Purpose: Germline pathogenic variants in the exonuclease domain (ED) of polymerases POLE and POLD1 predispose to adenomatous polyps, colorectal cancer (CRC), endometrial tumors, and other malignancies, and exhibit increased mutation rate and highly specific associated mutational signatures. The tumor spectrum and prevalence of POLE and POLD1 variants in hereditary cancer are evaluated in this study. Methods: POLE and POLD1 were sequenced in 2813 unrelated probands referred for genetic counseling (2309 hereditary cancer patients subjected to a multigene panel, and 504 patients selected based on phenotypic characteristics). Cosegregation and case-control studies, yeast-based functional assays, and tumor mutational analyses were performed for variant interpretation. Results: Twelve ED missense variants, 6 loss-of-function, and 23 outside-ED predicted-deleterious missense variants, all with population allele frequencies <1%, were identified. One ED variant (POLE p.Met294Arg) was classified as likely pathogenic, four as likely benign, and seven as variants of unknown significance. The most commonly associated tumor types were colorectal, endometrial and ovarian cancers. Loss-of-function and outside-ED variants are likely not pathogenic for this syndrome. Conclusions: Polymerase proofreading-associated syndrome constitutes 0.1-0.4% of familial cancer cases, reaching 0.3-0.7% when only CRC and polyposis are considered. ED variant interpretation is challenging and should include multiple pieces of evidence.

BackgroundOnly certain disseminated cells are able to grow in secondary organs to create a metastatic tumor. Under the hypothesis that the immune microenvironment of the host tissue may play an important role in this process, we have categorized metastatic samples based on their immune features.MethodsGene expression data of metastatic samples (n=374) from four secondary sites (brain, bone, liver and lung) were used to characterize samples based on their immune and stromal infiltration using gene signatures and cell quantification tools. A clustering analysis was done that separated metastatic samples into three different immune categories: high, medium and low.ResultsSignificant differences were found between the immune profiles of samples metastasizing in distinct organs. Metastases in lung showed a higher immunogenic score than metastases in brain, liver or bone, regardless of their primary site of origin. Also, they preferentially clustered in the high immune group. Samples in this cluster exhibited a clear inflammatory phenotype, higher levels of immune infiltrate, overexpression of programmed death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) pathways and upregulation of genes predicting clinical response to programmed cell death protein 1 (PD-1) blockade (T-cell inflammatory signature). A decision tree algorithm was used to select CD74 as a biomarker that identify samples belonging to this high-immune subtype of metastases, having specificity of 0.96 and sensitivity of 1.ConclusionsWe have found a group of lung-enriched metastases showing an inflammatory phenotype susceptible to be treated with immunotherapy.

Uveal melanoma is considered a rare disease but it is the most common intraocular malignancy in adults. Local treatments are effective, but the systemic recurrence rate is unacceptably high. Moreover, once metastasis have developed the prognosis is poor, with a 5-year survival rate of less than 5%, and systemic therapies, including immunotherapy, have rendered poor results. The tumour biology is complex, but angiogenesis is a highly important pathway in these tumours. Vasculogenic mimicry, the ability of melanomas to generate vascular channels independently of endothelial cells, could play an important role, but no effective therapy targeting this process has been developed so far. Angiogenesis modulates the tumour microenvironment of melanomas, and a close interplay is established between them. Therefore, combining immune strategies with drugs targeting angiogenesis offers a new therapeutic paradigm. In preclinical studies, these approaches effectively target these tumours, and a phase I clinical study has shown encouraging results in cutaneous melanomas. In this review, we will discuss the importance of angiogenesis in uveal melanoma, with a special focus on vasculogenic mimicry, and describe the interplay between angiogenesis and the tumour microenvironment. In addition, we will suggest future therapeutic approaches based on these observations and mention ways in which to potentially enhance current treatments.

