Ki-67 expression in melanoma. A potential method of risk assessment for the patient with a positive sentinel node.

Melanoma patients with a positive sentinel node biopsy generally proceed to regional lymph node dissection, though ultimately only around 20% have evidence of tumour in their "non-sentinel" nodes. A means to identify patients at high risk of non-sentinel node involvement could potentially spare a large number of patients a procedure with significant morbidity. The proliferation marker Ki-67 has been associated with tumour progression in primary melanoma but has not been extensively studied in metastases. The study aims to investigate Ki-67 in primary melanoma and lymph node metastases and investigate any relationship with disease progression. Tissue Arrays of primary melanoma (n=79) and lymph node metastases (n=92) were constructed from paraffin embedded tissue and Ki-67 expression examined by immunohistochemistry. Staining positivity and intensity were assessed and correlated with standard staging criteria and clinical outcome. High Ki-67 expression was associated with both Breslow thickness (chi(2)=8.54, p=0.035) and presence of ulceration (Fisher's Exact test p=0.003) in primary melanoma. In lymph node metastases high Ki-67 expression correlated with Nodal (N) Stage (chi(2)=8.193, p=0.0 17). High Ki-67 expression is associated with melanoma progression and multiple lymph node involvement. This might potentially form the basis of a risk analysis for patients with positive sentinel nodes.

Direct comparison of bromodeoxyuridine and Ki-67 labelling indices in human tumours.

Direct comparison of bromodeoxyuridine (BrdUrd) and Ki-67 labelling indices was achieved by selecting similar areas from serial sections of human tumours. Fifteen patients were selected who had been administered BrdUrd in vivo and both proliferation markers were assessed by immunohistochemistry. The data show a good correlation between both BrdUrd LI and MIB-1 LI and Tpot (calculated using the flow cytometry derived duration of S phase) and MIB-1 LI. The contribution of BrdUrd LI to growth fraction varied as a function of proliferation characteristics. In tumours with a high LI, the number of DNA synthesizing cells represented half the growth fraction, whilst in tumours with lower LI's ( < 10%) the ratio of DNA precursor labelled cells as a function of growth fraction fell to between 10% and 20%. Tpot showed a linear correlation with MIB-1/BrdUrd ratio with a slope approaching unity. It was apparent that both intra- and interpatient variation in proliferation index was greater for BrdUrd labelling than for MIB-1 expression.

bcl-2 expression in head and neck cancer: an enigmatic prognostic marker.

PURPOSE: The role of bcl-2 overexpression in cancer presents a paradox. In some tumor types, it is associated with favorable outcome, whereas in others the reverse is true. The purpose of this study was to explore the influence of bcl-2 in a large series of head and neck cancer patients treated in the CHART randomized trial. METHODS AND MATERIALS: Histologic material was obtained from 400 patients; bcl-2 expression was assessed by immunohistochemistry as either positive or negative cytoplasmic staining. RESULTS: Positivity of bcl-2 was recorded in 12.8% (9.5-16.5%, 95% confidence limits) of tumors. There were significant differences in positive tumors within different sites with nasopharynx showing the highest incidence (46.2%). A multivariate logistic regression analysis showed that bcl-2 was strongly associated with histologic dedifferentiation, as well as increasing N stage and female gender. In univariate analyses, bcl-2 positive patients had a lower locoregional relapse rate (RR 0.57, p = 0.02) and improved survival (RR 0.49, p = 0.004) compared to bcl-2 negative patients; this became more significant in multivariate analysis. CONCLUSION: These data demonstrate that bcl-2 overexpression is a marker of what is considered to be more advanced and aggressive disease yet it is associated with a more favorable outcome irrespective of the treatment schedule.

Observer study of the grading of dysplasia in ulcerative colitis: comparison with clinical outcome.

Patients with extensive ulcerative colitis are entered into surveillance programs that aim to detect premalignant changes. Biopsy specimens have been collected in the St Mark's Hospital (London) surveillance program over a 22-year-period. Specimens from patients reported as having dysplasia were reexamined. A total of 207 biopsy specimens from 86 patients were graded by five experienced pathologists according to the severity of the dysplasia. The overall agreement between the pathologists grading the specimens was poor; each pair agreed on between 42% and 65% of the slides. The best agreement was for slides that were said to show no dysplasia. Comparison with clinical outcome indicated that the pathologists most likely to diagnose dysplasia in patients with carcinoma were also likely to diagnose dysplasia in patients who did not go on to develop carcinoma. Calculating an average grade of dysplasia did not significantly improve diagnostic accuracy. Despite the findings of this interobserver study, dysplasia has been a successful marker in clinical practice. Pathologists should ensure that they have access to previous slides from the same patient and adequate clinical information before reporting biopsies as positive for dysplasia. An additional biopsy should usually be undertaken before surgery is considered.

