Colette Charpin

Gene Expression Profiling Shows Medullary Breast Cancer Is a Subgroup of Basal Breast Cancers

Medullary breast cancer (MBC) is a rare but enigmatic pathologic type of breast cancer. Despite features of aggressiveness, MBC is associated with a favorable prognosis. Morphologic diagnosis remains difficult in many cases. Very little is known about the molecular alterations involved in MBC. Notably, it is not clear whether MBC and ductal breast cancer (DBC) represent molecularly distinct entities and what genes/proteins might account for their differences. Using whole-genome oligonucleotide microarrays, we compared gene expression profiles of 22 MBCs and 44 grade III DBCs. We show that MBCs are less heterogeneous than DBCs. Whereas different molecular subtypes (luminal A, luminal B, basal, ERBB2-overexpressing, and normal-like) exist in DBCs, 95% MBCs display a basal profile, similar to that of basal DBCs. Supervised analysis identified gene expression signatures that discriminated MBCs from DBCs. Discriminator genes are associated with various cellular processes related to MBC features, in particular immune reaction and apoptosis. As compared with MBCs, basal DBCs overexpress genes involved in smooth muscle cell differentiation, suggesting that MBCs are a distinct subgroup of basal breast cancer with limited myoepithelial differentiation. Finally, MBCs overexpress a series of genes located on the 12p13 and 6p21 chromosomal regions known to contain pluripotency genes. Our results contribute to a better understanding of MBC and of mammary oncogenesis in general.

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.

Sixteen-Kinase Gene Expression Identifies Luminal Breast Cancers with Poor Prognosis

Breast cancer is a heterogeneous disease made of various molecular subtypes with different prognosis. However, evolution remains difficult to predict within some subtypes, such as luminal A, and treatment is not as adapted as it should be. Refinement of prognostic classification and identification of new therapeutic targets are needed. Using oligonucleotide microarrays, we profiled 227 breast cancers. We focused our analysis on two major breast cancer subtypes with opposite prognosis, luminal A (n = 80) and basal (n = 58), and on genes encoding protein kinases. Whole-kinome expression separated luminal A and basal tumors. The expression (measured by a kinase score) of 16 genes encoding serine/threonine kinases involved in mitosis distinguished two subgroups of luminal A tumors: Aa, of good prognosis and Ab, of poor prognosis. This classification and its prognostic effect were validated in 276 luminal A cases from three independent series profiled across different microarray platforms. The classification outperformed the current prognostic factors in univariate and multivariate analyses in both training and validation sets. The luminal Ab subgroup, characterized by high mitotic activity compared with luminal Aa tumors, displayed clinical characteristics and a kinase score intermediate between the luminal Aa subgroup and the luminal B subtype, suggesting a continuum in luminal tumors. Some of the mitotic kinases of the signature represent therapeutic targets under investigation. The identification of luminal A cases of poor prognosis should help select appropriate treatment, whereas the identification of a relevant kinase set provides potential targets.

CD105 expression is a marker of high metastatic risk and poor outcome in breast carcinomas. Correlations between immunohistochemical analysis and long-term follow-up in a series of 929 patients.

CD105 (endoglin) is expressed significantly in activated endothelial cells in culture and in tumor microvessels. Quantification of CD105 immunocytochemical expression that may be clinically relevant has not been accurately evaluated. We studied CD105 expression on frozen tissue sections by using immunohistochemical assays in a series of 929 patients and correlated the findings with long-term follow-up (median, 11.3 years). Univariate (Kaplan-Meier) analysis showed that the number of CD105+ microvessels (cutoff, 15 vessels) correlated significantly with poor overall survival among all patients (P = .001). This correlation was less significant in node-negative patients (P = .035). Marked CD105 expression also correlated with a high risk for metastasis among all patients (P = .006) and among node-negative patients (P = .001). Multivariate analysis (Cox model) identified CD105 immunodetection as an independent prognostic indicator. Our results suggest that immunohistochemical expression of CD105 has practical clinical relevance for identifying node-negative patients with a poor prognosis. Moreover, immunodetection of CD105 also may be considered a potential tool for selecting patients who could benefit from specific antiangiogenic therapy, using anti-CD105 conjugates.

Impact of menopausal hormone-replacement therapy on clinical and laboratory characteristics of breast cancer.

