p53 as an independent prognostic marker in lymph node-negative breast cancer patients.

BACKGROUND: At present, most decisions concerning the use of adjuvant therapy in lymph node-negative breast cancer patients are made on the basis of traditional factors such as tumor size, nodal status, and histopathologic features. However, prognostic factors are being investigated that could identify high-risk groups and that could better address treatment efforts for those patients. Identification of more accurate prognostic markers, such as the expression of the mutant p53 protein encoded by the p53 (also known as TP53) tumor suppressor gene, that are reproducible, easily assessable, and independent in predicting clinical outcome would have a beneficial impact on cancer treatment decisions. PURPOSE: Our purpose was to analyze the predictive relevance of mutant p53 protein expression on 6-year relapse-free and overall survival in node-negative breast cancer patients in relation to menopausal status, tumor size, cell kinetics, and estrogen receptor status. METHODS: Expression of mutant p53 protein was detected by an immunohistochemical technique using a 1:50 dilution of PAb1801 monoclonal antibody on paraffin-embedded tumor specimens obtained from 256 axillary lymph node-negative breast cancer patients, with long-term follow-up (median, 72 months). The [3H]thymidine labeling index, a measure of cell kinetics, was evaluated on histologic sections after fresh tumor tissue was labeled with [3H]thymidine. Estrogen receptor status was determined by the dextran-coated charcoal absorption technique. Statistical comparisons were made for levels of p53 protein expression, [3H]thymidine labeling index, estrogen receptor status, tumor size, and menopausal status with respect to 6-year relapse-free survival and overall survival. RESULTS: Overexpression of the p53 protein, defined as the presence of more than 5% positive cells, was detected in 113 (44%) of 256 tumors. Odds ratios (ORs) for multiple regression analysis of 6-year relapse-free survival were significantly higher for p53 (OR = 3.24; 95% confidence limits [CL] = 2.01-5.23) and [3H]thymidine labeling index (OR = 1.92; 95% CL = 1.19-3.12), both of which appeared to be the most relevant indicators of relapse, than for tumor size (OR = 1.49; 95% CL = 0.94-2.38) and estrogen receptor status (OR = 0.91; 95% CL = 0.55-1.51). Overexpression was found to be unrelated to menopausal status. CONCLUSIONS: Immunohistochemically detected p53 overexpression is an independent marker for shortened 6-year relapse-free and overall survival in node-negative patients with resectable breast cancers. Based on these findings, p53 overexpression should be used with other established prognostic factors, such as [3H]thymidine labeling index and estrogen receptor status, to further refine the prognostic assessment of node-negative breast cancer.

The Bcl-2 protein: a prognostic indicator strongly related to p53 protein in lymph node-negative breast cancer patients.

BACKGROUND: The bcl-2 gene (also known as BCL2) encodes for a mitochondrial protein thought to prevent apoptosis of normal cells. The protein has been detected by immunohistochemical procedures in hormonally regulated epithelia. PURPOSE: We analyzed the predictive relevance of Bcl-2 expression on 6-year relapse-free and overall survival in lymph node-negative breast cancers in relation to pathologic (tumor size) and biologic ([3H]thymidine-labeling index, p53 protein expression, and estrogen receptor [ER] status) features. METHODS: The expression of Bcl-2 and p53 was detected by immunohistochemistry on paraffin-embedded sections from 283 node-negative resectable breast cancers treated with local-regional therapy alone until relapse. The [3H]thymidine-labeling index was evaluated on histologic sections after incubation of fresh tumor tissue with [3H]thymidine, and ER content was determined by the dextran-coated charcoal absorption technique. RESULTS: A significantly higher fraction of Bcl-2-positive cells was observed in small, ER-positive, slowly proliferating, and p53-negative tumors than in large, ER-negative, rapidly proliferating, and p53-positive tumors. A stronger association was observed between Bcl-2 and p53 expression than between these variables and [3H]thymidine-labeling index. In univariate analysis, Bcl-2 and p53 expression, [3H]thymidine-labeling index, tumor size, and ER status were indicators for relapse-free and, with the exception of tumor size, overall survival within 6 years of surgery. In multivariate analysis, Bcl-2 failed to maintain its prognostic role for relapse-free and overall survival in the presence of p53 expression, whereas the [3H]thymidine-labeling index was still statistically significant as a predictor for both events. CONCLUSION: The predictive role of Bcl-2 expression on 6-year relapse-free and overall survival was mainly dependent on p53 expression.

