Validation of tumor-associated macrophage ferritin light chain as a prognostic biomarker in node-negative breast cancer tumors: A multicentric 2004 national PHRC study.

Novel prognostic biomarkers are imperatively needed to help direct treatment decisions by typing subgroups of node-negative breast cancer patients. Large screening of different biological compartments, such as the proteome, by means of high throughput techniques may greatly help scientists to find such markers. The present retrospective multicentric study included 268 node-negative breast cancer patients. We used a proteomic approach of SELDI-TOF-MS screening to identify differentially expressed cytosolic proteins with prognostic impact. The screening cohort was composed of 198 patients. Seventy supplementary patients were included for validation. Immunohistochemistry (IHC) and immunoassay (IA) were run to confirm the prognostic role of the marker identified by SELDI-TOF-MS screening. IHC was also used to explore links between selected marker and epithelial-mesenchymal transition (EMT)-like, proliferation and macrophage markers. Ferritin light chain (FTL) was identified as an independent prognostic marker (HR = 1.30-95% CI: 1.10-1.50, p = 0.001). Validation step by means of IHC and IA confirmed the prognostic value of FTL level. CD68 IHC showed that FTL was stored in tumor-associated macrophages (TAM), which exhibit an M2-like phenotype. We report here, first, the validation of FTL as a breast tumor prognostic biomarker in node-negative patients, and second, the fact that FTL is stored in TAM.

bc-GenExMiner: an easy-to-use online platform for gene prognostic analyses in breast cancer.

Gene prognostic meta-analyses should benefit from breast tumour genomic data obtained during the last decade. The aim was to develop a user-friendly, web-based application, based on DNA microarrays results, called "breast cancer Gene-Expression Miner" (bc-GenExMiner) to improve gene prognostic analysis performance by using the same bioinformatics process. bc-GenExMiner was developed as a web-based tool including a MySQL relational database. Survival analyses are performed with R statistical software and packages. Molecular subtyping was performed by means of three single sample predictors (SSPs) and three subtype clustering models (SCMs). Twenty-one public data sets have been included. Among the 3,414 recovered breast cancer patients, 1,209 experienced a pejorative event. Molecular subtyping by means of three SSPs and three SCMs was performed for 3,063 patients. Furthermore, three robust lists of stable subtyped patients were built to maximize reliability of molecular assignment. Gene prognostic analyses are done by means of univariate Cox proportional hazards model and may be conducted on cohorts split by nodal (N), oestrogen receptor (ER), or molecular subtype status. To evaluate independent prognostic impact of genes relative to Nottingham Prognostic Index and Adjuvant! Online, adjusted Cox proportional hazards models are performed. bc-GenExMiner allows researchers without specific computation skills to easily and quickly evaluate the in vivo prognostic role of genes in breast cancer by means of Cox proportional hazards model on large pooled cohorts, which may be split according to different prognostic parameters: N, ER, and molecular subtype. Prognostic analyses by molecular subtype may also be performed in three robust molecular subtype classifications.

A 38-gene expression signature to predict metastasis risk in node-positive breast cancer after systemic adjuvant chemotherapy: a genomic substudy of PACS01 clinical trial.

Currently, no prognostic gene-expression signature (GES) established from node-positive breast cancer cohorts, able to predict evolution after systemic adjuvant chemotherapy, exists. Gene-expression profiles of 252 node-positive breast cancer patients (median follow-up: 7.7 years), mostly included in a randomized clinical trial (PACS01), receiving systemic adjuvant regimen, were determined by means of cDNA custom array. In the training cohort, we established a GES composed of 38 genes (38-GES) for the purpose of predicting metastasis-free survival. The 38-GES yielded unadjusted hazard ratio (HR) of 4.86 (95% confidence interval = 2.76-8.56). Even when adjusted with the best two clinicopathological prognostic indexes: Nottingham prognostic index (NPI) and Adjuvant!, 38-GES HRs were 3.30 (1.81-5.99) and 3.40 (1.85-6.24), respectively. Furthermore, 38-GES improved NPI and Adjuvant! classification. In particular, NPI intermediate-risk patients were divided into 2/3 close to low-risk group and 1/3 close to high-risk group (HR = 6.97 [2.51-19.36]). Similarly, Adjuvant! intermediate-risk patients were divided into 2/3 close to low-risk group and 1/3 close to high-risk group (HR = 4.34 [1.64-11.48]). The 38-GES was validated on gene-expression datasets from three external node-positive breast cancer subcohorts (n = 224) generated from different microarray platforms, with HR = 2.95 (1.74-5.01). Moreover, 38-GES showed prognostic performance in supplementary cohorts with different lymph-node status and endpoints (1,040 new patients). The 38-GES represents a robust tool able to type systemic adjuvant treated node-positive patients at high risk of metastatic relapse, and is especially powerful to refine NPI and Adjuvant! classification for those patients.

