Elina Eriksson

Analysis of polypeptide expression in benign and malignant human breast lesions: down-regulation of cytokeratins.

Malignant progression of tumour cells is caused by the accumulation of genetic defects, which when combined will generate a large phenotypic diversity. Simultaneous quantitation of a large number of gene products in tumour cells is desirable, but difficult to achieve. We have here quantitated the levels of a number of abundant polypeptides in human breast carcinoma cells using two-dimensional gel electrophoresis (2-DE; PDQUEST). For this purpose, tumour cells were prepared from the tissue of 17 breast carcinomas. Fibroadenoma tissue was used as reference for benign cells. An increase of the spot density of the PCNA polypeptide was observed in rapidly proliferating tumour cells, confirming the validity of the procedures used. In the set of 24 polypeptide spots with known identity, decreases in cytokeratin and tropomyosin levels were observed. The levels of all cytokeratin forms resolved (CK7, CK8, CK15 and CK18) were significantly lower in carcinomas than in fibroadenomas. The levels of tropomyosin 2 and 3 were lower in carcinomas than in fibroadenomas. In contrast, the levels of some members of the stress protein family (pHSP60, HSP90 and calreticulin) were higher in carcinomas. Furthermore, changes in the expression of lactate dehydrogenase and GT-pi, but not in nm23, were observed. We conclude that simultaneous analysis of multiple polypeptides in human carcinomas can be achieved by 2-DE and may be useful in prognostic studies, and that malignant progression of breast carcinomas results in the decreased expression of cytokeratin polypeptides. This phenomenon must be considered in studies where cytokeratins are used as markers to identify the epithelial cell compartment in breast carcinomas.

Gelatinase A, Membrane Type 1 Matrix Metalloproteinase, and Extracellular Matrix Metalloproteinase Inducer mRNA Expression: Correlation with Invasive Growth of Breast Cancer

Invasive breast cancer varies widely in biologic aggressiveness, from fairly indolent tumors to rapidly disseminating carcinomas. Matrix metalloproteinases have enzymatic activity and assist in tumor invasion by degrading basement membranes and extracellular matrix. The extracellular matrix metalloproteinase inducer EMMPRIN is thought to stimulate fibroblasts to produce the zymogen pro-gelatinase A. The membrane type 1-matrix metalloproteinase (MT1-MMP) is thought to assist in tumor invasion and metastasis by activating pro-gelatinase A, which shows enhanced expression in various tumors. Overexpression of gelatinase A has shown to correlate with a malignant phenotype in many tumor forms. The aim of the study was to investigate the mRNA expression pattern of MT1-MMP, gelatinase A, and EMMPRIN in breast tumors. Formalin-fixed paraffin-embedded breast tissue samples from 18 patients operated on with breast-conserving surgery for invasive breast carcinoma <20 mm between 1977 and 1985 were analyzed using the mRNA in situ hybridization technique. Most of the patients were node-negative (15/18) and underwent postoperative irradiation to the breast (16/18). The median age at diagnosis was 52 years (21–83 years). At the time of the study 11 patients were alive, 4 without recurrence; 7 patients had been operated for ipsilateral breast tumor recurrences, and 2 had distant metastases. The median follow-up was 112 months (102–193 months). Seven patients died of disseminated breast cancer; their median follow-up was 43 months (22–116 months). 35S-labeled antisense and sense mRNA probes transcribed from linearized plasmids containing cDNA for the matrix metalloproteinases gelatinase A and MT1-MMP and the glycoprotein EMMPRIN were hybridized to 5 μm paraffin-embedded tissue sections. Several invasive carcinomas were surrounded by normal tissue and carcinoma in situ lesions. Gelatinase A, MT1-MMP, and EMMPRIN mRNA expression were detected in all of the carcinomas. The gelatinase A mRNA expression was mainly localized to stromal cells at moderate to high levels surrounding the invading carcinoma cells but was also seen in single cells at low levels in in situ lesions and in some normal glandular cells. MT1-MMP and EMMPRIN were expressed in all of the carcinomas and were mainly localized to tumor cells; but they were also seen to some extent in single cells at low levels in in situ lesions and in normal glandular cells. No differences in levels of expression for gelatinase A, MT1-MMP, or EMMPRIN were seen in patients who survived compared to patients who died from metastatic disease. The co-expression of gelatinase A, MT1-MMP, and EMMPRIN mRNA in invasive breast carcinoma supports the theory that these proteins interact and are important for the invasive phenotype in breast carcinoma. Hence EMMPRIN may be a central factor for stimulation of gelatinase A activation. Specific inhibitors for individual MMP members could in the future be target-specific events in breast tumor progression. Inhibition of EMMPRIN could be such a target.

