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Human apurinic endonuclease 1 (HAP1) plays a key role in the repair of baseless sites in DNA. HAP1 is also known to be a potent regulator of the binding activity of a number of transcription factors. We have examined the immunohistochemical expression of the HAP1 protein in normal colorectal mucosa, hyperplastic polyps, tubulovillous adenomas, and carcinomas. In normal colonic mucosa, the predominant staining was nuclear in the less differentiated cells located at the lower part of the crypt, but it was cytoplasmic in the more differentiated superficial colonic epithelium. HAP1 expression was nuclear in 3 of 30 adenomas (10%) and 5 of 44 carcinomas (11%), but it was cytoplasmic in 11 of 30 adenomas (37%) and 22 of 44 carcinomas (50%) and both nuclear and cytoplasmic in 16 of 30 adenomas (53%) and 17 of 44 carcinomas (39%). The observed staining in stromal fibroblasts and endothelial cells was nuclear, whereas that in macrophages was cytoplasmic. Our data indicate that HAP1 is expressed in different subcellular compartments during normal differentiation and that this pattern is disrupted in adenomas and carcinomas. The differential localization may be relevant to the two different proposed functions of HAP1.

Background: Thymidine phosphorylase (TP) catalyses the reversible phosphorylation of thymidine to thymine and 2-deoxyribose-1-phosphate. High expression of TP in cell lines potentiates the effects of the cytotoxic drugs 5-fluorouracil and methotrexate, both of which are used in the cyclophosphamide, 5-fluorouracil and methotrexate (CMF) treatment regimen of breast cancer., Patients and Methods: We therefore examined the expression of this enzyme in 328 invasive breast carcinomas using immunohistochemistry and assessed whether the expression of this enzyme by the tumour predicts patients response to CMF in node-positive patients., Results: Whereas no significant difference in either relapse-free survival (RFS) (P = 0.2) or overall survival (OS) (P = 0.07) was observed between TP-negative and -positive tumours in non-treated patients, there was a significant increase in both RFS (P = 0.02) and OS (P = 0.02) in patients treated with CMF in TP-positive compared with TP-negative tumours. A multivariate analysis of the 134 node-positive patients demonstrated that in ductal carcinomas, TP was an independent variable for OS., Conclusions: This pilot study suggests that patients with TP-positive tumours have a significant survival benefit when treated with CMF and supports the hypothesis that TP enhances tumour sensitivity to the anti-metabolites 5-fluorouracil and methotrexate.

The distribution of TAL-1 protein, an important vascular promoter in mice, has been examined immunohistochemically in a range of human vascular lesions and normal tissues. Formalin-fixed, paraffin-embedded vascular lesions including granulation tissue, haemangiomas, Kaposi's sarcomas, spindle cell haemangioendotheliomas, and angiosarcomas, were examined using a monoclonal antibody to recombinant TAL-1. Endothelial cells in all lesions gave positive immunostaining of variable intensity. Granulation tissue and spindle cell areas of the vascular tumours gave the strongest staining (nuclear and cytoplasmic). The better-differentiated endothelial cells within the tumours and resident well-formed vessels were less positive and some cells were in fact negative. The malignant endothelial cells in angiosarcomas showed less intense positive staining than KS cells. This study has shown TAL-1 protein expression in a range of reactive, benign, and malignant vascular lesions. Protein expression appears to be stronger in the spindle cell areas, perhaps reflecting greater expression in less-differentiated endothelial cells.

Platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) is an enzyme with angiogenic and cell motility properties. Moreover, it is involved in the transformation of fluoropyrimidines into active cytotoxic metabolites. In the present study, the expression of PD-ECGF in normal lung and lung cancer was immunohistochemically evaluated using the P-GF.44C monoclonal antibody. Alveolar and tumoural macrophages were invariably stained and were used as an internal control for assessment of the staining. Alveolar epithelium was always negative, whilst bronchiolar epithelium showed occasional positive reactivity. Normal lung and tumour endothelium was occasionally positive. Positive staining in more than 50 per cent of cells was observed in 23/71 squamous carcinomas (32 per cent), 16/38 (42 per cent) adenocarcinomas, and 2/6 (33 per cent) adenosquamous carcinomas. Differentiated areas and areas of squamous metaplasia were more strongly positive than other tumour areas. All 22 small cell carcinomas and one carcinoid tumour were negative. The present study provides a baseline for future studies in non-small cell lung cancer to correlate PD-ECGF expression with tumour vascularization, prognosis, and response to chemotherapy.

Angiogenesis, the formation of new blood vessels from the existing vascular network, has been demonstrated to be an important prognostic factor in invasive breast carcinoma. The switch to an angiogenic phenotype represents a growth-limiting step during carcinogenesis and might, in pre-invasive lesions, indicate the risk for developing an invasive phenotype. The discrepancy between modern therapy options for invasive breast carcinomas and the relatively aggressive treatment of in situ lesions underlines the need for such prognostic factors for ductal in situ breast carcinomas (DCIS). Patterns of vascularity were examined in 75 formalin-fixed, paraffin-embedded DCIS by immunohistochemical staining of vessels using antibodies against Factor VIII-related antigen. Histological classification was performed according to four different systems, based on architectural or cytonuclear features, or a combination of both. Two distinct vascular patterns were observed: a diffuse increase of stromal vascularity between duct lesions (pattern I), which was present alone in 8/75 (11 per cent), and a dense rim of microvessels adjacent to the basement membrane of individual ducts (pattern II), present alone in 12/75 (16 per cent). In total, 57 per cent (43/75) showed pattern 1 and 62 per cent (47/75) showed pattern II. There was a significant correlation between these patterns (P = 0.0001; chi 2 = 15.1), such that both were present in 35 (47 per cent). These different vascular patterns imply two angiogenic pathways: one pathway mediated by angiogenic factors released directly by tumour cells resulting in the rim of vessels and another generated indirectly via recruitment of accessory cells such as macrophage and endothelial cells, which themselves release other angiogenic factors, causing the increase of stromal vascularity. A significant increase in both stromal vascularity (pattern I) and the presence of a rim (pattern II) was observed in high-grade DCIS lesions (P = 0.005 and P = 0.037). Indeed, all the patient relapses occurred in these high-grade lesions, but due to the small number of patient events, no significant correlation of vascular pattern to survival was observed (P > 0.05). This study suggests that distinct patterns of vascularity in DCIS might be useful for identifying patients who are at risk of relapse.

