Hanneke W. Mensink

Gene expression profiling in uveal melanoma: two regions on 3p related to prognosis.

PURPOSE Although studies on uveal melanoma (UM) revealed prognostic significance of chromosomal aberrations, they resulted in classification errors in survival prediction. A robust prognostic classifier with strong predictive value and further insight in genes responsible for poor prognosis were obtained by performing a gene-expression profile in tumors of UM patients for which extensive clinical, histopathologic, cytogenetic, and follow-up data were available. Furthermore, the UM microarray expression data were compared with cytogenetic data. METHODS Gene-expression profiles of 46 UMs were obtained with microchip assays. Data were analyzed with cluster-analysis and predictive analysis of microarrays (PAM) software and validated with real-time PCR. The prognostic significance of UMs with specific molecular signatures was determined. Furthermore, LAP analysis resulted in the identification of differentially expressed chromosomal regions. RESULTS The primary UMs were classified in two distinct molecular classes with a strong prognostic value (P < 0.001; hazard ratio 7.7). Classifier gene sets for microarray class and disease-free survival were validated with real-time PCR, and the predictive value of the UM class marker set was validated with gene-expression profiles of tumors provided by other institutions, showing a sensitivity of 0.93 and specificity of 1.00 for class II tumors. A locally adaptive statistical procedure identified two regions on the short arm of chromosome 3 with decreased gene-expression in tumors with shorter disease-free survival. CONCLUSIONS Microarray classification outperforms known prognostic indicators for UM, such as clinical, histopathologic, and cytogenetic parameters. In addition, the identified regions with lower expressed genes on 3p could harbor genes that are responsible for the poor prognosis of patients with UM.

Chromosome 3 intratumor heterogeneity in uveal melanoma

PURPOSE To investigate the presence of focal or diffuse heterogeneity of monosomy 3 in uveal melanoma, by using fluorescence in situ hybridization (FISH). METHODS Direct interphase FISH in a series of 151 uveal melanomas revealed 82 tumors with loss of chromosome 3. Tumors with monosomy 3 were suspected to be heterogeneous if there were low percentages of monosomy 3, triploid clones, inconsistencies between FISH on centromere 3 and the long arm of chromosome 3, or discrepancies between fine-needle-aspiration biopsies (FNABs) and the main tumor. These tumors (n=16), all choroidal melanomas, were selected and analyzed for intratumor heterogeneity by using FISH on paraffin-embedded tissue sections. RESULTS Different sections of each tumor were evaluated with FISH: 6 tumors showed monosomy 3 in the same percentage throughout the tumor, and 10 showed multiple clones with different percentages of monosomy 3. However, these tumors did not show focal heterogeneity with respect to chromosome 3 status, and differences in monosomy 3 distribution between the base and apex of the tumor could not be identified. CONCLUSIONS Although a small number of uveal melanomas show heterogeneity for chromosome 3, it does not affect survival. In the presence of triploid clones, the loss of chromosome 3 is more difficult to interpret. In general, tumor biopsies in uveal melanoma provide an accurate prediction of the patients prognosis.

Multiplex ligation-dependent probe amplification equals fluorescence in-situ hybridization for the identification of patients at risk for metastatic disease in uveal melanoma.