Assessment of homogeneity in polypeptide expression in breast carcinomas shows widely variable expression in highly malignant tumors

We describe the results from a protein‐based approach to the study of heterogeneity in gene expression between human tumors. Cell preparations from 5 benign breast lesions, 5 potentially weakly malignant and 4 potentially highly malignant invasive ductal breast carcinomas were examined by 2‐dimensional gel electrophoresis (2‐DE) gels. Qualitative and quantitative differences were recorded by computerized analysis. Analysis of samples from different areas of the same tumor showed a high degree of similarity in the pattern of polypeptide expression. Analysis of 2 tumors and their metastases revealed similar 2‐DE profiles. In contrast, variations between different lesions with comparable histological characteristics were considerable. Greater differences in polypeptide expression were observed between potentially highly malignant carcinomas compared with comparisons of less malignant lesions. Our results show that malignant human breast carcinomas may be highly heterogeneous in their patterns of gene expression.

Expression of the c-erbB-2 proto-oncogene product and nuclear DNA content in benign and malignant human breast parenchyma

The expression of the c-erbB-2 proto-oncogene product was investigated immunohistochemically in 474 formalin-fixed and paraffin-embedded human breast tissue samples. The series included 32 benign and 26 hyperplastic lesions, 32 carcinomas in situ and 384 invasive breast carcinomas, 107 of which were less than 1 cm in diameter. Cytometric DNA assessments were performed on histopathologically or cytodiagnostically identified cell nuclei, using image analysis. C-erbB-2 immunoreactivity was not seen in normal parenchyma or in benign and hyperplastic lesions. Mammary carcinomas in situ were more frequently immunoreactive (59%) than invasive neoplasms (23%). Invasive tumours more than 1 cm in diameter immunoreacted more often (26%) than small invasive carcinomas (16%). C-erbB-2 expression in regional lymph node metastases was the same as in the corresponding primary tumours. Significant differences were observed between the c-erbB-2 expression in DNA diploid and aneuploid lesions; for carcinomas in situ the figures were 40% and 72%, respectively. Invasive carcinomas of DNA diploid type rarely showed c-erb-B-2 expression, irrespective of tumour size and nodal status (7–11%). DNA aneuploid tumours were more frequently immunoreactive with increasing levels during progression (32–41%). Our data indicate that genetically stable invasive mammary tumours seem rarely to express the c-erbB-2 protein, even during progression, whereas genetically unstable invasive neoplasms frequently show c-erbB-2 immunoreactivity which increases during tumour progression.

Laminin-5 gamma2 chain expression facilitates detection of invasive squamous cell carcinoma of the uterine cervix.

Summary:Because it has been suggested that laminin-5 can be used as a sensitive marker for epithelial cell invasion, specimens from patients with invasive squamous cell carcinoma of the uterine cervix with a previous history of preinvasive lesions were evaluated by a newly developed monoclonal antibody directed against the γ2 chain of laminin-5. Thirty-two archival paraffin specimens consisting of the matched preinvasive and invasive lesions from 15 women were evaluated to determine whether γ2 chain laminin-5 staining was present in lesions that progressed to invasive cancer. With the exception of one tumor (a small cell nonkeratinizing squamous carcinoma), all squamous cell carcinomas exhibited positive staining. Five of 17 preinvasive lesions also were immunoreactive for the laminin-5 protein. A blinded histologic reevaluation revealed invasion or lesions suspicious for invasion in four of five preinvasive lesions. Our findings suggest that laminin-5 determined by a monoclonal antibody facilitates the identification of invasive lesions that are difficult or impossible to identify on routinely stained histologic sections.

Prognostic value of the combined assessment of proliferating cell nuclear antigen immunostaining and nuclear DNA content in invasive human mammary carcinomas

The expression of theS-phase associated, nuclear protein proliferating cell nuclear antigen (PCNA) was investigated in routinely paraffin-embedded surgical specimens from 209 breast cancer patients. Cytometric DNA assessments were performed on fine-needle aspirates, upon which the primary diagnosis of breast cancer had been based. The mean clinical follow-up was 16 years (range 13–20 years). The percentage of PCNA immunoreactive tumour cells ranged between less than 5 to 60% (mean value 13.34%). There was a direct association between PCNA expression, high histological tumour grade (p<0.01), and DNA aneuploidy (p=0.009). In a subgroup of 22 patients with near-diploid DNA distribution patterns the PCNA expression yielded additional prognostic information. Patients with tumours of near-diploid DNA histograms and more than 20% of PCNA immunoreactive neoplastic cells had a significantly worse clinical course, than patients with neardiploid tumours containing less than 20% PCNA immunoreactive cells (p=0.0001). In contrast, the PCNA immunoreactivity did not yield additional prognostic information for patients with distinctly diploid or highly aneuploid tumour variants. In a multivariate analysis comprising all 209 patients, nodal status (p<0.01), tumour size (p<0.01), and DNA ploidy (p<0.01) were found to have significant prognostic effect. The findings indicate that carcinomas characterised by high proliferative activity and near-diploid DNA distribution patterns can show rapid tumour progression. The combined assessment of the PCNA immunoreactivity and of the nuclear DNA content in routinely processed surgical specimens of breast cancer patients appears to be of prognostic value.

