Abstract 2846: Molecular platforms for mutation analysis from whole blood derived clinical samples by nextgen sequencing
Authors: Strauss William M. , Klem Erich , Cuellar Maria , Simmons Jill , Winer-Jones Jessamine , Dempsey Paul W. 
Published: May 2015

Abstract
Both cell free DNA (cfDNA) and circulating tumor cells (CTC) represent important possible templates for mutation analysis of clinical samples with different theoretical advantages for a clinical test. cfDNA is easy to access and isolate, while CTC can provide both DNA as well as RNA for clinical testing. We have tested matched cfDNA and CTC DNA in a Next Generation sequencing test of clinical samples. Both cfDNA and CTC samples provided sufficient quantity for a direct sequencing clinical test. No whole genome amplification was required. The test consisted of coupling these purification technologies to an amplicon re-sequencing panel of 50 cancer-associated genes using a CLIA validated sequencing pipeline for single nucleotide variant (SNV) mutations with a sensitivity of 1%. Typically using this pipeline, blood borne cancer cells are isolated, extracted, sequenced and analyzed in 7 days. In the CLIA setting, the validated DNA sequencing process applied to breast cancer patient samples, has demonstrated useful and actionable clinical data. Results of SNV detection from known clinical positive control samples were quite definitive. We observed perfect concordance in True Positive (TP) SNV detection between the cfDNA and CTC templates. The only discordant results observed were in the detection of False Positives (FP). Using the identical software for SNV detection and variant calls, CTC templates exhibited ∼5x greater sensitivity in our sample set. cfDNA samples showed a much greater noise spectrum of FP. As blood samples are routinely fixed at collection, we evaluated whether fixation might contribute to the cfDNA observed high FP frequency. Our experimental results conclusively show that fixation did not contribute to increased FP observed with cfDNA. Whereas CTC samples showed zero FP over the reportable range, the cfDNA samples exhibited variable FP output ranging from 0 to &gt;10 FP per sample. To extend the capabilities of sequencing rare cells, several RNA sequencing panels have been developed that allow for both SNV, expression, and fusion transcript detection. Although these panels have not been successfully used with cell free templates, they have been successfully applied to rare cell templates and promise to be an important capability for clinical use of CTC in the future. Looking at both lung and breast cancer derived samples we have measured both SNV, CNV (expression based), and fusion transcripts. We describe simultaneous detection of SNV mutations (KRAS: G12S, STK11:Q37*, PIK3CA:E545K, EGFR:L858R, EGFR:T790M, TP53:R273H) as well as fusion transcripts events EML4-ALK.E6aA20.AB374361, and EML4-ALK.E13A20.AB462411. In summary, both cfDNA and CTC derived DNA provided concordant results in SNV mutation detection. cfDNA exhibited a much higher FP rate as compared to CTC DNA. We further demonstrate that rare cells can provide useful template for RNA sequencing.
Citation Format: William M. Strauss, Erich Klem, Maria Cuellar, Jill Simmons, Jessamine Winer-Jones, Paul W. Dempsey. Molecular platforms for mutation analysis from whole blood derived clinical samples by nextgen sequencing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2846. doi:10.1158/1538-7445.AM2014-2846