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 mutant and wild-type CYSLTR2 in primary and metastatic uveal melanoma. Results 1/16 nevi and 2/120 melanomas carried the CYSLTR2 mutation. The mutation was found in a subpopulation of 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 two CYSLTR2 mutant primary melanomas and one metastatic lesion from other cohorts. In CYSLTR2 wild-type melanomas, high expression of CYSLTR2 correlated to tumour inflammation, but expression originated from melanoma cells specifically. 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.

Scientific and clinical implications of genetic and cellular heterogeneity in uveal melanoma

“…As opposed to next generation sequencing, absolute quantification with digital PCR allows to detect small differences in clone fractions and is therefore able to differentiate between early events [26].…”

“…The important role of Gαq signalling in the development of uveal melanoma has been further supported by the identification of recurrent mutations in PLCB4 (p.D630, encoding phospholipase C β4) and CYSLTR2 (p.L129Q, encodinga Gαq-coupled receptor) [ 20 , 21 ]. These mutations are typically found in GNAQ and GNA11 wild-type tumours and provide an alternative way to activate the Gαq signalling pathway [ 20 – 26 ] (Fig. 3 ).…”

“…Still, each melanoma originates from a unique somatic genetic event, and this is most dramatically illustrated by patients with multiple choroidal nevi. Though they all present with a Gαq signalling mutation, the studies by Vader et al and Nell et al showed that by chance each nevus carries another specific variant [ 19 , 26 ]. After such driver mutation, additional genetic events are necessary to progress and with each additional event a possible heterogeneity emerges.…”

“…Since driver mutations (usually GNAQ/11 ) are present in all tumour cells of most UM, this is considered to be the primary event. As opposed to next generation sequencing, absolute quantification with digital PCR allows to detect small differences in clone fractions and is therefore able to differentiate between early events [ 26 ].…”

In Uveal Melanoma, Angiopoietin-2 but Not Angiopoietin-1 Is Increased in High-Risk Tumors, Providing a Potential Druggable Target

“…Moreover, the ANG-2-expressing macrophages and T cells were equally distributed among the immune infiltrate in the tumor. Furthermore, we tried to determine which cell type expressed ANG-2 by looking at single cell RNA data from eight primary UM (GSE139829) [43,44]. However, ANG-2 was expressed in a very low fraction (0-3%) of the cells per tumor, which was too marginal to draw any definite conclusions regarding the origin of ANG-2.…”