Tumour angiogenesis is an important prognostic factor in non-small cell lung cancer. Recently, EGFR and c-erbB-2 protein was found to regulate cell adhesion and the invasive growth of cancer through its association with the cadherin-catenin complex. The role of c-erbB-2 protein in cell migration has been also reported. In this study we investigate the combined role of tumoral neoangiogenesis and c-erbB-2/EGFR expression in the metastatic behaviour and prognosis of operable non-small cell lung cancer. 107 tumour samples from patients suffering from operable non small cell lung cancer were examined. EGFR and c-erbB-2 were not correlated with each other. C-erbB-2 expression was associated with low angiogenesis, approaching statistical significance in adenocarcinomas (p = 0.08). The absence of expression of both c-erbB-2 and EGFR oncogenes in tumours with high angiogenesis, was most frequently observed in node negative cases (p = 0.04). C-erbB-2 overexpression defined a subgroup of node negative patients with low angiogenesis and prognosis similar to patients with tumours bearing high angiogenesis. These findings support the hypothesis that expression of the erb genes is a mechanism activated in non-small cell lung cancer to enable cancer cell migration. This pathway seems to be activated mainly in tumours with poor vasculature presumably lading to an unfavourable intratumoral nutritional and oxygen ambience.

Thymidine phosphorylase (TP), also known as platelet-derived endothelial cell growth factor, has been implicated in bladder cancer angiogenesis. To examine its role more clearly, we have quantified and localized its expression using Western analysis and immunohistochemistry in a series of 105 bladder cancers. We have also assessed the relationship between TP expression and other tumor parameters including quantitative angiogenesis, p53 status, ploidy, and survival. By Western analysis, TP expression was 5-fold higher in tumors than in normal bladder samples (P < 0.02). Expression was 15-fold higher in invasive tumors than in normal bladder (P < 0.001) and 8-fold higher than in superficial tumors (P < 0.005). Immunohistochemistry of the tumors showed TP was present in the neoplastic epithelium in 27% of the tumors, in the inflammatory cells in 72% of the tumors, in stromal cells in 30% of the tumors, and in tumor-associated endothelium in 11% of the tumors. Expression by Western blotting and immunohistochemistry was significantly up-regulated in tumors compared with normal bladder (P < 0.05). Tumor cell TP expression correlated with tumor grade (P < 0.02), but there was no correlation between tumor cell TP expression and tumor stage (P = 0.46), ploidy (P = 0.52), p53 expression (P = 0.9), tumor vascularity (P = 0.8), relapse-free survival (P = 0.57), or overall survival (P = 0.94). TP protein is expressed in bladder cancers, and expression is associated with an aggressive phenotype. Because TP can activate a number of cytotoxic agents, it provides a potential therapeutic target in bladder cancer.

The aim of this study was to examine the immunohistochemical expression of p53 and bcl-2 in Kaposi's sarcoma and relate this with proliferation index (as measured by MIB-1 staining) and clinicopathological subtypes. Twenty formalin-fixed, paraffin-embedded cases of Kaposi's sarcoma were stained with commercially available antibodies to p53, bcl-2 and MIB-1, after pressure cooking antigen retrieval. All cases were strongly positive for bcl-2 with the majority containing more than 75% positive cells. In comparison, p53 expression was less striking. Eleven cases contained less than 24% (+1) of cells staining positively. Only two cases showed greater than 75% of positive cells, and both of these latter two lesions had metastasized. The MIB-1 staining in all cases of Kaposi's sarcoma was strongly positive, irrespective of clinicopathological type, in keeping with the highly proliferative nature of this lesion. Thus, we have demonstrated uniformly increased expression of bcl-2 protein in Kaposi's sarcoma irrespective of clinicopathological subtype and MIB-1 staining, while p53 expression is relatively less common, except in those cases which have metastasized. This may help identify those cases that will behave in a more aggressive manner. However, more cases need to be evaluated to verify this.

Tumour angiogenesis is an important factor for tumour growth and metastasis. Although some recent reports suggest that microvessel counts in non-small cell lung cancer are related to a poor disease outcome, the results were not conclusive and were not compared with other molecular prognostic markers. In the present study, the vascular grade was assessed in 107 (T1,2-N0,1) operable non-small cell lung carcinomas, using the JC70 monoclonal antibody to CD31. Three vascular grades were defined with appraisal by eye and by Chalkley counting: high (Chalkley score 7-12), medium (5-6), and low (2-4). There was a significant correlation between eye appraisal and Chalkley counting (P < 0.0001). Vascular grade was not related to histology, grade, proliferation index (Ki67), or EGFR or p53 expression. Tumours from younger patients had a higher grade of angiogenesis (P = 0.05). Apart from the vascular grade, none of the other factors examined was statistically related to lymph node metastasis (P < 0.0001). A univariate analysis of survival showed that vascular grade was the most significant prognostic factor (P = 0.0004), followed by N-stage (P = 0.001). In a multivariate analysis, N-stage and vascular grade were not found to be independent prognostic factors, since they were strongly related to each other. Excluding N-stage, vascular grade was the only independent prognostic factor (P = 0.007). Kaplan-Meier survival curves showed a statistically significant worse prognosis for patients with high vascular grade, but no difference was observed between low and medium vascular grade. These data suggest that angiogenesis in operable non-small cell lung cancer is a major prognostic factor for survival and, among the parameters tested, is the only factor related to cancer cell migration to lymph nodes. The integration of vascular grading in clinical trials on adjuvant chemotherapy and/or radiotherapy could substantially contribute in defining groups of operable patients who might benefit from cytotoxic treatment.

Aims: To examine the immunohistochemical distribution of thymidine phosphorylase (TP) in all clinicopathological subtypes of Kaposi sarcoma., Methods: Thirty two biopsy specimens of Kaposi sarcoma (29 patients) were studied. Six of these patients represented classic, six endemic, eight HIV associated, seven post-immunosuppression/transplant related, and two unclassified variants of Kaposi sarcoma. The average age was 49 years (range 22-83 years) and the male: female ratio 24:5. Four samples of angiosarcoma and one of spindle cell haemangio-endothelioma were stained in parallel. All specimens were fixed in formalin, embedded in paraffin wax and processed routinely. Immunohistochemistry was carried out using an antibody directed against CD31 (JC70) and the monoclonal antibody P-GF.44C against TP., Results: All biopsy specimens showed immunoexpression for TP. The spindle cell component stained more strongly than newly formed endothelium lined vessels and normal, resident vessels at a distance from the lesions., Conclusions: The strong immunoexpression of TP suggests up-regulation of TP and a role for TP in angiogensis in Kaposi sarcoma. The mechanism for the up-regulation of TP remains unknown, but viral infections may trigger it. The differential staining of the various cell components of Kaposi sarcoma also suggest that TP either plays a role in the differentiation and maturation of Kaposi sarcoma or is a reflection of such changes.

