Conjunctival melanoma (CM) is a rare but lethal form of cancer. Similar to cutaneous melanoma, CM frequently carries activating mutations in
and
. We studied whether CM as well as conjunctival benign ...and premalignant melanocytic lesions express targets in the mitogen-activated protein kinase (MAPK) and AKT pathways, and whether specific inhibitors can suppress CM growth
.
131 conjunctival lesions obtained from 129 patients were collected. The presence of
V600E mutation and expression of phosphorylated (p)-ERK and p-AKT were assessed by immunohistochemistry. We studied cell proliferation, phosphorylation, cell cycling and apoptosis in three CM cell lines using two BRAF inhibitors (Vemurafenib and Dabrafenib), a MEK inhibitor (MEK162) and an AKT inhibitor (MK2206).
The
V600E mutation was present in 19% of nevi and 26% of melanomas, but not in primary acquired melanosis (PAM). Nuclear and cytoplasmic p-ERK and p-AKT were expressed in all conjunctival lesions. Both BRAF inhibitors suppressed growth of both
mutant CM cell lines, but only one induced cell death. MEK162 and MK2206 inhibited proliferation of CM cells in a dose-dependent manner, and the combination of these two drugs led to synergistic growth inhibition and cell death in all CM cell lines.
ERK and AKT are constitutively activated in conjunctival nevi, PAM and melanoma. While BRAF inhibitors prohibited cell growth, they were not always cytotoxic. Combining MEK and AKT inhibitors led to more growth inhibition and cell death in CM cells. The combination may benefit patients suffering from metastatic conjunctival melanoma.
Purpose
Inflammation is a bad prognostic sign in Uveal Melanoma (UM), but it is as yet unclear how it is regulated. We set out to investigate the relation between microRNA’s and inflammation in UM ...and their potential regulation by chromosome 3 and 8.
Methods
The expression of 125 microRNA’s was determined using an Illumina HT12V4 array in 64 primary UM. Similarly, the mRNA expression of HLA‐A, HLA‐B and markers of tumor‐infiltrating lymphocytes (TIL) such as CD3E, CD4, CD8, and tumor‐associated macrophages (TAM), such as CD68 and CD163, was obtained from the array.
Results
When looking at all 64 UM tumors, expression levels of seven of the 125 miRNA’s that were analysed correlated with the presence of tumor‐infiltrating lymphocytes (TIL), tumor‐associated macrophages (TAM) and HLA Class I expression. Increased levels of miRNA 22 and 155 were associated with increased HLA expression and high numbers of TIL and TAM, while expression of miRNAs 7, 98, 125, 302 and 599 was associated with less infiltrate and a lower HLA expression. MiRNAs 22 (p ≤ 0.001) and 155 (p = 0.005) were upregulated in monosomy 3 tumors and expression of miRNAs 7 (p = 0.35), 98 (p = 0.26), 125 (p = 0.91) and 599 (p = 0.80) was not related to monosomy 3, while miR‐302 (p = 0.02) was slightly higher in D3 tumors. MiR‐22 was positively associated to chromosome 8q gain (p = 0.01) but miRNA’s 7 (p = 0.004), 98 (p = 0.002), 125 (p = 0.005), 302 (p = 0.02), and 599 (p ≤ 0.001) were all decreased in tumours with 8q gain.
Conclusions
We identified a set of microRNA’s associated with inflammatory markers (HLA Class I, TIL’s and TAM’s) in UM. The microRNA’s that are positively associated with inflammation are highly expressed in tumors with one copy of chromosome 3 and 8q gain while this is the opposite for microRNA’s negatively associated with inflammation. We propose a regulatory role for chromosome 3 and 8q abnormality in the control of inflammation‐related microRNA’s and suggest a potential involvement of these microRNA’s in the development of inflammation in UM.
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.
Activating Gα
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 CysLT
R) forms a rare alternative. The role of wild-type CysLT
R in uveal melanoma remains unknown.
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.
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.
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 CysLT
R an attractive therapeutic target in uveal melanoma.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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 (
= 0.008 and
= 0.004, respectively). Within BAP1-positive lesions (
= 20), 8q-gain did not relate to MVD (
= 0.51). A high MVD was associated with an increased expression of angiopoietin 2 (ANGPT2) (
= 0.041), Von Willebrand Factor (VWF) (
= 0.010), a decreased expression of vascular endothelial growth factor B (VEGF-B) (
= 0.024), and increased numbers of tumour-infiltrating macrophages (CD68+,
= 0.017; CD68+CD163+,
= 0.031) and lymphocytes (CD4+,
= 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.
