Abstract 4133: Comparison of qNPA vs qPCR on FFPE tissue.
Authors: Rounseville Matthew B. , Pechhold Klaus , Thompson Debrah , Schwartz Mark , Seligmann Bruce 
Published: August 2014

Abstract
Accurate, sensitive and robust multiplexed measurements of gene expression from formalin fixed paraffin embedded (FFPE) tissue or paraformaldehyde fixed samples are ideally required for clinical diagnostic tests and retrospective analysis of archived samples. We evaluated the measurement of gene expression from FFPE tissues using HTG Molecular's qNPA™ assay and made comparisons to qPCR. The results show that qNPA provides a highly sensitive, accurate, quantitative, and robustly reliable automated multiplexed assay of gene expression from FFPE, permitting the rapid development and launch of new diagnostic and research assays. qNPA uses a lysis-only, extraction-free protocol which measures up to 47 genes/sample. In contrast, qPCR requires RNA extraction/reverse transcription. Accuracy was determined by correlating measurements from matched frozen and fixed samples, determining the R2 correlation coefficient. Comparing frozen/FFPE cell pellets qNPA R2 = 0.97. Comparing frozen/FFPE pancreas tissue qNPA R2= 0.97 with a present call rate of 91%, compared to qPCR R2= 0.86 and a present call rate of just 17%, even using 26 times more FFPE sample amount than qNPA. Comparing fresh/paraformaldehyde fixed Islet cells, qNPA R2= 0.98, qPCR R2= 0.02, after staining for sorting by cytometry qNPA R2= 0.96, qPCR R2= 0.12. Thus, qNPA fixed tissue measurements were much more accurate than qPCR. In an additional comparison using matched breast cancer frozen compared with FFPE samples, qNPA measurements averaged R2=0.81, with 6% CV for triplicate measurements of separately processed samples. For &gt;95% of genes the expression levels measured by qNPA were independent of cold ischemic times of 0 to 16 hr. Measured levels are also independent of fixation from 4 to 72 hr. The robust performance of qNPA measurements from FFPE translated into quantitative consistency that has previously not been achievable using qPCR. Data from clinical FFPE samples submitted by a large number of HTG's clients was reviewed and for each set the number of samples tested, the average area tested/sample (cm2 area of a 5 micron thick section), the failure rate (FR), and the avg reproducibility (%CV for samples independently processed in triplicate) was determined for breast FFPE (300 samples, 0.3 cm2/sample, 1% FR, 10.5%CV), lung FFPE (700 samples, 0.3 cm2/sample, 0.5% FR, 9.5%CV), prostate FFPE (15 samples, 0.3 cm2/sample, 0% FR, 11%CV), colon FFPE (15 samples, 0.3 cm2/sample, 0% FR, 9%CV), ovary FFPE (15 samples, 0.3 cm2/sample, 0% FR, 10%CV) and lymphoma FFPE (300 samples, 0.3 cm2/sample, 0.5% FR, 8%CV). Thus, qNPA provides a highly accurate, reproducible, robust multiplexed measurement of gene expression from FFPE tissue that is not affected by wide variations in ischemic or fixation time. Fully automated qNPA on the EDGE platform is expected in Jan 2013. This system, with a turn-around time of less than 24 hours, will improve even further the robustness of extraction-free gene expression.
Citation Format: Matthew B. Rounseville, Klaus Pechhold, Debrah Thompson, Mark Schwartz, Bruce Seligmann. Comparison of qNPA vs qPCR on FFPE tissue. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4133. doi:10.1158/1538-7445.AM2013-4133