Aberrant methylation of p16(INK4a) and deletion of p15(INK4b) are frequentevents in human esophageal cancer in Linxian, China

p16(INK4a) and p15(INK4b) genes, which encode two functionally related CDK inhibitors, recently emerged as candidate tumor suppressor genes since they were both localized to 9p21, which frequently undergoes hemizygous and hom ozygous deletion in a variety of tumor types. To determine the mode of inac tivation of these two genes in human esophageal squamous cell carcinoma (ES CC), we performed multiple molecular analyses in 60 ESCC specimens from Lin xian, China using DNA methylation assay, LOH analysis, deletion screening a nd SSCP-sequencing. We observed that p16(INK4a) inactivation was predominan tly associated with aberrant methylation in the CpG island of its promoter region, whereas p15(INK4b) frequently had homozygous deletions. Compared wi th aberrant methylation, which occurred in 17 of 34 cases, homozygous delet ion of p16(INK4a) and LOH at its nearby D9S942 microsatellite marker were o bserved at a much lower frequency (17%), Intragenic mutation in p16(INK4a) gene was rare. In contrast, homozygous deletion in p15(INK4b) and LOH at th e nearby D9S171 marker were observed at frequencies of 35 and 47%, respecti vely, and the two events were significantly associated with each other. On the other hand, aberrant methylation of p15(INK4b) was relatively infrequen t (6/34) and occurred concomitantly with p16(INK4a) methylation. Among the 60 cases, only four contained a continuous homozygous deletion spanning bot h p16(INK4b) and p16(INK4a). Six cases were exclusively deleted at p16(INK4 a) and 17 exclusively deleted at p15(INK4b). LOH at D9S942 and D9S171 was a lso found to be mutually exclusive. Our results suggest that the alteration mode at 9p21 was not uniform, and the two genes were inactivated by distin ct mechanisms. Altogether, 68% of the samples harbor at least one type of a lteration in p16(INK4a) gene and 50% of the samples were altered in p15(INK 4b) gene, indicating that they are the frequent inactivating targets during ESCC development.