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Purpose: Neoadjuvant systemic therapy (NAC) is currently used in the treatment of stage II/III breast cancer. Pathological complete response as a surrogate endpoint for clinical outcomes is not completely validated for all subgroups of breast cancers. Therefore, there is a need for reliable predictive tests of the most effective treatment., Methods: We used a combination of predictive clinical, pathological, and gene expression-based markers of response to NAC in a prospective phase II multicentre randomized clinical trial in breast cancer patients, with a long follow-up (8 years). This study concerned the subpopulation of 188 patients with similar levels of pathological response rates to sequential epirubicin/cyclophosphamide and docetaxel to determine predictive marker of pCR and DFS. We used a set of 45 genes selected from high throughput analysis and a standardized RT-qPCR. We analyzed the predictive markers of pathological complete response (pCR) and DFS in the overall population and DFS the subpopulation of 159 patients with no pCR., Results: In the overall population, combining both clinical and genomic variables, large tumor size, low TFF1, and MYBL2 overexpression were significantly associated with pCR. T4 Stage, lymphovascular invasion, negative PR status, histological type, and high values of CCNB1 were associated with DFS. In the no pCR population, only lymphovascular invasion and high values of BIRC5 were associated with DFS., Conclusions: We confirm the importance of ER-related and proliferation genes in the prediction of pCR in NAC-treated breast cancer patients. Furthermore, we identified BIRC5 (survivin) as a main pejorative prognostic factor in patients with breast cancers with no pCR. These results also open perspective for predictive markers of new targeted therapies.

Several publications have suggested that histone deacetylase inhibitors (HDACis) could reverse the repression of estrogen receptor alpha (ERα) in triple-negative breast cancer (TNBC) cell lines, leading to the induction of a functional protein. Using different HDACis, vorinostat, panobinostat, and abexinostat, we therefore investigated this hypothesis in various human TNBC cell lines and patient-derived xenografts (PDXs). We used three human TNBC cell lines and three PDXs. We analyzed the in vitro toxicity of the compounds, their effects on the hormone receptors and hormone-related genes and protein expression both in vitro and in vivo models. We then explored intra-tumor histone H3 acetylation under abexinostat in xenograft models. Despite major cytotoxicity of all tested HDAC inhibitors and repression of deactylation-dependent CCND1 gene, neither ERα nor ERβ, ESR1 or ESR2 genes respectively, were re-expressed in vitro. In vivo, after administration of abexinostat for three consecutive days, we did not observe any induction of ESR1 or ESR1-related genes and ERα protein expression by RT-qPCR and immunohistochemical methods in PDXs. This observation was concomitant to the fact that in vivo administration of abexinostat increased intra-tumor histone H3 acetylation. These observations do not allow us to confirm previous studies which suggested that HDACis are able to convert ER-negative (ER-) tumors to ER-positive (ER+) tumors, and that a combination of HDAC inhibitors and hormone therapy could be proposed in the management of TNBC patients.

The purpose of this study was to investigate, in the context of a prospective node-positive-breast cancer trial HER2 containing-regimen (UNICANCER-PACS 04 trial), the predictive value of HER2, FCGRIIA, and FCGRIIIA gene polymorphisms for cardiac toxicity and efficacy of trastuzumab. We analyzed HER2-I655V, FCGR2A-H131R, and FCGR3A-V158F single nucleotide polymorphisms in patients in adjuvant setting treated by six courses of either fluorouracil 500 mg/m(2), epirubicin 100 mg/m(2) and cyclophosphamide 500 mg/m(2), or epirubicin 75 mg/m(2) and docetaxel 75 mg/m(2) every 3 weeks then randomly assigned, in case of HER2 overexpressing tumor, to either trastuzumab for 1 year or nothing. Left ventricular ejection fraction and clinical examination were monitored in each patient, seven times throughout the study to detect congestive heart failure or asymptomatic subclinical cardiac toxicity. All genotypes were analyzed in relation to cardiac toxicity, EFS, and OS. One hundred and thirty-two HER2-positive breast cancer patients were analyzed. The HER2-I655V genotype was significantly associated with cardiac toxicity (p = 0.025). The FCGR2A-131 H/H genotype was significantly correlated with a shorter EFS (p = 0.027). The FCGR3A-158 V/V genotype was not correlated with EFS nor OS. These results might be useful in making a treatment choice of HER2 blockers in adjuvant setting by with an increase in efficacy and decrease in toxicity.

