A new approach allowing an early prognosis in breast cancer: the ratio of estrogen receptor (ER) ligand binding activity to the ER-specific mRNA level.

We have performed a quantitative analysis of steady-state levels of ER-mRNA for 88 untreated, primary breast carcinomas. We compared the amount of specific mRNA with the amount of ER receptor measured, through ligand binding activity, by calculating the ratio R = [ER-protein/ER-mRNA]. This analysis showed that the relative level of ER-mRNA displayed a large range of values partly related to the concentration of ER-protein. We found a greater percentage of tumors with a high R ratio value in the tumor population containing elevated levels of ER-protein. A statistical analysis performed on a homogeneous population of 63 patients shows no correlation between the R ratio, lymph-node involvement and histological grade. However, R appears to be significantly related to the risk of relapse within a relatively short period of time following the first observation. An R value higher than 1.5 appears to constitute a significant and early prognostic factor of recurrence (P = 0.003).

Is the negative prognostic value of high oestrogen receptor (ER) levels in postmenopausal breast cancer patients due to a modified ER gene product?

Recently, it was found that, among post menopausal breast cancer patients receiving no adjuvant therapy, the highest oestrogen receptor (ER) levels (ER++) as opposed to the intermediate ER levels (ER+) indicated a poorer prognosis in terms of recurrence-free survival (Thorpe et al. Eur J Cancer 1993, 29A, 971-977). In the present study, we confirm, in a series of 218 node negative, postmenopausal patients in whom ER was determined using a one-dose saturating method, that ER+ tumours have a more negative effect on disease-free survival (DFS) than ER+ tumours (P = 0.02). In another series of 87 ER positive, postmenopausal patients, we found a significant correlation (P = 0.04) between the ER level and ER+R ratio (ER protein/ER-specific mRNA): the higher the ER level, the more numerous the high ER+R ratio cases (ER+R > 1.5), reflecting an imbalance between the ER protein level and ER-specific mRNA. From these results, we hypothesise that high ER levels related to a high ER+R ratio suggest the presence of a modified ER gene product.

Quantitative determination of c-erbB-2 in human breast tumours: potential prognostic significance of low values.

The purpose of this prospective multicentric study was to quantify the c-erbB-2 protein and investigate its relationship with DNA amplification and with various prognostic parameters of breast cancer. A total of 1062 primary operable human breast tumours were collected from six French anticancer centres. The c-erbB-2 protein was measured using an enzymoimmunoassay using two monoclonal antibodies directed against the extracellular domain of the protein. The results were expressed in arbitrary units/mg membrane protein (AU) after adjustment for the anticancer centre. A significant association was found between the dosage of the protein and DNA amplification (P = 0.0001). A value of 200 AU was found to maximise sensibility and specificity and was chosen as a cut-off for over-expression. Significant associations were found between c-erbB-2 values and oestrogen receptor (ER) (P = 0.01), progesterone receptor (PgR) (P = 0.0001) and histological grading (P = 0.01). The extreme high values (above the mean plus one standard deviation, S.D.) were significantly more numerous in ER- (P = 10(-16)), PgR- (P = 10(-14)) and grade III (P = 10(-8)) tumours. The extreme low values (below the mean minus one S.D.) were significantly more numerous in ER- (P = 10(-9)) and PgR- (P = 0.02) tumours. This prospective study confirms that high c-erbB-2 protein values are linked to poor prognostic factors and shows for the first time that low values are also linked to hormone receptor negative tumours, suggesting that these low values might also have a negative prognostic significance.

Characterization of two monoclonal antibodies against the COOH-terminal part of the human epidermal growth factor receptor and potential clinical use.