Summary
Patients with haematological malignancies are at risk for invasive fungal diseases (IFD). A survey was conducted in all Dutch academic haematology centres on their current diagnostic, ...prophylactic and therapeutic approach towards IFD in the context of azole‐resistance. In all 8 centres, a haematologist and microbiologist filled in the questionnaire that focused on different subgroups of haematology patients. Fungal prophylaxis during neutropaenia was directed against Candida and consisted of fluconazole and/or amphotericin B suspension. Mould‐active prophylaxis was given to acute myeloid leukaemia patients during chemotherapy in 2 of 8 centres. All centres used azole prophylaxis in a subset of patients with graft‐versus‐host disease. A uniform approach towards the diagnosis and treatment of IFD and in particular azole‐resistant Aspergillus fumigatus was lacking. In 2017, all centres agreed to implement a uniform diagnostic and treatment algorithm regarding invasive aspergillosis with a central role for comprehensive diagnostics and PCR‐based detection of azole‐resistance. This study (DB‐MSG 002) will re‐evaluate this algorithm when 280 patients have been treated. A heterogeneous approach towards antifungal prophylaxis, diagnosis and treatment was apparent in the Netherlands. Facing triazole‐resistance, consensus was reached on the implementation of a uniform diagnostic approach in all 8 centres.
MicroRNAs are known to play a role in the regulation of inflammation. As a high HLA Class I expression is associated with a bad prognosis in UM, we set out to determine whether any miRNAs were ...related to a high HLA Class I expression and inflammation. We also determined whether such miRNAs were related to the UM’s genetic status. The expression of 125 miRNAs was determined in 64 primary UM from Leiden. Similarly, the mRNA expression of HLA-A, HLA-B, TAP1, BAP1, and immune cell markers was obtained. Expression levels of 24 of the 125 miRNAs correlated with expression of at least three out of four HLA Class I probes. Four miRNAs showed a positive correlation with HLA expression and infiltration with leukocytes, 20 a negative pattern. In the first group, high miRNA levels correlated with chromosome 3 loss/reduced BAP1 mRNA expression, in the second group low miRNA levels. The positive associations between miRNA-22 and miRNA-155 with HLA Class I were confirmed in the TCGA study and Rotterdam cohort, and with TAP1 in the Rotterdam data set; the negative associations between miRNA-125b2 and miRNA-211 and HLA-A, TAP1, and CD4 were confirmed in the Rotterdam set. We demonstrate two patterns: miRNAs can either be related to a high or a low HLA Class I/TAP1 expression and the presence of infiltrating lymphocytes and macrophages. However, both patterns were associated with chromosome 3/BAP1 status, which suggests a role for BAP1 loss in the regulation of HLA expression and inflammation in UM through miRNAs.
Uveal melanoma (UM), a rare cancer of the eye, is characterized by initiating mutations in the genes G-protein subunit alpha Q (
), G-protein subunit alpha 11 (
), cysteinyl leukotriene receptor 2 (
...), and phospholipase C beta 4 (
) and by metastasis-promoting mutations in the genes splicing factor 3B1 (
), serine and arginine rich splicing factor 2 (SRSF2), and BRCA1-associated protein 1 (
). Here, we tested the hypothesis that additional mutations, though occurring in only a few cases ("secondary drivers"), might influence tumor development.
We analyzed all the 4125 mutations detected in exome sequencing datasets, comprising a total of 139 Ums, and tested the enrichment of secondary drivers in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that also contained the initiating mutations. We searched for additional mutations in the putative secondary driver gene protein tyrosine kinase 2 beta (
) and we developed new mutational signatures that explain the mutational pattern observed in UM.
Secondary drivers were significantly enriched in KEGG pathways that also contained
and
, such as the calcium-signaling pathway. Many of the secondary drivers were known cancer driver genes and were strongly associated with metastasis and survival. We identified additional mutations in
. Sparse dictionary learning allowed for the identification of mutational signatures specific for UM.
A considerable part of rare mutations that occur in addition to known driver mutations are likely to affect tumor development and progression.
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
-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.
In Uveal Melanoma (UM), an inflammatory phenotype is strongly associated with the development of metastases and with chromosome 3/BAP1 expression loss. As an increased expression of several Histone ...Deacetylases (HDACs) was associated with loss of chromosome 3, this suggested that HDAC expression might also be related to inflammation. We analyzed HDAC expression and the presence of leukocytes by mRNA expression in two sets of UM (Leiden and TCGA) and determined the T lymphocyte fraction through ddPCR. Four UM cell lines were treated with IFNγ (50IU, 200IU). Quantitative PCR (qPCR) was used for mRNA measurement of HDACs in cultured cells. In both cohorts (Leiden and TCGA), a positive correlation occurred between expression of HDACs 1, 3 and 8 and the presence of a T-cell infiltrate, while expression of HDACs 2 and 11 was negatively correlated with the presence of tumor-infiltrating macrophages. Stimulation of UM cell lines with IFNγ induced an increase in HDACs 1, 4, 5, 7 and 8 in two out of four UM cell lines. We conclude that the observed positive correlations between HDAC expression and chromosome 3/BAP1 loss may be related to the presence of infiltrating T cells.
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