bcl-2 was originally identified as an oncogene involved in follicular lymphomas as a result of chromosomal translocation (14;18). It is now believed that bcl-2 is implicated in the regulation of cell death by inhibiting apoptosis and that its expression is not restricted to haematopoietic cells, but is also present in many epithelial and mesenchymal tissues. Recent studies have analysed the expression of this molecule in a variety of non-lymphoid malignancies including lung, breast, prostate, and nasopharyngeal carcinomas and neuroblastoma. In the present study, 50 colorectal adenomas, 10 hyperplastic polyps, and 142 carcinomas, including 25 arising from pre-existing adenomas, were examined using an antibody detecting the bcl-2 protein product. In non-neoplastic mucosa, bcl-2 was expressed in the crypt cells only, whilst the more differentiated surface epithelial cells lacked any demonstrable bcl-2. Forty-one of the 50 adenomas (82 per cent) and 48 of the 142 carcinomas were positive for bcl-2 expression. All hyperplastic polyps were negative. A reciprocal relationship was found between bcl-2 reactivity and p53 overexpression, as detected by DO7 antibody, in approximately 65 per cent of the cases. The bcl-2-positive/p53-negative subgroup showed a strong correlation (P = 0.0056) with negative lymph node status (Dukes' A and B), implying a less aggressive pathway of neoplastic transformation.

An inconsistent association exists between EBV-LMP-1 and bcl-2 protein expression in Reed-Sternberg cells seen in Hodgkin's disease. In fact, many studies have concluded that there is no correlation between EBV-LMP and bcl-2 expression in Hodgkin's disease. We undertook an analysis of post-transplant lymphoproliferative disorders to explore the relationship between EBV-LMP and bcl-2 in Reed-Sternberg-like cells found in this condition, given the strong association between this disorder and EBV. Reed-Sternberg-like cells were found histologically in 11 of 28 cases of renal, heart and heart-lung post-transplant lymphoproliferative disorders. Formalin-fixed, paraffinembedded sections were stained with monoclonal antibodies to EBV-LMP-1 and bcl-2 proteins. Reed-Sternberg-like cells in all 11 cases co-expressed EBV-LMP and bcl-2. A similar relationship was noted with large, mononuclear cells and occasional small lymphoid cells. The staining pattern seen with both antibodies was of similar intensity and both displayed cytoplasmic Golgi accentuation. In the setting of post-transplant lymphoproliferative disorders. Reed-Sternberg-like cells exhibit strong co-expression of EBV-LMP-1 and bcl-2 proteins, supporting a positive correlation between them. This is in contrast to the findings in Hodgkin's disease. The reason for this discrepancy may be due to the iatrogenic immunosuppression and resultant severe EBV infection, together with other cellular events.

Although c-erbB-2 oncoprotein immunohistochemical expression has been thoroughly studied in a variety of human tumors, its prognostic significance remains unclear. Moreover, differences in assessment criteria further complicate the evaluation of c-erbB-2 as a prognostic marker. In the present study we examined the expression of c-erbB-2 protein in 107 patients suffering from operable (T 1,2-N0, 1 staged) non-small cell lung cancer (30 adenocarcinomas and 69 squamous cell carcinomas) treated with surgery alone. A 3-7 year of follow up (median 45 months) was available for all patients. Paraffin embedded sections were stained with the NCL-CB11 monoclonal antibody using the immunoperoxidase technique. Analysis was based on cytoplasmic reactivity as membrane staining was impossible to assess against this background. Strong positive cytoplasmic staining was identified in 20/107 (19%) of cases, weak in 30/107 (20%) and negative in 57/107 (53%). Results were correlated with patient variables (age,sex) and tumor parameters (T,N-stage, grade, histology, Ki67 proliferation index, p53 and EGFR expression). C-erbB-2 expression was not related to any of these factors. Although c-erbB-2 defined a worse prognosis, univariate analysis of survival did not confirm any statistically significant difference between the c-erbB-2 staining groups (p=0.5). T,N-stage were the only statistically significant prognostic variables. Any contribution of c-erbB-2 to the development of tumour aggressive behaviour in non-small cell lung cancer requires assessment in the specific subgroups of patients.

The recent demonstration that the murine anti-p53 monoclonal antibody PAb248 can identify human p53 in a variety of normal tissues proves that immunohistochemical detection does not necessarily indicate the presence of mutations. PAb248 can detect p53 protein in a cytoplasmic-perinuclear localization, not previously described. The present study presents the expression of this antibody in a series of 34 cases of Hodgkin's disease, comparing it with the antibodies CM1, PAb1801, and PAb240. In all cases, PAb248 showed uniform cytoplasmic-perinuclear staining in small and medium-sized lymphocytes, while it was constantly negative in Hodgkin, Reed-Sternberg (R-S/H) cells, and variants. This pattern of staining was the opposite to that observed with the antibodies CM1, PAb1801, and PAb240, where the staining was nuclear and restricted to the R-S/H cells, with the small lymphocytes being negative. p53 can be found in different conformations and localizations, with the cytoplasmic-perinuclear localization mainly, although not exclusively, being found in normal and reactive tissues and the nuclear localization being mainly expressed by neoplastic cells. These results give further support to the theory that the R-S/H cells are the neoplastic population in Hodgkin's disease, while the surrounding lymphocytes are reactive.

Sequential interaction of neoplastic cells with the endothelium of tumour neovasculature is believed to be a significant step in tumour metastasis. Increasing evidence suggests that inducible endothelial adhesion molecules are intimately involved in this process. An immunohistochemical approach was used to examine the expression of adhesion molecules in 14 normal controls and a series of 64 invasive breast carcinomas. Endothelium in normal breast showed constitutive expression of PECAM (100 per cent), ICAM-2 (100 per cent), and P-selectin (64 per cent); variable and focal expression of ICAM-1 (71 per cent); and only weak staining for E-selectin (21 per cent). No ICAM-3 or VCAM-1 expression was observed. Similarly to normal breast endothelium, widespread and intense immunoreactivity on the endothelium of tumour-associated vessels was seen for PECAM (100 per cent), ICAM-1 (69 per cent), and ICAM-2 (95 per cent). In contrast to normal tissues, E- and P-selectins showed increased intensity of staining (52 and 67 per cent of cases, respectively) and expression of E- and P-selectins was more prominent at the tumour periphery. ICAM-3 expression was increased on tumour endothelium (15 per cent of cases), but in common with VCAM-1 (10 per cent) expression was focal. A previously unreported finding was the immunoreactivity of the neoplastic epithelial cells for the non-epithelial lineage markers ICAM-1 (34 per cent), ICAM-3 (10.9 per cent), PECAM (1.6 per cent), and E- and P-selectins (7 and 37 per cent of cases, respectively). These findings show that tumour endothelium displays significant heterogeneity and can assume a pro-inflammatory phenotype, probably as a result of cytokine stimulation. Upregulation of adhesion molecules might contribute to changes in invasive phenotype by promoting endothelial cell adhesion and angiogenesis, as well as forming a substratum for tumour cells to assemble and attract macrophages.

