A primary melanoma and its asynchronous metastasis highlight the role of BRAF, CDKN2A, and TERT

Abstract

Background: Alterations in pathways including BRAF, CDKN2A, and TERT contribute to the development of melanoma, but the sequence in which the genetic alterations occur and their prognostic significance remains unclear. To clarify the role of these pathways, we analyzed a primary melanoma and its metastasis.

Methods: Immunohistochemistry for BRAF-V600E, Sanger sequencing of BRAF and the TERT promoter, fluorescence in-situ hybridization, and telomere analyses were performed on a primary melanoma and its asynchronous cerebellar metastasis. Using the log-rank test and Cox-proportional model, the cancer genome atlas (TCGA) cohort of melanomas was analyzed for the effect of BRAF mutation and CDKN2A loss on survival.

Results: The primary melanoma expressed mutant BRAF-V600E and possessed a homozygous deletion of CDKN2A. In addition to these early defects, the metastatic lesion also possessed evidence of aneuploidy and an activating mutation of the TERT promoter. In the TCGA melanoma cohort, there was a non-significant trend toward poor prognosis in early stage cutaneous melanoma patients with concomitant BRAF mutation and CDKN2A loss.

Conclusion: BRAF mutation and CDKN2A loss occurred early and TERT promoter mutation later in a case of lethal metastatic melanoma. The effects of these pathways on survival warrant further investigation in early stage cutaneous melanoma patients.

Keywords: BRAF; CDKN2A; FISH; TERT; melanoma.

Figure 1

Histology of primary and metastatic melanoma. (A) The primary cutaneous lesion is an asymmetric cellular tumor filling the dermis with lack of maturation at the base of the lesion. (B) The primary lesion also shows epithelial hyperplasia and some nests comprised of large and epithelioid cells with abundant cytoplasm (arrowheads). Inset highlights a nest of epitheliod melanocytes. (C) The metastatic cerebellar lesion shows a densely cellular neoplasm with zones of necrosis and hemorrhage. (D) There is marked pleomorphism at higher magnification. Arrows indicates an aberrant mitotic figure. Scale bar=200µm.

Figure 2

BRAF immunohistochemistry. (A) Melanoma biopsy shows diffuse cytoplasmic staining by the BRAF-V600E specific antibody (VE1) supporting the presence of underlying BRAF-V600E mutation. Inset shows individual B-Raf-mutant melanocytes invading the dermis. (B) Cerebellar lesion shows strong BRAF-V600E reactivity by the infiltrating tumor cells. Scale bar=200µm.

Figure 3

FISH analysis of tumors. (A) Representative image of 4 probe analysis of primary melanoma shows homozygous loss of CDKN2A. (B) Table summarizing the scoring of signal number from 25 cells. Average (Ave) indicates the mean number of foci per nuclei. (C) FISH from metastatic melanoma shows homozygous loss of CDKN2A but increased signals from all remaining probes suggested genomic instability and possible aneuploidy. (D) Table summarizing the scoring of the brain lesion.

Figure 4

TERT promoter and telomere analysis. (A) Sanger sequencing of the TERT promoter sequencing of the primary melanoma reveals a wild type promoter. The metastatic cerebellar lesion carried a C228T mutation in the TERT promoter. (B) Representative nuclei stained for TIF foci using 53BP1 to identify sites of DNA damage (red) and TTAGGG FISH to identify telomeres. Yellow signals indicate co-localization. Dashed lines outline the cell nucleus. Scale bar=2µm. (C) Quantitation of the percentage of cells with ≥4 TIFs per nucleus. Greater than 100 cells were scored for each replicate.

Figure 5

Kaplan-Meier survival curve demonstrates that early stage (Stage I and II) cutaneous melanoma patients with BRAF mutation and CDKN2A loss do not have a significantly worse prognosis compared to those without CDKN2A loss. Wald test: p = 0.032.