Quality assessment of a clinical next-generation sequencing melanoma panel within the Italian Melanoma Intergroup (IMI)

Antonio Cossu,Ulrich Pfeffer,Maria Colombino,Enrica Teresa Tanda,Virginia Andreotti,Antonella Manca,Lorenza Pastorino,Giuseppe Palmieri,Milena Casula,Irene Vanni,Carla Rozzo,Paola Ghiorzo,Marina Pisano,Panagiotis Paliogiannis,Bruna Dalmasso

doi:10.1186/s13000-020-01052-5

Abstract

BackgroundIdentification of somatic mutations in key oncogenes in melanoma is important to lead the effective and efficient use of personalized anticancer treatment. Conventional methods focus on few genes per run and, therefore, are unable to screen for multiple genes simultaneously. The use of Next-Generation Sequencing (NGS) technologies enables sequencing of multiple cancer-driving genes in a single assay, with reduced costs and DNA quantity needed and increased mutation detection sensitivity.MethodsWe designed a customized IMI somatic gene panel for targeted sequencing of actionable melanoma mutations; this panel was tested on three different NGS platforms using 11 metastatic melanoma tissue samples in blinded manner between two EMQN quality certificated laboratory.ResultsThe detection limit of our assay was set-up to a Variant Allele Frequency (VAF) of 10% with a coverage of at least 200x. All somatic variants detected by all NGS platforms with a VAF ≥ 10%, were also validated by an independent method. The IMI panel achieved a very good concordance among the three NGS platforms.ConclusionThis study demonstrated that, using the main sequencing platforms currently available in the diagnostic setting, the IMI panel can be adopted among different centers providing comparable results.

Highlights

Malignant melanoma is one of the most aggressive, drug-resistant human cancers, and its incidence has risen persistently during the last few decades, in the Caucasian population [1]
The Next-Generation Sequencing (NGS) analysis was performed using a specific multiple-gene panel constructed by the Italian Melanoma Intergroup, the IMI Somatic Panel, arranged in three primer pools, and designed using the Ion AmpliSeq Designer to explore the mutational status of selected regions (343 amplicons; amplicon range: 125–175 bp; coverage 100%) within the 25 genes reported as the most frequently mutated in melanomas by The Cancer Genome Atlas (TGCA) and successive NGS-based studies [5, 14]
We have developed a custom panel to screen hotspots in 25 genes for clinically relevant mutations in melanoma based on the available literature at the time of panel design, including information retrieved from The cancer genome atlas (TGCA) and available literature data on melanoma

Summary

Introduction

Malignant melanoma is one of the most aggressive, drug-resistant human cancers, and its incidence has risen persistently during the last few decades, in the Caucasian population [1]. Of great interest are the RAS-RAFMEK-ERK, PI3K/PTEN and c-Kit pathways, since patients harboring activating mutations in BRAF, NRAS and KIT genes could benefit of target treatment options or tailored combinations of target- and immuno-therapies. The identification of variants predictive of response or resistance to systemic treatments is already recommended today for proper management of advanced melanoma and molecular testing is a priority in determining the course of therapy. In case of a BRAF-wild type tumor, NRAS and c-KIT (mucosal and acrolentigenous primaries) testing should be performed Identification of somatic mutations in key oncogenes in melanoma is important to lead the effective and efficient use of personalized anticancer treatment. The use of Next-Generation Sequencing (NGS) technologies enables sequencing of multiple cancer-driving genes in a single assay, with reduced costs and DNA quantity needed and increased mutation detection sensitivity

Methods

Results

Discussion

Conclusion