Comparison of multiple tumor lesions from the same melanoma patients using next-generation sequencing (NGS) approaches

Background: Our group previously demonstrated a high consistency of BRAF and NRAS mutations between primary tumors and lymph node metastases in melanoma patients (JCO 2012; 30: 2522-9). To further verify the level of homogeneity during clonal expansion to lymph node sites, we here performed a more detailed analysis on a subset of those paired samples using a NGS technology. Methods: Genomic DNA was isolated from tumor tissues of 12 melanoma patients, using standard methods. Specimens were analyzed for mutations in 50 most common cancer genes with the Ion Torrent AmpliSeq Cancer Panel HotSpot V2 (CHPv2) on the Ion Torrent platform. All variants detected by NGS were confirmed through PCR-based Sanger sequencing. Results: Genomic DNA was isolated from paired metastatic lymph nodes either asynchronously (n = 8) or synchronously (n = 4) excised from melanoma patients with clinically-evident nodal involvement. Data from the runs on the Ion Ampliseq Cancer Hotspot panel indicated an average of about 1500 reads (range, 500-2000) per amplicon. Overall, one (8%) out of the 12 analyzed patients presented discrepancies in mutation patterns during disease progression to lymph nodes and between the distinct lymph nodal metastases. In particular, the differences were represented by BRAF and CDKN2A mutations, suggesting that changes - when occur (though in a very limited fraction of cases) - may affect the main genes controlling cell proliferation and survival in melanoma. Mutations in BRAF, NRAS, CDKN2A, and TP53 genes were confirmed to play a predominant role in melanoma pathogenesis. Conclusions: Even with a more comprehensive mutation analysis, our findings indicated a very low genetic heterogeneity in metastatic lymph nodes during disease progression in melanoma patients, confirming previously published data on BRAF and NRAS mutational status only.