Prospective blinded study of somatic mutation detection in cell-free DNA utilizing a targeted 54-gene next generation sequencing panel in metastatic solid tumor patients

Seung Tae Kim, Won-Suk Lee, Richard B Lanman, Stefanie Mortimer, Oliver A Zill, Kyoung-Mee Kim, Kee Taek Jang, Seok-Hyung Kim, Se Hoon Park, Joon Oh Park, Young Suk Park, Ho Yeong Lim, Helmy Eltoukhy, Won Ki Kang, Woo Yong Lee, Hee-Cheol Kim, Keunchil Park, Jeeyun Lee, AmirAli Talasaz, Seung Tae Kim, Won-Suk Lee, Richard B Lanman, Stefanie Mortimer, Oliver A Zill, Kyoung-Mee Kim, Kee Taek Jang, Seok-Hyung Kim, Se Hoon Park, Joon Oh Park, Young Suk Park, Ho Yeong Lim, Helmy Eltoukhy, Won Ki Kang, Woo Yong Lee, Hee-Cheol Kim, Keunchil Park, Jeeyun Lee, AmirAli Talasaz

Abstract

Sequencing of the mutant allele fraction of circulating cell-free DNA (cfDNA) derived from tumors is increasingly utilized to detect actionable genomic alterations in cancer. We conducted a prospective blinded study of a comprehensive cfDNA sequencing panel with 54 cancer genes. To evaluate the concordance between cfDNA and tumor DNA (tDNA), sequencing results were compared between cfDNA from plasma and genomic tumor DNA (tDNA). Utilizing next generation digital sequencing technology (DST), we profiled approximately 78,000 bases encoding 512 complete exons in the targeted genes in cfDNA from plasma. Seventy-five patients were prospectively enrolled between February 2013 and March 2014, including 61 metastatic cancer patients and 14 clinical stage II CRC patients with matched plasma and tissue samples. Using the 54-gene panel, we detected at least one somatic mutation in 44 of 61 tDNA (72.1%) and 29 of 44 (65.9%) cfDNA. The overall concordance rate of cfDNA to tDNA was 85.9%, when all detected mutations were considered. We collected serial cfDNAs during cetuximab-based treatment in 2 metastatic KRAS wild-type CRC patients, one with acquired resistance and one with primary resistance. We demonstrate newly emerged KRAS mutation in cfDNA 1.5 months before radiologic progression. Another patient had a newly emerged PIK3CA H1047R mutation on cfDNA analysis at progression during cetuximab/irinotecan chemotherapy with gradual increase in allele frequency from 0.8 to 2.1%. This blinded, prospective study of a cfDNA sequencing showed high concordance to tDNA suggesting that the DST approach may be used as a non-invasive biopsy-free alternative to conventional sequencing using tumor biopsy.

Trial registration: ClinicalTrials.gov NCT02067754.

Keywords: cell-free DNA (cfDNA); digital sequencing; genomic test.

Conflict of interest statement

CONFLICTS OF INTEREST

None declared except for RBL, SM, HE, AT who are employed by Guardant Health Inc.

Figures

Figure 1. STARD diagram
Figure 1. STARD diagram
Figure 2. A. Mutational profiles (clinically significant…
Figure 2. A. Mutational profiles (clinically significant variants, variants reported in COSMIC and other novel variants) detected in cfDNA for 61 advanced cancer patients with various tumor types and B. Details for genetic aberration analyzed in cfDNA and the concordance for comprehensive mutational profiles between tumor-tissue analysis and cfDNA
Figure 3. Patient monitoring with cfDNA during…
Figure 3. Patient monitoring with cfDNA during cetuximab-based treatment in metastatic colon cancer A. acquired resistance to cetuximab; B. primary resistance to cetuximab

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