title: Digital PCR validates 8q dosage as prognostic tool in uveal melanoma.
abstract: BACKGROUND
  Uveal melanoma (UM) development and progression is correlated with specific molecular changes. Recurrent mutations in GNAQ and GNA11 initiate UM development while tumour progression is correlated with monosomy of chromosome 3 and gain of chromosome 8q. Hence, molecular analysis of UM is useful for diagnosis and prognosis. The aim of this study is to evaluate the use of digital PCR (dPCR) for molecular analysis of UM.
  
  
  METHODS
  A series of 66 UM was analysed with dPCR for three hotspot mutations in GNAQ/GNA11 with mutation specific probes. The status of chromosomes 3 and 8 were analysed with genomic probes. The results of dPCR analysis were cross-validated with Sanger sequencing, SNP array analysis, and karyotyping.
  
  
  RESULTS
  Using dPCR, we were able to reconstitute the molecular profile of 66 enucleated UM. With digital PCR, GNAQ/GNA11 mutations were detected in 60 of the 66 UM. Sanger sequencing revealed three rare variants, and, combined, these assays revealed GNAQ/GNA11 mutations in 95% of UM. Monosomy 3 was present in 43 and chromosome 8 aberrations in 52 of the 66 UM. Survival analysis showed that increasing 8q copy numbers were positively correlated with metastasis risk.
  
  
  CONCLUSIONS
  Molecular analysis with dPCR is fast and sensitive. Just like the recurrent genomic aberrations of chromosome 3 and 8, hotspot mutations in GNAQ and GNA11 are effectively detected in heterogeneous samples. Increased sensitivity contributes to the number of mutations and chromosomal aberrations detected. Moreover, quantification of copy number with dPCR validated 8q dosage as a sensitive prognostic tool in UM, of which implementation in disease prediction models will further improve prognostication.
authors.affiliation: Department of Ophthalmology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Mieke
authors.initials: M
authors.lastname: Versluis
authors.affiliation: Department of Ophthalmology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: de Lange
authors.affiliation: Department of Ophthalmology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Sake I
authors.initials: SI
authors.lastname: van Pelt
authors.affiliation: Department of Clinical Genetics, Laboratory for Diagnostic Genome Analysis (LDGA), Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Claudia A L
authors.initials: CA
authors.lastname: Ruivenkamp
authors.affiliation: Department of Clinical Genetics, Laboratory for Diagnostic Genome Analysis (LDGA), Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Wilma G M
authors.initials: WG
authors.lastname: Kroes
authors.affiliation: Department of Ophthalmology, Leiden University Medical Center, Leiden, The Netherlands; Department of Ophthalmology, The Second Hospital of Jilin University, Changchun, China.
authors.forename: Jinfeng
authors.initials: J
authors.lastname: Cao
authors.affiliation: Department of Ophthalmology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Martine J
authors.initials: MJ
authors.lastname: Jager
authors.affiliation: Department of Ophthalmology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Gre P M
authors.initials: GP
authors.lastname: Luyten
authors.affiliation: Department of Ophthalmology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Pieter A
authors.initials: PA
authors.lastname: van der Velden
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002893:Chromosomes, Human, Pair 3 / Q000235:genetics; D002898:Chromosomes, Human, Pair 8 / Q000235:genetics*; D005260:Female; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D018628:Gene Dosage; D006801:Humans; D008297:Male; D008545:Melanoma / Q000175:diagnosis* / Q000235:genetics; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011379:Prognosis; D014604:Uveal Neoplasms / Q000175:diagnosis* / Q000235:genetics; D055815:Young Adult; D000098943:Uveal Melanoma
chemical_list: C554062:GNA11 protein, human; C554061:GNAQ protein, human; D044385:GTP-Binding Protein alpha Subunits; D043802:GTP-Binding Protein alpha Subunits, Gq-G11