MGMT Promoter Hypermethylation Research Articles

Dual MGMT inactivation by promoter hypermethylation and loss of chromosome 10q as relevant prognostic marker in glioblastoma

Abstract Background Epigenetic inactivation of MGMT by methylation of its promoter is a prognostic factor and is predictive of Temozolomid (TMZ) response in glioblastoma (GBM). MGMT is located on chromosome 10q26 and the loss of chromosome 10q is observed in 70% of GBM. In this study, we assessed the hypothesis that the dual inactivation of MGMT, by hypermethylation of MGMT promoter and by loss of chromosome 10q, may confer greater sensitivity to TMZ. Methods A total of 149 patients diagnosed with GBM, based on the WHO 2016 classification, and initially treated according to the Stupp's protocol between November 2016 and December 2018, were included in this multicentre retrospective study. Methylation status of MGMT promoter was evaluated by pyrosequencing and status of chromosome 10q was assessed by array comparative genomic hybridization. Overall survival (OS) and progression-free survival (PFS) were evaluated by the Kaplan-Meier method. Results The present study showed that GBM patients with chromosome 10q loss associated with hypermethylation of MGMT promoter had significantly longer OS (p = 0.0024) and PFS (p = 0.031). Indeed, median OS of patients with dual inactivation of MGMT was 21.5 months compared to 12 months, 8.2 months and 9.5 months for groups with single MGMT inactivation (hypermethylation or 10q loss) or none. Moreover, all long-term survivors with persistent response to TMZ treatment (OS > 30 months) displayed dual inactivation of MGMT. Conclusions To our knowledge, our study is the first to demonstrate the prognostic relevance of combining chromosome 10q loss factor and methylation of MGMT promoter factor in patients with GBM. Our data suggest that the molecular subgroup characterized by the dual inactivation of MGMT receives greater benefit from TMZ treatment. The results of our study may be of immediate clinical interest since chromosome 10q status and methylation of MGMT promoter are commonly determined in routine practice. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

Associations of MGMT promoter hypermethylation with squamous intraepithelial lesion and cervical carcinoma: A meta-analysis.

BackgroundIn this research, an meta-analysis was performed for assessment of the associations between O6-methyguanine-DNA methyltransferase (MGMT) promoter hypermethylation possessing low-grade intraepithelial lesion (LSIL), high-grade intraepithelial lesion (HSIL), cervical cancer (CC), and clinicopathological characters of CC.MethodsLiterature selection were conducted through searching PubMed, Web of science, EMBASE, China National Knowledge Infrastructure and Wanfang databases (up to November 2018). An assessment of associations between MGMT methylation and LSIL, HSIL, CC risk and clinicopathological characteristics was performed through pooled odds ratios (ORs) with relevant 95% confidence intervals (CIs). Subgroup analyses, meta-regressions and Galbraith plots were conducted to conduct an exploration on the possible sources of heterogeneity. The genome-wide DNA methylation array studies were extracted from Gene Expression Omnibus (GEO) databases for validation of these outcomes.ResultsIn this meta-analysis of 25 published articles, MGMT hypermethylation gradually elevated the rates among control group (12.16%), LSIL (20.92%), HSIL (36.33%) and CC (41.50%) specimens. MGMT promoter methylation was significant associated with the increased risk of LSIL by 1.74-fold (P<0.001), HSIL by 3.71-fold (P<0.001) and CC by 7.08-fold (P<0.001) compared with control. A significant association between MGMT promoter methylation with FIGO stage was also found (OR = 2.81, 95% CI: 1.79–4.41, p<0.001). The results of GEO datasets showed that 5 CpG sites in MGMT with a great diagnostic value for the screening of cervical cancer.ConclusionThe meta-analysis indicated the association between MGMT promoter hypermethylation and squamous intraepithelial lesion and cervical cancer. MGMT methylation detection might have a potential value to be an epigenetic marker for the clinical diagnosis of cervical cancer.

Combinational analysis of IDH1/IDH2 mutations, 1p/19q deletions and MGMT promoter methylation for molecular testing of glioma.

e13152 Background: Mutations in IDH1 and IDH2, co-deletion of 1p and 19q, and the hyper methylation of the MGMT promoter are the most reported genetic alterations in glioma tumors. Therefore, we designed a study to analyze the prevalence of these biomarkers in glioma, and the correlation of these biomarkers with diagnosis and prognosis. Methods: From September 2018 to January 2019, eighteen patients with primary glioma were prospectively enrolled. For each patient, freshly frozen tissue or FFPE samples were collected. DNA was extracted from these samples and sequenced to at least 5,000× coverage. IDH1/IDH2 mutations and 1p/19q deletions were detected from the sequencing data, whereas MGMT promoter methylation was evaluated by real-time fluorescence qPCR. Results: Of the eighteen patients, 44.4%(8/18) and 33.3%(6/18) harbored IDH1 /IDH2 mutations and 1p/19q deletions, respectively, and 72.2%(13/18) contained MGMT promoter hyper methylation. Within these patients, we found a correlation between IDH1/IDH2 mutation and 1p/19q deletion. Namely, among the 8 patients with a IDH1/IDH2 mutation, 75%(6/8) also contained a 1p/19q deletion, whereas none of the 10 patients with wide-type IDH1/IDH2 displayed the 1p/19q deletion. There was also a correlation between mutations at the loci and MGMT promoter hyper-methylation, specifically, 87.5%(7/8) of the patients with IDH1/IDH2 mutations also exhibited hyper-methylation in MGMT, whereas only hyper-methylation was observed in only 40% (4/10) of IDH1/IDH2 negative patients. Conclusions: From our preliminary result, IDH1/IDH2 mutations may be associated with 1p/19q deletion. To further verify this result, a larger, longitudinal study is ongoing at our institution.

