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The expression of estrogen (ER) and progesterone (PgR) receptors was analyzed in a retrospective series of 3000 patients who had operable primary breast cancer. Patients were stratified according to ER and PgR status and the study was focused on the two groups (ER-PgR+ and ER-PgR-) of patients whose tumors contained low levels of ER (< 15 fmol/mg protein), regarding potential response to endocrine therapy. The comparison of clinical or histological characteristics between ER-PgR+ and ER-PgR- patients was analyzed as well as the disease-related death and survival. The mean follow-up was 86.3 months. Among the 529 ER-patients, 62 were PgR+ (12%), whereas 467 were PgR- (88%). The ER-PgR+ and ER-PgR- populations represented 2% and 15.6% of the overall population, respectively. In ER- tumors, the PgR status was significantly related to: age, menopausal status, tumor size, SBR grade, and histological type, but not to the type of surgical treatment or to lymph node involvement. ER-PgR+ tumors had smaller size (64% T1 vs 43%) (p=0.004) and were more frequently grade I (28% vs 12%) than ER-PgR- tumors (p < 0.001). In addition, the patients with ER-PgR+ tumors were significantly younger (49.4 years vs 58.4 years; p < 0.0001), and were more frequently premenopausal (76% vs 36%, p < 0.001). The disease-free interval and the metastasis-free survival tended to be worse for ER-PgR- than for ER-PgR+ patients, but the difference was not statistically significant at 10 years. However, a small but significant difference in overall survival, in favor of the PgR+ group, was observed between the two groups during the first 5 years (p=0.03). We conclude that in combination with ER, PgR status defines a group of patients with clinical and biological specificity, which could be considered for specific endocrine therapy.

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There is no information available on the relation between response to chemotherapy and the high-risk phenotype assessed by uPA and/or PAI-1. The clinical situation of neoadjuvant chemotherapy provides a means of rapidly assessing the sensitivity of the primary tumour to cytotoxic drug regimens. The goal of the study was to assess prospectively the predictive value of PAI-1 for response to first-line chemotherapy. PAI-1 concentration was measured on hypertonic cytosolic extracts (0.4 M potassium chloride) by ELISA before chemotherapy on a drill biopsy sample of the tumour in 69 T2 and T3 breast cancer patients (median age 46 years). Oestrogen receptor (ER) (51% ER+), progesterone receptor (PR) (58% PR+), S-phase (median 4.0%) and ploidy were also assessed in the majority of cases. The clinical response to treatment was evaluated after four cycles of FAC or FEC regimen (5-fluorouracil, epidoxorubicin or doxorubicin and cyclophosphamide) (one cycle every 4th week). PAI-1 could be assayed in 29 post-chemotherapy surgical samples. The objective response rate (complete response plus partial response) was 59% (41 out of 69). PAI-1 expressed as gram of tissue (range 19-2370 ng g(-1) tissue) was highly correlated (r = 0.98) to PAI-1 expressed as mg protein (range 0.5-68 ng mg(-1) protein). No correlation between PAI-1 level and response could be observed, with any cut-off. The post- and pre-chemotherapy PAI-1 levels were correlated (r = 0.66). Of all biological parameters, only high S-phase (cut-off 5%) was slightly correlated (chi2 = 3.91, P = 0.05) to response. These data suggest that PAI-1 is not a predictive marker of response to chemotherapy in breast cancer and that its level is not altered by neoadjuvant chemotherapy.

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The biochemical assay for estrogen (ER) and progesterone receptors (PR) as a routine procedure in the clinical evaluation of human breast cancer is well established. Since there are various and complex phenotypic alterations in breast cancer, there is a need for a multiparametric assessment of the biological profile of breast tumours. However, multiparametric analysis requires a large amount of tissue and various methods of quantitative analysis involving expensive reagents. Thus, an evaluation of the diagnostic and prognostic applications of the measurement of mRNA expression by reverse transcription polymerase chain reaction (RT-PCR) has been initiated. A series of 105 surgical samples of breast cancer was assayed for ER and PR expression in parallel by semi-quantitative RT-PCR and standardized enzymoimmunoassays (EIA). 79 (75%) tumour samples were positive for ER expression by EIA, and 86 (82%) by RT-PCR. This shows a good concordance of the two methods (90%). In the case of PR expression 65 (62%) tumour samples were positive by EIA and only 53 (51%) samples by RT-PCR. In conclusion ER-RT-PCR appears to provide information concerning ER expression similar to ER-EIA, and may be an alternative to this assay. The information derived by PR-RT-PCR appears somewhat different from PR-EIA. We are currently evaluating the biological and clinical significance of this discrepancy.

