MGMT Hypermethylation Research Articles

Abstract 4893: Role of MGMT expression in the genomic landscape and immunogenicity in human colorectal carcinomas

Abstract Introduction: Colorectal cancer (CRC) is second most common cause of cancer-related mortality in the USA and most patients with advanced tumors lack effective therapeutic options. Epigenetic silencing resulting in reduced expression of O6-methylguanine-DNA methyltransferase (MGMT) has been described in diverse tumor types including CRC and it is linked with reduced DNA repair capability and increased sensitivity to alkylating agents such as temozolomide. However, the potential role and clinical significance of MGMT overexpression in cancer cells has not been studied. Methods: We used spatially resolved multiplex quantitative immunofluorescence (mQIF) analysis to concurrently measure the levels of MGMT protein, γH2AX and CD8+ tumor infiltrating lymphocytes (TILs) in a cohort of 111 CRCs and paired non-tumor colorectal mucosa from the same patient represented in tissue microarray format. To assess the genomic and immunologic context of MGMT expression, we studied the methylome, mutation profiling and mRNA expression of 526 CRCs from TCGA. The immunologic landscape of tumors was studied using GSEA and multiple transcriptomic-based tumor immune infiltration scores such as TIMER, CIBERSORT, etc. Finally, the genomic alterations and immunogenicity of SW620 human CRC cells (with baseline MGMT hypermethylation) with or without MGMT transfection were studied using in vitro co-culture preparations coupled to ultra-deep DNAseq and flow cytometry analysis. Results: On mQIF analysis, cancer-cell MGMT protein upregulation was identified in 73.9% of CRCs and was associated with reduced levels of γH2Ax in malignant cells and lower CD8+ TILs. 26.1% of cases showed MGMT protein downregulation. The genomic analysis of the TCGA cohort revealed that the mean nonsynonymous mutational load was significantly lower in MGMT-high than low tumors with marked differences in the type of variants and frequency of oncogenic mutations. The transcriptomic analysis revealed that MGMT overexpressing tumors showed lower immune infiltration than the MGMT-low group and reduced representation of immune associated signatures including interferon gamma response and TNF-alpha signaling. In contrast, DNA repair, TP53 pathways, were enriched. The exogenous expression of MGMT in SW620 cells reduced the number of nonsynonymous mutations by 15.6% (141 vs 119 mean mutations) and was also associated with low T-cell activation (CD69+/CD8+ T-cells) and cancer-cell elimination on allogenic co-cultures with human PBMCs. Conclusion: MGMT protein upregulation is common in human CRCs and is associated with reduced DNA damage response, distinct genomic features, and adaptive immune evasion. To our knowledge, this study is the first to report the biological and clinical characteristics of CRCs with MGMT overexpression that could support diagnostic and therapeutic developments. Citation Format: Barani Kumar Rajendran, Janie Zhang, Shruti Desai, Joanna Gibson, Jassim DiPalermo, Patricia LoRusso, Yong Kong, Hongyu Zhao, Michael Cecchini, Kurt Alex Schalper. Role of MGMT expression in the genomic landscape and immunogenicity in human colorectal carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4893.

ABERRANT METHYLATION OF CANCER-RELATED GENES IN VIETNAMESE BREAST CANCER PATIENTS: ASSOCIATIONS WITH CLINICOPATHOLOGICAL FEATURES.

Epigenetic alteration is one of the most common molecular changes identified in the progression of breast cancer (BC). To study the frequency and relation between methylation of BRCA1, MLH1, MGMT, GSTP1, APC, RASSF1A, p16, WIF, and EGFR and the clinicopathological features in Vietnamese BC patients. Methylation-specific polymerase chain reaction (MS-PCR) and SPSS 20.0 software were utilized in order to identify methylated frequency as well as evaluate its relationship with the patient's clinical features. In 162 BC cases, the methylation rates of the selected genes were 53.7%, 22.8%, 38.9%, 34.6%, 29.0%, 46.3%, 20.4%, 18.5%, and 28.4% respectively. In 32 cases of benign breast diseases (BBD) - 12.5%, 15.6%, 6.3%, 3.1%, 12.5%, 21.9%, 3.1%, 15.6% and 3.1%. BC samples displayed higher BRCA1, MGMT, GSTP1, APC, RASSF1A, WIF1, and p16 methylation levels than BBD samples (p < 0.001). Hypermethylation of BRCA1, GSTP1, and RASSF1A was predominant in the invasive ductal carcinoma, while hypermethylation of BRCA1, GSTP1, RASSF1A, WIF-1, and p16 was found to significantly correlate with lymph node metastasis (p < 0.05). Hypermethylation of BRCA1, MGMT, and GSTP1 was more common in stage III (p < 0.05) than in stages I/II, whereas MLH1 methylation was predominant in stage I and APC methylation was less common in stage III (p = 0.03). In addition, methylation of RASSF1A and EGFR was more frequent in younger patients (p < 0.01) than in elder patients. These data suggest that a gene panel (BRCA1/MGMT/GSTP1) can be used to support the diagnosis and screening of Vietnamese patients' BC with a sensitivity of 70%, and a specificity of 85%.