Impact of catechol-O-methyltransferase gene variants on methylation status of P16 and MGMT genes and their downregulation in colorectal cancer.

Globally, colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and the second most commonly diagnosed cancer in females, with 1.4 million new cases and almost 694 000 deaths estimated to have occurred in 2012. The development and progression of CRC is dictated by a series of alterations in diverse genes mostly proto-oncogenes and tumor suppressor genes. In this dreadful disease disturbances different from mutations called as epigenetic regulations are also taken into consideration and are thoroughly investigated. The present study was designed to analyze the promoter hypermethylation of CpG (cytosine, followed by guanine nucleotide) islands of cyclin-dependent kinase inhibitor 2A (P16) and O-methylguanine-DNA methyltransferase (MGMT) genes and its subsequent effect on the protein expression in CRC. The impact of the common functional polymorphism of the catechol-O-methyltransferase (COMT) gene, Val158Met, on promoter hypermethylation of P16 and MGMT genes in CRC was also investigated. The study included 200 CRC cases and equal numbers of normal samples. DNA was extracted using the kit method and methylation specific-PCR was performed for analysis of the promoter hypermethylation status. Total protein was isolated form all CRC cases and western blotting was performed for P16 and MGMT proteins. The COMT Val158Met polymorphism was analyzed by a PCR-restriction fragment length polymorphism assay. Epigenetic analysis showed that unlike other high-risk regions, the Kashmiri population has a different promoter hypermethylation profile of both P16 and MGMT genes, with frequent and significant promoter hypermethylation of both in CRC. The frequency of promoter hypermethylation of both genes was significantly higher in males and was insignificantly found to be higher in stage III/IV. The degree of P16 and MGMT promoter hypermethylation increased significantly with increasing severity of the lesion. We also found a significant correlation between P16 and MGMT promoter hypermethylation and loss of protein expression in CRC. A significant association was found between COMT polymorphism (homozygous variant) and P16 methylation status. Similar results were also found for MGMT hypermethylated cases.

PARP Inhibitors for Sensitization of Alkylation Chemotherapy in Glioblastoma: Impact of Blood-Brain Barrier and Molecular Heterogeneity.

Prognosis of patients with glioblastoma (GBM) remains dismal despite maximal surgical resection followed by aggressive chemo-radiation therapy. Almost every GBM, regardless of genotype, relapses as aggressive recurrent disease. Sensitization of GBM cells to chemo-radiation is expected to extend survival of patients with GBM by enhancing treatment efficacy. The PARP family of enzymes has a pleiotropic role in DNA repair and metabolism and has emerged as an attractive target for sensitization of cancer cells to genotoxic therapies. However, despite promising results from a number of preclinical studies, progress of clinical trials involving PARP inhibitors (PARPI) has been slower in GBM as compared to other malignancies. Preclinical in vivo studies have uncovered limitations of PARPI-mediated targeting of base excision repair, considered to be the likely mechanism of sensitization for temozolomide (TMZ)-resistant GBM. Nevertheless, PARPI remain a promising sensitizing approach for at least a subset of GBM tumors that are inherently sensitive to TMZ. Our PDX preclinical trial has helped delineate MGMT promoter hyper-methylation as a biomarker of the PARPI veliparib-mediated sensitization. In clinical trials, MGMT promoter hyper-methylation now is being studied as a potential predictive biomarker not only for response to TMZ therapy alone, but also PARPI-mediated sensitization of TMZ therapy. Besides the combination approach being investigated, IDH1/2 mutant gliomas associated with 2-hydroxygluterate (2HG)-mediated homologous recombination (HR) defect may potentially benefit from PARPI monotherapy. In this article, we discuss existing results and provide additional data in support of potential alternative mechanisms of sensitization that would help identify potential biomarkers for PARPI-based therapeutic approaches to GBM.

Preoperative MRI-radiomics features improve prediction of survival in glioblastoma patients over MGMT methylation status alone.

