Additive Role of Immune System Infiltration and Angiogenesis in Uveal Melanoma Progression

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

Uveal Melanoma, Angiogenesis and Immunotherapy, Is There Any Hope?

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

Genetic and Immune Changes Associated with Disease Progression under the Pressure of Oncolytic Therapy in A Neuroblastoma Outlier Patient

Little is known about the effect of oncolytic adenovirotherapy on pediatric tumors. Here we present the clinical case of a refractory neuroblastoma that responded positively to Celyvir (ICOVIR-5 oncolytic adenovirus delivered by autologous mesenchymal stem cells) for several months. We analyzed samples during tumor evolution in order to identify molecular and mutational features that could explain the interactions between treatment and tumor and how the balance between both of them evolved. We identified a higher adaptive immune infiltration during stabilized disease compared to progression, and also a higher mutational rate and T-cell receptor (TCR) diversity during disease progression. Our results indicate an initial active role of the immune system controlling tumor growth during Celyvir therapy. The tumor eventually escaped from the control exerted by virotherapy through acquisition of resistance by the tumor microenvironment that exhausted the initial T cell response

Recommendations for the classification of germline variants in the exonuclease domain of POLE and POLD1

BackgroundGermline variants affecting the proofreading activity of polymerases epsilon and delta cause a hereditary cancer and adenomatous polyposis syndrome characterized by tumors with a high mutational burden and a specific mutational spectrum. In addition to the implementation of multiple pieces of evidence for the classification of gene variants, POLE and POLD1 variant classification is particularly challenging given that non-disruptive variants affecting the proofreading activity of the corresponding polymerase are the ones associated with cancer. In response to an evident need in the field, we have developed gene-specific variant classification recommendations, based on the ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology) criteria, for the assessment of non-disruptive variants located in the sequence coding for the exonuclease domain of the polymerases.MethodsA training set of 23 variants considered pathogenic or benign was used to define the usability and strength of the ACMG/AMP criteria. Population frequencies, computational predictions, co-segregation data, phenotypic and tumor data, and functional results, among other features, were considered.ResultsGene-specific variant classification recommendations for non-disruptive variants located in the exonuclease domain of POLE and POLD1 were defined. The resulting recommendations were applied to 128 exonuclease domain variants reported in the literature and/or public databases. A total of 17 variants were classified as pathogenic or likely pathogenic, and 17 as benign or likely benign.ConclusionsOur recommendations, with room for improvement in the coming years as more information become available on carrier families, tumor molecular characteristics and functional assays, are intended to serve the clinical and scientific communities and help improve diagnostic performance, avoiding variant misclassifications

Potential Involvement of NSD1, KRT24 and ACACA in the Genetic Predisposition to Colorectal Cancer

The ALFRED (Allelic Loss Featuring Rare Damaging) in silico method was developed to identify cancer predisposition genes through the identification of somatic second hits. By applying ALFRED to ~10,000 tumor exomes, 49 candidate genes were identified. We aimed to assess the causal association of the identified genes with colorectal cancer (CRC) predisposition. Of the 49 genes, NSD1, HDAC10, KRT24, ACACA and TP63 were selected based on specific criteria relevant for hereditary CRC genes. Gene sequencing was performed in 736 patients with familial/early onset CRC or polyposis without germline pathogenic variants in known genes. Twelve (predicted) damaging variants in 18 patients were identified. A gene-based burden test in 1596 familial/early-onset CRC patients, 271 polyposis patients, 543 TCGA CRC patients and >134,000 controls (gnomAD, non-cancer), revealed no clear association with CRC for any of the studied genes. Nevertheless, (non-significant) over-representation of disruptive variants in NSD1, KRT24 and ACACA in CRC patients compared to controls was observed. A somatic second hit was identified in one of 20 tumors tested, corresponding to an NSD1 carrier. In conclusion, most genes identified through the ALFRED in silico method were not relevant for CRC predisposition, although a possible association was detected for NSD1, KRT24 and ACACA

Lung metastases share common immune features regardless of primary tumor origin

Background: Only 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. Methods: Gene 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. Results: Significant 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. Conclusions: We have found a group of lung-enriched metastases showing an inflammatory phenotype susceptible to be treated with immunotherapy