Resistance to endocrine therapy is a major complication of luminal breast cancer and studies of the biological features of hormonal resistance are limited by the lack of adequate preclinical models. The aim of this study is to establish and characterize a panel of primary human luminal breast carcinoma xenografts, and to evaluate their response to endocrine therapies. Four hundred and twenty-three tumor fragments obtained directly from patients have been grafted in the interscapular fatpad of Swiss nude mice. After stable engraftment with estradiol supplementation, xenografted tumors have been validated by conventional pathology and immunohistochemistry examination, and additional molecular studies. In vivo tumor growth and response to different endocrine treatments were evaluated. We have engrafted 423 tumors including 314 ER+ tumors, and 8 new luminal breast cancer xenografts have been obtained (2.5%). Tumor take was much lower for luminal tumors than for non-luminal tumors (2.5 vs. 24.7%, P < 0.0001), and was associated with two independent criteria, i.e., ER status (P < 0.0001) and a high grade tumor (P = 0.05). Histological and immunohistochemical analyses performed on patient's tumors and xenografts showed striking similarities in the tumor morphology as well as in the expression level of ER, PR, and HER2. Response to hormone therapy, evaluated in 6 luminal models, showed different sensitivities, thus exhibiting heterogeneity similar to what is observed in the clinic. We have established a panel of primary human luminal breast cancer xenografts, recapitulating the biological and clinical behaviors of patient tumors, and therefore suitable for further preclinical experiments.

Clinicians can use biomarkers to guide therapeutic decisions in estrogen receptor positive (ER+) breast cancer. One such biomarker is cellular proliferation as evaluated by Ki-67. This biomarker has been extensively studied and is easily assayed by histopathologists but it is not currently accepted as a standard. This review focuses on its prognostic and predictive value, and on methodological considerations for its measurement and the cut-points used for treatment decision. Data describing study design, patients' characteristics, methods used and results were extracted from papers published between January 1990 and July 2010. In addition, the studies were assessed using the REMARK tool. Ki-67 is an independent prognostic factor for disease-free survival (HR 1.05-1.72) in multivariate analyses studies using samples from randomized clinical trials with secondary central analysis of the biomarker. The level of evidence (LOE) was judged to be I-B with the recently revised definition of Simon. However, standardization of the techniques and scoring methods are needed for the integration of this biomarker in everyday practice. Ki-67 was not found to be predictive for long-term follow-up after chemotherapy. Nevertheless, high KI-67 was found to be associated with immediate pathological complete response in the neoadjuvant setting, with an LOE of II-B. The REMARK score improved over time (with a range of 6-13/20 vs. 10-18/20, before and after 2005, respectively). KI-67 could be considered as a prognostic biomarker for therapeutic decision. It is assessed with a simple assay that could be standardized. However, international guidelines are needed for routine clinical use.

Seven fulvestrant resistant cell lines derived from the estrogen receptor alpha positive MCF-7 human breast cancer cell line were used to investigate the importance of epidermal growth factor receptor (ErbB1-4) signaling. We found an increase in mRNA expression of EGFR and the ErbB3/ErbB4 ligand heregulin2 (hrg2) and a decrease of ErbB4 in all resistant cell lines. Western analyses confirmed the upregulation of EGFR and hrg2 and the downregulation of ErbB4. Elevated activation of EGFR and ErbB3 was seen in all resistant cell lines and the ErbB3 activation occurred by an autocrine mechanism. ErbB4 activation was observed only in the parental MCF-7 cells. The downstream kinases pAkt and pErk were increased in five of seven and in all seven resistant cell lines, respectively. Treatment with the EGFR inhibitor gefitinib preferentially inhibited growth and reduced the S phase fraction in the resistant cell lines concomitant with inhibition of Erk and unaltered Akt activation. In concert, inhibition of Erk with U0126 preferentially reduced growth of resistant cell lines. Treatment with ErbB3 neutralizing antibodies inhibited ErbB3 activation and resulted in a modest but statistically significant growth inhibition of two resistant cell lines. These data indicate that ligand activated ErbB3 and EGFR, and Erk signaling play important roles in fulvestrant resistant cell growth. Furthermore, the decreased level of ErbB4 in resistant cells may facilitate heterodimerization of ErbB3 with EGFR and ErbB2. Our data support that a concerted action against EGFR, ErbB2 and ErbB3 may be required to obtain complete growth suppression of fulvestrant resistant cells.

Breast cancer is one of the most frequently diagnosed cancers in women in France, but its aetiology remains unknown. Viruses including Epstein-Barr virus (EBV), a human equivalent of murine mammary tumour virus (MMTV) and human papillomavirus (HPV) have been detected in benign breast tissues and breast tumours and are considered to be involved in the aetiology of breast cancer. The aim of this study was to determine the prevalence of high-risk oncogenic HPVs in breast carcinoma from French patients. Fifty unselected DNA samples extracted from invasive breast carcinoma tissues were analysed. Polymerase chain reaction (PCR) was performed to amplify both consensus GP5+/GP6+HPV sequences and specific sequences for HPV types 16, 18, 33, 45 and 6-11. No HPV-DNA sequences were detected in our series of 50 samples. These data argue against the role of oncogenic HPV in the pathogenesis of breast cancer.

