A: Analysis of dilution series of indicated mutant EGFR variants spiked into 60 ng (â‰^18,180 genome equivalents) of WT DNA. Each data point represents one preparative within 6 independent dilutions series prepared and analyzed by two operators on two different instruments on three non-consecutive days for a total of 18 samples per dilution point. An analysis algorithm was applied to transform the mutant EGFR sequencing reads into the absolute mutant copies detected. The box-and-whisker plots show the median (center line), 25th and 75th percentiles (box) with the connecting "whiskers" extending from the first quartile minus 1.5 of the interquartile range (IQR, the third quartile less the first quartile) and the third quartile plus 1.5 of the IQR. A positive Spearman's correlation close to 1 indicates a strong, positive relationship between the absolute mutant EGFR copies detected and the absolute mutant EGFR copies per input. B: Inter-run reproducibility of the EGFR exon 19 deletions, L858R and T790M enrichment PCR-NGS assays for the dilution series shown in panel A. The Coefficient of Variation Percent (CV%) was calculated as the ratio of the standard deviation to the mean of the absolute EGFR copies detected within each absolute copy per input level and is reported as a percentage.
ARTICLE ABSTRACT
Purpose: Noninvasive drug biomarkers for the early assessment of tumor response can enable adaptive therapeutic decision-making and proof-of-concept studies for investigational drugs. Circulating tumor DNA (ctDNA) is released into the circulation by tumor cell turnover and has been shown to be detectable in urine.Experimental Design: We tested the hypothesis that dynamic changes in EGFR activating (exon 19del and L858R) and resistance (T790M) mutation levels detected in urine could inform tumor response within days of therapy for advanced non–small cell lung cancer (NSCLC) patients receiving osimertinib, a second-line third-generation anti-EGFR tyrosine kinase inhibitor.Results: Eight of nine evaluable NSCLC patients had detectable T790M-mutant DNA fragments in pretreatment baseline samples. Daily monitoring of mutations in urine indicated a pattern of intermittent spikes throughout week 1, suggesting apoptosis with an overall decrease in fragment numbers from baselines to day 7 preceding radiographic response assessed at 6 to 12 weeks.Conclusions: These findings suggest drug-induced tumor apoptosis within days of initial dosing. Daily sampling of ctDNA may enable early assessment of patient response and proof-of-concept studies for drug development. The modeling of tumor lysis through the day-to-day kinetics of ctDNA released into the blood and then into the urine is demonstrated in this proof-of-concept study in lung cancer patients receiving anti-EGFR tyrosine kinase inhibitors. This strategy may determine the specific clonal populations of cells which undergo apoptosis within the first week of therapy. This has important implications for developing combinational strategies to address inter- and intralesional heterogeneity and characterizing residual disease after initial drug exposure. Clin Cancer Res; 23(16); 4716–23. ©2017 AACR.
