RESUMO
BACKGROUND: To discover cancer specific DNA methylation markers, large-scale screening methods are widely used. The pharmacological unmasking expression microarray approach is an elegant method to enrich for genes that are silenced and re-expressed during functional reversal of DNA methylation upon treatment with demethylation agents. However, such experiments are performed in in vitro (cancer) cell lines, mostly with poor relevance when extrapolating to primary cancers. To overcome this problem, we incorporated data from primary cancer samples in the experimental design. A strategy to combine and rank data from these different data sources is essential to minimize the experimental work in the validation steps. AIM: To apply a new relaxation ranking algorithm to enrich DNA methylation markers in cervical cancer. RESULTS: The application of a new sorting methodology allowed us to sort high-throughput microarray data from both cervical cancer cell lines and primary cervical cancer samples. The performance of the sorting was analyzed in silico. Pathway and gene ontology analysis was performed on the top-selection and gives a strong indication that the ranking methodology is able to enrich towards genes that might be methylated. Terms like regulation of progression through cell cycle, positive regulation of programmed cell death as well as organ development and embryonic development are overrepresented. Combined with the highly enriched number of imprinted and X-chromosome located genes, and increased prevalence of known methylation markers selected from cervical (the highest-ranking known gene is CCNA1) as well as from other cancer types, the use of the ranking algorithm seems to be powerful in enriching towards methylated genes.Verification of the DNA methylation state of the 10 highest-ranking genes revealed that 7/9 (78%) gene promoters showed DNA methylation in cervical carcinomas. Of these 7 genes, 3 (SST, HTRA3 and NPTX1) are not methylated in normal cervix tissue. CONCLUSION: The application of this new relaxation ranking methodology allowed us to significantly enrich towards methylation genes in cancer. This enrichment is both shown in silico and by experimental validation, and revealed novel methylation markers as proof-of-concept that might be useful in early cancer detection in cervical scrapings.
RESUMO
We report three Dutch families with familial clustering of (pre)neoplastic cervical disease, review the literature on familial risks of cervical intraepithelial neoplasia (CIN) and cervical cancer, and discuss possible practical guidelines for women with a family history of cervical cancer. Daughters and sisters of women with cervical cancer have been reported to have a relative risk of 1.5-2.3 to develop this type of cancer. From a practical clinical point of view, we suggest that as in women with an increased non-genetic risk to develop cervical cancer (e.g. because of immunosuppressive therapy) increased surveillance to detect this tumour should be considered in women with an increased risk based on family history. Cessation of smoking should be advised. As the use of condoms at least prevents HPV re-infection its use can be recommended as a way to lower the cervical cancer risk. Future studies to determine the genetic contribution to the development of cervical cancer should include the paternal family history of cancer and, because genetic predisposition might express itself as a higher risk to develop precursors of cervical cancer, carcinoma in situ and CIN grade II-III.
