Immunohistochemical detection of p53 protein accumulation in head and neck cancer: correlation with p53 gene alterations.

The genetic and functional status of the p53 gene may be an important factor in guiding therapeutic strategies for patients with cancer. The purpose of this study was to determine whether p53 immunohistochemistry (IHC) accurately reflects the mutational status of the p53 gene and to determine whether p53 IHC independently predicts tumor responsiveness to radiation therapy for patients with HNSCC. p53 IHC was performed using the monoclonal antibody DO7 on tumors from 85 patients with HNSCC treated with primary or adjuvant radiation. The p53 status in all of these tumors was previously assessed by direct sequence analysis of exons 5 through 9: 49 tumors were p53 wild-type, and 36 harbored p53 gene mutations. All patients were well characterized with respect to locoregional recurrence, distant spread, and survival. Positive p53 staining was observed in 53 of the 85 cases (62%). Only 27 (51%) of these 53 IHC-positive cases harbored gene mutations in exons 5 through 9; 23 (72%) of the 32 IHC-negative cases did not harbor mutations. The overall correlation rate between IHC and sequencing was 59% (P < .04, chi2). Discordant results were observed for 35 (41%) cases, including 26 IHC-positive cases and 9 IHC-negative cases. In 7 of 9 cases, false-negative staining was due to a nonsense or splice-site mutation. p53 IHC was not predictive of overall survival (P = .37) or disease-free survival (P = .95). In a sizable number of cases, p53 IHC does not reflect the mutational status of the p53 gene. Specific types of alterations (eg, truncating mutations) and other factors may contribute to this poor correlation. Moreover, p53 IHC does not appear to be an independent predictor of tumor responsiveness to radiation in patients with HNSCC.     

Immunohistochemical study of p53 protein in human and animal pituitary tumors

In many human cancers, p53 gene mutations are frequently occurring genetic abnormalities, which may be detected by immunohistochemical staining for p53 protein. In the present study, p53 immunoreactivity was investigated in formalin-fixed, paraffin-embedded tissues from human and animal pituitary tumors, using the avidin-biotin-peroxidase complex technique. No p53 was detected in 3 nontumorous human adenohypophyses or in 40 human pituitary tumors including 5 GH cell adenomas, 10 PRL cell adenomas, 2 mixed GH cell-PRL cell adenomas, 2 acidophil stem cell adenomas, 8 ACTH cell adenomas, 1 TSH cell adenoma, 1 FSH/LH cell adenoma, 5 null cell adenomas, 5 oncocytomas, and 1 plurihormonal adenoma. Twenty nontumorous and hyperplastic pituitaries of hGRH transgenic mice and 8 tumors in these transgenic animals were immunonegative for p53. All pituitary tumors found in AVP/SV40 transgenic mice contained p53 immunoreactivity in the nuclei, while the nontumorous adenohypophysis of one such transgenic mouse was negative. It can be concluded that p53 mutations are apparently not involved in the pathogenesis of human pituitary adenomas or of the pituitary tumors which develop in hGRH transgenic mice. However, pituitary tumors in AVP/ SV40 transgenic mice are accompanied by p53 expression.     

Immunohistochemical detection of the p53 oncoprotein in tumours of melanocytic origin

We employed the polyclonal anti-p53 antibody NCL-CM1 to cultured cells and pathological tissues in order to investigate the expression of p53 oncoprotein in human malignant melanomas. The results in the cultured cells showed that the antigenic determinant was sensitive to formalin fixation, resulting in a lower reactivity than with fixation by alcohol. In pathological tissues, the expression of p53 oncoprotein increased with progression of the tumour. Among 79 melanomas 37 (47%) showed distinct nuclear labelling and the highest proportion of reactive cells was observed in metastatic melanomas (mean 4.8%). An immunocytochemical study also revealed the presence of mutant-type oncoprotein in human melanoma cell lines, which was recognized by monoclonal antibody P240, and we confirmed that the molecular weight of the antigens recognized by both antibodies was 53 kDa by Western blot analysis. Therefore, although the presence of point mutations in human melanomas is yet to be confirmed our data suggest that the antigen detected by NCL-CM1 is a mutanttype or a complex of mutant and wild-type p53 oncoproteins. This antibody may be useful in retrospective studies of tumours of melanocytic origin.     

