Implementation of an NGS panel for clinical practice in paraffin-embedded tissue samples from locally advanced and metastatic melanoma patients

Paola Castillo; Marta Marginet; Pedro Jares; Mireia García; Elena Gonzalvo; Ana Arance; Adriana García; Llucia Alos; Cristina Teixido

doi:10.37349/etat.2020.00006

Implementation of an NGS panel for clinical practice in paraffin-embedded tissue samples from locally advanced and metastatic melanoma patients

Explor Target Antitumor Ther. 2020;1(2):101-108. doi: 10.37349/etat.2020.00006. Epub 2020 Apr 28.

Authors

Paola Castillo¹, Marta Marginet¹, Pedro Jares¹, Mireia García¹, Elena Gonzalvo¹, Ana Arance², Adriana García¹, Llucia Alos¹, Cristina Teixido¹

Affiliations

¹ Department of Pathology, Hospital Clinic, IDIBAPS, University of Barcelona, 08036 Barcelona, Spain.
² Department of Medical Oncology, Hospital Clinic, IDIBAPS, University of Barcelona, 08036 Barcelona, Spain.

Abstract

Aim: Single biomarker diagnostic test of BRAFV600 locus in metastatic melanoma is mandatory for treatment decision; however, multiple-gene based techniques, such as targeted next-generation sequencing (NGS) are being used to maximize the number of patients that can benefit from a targeted therapy. The main objective of this study is to investigate whether an NGS panel could be adopted in routine clinical care for advanced melanoma.

Methods: Patients diagnosed with advanced melanoma at our center from 2017 to 2019 were included. Presence of genetic alterations was performed using two methods: real-time polymerase chain reaction-based Idylla test (Biocartis) and NGS with the oncomine solid tumor DNA kit (Thermo Fisher Scientific). Total genomic DNA was extracted from formalin-fixed and paraffin embedded samples for sequencing.

Results: A total of 155 samples were evaluated for molecular analysis but 40 samples (25.8%) were inadequate for sequencing. The clinical utility of BRAFV600 real-time polymerase chain reaction and targeted-NGS was compared in 29 samples and a very good concordance was observed (Kappa = 0.89, 95% confidence interval 0.68 ± 1.05). An oncogenic mutation by NGS was found in 75 samples (65%)-53% of whom were candidates for personalized therapies. The most prevalent mutated genes were BRAF (39%), TP53 (23%), and NRAS (14%). Other genes identified at lower incidence (< 5%) were: PIK3CA, ERBB4, CTNNB1, STK11, FGFR1, SMAD4, KRAS, FGFR3, PTEN and AKT. Co-occurrence of oncogenic mutations was detected in 40% of the samples. Among the mutations identified, TP53 was significantly more prevalent in men (men 31.8% versus women 12.2%, P = 0.03) and NRAS in women (men 9.1% versus women 24.4%, P = 0.03).

Conclusions: Targeted-NGS testing is a feasible technique to implement in the routine clinical practice. Based on our results, NGS has provided more information on target-genes than RT-PCR technique, maximizing the benefit for patients with advanced melanoma.

Keywords: BRAF; Idylla; Melanoma; next-generation sequencing; real-time polymerase chain reaction.