[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.

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.

Prognostic value of steroid receptors after long-term follow-up of 2257 operable breast cancers.

The prognostic value of oestrogen receptor (ER) and progesterone receptor (PR) was estimated through a multicentric study of 2257 operable breast cancer patients followed up for a median of 8.5 years. None of the patients had received adjuvant therapy. The series included 33.3% stage I patients, 57.1% stage II, 5.7% stage IIIa and 2.4% stage IIIb. At the end point of the study 589 metastases and 537 deaths from cancer were recorded. Receptor measurements were performed by radiolgand assay according to a uniform protocol. A total of 68.8% of the tumous were ER positive and 54.0% PR positive ( > or = 10 fmol mg-1 cytosol protein). In univariate analysis, ER and PR status (positive/negative) were of prognostic value (P < 0.001) for the disease-free interval (DFI), the metastases-free interval (MFI) and the overall survival (OS). The OS of the patients after a first metastasis was also significantly different between ER-positive and -negative tumours (P < 0.001). In multivariate analysis (Cox proportional hazard model, 1665 patients), only the ER status showed a significant difference (P < 0.01) between positive and negative groups regarding the DFI, MFI and OS. By using Cox non-proportional, time-dependent models, we show that the predictive value of ER status of the primary tumour decreases by approximately 20% per year, losing its significance after 8 years of follow-up. Overall, when compared with TNM and histological grading, ER and PR status have a low prognostic value, their major interest remaining solely in the domain of therapeutic decision.

Use of a polyclonal antibody for the determination of the prognostic value of c-erbB-2 protein over-expression in human breast cancer.

A rabbit-specific polyclonal antibody was obtained raised to a synthetic peptide corresponding to the 1238-1255 C-terminal predicted sequence of the c-erbB-2 protein. This antibody was used in an immunohistochemical procedure to detect the c-erbB-2 protein on a series of 88 paraffin-embedded human breast carcinomas. In 14/88 cases (16%) the c-erbB-2 protein was found to be overexpressed (immunohistochemical score > 1) with a good concordance with the previously determined mRNA level (79/88 cases: 90%). Prognostic significance of c-erbB-2 protein overexpression as detected by immunohistochemistry was tested by the log-rank test. The relative risk of relapse is higher for patients with an immunohistochemical score > 1 (p = 0.00002). In a multivariate analysis of the c-erbB-2 immunohistochemical score was the only powerful parameter (p < 1 x 10(-3). In conclusion, this antibody seems to be a valuable tool in estimating the c-erbB-2 protein regarded in our series as a parameter able to identify a subgroup of operable breast cancer patients with a high risk of relapse.

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.

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.

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.

Estrogen receptors (ER) in human breast cancer. The significance of a new prognostic factor based on both ER protein and ER mRNA contents.

BACKGROUND: The response to endocrine therapy is not entirely predictable from the estrogen receptor (ER) and progesterone receptor (PgR) status of primary breast tumors. The authors previously proposed a new prognostic factor, ER.R, which was based on both ER protein and mRNA levels. A previous analysis of 88 primary breast carcinomas showed that ER.R permits the identification of a subset of ER-positive women with a higher risk of early relapse. The purpose of the present study was to confirm the prognostic significance of ER.R. METHODS: Estrogen receptor protein levels were determined for 171 patients with primary breast cancer either by radio-ligand binding assay (ER-LBA) or enzyme immunoassay (ER-EIA). Estrogen receptor, pS2, and c-erbB-2 mRNA were measured by Northern blot analysis. RESULTS: ER.R factor is determined by calculating the ratio of the values (ER protein in fentomoles per milligram of total proteins) to (ER mRNA in picograms per 4 micrograms of total RNA). A cutoff value of 1.5 (protein levels measured by ER-LBA) or 3 (protein levels measured by ER-EIA) discriminate the two ER.R1 (lower ratio) and ER.R2 (higher ratio) subgroups, which present a significantly lower and higher risk of early relapse, respectively. No association was found between ER.R status and either PgR status or c-erbB-2 and pS2 expression. According to a Cox multivariate analysis for disease free survival, the two stronger factors in predicting a poor prognosis were c-erbB-2 overexpression and ER.R2. In the present analysis, ER.R2 was a stronger predictor of recurrence than was ER negativity. CONCLUSIONS: In accordance with the authors' first published data, the analysis of a larger population with a longer follow-up showed that ER.R2 keeps its significance to predict a poorer outcome for a patient, regardless of which assay was used to quantify ER.

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.

Epidermal growth factor receptors and prognosis in primary breast cancer.

A retrospective study was performed on 109 human breast tumors stored in liquid nitrogen in order to assess the prognostic value of epidermal growth factor receptor (EGF-R) (median patient follow-up 5 years). A significant inverse relationship was observed between EGF-R and both estrogen (ER) and progesterone receptors (PR). Univariate analysis showed a trend towards a shorter metastasis-free survival both in the overall population and in node-negative patients with EGF-R positive tumors. Multivariate analysis of the overall population showed that lymph-node involvement and PR status were the only significant variables in predicting metastasis-free survival. However, in patients receiving no adjuvant treatment (hormone therapy or chemotherapy). EGF was the only significant variable in the multivariate Cox analysis. No c-erbB-1 amplification was detected in these tumors.