Colonic pericrypt sheath cells: characterisation of cell type with new monoclonal antibody.

A new monoclonal antibody, PR 2D3, was raised against a crude homogenate of normal colorectal mucosa and found to react with the cells in the pericrypt sheath. It also reacted with smooth muscle throughout the body and, in specific sites, with those mesenchymal cells known as myofibroblasts. It did not react with cardiac or skeletal muscle, nor with fibroblasts. PR 2D3 is an IgG1 antibody and identifies a membrane component of about 140 K molecular weight. The pericrypt cells have been described as fibroblasts, but in view of the specificity of PR 2D3 for smooth muscle, and its selective staining of the colonic pericrypt cells, this cell type was re-examined for other smooth muscle properties. Ultrastructurally, the cells had many characteristics in common with smooth muscle and were identical with the myofibroblasts of the umbilical cord. On immunocytochemical examination they were found to contain desmin, myosin, and filamin. The confirmation that the pericrypt cells are myofibroblasts suggest that they have both contractile and secretory roles.

Reporting basal cell carcinoma: a survey of the attitudes of histopathologists.

AIMS: To investigate the histopathological reporting of basal cell carcinoma. METHODS: Methods of classification and attitudes to excision margins were ascertained from histopathologists in 130 centres; 82 replies were obtained (63% response rate). RESULTS: 24% of those replying did not use any classification system for basal cell carcinoma. The remainder (76%) used a wide variety of different classification systems. A small number (9%) of those questioned felt reporting on completeness of excision was not important. The majority of histopathologists considered the excision margin was worth reporting but there were differences in methods of processing and reporting biopsies. CONCLUSIONS: There is considerable variation in histopathological reporting of basal cell carcinoma. There is a need for uniformity of histopathological reporting to allow both improved management decisions and comparative audit of this extremely common skin cancer.

Brush cytology of the colon and rectum in ulcerative colitis: an aid to cancer diagnosis.

In a prospective study of 100 patients with ulcerative colitis, 82 of whom had extensive colitis, carcinoma and dysplasia were distinguished cytologically from reactive hyperplasia. Six patients had carcinoma complicating colitis and satisfactory samples were obtained from five; the cytological appearances were interpreted as carcinoma in three and as dysplasia in two. Seventy eight patients had not developed carcinoma or dysplasia; the cytological appearances were interpreted as negative for dysplasia in 75 and indefinite for dysplasia in three. In patients who had developed dysplasia the changes seemed to be more widespread on cytological rather than on histological examination. Brush cytology may complement histological assessment in patients with ulcerative colitis who have developed strictures or in whom there is a high suspicion of neoplastic change.

Increased expression of laminin/collagen receptor (VLA-1) on epithelium of inflamed human intestine.

Immunohistochemical techniques were used to investigate the epithelial expression of VLA-1 in inflammatory bowel disease in six patients with Crohn's disease, in four patients with ulcerative colitis, and in one patient with indeterminate colitis, and compared with that in the small intestine and colons of 10 normal controls. In normal small bowel VLA-1 was expressed on crypt epithelial cells and only weakly or not at all on surface epithelium. VLA-1 was again expressed weakly in normal colon, except in one case, a 1 year old child with diarrhoea but no histological abnormalities. In small and large intestine affected with Crohn's disease, ulcerative colitis, or indeterminate colitis, there was increased expression of VLA-1 on the basolateral aspects of crypt cells and de novo expression on surface epithelium. It is suggested that this is an adaptive response to prevent epithelial cell loss as a result of inflammation in the underlying lamina propria.

Histopathology and immunohistochemistry of the caecum in children with the Trichuris dysentery syndrome.