The expression of estrogen (ER) and progesterone (PgR) receptors was analyzed in a retrospective series of 3000 patients who had operable primary breast cancer. Patients were stratified according to ER and PgR status and the study was focused on the two groups (ER-PgR+ and ER-PgR-) of patients whose tumors contained low levels of ER (< 15 fmol/mg protein), regarding potential response to endocrine therapy. The comparison of clinical or histological characteristics between ER-PgR+ and ER-PgR- patients was analyzed as well as the disease-related death and survival. The mean follow-up was 86.3 months. Among the 529 ER-patients, 62 were PgR+ (12%), whereas 467 were PgR- (88%). The ER-PgR+ and ER-PgR- populations represented 2% and 15.6% of the overall population, respectively. In ER- tumors, the PgR status was significantly related to: age, menopausal status, tumor size, SBR grade, and histological type, but not to the type of surgical treatment or to lymph node involvement. ER-PgR+ tumors had smaller size (64% T1 vs 43%) (p=0.004) and were more frequently grade I (28% vs 12%) than ER-PgR- tumors (p < 0.001). In addition, the patients with ER-PgR+ tumors were significantly younger (49.4 years vs 58.4 years; p < 0.0001), and were more frequently premenopausal (76% vs 36%, p < 0.001). The disease-free interval and the metastasis-free survival tended to be worse for ER-PgR- than for ER-PgR+ patients, but the difference was not statistically significant at 10 years. However, a small but significant difference in overall survival, in favor of the PgR+ group, was observed between the two groups during the first 5 years (p=0.03). We conclude that in combination with ER, PgR status defines a group of patients with clinical and biological specificity, which could be considered for specific endocrine therapy.

The role of p53 in modulating apoptosis has suggested that it may affect efficacy of anti cancer agents. For this reason, we have evaluated p53 alterations in 282 consecutive patients with infiltrating node-negative breast cancer who underwent primary surgery and were randomized either to CMF (Cyclophosphamide 400 mg/m2, Fluorouracil 400 mg/m2, and Methotrexate 40 mg/m2) or control arm (no adjuvant therapy) from 1980 to 1989. p53 alterations were analyzed by immunohistochemistry using DO7 MoAb, revealed by immunoperoxidase technique, and quantitated in term of percentage of positive cells. We observed a positive staining in 24% of the tumors. Among them, 10% had a positive staining in more than 75% of the cells. There was a highly significant association between the proportion of positive cells and histologic grade of the infiltrating ductal carcinomas (p<0.004). However, there was no association with age, tumor size, hormone receptor content, or vascular embolism. There was a trend but no significant relationship between positive staining and overall survival either in each arm of the trial or in the overall population. Interestingly, we observed a higher relative risk of local relapse after conservative therapy in the boosted area in the group of mutated p53 (RR=4.41; p<0.0005). We conclude that, in this node-negative breast tumor population, alteration of p53 cannot predict the response to the chemotherapy. However, it may represent a useful marker of risk of local relapse and of radio resistance.

The Tn determinant (GalNAc alpha-O-Ser/Thr) is expressed by about 90% of human carcinomas, but is cryptic in most normal human tissues. A murine monoclonal antibody (MAb) 83D4, developed following immunization with human breast carcinoma cells, reacts with a Tn-related epitope. In the present study we characterized the glycoprotein antigen identified by 83D4 in the human breast carcinoma cell line MCF-7. We further showed that the 83D4 antigenic determinant is masked in human milk fat globule membranes (HMFGM), and can be exposed upon mild m-periodate treatment after desialylation. Western-blot analysis resolved the 83D4 antigen from MCF-7 into two main components of 120-190 kD and > 500 kD respectively. Non equilibrium pH gradient electrophoresis/SDS PAGE revealed the acidic nature of the reactive glycoproteins (pI 4.43-4.70). 83D4 antigenic activity resolved by CsCl gradient ultracentrifugation layered on a wide range of densities (1.30-1.46 g/ml) including typical densities of mucin-like glycoproteins but also lower densities. The amino acid composition of the antigen, relatively rich in serine but poor in threonine and proline, confirmed the divergence from other mucin-like carcinoma-associated glycoproteins. Dicarboxylic amino acids were abundant, accounting in part for the acidic nature of the molecules. ELISA and Western-blot analysis of the subcellular fractions from MCF-7 cells revealed that the 83D4 antigen is mainly contained in plasma membranes (85%) from which it may be resolved into two broad bands (slow and fast migrating components). These results provide information on a group of breast carcinoma associated glycoproteins related to but different from typical mucins, and provide data on alteration of O-glycosylation in tumor cells.