Mutation analysis in individual circulating tumor cells depicts intratumor heterogeneity in melanoma.

Circulating tumor DNA (ctDNA) is the cornerstone of liquid biopsy diagnostics, revealing clinically relevant genomic aberrations from blood of cancer patients. Genomic analysis of single circulating tumor cells (CTCs) could provide additional insights into intra-patient heterogeneity, but it requires whole-genome amplification (WGA) of DNA, which might introduce bias. Here, we describe a novel approach based on mass spectrometry for mutation detection from individual CTCs not requiring WGA and complex bioinformatics pipelines. After establishment of our protocol on tumor cell line-derived single cells, it was validated on CTCs of 33 metastatic melanoma patients and the mutations were compared to those obtained from tumor tissue and ctDNA. Although concordance with tumor tissue was superior for ctDNA over CTC analysis, a larger number of mutations were found within CTCs compared to ctDNA (p = 0.039), including mutations in melanoma driver genes, or those associated with resistance to therapy or metastasis. Thus, our results demonstrate proof-of-principle data that CTC analysis can provide clinically relevant genomic information that is not redundant to tumor tissue or ctDNA analysis.

Prognostic value of von Willebrand factor levels in patients with metastatic melanoma treated by immune checkpoint inhibitors.

BACKGROUND: An increased incidence of thrombotic complications associated with an increased mortality rate has been observed under immune checkpoint inhibition (ICI). Recent investigations on the coagulation pathways have highlighted the direct role of key coagulatory proteins and platelets in cancer initiation, angiogenesis and progression. The aim of this study was to evaluate the prognostic value of von Willebrand factor (vWF) and its regulatory enzyme a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), D-dimers and platelets in a cohort of patients with metastatic melanoma receiving ICI. METHODS: In a prospective cohort of 83 patients with metastatic melanoma, we measured the systemic levels of vWF-antigen (vWF:Ag), ADAMTS13 activity, D-dimers and platelets, before the beginning of the treatment (baseline), and 6, 12 and 24 weeks after. In parallel, we collected standard biological parameters used in clinical routine to monitor melanoma response (lactate deshydrogenase (LDH), S100). The impact of neutrophil-to-lymphocyte ratio (NLR) and C-reactive protein (CRP) on overall survival (OS) in patients receiving ICI was assessed. Univariable and multivariable Cox proportional models were then used to investigate any potential association of these parameters to clinical progression (progression-free survival (PFS) and OS). Baseline values and variations over therapy course were compared between primary responders and resistant patients. RESULTS: Patients with melanoma present with dysregulated levels of vWF:Ag, ADAMTS13 activity, D-dimers, LDH, S100 and CRP at the beginning of treatment. With a median clinical follow-up of 26 months, vWF:Ag interrogated as a continuous variable was significantly associated with PFS in univariate and multivariate analysis (HR=1.04; p=0.007). Lower values of vWF:Ag at baseline were observed in the primary responders group (median: 29.4 µg/mL vs 32.9 µg/mL; p=0.048) when compared with primary resistant patients. As for OS, we found an association with D-dimers and ADAMTS13 activity in univariate analysis and vWF:Ag in univariate and multivariate analysis including v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation and Eastern Cooperative Oncology Group (ECOG) performance status. Follow-up over the course of treatment depicts different evolution profiles for vWF:Ag between the primary response and resistance groups. CONCLUSIONS: In this prospective cohort, coagulatory parameters such as ADAMTS13 activity and D-dimers are associated with OS but baseline vWF:Ag levels appeared as the only parameter associated with response and OS to ICI. This highlights a potential role of vWF as a biomarker to monitor ICI response of patients with malignant melanoma.

Current and Future Clinical Applications of ctDNA in Immuno-Oncology.

Testing peripheral blood for circulating tumor DNA (ctDNA) offers a minimally invasive opportunity to diagnose, characterize, and monitor the disease in individual cancer patients. ctDNA can reflect the actual tumor burden and specific genomic state of disease and thus might serve as a prognostic and predictive biomarker for immune checkpoint inhibitor (ICI) therapy. Recent studies in various cancer entities (e.g., melanoma, non-small cell lung cancer, colon cancer, and urothelial cancer) have shown that sequential ctDNA analyses allow for the identification of responders to ICI therapy, with a significant lead time to imaging. ctDNA assessment may also help distinguish pseudoprogression under ICI therapy from real progression. Developing dynamic changes in ctDNA concentrations as a potential surrogate endpoint of clinical efficacy in patients undergoing adjuvant immunotherapy is ongoing. Besides overall ctDNA burden, further ctDNA characterization can help uncover tumor-specific determinants (e.g., tumor mutational burden and microsatellite instability) of responses or resistance to immunotherapy. In future studies, standardized ctDNA assessments need to be included in interventional clinical trials across cancer entities to demonstrate the clinical utility of ctDNA as a biomarker for personalized cancer immunotherapy.