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.

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.

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 μg 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.

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.

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.