Abstract 5549: Normal bronchial epithelial cells (NBEC) sample RNA quality characteristics that will yield reliable measurement of lung cancer risk test

Abstract

Abstract Lung cancer is the leading cause of cancer mortality in the United States with cigarette smoking the primary risk factor. Lung cancer has a low survival rate because it typically is at an advanced stage when first detected and treated. In previous studies a Lung Cancer Risk Test (LCRT) was identified comprising transcript abundance measurement of 14 key antioxidant, DNA repair and transcription factor genes measured in normal bronchial epithelial cells (NBEC) sampled after bronchoscopy. The LCRT promises to identify high-risk individuals who will develop lung cancer. This will enable even more focused selection for closer monitoring, further reduce risk of false positive findings and markedly reduce cost of implementation. An ongoing multi-institutional prospective cohort nested case control trial, funded by NCI grant RC2148572 is intended to test the validity of LCRT as an accurate test for lung cancer risk. More than twelve test sites are providing NBEC samples for the LCRT study. Accurate LCRT measurement will be dependent upon sample RNA quality. Thus, the purpose of this study is to identify cut-off criteria according to which NBEC RNA biospecimens can be expected to yield reliable quantitative, reverse-transcriptase polymerase chain reaction (qRT-PCR) results for the genes comprised by the LCRT. RNA quality of each collected NBEC sample was measured using three parameters: quantity, purity and integrity. RNA integrity was measured by one of two tests, GAPD and GUSB. Each test used one reverse primer and two forward primers and a sequence specific fluorescent labeled probe for signal detection. The GAPD test yielded 70bp and 230bp products and the GUSB test yielded 60bp and 120bp products. The integrity of each unknown RNA sample was determined by dividing the yield of longer product by that of shorter product. Of 126 NBEC samples assessed so far, 77.8% provided > 500 ng total RNA, 94.4% had A260/280 values > 1.5 and 95% gave integrity test values > 0.5. To determine the specific cut-off threshold for each NBEC RNA quality criterion above which LCRT results will be reliable, we established an A549 cell line RNA integrity reference model by incubating cell populations at room temperature for different amounts of time following cytolysis, generating samples with varying decreasing integrity. The LCRT will be measured in these intentionally degraded RNA samples, allowing comparison between integrity levels and LCRT values. The integrity level above which measurement of each gene comprised by the LCRT can be expected to be reliable and accurate will be determined. 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 5549. doi:1538-7445.AM2012-5549