Metastatic uveal melanoma (UM) is a rare, but often lethal, form of ocular cancer arising from melanocytes within the uveal tract. UM has a high propensity to spread hematogenously to the liver, with up to 50% of patients developing liver metastases. Unfortunately, once liver metastasis occurs, patient prognosis is extremely poor with as few as 8% of patients surviving beyond two years. There are no standard-of-care therapies available for the treatment of metastatic UM, hence it is a clinical area of urgent unmet need. Here, the clinical relevance and therapeutic potential of cysteinyl leukotriene receptors (CysLT1 and CysLT2) in UM was evaluated. High expression of CYSLTR1 or CYSLTR2 transcripts is significantly associated with poor disease-free survival and poor overall survival in UM patients. Digital pathology analysis identified that high expression of CysLT1 in primary UM is associated with reduced disease-specific survival (p = 0.012; HR 2.76; 95% CI 1.21–6.3) and overall survival (p = 0.011; HR 1.46; 95% CI 0.67–3.17). High CysLT1 expression shows a statistically significant (p = 0.041) correlation with ciliary body involvement, a poor prognostic indicator in UM. Small molecule drugs targeting CysLT1 were vastly superior at exerting anti-cancer phenotypes in UM cell lines and zebrafish xenografts than drugs targeting CysLT2. Quininib, a selective CysLT1 antagonist, significantly inhibits survival (p < 0.0001), long-term proliferation (p < 0.0001), and oxidative phosphorylation (p < 0.001), but not glycolysis, in primary and metastatic UM cell lines. Quininib exerts opposing effects on the secretion of inflammatory markers in primary versus metastatic UM cell lines. Quininib significantly downregulated IL-2 and IL-6 in Mel285 cells (p < 0.05) but significantly upregulated IL-10, IL-1β, IL-2 (p < 0.0001), IL-13, IL-8 (p < 0.001), IL-12p70 and IL-6 (p < 0.05) in OMM2.5 cells. Finally, quininib significantly inhibits tumour growth in orthotopic zebrafish xenograft models of UM. These preclinical data suggest that antagonism of CysLT1, but not CysLT2, may be of therapeutic interest in the treatment of UM.

Melanoma is a malignant neoplasia that is highly resistant to chemotherapy and radiotherapy and is associated with poor prognosis in advanced stage. Targeting melanoma that harbors the common BRAF V600E mutation with kinase inhibitors, such as vemurafenib, reduces tumor burden, but these tumors frequently acquire resistance to these drugs. We previously proposed that T-type calcium channel (TTCC) expression may serve as a biomarker for melanoma progression and prognosis, and we showed that TTCC blockers reduce migration and invasion rates because of autophagy blockade only in BRAF V600E -mutant melanoma cells. Here, we demonstrated that high expression of the TTCC Cav3.1 isoform is related to autophagic status in vemurafenib-resistant BRAF V600E -mutant melanoma cells and human biopsies, and in silico analysis revealed an enrichment of Cav3.1 expression in post-treatment melanomas. We also demonstrated that the TTCC blocker mibefradil induces apoptosis and impairs migration and invasion via inhibition of autophagy in resistant melanoma cells and mouse xenograft models. Moreover, we identified an association between PTEN status and Cav3.1 expression in these cells as a marker of sensitivity to combination therapy in resistant cells. Together, our results suggest that TTCC blockers offer a potential targeted therapy in resistant BRAF V600E -mutant melanoma and a therapeutic strategy to reduce progression toward BRAF inhibitor resistance.

Uveal melanoma (UM) is a malignant tumor that arises in the melanocytes of the uveal tract. It is the most frequent eye cancer, and despite new therapeutic approaches, prognosis is still poor, with up to 50% of patients developing metastasis with no efficient treatment options available. In contrast to cutaneous melanoma, UM is considered an “immune-cold” tumor due to the low mutational burden and the unique immunosuppressive microenvironment. To gain insight into the role of the UM microenvironment in regard to prognosis and metastatic progression, we have performed a pool analysis characterizing the UM microenvironment by using a bioinformatic approach. A variety of scores based on gene expression measuring stromal infiltration were calculated and used to assess association with prognosis. As a result, the highest immune and stromal scores were associated with poor prognosis. Specifically, stromal cells (fibroblasts and endothelial cells), T cells CD8+, natural killer (NK) cells, and macrophages M1 and M2 infiltration were associated with poor prognosis. Contrary to other tumors, lymphocytic infiltration is related to poor prognosis. Only B cells were associated with more favorable prognosis. UM samples scoring high in both angiogenesis (Angio) and antigen presentation (AP) pathways showed a poor prognosis suggesting an additive role of both functions. Almost all these tumors exhibited a chromosome 3 monosomy. Finally, an enrichment analysis showed that tumors classified as high Angio-high AP also activated metabolic pathways such as glycolysis or PI3K-AKT-MTOR. In summary, our pool analysis identified a cluster of samples with angiogenic and inflammatory phenotypes exhibiting poor prognosis and metabolic activation. Our analysis showed robust results replicated in a pool analysis merging different datasets from different analytic platforms.

