Biological and clinical aspects of prolactin receptors (PRL-R) in human breast cancer

PRL has a definite activity in the induction and promotion of mouse and in the growth of rat mammary tumors. We and others have found that human PRL or growth hormone (GH) had a growth promoting effect on human mammary cancer cells. It has been shown that prolactin receptors (PRL-R) which are specific for all lactogenic hormones (hPRL, hGH, hPL) are present on mammary cancer cells in long-term tissue culture and also in tumor biopsies. We found that 43% of the tumors had free PRL-R (FPRL-R) and that 72% had total PRL-R (TPRL-R) which have been desaturated in vitro. A significant correlation (Spearman test) was found between PRL-R (especially TPRL-R) on the one hand, estradiol (P<0.001) and progesterone receptors (P<0.01) on the other. The demonstration of PRL-induced proteins (PIP) might be a better sign of PRL sensitivity than the existence or PRL-R; PIP have been found by Northern blot analysis in 47% of 70 breast cancers. Overall survival (OS) and relapse-free survival (RFS) analysis with a median duration of follow-up of 5.3 yr showed that TPRL-R had a significant prognostic value only in node positive patients (χ²=5.61, P=0.02). Neither FPRL-R or TPRL-R were a significant prognostic factor when studied by Cox analysis. This confirms our previous results. Since at least some human mammary cancers appear to be PRL-dependent we carried out a multicenter randomized trial comparing as the first hormonal treatment tamoxifen (TAM) (30 mg/day)+bromocriptin (B) (5 mg/day) vs TAM + placebo. 171 patients entered this trial. No difference was observed between the two groups in response rates, duration of response or survival.

Recent studies are thus in favor of a role of lactogenic hormones during the course of breast cancer. However no improvement in therapy has been observed yet. The combination of drugs to achieve a total anti-lactogenic treatment, the use of anti-PRL-R antibodies are interesting areas of research; the recent cloning of PRL-R and GH receptors may open new clinical perspectives.     

Time-resolved fluorescence resonance energy transfer (TR-FRET) to analyze the disruption of EGFR/HER2 dimers: a new method to evaluate the efficiency of targeted therapy using monoclonal antibodies

In oncology, simultaneous inhibition of epidermal growth factor receptor (EGFR) and HER2 by monoclonal antibodies (mAbs) is an efficient therapeutic strategy but the underlying mechanisms are not fully understood. Here, we describe a time-resolved fluorescence resonance energy transfer (TR-FRET) method to quantify EGFR/HER2 heterodimers on cell surface to shed some light on the mechanism of such therapies. First, we tested this antibody-based TR-FRET assay in NIH/3T3 cell lines that express EGFR and/or HER2 and in various tumor cell lines. Then, we used the antibody-based TR-FRET assay to evaluate in vitro the effect of different targeted therapies on EGFR/HER2 heterodimers in the ovarian carcinoma cell line SKOV-3. A simultaneous incubation with Cetuximab (anti-EGFR) and Trastuzumab (anti-HER2) disturbed EGFR/HER2 heterodimers resulting in a 72% reduction. Cetuximab, Trastuzumab or Pertuzumab (anti-HER2) alone induced a 48, 44, or 24% reduction, respectively. In contrast, the tyrosine kinase inhibitors Erlotinib and Lapatinib had very little effect on EGFR/HER2 dimers concentration. In vivo, the combination of Cetuximab and Trastuzumab showed a better therapeutic effect (median survival and percentage of tumor-free mice) than the single mAbs. These results suggest a correlation between the extent of the mAb-induced EGFR/HER2 heterodimer reduction and the efficacy of such mAbs in targeted therapies. In conclusion, quantifying EGFR/HER2 heterodimers using our antibody-based TR-FRET assay may represent a useful method to predict the efficacy and explain the mechanisms of action of therapeutic mAbs, in addition to other commonly used techniques that focus on antibody-dependent cellular cytotoxicity, phosphorylation, and cell proliferation.     

Measurement of mRNA of 11 biomarkers by RT-PCR to detect lymph node involvement in cervical cancer

Lymph node metastases are a major prognostic factor in cervical carcinomas. The aim of this study was to characterize the expression of 11 markers in cervical tumors and negative lymph nodes and to determine which ones could be helpful for improving the specificity of molecular diagnosis of nodal involvement. Using TaqMan RT-PCR, we studied the expression of CK19, MUC1, HER1-HER4, VEGF, VEGF-C, uPA, MMP9, and PRAD1 in uterine cervical tumors and in histologically nonmetastatic lymph nodes of 8 patients diagnosed with locally advanced cervical cancer. We observed that CK19, MUC1, HER1-HER3, uPA, and VEGF had a significantly higher expression in cervical tumors than in the negative nodes, whereas VEGF-C expression level was higher in the negative nodes than in the tumors. PRAD1 harbored similar expression levels in the tumors and in the negative nodes. Interestingly, 1 of the 4 patients who presented a clinical recurrence, showed elevated HER1, HER2, uPA, and VEGF in the histologically negative nodes. Our results suggest that CK19, MUC1, HER1-3, uPA, and VEGF are biomarkers that have a higher expression in tumoral cervical tissues compared with the negative lymph nodes and could be useful to diagnose nodal involvement in uterine cervical carcinoma. Our results should encourage us in continue to investigate a greater number of patients, including patients with histologically involved nodes.     