Two monoclonal antibodies of the IgG2 subclass, designated A 01 and B 11, were prepared against two synthetic peptides corresponding to the COOH-terminal sequence of the human epidermal growth factor receptor (EGF-R), in order to detect EGF-R in a radioimmunometric assay and by immunohistochemistry. Characterization of these Mabs showed that they recognized two different eptiopes on the original peptides with Kd of 1.7 x 10(-8) M and 1.3 x 10(-7) M, respectively, without crossreaction. The A 431 antigen recognized by A 01 and B 11 had an apparent molecular weight of approximately 170,000 and was able to specifically link to EGF. Thus, A 01 and B 11 are directed against an antigenic site on the human EGF-R. With Western blot analysis and immunostaining, A 01 was shown to be EGF-R specific. In addition to the EGF-R, B 11 recognized two unidentified soluble proteins present in the cytoplasm of the SKBR-3 cell line but different from the c-erb B-2 oncoprotein expressed by these cells. Mabs A 01 and B 11 were used in an IRMA for the determination of EGF-R using the A 431 cell line as a source of EGF-R. Mab A 01 was also shown to be a useful tool for immunohistochemical detection of EGF-R.

Human estrogen receptor messenger RNA variants in both normal and tumor breast tissues.

By using the PCR-SSCP technique we characterized various ER-specific RNA species present in a series of primary breast cancers, as well as in cell lines established from breast carcinomas and in mammary gland tissues from healthy specimens. A series of six truncated messenger RNAs generated by alternative splicing was characterized. These RNAs correspond to specific deletions of one (exons 2-7, except exon 6) or two (exons 3 + 4) exons. All these RNA variants are observed in each one of the analyzed RNAs, regardless of origin. In addition, the relative amount of these different variants in ER + tumors is comparable to that measured in ER - tumors and healthy mammary gland tissues. This data suggests that tumor progression is not related to the emergence of any of the ER mRNA variants.

Systematic search for 1 alpha,25-dihydroxy-vitamin D3 receptors in human breast carcinomas.

We looked systematically for the presence of receptor like binding sites for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) in the cytosol from 22 breast cancers. Cytosols were centrifuged on 5-20% sucrose gradients after labeling with tritiated 1,25 (OH)2D3 (3H-1,25(OH)2D3 or 25-hydroxyvitamin D3 (25(OH)D3 alone or in the presence of a large excess of these unlabeled sterols. Binding sites for 1,25 (OH)2D3 migrating in the 3.5-3.7 S region were found in 7 out of 22 cancers, while 5.5-6.5 binding sites for 25 (OH)D3 were found in all cytosols. In a patient in whom cytosol containing a 3.5-3.7 S binding site was in sufficient amount, quantification of 1,25 (OH)2D3 binding indicated a KD of 0.28 nM and a maximal binding capacity of 0.15 pmol/mg prot. No relation was found between the presence or the absence of 1,25 (OH)2D3 binding sites and the histological type, the extension of the cancer, the presence of radiological or histological calcifications, the amount of estrogen or progesterone receptors, the plasma calcium or phosphate concentration and the emergence of metastases after 1 year. The significance of the presence of receptor-like binding sites for 1,25-(OH)2D3 in one third of breast cancers remains therefore unknown at the present time.

Soft agar clonogenic assay in human breast cancer.

Relationship between histology, cloning efficiencies and estrogen receptors was studied in the group of breast cancer patient. Mechanical disaggregation gave poor cellular yields since only in 66% cells were ready for the bioassay. Comparison between clonogenic assay, histology and estrogen status of human breast cancer is of a limited value only, because of a small number of patients in our study.

[Presence of free retinol receptor (cRBP) and retinoic acid receptor (cRABP) in human skin tumors]. / Présence du récepteur libre au rétinol (cRBP) et du récepteur à l'acide rétinoïque (cRABP) dans les tumeurs cutanées humaines.

The concentrations of cellular retinoic acid binding protein (cRABP) and free cellular retinol binding protein (cRBP) were determined by ultracentrifugation in sucrose gradient in the cytosol of 41 human skin tumours (14 melanomas, 19 basal cell carcinomas, 8 squamous cell carcinomas). cRBP was found respectively in 36%, 42% and 37% of the studied samples. On the contrary, cRABP was more frequently found in carcinomas (89% in basal cell carcinomas and 100% in squamous cell carcinomas) than in melanomas (21%) (p less than 0.001). These results are discussed according to the different embryologic origin of carcinomas and melanomas. Furthermore, the better efficiency of synthetic retinoids in carcinomas than in melanomas should be explained by a different way of action in these 2 kinds of tumours.