Institutional Validation Study Inferring 2% MGMT Methylation Positive Impact on Survival in Newly Diagnosed Glioblastoma (GBM) Patients

O6-methylguanine DNA methyltransferase (MGMT) MATERIALS/METHODS: is a well-established prognostic factor in patients with newly diagnosed glioblastoma (GBM). However, there is no consensus on a standardized method of threshold for MGMT testing. Previous studies have reported levels of as little as 1-3% to confer better prognosis. This study reports a single institutional experience of determining methylation status via methylation-sensitive high-resolution melting (MS-HRM). Previous literature suggests 10% cutoff for MGMT methylation using MS-HRM. We hereby report clinical outcomes using a lower threshold of 2%. GBM patients treated at our institution retrospectively reviewed between the years 2013 and 2022 were included in the study. Patients who received hypofractionated radiation (<60 Gy) were excluded. All patients had MS-HRM test for MGMT methylation status. A real-time PCR assay was used to amplify a 62 base-pair region of MGMT for both methylated and unmethylated alleles. PCR products underwent HRM analysis and the fraction of methylated DNA was determined by comparison with a standard curve. Clinical data were collected retrospectively. Kaplan-Meier and log-rank tests were performed to compare survival. A total of 181 patients with newly diagnosed GBM were initially included in this study. 42 patients treated with hypofractionated radiation were excluded. All patients received concurrent and maintenance temozolomide. Median age was 61.5 years. A total of 84 patients had MGMT methylation levels <2%, and 55 patients had MGMT methylation level ≥ 2% with a median methylation level of 28.5% (Range 0.8%-100%). Patients with MGMT methylation level ≥ 2% had an improved median overall survival (25.1 vs 16.0 months; p = 0.006) and improved median progression free survival (11.3 vs 7.9 months; p = 0.017). In a multivariable mode that included age, use of tumor-treating fields, KPS, sex, and BMI, only age, KPS, and MGMT remained significant. Our institutional review confirmed low level of MGMT hypermethylation (≥ 2%) predicts improved outcome in patients with newly diagnosed GBM. Further investigation on optimal cut off level for MGMT methylation is still warranted.

A Comprehensive Review of miRNAs and Their Epigenetic Effects in Glioblastoma.

Glioblastoma is the most aggressive form of brain tumor originating from glial cells with a maximum life expectancy of 14.6 months. Despite the establishment of multiple promising therapies, the clinical outcome of glioblastoma patients is abysmal. Drug resistance has been identified as a major factor contributing to the failure of current multimodal therapy. Epigenetic modification, especially DNA methylation has been identified as a major regulatory mechanism behind glioblastoma progression. In addition, miRNAs, a class of non-coding RNA, have been found to play a role in the regulation as well as in the diagnosis of glioblastoma. The relationship between epigenetics, drug resistance, and glioblastoma progression has been clearly demonstrated. MGMT hypermethylation, leading to a lack of MGMT expression, is associated with a cytotoxic effect of TMZ in GBM, while resistance to TMZ frequently appears in MGMT non-methylated GBM. In this review, we will elaborate on known miRNAs linked to glioblastoma; their distinctive oncogenic or tumor suppressor roles; and how epigenetic modification of miRNAs, particularly via methylation, leads to their upregulation or downregulation in glioblastoma. Moreover, we will try to identify those miRNAs that might be potential regulators of MGMT expression and their role as predictors of tumor response to temozolomide treatment. Although we do not impact clinical data and survival, we open possible experimental approaches to treat GBM, although they should be further validated with clinically oriented studies.

