Amplicon-based next-generation sequencing of plasma cell-free DNA for detection of driver and resistance mutations in advanced non-small cell lung cancer

Abstract

Background: Genomic analysis of plasma cell-free DNA is transforming lung cancer care; however, available assays are limited by cost, turnaround time, and imperfect accuracy. Here, we study amplicon-based plasma next-generation sequencing (NGS), rather than hybrid-capture-based plasma NGS, hypothesizing this would allow sensitive detection and monitoring of driver and resistance mutations in advanced non-small cell lung cancer (NSCLC).

Patients and methods: Plasma samples from patients with NSCLC and a known targetable genotype (EGFR, ALK/ROS1, and other rare genotypes) were collected while on therapy and analyzed blinded to tumor genotype. Plasma NGS was carried out using enhanced tagged amplicon sequencing of hotspots and coding regions from 36 genes, as well as intronic coverage for detection of ALK/ROS1 fusions. Diagnostic accuracy was compared with plasma droplet digital PCR (ddPCR) and tumor genotype.

Results: A total of 168 specimens from 46 patients were studied. Matched plasma NGS and ddPCR across 120 variants from 80 samples revealed high concordance of allelic fraction (R2 = 0.95). Pretreatment, sensitivity of plasma NGS for the detection of EGFR driver mutations was 100% (30/30), compared with 87% for ddPCR (26/30). A full spectrum of rare driver oncogenic mutations could be detected including sensitive detection of ALK/ROS1 fusions (8/9 detected, 89%). Studying 25 patients positive for EGFR T790M that developed resistance to osimertinib, 15 resistance mechanisms could be detected including tertiary EGFR mutations (C797S, Q791P) and mutations or amplifications of non-EGFR genes, some of which could be detected pretreatment or months before progression.

Conclusions: This blinded analysis demonstrates the ability of amplicon-based plasma NGS to detect a full range of targetable genotypes in NSCLC, including fusion genes, with high accuracy. The ability of plasma NGS to detect a range of preexisting and acquired resistance mechanisms highlights its potential value as an alternative to single mutation digital PCR-based plasma assays for personalizing treatment of TKI resistance in lung cancer.

Figure 1.

(A) Focusing on nine cases of EGFR-mutant non-small cell lung cancer (NSCLC) with the lowest tumor DNA shed (< 1.1% AF), sensitivity appears better with amplicon-based plasma NGS compared with plasma ddPCR. *Two T790M mutations were detected with ddPCR but not NGS, though the ddPCR signal was below the level for clinical reporting and may have been a false positive (supplementary Figure S2, available at Annals of Oncology online). AF, allelic fraction. (B) Quantitative concordance was high (R² = 0.95) across 120 EGFR variants from 80 specimens detected both with plasma NGS and plasma ddPCR. (C) Detection of a range of fusions and rare genotypes using amplicon-based plasma NGS. One apparent false positive (*) secondarily tested negative by ddPCR for both KRAS and BRAF mutations. (D) Three examples where fusion detection permits the determination of fusion partner and breakpoint.

Figure 2.

(A and B) Acquired gene amplifications detected in plasma cfDNA at the time of resistance to osimertinib. (A) Acquired ERBB2 and MYC amplification (driver AF 63%). (B) Acquired ERBB2 amplification and TP53 deletion (driver AF 38%). (C) EGFR amplification and acquired MET amplification (driver AF 22%). (D–G) Early detection of mechanisms of resistance to osimertinib through serial NGS of cfDNA in four patients. (D and E) Two cases with mixed response in plasma (complete clearance of T790M, incomplete response of the driver) had loss of T790M at the time of resistance. Possible competing resistance mutations (D: BRAF V600E; E: PIK3CA E545K) coexisting with T790M at baseline can be detected pretreatment. (F and G) In two patients with maintained T790M at resistance, serial plasma NGS can detect acquired tertiary EGFR mutations several months before clinical progression (F: 3 months; G: 7.5 months). In one patient (F), plasma NGS could also detect a competing KRAS Q61K mutation at low AF at baseline that with increased AF at resistance.