A gene called deleted in colon cancer (DCC) has been identified on a region of chromosome 18, which is deleted in 70% of colorectal cancers. The DCC gene encodes a protein belonging to the immunoglobulin superfamily with similarity to the N-CAM transmembrane proteins and is a putative tumor-suppressor gene. Alternative splicing of transcripts of transmembrane proteins, including N-CAM, is known to occur, resulting in different isoforms of the protein. Using five antibodies against the DCC gene product (three monoclonal antibodies raised in our laboratory, one commercially available antibody, and a rabbit polyclonal antibody), we have demonstrated by immunostaining a DCC protein isoform in reticuloendothelial cells in human thymus, tonsil, and lymph node. This can be distinguished from another isoform described in normal colonic epithelium, because this latter is not demonstrable with the antibodies we have used. It could not be detected in normal colonic epithelium, polyps or colorectal carcinomas. This restrictive distribution suggests that not all DCC gene products are important in colonic cancer.

We studied by immunohistochemistry the HLA-allelic, beta 2-microglobulin, and TAP-1 expression in primary breast carcinomas and related lymph node metastases. Thirty-three of the primary tumors and 44% of the lymph node metastases had a complete HLA class I loss. The higher incidence of antigenic loss in metastatic tumors suggests that recognition of HLA class I antigens by the host immunity could have an important role in the metastatic evolution of breast cancer. We observed a simultaneous defective expression of all three components involved in HLA class I expression. Since the controlling genes of heavy chain and TAP-1 are located in different chromosome than beta 2-microglobulin, it could be that a common factor exists regulating HLA class I antigenic expression. Five of 25 (20%) primary and metastatic tumors from HLA-A2-positive individuals also had a selective loss. The high incidence of HLA class I loss in breast cancer patients shows that adjuvant immunotherapy to induce HLA class I expression could be of value in a subgroup of patients.

In some studies of breast cancer, quantitation of immunohistochemically highlighted microvessel 'hot spots' has been shown to be a powerful prognostic tool. However, the antibody used, the number and size of the 'hot spots' assessed, and the stratification of patients into high and low vascular groups vary between studies. Furthermore, little is known about the relationship between microvessel density and other vascular parameters. These uncertainties and the laborious nature of the technique make it unsuitable for diagnostic practice. Both manual and computerized image analysis techniques were used in this study to examine the relationship between microvessel density and the vascular parameters in different sized microscopic fields in a pilot series of 30 invasive breast carcinomas. Automated pixel analysis of immunohistochemical staining, Chalkley point counting, and observer subjective vascular grading were also assessed as more rapid methods of measuring tumour vascularity. A Chalkley count was also performed on a further 211 invasive breast carcinomas. Significant correlations were observed between manual microvessel density and luminal perimeter (r = 0.6, P = 0.0004), luminal area (r = 0.56, P = 0.002), and microvessel number (r = 0.57, P = 0.0009) by computerized analysis. There were also significant correlations between the microscopic hot spots of 0.155 mm2 and 0.848 mm2 for microvessel number (r = 0.81, P < 0.00005), luminal perimeter (r = 0.78, P < 0.00005), and luminal area (r = 0.65, P = 0.0001). In addition, a significant correlation was observed between microvessel density and both subjective vascular grade (P = 0.002) and Chalkley count (P = 0.0001). A significant reduction in overall survival was observed between patients stratified by Chalkley count in both a univariate (P = 0.02) and a multivariate (P = 0.05) analysis in the 211 invasive breast carcinomas. These findings show that Chalkley counting is a rapid method of quantifying tumour angiogenesis and gives independent prognostic information which might be useful in diagnostic practice.

Apoptosis is an important determinant of tumour growth which can be regulated by the bcl-2 and p53 genes. This study examines the relationship between apoptosis, growth fraction (Ki-67 immunolabelling index), and accumulation of the bcl-2 and p53 proteins in a spectrum of cerebral astrocytic tumours (n = 81), including fibrillary astrocytomas (n = 16), anaplastic astrocytomas (n = 19), and glioblastomas (n = 46). Median apoptosis indices (AIs) increased across this spectrum of tumours, and a significant (P < 0.0001) correlation was demonstrated between AI and Ki-67 labelling index (LI). Immunolabelling with the bcl-2 antibody was found in 44% of fibrillary astrocytomas, 42% of anaplastic astrocytomas, and 28% of glioblastomas. It was also found in the vascular endothelial proliferation typically seen in glioblastomas, and in the giant, multinucleated cells of some glioblastomas. No clear relationship between AI and bcl-2 accumulation was evident. Immunolabelling with the p53 antibody was found in 56% of fibrillary astrocytomas, 79% of anaplastic astrocytomas, and 50% of glioblastomas. No clear relationship between AI and patterns of p53 immunolabelling was evident. Equal proportions of p53-positive tumours were bcl-2 positive and bcl-2 negative, but a small proportion of p53-negative tumours was bcl-2 positive. The correlation between AI and Ki-67 LI is in line with findings in other malignant tumours. We suggest that the regulation of apoptosis in astrocytic tumours is too complex for a clear association between AI and bcl-2 and p53 protein expression to be demonstrated.

