Uveal Melanomas with SF3B1 Mutations: A Distinct Subclass Associated with Late-Onset Metastases.

PURPOSE: To investigate the prevalence and prognostic value of SF3B1 and EIF1AX mutations in uveal melanoma (UM) patients. DESIGN: Case series. PARTICIPANTS: Cohort of 151 patients diagnosed with and treated for UM. METHODS: SF3B1 and EIF1AX mutations in primary tumors were investigated using whole-exome sequencing (n = 25) and Sanger sequencing (n = 151). For the detection of BAP1 mutations, a previously reported cohort of 90 patients was extended using BAP1 sequencing or immunohistochemistry. MAIN OUTCOME MEASURES: The status of SF3B1, EIF1AX, and BAP1 in tumors of patients were correlated to clinical, histopathologic, and genetic parameters. Survival analyses were performed for patients whose tumors had SF3B1, EIF1AX, and BAP1 mutations. RESULTS: Patients with tumors harboring EIF1AX mutations rarely demonstrated metastases (2 of 28 patients) and overall had a longer disease-free survival (DFS; 190.1 vs. 100.2 months; P < 0.001). Within the patient group with disomy 3, UM patients with an SF3B1 mutation had an increased metastatic risk compared with those without an SF3B1 mutation (DFS, 132.8 vs. 174.4 months; P = 0.008). Patients with such a mutation were more prone to demonstrate late metastases (median, 8.2 years; range, 23-145 months). Patients with UM and loss of BAP1 expression had a significantly decreased survival (DFS, 69.0 vs. 147.9 months; P < 0.001). CONCLUSIONS: According to our data, patients with UM can be classified into 3 groups, of which EIF1AX-mutated tumors and tumors without BAP1, SF3B1, or EIF1AX mutations are associated with prolonged survival and low metastatic risk, SF3B1-mutated tumors are associated with late metastasis, and tumors with an aberrant BAP1 are associated with an early metastatic risk and rapid decline in patient DFS.

Targeted Next Generation Sequencing reveals previously unidentified TSC1 and TSC2 mutations.

BACKGROUND: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in TSC1 and TSC2. Conventional DNA diagnostic screens identify a TSC1 or TSC2 mutation in 75 - 90% of individuals categorised with definite TSC. The remaining individuals either have a mutation that is undetectable using conventional methods, or possibly a mutation in another as yet unidentified gene. METHODS: Here we apply a targeted Next Generation Sequencing (NGS) approach to screen the complete TSC1 and TSC2 genomic loci in 7 individuals fulfilling the clinical diagnostic criteria for definite TSC in whom no TSC1 or TSC2 mutations were identified using conventional screening methods. RESULTS: We identified and confirmed pathogenic mutations in 3 individuals. In the remaining individuals we identified variants of uncertain clinical significance. The identified variants included mosaic changes, changes located deep in intronic sequences and changes affecting promoter regions that would not have been identified using exon-only based analyses. CONCLUSIONS: Targeted NGS of the TSC1 and TSC2 loci is a suitable method to increase the yield of mutations identified in the TSC patient population.

Clinical significance of immunohistochemistry for detection of BAP1 mutations in uveal melanoma.

Uveal melanoma is a lethal cancer with a strong propensity to metastasize. Limited therapeutic options are available once the disease has disseminated. A strong predictor for metastasis is the loss of chromosome 3. Inactivating mutations in BAP1 encoding the BRCA1-associated protein 1 and located on chromosome 3p21.1, have been described in uveal melanoma and other types of cancer. In this study, we determined the prevalence of somatic BAP1 mutations and examined whether these mutations correlate with the functional expression of BAP1 in uveal melanoma tissue and with other clinical, histopathological and chromosomal parameters. We screened a cohort of 74 uveal melanomas for BAP1 mutations, using different deep sequencing methods. The frequency of BAP1 mutations in our study group was 47%. The expression of BAP1 protein was studied using immunohistochemistry. BAP1 staining was absent in 43% of the cases. BAP1 mutation status was strongly associated with BAP1 protein expression (P<0.001), loss of chromosome 3 (P<0.001), and other aggressive prognostic factors. Patients with a BAP1 mutation and absent BAP1 expression had an almost eightfold higher chance of developing metastases compared with those without these changes (P=0.002). We found a strong correlation between the immunohistochemical and sequencing data and therefore propose that, immunohistochemical screening for BAP1 should become routine in the histopathological work-up of uveal melanoma. Furthermore, our analysis indicates that loss of BAP1 may be particularly involved in the progression of uveal melanoma to an aggressive, metastatic phenotype.

