Application of Circulating Cell-Free Tumor DNA Profiles for Therapeutic Monitoring and Outcome Prediction in Genetically Heterogeneous Metastatic Melanoma

Abstract

Purpose: Circulating cell-free tumor DNA (ctDNA) reflects the heterogeneous spectrum of tumor-specific mutations, especially in systemic disease. We validated plasma-based assays that allow the dynamic quantitative detection of ctDNA as a prognostic biomarker for tumor load and prediction of therapy response in melanoma.

Materials and methods: We analyzed plasma-derived ctDNA from a large training cohort (n = 96) of patients with advanced-stage melanoma, with assays for the BRAF ^V600E and NRAS ^Q61 driver mutations as well as TERT ^C250T and TERT ^C228T promoter mutations. An independent patient cohort (n = 35) was used to validate the utility of ctDNA monitoring under mitogen-activated protein kinase-targeted or immune checkpoint therapies.

Results: Elevated plasma ctDNA level at baseline was an independent prognostic factor of disease progression when compared with serum S100 and lactate dehydrogenase levels in multivariable analyses (hazard ratio [HR], 7.43; 95% CI, 1.01 to 55.19; P = .05). The change in ctDNA levels during therapy correlated with treatment response, where increasing ctDNA was predictive for shorter progression-free survival (eg, for BRAF ^V600E ctDNA, HR, 3.70; 95% CI, 1.86 to 7.34; P < .001). Increasing ctDNA levels predicted disease progression significantly earlier than did routine radiologic scans (P < .05), with a mean lead time of 3.5 months. NRAS-mutant ctDNA was detected in a significant proportion of patients with BRAF-mutant tumors under therapy, but unexpectedly also at baseline. In vitro sensitivity studies suggested that this represents higher-than-expected intratumoral heterogeneity. The detection of NRAS ^Q61 ctDNA in baseline samples of patients with BRAF ^V600E mutation who were treated with mitogen-activated protein kinase inhibitors significantly correlated with shorter progression-free survival (HR, 3.18; 95% CI, 1.31 to 7.68; P = .03) and shorter overall survival (HR, 4.08; 95% CI, 1.57 to 10.58; P = .01).

Conclusion: Our results show the potential role of ctDNA measurement as a sensitive monitoring and prediction tool for the early assessment of disease progression and therapeutic response in patients with metastatic melanoma.

FIG 1.

Basic characteristics of patients with melanoma who have BRAF^V600E, NRAS^Q61, and TERT^prom mutations. Overview of (A) the training cohort (n = 96 patients with stage III and IV disease) and (B) the validation cohort (n = 35 patients with stage III and IV disease). In the upper panels, the demographic and tumor characteristics are represented. The middle panels show mutations detected by DNA sequencing in respective tumor samples. The lower panels correspond to respective plasma samples analyzed with droplet digital polymerase chain reaction. Radiologic tumor-load information at baseline is represented at the bottom of the panels. See also the Data Supplement. AJCC, American Joint Committee on Cancer; ID, identification; TL, tumor load.

FIG 2.

Correlation of baseline circulating cell-free tumor DNA (ctDNA) levels with metastatic stage and routine serum markers in the training cohort. Correlation of baseline BRAF^V600E ctDNA levels with increasing metastatic tumor load in (A) lymph nodes (N1, n = 3; N2, n = 3; and N3, n = 14) and (B) organs (M1a, n = 5; M1b, n = 5; M1c, n = 41). (C) Correlation of NRAS^Q61 ctDNA levels with organ metastasis (M1a, n = 3; M1b, n = 2; M1c, n = 11). (D) Correlation of mutant TERT^prom ctDNA levels with metastatic progression from locoregional (stage IIIB, n = 2; or IIIC, n = 6) to systemic disease (stage IV; n = 14) in patients with TERT^prom-mutated melanomas. (E) Correlation of baseline serum lactate dehydrogenase (LDH) and S100 levels with metastatic progression in BRAF^V600E-positive patients (gray line, upper limit of normal). Box-and-whisker plots represent median values and interquartile range. Blue dots represent mean values. Welch t test was used to calculate statistical significance. (F-K) Correlation of serum LDH and S100 levels with BRAF^V600E (n = 274 and n = 216, respectively), NRAS^Q61 (n = 97 and n = 67, respectively), and mutant TERT^prom (n = 152 and n = 124, respectively) ctDNA levels in the training cohort. Sample pairs were analyzed for the Spearman correlation coefficient (ρ). The upper limit of normal (gray line) for LDH is 247 IU/L and for S100 is 0.15 µg/L. AJCC, American Joint Committee on Cancer.

FIG 3.

