Showing papers in "The Journal of Pathology in 1991"

A comparison of immunohistochemical markers of cell proliferation with experimentally determined growth fraction.

Abstract: The relationship between immunoreactivity for cell proliferation markers (Ki67 and PCNA) and the growth fraction as determined by the fraction of labelled mitoses method was assessed in xenograft tumours grown from the LoVo cell line in nude mice. FLM curves were constructed by injecting tritiated thymidine and then preparing autoradiographs from the tumours. From this data an estimate of growth fraction and cell cycle time were made. Using frozen material from the same tumours, the Ki67 index was determined by immunostaining. PCNA staining was determined in the fixed material which had been used for the autoradiographs. The results show that Ki67 staining follows the same trend as the FLM-determined growth fraction as the tumour increases in size and the rate of growth decreases. However the Ki67 index does produce a consistent overestimate of the growth fraction in this in-vivo system, as compared to observations in cell culture. PCNA staining showed virtually 100 per cent positive staining in all the tumours, which is likely to reflect the long half-life of the antigen, compared to the very fast cell-cycle time of the xenograft tumours. These results show that staining with proliferation markers is not a precise determinant of growth fraction. Ki67 staining is a method that can be usefully applied as an operational marker of cell proliferation, but should not be used uncritically. Further caution is necessary in the use of PCNA. The findings also demonstrate the need to use a range of methods when assessing a new proliferation marker.

Insulitis in type 1 (insulin‐dependent) diabetes mellitus in man—macrophages, lymphocytes, and interferon‐γ containing cells

Abstract: This study sought to determine, firstly, the relative frequency of lymphocytes and macrophages and, secondly, the percentage of lymphocytes containing interferon-gamma in inflamed islets (insulitis) of patients with type 1 (insulin-dependent) diabetes. Autopsy pancreases of 12 patients who had died of recent-onset type 1 diabetes and one pre-diabetic patient who had died of cardiomyopathy were examined immunohistochemically. In the 87 islets that were studied, the lymphocyte macrophage ratio was 9.7:1 and approximately 40 per cent of the lymphocytes contained interferon-gamma. Interferon-gamma release in the insulitis process may be involved in the pathogenesis of type 1 diabetes.

Transforming growth factor alpha and epidermal growth factor in human pancreatic cancer.

Abstract: Overexpression of the epidermal growth factor receptor (EGFR) has been reported as an important molecular abnormality in human pancreatic cancer. There is in vitro evidence that simultaneous overproduction of one of its ligands, transforming growth factor alpha (TGF-alpha), might result in an autocrine loop with an increased proliferation signal. We analysed by immunocytochemical staining a retrospective series of human pancreatic cancers, chronic pancreatitis, and normal fetal and adult pancreatic tissues for the presence of TGF-alpha and epidermal growth factor (EGF). Ductal epithelial cells showed TGF-alpha immunoreactivity in both normal tissue and chronic pancreatitis, and 95 per cent of tumours showed strong immunoreactivity. In contrast, EGF immunoreactivity was not found in normal pancreas, but was expressed in 12 per cent of pancreatic carcinomas. Well-defined areas of EGF immunoreactivity in exocrine ducts showing reactive changes in pancreatitis might represent a benign response to tissue damage similar to that previously described in the gastric mucosa.

p53 expression in human breast cancer related to survival and prognostic factors: an immunohistochemical study.

Abstract: In a study of 90 breast cancer patients, tumour p53 protein expression was determined by immunohistochemistry using the monoclonal antibody PAb1801. Patient lymph node status and Bloom's grade were determined, and both oestrogen and progesterone status assessed, also by immunohistochemistry. Lymph node status, tumour grade, and progesterone receptor status all had a significant influence on survival. Patients with p53-positive tumours showed poorer survival but this did not achieve significance. p53 protein expression showed a significant relationship to high tumour grade and a weak correlation with negative oestrogen receptor status. The data suggest taht p53 protein expression may be a marker of more aggressive carcinomas but that the prognostic power of expression is likely to be weak and unlikely, therefore, to be of clinical value. The results do not resolve whether detectable p53 protein expression represents a random product of dedifferentiation, or an important feature of the malignant phenotype, playing a key role in tumour behaviour. The number of patients in our study is small, however, and investigation of a larger series is clearly indicated.

