NGS Analysis of Liquid Biopsy (LB) and Formalin-Fixed Paraffin-Embedded (FFPE) Melanoma Samples Using Oncomine™ Pan-Cancer Cell-Free Assay

Abstract

Next-generation sequencing (NGS) in liquid biopsies may contribute to the diagnosis, monitoring, and personalized therapy of cancer through the real-time detection of a tumor’s genetic profile. There are a few NGS platforms offering high-sensitivity sequencing of cell-free DNA (cfDNA) samples. The aim of this study was to evaluate the Ion AmpliSeq HD Technology for targeted sequencing of tumor and liquid biopsy samples from patients with fourth-stage melanoma. Sequencing of 30 samples (FFPE tumor and liquid biopsy) derived from 14 patients using the Oncomine™ Pan-Cancer Cell-Free Assay was performed. The analysis revealed high concordance between the qPCR and NGS results of the BRAF mutation in FFPE samples (91%), as well as between the FFPE and liquid biopsy samples (91%). The plasma-tumor concordance of the non-BRAF mutations was 28%. A total of 17 pathogenic variants in 14 genes (from 52-gene panel), including TP53, CTNNB1, CCND1, MET, MAP2K1, and GNAS, were identified, with the CTNNB1S45F variant being the most frequent. A positive correlation between the LDH level and cfDNA concentration as well as negative correlation between the LDH level and time to progression was confirmed in a 22-patient cohort. The analysis showed both the potential and limitations of liquid biopsy genetic profiling using HD technology and the Ion Torrent platform.

Angiogenesis Still Plays a Crucial Role in Human Melanoma Progression

“…In recent years, Next-Generation Sequencing (NGS) has allowed us to make progress in clinical applied research, particularly in the context of melanoma, with particular molecular techniques that have contributed strongly to clarifying and discovering new important characteristics of the TME [115,116]. Among these, single-cell RNA sequencing has allowed us to characterize in detail not only the different cell populations present in the TME but also their functional activation/deactivation status, allowing us to examine the dynamism of the TME in greater detail [117].…”

“…For example, it is now clear that in TME (beyond T and B cells with some activation states such as dysfunctional, exhausted, activated), M1 and M2 macrophages, N1 and N2 neutrophils, M1 and M2 myeloid-derived suppressor cells (MDSC), and dendritic cells are also present. While alternatively activated myeloid cells are anti-inflammatory and promote tumor growth, classically activated neutrophils, macrophages, and MDSCs exhibit pro-inflammatory and anti-tumor properties [110][111][112][113][114][115][116][117][118][119][120][121][122]. Finally, CAFs undergo phenotypic switching, further adding to the complexity of ITH [123].…”

Current and Future Perspectives of Cell-Free DNA in Liquid Biopsy

“…In addition, Olbryt et al performed the sequencing of formalin-fixed paraffin-embedded (FFPE) tumor and cfDNA samples derived from melanoma patients. The analysis revealed high concordance between the real-time quantitative PCR (qPCR) and NGS results of the BRAF (B-Raf proto-oncogene, serine/threonine kinase) mutation in FFPE samples (91%), as well as between the FFPE and cfDNA samples (91%) [ 47 ]. It is not only mutations of cfDNA that can be applied to cancer diagnosis, as fragment size of cfDNA [ 48 , 49 ], DNA methylation [ 24 , 50 , 51 , 52 , 53 ], and end coordinate [ 54 , 55 ] can also be used for the diagnosis of cancer.…”