Caecal biopsy specimens from Jamaican children with the Trichuris dysentery syndrome (TDS) and age matched Jamaican controls were investigated by immunohistochemistry and by light microscopy. Biopsy specimens from all children (with TDS and controls) showed a mild to moderate increase in inflammatory cells. Except in the vicinity of the worm, where the epithelium was flattened, there was no other epithelial abnormality. Compared with controls, children with TDS had increased IgM lamina propria plasma cells and decreased intraepithelial T cells. There was also an increase in crypt epithelial cell proliferation. Lamina propria T cells (both activated and non-activated) were no more common in children with the Trichuris syndrome than controls. Epithelial cell HLA-DR and VLA-1 expression (which are increased in other colitides) were the same in both groups. Despite the presence of large worm burdens and chronic dysentery, therefore, only minor changes were seen in the caecal mucosa of children with TDS.

Investigation into the value of Papanicolaou stained cervical smears for the diagnosis of chlamydial cervical infection.

Forty five (37%) of 121 female contacts of men with non-gonococcal urethritis or gonorrhoea were chlamydia positive, as judged by isolation or by detecting elementary bodies in smears with a fluorescein labelled chlamydial monoclonal antibody. Only six (13%) of these, however, had Papanicolaou stained smears in which there were inclusion like changes suggestive of chlamydial infection. Furthermore, of 15 patients who had such cytological changes, chlamydiae were detected in only six and the abnormalities were found also in Papanicolaou stained smears from 10 (13%) of the 76 chlamydia negative patients. Modifying the Papanicolaou stained smears by including endocervical material did not increase sensitivity. In addition, destaining and restaining them with the monoclonal antibody was time consuming and the results were unreliable. The staining of cervical smears with Papanicolaou reagent is a technique of low sensitivity and specificity for diagnosing or screening for chlamydial cervical infection and cannot be recommended.

Cellular proliferation characteristics of basal cell carcinoma: relationship to clinical subtype and histopathology.

This study investigates the proliferation characteristics of 81 primary basal cell carcinomas (BCC) using detection of the Ki-67 antigen by immunohistochemistry. The tumours were classified into distinct sub-types based on their histological growth pattern and differentiation status. The mean Ki-67 growth fraction was 0.293 and this was found to vary between the different growth patterns, with morpheic, infiltrating and superficial tumours showing the highest levels of proliferation at 0.373, 0.351 and 0.335, respectively; the nodular and micronodular growth patterns were significantly lower at 0.248 and 0.232, respectively. No overall association was seen between proliferation and differentiation status although certain histological growth patterns such as nodular showed a greater propensity to differentiate. Proliferation was related to tumour size, with larger lesions exhibiting higher growth fractions although this may have also been related to tumour subtype as infiltrating and morpheic tumours tended to present with larger tumour diameters. The spatial distribution of proliferating cells by Ki-67 labelling was not related to tumour subtype, differentiation or growth fraction. These studies have shown BCC to possess proliferative characteristics akin to other solid tumours commonly regarded as more rapidly dividing. There was an association between growth fraction and tumour subtype consistent with higher proliferation in the lesions considered to be more aggressive.

C-myc oncogene expression in human melanoma and its relationship with tumour antigenicity.

Melanoma produces specific tumour antigens which are capable of eliciting an immune response. However, this tumour evades the immune system, in part, by downregulation of class I HLA antigens on the cell surface, which are required for T cell recognition. It has been suggested that the oncogene c-myc may have a role in effecting this change in vitro, however, the relationship between oncoprotein level and tumour antigenicity has not been established in human tumours. This study measured c-myc oncoprotein in 94 melanoma specimens (46 primary tumours and 48 regional metastases) using flow cytometry and evaluated class I HLA expression with immunohistochemistry. C-myc expression was found in 91 tumours (96%) with higher expression in metastases than primary melanomas (P<0.005). Class I HLA expression was found to show great variation although metastases showed less antigenicity than primary tumours (P<0.01). Analysis of the relationship between these two parameters revealed a highly significant correlation in both primary (P<0.01) and metastatic disease (P<0.01), with high oncoprotein being associated with down regulation of cell surface antigens. Knowledge of the control of tumour antigenicity is likely to provide an objective platform for the development of new strategies for immunotherapy.

An analysis of p16 protein expression in sporadic malignant melanoma.

