Uveal Melanoma: GNAQ and GNA11 Mutations in a Greek Population

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Uveal Melanoma: GNAQ and GNA11 Mutations in a Greek Population ANTICANCER RESEARCH 37 : 5719-5726 (2017) doi:10.21873/anticanres.12010 Uveal Melanoma: GNAQ and GNA11 Mutations in a Greek Population FILIPPOS PSINAKIS 1, ANASTASIA KATSELI 2, CHRYSSANTHY KOUTSANDREA 1, KONSTANTINA FRANGIA 3, LINA FLORENTIN 2, DESPINA APOSTOLOPOULOU 2, KONSTANTINA DIMAKOPOULOU 4, DIMITRIOS PAPAKONSTANTINOU 5, ELENI GEORGOPOULOU 6 and DIMITRIOS BROUZAS 1 11st Department of Ophthalmology, National and Kapodistrian University of Athens, Athens, Greece; 2ALFA LAB, Molecular Biology and Cytogenetics Center, Leto Maternity Hospital, Athens, Greece; 3HistoBio Diagnosis Pathology Center, Athens, Greece; 4Department of Hygiene, Epidemiology and Medical Statistics, Medical School, National and Kapodistrian University of Athens, Athens, Greece; 5Department of Opthalmology, University of Athens, Georgios Gennimatas General Hospital, Athens, Greece; 6Private Practice, Athens, Greece Abstract. Background/Aim: Uveal melanoma is the most Uveal melanoma (UM) is the most common primary common primary adult intraocular malignancy. It is known malignancy of the eye, arising from melanocytes of the to have a strong metastatic potential, fatal for the vast choroid, ciliary body and iris. The disruption of specific majority of patients. In recent years, meticulous cytogenetic signaling pathways is considered to be involved in its and molecular profiling has led to precise prognostication, tumorigenesis. One of the main known pathways is mitogen- that unfortunately is not matched by advancements in activated protein kinase (MAPK)/ERK, known to be adjuvant therapies. G Protein subunits alpha Q (GNAQ) and disturbed as a result of G protein subunit alpha Q ( GNAQ ) alpha 11 (GNA11) are two of the major driver genes that or subunit alpha 11 ( GNA11 ) mutations (1). These mutations contribute to the development of uveal melanoma. appear to play a major role in the development of UM. Understanding their prognostic significance can allow GNAQ and GNA11 are proto oncogenes that encode tailored management and facilitate their use in the on-going closely related Gq alpha subunits of the heterotrimeric Gq quest of targeted uveal melanoma therapies. Materials and protein that activates phospholipase C, leading to a series of Methods: Formalin-fixed, paraffin-embedded specimens were downstream signaling effects, one of which is the activation obtained from 47 patients of Greek origin, with uveal of the MAPK growth signaling pathway. Activation of Gq is melanoma. GNAQ and GNA11 genes were screened for normally terminated by a GTPase activity intrinsic to the G- mutations in exons 4 and 5, by polymerase chain reaction alpha subunit. However, mutations that occur in UM at and Sanger sequencing. Results: The overall mutation amino acid residues glutamine-209 and arginine-183 disable frequency of GNAQ/GNA11 genes was 42.4%. A novel the GTPase activity and prevent inactivation of the protein, mutation c.625_626delinsGC was identified in GNA11. No leading to constitutive activation of the MAPK pathway and correlation was observed between the mutation status and other growth pathways (2). metastasis occurrence or overall survival time of patients. The GNAQ and GNA11 mutations at glutamine-209 and Conclusion: Mutations in GNAQ and GNA11 genes in this arginine-183 are somatic and mutually exclusive (3), with Greek population present frequencies that qualify them as glutamine-209 mutations being much more frequent (4). potential targets for customized therapy. They are found in benign uveal nevi and in the vast majority of UM, with a reported frequency of 80-88% (3, 5). This suggests that perhaps they are initiating events and are not sufficient for full malignant transformation (6). Correspondence to: Anastasia Katseli, ALFA LAB, Molecular Discovering these mutations is part of the major recent Biology and Cytogenetics Center, Leto Maternity Hospital, Athens, Greece. Tel: +30 6948858103, Fax: +30 2106902083, e-mail: advances in molecular genetics that have enabled the gradual katseli.anastasia@gmail.com construction of a detailed molecular landscape of UM. Better understanding of underlying genetic and molecular Key Words: Uveal melanoma, GNAQ, GNA11, Sanger-sequencing. abnormalities implicated in the development and progression 5719 ANTICANCER RESEARCH 37 : 5719-5726 (2017) Table I. Clinicopathological data of the patients included in this study. Parameter Value Total patients 33 Age at enucleation, years 62±12.7 (39-88) Male/female 15 (45.45%)/18 (54.55%) Histopathological cell type/cellular type Spindle 5 (15.15%) Mixed 23 (69.7%) Epithelioid 2 (6.06%) Necrotic 2 (6.06%) Unknown 1 (3.03%) Tumor location Figure 1. Polymerase chain reaction products of exons 4 and 5 of G