Mutational analysis of selected genes in the TGFbeta, Wnt, pRb, and p53 pathways in primary uveal melanoma.
OBJECTIVE
It is known that the pRb pathway cell-cycle inhibitor p16(INK4A) plays a significant role in cutaneous melanoma and that alteration of p16(INK4A), which resides within the 9p21-22 locus that also contains p15(INK4B) and p14(ARF), may occur in up to one third of uveal melanomas. The absence of TGFbeta responsiveness noted in cultured uveal melanoma cells also suggests that the TGFbeta pathway plays a role in the formation of this tumor. Therefore, mutational screening was performed in several key genes in tumor-suppressor pathways that are known to be altered in some uveal melanomas.


METHODS
Using denaturing high-performance liquid chromatography (DHPLC) analysis and DNA sequencing, a series of 67 uveal melanomas were screened for inactivating mutations in the TGFbeta pathway members Smad4 and TGFbeta receptor type 2 (TGFbetaR2), the downstream cell-cycle inhibitor p15(INK4B), and the cell-cycle inhibitors p14(ARF) and p16(INK4A). p16(INK4A) was also investigated for promoter hypermethylation. Mutational analysis was also performed on the Wnt pathway gene beta-catenin, known to be mutated in approximately one quarter of cutaneous melanoma cell lines.


RESULTS
Polymorphisms in p16(INK4A) were detected in 3 of 50 samples, but no inactivating mutations were detected in any of the genes screened. Promoter hypermethylation of p16(INK4A) was detected in 5 of 55 tumors, and loss of heterozygosity of the p16(INK4A) locus was detected in 5 of 16 tumors.


CONCLUSIONS
Most primary uveal melanomas do not appear to contain somatic mutations in Smad4, TGFbetaR2, p14(ARF), p15(INK4B), p16(INK4A), or beta-catenin. However, methylation of the p16(INK4A) promoter and loss of heterozygosity of the p14(ARF)-p16(INK4A) locus occurs in some tumors.
Centre for Cutaneous Research, St Bartholomew's and the London School of Medicine and Dentistry, Queen Mary College, University of London, London, United Kingdom.
Scott C
SC
Edmunds
David P
DP
Kelsell
John L
JL
Hungerford
Ian A
IA
Cree
D018797:Cell Cycle Proteins / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics; D019175:DNA Methylation; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000032:analysis; D005260:Female; D006801:Humans; D019656:Loss of Heterozygosity; D008297:Male; D008545:Melanoma / Q000235:genetics*; D008875:Middle Aged; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics; D016212:Transforming Growth Factor beta / Q000235:genetics*; D025803:Tumor Suppressor Protein p14ARF / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*; D025521:Tumor Suppressor Proteins; D014604:Uveal Neoplasms / Q000235:genetics*; D051153:Wnt Proteins; D029961:Zebrafish Proteins
C493966:CDKN2B protein, human; D018797:Cell Cycle Proteins; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004273:DNA, Neoplasm; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D016212:Transforming Growth Factor beta; D025803:Tumor Suppressor Protein p14ARF; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D051153:Wnt Proteins; D029961:Zebrafish Proteins