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Objective To ascertain the influence of hormone replacement therapy on clinical and biologic prognostic factors of breast cancer. Methods Between 1976–1992, we treated 1081 postmenopausal women for breast cancer at our institution. Of these, 68 were undergoing postmenopausal hormone replacement therapy at the time of diagnosis. These patients were compared with a matched control group of 272 breast cancer patients who had not undergone prior hormone replacement therapy. Results Patients who developed breast cancer during hormone replacement therapy had fewer locally advanced cancers (large tumors and extensive lymph node involvement) and more well-differentiated cancers (infiltrating lobular cancers and grade 1 cancer). The number of patients with estradiol or progesterone receptors was lower in the hormone-treated group. Metastasis-free survival curves showed a tendency ( P = .05) for better prognosis in hormone-treated patients both overall and in stage T2. Conclusions Hormone replacement therapy per se does not affect the prognosis of breast cancer. Regular surveillance during hormone replacement therapy reduces the number of locally advanced cancers and thus improves the survival rate. The higher number of well-differentiated cancers and the distribution of hormone receptivity may reflect interaction between neoplastic tissue and exogenous hormones.

This study was carried out with three aims: to assess the variations of receptors for beta-oestradiol and progesterone, and for epidermal growth factor (EGF), alpha-fibroblast growth factor (FGF) and beta-FGF, during a spontaneous cycle in normo-ovulatory patients, 2 days before, and 2 and 7 days after the day of ovulation; to compare the control group of patients with those given ovulation induction using a multiparametric approach; and to study the role of luteal supplementation with progesterone in two groups of patients treated by a luteinizing hormone-releasing hormone agonist plus human menopausal gonadotrophin. The results were assessed by biochemical analyses and by immunohistochemistry. In spontaneous cycles, a decrease in steroid receptors occurred during the luteal phase. There was an increase in EGF and EGF receptors which was induced by oestradiol secretion, and an increase during the luteal phase of alpha-FGF and beta-FGF, which are considered to be angiogenic factors. Comparisons between spontaneous and induced cycles showed a few variations in the endometrium but no differences were related to the type of ovulation induction.

Both DNA ploidy and S-phase ploidy are promising prognostic factors for node-negative breast cancer patients. Based largely on the analysis of one large study, much of the reported problems with these factors have been caused by some unappreciated complexities in categorizing DNA ploidy into low- and high-risk groups and the lack of some necessary adjustments to eliminate unwanted correlations between DNA S-phase and ploidy. When both DNA ploidy and S-phase are compensated properly, they become independent prognostic markers, forming a powerful prognostic model.

The lack of a standardized methodology for quantifying DNA ploidy and S-phase fraction (SPF) by flow cytometry is hindering routine use of these markers in breast cancer management. In a retrospective clinical multicenter study, we validated a standardized flow cytometry protocol. We tested 633 frozen T(1)T(2), N(0)N(1), M(0) breast tumors obtained in four institutions. Cell preparation was standardized, and precise rules for data interpretation were followed. Three SPF classes were defined on the basis of tertiles after adjustment for ploidy. DNA aneuploidy was observed in 61.0% of cases. No significant difference was observed among centers. Aneuploidy and high SPF were associated with large tumor size, node involvement, high histological grade, and hormone receptor negativity. In the overall population (median follow-up, 69 months), patients with medium and high SPF values had shorter disease-free survival (DFS) than those with low SPF values (P0.0001). Ploidy had no significant influence. By Cox analysis, SPF, pN, and estrogen receptor status were independent predictors of DFS (P = 0.0002, P = 0.001, and P = 0.05). In node-negative patients, SPF was the only predictor of DFS (P = 0.01), whereas in node-positive patients, the risk of relapse increased with both high SPF (P = 0.003) and estrogen receptor negativity (P = 0.004). Low SPF values distinguished grade II tumors with a particularly good outcome. Our results strongly support the use of SPF in multicenter studies and clinical trials and suggest that node-negative patients with slowly proliferating tumors do not require systemic adjuvant therapy.

