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CD44 is a family of cell surface transmembrane glycoproteins members which differ in the extracellular part by sequences derived by alternative splicing of 10 variant exons (v1– v10). CD44 proteins containing such variant sequences have been implicated in tumor metastasis formation. Here, we have evaluated the expression of CD44 variants by immuno-histochemistry in primary breast cancer samples of 237 node-negative and 230 node-positive patients. For the analysis of samples derived from node-negative patients, the exon-specific antibodies used were DIII, vff7 and vff18 (v6), vff17 (v7/v8), fw11.24 (v9) and vff16 (v10). With the different antibodies which recognize v6 epitopes, the majority of tumors were positively stained (≥65% of the tumors) with varying intensities. Thirty-nine percent of the tumors were positively stained with the antibody vff16, and approximately half of the tumors with the antibodies vff17 and fw11.24. The expression of CD44 v6 epitopes in tumors from node-negative patients was associated with a favorable prognosis, both upon univariate and multivariate analysis. The expression of CD44 v7/8, v9 or v10 epitopes was not significantly related with relapse-free survival. Samples from node-positive patients were only examined with the antibodies vff7, vff17 and vff18. The staining with none of these antibodies was correlated with the length of relapse-free survival of the patients. Our data suggest that, generally, the usefulness of knowledge of CD44 variant expression is of limited value for assessing the risk of relapse in patients with primary breast cancer. However, the expression of exon v6 of CD44 may be a marker to identify patients with a relatively favorable prognosis in node-negative patients. Int. J. Cancer (Pred. Oncol.) 84:209–215, 1999. © 1999 Wiley-Liss, Inc.

The urokinase-type plasminogen activator (uPA) may be considered as a key enzyme in the processes of cancer cell invasion and metastasis. Evidence has been presented that, in breast stroma, uPA is expressed predominantly by myofibroblasts located at the invasive areas of the tumor. To examine whether transforming growth factor type-1 (TGF beta(1)) produced by breast-carcinoma cells is a candidate responsible for the induction of uPA-producing myofibroblasts, we studied in vitro the capacity of normal and tumor-derived human breast fibroblasts to express uPA and the myofibroblast marker alpha-smooth-muscle actin in response to TGF beta(1). Next, we compared these influences with those elicited by factor(s) released by epithelial-cancer cells. In all 8 fibroblast strains tested, TGF beta(1) induced a similar concentration-dependent increase in the fraction of alpha-smooth-muscle-actin-positive fibroblasts. While uPA expression was decreased by TGF beta(1) in most of the fibroblast strains, 2 strains were relatively insensitive to TGF beta(1) in this respect. Although factors present in media conditioned by non-uPA-producing epithelial-tumor cells could trigger fibroblasts to become potent producers of uPA, the TGF beta(1) content of the conditioned media were linked to the differential effects of externally added TGF beta(1) with respect to uPA expression. The data demonstrate that, although fibroblasts may utilize TGF beta(1) secreted by tumor cells to differentiate into myofibroblasts, tumor cells secrete factor(s) other than TGF beta(1) ultimately responsible for the generation of powerful uPA-producing fibroblasts.

As in many human malignancies, TP53 mutations are the most common genetic alterations in malignant human ovarian tumours. An approach often used in the determination of TP53 status is immunohistochemical staining of the protein. Non-missense mutations, especially those of the null type, causing premature termination codons and resulting in truncated proteins, may often not be detectable by immunohistochemistry. Therefore, current estimates of TP53 alterations in ovarian cancer may be inaccurate. By using polymerase chain reaction-single strand conformation polymorphism analysis and sequencing techniques, we have found a high prevalence of TP53 non-missense mutations in exons 5-8 in ovarian tumour specimens from patients from the southwestern part of The Netherlands. Twenty-nine of 64 tumours showed mutations, of which 10 were non-missense mutations. The majority (9 of 10) of these non-missense mutations, including 7 nonsense mutations and 2 frameshift deletions, were null type mutations and could not be detected by immunohistochemical staining. Five of the 7 nonsense mutations were mutations at codon 213 (Arg-->Stop). The nature of the high prevalence of this nonsense mutation in our series of ovarian carcinomas remains unknown. In addition to the 9 null type mutations, a splice junction mutation was encountered. In conclusion, we have observed a high prevalence (13%) of ovarian tumours with null type mutations in exons 5-8 that did not result in immunostaining. Our data suggest that, especially in ovarian cancer, immunological assessment of TP53 is not an adequate tool to study TP53 alteration. A frequent nonsense mutation at codon 213 in 5 (8%) of 64 tumour specimens represents an important finding.