Aim-To investigate the expression of p53 protein in invasive squamous cell carcinoma (SCC) of the larynx and dysplasia in relation to histological grade and tobacco smoking.Method-Paraffin wax embedded tissue sections from 41 cases of invasive SCC of the larynx, 28 cases of dysplasia and 14 control laryngeal biopsy specimens were studied immunohistochemically using two anti-p53 monoclonal antibodies (DO7 and 1801). The Streptavidin/horseradish peroxidase method was used after microwave antigen retrieval and a semiquantitative method was applied to assess the extent of p53 expression.Results-Of the cases of invasive SCC of the larynx, 78% (32/41), regardless of histological grade, overexpressed p53 compared with only 30% (eight of 28) of cases of mild dysplasia. A gradual increase in p53 expression from mild to severe dysplasia (60%) was observed, and only three of 14 control biopsy specimens of laryngeal nodules showed occasional weakly positive basal cells.Conclusion-The gradual increase in p53 expression from mild to severe dysplasia to invasive SCC indicates that p53 overexpression is an early event in laryngeal carcinogenesis which may lead to invasive malignancy. p53 overexpression may be related to environmental factors as most of the patients smoked tobacco. Microwave postfixation may be essential for the reliable detection of p53.

Angiogenesis is the formation of new blood vessels from the existing vascular bed. It is a complex multi-step process controlled by a number of angiogenic factors. One such factor is platelet-derived endothelial cell growth factor (PD-ECGF), recently shown to be thymidine phosphorylase (TP), which is angiogenic in several in vivo assays and tumour systems. PD-ECGF/TP catalyses the reversible phosphorylation of thymidine to deoxyribose-1-phosphate and thymine. Since PD-ECGF/TP has an important role in cellular metabolism and in angiogenesis and its expression has been only partially characterized, we raised a monoclonal antibody against recombinant PD-ECGF/TP and used an immunohistochemical approach to examine the expression of PD-ECGF/TP in a comprehensive range of normal human tissues. The clone P-GF44.C, which recognizes recombinant PD-ECGF/TP and cell lysates transfected with a plasmid expressing PD-ECGF/TP cDNA on Western blotting, was selected for its ability to stain routinely processed tissue. Staining was observed in both the cytoplasm and/or the nucleus. Immunoreactivity was strongly expressed by macrophages, stromal cells, glial cells, and some epithelia. Gastrointestinal epithelium, smooth muscle, adrenal, lung, and testis were negative. Although endothelial cell expression was observed, there was no correlation with sites of new vessel growth. This pattern of expression suggests tight PD-ECGF/TP regulation and that cellular thymidine pools may serve to control its different functions. Thus, in the nucleus it might modulate the pool for DNA synthesis, whilst in the cytoplasm it could control other effects through different enzyme systems. The high expression present in macrophages and skin might be important for total body thymidine homeostasis.

Aims: To examine thymomas for proteins encoded by oncogenes and to determine whether their presence correlates with tumour growth and associated myasthenia gravis., Methods: Sections of 24 thymomas were incubated with anti-EGF receptor (EGF-R), anti-Ki67 antigen, anti-p53, and anti-bcl-2 antibodies, and then stained using the alkaline phosphatase/anti-alkaline phosphatase (APAAP) technique. Cell suspensions and epithelial cell cultures from some of the tumours were also studied., Results: Whereas EGF-R expression was not detected in any of the controls (but only in a 20 week old fetus), it was detected in neoplastic epithelial cells of all thymomas, and was most strongly expressed in metastases and in samples from donors with severe myasthenia gravis. Ki67 labelling was also increased, especially in the larger thymomas. Epithelial expression of both of these markers was confirmed in fresh cell suspensions and monolayer cultures from the five available cases. In contrast, p53 and bcl-2 were not detected in the neoplastic cells, but bcl-2 was present in the intermingling thymocytes., Conclusions: Neoplastic thymoma cells express EGF-R and Ki67, but there is no concomitant increase in the expression of p53 and bcl-2 proteins. Increased EGF-R expression may result in increased proliferation of neoplastic cells and also in myasthenia gravis. Measurement of EGF-R concentrations may be of prognostic value. The bcl-2 staining pattern in T lymphocytes illustrates the broad spectrum of maturational stages in thymoma lymphocytes.

CD30 positive anaplastic large cell lymphoma (ALCL) is a type of non-Hodgkin's lymphoma associated with a specific chromosome translocation between chromosomes 2 and 5. Recent molecular characterization of the translocation breakpoint has identified a gene fusion between NPM (nucleophosmin) and ALK (anaplastic lymphoma kinase). Using a DNA hybridization technique, the NPM rearrangement was found among 5/5 ALCL samples. We have developed a PCT methodology which has enabled the detection of the NPM-ALK rearrangements amongst seven t(2;5)(p23;q35) ALCL cases based on a long-range PCR of genomic DNA. The rapidity and robustness of this method may have diagnostic applications for ALCL.

The p53 tumour suppressor gene contributes to the regulation of DNA repair and the initiation of apoptosis. Mutations of this gene and nuclear accumulation of its protein product are features of a large variety of tumours. A histological spectrum of meningiomas, including anaplastic examples and a meningosarcoma, was analysed for accumulation of the p53 protein and mutations in exons 4-9 of the p53 gene. No mutations were found, but 9/34 (26%) tumours showed accumulation of the p53 protein. The p53-positive meningiomas came from across the histological spectrum and were not restricted to the anaplastic group. The accumulation of wild-type p53 in a proportion of meningiomas may reflect this gene's role in DNA repair, or its enhanced stability when bound to cellular proteins such as the mdm-2 gene product.

Three cases of Hodgkin's disease presenting primarily in the lung are described. All 3 patients presented with respiratory symptoms and chest X-rays revealed discrete masses within the lung parenchyma. CT examination of the mediastinum did not reveal regional or generalized lymphadenopathy at the time of diagnosis in 2 of the cases. A diagnosis of Hodgkin's disease was made on open lung biopsies and despite aggressive chemotherapy, 2 patients died within 2 yrs. The histological and immunocytochemical features of these cases were typical of that expected in Hodgkin's disease. However, the stated indolent course of primary pulmonary Hodgkin's disease is not invariable. Those patients with bilateral interstitial disease and systemic symptoms have a poor prognosis. Furthermore, it is difficult to exclude definitely lymph node involvement (although not enlarged) at the time of diagnosis. Since several cases described in the literature have concurrent and/or subsequent nodal involvement, the entity of primary pulmonary Hodgkin's disease without lymph node involvement is exceedingly rare.

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.

Platelet-derived endothelial cell growth factor was previously identified as the sole angiogenic activity present in platelets; it is now known to be thymidine phosphorylase (TP). The effect of TP on [methyl-3H]thymidine uptake does not arise from de novo DNA synthesis and the molecule is not a growth factor. Despite this, TP is strongly angiogenic in a rat sponge and freeze-injured skin graft model. Neutralizing antibodies and site-directed mutagenesis confirmed that the enzyme activity of TP is a condition for its angiogenic activity. The level of TP was found to be elevated in human breast tumors compared to normal breast tissue (P < 0.001). Overexpression of TP in MCF-7 breast carcinoma cells had no effect on growth in vitro but markedly enhanced tumor growth in vivo. These data and the correlation of expression in tumors with malignancy identify TP as a target for antitumor strategies.