Molecular screening of interleukin-6 gene promoter and influence of -174G/C polymorphism on breast cancer.

Interleukin-6 (IL-6) is a cytokine involved in different physiologic and pathophysiologic processes including carcinogenesis. In 2003, a single nucleotide polymorphism (-174G/C) of the IL-6 gene promoter has been linked to breast cancer prognosis in node-positive (N+) breast cancer patients. Since, different studies have led to conflicting conclusions about its role as a prognostic and/or diagnostic marker. The primary aim of our study was to investigate the link between -174G/C polymorphism and breast cancer risk on the one hand, and -174G/C polymorphism and prognosis in different groups of patients: sporadic N+breast cancers (n=138), sporadic N- breast cancers (n=95) and familial breast cancer (n=60) on the other hand. The variables of interest were disease-free survival and overall survival. The secondary aim of the study was to screen IL-6 gene promoter using direct sequencing to identify new polymorphisms in our French Caucasian breast cancer population. No association or trend of association between -174G/C polymorphism of IL-6 gene promoter gene and breast cancer diagnosis or prognosis was shown, even in meta-analyses. Furthermore, we have identified four novel polymorphic sites in the IL-6 gene promoter region: -764G-->A, -757C-->T, -233T-->A, 15C-->A.

Pooled analysis of prognostic impact of urokinase-type plasminogen activator and its inhibitor PAI-1 in 8377 breast cancer patients.

BACKGROUND: Urokinase-type plasminogen activator (uPA) and its inhibitor (PAI-1) play essential roles in tumor invasion and metastasis. High levels of both uPA and PAI-1 are associated with poor prognosis in breast cancer patients. To confirm the prognostic value of uPA and PAI-1 in primary breast cancer, we reanalyzed individual patient data provided by members of the European Organization for Research and Treatment of Cancer-Receptor and Biomarker Group (EORTC-RBG). METHODS: The study included 18 datasets involving 8377 breast cancer patients. During follow-up (median 79 months), 35% of the patients relapsed and 27% died. Levels of uPA and PAI-1 in tumor tissue extracts were determined by different immunoassays; values were ranked within each dataset and divided by the number of patients in that dataset to produce fractional ranks that could be compared directly across datasets. Associations of ranks of uPA and PAI-1 levels with relapse-free survival (RFS) and overall survival (OS) were analyzed by Cox multivariable regression analysis stratified by dataset, including the following traditional prognostic variables: age, menopausal status, lymph node status, tumor size, histologic grade, and steroid hormone-receptor status. All P values were two-sided. RESULTS: Apart from lymph node status, high levels of uPA and PAI-1 were the strongest predictors of both poor RFS and poor OS in the analyses of all patients. Moreover, in both lymph node-positive and lymph node-negative patients, higher uPA and PAI-1 values were independently associated with poor RFS and poor OS. For (untreated) lymph node-negative patients in particular, uPA and PAI-1 included together showed strong prognostic ability (all P<.001). CONCLUSIONS: This pooled analysis of the EORTC-RBG datasets confirmed the strong and independent prognostic value of uPA and PAI-1 in primary breast cancer. For patients with lymph node-negative breast cancer, uPA and PAI-1 measurements in primary tumors may be especially useful for designing individualized treatment strategies.

Pooled analysis of prognostic impact of uPA and PAI-1 in breast cancer patients.

In this report we present an extension of the pooled analysis of the prognostic impact of urokinase-type plasminogen activator (uPA) and its inhibitor PAI-I in breast cancer patients. We analyzed a different endpoint, metastasis-free survival (MFS). We checked the consistency of the estimates for uPA and PAI-1 for relapse-free survival (RFS) and MFS exploring possible sources of heterogeneity. Nodal status, the most important prognostic factor for breast cancer, introduced heterogeneity in the uPA/PAI-1 survival analyses, reflecting the interaction between nodal status and uPA/PAI-1. The estimates for uPA and PAI-1 were found to be consistent, even when a different transformation of their values was used. The heterogeneity of the separate data sets decreased if the levels of uPA and PAI-1 were ranked, data sets were pooled, and the analyses corrected for the base model that included all traditional prognostic factors, and stratified by data set. We conclude that uPA and PAI-1 are ready to be used in the clinic to help classify breast cancer patients into high and low risk groups.

bc-GenExMiner 3.0: new mining module computes breast cancer gene expression correlation analyses.