Chorioid 28 (84.85%) protein subunit alpha 11 (GNA11) and alpha Q (GNAQ) genes, after Iris 1 (3.03%) visualization on ethidium bromide-stained agarose gel. Unknown 4 (12.12%) Metastasis (liver) 8 (24.24) Unknown 3 (9.1%) Survival time post-enucleation, months 48±30.4 each gene). All PCR primers (Table II) were designed to include an Data are the mean±SD (range) or n (%). M13 sequence tail to simplify the sequencing reaction setup. PCR reactions for each target were performed with 50-200 ng of genomic DNA as starting material in a 30 μl reaction mix with 500 μM dNTPs (Fermentas-Thermo Scientific Life Science, Milan, of UM provides a great opportunity for improved patient Italy), 0.35 μM primers, 2 mM MgCl 2, 1X Buffer and 2.5 U management and the development of targeted therapies. The Qiagen HotStarTaq DNA Polymerase (Qiagen, Hilden, Germany) estimation of the presence of GNAQ and GNA11 mutations on a Veriti 96-well Thermal Cycler (Applied Biosystems, Life Technologies Corporation, Foster City, CA, USA). Thermal cycling in Greek patients with UM will hopefully contribute to this conditions were as follows: 95˚C for 5 min, followed by 40 cycles ongoing effort. of 95˚C for 40 s, 60˚C for 40 s, 72˚C for 60 s, and a final extension at 72˚C for 10 min. Gel electrophoresis followed in order to ensure Materials and Methods the presence of amplicons in each reaction (Figure 1). Nucleofast96 PCR Plates (Macherey-Nagel, Düren, Germany) were used to This study was conducted at the First Department of Ophthalmology purify the amplification products. of the National and Kapodistrian University of Athens in association with ALFA LAB (Molecular Biology & Cytogenetics Center) and DNA sequencing. Mutational screening was carried out by direct HistoBio Diagnosis Pathology Center. In total, 47 tumor samples sequencing of fragments obtained by PCR using an ABI3130xl or were collected between 1996 and 2016 from patients of Greek ABI3500 Genetic Analyzer (Applied Biosystems, Life Technologies origin with UM, arising mainly from the choroid (Table I). Written Corporation). Sequencing data were analyzed using Sequencing informed consent was obtained from all patients and this study was Analysis v5.3.1 or SeqA6 software, respectively, (Applied approved by the Medical Ethics Committee of the University of Biosystems, Life Technologies Corporation) and Mutation Surveyor Athens (8649/9-4-2012). software (SoftGenetics, State College, PA, USA). Big Dye Terminator v1.1 Cycle Sequencing Kit (Applied Tumor tissue samples and DNA extraction. UM tissue specimens were Biosystems, Life Technologies Corporation) was used to set-up the formalin-fixed and embedded in paraffin blocks. A pathologist marked sequencing reactions, according to the manufacturer’s instructions. the most dense tumor area, which was subsequently macrodissected. The purified PCR products were used as templates. The sequencing These specimens were carefully selected to contain 80-100% neoplastic reaction was performed using the M13 forward and reverse primer cells. Paraffin-embedded tissue specimens (two 5- μm-thick sections cut sequences and the following program on the thermocycler: 30 cycles from each paraffin block) were retrieved from the Pathology of a 10-s denaturation at 96˚C, annealing of the primer at 50˚C for 5 Department of HistoBio Diagnosis Laboratory. DNA was extracted s, and the extension step at 60˚C for 4 min. The sequencing products from formalin-fixed, paraffin-embedded (FFPE) tissue sections, using were purified before capillary electrophoresis to remove MagCore Genomic DNA-FFPE One-Step Kit (RBC Bioscience, New unincorporated ddNTPs using Sephadex columns (Illustra Sephadex Taipei City, Taiwan) with an Automated Nucleic Acid Extractor G-50/fine DNA grade; GE Healthcare, Chicago, IL, USA). MagCore HF16 (RBC Bioscience, New Taipei City, Taiwan). DNA concentration and quality were evaluated using a Nanodrop2000 Statistical analysis. The distribution of all continuous and categorical spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). variables is described by reporting the mean (standard deviation; SD) values; range and frequency and percentage, respectively. The Amplification step. Polymerase chain reactions (PCR) were set-up and samples were analyzed all together and were further analyzed after optimized for the two genes on chromosome 9, GNAQ (NM_002072) being subdivided into two groups depending on the storage and GNA11 (NM_002067) and four target sequences (exons 4, 5 of preservation time of FFPE specimens:group A included samples 5720 Psinakis et al : GNAQ and GNA11 Mutations in a Greek Uveal Melanoma Population Table II. Primer sequences used for mutation analysis of G protein subunit alpha 11 (GNA11) and alpha Q (GNAQ) genes. Gene Exon Primer sequence PCR product (bp) GNA11 4 5’ M13F tail-GTGCTGTGTCCCTGTCCTG
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