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Bromodomain and extra-terminal tail (BET) proteins have been identified as potential epigenetic targets in cancer, including glioblastoma. These epigenetic modifiers link the histone code to gene transcription that can be disrupted with small molecule BET inhibitors (BETi). With the aim of developing rational combination treatments for glioblastoma, we analyzed BETi-induced differential gene expression in glioblastoma derived-spheres, and identified 6 distinct response patterns. To uncover emerging actionable vulnerabilities that can be targeted with a second drug, we extracted the 169 significantly disturbed DNA Damage Response genes and inspected their response pattern. The most prominent candidate with consistent downregulation, was the O-6-methylguanine-DNA methyltransferase (MGMT) gene, a known resistance factor for alkylating agent therapy in glioblastoma. BETi not only reduced MGMT expression in GBM cells, but also inhibited its induction, typically observed upon temozolomide treatment. To determine the potential clinical relevance, we evaluated the specificity of the effect on MGMT expression and MGMT mediated treatment resistance to temozolomide. BETi-mediated attenuation of MGMT expression was associated with reduction of BRD4- and Pol II-binding at the MGMT promoter. On the functional level, we demonstrated that ectopic expression of MGMT under an unrelated promoter was not affected by BETi, while under the same conditions, pharmacologic inhibition of MGMT restored the sensitivity to temozolomide, reflected in an increased level of g-H2AX, a proxy for DNA double-strand breaks. Importantly, expression of MSH6 and MSH2, which are required for sensitivity to unrepaired O6-methylGuanin-lesions, was only briefly affected by BETi. Taken together, the addition of BET-inhibitors to the current standard of care, comprising temozolomide treatment, may sensitize the 50% of patients whose glioblastoma exert an unmethylated MGMT promoter.

Purpose: The methylation status of the O6-methylguanine DNA methyltransferase (MGMT) gene promoter is predictive for benefit from temozolomide in glioblastoma (GBM). A clinically optimized cutoff was sought allowing patient selection for therapy without temozolomide, while avoiding to withhold it from patients who may potentially benefit. Experimental Design: Quantitative MGMT methylation-specific PCR data were obtained for newly diagnosed patients with GBM screened or treated with standard radiotherapy and temozolomide in four randomized trials. The pooled dataset was randomly split into a training and test dataset. The unsupervised cutoff was obtained at a 50% probability to be (un)methylated. ROC analysis identified an optimal cutoff supervised by overall survival (OS). Results: For 4,041 patients valid MGMT results were obtained, whereof 1,725 were randomized. The unsupervised cutoff in the training dataset was 1.27 (log2[1,000 × (MGMT+1)/ACTB]), separating unmethylated and methylated patients. The optimal supervised cutoff for unmethylated patients was −0.28 (AUC = 0.61), classifying “truly unmethylated” (≤−0.28) and “gray zone” patients (>−0.28, ≤1.27), the latter comprising approximately 10% of cases. In contrast, for patients with MGMT methylation (>1.27) more methylation was not related to better outcome. Both methylated and gray zone patients performed significantly better for OS than truly unmethylated patients [HR = 0.35, 95% confidence interval (CI), 0.27–0.45, P < 0.0001; HR = 0.58, 95% CI, 0.43–0.78, P < 0.001], validated in the test dataset. The MGMT assay was highly reproducible upon retesting of 218 paired samples (R2 = 0.94). Conclusions: Low MGMT methylation (gray zone) may confer some sensitivity to temozolomide treatment, hence the lower safety margin should be considered for selecting patients with unmethylated GBM into trials omitting temozolomide.