We have investigated the relationship between the p53 genotype and phenotype in a series of 22 high-grade non-Hodgkin's lymphomas (NHLs) in which we sequenced the p53 gene open reading frame (exons 2-11). Immunostaining for p53 was already available for these cases. Mutations were found in 10/22 cases (45%) and 3/10 were in exons 4 or 10 outside the classic 'hot spot' regions (exons 5-8). Comparison with immunostaining indicated that, besides cases with the 'expected' patterns (in which gene mutation and protein detection were either both present or both absent) there were also cases in which p53 protein was detected in the absence of any mutation and those with a mutant gene in which the protein was undetectable. These data show that: (1) in high-grade NHLs mutations frequently occur outside the classic hot spot regions and (2) staining for p53 is not predictive of the status of the gene, i.e. whether or not a mutation is present. Therefore in order to document p53 involvement in lymphoid tumours it is necessary both to sequence at least the whole translated open reading frame of the gene and to show evidence of protein expression by immunostaining.

Bcl-2 protein expression was studied in a series of 58 MALT lymphomas using a monoclonal antibody which recognises this protein in routinely processed paraffin embedded tissue. Thirty-three of 58 cases showed heterogeneity for bcl-2 expression, 18 of 58 cases were bcl-2 positive and 7 of 58 were bcl-2 negative. High grade and low grade MALT lymphomas showed different patterns of staining. All 21 low grade tumours were positive for bcl-2, though in seven cases only a proportion of the neoplastic cells expressed this protein. In the 37 high grade tumours the majority of the neoplastic cells were negative with seven cases showing no reactivity at all. These findings give further support to the theory that MALT lymphomas differ in pathogenesis to nodal lymphomas and suggest that the good prognosis of MALT lymphomas may partly be explained by the fact that they maintain a normal pattern of bcl-2 expression.

A new classification of lymphoid neoplasms, mostly based on existing terminology, is proposed by the International Lymphoma Study Group. The proposed classification was reached through a consensus of the members, despite their diverse backgrounds, and consists of a listing of currently recognized clinicopathological entities. These tumours are divided into three major categories: B-cell neoplasms, T-cell and postulated natural killer cell neoplasms, and Hodgkin's disease. The characterization of each entity is based on a synthesis of all available information. This concept departs from a purely morphological approach to lymphoma classification, which is considered to be inadequate, because many biologically distinctive lymphoma types can exhibit a broad and overlapping morphological spectrum. Some entities are provisional, pending further data to confirm that their recognition is reproducible. The salient clinicopathological features of each entity are summarized in this review.

The sequestration of parasitized erythrocytes in the microvasculature of vital organs is central to the pathogenesis of severe Plasmodium falciparum malaria. This process is mediated by specific interactions between parasite adherence ligands and host receptors on vascular endothelium such as intercellular adhesion molecule-1 (ICAM-1) and CD36. Using immunohistochemistry we have examined the distribution of putative sequestration receptors in different organs from fatal cases of P. falciparum malaria and noninfected controls. Receptor expression and parasite sequestration in the brain were quantified and correlated. Fatal malaria was associated with widespread induction of endothelial activation markers, with significantly higher levels of ICAM-1 and E-selectin expression on vessels in the brain. In contrast, cerebral endothelial CD36 and thrombospondin staining were sparse, with no evidence for increased expression in malaria. There was highly significant co-localization of sequestration with the expression of ICAM-1, CD36, and E-selectin in cerebral vessels but no cellular inflammatory response. These results suggest that these receptors have a role in sequestration in vivo and indicate that systemic endothelial activation is a feature of fatal malaria.

Presentation of endogenous antigenic peptides to cytotoxic T lymphocytes (CTLs) is mediated by the major histocompatibility complex (MHC) class I molecules. These antigenic peptides derived from the cytoplasmic protein pool are transported by the recently described MHC-encoded transporters (TAP1 and TAP2) into a pre-Golgi region where they take part in the assembly of MHC class I molecules. Using an affinity-purified polyclonal antibody (AK1.7) for TAP1, we analyzed 81 colorectal carcinomas, 32 adenomas, and the respective nonneoplastic mucosa. Loss of the transporter molecule (TAP1) was observed in 14% (11 of 81) of the carcinomas, either complete (7 of 11) or focal (4 of 11), whereas adenomas and normal mucosa were always positive. This study adds further information to the understanding of the mechanisms related to the defective presentation of the MHC class I molecules by tumor cells.

At least 20 different isoforms of the human CD44 lymphocyte-homing receptor/hyaluronan receptor have been described to date that arise from the differential splicing of up to 10 alternative exons (termed v1-v10) encoding the membrane-proximal extracellular domain. Although numerous analyses at the mRNA level have indicated tissue-specific expression of CD44 variants, few analyses have been performed at the protein level because of limited availability of suitable monoclonal antibodies. Recently, however, exon-specific monoclonal antibodies have been generated using bacterial fusion proteins, and these have been reported to detect high levels of vCD44 containing the v6 exon on human tumors. Together with earlier evidence linking this particular exon with tumor metastasis in the rat, these latter experiments have led to the interpretation that v6 splice variants play a causative role in tumor dissemination. In this paper we describe the use of a new and comprehensive panel of CD44 exon-specific monoclonal antibodies generated against a recombinant CD44(v3-10)-immunoglobulin chimera to study vCD44 expression in a large number of normal and neoplastic tissues. We show that the expression of vCD44 varies greatly among different human tumors and that some express either very low levels of vCD44 or no CD44 at all. Furthermore, we demonstrate that expression is not limited to isoforms containing the v6 exon but includes variants carrying v3, v4/5, and v8/9. Additionally, normal epithelial tissues are shown to express considerable levels of these same vCD44 isoforms. Such results argue against a ubiquitous role for vCD44 isoforms in promoting tumor growth and metastasis.

Monoclonal antibodies (MAbs) were raised in mice against a bacterial fusion protein composed of the intracellular serine/threonine kinase domain of the type-2 activin receptor, ACTR2, fused to glutathione S-transferase. Three MAbs with high affinity toward the ACTR2 kinase domain were isolated, one of which recognized specifically ACTR2 expressed transiently in vascular endothelial cells. These reagents should be of use in the elucidation of mechanisms of transmembrane signaling by this member of the emerging receptor serine threonine kinase family.