Comparison of immunochemical and radioligand binding assays for estrogen receptors in human breast tumors.

We have compared a new enzyme immunoassay (EIA) for estrogen receptors (ER) with our conventional radioligand binding assays (multipoint dextran-coated charcoal assay for cytoplasmic ER and hydroxylapatite exchange assay for nuclear ER). Cytoplasmic ERs were measured in 76 human breast cancer specimens by EIA and by five-point Scatchard analysis. The correlation between the two assays yielded a straight line with a slope of 0.92 (r = 0.95; P less than 0.001); conversely, in 31 nuclear salt extracts, linear regression analysis of hydroxylapatite exchange assay data with EIA showed a clear correlation (r = 0.93; P less than 0.001) but a slope of 1.7, demonstrating that EIA detects more ER sites. The binding of the antibody to the cytoplasmic ER molecules was investigated by sucrose density gradient analysis, which showed that EIA recognizes both cytoplasmic forms (9 and 3S), but does not distinguish between them. Advantages and drawbacks of this method are discussed with respect to its application for routine receptor determination for clinical management of breast cancer patients.

3H-thymidine-labeling index as a prognostic indicator in node-positive breast cancer.

The prognostic relevance of 3H-thymidine-labeling index (3H-TdR-LI) was retrospectively evaluated in 523 women with resectable node-positive breast cancer given adjuvant combination chemotherapy consisting of cyclophosphamide, methotrexate, and fluorouracil (CMF) +/- Adriamycin (doxorubicin; Farmitalia, Carlo Ezba, Italy). The 5-year relapse-free survival (RFS) and overall survival (OS) rates were significantly higher for patients with slowly proliferating tumors (3H-TdR-LI less than or equal to 2.8%) compared with rapidly proliferating tumors (RFS, 66% v 50%, P = .0007; OS, 85% v 73%, P = .0012). In the analysis of RFS, 3H-TdR-LI provided prognostic information independent of axillary node involvement, tumor size, and estrogen receptor (ER) status, with an estimated lower hazard ratio compared with the degree of nodal involvement, but equivalent to that of the other indicators. Conversely, nodal involvement was found to interact with 3H-TdR-LI and receptors on survival. Present findings confirm that tumor cell kinetics represents a prognostic indicator also in an adjuvant situation. 3H-TdR-LI can substantially contribute to a more precise definition of high-risk patients within subsets having the traditionally favorable prognostic characteristics.

Cell kinetics as a predictive factor in node-positive breast cancer treated with adjuvant hormone therapy.

PURPOSE: The fraction of cells that incorporate 3H-thymidine (3H-dT labeling index [3H-dT LI]) proved to be a prognostic indicator in patients with node-negative and node-positive resectable breast cancers treated with locoregional treatment alone or with adjuvant combination chemotherapy. In this study, we assessed the prognostic role of 3H-dT LI alone and in association with other pathologic and biologic variables in a series of 249 women with node-positive breast cancers treated with adjuvant endocrine therapy. PATIENTS AND METHODS: All patients were postmenopausal, had resectable estrogen receptor-positive (ER+) tumors, and had received tamoxifen for at least 1 year after radical or conservative surgery plus radiotherapy. The median follow-up duration was 48 months. RESULTS: The 4-year relapse-free survival (RFS) rates were significantly lower for patients with large tumors (> 2 cm), with more than three positive lymph nodes, with low (< 150 fmol/mg proteins) ER content, without progesterone receptors (PgRs), or with rapidly proliferating tumors. 3H-dT LI provided prognostic information independent of axillary node involvement, ER content, PgR status, and tumor size, with an estimated odds ratio (OR) higher than that of tumor size, lymph node involvement, or ER concentration. In addition, 3H-dT LI and PgR in association were able to identify patients with different risks of relapse within subsets of tumors with less or more than three positive nodes. CONCLUSION: 3H-dT LI provides prognostic information complementary to PgR, tumor size, lymph node involvement, and ER content in the prediction of RFS of postmenopausal patients with node-positive, ER + resectable tumors treated with adjuvant hormone therapy.

