Immunohistochemical p16INK4a Analysis of Archival Tumors with Deletion, Hypermethylation, or Mutation of the CDKN2/MTS1 Gene

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

doi:10.1097/00129039-200003000-00011

Cited by 49 publications

(43 citation statements)

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“…The experiments were carried out in a Shandon Sequenza immunostainer. The immunohistochemical assay for detecting p16 INK4a in fixed and paraffin-embedded tissues has been described in detail elsewhere (4,5,14). Briefly, after antigen retrieval in 0.1 M EDTA pH8.0 (20 minutes at 95 to 100°C), the sections were reacted with the antip16 monoclonal antibody at 1 g/mL at 4°C overnight.…”

Section: Immunohistochemistrymentioning

confidence: 99%

“…Briefly, after antigen retrieval in 0.1 M EDTA pH8.0 (20 minutes at 95 to 100°C), the sections were reacted with the antip16 monoclonal antibody at 1 g/mL at 4°C overnight. Some of the p16 INK4a staining data had been included in two earlier studies (5,12). For p14 ARF IHC, we initially tested all five antibodies (see Results).…”

Section: Immunohistochemistrymentioning

confidence: 99%

“…Several cell lines and tumors with known homozygous INK4a deletions served as external negative controls. A specimen was considered positive for p16 INK4A or p14 ARF if there was nuclear staining above any cytoplastic background; cytoplasmic staining itself was disregarded (5). If the cells of interest failed to show distinct nuclear reactivity, the specimen was considered negative for the respective protein.…”

Section: Immunohistochemistrymentioning

confidence: 99%

“…The frequency of p16 INK4a inactivation in human neoplasia rivals that of p53. We previously demonstrated that immunohistochemistry (IHC) is a sensitive and specific method of detecting the absence of functional p16 INK4a in formalin-fixed, paraffin-embedded tumors, whatever the underlying mechanism (4,5). In contrast, p14 ARF primarily acts as a p53 agonist by inhibiting the MDM2-mediated degradation of the latter (6,7).…”

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utImmunohistochemical Detection of the Alternate INK4a-Encoded Tumor Suppressor Protein p14ARF in Archival Human Cancers and Cell Lines Using Commercial Antibodies: Correlation with p16INK4a Expression

2001

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The INK4a locus encodes two structurally unrelated tumor suppressor proteins, p16 INK4a and p14 ARF . Although the former is one of the most common targets for inactivation in human neoplasia, the frequency of p14 ARF abrogation is not established. We have developed an immunohistochemical assay that allows the evaluation of p14 ARF expression in formalin-fixed, paraffin-embedded tissues, using commercially available antibodies. p14 ARF positive cells showed nuclear/nucleolar staining, which was absent in all cell lines and tumors with homozygous deletions of the INK4a gene. The assay was applied to 34 paraffin-embedded cell buttons, 30 non-small cell lung cancers and 28 pancreatic carcinomas, and the staining results were correlated with p16 INK4a expression. Loss of p14 ARF expression was common but less frequent than down-regulation of p16 INK4a (53% versus 76% of all specimens). The p14 ARF and p16 INK4a expression pattern was concordant in 65 of 92 cases (71%). Significantly, 24 cases were p16 INK4a ؊/p14 ARF ؉, while the opposite staining pattern was observed in three cases, consistent with the notion that the two proteins have nonredundant functions. The immunohistochemical assay described here may facilitate studies on the prevalence and significance of aberrant p14 ARF expression in human tumors.

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Section: Immunohistochemistrymentioning

confidence: 99%

Section: Immunohistochemistrymentioning

confidence: 99%

Section: Immunohistochemistrymentioning

confidence: 99%

mentioning

confidence: 99%

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utImmunohistochemical Detection of the Alternate INK4a-Encoded Tumor Suppressor Protein p14ARF in Archival Human Cancers and Cell Lines Using Commercial Antibodies: Correlation with p16INK4a Expression

2001

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“…Using the p16 polyclonal antibody, seven previously unevaluable breast cancers were found to be positive and three were negative. Because of our recent observation that the p16 polyclonal antibody may give false positive staining results (14), 37 of 45 cases that were positive for both p16 and pRB on initial analysis were reanalyzed with the specific antip16 monoclonal antibody. Thirty-one tumors remained p16 positive, three were negative, and three were inconclusive.…”

Section: Staining Patternsmentioning

confidence: 99%

Abnormal Expression of Cell Cycle Regulatory Proteins in Ductal and Lobular Carcinomas of the Breast

