Detection of Therapeutically Targetable Driver and Resistance Mutations in Lung Cancer Patients by Next-Generation Sequencing of Cell-Free Circulating Tumor DNA

Abstract

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.

©2016 American Association for Cancer Research.

Figures

Figure 1

Overview of study for the detection of therapeutically targetable mutations in the tissue and plasma of patients with advanced lung cancer by next generation sequencing.

Figure 2

Variant detection in ctDNA. 275 total mutations were detected in the ctDNA of 86 out of 102 patients. Shown here is the frequency of variants by gene for the 219 variants detected in the 16 most commonly mutated genes.

Figure 3

Mutational analysis for 50 patients for whom both plasma and tissue samples were obtained and sequenced. A. Detailed summary of all variants covered and called by both the tDNA and ctDNA platforms. The EGFR row includes the variants: L858R, G719A, L861Q, Exon 19 deletions, and Exon 20 insertions. Gene names with a grey background are known resistance mutations to EGFR-targeted therapy. Tumor DNA was sequenced using the 47-gene TSACP panel, except for those patient numbers with an asterisk indicating that the 20-gene panel was used due to low input DNA. Genes with two variants identified by either platform are denoted with a diagonal line. B. Allelic fractions for all variants detected in tDNA and ctDNA were compared. C. Concordance of all variant calls for tDNA and ctDNA by biopsy site (primary tumor, n=19; metastatic tumor, n=14) for samples obtained within 6 months. D. Concordance of actionable EGFR mutations detected in tDNA and ctDNA in relation to time interval between tissue biopsy and blood draw (≤ 2 weeks, n=9; ≤2 months, n=12; ≤6 months, n=14; > 6 months, n=10). AF (%) = Allelic Fraction.

Figure 4

Use of ctDNA to predict survival. Patient survival was calculated as the number of months since date of metastatic diagnosis, and then compared to ctDNA measurements (n=98 patients with metastatic disease). A. Kaplan Meier survival curve and log-rank test dichotomized around a threshold of ≥ 3 mutations. B. Kaplan Meier survival curve and log-rank test dichotomized around a threshold of ≥ 3 ng/μL of cfDNA detected.

Figure 5. Serial ctDNA testing

Real-time molecular monitoring of patients on therapy with time depicted on the x-axis as months since first plasma-based ctDNA analysis. The black arrows above each panel indicate targeted agents administered to patient over the indicated time periods. Below each panel are imaging results at the indicated time point. A. Patient #72 was treated with crizotinib following identification of EML4-ALK fusion in ctDNA but not tDNA. Imaging shows response to therapy correlating with decrease in plasma-based EML4-ALK AF. B. ctDNA analysis for Patient #82 confirmed EGFR (G719A) mutation and identified the emergence of EGFR (T790M) mutation that was not detected in tDNA several months prior. Serial ctDNA sampling shows AF of detected variants correlating with response to therapy. C. ctDNA analysis identified the presence of an EGFR (T790M) mutation at Month 0 that was not detected in a tDNA sample obtained 6 days prior. Following imaging- and ctDNA- confirmed disease progression, patient #15 was started on osimertinib with good clinical response. AF (%) = Allelic Fraction, Pre = Pre-Therapy, PD = Progressive Disease, PR = Partial Response.