Time-dependent relevance of steroid receptors in breast cancer.

PURPOSE: To analyze the time-dependent prognostic role of the investigated variables, considered, when appropriate, on a continuous scale, for the purpose of evaluating and describing the interrelationships between clinically relevant patient and tumor characteristics (age, size and histology, and estrogen receptor [ER] and progesterone receptor content) and the risk of new disease manifestation. PATIENTS AND METHODS: We applied a flexible statistical model to a case series of 1,793 patients with axillary lymph node-negative breast cancer with a minimal potential follow-up of 10 years. To avoid a potential confounding effect of adjuvant treatment, only patients given local-regional therapy until relapse were considered. RESULTS: ER content and tumor size (adjusted for all the other covariates) showed a time-dependent relationship with the risk of new disease manifestations. In particular, ER content failed to show a prognostic effect within the first years of follow-up; thereafter, a positive association with risk of relapse was observed. For tumor size, within the first years of follow-up, the risk of relapse was directly related to size for only tumors up to 2.5 cm in diameter; thereafter, the impact on prognosis progressively decreased. CONCLUSION: The availability of a long follow-up on a large breast cancer series, as well as the use of innovative statistical approaches, allowed us to explore the functional relation between steroid receptors and clinical outcome and to generate a hypothesis on the involvement of ER in favoring long-term metastasis development.

Biologic and clinicopathologic factors as indicators of specific relapse types in node-negative breast cancer.

PURPOSE AND METHODS: We evaluated, in 1,800 patients with node-negative tumors treated with locoregional therapy until relapse, the competitive risks for different types of metastasis by cell proliferation (3H-thymidine labeling index [3H-dT LI]), estrogen receptors (ERs), and progesterone receptors (PgRs), and by the integration of biologic and clinicopathologic information. RESULTS: Hormone receptor status and proliferative activity of the primary tumor were not indicative of contralateral failures. Hormone receptors failed to predict the 8-year incidence of locoregional recurrence, but they were significant indicators of distant metastasis and overall survival. The latter finding was confirmed even in multivariate analysis. Conversely, cell proliferation predicted both locoregional and distant metastases and survival, regardless of patient age, tumor size, and ER and PgR status. Recursive partitioning and amalgamation analysis ascribed to cell proliferation an important prognostic role for locoregional recurrence together with patient age and tumor size. CONCLUSION: Biologic markers, in particular cell proliferation, provide information for the different types of relapse and could complement the predictive role of pathologic staging.

Effect of sodium butyrate on human breast cancer cell lines.

We have investigated the effects exerted by sodium butyrate (NaBu) on the growth and cell cycle perturbations of four human breast cancer cell lines (MCF7, T47D, MDA-MB231 and BT20) with different steroid receptor profiles. Moreover, since one of the supposed mechanisms of action for NaBu activity involves the induction of apoptosis, we have studied the effects of NaBu on DNA fragmentation by agarose gel electrophoresis and flow cytometry. In all investigated cell lines, NaBu exerted a time- and dose-dependent inhibition of growth and caused a maximum inhibitory effect (85% to 90%) at the concentration of 2.5 mM. The inhibition was already evident after 3 days of treatment. The antiproliferative effect of NaBu was associated with a persistent block of cells in the G2M phase. The block was associated with apoptosis only in oestrogen-receptor positive cell lines. The inhibiting effect of NaBu in hormone-dependent and independent cell lines and its ability to induce apoptosis through a cell cycle perturbation in hormone-dependent cell lines may have important implications in the treatment of human tumours including breast cancer.

