Immunohistochemical p16(INK4a) analysis of archival tumors with deletion, hypermethylation, or mutation of the CDKN2/MTS1 gene - A comparison of fourcommercial antibodies

The MTS1/CDKN2/p16 gene encoding the p16(INK4a) tumor-suppressor protein is commonly inactivated by homozygous deletion Or hypermethylation of the pro moter in a wide range of human malignancies. In select tumor types, includi ng pancreatic adenocarcinomas, intragenic mutations are found in a signific ant percentage of cases. The immunoreactivity of mutant p16 proteins has no t been comprehensively studied. Moreover, the immunohistochemical propertie s of commercially available antibodies have not been described in detail. W e studied 35 pancreatic adenocarcinomas with a molecularly defined p16 stat us (16 homozygous deletions, 3 hypermethylated cases, and 16 tumors with an intragenic mutation in one allele associated with loss of the second allel e). In addition, we studied nine cell lines (three homozygous deletions, th ree hypermethylated lines, and three intragenic mutations). Paraffin sectio ns of the tumors and cell blocks were reacted with four different anti-p16 antibodies: polyclonal and monoclonal (clone G175-405) antibodies from Phar Mingen, monoclonal antibody DCS-50 from Oncogene Science, and monoclonal an tibody ZJ11 from Neo-Markers. Optimal staining conditions were established for each antibody. The pancreatic carcinomas with homozygous p16 deletions were largely devoid of nuclear staining (admixed nonneoplastic cells served as internal positive controls); only one adenocarcinoma each reacted with DCS-50 and the polyclonal antibody, and five were positive with ZJ11, sugge sting that nonspecific nuclear staining can occur under certain conditions. Antibody DCS-50 produced nuclear staining in all three hypermethylated car cinomas, whereas G175-405 stained none of them. Three of the four antibodie s produced nuclear immunoreactivity in 7 to 14 of the 16 carcinomas carryin g p16 mutations; G175-405 showed only weak reactivity in one case; Cytoplas mic staining was present in all carcinomas and cell lines and with all anti bodies and therefore cannot be considered specific; it was strongest with G 175-405. Thus, we found antibody G175-405 to be the most specific, and mono clonals DCS-50 and ZJ11 the least specific for wild-type p16. However, the former tends to give stronger cytoplasmic background staining. For tumor ty pes in which p16 mutations are uncommon, the PharMingen polyclonal antibody may be a suitable alternative.