Ryan S. Alden

PURPOSE: Third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have demonstrated potent activity against TKI resistance mediated by EGFR T790M. We studied whether noninvasive genotyping of cell-free plasma DNA (cfDNA) is a useful biomarker for prediction of outcome from a third-generation EGFR-TKI, osimertinib. METHODS: Plasma was collected from all patients in the first-in-man study of osimertinib. Patients who were included had acquired EGFR-TKI resistance and evidence of a common EGFR-sensitizing mutation. Genotyping of cell-free plasma DNA was performed by using BEAMing. Plasma genotyping accuracy was assessed by using tumor genotyping from a central laboratory as reference. Objective response rate (ORR) and progression-free survival (PFS) were analyzed in all T790M-positive or T790M-negative patients. RESULTS: Sensitivity of plasma genotyping for detection of T790M was 70%. Of 58 patients with T790M-negative tumors, T790M was detected in plasma of 18 (31%). ORR and median PFS were similar in patients with T790M-positive plasma (ORR, 63%; PFS, 9.7 months) or T790M-positive tumor (ORR, 62%; PFS, 9.7 months) results. Although patients with T790M-negative plasma had overall favorable outcomes (ORR, 46%; median PFS, 8.2 months), tumor genotyping distinguished a subset of patients positive for T790M who had better outcomes (ORR, 69%; PFS, 16.5 months) as well as a subset of patients negative for T790M with poor outcomes (ORR, 25%; PFS, 2.8 months). CONCLUSION: In this retrospective analysis, patients positive for T790M in plasma have outcomes with osimertinib that are equivalent to patients positive by a tissue-based assay. This study suggests that, upon availability of validated plasma T790M assays, some patients could avoid a tumor biopsy for T790M genotyping. As a result of the 30% false-negative rate of plasma genotyping, those with T790M-negative plasma results still need a tumor biopsy to determine presence or absence of T790M.

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.

PURPOSE: Tumor genotyping is a powerful tool for guiding non-small cell lung cancer (NSCLC) care; however, comprehensive tumor genotyping can be logistically cumbersome. To facilitate genotyping, we developed a next-generation sequencing (NGS) assay using a desktop sequencer to detect actionable mutations and rearrangements in cell-free plasma DNA (cfDNA). EXPERIMENTAL DESIGN: An NGS panel was developed targeting 11 driver oncogenes found in NSCLC. Targeted NGS was performed using a novel methodology that maximizes on-target reads, and minimizes artifact, and was validated on DNA dilutions derived from cell lines. Plasma NGS was then blindly performed on 48 patients with advanced, progressive NSCLC and a known tumor genotype, and explored in two patients with incomplete tumor genotyping. RESULTS: NGS could identify mutations present in DNA dilutions at ≥ 0.4% allelic frequency with 100% sensitivity/specificity. Plasma NGS detected a broad range of driver and resistance mutations, including ALK, ROS1, and RET rearrangements, HER2 insertions, and MET amplification, with 100% specificity. Sensitivity was 77% across 62 known driver and resistance mutations from the 48 cases; in 29 cases with common EGFR and KRAS mutations, sensitivity was similar to droplet digital PCR. In two cases with incomplete tumor genotyping, plasma NGS rapidly identified a novel EGFR exon 19 deletion and a missed case of MET amplification. CONCLUSIONS: Blinded to tumor genotype, this plasma NGS approach detected a broad range of targetable genomic alterations in NSCLC with no false positives including complex mutations like rearrangements and unexpected resistance mutations such as EGFR C797S. Through use of widely available vacutainers and a desktop sequencing platform, this assay has the potential to be implemented broadly for patient care and translational research.

Abstract Amplified and/or mutated MET can act as both a primary oncogenic driver and as a promoter of tyrosine kinase inhibitor (TKI) resistance in non–small cell lung cancer (NSCLC). However, the landscape of MET-specific targeting agents remains underdeveloped, and understanding of mechanisms of resistance to MET TKIs is limited. Here, we present a case of a patient with lung adenocarcinoma harboring both a mutation in EGFR and an amplification of MET, who after progression on erlotinib responded dramatically to combined MET and EGFR inhibition with savolitinib and osimertinib. When resistance developed to this combination, a new MET kinase domain mutation, D1228V, was detected. Our in vitro findings demonstrate that METD1228V induces resistance to type I MET TKIs through impaired drug binding, while sensitivity to type II MET TKIs is maintained. Based on these findings, the patient was treated with erlotinib combined with cabozantinib, a type II MET inhibitor, and exhibited a response. Significance: With several structurally distinct MET inhibitors undergoing development for treatment of NSCLC, it is critical to identify mechanism-based therapies for drug resistance. We demonstrate that an acquired METD1228V mutation mediates resistance to type I, but not type II, MET inhibitors, having therapeutic implications for the clinical use of sequential MET inhibitors. Cancer Discov; 6(12); 1334–41. ©2016 AACR. See related commentary by Trusolino, p. 1306. This article is highlighted in the In This Issue feature, p. 1293

BACKGROUND: Noninvasive genotyping using plasma cell-free DNA (cfDNA) has the potential to obviate the need for some invasive biopsies in cancer patients while also elucidating disease heterogeneity. We sought to develop an ultra-deep plasma next-generation sequencing (NGS) assay for patients with non-small-cell lung cancers (NSCLC) that could detect targetable oncogenic drivers and resistance mutations in patients where tissue biopsy failed to identify an actionable alteration. PATIENTS AND METHODS: Plasma was prospectively collected from patients with advanced, progressive NSCLC. We carried out ultra-deep NGS using cfDNA extracted from plasma and matched white blood cells using a hybrid capture panel covering 37 lung cancer-related genes sequenced to 50 000× raw target coverage filtering somatic mutations attributable to clonal hematopoiesis. Clinical sensitivity and specificity for plasma detection of known oncogenic drivers were calculated and compared with tissue genotyping results. Orthogonal ddPCR validation was carried out in a subset of cases. RESULTS: In 127 assessable patients, plasma NGS detected driver mutations with variant allele fractions ranging from 0.14% to 52%. Plasma ddPCR for EGFR or KRAS mutations revealed findings nearly identical to those of plasma NGS in 21 of 22 patients, with high concordance of variant allele fraction (r = 0.98). Blinded to tissue genotype, plasma NGS sensitivity for de novo plasma detection of known oncogenic drivers was 75% (68/91). Specificity of plasma NGS in those who were driver-negative by tissue NGS was 100% (19/19). In 17 patients with tumor tissue deemed insufficient for genotyping, plasma NGS identified four KRAS mutations. In 23 EGFR mutant cases with acquired resistance to targeted therapy, plasma NGS detected potential resistance mechanisms, including EGFR T790M and C797S mutations and ERBB2 amplification. CONCLUSIONS: Ultra-deep plasma NGS with clonal hematopoiesis filtering resulted in de novo detection of targetable oncogenic drivers and resistance mechanisms in patients with NSCLC, including when tissue biopsy was inadequate for genotyping.