Immunohistochemical detection of p53 and c-myc proteins in canine mammary tumours

The objectives of this study were to detect by immunohistochemical means, nuclear accumulations of p53 and c-myc proteins in mammary tumours of dogs. Moderate or intense p53 protein nuclear labelling was shown by each of five osteosarcomas. In contrast, focal immunoreactivity was shown by three of five adenocarcinomas and two of three malignant myoepitheliomas. Six benign mixed tumours and three myoepitheliomas showed no detectable immunoreactivity. On the other hand, three patterns of c-myc protein nuclear reactivity were observed in these tumours. Osteosarcomas, adenocarcinomas, malignant myoepitheliomas and myoepitheliomas showed intense or moderate reactivity. In benign mixed tumours, the epithelial component showed moderate or intense reactivity, and the myoepithelial component showed focal or moderate reactivity. These results demonstrated that p53 protein was expressed only in the osteosarcomas, but that c-myc expression was detectable in both the epithelial and myoepithelial components.     

Immunohistochemical expression of p53 proteins in Wilms' tumour: a possible association with the histological prognostic parameter of anaplasia

Wilms' tumour (nephroblastoma) has been associated with chromosomal abnormalities at the 11p13, 11p15 and 16q regions. A study into the possibility of mutations occurring within p53, the ubiquitous adult tumour suppressor gene, in Wilms' tumour was carried out. Thirty-eight ca ses were studied. Of these 36 were categorised into the favourable histology group and two into the unfavourable histology group based on the National Wilms' Tumour Study criteria. Archival formalin-fixed, paraffin-embedded tissue sections from each case were stained with a polyclonal (AB565:Chemicon) and a monoclonal (DO7:Dako) antibody raised against p53 protein using a peroxidase-labelled streptavidin biotin kit (Dako). 'Cure' (disease-free survival of 60 months or longer) was documente d in 39% of cases with favourable histology tumours. Eleven percent in this group succumbed to the disease. Both cases with unfavourable histology died. Four out of 36 (11%) tumours with favourable histology demonstrated weak to moderate staining with both AB565 and DO7 in more than 75% of tumour cells. In contrast, p53 protein expression in unfavourable histology tumours was significantly increased compared with the favourable histology group ( P  = 0.021) with both cases demons trating immunopositivity in >75% of tumour cells when stained with AB565 and DO7. The intensity of staining ranged from moderate to strong in both cases. It appears from this preliminary study that the immunohistochemical expression of p53 protein in Wilms' tumour, presumably a result of mutation in the p53 tumour suppressor gene, correlates with histological classification, histological categorisation being one of the useful features in the prognostic assessment of Wilms' tumours.     

Expression of c-Myc and p53 correlates with clinical outcome in diffuse large B-cell lymphomas

We performed a retrospective immunohistochemical study of the relationships between clinical manifestations and outcomes of diffuse large B-cell lymphoma (DLBCL) and expression of oncogenic proteins in 21 cases of DLBCL at various clinical stages. Cases of nodal origin expressed p53 more often and presented with high clinical stage more frequently than those of extranodal origin. Expression of c-Myc or p53, but not Bcl-6, Bcl-2, or Bcl-1, showed a statistically significant positive correlation with high clinical stage at presentation and with high or high-intermediate risk. Coexpression of c-Myc and p53 occurred in 7 of 12 patients with high clinical stage but was absent in patients with low clinical stage; coexpression was more frequent in patients with high or high-intermediate risk than in patients with low or low-intermediate risk. Four patients with this coexpression pattern demonstrated an unusually aggressive clinical course (median survival, 7 months). Coexpression of c-Myc and p53 seems to be a better indicator than the MIB1 proliferative index for identification of a cohort of aggressive disease in patients with DLBCL.     

p53 expression in colorectal tumors.

