Expression of p21waf1/Cip1, MDM2 and p53 in vivo: analysis of cytological preparations.

The aim of this study was to test the hypothesis that expression of p21waf1/Cip1 and MDM2 could be used as indicators of the activity of wild-type p53 which can transcriptionally activate the p21waf1/Cip1 and mdm2 genes. In cytological preparations of serous fluids, the expression of p53, p21waf1/Cip1 and MDM2 protein was assessed by immunohistochemistry. A series of 50 cases was assessed for both p53 and p21waf1/Cip1 expression and a subset of 37 cases had sufficient material for analysis of MDM2. In samples in which there were reactive mesothelial cells (n = 48) there was general concordance between p53 and p21waf1/Cip1 expression, but in nine cases p21waf1/Cip1 was expressed in the absence of detectable p53. Similarly, MDM2 expression was not correlated with p53 in 15 of 31 cases. p21waf1/Cip1 was correlated with MDM2 in 24 of 31 cases, while in the remaining seven, MDM2 was expressed without detectable p21waf1/Cip1 immunoreactivity. In samples with neoplastic cells (n = 18) the presence of p21waf1/Cip1 and MDM2 expression was always associated with p53 expression. Polymorphonuclear leucocytes frequently showed p21waf1/Cip1 immunoreactivity, and this was confirmed by immunoblotting of peripheral blood polymorphonuclear leucocytes. These data indicate that in general p21waf1/Cip1 expression correlates with p53 expression in reactive mesothelial cells, consistent with a known mechanism of regulation. However, in reactive mesothelial cells, MDM2 expression is perhaps dissociated from p53 expression, contrary to current models of MDM2 regulation. Finally, in addition to many normal tissues, it is likely that in reactive mesothelial cells and some tumours p21waf1/Cip1 expression is not dependent on the presence of wild-type p53 protein. In conclusion, p53 status cannot be reliably predicted based only on p21waf1/Cip1 or MDM2 expression.

Antigen retrieval: p53 staining in benign, pre-malignant and malignant tissues of the larynx.

Antigen retrieval techniques have been reported to increase p53 detection. Using an antigen retrieval technique applied to immunohistochemistry, a study was performed on 67 laryngeal lesions (21 benign, 16 carcinoma in situ, 30 squamous cell carcinoma). p53 staining was observed in 30% of carcinoma in situ specimens and 53% of squamous cell carcinomas but was not detected prior to antigen retrieval in any benign lesion. However, over-expression of p53 was identified in 92.5% of benign lesions after antigen retrieval using the microwave oven heating. There was also increased p53 staining in both the carcinoma in situ (43.7%) and squamous cell carcinomas (30.0%) after antigen retrieval. We conclude that antigen retrieval using microwave oven heating increases immunohistochemical detection of p53 such that positive staining is observed in benign conditions. We postulate that this apparent over-expression is a manifestation of the wild-type protein, which may be found in more evidence in basal cells than suprabasal cells. Our results thus offer a cautionary note to such studies involving squamous cell cancers that attempt to correlate p53 over-expression with clinical parameters.

The use of antigen retrieval for immunohistochemical detection of p53 over-expression in malignant and benign oral mucosa: a cautionary note.

The tumour suppressor gene p53 is frequently mutated in neoplasia. Since mutant p53 protein is often over-expressed, mutation can be indirectly detected by immunocytochemical techniques. As microwave antigen retrieval is becoming a widespread method for increasing the antigenicity of paraffin sections, we investigated the application of this technique to p53 immunohistochemical staining of oral mucosa specimens. Paraffin sections of 22 squamous cell carcinomas (SCC) and 36 benign lesions were immunohistochemically stained with and without antigen retrieval. Without antigen retrieval p53 over-expression was observed in 6/22 SCC and 1/36 benign lesions. Following antigen retrieval positive staining was observed in 15/22 SCC and 35/36 benign lesions. Staining in benign lesions was confined to basal and parabasal cells and could reflect normal functioning of wild-type p53. We conclude that antigen retrieval increases the sensitivity of p53 immunoreactivity, but such staining is not specific for malignancy and should be interpreted with caution.

Clinical utility of the immunocytochemical detection of p53 protein in cytological specimens.

