Véronique Becette

Identification of molecular apocrine breast tumours by microarray analysis

Previous microarray studies on breast cancer identified multiple tumour classes, of which the most prominent, named luminal and basal, differ in expression of the oestrogen receptor α gene (ER). We report here the identification of a group of breast tumours with increased androgen signalling and a ‘molecular apocrine’ gene expression profile. Tumour samples from 49 patients with large operable or locally advanced breast cancers were tested on Affymetrix U133A gene expression microarrays. Principal components analysis and hierarchical clustering split the tumours into three groups: basal, luminal and a group we call molecular apocrine. All of the molecular apocrine tumours have strong apocrine features on histological examination (P=0.0002). The molecular apocrine group is androgen receptor (AR) positive and contains all of the ER-negative tumours outside the basal group. Kolmogorov–Smirnov testing indicates that oestrogen signalling is most active in the luminal group, and androgen signalling is most active in the molecular apocrine group. ERBB2 amplification is commoner in the molecular apocrine than the other groups. Genes that best split the three groups were identified by Wilcoxon test. Correlation of the average expression profile of these genes in our data with the expression profile of individual tumours in four published breast cancer studies suggest that molecular apocrine tumours represent 8–14% of tumours in these studies. Our data show that it is possible with microarray data to divide mammary tumour cells into three groups based on steroid receptor activity: luminal (ER+ AR+), basal (ER− AR−) and molecular apocrine (ER− AR+).

A stroma-related gene signature predicts resistance to neoadjuvant chemotherapy in breast cancer

To better understand the relationship between tumor-host interactions and the efficacy of chemotherapy, we have developed an analytical approach to quantify several biological processes observed in gene expression data sets. We tested the approach on tumor biopsies from individuals with estrogen receptor–negative breast cancer treated with chemotherapy. We report that increased stromal gene expression predicts resistance to preoperative chemotherapy with 5-fluorouracil, epirubicin and cyclophosphamide (FEC) in subjects in the EORTC 10994/BIG 00-01 trial. The predictive value of the stromal signature was successfully validated in two independent cohorts of subjects who received chemotherapy but not in an untreated control group, indicating that the signature is predictive rather than prognostic. The genes in the signature are expressed in reactive stroma, according to reanalysis of data from microdissected breast tumor samples. These findings identify a previously undescribed resistance mechanism to FEC treatment and suggest that antistromal agents may offer new ways to overcome resistance to chemotherapy.

Retraction--Validation of gene signatures that predict the response of breast cancer to neoadjuvant chemotherapy: a substudy of the EORTC 10994/BIG 00-01 clinical trial.

BACKGROUNDnWe have previously described gene-expression signatures that predict growth inhibitory and cytotoxic effects of common chemotherapeutic drugs in vitro. The aim of this study was to confirm the validity of these gene-expression signatures in a large series of patients with oestrogen-receptor-negative breast tumours who were treated in a phase III neoadjuvant clinical trial.nnnMETHODSnThis trial compares a non-taxane regimen (fluorouracil, epirubicin, and cyclophosphamide [FEC] for six cycles) with a taxane regimen (docetaxel for three cycles followed by epirubicin plus docetaxel [TET] for three cycles) in women with oestrogen-receptor-negative breast cancer. The primary endpoint of the study is the difference in progression-free survival based on TP53 status and will be reported later. Predicting response with gene signatures was a planned secondary endpoint of the trial and is reported here. Pathological complete response, defined as complete disappearance of the tumour with no more than a few scattered tumour cells detected by the pathologist in the resection specimen, was used to assess chemosensitivity. RNA was prepared from sections of frozen biopsies taken at diagnosis and hybridised to Affymetrix X3P microarrays. In-vitro single-agent drug sensitivity signatures were combined to obtain FEC and TET regimen-specific signatures. This study is registered on the clinical trials site of the US National Cancer Institute website http://www.clinicaltrials.gov/ct/show/NCT00017095.nnnFINDINGSnOf 212 patients with oestrogen-receptor-negative tumours assessed, 87 patients were excluded. 125 oestrogen-receptor-negative tumours (55 that showed pathological complete responses) were tested: 66 in the FEC group (28 that showed pathological complete responses) and 59 in the TET group (27 that showed pathological complete responses). The regimen-specific signatures significantly predicted pathological complete response in patients treated with the appropriate regimen (p<0.0001). The FEC predictor had a sensitivity of 96% (27 of 28 patients [95% CI 82-99]), specificity of 66% (25 of 38 patients [50-79]), positive predictive value (PPV) of 68% (27 of 40 patients [52-80]), and negative predictive value (NPV) of 96% (25 of 26 patients [81-99]). The TET predictor had a sensitivity of 93% (25 of 27 patients [77-98]), specificity 69% (22 of 32 patients [51-82]), PPV of 71% (25 of 35 patients [55-84]), and NPV of 92% (22 of 24 patients [74-98]). Analysis of tumour size, grade, nodal status, age, and regimen-specific signatures showed that the genomic signatures were the only independent variables predicting pathological complete response at p<0.01. Selection of patients with these signatures would increase the proportion of patients with pathological complete responses from 44% to around 70% in the patients studied here.nnnINTERPRETATIONnWe have validated the use of regimen-specific drug sensitivity signatures in the context of a multicentre randomised trial. The high NPV of both signatures may allow early selection of patients with breast cancer who should be considered for trials with new drugs.