Prognostic value of O6-methylguanine-DNA methyltransferase hypermethylation and expression in head and neck cancer: A systematic review and meta-analysis

O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that maintains the stability of genetic information. MGMT is a strong prognostic biomarker in patients with glioblastoma. However, the effect of its gene hypermethylation and expression on the survival rate of head and neck cancer (HNC) patients is still disputed. Therefore, we conducted a meta-analysis to evaluate the prognostic value of MGMT hypermethylation and expression in HNC patients. This meta-analysis was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses 2020 guidelines and was registered at the International Prospective Register of Systematic Reviews (CRD42021274728). Literature related to the survival rate of HNC patients and MGMT was systematically searched in PubMed, Embase, The Cochrane Library and Web of Science electronic databases (published from inception to February 1, 2023). The association was evaluated by the combined hazard ratio (HR) and related 95% confidence interval (CI). Two authors independently screened all records and extracted the data. The certainty of evidence was assessed using the Grading of Recommendations Assessment, Development and Evaluation system. All of the statistical tests used in this meta-analysis were conducted with Stata 12.0 software. We included 5 studies with 564 HNC patients for the meta-analysis. All of the included patients were primary tumors and underwent surgical resection without prior radiotherapy or chemotherapy therapy. No significant heterogeneity was noted between MGMT and overall survival, MGMT and disease-free survival, and a fixed-effects model was used. HNC patients with MGMT hypermethylation and low expression had a poor prognosis, with pooled HR for overall survival (HR = 1.23, 95% CI: 1.10-1.38, P < .001) and disease-free survival (HR = 2.28, 95% CI: 1.45-3.58, P < .001). Subgroup analysis stratified by molecular abnormalities, such as hypermethylation or low expression, showed similar results. The insufficient number of trials included in our study encountered high risk of bias and may increase the deviation of the final meta-analysis results. HNC patients with MGMT hypermethylation and low expression were more likely to exhibit poorer survival. MGMT hypermethylation and low expression can predict survival in patients with HNC.

Establishing a glutamine metabolism-based model for predicting the prognosis of low-grade glioma.

Background: The natural history of patients with low-grade glioma (LGG) varies widely, but most patients eventually deteriorate, leading to poor prognostic outcomes. We aim to develop biological models that can accurately predict the outcome of LGG prognosis. Methods: Prognostic genes for glutamine metabolism were searched by univariate Cox regression, and molecular typing was constructed. Functional enrichment analysis was done to evaluate potential prognostic-related pathways by analyzing differential genes in different subtypes. Enrichment scores of specific gene sets in different subtypes were measured by gene set enrichment analysis. Different immune infiltration levels among subtypes were calculated using algorithms such as CIBERSORT and ESTIMATE. Gene expression levels of prognostic-related gene signatures of glutamine metabolism phenotypes were used to construct a RiskScore model. Receiver operating characteristic curve, decision curve and calibration curve analyses were used to evaluate the reliability and validity of the risk model. The decision tree model was used to determine the best predictor variable ultimately. Results: We found that C1 had the worst prognosis and the highest level of immune infiltration, among which the highest macrophage infiltration can be found in the M2 stage. Moreover, most of the pathways associated with tumor development, such as MYC_TARGETS_V1 and EPITHELIAL_MESENCHYMAL_TRANSITION, were significantly enriched in C1. The wild-type IDH and MGMT hypermethylation were the most abundant in C1. A five-gene risk model related to glutamine metabolism phenotype was established with good performance in both training and validation datasets. The final decision tree demonstrated the RiskScore model as the most significant predictor of prognostic outcomes in individuals with LGG. Conclusion: The RiskScore model related to glutamine metabolism can be an exceedingly accurate predictor for LGG patients, providing valuable suggestions for personalized treatment.

