Pierre Marie Martin

Influence of pregnancy on the outcome of breast cancer: A case‐control study

The relationship between pregnancy and the outcome of breast cancer remains controversial. The purpose of this study was to determine the prognostic value of pregnancy at the time of diagnosis of primary infiltrating breast cancer. In a retrospective multi‐center study we compared a group of 154 patients presenting pregnancy‐associated (PA) breast cancer with a control group of 308 patients presenting non‐pregnancy‐associated (non‐PA) breast cancer. Classic prognostic factors, treatment modalities, disease‐free survival and overall survival were compared in the 2 groups. The relative importance of pregnancy was assessed by Cox multivariate analysis. There was a significantly higher proportion of inflammatory breast cancer, large tumors and negative receptor status in the PA group. Five‐year recurrence‐free survival, metastasis‐free survival and overall survival were lower both in the whole PA group and in the PA sub‐group excluding patients with inflammatory breast cancer than in the corresponding non‐PA groups. According to clinical stage, histoprognostic grade and microscopic lymph‐node involvement, probability of 5‐year metastasis‐free survival and overall survival was lower in the PA group. Outcome was significantly poorer after chemotherapy for patients in the PA sub‐group than in the non‐PA sub‐group. Multivariate analysis demonstrated that pregnancy was an independent and significant prognostic factor. Pregnancy has an adverse effect on the outcome of breast cancer. Concurrent or recent pregnancy should be taken into account in the development of new systemic therapies. Our findings have important implications for further research into the basic mechanisms of cancer. Int. J. Cancer 72:720–727, 1997.

Immunohistochemical detection of laminin in 98 human breast carcinomas: A light and electron microscopic study

The distribution of laminin was studied in 98 breast carcinomas with antilaminin and the avidin-biotin-peroxidase complex method. Laminin was observed within vascular and epithelial basement membranes. Laminin displayed a continuous linear pattern in intraductal carcinomas, and it was heterogeneously distributed, with a discontinuous linear pattern, in invasive carcinomas. No intracellular laminin staining was detected. Electron microscopic study showed laminin immunostaining in the lamina densa of basement membranes in nonneoplastic breast tissue. In tumors, laminin immunostaining frequently revealed multilayered basement membranes and abnormal multilayered basement membranes in blood vessels in the tumor stroma. These data suggest that laminin immunostaining, as a new approach to the heterogeneous basement membrane changes occurring in carcinomas, should permit better understanding of cell diffusion processes and of stroma-tumor cell interactions. The consistent extracellular distribution of laminin in contact with the stroma indicates that the latter plays an important role in the assembly of basement membrane components.

Subcellular compartmentalization of estrogen receptors in human breast cancer cells

Abstract In MCF-7 human breast cancer cells about 75% of the cells total estrogen receptor sites were unexpectedly found in nuclei not bound with estrogens [13]. These findings were based on studies with crude nuclei and it was originally proposed that these unfilled nuclear estrogen receptors might be biologically active in the absence of estrogen [23]. In order to examine the subcellular distribution of receptor more critically, we have prepared highly purified MCF-7 nuclei, assessed their purity by electron microscopy RNA DNA and DNA protein ratios, and compared their estrogen receptor content with less pure subcellular fractions. In estrogen-withdrawn cells the percentage of the total unfilled receptors found in nuclear fractions (Rn) decreased dramatically from 30% (1.81 pmol/mg DNA) in the crudest preparation to only 3% (0.19 pmol/mg DNA) in highly purified nuclei. (This decrease is not due to an irreversible loss of receptor sites, as receptors lost from nuclei were recovered in the cytosol.) By contrast, after 1 h incubation in vivo with estradiol, the number of estrogen-filled receptor sites (RnE) found in the crudest nuclear preparation (3.28 pmol/mg DNA) and in the most highly purified nuclei (2.66 pmol/mg DNA) were nearly the same. It is very likely that the high level of unfilled receptors found in crude nuclei is a contamination of the preparation with cytoplasmic receptor. 1. 1. Electron microscopy shows gross contamination of crude nuclei with whole cells and cytoplasmic attachments. Purification of nuclei removes attaching cytoplasm which is parallelled by a dramatic decrease in unfilled receptor sites contained in the nuclear preparations. 2. 2. Multiple extractions with Tris buffer (without KCl) releases as much as 72% of the receptor contained in crude nuclei and treatment with 0.2% Triton X-100 extracts more than half. 3. 3. Tris-extractable receptor from crude nuclei sediments identically with cytosol receptor as a single 8S peak on low salt sucrose gradients. 4. 4. Unfilled receptor does not bind in vitro to purified nuclei. Although we cannot entirely exclude the possibility that unfilled receptors are nuclear in origin but may leak from nuclei during cell fractionation, these results are consistent with the localization of unfilled receptor largely in the cytoplasm of MCF-7 cells, as found in normal target tissues such as the rat uterus.

