PRAME Expression: A Target for Cancer Immunotherapy and a Prognostic Factor in Uveal Melanoma.

Purpose: Uveal melanoma (UM) is a rare disease with a high mortality, and new therapeutic options are being investigated. Preferentially Expressed Antigen in Melanoma (PRAME) is a cancer testis antigen, expressed in the testis, but also in cancers, including uveal melanoma. PRAME is considered a target for immune therapy in several cancers, and PRAME-specific T cell clones have been shown to kill UM cells. Methods: We studied the literature on PRAME expression in hematological and solid malignancies, including UM, and its role as a target for immunotherapy. The distribution of tumor features was compared between PRAME-high and PRAME-low UM in a 64-patient cohort from the Leiden University Medical Center (LUMC) and in the Cancer Genome Atlas (TCGA) cohort of 80 cases and differential gene expression analysis was performed in the LUMC cohort. Results: PRAME is expressed in many malignancies, it is frequently associated with a negative prognosis, and can be the target of T cell receptor (TCR)-transduced T cells, a promising treatment option with high avidity and safety. In UM, PRAME is expressed in 26% to 45% of cases and is correlated with a worse prognosis. In the LUMC and the TCGA cohorts, high PRAME expression was associated with larger diameter, higher Tumor-Node-Metastasis (TNM) stage, more frequent gain of chromosome 8q, and an inflammatory phenotype. Conclusions: We confirm that PRAME is associated with poor prognosis in UM and has a strong connection with extra copies of 8q. We show that PRAME-specific immunotherapy in an adjuvant setting is promising in treatment of malignancies, including UM.

Tumor-Infiltrating T Cells Can Be Expanded Successfully from Primary Uveal Melanoma after Separation from Their Tumor Environment.

Purpose: To evaluate whether expanded tumor-infiltrating lymphocytes (TILs) can be obtained from primary uveal melanoma (UM) for potential use as adjuvant treatment in patients at risk of developing metastatic disease. Design: Experimental research study. Participants: Freshly obtained primary UM from 30 patients. Methods: Three different methods were used to expand TILs: (1) direct culture from small fragments of fresh tumor tissue, (2) single-cell tissue preparation by enzymatic digestion and subsequent enrichment of mononuclear cells, and (3) selection of CD3+ T cells using magnetic beads. Surface expression of costimulatory and inhibitory T-cell markers and T-cell reactivity against autologous tumor cells was assessed. Clinical, histopathologic, genetic, and immunologic characteristics of the tumors were compared with the capacity to expand TILs and with their reactivity against autologous tumor cells. Main Outcome Measures: The feasibility of expanding TILs from primary UM, testing their reactivity to autologous UM cells, and evaluating the impact of an immunomodulatory environment. Results: Direct culture of tumor parts led to successful TIL culture in 4 of 22 tumors (18%), enrichment of mononuclear cells gave rise to TILs in 5 of 12 tumors (42%), while preselection of CD3+ T cells with magnetic beads resulted in TIL expansion in 17 of 25 tumors (68%). In 8 of 17 tumors (47%), the TIL cultures comprised UM-reactive T cells. The presence of UM-reactive T cells among TILs was not related to clinical, histologic, genetic, or immunological tumor characteristics. Interestingly, RNA-Seq analysis showed that approximately half of the UM tumors displayed an increased expression of immunomodulatory molecules related to T-cell suppression, such as galectin 3, programmed death-ligand 1, cytotoxic T-lymphocyte-associated protein 4, indoleamine 2,3-dioxygenase 1, and lymphocyte activating 3, potentially explaining why T cells require optimal removal of tumor components for expansion. Conclusions: The need to separate TILs from their tumor microenvironment for their successful expansion and the presence of UM-reactive T cells among TILs suggests that these UM-reactive T cells are strongly suppressed in vivo and that UM is immunogenic. These findings indicate that adoptive TIL therapy could be an option as an adjuvant treatment in primary UM patients at high risk of developing metastatic disease.

Soluble HLA in the Aqueous Humour of Uveal Melanoma Is Associated with Unfavourable Tumour Characteristics.