Inactivation of p16 tumour suppressor gene has been reported frequently in melanoma cell lines, and mutations have been detected in familial melanoma kindreds. The aim of this study was to assess the role of p16 inactivation in melanocytic progression by measuring the level of p16 protein in a range of sporadic, benign and malignant melanocytic lesions. Using dual parameter flow cytometry, p16 protein expression was measured in 30 benign melanocytic naevi, 38 primary and 51 metastatic melanomas. A high level of p16 expression was demonstrated in benign melanocytic naevi (96% median nuclear positivity), with a significant reduction in primary melanomas (69%, P < 0.001). The median nuclear positivity of primary melanomas was significantly higher (P < 0.03) than the level of expression in metastatic lesions (median positivity 37%). A progressive loss of p16 expression was demonstrated from benign melanocytic naevi through to primary and metastatic lesions. These data suggest that loss of p16 protein expression is not only associated with the early transformation of benign lesions, but also with the later stages of malignant progression.

Bcl-2 expression correlates with favourable outcome in head and neck cancer treated by accelerated radiotherapy.

The aim of this study was to investigate bcl-2 expression in head and neck cancer patients and to investigate its correlation with biological and clinical characteristics and outcome of accelerated radiotherapy. A series of 93 patients with squamous cell carcinoma of the head and neck who had been uniformly treated with continuous hyperfractionated accelerated radiation treatment (CHART) were investigated. These patients had also been injected with bromodeoxyuridine (BrdUrd) to measure cell kinetic parameters using flow cytometry (FCM) and their p53 protein status had also previously been described. Bcl-2 expression was assessed using immunohistochemistry. Sixteen of the 93 (17.2%) patients stained positively for bcl-2 proto-oncogene. The percentage of positive tumour cells within the specimens was highly variable, ranging from a few percent to complete positivity. Bcl-2 positivity was correlated with improved local control (p > 0.0016) and survival (p > 0.012) in comparison with non-expressing tumours. There was no correlation between bcl-2 expression and histological grade, T stage or site but overexpressors were almost exclusively node negative. The significance of bcl-2 was reduced when node negative tumours were analysed alone. There was no correlation of bcl-2 with p53 expression but there was a trend for overexpression to be associated with diploidy and rapidly proliferating tumours. These data suggest that bcl-2 expression in head and neck cancer is not associated with disease progression.

High levels of hCG in choriocarcinoma can result in renal failure and a false-negative pregnancy test in men.

The diagnosis of choriocarcinoma was delayed because a urinary pregnancy test for hCG was falsely negative. This man's hCG rose to 25,000,000 iu/l. He developed renal impairment, probably due to precipitation of hCG in his renal rubules.

Measurement of invasive potential provides an accurate prognostic marker for giant cell tumour of tendon sheath.

Giant cell tumours of tendon sheath vary from solitary nodules to a multinodular variety that exhibits local infiltration. Recent advances in molecular oncology have defined a gene, nm23, expressed in normal cells which is responsible for inhibiting infiltration. The aim of this study was to investigate the expression of nm23 in a series of 52 giant cell tumours using immunohistochemistry and to assess its prognostic potential. nm23 expression was absent in 21%, of tumours and this was associated with a highly significant risk of local recurrence (P<0.0001). Multivariate analysis of outcome showed nm23 expression to be more reliable than other clinicopathological parameters for predicting outcome. This immunohistochemical test for nm23 is easily performed on standard paraffin sections and is recommended as an accurate prognostic marker for giant cell tumours of tendon sheath.

Stratification of patients by melanoma cell adhesion molecule (MCAM) expression on the basis of risk: implications for sentinel lymph node biopsy.

BACKGROUND: The usual indication for sentinel lymph node biopsy (SLNB) in melanoma is a primary tumour >1mm thickness but under these criteria less than 20% of SLNBs are positive. Of those patients with a negative sentinel node (SN) over 10% will have disease recurrence within 3 years. A more accurate delineation of candidate patients for SLNB and risk profile for negative SN patients is therefore desirable. Melanoma cell adhesion molecule (MCAM) is a predominant cell adhesion molecule of melanomas and its expression has been implicated in tumour progression and metastasis. AIMS: To compare MCAM expression in primary and metastatic melanoma and to investigate if MCAM expression in patients meeting the criteria for SLNB correlated with patient outcome. METHODS: Tissue arrays of primary (n=78) and metastatic (n=92) melanomas were constructed from archived paraffin embedded tissue and MCAM expression detected by immunohistochemistry. Staining positivity and intensity were assessed by visual scoring and correlated with clinical outcome. RESULTS: In patients meeting the current criteria for SLNB, Cox multivariate analysis showed both MCAM expression positivity and intensity were independently predictive of survival (P=0.007) and development of lymph node disease (P=0.01) in primary melanoma over and above established markers of prognosis, such as Breslow thickness. MCAM-negative patients had a 5-year survival of 92% compared with 40% for MCAM positive. CONCLUSIONS: Measurement of MCAM expression represents a potential method to stratify SLNB patients on the basis of risk. This would have considerable benefits in terms of both cost and patient morbidity.