BackgroundGlioblastoma (GBM) is the most common malignant central nervous system tumor, and MGMT promoter hypermethylation in this tumor has been shown to be associated with better prognosis. We evaluated the capacity of radiomics features to add complementary information to MGMT status, to improve the ability to predict prognosis.Methods159 patients with untreated GBM were included in this study and divided into training and independent test sets. 286 radiomics features were extracted from the magnetic resonance images acquired prior to any treatments. A least absolute shrinkage selection operator (LASSO) selection followed by Kaplan-Meier analysis was used to determine the prognostic value of radiomics features to predict overall survival (OS). The combination of MGMT status with radiomics was also investigated and all results were validated on the independent test set.ResultsLASSO analysis identified 8 out of the 286 radiomic features to be relevant which were then used for determining association to OS. One feature (edge descriptor) remained significant on the external validation cohort after multiple testing (p=0.04) and the combination with MGMT identified a group of patients with the best prognosis with a survival probability of 0.61 after 43 months (p=0.0005).ConclusionOur results suggest that combining radiomics with MGMT is more accurate in stratifying patients into groups of different survival risks when compared to with using these predictors in isolation. We identified two subgroups within patients who have methylated MGMT: one with a similar survival to unmethylated MGMT patients and the other with a significantly longer OS.

Decreased Frequency of MGMT Promoter Hypermethylation in Locally Relapsed Versus Locally Controlled p16 Negative Head and Neck Squamous Cell Carcinoma Patients after Chemoradiotherapy

The O6-methylguanine-DNA methyltransferase (MGMT) gene plays a role in DNA damage repair. MGMT promoter hypermethylation (MGMT -HM), which leads to silencing of the gene, predicts for improved treatment response in glioblastoma patients treated with chemotherapy and/or radiotherapy. The aim of this study is to determine the frequency of MGMT -HM in head and neck squamous cell carcinoma (HNSCC) patients in relation to p16 status and outcomes after chemoradiotherapy. Pre-treatment tumor blocks from 58 primary HNSCC patients treated with chemoradiotherapy with a median age of 60 years (range 36-85) diagnosed between the years 1993 and 2015 were identified for MGMT pyrosequencing using a commercially available kit. Eighty-three percent of patients were male and 17% female. HPV infection status was independently ascertained by p16 immunohistochemistry. After institutional review board approval, clinicopathological characteristics and treatment outcomes data were extracted from patients’ charts. Among the analyzed HNSCC patients, MGMT -HM was identified in 38% (22/58) and p16 positivity was identified in 64% (37/58) of cases. The frequency of MGMT -HM was 38% in both p16 positive (14/37) and p16 negative (8/21) patients. With a median follow-up of 3.7 years (range 0.4-13.9 years), 17 (29%) local relapses were observed overall. Local control was observed in 86% (32/37) of p16 positive patients. The frequency of MGMT -HM was similar between locally controlled and locally relapsed p16 positive patients at 38% (12/32) and 40% (2/5), respectively. Conversely, local control was observed in 43% (9/21) of p16 negative patients. The frequency of MGMT -HM was more than double in locally controlled p16 negative patients than in locally relapsed p16 negative patients at 56% (5/9) and 25% (3/12), respectively. As expected, p16 negative HNSCC patients demonstrated increased local relapses after chemoradiotherapy. The rates of MGMT -HM were similar between p16 positive and p16 negative HNSCC patients. To our knowledge, this is the first report demonstrating a decreased frequency of MGMT -HM in p16 negative HNSCC patients who experienced local relapse. Accordingly, the potential role of MGMT -HM in modulating differential treatment outcomes in p16 negative HNSCC patients needs to be further investigated.

Predicting cervical intraepithelial neoplasia recurrence in HIV-infected and -noninfected women by detecting aberrant promoter methylation in the CDH1, TIMP3, and MGMT genes.

Aberrant DNA methylation is present in virtually all types of human cancer. There is no clear evidence that methylation status can predict bad prognosis in patients with CIN recurrence in HIV infected. This study evaluates the relationship between aberrant methylation of CpG islands of CDH1, TIMP3 and MGMT genes and CIN recurrence in HIV-infected and -noninfected women. This is a nested case-control study involving 33 cases with CIN recurrence and 114 controls without recurrence, HIV infected and noninfected, treated with LEEP, between 1999 and 2004. Recurrence diagnosis was established after biopsy. Genes methylation profile was assessed by MSP-PCR technique in formalin-fixed, paraffin-embedded cone specimens. Statistical analysis was performed to compare categorical variables, using χ2 test with Yates correction and Fisher's exact test. Multivariate analysis was carried out using logistic regression. CIN recurrence was more frequent in women with glandular involvement (OR 11.6; 95% CI 2.93-45.89) and compromised surgical margins (OR 2.5; 95% CI 0.87-7.27) in the cervical cone and in HIV-infected women (OR 2.47; 95% CI 0.87-7.05). One methylated allele of CDH1, TIMP3 and MGMT genes was present in 87.9% women with CIN recurrence. Promoter hypermethylation of TIMP3 and MGMT was detected in women with CIN recurrence and without CIN recurrence independent of HIV infection with significant difference between groups (p = 0.04 and p = 0.02, respectively). CIN recurrence was associated with glandular involvement and compromised margins in cone biopsy and HIV infection. The presence of CpG islands hemimethylation in TIMP3 and MGMT genes is a promising triage method in CIN recurrence.