Metastatic uveal melanoma (MUM) is characterized by poor patient survival. Unfortunately, current treatment options demonstrate limited benefits. In this study, we evaluate the efficacy of ACY-1215, a histone deacetylase inhibitor (HDACi), to attenuate growth of primary ocular UM cell lines and, in particular, a liver MUM cell line in vitro and in vivo, and elucidate the underlying molecular mechanisms. A significant (p = 0.0001) dose-dependent reduction in surviving clones of the primary ocular UM cells, Mel270, was observed upon treatment with increasing doses of ACY-1215. Treatment of OMM2.5 MUM cells with ACY-1215 resulted in a significant (p = 0.0001), dose-dependent reduction in cell survival and proliferation in vitro, and in vivo attenuation of primary OMM2.5 xenografts in zebrafish larvae. Furthermore, flow cytometry revealed that ACY-1215 significantly arrested the OMM2.5 cell cycle in S phase (p = 0.0001) following 24 h of treatment, and significant apoptosis was triggered in a time- and dose-dependent manner (p < 0.0001). Additionally, ACY-1215 treatment resulted in a significant reduction in OMM2.5 p-ERK expression levels. Through proteome profiling, the attenuation of the microphthalmia-associated transcription factor (MITF) signaling pathway was linked to the observed anti-cancer effects of ACY-1215. In agreement, pharmacological inhibition of MITF signaling with ML329 significantly reduced OMM2.5 cell survival and viability in vitro (p = 0.0001) and reduced OMM2.5 cells in vivo (p = 0.0006). Our findings provide evidence that ACY-1215 and ML329 are efficacious against growth and survival of OMM2.5 MUM cells.

Uveal melanoma (UM) is a rare, but often lethal, form of ocular cancer arising from melanocytes within the uveal tract. UM has a high propensity to spread hematogenously to the liver, with up to 50% of patients developing liver metastases. Unfortunately, once liver metastasis occurs, patient prognosis is extremely poor with as few as 8% of patients surviving beyond two years. There are no standard-of-care therapies available for the treatment of metastatic uveal melanoma, hence it is a clinical area of urgent unmet need. Here, the clinical relevance and therapeutic potential of cysteinyl leukotriene receptors (CysLT1 and CysLT2) in UM was evaluated. High expression of CYSLTR1 or CYSLTR2 transcripts is significantly associated with poor disease-free survival and poor overall survival in UM patients. Digital pathology analysis identified high expression of CysLT1 in primary UM is associated with reduced disease-specific survival (p = 0.012) and overall survival (p = 0.011). High CysLT1 expression shows a statistically significant (p = 0.041) correlation with ciliary body involvement, a poor prognostic indicator in UM. Small molecule drugs targeting CysLT1 were vastly superior at exerting anti-cancer phenotypes in UM cell lines and zebrafish xenografts than drugs targeting CysLT2. Quininib, a selective CysLT1 antagonist, significantly inhibits survival (p < 0.0001), long-term proliferation (p < 0.0001), and oxidative phosphorylation (p < 0.001), but not glycolysis, in primary and metastatic UM cell lines. Quininib exerts opposing effects on the secretion of inflammatory markers in primary versus metastatic UM cell lines. Quininib significantly downregulated IL-2 and IL-6 in Mel285 cells (p < 0.05), but significantly upregulated IL-10, IL-1β, IL-2 (p < 0.0001), IL-13, IL-8 (p < 0.001), IL-12p70 and IL-6 (p < 0.05) in OMM2.5 cells. Finally, quininib significantly inhibits tumour growth in orthotopic zebrafish xenograft models of UM. These preclinical data suggest that antagonism of CysLT1, but not CysLT2, may be of therapeutic interest in the treatment of UM.