In uveal melanoma, loss of chromosome 3 and gain of chromosome 8q are associated with a high risk of metastasis. In this study, we validated the use of multiplex ligation-dependent probe amplification (MLPA) in detecting patients at risk for metastatic disease in comparison with the predictive power of fluorescence in-situ hybridization (FISH). For 64 uveal melanoma samples, the MLPA results of chromosome 3 and 8 were compared with the results obtained by FISH. For seven samples, a single nucleotide polymorphism array was performed to clarify discrepancies. Clinical information together with the histopathology and chromosomal aberrations of chromosomes 1, 3, 6, and 8 were evaluated for correlation with the patients’ prognosis. Loss of chromosome 3, loss or gain of 8p, and gain of 8q, found with MLPA, correlated with a significantly lower disease-free survival (P<0.001). On the basis of the clinical outcome, 12 patients would have been classified incorrectly using MLPA results of chromosomes 3 and 8. FISH results led to the same incorrect classification. Four patients with abnormalities of chromosomes 3 and 8 in the tumor, detected with MLPA, are still alive without metastasis. Eight patients without concurrent aberrations of chromosomes 3 and 8 in the tumors died due to metastasis. The sensitivity of MLPA to detect patients at risk for metastatic disease is higher than with the results obtained with FISH (0.795 vs. 0.692). The specificity is equal for both techniques (0.840). MLPA is able to detect patients at risk for metastasis using the results for chromosomes 3 and 8. There is no significant difference in the predictive power of MLPA compared with FISH.

Molecular cytogenetic analysis of archival uveal melanoma with known clinical outcome

Uveal melanoma (UM) is the most common primary intraocular tumor in the Western world. Cytogenetically, this tumor is characterized by typical chromosomal aberrations such as loss of 1p, 3, and 6q, and gain of 6p and 8q. Routinely, karyotyping and fluorescent in situ hybridization (FISH) on fresh tumor-biopsies are used to identify chromosomal changes. In addition, archival UM samples can be examined using comparative genomic hybridization (CGH). In the presented study, we used CGH on a series of 46 archival uveal melanomas to identify chromosomal changes. In 44 tumors aberrations were present and classic prognostic markers as loss of 1p (12 tumors, 26.1%), monosomy 3 (26 tumors, 56.5%), loss of 6q (10 tumors, 21.7%), and gain of chromosome arm 8q (27 tumors, 58.7%) were observed. Gain of chromosome arms 18q or 21q was found in three UMs. Multiplex ligation-dependent probe amplification (MLPA), a novel technique in UM, was performed to verify this low number of chromosome 18 and 21 abnormalities, but we could not confirm the previously reported gain of 18q11.2 and 21q11.2 as poor prognostic factors in UM.

Long Overall Survival After Dendritic Cell Vaccination in Metastatic Uveal Melanoma Patients

PURPOSE To assess the safety and efficacy of dendritic cell vaccination in metastatic uveal melanoma. DESIGN Interventional case series. METHODS We analyzed 14 patients with metastatic uveal melanoma treated with dendritic cell vaccination. Patients with metastatic uveal melanoma received at least 3 vaccinations with autologous dendritic cells, professional antigen-presenting cells loaded with melanoma antigens gp100 and tyrosinase. The main outcome measures were safety, immunologic response, and overall survival. RESULTS Tumor-specific immune responses were induced with dendritic cell vaccination in 4 (29%) of 14 patients. Dendritic cell-vaccinated patients showed a median overall survival with metastatic disease of 19.2 months, relatively long compared with that reported in the literature. No severe treatment-related toxicities (common toxicity criteria grade 3 or 4) were observed. CONCLUSIONS Dendritic cell vaccination is feasible and safe in metastatic uveal melanoma. Dendritic cell-based immunotherapy is potent to enhance the hosts antitumor immunity against uveal melanoma in approximately one third of patients. Compared with other prospective studies with similar inclusion criteria, dendritic cell vaccination may be associated with longer than average overall survival in patients with metastatic uveal melanoma.