Targeted genetic and epigenetic profiling of esophageal adenocarcinomas and non-dysplastic Barrett’s esophagus

BackgroundDespite the efforts to describe the molecular landscape of esophageal adenocarcinoma (EAC) and its precursor lesion Barrett’s esophagus (BE), discrepant findings are reported. Here, we investigated the prevalence of selected genetic (TP53 mutations and microsatellite instability (MSI) status) and epigenetic (DNA promoter hypermethylation of APC, CDKN2A, MGMT, TIMP3 and MLH1) modifications in a series of 19 non-dysplastic BE and 145 EAC samples. Additional biopsies from adjacent normal tissue were also evaluated. State-of-the-art methodologies and well-defined scoring criteria were applied in all molecular analyses.ResultsOverall, we confirmed frequent TP53 mutations among EAC (28%) in contrast to BE, which harbored no mutations. We demonstrated that MSI and MLH1 promoter hypermethylation are rare events, both in EAC and in BE. Our findings further support that APC, CDKN2A, MGMT and TIMP3 promoter hypermethylation is frequently seen in both lesions (21–89%), as well as in a subset of adjacent normal samples (up to 12%).ConclusionsOur study further enlightens the molecular background of BE and EAC. To the best of our knowledge, this is one of the largest studies addressing a targeted analysis of genetic and epigenetic modifications simultaneously across a combined series of non-dysplastic BE and EAC samples.

Accelerated hyper-versus normofractionated radiochemotherapy with temozolomide in patients with glioblastoma: a multicenter retrospective analysis

Background and purposeThe standard treatment of glioblastoma patients consists of surgery followed by normofractionated radiotherapy (NFRT) with concomitant and adjuvant temozolomide chemotherapy. Whether accelerated hyperfractionated radiotherapy (HFRT) yields comparable results to NFRT in combination with temozolomide has only sparsely been investigated. The objective of this study was to compare NFRT with HFRT in a multicenter analysis.Materials and methodsA total of 484 glioblastoma patients from four centers were retrospectively pooled and analyzed. Three-hundred-ten and 174 patients had been treated with NFRT (30 × 1.8 Gy or 30 × 2 Gy) and HFRT (37 × 1.6 Gy or 30 × 1.8 Gy twice/day), respectively. The primary outcome of interest was overall survival (OS) which was correlated with patient-, tumor- and treatment-related variables via univariable and multivariable Cox frailty models. For multivariable modeling, missing covariates were imputed using multiple imputation by chained equations, and a sensitivity analysis was performed on the complete-cases-only dataset.ResultsAfter a median follow-up of 15.7 months (range 0.8–88.6 months), median OS was 16.9 months (15.0–18.7 months) in the NFRT group and 14.9 months (13.2–17.3 months) in the HFRT group (p = 0.26). In multivariable frailty regression, better performance status, gross-total versus not gross-total resection, MGMT hypermethylation, IDH mutation, smaller planning target volume and salvage therapy were significantly associated with longer OS (all p < 0.01). Treatment differences (HFRT versus NFRT) had no significant effect on OS in either univariable or multivariable analysis.ConclusionsSince HFRT with temozolomide was not associated with worse OS, we assume HFRT to be a potential option for patients wishing to shorten their treatment time.

Prognostic value of methylation status of MGMT gene promoter in patients with glioblastoma after combination treatment.

e14025 Background: The effectiveness of radiation therapy in the classical and stereotactic treatment regimens of the primary glioblastoma multiforme was analyzed and compared. Methods: 87 patients with primary glioblastoma multiforme were hospitalized during the study period. 17 patients of them were in the treatment group (stereotactic radiotherapy, dose per fraction 2 Gy, 60 Gy total dose, CTV 2.0 cm, PTV 0.1 cm - group 1), and 70 patients were in the control group (conventionally fractionated radiotherapy, dose per fraction 2 Gy, 60 Gy total dose, CTV 2.0 cm, PTV 0.5 cm - group 2). Patients of both groups were treated with alkylating agents as the third stage of complex treatment. MGMT methylation was evaluated by pyrosequencing. Results: The use of stereotactic radiation therapy allowed to conduct a full course of treatment without interruption, whereas in the second group, due to the development of adverse reactions, the course was interrupted for 7 patients (10%). The advantage of stereotactic radiation therapy was a less pronounced increase and rapid regression of neurological deficits during treatment, manifested by better local control for 6 months. Hypermethylation of MGMT was detected in 39% of glioblastoma cases and was not detected in non-tumor tissue. The presence of promoter methylation disrupts DNA repair and is associated with longer survival in glioblastoma patients receiving alkylating agents. Note that the median survival of patients with MGMT hypermethylation in the group 1 was 285 days (IQR = 323.75), and in the group without hypermethylation was significantly more than 338.5 days (IQR = 476) (p = 0.045). A similar trend was observed in the second group: in patients with hypermethylated MGMT, the median survival was 271 days (IQR = 337), the result is different from patients without hypermethylated MGMT – 342 days (IQR = 493) (p = 0.039). Conclusions: As a result, the application of the stereotactic approach of radiation therapy in the complex treatment of patients with glioblastoma and their classification by the MGMT methylation status with a cut-off point of 10% has the basis for improving the effectiveness and tolerability of radiation therapy and contributes to the overall survival of patients.

