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Simple Summary: Circulating tumour DNA (ctDNA) has been shown to be an informative biomarker in melanoma. Here we analysed plasma ctDNA in a real-world metastatic melanoma cohort. We found the kinetics of ctDNA decline are delayed in patients treated with immunotherapy compared to those receiving MAPK inhibitors. Nonetheless, decreasing ctDNA levels within 12 weeks of immunotherapy or BRAF/MEK inhibitors was strongly concordant with treatment response and significantly associated with longer progression-free survival (PFS). Furthermore, exploratory analysis of nine patients commencing anti-PD-1 therapy showed a trend of high tumour mutational burden and neoepitope load in responders compared to non-responders. The results support the use of ctDNA as a dynamic biomarker for assessment of response in melanoma patients. In this study, we evaluated the predictive value of circulating tumour DNA (ctDNA) to inform therapeutic outcomes in metastatic melanoma patients receiving systemic therapies. We analysed 142 plasma samples from metastatic melanoma patients prior to commencement of systemic therapy: 70 were treated with BRAF/MEK inhibitors and 72 with immunotherapies. Patient-specific droplet digital polymerase chain reaction assays were designed for ctDNA detection. Plasma ctDNA was detected in 56% of patients prior to first-line anti-PD1 and/or anti-CTLA-4 treatment. The detection rate in the immunotherapy cohort was comparably lower than those with BRAF inhibitors (76%, p = 0.0149). Decreasing ctDNA levels within 12 weeks of treatment was strongly concordant with treatment response (Cohen's k = 0.798, p < 0.001) and predictive of longer progression free survival. Notably, a slower kinetic of ctDNA decline was observed in patients treated with immunotherapy compared to those on BRAF/MEK inhibitors. Whole exome sequencing of ctDNA was also conducted in 9 patients commencing anti-PD-1 therapy to derive tumour mutational burden (TMB) and neoepitope load measurements. The results showed a trend of high TMB and neoepitope load in responders compared to non-responders. Overall, our data suggest that changes in ctDNA can serve as an early indicator of outcomes in metastatic melanoma patients treated with systemic therapies and therefore may serve as a tool to guide treatment decisions. [ABSTRACT FROM AUTHOR]

In recent years, circulating tumor DNA (ctDNA) has emerged as a promising prognostic and monitoring biomarker of various cancers, including melanoma. However, sensitive methods are required for its preservation, isolation, and detection. Here we describe a sensitive method for plasma ctDNA isolation using a column-based extraction kit, followed by quantification using a single mutational target with a droplet digital PCR system. This sensitive protocol has been successfully used to quantify diverse mutations present in plasma-derived ctDNA from cancer patients. The full procedure, from blood processing to the analysis of results, takes approximately a day of work.

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., (©2020 American Association for Cancer Research.)

The analysis of plasma circulating tumour nucleic acids provides a non-invasive approach to assess disease burden and the genetic evolution of tumours in response to therapy. BRAF splicing variants are known to confer melanoma resistance to BRAF inhibitors. We developed a test to screen cell-free RNA (cfRNA) for the presence of BRAF splicing variants. Custom droplet digital PCR assays were designed for the detection of BRAF splicing variants p61, p55, p48 and p41 and then validated using RNA from cell lines carrying these variants. Evaluation of plasma from patients with reported objective response to BRAF/MEK inhibition followed by disease progression was revealed by increased circulating tumour DNA (ctDNA) in 24 of 38 cases at the time of relapse. Circulating BRAF splicing variants were detected in cfRNA from 3 of these 38 patients; two patients carried the BRAF p61 variant and one the p55 variant. In all three cases the presence of the splicing variant was apparent only at the time of progressive disease. BRAF p61 was also detectable in plasma of one of four patients with confirmed BRAF splicing variants in their progressing tumours. Isolation and analysis of RNA from extracellular vesicles (EV) from resistant cell lines and patient plasma demonstrated that BRAF splicing variants are associated with EVs. These findings indicate that in addition to plasma ctDNA, RNA carried by EVs can provide important tumour specific information., Competing Interests: CONFLICTS OF INTEREST The following authors have received travel support from: MAK [Merck Sharp and Dohme (MSD), Bristol-Myers Squibb (BMS) and Merck Serono], TMM [BMS, Novartis, AstraZeneca (AZ)] and ESG [MSD]. The following authors sit on advisory boards for: TMM [BMS, MSD, Novartis, AZ]; MSC [Sanofi, Nektar, Merck Serono BMS, MSD, Novartis, Amgen, Pierre Fabre, Ideaya]; AMM [BMS, MSD, Novartis, Roche, Pierre-Fabre], GVL [Aduro, Amgen, Array, BMS, MSD, Novartis, Pierre-Fabre, Oncosec, Roche] and MM [BMS, AZ, Roche, MSD]., (Copyright: © 2020 Clark et al.)

Circulating Tumour Cells (CTCs) are promising cancer biomarkers. Several methods have been developed to isolate CTCs from blood samples. However, the isolation of melanoma CTCs is very challenging as a result of their extraordinary heterogeneity, which has hindered their biological and clinical study. Thus, methods that isolate CTCs based on their physical properties, rather than surface marker expression, such as microfluidic devices, are greatly needed in melanoma. Here, we assessed the ability of the slanted spiral microfluidic device to isolate melanoma CTCs via label-free enrichment. We demonstrated that this device yields recovery rates of spiked melanoma cells of over 80% and 55%, after one or two rounds of enrichment, respectively. Concurrently, a two to three log reduction of white blood cells was achieved with one or two rounds of enrichment, respectively. We characterised the isolated CTCs using multimarker flow cytometry, immunocytochemistry and gene expression. The results demonstrated that CTCs from metastatic melanoma patients were highly heterogeneous and commonly expressed stem-like markers such as PAX3 and ABCB5. The implementation of the slanted microfluidic device for melanoma CTC isolation enables further understanding of the biology of melanoma metastasis for biomarker development and to inform future treatment approaches., Competing Interests: CONFLICTS OF INTEREST All authors, except for M.H.F., declare no conflicts of interest. M.H.F. is inventor or co-inventor of US and international patents assigned to Brigham and Women’s Hospital and/or Boston Children's Hospital, Boston, MA, and licensed to Ticeba GmbH (Heidelberg, Germany) and Rheacell GmbH & Co. KG (Heidelberg, Germany). M.H.F. serves as a scientific advisor to Ticeba GmbH and Rheacell GmbH & Co. KG. and participates in corporate sponsored research collaborations with Rheacell GmbH & Co. KG.