Recent evidence indicates that membrane-bound immunoglobulin on B lymphocytes is associated with a molecule which comprises the products of the mb-1 and B29 genes. This molecule is a highly specific marker for B-cells, presumably because of its central functional role in antigen triggering, and has recently been clustered as CD79a at the 5th Leucocyte Workshop. Recently there has been controversy surrounding reports of B-cell antigen expression by Reed-Sternberg and related cells, and we have therefore studied 108 cases of Hodgkin's disease immunohistochemically using a novel antibody which detects mb-1 protein in paraffin sections. The results were compared with those achieved using antibody L26 to detect CD20. The mb-1 protein was present in the neoplastic cells in all 14 cases of lymphocyte predominance Hodgkin's disease studied, and CD20 immunoreactivity was also found in seven of the eight cases of this subtype studied. Of the non-lymphocyte predominance cases, 20% (19/94) expressed mb-1 and 30% (20/67) CD20 in the Reed-Sternberg cells, but the cells positive for either of these two markers usually constituted only a very small proportion of the neoplastic population. However, in occasional cases (one of 94 for mb-1 and five of 67 for CD20), more than 50% of the neoplastic cells expressed one or both B-cell antigens. These results confirm the B-cell origin of the neoplastic cells in lymphocyte predominance Hodgkin's disease, but they also indicate that, contrary to our previous study, mb-1 expression may occasionally be found in what appears, on histological grounds, to be other types of Hodgkin's disease.

The reproductive cycle in eukaryotic cells is partly controlled by the p34 protein kinase, the product of the cdc2 gene. We report here the tissue reactivity of four new anti-cdc2 monoclonal antibodies in relation to the known proliferation markers Ki-67 and JC1. In tissues where proliferation occurs, germinal centres in the tonsil, basal layers of tonsular epithelium and skin, cortex of the thymus, seminiferous tubules of the testis and epithelium of the colon, the anti-cdc2 antibodies gave positive nuclear staining as did the proliferation markers. The percentage of positive cells was, however, lower with the cdc2 antibodies. Given the role of the cdc2 gene at specific points of the cell cycle, these antibodies are potentially useful as markers of different phases of the cell cycle and may help to detect abnormalities in cell cycle control in disease.

Aims: To describe the distribution of the recently cloned human leucocyte adhesion molecule ICAM-3 in normal and neoplastic tissues and cell lines., Methods: A panel of four monoclonal antibodies to ICAM-3 were used to stain cell lines and sections of human lymphoid tissues using the alkaline phosphatase-anti-alkaline phosphatase immunocytochemical method (APAAP)., Results: In peripheral blood ICAM-3 was detected on monocytes, granulocytes, and most lymphocytes. In sections of human lymphoid tissue the antigen was also found on most lymphocytes, but many of the proliferating B cells found in the germinal centres of secondary lymphoid follicles were ICAM-3 negative. ICAM-3 was also found on neoplastic white cells (in chronic lymphocytic leukaemia, hairy cell leukaemia, acute and chronic myeloid leukaemia, and multiple myeloma) with the exception of Reed-Sternberg cells in Hodgkin's disease, many of which were negative. ICAM-3 was consistently absent from cells and tissues of non-haemopoietic origin. Endothelium (which expresses ICAM-1) was negative for ICAM-3, with the exception of vessels in some neoplastic lymphoid samples which showed variable staining for ICAM-3., Conclusions: These findings suggest that ICAM-3 is essentially restricted to the haemopoietic system and is reciprocal in its expression to ICAM-1, in that it is present on resting cells and its level falls as a result of cell activation.

Aims: To provide a detailed knowledge of the distribution of the CD13 molecule, also known as the protease aminopeptidase-N, on both normal tissues and malignant neoplasms of epithelial and lymphoid origin., Methods: CD13 antigen was examined by immunocytochemistry, using a recently produced antibody (VS5E) alongside a commercially available anti-CD13 monoclonal antibody. The VS5E recognising CD13 was produced by immunising a doxorubicin resistant breast cancer cell line (MCF-7-ADr). A striking feature of this antibody was that it stained the doxorubicin resistant cells but not the parental cell line. Both antibodies were tested on a broad range of normal tissues and three common types of epithelial malignancy (colon n = 28, lung n = 30, breast n = 35), and 12 cases of Hodgkin's and 52 of non-Hodgkin's lymphomas., Results: CD13 was expressed on many tissue and cell types outside the haematopoietic system. In particular it was present on breast epithelium and in 20% (seven of 35) of breast carcinomas, but absent in normal and neoplastic colonic and bronchial tissues and lymphomas., Conclusions: This study provides not only detailed information about the expression of the CD13 antigen, but also raises the important possibility that CD13 expression may correlate with drug resistance in breast carcinomas.

The role of bcl-2 expression in solid tumours is as yet undefined. It was, therefore, the purpose of this study to investigate expression of bcl-2 protein in 111 human breast carcinomas using immunohistochemistry and the monoclonal antibody bcl-2 124. Expression was then compared with the established indicators of prognosis and biological behaviour in malignant breast disease. No relationship could be observed between bcl-2 and node status, tumour size, differentiation, type or age at excision. However, a strong positive relationship was seen between bcl-2 and oestrogen receptor (ER), with 70 of 88 (80%) bcl-2-positive tumours being ER positive also, compared with seven of 23 (30%) bcl-2-negative tumours being ER positive (P < 0.0001). The converse was found when bcl-2 was compared with epidermal growth factor receptor (EGFR). A strong negative relationship was observed, with 26 of 88 (30%) bcl-2-positive tumours being EGFR positive, compared with 16 of 23 (70%) bcl-2-negative tumours being EGFR positive (P = 0.001), raising the possibility that bcl-2 is an ER-regulated gene. An inverse relationship was also found between bcl-2 and the oncogenes c-erbB-2 and p53. Thus, loss of bcl-2 expression in breast cancer is associated with a range of molecular markers of poor prognosis and may define part of an ER-negative, EGFR-positive phenotype.