Thymidylate synthase (TS) is a crucial target for 5-fluorouracil (5-FU) in the de novo pathway of pyrimidine synthesis, which is necessary for DNA synthesis. Thymidine kinase (TK) plays a key role in the complementary or alternative salvage pathway of pyrimidine synthesis in acute or pathological tissue stress. In the present study, the activity levels of TS and TK were determined in 257 primary breast tumors of patients who received tamoxifen as first-line systemic therapy after diagnosis of advanced disease. In 155 (60%) responding patients, the median response duration was 23 months for tumors with low TK activity, 15 months for tumors with intermediate TK activity, and 13 months for tumors with high TK activity (P = 0.003). In Cox multivariate analysis corrected for classical predictive factors including estrogen receptor and progesterone receptor, patients with intermediate and high levels of TK activity in their tumors showed a rapid disease progression (P = 0.0002) and an early death (P = 0.002) after start of tamoxifen treatment. Tumor TS activity levels were not significantly associated with the efficacy of tamoxifen treatment. In 121 patients who became resistant to tamoxifen or additional endocrine treatments and who received 5-FU-containing polychemotherapy, tumor TK activity was not significantly related to the efficacy of chemotherapy. Of the 13 patients with low tumor TS activity, only 1 (8%) responded favorably, whereas 46% (43 of 93) of those with intermediate and 73% (11 of 15) of those with high TS activity responded (P = 0.001). In Cox multivariate regression analysis in which TS was the only significant variable, intermediate and high TS activities were associated with a slow disease progression (P = 0.005) and prolonged survival (P = 0.016) on chemotherapy. In conclusion, for patients with recurrent breast cancer, high tumor TK activity is a significant marker of poor clinical outcome on tamoxifen therapy. Elevated tumor TS activity predicts a favorable outcome for 5-FU-containing polychemotherapy when applied after tumor progression on endocrine therapy.

S-phase fraction (SPF) is a reference for cell-kinetic analysis. In this study, the links between SPF and the essential enzymes participating in the pyrimidine synthesis were investigated in breast cancer and their relationships with the natural history of the disease were compared. We measured thymidine kinase (TK) for salvage synthesis, thymidylate synthase (TS) for de novo synthesis and thymidylate kinase (TMK), which is required for both pathways. Our study population consisted of 211 premenopausal women with node-negative tumors. SPF was assessed prospectively by flow cytometry, whereas enzyme activities were measured retrospectively in cytosols using radioenzymatic methods. Among the enzymes analyzed, only TK demonstrated a strong correlation with SPF (r(s) = 0.59). In univariate analysis, high SPF and high levels of TK were associated with increased risk of developing distant recurrences (p0.001). Correlations with other prognostic factors (histological grade, steroid receptors, DNA ploidy status, urokinase plasminogen activator and plasminogen activator inhibitor type 1) confirmed a parallel association of SPF and TK with the most aggressive tumors. In contrast, TS and TMK were not associated with prognosis. After adjustment for SPF, the risk of relapse increased significantly with TK values. Subgroup analysis showed that additional information was provided by TK in the tumors with low SPF. When urokinase plasminogen activator (uPA) was a candidate variable in multivariate analysis, TK remained significant. Combined with SPF and uPA, TK could be useful to define premenopausal node-negative patients with rapidly proliferating tumors at a high risk of metastatic disease.

High thymidine kinase (TK) activity in cancer cells could counteract adjuvant chemotherapy directed at the inhibition of de novo DNA synthesis. TK is an independent prognostic factor in breast cancer patients receiving adjuvant chemotherapy.In this paper, we describe the effects of extraction and dilution buffer composition on TK enzymatic activity values obtained in breast cancer cytosols with the Prolifigen serum TK-REA kit (Sangtec Medical, Sweden).The addition of MgCl2 and ATP early in the assay, preferably during the extraction of tumor tissue, seems critical to stabilise the enzyme. Furthermore, the use of normal calf serum to dilute both standards and samples is necessary to obtain satisfactory parallelism between TK values in serial dilutions of breast cancer cytosols.Based on the data reported here, the manufacturer has changed the cytosol diluent composition and is adding a specific cytosol assay insert to the Prolifigen TK-REA kit. As evidenced by the laboratory reproducibility, these modifications to the serum assay led to an adequate, standardized protocol for analyzing TK activity in breast tumor cytosols.