MGMT-Methylation in Non-Neoplastic Diseases of the Central Nervous System.

Quantifying O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation plays an essential role in assessing the potential efficacy of alkylating agents in the chemotherapy of malignant gliomas. MGMT promoter methylation is considered to be a characteristic of subgroups of certain malignancies but has also been described in various peripheral inflammatory diseases. However, MGMT promoter methylation levels have not yet been investigated in non-neoplastic brain diseases. This study demonstrates for the first time that one can indeed detect slightly enhanced MGMT promoter methylation in individual cases of inflammatory demyelinating CNS diseases such as multiple sclerosis and progressive multifocal leucencephalopathy (PML), as well as in other demyelinating diseases such as central pontine and exptrapontine myelinolysis, and diseases with myelin damage such as Wallerian degeneration. In this context, we identified a reduction in the expression of the demethylase TET1 as a possible cause for the enhanced MGMT promoter methylation. Hence, we show for the first time that MGMT hypermethylation occurs in chronic diseases that are not strictly associated to distinct pathogens, oncogenic viruses or neoplasms but that lead to damage of the myelin sheath in various ways. While this gives new insights into epigenetic and pathophysiological processes involved in de- and remyelination, which might offer new therapeutic opportunities for demyelinating diseases in the future, it also reduces the specificity of MGMT hypermethylation as a tumor biomarker.

Improved local control in p16 negative oropharyngeal cancers with hypermethylated MGMT

IntroductionPatients with oropharyngeal cancers that are p16 negative (p16−) have worse outcomes than those who are p16 positive (p16+) and there is an unmet need for prognostic markers in this population. O6-Methylguanine (O6-MG)-DNA-methyltransferase (MGMT) gene methylation has been associated with response to chemoradiotherapy (CRT) in glioblastoma. We sought to find if MGMT promoter methylation was associated with outcomes of locally advanced oropharyngeal and oral cavity squamous cell carcinoma (OOSCC) in patients treated with definitive concurrent CRT. MethodsPatients were identified with primary OOSCC, known p16 status, retrievable pre-treatment biopsies, and at least 6 months of follow-up who received definitive concurrent CRT from 2004 to 2015. Biopsies were tested for MGMT hypermethylation (MGMT+) using a Qiagen pyrosequencing kit (Catalog number 970061). Outcomes were subsequently recorded and analyzed. ResultsFifty-eight patients were included with a median follow up of 78 (range 6–196) months. Fourteen patients (24.1%) had oral cavity cancer and 44 (75.9%) had oropharyngeal cancer. A significant difference was found for local recurrence free survival (LRFS) by combined MGMT and p16 status (p = 0.0004). Frequency of LR in MGMT+/p16+, MGMT+/p16−, MGMT−/p16+, and MGMT−p16− patients was 14.3%, 14.3%, 13.0%, and 69.2%, respectively (p = 0.0019). A significant difference was not found for distant recurrence free survival (p = 0.6165) or overall survival (p = 0.1615). LRFS remained significant on analysis restricted to oropharyngeal cancer patients (p-value = 0.0038). ConclusionPatients who are p16− and MGMT+ with oropharyngeal and oral cavity squamous cell carcinoma have significantly better LC with definitive CRT than those who are p16− and MGMT−. Prospective studies are needed to verify these findings.

NCMP-17. MISMATCH REPAIR MUTATIONS AND THE CENTRAL NERVOUS SYSTEM: A CASE SERIES OF GERMLINE MUTATIONS AND CNS MALIGNANCY

