[High-throughput quantification of tissue microarrays: identification of candidate target proteins in inflammatory breast cancer]. / La lecture automatisée à haut débit des micro-biopsies en pathologie: mise en évidence de la surexpression de c-Met et PI3K dans les cancers du sein inflammatoire.

Inflammatory breast carcinoma (IBC) is a rare but very aggressive tumour phenotype. Increased c-Met protein expression correlates with reduced survival and a higher metastatic risk in many human malignancies, including breast cancer Several studies have shown that c-Met protein is targetable by specific drugs. Here we compared c-Met expression in IBC (n = 41) and non IBC (n = 480). Two microarrays of IBC and non IBC tissues were constructed and standardized. C-Met, P13K and E-cadherin were immunodetected (Ven-tana Benchmark Autostainer) on serial sections. The results were quantified with an automated image analysis device (SAMBA Technologies) by immunoprecipitate densitometry of each core section (0.6 microns thick). We found that (i) c-Met is significantly overexpressed in IBC compared to non IBC (p < 0. 001), (ii) P13K is also overexpressed (p < 0.001) in IBC, suggesting that overexpressed c-Met is functionally active, at least through the PI3K signal transduction pathway ; and (iii) E-cadherin is paradoxically overexpressed in IBC. We conclude that c-Met may constitute a target for specific therapy in patients with poor-prognosis malignancies like IBC Automated image analysis of TMA is a valuable tool for high-throughput quantification of the immunohistochemical expression of the tumor proteome.

Overexpression of c-Met and of the transducers PI3K, FAK and JAK in breast carcinomas correlates with shorter survival and neoangiogenesis.

c-Met is responsible for cell motility and tumour spreading. c-Met expression and signal transducers reflecting c-Met functionality were investigated in breast carcinomas, in correlation with patient outcome and tumour vasculature. Tissue microarrays of 930 breast carcinomas were constructed, categorised according to patients' follow-up (4- to 10-year follow-up; median, 6.5 years). Standardised immunocytochemical procedures were performed using anti-c-Met, -PI3K, -FAK, -JAK, and -CD146, -FYN and an automated autostainer (Ventana). High-throughput densitometry measuring the extent of immunoprecipitates was assessed by image analysis (SAMBA). c-Met overexpression correlated with poor survival along with PI3K and FAK reflecting c-Met functionality and CD146 and FYN expression in endothelial cells. Automated quantification of immunocytochemical precipitates using image analysis was shown to provide an objective means of measuring cellular proteins that are potentially relevant for current practice in pathological diagnosis and for specific therapy combining inhibitors of both c-Met and downstream transducer pathways, and of tumour angiogenesis.

Poor prognosis in breast carcinomas correlates with increased expression of targetable CD146 and c-Met and with proteomic basal-like phenotype.

Genomic studies have led to new taxonomic classifications of breast carcinomas. Proteomic investigations using tissue microarrays have yielded complementary results and are useful in identifying potential molecular targets for specific therapies. Searching for new drug targets is particularly important for tumors of poor prognosis, such as breast tumors that lack estrogen receptors and HER2 amplification; in these tumors, certain molecules probably play a significant role in tumor spreading through the stromal microvasculature. We investigated 930 breast carcinomas categorized according to patients' survival (range of follow-up = 4-10 years; median follow-up = 6.5 years) using (1) automated immunohistochemical procedures (Ventana, Cedex, France) with tissue microarrays (Alphelys, Plaisir, France) and (2) quantification of immunoprecipitates assessed by automated image analysis densitometry (SAMBA, Meylan, France). Expression of c-Met and CD146 and that of signaling transducers PI3K, FAK, and FYN were compared in living and deceased patients. Expression of some proteins recently reported to be characteristic of basal cell carcinomas was also assessed, namely, CK5-6, caveolin-1, carbonic anhydrase IX, p63, and CD117; these also constitute potential targets for therapies for aggressive tumors. Overexpression of these proteins was observed in deceased or metastatic patients (P < .01 to P < .00001), particularly node-negative patients (except for FYN, p63, and CD146). c-Met and CD146 are involved in tumor spreading, and our results suggest that they probably play an important role in patients' death, along with other proteins involved in hypoxia (carbonic anhydrase IX) and other cell functions or structures (caveolin-1, CD117, CK5-6, and p63) that are expressed in an aggressive subtype of basal cell carcinoma for which no specific therapy is available.

Effects of progestins of human proliferative endometrium: an in vitro model of potential clinical relevance.

Endometrium biopsy is a useful indicator of endometrium proliferation and is clinically relevant to diagnose cell proliferation and to evaluate response to progestin treatment and to monitor hormone replacement therapy. The aim of our study was to investigate the in vitro effects of progesterone and synthetic progestins on endometrium explants with a particular focus on estradiol receptor (ER) and progesterone receptor (PR) expression which reflects through cell secretion the hormone treatment efficiency. Most widely used progestagens belonging to three distinctive groups were investigated, i.e, medroxyprogesterone acetate (MPA), norethindrone acetate (NOR) and nomegestrol acetate (TX) which are respectively pregnane, 19-nortestosterone and norpregnane derivatives. We used organ culture from human proliferative endometrium, in which tissue integrity, particularly gland/stroma relationships are preserved. Progestins induce epithelial cell secretion and most effects were observed at the highest concentration tested (10(-7) M) and by TX and MPA on homogeneous and on heterogeneous (including also secretory glands) proliferative endometrium respectively. In these conditions, ER as well as PR expression were decreased on both glandular and stromal cells. In contrast, progesterone at 10(-7) M significantly decreased only PR, in glands and in stroma of homogeneous proliferative endometrium, and just in stroma of heterogeneous endometrium. NOR exhibited less effects. At lower concentrations (10(-8) M, 10(-9) M), significantly less effects were observed by synthetic progestins on proliferative endometrium. The experiments show that the different types of progestins do not exhibit in vitro similar effects. Since progestins variably act on proliferative endometrium, the exposure of endometrium explants to progestins may be a useful tool to predict clinical response to hormone therapy (individual "hormonogram") and to monitor endometrium proliferation.