As part of a clinical research project, proliferative parameters were studied in primary breast cancer: standardization and technical validation of thymidine kinase (TK), thymidylate synthase (TS) and protein tyrosine kinase (PTK) are described. A total of 633 frozen tumor specimens, available in four institutions, was analyzed in three flow cytometry laboratories for DNA content and percentage of S-phase cells (%S) measurement. 1) The standardization step consisted in developing a common protocol for sample preparation; then, common cell suspensions were analyzed in order to perform an inter-laboratory control. Objective guidelines were elaborated to interpret DNA histograms in breast carcinoma. 2) DNA-aneuploidy was observed in 61% of cases of the retrospective series. Compared with DNA-aneuploid tumors, mean %S was significantly lower in case of DNA-diploidy (respectively: 6.4% and 2.2%, p0.001). When compared between the four institutions, %S distributions did not differ significantly. 3) %S is strongly correlated with TK, TS and PTK and high percentages were also observed in high grade tumors or tumor without hormone receptors. These results show that a standardization in using flow cytometers and DNA software allows multicenter studies.

Retrospective analysis of results of treatment of 132 subclinical ductal carcinomas in situ, non-palpable.Patients were treated with limited surgery and 70 Gy radiation therapy (70 Gy).With a median follow-up of 7 years, the total recurrence rate was 6%, and the actuarial rate at 5 years 4% and at 10 years 13% at. These have no influence on recurrence on the specific actuarial survival rate which was 100% at 10 years. In spite of five infiltrating recurrences of seven, no metastasis appeared 48 months after the salvage surgery. The global rate of breast preservation was 92% at 7 years.Therapeutic indications were developed taking into account the present analysis and a literature review (2,338 in situ ductal carcinomas, palpable or not, treated with conservative surgery, with or without adjuvant radio-therapy).

Thymidine kinase (TK) and thymidylate synthase (TS) play a key role in, respectively, the salvage and the de novo DNA synthesis pathways. TS is a crucial target for 5-fluorouracil(5-FU) and may also influence methotrexate(MTX) efficiency. Tyrosine kinase(TPK) has been associated with the cytoplasmic domain of growth factor receptors as well as oncoproteins. We investigated whether TK, TS and TPK are predictive factors for drug sensitivity evaluated in terms of relapse-free improvement in breast-cancer patients receiving adjuvant chemotherapy. TK, TS and TPK activities were determined in the cytosols of 154 node-positive primary breast cancers. All patients received 5-FU containing adjuvant chemotherapy. Measurements were performed using radioenzymatic methods. The levels of TK were correlated with those of TS and TPK. The levels of TS and TPK were less strongly correlated with each other. High TK levels were more often found in larger tumours, and the levels of both TK and TPK were negatively correlated with those of PgR. Patients whose tumours contained high levels of TK had increased risks of relapse and death. TS was not of prognostic value, while a high level of TPK was associated with early death. In Cox analysis, TK and TPK retained their independent prognostic value. While target enzyme activities on the de novo DNA synthesis pathway could determine response to anti-metabolics mainly inhibiting this pathway, high activities on the alternative salvage pathway could circumvent induced growth inhibition.

All French laboratories that routinely assay estradiol (ER) and progesterone (PR) receptors participate in the European EORTC quality control program based on twice-yearly analysis of 5 cytosolic preparations. This system has considerably reduced inter-laboratory variations, but does not cover all aspects of these assays. Analysis of receptor value distributions is also crucial to ensure that receptor measurements remain stable with time, independently of the laboratory and assay method. This study involved 83907 receptor assays carried out in the last 17 years by 17 laboratories belonging to the French Study Group on Tissue and Molecular Biopathology. The assays were based on radioligand binding (RLA) or immunoenzymology (EIA). For each laboratory, the medians and positivity rates were analysed according to two totally objective criteria, the patient's age and the year of assay, and according to histological grade and histological type of the tumor in order to verify the correlations classically described. Age-related distributions varied little between laboratories, compared with data published by 7 European EORTC laboratories [1]. The results remained relatively stable with time in the RLA method for ER and PR, and in the EIA method for PR. Median ER-EIA data showed a marked increase between 1987 and 1989, mainly due to changes in the quality of Abbott reagents during this period. Otherwise, this analysis confirms previous pathophysiological observations.