A multiparametric analysis of endometrial estrogen and progesterone receptors after the postovulatory administration of mifepristone.

A double-blind randomized study was performed in two groups of eight normally cycling patients: group I received 10 mg/d of RU486 for 4 days from the date of ovulation and group II received a placebo. On day +5, cytosol and endometrial estrogen receptors (ERs), and progesterone receptors (PRs) were analyzed by radioligand binding assay as well as by enzyme immunochemistry. Histologic studies showed that all the endometria of group I were abnormal (luteal insufficiency and/or E/P imbalance). The nuclear PR levels were significantly higher in group I (843 +/- 422 fmol/mg) deoxyribonucleic (DNA) compared with 482 +/- 232 fmol/mg DNA in group II. Immunohistochemical study showed that ER and PR staining was higher for both glands and stroma in group I (52% and 72% for the respective receptors), compared with the receptor-immunostained surface observed in group II, which was reduced to 40% for ER and to 4% for PR. This study demonstrates that RU486 administered in the immediate postovulatory period blocks normal tissue evolution in the follicular phase as well as the processing of PR.

Genomic analysis of the 67-kDa laminin receptor in normal and pathological tissues: Circumstantial evidence for retroposon features

We have cloned two cDNAs for the human 67-kDa laminin receptor (LR). In the present report we show that these clones hybridize to many restriction fragments in Southern experiments in human. This particular pattern is accounted for by the presence of up to 16 and 21 copies of the laminin receptor gene per haploid genome in human and mouse, respectively. In contrast, a single gene copy is found in chicken. Chromosomal localization reveals four main loci: LAMRP1, laminin receptor pseudogene 1 (Chr 3); LAMRP2, laminin receptor pseudogene 2 (Chr 12); LAMRP3, laminin receptor pseudogene 3 (Chr 14); LAMRP4, laminin receptor pseudogene 4 (Chr X). Comparison of our experimental results to the known features of processed retropseudogenes enabled us to conclude that the LR gene belongs to a retroposon family in mammals.

Early alterations at the plasma membrane of breast cancer cell lines in response to estradiol and hydroxytamoxifen

The time course of the early stage of estradiol-17 beta (E2) and hydroxytamoxifen (OHTAM) action at the plasma membrane of hormone-responsive MCF-7 and non-responsive MDA-MB-231 (MDA) breast cancer cell lines was investigated using scanning electron microscopy (SEM), electron probe X-ray microanalysis and microelectrophysiology analysis. SEM showed a marked increase in the density and the length of microvilli (MV) on MCF-7 cells treated with 1 nM estradiol for 1 min. This membrane response disappeared at 5 min. No early effect was obtained with OHTAM, but both compounds produced a similar surge of heterogeneous MV at 15 min of treatment. The morphological change induced by E2 subsided at 60 min, whereas that of OHTAM persisted. X-ray microanalysis and computer determination of peak/background ratios permitted the demonstration that these morphological alterations were concomitant with a rise in the intracellular level of potassium. Microelectrophysiology analysis showed a sharp transitory decrease in the membrane potential of MCF-7 cells in response to estradiol. In the estrogen-insensitive MDA cells, the hormone did not modify the membrane potential and K levels decreased at 1 and 5 min before rising again to control levels at minute 15 when MV appeared. With OHTAM, potassium decreased significantly at 60 min of treatment. These initial and transitory changes in surface morphology paralleled by alterations in potassium level may be consistent with the occurrence of estrogen membrane receptors on target cells, a new aspect of steroid hormone action.

