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

Joseph Geradts, Ralph H Hruban, Mieke Schutte, Scott E Kern, Robert Maynard

Research output: Contribution to journalArticle

Abstract

The MTS1/CDKN2/p16 gene encoding the p16(INK4a) tumor-suppressor protein is commonly inactivated by homozygous deletion or hypermethylation of the promoter in a wide range of human malignancies. In select tumor types, including pancreatic adenocarcinomas, intragenic mutations are found in a significant percentage of cases. The immunoreactivity of mutant p16 proteins has not been comprehensively studied. Moreover, the immunohistochemical properties of commercially available antibodies have not been described in detail. We studied 35 pancreatic adenocarcinomas with a molecularly defined p16 status (16 homozygous deletions, 3 hypermethylated cases, and 16 tumors with an intragenic mutation in one allele associated with loss of the second allele). In addition, we studied nine cell lines (three homozygous deletions, three hypermethylated lines, and three intragenic mutations). Paraffin sections of the tumors and cell blocks were reacted with four different anti- p16 antibodies: polyclonal and monoclonal (clone G175-405) antibodies from PharMingen, monoclonal antibody DCS-50 from Oncogene Science, and monoclonal antibody 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, suggesting that nonspecific nuclear staining can occur under certain conditions. Antibody DCS-50 produced nuclear staining in all three hypermethylated carcinomas, whereas G175-405 stained none of them. Three of the four antibodies produced nuclear immunoreactivity in 7 to 14 of the 16 carcinomas carrying p16 mutations; G175-405 showed only weak reactivity in one case. Cytoplasmic staining was present in all carcinomas and cell lines and with all antibodies and therefore cannot be considered specific; it was strongest with G175-405. Thus, we found antibody G175-405 to be the most specific, and monoclonals DCS-50 and ZJ11 the least specific for wild-type p16. However, the former tends to give stronger cytoplasmic background staining. For tumor types in which p16 mutations are uncommon, the PharMingen polyclonal antibody may be a suitable alternative.

Original languageEnglish (US)
Pages (from-to)71-79
Number of pages9
JournalApplied Immunohistochemistry
Volume8
Issue number1
DOIs
StatePublished - 2000

Fingerprint

p16 Genes
Mutation
Antibodies
Staining and Labeling
Neoplasms
Adenocarcinoma
Carcinoma
Alleles
Monoclonal Antibodies
Tumor Suppressor Proteins
Cell Line
Mutant Proteins
Oncogenes
Paraffin
Anti-Idiotypic Antibodies
Clone Cells

Keywords

  • Antibodies
  • Immunohistochemistry
  • p16
  • Specificity

ASJC Scopus subject areas

  • Anatomy
  • Medical Laboratory Technology

Cite this

@article{e1c4ae2c266e41e4a66147cb8039f3c2,
title = "Immunohistochemical p16(INK4a) analysis of archival tumors with deletion, hypermethylation, or mutation of the CDKN2/MTS1 gene: A comparison of four commercial antibodies",
abstract = "The MTS1/CDKN2/p16 gene encoding the p16(INK4a) tumor-suppressor protein is commonly inactivated by homozygous deletion or hypermethylation of the promoter in a wide range of human malignancies. In select tumor types, including pancreatic adenocarcinomas, intragenic mutations are found in a significant percentage of cases. The immunoreactivity of mutant p16 proteins has not been comprehensively studied. Moreover, the immunohistochemical properties of commercially available antibodies have not been described in detail. We studied 35 pancreatic adenocarcinomas with a molecularly defined p16 status (16 homozygous deletions, 3 hypermethylated cases, and 16 tumors with an intragenic mutation in one allele associated with loss of the second allele). In addition, we studied nine cell lines (three homozygous deletions, three hypermethylated lines, and three intragenic mutations). Paraffin sections of the tumors and cell blocks were reacted with four different anti- p16 antibodies: polyclonal and monoclonal (clone G175-405) antibodies from PharMingen, monoclonal antibody DCS-50 from Oncogene Science, and monoclonal antibody 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, suggesting that nonspecific nuclear staining can occur under certain conditions. Antibody DCS-50 produced nuclear staining in all three hypermethylated carcinomas, whereas G175-405 stained none of them. Three of the four antibodies produced nuclear immunoreactivity in 7 to 14 of the 16 carcinomas carrying p16 mutations; G175-405 showed only weak reactivity in one case. Cytoplasmic staining was present in all carcinomas and cell lines and with all antibodies and therefore cannot be considered specific; it was strongest with G175-405. Thus, we found antibody G175-405 to be the most specific, and monoclonals DCS-50 and ZJ11 the least specific for wild-type p16. However, the former tends to give stronger cytoplasmic background staining. For tumor types in which p16 mutations are uncommon, the PharMingen polyclonal antibody may be a suitable alternative.",
keywords = "Antibodies, Immunohistochemistry, p16, Specificity",
author = "Joseph Geradts and Hruban, {Ralph H} and Mieke Schutte and Kern, {Scott E} and Robert Maynard",
year = "2000",
doi = "10.1097/00022744-200003000-00011",
language = "English (US)",
volume = "8",
pages = "71--79",
journal = "Applied Immunohistochemistry and Molecular Morphology",
issn = "1541-2016",
publisher = "Lippincott Williams and Wilkins",
number = "1",

