Abstract A70: Epigenome and genome alterations in platinum resistant ovarian tumors.
Authors: Fang Fang , Cardenas Horacio , Miller Dave , Buechlein Aaron , Yu Qing , Liu Yunlong , Jiang Guanglong , Taverna Pietro , Keer Harold , Rusch Doug , Matei Daniela , Nephew Kenneth P. 
Published: May 2016

Purpose: Epigenetic changes, particularly in DNA methylation, have been implicated in acquired resistance to platinum in ovarian cancer (OC). The goal of the current study was to analyze and integrate global RNA expression and DNA methylation profiles of platinum resistant tumors compared to untreated, platinum-sensitive ovarian tumors, as well as to measure genomic and epigenomic changes induced by guadecitabine (SGI-110) in tumors.

Methods: An ongoing phase I/II multi-institutional clinical trial uses the novel DNA methyltransferase (DNMT) inhibitor guadecitabine to re-sensitize recurrent platinum resistant OC to carboplatin. Patients enrolled in this trial had recurrent platinum resistant OC. Tumor biopsies were collected at baseline and after two cycles of guadecitabine administered daily for 5 days at a low (30mg/m2) dose (28 days per cycle). RNA and DNA were extracted from 48 and 57 baseline tumors and analyzed for next generation sequencing approaches to interrogate transcriptomes (RNA-seq) and methylomes (Infinium Human Methylation450 (HM450) arrays), respectively. Differential gene expression and DNA methylation profiles were generated and used for Ingenuity Pathway Analysis (IPA) to identify the top altered pathways in response to guadecitabine. Expression of DNMTs was examined by real-time RT-PCR and immunohistochemistry. LINE1 methylation and promoter methylation of selected genes (MAGE-A2, MAGE-A3, MAGE-A11, NY-ESO, RASSF1, MLH1, and HOXA11) were quantified by pyrosequencing before and after guadecitabine treatment (n=12 paired samples).

Results: Analysis of a limited number of paired samples before and after treatment (n=8) revealed significant changes in global gene expression profiles induced by guadecitabine, with 960 altered genes representing immunopathway enrichment including cytokine production in macrophages and T helper cells by IL-17A and IL-17F, granulocyte /agranulocyte adhesion and inflammation, IL-8 signaling, p38 MAPK signaling, cAMP-mediated signaling, and innate immunity. Epigenetic profiling using HM450 revealed extensive methylation changes when comparing recurrent platinum resistant ovarian tumors (n=42) to primary, untreated ovarian cancer specimens analyzed as part of the TCGA project (n=10). Six hundred and four promoters were significantly differentially methylated (adjusted p&lt;0.05, absolute methylation changes β&gt;0.2), among which, 498 and 106 were hypermethylated or hypomethylated respectively in recurrent platinum resistant ovarian tumors. IPA analysis of baseline tumor transcriptome and methylome demonstrated significant enrichment in a wide range of pathways associated with cancer, stem cells, inflammation and the immune system. DNMT1, 3A, and 3B mRNA levels in the tumors were highly variable (n=19). Analysis of a limited number of paired samples (n=7) revealed no significant changes in global methylation or in DNMT expression levels induced by treatment with guadecitabine (adjusted p&gt;0.05). However, the DNMT inhibitor induced significant methylome alterations in selected patients. Significant hypomethylation of MAGE-A3 and MAGE–A11 promoters (p&lt;0.05) was detected. Correlations between methylation changes and clinical outcomes are being explored.

Conclusions: These data suggest that treatment with the DNMT inhibitor guadecitabine induces a reactivation of immune responses in human OC. Correlations between methylation changes and expression profiles are being explored.

Citation Format: Fang Fang, Horacio Cardenas, Dave Miller, Aaron Buechlein, Qing Yu, Yunlong Liu, Guanglong Jiang, Pietro Taverna, Harold Keer, Doug Rusch, Daniela Matei, Kenneth P. Nephew. Epigenome and genome alterations in platinum resistant ovarian tumors. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A70.