title: High throughput mass spectrometry-based mutation profiling of primary uveal melanoma.
abstract: OBJECTIVE
  We assessed for mutations in a large number of oncogenes and tumor suppressor genes in primary uveal melanomas using a high-throughput profiling system.
  
  
  METHODS
  DNA was extracted and purified from 134 tissue samples from fresh-frozen tissues (n = 87) or formalin-fixed, paraffin-embedded tissues (n = 47) from 124 large uveal melanomas that underwent primary treatment by enucleation. DNA was subjected to whole genome amplification and MALDI-TOF mass spectrometry-based mutation profiling (>1000 mutations tested across 120 oncogenes and tumor suppressor genes) using the OncoMap3 platform. All candidate mutations, as well as commonly occurring mutations in GNAQ and GNA11, were validated using homogeneous mass extension (hME) technology.
  
  
  RESULTS
  Of 123 samples, 97 (79%, representing 89 unique tumors) were amplified successfully, passed all quality control steps, and were assayed with the OncoMap platform. A total of 58 mutation calls was made for 49 different mutations across 26 different genes in 34/98 (35%) samples. Of 91 tumors that underwent hME validation, 83 (91%) harbored mutations in the GNAQ (47%) or GNA11 (44%) genes, while hME validation revealed two tumors with mutations in EGFR. These additional mutations occurred in tumors that also had mutations in GNAQ or GNA11.
  
  
  CONCLUSIONS
  The vast majority of primary large uveal melanomas harbor mutually-exclusive mutations in GNAQ or GNA11, but very rarely have the oncogenic mutations that are reported commonly in other cancers. When present, these other mutations were found in conjunction with GNAQ/GNA11 mutations, suggesting that these other mutations likely are not the primary drivers of oncogenesis in uveal melanoma.
authors.affiliation: Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114-3096, USA.
authors.forename: Anthony B
authors.initials: AB
authors.lastname: Daniels
authors.forename: Joo-Eun
authors.initials: JE
authors.lastname: Lee
authors.forename: Laura E
authors.initials: LE
authors.lastname: MacConaill
authors.forename: Emanuele
authors.initials: E
authors.lastname: Palescandolo
authors.forename: Paul
authors.initials: P
authors.lastname: Van Hummelen
authors.forename: Scott M
authors.initials: SM
authors.lastname: Adams
authors.forename: Margaret M
authors.initials: MM
authors.lastname: DeAngelis
authors.forename: William C
authors.initials: WC
authors.lastname: Hahn
authors.forename: Evangelos S
authors.initials: ES
authors.lastname: Gragoudas
authors.forename: J William
authors.initials: JW
authors.lastname: Harbour
authors.forename: Levi A
authors.initials: LA
authors.lastname: Garraway
authors.forename: Ivana K
authors.initials: IK
authors.lastname: Kim
mesh_terms: D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000235:genetics; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D005784:Gene Amplification; D020869:Gene Expression Profiling; D006801:Humans; D008545:Melanoma / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D009857:Oncogenes / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D025521:Tumor Suppressor Proteins / Q000235:genetics; D014604:Uveal Neoplasms / Q000235:genetics*
chemical_list: D004273:DNA, Neoplasm; C554062:GNA11 protein, human; C554061:GNAQ protein, human; D044385:GTP-Binding Protein alpha Subunits; D025521:Tumor Suppressor Proteins; D043802:GTP-Binding Protein alpha Subunits, Gq-G11