The expression of the nuclear phosphoprotein p53 was studied immunohistochemically in a series of 150 benign and malignant colorectal tumors. Using monoclonal antibody PAb1801, tumors divided unequivocally into two groups on the basis of immunohistochemistry. Forty of the carcinomas (46.5%) showed positive staining but only 4 of the adenomas (8.7%) were positive (P less than 0.001). The few positive adenomas always showed moderate or severe dysplasia. Metaplastic polyps (n = 9) and small familial adenomatous polyposis-related adenomas (n = 9) were uniformly negative. Carcinomas with p53 expression did not differ from those without in terms of site, differentiation or the prognostic indicators of Dukes' stage, DNA ploidy, or tumor histology. The improved morphologic resolution available in periodate lysine paraformaldehyde dichromate (PLPD)-fixed, paraffin-embedded tissue permitted several conclusions to be made: p53 is confined to neoplastic nuclei; staining in positive tumors is heterogeneous and often more marked at the infiltrative margins; and staining intensity is dramatically reduced in mitotic cells. It is concluded that expression of immunohistochemically detectable p53 (probably representing mutated forms of the protein) occurs in some adenomas around the time of transition to carcinoma. Therefore there is an association with the appearance of infiltrative behavior but not with degree of tumor progression (including metastasis) at the time of resection.     

Immunohistochemical inactivation of p14ARF concomitant with MDM2 overexpression inversely correlates with p53 overexpression in oral squamous cell carcinoma

The CDKN2 gene encodes two structurally different proteins: a cyclin-dependent kinase inhibitor called p16, which regulates retinoblastoma protein (pRb)-dependent G1 arrest, and a cell cycle inhibitor designated p14ARF, which arrests cell growth in G1-S and also in G2-M. Whereas inactivation of p16 has been described as a frequent event in various cancers, including oral cancer, the current function of p14ARF is still poorly understood. A physical association between p14ARF and MDM2 blocks MDM2-induced p53 degradation, resulting in increased levels of p53, which in turn leads to cell cycle arrest. The present study immunohistochemically examined the expression of p16 and p14ARF together with pRb, MDM2 and p53 status in a series of oral cancers. The results showed that p14ARF was frequently absent in the oral cancers (15/37, 41%) as was p16 immunostaining. Concomitant immunopositivity for p14ARF and MDM2 overexpression was frequently observed in a subset of the cancers, whereas an inverse correlation between p14ARF and MDM2 expression and the diffuse staining of p53 was clearly detected. Moreover, the results showed that in most cases of oral cancer (35/37, 95%) at least one protein was altered, and lymph node metastasis was more frequent in the tumors with alterations in both the p16/pRb and p14ARF/p53 pathway (8/16, 50%) than in the tumors with one or no alteration of these two major pathways.     

Immunohistochemical study of p53 in human lung carcinomas.

Immunohistochemical analysis of p53 protein was carried out on 95 lung carcinomas from all histological types, including 60 primary tumors, 35 lymph node metastases, and 36 corresponding nude mice xenografts, using four antibodies: PAb240 specific for some mutant conformations; PAb421, PAb1801, and CM1 reactive with most of the forms of p53. Nuclear staining with at least two of those four antibodies revealed the presence of an accumulated protein, considered as indicative of a missense mutation in the p53 gene, in 50% of primary tumors of all histological types, except carcinoids. Some defect of messenger RNA expression was detected by Northern blot analysis in an additional 26% of tumors. p53 immunophenotype of the original tumor was fairly maintained on nude mice. p53 accumulation was not correlated with survival, but with disease extension (P = 0.01). Finally, immunohistochemical analysis allowed the recognition of p53 mutant immunophenotype in 41% of tumors where p53 DNA and messenger RNA were apparently normal, using standard molecular biology. Thus, this method provides a rapid and efficient approach for studying p53 mutations leading to an accumulated protein in lung tumors cells.     