Microarray gene expression profiling and analysis of bladder cancer supports the sub-classification of T1 tumours into T1a and T1b stages.

To try and identify a molecular signature for pathological staging and/or grading. through microarray analysis.

Topographical analysis of spatial patterns generated by a cellular automaton model of the proliferation of a cancer cell line in vitro

A well‐suited model to simulate cellular population dynamics is the two‐dimensional cellular automaton model, which consists of a lattice of sites, the value ai,j of each site being updated in discrete time steps according to an identical deterministic rule depending on a neighbourhood of sites around it. A cellular automaton is described which mimics cell population proliferation by replacing the site values by the age and the cycle phase of cells. The model takes into account the size of the cells. It is used to simulate the proliferation of the human breast cancer cell line MCF‐7 and the results of the simulation are compared with experimental data obtained from a light microscopic image analysis of the proliferation process. The initial configuration of the cellular automaton is obtained from the discretization of the results of the initial stage of the image processing. After each day of proliferation the pattern obtained from the simulation is compared to the experimental result of the corresponding image analysis. The comparison is made from a topographical point of view through the concept of the minimal spanning tree graph. The agreement between experiment and model is a good starting point to complex models such as cell proliferation under growth effectors or drugs.

Prognostic assessment of PTK activity in T1-T2, N0-N1, M0 breast cancer: a multicentric retrospective study.

Protein tyrosine kinases (PTKs) play a major role in the transduction of intracellular mitogenic signal. PTKs are also involved in the process of cellular transformation. A number of studies have reported increased PTK activities in cytosolic fractions from human breast carcinoma. However, the possible pronostic value of these activities is difficult to establish from these studies, mostly conducted on limited numbers of patients. In order to clear up the issue, we have investigated a large series of patients with a long follow-up, using a retrospective multicentric study (894 breast cancers T1-T2, N0-N1, M0; median follow-up: 67 months). PTKs were measured using a radioenzymatic assay as described in our previously report. We confirmed the already observed correlation between PTK activities and Scarff–Bloom grading (p > 10−5), negative estrogen receptor (ER), and progesterone receptor (PR) status. By contrast, we found in this study a correlation between PTK values and clinical nodal status (p = 0.00027) not showed in our precedent analysis. In Cox multivariate analysis, PTK activity does not emerge as a significant pronostic parameter. On the other hand, tumor PTK activity assay may prove of great interest in clinical research using newly developed tyrosine kinase inhibitors in order to assess their biological impact and eventually to predict the responsiveness to these new therapeutic agents.

Morphological evidence for a subpopulation selection effect by estrogen and antiestrogen treatments in the heterogeneous MCF‐7 cell line

Recently, we developed a method to quantitatively study tumour cell heterogeneity in terms of both nuclear size and estrogen receptor (ER) content by image cytometry. The method, previously used to analyse the proliferation of the breast cancer cell line MCF‐7, was applied here to analyse the growth of this cell line under estradiol (E2), hydroxytamoxifen (OH‐TAM), and both E2 and OH‐TAM treatments. The method extracts characteristic parameters of single nuclei and features that measure the global and local organisation of the cells in their growing phase. Modifications of the heterogeneity of the cell line are emphasised through phenotypic changes and modifications of the spatial organisation of the cells. The hormone (E2) generates a very fast growth of cells with small nuclei that became ER negative in the long term. The antihormone (OH‐TAM) produces a gradual selection of ER negative or poorly positive cells with large nuclei. These modifications are reversed when E2 and OH‐TAM are simultaneously used. Moreover, estradiol induces a permissive context of proliferation, whereas hydroxytamoxifen acts only on some subpopulations. The combination of cell count, cytomorphology, and cell organisation revealed the magnitude of the potential of structuration of hormones or antihormones on in vitro growing cells.