Genomic Heterogeneity Underlies Mixed Response to Tropomyosin Receptor Kinase Inhibition in Recurrent Glioma

Glioblastoma is the most common malignant primary brain tumor in adults. Despite aggressive initial treatment, recurrence is inevitable and almost always fatal. Treatment options for recurrent disease are limited and do not substantially improve survival. In recent years, molecular characterization of gliomas has revealed recurrent somatic mutations, structural rearrangements, and epigenetic alterations. These genomic alterations have implications for prognosis and response to treatment. IDH1/2 mutations are associated with a more favorable prognosis among glioblastomas, whereas tumors with MGMT promoter hypermethylation are more likely to respond to treatment with alkylating agents.1,2 The pattern of genomic alterations can also provide insight into the origin and evolution of the tumor. For example, the presence of coexistent mutations in IDH1 and ATRX suggest a secondary glioma that has progressed from a lower-grade glioma.3-5 In addition to informing prognosis and diagnostic classification, some genomic alterations in glioma represent potential therapeutic targets. Specifically, recurrent alterations in one of several receptor tyrosine kinases (RTKs), including EGFR, PDGFRA, MET, and FGFR1/2/3, have been observed in the majority of glioblastomas.6 Unfortunately, efforts to develop genomically targeted therapy for the treatment of glioblastoma have been unsuccessful to date.7 It remains unknown the extent to which the lack of activity of targeted therapy may be attributed to so-called key alterations that do not represent true clonal drivers that are critical to tumor growth and progression compared with the simple inability of existing drugs to achieve therapeutic exposures within the CNS. In addition to these more common genomic alterations, less common fusions that involve the kinase domain of tropomyosin receptor kinases (TRKs; A/B/C) encoded by the genes, NTRK1, NTRK2, and NTRK3 have also been identified in gliomas.8-11 In other cancer types, selective inhibition of these TRK fusions has resulted in remarkable efficacy12; however, it remains unknown whether this therapeutic strategy is effective in primary brain tumors. Here, we report the case of a young woman with recurrent glioblastoma treated with larotrectinib, a first-in-class, potent, highly selective TRK inhibitor. To our knowledge, this is the first report of the use of a TRK inhibitor in a patient with a TRK fusion–positive glioblastoma.

O6-Methyguanine-DNA Methyl Transferase (MGMT) Promoter Methylation in Serum DNA of Iranian Patients with Colorectal Cancer

Introduction:Colorectal cancer (CRC) is a leading cause of cancer deaths worldwide but current molecular targeted therapy is not providing major success in CRC treatment, so early detection by non-invasive methods continues to be vital. Aberrant methylation of CpG islands in promoter regions is associated with inactivation of various tumor suppressor genes. O6-methyguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that removes mutagenic and cytotoxic adducts from O6-guanine in DNA. Aberrant hypermethylation of the MGMT promoter has been associated with lack of mRNA expression, with concomitant loss of protein content and enzyme activity.AIM:Our aim was to determine whether MGMT promoter methylation might be detectable in circulating free DNA in the serum of CRC patients and normal individuals using a methylation specific (MSP) polymerase chain reaction (PCR) method.Methods:A total of 70 subjects were enrolled in the study. Of these, 30 patients who were diagnosed previously as untreated colon adenocarcinoma by a gastroenterologist and the other 40 were nearly age-matched individuals who had a normal colonoscopic evaluation (except for hemorrhoids or fissures) and normal pathologic reports. After bisulphite modification of DNA, serum samples were examined for MGMT promoter methylation using MSP.Results:Ninety percent of CRC patients had MGMT promoter hypermethylation as compared to no methylation in normal subjects’ serum. Most of the cancers were stage П and moderately differentiated adenocarcinomas; nearly 60% were found in the left colon. No statistically significant correlation was found between the promoter methylation status and gender and age.Discussion and Conclusions:MGMT hypermethylation can be detected in free circulating DNA in serum of CRC patients and can be used “as a clinical biomarker” for early diagnosis and prognostic assessment of the disease. Our data confirm previous studies indicating utility for free circulating DNA as a serum biomarker for early detection, diagnosis and monitoring of CRC patients.

MGMT gene promoter methylation and its correlation with clinicopathological parameters in glioblastomas.

MGMT (O6-methyl guanine DNA methyl transferase) promoter hypermethylation is a prognostic and predictive biomarker for glioblastomas (GBM). To evaluate the frequency of MGMT methylation status in a single institute series of 134 GBMs and correlate it with clinical (age, sex, location, survival) and other molecular parameters [such as p53 expression, alpha thalassemia/mental retardation syndrome X-linked (ATRX) expression, isocitrate dehydrogenase (IDH) 1R132H mutation, and epidermal growth factor receptor (EGFR) gene amplification]. One hundred and thirty-four GBMs were evaluated by methylation-specific polymerase chain reaction (MSP) for MGMT promoter methylation status. The results were correlated with the above mentioned clinicopathological parameters. MGMT gene promoter methylation was identified in 49.2% (66/134) GBMs, and was significantly associated with IDH1R132H mutation (14/66; 21%; P - value, 0.01) and ATRX loss (15/66; 23%; P - value, 0.01). Confluent necrosis was found to be significantly associated with MGMT unmethylation status (P - value: 0.002). Multivariable logistic regression analysis showed confluent necrosis as a single independent predictor (odds ratio [OR], 2.5; confidence interval [CI], 1.0-5.8; P - value, 0.04) of MGMT unmethylation status among all the parameters studied. The frequency of MGMT promoter methylation in GBMs was 49.2%, which was significantly associated with IDHR132H mutation and ATRX loss. In addition, the presence of confluent necrosis was significantly associated with MGMT unmethylation and was found to be an independent predictor of the same.