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Both preoperative cytological (FNS) and tumorectomy surgical specimens of the same tumor were obtained from 89 patients with breast cancer. Estrogen and progesterone receptors were assayed on cytosolic extracts by enzyme immunoassay (EIA), in 89 patients for estrogen and in 68 patients for progesterone. Both concentrations were highly correlated between the FNS and the corresponding surgical sample, but FNS values yielded 2 to 3 times higher. With cut-off values of 250 fmol/mg DNA, corresponding to 15 fmol/mg cytosol protein for surgical samples and 500 fmol/mg DNA for FNS, the receptor status was concordant between the types of samples in 84 of 89 (94%) and 59 of 68 (86.8%) patients for estrogen and progesterone, respectively. Sixty-seven carcinomas were estrogen positive, 17 estrogen negative, 38 progesterone positive, and 21 were progesterone negative in both samples. The authors conclude that estrogen and progesterone EIAs can be performed on FNS provided by properly trained pathologists, with a control of their cellularity by DNA assay.

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The urokinase type plasminogen activator (u-PA) and the plasminogen activator inhibitor-1 (PAI-1) are among the best second-generation prognostic tissue factors in breast cancer. However, different extraction procedures and assay kits are used in different laboratories. A total of 79 breast tumour tissues stored in liquid nitrogen were analysed in this study. We compared u-PA and PAI-1 levels determined with the American Diagnostics (AD) kit after various extraction procedures. The median cytosolic extraction yield in the presence of 0.4 mol/l KCl, calculated relative to extraction in the presence of 10 ml/l Triton X100 when adapted to standard laboratory working hours (incubation for 2 h instead of 12 h) was 74.4% for u-PA and 85.8% for PAI-1. In addition, the correlations were acceptable. Cytosolic extracts prepared with KCl could permit optimal u-PA and PAI-1 assays while also enabling hormone receptors to be determined with the same specimens. Further studies with clinical data are now necessary to determine the prognostic relevance of this extraction procedure.

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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.

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The expression of estrogen (ER) and progesterone (PR) receptors was assayed by steroid binding in a series of 95 malignant breast tumors, for which the analysis of chromosome aberrations was performed and allowed the reconstruction of their chromosomal evolution. It was shown that breast tumors undergo a progressive loss of chromosomes, with occasionally one and rarely two endoreduplications. Chromosome losses were often the consequence of rearrangements, and the rate of rearranged chromosomes, which increases progressively, appeared as a possible indicator of tumor progression. The distribution of ER and PR values in the sample of 95 tumors was compared to that of a larger control series of consecutive cases: 598 for ER and 460 for PR. The similarities of the distributions indicated that the sample of 95 tumors was representative of the general population of breast cancers. The levels of ER and PR expression were very strongly and negatively correlated to the rate of rearranged chromosomes, but not to the modal number of chromosomes. However, when tumors having either undergone endoreduplication or not (greater than 50 or less than 51 chromosomes, respectively) were considered separately, a significant correlation between ER and PR expression and chromosome number was found within each group. Finally, breast cancers were subdivided into 4 stages of cytogenetic evolution, from the least to the most evolved: stage 1: less than or equal to 50 chromosomes, less than 25% rearranged chromosomes; stage 2: greater than 50 chromosomes, less than 25% rearranged chromosomes; stage 3: less than or equal to 50 chromosomes, greater than 25% rearranged chromosomes; stage 4: greater than 50 chromosomes, greater than 25% rearranged chromosomes.(ABSTRACT TRUNCATED AT 250 WORDS)

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A method was developed for the detection of progesterone receptors (PgR) by flow cytometry (FCM) in cell suspensions obtained from mechanically dispersed fragments of operated breast cancers. Two monoclonal antibodies were tested for sensitivity and specificity on four breast cancer cell lines of known PgR expression and a calibration curve thus established. A simple procedure was used to calculate the level of PgR expression, taking into account the relative displacement of total cellular fluorescence compared to nonspecific fluorescence for each sample and the average DNA content of the cells derived from the corresponding histograms. The PgR-specific immunofluorescence of the tumor specimens measured in arbitrary units (channels) was then transformed to fmoles/mg DNA by comparison with the calibration curve. The FCM-derived results were compared with those of a conventional immunoenzymatic PgR assay on 30 surgical samples. PgR content ranged from 10 to 22,000 fmoles/mg DNA and linear regression analysis yielded a good correlation (r = 0.86). With a threshold of positivity of 300 fmoles/mg DNA, the two methods concurred for 28 of 30 tumors (93%). Nine specimens were analyzed repeatedly, showing good reproducibility. This method could prove to be more useful than the biochemical assays on homogenates, since it allows the simultaneous analysis of receptor expression in individual cells and of DNA index (ploidy).

