SMARCB1 protein and mRNA loss is not caused by promoter and histone hypermethylation in epithelioid sarcoma

About 10% of epithelioid sarcomas have biallelic mutation of the SMARCB1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily b, member 1) gene resulting in a lack of this nuclear protein. It has been suggested that SMARCB1 may be silenced by epigenetic changes in the remaining 90% of tumors. Thus, we hypothesized that the promoter of SMARCB1 is hypermethylated. We also examined SMARCB1 mRNA level to determine if a post-translational change was possible. Thirty-six cases of epithelioid sarcomas were studied. Immunohistochemistry and mutation analysis of the SMARCB1 gene were performed to select appropriate cases. Methylation status was assessed by methylation-specific PCR. Laser capture microdissection of tumor cells followed by real-time PCR was applied to examine the expression of SMARCB1 mRNA. Of 36 epithelioid sarcomas, 31 (86%) displayed a lack of SMARCB1 nuclear protein. In all, 4 (13%) of 31 SMARCB1-negative cases harbored biallelic deletion while 9 (33%) cases showed single-allelic deletion. One (4%) frameshift deletion of exon 3 and one point mutation of exon 7 were also found. In 16 (59%) cases, both alleles were intact. Altogether, 25/31 (81%) SMARCB1-negative cases had at least one intact allele. None of these cases demonstrated promoter hypermethylation. Low levels of SMARCB1 mRNA were found in all cases with tumor tissue extracted RNA (because of the minimal normal cell contamination) but no mRNA could be detected in laser dissected cases (containing only tumor cells). Enhancer of zeste homolog 2 (EZH2) overexpression was not characteristic of epithelioid sarcoma. Thus, loss of SMARCB1 expression in epithelioid sarcoma is caused neither by DNA hypermethylation nor by post-translational modifications. Most likely it is the microRNA destruction of SMARCB1 mRNA but further investigations are needed to elucidate this issue.Modern Pathology advance online publication, 23 November 2012; doi:10.1038/modpathol.2012.190

<it>MGMT</it>, <it>GATA6</it>, <it>CD81</it>, <it>DR4</it>, and <it>CASP8</it> gene promoter methylation in glioblastoma

<p>Abstract</p> <p>Background</p> <p>Methylation of promoter region is the major mechanism affecting gene expression in tumors. Recent methylome studies of brain tumors revealed a list of new epigenetically modified genes. Our aim was to study promoter methylation of newly identified epigenetically silenced genes together with already known epigenetic markers and evaluate its separate and concomitant role in glioblastoma genesis and patient outcome.</p> <p>Methods</p> <p>The methylation status of <it>MGMT</it>, <it>CD81</it>, <it>GATA6</it>, <it>DR4</it>, and <it>CASP8</it> in 76 patients with primary glioblastomas was investigated. Methylation-specific PCR reaction was performed using bisulfite treated DNA. Evaluating glioblastoma patient survival time after operation, patient data and gene methylation effect on survival was estimated using survival analysis.</p> <p>Results</p> <p>The overwhelming majority (97.3%) of tumors were methylated in at least one of five genes tested. In glioblastoma specimens gene methylation was observed as follows: <it>MGMT</it> in 51.3%, <it>GATA6</it> in 68.4%, <it>CD81</it> in 46.1%, <it>DR4</it> in 41.3% and <it>CASP8</it> in 56.8% of tumors. Methylation of <it>MGMT</it> was associated with younger patient age (p < 0.05), while <it>CASP8</it> with older (p < 0.01). <it>MGMT</it> methylation was significantly more frequent event in patient group who survived longer than 36 months after operation (p < 0.05), while methylation of <it>CASP8</it> was more frequent in patients who survived shorter than 36 months (p < 0.05). Cox regression analysis showed patient age, treatment, <it>MGMT</it>, <it>GATA6</it> and <it>CASP8</it> as independent predictors for glioblastoma patient outcome (p < 0.05). <it>MGMT</it> and <it>GATA6</it> were independent predictors for patient survival in younger patients’ group, while there were no significant associations observed in older patients’ group when adjusted for therapy.</p> <p>Conclusions</p> <p>High methylation frequency of tested genes shows heterogeneity of glioblastoma epigenome and the importance of <it>MGMT</it>, <it>GATA6</it> and <it>CASP8</it> genes methylation in glioblastoma patient outcome.</p