Feasibility of real-time molecular profiling for patients with newly diagnosed glioblastoma without MGMT promoter hypermethylation-the NCT Neuro Master Match (N2M2) pilot study.

O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status is a predictive biomarker in glioblastoma patients. Glioblastoma without hypermethylated MGMT promoter is largely resistant to treatment with temozolomide. These patients are in particular need of new treatment approaches, which are offered by biomarker-driven clinical trials with targeted drugs based on molecular characterization of individual tumors. In preparation for an upcoming clinical study, a comprehensive molecular profiling approach was undertaken on tissues from 43 glioblastoma patients harboring an unmethylated MGMT promoter at diagnosis. The diagnostic pipeline covered various levels of molecular characteristics, including whole-exome sequencing, low-coverage whole-genome sequencing, RNA sequencing, as well as microarray-based gene expression profiling and DNA methylation arrays. Complex multilayer molecular diagnostics were feasible in this setting with a median turnaround time of 4-5 weeks from surgery to the molecular tumor board. In 35% of cases, potentially relevant therapeutic decisions were derived from the data. Alterations were most frequently found in receptor tyrosine kinases, members of the phosphoinositide 3-kinase/Akt/mechanistic target of rapamycin and mitogen-activated protein kinase pathway as well as cell cycle control and p53 regulation cascades. Individual tumors harbored clonal alterations such as oncogenic fusions of tyrosine kinases which constitute promising targets for targeted therapies. A prioritization algorithm is proposed to allocate patients with multiple targets to the potentially best treatment option. With this feasibility study, a comprehensive molecular profiling approach for patients with newly diagnosed glioblastoma harboring an unmethylated MGMT promoter is presented. Analyses in this pilot cohort serve as a basis for trials based on targetable alterations and on the question of allocation of patients to the best treatment arm.

MGMT Promoter Hypermethylation is a Common Event in Head and Neck Squamous Cell Carcinoma

Promoter hypermethylation of the DNA repair O6-methylguanine-DNA methyltransferase (MGMT) gene is an independent predictor of response to chemoradiotherapy and survival in gliomas and lymphomas. We used pyrosequencing to determine the rate of MGMT promoter hypermethylation (MGMT-HM) in head and neck squamous cell carcinoma (HNSCC). Pre-treatment tumor blocks from 153 primary HNSCC patients with a median age of 61 years (range 32-90) diagnosed between the years 1993 and 2015 were identified for MGMT pyrosequencing using a commercially available kit. Seventy-three percent of patients were male and 27% female. Seventy-eight percent were tobacco users. Human Papilloma Virus (HPV) infection status was independently ascertained by p16 immunohistochemistry. After IRB approval, clinicopathological characteristics were extracted from patient’s charts, including age, gender, site of disease, and tobacco history. Overall, MGMT-HM was identified in 36% of the patients. In the 64, 79, and 10 patients with oral cavity, oropharynx, and larynx/hypopharynx primaries, MGMT-HM was seen in 36%, 33%, and 60%, respectively. Overall, HPV positivity was seen in 51% of the patients. In the patients with oropharynx, oral cavity, and larynx/hypopharynx primaries, HPV positivity was seen in 28%, 73%, and 20%, respectively. Among all HPV positive and negative patients, 31% and 41% had MGMT-HM, respectively. Among all tobacco users and non-users, 33% and 45% had MGMT-HM, respectively. Among oral cavity HPV positive and HPV negative patients, 28% and 39% had MGMT-HM, respectively. Among oral cavity tobacco users and non-users, 29% and 53% had MGMT-HM, respectively. Among oropharynx HPV positive and negative patients, 31% and 38% had MGMT-HM, respectively. Among oropharynx tobacco users and non-users, 32% and 36% had MGMT-HM, respectively. MGMT-HM is a common event in HNSCC and seems to be unrelated to tumor sub-site or HPV status. The frequency of MGMT-HM was observed to be higher in patients who did not use tobacco. It remains to be seen if MGMT-HM plays a role in the differential response to chemoradiotherapy along with HPV status in HNSCC.