Epstein–Barr virus and carcinomas: Undifferentiated carcinomas but not squamous cell carcinomas of the nasopharynx are regularly associated with the virus

Abstract: The Epstein-Barr virus (EBV) is consistently associated with undifferentiated nasopharyngeal carcinoma (NPC). There is, however, conflicting evidence as to whether squamous cell NPCs are also EBV-associated. Moreover, it has been proposed that other epithelial tumours, particularly thymomas and thymic carcinomas, should be included in the group of EBV-associated neoplasias. However, since the viral DNA in these studies was demonstrated only in extracted DNA, the cellular origin of the viral DNA is uncertain. We have therefore investigated 152 epithelial tumours from various sites for the presence of EBV-DNA by in situ hybridization with 35S-labelled probes. Sixty-eight of 77 undifferentiated NPCs showed an EBV-specific autoradiographic signal, thus confirming the strong association of this tumour type with EBV even in geographical areas where undifferentiated NPC is not endemic. None of eight squamous cell NPCs showed an EBV-specific signal. All of 15 carcinomas with a similar morphology to undifferentiated NPC but from different anatomic sites (thymus, tonsil, breast) were EBV-negative as were 9 thymomas, 26 squamous cell carcinomas of the palatine tonsil, and 14 cervical carcinomas. Our results therefore suggest a unique association of EBV with undifferentiated NPC and support concepts assigning different biological properties to undifferentiated NPC as compared with squamous cell NPC.

Apoptosis induced by mild hyperthermia in human and murine tumour cell lines: A study using electron microscopy and DNA gel electrophoresis

Abstract: Mild hyperthermia is known to enhance apoptosis in a range of normal and neoplastic cell populations. Studies of tumours previously shown to respond to heating in this manner might be expected to provide insights not only into the mechanism of hyperthermic cell killing, but also into the apoptotic process in general. In the present study, cell death induced by 43°C heating for 30 min in two human Burkitt's lymphoma lines, BM 13674 and WW1, and in murine mastocytoma P‐815 × 2·1 was found to be exclusively apoptotic in type, identification being based on light and electron microscopic appearances and on the presence of internucleosomal cleavage of DNA into fragments that are multiples of 180–200 base pairs, which was demonstrated by agarose gel electrophoresis. The heat‐induced apoptosis was prevented by the presence of zinc sulphate, an inhibitor of the endonuclease considered to be responsible for the DNA cleavage, but was not suppressed by the protein synthesis inhibitor cycloheximide. The findings question the validity of the widely held view that active protein synthesis is an invariable prerequisite for the execution of apoptosis. It is suggested that an inositol triphosphate‐mediated increase in cytosolic Ca, resulting from limited membrane damage, might be the critical event responsible for activation of apoptosis by mild hyperthermia. Copyright

Expression of p53 protein in infiltrating and in-situ breast carcinomas.

Abstract: Five antibodies directed against the whole or part of p53 protein have been used to detect the protein immunohistochemically in 70 infiltrating breast carcinomas and 10 ductal carcinomas in situ. Mutations are known to occur in different conserved domains, and the antibodies employed spanned the expected sites. p53 protein was identified in 53 per cent of infiltrating carcinomas using the antibodies PAb 240, PAb 1801, C19, and JG8. The antibody PAb 421 detected the protein in 31.5 per cent; all positive with the other antibodies. Well-differentiated oestrogen receptor-positive tumours had a low incidence of p53 detection. Variation in the percentage of reactivity was seen between carcinomas and in some cases between different antibodies in the same cancer. Those carcinomas with a high percentage of positive cells with all antibodies were more likely to have metastasized to nodes, be at an advanced stage, and be oestrogen receptor-negative/epidermal growth factor receptor-positive. There was no significant correlation with c-erbB-2 protein expression or retinoblastoma protein loss. p53 protein was detected in a high proportion of cells in three of the six comedo ductal carcinomas in situ studied but either not at all or at a lower level in tumours of the cribriform type. p53 mutations are common in breast carcinomas, but heterogeneity within individual tumours is frequent. Marked expression of p53 appears to relate to tumour progression.

Necrotizing myopathy in critically-ill patients.

Abstract: Skeletal muscle wasting is commonly observed in critically-ill patients and has been attributed to catabolic fibre atrophy and to neuropathy. This study describes the occurrence of a necrotizing myopathy in 15 out of 31 critically-ill patients who had percutaneous biopsies taken from the tibialis anterior muscles. While most cases showed necrosis of isolated fibres, 5 of the 12 patients who had serial biopsies showed progressive necrosis of up to 95 per cent of the fibres. One other case showed infarction and one case had staphylococcal vasculitis. Atrophy of type 1 and/or type 2 fibres was documented by morphometry in 12 cases. Myoglobin-containing casts were demonstrated immunohistochemically in renal tubules on either biopsy or necropsy material in 5 out of 7 cases. The presence of muscle necrosis was a clinically unexpected finding which may contribute to weakness, complicate the interpretation of tissue biochemistry and energy balance studies, and potentiate renal failure. The necrosis is probably multifactorial in origin, with ischaemia and sepsis contributing factors.