[Action of cytotoxic drugs on normal hepatic tissue in the baboon. Immediate and late lesions]. / Action de médicaments cytotoxiques sur le tissu hépatique normal du babouin. Lésions immédiates et tardives

The perfusion of cytotoxic drugs in the treatment of inoperable tumours of liver is used even in children. The experiment described here was planned to study the effect of three cytotoxic drugs (5. Fluorouracil, Méthotrexate and S.P.I.) on the liver of healthy baboons. The drugs were introduced by perfusion through the hepatic artery. Liver biopsies were performed at the end of the treatment and 15 and 60 days later. In order to avoid acute toxic effects, low doses of the drugs must be given during the first five days. The therapeutic dose can then be increased and maintained for variable periods. Of the three products tested, the toxicity increases in the following order: S.P.I., 5 F U. and Methotrexate.

[Enzymo-immunoassay of progesterone receptors in human breast lesions and tumors. Comparison with a biochemical method]. / Enzymo-immunodosage des récepteurs à la progestérone dans les lésions et tumeurs mammaires humaines comparison à la méthode biochimique.

The determination of progesterone receptors (RP) was performed on 80 benign and malignant human breast tumors with a single saturating dose method (10 nM) using dextran-coated charcoal (PR-Bio) and an enzymo-immunoassay (PgR-EIA). There was a significant correlation between the 2 methods qualitatively (P less than 0.001) and quantitatively (r = 0.79). However the results were significantly higher using the PgR-EIA method than the PR-Bio method (P = 0.04) with a regression line Y = 0.81 x +0.58.

Combined overexpression of c-erbB-2 protein and epidermal growth factor receptor (EGF-R) could be predictive of early and long-term outcome in human breast cancer: a pilot study.

In order to determine the prognostic value of c-erbB-2 protein and Epidermal Growth Factor Receptor (EGF-R), we used an immunohistochemical procedure with specific antibodies on paraffin-embedded material from a series of 73 operable breast cancer carcinomas. c-erbB-2 protein (c-erbB-2 score > 1) was overexpressed in 10/73 cases (14%) and EGF-R (EGF-R ratio > 1) in 42/73 cases (58%). c-erbB-2 overexpression was correlated with tumour size (P < 0.02) and lymph-node involvement (P = 0.05) whereas EGF-R overexpression did not correlate with any of the variables tested. The relative risk of relapse was respectively 1 vs 4.5 (P = 0.001) for patients with a negative (0-1) or positive (> 1) c-erbB-2 score and 1 vs 3 for patients with an EGF-R ratio < or = 1 and > 1 (P = 0.03). Moreover, c-erbB-2 protein overexpression is more specifically an early factor of poor prognosis whereas EGF-R overexpression is a long-term factor of poor prognosis. Patients with an early good prognosis (c-erbB-2 score = 0-1) are found to relapse with time when EGF-R is overexpressed. In a multivariate analysis including axillary lymph-node status, histological grade, tumour size, ER status, c-erbB-2 score, EGF-ratio and hormonal treatment, c-erbB-2 overexpression was the most powerful parameter (P = 0.001) followed by EGF-R overexpression (P = 0.02). We concluded that, in our series, the combined determination of c-erbB-2 protein and EGF-R appeared to be a prognostic indicator whereby both early and long term prognosis could be determined in breast cancer patients.

[Long-term prognostic role of steroid receptors in cancer of the breast]. / Rôle pronostique à long terme des récepteurs stéroïdiens dans le cancer du sein.

