Abstract 1383: NGS-based measurement of gene expression of 2560 oncology-related biomarkers in formalin-fixed, paraffin-embedded (FFPE) tissues
Authors: Reinholz Monica M. , Thompson Debrah M. , Botros Ihab , Rounseville Matt , Roche Patrick C. 
Published: July 2016

Abstract
Background: HTG Molecular (HTG) developed a targeted Next Generation Sequencing (NGS)-based gene expression assay that measures mRNA levels of 2,560 genes (2,532 oncology-related biomarker genes). The HTG EdgeSeq Oncology Biomarker Panel Assay (OBP) is based on a novel derivative of our quantitative nuclease protection chemistry (qNPA) that enables extraction-free quantitation (detection) of mRNA from variety of sample types including formalin-fixed, paraffin embedded (FFPE) tissue.
Purpose: To determine (1) linearity across a range of sample inputs, (2) recommended sample input amounts for FFPE, cells, and extracted RNA, and (3) reproducibility of the HTG EdgeSeq Oncology Biomarker Panel Assay in measuring the mRNA expression of 2,560 genes.
Methods: Lysates of 5 micron sections of FFPE tissues (lung, breast, prostate, and colon carcinoma; melanoma; 25 mm2 to 0.78 mm2 per reaction), THP-1 and HCC78 cell lines (7500 cells to 234 cells per reaction), and Universal RNA (URNA; 25 ng to 1.56 ng per reaction) were used for the linearity and sample input studies. URNA (25 ng per reaction) was used to demonstrate reproducibility of the assay across multiple days and processors (one processor across three days and three processors on one day). Sequencing libraries were generated from the qNPA reactions and run on an Illumina MiSeq sequencing platform. The HTG EdgeSeq Parser was used for post-sequencing data processing. Linear regression (R2) and Pearson correlation coefficients (r) were used to assess linearity and reproducibility of the assay.
Results: The R2 for linearity across four concentration points for lung FFPE tissue (6.25-0.78 mm2), cell lines (1875-234 cells), and URNA (12.5-1.56 ng) were &gt;0.97, 0.99, and 0.99, respectively. The (r) between low (1.56 mm2) and high (12.5 mm2) sample inputs for each FFPE tissue type was &gt; 0.98. The (r) for intra-run, inter-day, and inter-run reproducibility were &gt; 0.95, &gt; 0.98, and &gt; 0.98 respectively. In addition, differential expression of tissue-specific genes was identified in the respective FFPE tissues, including NKX2 and MUC1 in lung, ERBB2 in breast, NKX3, KLK2, and KLK3 in prostate, and SPP1 and PRAME in melanoma.
Conclusions: The HTG EdgeSeq Oncology Biomarker Panel Assay for a 2,560-gene panel of oncology-related biomarkers is linear over a wide range of sample inputs, can comprehensively analyze very small, clinically relevant tissues, and is highly reproducible. The demonstrated performance of the assay in breast, lung, colon, and prostate cancer and melanoma FFPE samples enables multiplex oncology biomarker profiling of these and other malignant neoplasms.
Citation Format: Monica M. Reinholz, Debrah M. Thompson, Ihab Botros, Matt Rounseville, Patrick C. Roche. NGS-based measurement of gene expression of 2560 oncology-related biomarkers in formalin-fixed, paraffin-embedded (FFPE) tissues. [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 1383.