Low expression of β1, α2 and α3 subunits of VLA integrins in malignant mammary tumours

Abstract: The Very Late Antigens (VLAs) are alpha beta heterodimeric transmembrane proteins mediating cell-substratum as well as cell-cell interactions. Changes in their expression and/or function seem to occur in a number of invasive carcinomas and may at least in part explain their abnormal patterns of growth and differentiation. Using monoclonal antibodies to the beta 1 (DH12, A1A-5), alpha 2 (B1.515) and alpha 3 (E1.56) chains, VLA-2 (alpha 2 beta 1) and VLA-3 (alpha 3 beta 1) were studied on cryostat sections of three fibroadenomas and 43 invasive breast carcinomas (29 ductal, 14 lobular) by the avidin-biotin complex immunoperoxidase technique. In non-neoplastic breast tissue and in fibroadenomas VLA-2 and VLA-3 were expressed by myoepithelial cells and on the basolateral surface of the luminal cells. There was weak or absent expression of alpha 2, alpha 3 and the common beta 1 chain in the majority of invasive carcinomas compared to the adjacent normal breast epithelium and preinvasive (in-situ) carcinomas. In addition, the expression of the alpha 2 chain of VLA-2 was reduced significantly (P less than 0.005) in the poorly differentiated ductal breast carcinomas (Grade III) compared to the well (Grade I) and moderately (Grade II) differentiated ductal tumours. These data give further evidence that loss or down-regulation of VLA-2 and VLA-3 occur relatively frequently in invasive cancers, and, at least in the invasive ductal breast carcinomas. Loss of an extracellular matrix receptor controlling growth and differentiation seems to be one of the abnormalities underlying the progression towards an undifferentiated morphology.

Gastrointestinal stromal tumours: correlation of immunophenotype with clinicopathological features.

Abstract: Stromal tumours of the gastrointestinal tract remain a persistent source of controversy with regard to both their proposed lines of differentiation and the difficulty in predicting their biological behaviour. We have examined 60 cases with a panel of seven antibodies directed at the identification of smooth muscular or neural differentiation. In our hands, 36 per cent of cases showed neural differentiation (although only 6.6 per cent expressed S-100 protein); 31 per cent appeared smooth muscular; 20 per cent manifested bidirectional differentiation; and 13 per cent were negative for all the markers used. Histological appearances do not reliably reflect immunophenotype. We have attempted to correlate immunophenotype with the site of the lesions and with their clinical behaviour. Mean follow-up of 5 years was obtained in 42 cases. Tumours with a neural phenotype have the best prognosis. Gastric tumours expressing both desmin and smooth muscle actin (in the absence of other markers) with up to 4 mitoses per 30 HPF behave in a benign fashion. Larger studies are required to substantiate the value of immunophenotyping this complex group of tumours.

Translocation (11;14): a cytogenetic anomaly associated with B-cell lymphomas of non-follicle centre cell lineage.

Abstract: Nine patients with t(11;14) and B non-Hodgkin's lymphomas composed of small to intermediately sized cells with irregular nuclei are described. Immunophenotyping was performed on seven cases, which were M+, D- with light chain restriction, CD5+, CD10-, and CD20+, suggesting that they were non-follicle centre cell lymphomas. The translocation (11;14) (in three cases the only cytogenetic anomaly) was associated with rearrangement of bcl-1 in four of the five cases investigated. Translocation (11;14) has been described in an apparently heterogeneous group of low-grade lymphoid malignancies which we suggest have a non-follicle centre cell lineage in common. This translocation may be associated with these lymphomas in the same way that t(14;18) is associated with follicle centre cell lymphomas.

Expression of Epstein-Barr virus replicative proteins in AIDS-related non-Hodgkin's lymphoma cells.

