Comparison of BRAF Mutation Screening Strategies in a Large Real-Life Series of Advanced Melanoma Patients.

Maria Colombino,Carla Rozzo,Maria Antonietta Fedeli,Antonella Manca,Marina Pisano,Antonio Cossu,Maria Cristina Sini,Grazia Palomba,Corrado Caracò,Giuseppe Palmieri,Paolo A Ascierto,Amelia Lissia,Milena Casula,Valentina Doneddu,Panagiotis Paliogiannis

doi:10.3390/jcm9082430

Abstract

Malignant melanoma (MM) is one of the deadliest skin cancers. BRAF mutation status plays a predominant role in the management of MM patients. The aim of this study was to compare BRAF mutational testing performed by conventional nucleotide sequencing approaches with either real-time polymerase chain reaction (rtPCR) or next-generation sequencing (NGS) assays in a real-life, hospital-based series of advanced MM patients. Consecutive patients with AJCC (American Joint Committee on Cancer) stage IIIC and IV MM from Sardinia, Italy, who were referred for molecular testing, were enrolled into the study. Initial screening was performed to assess the mutational status of the BRAF and NRAS genes, using the conventional methodologies recognized by the nationwide guidelines, at the time of the molecular classification, required by clinicians: at the beginning, Sanger-based sequencing (SS) and, after, pyrosequencing. The present study was then focused on BRAF mutation detecting approaches only. BRAF wild-type cases with available tissue and adequate DNA were further tested with rtPCR (Idylla™) and NGS assays. Globally, 319 patients were included in the study; pathogenic BRAF mutations were found in 144 (45.1%) cases examined with initial screening. The rtPCR detected 11 (16.2%) and 3 (4.8%) additional BRAF mutations after SS and pyrosequencing, respectively. NGS detected one additional BRAF-mutated case (2.1%) among 48 wild-type cases previously tested with pyrosequencing and rtPCR. Our study evidenced that rtPCR and NGS were able to detect additional BRAF mutant cases in comparison with conventional sequencing methods; therefore, we argue for the preferential utilization of the aforementioned assays (NGS and rtPCR) in clinical practice, to eradicate false-negative cases and improve the accuracy of BRAF detection.

Highlights

Malignant melanoma (MM) is the deadliest form of skin cancer; it is estimated to affect more than 100,000 individuals, causing approximately 7000 deaths in the United States (US) in 2020 [1]
Our study evidenced that real-time polymerase chain reaction (rtPCR) and next-generation sequencing (NGS) were able to detect additional BRAF mutant cases in comparison with conventional sequencing methods; we argue for the preferential utilization of the aforementioned assays (NGS and rtPCR) in clinical practice, to eradicate false-negative cases and improve the accuracy of BRAF detection
The discovery and description of the crystal structure of the mutated BRAF protein [10] led to the development of several specific inhibitors, such as vemurafenib, dabrafenib, and encorafenib, which have been approved both in the US and Europe for the treatment of advanced (AJCC stage IIIC and stage IV) MM-harboring BRAF V600 mutations [11,12,13,14]

Summary

Introduction

Malignant melanoma (MM) is the deadliest form of skin cancer; it is estimated to affect more than 100,000 individuals, causing approximately 7000 deaths in the United States (US) in 2020 [1]. In the last few years, new therapeutic options have revolutionized the treatments of patients with III/IV American Joint Committee on Cancer (AJCC) stage melanomas They include therapies targeted to specific genetic tumor mutations, as well as immunotherapy with immune checkpoint inhibitors (ICIs) [5,6]. The discovery and description of the crystal structure of the mutated BRAF protein [10] led to the development of several specific inhibitors, such as vemurafenib, dabrafenib, and encorafenib, which have been approved both in the US and Europe for the treatment of advanced (AJCC stage IIIC and stage IV) MM-harboring BRAF V600 mutations [11,12,13,14] These BRAF inhibitors, administered in combination with MEK inhibitors, resulted in rapid therapeutic responses, with significant improvements in both progression-free and overall survival in large fractions of metastatic MM patients [2,15]

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