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. 2023 Feb 21;24(5):4302.
doi: 10.3390/ijms24054302.

Whole-Exome Sequencing and cfDNA Analysis Uncover Genetic Determinants of Melanoma Therapy Response in a Real-World Setting

Irene Vanni  1 , Lorenza Pastorino  1   2 , Enrica Teresa Tanda  2   3 , Virginia Andreotti  1 , Bruna Dalmasso  1 , Nicola Solari  4 , Matteo Mascherini  5 , Francesco Cabiddu  6 , Antonio Guadagno  6 , Simona Coco  7 , Eleonora Allavena  2 , William Bruno  1   2 , Gabriella Pietra  8   9 , Michela Croce  10 , Rosaria Gangemi  10 , Michele Piana  11   12 , Gabriele Zoppoli  2   13 , Lorenzo Ferrando  2   13 , Francesco Spagnolo  3   14 , Paola Queirolo  15 , Paola Ghiorzo  1   2
Affiliations

Whole-Exome Sequencing and cfDNA Analysis Uncover Genetic Determinants of Melanoma Therapy Response in a Real-World Setting

Irene Vanni et al. Int J Mol Sci. .

Abstract

Although several studies have explored the molecular landscape of metastatic melanoma, the genetic determinants of therapy resistance are still largely unknown. Here, we aimed to determine the contribution of whole-exome sequencing and circulating free DNA (cfDNA) analysis in predicting response to therapy in a consecutive real-world cohort of 36 patients, undergoing fresh tissue biopsy and followed during treatment. Although the underpowered sample size limited statistical analysis, samples from non-responders had higher copy number variations and mutations in melanoma driver genes compared to responders in the BRAF V600+ subset. In the BRAF V600- subset, Tumor Mutational Burden (TMB) was twice that in responders vs. non-responders. Genomic layout revealed commonly known and novel potential intrinsic/acquired resistance driver gene variants. Among these, RAC1, FBXW7, GNAQ mutations, and BRAF/PTEN amplification/deletion were present in 42% and 67% of patients, respectively. Both Loss of Heterozygosity (LOH) load and tumor ploidy were inversely associated with TMB. In immunotherapy-treated patients, samples from responders showed higher TMB and lower LOH and were more frequently diploid compared to non-responders. Secondary germline testing and cfDNA analysis proved their efficacy in finding germline predisposing variants carriers (8.3%) and following dynamic changes during treatment as a surrogate of tissue biopsy, respectively.

Keywords: BRAF V600; circulating free DNA; germline pathogenic variants; immunotherapy; loss of heterozygosity; melanoma; targeted therapy; tumor mutational burden; tumor ploidy; whole-exome sequencing.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
BRAF V600 mutation dynamic changes in longitudinal samples from non-responding patients. The figure shows BRAF V600 levels in circulating tumor DNA assessed longitudinally in three non-responding patients (#60 (A), #8 (B), and #62 (C)) by next-generation sequencing and/or droplet digital PCR compared with whole-exome sequencing data in fresh tumor tissue. Abbreviations: AF: Allele Frequency; WES: Whole-Exome Sequencing; ddPCR: droplet digital PCR; cfDNA: circulating free DNA; TF: Fresh Tissue.
Figure 1
Figure 1
BRAF V600 mutation dynamic changes in longitudinal samples from non-responding patients. The figure shows BRAF V600 levels in circulating tumor DNA assessed longitudinally in three non-responding patients (#60 (A), #8 (B), and #62 (C)) by next-generation sequencing and/or droplet digital PCR compared with whole-exome sequencing data in fresh tumor tissue. Abbreviations: AF: Allele Frequency; WES: Whole-Exome Sequencing; ddPCR: droplet digital PCR; cfDNA: circulating free DNA; TF: Fresh Tissue.
Figure 2
Figure 2
Concordance analysis between circulating free DNA and tumor tissue. The figure shows the concordance between cfDNA (yellow circle) and tumor tissue (blue circle) when considering only BRAF p.Val600Glu and KIT p.Lys642Glu hotspot mutations (A), all hotspot SNVs/indels (B), all hotspot CNVs (C). Abbreviations: SNVs: Single Nucleotide Variants; indels: insertions/deletions; CNVs: Copy Number Variations.
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