Abstract 3195: Formalin-fixed and paraffin-embedded (FFPE) DNA recovery for high-throughput genotyping of lung cancer tissues
Authors: Galindo Hector , Solis Luisa , Fujimoto Junya , Hanson Nana E. , McDowell Christina , Riquelme Erick , Tang XiMing , Behrens Carmen , Lee Jack J. , Kim Edward S. , Wistuba Ignacio I. , Erickson Heidi S. 
Published: August 2012

Abstract
Tumor biomarker evaluation, such as EGFR or KRAS mutation status, is a main factor in non-small cell lung carcinoma (NSCLC) treatment decisions. For most NSCLC patients, tumor-specific biomarkers have not been identified for effective therapy. This has been due in part to difficulties obtaining the DNA quantity and quality from archival FFPE clinical specimens necessary for high-throughput (HT) mutation analysis. Activating mutations and copy number variation (CNV) analysis of key lung cancer-related oncogenes in NSCLC FFPE tissues will allow for identification of subsets of patients with oncogenotypes of which treatment can be personalized. Thus, development of improved DNA recovery methods from FFPE tissues is imperative. The goal of this study was to improve DNA recovery from archival specimens for analyses by HT genotyping technologies. DNA was extracted/isolated from 2 normal human epithelial bronchial/4 NSCLC cell lines and 389 resected FFPE NSCLC tissues using improved SPRI-TE/DNA cleanup and compared to standard method (SM). Detailed tissue characteristics [whole tissue (WT), manual microdissected (MMD), artificial core needle biopsy (aCNB)], thickness (10 or 5 μm), tumor area (cm2), % tumor cells, and % fibrotic/necrotic tissue was recorded. DNA recovery quantity, quality and amplification were assessed. HT genotyping methodologies for analysis of FFPE DNA included 1) aCGH/qPCR for CNV, 2) DNA amplification for sequencing, and 3) Sanger, pyrosequencing, and MALDI-TOF MS SNP analysis for KRAS mutation status. Data were analyzed by unpaired t-test with Welch's correction, one-way ANOVA, and linear regression model for multivariable analyses. Statistical significance was defined as p≤0.05. DNA recovery using improved SPRI-TE/DNA cleanup was 5X higher than SM. DNA recovery was significantly higher (p≤0.05) from: a) 2 x 10 μm sections WT vs. MMD or aCNBs, b) tissue area &gt;1 cm2, and c) decreasing % fibrosis. aCGH was successfully performed using DNA from FFPE NSCLC primary tumors (PT) and brain metastases (BM), identified multiple chromosomal regions with significant CNV. BM demonstrated significantly higher frequency of CN gains than PT in 3 chromosomal regions: 8q24.3 (100% vs. 40%), 19q13.33 (90% vs. 20%), and 20q13.12 (60% vs. 0%). DNA amplification (WGA vs. matched non-WGA) was evaluated for sequencing and resulted in correct mutation calls (p≤0.05). KRAS mutation analysis by 3 methods resulted in 100% identical mutation calls. Using our improved SPRI-TE/DNA cleanup, recovery of quantities and qualities of DNA from FFPE NSCLC tissues for precise HT genotyping analysis was achieved. Best predictor of optimal DNA quantity recovery is tumor area &gt;1 cm2. FFPE NSCLC tissue DNA is suitable for use in HT genotyping assays and discovery of oncogene-specific genotypes which could be applied to personalized medicine. This work was supported by The V Foundation.
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 3195. doi:1538-7445.AM2012-3195