Frequent contribution of T cell clonotypes with public TCR features to the chronic response against a dominant EBV-derived epitope: application to direct detection of their molecular imprint on the human peripheral T cell repertoire

In an attempt to provide a global picture of the TCR repertoire diversity of a chronic T cell response against a common Ag, we performed an extensive TCR analysis of cells reactive against a dominant HLA-A2-restricted EBV epitope (hereafter referred to as GLC/A2), obtained after sorting PBL or synovial fluid lymphocytes from EBV-seropositive individuals using MHC/peptide multimers. Although TCR beta-chain diversity of GLC/A2+ T cells was extensive and varied greatly from one donor to another, we identified in most cell lines several recurrent Vbeta subsets (Vbeta2, Vbeta4, and Vbeta16 positive) with highly conserved TCRbeta complementarity-determining region 3 (CDR3) length and junctional motifs, which represented from 11 to 98% (mean, 50%) of GLC/A2-reactive cells. While TCR beta-chains expressed by these subsets showed limited CDR1, CDR2, and CDR3 homology among themselves, their TCR alpha-chains comprised the same TCRAV region, thus suggesting hierarchical contribution of TCR alpha-chain vs TCR beta-chain CDR to recognition of this particular MHC/peptide complex. The common occurrence of T cell clonotypes with public TCR features within GLC/A2-specific T cells allowed their direct detection within unsorted PBL using ad hoc clonotypic primers. These results, which suggest an unexpectedly high contribution of public clonotypes to the TCR repertoire against a dominant epitope, have several implications for the follow-up and modulation of T cell-mediated immunity.     

egc, a highly prevalent operon of enterotoxin gene, forms a putative nursery of superantigens in Staphylococcus aureus

The recently described staphylococcal enterotoxins (SE) G and I were originally identified in two separate strains of Staphylococcus aureus. We have previously shown that the corresponding genes seg and sei are present in S. aureus in tandem orientation, on a 3.2-kb DNA fragment (Jarraud, J. et al. 1999. J. Clin. Microbiol. 37:2446-2449). Sequence analysis of seg-sei intergenic DNA and flanking regions revealed three enterotoxin-like open reading frames related to seg and sei, designated sek, sel, and sem, and two pseudogenes, psi ent1 and psi ent2. RT-PCR analysis showed that all these genes, including seg and sei, belong to an operon, designated the enterotoxin gene cluster (egc). Recombinant SEG, SEI, SEK, SEL, and SEM showed superantigen activity, each with a specific V beta pattern. Distribution studies of genes encoding superantigens in clinical S. aureus isolates showed that most strains harbored such genes and in particular the enterotoxin gene cluster, whatever the disease they caused. Phylogenetic analysis of enterotoxin genes indicated that they all potentially derived from this cluster, identifying egc as a putative nursery of enterotoxin genes.     

Prenatal diagnosis of Niemann-Pick type C disease: current strategy from an experience of 37 pregnancies at risk.

Thirty-seven pregnancies at risk for Niemann-Pick type C disease were monitored by study of cultured amniotic fluid cells (8 cases) or chorionic villus cells (29 cases) in 23 couples over the period 1984-91. An early protocol combined determination of sphingomyelinase activity with electron microscopy. The current strategy, based on the demonstration of specific abnormalities in intracellular processing of exogenous cholesterol, combines the study of the early phase (first 6 h) of LDL-induced cholesteryl ester formation and the histochemical evaluation (filipin staining after 24 h of LDL uptake) of the LDL-induced accumulation of unesterified cholesterol. Thirteen fetuses were predicted to be affected. Confirmation of the diagnosis was made by study of cholesterol processing in fetal skin fibroblast cultures and/or by demonstration of a characteristic lipid storage in fetal liver, already present at 14 w gestation. Definition of the biochemical phenotype (classical, variant, or intermediate) of the index case, with regard to cholesterol-processing abnormalities, is an absolute prerequisite to adequate genetic counseling in a given family. Prenatal diagnosis has now proved a safe procedure in the predominant (approximately 85%) group of families with the classical phenotype.