Abstract 4499: Integration of targeted RNA sequencing to targeted DNA sequencing for the characterization of clinically relevant variants in a population of thousands of patients treated on clinical trial
Authors: Lee Woochang , Jo Heejoon , Yin Xiaoying , Eberhard David A. , Patel Nirali M. , Hayward Michele C. , Salazar Ashley H. , Parker Joel S. , Kim William Y. , Earp Henry S. , Sharpless Norman E. , Hayes David N. 
Published: July 2016

Abstract
Background: Targeted sequencing has become common in the care of some patients with cancer, both for the detection of standard of care clinical mutations as well as in the care of patients with advanced disease who are looking for clinical trials or other non-standard therapies. Many patients have mutations detected, although many variants are of unknown significance, and many patients have no mutations at all. We added RNA targeted sequencing along with DNA targeted sequencing to add utility of targeted sequencing strategy in cancer genomics and assessed the performances of RNA sequencing analysis.
Methods: Genomic sequencing of investigative biomarkers was prospectively offered to selected patients. DNA and RNA libraries were prepared separately from a retrieved archival FFPE tumor sample or a fresh frozen tumor sample from each patient. Relevant targets were enriched by custom designed Agilent SureSelect hybrid capture baits using standard protocols. Samples were sequenced on Illumina HiSeq 2000/2500 platforms. We compared somatic variants detected by DNA alone to those detected by DNA plus RNA using the UNCeqR algorithm and software.
Results: From a population of 2200 patients consented as part of LCCC1108 (NCT01457196), a subset of 300 patients was selected at random for RNA profiling. Selected patients were 7 to 83 years old (median, 54) and the cases included more than 20 cancer types including uterus, breast, ovary, and thyroid etc. And stages of cancers were as follows; I, 34.6%, II, 16.8%, III, 25.2%, and IV, 23.4% respectively. Sequencing of RNA was successful in 90% of specimens using 2.5 ug of RNA. RNA sequencing could detect 97.5% of sequence variants called by DNA sequencing and detect 20% more variants than DNA sequencing. Also 97.9% of variants showed higher mutant allele fraction (MAF) in RNA sequencing than DNA sequencing. We further interrogated the impact of certain classes of mutations on transcript structure and abundance. Specifically, we observed that splice site mutations and indels were associated with detectable alterations in the full length transcripts of their respective genes as well as overall gene abundance, with nonsense and frameshift mutations associated with decreased relative expression of the transcript relative to controls. We also observed that gene amplification of EGFR and ERBB2 was reflected in the RNA sequencing as increased gene expression with statistical significance by Fishers’ exact test (P &lt; 0.05), which suggests that RNA can be used as a surrogate for known protein and nucleic quantitative assays.
Conclusion: Using clinical samples, including relatively small quantities, we were able to confirm that nucleotide variants detected at DNA level leads to significant alteration at the transcription level and to have additional information potentially helpful for better management of cancer patients.
Citation Format: Woochang Lee, Heejoon Jo, Xiaoying Yin, David A. Eberhard, Nirali M. Patel, Michele C. Hayward, Ashley H. Salazar, Joel S. Parker, William Y. Kim, Henry S. Earp, Norman E. Sharpless, David N. Hayes. Integration of targeted RNA sequencing to targeted DNA sequencing for the characterization of clinically relevant variants in a population of thousands of patients treated on clinical trial. [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 4499.