Torben Haerslev

Correlation of growth fraction by Ki-67 and proliferating cell nuclear antigen (PCNA) immunohistochemistry with histopathological parameters and prognosis in primary breast carcinomas.

The immunoreactivity for Ki-67 and PCNA was investigated in 487 patients with primary breast carcinomas using MIB-1 (Immunotech, France) and PC-10 (DAKO, Denmark) as primary antibodies. Formalin-fixed and paraffin-embedded tissue sections were used. The immunoreactivity for Ki-67 and PCNA was found to be independent of the length of fixation if the sections were pretreated in a microwave oven in citrate buffer and distilled water, respectively. The immunostaining was evaluated semiquantitatively.High Ki-67 score (more than 1% positive tumour cells) and PCNA over-expression (more than 25% positive tumour cells) were correlated with clinicopathological parameters such as large tumour size, high histological grade (poor differentiation), and absence of steroid hormone receptors, which are parameters of an aggressive phenotype of the tumour.In univariate analysis of survival data, both Ki-67 and PCNA were parameters of a poor overall survival in both lymph node-positive and -negative patients.In multivariate analysis using a Cox model stratified by nodal status, Ki-67 and PCNA failed to be of prognostic significance whereas classical histopathological parameters such as tumour size and histological grade turned out to be of independent prognostic significance in both lymph node-positive and -negative patients, while progesterone receptors were of independent prognostic significance only in lymph node-positive patients.

An immunohistochemical study of p53 with correlations to histopathological parameters, c-erbB-2, proliferating cell nuclear antigen, and prognosis

In an immunohistochemical study of 490 primary breast cancer patients with a follow-up period of more than 10 years, we found that p53 was not a prognostic factor for disease-free or overall survival among the whole cohort or among lymph node-positive or -negative patients. In a multiple logistic regression model classical histopathological parameters, such as lymph node status, number of mitoses, histological grade, and absence of progesterone receptors, were independent, poor prognostic predictors. In univeriate analysis p53 immunoreactivity was positively correlated with the absence of tubule formation, high histological grade (poor differentiation), absence of estrogen receptors (ER), and a high proliferating cell nuclear antigen (PCNA) score (ie, parameters indicative of an aggressive phenotype). The lack of prognostic significance may be attributable partly to the method used, because immunohistochemistry underdetects rather than overdetects p53 protein. No correlation between p53 and c-erbB-2-oncoprotein was demonstrated.

Immunohistochemical study of estrogen receptors in primary breast carcinomas and their lymph node metastases including comparison of two monoclonal antibodies

Estrogen receptors (ER) status was investigated in 101 primary breast carcinomas and their axillary lymph node metastases to determine if the malignant cells retained or changed this phenotypic feature during the metastatic process. Immunohistochemistry with the ER‐ICA kit (Abbott Laboratory) on formalin‐fixed paraffin‐embedded tissue was used (paraffin ER‐ICA). The ER status in primary and secondary tumours was concordant in 80 patients (79%) and discordant in 21 (21%). Eighteen of these twenty‐one patients had ER‐positive primary tumours and ER‐negative lymph node metastases. This discordance, which may be due to loss of ERs in the metastatic cells or tumour heterogeneity, could explain the well‐known failure of endocrine treatment in some of the patients with ER‐positive primary tumours. A new monoclonal antibody ID5 (DAKO) against ERs was applied on formalin‐fixed paraffin‐embedded tissue from 83 of these 101 primary carcinomas. These analyses and paraffin ER‐ICA analyses were compared to prior analyses of the same 83 tumours using the ER‐ICA kit on fresh frozen tissue (“gold standard”, frozen ER‐ICA). Kappa coefficient, sensitivity and specificity were 0.74, 0.96 and 0.75 for ID5 antibody, and 0.59, 0.72 and 0.96 for ER‐ICA antibody on paraffin sections.

Metallothionein expression in basaloid proliferations overlying dermatofibromas and in basal cell carcinomas

Basaloid proliferations overlying dermatofibromas which morphologically resemble superficial basal cell carcinomas have been interpreted as both reactive/regressive and frankly malignant. Metallothioneins (MTs) are low‐molecular‐weight proteins with a selective binding affinity for heavy metal ions. MTs has been proposed to represent a biological marker of carcinogenesis and, in a variety of human tumours, a correlation between immunohistochemically overexpresstion of MT and aggressive clinical behaviour has been shown. In order to clarify the nature of basaloid proliferations overlying dermatofibromas, we examined, immunohistochemically, 10 dermatofibromas with overlying simple hyperplasia, 16 dermatofibromas with overlying basaloid proliferation, and 35 basal cell carcinomas, for expression of MT.

The prognostic significance of immunohistochemically detectable metallothionein in primary breast carcinomas

Metallothionein (MT) is a low‐molecular‐weight protein with a high affinity for group II metal ions, especially zinc and copper. MT serves as an intracellular reservoir of these ions, but may also be involved in the detoxification of certain toxic metalions such as cadmium. In addition, high MT contents might protect tumour cells from alkylating agents and irradiation. The aim of this study was to evaluate the prognostic significance of immunohistochemically detected MT overexpression in patients with primary breast carcinoma: 478 patients with primary breast carcinoma diagnosed during the period 1980–1985 were included. Formalin‐fixed and paraffin‐embedded tissue was used. Immuno‐reactivity for MT was found to be independent of the length of formalin fixation if the sections were microwave processed before incubation with the primary antibody. Patients were divided into two groups: those with MT overexpression (more than 10% positive tumour cells) and those with low expression (less than 10% positive tumour cells). MT overexpression was correlated with postmenopausal status, large tumour size, presence of lymph node metastases, high number of mitoses, severe nuclear pleomorphism, high histological grade (poor differentiation), and absence of PgR. In univariate analysis of survival data, MT overexpression was a predictor of poor overall survival in the entire group of patients. In multivariate analysis, MT overexpression failed to be of prognostic significance, whereas classical histopathological parameters such as tumour size, histological grade, and PgR were of independent prognostic significance.

