RESUMO
Cholangiocarcinoma in northeast Thailand is associated with liver fluke infection. Mechanisms of inactivation of the p15(INK4b), p16(INK4a), and p14(ARF) have been reported in many human cancers but have not hitherto been studied in liver fluke-related cholangiocarcinoma, particularly genetic and epigenetic effects on protein expression. We investigated loss of heterozygosity and microsatellite instability and performed fine mapping of the chromosomal region 9p21-pter in 94 microdissected cholangiocarcinoma samples using polymerase chain reaction based-microsatellite markers. Methylation and protein expression of p14(ARF), p15(INK4b), and p16(INK4a) was determined using methylation-specific polymerase chain reaction and immunohistochemistry, respectively. Genetic and epigenetic alterations, including loss of protein expression, were correlated with clinicopathological data. Fine mapping at 9p21-pter showed a distinctive region between D9S286 and D9S1752 of common loss. Methylation frequency was 40.2% for p14(ARF), 48.9% for p15(INK4b), and 28.3% for p16(INK4a). Loss of protein expression of p14(ARF), p15(INK4b), and p16(INK4a) was 30.9%, 58%, and 81.5%, respectively. Both p14(ARF) methylation and allelic loss at 9p21 were associated with loss of p14(ARF) expression. Poor prognosis was associated with loss of p16(INK4a) expression. In conclusion, mechanisms of inactivation of p14(ARF), p15(INK4b), and p16(INK4a) in liver fluke-related cholangiocarcinoma are preferentially different, by which epigenetic event being the main mechanism of p14(ARF), whereas p16(INK4a) and p15(INK4b) inactivation occurs through genetic and both genetic and epigenetic events, respectively.
Assuntos
Colangiocarcinoma/genética , Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Fasciolíase/complicações , Inativação Gênica/fisiologia , Genes p16/fisiologia , Proteína Supressora de Tumor p14ARF/genética , Adulto , Idoso , Mapeamento Cromossômico , Metilação de DNA , Epigênese Genética , Feminino , Humanos , Imuno-Histoquímica , Perda de Heterozigosidade , Masculino , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Família Multigênica , PrognósticoRESUMO
Cholangiocarcinoma is a crucial health problem in Northeast Thailand where liver fluke infection is endemic. However, molecular genetic and epigenetic mechanisms involved in carcinogenesis of this cancer remain unclear. We attempted to study genetic and epigenetic alterations of hMLH1 gene in 65 intrahepatic cholangiocarcinoma using polymerase chain reaction (PCR) based microsatellite marker D3S1611 and methylation-specific PCR, respectively. Of 65 cases, 29 (44.6%) showed hypermethylation of hMLH1 promoter. Loss of heterozygosity (LOH) of hMLH1 was detected in 12 of 51 informative cases (23.5%). Five out of 29 (17.2%) methylated cases demonstrated LOH. Aberrant methylation of hMLH1 promoter was significantly associated with poorly differentiated type (P=0.013). Our study suggests that both genetic and epigenetic mechanisms cause the inactivation of hMLH1 where epigenetic is a major event resulting in mismatch repair deficiency and contributing to carcinogenesis of liver fluke related cholangiocarcinoma. Since, gene silencing by methylation is an early event in carcinogenesis, promoter hypermethylation of hMLH1 may be a molecular targeted therapy and prevention of liver fluke related cholangiocarcinoma.