Abstract: Epstein-Barr virus (EBV) infection in lymphoproliferative lesions has been assumed to be strictly latent. In order to investigate the possible occurrence of EBV replication in AIDS-related lymphoma (ARL) cells, we studied 13 cases by immunohistology using monoclonal antibodies to the EBV-encoded switch-protein BZLF1, early antigens (EAs), late replicative proteins [virus capsid antigens (VCAs) and membrane antigens (MAs)], and to the latent proteins EB nuclear antigen 2 (EBNA 2) and latent membrane protein (LMP). EBV genomes were detected by in situ hybridization. EBV genomes and/or gene products were demonstrated in ten cases, including all immunoblast-rich lymphomas, two Burkitts lymphomas, and a T-cell anaplastic large-cell lymphoma. The BZLF1 protein, which disrupts latency in B cells, was identified in six (60 per cent), and EAs in four (40 per cent) of the EBV-positive ARL. Only one lymphoma (10 per cent) expressed VCAs and MAs. EBNA 2 and LMP were detected in three (30 per cent) and eight (80 per cent) of EBV-positive cases, respectively. EBV DNA was detected in lymphoma cells in 7 of 12 (58 per cent) cases. The most important finding of this study was frequent spontaneous activation of latent EBV in ARL. Production of complete virus, however, was either aborted, or tumour cells expressing late productive cycle proteins (VCA, MA) were rapidly cleared from tissues. It is suggested that host factors that normally inhibit replication of EBV are deficient in AIDS patients.

Characterization of integrin chains in normal and neoplastic human pancreas.

Abstract: Integrins are a complex family of non-covalently linked heterodimeric glycoproteins which function as cell adhesion molecules, interacting with extracellular matrix molecules such as laminin, fibronectin, vitronectin, and collagen, and also having a role in intercellular adhesion. Each integrin subfamily is characterized by a common beta chain associated with variable alpha chains. We have examined, using immunohistological methods, the expression of the VLA (very late activation) family comprising beta 1 in association with alpha 1-6, and also alpha 6 in association with beta 4, the LFA beta chain beta 2, and the vitronectin receptor, in association with beta 1 or beta 5 and as the complex alpha v beta 3. Cryostat sections of normal pancreas, pancreatic adenocarcinomas, and ampullary tumours were studied together with six pancreatic carcinoma cell lines. Normal pancreas showed expression of beta 1 in all parenchyma. alpha 2 and alpha 6 had a similar distribution whereas alpha 3 expression was confined to ducts, including the very smallest radicles. Staining along the basement membranes of ducts was seen with beta 4 and the anti-vitronectin alpha v chain receptor antibody 13C2. Islet cells failed to stain with any antibody. No staining of epithelial components was seen with antibodies to alpha 1, alpha 4, alpha 5, or to the alpha v beta 3 form of the vitronectin receptor (beta 3 and alpha v beta 3 using the antibody 23C6). Pancreatic adenocarcinomas and ampullary tumours showed expression of alpha 2, alpha 3, alpha 6, beta 1, beta 4, and the vitronectin receptor (alpha v associated with beta 1 or beta 5).(ABSTRACT TRUNCATED AT 250 WORDS)

AgNOR area in interphase nuclei of human tumours correlates with the proliferative activity evaluated by bromodeoxyuridine labelling and Ki-67 immunostaining.

Abstract: The area of silver-stained proteins associated with interphase nucleolar organizer regions (AgNORs) was compared with labelling data obtained by bromodeoxyuridine (BrdU) incorporation and Ki-67 immunostaining in 25 tumours of different origins and two non-neoplastic lesions of the thyroid. Our data demonstrate a highly significant correlation between the mean area occupied by the AgNOR proteins measured by an image processing system and the proliferative indices evaluated by BrdU labelling (r = 0.89, P less than 0.001) and Ki-67 immunostaining (r = 0.86, P less than 0.001). AgNOR protein area measurement is therefore proposed as a simple, inexpensive, and reliable method of evaluating the proliferative activity in routinely processed tumour samples.

Human papillomavirus 6, 11, and 16 in laryngeal papillomas.

Abstract: Twenty-seven cases of benign laryngeal papillomas, both single and multiple variants, were analysed for human papillomavirus (HPV) by DNA slot-blot hybridization chiefly to determine the pattern of infection in Hong Kong Chinese. DNA was extracted from paraffin blocks of formalin-fixed tissue and probed separately for HPV 6, 11, 16, and 18. Sixteen cases (59 per cent) showed the presence of at least one of these four HPV genomes. Thirteen cases (48 per cent) were positive for HPV 11 only. Three other cases (11 per cent) showed triple positivity for HPV 6, 11, and 16. None were positive for HPV 18. The predominance of HPV 11 infection contrasts with other series which have shown either an almost equal distribution of HPV 6 and 11 or a predominance of HPV 6. The finding of HPV 16 in three cases was unexpected. Using the polymerase chain reaction (PCR) with primers complementary to the upstream regulatory region of the HPV 16 viral DNA, the presence of HPV 16 genome was confirmed in all three cases. As the number of HPV 16-positive cases in this study is small, analysis of more cases using fresh biopsy material and a wider range of HPV type-specific PCR primers is warranted to determine the relative incidence of HPV subtypes in these benign laryngeal papillomas.