TP53 status for prediction of sensitivity to taxane versus non-taxane neoadjuvant chemotherapy in breast cancer (EORTC 10994/BIG 1-00): a randomised phase 3 trial

BACKGROUNDnTP53 has a crucial role in the DNA damage response. We therefore tested the hypothesis that taxanes confer a greater advantage than do anthracyclines on breast cancers with mutated TP53 than in those with wild-type TP53.nnnMETHODSnIn an open-label, phase 3 study, women (age <71 years) with locally advanced, inflammatory, or large operable breast cancers were randomly assigned in a 1:1 ratio to either a standard anthracycline regimen (six cycles of intravenous fluorouracil 500 mg/m², epirubicin 100 mg/m², and cyclophosphamide 500 mg/m² every 21 days [FEC100], or fluorouracil 600 mg/m², epirubicin 75 mg/m², cyclophosphamide 900 mg/m² [tailored FEC] starting on day 1 and then every 21 days) or a taxane-based regimen (three cycles of docetaxel 100 mg/m², intravenously infused over 1 h on day 1 every 21 days, followed by three cycles of intravenous epirubicin 90 mg/m² and docetaxel 75 mg/m² on day 1 every 21 days [T-ET]) at 42 centres in Europe. Randomisation was by use of a minimisation method that stratified patients by institution and initial tumour stage. The primary endpoint was progression-free survival (PFS) according to TP53 status. Analysis was by intention to treat. This is the final analysis of this trial. The study is registered with ClinicalTrials.gov, number NCT00017095.nnnFINDINGSn928 patients were enrolled in the FEC group and 928 in the T-ET group. TP53 status was not assessable for 183 (20%) patients in the FEC group and 204 (22%) patients in the T-ET group mainly because of low tumour-cell content in the biopsy. 361 primary endpoint events were recorded in the FEC group and 314 in the T-ET group. In patients with TP53-mutated tumours, 5-year PFS was 59·5% (95% CI 53·4-65·1) in the T-ET group (n=326) and 55·3% (49·2-60·9) in the FEC group (n=318; hazard ratio 0·84, 98% CI 0·63-1·14; p=0·17). In patients with TP53 wild-type tumours, 5-year PFS was 66·8% (95% CI 61·4-71·6) in the T-ET group (n=398) and 64·7% (59·6-69·4) in the FEC group (n=427; 0·89, 98% CI 0·68-1·18; p=0·35). For all patients, irrespective of TP53 status, 5-year PFS was 65·1% (95% CI 61·6-68·3) in the T-ET group and 60·8% (57·3-64·2) in the FEC group (0·85, 98% CI 0·71-1·02; p=0·035). At the sites using FEC100 versus T-ET, the most common grade 3 or 4 adverse events were febrile neutropenia (75 [9%] of 803 vs 173 [21%] of 809, respectively), and neutropenia (653 [81%] vs 730 [90%], respectively). At the sites using tailored FEC versus T-ET, the most common grade 3 or 4 adverse events were febrile neutropenia (ten [8%] of 118 vs 26 [22%] of 116, respectively), and neutropenia (100 [85%] vs 115 [99%], respectively). Two patients died of toxicity during or within 30 days of chemotherapy completion and without disease relapse (one in each group).nnnINTERPRETATIONnAlthough TP53 status was prognostic for overall survival, it was not predictive of preferential sensitivity to taxanes. TP53 status tested by use of the yeast assay in this patient population cannot be used to select patients for an anthracycline-based chemotherapy versus a taxane-based chemotherapy.nnnFUNDINGnUS National Cancer Institute, La Ligue Nationale Contre le Cancer, European Union, Pharmacia, and Sanofi-Aventis.