Abstract Our understanding of genetic predispositions for malignancy is continually evolving. One family of germline mutations well described in the literature is that of the DNA mismatch repair mechanism (MMR). Lynch syndrome (LS) is due to a loss of function mutation of several MMR genes- MSH2, MLH1, MSH6, and PMS2. Germline MMR mutations lead to microsatellite instability and loss of genomic integrity resulting in an increased risk for various cancers (colorectal, genitourinary, etc). LS may be as common as 1 in 400 people and some MMR mutations have been associated with gliomas. There is a paucity of information regarding frequency of glioma subtypes as well as tumor genetic and molecular characteristics which have important clinical implications. We describe a case series of 6 individuals with germline MMR mutations and brain tumors. Those with MSH2 and PMS2 mutations (n=3) developed glioblastomas at a mean age at diagnosis of 48 years. These tumors expressed MGMT hyper-methylation and high tumor mutational burden. Only one had IDH-1 mutation. Those with MLH1 mutations (n=3), did not develop gliomas. This raises the question of differential glioma subtype development based on MMR gene. It also highlights the possibility of Lynch-associated gliomas having more favorable treatment response due to MGMT methylation and potential response to immunotherapy based on high tumor mutational burden. Though the sample size is small, there appears to be a preponderance of women compared to men (5:1 respectively). Larger studies are needed to verify CNS involvement in germline MMR mutations. In doing so, we hope to identify factors that may influence clinical management and lead to a better understanding of treatment response and disease prognosis.

The distribution of isocitrate dehydrogenase mutations, O6-methylguanine-DNA methyltransferase promoter methylation, and 1p/19q codeletion in different glioma subtypes and their correlation with glioma prognosis in Taiwanese population: A single center study

The molecular characteristics of glioma patients, such as 1p/19q codeletion, MGMT promoter region methylation, and IDH1/IDH2 mutations, carry important information for tumor classification, biological behavior, and the prediction of patient survival. We sought to address the clinical characteristics of brain tumor patients in the Taiwanese population. Our aim was to compare the H&E-based classification and the 2016 WHO classification in glioma patients. 192 patients were recruited in this study including glioblastomas (GBMs) and lower-grade gliomas (LGGs). We performed assays to determine the statuses of 1p/19q codeletion, MGMT promoter methylation, and IDH mutations in tumors and assessed their associations with survival time. We also compared the difference between H&E and WHO 2016 classification. Using the H&E classification, the overall survival time (OST) was associated with IDH status in astrocytoma and oligoastrocytoma patients but not in oligodendroglioma and GBM patients. However, OST showed no significant correlation with MGMT promoter methylation in all the groups. Furthermore, no significant association was observed between 1p/19q codeletion and OST in GBM patients. Using the WHO 2016 classification, our results indicated that astrocytoma and oligodendroglioma patients with mutant IDH showed a significantly prolonged OST than patients with wild-type IDH. However, only oligodendroglioma patients with MGMT hypermethylation demonstrated statistically significant difference. Prognostic factors, including the age at diagnosis, IDH mutations, MGMT promoter methylation, and 1p/19q codeletion, could independently predict patient survival. Based on the WHO 2016 classification of glioma, the new histological groups provide a better and objective method to categorize glioma patients and predict patient survival.

Associations among smoking, MGMT hypermethylation, TP53-mutations, and relapse in head and neck squamous cell carcinoma.

BackgroundEpigenetic silencing of the O6-methylguanine-DNA methyltransferase (MGMT) DNA repair enzyme via promoter hypermethylation (hmMGMT) may increase mutations in the TP53 oncosuppressor gene and contribute to carcinogenesis. The effects of smoking, which is a risk factor for head and neck squamous cell carcinoma (HNSCC), were investigated to determine whether they up- or down-regulate hmMGMT. Additionally, the impact of hmMGMT and disruptive TP53-mutations on relapse was investigated in patients with HNSCC.MethodsThis study included 164 patients with HNSCC who were negative for both p16 protein expression and human papilloma virus infection. The association of smoking and hmMGMT was investigated using multiple logistic regression analysis. Competing risk regression was used to evaluate the effects of hmMGMT and TP53-mutations in exon 2 to 11 on relapse of HNSCC.ResultshmMGMT was observed in 84% of the 164 patients. TP53-mutations, specifically, G:C>A:T transition, were more frequent in patients with hmMGMT (32%) than in those without hmMGMT (8%). The frequency of disruptive TP53-mutations was not significantly different between groups. Compared with nonsmoking, heavy smoking of 20 pack-years or more was significantly associated with decreased hmMGMT (adjusted odds ratio, 0.08; 95% CI, 0.01 to 0.56; P = 0.01). Patients who had both hmMGMT and disruptive TP53-mutations showed a significantly higher relapse rate than all other patients (subdistribution hazard ratio, 1.77; 95% CI, 1.07 to 2.92; P = 0.026).ConclusionsIt was found that hmMGMT was suppressed by heavy smoking, and hmMGMT combined with disruptive TP53-mutations may indicate a poor prognosis in patients with HNSCC.