A series of 14 new monoclonal antibodies to keratins: Characterization and value in diagnostic histopathology

Abstract: A series of 14 new mouse monoclonal antibodies (MAbs) to keratins is described and the data suggesting their potential value in the differential diagnosis of human tumours are reported. The specificities of individual MAbs of the 'C-series' presented here range from monospecificity for keratin No. 7 (MAbs C-18, C-35, C-62, and C-68), keratin No. 8 (MAbs C-15, C-43, and C-15), and keratin No. 18 (MAbs C-04 and C-08) up to the broadly reacting 'pan-keratin' MAb C-11, with the target epitopes of the remaining four MAbs being shared by different pairs of keratin polypeptides. The results of the biochemical characterization of the MAbs, together with their immunohistochemical staining patterns on frozen as well as on paraffin sections of normal human tissues, suggest that they represent a significant contribution to the growing list of anti-keratin MAbs applicable in both research and routine diagnostic pathology. The immunohistochemical examination of a wide range of human neoplasms with the new MAbs not only confirmed their value in making distinctions between carcinomas, on the one hand, and lymphomas, and gliomas, on the other, but also verified the possibility of more subtle subdivisions within the group of adenocarcinomas and their metastases. Furthermore, the identification of small subsets of breast carcinomas with decreased levels or apparent loss of the keratin No. 7 polypeptide and some cases of stomach carcinoma with apparently induced expression of this keratin suggests that such 'exceptions' must be considered when using keratin spectra as one of the criteria in differential diagnosis.

Immunohistochemical demonstration of keratin 7 in routinely fixed paraffin-embedded human tissues.

Abstract: The immunoreactivity of OV-TL 12/30, a monoclonal anti-keratin 7 antibody (Mab), was investigated on frozen as well as paraffin-embedded human tissues. Its reactivity patterns were compared with another well-characterized monoclonal antibody to keratin 7 (RCK 105), and with broadly cross-reacting monoclonal (OV-TL 12/5) as well as polyclonal (pKer) keratin antisera. In frozen sections of normal and malignant human tissues both keratin 7 Mabs gave similar staining patterns. The immunoreactivity for OV-TL 12/30 and the polyclonal antibody (pKer) in tissue sections fixed in 4 per cent formalin or Bouin solution, was completely restored when pretreated with 0.1 per cent pronase, 0.1 per cent trypsin in phosphate-buffered saline (PBS) or with 0.5 per cent pepsin in 0.01 N HCl. Except for loss of immunoreactivity on human normal stomach surface epithelium and glandular mucous cells, Mab OV-TL 12/30 reacted strongly positive with essentially all those formalin- or Bouin-fixed paraffin-embedded tissues that had been shown to stain in non-fixed, frozen sections. In addition to the good correlation in human tissues, a complete correlation between the reactivity on frozen and paraffin-embedded human carcinomas (n = 86) was found as well. While both RCK 105 (anti-keratin 7) and OV-TL 12/5 (anti-keratin 5, 7, 14, 19) did not stain on paraffin-embedded sections, the polyclonal control antiserum (pKer) lost immunoreactivity in some cell types (e.g. mucous cells in compound glands, hepatocytes, pancreatic acinar cells, and proximal and distal convoluted tubules of the kidney). Our study shows that the keratin 7 Mab OV-TL 12/30 is an excellent marker for tumour histopathology since it is reactive in paraffin-embedded formalin-fixed human tissues.

Apoptosis in cultured rat hepatocytes: The effects of tumour necrosis factor α and interferon γ

Abstract: We investigated the cytotoxic effects of tumour necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) on rat hepatocytes in culture. Under phase contrast microscopy, we found a small number of dying hepatocytes in control cultures, each having been transformed into a cluster of small spheres. Under transmission electron microscopy, these cells showed the characteristics of apoptosis. TNF alpha and a combination of TNF alpha and IFN gamma exerted a cytotoxic effect, whereas IFN gamma showed no significant cytotoxicity when assessed by neutral red assay and by measuring LDH activity in culture medium. Under phase contrast microscopy, the number of apoptotic cells increased with the addition of either TNF alpha or IFN gamma, and markedly with the addition of both. DNA extracted from apoptotic cells cultured with TNF alpha and IFN gamma was fragmented, and a set of bands of the '200 bp ladder', which is characteristic of the DNA of apoptotic cells, was observed in agarose gel electrophoresis. These findings indicate that cultured hepatocytes die from apoptosis. TNF alpha killed cultured rat hepatocytes by increasing apoptosis, and this effect was potentiated by the addition of IFN gamma, which by itself was also weakly cytotoxic.