}

TY - JOUR

T1 - Immunohistochemical p16(INK4a) analysis of archival tumors with deletion, hypermethylation, or mutation of the CDKN2/MTS1 gene

T2 - A comparison of four commercial antibodies

AU - Geradts, Joseph

AU - Hruban, Ralph H

AU - Schutte, Mieke

AU - Kern, Scott E

AU - Maynard, Robert

PY - 2000

Y1 - 2000

N2 - The MTS1/CDKN2/p16 gene encoding the p16(INK4a) tumor-suppressor protein is commonly inactivated by homozygous deletion or hypermethylation of the promoter in a wide range of human malignancies. In select tumor types, including pancreatic adenocarcinomas, intragenic mutations are found in a significant percentage of cases. The immunoreactivity of mutant p16 proteins has not been comprehensively studied. Moreover, the immunohistochemical properties of commercially available antibodies have not been described in detail. We studied 35 pancreatic adenocarcinomas with a molecularly defined p16 status (16 homozygous deletions, 3 hypermethylated cases, and 16 tumors with an intragenic mutation in one allele associated with loss of the second allele). In addition, we studied nine cell lines (three homozygous deletions, three hypermethylated lines, and three intragenic mutations). Paraffin sections of the tumors and cell blocks were reacted with four different anti- p16 antibodies: polyclonal and monoclonal (clone G175-405) antibodies from PharMingen, monoclonal antibody DCS-50 from Oncogene Science, and monoclonal antibody 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, suggesting that nonspecific nuclear staining can occur under certain conditions. Antibody DCS-50 produced nuclear staining in all three hypermethylated carcinomas, whereas G175-405 stained none of them. Three of the four antibodies produced nuclear immunoreactivity in 7 to 14 of the 16 carcinomas carrying p16 mutations; G175-405 showed only weak reactivity in one case. Cytoplasmic staining was present in all carcinomas and cell lines and with all antibodies and therefore cannot be considered specific; it was strongest with G175-405. Thus, we found antibody G175-405 to be the most specific, and monoclonals DCS-50 and ZJ11 the least specific for wild-type p16. However, the former tends to give stronger cytoplasmic background staining. For tumor types in which p16 mutations are uncommon, the PharMingen polyclonal antibody may be a suitable alternative.

AB - The MTS1/CDKN2/p16 gene encoding the p16(INK4a) tumor-suppressor protein is commonly inactivated by homozygous deletion or hypermethylation of the promoter in a wide range of human malignancies. In select tumor types, including pancreatic adenocarcinomas, intragenic mutations are found in a significant percentage of cases. The immunoreactivity of mutant p16 proteins has not been comprehensively studied. Moreover, the immunohistochemical properties of commercially available antibodies have not been described in detail. We studied 35 pancreatic adenocarcinomas with a molecularly defined p16 status (16 homozygous deletions, 3 hypermethylated cases, and 16 tumors with an intragenic mutation in one allele associated with loss of the second allele). In addition, we studied nine cell lines (three homozygous deletions, three hypermethylated lines, and three intragenic mutations). Paraffin sections of the tumors and cell blocks were reacted with four different anti- p16 antibodies: polyclonal and monoclonal (clone G175-405) antibodies from PharMingen, monoclonal antibody DCS-50 from Oncogene Science, and monoclonal antibody 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, suggesting that nonspecific nuclear staining can occur under certain conditions. Antibody DCS-50 produced nuclear staining in all three hypermethylated carcinomas, whereas G175-405 stained none of them. Three of the four antibodies produced nuclear immunoreactivity in 7 to 14 of the 16 carcinomas carrying p16 mutations; G175-405 showed only weak reactivity in one case. Cytoplasmic staining was present in all carcinomas and cell lines and with all antibodies and therefore cannot be considered specific; it was strongest with G175-405. Thus, we found antibody G175-405 to be the most specific, and monoclonals DCS-50 and ZJ11 the least specific for wild-type p16. However, the former tends to give stronger cytoplasmic background staining. For tumor types in which p16 mutations are uncommon, the PharMingen polyclonal antibody may be a suitable alternative.

KW - Antibodies

KW - Immunohistochemistry

KW - p16

KW - Specificity

UR - http://www.scopus.com/inward/record.url?scp=0034153642&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034153642&partnerID=8YFLogxK

U2 - 10.1097/00022744-200003000-00011

DO - 10.1097/00022744-200003000-00011

M3 - Article

C2 - 10937052

AN - SCOPUS:0034153642

VL - 8

SP - 71

EP - 79

JO - Applied Immunohistochemistry and Molecular Morphology

JF - Applied Immunohistochemistry and Molecular Morphology

SN - 1541-2016

IS - 1

ER -