Prospective Validation of Rapid Plasma Genotyping for the Detection of EGFR and KRAS Mutations in Advanced Lung Cancer

Abstract

Importance: Plasma genotyping of cell-free DNA has the potential to allow for rapid noninvasive genotyping while avoiding the inherent shortcomings of tissue genotyping and repeat biopsies.

Objective: To prospectively validate plasma droplet digital PCR (ddPCR) for the rapid detection of common epidermal growth factor receptor (EGFR) and KRAS mutations, as well as the EGFR T790M acquired resistance mutation.

Design, setting, and participants: Patients with advanced nonsquamous non-small-cell lung cancer (NSCLC) who either (1) had a new diagnosis and were planned for initial therapy or (2) had developed acquired resistance to an EGFR kinase inhibitor and were planned for rebiopsy underwent initial blood sampling and immediate plasma ddPCR for EGFR exon 19 del, L858R, T790M, and/or KRAS G12X between July 3, 2014, and June 30, 2015, at a National Cancer Institute-designated comprehensive cancer center. All patients underwent biopsy for tissue genotyping, which was used as the reference standard for comparison; rebiopsy was required for patients with acquired resistance to EGFR kinase inhibitors. Test turnaround time (TAT) was measured in business days from blood sampling until test reporting.

Main outcomes and measures: Plasma ddPCR assay sensitivity, specificity, and TAT.

Results: Of 180 patients with advanced NSCLC (62% female; median [range] age, 62 [37-93] years), 120 cases were newly diagnosed; 60 had acquired resistance. Tumor genotype included 80 EGFR exon 19/L858R mutants, 35 EGFR T790M, and 25 KRAS G12X mutants. Median (range) TAT for plasma ddPCR was 3 (1-7) days. Tissue genotyping median (range) TAT was 12 (1-54) days for patients with newly diagnosed NSCLC and 27 (1-146) days for patients with acquired resistance. Plasma ddPCR exhibited a positive predictive value of 100% (95% CI, 91%-100%) for EGFR 19 del, 100% (95% CI, 85%-100%) for L858R, and 100% (95% CI, 79%-100%) for KRAS, but lower for T790M at 79% (95% CI, 62%-91%). The sensitivity of plasma ddPCR was 82% (95% CI, 69%-91%) for EGFR 19 del, 74% (95% CI, 55%-88%) for L858R, and 77% (95% CI, 60%-90%) for T790M, but lower for KRAS at 64% (95% CI, 43%-82%). Sensitivity for EGFR or KRAS was higher in patients with multiple metastatic sites and those with hepatic or bone metastases, specifically.

Conclusions and relevance: Plasma ddPCR detected EGFR and KRAS mutations rapidly with the high specificity needed to select therapy and avoid repeat biopsies. This assay may also detect EGFR T790M missed by tissue genotyping due to tumor heterogeneity in resistant disease.

Figure 1

(A) The sensitivity of plasma ddPCR for the detection of EGFR and KRAS mutations increases directly with the number of metastatic sites present in a given patient (p<0.001). (B) Dynamic range of plasma genotyping using a validated ddPCR-based assay. Wide dynamic range and the absence of false positives are noted for the detection of KRAS G12X and EGFR sensitizing mutations. A small number of false positives are seen with the EGFR T790M assay – potentially secondary to tumor heterogeneity with respect to acquired resistance mechanisms (n=174).

Figure 2

Distinct patterns of ddPCR plasma response emerge in patients undergoing serial plasma genotyping at 2 weeks and 6 weeks post-treatment. Mutant cfDNA was observed to either become undetectable at 2 weeks (A), decrease and then become undetectable at 6 weeks (B), decrease progressively but remain detectable at 6 weeks (C), increase initially and then decrease at 6 weeks (D), decrease at 2 weeks and then rebound at 6 weeks (E), or progressively increase (F). Patients with complete resolution of mutant cfDNA (A-B) exhibited a treatment discontinuation rate of 0% (0/23) and 4% (1/23) at initial and second restaging CT scans. Alternatively, patients without complete resolution (C-F) had a treatment discontinuation rate of 33% (9/27) at initial re-imaging and 56% (15/27) at second re-imaging assessment. Patient genotypes included EGFR sensitizing alone (--), EGFR sensitizing in the presence of T790M (--) and KRAS G12X (--).

Figure 3

Case Report: A patient in their eighties with metastatic EGFR mutant NSCLC developed acquired resistance to erlotinib. Symptomatic progression of pulmonary and bone metastases were noted (primary lung lesion labelled). Empiric single-agent chemotherapy or best supportive care alone were considered given the patient’s age and comorbidities. However, plasma ddPCR was performed and the result returned the next day revealing 806 copies/mL of EGFR T790M. The patient underwent rebiopsy which confirmed EGFR T790M and the patient was able to start therapy with osimertinib – a novel third generation EGFR kinase inhibitor with excellent clinical and radiographic response. Importantly, the plasma ddPCR T790M result was returned 24 days before the results of the repeat tissue biopsy were available.