Purpose: EORTC 26082 assessed the activity of temsirolimus in patients with newly diagnosed glioblastoma harboring an unmethylated O6 methylguanine-DNA-methyltransferase (MGMT) promoter. Experimental Design: Patients (n = 257) fulfilling eligibility criteria underwent central MGMT testing. Patients with MGMT unmethylated glioblastoma (n = 111) were randomized 1:1 between standard chemo-radiotherapy with temozolomide or radiotherapy plus weekly temsirolimus (25 mg). Primary endpoint was overall survival at 12 months (OS12). A positive signal was considered >38 patients alive at 12 months in the per protocol population. A noncomparative reference arm of 54 patients evaluated the assumptions on OS12 in a standard-treated cohort of patients. Prespecified post hoc analyses of markers reflecting target activation were performed. Results: Both therapies were administered per protocol with a median of 13 cycles of maintenance temsirolimus. Median age was 55 and 58 years in the temsirolimus and standard arms, the WHO performance status 0 or 1 for most patients (95.5%). In the per protocol population, 38 of 54 patients treated with temsirolimus reached OS12. The actuarial 1-year survival was 72.2% [95% confidence interval (CI), 58.2–82.2] in the temozolomide arm and 69.6% (95% CI, 55.8–79.9) in the temsirolimus arm [hazard ratio (HR) 1.16; 95% CI, 0.77–1.76; P = 0.47]. In multivariable prognostic analyses of clinical and molecular factors, phosphorylation of mTORSer2448 in tumor tissue (HR 0.13; 95% CI, 0.04–0.47; P = 0.002), detected in 37.6%, was associated with benefit from temsirolimus. Conclusions: Temsirolimus was not superior to temozolomide in patients with an unmethylated MGMT promoter. Phosphorylation of mTORSer2448 in the pretreatment tumor tissue may define a subgroup benefitting from mTOR inhibition. Clin Cancer Res; 22(19); 4797–806. ©2016 AACR.

Purpose The clinical consequences of PMS2 germline mutations are poorly understood compared with other Lynch-associated mismatch repair gene (MMR) mutations. The aim of this European cohort study was to define the cancer risk faced by PMS2 mutation carriers. Methods Data were collected from 98 PMS2 families ascertained from family cancer clinics that included a total of 2,548 family members and 377 proven mutation carriers. To adjust for potential ascertainment bias, a modified segregation analysis model was used to calculate colorectal cancer (CRC) and endometrial cancer (EC) risks. Standardized incidence ratios (SIRs) were calculated to estimate risks for other Lynch syndrome–associated cancers. Results The cumulative risk (CR) of CRC for male mutation carriers by age 70 years was 19%. The CR among female carriers was 11% for CRC and 12% for EC. The mean age of CRC development was 52 years, and there was a significant difference in mean age of CRC between the probands (mean, 47 years; range, 26 to 68 years) and other family members with a PMS2 mutation (mean, 58 years; range, 31 to 86 years; P < .001). Significant SIRs were observed for cancers of the small bowel, ovaries, breast, and renal pelvis. Conclusion CRC and EC risks were found to be markedly lower than those previously reported for the other MMR. However, these risks embody the isolated risk of carrying a PMS2 mutation, and it should be noted that we observed a substantial variation in cancer phenotype within and between families, suggesting the influence of genetic modifiers and lifestyle factors on cancer risks.

BACKGROUND:Genome-wide association studies have identified at least 15 independent common genetic variants associated with colorectal cancer (CRC) risk. The aim of this study was to investigate whether 11 of these variants are associated with CRC risk for carriers of germline mutations in DNA mismatch repair (MMR) genes.METHODS:A total of 927 MMR gene mutation carriers (360 MLH1, 442 MSH2, 85 MSH6 and 40 PMS2) from 315 families enrolled in the Colon Cancer Family Registry, were genotyped for the single nucleotide polymorphisms (SNPs): rs16892766 (8q23.3), rs6983267 (8q24.21), rs719725 (9p24), rs10795668 (10p14), rs3802842 (11q23.1), rs4444235 (14q22.2), rs4779584 (15q13.3), rs9929218 (16q22.1), rs4939827 (18q21.1), rs10411210 (19q13.1) and rs961253 (20p12.3). We used a weighted Cox regression to estimate CRC risk for homozygous and heterozygous carriers of the risk allele compared with homozygous non-carriers as well as for an additive per allele model (on the log scale).RESULTS:Over a total of 40,978 person-years observation, 426 (46%) carriers were diagnosed with CRC at a mean age of 44.3 years. For all carriers combined, we found no evidence of an association between CRC risk and the total number of risk alleles (hazard ratio [HR] per risk allele=0.97, 95% confidence interval [CI]=0.88-1.07, p=0.52).CONCLUSIONS:We found no evidence that the SNPs associated with CRC in the general population are modifiers of the risk for MMR gene mutation carriers overall, and therefore any evidence of proven clinical utility in Lynch syndrome.