Effect of supplementation with zinc and other micronutrients on malaria in Tanzanian children: a randomised trial.

BACKGROUND: It is uncertain to what extent oral supplementation with zinc can reduce episodes of malaria in endemic areas. Protection may depend on other nutrients. We measured the effect of supplementation with zinc and other nutrients on malaria rates. METHODS AND FINDINGS: In a 2×2 factorial trial, 612 rural Tanzanian children aged 6-60 months in an area with intense malaria transmission and with height-for-age z-score≤-1.5 SD were randomized to receive daily oral supplementation with either zinc alone (10 mg), multi-nutrients without zinc, multi-nutrients with zinc, or placebo. Intervention group was indicated by colour code, but neither participants, researchers, nor field staff knew who received what intervention. Those with Plasmodium infection at baseline were treated with artemether-lumefantrine. The primary outcome, an episode of malaria, was assessed among children reported sick at a primary care clinic, and pre-defined as current Plasmodium infection with an inflammatory response, shown by axillary temperature ≥37.5°C or whole blood C-reactive protein concentration ≥ 8 mg/L. Nutritional indicators were assessed at baseline and at 251 days (median; 95% reference range: 191-296 days). In the primary intention-to-treat analysis, we adjusted for pre-specified baseline factors, using Cox regression models that accounted for multiple episodes per child. 592 children completed the study. The primary analysis included 1,572 malaria episodes during 526 child-years of observation (median follow-up: 331 days). Malaria incidence in groups receiving zinc, multi-nutrients without zinc, multi-nutrients with zinc and placebo was 2.89/child-year, 2.95/child-year, 3.26/child-year, and 2.87/child-year, respectively. There was no evidence that multi-nutrients influenced the effect of zinc (or vice versa). Neither zinc nor multi-nutrients influenced malaria rates (marginal analysis; adjusted HR, 95% CI: 1.04, 0.93-1.18 and 1.10, 0.97-1.24 respectively). The prevalence of zinc deficiency (plasma zinc concentration <9.9 µmol/L) was high at baseline (67% overall; 60% in those without inflammation) and strongly reduced by zinc supplementation. CONCLUSIONS: We found no evidence from this trial that zinc supplementation protected against malaria. TRIAL REGISTRATION: ClinicalTrials.gov NCT00623857

Retinal haemangioblastomas in von Hippel-Lindau germline mutation carriers: progression, complications and treatment outcome.

PURPOSE: Evaluation of phenotype and treatment outcome of retinal haemangioblastomas (RH) in von Hippel-Lindau (VHL) disease and correlation of these features with the genotype of VHL germline mutation carriers. METHODS: Retrospective analysis of a longitudinal cohort of 21 VHL germline mutation carriers and RH. Clinical and genetic data were obtained to analyse the correlation of genotype with phenotype and treatment outcomes. RESULTS: All patients were categorized in two genotypic categories: missense mutations (MM) and truncating mutations (TM). Mean follow-up duration was 16.3 years and did not differ significantly between mutation groups (p = 0.383). Missense mutations (MM) carriers (n = 6) developed more progression-related complications compared to TM carriers (n = 15) (p = 0.046). Vitreoretinal surgery was more often applied in MM carriers (p = 0.036). Moderate (visual acuity (VA)20/80 to 20/200) to severe (VA < 20/200) visual impairment was observed in 53.3% of the eyes of MM carriers and 28.1% of the eyes of TM carriers at last recorded visit. CONCLUSION: Missense mutations in VHL patients seem to have a higher prevalence of progression-related complications. Missense mutations (MM) carriers required therefore more often vitreoretinal surgical treatment with a worse treatment outcome. Genetic analysis may play a role in determining a pro-active treatment strategy and prognosis for RH.

Metastatic disease in uveal melanoma: importance of a genetic profile?

