p16INK4a promoter methylation and 9p21 allelic loss in colorectal carcinomas: relation with immunohistochemical p16INK4a expression and with tumor budding

SummaryIn colorectal carcinomas, p16INK4a inactivation is known to occur by allelic loss and by promoter methylation, but mutations are rare. p16INK4a is up-regulated in tumor buds, and the consequent shutdown of proliferation may be a prerequisite for tumor budding. Fifty-seven colorectal carcinomas from a consecutive series were investigated. Using DNA from tissue homogenates, p16INK4a promoter methylation was seen in 17 of 57 tumors by methylation-specific polymerase chain reaction, and this could be confirmed using DNA from laser-capture microdissected material in 16 of these cases. A total loss of immunohistochemical p16INK4a expression was seen in 6 of 17 tumors with promoter methylation. Quantification of immunohistochemical p16INK4a expression for the remaining 11 cases revealed statistically lower frequencies of expression as compared with cases without p16INK4a promoter methylation. 9p21 allelic loss was observed in 9 cases, but p16INK4a expression in these carcinomas was not reduced. Attempted linear regression of p16INK4a expression in tumor buds on the degree of tumor budding, as counted on pan-cytokeratin immunostains, did not show a correlation. p16INK4a promoter methylation can completely abrogate p16INK4a expression in colorectal carcinomas. In many cases, however, it has an appreciable but only modulatory influence on p16INK4a expression. Possibly, methylations are heterozygous, and/or mosaic in colorectal carcinomas and/or methylations are not totally stable but can be lost between carcinoma cell replication cycles. Up-regulation of p16INK4a does not seem to be a strict requirement for tumor budding, hence, the absence of a correlation.