Immunohistochemical analysis of p53 expression in malignant lymphomas.

p53 is a tumor suppressor gene that commonly undergoes mutations in human tumors, including lymphomas. Because p53 mutations are not restricted to a single locus, immunohistochemistry is useful to detect p53 expression and correlate this finding with lymphoma phenotype. Cryostat sections from 125 cases of lymphoma were analyzed for p53 expression using three different monoclonal antibodies (pAb 421, 1801, 240) which react with human cellular p53 and a common conformational epitope on mutant p53. A control antibody (pAb 246) reacts only with wild type p53 of murine origin and was negative in all cases. Tissue from 29 cases of lymphoid hyperplasia, including six from human immunodeficiency virus-positive (HIV+) patients, were negative for p53. p53 was predominantly localized in nuclei of high-grade lymphomas, including 14 of 46 cases of B cell immunoblastic lymphomas and two of five T cell immunoblastic lymphomas. p53 expression was relatively common in lymphomas from HIV+ patients, and unusual in intermediate and low-grade lymphomas of follicular center cell type. Low-grade lymphoma of small lymphocytic type disclosed p53+ large cells (paraimmunoblasts) that may play a role in tumor progression in this lymphoma subtype. p53 was also strongly expressed in the nuclei of Reed Sternberg cells from 19 of 37 cases of Hodgkin's disease, including six cases of mixed cellularity, and 13 cases of nodular sclerosing type. Immunohistochemical staining is a rapid method to identify p53 expression in lymphomas.     

Immunohistochemical analysis of p53 protein expression in benign and malignant skin tumors using a panel of anti-p53 antibodies.

The expression of p53 in a variety of benign and malignant skin lesions has been first assessed in frozen sections and then compared with the results obtained in corresponding paraffin-embedded sections using various immunohistochemical staining methods with a panel of anti-p53 antibodies. Of the 48 benign and malignant skin lesions studied, 46(96%) had corresponding paraffin sections and immunohistochemical results obtained with DO7 on frozen and paraffin sections were concordant in 97%, qualitatively. Using streptavidin-biotin complex method, p53 was identified in 33% of dysplastic squamous lesions, 50% of squamous cell carcinomas (SCCs) and 36% of basal cell carcinomas (BCCs) on frozen section, whereas 25% of dysplastic squamous lesions, 40% of SCCs, and 32% of BCCs showed p53 positivity on paraffin-embedded sections. In frozen sections, the same regions of each specimen exhibited similar topographic patterns of positive immunoreactivity with both monoclonal antibodies, PAb 1801 and DO7. In contrast, immunohistochemical staining with polyclonal antibody, CM-1, gave poor morphologic resolution, although effective in paraffin-embedded sections.     

Infrequent p53 gene mutations and lack of p53 protein expression in clear cell sarcoma of the kidney: immunohistochemical study and mutation analysis of p53 in renal tumors of unfavorable prognosis.

A high prevalence of p53 gene mutation and protein expression has been found in the anaplastic variant of Wilms' tumor (WT), known to be associated with poor outcome. However, there are very few studies of p53 alterations in the other two rare and highly malignant renal tumors in childhood, in other words, clear cell sarcoma of the kidney (CCSK) and malignant rhabdoid tumor of the kidney (MRTK). Overexpression of p53 protein has been detected in eight CCSKs in one study, and in two in another, yet no molecular correlation with p53 gene mutations has been carried out. Our study is the first molecular analysis concerning p53 in CCSK. We investigated eight cases of CCSK and one case of MRTK for p53 protein expression by immunohistochemical staining. All were analyzed for p53 mutations in the region of exons 4 to 8 by polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) method and DNA sequencing analysis. By histological study, no CCSK showed anaplastic features. None expressed p53 protein, but two harbored p53 mutations. One was in exon 5, with a base pair insertion between codons 162 to 163 causing frameshift alteration in amino acid. Another was a silent CTC-->CTT transversion in codon 289 of exon 8. The case of MRTK did not show any alterations of p53 protein or gene. Our result indicates that p53 alterations are infrequent in CCSK and do not seem to be primary genetic events in the pathogenesis of CCSK.     