Uveal Melanoma Cell Line Proliferation Is Inhibited by Ricolinostat, a Histone Deacetylase Inhibitor

Uveal melanoma (UM) is the most common adult eye cancer. UM originates in the iris, ciliary body or choroid (collectively known as the uvea), in the middle layer of the eye. This first or primary UM is treated by targeting cancer cells using ocular radiation implants or by surgical removal of the eye. However, when UM spreads to the liver and other parts of the body, patients have a poor survival prognosis. Unfortunately, there are no effective treatment options for UM that has spread. Our aim is to help identify effective treatments for UM. In our study, we identified that the drug ACY-1215 prevents the growth of cells derived from UM in the eye and a UM that spread to the liver. Our pre-clinical study uncovered a potential treatment approach for advanced UM. 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

Driver mutations in <i>GNAQ</i> and <i>GNA11</i> genes as potential targets for precision immunotherapy in uveal melanoma patients

Uveal melanoma (UM) is the most common ocular malignancy in adults. Nearly 95% of UM patients carry the mutually exclusive mutations in the homologous genes GNAQ (amino acid change Q209L/Q209P) and GNA11 (aminoacid change Q209L). UM is located in an immunosuppressed organ and does not suffer immunoediting. Therefore, we hypothesize that driver mutations in GNAQ/11 genes could be recognized by the immune system. Genomic and transcriptomic data from primary uveal tumors were collected from the TCGA-UM dataset (n = 80) and used to assess the immunogenic potential for GNAQ/GNA11 Q209L/Q209P mutations using a variety of tools and HLA type information. All prediction tools showed stronger GNAQ/11 Q209L binding to HLA than GNAQ/11 Q209P. The immunogenicity analysis revealed that Q209L is likely to be presented by more than 73% of individuals in 1000 G databases whereas Q209P is only predicted to be presented in 24% of individuals. GNAQ/11 Q209L showed a higher likelihood to be presented by HLA-I molecules than almost all driver mutations analyzed. Finally, samples carrying Q209L had a higher immune-reactive phenotype. Regarding cancer risk, seven HLA genotypes with low Q209L affinity show higher frequency in uveal melanoma patients than in the general population. However, no clear association was found between any HLA genotype and survival. Results suggest a high potential immunogenicity of the GNAQ/11 Q209L variant that could allow the generation of novel therapeutic tools to treat UM like neoantigen vaccinations

Differences in genome, transcriptome, miRNAome, and methylome in synchronous and metachronous liver metastasis of colorectal cancer

Despite distant metastases being the critical factor affecting patients' survival, they remain poorly understood. Our study thus aimed to molecularly characterize colorectal cancer liver metastases (CRCLMs) and explore whether molecular profiles differ between Synchronous (SmCRC) and Metachronous (MmCRC) colorectal cancer. This characterization was performed by whole exome sequencing, whole transcriptome, whole methylome, and miRNAome. The most frequent somatic mutations were in APC, SYNE1, TP53, and TTN genes. Among the differently methylated and expressed genes were those involved in cell adhesion, extracellular matrix organization and degradation, neuroactive ligand-receptor interaction. The top up-regulated microRNAs were hsa-miR-135b-3p and -5p, and the hsa-miR-200-family while the hsa-miR-548-family belonged to the top down-regulated. MmCRC patients evinced higher tumor mutational burden, a wider median of duplications and deletions, and a heterogeneous mutational signature than SmCRC. Regarding chronicity, a significant down-regulation of SMOC2 and PPP1R9A genes in SmCRC compared to MmCRC was observed. Two miRNAs were deregulated between SmCRC and MmCRC, hsa-miR-625-3p and has-miR-1269-3p. The combined data identified the IPO5 gene. Regardless of miRNA expression levels, the combined analysis resulted in 107 deregulated genes related to relaxin, estrogen, PI3K-Akt, WNT signaling pathways, and intracellular second messenger signaling. The intersection between our and validation sets confirmed the validity of our results. We have identified genes and pathways that may be considered as actionable targets in CRCLMs. Our data also provide a valuable resource for understanding molecular distinctions between SmCRC and MmCRC. They have the potential to enhance the diagnosis, prognostication, and management of CRCLMs by a molecularly targeted approach