Abstract LB-221: Differential aberrant DNA methylation predicts for Tarceva response in NSCLC
Authors: Shames David S. , Januario Tom , Holcomb Thomas , Soriano Robert , Wu Thomas , Modrusan Zora , Seshagiri Somasekar , Egan Brian , Hampton Garret M. , Amler Lukas C. , Yauch Robert L. 
Published: February 2011

Recent phase III clinical trials have shown that some non-small cell lung cancer (NSCLC) patients without epidermal growth factor receptor mutations derive clinical benefit from erlotnib, a small molecule EGFR inhibitor. Currently there is no way to identify this important patient subset. In an effort to explore whether aberrant DNA methylation profiles could be used to better define the subset of patients with NSCLC who might respond to erlotinib, we have undertaken an integrated genomics approach employing both gene expression profiling and methylated DNA immunoprecipitation (meDIP). In the initial phase of this study, we used a series of twelve NSCLC cell lines without EGFR mutations, which we defined in a previous report as either sensitive or resistant to erlotinib, based on an in vitro microtiter assay (cell titer glow). We treated each cell line with the DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-azadC) qod for 6 days and confirmed gene induction using quantitative RT-PCR (QPCR). We then performed gene expression profiling on the mock treated (DMSO) and 5-azadC (1 M) treated cell lines using the Affymetrix U133P 2.0 chip. Consistent with previous reports we found that the expression levels of large numbers of genes are affected by 5-azadC treatment, and in both directions. In addition, since it is known that 3′ biased gene expression arrays can miss expression changes in alternatively spliced (and methylated) variants of genes (such as RASSF1A and p16), we sought to improve our ability to discover differentially methylated regions (DMR) by using meDIP in parallel with gene expression profiling.

We performed meDIP on DNA prepared from cell pellets made at the same time as we did the 5-azadC treatments. We confirmed successful immunoprecipitation and enrichment of methylated DNA using QPCR for several targets including the imprinted H19 locus, as well as several others. Analysis of these data suggests that thousands of loci are differentially methylated between the twelve cell lines, and that there are several hundred DMRs which differentiate between Tarceva sensitive and resistant NSCLC lines.

While there was overlap between the meDIP and gene expression data, there were many genes that appeared only on one or the other platform. However, we confirmed that both approaches identify loci that are differentially methylated by direct sodium bisulfite sequencing between the sensitive and resistant cell lines.

Subsequent validation of the DMRs in a broader panel of NSCLC lines shows that our approach has successfully identified markers which differentiate between Tarceva sensitive and resistant cell lines. Future studies will determine whether these DMRs can distinguish between tumors that respond to erlotnib and those that are resistant.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-221.