Abstract B140: Genomic profiling of over 5,000 consecutive cancer patients with a CLIA-certified cell-free DNA NGS test: Analytic and clinical validity and utility
Authors: Banks Kimberly C. , Mortimer Stefanie A. W. , Zill Oliver A. , Lanman Richard B. , Eltoukhy Helmy , Talasaz AmirAli 
Published: May 2016

Abstract
Background: Analysis of cell-free circulating tumor DNA (ctDNA) by next-generation sequencing (NGS) enables non-invasive real-time testing. The Guardant360® (G360) ctDNA panel includes all NCCN-recommended solid tumor genomic targets and sequences complete exons of 68 genes for single nucleotide variants (SNVs), and fusions, amplifications and indels for a subset. We conducted updated validity studies for this larger panel.
Methods: Analytical sensitivity was assessed via serial dilution studies of cell-free DNA (cfDNA) with known alterations spiked into healthy cfDNA background. SNVs detected with G360 were compared to whole-exome sequencing at an outside lab for analytic specificity. Tissue NGS for fusions, SNVs, indels, and/or amplifications was compared to G360 results. Consecutive G360 tests over 12 months were analyzed for assay success rate, yield, mutant allele fractions (MAF), and actionability.
Results: Serial dilution studies confirmed the previously reported 0.1% lower limit of detection. The accuracy and analytic specificity studies were 98.6% concordant, with 3 putative false positives. Sanger sequencing by a 3rd lab for these discordant calls confirmed the G360 calls as true positives, verifying the previously reported &gt;99.9999% specificity. Retrospective review of outside tumor test reports of 56 patients with varied genomic alterations found 90% concordance with G360 (77/86 variants: 4/4 fusions, 9/9 indels, 13/17 amps, 51/56 SNVs). Of 5,491 tests, 99.5% were successfully reported with somatic alterations found in 76% [median MAF 0.42% (range 0.1-92.8%)]. Lung, breast and colon cancers were most common (see Table). Overall, 10% of patients had &gt;1 on-label matched therapy identified, 54% off-label, and 77% had clinical trial options. On-label matched therapy rates rose for lung adenocarcinoma (15%), breast (16%) and colon (16%) cancers (Table 1).
Conclusion: The updated G360 test achieves single-molecule sensitivity to 0.1% MAF and analytical specificity of &gt;99.9999%, as reported previously. Clinical concordance of plasma to tissue-based NGS across all four types of genomic alterations was high, and actionability rose modestly, especially in lung, breast and colon cancer. Clinical utility included biopsy avoidance, rescue of tissue samples with insufficient DNA and tissue-false negatives due to tumor evolution and heterogeneity.
Table 1Cancer Type% of 4,888 unique patients% with ctDNA alterations identified% with on-label therapy options identified% with off-label therapy options identified% with clinical trial options identifiedMost commonly involved gene2nd most commonly involved gene3rd most commonly involved geneLung - ALL33%82%10%52%78%TP53 (55%)KRAS (18%)EGFR (18%)Lung adenocarcinoma11%82%15%56%78%TP53 (51%)EGFR (23%)KRAS (19%)Breast15%74%16%58%79%TP53 (43%)PIK3CA (28%)ERBB2/ESR1 (10%) / (9%)CRC12%82%16%72%88%TP53 (61%)APC (38%)KRAS (38%)PDAC7%70%3%57%72%TP53 (47%)KRAS (42%)PIK3CA (6%)Ovarian3%81%1%42%81%TP53 (69%)PIK3CA (15%)KRAS (11%)CUP3%85%0%64%83%TP53 (40%)KRAS (24%)PIK3CA (14%)All100%76%10%54%77%TP53 (50%)KRAS (17%)PIK3CA (14%)
Citation Format: Kimberly C. Banks, Stefanie A. W. Mortimer, Oliver A. Zill, Richard B. Lanman, Helmy Eltoukhy, AmirAli Talasaz. Genomic profiling of over 5,000 consecutive cancer patients with a CLIA-certified cell-free DNA NGS test: Analytic and clinical validity and utility. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B140.