Current studies of tumor angiogenesis rely on the concept that endothelium proliferates 30-40 times faster in tumors than in normal tissues. This evidence is based on histological autoradiographic data largely from animal studies. To assess endothelial cell proliferation in human cancer we used the more sensitive and specific technique of immunohistochemistry. We measured the frequency and distribution of endothelial cell proliferation and examined their relationship to tumor cell proliferation. For the first time, we also correlated endothelial and tumor cell proliferation with tumor vascularity. Twenty breast carcinomas from patients exposed to bromodeoxyuridine 3-8 h prior to surgery were double immunostained using antibodies to CD31 (as a marker of endothelium) and bromodeoxyuridine (as a marker of proliferation). The labeling index (LI) for both tumor and endothelial cells was determined and tumor vascularity was assessed by counting the number of CD31 positive vessels. Endothelial cell proliferation was predominantly at the tumor periphery while tumor cell proliferation occurred throughout the lesion. The mean LIs for endothelium and tumor were 2.2% (range, 0.8-5.3) and 7.3% (range, 1.3-17.1), respectively. There was no correlation between tumor and endothelial cell LI (P = 0.414) or between the tumor LI or endothelial cell LI and tumor vascularity (P = 0.08 and P = 0.39, respectively). These findings suggest that previous studies in animal tumors have significantly overestimated endothelial cell proliferation and that its importance in tumor angiogenesis may be related more to continual remodeling and migration of vessels than to proliferation alone.

Background: The proto-oncogene bcl-2 encodes a protein that inhibits programmed cell death (apoptosis). The protein is expressed in basal cells in normal human epithelium, but no data are available on the frequency or clinical importance of its expression in carcinoma. We studied bcl-2 expression in patients with non-small-cell lung carcinoma and correlated this phenomenon with survival., Methods: Immunochemical analysis with a monoclonal antibody specific for bcl-2 was used to detect the protein in tumor samples from 122 patients undergoing surgery for squamous-cell carcinoma (80 patients) or adenocarcinoma (42 patients). The possibility that bcl-2 expression correlated with survival was investigated with use of the log-rank test, hazard ratios, and their confidence intervals., Results: We detected bcl-2 protein in 25 percent of squamous-cell carcinomas (20 of 80) and 12 percent of adenocarcinomas (5 of 42). In adjacent normal respiratory epithelium, bcl-2 was expressed only in basal cells. Survival at five years was higher among patients with bcl-2-positive tumors, both in the group as a whole (P < 0.1) and in the group with squamous-cell carcinoma (P < 0.02). Patients 60 years of age or older who had bcl-2-positive tumors had the best prognoses, both in the group as a whole (P < 0.02) and in the group with squamous-cell carcinoma (P < 0.01)., Conclusions: The proto-oncogene bcl-2 is abnormally expressed in some lung carcinomas, and its expression may have prognostic importance.

Background: Endothelial cells are important in initiating adhesive processes between circulating cells and extracellular structures and changes in their distribution are believed to be important in many pathologic conditions. Since little is known about the detailed distribution of adhesion molecules in human endothelium in different sites and circumstances, the present study has undertaken a detailed analysis of 5 of the putative most important adhesion molecules on a wide range of normal tissue endothelium. We have compared this reactivity with that seen in a comprehensive range of vascular tumors both benign and malignant., Experimental Design: Fresh samples of a wide range of normal tissues and vascular tumors were stained by antibodies against the following adhesion molecules; intercellular adhesion molecule-1 (CD54), E-selectin (endothelial cell adhesion molecule-1), vascular cell adhesion molecule-MUC-1, P-selectin (platelet activation-dependent granule to external membrane protein, CD62) and MUC-18 using either the APAAP immuno-alkaline phosphatase or an immunoperoxidase method., Results: Labeling of the endothelial cells in different normal tissues with intercellular adhesion molecule-1, P-selectin, and MUC-18 was heterogeneous both in terms of vessel size and strength of staining. Vascular cell adhesion molecule-1 and E-selectin were largely absent. The vascular tumors were likewise variable in their staining patterns which frequently differed from the immunophenotype of the reactive vessels surrounding the tumor., Conclusions: This study demonstrates that the expression of adhesion molecules of the immunoglobulin and selectin family on normal tissue endothelium, and vascular tumors is much less predictable than that obtained with other vascular markers such as F8 RA, CD31, CD34, and CD36. Adhesion molecules show considerable heterogeneity of expression on vascular endothelium which presumably reflects their varied functions on different types of vessel. In general their expression is markedly reduced on vascular tumors.

It has been proposed that wild-type p53 cell-regulating functions are annulled in human cervical carcinomas, either by mutations in the human papillomavirus (HPV)-negative cases or as a consequence of their complexing with HPV E6. The aim of this study was to test this hypothesis on 39 fresh cervical biopsies by p53 immunocytochemistry (ICC) with antibody PAb 240 and with NISH (non-isotopic in situ hybridization) and PCR (polymerase chain reaction) for HPV detection. p53 protein was present in the basal layer of pure wart virus infection; the basal to middle third of CIN (cervical intraepithelial neoplasia); in 19/22 (86 per cent) HPV-positive cervical carcinomas, ten of which contained integrated HPV; and in 4/8 (50 per cent) HPV-negative cervical carcinomas. Dual detection of p53 antigen and HPV 16 DNA in the same sections demonstrated either p53 protein or integrated HPV 16 alone in the majority of cells. Co-localization of both signals was only evident in isolated cells. These data suggest that PAb 240 immunoreactivity is not mutant-specific. They are, however, consistent with the conformation hypothesis which proposes that wild-type p53 changes from a suppressor (PAb 240-negative) to a promoter (PAb 240-positive) form during cell growth response. Hence, according to this hypothesis, p53 protein expression may represent either the wild-type promoter form or mutant p53 protein, both of which share the same conformation. This may explain co-localization of p53 and HPV in some tumours. However, the absence of p53 protein in 50 per cent HPV-negative squamous cell carcinomas suggests that not all HPV-negative tumours accumulate p53 protein.

In view of their reported reciprocal effects on apoptosis, the expression of p53 and bcl-2 proteins was studied in 46 cases of Hodgkin's disease by immunocytochemical labeling. We found p53 protein in Reed-Sternberg cells and their mononuclear variants in 16 of the 46 cases (34.7%) of Hodgkin's disease, mainly in a nuclear pattern. This restricted expression on Reed-Sternberg cells and variants supports their neoplastic nature. This overexpression of p53 protein in one third of Hodgkin's disease cases is similar to that seen in many other human malignancies. bcl-2 protein was present in mantle zone B cells and scattered T cells in all cases, and in 17 cases (37.7%) of Hodgkin's disease in Reed-Sternberg cells and their mononuclear variants. Six cases coexpressed both proteins, whereas in 18 cases neither was identified. There is no apparent relationship between p53 and bcl-2 protein expression, and on the basis of the present results there is no reason to suppose that they have any particular complementary effects on the neoplastic transformation in Hodgkin's disease.