468P - Activity of temozolomide (TMZ) in patients (PTS) with malignant pheochromocytoma or paraganglioma (MPP): A mono-institutional retrospective study

Background: MPP are very rare neuroendocrine tumors and, currently, there is no standard chemotherapy for their treatment. TMZ showed some benefit in digestive neuroendocrine tumors. We investigate TMZ activity in PTS with MPP. Methods: Retrospectively, we evaluated TMZ activity in MPP PTS treated at our oncological center, Veneto Institute of Oncology, from January 2015 to March 2017. The inclusion criteria were: pathological diagnosis of MPP, ECOG PS 0-2, no haemopoietic, renal and hepatic abnormalities. TMZ schedule was 150-200mg/m2 for 5 consecutive days every 28 days until progression disease or unacceptable toxicity. CT scan and urinary concentrations of metanephrines were performed every 12 wks; evaluation of tumor response was performed according to RECIST 1.1 criteria. Germinal mutational analysis of the genes of susceptibility to pheochromocytoma/paraganglioma (SDHx, MAX, TMEM127, RET, VHL, FH) was performed. Aberrant hypermethylation of MGMT promoter was analyzed on DNA obtained from surgical tissue. Median OS and PFS were estimated by the Kaplan-Meier method. Toxicity was evaluated by CTCAE v.4. Results: We enrolled 12 consecutive PTS; 7 were males; ECOG PS was 1 and 2 in 9 and 3 PTS. MAX gene was mutated in 1 PT. SDHB gene was mutated in 2 PTS. MGMT promoter was methylated in 1 patient. No other genetic mutations were found. 5 PTS were already treated with a prior chemotherapy (3 sunitinib, 1 capecitabine, 1 dacarbazine) and 4 with a prior MIBG. 9 PTS were evaluable for response: 2 PTS had a partial response, 5 stable disease, 2 progressive disease. Median follow up was 9.2ms (range 1.1- 28ms). 2 PTS received TMZ for more than 2 years and other 2 PTS for more than 1 year. Median PFS and OS were not reached (95% CI = 3.4ms-n.a.; 6.2ms-n. a, respectively). Urinary metanephrines levels seem to correlate with response. Hypertension descreased significantly in 5 PTS during TMZ treatment. No grade 3-4 toxicity was recorded. Conclusions: TMZ is an active and safe treatment for MPP, regardless of previous treatment. A prospective phase II study is ongoing. Legal entity responsible for the study: Giuseppe Lombardi Funding: None Disclosure: All authors have declared no conflicts of interest.

Short article: Evaluation of O6-methylguanine-DNA methyltransferase as a predicting factor of response to temozolomide-based chemotherapy in well-differentiated metastatic pancreatic neuroendocrine tumors.

Temozolomide (TMZ) is an alkylating agent frequently used in well-differentiated metastatic pancreatic neuroendocrine tumors (PNETs) with very variable responses. O-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme whose loss of expression has been suggested to be predictive of response to TMZ in various human tumors. We evaluated the predictive value of MGMT status, assessed by immunohistochemistry (IHC) and methylation-specific PCR (MS-PCR), in well-differentiated metastatic PNETs treated by a TMZ-based chemotherapy. All patients with metastatic PNETs treated with TMZ-based chemotherapy between 2010 and 2016 in two academic centers, for whom the tumor samples were available, were included. Clinical data were collected and the MGMT status of the tumors was analyzed using MS-PCR and IHC. Twenty-two patients (nine men, median age 61 years) were included. The loss of MGMT protein expression detected by IHC was observed in 13 (59%) patients and MGMT promoter hypermethylation was detected by MS-PCR in three (15%) out of 20 interpretable cases. MGMT status did not correlate significantly with the best radiological response according to the Response Evaluation Criteria In Solid Tumors criteria or with progression-free survival. There was no correlation between MGMT protein expression and MGMT gene promoter methylation. These results indicate that a deficient MGMT status in PNETs, determined by loss of protein expression in IHC or by the presence of MGMT gene promoter methylation measured by MS-PCR, is not associated with a better response to TMZ-based chemotherapy and cannot be used as a predictive marker to lead treatment decisions.

Pooled Analysis of Clinical Outcome of Patients with Chemorefractory Metastatic Colorectal Cancer Treated within Phase I/II Clinical Studies Based on Individual Biomarkers of Susceptibility: A Single-Institution Experience

BackgroundPatients with metastatic colorectal cancer (mCRC) refractory to standard therapies have a poor prognosis. In this setting, recruitment into clinical trials is warranted, and studies driven by selection according to individual tumor molecular characteristics are expected to provide added value.ObjectiveWe retrospectively analyzed data from patients with mCRC refractory to or following failure of standard therapies who were enrolled into phase I/II clinical studies at the Niguarda Cancer Center based on the presence of a specific molecular profile expected to represent the target of susceptibility to the experimental drug(s).Patients and MethodsFrom June 2011 to May 2016, 2044 patients with mCRC underwent molecular screening. Eighty patients (3.9%) were enrolled in ad hoc studies; the median age was 60 years (range 36–86) and the median number of previous treatment lines was five (range 2–8). Molecular characteristics exploited within these studies were MGMT promoter hypermethylation (48.7%), HER2 amplification (28.8%), BRAF V600E mutation (20%), and novel gene fusions involving ALK or NTRK (2.5%).ResultsOne patient (1%) had RECIST (Response Evaluation Criteria In Solid Tumors) complete response (CR), 13 patients (16.5%) experienced a partial response (PR), and 28 (35%) stable disease (SD). Median progression-free survival (PFS) was 2.8 months (range 2.63–3.83), with 24% of patients displaying PFS >5 months. Median growth modulation index (GMI) was 0.85 (range 0–15.61) and 32.5% of patients had GMI >1.33. KRAS exon 2 mutations were found in 38.5% of patients, and among the 78 patients with known KRAS status, those with wild-type tumors had longer PFS than those with mutated tumors (3.80 [95% CI 2.80–5.03] vs. 2.13 months [95% CI 1.77–2.87], respectively, p = 0.001). Median overall survival (OS) was 7.83 months (range 7.17–9.33) for all patients, and patients with KRAS wild-type tumors had longer OS than those with mutated tumors (7.83 [95% CI 7.33–10.80] vs. 7.18 months [95% CI 5.63–9.33], respectively, p = 0.06).ConclusionsThis single-institution retrospective study indicates that in a heavily pretreated population approximately 4% of mCRC tumors display a potential actionable molecular context suitable for therapeutic intervention. Application of molecular selection is challenging but improves clinical outcome even in later lines of treatment.