Prognostic relevance of cathepsin D versus oestrogen receptors in node negative breast cancers.

The concentration of total cathepsin D in cytosols of 199 node negative women with primary breast cancer in a 10-year retrospective cohort was assayed. Cathepsin D status alone was unable to predict disease-free or overall survival. However, those patients with receptor positive tumours who were cathepsin D positive had shorter [corrected] disease-free (P = 0.02) and overall survival (P = 0.01) than cathepsin D negative patients. Therefore, measurement of cathepsin D appears to provide additional prognostic information for the prediction of disease-free and overall survival in patients with node negative breast cancer.

Prognostic relevance of pS2 status in association with steroid receptor status and proliferative activity in node-negative breast cancer.

Expression of the oestrogen-regulated pS2 protein was investigated on paraffin-embedded sections of primary breast tumours from 200 node-negative patients. Immunoreactivity was observed in 56% of the cases. pS2 expression was inversely correlated with tumour size and proliferative activity, whereas a direct correlation was observed with steroid receptor. 5-year relapse free survival was influenced by tumour size (P = 0.02), oestrogen receptor status (P less than 0.05), and proliferative activity (P less than 0.01). No difference in relapse-free survival was observed between patients subdivided according to pS2 expression alone. However, among patients with oestrogen-receptor-negative tumors, pS2 expression predicted a shorter relapse-free survival.

Biological characterisation of primary and metachronous lesions in breast cancer patients.

Proliferative activity, evaluated as [3H]thymidine labelling index ([3H]dT LI), and hormone receptors were determined on 97 primary breast cancers and on metachronous lesions from the same patient. Overall, the [3H]dT LI of metachronous lesions was significantly higher than that of the primary tumour (P = 0.003). Hormone receptor profiles of the two lesions were similar in about 75% of the cases; disagreements were mainly due to a disappearance of hormone receptors in metachronous lesions. In contralateral tumours, [3H]dT LI and hormone receptors were unrelated to those of the relative primary lesion. In this series of relapsing patients, [3H]dT LI was unrelated to hormone receptor status in the primary tumour, but it was higher in the metachronous lesions from patients with hormone receptor-negative primary tumours. For patients given no systemic therapy between surgery and relapse, the time to develop local-regional recurrences or contralateral tumours was inversely related to the [3H]dT LI of the metachronous lesions.

Interaction between hormone-dependent and hormone-independent human breast cancer cells.

We developed two different models based on in vitro co-culture of hormone-dependent and hormone-independent cell lines to simulate the cell population heterogeneity of human breast cancer tumours. Oestrogen-dependent (MCF-7, ZR 75.1) and oestrogen-independent cell lines (MDAMB-231 BT-20) were grown under serum-free conditions. Co-culture of hormone-dependent and hormone-independent cell lines resulted in an increased cell yield compared to single cell cultures carried out at the same seeding ratios. Such an increase was not affected by addition of oestradiol and single growth factors (EGF, bFGF and IGF-I). These results allow us to conclude that in a heterogeneous cell population like human breast tumours, interaction between hormone-dependent and hormone-independent cell lines may result in a complex regulation of cell growth.

Expression of P-glycoprotein in breast cancer tissue and in vitro resistance to doxorubicin and vincristine.