Elevated levels of expression of the urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 have shown to be related to poor prognosis in a variety of cancer types. In the present study, cytosolic levels of uPA and PAI-1 were determined with enzyme-linked immunosorbent assays in cytosols prepared from 244 human ovarian tissues of different histological sub-types. Both uPA and PAI- I were significantly associated with the malignant progression of ovarian tissues; the levels were increased going from normal tissue, via benign and borderline adenomas, to primary and metastatic adenocarcinomas. For the 90 patients (34 early-stage and 56 patients with advanced disease) from whom the primary adenocarcinoma tissues were examined, uPA and PAI- I levels were evaluated for their association with clinicopathological parameters and with progression-free and overall survival. Neither uPA nor PAI-1 were significantly associated with the age of the patient, FIGO stage, tumor grade, tumor rest, the presence of ascites, or with progression-free or overall survival. On the other hand, age, FIGO stage/tumor rest and the presence of ascites, were significantly related to the length of both progression-free and overall survival in univariate analyses. Tumor grade was of prognostic significance in the analysis for progression-free survival, but not for overall survival. After adjustment for FIGO stage/tumor rest, only age retained its prognostic significance, in the analysis for progression-free survival and in that for overall survival.

A stromal fibroblast-mediated paracrine regulation of epithelial tumor cell proliferation and differentiation plays an important role in the development and progression of breast tumors. We have studied the paracrine growth regulation of various phenotypically different breast cancer cell lines using conditioned serum-free media (C-SFM) from primary breast fibroblasts. Fibroblast cultures were established from malignant primary tumors and adjacent normal breast tissue, benign fibroadenomas, cosmetic reduction mammoplasties and breast skin tissues. All fibroblast-conditioned media were shown to stimulate the proliferation of breast cancer cell lines. However, the C-SFM-induced MCF-7 proliferative response was shown to be significantly higher than the proliferative response observed with any of the other cell lines tested. More importantly, the MCF-7 proliferative response obtained with malignant tumor tissue fibroblast C-SFM was shown to be significantly higher than the response to C-SFM from paired (and unpaired) normal adjacent breast tissue fibroblasts. The MCF-7 proliferative response to fibroblast C-SFM from normal tissue (adjacent to the tumor) was further shown to be comparable to the MCF-7 response using benign or reduction mammoplastic tissue fibroblast C-SFM. In addition, we show that IGFs are only partly responsible for the observed proliferative effect of the C-SFMs, while EGF, TGF alpha and basic-FGF are shown not to be involved. We conclude that stromal fibroblasts can differentially regulate breast cancer cell proliferation. Both the fibroblast's tissue source as well as the target tumor cell's phenotype will determine the extent of the proliferative response.

The prognostic value of epidermal growth factor receptor (EGFR) expression and its biological role in estrogen receptor-positive (ER+) and ER-negative (ER−) primary breast cancer is controversial. In this study, distributions of ER, progesterone receptor and EGFR have been established using immunohisto-chemistry in both primary breast tumors and their matched axillary lymph node metastases of 26 patients or their matched distant metastases of 2 patients. In addition, 5 patients with bilateral breast cancer were studied. ER+ tumor cells were detected in 22 (69%) and EGFR+ tumor cells were detected in 11 (34%) primary breast carcinomas. Expression of ER and EGFR was inverse regarding the individual tumor cells in both primary tumors and metastases. Relationship of EGFR expression with poorly differentiated and large breast tumors was observed. Furthermore, primary tumors with a predominant lobular component were ER+ and, with one exception, EGFR−. Invasive ductal carcinomas were more frequently EGFR+. No apparent differences in receptor expression were observed between primary tumors and lymph node metastases or chro-nously or metachronously occurring bilateral breast cancers. Only one ER+ primary tumor showed a switch to EGFR expression in the involved lymph node. Our study shows that a shift in receptor phenotype between primary tumors and lymph node metastases is a rare event and, thus, additional analyses of involved lymph nodes will not likely serve as a better predictor for response to anti-estrogen therapy. We conclude that expression of EGFR is not a prerequisite for development of metastases. © 1995 Wiley-Liss, Inc.

Ki-67 immunoreactivity was studied in relation to immunohistochemically assessed expression of epidermal-growth-factor receptor (EGFR), estrogen receptor (ER) progesterone receptor (PgR) and cytosolic levels of cathepsin D in advanced human ovarian adenocarcinomas, borderline and benign cystadenomas and normal ovaries. A significantly higher number of Ki-67-positive cells were found in metastatic tumors vs. primary adenocarcinomas and in the total group of adenocarcinomas vs. benign/borderline cystadenomas. Cathepsin-D levels were also significantly higher in metastatic tumors than in primary adenocarcinomas, which in turn presented higher levels than were found in normal ovaries. However, no significant difference was observed between cathepsin-D levels in malignant adenocarcinomas and borderline/benign cystadenomas. Immunohistochemically assessed expression of ER and PgR was detected in variable percentages of epithelial tumor cells, and stromal cells were occasionally positive as well. In the group of primary adenocarcinomas, 46% were ER-positive and 34% were PgR-positive, although there was no significant difference between primary and metastatic lesions with respect to ER or PgR expression. Concordance between immunohistochemically assessed ER or PgR data and cytosolic ER and PgR levels measured with enzyme immunoassay was relatively low. EGFR, immunohistochemically assessed with MAb-EGFRI, was positive in 76% of the primary and in 78% of the metastatic adenocarcinomas. A strong positive association was detected between ER and PgR, and EGFR was observed to present a weak positive correlation with Ki-67 and ER. Cathepsin-D levels were not found to be significantly correlated with the expression of ER, PgR, EGFR or Ki-67. © 1994 Wiley-Liss, Inc.

