Methylation of the p16(Ink4 alpha) tumor suppressor gene 5 '-CpG island inbreast cancer

Methylation of the p16(lnk4a) turner suppressor gene 5' CpG island was anal yzed in 104 primary breast cancer specimens using Southern blotting and met hylation specific polymerase chain reaction (PCR) (MSP). Eight and four tum ors, respectively, showed methylation, and all MSP positive tumors were det ected by Southern blotting. To investigate possible methylation not detecta ble by these methods, bisulphite genomic sequencing was performed in 220 cl ones from 14 selected tumors. Absent methylation or methylation of single C pG dinucleotides prevailed in all tumors, but of the MSP positive tumors, t hree contained alleles with methylation of 31 or 32 of the 32 analyzed CpG dinucleotides in the island. Partially methylated alleles were also observe d. In a group with low p16(lnk4a) expression determined by Western blotting , foul randomly selected tumors contained several identical clones with met hylation of 15 CpG dinucleotides by bisulphite genomic sequencing but with a methylation pattern that did not support detection by either Southern blo tting or MSP, increasing the potential significance of p16(lnk4a) methylati on in breast cancer. (C) 2001 Elsevier Science Ireland Ltd. All rights rese rved.