Expression of P-glycoprotein was evaluated by C219 monoclonal antibody immunoblots in 34 previously untreated and 14 pretreated breast cancers and in benign breast lesions or histologically normal breast glands. P-glycoprotein was not detectable in the few cases of normal or benign tissue. P-glycoprotein was expressed in the 170 kD areas of 29% (10/34) of untreated and 64% (9/14) of previously treated tumours (P = 0.02). In treated tumours, high intensity expression was observed more frequently than in untreated breast cancer (40% vs. 9%). Moreover, there was a significant association between P-glycoprotein expression and in vitro resistance to doxorubicin and vincristine. Simultaneous resistance was observed in all of the P-glycoprotein positive and in only 56% of the P-glycoprotein negative tissues (P less than 0.01). Some aspects of the typical multidrug resistant phenotype, such as P-glycoprotein expression and simultaneous resistance to doxorubicin and vincristine, could be detected in small subsets of breast cancer patients. No relation between P-glycoprotein expression and the type of previous clinical treatment was observed.

Is steroid receptor profile in contralateral breast cancer a marker of independence of the corresponding primary tumour?

We compared oestrogen receptor (ER) and progesterone receptor (PgR) profiles between primary and corresponding contralateral breast cancer (CBC) to investigate whether CBC should be considered relapse of a primary or as a feature of the multicentric origin of breast cancer. We adjusted for patient age, menopausal status, histology and adjuvant therapy. In spite of the general application of a cut-off value to dichotomise ER and PgR, we considered them as continuous variables. Moreover, we considered as synchronous cancers only simultaneously occurring lesions. For 399 patients, ER and PgR receptor levels in primary and CBC did not differ significantly, but were significantly correlated within the same patient. The correlation was higher for synchronous than for metachronous lesions when considering ER, but not PgR. The correlation between ER and PgR levels in the same tumour (primary or CBC) appeared stronger than the correlation of either receptor type (ER or PgR) between primary and CBC. Age, histology and adjuvant treatment affected ER concentration, whereas age, menopausal status and histology affected PgR concentration. The analysis indicated that primary and CBC tend to be characterised by a similar steroid receptor profile. The finding may support the hypothesis of CBC as a second primary arising in a common predisposing milieu, rather than a primary-dependent contralateral lesion. In this light, the clinical management of patients with a bilateral breast cancer should be similar to that of a unilateral breast cancer.

Genistein in the control of breast cancer cell growth: insights into the mechanism of action in vitro.

Genistein significantly inhibited cell growth (IC50 around 10 microM) of MCF-7, MDAMB-231 and HBL-100 cell lines, but not of skin-derived fibroblasts and counteracted the growth-stimulatory effects exerted by estradiol and growth factors. It abolished the paracrine stimulation observed in MCF-7 cells in co-culture with MDAMB-231 or fibroblasts. Genistein-treated cells accumulated in the S and G2/M phases of the cell cycle and underwent apoptosis. Genistein decreased tyrosine phosphorylation induced upon treatment with transforming growth factor-alpha. Finally, genistein bound the estrogen receptor (ER) (relative affinity constant Kd = 4 nM), induced pS2 and cathepsin-D transcription and increased nuclear ER levels.

Influence of culture conditions on the estrogenic cell growth stimulation of human breast cancer cells.

17 beta-Estradiol is a potent mitogen for hormone-dependent cell lines (MCF-7, T47D and ZR 75.1). However, the degree of hormone sensitivity is very much influenced by culture conditions. In order to understand which factors modulate estrogenic effects on cell growth, four different culture conditions were used: (a) medium with dextran-coated charcoal-treated fetal calf serum (DCC-FCS); (b) medium with dextran-coated charcoal-treated growth factor-inactivated serum (DCC-FCSd); (c) serum-free medium, after a 24-h incubation with serum to allow cell attachment; and (d) serum-free medium on collagen IV-treated plates. In all cell lines the highest cell growth stimulation was achieved when estradiol was added in the presence of 5% DCC-FCS, whereas reducing or removing serum from the culture medium resulted in a decrease in cell proliferation stimulation. We postulate that serum contains some still unknown components able to modulate the degree of estrogenic action in endocrine-dependent breast cancer cell lines.

Estrogen-receptor status and response to chemotherapy in early and advanced breast cancer.