Mutation of SF3B1 has been identified in low-grade uveal melanoma with a good prognosis. In this study, we compare chromosomal aberrations and gene mutations between a primary uveal melanoma and its multiple hepatic and peripancreatic metastases. DNA was isolated from a large primary uveal melanoma after fractionated stereotactic radiotherapy and three distinct metastases (two liver samples and one peripancreatic lymph node) to perform single-nucleotide polymorphism array and fluorescent in-situ hybridization. We analyzed mutations in uveal melanoma target genes BAP1, GNAQ, GNA11, SF3B1, and EIF1AX. The primary tumor showed no abnormalities in chromosome 3, whereas metastases showed deletion of at least 3q12.1-q24 and the BAP1 gene was not mutated. All samples indicated the following consistent chromosomal aberrations: loss of 1p, gain of 6p, and gain of 8q. Subsequently, heterozygous SF3B1 and heterozygous GNA11 mutations were observed. The metastases showed more genetic aberrations than the primary tumor and may therefore represent the genetic status of the tumor before irradiation, whereas the current primary tumor shows presumably irradiation artifacts. An early occurring mutation in GNA11 was observed in all samples. The SF3B1 mutation seems to predispose for late metastatic disease in the absence of a BAP1 mutation.

Clinical and etiological heterogeneity in patients with tracheo-esophageal malformations and associated anomalies.

Esophageal Atresia (EA) is a severe developmental defect of the foregut that presents with or without a Tracheo-Esophageal Fistula (TEF). The prevalence of EA/TEF over time and around the world has been relatively stable. EA/TEF is manifested in a broad spectrum of anomalies: in some patients it manifests as an isolated atresia or fistula, but in over half it affects several organ systems. While the associated malformations are often those of the VACTERL spectrum (Vertebral, Anorectal, Cardiac, Tracheo-Esophageal, Renal and Limb), many patients are affected by other malformations, such as microcephaly, micrognathia, pyloric stenosis, duodenal atresia, a single umbilical artery, and anomalies of the genitourinary, respiratory and gastrointestinal systems. Though EA/TEF is a genetically heterogeneous condition, recurrent genes and loci are sometimes affected. Tracheo-Esophageal (TE) defects are in fact a variable feature in several known single gene disorders and in patients with specific recurrent Copy Number Variations and structural chromosomal aberrations. At present, a causal genetic aberration can be identified in 11-12% of patients. In most, EA/TEF is a sporadic finding; the familial recurrence rate is low (1%). As this suggests that epigenetic and environmental factors also contribute to the disease, non-syndromic EA/TEF is generally believed to be a multifactorial condition. Several population-based studies and case reports describe a wide range of associated risks, including age, diabetes, drug use, herbicides, smoking and fetal alcohol exposure. The phenotypical and genetic heterogeneity seen in EA/TEF patients indicates not one underlying cause, but several. Unraveling the complex multifactorial and heterogeneous etiology of EA/TEF and associated features will require large cohorts of patients. Combined statistical analysis of component findings, genome sequencing, and genome wide association studies will elucidate new causal genetic defects and predisposing loci in the etiology within specific sub-populations. Improved knowledge of environmental risk factors, genetic predisposition and causal genetic syndromes may improve prediction and parental counseling, and prevent co-morbidity.

The prognostic value of extraocular extension in relation to monosomy 3 and gain of chromosome 8q in uveal melanoma.

PURPOSE: To identify the prognostic value of extraocular extension in enucleated uveal melanoma (UM) patients and to correlate extraocular extension to chromosomal aberrations, metastasis-free survival (MFS), and clinico-histopathological risk factors. METHODS: Retrospective study of patients with UM treated with enucleation between 1987 and 2011. Melanoma-related metastasis and death were recorded. Statistical analysis (log-rank test or Cox regression analysis) was performed to correlate MFS with tumor characteristics, extraocular extension, episcleral diameter of the extraocular extension, cell type, extracellular matrix patterns, inflammation, loss of chromosome 3, and gain of chromosome 8q. RESULTS: In 43 (12%) of 357 patients, extraocular extension was observed. In this subset of patients, we noted a reduced survival of 70 months (105.5 months, P = 0.010) compared with patients without extraocular extension (175.8 months). Patients with gain of chromosomal region 8q in UM with extraocular extension had an increased risk of metastatic disease (P < 0.001). In multivariate Cox proportional hazard analysis, largest basal tumor diameter (P = 0.001), extracellular matrix patterns (P = 0.009), episcleral diameter of the extraocular extension (P = 0.016), loss of chromosome 3 (P < 0.001), and gain of 8q (P < 0.001) were independent predictors for MFS. CONCLUSIONS: Larger episcleral diameter of the extraocular extension and additional gain of chromosome 8q in extraocular extension UM correlates to a worse prognosis. MFS is significantly reduced in UM with a large basal tumor diameter, extracellular matrix patterns, loss of chromosome 3, and gain of chromosome 8q.

