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

Abstract

Background: Identification 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.

Methods: We 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.

Results: The 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.

Conclusion: This 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.

Keywords: BRAF; Gene panel testing; Melanoma; Next generation sequencing (NGS); Quality controls; Somatic mutations; Target therapy.

Fig. 1

PGM™ platform low-covered regions. The figure shows for amplicons with a coverage lower than 200x in at least two tumor samples. The histograms report on the x axis the amplicons name not covered 200x in at least two sample for all case and in y axis the amplicon coverage

Fig. 2

Proton™ platform low-covered regions. The figure shows amplicons with a coverage lower than 200x in at least two tumor samples. The histograms report on the x axis the amplicons name not covered 200x in at least two sample for all case and in y axis the amplicon coverage

Fig. 3

MiSeq™ Illumina platform low-covered regions. The figure shows amplicons with a coverage lower than 200x in at least two tumor samples. The histograms report on the x axis the amplicons name not covered 200x in at least two sample for all case and in y axis the amplicon coverage

Fig. 4

Venn Diagram of 126 exonic genetic variants called using the three different NGS platforms regardless of coverage and allele frequency (a) and of 37 exonic genetic variants called using the three different NGS platforms with an VAF > 10% (b)