Hypermethylation status of DAPK, MGMT and RUNX3 in HPV negative oral and oropharyngeal squamous cell carcinoma.

Squamous cell carcinoma of the oral cavity and oropharynx is the sixth most common type of cancer in the world. During tumorigenesis, gene promoter hypermethylation is considered an important mechanism of transcription silencing of tumor suppressor genes, such as DAPK, MGMT and RUNX3. These genes participate in signaling pathways related to apoptosis, DNA repair and proliferation whose loss of expression is possibly associated with cancer development and progression. In order to investigate associations between hypermethylation and clinicopathological and prognostic parameters, promoter methylation was evaluated in 72 HPV negative oral and oropharyngeal tumors using methylation-specific PCR. Hypermethylation frequencies found for DAPK, MGMT and RUNX3 were 38.88%, 19.44% and 1.38% respectively. Patients with MGMT hypermethylation had a better 2-year overall survival compared to patients without methylation. Being MGMT a repair gene for alkylating agents, it could be a biomarker of treatment response for patients who are candidates for cisplatin chemotherapy, predicting drug resistance. In view of the considerable levels of hypermethylation in cancer cells and, for MGMT, its prognostic relevance, DAPK and MGMT show potential as epigenetic markers, in a way that additional studies may test its viability and efficacy in clinical management.

GEN-06 CLINICAL COURSE AFTER TUMOR RECURRENCE OF MGMT HYPERMETHYLATED GLIOBLASTOMA

MGMT methylation in glioblastoma is a biomarker for determining treatment responsiveness and predicting prognosis. We analyzed whether there were differences in the prognosis between glioblastoma with MGMT hypermethylation and other glioblastomas after tumor recurrence. We enrolled 184 patients who underwent radiation therapy and temozolomide chemotherapy after tumor resection for newly diagnosed glioblastoma. MGMT methylation was quantitatively analyzed using methylation-specific high resolution melting analysis. The cut-off value for MGMT methylation had a difference of 35% from the previous values. The subjects were split into three groups according to their MGMT methylation levels, 122 in the low (L) methylation group (levels of 0–34%), 40 in the medium (M) methylation group (levels of 35–69%), and 22 in the high (H) methylation group (levels of 70% or more). We mainly focused on and compared the progression after recurrence. The progression-free survival (PFS) rate and overall survival (OS) rate were significantly longer in the M and H groups than in the L group. There was no difference in PFS between group M and group H, but OS was significantly longer in group H. The details of treatment for the 16 of 22 patients who had recurrences in group H are as follows: temozolomide, n = 1; bevacizumab, n = 8; investigational drugs (peptide vaccines and immune checkpoint inhibitors), n = 3; and supportive care, n = 4. The median survival rate for these 16 patients after recurrence was 18 months. Even patients who received only supportive care had a median survival time ranging between 9 and 17 months. Our results indicate that MGMT hypermethylation in glioblastoma is effective to a certain degree with other treatments even after recurrence. Even patients who underwent only supportive care survived for a relatively longer period of time. Biologically, MGMT hypermethylation may be associated with a moderately slow-growing tumor.

Aberrant epigenetic inactivation of RASSF1A and MGMT gene and genetic mutations of KRAS, cKIT and BRAF in Indian testicular germ cell tumours

Testicular germ cell tumor (TGCT) development may involve a series of modification at epigenetic and genetic level which may act synergistically and transform the primordial gonocyte. This study evaluated the frequency and distribution pattern of RASSF1A/MGMT gene methylation and KRAS, BRAF and cKIT gene mutation in Indian TGCT patient, and their correlation with clinicopathological features. Forty-one TGCT tumors were used to investigate hypermethylation of RASSF1A and MGMT gene and mutations of KRAS codon 12/13, BRAF V600E and cKIT exon 17 mutations. RASSF1A and MGMT methylation was noted in 58.5% and 10% of the TGCTs. A higher frequency of RASSF1A methylation was noted in seminomas (71%, 17/24), while MGMT methylation was frequent in mixed tumors (23%, 3/13). Interestingly, 3 of 41 cases showed concurrent methylation of both the genes. The absence of tumor necrosis was significantly associated with increased frequency of MGMT hypermethylation (30% vs. 3%, p = 0.03). KRAS mutation was identified in 17% (7/41), while none showed BRAF and cKIT mutation. KRAS mutations were predominantly found in codon 12 with G12V as the most recurrent mutations. Mixed germ tumors tends to show increased frequency of KRAS mutation (31%, 4/13), followed by pure seminomas (4%, 1/24). Interestingly, KRAS mutation rate was significantly higher in metastatic tumors in comparison to primary tumors (100% vs. 13%, p = 0.02). No other association of RASSF1A/MGMT/KRAS alterations with other clinicopathological features was noted. In conclusion, these data support the notion that the cancer-associated alterations in the RASSF1, MGMT and KRAS gene may significantly contribute to TGCT pathogenesis.