Dissemination Risk Index Based on Plasminogen Activator System Components in Primary Breast Cancer

PURPOSEnTo study interactions between disease-free survival (DFS) and four components of the plasminogen activator system: urokinase-type plasminogen activator (uPA), its two inhibitors (PAI-1 and PAI-2), and its membrane receptor uPAR.nnnPATIENTS AND METHODSnWe conducted a retrospective study of 499 primary breast cancer patients (median follow-up, 6 years). uPA, PAI-1, and PAI-2 were determined on cytosols and uPAR on solubilized pellets, using enzyme-linked immunoadsorbent assay kits (American Diagnostica, Greenwich, CT). Classical univariate and multivariate statistical methods were used together with multiple correspondence analysis to graphically examine interactions between the variables and outcome.nnnRESULTSnBy univariate analysis, higher uPA and PAI-1 values were significantly related to shorter DFS (P =.002; P <.00002). PAI-2 was not significantly related to DFS, although patients with high and very low PAI-2 values had a longer DFS. Multiple correspondence analysis showed the parallel impact of uPA and PAI-1 on outcome, and the clearly different behavior of PAI-2 compared with PAI-1. The prognostic contribution of uPAR seemed weak by both methods. A dissemination risk index [uPA x PAI-1/(PAI-2 + 1)], taking into account the modulation of uPA proteolytic activity by the ratio of its two inhibitors, was then tested. Dissemination risk index was selected as an independent variable in the Cox model in the overall population (P <.000001) and in node-positive patients (P <.00001). It was the only variable selected in node-negative patients (P =. 003).nnnCONCLUSIONnA dissemination risk index determined on primary tumor and taking into account the different effects of PAI-1 and PAI-2 on uPA can be of major help in clinical management of breast cancer, particularly in node-negative patients.

Combination of COX-2 expression and PIK3CA mutation as prognostic and predictive markers for celecoxib treatment in breast cancer

COX-2 expression level and prognostic value are still a matter of debate in breast cancer (BC). We addressed these points in the context of PIK3CA mutational status. Based on an interesting study of aspirin efficacy in colorectal cancer, we hypothesized that celecoxib antitumoral activity may be restricted to PIK3CA mutated BC. COX-2 mRNA expression was analyzed in 446 BC samples and in 61 BC patient-derived xenografts (PDX) using quantitative RT-PCR. The prognostic impact of COX-2 expression level was assessed independently and according to PIK3CA mutational status in our cohort and in a validation set of 817 BC. The antitumoral activity of celecoxib was tested in two triple-negative (TN) PDX with a PIK3CA wild-type (wt) or mutated genotype. COX-2 mRNA was overexpressed in 2% of BC and significantly associated with TN subtype. Metastasis-free survival (MFS) was significantly better in patients with high COX-2 expression level, the prognosis of whom was similar to patients with PIK3CA mutations. TCGA validation cohort confirmed that patients with low COX-2 expression PIK3CA wt tumors had the worse disease-free survival (DFS) compared to all other subgroups. Celecoxib had a significant antitumoral effect in PIK3CA mutated PDX only. Celecoxib antitumoral activity involved S6 ribosomal protein and AKT phosphorylation. Low expression of COX-2 has a significant negative impact on the MFS/DFS of BC patients. Antitumoral effect of celecoxib is restricted to PIK3CA mutated PDX. These results suggest that PIK3CA mutation may be a new predictive biomarker for celecoxib efficacy.