Hormone‐replacement therapy (HRT) is widely used by post‐menopausal women. Although this treatment may slightly increase the incidence of breast cancer, more and more cases are diagnosed while women are taking HRT. The purpose of this study was to ascertain the influence of HRT on prognostic factors and outcome of breast cancer. Data on all breast‐cancer patients, including precise information on HRT, was prospectively and systematically recorded in a data base. From 1985 to 1995, 1379 post‐menopausal women fulfilled the eligibility criteria for this study. All were treated by us (P.B. and L.P.) in our ward of a large public hospital of Marseilles, France. The clinical features, laboratory findings and survival rates in 142 HRT users who developed breast cancer while being treated were compared with those of 284 matched never user breast‐cancer patients. Patients who developed breast cancer during HRT had fewer locally advanced cancers and smaller and better‐differentiated cancers. Lymph‐node involvement was significantly less frequent in the user group than in the non‐user group (non‐significant). Estradiol receptivity was both qualitatively and quantitatively lower in users. There was no significant difference with regard to recurrence and metastasis‐free survival and overall survival. We conclude that HRT does not affect the prognosis of breast cancer. Regular surveillance during HRT allows early detection of smaller lesions. The higher number of well‐differentiated cancers and the distribution of hormone receptivity may reflect interaction between neoplastic tissue and exogenous hormones. Int. J. Cancer (Pred. Oncol.) 79:278–282, 1998.© 1998 Wiley‐Liss, Inc.

E‐cadherin quantititive immunocytochemical assays in breast carcinomas

The reduction of E‐cadherin expression, which is involved in the initial step of invasion and metastasis of cancer, was investigated in 218 human breast carcinomas. Quantitative immunohistochemical assays (ICAs) were performed on frozen sections. Quantitation was assessed by processing digitized microscopic images of immunoreactions using a computerized system of image analysis (SAMBA). The results were correlated with clinicopathological data and quantitative immunodetection of other molecules. E‐cadherin expression was significantly (P<0·001) stronger in ductal carcinomas than in lobular carcinomas and stronger (P<0·01) in low grades than in high grades, but E‐cadherin was independent of lymph node status and tumour size. Also an inverse significant (P<0·01) relationship was observed between E‐cadherin expression on tissue sections and positive immunoreactions with anti‐P53, MIB1 (growth fraction), and anti‐c‐erb‐B2 product. Conversely, strong positive and anti‐E‐cadherin immunoreactions correlated with strong positive anti‐ER and anti‐PR immunoreactions (P<0·01). No relationship was observed between E‐cadherin and the results of quantitative ICAs of cathepsin D, CD31, and P‐glycoprotein, assessed on consecutive sections from the same frozen tissue samples. The results show that preserved E‐cadherin expression correlates with high degree of tumour differentiation, low proliferative activity, and low expression of prognostic markers. The deregulation of E‐cadherin is independent of other steps of tumour invasion, such as protease digestion of extracellular matrix and angiogenesis.

A signature predictive of disease outcome in breast carcinomas, identified by quantitative immunocytochemical assays

Quantitative immunocytochemical assays of 1,200 breast carcinomas were assessed after construction of tissue microarrays. A total of 42 markers were evaluated for prognostic significance by univariate log rank test (mean follow‐up, 79 months), using quantitative scoring by an image analysis device and specific software. Complete data were obtained for 924 patients, for whom 27 of the 42 markers proved to be significant prognostic indicators. Analysis of these 27 markers by logistic regression showed that 18 (cMet, CD44v6, FAK, moesin, caveolin, c‐Kit, CK14, CD10, P21, P27, pMAPK, pSTAT3, STAT1, SHARP2, FYN, ER, PgR and c‐erb B2), and 15 when ER, PgR and c‐erb B2 were excluded, were 80.52% and 78.9% predictive of disease outcome, respectively. The immunocytochemical assays on 4 micron thick sections of fixed tissue are easy to handle in current practice and are cost‐effective. Quantitative densitometric measurement of immunoprecipitates by computer‐assisted devices from digitized microscopic images allows standardized high‐throughput “in situ” molecular profiling within tumors. It is concluded that this 15 marker immunohistochemical signature is suitable for current practice, since performed on paraffin sections of fixed tumor samples, and can be used to select patients needing more aggressive therapy, since this signature is about 80% predictive of poor clinical outcome. Also, the markers included in the signature may be indicative of tumor responsiveness to current chemotherapy or suggest new targets for specific therapies.