Metastatic disease in polyploid uveal melanoma patients is associated with BAP1 mutations

PURPOSE Most of the uvea melanoma (UM) display a near-diploid (normal, -2N) karyotype with only a few chromosomal changes. In contrast to these simple aberrations 18% of the UM samples show a polyploid character (>2N) and this was associated with an unfavorable prognosis. This study attempts to gain insight in the prognostic value of polyploidy in UM. METHODS In 202 patients the ploidy status of the UM was determined using cytogenetic analysis, fluorescence-in-situ-hybridization (FISH), multiplex ligation dependent probe amplification (MLPA), and/or single nucleotide polymorphism (SNP) array analysis. Immunohistochemistry was used to determine the BAP1 expression and mutation analyses of BAP1 (coding regions) and the mutation hotspots for the SF3B1, EIF1AX, GNAQ, and GNA11 genes was carried out using Sanger sequencing or whole-exome sequencing. RESULTS Twenty-three patients had a polyploid UM karyotype (11.4%). Patients with a polyploid tumor had larger tumors (15.61 vs. 13.13 mm, P = 0.004), and more often loss of heterozygosity of chromosome 3 (P = 0.003). No difference in occurrence of mutations between polyploid and diploid tumors was observed for BAP1, SF3B1, EIF1AX, GNAQ, and GNA11. Polyploidy did not affect survival (P = 0.143). BAP1 deficiency was the only significant independent prognostic predictor for patients with polyploid tumors, with a 16-fold increased hazard ratio (HR 15.90, P = 0.009). CONCLUSIONS The prevalence of mutations in the UM related genes is not different in polyploid UM compared with diploid UM. Moreover, similar to patients with diploid UM, BAP1 mutation is the most significant prognostic predictor of metastasis in patients with polyploid UM.

Genetics of uveal melanoma

In this review, we discuss the genetic features of uveal melanoma and the different available genetic and genomic tests. Despite improvements in diagnosis and treatment of this intraocular tumor, there has not been a change in survival in the past few decades. Insights into the molecular changes in uveal melanoma have provided us with the opportunity to assess the risk of metastatic disease for individual patients. There are few treatments available for metastasized uveal melanoma and, in a relatively short time-frame, patients with this condition die because of this metastasis. The focus of research may need to shift towards adjuvant therapies for high-risk uveal melanomas.

In situ adenocarcinoma ex pleomorphic adenoma of the lacrimal gland.

A case of an in situ adenocarcinoma of the lacrimal gland that developed in a recurring pleomorphic adenoma is presented. Six years after incomplete resection of a pleomorphic adenoma, the patient complained of a progressive swelling of her right upper eyelid without pain or diplopia. An orbital computerized tomography scan showed an inhomogeneous mass in the right lacrimal gland region without bone involvement. After local excision of the in situ adenocarcinoma ex pleomorphic adenoma, the tumour has been in control for over 6 years.

Adjuvant Dendritic Cell Vaccination in High-Risk Uveal Melanoma.

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults with an annual incidence of 4 to 10 per million in the white population. The 5-year overall survival (OS) rate is approximately 70% to 80%. Up to 50% of patients with UM develop metastases, usually after a long disease-free interval (2-5 years). If metastatic disease is present, the prognosis is dismal with a 1-year OS rate of 10% to 40%. Currently, no effective systemic treatment improving OS is available for patients with metastatic UM, nor has any adjuvant treatment shown survival benefit. Our research group and others have performed several prospective dendritic cell (DC) vaccination studies in patients with cutaneous melanoma showing little toxicity and promising immunologic and clinical results. [...]

Concurrent Myopathy in Patients with Graves’ Orbitopathy

The extraocular muscles may be affected by various disorders, including inflammatory myopathy, myotonic dystrophy, mitochondrial disease, vitamin E deficiency and neoplasia (Kaminski et al., 2002; Schoser & Pongratz, 2006). The distinct properties of the extraocular muscles compared to other striated muscles–a more complex myosin expression, higher metabolic rate, and a higher maximum firing frequency–explain their frequent involvement in conditions such as myasthenia gravis, chronic progressive external ophthalmoplegia (CPEO), and Graves’ orbitopathy (GO) (Yu Wai Man et al., 2005). The clinical spectrum of these various myopathies are diverse. This report describes four patients whose extraocular muscles were affected by more than one disorder simultaneously. The concurrence of GO and a specific neuromuscular disorder seems extremely rare, but may be underreported.