Antipeptide antibodies against the pNR-2 oestrogen-regulated protein of human breast cancer cells and detection of pNR-2 expression in normal tissues by immunohistochemistry.

Abstract: Five peptides, corresponding to regions of the predicted protein sequence of the oestrogen-regulated pNR-2 protein which is expressed in oestrogen-responsive human breast cancer cells, were synthesized. Two peptides were immunogenic in rabbits and antisera against one peptide reacted with the pNR-2 protein in sections of formalin-fixed, paraffin-embedded breast tumour. There was a significant correlation between the extent of pNR-2 protein expression detected by immunohistochemistry and pNR-2 mRNA levels determined by hybridization with a cDNA probe in a series of primary breast tumours. pNR-2 expression was assessed immunohistochemically in a panel of normal tissues. Expression was detected in normal breast, small intestine, and stomach (body and antrum).

Nucleolar organizer regions and prognosis in renal cell carcinoma.

Abstract: The prognostic significance of nucleolar organizer regions (NORs) in renal cell carcinoma (RCC) was evaluated. NORs were quantified in a series of 182 cases of RCC using the silver-colloid method. The cases were staged according to Robson's method (48 stage I, 26 stage II, 33 stage III, 75 stage IV) and mean NOR numbers for each tumour were correlated with survival over a 5-year period. Localized tumours (stages I and II) with low NOR numbers had an almost 100 per cent 5-year survival. Those patients with clinical evidence of metastases at presentation showed a high mortality, although those with low numbers of NORs had a significantly increased disease-free interval. Statistical analysis using the log rank test indicated NORs to be a significant predictor of survival over the whole series (P = 0.0001) and within each of Robson's stages (P = 0.0008 stage I, P = 0.0154 stage II, P = 0.0009 stage III, P = 0.0001 stage IV). Analysis of data using Cox's proportional hazard model showed mean NOR numbers to be independent of stage as a predictor of survival.

Helicobacter pylori in gastric biopsy specimens. Comparison of culture, modified giemsa stain, and immunohistochemistry. A retrospective study.

Abstract: Antral biopsy specimens of 302 different endoscopic Investigations of 200 patients with non-ulcer dyspepsia were studied for the presence of Helicobacter pylori in order to determine the most sensitive detection method. Part of the biopsy was cultured, and part stained using a modification of the Giemsa stain, and with an immunoperoxidase technique using a polyclonal rabbit anti-H. pylori antiserum. Cross-reactivity of this antiserum with other Campylobacter species was minimal. Material from 244 investigations was studied using all three detection methods. Culture was positive in 44 per cent, Giemsa in 78 per cent, and immunoperoxidase in 89 per cent of these biopsy specimens. Only five positive Giemsa stains with negative immunoperoxidase stain were found, whereas in 32 cases a negative Giemsa stain with a positive immunoperoxidase stain was seen. In the latter cases, the bacterial load was very low. The specimens revealed bacteria only sporadically, always confined to the deep layers of the gastric pits. Culture results correlated significantly with the bacterial load observed in the Giemsa stain. It is concluded that culture of H. pylori is the least sensitive detection method, whereas immunoperoxidase staining is the most sensitive. For daily practice the modified Giemsa stain, however, appears to be sufficient to diagnose the presence of the micro-organism.

The vascularity of primary cutaneous melanoma.

Abstract: In primary cutaneous malignant melanoma, the vascularity of the dermis immediately deep to the lesion may relate to tumour aggressiveness and to prognosis. These newly formed dermal vessels are incorporated into the melanoma to form the tumour microcirculation. We have assessed the percentage vascular volume in a series of primary melanomas in order to investigate the relationship between tumour vascularity and maximum tumour thickness. For the 64 melanomas included in this study, there appeared to be a significant relationship between the percentage vascular volume and the maximum tumour thickness. This relationship was not influenced by the presence of necrosis, vascular invasion, regression, or lymphocytic infiltrate, nor by the growth phase of the tumour. However, the percentage vascular volume was very low in the occasional thick melanoma, at least one of which was associated with prolonged survival. It seems possible that a low tumour vascularity could correlate with a relatively favorable outcome in cutaneous melanoma.

