Abstract LB-263: Analysis of microenvironment effects on pancreatic cancer biomarker expression using a novel method for FFPE RNA extraction
Authors: Hoover Malachia , Adamian Yvess , Brown Mark , Maawy Ali A. , Hoffman Robert , Bouvet Michael , Doebler Robert , Kelber Jonathan A. 
Published: July 2016

Abstract
Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths. There are virtually no biomarkers to aid in early detection or predicting therapy response, newly diagnosed patients have less than a 7% 5-year survival rate and the median survival from the time of diagnosis is less than 12 months. Next-Generation Sequencing (NGS) together with in vitro/in vivo functional studies is likely to identify and validate new biomarkers of PDAC onset, progression and therapy resistance. Importantly, the substantial archives of formalin-fixed, paraffin-embedded (FFPE) samples from PDAC patients are likely to be a rich resource for linking molecular signatures to relevant clinical data. However, NGS methods on FFPE samples are severely hindered because extracting high-quality nucleic acid material from these samples is time-consuming and inefficient. We have sought to develop novel methods for improving the extraction of high-quality RNA from FFPE samples within the context of commercially-available FFPE RNA kits and protocols. Together with researchers and clinicians at Claremont BioSolutions, AntiCancer Inc. and UCSD our laboratory has developed a novel nucleic acid extraction method that significantly increases RNA yield and integrity from PDAC cell line and Patient-Derived Xenograft FFPE samples. By briefly (&lt;5 minutes) integrating the newly designed Claremont BioSolutions’ microhomogenizer (mH) tool within the commercially available Qiagen FFPE RNA extraction protocol, RNA recovery from these samples is increased by approximately 3-fold while maintaining standard 260/280 ratios (2.03 +/- 0.02 w/ mH step) and high RNA Quality Index (RQI) values (7.3 +/- 0.6 w/ mH step). Bioanalyzer testing further demonstrated that the mH-purified FFPE RNA was longer. Previous studies have revealed that PDAC cell gene expression signatures vary significantly when cells are propagated in vitro versus in vivo as subcutaneous or orthotopic xenografts. Notably, we found that the previously published expression patterns for KRas dependency genes within these three microenvironments were most accurately reproduced when extracting PDAC FFPE RNA with our mH-based method. Finally, we used our mH-based method to test the effects of the in vivo tumor microenvironment (TME) on the expression trends of a panel of novel PDAC biomarkers. In this regard, we demonstrate that PEAK1 and MST1R expression levels are decreased and increased, respectively, by over 100 fold in the orthotopic microenvironment relative to the subcutaneous microenvironment. These results reveal the critical nature of the tumor microenvironment when evaluating the clinical relevance of new biomarkers in cell lines or patient-derived samples. Furthermore, this new mH-based FFPE RNA extraction method has the potential to positively impact the FFPE-RNA-NGS workflow for cancer biomarker identification/validation.
Citation Format: Malachia Hoover, Yvess Adamian, Mark Brown, Ali A. Maawy, Robert Hoffman, Michael Bouvet, Robert Doebler, Jonathan A. Kelber. Analysis of microenvironment effects on pancreatic cancer biomarker expression using a novel method for FFPE RNA extraction. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-263.