The method of claim 17 , wherein the specimen is a surgical specimen and adjuvant chemotherapy or adjuvant radiation therapy is recommended to the patient.
21. A method for identifying, in a test sample, cervical cancer or its precursor, or predisposition to cervical cancer and performing screening or treating, said method comprising: providing a test sample comprising cervical cells or nucleic acids from cervical cells; detecting in said test sample methylation of at least one gene selected from the group consisting of EPB41L3, JAM3, and TERT 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 by amplifying the contacted portion and detecting an amplification product, the amplification product comprising at least one of SEQ ID NO:309, SEQ ID NO:310, SEQ ID NO:331, and SEQ ID NO:384; wherein methylation 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, wherein
if the at least one gene is methylated, refer the woman for colposcopy; or
if the at least one gene is unmethylated, refer the woman to screening for the presence of hr-HPV; or
if the at least one gene is methylated, refer the woman for cervical resection.
22. The method of claim 21 , wherein the step of detecting a product comprises amplifying the contacted portion 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.
23. The method of claim 21 , wherein the step of detecting a product comprises amplifying the contacted portion 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.