Anna E. Koopmans

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.

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

BackgroundTuberous 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.MethodsHere 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.ResultsWe 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.ConclusionsTargeted NGS of the TSC1 and TSC2 loci is a suitable method to increase the yield of mutations identified in the TSC patient population.

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.

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.

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.

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

Evaluation of: Gerami P, Li G, Pouryazdanparast P et al. A highly specific and discriminatory FISH assay for distinguishing between benign and malignant melanocytic neoplasms. Am. J. Surg. Pathol. 36(6), 808–817 (2012). 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.

Diagnosis and Management of Uveal Melanoma

Uveal melanoma (UM) is the most common cause of primary eye cancer in the developed world. UM may arise in the iris, ciliary body or choroid. Choroidal melanomas are the most frequent and usually display a discoid, domeor mushroom-shaped growth pattern. Diagnosis is based on a clinical examination with the slit lamp and indirect ophthalmoscope together with ultrasonography of the eye. Despite improvements of primary treatment and a shift towards more conservative eye treatments, survival has not improved during the past decades. Aims of conservative treatment of UM are to elucidate the tumour and to retain the eye and in some cases the visual function. Primary treatment of UM consists of several options such as brachytherapy with either ruthenium-106 (Ru-106) and iodine-125 (I-125) isotopes, transpupillary therapy, proton beam therapy, stereotactic radiotherapy, photodynamic therapy and surgical excision. Enucleation must be reserved for patients with large or advanced tumours, or when extraocular extension is present.