Two distinct pathways of p16 gene inactivation in gallbladder cancer.
OBJECTIVE
To examine the mechanism of inactivation of the p16 gene in gallbladder cancer, and to investigate p16 alterations and their correlation with clinicopathological features.


METHODS
Specimens were collected surgically from 51 patients with gallbladder cancer. We evaluated the status of protein expression, loss of heterozygosity (LOH), homozygous deletion and promoter hypermethylation using immunohistochemistry, microsatellite analysis, quantitative real-time polymerase chain reaction (PCR) and methylation-specific PCR, respectively. In addition, mutations were examined by direct DNA sequencing.


RESULTS
Homozygous deletions of the p16 gene exon2, LOH at 9p21-22, p16 promoter hypermethylation, and loss of p16 protein expression were detected in 26.0% (13/50), 56.9% (29/51), 72.5% (37/51) and 62.7% (32/51), respectively. No mutations were found. LOH at 9p21 correlated with the loss of p16 protein expression (P < 0.05). Homozygous deletion of the p16 gene, a combination LOH and promoter hypermethylation, and multiple LOH at 9p21 were significantly correlated with the loss of p16 protein expression (P < 0.05). LOH at 9p21 and promoter hypermethylation of the p16 gene were detected in 15.4% (2/13) and 92.3% (12/13) of the tumors with homozygous deletion of the p16 gene, respectively. P16 alterations were not associated with clinicopathological features.


CONCLUSIONS
Our results suggest that LOH and homozygous deletion may be two distinct pathways in the inactivation of the p16 gene. Homozygous deletion, a combination of LOH and promoter hypermethylation, and multiple LOH are major mechanisms of p16 inactivation in gallbladder cancer.
Tadokoro
Hiroyuki
H
Department of Gastroenterology, Juntendo Tokyo Koto Geriatric Medical Center, 3-3-20 Shinsuna, Koto-ku, Tokyo 136-0075, Japan. heroyou27@yahoo.co.jp
Shigihara
Takako
T
Ikeda
Tomomi
T
Takase
Masaru
M
Suyama
Masafumi
M
D000230:Adenocarcinoma / Q000737:chemistry / Q000235:genetics* / Q000473:pathology; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D018196:Carcinoma, Adenosquamous / Q000737:chemistry / Q000235:genetics* / Q000473:pathology; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000032:analysis / Q000235:genetics*; D019175:DNA Methylation; D004252:DNA Mutational Analysis; D005091:Exons; D005260:Female; D005706:Gallbladder Neoplasms / Q000737:chemistry / Q000235:genetics* / Q000473:pathology; D017353:Gene Deletion; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D019942:Genes, p16; D006720:Homozygote; D006801:Humans; D007150:Immunohistochemistry; D019656:Loss of Heterozygosity; D008297:Male; D018895:Microsatellite Repeats; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic
D019941:Cyclin-Dependent Kinase Inhibitor p16