Promoter methylation in the PTCH gene in cervical epithelial cancer and ovarian cancer tissue as studied by eight novel Pyrosequencing assays

DNA methylation status in the CpG sites of promoter regions in cancer-related genes, such as PTCH, has traditionally been investigated using either dye-terminator sequencing or methylation-specific PCR. We aimed to study the PTCH gene promoter methylation in gynecological cancers, with a method that gives a quantitative measure of the methylation status of the promoter region of the studied gene, and for this purpose, we designed novel Pyrosequencing based assays. Bisulfite-treated genomic DNA (bsDNA)was amplified by standard PCR and applied to novel Pyrosequencing assays, in order to measure the methylated fraction(%) at each CpG site of the PTCH gene promoter. We analyzed 22 squamous cell cervical cancer tissue specimens (11 with good and 11 with poor outcomes after radiotherapy)and 5 ovarian cancer tissue specimens matched with 5 normal ovarian tissue specimens. Six optimized PCR protocols which generated 8 Pyrosequencing assays covering 63 CpG sites in the promoter regions 1 and 2 as well as the previously unanalyzed promoter region 3 in the PTCH gene were developed. The 27 tumor tissue specimens and 5 normal tissues did not show any methylation within any of the 63 CpG sites. Our data suggest that methylation of the PTCH promoter is not a high-prevalence feature of squamous cell cervical cancer or ovarian cancer, but Pyrosequencing assays are a good method for studying promoter methylation

Whole genome expression profiling of blood cells in ovarian cancer patients : prognostic impact of the CYP1B1, MTSS1, NCALD, and NOP14 genes

Ovarian cancer patients with different tumor stages and cell differentiation might be distinguished from each other by gene expression profiles in whole blood cell mRNA by the Affymetrix Human Gene 1.0 ST Array. We also examined if there is any association with other clinical variables, response to therapy, and residual tumor burden after surgery. Patients were divided into two groups, one with poor prognosis, advanced stage and poorly differentiated tumors (n = 22), and one group with good prognosis, early stage and well-to medium differentiated tumors (n = 11). Six genes were found to be differentially expressed: the PDIA3, LYAR, NOP14, NCALD and MTSS1 genes were down-regulated and the CYP1B1 gene expression was up-regulated in the poor prognosis group, all with p value <0.05, adjusted for mass comparison. In survival analyses, CYP1B1, MTSS1, NCALD and NOP14 remained significantly different (p<0.05). Patient groups did not differ in any transcript related to acute phase or immune responses. This minimal gene expression signature of prognostic ovarian cancer-related genes opens up an avenue for more practicable monitoring of ovarian cancer patients by simple peripheral blood tests, which may evolve into a tool to guide selection of curative and postoperative supportive therapies

Whole genome expression profiling of blood cells in ovarian cancer patients -Prognostic impact of the CYP1B1, MTSS1, NCALD, and NOP14 genes

This is the published version of a paper published in OncoTarget. Citation for the original published paper (version of record): Isaksson, H., Sorbe, B., Nilsson, T. (2014) Whole genome expression profiling of blood cells in ovarian cancer patients: prognostic impact of the CYP1B1, MTSS1, NCALD, and NOP14 genes. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. OncoTarget ABSTRACT Ovarian cancer patients with different tumor stages and cell differentiation might be distinguished from each other by gene expression profiles in whole blood cell mRNA by the Affymetrix Human Gene 1.0 ST Array. We also examined if there is any association with other clinical variables, response to therapy, and residual tumor burden after surgery. Patients were divided into two groups, one with poor prognosis, advanced stage and poorly differentiated tumors (n = 22), and one group with good prognosis, early stage and well-to medium differentiated tumors (n = 11). Six genes were found to be differentially expressed: the PDIA3, LYAR, NOP14, NCALD and MTSS1 genes were down-regulated and the CYP1B1 gene expression was up-regulated in the poor prognosis group, all with p value <0.05, adjusted for mass comparison. In survival analyses, CYP1B1, MTSS1, NCALD and NOP14 remained significantly different (p<0.05). Patient groups did not differ in any transcript related to acute phase or immune responses. This minimal gene expression signature of prognostic ovarian cancer-related genes opens up an avenue for more practicable monitoring of ovarian cancer patients by simple peripheral blood tests, which may evolve into a tool to guide selection of curative and postoperative supportive therapies

Hypermethylation of promoter regions of the APC1A and p16INK4a genes in relation to prognosis and tumor characteristics in cervical cancer patients

Hypermethylation of the O6-MGMT, p14ARF, p16INK4a, RASSF1A and APC1A genes are unfavourable prognostic markers in colorectal cancer (CRC). We hypothesized that they could be related to prognosis also in cervical cancer. Methylation was studied in DNA extracts from surgical specimens of cancer tissue by novel pyrosequencing methods. In 109 patients (90 squamous cell carcinomas, 19 adenocarcinomas), we found that hypermethylation of the APC1A gene promoter occurred in 8.3% of patients, and of p16INK4a in 1.8%. APC1A hypermethylation was significantly related to more advanced FIGO stage of the tumor (P=0.013), larger tumor diameter (P=0.049) and distant recurrence-free survival (P=0.0007), but not with locoregional recurrence rate, age, HPV status, DNA ploidy, tumor grade or malignancy grading score. We conclude that methylation of the APC1A promoter in cervical cancer, as diagnosed by pyrosequencing, is significantly related to major biological characteristics of the tumor, and may be a new predictor of poor prognosis in cervical cancer