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Total amount of cfDNA extracted from each patient sample

Available therapies and clinical trials

Allelic fractions for all variants detected in tDNA and ctDNA* in patients for whom both plasma and tissue samples were obtained (n=50)

Summary of driver and resistance mutations in EGFR and ALK detected in ctDNA, tDNA, and both ctDNA and tDNA

Tissue next-generation sequencing gene coverage. A: Illumina TruSeq panel (47 genes), and B: Penn Precision Panel (20 genes)

Analysis of discordant EGFR driver and resistance mutations detected in tDNA or ctDNA

All mutations detected in patient ctDNA (n=102)

Circulating tumor DNA next-generation sequencing gene coverage

Purpose: The expanding number of targeted therapeutics for non–small cell lung cancer (NSCLC) necessitates real-time tumor genotyping, yet tissue biopsies are difficult to perform serially and often yield inadequate DNA for next-generation sequencing (NGS). We evaluated the feasibility of using cell-free circulating tumor DNA (ctDNA) NGS as a complement or alternative to tissue NGS.Experimental Design: A total of 112 plasma samples obtained from a consecutive study of 102 prospectively enrolled patients with advanced NSCLC were subjected to ultra-deep sequencing of up to 70 genes and matched with tissue samples, when possible.Results: We detected 275 alterations in 45 genes, and at least one alteration in the ctDNA for 86 of 102 patients (84%), with EGFR variants being most common. ctDNA NGS detected 50 driver and 12 resistance mutations, and mutations in 22 additional genes for which experimental therapies, including clinical trials, are available. Although ctDNA NGS was completed for 102 consecutive patients, tissue sequencing was only successful for 50 patients (49%). Actionable EGFR mutations were detected in 24 tissue and 19 ctDNA samples, yielding concordance of 79%, with a shorter time interval between tissue and blood collection associated with increased concordance (P = 0.038). ctDNA sequencing identified eight patients harboring a resistance mutation who developed progressive disease while on targeted therapy, and for whom tissue sequencing was not possible.Conclusions: Therapeutically targetable driver and resistance mutations can be detected by ctDNA NGS, even when tissue is unavailable, thus allowing more accurate diagnosis, improved patient management, and serial sampling to monitor disease progression and clonal evolution. Clin Cancer Res; 22(23); 5772–82. ©2016 AACR.

Purpose: The expanding number of targeted therapeutics for non–small cell lung cancer (NSCLC) necessitates real-time tumor genotyping, yet tissue biopsies are difficult to perform serially and often yield inadequate DNA for next-generation sequencing (NGS). We evaluated the feasibility of using cell-free circulating tumor DNA (ctDNA) NGS as a complement or alternative to tissue NGS. Experimental Design: A total of 112 plasma samples obtained from a consecutive study of 102 prospectively enrolled patients with advanced NSCLC were subjected to ultra-deep sequencing of up to 70 genes and matched with tissue samples, when possible. Results: We detected 275 alterations in 45 genes, and at least one alteration in the ctDNA for 86 of 102 patients (84%), with EGFR variants being most common. ctDNA NGS detected 50 driver and 12 resistance mutations, and mutations in 22 additional genes for which experimental therapies, including clinical trials, are available. Although ctDNA NGS was completed for 102 consecutive patients, tissue sequencing was only successful for 50 patients (49%). Actionable EGFR mutations were detected in 24 tissue and 19 ctDNA samples, yielding concordance of 79%, with a shorter time interval between tissue and blood collection associated with increased concordance (P = 0.038). ctDNA sequencing identified eight patients harboring a resistance mutation who developed progressive disease while on targeted therapy, and for whom tissue sequencing was not possible. Conclusions: Therapeutically targetable driver and resistance mutations can be detected by ctDNA NGS, even when tissue is unavailable, thus allowing more accurate diagnosis, improved patient management, and serial sampling to monitor disease progression and clonal evolution. Clin Cancer Res; 22(23); 5772–82. ©2016 AACR.