Abstract 3148A: MYOD1, hTERT, and RASSF1A gene promoter methylation is useful for prediction of outcome/ prognostication in patients with invasive cervical cancer

Abstract

Abstract Background: Cervical cancer is the leading cause of cancer related mortality in women worldwide including India. DNA methylation, an epigenetic event regulates gene expression and chromatin structure. We hypothesized that the promoter methylation of genes involved in tumor suppression, DNA repair, cell cycle and apoptosis may affect response to radiation therapy and therefore the outcome of women with invasive cervical cancer. Objective: To perform DNA methylation profiling of BRCA1, RASSF1A, MLH1, ESR1, MYOD1 and hTERT genes in tissue samples of invasive cervical cancer and correlate the methylation pattern to radiation response and clinical outcome. Methodology: After obtaining informed consent, patients with invasive cervical cancer who underwent chemo-radiation as per protocol and with a minimum of 2 years follow-up after completion of treatment were included in the analysis [N=91; 2008-11]. The patients were followed up 3 monthly for evidence of local or systemic disease and their outcome documented. 60 patients were alive and well with No evidence of disease [NED] at the end of 24 months whereas 31 patients had evidence of disease [ED]. Out of these 31 patients, 3 died of disease, 9 had evidence of systemic disease [SED] and 19 patients had local disease recurrence [LED]. Genomic DNA was extracted from the snap frozen cervical biopsy tissues using TRIzol reagent, modified using the EZDNA methylation kit and promoter methylation of the individual genes determined by Real Time Polymerase Chain Reaction using Taqman probe chemistry using previously published primer and probe sets and appropriate controls. The probes were labelled with 5′-FAM and 3′-BHQ respectively. The housekeeping gene α-ACTIN was taken as the reference gene. Statistical analysis: Pearson's Chi-square test with Yates correction and Step-wise Logistic Regression was performed using the SPSS package version 18.0. A p-value <0.05 was considered as statistically significant. Results: In invasive cervical cancer, BRCA1, RASSF1A, MLH1, MYOD1, ESR1 and hTERT gene promoters were methylated in 51%, 47%, 40%, 51%, 38% and 60% cases respectively. Comparison of the NED vs LED groups revealed that methylated MYOD1 and hTERT genes in combination predicted for better outcome (Chi-Square test, p=0.03). Further logistic regression analysis revealed that methylation of RASSF1A, MYOD1 and hTERT genes predicted for better outcome following radiation therapy indicating that these genes play a role in determining radiation responsiveness of a tumor. The cross-classification table showed that 80% of the cases could be correctly classsified by the logistic model. Conclusion: A panel of 3 genes which includes MYOD1, hTERT and RASSF1A may be useful to predict the outcome of invasive cervical carcinoma patients treated with standard chemo-radiation therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3148A. doi:1538-7445.AM2012-3148A