A high HLA expression in uveal melanoma (UM) is part of the prognostically unfavorable inflammatory phenotype. We wondered whether the presence of soluble HLA (sHLA) in the aqueous humour is associated with clinical, histopathological or genetic tumour characteristics, and represents tumour HLA expression and intratumoural inflammation. Aqueous humour from 108 UM patients was analysed for the presence of sHLA, using a Luminex assay specific for HLA Class I. Clinical and genetic parameters were compared between sHLA-positive and negative eyes. A qPCR analysis was performed on tumour tissue using a Fluidigm assay. In 19/108 UM-containing eyes, the sHLA level in the aqueous was above the detection limit. Tumours in sHLA-positive eyes were significantly larger, more frequently involved the ciliary body, and more often showed monosomy 3, gain of chromosome 8q and loss of BAP1 staining. Melanoma-related survival was worse in patients with sHLA-positive aqueous humour. sHLA in the aqueous did not represent the tumour's HLA expression and did not relate to immune cell infiltration in the tumour. We conclude that UM-containing eyes may contain sHLA in the aqueous humour, where it is a prognostically-unfavourable sign and may influence local immune responses.

Tumour Angiogenesis in Uveal Melanoma Is Related to Genetic Evolution.

Increased angiogenesis is associated with a higher metastasis- and mortality rate in uveal melanoma (UM). Recently, it was demonstrated that genetic events, such as 8q-gain and BAP1-loss, influence the level of immune infiltrate. We aimed to determine whether genetic events, and specific cytokines, relate to angiogenesis in UM. Data from UM patients who underwent enucleation between 1999 and 2008 were analysed. Microvascular density (MVD) and the presence of infiltrating immune cells were determined with immunohistochemistry (IHC) and immunofluorescence in 43 cases. Chromosome status, BAP1 IHC and mRNA expression of angiogenesis-related genes were known in 54 cases. Tumours with monosomy 3/BAP1-loss showed a higher MVD compared to tumours with disomy 3/normal BAP1 expression (p = 0.008 and p = 0.004, respectively). Within BAP1-positive lesions (n = 20), 8q-gain did not relate to MVD (p = 0.51). A high MVD was associated with an increased expression of angiopoietin 2 (ANGPT2) (p = 0.041), Von Willebrand Factor (VWF) (p = 0.010), a decreased expression of vascular endothelial growth factor B (VEGF-B) (p = 0.024), and increased numbers of tumour-infiltrating macrophages (CD68+, p = 0.017; CD68+CD163+, p = 0.031) and lymphocytes (CD4+, p = 0.027). Concluding, vascular density of UM relates to its genetic profile: Monosomy 3 and BAP1-loss are associated with an increased MVD, while an early event (gain of 8q) is not independently related to MVD, but may initiate a preparation phase towards development of vessels. Interestingly, VEGF-B expression is decreased in UM with a high MVD.

Ischemia Is Related to Tumour Genetics in Uveal Melanoma.

Hypoxia-inducible factor 1-alpha (HIF1a) and its regulator von Hippel-Lindau protein (VHL) play an important role in tumour ischemia. Currently, drugs that target HIF1a are being developed to treat malignancies. Although HIF1a is known to be expressed in uveal melanoma (UM), it is as yet unknown which factors, such as tumour size or genetics, determine its expression. Therefore, we aimed to determine which tumour characteristics relate to HIF1a expression in UM. Data from 64 patients who were enucleated for UM were analysed. Messenger RNA (mRNA) expression was determined with the Illumina HT-12 v4 chip. In 54 cases, the status of chromosomes 3 and 8q, and BRCA1-associated protein 1 (BAP1) protein expression (immunohistochemistry) were determined. Findings were corroborated using data of 80 patients from the Cancer Genome Atlas (TCGA) study. A significantly increased expression of HIF1a, and a decreased expression of VHL were associated with monosomy 3/loss of BAP1 expression. The relationship between BAP1 loss and HIF1a expression was independent of chromosome 3. The largest basal diameter and tumour thickness showed no relationship with HIF1a. HIF1a expression related to an increased presence of infiltrating T cells and macrophages. From this study, we conclude that HIF1a is strongly related to tumour genetics in UM, especially to loss of BAP1 expression, and less to tumour size. Tumour ischemia is furthermore related to the presence of an inflammatory phenotype.

PRAME as a Potential Target for Immunotherapy in Metastatic Uveal Melanoma.

