Abstract 5348: Reliable gene expression profiling from smaller and H&E stained clinical FFPE specimens using EdgeSeq platform
Authors: Qi Zhenhao , Wang Lisu , Desai Keyur , Cogswell John , Stern Mark , Lawson Byron , LaFleur Bonnie , Vitazka Patrik 
Published: July 2017

Abstract
Background: The scarcity of biological specimens and suboptimal quality of collected material, make clinical biomarker studies challenging. The EdgeSeq system, a novel genomics analysis platform which combines quantitative nuclease protection assay (qNPA) technology with next-generation sequencing (NGS), has the potential to mitigate the aforementioned challenges. The technology allows for a limited sample input and delivers high quality gene expression profiling data from challenging material such as FFPE tissues. However, there is further unmet need toward even smaller tissue input or in taking advantage of archival stained FFPE slides to fill the data gap, specifically for studying tumor microenvironment in the oncology clinical trials
Methods: We performed EdgeSeq analysis on six triplets of unstained, H&E stained and IHC stained slides from non-small cell lung cancer (NSCLC) patients tumor tissue using Oncology biomarker panel (OBP). Next, we extended the EdgeSeq OBP analysis to 44 pairs of unstained and H&E stained slides from NSCLC to confirm the good correlation. To assess feasibility of limited tissue input, we first optimized bead-to-DNA volume ratio during NGS library cleanup by testing 4 ratios of bead-to-DNA from 2.5 to 1.0. Next, crude lysate prepared from small cell lung cancer (SCLC) FFPE tissue was 2-fold diluted from equivalent tissue area of 5mm2 to 0.08 mm2, and was subjected to EdgeSeq OBP profiling using the optimal bead-to-DNA ratio. Finally, correlation of gene expression profiles between stained and unstained pairs were calculated.
Results: Within the six triplets of unstained, H&E stained and IHC stained slides, the gene expression profiles from H&E slides showed good correlation with the profiles from matched unstained FFPE. An extended NSCLC sample set of 44 pairs of unstained and H&E stained FFPE further confirmed overall good correlation with 84% (37 out of 44 ) pairs with Pearson correlation coefficients of 0.9 or higher. In assessing the limited tissue input, crude lysate was diluted to as little as equivalent tissue area of 0.08mm2. The tissue area as small as 0.31 mm2 resulted in Pearson correlation coefficients of 0.9 with 5mm2 surface area input.
Conclusion: We demonstrated feasibility of using EdgeSeq to generate reliable gene expression data from H&E stained FFPE tissues. In addition, we showed that EdgeSeq platform can be used to generate reliable expression data from crude FFPE tissue lysate equivalent to surface area as low as 0.31 mm2 (size of larger laser microdissection tile or half of a tissue microarray core). The data show that EdgeSeq can be considered as a platform of choice to analyze specimens of suboptimal quality or of limited quantity and thus may help to fill the biomarker data gap in the oncology clinical trials, including enabling research into patterns of interactions and mechanisms of response and resistance of immune cell engagement with tumor at the loco-regional level.
Citation Format: Zhenhao Qi, Lisu Wang, Keyur Desai, John Cogswell, Mark Stern, Byron Lawson, Bonnie LaFleur, Patrik Vitazka. Reliable gene expression profiling from smaller and H&E stained clinical FFPE specimens using EdgeSeq platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5348. doi:10.1158/1538-7445.AM2017-5348