Digital PCR quantification of MGMT methylation refines prediction of clinical benefit from alkylating agents in glioblastoma and metastatic colorectal cancer.

BACKGROUND: O(6)-methyl-guanine-methyl-transferase (MGMT) silencing by promoter methylation may identify cancer patients responding to the alkylating agents dacarbazine or temozolomide. PATIENTS AND METHODS: We evaluated the prognostic and predictive value of MGMT methylation testing both in tumor and cell-free circulating DNA (cfDNA) from plasma samples using an ultra-sensitive two-step digital PCR technique (methyl-BEAMing). Results were compared with two established techniques, methylation-specific PCR (MSP) and Bs-pyrosequencing. RESULTS: Thresholds for MGMT methylated status for each technique were established in a training set of 98 glioblastoma (GBM) patients. The prognostic and the predictive value of MGMT methylated status was validated in a second cohort of 66 GBM patients treated with temozolomide in which methyl-BEAMing displayed a better specificity than the other techniques. Cutoff values of MGMT methylation specific for metastatic colorectal cancer (mCRC) tissue samples were established in a cohort of 60 patients treated with dacarbazine. In mCRC, both quantitative assays methyl-BEAMing and Bs-pyrosequencing outperformed MSP, providing better prediction of treatment response and improvement in progression-free survival (PFS) (P < 0.001). Ability of methyl-BEAMing to identify responding patients was validated in a cohort of 23 mCRC patients treated with temozolomide and preselected for MGMT methylated status according to MSP. In mCRC patients treated with dacarbazine, exploratory analysis of cfDNA by methyl-BEAMing showed that MGMT methylation was associated with better response and improved median PFS (P = 0.008). CONCLUSIONS: Methyl-BEAMing showed high reproducibility, specificity and sensitivity and was applicable to formalin-fixed paraffin-embedded tissues and cfDNA. This study supports the quantitative assessment of MGMT methylation for clinical purposes since it could refine prediction of response to alkylating agents.

Global DNA hypomethylation in prostate cancer development and progression: a systematic review.

BACKGROUND: The role of global DNA methylation in prostate cancer (PCa) remains largely unknown. Our aim was to summarize evidence on the role of global DNA hypomethylation in PCa development and progression. METHODS: We searched PubMed through December 2013 for all studies containing information on global methylation levels in PCa tissue and at least one non-tumor comparison tissue and/or studies reporting association between global methylation levels in PCa tissue and survival, disease recurrence or at least one clinicopathological prognostic factor. We summarized results using non-parametric comparisons and P-value summary methods. RESULTS: We included 15 studies in the review: 6 studies with both diagnostic and prognostic information, 5 studies with only diagnostic information and 4 studies with only prognostic information. Quantitative meta-analysis was not possible because of the large heterogeneity in molecular techniques, types of tissues analyzed, aims and study designs. Summary statistical tests showed association of DNA hypomethylation with PCa diagnosis (P<0.006) and prognosis (P<0.001). Restriction to studies assessing 5-methylcytosine or long interspersed nucleotide element-1 revealed results in the same direction. Analyses restricted to specific clinicopathological features showed association with the presence of metastasis and tumor stage in all tests with P<0.03, and no association with Gleason score (all tests P>0.1 except for the weighted Z-test, P=0.05). CONCLUSION: DNA hypomethylation was associated with PCa development and progression. However, due to the heterogeneity and small sample sizes of the included studies, along with the possibility of publication bias, this association requires additional assessment.

Malignant fibrous histiocytoma associated with a meningothelial meningioma.

A 72-year-old woman was operated for a left parietal tumor of the meninges. The symptomatology began 22 years earlier with a right hemiparesis. The histological examination of the tumor showed a proliferation of meningothelial cells with whorl formation, associated with a pleomorphic proliferation of a malignant fibrous histiocytoma. The meningothelial meningioma showed a typical aspect, with nuclear inclusions and absence of mitotic activity. The second tumor component showed a storiform architecture with giant multinucleated cells, necrosis and many typical and atypical mitoses. There was also an inflammatory component with infiltrates of lymphocytes. This tumor component at the border with the meningioma appeared to arise from the septa of the meningioma with many prongs merging into one large and pleomorphic histiocytomatous tumor. The dual histological aspect and the case are discussed in the light of what is already known in the literature. The woman has been irradiated after surgery and she is doing well 6 months after operation.

Distribution of activated caspase-3 in relation with apoptosis in human malignant gliomas.

Activated caspase-3has been immunohistochemically studied in 30glioblastomas. Its distribution has been compared with that of apoptotic nuclei demonstrated by terminal dUTP nick-end labeling (TUNEL) and morphology. The best procedure for the demonstration of caspase-3 requires formalin fixation, followed by Carnoy fixation, with microwave irradiation. The number of positive cells is lower than that of apoptotic nuclei shown by TUNEL technique, especially in perinecrotic pseudo-palisadings, and there are also qualitative variations. Positive staining occurs in nuclei, cytoplasms or in both cell compartments. The interpretation of Caspase-3 positive staining is based on its crucial position in the final pathway to apoptosis and on the mechanisms by which it cleaves cytoplasmic and nuclear proteins among which inhibitory/caspase-activated DNase system is included.