Importance: Uveal melanoma (UM) is an intraocular primary malignant neoplasm that often gives rise to metastatic disease for which there are no effective therapies. A substantial proportion of UMs express the cancer-testis antigen PRAME (preferentially expressed antigen in melanoma), which can potentially be targeted by adoptive T-cell therapy. Objective: To determine whether there may be a rationale for PRAME-directed T-cell therapy for metastatic UM. Design, Setting, and Participants: An experimental study using a retrospective cohort of 64 patients with UM (median follow-up, 62 months) was conducted from January 8, 2015, to November 20, 2016, at the Leiden University Medical Center. Clinical, histopathologic, and genetic parameters were compared between 64 PRAME-positive and PRAME-negative UMs. HLA class I restricted, PRAME-specific T cells were stimulated with UM cell lines to measure their antigen-specific reactivity against these cell lines, which were analyzed for PRAME expression by real-time quantitative polymerase chain reaction. Uveal melanoma metastases from 16 unrelated patients were assessed for PRAME expression by messenger RNA fluorescence in situ hybridization and for HLA class I expression by immunofluorescence staining. Main Outcomes and Measures: Interferon γ production for antigen-specific reactivity and detection of PRAME and HLA class I expression in primary and metastatic UM. Results: Of the 64 patients in the study (31 women and 33 men; mean [SD] age at the time of enucleation, 60.6 [15.6] years), PRAME expression was negative in 35 primary UMs and positive in 29 primary UMs. Positive PRAME expression was associated with a high largest basal diameter (15.0 vs 12.0 mm; P = .005), ciliary body involvement (59% vs 26%; P = .008), and amplification of chromosome 8q (66% vs 23%; P = .002). PRAME-specific T cells reacted against 4 of 7 UM cell lines, demonstrating that T-cell reactivity correlated with PRAME expression. Metastatic UM samples were positive for PRAME messenger RNA in 11 of 16 patients and for HLA class I in 10 of 16 patients, with 8 of 16 patients demonstrating coexpression of both PRAME and HLA class I. Conclusions and Relevance: PRAME is expressed in many primary and metastatic UMs, and about half of the metastatic UMs coexpress PRAME and HLA class I. The finding that PRAME-specific T cells in this study reacted against PRAME-positive UM cell lines suggests a potential role for PRAME-directed immunotherapy for selected patients with metastatic UM.

Genetic evolution of uveal melanoma guides the development of an inflammatory microenvironment.

Uveal melanoma (UM) is characterized by a number of genetic aberrations that follow a certain chronology and are tightly linked to tumor recurrence and survival. Loss of chromosome 3, bi-allelic loss of BAP1 expression, and gain in chromosome 8q have been associated with metastasis formation and death, while loss of chromosome 3 has been associated with the influx of macrophages and T cells. We used a set of genetically-classified UM to study immune infiltration in the context of their genetic evolution. We show in two independent cohorts that lack of BAP1 expression is associated with an increased density of CD3+ T cells and CD8+ T cells. The presence of extra copies of chromosome 8q in disomy 3 tumors with a normal BAP1 expression is associated with an increased influx of macrophages (but not T cells). Therefore, we propose that the genetic evolution of UM is associated with changes in the inflammatory phenotype. Early changes resulting in gain of chromosome 8q may activate macrophage infiltration, while sequential loss of BAP1 expression seems to drive T cell infiltration in UM.

PRAME as an Independent Biomarker for Metastasis in Uveal Melanoma.

PURPOSE: Uveal melanoma (UM) can be classified by gene expression profiling (GEP) into Class 1 (low metastatic risk) and Class 2 (high metastatic risk), the latter being strongly associated with mutational inactivation of the tumor suppressor BAP1. Nevertheless, a small percentage of Class 1 tumors give rise to metastatic disease. The purpose of this study was to identify biomarkers of metastasis in Class 1 tumors. EXPERIMENTAL DESIGN: A total of 389 consecutive patients with UM were assigned to Class 1 or Class 2 using a prospectively validated 12-gene prognostic classifier. Selected tumors were further analyzed using global GEP and single nucleotide polymorphism microarrays. PRAME (preferentially expressed antigen in melanoma) mRNA expression was analyzed in 64 Class 1 tumors by qPCR. RESULTS: Among Class 1 UMs, the most significant predictor of metastasis was PRAME mRNA expression (P = 0.0006). The 5-year actuarial rate of metastasis was 0% for Class1(PRAME-), 38% for Class1(PRAME+), and 71% for Class 2 tumors. Median metastasis-free survival for Class1(PRAME+) patients was 88 months, compared to 32 months for Class 2 patients. Findings were validated using three independent datasets, including one using disomy 3 to identify low-risk UM. Chromosome copy number changes associated with Class1(PRAME+) tumors included gain of 1q, 6p, 8q, and 9q and loss of 6q and 11q. PRAME expression was associated with larger tumor diameter (P = 0.05) and SF3B1 mutations (P = 0.003). CONCLUSIONS: PRAME is an independent prognostic biomarker in UM, which identifies increased metastatic risk in patients with Class 1 or disomy 3 tumors. This finding may further enhance the accuracy of prognostic testing and precision medicine for UM.

