Abstract 794: Targeted-therapy related molecular biomarker characterization in NSCLC smokers and never-smokers by a novel qRT-PCR for microdissected tissues methodology
Authors: Yan Wusheng , Shih Joanna H. , Raso M. Gabriela , Rodriguez-Canales Jaime , Behrens Carmen , Papadimitrakopoulou Vali A. , Herbst Roy S. , Emmert-Buck Michael R. , Wistuba Ignacio I. , Erickson Heidi S. 
Published: February 2011

Abstract
Several studies have focused on the identification of biologic markers that predict early recurrence and death in patients with non-small cell lung cancer (NSCLC); however, few studies have focused on indicators of outcome in ever-smoker and never-smoker lung cancer patients. We hypothesize that NSCLC primary tumors from smokers and never-smokers differ significantly in the expression of molecular abnormalities and their corresponding biomarkers. We also believe that the different pattern of molecular pathway abnormalities in NSCLCs based on smoking status may explain different response to targeted therapy in both patients’ populations.
To test our hypotheses, and better understand the results of biomarker analysis in molecularly targeted clinical trials, we developed a novel qRT-PCR pre-amplification methodology for gene expression analysis of microdissected (MD) tissues to determine and compare the gene expression patterns of targeted therapy related molecular biomarkers and pathways from surgically resected tissue specimens obtained from NSCLC patients. Previously only 9 genes of interest could be analyzed from a typical MD sample (Erickson et al. 2009 Nature Protocols). Our new methodology allows for the use of 8 ng of total RNA for the analysis of up to 24 genes of interest or 24 ng of total RNA for the analysis of up to 72 genes of interest.
We then applied this methodology to 40 MD stage I/II NSCLC adenocarcinoma tissues from tumor epithelium and matched normal bronchial epithelium from 20 smokers and 20 never-smokers for EGFR, KRAS, HER2N, BRAF, PIK-3, IGFR-1, LKB-1, mTOR, and AKT analysis. An average of 77.3 ng of total RNA (average RIN = 5.0) was recovered from ∼5000 MD epithelial cells per tissue sample. Gene expression was analyzed by relative quantitation using the 2−ΔΔCt method. Results show that analysis of the expression of multiple genes using a pre-amplification step is a feasible approach for small tissue specimens obtained from clinical trials with advanced lung cancer patients. Preliminary analysis revealed distinct gene expression patterns in smokers compared to never-smokers. Future plans include correlating gene expression with gene mutation, copy number variation, and immunohistochemistry analysis of these tissue specimens.
This novel qRT-PCR methodology for gene expression analysis of MD tissues will be used to analyze fine needle aspiration and core needle biopsy tissue specimens from biomarker-driven clinical trial patients (e.g. BATTLE) for validation of profiles and analysis of multiple molecular pathway targets that can predict response to chemotherapy and targeted therapy.
This work was funded by Department of Defense BATTLE Lung Cancer Program Initiative W81XWH-06-1-0303, Cohen-Reinauch BATTLE-2 Fund, and National Cancer Institute Intramural Program.
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 794.