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Platelet-derived growth factor (PDGF) is thought to play some role in the genesis of fibrosis associated with myeloproliferative disorders. In addition, transforming growth factor-beta (TGF-beta) has been confirmed to promote fibrotic process. Both PDGF and TGF-beta have been shown to cooperate with epidermal growth factor (EGF) in regulating the growth of human marrow fibroblasts. All three are contained in platelet alpha-granules. We report the results of a study in patients with myelofibrosis with myeloid metaplasia (MMM). We evaluated PDGF, TGF-beta and EGF-like activities in circulating platelets from patients compared to healthy subjects. In contrast to EGF-like intraplatelet levels which were similar in patients and in normal donors (1-4 ng/10(9) platelets), we found constantly higher values for both PDGF and TGF-beta in MMM patients. In both radioimmunoassay (RIA) and assay for mitogenic activity on human bone marrow fibroblasts, PDGF levels were increased on the average 2-3.5-fold over the levels found in normal donors (P less than 0.01 and P less than 0.001, respectively). PDGF serum levels in patients were consistent with those found in platelets. In platelet-poor plasma (PPP), PDGF concentrations were undetectable or congruent to 2 ng/ml in patients and in control donors as well. The total TGF-beta activity in platelet lysates, determined using a competitive radioreceptor binding assay on Swiss 3T3 mouse cells and an inhibition growth assay on CCL64 cells, was found 2-3-fold increased in patients with MMM as compared to control subjects (P less than 0.003). These results emphasize that, not only PDGF, but also TGF-beta are implicated in the myelofibrosis with myeloid metaplasia.

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The epidermal growth factor receptor (EGF-R) is currently being investigated in human clinical oncology, and particularly in breast cancer, as a potential prognostic factor and a biological target for therapy. As an alternative to the 125I-EGF binding assay, we propose a sensitive immuno-enzymetric assay (IEMA) suitable for EGF-R assay in breast cancer. The assay is performed on solubilized extracts of the 105,000 g pellet of a tumor homogenate, allowing estrogen (ER) and progesterone (PR) assays to be made on the cytosol. The IEMA is performed on 96-well plates coated with the monoclonal anti-EGF-R antibody RI, through an anti-mouse IgG2b bridge. Trapped EGF-R in the samples is covered by a second monoclonal antibody (MAb), 528, and revealed by an anti-IgG2a-peroxidase complex. The sensitivity is 1 fmol/mg membrane protein, and the asay can be performed on tissue samples down to 50 mg. Two hundred and twenty primary ductal breast carcinomas assayed by this method showed a log normal distribution with a modal value of 8 fmol/mg prot., a mean at 18 and a median at 13 fmol/mg prot. EGF-R-rich tumors (greater than 20 fmol/mg prot.) were highly correlated with the absence of estrogen receptors and/or with a high histological grade (SBR III). Our data demonstrate the validity of the IEMA assay of EGF-R in human breast tumors.

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An immunoenzymetric assay (IEMA) for the human epidermal growth factor receptor (EGFR) solubilized with nonionic detergent has been developed using two commercially available monoclonal antibodies (MoAb) and tested on breast tumor samples. The first MoAb (R1), immunoadsorbed on a solid phase, is used to immobilize solubilized EGFR. A second MoAb (528) binds to the immobilized EGFR and is revealed with o-phenylenediamine by a peroxidase-linked goat antimouse IgG2a. The detection limit is 2.5 fmol/ml, corresponding to 1-2.5 fmol/mg membrane protein which allows a determination of EGFR from as low as 100-200 micrograms of membrane proteins. The IEMA was linear for serial sample dilutions in a large range of EGFR concentrations. The recovery of increasing quantities of EGFR added to clinical samples ranged from 82 to 107%. We found a high reproducibility (r = 0.97) between two successive assays of 36 breast tumor samples. The EGFR content measured by this method in 50 breast tumor samples correlated (r = 0.95) with the values obtained by a radioligand assay on crude membrane preparations. This sensitive, accurate, reproducible, time and tissue quantity efficient IEMA appears suitable for biological and clinical studies of the role of EGFR in malignant pathology.

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A methodology, originally built up in our laboratory, for the simultaneous determination of estrogen (ER) and progesterone (PR) receptors on small samples of tumor tissues, has been adapted to fine needle aspirates (FNA) of breast tumors. The method is based on a simultaneous incubation of aliquots of high-salt (0.4 M KCl) cytosols with tritiated estrogen and progestin tags. The mixture of receptor-bound ligands is isolated by dextran-coated charcoal and extracted by ethanol. The extracted ligands are separated quantitatively by HPLC and counted by liquid scintillation. FNA provides sufficient cellular material for ER and/or PR assay by single point (5 nM) dextran-coated charcoal (DCC) assay. FNA samples, being contaminated by blood, [3H] R 2858 and [3H] O 2058 are appropriate tags for ER and PR respectively, giving lowest non-specific binding. Sixty-five simultaneous estrogen and progestin receptor determinations on FNA at time of diagnosis were compared to the individual assays performed on the surgical sample obtained from the same patients a few weeks after diagnosis. The correlation between the 2 sets of determinations is excellent in 60 cases with sufficient FNA cellularity. This correlation validates the reliability of estrogen and progestin receptor determination on FNA in the majority of clinical cases.

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