Epigenetic markers and related methods and means for the detection and management of certain cancers
The present invention relates to methods of determining the presence or absence of certain cancers in a human individual, as well as to related methods to determine the response to therapy against certain cancers in a human individual, in particular ovarian cancer in a woman. Such methods are based on the detection—from (eg cell-free) DNA of said human individual—of one or more methylated (or un-methylated) CpGs being associated with differentially methylated regions (DMRs) of the present invention; such as methylation (or un-methylation) at one or more or all of certain CpGs being associated with such DMRs. Accordingly, such methods have diagnostic, prognostic and/or predictive utility for detecting or managing certain cancers in women or men, in particular ovarian cancer in women. The present invention further relates to nucleic acids comprising certain sequences that may be detected during the method, or nucleic acids (such as probes and/or primers) that are useful to detect such sequences, as wells as compositions, kits, computer program products and other aspects that are useful for or related to the practice or application of such methods.
1. A method of determining the presence of, or a reduced response to a carboplatin therapy against, a cancer in a human individual, said method comprising the steps:
isolating cell-free DNA from a serum or plasma sample from said human individual;
treating said cell-free DNA with bisulfite that differentially modifies said cell-free DNA based on the methylation status of CpGs in the cell-free DNA;
bisulfite sequencing said modified cell-free DNA;
determining, in at least one molecule of said cell-free DNA using a computer, the methylation status at 7 CpGs located at positions 37, 42, 44, 60, 68, 77 and 86 within the nucleotide sequence of SEQ ID NO:3 (DMR #228) or a 100% complementary sequence of said nucleotide sequence and optionally at 11 CpGs located at positions 28, 31, 34, 48, 54, 67, 70, 82, 88, 101 and 104 within the nucleotide sequences of SEQ ID NO:4 (DMR #144), or a 100% complementary sequence of said nucleotide sequence(s);
determining the presence of the cancer in said human individual, or the reduced response to said carboplatin therapy against the cancer in said human individual when at least one molecule of said cell-free DNA is methylated at each of said 7 CpGs within the nucleotide sequence of SEQ ID NO: 3 and optionally at each of said 11 CpGs within the nucleotide sequence of SEQ ID NO: 4; and
treating the cancer in said human individual by (i) administering a chemotherapeutic agent selected from the group consisting of paclitaxel, docetaxel, cisplatin, liposomal doxorubicin, gemcitabine, trabectedin, etoposide, cyclophosphamide an angiogenesis inhibitor and a PARP inhibitor, or (ii) by performing tumor de-baulking surgery;
wherein: (a) when the human individual is a woman the cancer is selected from the group consisting of: breast cancer, lung cancer, colon cancer, pancreatic cancer, uterine (endometrial) cancer, cervical cancer and ovarian cancer; or (b) when the human individual is a man the cancer is selected from the group consisting of: lung cancer, colon cancer, pancreatic cancer and prostate cancer.
2. The method of claim 1 , wherein the method further comprises determining the methylation status at one or more CpGs located within at least one nucleotide sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, and a 100% complementary sequence of said nucleotide sequence(s).
3. The method of claim 1 , wherein the methylation status of said CpG(s) is determined in multiple molecules of said cell-free DNA.
4. The method of claim 3 , wherein a fraction or ratio of, or an absolute number of, cell-free DNA molecules in said sample having said methylated CpG(s) located within said nucleotide sequence of SEQ ID NO:3, and/or having a pattern of methylation for the nucleotide sequence of SEQ ID NO:65, is estimated.
5. The method of claim 1 , wherein the method is for determining the reduced response of a human individual suffering from a cancer to a carboplatin therapy against said cancer.
6. The method of claim 1 , further comprising using a primer pair for amplifying the nucleic acid sequence of SEQ ID NO:65 or a 100% complementary sequence of said nucleotide sequence.
7. The method of claim 1 , wherein the human individual is a woman and the cancer is selected from the group consisting of: breast cancer, uterine (endometrial) cancer, and ovarian cancer.
8. The method of claim 7 , wherein the cancer is ovarian cancer.
9. The method of claim 1 , wherein the carboplatin therapy is carboplatin-based neoadjuvant chemotherapy.
10. A method of treating cancer in a human individual previously treated with carboplatin comprising administering a chemotherapeutic agent to said human individual, wherein said chemotherapeutic agent is administered to the human individual within about 3 months of said human individual having been determined to have a reduced response to a carboplatin therapy by the method of claim 1 , and wherein the chemotherapeutic agent is selected from the group consisting of: paclitaxel, docetaxel, cisplatin, liposomal doxorubicin, gemcitabine, trabectedin, etoposide, cyclophosphamide an angiogenesis inhibitor and a PARP inhibitor.
11. The method of claim 10 , wherein the human individual is a woman and the cancer is selected from the group consisting of: breast cancer, uterine (endometrial) cancer, and ovarian cancer.
12. The method of claim 11 , wherein the cancer is ovarian cancer.
13. The method of claim 10 , wherein the carboplatin therapy is carboplatin-based neoadjuvant chemotherapy.