Abstract LB-046: Evaluation of an assay for on-demand and easy assessment of MGMT gene promoter methylation in glioblastoma patients

Abstract Background: Epigenetic silencing of the O6-methylguanine DNA methyltransferase (MGMT) gene by promoter methylation is observed in 40 to 50% of glioblastomas and is associated with improved overall survival on radiation and/or temozolamide therapy. Currently, pyrosequencing is commonly used to assess the methylation status of the MGMT gene in formalin-fixed, paraffin-embedded (FFPE) samples. We examined the concordance of the recently developed GeneXpert (GX) MGMT Methylation RUO Assay with pyrosequencing in three independent glioblastoma cohorts. Patients and methods: The GX MGMT Methylation RUO Assay is a research use only, cartridge-based methylation specific PCR assay performed on the GeneXpert® Instrument (Cepheid) that automates DNA purification, bisulfite conversion, and methylation-specific PCR after sample preparation. Cartridge results are reported in less than 4 hours as delta cycle threshold (dCt) measurements (dCt = beta-actin/ACTB Ct - MGMT Ct). The MGMT methylation status was assessed on FFPE tumor blocks from 262 glioblastoma patients obtained from OHSU (n=63), MUV (n=96), and KUL (n=103). Two adjacent sections from each block were prepared, one 4 µm section for H&amp;E staining to determine the % tumor cell content/tumor area and one 4 µm section for lysate preparation and GX testing. Both GX MGMT Methylation testing and pyrosequencing results were correlated with overall survival of the patients in the MUV cohort. Results: All of the 262 (100%) glioblastoma samples tested yielded valid results for both GX MGMT Methylation testing and pyrosequencing. The cut-offs were set at dCt -6.2 for the GX MGMT Methylation RUO Assay and 8% methylation for pyrosequencing. The concordance rate between GX MGMT Methylation testing and pyrosequencing was 92% (58/63 OHSU samples, 92% (88/96 MUV samples) and 83% (85/103 KUL samples). The overall concordance rate was 88% (231/262 samples). Sensitivity and specificity was 84% (27/32) and 100% (31/31) for OHSU samples, 89% (33/37) and 93% (55/59) for MUV samples, and 70% (31/44/) and 92% (54/59) for KUL samples, respectively. Overall sensitivity was 81% (91/113) and overall specificity was 94% (140/149). Overall survival data were available for 95 MUV patients. Patients with MGMT promoter hypermethylation based on pyrosequencing had a significantly longer overall survival compared to patients without hypermethylation (median 16 vs. 11 months, p = 0.001). Similar results were observed with the GX MGMT Methylation RUO Assay (median 15 vs. 11 months, p = 0.005). Conclusion: GX MGMT Methylation testing is highly reproducible and shows good concordance with pyrosequencing in three independent cohorts of glioblastoma patients. Our results suggest that standardized, simplified, and on-demand testing of MGMT promoter methylation by the GX MGMT Methylation RUO Assay is feasible. Citation Format: Martin Filipits, Anita Brandstetter, Matthias Preusser, Johannes Hainfellner, Sabine Spiegl-Kreinecker, Edwin W. Lai, Kriszten Kocmond, Andrew Kohlway, Jodi Weidler, Michael Bates, Christopher Corless. Evaluation of an assay for on-demand and easy assessment of MGMT gene promoter methylation in glioblastoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-046. doi:10.1158/1538-7445.AM2017-LB-046

Importance of biomarkers in glioblastomas patients receiving local BCNU wafer chemotherapy