Long‐term survival in breast cancer related to overexpression of the c‐erbB‐2 oncoprotein: An immunohistochemical study using monoclonal antibody NCL‐CB11

Abstract: In a previous series we have shown poor short-term (3-5 years) survival for patients with tumours overexpressing the c-erbB-2 oncoprotein. In this study we employed archival paraffin-embedded tissue from patients who underwent mastectomy 10-12 years prior to assessment (n = 187). Immunohistochemical staining was carried out by an indirect immunoperoxidase technique using the novel monoclonal antibody NCL-CB11. Tumours were scored according to intensity of membrane staining. Patient and tumour information was obtained by scrutiny of clinical records. Survival analysis was carried out for both time to relapse and time to death, using the log rank test. Patients with tumours demonstrating intense membrane staining had a poor prognosis compared with the rest, with a steeply sloped survival curve over the first 4 years; the survival difference was still evident at 12 years follow-up (P less than 0.001). The survival advantage for c-erbB-2 negative patients was maintained in lymph node negative patients (P less than 0.001). However, c-erbB-2 status did not influence survival in the node positive group, where all patients had a uniformly poor outlook. These results applied to both time to relapse and time to death. In conclusion, c-erbB-2 status, determined using NCL-CB11, is a powerful prognostic indicator, defining in particular node negative patients with a particularly poor prognosis, and for whom alternative therapeutic strategies may be appropriate.

An outbreak of aflatoxicosis and boric acid poisoning in Malaysia: A clinicopathological study

Abstract: An outbreak of food poisoning resulting in 13 deaths in children occurred in Malaysia during the Chinese Festival of the Nine-Emperor Gods in 1988. The offending food was a Chinese noodle called 'Loh See Fun' (LSF). The source was traced to a factory where a banned food preservative was added to make the LSF. The food poisoning was attributable to aflatoxins and boric acid. The clinical features included vomiting, pyrexia, diarrhoea, abdominal pain, anorexia, giddiness, seizures, and eventual coma. Initially, many presented with a Reye-like syndrome. Eleven post-mortem examinations were performed. The pathological findings included extensive coagulative necrosis of the liver with proliferative 'ductal/ductular metaplasia of the hepatocytes'. Giant cell formation, central vein sclerosis, bile stasis, and steatosis were also noted. There was presence of acute tubular necrosis, superficial upper gastrointestinal erosions, and ensuing encephalopathy. The eventual cause of death is acute hepatic and renal failure.

Co‐ordinate expression of the alpha‐6 integrin laminin receptor sub‐unit and laminin in breast cancer

Abstract: Interactions between cells and extracellular matrices are mediated in part by a family of heterodimeric molecules known as integrins. We have investigated, using immunohistology, the distribution of six integrin alpha sub-units in normal breast tissue and 26 breast carcinomas. Alpha-1 integrin (collagen/laminin receptor sub-unit) was detected in myoepithelium, but not in luminal epithelium nor in most (20/26) carcinomas. Its expression on fibroblasts was enhanced in desmoplastic stroma. Both benign and malignant epithelium showed uniform positive staining for alpha-2 (collagen receptor sub-unit) and for alpha-3 (collagen/fibronectin/laminin receptor sub-unit). All epithelium was negative for alpha-4 (sub-unit of a fibronectin receptor). Epithelial staining for alpha-5 (fibronectin receptor sub-unit) was weak in all samples. Alpha-6 (sub-unit of two integrin laminin receptors) showed conspicuous changes in all invasive carcinomas. In normal tissues, there was weak staining of epithelial cytoplasm with alpha-6 antibody and moderate cell membrane staining. Strongest staining was present in a basement membrane distribution. In carcinomas, loss of cytoplasmic and cell membrane staining was variable, but basal membrane staining was diminished or absent in all tumours. Loss of basal membrane staining for alpha-6 integrin corresponded closely to loss of immunoreactivity for its ligand laminin in invasive breast cancer.

Histopathological grading of soft tissue tumours. Prognostic significance in a prospective study of 278 consecutive cases.

Abstract: A consecutive 10-year series of 278 soft tissue sarcomas was prospectively graded, using a system based on the number of mitoses and taking into account parameters such as cellularity, anaplasia, necrosis, and histogenetic type and subtype of tumour. Prognostic factors in relation to metastasis-free survival were studied by uni- and multivariate analysis. Fifty-seven (20·5 per cent) were low-grade tumours, 43 (15·5 per cent) were intermediate, and 178 (64 per cent) were high grade. High-grade tumours were divided into two groups; 80 (29 per cent) grade 3A (=5–20 mitoses per 10 high power fields (HPF)) and 78 grade 3B (28 per cent) (=more than 20 mitoses/10 HPF); 10 HPF corresponds to 2·5 mm2. Twenty (7·2 per cent) high-grade tumours could not be further subdivided. Grading was found to be the prognostic factor associated with the strongest predictive value. Five-year survival in low-grade and intermediate tumours (95 and 86 per cent, respectively) differed significantly (P < 0·0001) from high grade (50 per cent) and (P = 0·0018) between grade 3A (64 per cent) and grade 3B (41 per cent). Other prognostic indicators of importance in high-grade tumours were age, local recurrence at presentation (primary operation outside the Centre), and localization (superficial vs. deep).