In the important cytopathological distinction between benign and malignant lesions, there is always a residue of suspicious cases which cannot be satisfactorily diagnosed. Overexpression of the p53 tumor suppressor gene product has been consistently correlated with p53 missense gene mutation and is associated with malignancy. Therefore, assessment of p53 expression may assist in the cytopathological diagnosis of malignancy. Immunohistological assessment of p53 expression has been performed on a prospective series of 1333 nongynecological cytological specimens in the setting of a teaching hospital group. Evaluation of p53 staining was performed without knowledge of cytological diagnosis. Resultant p53 expression data were correlated with cytological diagnosis and clinical information. Of the 999 assessable cases, 956 had a clear cytological diagnosis. In these, p53 overexpression occurred in 108 cases of which 86 were malignant lesions. Of the 848 p53-negative cases, 119 were in fact neoplasms. The false positives were predominantly (19 of 22) reactive mesothelial proliferations, and overexpression occurred in only a small proportion of cells. While the sensitivity of p53 overexpression is low (p53 overexpression only occurring in 41.9% of tumors), the overall specificity is 97%. In the 43 cytopathologically suspicious cases, 7 were p53 positive, all of which proved to be malignant. In this prospective unselected series, we have conclusively demonstrated a close correlation between overexpression of p53 protein and neoplasia. Furthermore, we have shown the possible utility of p53 immunostaining in cytopathology. While there are important technical caveats and some cytological specimens are suboptimal for immunostaining, the data indicate that assessment of p53 is a valuable adjunct to morphological assessment in the analysis of cytopathologically suspicious cases.

The immunocytochemical detection of p53 protein in cytological specimens: technical considerations.

The p53 gene encodes a 393 amino acid nuclear phosphoprotein that appears to act as a cell cycle checkpoint, possibly by transactivating other target genes. Abnormalities of the p53 gene are common in a wide range of human tumours and are associated in many cases with immunologically detectable p53 protein. Detection of p53 immunoreactivity is uncommon in normal cells, but is frequently seen in neoplasia. Here we define the optimum conditions for the detection of p53 immunoreactivity in cytological material, including fixation and storage. Immersion in acetone-methanol for 10 min is optimal, and after air drying, smears or cytospin preparations can be stored at -70 degrees C for at least 6 months. We describe the range of controls necessary, including the use of positive control cell lines with known mutations of the p53 gene and defined abnormalities of p53 protein. Negative controls should include cell lines (or strains) with no p53 abnormality as well as the conventional negative immunological controls. It is only with these technical caveats and controls that p53 immunoreactivity can be performed reliably on cytological specimens.

Electron microscopic changes in Enterocytozoon bieneusi following treatment with albendazole.

AIM: To identify and describe electron microscopic changes occurring in Enterocytozoon bieneusi in patients treated with albendazole. METHODS: Eighteen HIV seropositive patients with E bieneusi infection of the small intestine were treated with albendazole 400 mg twice a day for one month. Duodenal biopsy specimens obtained before and at the end of treatment were examined electron microscopically by a pathologist who was unaware of the clinical response. A semiquantitative assessment of the parasite load and description of the parasite morphology was made. RESULTS: A complete resolution of diarrhoea occurred in nine patients and a further three had a greater than 50% reduction in baseline stool frequency or volume. Three of the non-responders were also infected with cryptosporidium. There was a reduction in parasite load in those responding to treatment and an increase in abnormal forms in both responders and non-responders. CONCLUSION: The clinical response to albendazole treatment seen in some patients with small intestine microsporidiosis may be due to damage to the developmental stages, causing a partial inhibition of parasite reproduction.

Using microcomputers to improve the timeliness, accuracy, and accessibility of clinical data.

This article presents a microcomputer system as a solution to achieve timely, accurate and accessible clinical data. The article describes the components of the clinical data system as well as its functionality. The advantages of automated encoders for coding, editing and grouping of clinical data are outlined, as well as the advantages of relational database software for clinical data management. Clinical data support a myriad of activities within the health care facility. The microcomputer system provides the departmental user with flexibility and customization to meet and manage the ever-changing clinical data requirements. Client hospital experience demonstrate, not only an increase in the accuracy and availability of data, but also an increase in the productivity of the data collection process itself.