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CONTEXT AND AIMS:Breast cancer prognosis and predictive biomarkers development would allow sparing some patients from chemotherapy or identifying patients for whom chemotherapy would be indicated. In this context, in 2009, the French National Cancer Institute, a National Health and Science Agency dedicated to cancer, in collaboration with the French society of senology and breast pathology (SFSPM) published a report on the assessment of the prognostic and the predictive clinical validity of tissular biomarkers, uPA/PAI-1, Oncotype DX™ and MammaPrint(®), in breast cancer management. They concluded that only the uPA/PAI-1 prognosis value reached the highest level of evidence (LOE I according to Hayes 1998 classification). In 2012, it was decided to update this report since new data have emerged and because information disparities among clinicians have been identified. This article aims to present the main conclusions together with the levels of evidence associated with those conclusions.METHODS:The updating process was based on literature published since 2009 appraisal and on multidisciplinary and independent experts' opinion. The levels of evidence (LOE) used are those of the classification defined by Simon in 2009 (updated Hayes 1998 classification): LOE IA and LOE IB: high level of evidence; LOE IIB and LOE IIC: intermediate level of evidence; LOE IIIC and LOE IV-VD: low level of evidence.CONCLUSIONS:Among patients without lymph-node involvement, uPA/PAI-1, invasion process biomarkers, reach the highest level of evidence for 10 years recurrence free survival prognosis (LOE IA according to Simon). The predictive value to anthracyclins chemotherapy remains to be confirmed. Oncotype DX™ and MammaPrint(®) prognosis and predictive value do not reach the LOE I level. This updating' process confirms the 2009 levels of evidence for all the three biomarkers prognosis value. Besides, concerning Oncotype DX™ and MammaPrint(®), new data do not allow to conclude neither to their complementary clinical information to other clinicopathological existing biomarkers nor to a favorable cost-efficiency ratio in therapeutic decision making and this because of the methodological weakness and uncertainty that are identified in the selected studies. Practically, beyond the prognosis and predictive biomarkers validity, the clinical utility of a new biomarker for chemotherapy indication depends on its clinical added information with regard to validated biomarkers (HR, HER2 and Ki67) and to clinicopathological parameters. Since they are the sole validated biomarkers of the invasion process, uPA/PAI-1 could complete clinical information of other clinicopathological factors and consequently could confer an added clinical value. However, data concerning the impact of this information on chemotherapy clinical indication are lacking.Copyright © 2014. Published by Elsevier Masson SAS., IntroductionDans le cancer du sein, le développement des marqueurs biologiques pronostiques ou prédictifs a pour objectif de mieux identifier les patientes pour lesquelles un traitement par chimiothérapie pourrait être évité ou a contrario indiqué. Dans ce contexte, en 2009, l’Institut national du cancer (INCa), agence sanitaire et scientifique de l’État chargée de coordonner les actions de lutte contre le cancer, avait publié en partenariat avec la Société française de sénologie et de pathologie mammaire un rapport sur l’état des connaissances relatives aux biomarqueurs uPA/PAI-1, Oncotype DX™ et MammaPrint® dans la prise en charge du cancer du sein. Ce rapport avait montré que seule la valeur pronostique d’uPA/PAI-1 atteignait le plus haut niveau de preuve (LOE I selon la grille de Hayes 1998). En 2012, devant la parution de nouvelles publications et la divergence des messages diffusés sur les signatures moléculaires, il a été décidé d’actualiser le rapport de 2009. Cet article présente les principales conclusions accompagnées de leurs niveaux de preuve.MéthodeLe processus de mise à jour s’est appuyé sur l’analyse des données publiées depuis la recherche bibliographique de 2009, complétée par l’avis d’un groupe de travail multidisciplinaire indépendant. Les niveaux de preuve employés sont ceux de la classification définie par Simon en 2009 (grille de Hayes 1998 après mise à jour) : LOE IA et LOE IB : niveau de preuve élevé ; LOE IIB and LOE IIC : niveau de preuve intermédiaire ; LOE IIIC and LOE IV-VD : niveau de preuve faible.ConclusionsChez les patientes sans envahissement ganglionnaire (pN0), uPA/PAI-1, marqueurs d’invasion, ont un niveau de preuve élevé (LOE IA selon Simon) pour la valeur pronostique de la survie sans récidive à 10 ans. Il reste à confirmer leur valeur prédictive de réponse aux anthracyclines. Pour Oncotype DX™ et MammaPrint®, les valeurs pronostique et prédictive n’ont pas atteint à ce jour le niveau de preuve LOE I. Ce travail confirme les niveaux de preuve précédemment établis dans le rapport de 2009. Par ailleurs, les données ne permettent pas de conclure à une valeur ajoutée de Oncotype DX™ et MammaPrint® par rapport aux outils existants. Les données médico-économiques ne permettent pas de statuer sur le rapport coût/efficacité des stratégies utilisant ces tests dans la décision thérapeutique compte tenu d’un niveau de qualité insuffisant pour la plupart des études et d’une forte incertitude mise en évidence par les quelques études bien menées. En pratique, au-delà des niveaux de preuve attribuables à la valeur pronostique et prédictive d’un biomarqueur, l’utilité clinique d’un nouveau marqueur dans l’aide à la prescription d’une chimiothérapie repose sur sa valeur ajoutée par rapport aux marqueurs validés (RH, HER2 et les marqueurs de prolifération comme Ki67) et aux critères anatomo-cliniques. Puisqu’ils sont les seuls marqueurs validés à témoigner du processus d’invasion, uPA/PAI-1 peuvent apporter une information complémentaire et donc avoir une valeur ajoutée par rapport aux marqueurs existants. Les données de la littérature manquent pour apprécier le poids de cette valeur ajoutée dans la décision de prescrire ou non une chimiothérapie.

