Involvement of mutant and wild-type CYSLTR2 in the development and progression of uveal nevi and melanoma

Abstract

Background Activating Gαq signalling mutations are considered an early event in the development of uveal melanoma. Whereas most tumours harbour a mutation in GNAQ or GNA11, CYSLTR2 (encoding G-protein coupled receptor CysLT2R) forms a rare alternative. The role of wild-type CysLT2R in uveal melanoma remains unknown. Methods We performed a digital PCR-based molecular analysis of benign choroidal nevi and primary uveal melanomas. Publicly available bulk and single cell sequencing data were mined to further study wild-type and mutant CYSLTR2. Results 1/16 nevi and 2/120 melanomas carried the CYSLTR2 mutation. The mutation was subclonal in the nevus, while being clonal in both melanomas. In the melanomas, secondary, subclonal CYSLTR2 alterations shifted the allelic balance towards the mutant. The resulting genetic heterogeneity was confirmed in distinct areas of both tumours. At the RNA level, further silencing of wild-type and preferential expression of mutant CYSLTR2 was identified, which was also observed in 2/3 CYSLTR2 mutant melanomas from the TCGA cohort. In CYSLTR2 wild-type melanomas, high expression of CYSLTR2 originated from melanoma cells and correlated to tumour inflammation. Conclusions Our findings suggest that CYSLTR2 is involved in both early and late development of uveal melanoma. Whereas the CYSLTR2 p.L129Q mutation is likely to be the initiating oncogenic event, various mechanisms further increase the mutant allele abundance during tumour progression. This makes mutant CysLT2R an attractive therapeutic target in uveal melanoma. In GNAQ, GNA11 and PLCB4 mutant melanomas, expression of wild-type CYSLTR2 possibly facilitates an interaction with immune cells in the microenvironment and may also have therapeutic potential.

Estimation of the timing of BAP1 mutation in uveal melanoma progression

“…UM can occur de novo or through malignant transformation of a nevus. Studies have shown that choroidal nevi, similar to UM, can harbor G-protein coupled receptor (GPCR) mutations in GNA11 , GNAQ , and CYSLTR2 24 , 25 . Our computational analyses assume that the initial cell is a malignant cell capable of losing the BAP1 gene, whether it formed de novo or growing in a nevus.…”

Identification of clinically-relevant genetic alterations in uveal melanoma using RNA sequencing

“…Digital PCR was performed to measure the copy number values of chromosome 3 and 8q and to confirm mutations in GNAQ, GNA11, EIF1AX and SF3B1. These experiments were carried out using the QX200 Droplet Digital PCR System (Bio-Rad Laboratories, Hercules, USA) following established protocols [22,27,[29][30][31], and two new assays listed in Supplementary Table 1. The presence and pathogenicity of BAP1 alterations was confirmed by targeted Sanger or nextgeneration sequencing and an immunohistochemical staining, both performed as routine diagnostic analyses in two ISO accredited laboratories (Departments of Clinical Genetics and Pathology, LUMC) [16,32].…”

“…As another example, in three largescale genomic studies, a variety of complex BAP1 alterations were only identified after using RNA sequencing data in addition to DNA-derived data [21,25,26]. Finally, CYSLTR2 mutant uveal melanomas recurrently showed silencing of the wild-type allele and preferential expression of the p.L129Q mutation [27]. These examples emphasise the value of investigating RNA in addition to DNA to identify the presence and consequences of certain genetic alterations.…”