Prevalence and implications of TERT promoter mutation in uveal and conjunctival melanoma and in benign and premalignant conjunctival melanocytic lesions.

PURPOSE: Hot-spot mutations in the promoter region of telomerase reverse transcriptase (TERT promoter mutations) occur frequently in cutaneous and conjunctival melanoma and are exceedingly rare in uveal melanoma. No information is available on the presence of these mutations in the conjunctival melanocytic precursor lesion primary acquired melanosis (PAM). We tested a cohort of uveal and conjunctival melanomas as well as conjunctival benign and premalignant melanocytic lesions for TERT promoter mutations in order to elucidate the role of these mutations in tumor progression. METHODS: TERT promoter mutation analysis on fresh tumor DNA and DNA from formalin-fixed, paraffin-embedded specimens was performed by SNaPshot analysis in 102 uveal melanomas, 39 conjunctival melanomas, 26 PAM with atypia, 14 PAM without atypia, and 56 conjunctival nevi. RESULTS: Mutations of the TERT promoter were not identified in conjunctival nevi or PAM without atypia, but were detected in 2/25 (8%) of PAM with atypia and 16/39 (41%) of conjunctival melanomas. A single TERT promoter mutation was detected in 102 uveal melanomas (1%). CONCLUSIONS: We present the second documented case of TERT promoter mutation in uveal melanoma. In comparison with other types of melanoma, TERT promoter mutations occur at extremely low frequency in uveal melanoma. TERT promoter mutations are frequent in conjunctival melanoma and occur at lower frequency in PAM with atypia but were not detected in benign conjunctival melanocytic lesions. These findings favor a pathogenetic tumor progression role for TERT promoter mutations in conjunctival melanocytic lesions.

Chemokine receptor CCR7 expression predicts poor outcome in uveal melanoma and relates to liver metastasis whereas expression of CXCR4 is not of clinical relevance.

PURPOSE: To examine the prognostic relevance of expression of the chemokine receptors CCR7 and CXCR4 and its ligand CXCL12 in uveal melanoma in nonmetastatic and metastatic patients with correlation to liver metastasis and overall survival. METHODS: Primary uveal melanoma specimens from 19 patients with correlating liver metastasis specimens and 30 primary uveal melanoma specimens of patients without metastasis were collected between the years 1988 and 2008. Expression of CCR7, CXCR4, and CXCL12 were studied using immunohistochemistry. Single nucleotide polymorphism (SNP) arrays were used to examine gains or losses of chromosomes 1, 3, 6, and 8 and the regions of CCR7 (17q12-q21.2), CXCR4 (2q21), and CXCL12 (10q11.1) genes. RESULTS: Strong cytoplasmic staining for CCR7 correlated with the presence of epithelioid cells (P = 0.037), tumor thickness (P = 0.011), lymphocytic infiltration (P = 0.041), and necrosis (P = 0.045). Nuclear staining for CXCR4 correlated with lymphocytic infiltration (P = 0.017). CXCL12 showed no correlation to histologic parameters. Single nucleotide polymorphism analyses showed no copy number variations in the regions of CCR7, CXCR4, or CXCL12. Strong expression of CCR7 was observed in 76% of the metastatic patients and 0% of nonmetastasis patients. In multivariate analysis, CCR7 staining was inversely correlated to overall survival and disease-free survival, whereas CXCR4 nuclear staining was not. CONCLUSIONS: Our data suggest that CCR7 plays a role in uveal melanoma metastasis and is associated with poor survival. CCR7 and its involved related pathways are of prognostic value in uveal melanoma and may prove to be a target for therapeutic intervention.

Multicolor FISH with improved sensitivity and specificity in the diagnosis of malignant melanoma.

The diagnosis of malignant melanoma remains a challenging aspect in the field of pathology. In the last years, FISH has become an important tool for the diagnosis of melanocytic tumors in addition to conventional microscopy. Benign and malignant melanomas can be discriminated using a four-probe FISH assay targeting 6p25, 6q23, Cep6 and 11q13. Gerami et al. proposed to refine this current probe set with the incorporation of chromosome 8q24 and 9p21 probes into the FISH assay, and hereby increase sensitivity and specificity for distinguishing between benign and malignant melanoma and improve the detection of spitzoid melanomas. In this article, the authors evaluate this newly-defined multicolor FISH probe set for the diagnosis of malignant melanoma. Optimizing diagnostic tests in malignant melanoma are important for current and future management of patients with melanocytic proliferations.