Clinical validation of droplet digital PCR assays in detecting BRAFV600-mutant circulating tumour DNA as a prognostic biomarker in patients with resected stage III melanoma receiving adjuvant therapy (COMBI-AD): a biomarker analysis from a double-blind, randomised phase 3 trial

Abstract

Background

Cell-free, circulating tumour DNA (ctDNA) is an established measure of minimal residual disease; however, it is not utilised in melanoma management. We investigated whether ctDNA measurements could predict survival outcomes during adjuvant targeted therapy or placebo treatment in stage III melanoma, thereby identifying patients at high risk and low risk of recurrence.

Methods

Analytically validated mutation-specific droplet digital PCR assays were used to measure BRAF^V600E or BRAF^V600K ctDNA in patients aged 18 years or older who were enrolled in the COMBI-AD trial, which was a double-blind, randomised, phase 3 study of oral dabrafenib (150 mg twice daily) plus oral trametinib (2 mg once daily) combination therapy versus two matched placebos in resected BRAF^V600-mutant stage III melanoma. Patients were screened for enrolment between Jan 31, 2013, and Dec 11, 2014, had an Eastern Cooperative Oncology Group performance status of 0 or 1, and were randomly assigned (1:1) to the two treatment groups. The primary endpoint was recurrence-free survival, and the results from final analysis have been previously published and will not be described here. Biomarker analysis was a prespecified exploratory endpoint and performed in the intention-to-treat population. We compared associations between survival outcomes and baseline (post-resection) ctDNA copies per mL, tumour mutational burden and interferon gamma (IFNG) gene expression. In a subset of patients, ctDNA quantities during follow-up or at recurrence were measured. The trial is registered with ClinicalTrials.gov, NCT01682083, and has been completed.

Findings

Baseline plasma samples were available for 597 of 870 patients (331 male patients and 266 female patients) and samples for assessing the ctDNA positivity rate at landmark follow-up timepoints of 3 months, 6 months, 9 months, and 12 months after treatment initiation were available for 94 of 870 patients. Additionally, samples were available from 118 of 870 patients within a 2-month timeframe before or after clinical or radiographic recurrence. Median follow-up for the biomarker analyses was 60 months (IQR 39–66) in the combination therapy group and 58 months (21–66) for the placebo group. ctDNA was detectable in 79 (13%) of 597 baseline samples. ctDNA positivity rate and mutant copies per mL plasma were significantly higher in patients with higher disease substages. As a binary variable, ctDNA detection was associated with worse recurrence-free survival (placebo group: median 3·71 months [95% CI 2·39–6·89] vs 24·41 months [17·28–43·13]; hazard ratio [HR] 2·91 [95% CI 1·99–4·25], p<0·0001); combination therapy group: median 16·59 months [95% CI 12·02–26·80] vs 68·11 months [50·36–not reached]; HR 2·98 [1·95–4·54], p<0·0001) and overall survival (placebo group: median 33·90 months [13·96–not reached] vs not reached; HR 3·35 [2·01–5·55], p<0·0001); combination therapy group: median 40·31 months [24·90–not reached] vs not reached; HR 4·27 [2·50–7·27], p<0·0001) in the placebo group and combination therapy groups. Baseline ctDNA was more strongly associated with survival outcomes than IFNG gene expression or tumour mutational burden. Patients with adverse longitudinal ctDNA kinetics (molecular relapse or persistently positive) had markedly shorter median recurrence-free survival (8·31 months [95% CI 5·39–12·20] and 5·32 months [2·79–not reached], respectively) compared with patients with favourable kinetics (ie, undetectable after positive baseline result: 19·25 months [16·39–not reached]; and durable undetectable: not reached [38·44–not reached], p<0·0001).

Interpretation

Droplet digital PCR measurements of ctDNA to assess minimal residual disease before adjuvant targeted therapy and during follow-up can identify patients at high risk of early recurrence. Additional studies using ctDNA measurements to guide therapeutic interventions might lead to improvements in the management of resected stage III melanoma.

Funding

Novartis.