p16 and hMLH1 hypermethylation in synchronous endometrial and ovarian carcinomas
Department of Human Morphology, University of Insubria and Ospedale di Circolo, Varese, Italy
AIM: The pathogenesis and the molecular profile of synchronous primary endometrial and ovarian carcinomas are completely unknown, although the coexistence of the two malignancies as independent tumors is a fairly common and well-known phenomenon. The aim of our study was to assess the incidence of a microsatellite instability (MSI) phenotype in a series of synchronous primary endometrial and ovarian carcinomas evaluating also the frequency of hypermethylation and/or loss of expression of hMLH1, hMSH2 and p16INK4a genes. METHODS: The MSI status of 33 tumors (19 ovarian and 14 endometrial carcinomas) from 14 patients was established by a fluorescent pentaplex PCR at Bat-26, Bat-25, NR-21, NR-22, NR-24 mononucleotide markers using an automated DNA sequencer (Applied Biosystems 310). DNA methylation patterns in CpG islands of hMLH1 and p16INK4a genes were determined by Methylation-Specific PCR (MSP) on tumor DNA subjected to bisulfite modification. Immuohistochemical expression of hMLH1 and hMSH2 genes were evaluated in all cases using monoclonal antibodies. RESULTS: Six patients showed simultaneous presence of MSI in both endometrial and ovarian tumors, therefore a total of 14 (6 endometrial and 8 ovarian carcinomas) of 33 tumors (42%) were scored as MSI. The remaining 19 tumors from 8 patients were microsatellite stable (MSS). All MSI tumors showed both a hMLH1 hypermethylation and lack of immunohistochemical expression of the protein. On the contrary, no MSS tumor exhibited an abnormal methylated pattern or reduced expression of hMLH1. The immunohistochemical expression of hMSH2 was normal in all the 33 tested tumors. The additional hypermethylation analysis of p16INK4a revealed an abnormal methylated pattern of the gene in 12 of 27 tumors analysed (44%; 6 endometrial and 6 ovarian carcinomas, regardless of the MSI status). Of these hypermethylated cases, 6 tumors exhibited MSI (50% of the MSI tumors tested) and 6 tumors were MSS (40% of the MSS tumors tested). CONCLUSIONS: This work demonstrates a high frequency of MSI and de novo methylation of specific gene promoters, such as hMLH1 and p16, in a series of synchronous ovarian and endometrial tumors. Secondly, the very similar molecular profiles observed in synchronous tumors, fulfilling conventional clinico-pathological criteria for independent primary malignancies, suggest that common carcinogenic agents and/or unknown genetic predisposing events may induce tumorigenesis in two embryologically related tissues, as a result of field effect.
Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in poster session 893 (Molecular pathology).