Evaluation of ER, PgR, HER-2 and Ki-67 as predictors of response to neoadjuvant anthracycline chemotherapy for operable breast cancer.

Primary systemic therapy (PST) for operable breast cancer enables the identification of in vivo biological markers that predict response to treatment. A total of 118 patients with T2-4 N0-1 M0 primary breast cancer received six cycles of anthracycline-based PST. Clinical and radiological response was assessed before and after treatment using UICC criteria. A grading system to score pathological response was devised. Diagnostic biopsies and postchemotherapy surgical specimens were stained for oestrogen (ER) and progesterone (PgR) receptor, HER-2 and cell proliferation (Ki-67). Clinical, radiological and pathological response rates were 78, 72 and 38%, respectively. There was a strong correlation between ER and PgR staining (P < 0.0001). Higher Ki-67 proliferation indices were associated with PgR- tumours (median 28.3%, PgR+ 22.9%; P = 0.042). There was no relationship between HER-2 and other biological markers. No single pretreatment or postchemotherapy biological parameter predicted response by any modality of assessment. In all, 10 tumours changed hormone receptor classification after chemotherapy (three ER, seven PgR); HER-2 staining changed in nine cases. Median Ki-67 index was 24.9% before and 18.1% after treatment (P = 0.02); the median reduction in Ki-67 index after treatment was 21.2%. Tumours displaying >75% reduction in Ki-67 after chemotherapy were more likely to achieve a pathological response (77.8 vs 26.7%, P = 0.004).

Control of differentiation in human colorectal carcinoma cell lines: epithelial-mesenchymal interactions.

Mesenchymal elements have been investigated for their effects on the growth and differentiation of seven human colorectal carcinoma-derived cell lines. Epithelial cells were cultured as monolayers on plastic; they were also grown on fibroblast lawns and in collagen matrices, with and without fibroblasts. In each case, differentiation was assessed morphologically with monoclonal antibodies directed against components of normal goblet and columnar cells. The results were compared with those obtained when the cell lines were grown in vivo as xenografts in athymic mice. The xenografts allowed the greatest potential for differentiation, although two cell lines showed little or no response to mesenchyme either in vivo or in vitro. The presence of fibroblasts induced branched structures in all the remaining lines when these were cultured in collagen matrices. The collage matrix alone induced the formation of well-defined glandular structures in SW1222 cells, reminiscent of those seen in SW1222 xenografts and normal colonic crypts. Epithelial response to mesenchymal factors may require specific receptors, the expression of which dictates phenotype. Isolation and analysis of such receptors and factors could lead to clarification of the mechanisms underlying normal tissue morphogenesis and the growth and spread of neoplastic cells.

Monoclonal antibodies to human colorectal epithelium: markers for differentiation and tumour characterization.

A panel of monoclonal antibodies (MAbs) has been obtained, directed against determinants of normal human colorectal epithelium. BALB/c mice were immunized and boosted with mucosal scrapings and cell membrane preparations from normal large intestine. In one case boosting was also performed with HT29 colon carcinoma cells. Hybridoma supernatants were screened immunohistochemically on frozen sections of normal colorectal epithelium, leading to the selection of 12 MAbs which recognized determinants of the major epithelial cell lineages. These antibodies fall into two groups: group 1 antibodies react with mucus constituents, with or without other cell components; group 2 antibodies react only with non-mucus components of cells. The normal tissue distribution of the antibody panel has been characterized immunohistochemically. Three of the mucus-reactive group-1 antibodies, PR.4D4, PR.5D5 and PR.3A5, and also PR.1A3 of group 2, have a very restricted distribution. In all 4 cases, their reactivity outside the gastrointestinal tract is mainly confined to tracheal epithelium. A series of benign and malignant colorectal tumours has been studied with the antibody panel. The antibodies of group 1, and PR.1A3 from group 2, show a marked heterogeneity in their reactions with malignant cells and seem to be defining patterns of functional differentiation, independently of standard histological criteria. The reactivity of 6 colorectal carcinoma cell lines has also been assessed with the antibodies. The group-1 antibodies and PR.1A3 identify those cell lines which have retained some capacity for differentiation.