Quantifying T cells accurately in a variety of tissues of benign, inflammatory, or malignant origin can be of great importance in a variety of clinical applications. Flow cytometry and immunohistochemistry are considered to be gold-standard methods for T-cell quantification. However, these methods require fresh, frozen, or fixated cells and tissue of a certain quality. In addition, conventional and droplet digital PCR (ddPCR), whether followed by deep sequencing techniques, have been used to elucidate T-cell content by focusing on rearranged T-cell receptor (TCR) genes. These approaches typically target the whole TCR repertoire, thereby supplying additional information about TCR use. We alternatively developed and validated two novel generic single duplex ddPCR assays to quantify T cells accurately by measuring loss of specific germline TCR loci and compared them with flow cytometry-based quantification. These assays target sequences between the Dδ2 and Dδ3 genes (TRD locus) and Dβ1 and Jβ1.1 genes (TRB locus) that become deleted systematically early during lymphoid differentiation. Because these ddPCR assays require small amounts of DNA instead of freshly isolated, frozen, or fixated material, initially unanalyzable (scarce) specimens can be assayed from now on, supplying valuable information about T-cell content. Our ddPCR method provides a novel and sensitive way for quantifying T cells relatively fast, accurate, and independent of the cellular context.

: Uveal melanoma progression can be predicted by gene expression profiles enabling a clear subdivision between tumors with a good (class I) and a poor (class II) prognosis. Poor prognosis uveal melanoma can be subdivided by expression of immune-related genes; however, it is unclear whether this subclassification is justified; therefore, T cells in uveal melanoma specimens were quantified using a digital PCR approach. Absolute T-cell quantification revealed that T-cell influx is present in all uveal melanomas associated with a poor prognosis. However, this infiltrate is only accompanied by differential immune-related gene expression profiles in uveal melanoma with the highest T-cell infiltrate. Molecular deconvolution of the immune profile revealed that a large proportion of the T-cell-related gene expression signature does not originate from lymphocytes but is derived from other immune cells, especially macrophages. Expression of the lymphocyte-homing chemokine CXCL10 by activated macrophages correlated with T-cell infiltration and thereby explains the correlation of T-cell numbers and macrophages. This was validated by analysis of CXCL10 in uveal melanoma tissue with high T-cell counts. Surprisingly, CXCL10 or any of the other genes in the activated macrophage-cluster was correlated with reduced survival due to uveal melanoma metastasis. This effect was independent of the T-cell infiltrate, which reveals a role for activated macrophages in metastasis formation independent of their role in tumor inflammation. IMPLICATIONS: The current report uses an innovative digital PCR method to study the immune environment and demonstrates that absolute T-cell quantification and expression profiles can dissect disparate immune components.

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.

The telomerase reverse transcriptase (TERT) gene is responsible for telomere maintenance in germline and stem cells, and is re-expressed in 90% of human cancers. CpG methylation in the TERT promoter (TERTp) was correlated with TERT mRNA expression. Furthermore, two hotspot mutations in TERTp, dubbed C228T and C250T, have been revealed to facilitate binding of transcription factor ETS/TCF and subsequent TERT expression. This study aimed to elucidate the combined contribution of epigenetic (promoter methylation and chromatin accessibility) and genetic (promoter mutations) mechanisms in regulating TERT gene expression in healthy skin samples and in melanoma cell lines (n = 61). We unexpectedly observed that the methylation of TERTp was as high in a subset of healthy skin cells, mainly keratinocytes, as in cutaneous melanoma cell lines. In spite of the high promoter methylation fraction in wild-type (WT) samples, TERT mRNA was only expressed in the melanoma cell lines with either high methylation or intermediate methylation in combination with TERT mutations. TERTp methylation was positively correlated with chromatin accessibility and TERT mRNA expression in 8 melanoma cell lines. Cooperation between epigenetic and genetic mechanisms were best observed in heterozygous mutant cell lines as chromosome accessibility preferentially concerned the mutant allele. Combined, these results suggest a complex model in which TERT expression requires either a widely open chromatin state in TERTp-WT samples due to high methylation throughout the promoter or a combination of moderate methylation fraction/chromatin accessibility in the presence of the C228T or C250T mutations.

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