Circulating tumor DNA to monitor treatment response and detect acquired resistance in patients with metastatic melanoma

Abstract

Repeat tumor biopsies to study genomic changes during therapy are difficult, invasive and data are confounded by tumoral heterogeneity. The analysis of circulating tumor DNA (ctDNA) can provide a non-invasive approach to assess prognosis and the genetic evolution of tumors in response to therapy. Mutation-specific droplet digital PCR was used to measure plasma concentrations of oncogenic BRAF and NRAS variants in 48 patients with advanced metastatic melanoma prior to treatment with targeted therapies (vemurafenib, dabrafenib or dabrafenib/trametinib combination) or immunotherapies (ipilimumab, nivolumab or pembrolizumab). Baseline ctDNA levels were evaluated relative to treatment response and progression-free survival (PFS). Tumor-associated ctDNA was detected in the plasma of 35/48 (73%) patients prior to treatment and lower ctDNA levels at this time point were significantly associated with response to treatment and prolonged PFS, irrespective of therapy type. Levels of ctDNA decreased significantly in patients treated with MAPK inhibitors (p < 0.001) in accordance with response to therapy, but this was not apparent in patients receiving immunotherapies. We show that circulating NRAS mutations, known to confer resistance to BRAF inhibitors, were detected in 3 of 7 (43%) patients progressing on kinase inhibitor therapy. Significantly, ctDNA rebound and circulating mutant NRAS preceded radiological detection of progressive disease. Our data demonstrate that ctDNA is a useful biomarker of response to kinase inhibitor therapy and can be used to monitor tumor evolution and detect the early appearance of resistance effectors.

Keywords: MAPK inhibition; acquired resistance; ctDNA; immunotherapy; melanoma.

Figure 1. Baseline ctDNA association with response to treatment and PFS

Association of baseline ctDNA concentrations with A. and B. response to treatment and C. and D. 6 months PFS. Median with interquartile range is indicated on each data set. Contingency tables with corresponding Fisher's exact test p-value are indicated below each graph. Kaplan-Meier plots of PFS probabilities according to baseline ctDNA concentrations of E. cases treated with targeted therapies (n = 29) and F. immunotherapies (n = 19). Cox regression p-values, Hazard ratio (HR) and confidence intervals are indicated for each plot.

Figure 2. Change in ctDNA levels after therapy initiation relative to baseline

Plasma samples were tested for ctDNA at baseline and between 4 to 8 weeks after therapy initiation (follow up). Cases are colour coded according to therapy, and grouped by therapy type and response. * indicates two patients that were classified as non-responders but had stable disease for at least 6 months.

Figure 3. Monitoring ctDNA in plasma during clinical disease course

Levels of BRAF and NRAS mutated ctDNA in plasma collected longitudinally from five melanoma patients during treatment with A.-D. dabrafenib and trametinib combination therapy, followed by immunotherapies and E. during ipilimumab treatment. Clinical outcomes revealed by CT and/or PET scans are indicated at each assessment time (arrows). Patient death is indicated by a red cross (†). A. PET scan images at four clinical assessment time points for comparison with ctDNA levels.