Expression of cytokeratin and erbB‐2 oncoprotein in Paget's disease of the nipple

The occurrence of epithelial membrane antigen (EMA), low molecular weight cytokeratin (CK) and erbB‐2 oncoprotein was investigated in twenty‐eight cases of Pagets disease of the nipple (PD) to determine their diagnostic usefulness. The ABC technique with monoclonal antibodies was used on formalin‐fixed, paraffin‐embedded tissue. The Pagets cells showed positive immunoreactivity for all three antigens investigated in a high percentage of PD. Immunoreactivity for CK and erbB‐2 oncoprotein was restricted to the Pagets cells, whereas EMA in some cases also stained the adjacent keratinocytes. Since CK and/or erbB‐2 oncoprotein occurred in 93% of the cases, we conclude that demonstration of both antigens is useful in the diagnosis of PD. ErbB‐2 oncoprotein was also found to be expressed in a high percentage of the underlying intraductal and invasive carcinomas.

Microwave processing for immunohistochemical demonstration of proliferating cell nuclear antigen (PCNA) in formalin-fixed and paraffin-embedded tissue

The effect of microwave irradiation on the immunoreactivity of proliferating cell nuclear antigen (PCNA) in five primary breast carcinomas, a tonsil with hyperplasia, and a seminoma of the testis was evaluated in formalin‐fixed and paraffin‐embedded sections using the monoclonal antibody PC‐10. The tissues were fixed in 4% buffered formaldehyde for 6, 24, 28, 72 h, and 1 week. Before incubation with the primary antibody, sections were microwaved for × 2 5 min in distilled water, in citrate buffer, or they were processed routinely without microwave irradiation. In sections microwaved in distilled water the immunoreactivity was found to be independent of the length of fixation. PCNA immunoreactivity in sections microwaved twice in citrate buffer was not satisfactory. The PCNA immunoreactivity in sections which were routinely processed without microwave irradiation was markedly decreased after 48 h of fixation, and was nearly absent in sections fixed for up to 1 week. In sections processed without microwave irradiation the optimal dilution of the primary antibody was 1:10, whereas in sections microwaved twice in distilled water the optimal dilution was 1:600. The morphology of the tissue was well preserved after microwave processing. We therefore recommend microwave processing in distilled water for × 2 5 min for demonstration of PCNA in formalin‐fixed and paraffin‐embedded sections using the monoclonal antibody PC‐10, since this technique yields optimal staining reactions independent of the length of fixation.

Bcl‐2 overexpression in basaloid proliferations overlying dermatofibromas and basal cell carcinomas

Basaloid proliferations overlying dermatofibromas resembling superficial basal cell carcinomas have been interpreted both as reactive/regressive and frankly malignant. Basal cell carcinoma is a slow‐growing tumour, which so far has been regarded as an actively proliferating lesion with a high apoptotic activity. We examined immunohistochemically 6 dermatofibromas with overlying simple hyperplasia, 12 dermatofibromas with overlying basaloid proliferations, and 24 basal cell carcinomas for expression of Ki‐67 protein and bcl‐2 protein. The Ki‐67 labelling index represents an estimate of proliferative activity. Bcl‐2 protein suppresses apoptosis. The Ki‐67 labelling indexes of basaloid proliferations, basal cell carcinomas, and normal epidermis were similar (11–15%, p<0.05, Mann‐Whitney test). Bcl‐2 protein was expressed in all cells of basaloid proliferations, similar to the expression pattern in basal cell carcinomas. We suggest that basaloid proliferations overlying dermatofibromas might have achieved a phenotype that equals an early stage of BCC carcinogenesis.

c‐erbB‐2 oncoprotein is not an independent prognostic parameter in primary breast carcinoma. An immunohistochemical study

The prognostic significance of c‐erbB‐2 oncoprotein expression was studied using the monoclonal antibody, anti‐c‐erbB‐2 (CB‐11, BioGenex) and the avidin‐biotin‐complex (ABC) technique. Four hundred and ninety patients with primary breast carcinoma diagnosed at Gentofte Hospital in the period 1980–1985 were included. Information about treatment, relapse‐free period and overall survival was obtained from the Danish Breast Cancer Co‐operative Group (DBCG). The mean follow‐up period was more than 10 years. Fifteen per cent of the tumours showed positive immunoreactivity for c‐erbB‐2. Oncoprotein expression was correlated with presence of lymph node metastases, type of tumour, high number of mitoses, severe nuclear pleomorphism, high histological grade (poor differentiation), and absence of steroid hormone receptors. By univariate analysis, expression of c‐erbB‐2 oncoprotein, was correlated with poorer overall survival and shorter disease‐free period in the entire cohort and in patients with lymph metastases, but not in the group of patients without lymph node metastases. By multivariate analysis, c‐erbB‐2 failed to be an independent prognostic marker for either disease‐free period or overall survival, whereas classical histopathological parameters such as presence of lymph node metastases, high number of mitoses, high histological grade (poor differentiation) and absence of progesterone receptors turned out to be of independent prognostic significance.