Molecular and genetic diversity in the metastatic process of melanoma

Abstract

Diversity between metastatic melanoma tumours in individual patients is known; however, the molecular and genetic differences remain unclear. To examine the molecular and genetic differences between metastatic tumours, we performed gene-expression profiling of 63 melanoma tumours obtained from 28 patients (two or three tumours/patient), followed by analysis of their mutational landscape, using targeted deep sequencing of 1697 cancer genes and DNA copy number analysis. Gene-expression signatures revealed discordant phenotypes between tumour lesions within a patient in 50% of the cases. In 18 of 22 patients (where matched normal tissue was available), we found that the multiple lesions within a patient were genetically divergent, with one or more melanoma tumours harbouring 'private' somatic mutations. In one case, the distant subcutaneous metastasis of one patient occurring 3 months after an earlier regional lymph node metastasis had acquired 37 new coding sequence mutations, including mutations in PTEN and CDH1. However, BRAF and NRAS mutations, when present in the first metastasis, were always preserved in subsequent metastases. The patterns of nucleotide substitutions found in this study indicate an influence of UV radiation but possibly also DNA alkylating agents. Our results clearly demonstrate that metastatic melanoma is a molecularly highly heterogeneous disease that continues to progress throughout its clinical course. The private aberrations observed on a background of shared aberrations within a patient provide evidence of continued evolution of individual tumours following divergence from a common parental clone, and might have implications for personalized medicine strategies in melanoma treatment.

Keywords: BRAF; NRAS; deep sequencing; gene expression; melanoma.

Figure 1

Study design depicting the molecular and genetic analysis of melanoma metastatic tumours. (A) Flow chart of the main methods used in the study. (B) Bar plot showing numbers of somatic mutations in multiple metastases from 22 patients. Each melanoma tumour was classified into the four gene expression phenotypes: pigmentation, green; proliferative, red; high-immune, orange; or normal-like, blue, and coloured accordingly. (C) Bar plot showing numbers of private copy number aberrations (CNAs) and mutations per patient. Private mutations within each patient were defined as being present in only one of the analysed tumours in the patient

Figure 2

The mutational landscape of metastatic melanoma tumours: number of mutations (including shared and private somatic mutations) in the tumours of 22 patients depicted as Venn diagrams. The circle colours represent gene expression phenotypes: pigmentation, green; proliferative, red; high-immune, orange; or normal-like, blue. M1, metastatic lesion 1; M2, metastatic lesion 2; M3, metastatic lesion 3

Figure 3

Molecular heterogeneity in patient 1. (A) Expression heat map of the subtype-specific genes in the melanoma metastases: red, over-expressed genes; green, down-regulated genes. (B) Microphthalmia-associated transcription factor (MITF) immunohistochemistry of metastatic tumour sections from patient 1. Cell nuclei are shown in blue. M1, metastatic lesion 1; M2, metastatic lesion 2; M3, metastatic lesion 3

Figure 4

Molecular and genetic heterogeneity in patient 2. (A) Expression heat map of subtype-specific genes in the melanoma metastases. (B) Microphthalmia-associated transcription factor (MITF) immunohistochemistry in both metastases. (C) Mutated genes (blue) identified in metastases (upper panel). Genome-wide copy number profiles of M1 (black) and M2 (green) are superimposed onto each other and illustrate differences between the two metastases (lower panel)

Figure 5

Genetic heterogeneity in patient 1. (A) Mutated genes (upper panel, blue) and corresponding copy number (lower panel) in the three metastases from patient 1. Copy number: white = 2, green = 1, orange = 3, red = 4 inferred from GAP results. (B) Progression model: (upper panel) Somatic mutations (exonic, splice-site and intronic) plotted by log₂ copy number (x axis) versus variant allele frequency (y axis); colours are indicated in the legend; (lower panel) the common precursor clone (dotted circle on the far left) contained 63 exonic or splice site mutations and a rearrangement involving chromosomes 7 and 15, all found in all three metastases. M1 harboured two private mutations and another genomic rearrangement, between chromosomes 3 and 4. Hypothetical common predecessor of M2 and M3 (smaller dotted circle) developed from the precursor clone and contained two additional mutations. Finally, M2 acquired eight additional private exonic mutations, and M3 37 additional private mutations. Red dotted line represents time, with diagnoses and treatments indicated; LN, lymph node; SC, subcutaneous; met, metastasis; DTIC, dacarbazine. (C) Sequence context of shared (all three metastases, left) and M3 private (right) mutations demonstrates clear pattern differences