Background: In our previous experience (JCO 2012;30:2522-9), we demonstrated a high consistency of BRAF and NRAS mutations between primary tumors and lymph node metastases in melanoma patients. A subset of paired samples of multiple asynchronous lymph node metastases from that series was here screened mainly using NGS technologies in order to better elucidate the existence of a real genetic homogeneity during clonal expansion to lymph node sites. Methods: Genomic DNA was isolated from frozen tissues of paired asynchronous lymph nodes, ascertained for presence of melanoma metastases (at least 80% of metastatic cells), using standard methods. Specimens from 12 patients were analyzed for mutations in 50 most common cancer genes with the Ion Torrent AmpliSeq Cancer Panel HotSpot.V2 (CHPv2) on the Ion PGMsequencer. All variants detected by NGS were confirmed through PCR-based Sanger sequencing. Paraffinembedded tumor tissues from the same series were evaluated by fluorescence in situ hybridization (FISH) assays, using probes specific for CyclinD1/EGFR genes or 9p21 locus and correspondent control centromeres. Results: Overall, only one (8%) of the 12 analyzed patients presented discrepancies in mutation patterns between the two distinct lymph nodal metastases developed in different times. In particular, the differences were represented by the BRAF-V600E and the CDKN2A-R80X mutations, suggesting that changes, when occur (though in a limited fraction of cases), they affect the main genes controlling cell proliferation and survival involved in melanomagenesis. No discrepancy was observed by FISH analysis into the same series. Considering the total number of analyzed samples, we detected the following mutations: BRAF-V600E (54% of cases), KDR-Q472H (50%), TP53-P72R (42%), cKIT-M541L (25%), NRAS-Q61R/K (25%). Conclusions: Our findings indicated a very low genetic heterogeneity during lymph node dissemination in melanoma patients, confirming the previously published data. Mutations in BRAF, NRAS, and cKIT genes were further confirmed to play a predominant role in melanoma pathogenesis.
Evaluation of genetic heterogeneity in paired lymph node metastases from melanoma patients using next-generation sequencing (NGS) approaches
Palmieri Giuseppe;Sini MariaCristina;Manca Antonella;Colombino Maria;Casula Milena
2015
Abstract
Background: In our previous experience (JCO 2012;30:2522-9), we demonstrated a high consistency of BRAF and NRAS mutations between primary tumors and lymph node metastases in melanoma patients. A subset of paired samples of multiple asynchronous lymph node metastases from that series was here screened mainly using NGS technologies in order to better elucidate the existence of a real genetic homogeneity during clonal expansion to lymph node sites. Methods: Genomic DNA was isolated from frozen tissues of paired asynchronous lymph nodes, ascertained for presence of melanoma metastases (at least 80% of metastatic cells), using standard methods. Specimens from 12 patients were analyzed for mutations in 50 most common cancer genes with the Ion Torrent AmpliSeq Cancer Panel HotSpot.V2 (CHPv2) on the Ion PGMsequencer. All variants detected by NGS were confirmed through PCR-based Sanger sequencing. Paraffinembedded tumor tissues from the same series were evaluated by fluorescence in situ hybridization (FISH) assays, using probes specific for CyclinD1/EGFR genes or 9p21 locus and correspondent control centromeres. Results: Overall, only one (8%) of the 12 analyzed patients presented discrepancies in mutation patterns between the two distinct lymph nodal metastases developed in different times. In particular, the differences were represented by the BRAF-V600E and the CDKN2A-R80X mutations, suggesting that changes, when occur (though in a limited fraction of cases), they affect the main genes controlling cell proliferation and survival involved in melanomagenesis. No discrepancy was observed by FISH analysis into the same series. Considering the total number of analyzed samples, we detected the following mutations: BRAF-V600E (54% of cases), KDR-Q472H (50%), TP53-P72R (42%), cKIT-M541L (25%), NRAS-Q61R/K (25%). Conclusions: Our findings indicated a very low genetic heterogeneity during lymph node dissemination in melanoma patients, confirming the previously published data. Mutations in BRAF, NRAS, and cKIT genes were further confirmed to play a predominant role in melanoma pathogenesis.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