International audience; This study assessed the prognostic value of several markers involved in gliomagenesis, and compared it with that of other clinical and imaging markers already used. Four-hundred and sixteen adult patients with newly diagnosed glioma were included over a 3-year period and tumour suppressor genes, oncogenes, MGMT and hTERT expressions, losses of heterozygosity, as well as relevant clinical and imaging information were recorded. This prospective study was based on all adult gliomas. Analyses were performed on patient groups selected according to World Health Organization histoprognostic criteria and on the entire cohort. The endpoint was overall survival, estimated by the Kaplan-Meier method. Univariate analysis was followed by multivariate analysis according to a Cox model. p14(ARF), p16(INK4A) and PTEN expressions, and 10p 10q23, 10q26 and 13q LOH for the entire cohort, hTERT expression for high-grade tumours, EGFR for glioblastomas, 10q26 LOH for grade III tumours and anaplastic oligodendrogliomas were found to be correlated with overall survival on univariate analysis and age and grade on multivariate analysis only. This study confirms the prognostic value of several markers. However, the scattering of the values explained by tumour heterogeneity prevents their use in individual decision-making.

// Alboukadel Kassambara 1,2 , Claire Gourzones-Dmitriev 2 , Surinder Sahota 3 , Thierry Reme 1,2 , Jerome Moreaux 1 , Hartmut Goldschmidt 4 , Angelos Constantinou 5 , Philippe Pasero 5 , Dirk Hose 4 , Bernard Klein 1,2,6 1 CHU Montpellier, Institute of Research in Biotherapy, Montpellier, F-34295 FRANCE; 2 INSERM, U1040, Montpellier, F-34197 France; 3 Cancer Sciences Unit, Faculty of Medicine, University of Southampton, UK 4 Medizinische Klinik V, Universitaetsklinikum Heidelberg, Heidelberg D-69120 GERMANY; 5 Institute of Human Genetics, CNRS-UPR1142, Montpellier F-34396 FRANCE; 6 Universite MONTPELLIER1, UFR Medecine Correspondence: Bernard Klein, email: // Keywords : DNA repair, Multiple Myeloma, prognosis Received : December 24, 2013 Accepted : February 23, 2014 Published : February 24, 2014 Abstract DNA repair is critical to resolve extrinsic or intrinsic DNA damage to ensure regulated gene transcription and DNA replication. These pathways control repair of double strand breaks, interstrand crosslinks, and nucleotide lesions occurring on single strands. Distinct DNA repair pathways are highly inter-linked for the fast and optimal DNA repair. A deregulation of DNA repair pathways may maintain and promote genetic instability and drug resistance to genotoxic agents in tumor cells by specific mechanisms that tolerate or rapidly bypass lesions to drive proliferation and abrogate cell death. Multiple Myeloma (MM) is a plasma cell disorder characterized by genetic instability and poor outcome for some patients, in which the compendium of DNA repair pathways has as yet not been assessed for a disease-specific prognostic relevance. We design a DNA repair risk score based on the expression of genes coding for proteins involved in DNA repair in MM cells. From a consensus list of 84 DNA repair genes, 17 had a bad prognostic value and 5 a good prognostic value for both event-free and overall survival of previously-untreated MM patients. The prognostic information provided by these 22 prognostic genes was summed within a global DNA repair score ( DR Score) to take into account the tight linkage of repair pathways. DR score was strongly predictive for both patients’ event free and overall survivals. Also, DR score has the potential to identify MM patients whose tumor cells are dependent on specific DNA repair pathways to design treatments that induce synthetic lethality by exploiting addiction to deregulated DNA repair pathways.

The methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) gene is an important predictive biomarker for benefit from alkylating agent therapy in glioblastoma. Recent studies in anaplastic glioma suggest a prognostic value for MGMT methylation. Investigation of pathogenetic and epigenetic features of this intriguingly distinct behavior requires accurate MGMT classification to assess high throughput molecular databases. Promoter methylation-mediated gene silencing is strongly dependent on the location of the methylated CpGs, complicating classification. Using the HumanMethylation450 (HM-450K) BeadChip interrogating 176 CpGs annotated for the MGMT gene, with 14 located in the promoter, two distinct regions in the CpG island of the promoter were identified with high importance for gene silencing and outcome prediction. A logistic regression model (MGMT-STP27) comprising probes cg1243587 and cg12981137 provided good classification properties and prognostic value (kappa = 0.85; log-rank p