Genetic instability promotes the acquisition of chromosomal imbalances in T1b and T1c breast adenocarcinomas

In order to evaluate biological and genetic properties of early breast carcinomas we analyzed microdissected tissue from 33 primary breast carcinomas stage T1b and T1c with respect to the nuclear DNA content, the expression pattern of Ki‐67, cyclin A, p27KIP1, p53 and p21WAF1, and chromosomal gains and losses. The results show that T1b carcinomas (6–10 mm, n=17) were frequently near‐diploid (53%) with low proliferative activity and staining patterns of p53 and p21WAF1 that suggest the presence of wild type protein. The majority (12/16) of the T1c tumors (11–20 mm), however, was aneuploid, and proliferative activity and p53 expression were increased. Larger tumor size correlated with an increasing number of chromosomal copy number changes and in particular with regional amplifications. High level copy number increases (amplifications), however, were found exclusively in the aneuploid tumors. Amplification events correlated with elevated cyclin A and reduced p27 expression, respectively. Our results suggest that the sequential acquisition of genomic imbalances during tumor progression is accelerated in aneuploid tumors, and may contribute to the increased malignancy potential.

Immunoreactivity with monoclonal antibody A-80 and nuclear DNA content in benign and malignant human breast disease

Immunohistochemical analysis with the monoclonal antibody (MoAb) A-80, recognizing a tumor-associated cytoplasmic mucin-type glycoprotein, and cytometric nuclear DNA assessment were performed on 314 surgical specimens of the human mammary gland. The series included 36 benign conditions, 34 epithelial hyperplasias, 40 carcinomas in situ, and 204 primary invasive carcinomas. Normal breast parenchyma, benign tumors, and other nonmalignant lesions were all of DNA diploid (euploid) type and rarely expressed the A-80 glycoprotein. Differences in MoAb A-80 immunoreactivity and nuclear DNA content were noted among subtypes of epithelial hyperplasias. Fifteen of 34 epithelial hyperplasias were of DNA aneuploid type and the majority were A-80 immunoreactive. Of these 15 immunoreactive aneuploid epithelial hyperplasias, atypical intraductal hyperplasia was the most common subgroup. None of the 19 epithelial hyperplasias of DNA euploid type immunoreacted. Most of the intraductal (33 of 40) and invasive (180 of 204) carcinomas immunostained with MoAb A-80. The majority of the A-80 immunoreactive malignant tumors were of DNA aneuploid type (26 of 33 carcinomas in situ and 108 of 180 invasive mammary carcinomas). The results suggest that expression of the A-80 glycoprotein occurs at an early stage of malignant transformation. Genetically stable (euploid) mammary tumors seem to immunoreact with MoAb A-80 less frequently than genetically unstable (aneuploid) tumor variants. Combined analysis with MoAb A-80 and of nuclear DNA content in premalignant and malignant mammary lesions could be a useful tool of differential diagnostic and prognostic value.

Immunohistochemical expression of the mucin-type glycoprotein A-80 and prognosis in human breast cancer

Immunohistochemical expression of the tumour associated mucin-type glycoprotein A-80 was investigated in a series of 173 breast cancer patients with a clinical follow-up between 13 and 19 years. A routine immunoperoxidase technique was used in formalin-fixed, paraffin-embedded surgical tumour specimens. One hundred and fifty of 173 tumours (87%) immunostained with MAb A-80. The degree of A-80 immunoreactivity was related to the tumour grade but not to lymph node status, tumour size, or nuclear DNA distribution pattern. In univariate analysis the degree of A-80 expression was found to be of significant prognostic value both in node negative and in node positive breast cancer patients (P = 0.03). Patients with non-A-80 immunoreactive tumours had significant longer distant metastases-free survival times and fewer relapses than women with carcinomas composed of A-80 immunoreactive tumour cells. This prognostic value was reduced in a multivariate analysis, including lymph node status, tumour size, and nuclear DNA distribution pattern, but retained borderline significance (P = 0.08). In conclusion, the findings of this study indicate that expression of the mucin-type glycoprotein A-80 as determined by immunohistochemistry seems to be related to clinical outcome in breast cancer patients.

Tumorzell-Proliferation und Prognose beim Mammakarzinom

Das klinische Verhalten von Mammakarzinomen ist oft schwierig vorherzusagen. Daher wurden und werden viele Versuche unternommen, um in Erganzung zu den herkommlichen histopathologischen Kriterien weitere zuverlassige und reproduzier¬bare Prognoseparameter zu finden. In diesem Zusammenhang zeigte sich, das sowohl der cytometrisch gemessene Kern-DNS Gehalt, als auch zellkinetische Daten nutzliche Informationen beinhalten konnen [1].