Geradts

Ingram

2000

Modern Pathology

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In a previous study, we demonstrated that the G1 cell cycle checkpoint in carcinomas of the breast is frequently abrogated by loss of p16, the product of the CDKN2/INK4A gene, and, to a lesser extent, by loss of pRB, the product of the retinoblastoma gene. The purpose of the present study was to determine whether other mechanisms of cell cycle deregulation exist in breast cancers which have retained RB and p16 function. Paraffin sections of 81 invasive breast carcinomas (49 ductal, 26 lobular, 6 mixed) were reacted with monoclonal antibodies against cyclin D1 and p53, using optimized immunohistochemical staining protocols. The staining results were correlated with the expression of p16 and pRB, and with a variety of pathological parameters and DNA ploidy. Twenty-five tumors (31%) accumulated (presumably mutant) p53 and 28 (35%) overexpressed cyclin D1; 7 carcinomas (not including any pure lobular cancers) abnormally expressed both proteins. p53 accumulation correlated with nuclear, mitotic, and overall grade, but not with tumor size, lymph node involvement, or DNA ploidy. Overexpression of cyclin D1 was not associated with any of the patho-biological variables. There was an inverse correlation between loss of p16 and high levels of p53, but not cyclin D1. The G1 cell cycle checkpoint, which is controlled by RB, cyclin D1, and p16, was abrogated in 65% of carcinomas, and only p53 was abnormal in an additional 17%. The number of abnormally expressed genes correlated with mitotic activity and overall tumor grade, but not with tumor histology, size, or nodal status, suggesting that cell cycle deregulation is an early event in breast tumorigenesis. Only 18% of the carcinomas showed a normal level of expression of the four genes tested, and p16 appeared to be the most common target of cell cycle deregulation. These data point to the importance of cell cycle regulatory protein abnormalities in human breast cancer.KEY WORDS: Breast cancer, Cell cycle, Cyclin D1, Immunohistochemistry, p16, p53, Pathology. Mod Pathol 2000;13(9):945-953It is generally accepted that several pathways need to be abrogated in the genesis of a malignant neoplasm. There is an increasing body of evidence that deregulation of one or more of the cell cycle checkpoints is among the most common abnormalities in human neoplasia. We have been particularly interested in the genes controlling the late G1 restriction point. Possibly the most critical component of the latter is pRB, the product of the retinoblastoma gene. Hyperphosphorylation, and hence inactivation of pRB is catalyzed by cyclin dependent kinases (CDKs), most notably CDK4 and 6. The CDKs are activated by binding to their respective cyclins, including cyclin D1 (1). They are inhibited by CDK inhibitors including p15 INK4B and p16 INK4A (specific for CDK4/6) and p21 WAF1 . The latter, in turn, is partially under the transcriptional control of p53 (2). We recently demonstrated that abnormal expression of the genes controlling the G1 restriction point is a very common occurrence in carcinomas ...

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A comparison of the clinical utility of p16INK4a immunolocalization with the presence of human papillomavirus by hybrid capture 2 for the detection of cervical dysplasia/neoplasia

Holladay

Logan

Arnold

et al. 2006

Cancer

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BACKGROUND. Evidence suggests that overexpression of p16INK4a protein indicates infection and genomic integration of high‐risk human papillomavirus (HR HPV) and predicts progression to cervical high‐grade squamous intraepithelial lesions (HSILs) and carcinoma. The authors compared the ability of p16INK4a and HR HPV detection by Hybrid Capture 2 (HC2) to detect the presence of significant cervical disease. METHODS. Four hundred ThinPrep® specimens (100 each in 4 categories: 100 specimens that were negative for intraepithelial lesions, 100 specimens of atypical squamous cells of undetermined significance [ASC‐US], 100 specimens of low‐grade squamous intraepithelial lesions [LSILs], and 100 specimens of HSILs) were analyzed. p16INK4a protein was immunolocalized using a specific monoclonal antibody, and the detection of HR HPV in all 400 specimens was determined using HC2. RESULTS. p16INK4a was found to be positive in 78% of HSIL specimens, 42% of LSIL specimens, and 36% of ASC‐US specimens; whereas HC2 was positive in 92% of HSIL specimens, 81% of LSIL specimens, and 45% of ASC‐US specimens. In the HSIL category, the sensitivity, which was calculated using Grade 2 or greater cervical intraepithelial neoplasia as the endpoint, was 78% (50 of 66 specimens) for p16INK4a and 91% (60 of 66 specimens) for HC2. For LSIL, the sensitivity was 75% (3 of 4 specimens) for p16INK4a and 100% (4 of 4 specimens) for HC2. In the ASC‐US category, the sensitivity was 89% (8 of 9 specimens) for p16INK4a and 100% (9 of 9 specimens) for HC2. Overall, the sensitivity for HSIL was 92% for HC2 and 78% for p16INK4a. The specificity for HC2 was 8.3% for HSIL, 16.9% for LSIL, and 48.7% for ASC‐US; whereas the specificity for p16INK4a was 25% in HSIL, 59.1% in LSIL, and 68.4% in ASC‐US. The overall specificity was 25% for HC2 and 56% for p16INK4a. CONCLUSIONS. Although both p16INK4a and HC2 may aid in the clinical management of patients with clinically significant lesions, HC2 was found to have greater sensitivity, and p16INK4a greater specificity. The labeling of normal cells and bacteria may preclude the use of p16INK4a in automated screening or nonmorphologic assays. Cancer (Cancer Cytopathol) 2006. © 2006 American Cancer Society.

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Immunohistochemical p16INK4a Analysis of Archival Tumors with Deletion, Hypermethylation, or Mutation of the CDKN2/MTS1 Gene

Cited by 49 publications

References 26 publications

utImmunohistochemical Detection of the Alternate INK4a-Encoded Tumor Suppressor Protein p14ARF in Archival Human Cancers and Cell Lines Using Commercial Antibodies: Correlation with p16INK4a Expression

utImmunohistochemical Detection of the Alternate INK4a-Encoded Tumor Suppressor Protein p14ARF in Archival Human Cancers and Cell Lines Using Commercial Antibodies: Correlation with p16INK4a Expression

Abnormal Expression of Cell Cycle Regulatory Proteins in Ductal and Lobular Carcinomas of the Breast

A comparison of the clinical utility of p16INK4a immunolocalization with the presence of human papillomavirus by hybrid capture 2 for the detection of cervical dysplasia/neoplasia

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