Immunohistochemical analysis of p53 expression in anal squamous neoplasia.

AIMS--To determine the pattern of expression of the p53 tumour suppressor gene product in anal squamous neoplasia, and to determine if this could be used as a marker of disease progression. The association between p53 expression and human papillomavirus (HPV) 16 DNA status of the anal lesions was also investigated. METHODS--The presence and localisation of the p53 protein in formalin fixed, paraffin wax embedded specimens of anal squamous epithelium (normal and neoplastic) was examined using immunohistochemical staining with a panel of two monoclonal antibodies (DO-1, DO-7) and one polyclonal antibody (CM-1). Thirty nine normal anal epithelia, 14 anal intraepithelial neoplasia (AIN) grade 1, seven AIN 2, and 20 AIN 3 specimens were obtained from patients without demonstrable invasive disease; twelve AIN 3 specimens adjacent to invasive disease and 34 anal squamous cancers were also examined. Genomic DNA from all 126 specimens was extracted and analysed for HPV 16 DNA using the polymerase chain reaction (PCR). RESULTS--Nuclear p53 was strongly expressed in 67% (23/34) of invasive anal squamous tumours, 75% (9/12) of AIN 3 specimens adjacent to invasive disease, and in 60% (12/20) of AIN 3 specimens obtained from patients without demonstrable invasive disease. Two of the patients in the latter group with positively staining specimens subsequently developed invasive tumours which had staining characteristics similar to those of the AIN 3 specimens. p53 protein was expressed in very low concentrations in low grade AIN and not at all in normal anal squamous epithelium. In those specimens which stained positively for p53, HPV 16 DNA sequences were detected in 69.5% (16/23) of invasive disease, 77.7% (7/9) of AIN 3 adjacent to invasive disease, 75% (9/12) of AIN 3 obtained from patients without demonstrable invasive disease, 33.3% (2/6) of AIN 2, and in 40% (2/5) of AIN 1. There was no significant correlation between p53 immunostaining and HPV 16 DNA status (p < 0.05). CONCLUSIONS--Aberrant expression of the p53 gene product is probably involved in the pathogenesis of anal squamous neoplasia. Long term follow up studies of all patients with AIN are required to determine if this could be used as a marker of likely disease progression from high grade AIN to invasive disease. There does not seem to be an association between the presence or absence of HPV 16 DNA sequences and mutant p53 proteins in anal squamous neoplasia.     

Immunohistochemical detection of p53 and Bcl-2 proteins in Hashimoto's thyroiditis and primary thyroid lymphomas.

AIMS--To investigate whether immunohistochemical staining using p53 and/or bcl-2 distinguishes between florid Hashimoto's thyroiditis and low grade mucosa associated lymphoid tissue (MALT) lymphoma of the thyroid. METHODS--Ten cases of Hashimoto's thyroiditis and eight of primary thyroid lymphoma were stained with monoclonal antibodies directed against p53 and bcl-2. RESULTS--In Hashimoto's thyroiditis most small lymphoid cells in mantle zones, within the thyroid parenchyma and in lymphoepithelial lesions expressed bcl-2 protein. Very occasional centroblasts in reactive germinal centres were positive for p53, but all other lymphoid cells from cases of Hashimoto's disease were negative for p53. In diffuse, low grade lymphomas bcl-2 protein was uniformly expressed by most tumour cells. However, low grade lymphomas with a follicular pattern did not express bcl-2. The diffuse, low grade lymphomas were negative for p53, while occasional larger cells in the follicular subtype were positive. Both high grade lymphomas were bcl-2 negative but strongly p53 positive. CONCLUSIONS--This study indicates that there is an inverse correlation between p53 and bcl-2 immunostaining in thyroid lymphomas (low grade lymphomas: bcl-2 positive, p53 negative; high grade lymphomas: bcl-2 negative, p53 positive). Furthermore, immunohistochemical staining for bcl-2 and p53 proteins does not distinguish florid Hashimoto's thyroiditis from diffuse, low grade thyroid lymphoma.     

