Plasma Circulating Tumor DNA Levels for the Monitoring of Melanoma Patients: Landscape of Available Technologies and Clinical Applications

Abstract

Melanoma is a cutaneous cancer with an increasing worldwide prevalence and high mortality due to unresectable or metastatic stages. Mutations in BRAF, NRAS, or KIT are present in more than 60% of melanoma cases, but a useful blood-based biomarker for the clinical monitoring of melanoma patients is still lacking. Thus, the analysis of circulating tumor cells (CTCs) and/or cell-free circulating tumor DNA (ctDNA) analysis from blood (liquid biopsies) appears to be a promising noninvasive, repeatable, and systemic sampling tool for detecting and monitoring melanoma. Here, we review the molecular biology-based strategies used for ctDNA quantification in melanoma patients, as well as their main clinical applications. Droplet digital PCR (ddPCR) and next generation sequencing (NGS) technologies appear to be two versatile and complementary strategies to study rare variant mutations for the detection and monitoring of melanoma progression. Among the different clinical uses of ctDNA, we highlight the assessment of molecular heterogeneity and the identification of genetic determinants for targeted therapy as well as the analysis of acquired resistance. Importantly, ctDNA quantification might also be a novel biomarker with a prognostic value for melanoma patients.

Detection of cell‐free circulating BRAF V 600E by droplet digital polymerase chain reaction in patients with and without melanoma under dermatological surveillance

“…For diagnostic purposes, in addition to identifying factors involved in cf BRAF V600E detection, it is necessary to determine the sensitivity and specificity of the test to establish a positivity threshold. The ddPCR technology is one of the most relevant and sensitive techniques for quantifying rare mutations in cfDNA from patients with cancer, with a detection sensitivity of approximately 0·01% . Currently, there is no consensus as to the cf BRAF V600E positivity threshold in patients with melanoma.…”

“…9,10 Cutaneous melanoma is one of the neoplasms in which ctDNA characterization has gained interest. 11 Advanced cutaneous melanoma is one of the most aggressive and treatmentresistant human cancers. 12 Oncogenic BRAF mutations are detected in approximately 50% of melanomas.…”

“…The ddPCR technology is one of the most relevant and sensitive techniques for quantifying rare mutations in cfDNA from patients with cancer, 1,38,39 with a detection sensitivity of approximately 0Á01%. 11,40 Currently, there is no consensus as to the cfBRAF V600E positivity threshold in patients with melanoma. For instance, different studies evaluating the prognostic role of plasma cfBRAF V600E analysis established the cut off as 1 copy mL À1 to detect relapse after surgical resection in patients with stage III melanoma 26 and to detect disease progression in patients with unresectable stage IIIC/IV melanoma.…”

A field guide for cancer diagnostics using cell‐free DNA: From principles to practice and clinical applications

“…While the template molecules are separated in individual reaction chambers in digital approaches, the ddPCR utilizes oil emulsion droplets for physical separation of individual molecules and reactions. The analysis enables absolute quantification of mutant and wild type molecules in cfDNA and allows for the detection of allele frequencies as low as 0.01% . Both dPCR and ddPCR share a lower sensitivity toward inhibitors in the sample that may make qPCR analysis less reliable .…”

“…Although dPCR and ddPCR are cheaper and have lower turnaround times than NGS‐based approaches when testing for single mutations only, the main disadvantages are that mutations have to be tested sequentially and that detection of previously unknown mutations is impossible . As all dPCR technologies require only low quantities of template molecules, single molecule detection is advantageous for the analysis of limited amount of cfDNA alleles in plasma.…”