Human breast cancer: identification of populations with a high risk of early relapse in relation to both oestrogen receptor status and c-erbB-2 overexpression.

We recently defined a new early prognostic factor, the ER+(R) status, which permits the discrimination of a group presenting a high risk of early relapse among the ER+ patients. This group was referred to as ER+(R2) in contrast to ER+(R1) which corresponded to the group of ER+ patients having a lower risk of early relapse. Taking into account the whole population including the ER- and inflammatory tumours, we have extended this view and showed that ER+(R) status is a significant predictor of disease-free survival. Determination of c-erbB-2 mRNA levels in the same series of tumours showed that high expression of c-erbB-2 mRNA is significantly correlated with ER-, inflammatory tumours and with lymph nodes involvement. Moreover, a multivariate analysis showed that c-erbB-2 mRNA overexpression was a significant predictor of early relapse (P = 0.02), as significant as ER negativity and ER+(R2). For ER+ patients a high level of c-erbB-2 mRNA constitutes a higher risk of relapse for both ER+(R1) and ER+(R2) patients. However, in the case of ER- patients, early relapses were strongly correlated with c-erbB-2 overexpression. The counterpart of this observation is that ER- patients with no overexpression of c-erbB-2 constitute a group with a relatively good prognosis.

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).

Human breast tumors: a comparison between the biochemical method of measuring estrogen and progesterone receptors and that of an immunohistochemical method.

In a series of 94 human mammary carcinomas, the determination of total estrogen (ER) and progesterone (PR) receptors by a single saturating dose method (5 nM for ER, 10 nM for PR) using dextran-coated charcoal was compared to an immunohistochemical method utilizing ER monoclonal antibody (ER-ICA test). There was a good correlation expressed in positive terms between the ER-ICA test and the biochemical determination of ER (94% of concordance) with a statistical value of P less than 0.01 being found between the concentration of ER (biochemical) and the percentage of labeled cells (ER-ICA). The ER-ICA test complements the ER and PR (biochemical) and is particularly useful for ER determinations on small tumor specimens as no additional tissue other than that from the biopsy is required.

[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.

Markers in breast cancer: does CEA add to the detection by CA 15.3?

211 patients with various stages of breast cancer were studied by both the CA 15.3 and CEA markers to assess whether the latter may increase the screening sensitivity of the former. While both markers were equally specific, CA 15.3 was seen to be much more sensitive than CEA (p less than 0.0001). Also, the addition of the CEA did not add appreciably (7%) to positive detection by CA 15.3. There appears to be no advantage to including CEA in a marker panel to follow the course of breast carcinoma.

Epidermal growth factor receptor in human breast cancers: correlation with estrogen and progesterone receptors.

Epidermal growth factor receptor (EGFR), determined by the Scatchard curve method, was found in 22 cases of a random series of 100 patients with breast carcinoma. Two groups of patients were identified, one (n = 16) with a low concentration (0-50 fm/mg protein) of EGFR but with a high affinity (Kd = 3.2 nM), and the other (n = 6) with a high concentration (90-210 fm/mg protein) of EGFR but with a lower affinity (Kd = 6.3 nM). A significant inverse relationship was found between the presence of EGFR and receptors for estrogen (p less than 0.001) and progesterone (p = 0.001). EGFR was found in no (0/8) tumors with Grade I histoprognostic grade, 17% (10/58) Grade II, and 38% (11/29) Grade III (p less than 0.05). EGFR is present therefore in poorly differentiated tumors and associated with other factors of poor prognosis. Our in vivo analyses confirm results found in tissue culture derived from human breast carcinoma cells.

[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.

Immunohistochemical distribution of the 52-kDa protein in mammary tumors: a marker associated with cell proliferation rather than with hormone responsiveness.

We have previously described a secreted glycoprotein of mol. wt 52,000 (52-kDa protein) which is induced by estrogen in some human breast cancer cell lines. This protein has been identified as the proenzyme of a lysosomal cathepsin-D-like protease which is secreted in large proportions in breast cancer cells. To determine which information may be generated by this marker when detected in mammary tumors, in comparison with hormone receptors, we used monoclonal antibodies interacting specifically with the 52-kDa protein and its related cellular processed products (mols. wts 48 and 34 kDa). A high concentration of this protein has been shown in proliferative ductal mastopathies and cysts, suggesting its value in detecting high-risk mastopathies. We now present the immunoperoxidase distribution of this protein in breast carcinoma compared to the cytosolic hormone receptors assayed in parallel. In 232 breast cancers, no correlation was found between the cellular 52-kDa protein content and cytosolic estrogen or progesterone receptor concentrations. This absence of correlation was also shown by the constitutive production of this protein by estrogen-receptor-negative breast cancer cell lines and confirmed by double immunostaining of breast cancer cell aspirates showing a dissociation between the cytoplasmic staining of this 52-kDa lysosomal protease and the nuclear staining of the estrogen receptor. These clinical results, associated with the in vitro mitogenic and proteolytic activities of this protein, strongly suggest that the 52-kDa protein staining in tissue is associated with tumor proliferation and/or invasion, rather than with hormone responsiveness.