Immunohistochemical and SSCP analysis of p53 in malignant lymphomas.

We immunohistochemically investigated Epstein-Barr virus (EBV)-positive and negative 31 malignant lymphomas (MLs) for p53 protein using a monoclonal antibody which is expressed on a wild type and mutant human p53 protein. We evaluated the presence of mutations in exons 5 to 8 of the p53 gene using single-strand confirmation polymorphism analysis. Overexpression of p53 was detected in 13 out of 31 cases (41.9%) of MLs. However, we have documented the presence of structural alterations of the p53 gene in six cases of MLs. The presence of EBV infection in MLs was statistically unrelated to p53 protein overexpression. Excellent correlation was found between p53 immunoreactivity and histologic types of MLs. Even though the reason for discrepancy between p53 gene mutation and p53 protein overexpression remains unclear, p53 protein overexpression may be involved in the process of malignant transformation regardless of EBV infection in MLs.     

Immunohistochemical expression of heparan sulfate correlates with stromal cell proliferation in breast phyllodes tumors.

Phyllodes tumors are fibroepithelial neoplasms typified by stromal proliferation. We have previously shown the role of pathologic parameters and the prognostic significance of p53 and CD117 protein expression in these tumors. In this study, we evaluated the expression of heparan sulfate, which has been implicated in many biological processes such as cell adhesion, embryogenesis, and tumorigenesis (including malignant transformation of mammary cells) in 232 breast phyllodes tumors. We used a monoclonal antibody, 10E4, to examine the localization of heparan sulfate in phyllodes tumors by immunohistochemistry. The immunoreactivity of both epithelial and stromal components was examined and analyzed with pathological parameters and other immunohistochemical markers, including p53, MIB1, bcl2, and CD117. Stromal 10E4 expression was significantly associated with tumor grade, stromal p53, and MIB1 expression in proliferating cells, suggesting that heparan sulfate may participate in malignant tumor growth.     

Immunohistochemical detection of the ras oncogene product p21 in advanced ovarian cancer. Lack of correlation with clinical outcome

The monoclonal antibody RAP 5 immunoreactive with the ras gene product p21 was used in an immunohistochemical study of 57 patients with advanced ovarian cancer and in 28 normal ovaries. The pattern of the staining of various tumor specimens was similar to the germinal epithelium of normal ovaries, whereas the intensity of staining was more enhanced in carcinomas than in normal ovaries. However, we found a lack of correlation among staining intensity of RAP 5 and the histologic type, the histologic grade, the ploidy class, and the clinical outcome.     

Immunohistochemical detection of dUTPase in intracranial tumors

The nuclear isoform of deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase, OMIM *601266, EC 3.6.1.23) is immunohistochemically detectable in all proliferating tissues and may thus be a useful adjunct for the grading of tumors analogous to Ki-67 labeling. A hundred and twenty-seven human intracranial tumors, including 56 astrocytomas, 12 oligodendrogliomas, 8 oligoastrocytomas, 34 meningiomas, 7 ependymomas, and 10 metastatic carcinomas, were stained using the monoclonal rat anti-human dUTPase antibody (clone 3E6) with formalin-fixed and paraffin-embedded tissue. The labeling indices were compared with those obtained with the proliferation marker Ki-67 on parallel tissue sections. All tumors contained dUTPase-positive nuclei, whereas the percentage of positive tumor cells generally increased with grade of malignancy. Meningiomas of higher grades, i.e., World Health Organization (WHO) grades II and III, contained additional cells with cytoplasmic reactivity. There were usually fewer dUTPase- than Ki-67-positive nuclei detectable. Unlike Ki-67, dUTPase was not detectable in mitotic figures. Labeling indices for dUTPase, but not for Ki-67, showed significant differences between all 3 WHO grades of diffuse astrocytomas. In summary, dUTPase staining provides a useful measure of cell proliferation distinct from that offered by Ki-67 labeling. It proved particularly useful for the evaluation of diffuse astrocytomas.