The expression of oestrogen (ER), progesterone (PR) and androgen (AR) receptors in female breast cancer was investigated by immunohistochemistry on snap-frozen tissue specimens of a series of 100 breast cancers. For detection of the AR we used a recently developed mouse monoclonal antibody specific for the N-terminal domain of the human AR. Expression of AR was compared with that of ER and PR as well as with tumour grade and age. Of the breast cancers investigated, 76% were AR-positive. This high percentage corresponds well with previous data on AR expression in breast cancer determined with ligand-binding assays. In 53% of the tumours AR, ER and PR were present, while 9% of the tumours were positive for AR and negative for ER and PR. In 13% of the tumours no ER, PR or AR expression was seen; these were all grade-III tumours. A positive correlation was found between age and ER expression, but no correlation was seen between age and PR or AR. Future studies should establish the prognostic value of the combination ER, PR and AR determinations on female breast cancer with regard to biological behaviour and response rate to hormonal therapy. © 1992 Wiley-Liss, Inc.

Receptor status and gene amplification were studied in advanced human ovarian adenocarcinoma tissues, borderline and benign ovarian tumours and normal ovarian tissues. Sixty-five percent (53/82) of ovarian adenocarcinomas, 57% (8/14) of benign/borderline tumours and only 31% (5/16) of normal ovarian tissues studied showed specific 125I-EGF (epidermal growth factor) binding (median: 17; 10; and 0 fmol EGF-R/mg protein, respectively) and a significant increase in progesterone receptor (PgR) levels was observed in these groups (median: 5; 33; and 152 fmol/mg protein, respectively). No correlations were observed between the levels of EGF-R and the levels of either oestrogen receptors (ER) or PgR. All membrane samples of 25 adenocarcinomas studied by Scatchard analysis were positive for insulin-like growth-factor-1 receptors (IGF-1 -R) and contained higher IGF-I-R levels than membranes of 10 normal ovarian tissues, of which 9 were positive (median: 64 and 26 fmol IGF-I-R/mg membrane protein, respectively). Also, as measured by autoradiography, 37 adenocarcinoma tissues showed a higher expression of IGF-I-R (1.5+ to 4+) than sections derived from 10 normal ovarian tissues (I +). 125I-IGF-I binding was predominantly associated with epithelial tumour cells, the surrounding connective tissue was negative and in several samples high expression of IGF-I-R was found in areas of tumour necrosis. Southern blot analysis of DNAs isolated from 25 ovarian adenocarcinomas revealed no amplification of the IGF-I -R or the EGF-R gene. The HER2/neu gene was amplified only in 2 out of 3 histologically confirmed endometrioid adenocarcinomas studied but not in 22 other tumours. An amplification of the c-myc gene was observed in 28% (7/25) of the tumours. All c-myc-amplified tumours were PgR-negative. No rearrangement was observed for any of the genes studied. In conclusion, ovarian adenocarcinoma tissues show a decrease in PgR levels and an increased expression of IGF-I-R and EGF-R, in the absence of gene amplification, when compared to benign/borderline ovarian tumours and normal ovarian tissues. In addition, amplification of the c-myc and HER2/neu genes, without rearrangement of these genes, occurs in a minority of these tumours. © 1992 Wiley-Liss, Inc.

CD44 is a family of cell surface transmembrane glycoproteins members which differ in the extracellular part by sequences derived by alternative splicing of 10 variant exons (v1-v10). CD44 proteins containing such variant sequences have been implicated in tumor metastasis formation. Here, we have evaluated the expression of CD44 variants by immuno-histochemistry in primary breast cancer samples of 237 node-negative and 230 node-positive patients. For the analysis of samples derived from node-negative patients, the exon-specific antibodies used were DIII, vff7 and vff18 (v6), vff17 (v7/v8), fw11.24 (v9) and vff16 (v10). With the different antibodies which recognize v6 epitopes, the majority of tumors were positively stained (or = 65% of the tumors) with varying intensities. Thirty-nine percent of the tumors were positively stained with the antibody vff16, and approximately half of the tumors with the antibodies vff17 and fw11.24. The expression of CD44 v6 epitopes in tumors from node-negative patients was associated with a favorable prognosis, both upon univariate and multivariate analysis. The expression of CD44 v7/8, v9 or v10 epitopes was not significantly related with relapse-free survival. Samples from node-positive patients were only examined with the antibodies vff7, vff17 and vff18. The staining with none of these antibodies was correlated with the length of relapse-free survival of the patients. Our data suggest that, generally, the usefulness of knowledge of CD44 variant expression is of limited value for assessing the risk of relapse in patients with primary breast cancer. However, the expression of exon v6 of CD44 may be a marker to identify patients with a relatively favorable prognosis in node-negative patients.