We recently developed a user-friendly web-based application called bc-GenExMiner (http://bcgenex.centregauducheau.fr), which offered the possibility to evaluate prognostic informativity of genes in breast cancer by means of a 'prognostic module'. In this study, we develop a new module called 'correlation module', which includes three kinds of gene expression correlation analyses. The first one computes correlation coefficient between 2 or more (up to 10) chosen genes. The second one produces two lists of genes that are most correlated (positively and negatively) to a 'tested' gene. A gene ontology (GO) mining function is also proposed to explore GO 'biological process', 'molecular function' and 'cellular component' terms enrichment for the output lists of most correlated genes. The third one explores gene expression correlation between the 15 telomeric and 15 centromeric genes surrounding a 'tested' gene. These correlation analyses can be performed in different groups of patients: all patients (without any subtyping), in molecular subtypes (basal-like, HER2+, luminal A and luminal B) and according to oestrogen receptor status. Validation tests based on published data showed that these automatized analyses lead to results consistent with studies' conclusions. In brief, this new module has been developed to help basic researchers explore molecular mechanisms of breast cancer. DATABASE URL: http://bcgenex.centregauducheau.fr

Identification of potential prognostic biomarkers for node-negative breast tumours by proteomic analysis: a multicentric 2004 national PHRC study.

We used a 2D-electrophoresis (2-DE) proteomic approach to identify novel biomarkers in node-negative breast cancers. This retrospective study focused on a population of patients with ductal pN0M0 tumours. A subset of patients who developed metastases and in whose tumours were found high levels of uPA and PAI-1 (metastatic relapse, MR: n=20) were compared to another subset in whom no metastatic relapse occurred and whose tumours were found to have low levels of uPA and PAI-1 (no relapse, NR: n=21). We used a 2-DE coupled with MS approach to screen cytosol fractions using two pH-gradient scales, a broad scale (3.0-11.0) and a narrower scale focussing in on a protein rich region (5.0-8.0). This study was conducted on 41 cytosol specimens analyzed in duplicate on two platforms. The differential analysis of more than 2,000 spots in 2-DE gels, obtained on the two platforms, allowed the identification of 13 proteins which were confirmed by western blotting. Two proteins, GPDA and FABP4 were down-regulated in the MR subset whereas all the others were up-regulated. An in silico analysis revealed that GMPS (GUAA), GAPDH (G3P), CFL1 (COF1) and FTL (FRIL), the most informative genes, displayed a proliferation profile (high expression in basal-like, HER2+ and luminal B molecular subtypes). Inversely, similar to FABP4, GPD1 [GPDA] displayed a high expression in luminal A subtype, a profile characteristic of tumour suppressor genes. Despite the small size of our cohort, the 2-DE analysis gave interesting results which were confirmed by the in silico analysis showing that some of the corresponding genes had a strong prognostic impact in breast cancer, mostly because of their link with proliferation: GMPS, GAPDH, FTL and GPD1. A validation phase on a larger cohort is now needed before these biomarkers could be considered for use in clinical practice.

Surface-enhanced laser desorption/ionization time of flight mass spectrometry protein profiling identifies ubiquitin and ferritin light chain as prognostic biomarkers in node-negative breast cancer tumors.

Novel prognostic biomarkers are imperatively needed to help direct treatment decisions by typing subgroups of node-negative breast cancer patients. The current study has used a proteomic approach of SELDI-TOF-MS screening to identify differentially cytosolic expressed proteins with a prognostic impact in 30 node-negative breast cancer patients with no relapse versus 30 patients with metastatic relapse. The data analysis took into account 73 peaks, among which 2 proved, by means of univariate Cox regression, to have a good cumulative prognostic-informative power. Repeated random sampling (n = 500) was performed to ensure the reliability of the peaks. Optimized thresholds were then computed to use both peaks as risk factors and, adding them to the St. Gallen ones, improve the prognostic classification of node-negative breast cancer patients. Identification of ubiquitin and ferritin light chain (FLC), corresponding to the two peaks of interest, was obtained using ProteinChip LDI-Qq-TOF-MS. Differential expression of the two proteins was further confirmed by Western blotting analyses and immunohistochemistry. SELDI-TOF-MS protein profiling clearly showed that a high level of cytosolic ubiquitin and/or a low level of FLC were associated with a good prognosis in breast cancer.