The value of estrogen receptor (ER) status in the prediction of tumor response to combination chemotherapy was retrospectively analyzed in breast cancer patients selected for prospective controlled trials of chemotherapy (85 with advanced disease and 256 with operable tumors). All patients were previously untreated with either chemotherapy or endocrine therapy, and in all instances drug therapy was applied at the time of primary treatment. The ER status was considered positive in 54% of women with advanced disease and in 70% of women with operable breast tumor and positive axillary nodes, respectively. About 12% of patients were considered to have borderline ER. The response to drug therapy (complete plus partial remission in advanced breast cancer and 3-year relapse-free survival after mastectomy, respectively) was not significantly different between ER+ and ER- tumors. The comparative results of ER+ vs ER- patients were similar whether the cutoff point for ER+ t-mors was greater than 5 or greater than 10 fmol/mg cytosol protein. The present results indicate that in advanced and early breast cancer combination chemotherapy is effective regardless of ER status. Therefore, in the presence of ER+ tumors there is no reason to delay the early administration of effective chemotherapy. This is particularly true both in the presence of rapidly progressing metastatic disease and in the adjuvant setting.

Evaluation of cardiac activity and pharmacokinetic analysis of 3H-adriamycin in patients pretreated with beta-methyldigoxin.

The consequences at the cardiac level of adriamycin treatment alone or in association with the cardiac glycoside beta-methyldigoxin, were evaluated with reference to the PEP/LVET ratio, heart rate, and minimum blood pressure. The variation usually seen in the PEP/LVET ratio when adriamycin is administered alone was not observed when pretreatment with beta-methyldigoxin was also given. A similar situation is found with variations in blood pressure and heart rate. From a pharmacokinetic point of view, this treatment scheme does not seem to affect the general behavior of the antibiotic.

Effect of medroxyprogesterone acetate on the growth of mouse transplanted tumors: relation with hormone sensitivity.

The effect of high and low doses of medroxyprogesterone acetate (MPA) was investigated on three transplanted murine tumors (MXT mammary carcinoma, colon 38, and colon 26) in relation to receptor status and sensitivity of the tumors to ovariectomy and treatment with dexamethasone. MPA had no inhibitory activity on the growth of these tumors. It had no effect on the ovarian-sensitive MXT tumor; it significantly enhanced the growth of an MXT tumor line, selected through serial transplantations, which was stimulated also in ovariectomized animals. MPA, as well as ovariectomy, stimulated the growth of the colon 38 tumor, but this hormone sensitivity was lost during serial transplantations. No correlation was found between the effects of MPA and ovariectomy and the steroid receptor status of these tumors. MPA effects on these tumors do not seem contingent upon a glucocorticoid-like action since dexamethasone was highly effective on all the tested tumors. The combined treatment of the colon 26 tumor with a cytotoxic drug, 4'-deoxydoxorubicin, and MPA, which administered alone stimulated tumor growth and increased life span, caused a slight increase in the life span compared to single agents alone.

The effect of alpha-, beta- and gamma-interferon on the growth of breast cancer cell lines.

We investigated the antiproliferative effect of different concentrations (10, 100, 1000 IU/ml) of alpha-, beta- and gamma-interferons (IFN) on breast cancer cell lines. Cell lines were treated with IFN, in the absence or in the presence of estradiol for 9 days, and the effect on growth was evaluated as variation in DNA content. Inhibitory effect varied as a function of the type of interferon and cell line. Alpha-IFN and gamma-IFN were effective only at 1000 IU/ml and not in all cell lines, whereas a maximum effect was observed for beta-IFN regardless of the steroid receptor status of cell lines. In fact, a significant growth inhibition was observed at the intermediate concentration of 100 IU/ml in all but MDA-MB231 cell lines. Moreover, in both estrogen-receptor positive cell lines, beta-IFN counteracted growth stimulation induced by estradiol and showed a strong antiestrogenic activity. In conclusion, our results show that beta-IFN is the most active among the IFNs tested and suggest its usefulness in the treatment of all breast cancers, irrespective of their steroid receptor status.