As in many human malignancies, TP53 mutations are the most common genetic alterations in malignant human ovarian tumours. An approach often used in the determination of TP53 status is immunohistochemical staining of the protein. Non-missense mutations, especially those of the null type, causing premature termination codons and resulting in truncated proteins, may often not be detectable by immunohistochemistry. Therefore, current estimates of TP53 alterations in ovarian cancer may be inaccurate. By using polymerase chain reaction-single strand conformation polymorphism analysis and sequencing techniques, we have found a high prevalence of TP53 non-missense mutations in exons 5-8 in ovarian tumour specimens from patients from the southwestern part of The Netherlands. Twenty-nine of 64 tumours showed mutations, of which 10 were non-missense mutations. The majority (9 of 10) of these non-missense mutations, including 7 nonsense mutations and 2 frameshift deletions, were null type mutations and could not be detected by immunohistochemical staining. Five of the 7 nonsense mutations were mutations at codon 213 (Arg--Stop). The nature of the high prevalence of this nonsense mutation in our series of ovarian carcinomas remains unknown. In addition to the 9 null type mutations, a splice junction mutation was encountered. In conclusion, we have observed a high prevalence (13%) of ovarian tumours with null type mutations in exons 5-8 that did not result in immunostaining. Our data suggest that, especially in ovarian cancer, immunological assessment of TP53 is not an adequate tool to study TP53 alteration. A frequent nonsense mutation at codon 213 in 5 (8%) of 64 tumour specimens represents an important finding.

Elevated levels of expression of the urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 have shown to be related to poor prognosis in a variety of cancer types. In the present study, cytosolic levels of uPA and PAI-1 were determined with enzyme-linked immunosorbent assays in cytosols prepared from 244 human ovarian tissues of different histological sub-types. Both uPA and PAI-1 were significantly associated with the malignant progression of ovarian tissues; the levels were increased going from normal tissue, via benign and borderline adenomas, to primary and metastatic adenocarcinomas. For the 90 patients (34 early-stage and 56 patients with advanced disease) from whom the primary adenocarcinoma tissues were examined, uPA and PAI-1 levels were evaluated for their association with clinicopathological parameters and with progression-free and overall survival. Neither uPA nor PAI-1 were significantly associated with the age of the patient, FIGO stage, tumor grade, tumor rest, the presence of ascites, or with progression-free or overall survival. On the other hand, age, FIGO stage/tumor rest and the presence of ascites, were significantly related to the length of both progression-free and overall survival in univariate analyses. Tumor grade was of prognostic significance in the analysis for progression-free survival, but not for overall survival. After adjustment for FIGO stage/tumor rest, only age retained its prognostic significance, in the analysis for progression-free survival and in that for overall survival.

A stromal fibroblast-mediated paracrine regulation of epithelial tumor cell proliferation and differentiation plays an important role in the development and progression of breast tumors. We have studied the paracrine growth regulation of various phenotypically different breast cancer cell lines using conditioned serum-free media (C-SFM) from primary breast fibroblasts. Fibroblast cultures were established from malignant primary tumors and adjacent normal breast tissue, benign fibroadenomas, cosmetic reduction mammoplasties and breast skin tissues. All fibroblast-conditioned media were shown to stimulate the proliferation of breast cancer cell lines. However, the C-SFM-induced MCF-7 proliferative response was shown to be significantly higher than the proliferative response observed with any of the other cell lines tested. More importantly, the MCF-7 proliferative response obtained with malignant tumor tissue fibroblast C-SFM was shown to be significantly higher than the response to C-SFM from paired (and unpaired) normal adjacent breast tissue fibroblasts. The MCF-7 proliferative response to fibroblast C-SFM from normal tissue (adjacent to the tumor) was further shown to be comparable to the MCF-7 response using benign or reduction mammoplastic tissue fibroblast C-SFM. In addition, we show that IGFs are only partly responsible for the observed proliferative effect of the C-SFMs, while EGF, TGF alpha and basic-FGF are shown not to be involved. We conclude that stromal fibroblasts can differentially regulate breast cancer cell proliferation. Both the fibroblast's tissue source as well as the target tumor cell's phenotype will determine the extent of the proliferative response.