417P - Effect of cumulative dose of maintenance temozolomide on overall survival in patients with high grade glia tumours: A single institution analysis

BackgroundStandard of care of high grade glia tumors is represented by concomitant temozolomide (TMZ) and radiotherapy (RT), followed by maintenance TMZ. In clinical practice, administering the full programmed cumulative dose of treatment is often a challenge. The effect of different cumulative doses of maintenance TMZ on median overall survival (mOS) is yet not known. MethodsWe retrospectively analyzed patients with histological diagnosis of high grade glia tumors (91% grade IV, 7% grade III, 2% others) treated at our institution with RT plus concomitant TMZ, followed by maintenance TMZ (according to the trial by Stupp et al.), between February 2012 and April 2019. We planned 6 cycles of maintenance TMZ in patients without MGMT hypermethylation and 12 cycles in those with MGMT hypermethylation. The main objective of our research was to study the effect of cumulative dose of maintenance TMZ on mOS in patients without MGMT hypermethylation. We also focused on time from diagnosis to beginning of chemoradiotherapy and its potential effect on mOS in our whole cohort. We performed statistical analysis with SPSS, using Kaplan-Meier curves. ResultsWe enrolled 114 patients, with median age of 63 years (IQR 54-69 y). MGMT was hypermethylated in 35 (30%) patients, not hypermethylated in 56 (49%) patients, unknown in 24 (21%) patients. In our cohort, mOS was 14 months, in line with previously reported data. In patients without MGMT hypermethylation, mOS was 23,4 months when cumulative dose of maintenance TMZ was>= 4500mg/m2 versus 11 months when cumulative dose was < 4500mg/m2(HR 0,23 [0,10-0,54, CI 95%], p<0,001). When considering our entire cohort, chemoradiotherapy was started in a median of 44 days from diagnosis (IQR 35-56 days); mOS was not statistically different in patients who started therapy over 42 days after diagnosis (HR 0,76 [0,44-1,33, CI 95%], p 0,35). ConclusionsOur study shows that in patients with high grade glia tumors without MGMT hypermethylation a cumulative dose of maintenance TMZ < 4500mg/m2negatively impacts on mOS compared to a cumulative dose of>= 4500mg/m2. Moreover time elapsed from diagnosis to beginning of treatment does not significantly influence mOS. Legal entity responsible for the studyDepartment of Medical Oncology, ASST-Settelaghi, Varese (Italy). FundingHas not received any funding. DisclosureAll 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.

Rectal Aberrant Crypt Foci in Humans Are Not Surrogate Markers for Colorectal Cancer Risk.

INTRODUCTION:Over the past 20 years, aberrant crypt foci (ACF) have emerged as potential precursors and biomarkers for colorectal cancer (CRC). However, data regarding their molecular pathogenesis, as well as their endoscopic and histological identification, remain inconsistent.METHODS:A wide cohort of ACF from 100 control subjects and 100 case patients, including patients with adenoma and CRC, were characterized for endoscopic, morphologic, and molecular features.RESULTS:We observed that among all the endoscopic features evaluated, only the number of large ACF correlated with CRC risk (P = 0.003), whereas the histological classification, as assessed by 2 different pathologists, was inconsistent and did not differ between control and case patients. Moreover, only a few APC and BRAF mutations and no microsatellite instability were detected in our samples. KRAS mutations were detected in 16.3% of ACF samples, which also exhibited increased MGMT hypermethylation. However, none of those events were found to be predictive of CRC risk.DISCUSSION:Although ACF might be preneoplastic lesions of the colon, they are not suitable biomarkers for assessing CRC progression.