We screened for the prognostic value of estrogen receptor (ER) and progesterone receptor (PR) through a multicentric study of 2,257 operable breast cancer patients who did not received adjuvant therapy. Three hundred and seven local-regional recurrences, 105 metachronous contralateral breast cancer, 589 metastases and 537 deaths from cancer had been diagnosed with a median follow-up of 8.5 years. A total of 69% of the tumors were ER positive and 54% PR positive. For statistical analysis, 1,665 patients were studied because of complete clinical and biological data. In univariate analysis, ER and PR status were of prognostic value for the metastases-free interval (MFI) and the overall survival (OS). In multivariate analysis (Cox proportional hazard model), only the ER status showed a significant difference between positive and negative groups regarding the MFI and OS. By using Cox regression model with time-dependent covariates, we show that the predictive value of ER status of the primary tumor decreases by approximately 20% per year, losing its significance after 8 years of follow-up. These results show that ER and PR status have a relatively limited predictive value and their major interest remain in the domain of therapeutic decision.

[Analysis of hormone receptors in the treatment of breast neoplasms in women]. / Utilisation du dosage des récepteurs hormonaux dans le traitement des cancers du sein chez la femme.

PIP: Since only about 1/3 of breast cancers respond to hormonotherapy, it seems important to have available the means of selecting those tumors susceptible of responding to this type of treatment. It has been ascertained that the presence of hormone receptors in cancerous cells is a necessary condition for successful hormone therapy, and that there must be a quantitative relationship between receptors concentration and successful therapy. This relationship has in fact been proven by several studies, and it appears that when therapy is 80% successful the concentration of receptors is very high. It has also been shown that the presence of receptors in primitive tumors constitutes a factor for favorable prognosis, independently of other factors. This will allow the selection of high risk patients to be administered hormonotherapy without delay. It is hoped that better techniques will be discovered to permit a better measurement of hormone receptor dosage.^ieng

[Estrogen receptors in cancer of the breast. Comparison of 2 methods]. / Récepteurs estrogéniques dans les cancers du sein. Comparaison de deux méthodes.

The dosage of estrogenic receptors (ER) was performed on 80 cytosols of human breast carcinomas "immediately operable", by using a methode consisting of a single saturating dose (5 nM) with carbon-dextran and a new immuno-enzymatic method commercialized by ABBOTT laboratories using anti-ER monoclonal antibodies. The concordance is excellent between the two methods in terms of positivity. The results are significantly higher with the ER-IEA method than with the biochemical method (p. less than 1.10(-5]. The regression of the ER-IEA method as compared to the biochemical method is linear with a regression line: Y (ER-IEA) = 0.82 X (ER-Biochem) + 0.76 (logo10 conc. fmoles/g tissue). The correlation is good between the two methods (r = 0.90). A significant relationship (p less than 0.01) has also been observed with the histological grade of the carcinoma. Finally, the immuno-enzymatic technique seems particularly well adapted to small biopsies and slightly positive tumors.

[Multiple leiomyomatous pulmonary nodules in women. Apropos of 2 cases of metastasizing benign leiomyoma]. / Nodules léiomyomateux pulmonaires multiples de la femme. A propos de 2 observations de léiomyome bénin métastasiant.

Two cases of multiple pulmonary leiomyomas in women aged 51 and 45 years respectively are reported. The lesions were discovered 21 and 11 years respectively after hysterectomy for uterine fibromyoma without abnormal histological features. The pulmonary nodules were bilateral and either stable or growing slowly. They were revealed by non-specific symptoms. At surgical biopsy the histological picture was one of benign leiomyoma without mitosis. In one of the 2 patients a pulmonary nodule was explored for oestrogen and progesterone receptors with positive results. The various theories on the origin of these multiple leiomyomas are reviewed. Initially regarded as malformative tumours, they were later interpreted as metastases of uterine leiomyomas with a potential for dissemination. The finding of hormone receptors demonstrates a relationship between these pulmonary lesions, uterine leiomyomas and other multifocal leiomyomatous diseases, including lymphangioleiomyomatosis. The slow but benign course of these tumours differentiates them from metastases of low-malignancy leiomyosarcomas.