Noninvasive detection of response and resistance in EGFR-mutant lung cancer using quantitative next-generation genotyping of cell-free plasma DNA

Abstract

Purpose: Tumor genotyping using cell-free plasma DNA (cfDNA) has the potential to allow noninvasive assessment of tumor biology, yet many existing assays are cumbersome and vulnerable to false-positive results. We sought to determine whether droplet digital PCR (ddPCR) of cfDNA would allow highly specific and quantitative assessment of tumor genotype.

Experimental design: ddPCR assays for EGFR, KRAS, and BRAF mutations were developed using plasma collected from patients with advanced lung cancer or melanoma of a known tumor genotype. Sensitivity and specificity were determined using cancers with nonoverlapping genotypes as positive and negative controls. Serial assessment of response and resistance was studied in patients with EGFR-mutant lung cancer on a prospective trial of erlotinib.

Results: We identified a reference range for EGFR L858R and exon 19 deletions in specimens from KRAS-mutant lung cancer, allowing identification of candidate thresholds with high sensitivity and 100% specificity. Received operative characteristic curve analysis of four assays demonstrated an area under the curve in the range of 0.80 to 0.94. Sensitivity improved in specimens with optimal cfDNA concentrations. Serial plasma genotyping of EGFR-mutant lung cancer on erlotinib demonstrated pretreatment detection of EGFR mutations, complete plasma response in most cases, and increasing levels of EGFR T790M emerging before objective progression.

Conclusions: Noninvasive genotyping of cfDNA using ddPCR demonstrates assay qualities that could allow effective translation into a clinical diagnostic. Serial quantification of plasma genotype allows noninvasive assessment of response and resistance, including detection of resistance mutations up to 16 weeks before radiographic progression.

Figure 1

Plasma genotyping using droplet digital PCR (ddPCR). Cell free DNA (cfDNA) is extracted from a plasma specimen and emulsified with oil into thousands of droplets, each containing approximately 0-1 molecules of target DNA. PCR is performed to endpoint in each droplet. These droplets are run through a flow cytometer, where droplets containing mutant and wildtype DNA emit different colored signals. The count of these signals allows quantification of allelic prevalence.

Figure 2

Detection of mutant alleles in gold standard positive and negative populations, using assays for EGFR L858R (A), EGFR exon 19 deletion (B), and KRAS G12C (C). Receiver operating characteristic (ROC) curves are also shown (D,E,F). By studying plasma from lung cancer patients with non-overlapping genotypes, a “reference range” for each assay can be identified. Dashed lines indicate one candidate threshold for positive with a very high specificity and acceptable sensitivity

Figure 3

Plasma DNA quantification to optimize sensitivity. Studying genotype concentration in gold standard positive cases, the false negative results all have either low or very high levels of LINE-1. Sensitivity is 81% above the median LINE-1 concentration of 168 ng/mL. Circles represents EGFR-mutant cases and squares represents KRAS-mutant cases.

Figure 4

Serial measurement of plasma genotype for disease monitoring. A wide dynamic range is seen in some cases (A, B). Decreases in plasma genotype can be seen both in cases of objective tumor shrinkage (A, D) and in cases of symptomatic response with no measurable disease (B, C). Concurrent EGFR L858R (A, solid line) and T790M (A, dashed line) mutations trend in parallel.

Figure 5

Plasma levels of mutant EGFR in 9 patients receiving first-line erlotinib until objective disease progression (PD) by RECIST. In all patients, plasma levels of the EGFR sensitizing mutation (solid line) drop in response to treatment, with 8 patients (B-I) having a complete plasma response. In 6 patients, plasma genotype levels reemerge up to 4 months prior to PD, and a lower concentration of T790M (dashed line) is also detected. In 3 patients (G-I), plasma genotype was not detected at time of PD; all 3 had indolent progression in the chest only.