Monitoring Daily Dynamics of Early Tumor Response to Targeted Therapy by Detecting Circulating Tumor DNA in Urine

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.

Figure 1. Dynamic changes in urinary ctDNA with time-points preceding osimertinib drug administration, time points immediately post administration, and several time-points later in therapy

Subjects 1 and 16. Data points of ctDNA copies suggest a significant change from baseline after osimertinib drug administration, and this is represented as a spike during the first week of therapy.

Figure 2. Daily dynamics of ctDNA EGFR mutation levels on second line osimertinib

Urine samples were collected from patients prior to osimertinib treatment as baseline and daily on treatment. A consistent pattern was observed with spikes during the first week followed by an overall decrease in the numbers of copies by day 7. Data points are mutant EGFR copies per 100,000 genome equivalents detected. Dashed lines indicate clinical detection cut-offs for the EGFR activating mutations (L858R = red, exon 19 deletions = green) and T790M (blue).

Figure 3. Quantification of EGFR mutation levels in urine of patients with NSCLC before and after 1 and 2 weeks of osimertinib

Urine samples were collected from patients prior to osimertinib and at week 1 or week 2 time points on treatment. T790M ctDNA and corresponding EGFR L858R or exon 19 deletion levels are shown as copies per 100,000 genome equivalents (1A, B) or as percent of respective baselines (1C, D). A significant relative decrease in T790M mutation signal from baseline was observed at week 1 and 2 on treatment (one-sided p-values of 0.014 and 0.045, respectively, using Wilcoxon’s Test) (1C). Similar patterns were observed for the activating mutations L858R, exon 19 deletions at week 1 and week 2.

Figure 4. Potential applications of early response monitoring in EGFR-mutant NSCLC

Strategies that evaluate the daily kinetic changes in ctDNA during the first week of therapy will be explored to characterize response assessments based on the size, presence, or absence of peaks in ctDNA. A larger prospective analysis is underway to verify this in a larger cohort of patients with variable responses.