The proteome, first formalized in 1995, designs all the proteins expressed by the genome of a cell, tissu, or organ at a defined time. Proteomic analysis leads to a description of the regulation of gene expression by the study of proteins and of their post-translational modifications. Proteomic analysis is based on three technologies: 1) Two-dimensional electrophoresis allowing the separation of thousands of proteins from a single mixture; 2) mass spectrometry allowing the characterization of picoquantities of polypeptides and providing data on post-translational modifications; 3) Bioinformatic which is required for the quantification of protein level and for the constitution of databases of protein expression profiles. Complementing the methods of the genomics, the use of proteomic analysis is widely spreading in the fields of fundamental biology, biomedicine and pharmacology for the identification of new biological markers and therapeutic targets.

The BRCA1 gene modification is responsible for an autosomal dominant syndrome of inherited early onset breast and/or ovarian cancer. This gene is estimated to account for almost half of inherited breast cancers and three quarters of inherited breast/ovarian cancers. This suggests that about 1 out of 500 women may carry BRCA1 mutation. The BRCA1 gene was isolated by positional cloning in 1994. More than 100 different mutations have been found in the germline of affected individuals. We looked by systematic sequencing at BRCA1 germline mutations in 36 patients treated at the Centre Oscar-Lambret for breast and/or ovarian cancer and that belonged to high risk families. We have found 24 mutations: 9 true mutations inducing modifications of the BRCA1 protein (BRCA1+), 5 mutations with unknown consequences on the BRCA1 protein and 10 mutations corresponding to polymorphisms that had been previously described. All the BRCA1+ cases had a HPG3 tumor. The median age of discovery and the receptor positivity percentage are lower in hereditary breast cancer than in the standard population of the breast cancers treated in our center. Consequently, BRCA1 mutations are associated to parameters thought to be of bad prognosis.

We investigated binding characteristics of bFGF in membranes prepared from 4 human breast cancer cell lines (MCF-7, T-47D, BT-20 and MDA-MB-231) and 38 primary breast cancer biopsies. Results of competitive binding experiments were analysed using the "Ligand" program to determine binding site concentrations and affinities. bFGF mitogenic activity was also measured by [3H]-thymidine incorporation into DNA of breast cancer cell lines. The presence of high-affinity binding sites was demonstrated in each cell type (Kd: 0.5 nM). The presence of these high-affinity binding sites was confirmed by saturation experiments. A second class of low-affinity binding sites was detected in the 2 hormono-independent cells (BT-20: Kd = 2.9 nM; MDA-MB-231: Kd = 2.7 nM). bFGF stimulated the proliferation of MCF-7, 7-47D, BT-20 and not of MDA-MB-231 cell lines. In breast cancer biopsies, binding sites were detectable in 36/38 cases; high-affinity binding sites (Kd1 nM) were present in 19/39 cases and low-affinity binding sites (Kd2 nM) were present in 29/36 cases (the 2 classes of binding sites were present in 12 biopsies). No relation between FGF binding sites and node involvement nature or grade of tumor was evidenced. Negative correlations (Spearman test) were found between total bFGF binding site concentrations and estradiol receptor concentrations (P = 0.05) or progesterone receptor concentrations (P = 0.009). The demonstrations of 1), bFGF specific binding sites in breast cancer membranes; and 2) bFGF growth stimulation of some breast cancer cell lines, indicate that this factor could be involved in the growth of most breast cancers, and could act (among other factors) directly on the growth of cancer cells.

The kinetics of intratumoral uptake of cisplatin, carboplatin (CBDCA) and TNO-6 were studied in vitro in fragments of tumors of the head and neck maintained in culture medium under conditions simulating therapeutic conditions of chemo-embolization. Cisplatin and TNO-6 are more rapidly taken up than CBDCA (carboplatin). With each of these compounds, after 24 hours a fraction of the platinum taken up is not irreversibly bound to the tissue. The rate of uptake and the proportion of platinum irreversibly bound depends a great deal on the tumor under study. The characteristics of the diffusion indicate that TNO-6 is better used with chemo-embolization than with systemic injection. The opposite finding is seen with carboplatin. Cisplatin appears to to be about equally well-suited for either method of injection.

The binding characteristics of IGF1 on membranes prepared from 5 human breast cancer cell lines were investigated in detail. The presence of one class of high affinity IGF1 binding sites was demonstrated (BT-20: n = 230 fmol/mg protein, Ka = 0.7 nM-1; MCF-7: n = 124 fmol/mg protein, Ka = 1 nM-1; T-47D: n = 61 fmol/mg protein, Ka = 1.1 nM-1; HBL-100: n = 18 fmol/mg protein, Ka = 3.2 nM-1; MDA-MB-231: n = 7 fmol/mg protein, Ka = 2.8 nM-1). Chemical cross-linking of 125I IGF1 to breast cancer cell membranes then sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed one major band of relative molecular weight 130000. The specificity of these receptors was studied: native or recombinant IGF1 had the same potency to inhibit 125I IGF1 binding; IGF2 was able to compete for binding, whereas insulin competed with a potency lower than 1/100 that of IGF1. These characteristics of IGF1 binding sites in breast cancer cell membranes correspond to the previously described binding unit of type I IGF receptors (IGF1-R). Finally we determined that for a routinely used standard assay, it was necessary to incubate for 5 h at 4 degrees C a high amount of membrane protein (400 micrograms) and 200,000 cpm of tracer. Considering the known effect of IGF1 on breast cancer cell multiplication, it is tempting to suggest that this factor might play a major role in the growth of breast cancer: the measurement of IGF1-R, using this standardized method, will give an assessment of these tumors IGF1 sensitivity; it can be performed on the membrane fraction obtained when preparing cytosol for steroid receptor assay.