Health visitors and lay people have been working together in Maidenhead to support women suffering from postnatal illness. Alison Jones, Tricia Watts and Sybil Romain describe how the group supplements medical and psychiatric services, by tackling the isolation women with postnatal depression can feel, and helping them through until the illness ends.

Seven laboratories of the EORTC Receptor Study Group reported the distribution of oestrogen (ER) and progesterone receptors (PR) routinely assayed in breast cancer cytosols. A low interlaboratory variability was demonstrated for the median values, and for the frequency of positive tumours as measured by enzyme immunoassay (EIA). Larger variations were found for the frequency of positive tumours, as measured by radioligand binding assay (RLA). They are probably due to differences in the cut-off levels and in the sensitivity of the assay. Analysis of the variability over time clearly demonstrated that the ER-EIA values initially increased compared with RLA. A possible source of variations could be the calibration drift in the ER-EIA kit. In conclusion, quality assessment of steroid receptors should be monitored by comparison of both common standards and distributions routinely obtained in each laboratory. In-house analysis over time is also essential for reagent survey.

A new method (enzyme-ligand immunoassay, ELIA) is described for the estimation of estrogen (ER) and progesterone (PR) receptors in microsamples of human breast cancer tissue. The technique, based on the nonisotopic measurement of receptor-bound estradiol and progesterone, involves three steps: (a) simultaneous saturation of active receptors with their respective authentic ligands, (b) heat treatment of the cytosol to release the steroids from their cognate receptors before or after absorption with dextran-coated charcoal, and (c) measurement of both steroids present in the cytosol by a modified competitive-inhibition enzyme immunoassay. The useful range of the method was 10-4000 pmol/L for ER and 6.5-1000 pmol/L for PR. The correlation coefficient (r) between the one-point and Scatchard plot analysis was 0.95 for ER and 0.99 for PR. Comparison of the one-point ELIA and expected values with the radioligand binding assay (RLBA) results for EORTC samples gave r = 0.88 and 0.99 for ER and PR, respectively. Further comparison of the one-point ELIA with RLBA and with a commercial enzyme immunoassay, in blind testing of cancer tissue microsamples from 70 patients, gave good agreement for ER with r = 0.95-0.97 and concordance of 92.9-94.4% (cutoff, 15 pmol/g protein) against the other two methods. The results were more disperse in all three methods for PR estimation, the assay correlating perhaps better with the enzyme immunoassay (r = 0.90) at a concordance of 89.4% (same cutoff value).

Pilot retrospective studies have pointed to the prognostic value of thymidine kinase (TK) in breast cancer. We studied the Prolifigen TK-REA assay (Sangtec Medical, Sweden), usually applied to serum, for TK analysis in breast cancer cytosols. Reproducibility was good, provided that small volume pipetting was performed carefully. The TK assay was not influenced by the short-term storage of cytosols in liquid nitrogen or at -80 degrees C. However, some steps appeared critical for good laboratory practice. The TK level was affected by thawing the cytosols more than twice. Tumour storage in liquid nitrogen should be preferred over storage at -80 degrees C. The components of the homogenisation buffer, especially sodium molybdate and KCl can have a marked influence on results. Finally, linearity problems arose with some cytosols. Thus, although assay of TK in cytosols is apparently simple, care must be taken in practice. The TK-REA kit should be standardised before widespread use in breast cancer.

Among the great many prognostic factors currently available in breast cancer, three classes of tissue biological parameters appear to be the most reliable in the establishment of a clinical decision flowchart, when they will have been technically and clinically validated: parameters of hormone dependence, tumour aggressiveness and invasion and parameters of proliferation. This article discusses the difficulties encountered in the evaluation of some of these parameters (hormone receptors by various methodological approaches, proteases, enzymes involved in cell proliferation), with emphasis on standardisation of techniques, development of quality controls, clinical validation and objective information of the medical and scientific communities.