EMT-associated factors promote invasive properties of uveal melanoma cells.

PURPOSE: Transcription factors regulating the epithelial-to-mesenchymal transition (EMT) program contribute to carcinogenesis and metastasis in many tumors, including cutaneous melanoma. However, little is known about the role of EMT factors in the growth and metastatic dissemination of uveal melanoma cells. Here, we analyzed the expression and functions of the EMT factors ZEB1, Twist1, and Snail1 in uveal melanoma cell lines and primary tumors. METHODS: ZEB1, Twist1, and Snail1 mRNA levels were measured using qPCR in five uveal melanoma cell lines and in 30 primary tumors. Gene expression was used to determine class 1 and class 2 signatures in the primary tumors. Short hairpin RNA was used to downregulate the expressions of the EMT factors; then, growth and transwell invasion assays were performed. RESULTS: ZEB1, Twist1, and Snail1 were expressed in all five uveal melanoma lines, with ZEB1 having the highest protein levels. ZEB1 mRNA was significantly elevated in highly metastatic class 2 primary tumors for which survival data were not available, whereas a high gene expression of Twist1 was associated with a worse prognosis in a separate tumor cohort analyzed by expression profiling. The genetic downregulation of ZEB1 in OCM1, OMM1, and 92.1 resulted in a more than 50% reduction in invasion, but only suppressed growth in OMM1 cells. Suppression of Twist1 in Mel290 and OMM1 reduced growth and invasion by more than 50%. The downregulation of Snail1 in the 92.1 cell line reduced invasion by 50%, but did not interfere with growth. CONCLUSIONS: The downregulation of ZEB1, Twist1, and Snail1 reduces the invasive properties of uveal melanoma cells, and the elevated mRNA levels of ZEB1 and Twist1 are associated with a more aggressive clinical phenotype in uveal melanoma samples. Therefore, these factors could represent new therapeutic targets in patients with ocular melanoma.

Contrast-induced acute kidney injury and clinical outcomes after intra-arterial and intravenous contrast administration: risk comparison adjusted for patient characteristics by design.

BACKGROUND: Direct comparisons between risk of contrast induced acute kidney injury (CI-AKI) after intra-arterial versus intravenous contrast administration are scarce. We estimated and compared the risk of CI-AKI and its clinical course after both modes of contrast administration in patients who underwent both. METHODS: One hundred seventy patients who received both intra-arterial and intravenous contrast injections within one year between 2001 and 2010 were included. Primary outcome was occurrence of CI-AKI. Secondary outcomes were duration of hospital stay, the need for dialysis, recovery of renal function, and mortality. RESULTS: The risk of CI-AKI was 24/170 (14.0%, 95% CI 9.6-20.2) after intra-arterial contrast injection versus 20/170 (11.7%, 95% CI 7.7-17.5) after intravenous contrast administration, which led to a relative risk of 1.2 (95% CI 0.7-2.1). None of the patients had a need for dialysis. Median duration of hospital stay in CI-AKI patients was 15.0 days (2.5-97.5, percentile 1-92) after intra-arterial and 15.5 days (2.5-97.5, percentile 0-38) after intravenous contrast procedures. Renal function recovered after CI-AKI in 13/24 after intra-arterial and in 10/20 patients after intravenous contrast administration. Mortality risks in CI-AKI patients were slightly higher than in non-CI-AKI patients, hazard ratios 1.6 (95% CI 0.7-3.7) for intra-arterial and 1.7 (95% CI 0.7-4.4) for intravenous contrast administration, adjusted for confounders. CONCLUSION: The risk of CI-AKI, and its clinical course was similar after intra-arterial and intravenous contrast media administration, after adjustment by design for patient-related risk factors.