Tumor Neoangiogenesis by CD31 and CD105 Expression Evaluation in Breast Carcinoma Tissue Microarrays

Purpose: The aim of the study was to evaluate CD31 and CD105 immunohistochemical expressions in tissue microarrays from 360 breast carcinomas. Study design: Computerized (ACIS/Chromavision) assisted image analysis was performed to compare immunoreactions in tissue microarrays with those in current paraffin and frozen sections. We also aimed to determine the CD105 and CD31 prognostic significance and relevance in routine practice by correlating results of immunodetections with patients’ (n = 360) outcome (14.3-year follow-up). Results: The results show (a) that in tissue microarrays, the CD31 and CD105 expression quantified by image analysis device did not correlate with the measurements assessed on routine paraffin sections; (b) that CD105 expression is endowed of a prognostic significance in paraffin sections in terms of overall survival (P < 0.01), whereas in contrast, CD31 on paraffin sections did not correlate with patients overall survival; (c) that semiquantitative analysis of CD105 expression correlated with the image analysis measurements in frozen sections (ρ = 0.671, P < 0.01) and paraffin (ρ = 0.824, P < 0.01) sections. However, paraffin sections were less immunostained than frozen ones. Conclusions: It is concluded (a) that CD105 may be suitable in paraffin sections to evaluated neoangiogenesis; and (b) that tissue microarrays are not suitable substrates for neoangiogenesis evaluation as a prognostic indicator in breast carcinomas, in contrast to current tissue sections.

High-Resolution Comparative Genomic Hybridization of Inflammatory Breast Cancer and Identification of Candidate Genes

Background Inflammatory breast cancer (IBC) is an aggressive form of BC poorly defined at the molecular level. We compared the molecular portraits of 63 IBC and 134 non-IBC (nIBC) clinical samples. Methodology/Findings Genomic imbalances of 49 IBCs and 124 nIBCs were determined using high-resolution array-comparative genomic hybridization, and mRNA expression profiles of 197 samples using whole-genome microarrays. Genomic profiles of IBCs were as heterogeneous as those of nIBCs, and globally relatively close. However, IBCs showed more frequent “complex” patterns and a higher percentage of genes with CNAs per sample. The number of altered regions was similar in both types, although some regions were altered more frequently and/or with higher amplitude in IBCs. Many genes were similarly altered in both types; however, more genes displayed recurrent amplifications in IBCs. The percentage of genes whose mRNA expression correlated with CNAs was similar in both types for the gained genes, but ∼7-fold lower in IBCs for the lost genes. Integrated analysis identified 24 potential candidate IBC-specific genes. Their combined expression accurately distinguished IBCs and nIBCS in an independent validation set, and retained an independent prognostic value in a series of 1,781 nIBCs, reinforcing the hypothesis for a link with IBC aggressiveness. Consistent with the hyperproliferative and invasive phenotype of IBC these genes are notably involved in protein translation, cell cycle, RNA processing and transcription, metabolism, and cell migration. Conclusions Our results suggest a higher genomic instability of IBC. We established the first repertory of DNA copy number alterations in this tumor, and provided a list of genes that may contribute to its aggressiveness and represent novel therapeutic targets.

Hypoxia inducible factor 1α gene (HIF-1α) splice variants: potential prognostic biomarkers in breast cancer

BackgroundHypoxia-inducible factor 1 (HIF-1) is a master transcriptional regulator of genes regulating oxygen homeostasis. The HIF-1 protein is composed of two HIF-1α and HIF-1β/aryl hydrocarbon receptor nuclear translocator (ARNT) subunits. The prognostic relevance of HIF-1α protein overexpression has been shown in breast cancer. The impact of HIF-1α alternative splice variant expression on breast cancer prognosis in terms of metastasis risk is not well known.MethodsUsing real-time quantitative reverse transcription PCR assays, we measured mRNA concentrations of total HIF-1α and 4 variants in breast tissue specimens in a series of 29 normal tissues or benign lesions (normal/benign) and 53 primary carcinomas. In breast cancers HIF-1α splice variant levels were compared to clinicopathological parameters including tumour microvessel density and metastasis-free survival.ResultsHIF-1α isoforms containing a three base pairs TAG insertion between exon 1 and exon 2 (designated HIF-1αTAG) and HIF-1α736mRNAs were found expressed at higher levels in oestrogen receptor (OR)-negative carcinomas compared to normal/benign tissues (P = 0.009 and P = 0.004 respectively). In breast carcinoma specimens, lymph node status was significantly associated with HIF-1αTAGmRNA levels (P = 0.037). Significant statistical association was found between tumour grade and HIF-1αTAG(P = 0.048), and total HIF-1α (P = 0.048) mRNA levels. HIF-1αTAGmRNA levels were also inversely correlated with both oestrogen and progesterone receptor status (P = 0.005 and P = 0.033 respectively). Univariate analysis showed that high HIF-1αTAGmRNA levels correlated with shortened metastasis free survival (P = 0.01).ConclusionsOur results show for the first time that mRNA expression of a HIF-1αTAGsplice variant reflects a stage of breast cancer progression and is associated with a worse prognosis.See commentary: http://www.biomedcentral.com/1741-7015/8/45