BackgroundTo assess the influence of molecular markers with potential prognostic value to groups of patients with newly diagnosed glioblastoma patients were examined: group A with 36 patients (surgical resection plus standard combined chemoradiotherapy) and group B with 36 patients (surgical resection, standard combined chemoradiotherapy plus carmustine wafer implantation).Our aim was to determine chromosomal alterations, methylation status of MGMT, p15, and p16 (CDKN2A) in order to analyse the influence on patient survival time as well as radio- and chemotherapy responses. Promoter hypermethylation of MGMT, p16, and p15 genes were determined by MS-PCR. Comparative genomic hybridisation (CGH) analyses were performed with isolated, labelled DNA of each tumor to detect genetic alterations.ResultsAge of onset of the disease showed a significant effect on overall survival (OS) (p < 0.0001). Additional treatment with carmustine wafer (group B) compared to the control group (group A) did not result in improved OS (p = 0.562). Patients with a methylated MGMT promotor showed a significant longer OS compared to those patients with unmethylated MGMT promotor (p = 0.041). Subgroup analyses revealed that patients with methylated p15 showed a significant shorter OS when administered to group B rather than in group A (p = 0.0332). In patients additionally treated with carmustine wafer an amplification of 4q12 showed a significant impact on a reduced OS (p = 0.00835). In group B, a loss of 13q was significantly associated with a longer OS (p = 0.0364). If a loss of chromosome 10 occurred, patients in group B showed a significantly longer OS (p = 0.0123).ConclusionA clinical benefit for the widespread use of additional carmustine wafer implantation could not be found. However, carmustine wafer implantation shows a significantly improved overall survival if parts of chromosome 10 or chromosome 13 are deleted. In cases of 4q12 amplification and in cases of a methylated p15 promotor, the use of carmustine wafers is especially not recommended.The MGMT promoter methylation is a strong prognostic Biomarker for benefit from temozolomide and BCNU chemotherapy.

Promoter hypermethylation of MGMT gene may contribute to the pathogenesis of gastric cancer: A PRISMA-compliant meta-analysis.

Beckground:The association of MGMT (O6-methyguanine deoxyribonucleic acid methyltransferase) promoter hypermethylation with gastric cancer (GC) risk has been studied extensively, but the results remained unclear. Here, we performed a meta-analysis to evaluate whether promoter hypermethylation of the MGMT gene contributed to gastric pathogenesis.Methods:Relevant studies were identified by retrieving the PubMed, Web of Science, Embase, and China National Knowledge Infrastructure (CNKI) databases. The Newcastle–Ottawa Scale was applied to assess methodological quality of the included studies. Pooled odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated to evaluate the association of MGMT promoter hypermethylation with gastric pathogenesis. Moreover, STATA 12.0 software was used to summarize the extracted data in this meta-analysis.Results:Seventeen studies, comprising 1736 cases and 1291 controls, were included in this meta-analysis. The frequency of MGMT promoter hypermethylation in the GC group (32.97%) was significantly higher than those in the control group (18.00%) (OR = 2.83, CI = 1.93–4.15, P < .05). When stratified by cancer subtype, the results indicated that the frequency of MGMT promoter hypermethylation was significantly higher in gastric adenocarcinoma than in control group (OR = 3.47, CI = 1.06–11.35, P < .05). In addition, MGMT promoter hypermethylation significantly promoted distant metastasis and lymph node (LN) metastasis of gastric tumor (for distant metastasis, OR = 4.22, CI = 2.42–7.37, P < .05; for LN metastasis, OR = 1.56, CI = 1.14–2.13, P < .05). A significant association between MGMT promoter hypermethylation and TNM-stage was also found in the present meta-analysis (OR = 2.70, CI = 1.79–4.08, P < .05).Conclusion:The results of this meta-analysis suggested that MGMT gene-promoter hypermethylation was significantly associated with an increased risk of GC, especially in Asians. Furthermore, MGMT gene-promoter hypermethylation might be correlated with the distant metastasis and LN metastasis of GC.

The Association Between MGMT Promoter Methylation and Patients with Gastric Cancer: A Meta-Analysis.

Several previous studies have suggested that MGMT promoter methylation is significantly associated with gastric cancer, but the results were not consistent. Hence, we conducted a systematic meta-analysis to explore the potential correlation of MGMT promoter methylation with gastric cancer and its clinicopathologic characteristics. Searches of PubMed, EMBASE, Web of Science, Cochrane Library, and Chinese National Knowledge Infrastructure (CNKI) literature databases were conducted to identify relevant studies published in English or Chinese before July 1, 2016. The meta-analysis was performed using Stata 12.0 software. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to assess the association between MGMT promoter methylation and gastric cancer. We also conducted a subgroup analysis and metaregression to explore sources of heterogeneity. We identified 12 articles that met the inclusion criteria. The 12 articles described 14 studies that included 1571 tumor tissues and 1243 controls. The meta-analysis results demonstrated that the frequency of MGMT promoter methylation was higher in gastric cancer tissues compared with adjacent tissues and normal tissues (OR = 4.06, 95% CI: 2.55-6.46, p < 0.001; OR = 8.85, 95% CI: 1.15-68.23, p = 0.036; respectively). An assessment of the correlation between MGMT promoter methylation and clinicopathological characteristics indicated that MGMT promoter hypermethylation was significantly associated with tumor-node-metastasis stage, lymph node metastasis, and distant metastasis (OR = 2.11, 95% CI: 1.18-3.75, p = 0.011; OR = 1.99, 95% CI: 1.47-2.68, p < 0.001; and OR = 3.60, 95% CI: 2.17-5.95, p < 0.001; respectively). Our findings provide evidence that MGMT promoter methylation could play an important role in gastric carcinogenesis and may serve as an important biomarker for gastric cancer progression.