Changes in the BRAF^V600E circulating cell-free tumor DNA (ctDNA) levels correlate with therapy response and progression-free survival (PFS). Changes in mean BRAF^V600E ctDNA levels after therapy initiation relative to baseline (BL) in patients who received (A) immune checkpoint inhibition therapy (n = 18 patients) and (B) signaling targeted therapy (n = 33). Follow-up (FU) sampling was performed every 4 to 6 weeks. (*) P < .01, (†) P < .001, and (‡) P < .001 from unpaired t test. The data represent mean ± SEM. Kaplan-Meier plots are for PFS of the same patients with melanoma as assessed by routine radiologic scans. (C) Immune checkpoint inhibition (patients with ctDNA decrease [n = 6] v increase [n = 12]) and (D) signaling targeted therapy (patients with ctDNA decrease [n = 12] v increase [n = 21]). Categorization into decrease versus increase was based on the ctDNA change at the second sampling time point relative to BL. The hazard ration (HR) is indicated for ctDNA increase. The P value was determined by the log-rank test. (E, F) Scatter dot plots of BRAF^V600E ctDNA levels of responders versus nonresponders grouped according to (E) radiologic response 10 weeks after receiving any therapy or (F) radiologic PFS at 6 months of therapy (Mann-Whitney U test). Points represent individual patients; median with interquartile range is indicated for each plot. Gray lines indicate ctDNA thresholds as determined by receiver operating characteristic analyses (Data Supplement).

FIG 4.

Changes of NRAS^Q61 and TERT^prom circulating cell-free tumor DNA (ctDNA) levels correlated with therapy response and progression-free survival (PFS). Changes in mean NRAS^Q61 and TERT^prom ctDNA levels after therapy initiation relative to baseline (BL) in patients who received (A, C) immune checkpoint inhibition therapy, (n = 10 and n = 12, respectively) and (B, D) signaling targeted therapy, (n = 7 and n = 10, respectively). Follow-up (FU) sampling was performed every 4 to 6 weeks. (*) P < .05, (†) P < .01, (‡) P < .001, and (§) P < .001 from unpaired t test. The data are reported as mean ± SEM. Scatter dot plots of (E) NRAS^Q61 and (F) TERT^prom ctDNA levels of patients assessed at week 10 after receiving any therapy, grouped according to radiologic PFS at 6 months (Mann-Whitney U test). Points represent individual patients; median with interquartile range is indicated for each plot. Gray lines indicate ctDNA thresholds as determined by receiver operating characteristic analyses (Data Supplement).

FIG 5.

Validation cohort and time gain in assessment of disease progression. (A-D) circulating cell-free tumor DNA (ctDNA) dynamics correlated with clinical outcome in the validation cohort. (A) Changes in mean ctDNA levels after therapy initiation relative to baseline (BL) from patients who received immune checkpoint inhibition therapy (n = 35 patients). Follow-up (FU) sampling was performed every 6 weeks. (*) P < .05, (†) P < .01, and (‡) P < .001 from unpaired t test. The data are reported as mean ± SEM. (B) Kaplan-Meier plot for progression-free survival (PFS) of the same patients with melanoma who received immune checkpoint inhibition therapy who were assessed by routine radiologic scans (patients with ctDNA decrease [n = 15] v increase [n = 20]). Categorization into decrease versus increase was based on the ctDNA change at the second sampling time point relative to BL. Hazard ratio (HR) is indicated for ctDNA increase. The P value was determined by the log-rank test. (C-D) Scatter dot plots of BRAF^V600E, NRAS^Q61, and TERT^prom ctDNA levels of responders versus nonresponders, grouped according to (C) radiologic response 12 weeks after receiving any therapy or (D) radiologic PFS at 6 months of therapy (Mann-Whitney U test). Points represent individual patients; median with interquartile range is indicated for each data set. ctDNA as an early predictive parameter for therapy response and failure (data from training cohort). (E) Decreasing BRAF^V600E ctDNA levels (as compared with the last sampling time point) preceded radiologic detection of response in 12 of 15 responders with an average lead-time window of 1.5 months (range, 0.023 to 3.45 months). Wilcoxon signed-rank test P values are reported. (F) Increasing BRAF^V600E ctDNA levels preceded radiologic progression in 31 of 36 nonresponders, with an average lead-time window of 3.5 months (range, 0.23 to 18.86 months). CT, computed tomography; MRI, magnetic resonance imaging; US, ultrasound.

FIG 6.

Detection of NRAS^Q61 at baseline (BL) is a predictor of worse clinical outcome in patients treated with mitogen-activated protein kinase (MAPK) inhibitors. Kaplan-Meier plots for (A) radiologic progression-free survival (PFS) and (B) overall survival (OS) of patients with BRAF^V600E-positive tumors who received MAPK signaling targeted therapy (n = 21 with positive NRAS^Q61 detection in BL plasma v n = 12 without detection). Hazard ratio (HR) is reported for circulating cell-free tumor DNA (ctDNA) detected. P values were determined by the log-rank test. (C) Overview of 51 BRAF^V600E-, NRAS^Q61-, and TERT^prom-matched tumor tissue and plasma samples of patients who had available tumor biopsy specimen at therapy BL. Tumor samples were routinely analyzed for BRAF^V600E, NRAS^Q61, and TERT^prom mutations with amplicon-based next-generation sequencing (NGS; Fig. 1) and afterward reanalyzed by ddPCR. Plasma was sampled at BL (ie, before systemic therapy initiation) and analyzed for ctDNAs by droplet digital polymerase chain reaction (ddPCR).