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

Metastatic melanoma is a highly heterogeneous tumour, thus methods to analyse tumour-derived cells circulating in blood should address this diversity. Taking this into account, we analysed, using multiparametric flow cytometry, the co-expression of the melanoma markers MCAM and MCSP and the tumour initiating markers ABCB5, CD271 and RANK in individual circulating tumour cells (CTCs) from 40 late stages (III-IV) and 16 early stages (I-II) melanoma patients. CTCs were heterogeneous within and between patients, with limited co-expression between the five markers analysed. Analysis of patient matched blood and metastatic tumours revealed that ABCB5 and RANK subpopulations are more common amongst CTCs than in the solid tumours, suggesting a preferential selection for these cells in circulation. Pairwise comparison of CTC subpopulations longitudinally before and 6-13 weeks after treatment initiation, showed that the percentage of RANK⁺ CTCs significantly increased in the patients undergoing targeted therapy (N=16, P<0.01). Moreover, the presence of ≥5 RANK⁺ CTCs in blood of patients undergoing targeted therapies was prognostic of shorter progression free survival (HR 8.73, 95% CI 1.82-41.75, P<0.01). Taken together our results provide evidence of the heterogeneity amongst CTC subpopulations in melanoma and the differential response of these subpopulations to targeted therapy.

Background Multimarker quantitative real‐time polymerase chain reaction (qRT‐PCR) represents an effective method for detecting circulating tumour cells in the peripheral blood of patients with melanoma. Objectives To investigate whether the phenotype of circulating melanoma cells represents a useful indicator of disease stage, recurrence and treatment efficacy. Methods Peripheral blood was collected from 230 patients with melanoma and 152 healthy controls over a period of 3 years and 9 months. Clinical data and blood samples were collected from patients with primary melanoma (early stages, 0–II, n = 154) and metastatic melanoma (late stages, III–IV, n = 76). Each specimen was examined by qRT‐PCR analysis for the expression of five markers: MLANA, ABCB5, TGFβ2, PAX3d and MCAM. Results In total, 212 of the patients with melanoma (92%) expressed markers in their peripheral blood. Two markers, MLANA and ABCB5, had the greatest prognostic value, and were identified as statistically significant among patients who experienced disease recurrence within our study period, being expressed in 45% (MLANA) and 49% (ABCB5) of patients with recurrence (P = 0·001 and P = 0·031, respectively). For patients administered nonsurgical treatments, MCAM expression correlated with poor treatment outcome. Conclusions Circulating tumour cells were detectable at all stages of disease and long after surgical treatment, even when patients were considered disease free. Specifically, expression of ABCB5 and MLANA had significant prognostic value in inferring disease recurrence, while MCAM expression was associated with poor patient outcome after treatment, confirming multimarker qRT‐PCR as a potential technique for monitoring disease status.

Background: PD-1 inhibitors are routinely used for the treatment of advanced melanoma. This study sought to determine whether PD-L1 expression on circulating tumor cells (CTCs) can serve as a predictive biomarker of clinical benefit and response to treatment with the PD-1 inhibitor pembrolizumab. Methods: Blood samples were collected from patients with metastatic melanoma receiving pembrolizumab, prior to treatment and 6–12 weeks after initiation of therapy. Multiparametric flow cytometry was used to identify CTCs and evaluate the expression of PD-L1. Results: CTCs were detected in 25 of 40 patients (63%). Patients with detectable PD-L1+ CTCs (14/25, 64%) had significantly longer progression-free survival (PFS) compared with patients with PD-L1− CTCs (26.6 months vs. 5.5 months; p = .018). The 12-month PFS rates were 76% versus 22% in the PD-L1+ versus PD-L1− CTCs groups (p = .012), respectively. A multivariate linear regression analysis confirmed that PD-L1+ CTC is an independent predictive biomarker of PFS (hazard ratio, 0.229; 95% confidence interval, 0.052–1.012; p = .026). Conclusion: Our results reveal the potential of CTCs as a noninvasive real-time biopsy to evaluate PD-L1 expression in patients with melanoma. PD-L1 expression on CTCs may be predictive of response to pembrolizumab and longer PFS.

Purpose: We evaluated the predictive value of pretreatment ctDNA to inform therapeutic outcomes in patients with metastatic melanoma relative to type and line of treatment. Experimental Design: Plasma circulating tumor DNA (ctDNA) was quantified in 125 samples collected from 110 patients prior to commencing treatment with immune checkpoint inhibitors (ICIs), as first- (n = 32) or second-line (n = 27) regimens, or prior to commencing first-line BRAF/MEK inhibitor therapy (n = 66). An external validation cohort included 128 patients commencing ICI therapies in the first- (N = 77) or second-line (N = 51) settings. Results: In the discovery cohort, low ctDNA (≤20 copies/mL) prior to commencing therapy predicted longer progression-free survival (PFS) in patients treated with first-line ICIs [HR, 0.20; 95% confidence interval (CI) 0.07–0.53; P < 0.0001], but not in the second-line setting. An independent cohort validated that ctDNA is predictive of PFS in the first-line setting (HR, 0.42; 95% CI, 0.22–0.83; P = 0.006), but not in the second-line ICI setting. Moreover, ctDNA prior to commencing ICI treatment was not predictive of PFS for patients pretreated with BRAF/MEK inhibitors in either the discovery or validation cohorts. Reduced PFS and overall survival were observed in patients with high ctDNA receiving anti–PD-1 monotherapy, relative to those treated with combination anti–CTLA-4/anti–PD-1 inhibitors. Conclusions: Pretreatment ctDNA is a reliable indicator of patient outcome in the first-line ICI treatment setting, but not in the second-line ICI setting, especially in patients pretreated with BRAF/MEK inhibitors. Preliminary evidence indicated that treatment-naïve patients with high ctDNA may preferentially benefit from combined ICIs.