‘Neuroendocrine’ differentiation in primary neoplasms of the liver

Abstract: Thirty primary liver neoplasms (16 hepatocellular, nine biliary, and five epithelioid haemangioendotheliomas) were studied for the expression of the general 'neuroendocrine' markers, neurone specific enolase (NSE) and protein gene product 9.5 (PGP 9.5). Grimelius silver staining for neurosecretory granules and immunostaining for S100 protein, HMB-45, vasoactive intestinal polypeptide (VIP), and calcitonin were also performed. Eleven of the 16 hepatocellular carcinomas stained positively for PGP 9.5, four for NSE, six for HMB-45, and two for S100 protein. Seven exhibited granular staining by the Grimelius method; eight showed immunostaining for VIP, and two for calcitonin. Three of the five haemangioendotheliomas demonstrated positive immunostaining for PGP 9.5, and two for NSE; of the nine biliary carcinomas, two showed staining for PGP 9.5 and NSE, and four contained cells staining with the Grimelius technique. Primary neoplasms of liver may show 'neuroendocrine' differentiation and this aspect of their phenotypic expression has to be considered before predicting the site of origin of a tumour in the liver.

Phenotypic modulation in lipocytes in experimental liver fibrosis.

Abstract: The presence of a-smooth muscle actin (smA)-positive cells has recently been reported in the fibrotic liver. Lipocytes have been considered to play important roles in hepatic fibrosis. However, the relation of the a-smA-positive cells and lipocytes has not been determined. The biological implication of a-smA expression remains unknown. To study these questions, we carried out double immunofluorescent staining of a-smA and desmin (a marker for lipocytes), or a-smA and collagen, and double immunohistochemical staining of a-smA and 5-bromo-2'-deoxyuridine (BrdUrd) in carbon tetrachloride-induced fibrotic rat livers. In normal and control livers, a-smA-positive cells were not seen in the lobules, whereas scattered desmin-positive cells were present. With the development of hepatic fibrosis, a-smA was expressed only in a portion of desmin-positive cells located predominantly around collagen bundles. A number of a-smA-positive cells in the lobules were labelled with BrdUrd. These results suggest phenotypic modulation in lipocytes and differentiation of lipocytes towards myofibroblast-like cells, since a-smA is expressed with desmin in myofibroblasts in scar tissue. The expression of a-smA may be related to events of the fibrotic process, such as tissue contraction or fibrogenesis per se.

Variations in the occurrence of silver‐staining nucleolar organizer regions (AgNORs) in non‐proliferating and proliferating tissues

Abstract: Previous studies on the subject of silver-staining nucleolar organizer regions (AgNORs) as indicators of precise proliferative status of tissues have sometimes resulted in ambiguity. The studies, however, have most frequently addressed themselves to the prognosis of neoplasias, with the aim of using AgNORs principally to distinguish between benign and malignant tumours. This investigation was to determine a base-line relationship of AgNOR clusters to proliferation and thus concentrated on normally proliferative tissues and conditionally renewing tissues after appropriate stimulation. Two murine transplantable tumours were also examined as examples of frank malignancy. As an example of the former, variations in AgNOR clusters were noted in the small intestine of man, mouse, and rat. The conditionally renewing systems of liver, prostate, and salivary glands were stimulated into proliferation by two-thirds partial hepatectomy, castration followed by treatment with testosterone, and isoproterenol treatment, respectively, in rat models; the murine sarccma SaF and carclnoma CaNT provided relatively simple malignant tumours for AgNOR investigation. Proliferation was monitored by noting labelling indices after injection with bromodeoxyuridine (BrdUrd) in vivo followed by immunocytochemical visualization of S-phase cells. In all tissues, an increase in the size of AgNOR clusters rather than their number correlated positively with elevated labelling, particularly with the emergence of silver-staining regions of 2–3 μm visible diameter. Thus, increased AgNOR cluster size (diameter) as representative of AgNOR cluster/nucleolus volume was found to be dependent on proliferative activity in a range of normal and neoplastic tissues.