Inflammatory breast carcinoma has to be defined by accurate clinical and pathological criteria. The prognosis is very poor and improvements made by chemotherapy are limited to the increase in the disease-free period and overall survival for a few months.

This study concerns the correlation between ER and PR status, menopausal status and the effect of adjuvant hormonotherapy in high risk primary breast cancer patients. We have compared the results obtained in a randomized trial (Institut Paoli-Calmettes, Marseille) with those of a historic series (Centre René Huguenin, Saint-Cloud). The patients presented the same clinical and histological criteria and received identical therapeutic protocols (chemotherapy and/or hormonotherapy). Compared with patients receiving no adjuvant treatment, it appeared that: 1) in post-menopausal patients, a significant detrimental effect of tamoxifen was found in ER-negative patients, while in ER-positive patients hormonal treatment was well correlated with both the presence and level of steroid receptors; 2) in pre-menopausal patients, hormonal therapy (oophorectomy + tamoxifen) appeared to be mediated by a complex mechanism involving more than an ER-positive cell population. In the light of the published results, the present findings underline the importance of reevaluating the indications of hormonotherapy in terms of hormone receptors and menopausal status. They also indicate the importance of biological factors in the evaluation of response to therapy. They can identify, subsets of patients in whom a given therapeutic protocol is detrimental, even though it may be beneficial for the overall population. heterogeneity in response to therapy among patient subsets is one of the most important problems which confronts medical statisticians and clinical investigators.

To test the qualities of two assays in the same laboratory on the same tumours, a single-point dextran-coated charcoal radioligand binding assay (RLA-DCC) and the Abbott enzyme immunoassay (EIA) were used to perform oestrogen receptor determinations on cytosols from 1841 breast cancers over a 2-year period. Statistical analysis of the data was performed by the Passing-Bablock linear regression technique. The final regression curve between EIA (y) and RLA-DCC (x) yielded y = 1.187 × fmol/mg of protein. However, a high variability in this correlation was observed from 1986 to 1988. This variability could be explained by calibration problems in the immunoassay kits and changes in our technical team. The binding assay appears to be more sensitive to the technicians' experience than the immunoassay. Other technical points are discussed, particularly cytosol preparation and KCl presence or absence in the homogenisation buffer. Finally, the Passing-Bablock and the least squares regression procedures are compared. The conditions allowing optimal correlation and routine determination reliability are defined and the correlation variability is discussed.

The total amount of cathepsin-D (mature forms and pro-enzyme especially) in the cytosol of 88 breast cancers including 85 primary cancers and 11 axillary lymph node metastasis was measured by way of immuno-radiometric assay. Maximum follow-up is 58 months. Cathepsin-D was found to be independent of clinical and biological parameters including axillary lymph node involvement and oestradiol receptors (RE). Univariate analysis has pointed out a significant linkage between overall survival and cathepsin-D, using a cut-off level of 30 pmol/mg protein which is the most discriminating value. Cathepsin-D appeared to be particularly useful in lymph-node-positive and RE-negative patients but it was not significant in the node-negative population. Multivariate analysis of the overall survival (Cox model) revealed that it was strongly related to oestradiol receptor, lymph node involvement and cathepsin-D.

We analyzed erbB -2 gene amplification in 170 primary breast carcinomas. Thirty-one percent of tumors exhibited additional copies of erbB -2 gene. Chi-square analysis did not elicit any association between gene amplification and either menopausal or node status. A slight trend was observed with respect to the SBR grading. In contrast, significant correlation was associated with the age of patients and we found a strong relation with the intratumoral steroid receptor status. erbB -2 amplification significantly occurs in tumors whose estrogen receptor and progesterone receptor were below the cut-off value or absent and tumors with dissociated estrogen receptor and progesterone receptor status were revealed as entities similar to both estrogen receptor and progesterone receptor negative tumors. erbB -2 gene/hormonal receptors/breast cancer