Immunomarker Studies of Fine-Needle Cytopuncture Cell Blocks for Tumor Response Prediction After Preoperative Chemotherapy and Prognosis in Operable Nonmetastatic Primary Breast Carcinoma

Abstract:u2002 Neo‐adjuvant chemotherapy of breast cancer provides an opportunity to evaluate predictive factors at initial tumor biopsy. We evaluated these factors on cell blocks obtained by diagnostic fine‐needle cytopuncture (FNC), with respect to tumor regression and outcome. A prospective study (1996–2003, median follow‐up 82u2003months) involved 163 patients with breast carcinoma (T2u2003≥3u2003cm, T3, T4 noninflammatory) diagnosed by means of FNC. Malignancy, cytologic grade, and the presence of lymphocytes were determined on cytologic smears. Ki67, estrogen receptor (ER), progesterone receptor (PgR), HER2, and p53 expression was assessed on cell blocks by means of immunohistochemistry. All the patients received anthracycline‐based chemotherapy. A combined clinical and pathologic tumor regression score was calculated. Twelve cases (7.5%) showed a complete regression, 72 cases (44%) a partial regression and 79 cases (48.5%) no regression. Factors predictive of regression were high grade, presence of lymphocytes, pN0, high Ki67 expression, hormone receptor negativity, and the “triple negative” phenotype. In univariate analysis 5‐year metastasis‐free survival rate (MFS) correlated with cytologic grade, pN, ER, and p53 status, while overall survival (OS) correlated with cytologic grade, type of surgery, pN, and ER status. In multivariate analysis, MFS was significantly influenced by the regression score, Ki67, age, ER status, pN, HER2, and initial tumor size. Except for age, the same parameters correlated with OS. FNC with the cell block technique is a rapid, minimally invasive, reliable, and inexpensive method for analyzing predictive biomarkers, and may thus be useful in the management of breast cancer patients requiring neo‐adjuvant chemotherapy.

Lymphovascular invasion after neoadjuvant chemotherapy is strongly associated with poor prognosis in breast carcinoma

PurposeFew studies evaluated the prognostic value of the presence of lymphovascular invasion (LVI) after neoadjuvant chemotherapy (NAC) for breast cancer (BC).MethodsThe association between LVI and survival was evaluated in a cohort of BC patients treated by NAC between 2002 and 2011. Five post-NAC prognostic scores (ypAJCC, RCB, CPS, CPSxa0+xa0EG and Neo-Bioscore) were evaluated and compared with or without the addition of LVI.ResultsOut of 1033 tumors, LVI was present on surgical specimens in 29.2% and absent in 70.8% of the cases. Post-NAC LVI was associated with impaired disease-free survival (DFS) (HR 2.54; 95% CI 1.96–3.31; Pxa0<xa00.001), and the magnitude of this effect depended on BC subtype (Pinteractionxa0=xa00.003), (luminal BC: HR 1.83; Pxa0=xa00.003; triple negative BC: HR 3.73; Pxa0<xa00.001; HER2-positive BC: HR 6.21; Pxa0<xa00.001). Post-NAC LVI was an independent predictor of local relapse, distant metastasis, and overall survival; and increased the accuracy of all five post-NAC prognostic scoring systems.ConclusionsPost-NAC LVI is a strong independent prognostic factor that: (i) should be systematically reported in pathology reports; (ii) should be used as stratification factor after NAC to propose inclusion in second-line trials or adjuvant treatment; (iii) should be included in post-NAC scoring systems.

P53 functional assay in yeast: evaluation in 1856 patients in a large prospective clinical trial (EORTC 10994/BIG 00-01).

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 AbstractsnnAbstract #1067 nnBackground:Although mutations in the p53 gene can occur in 20-30% of sporadic breast cancer, and are associated with other poor prognostic factors, there is no clear evidence as to whether p53 status should influence therapy. Immunohistochemistry has been demonstrated to be an inferior method compared with sequencing, but the optimal technique to assess the functional p53 status is not known. The yeast test is a functional assay which tests the transcriptional competence of p53, and detects biologically important mutations. Here we present the results of p53 evaluation using this method in a prospective study. Material and methods: EORTC 10994/BIG 00-01 trial is a randomized trial comparing the neoadjuvant use of an anthracycline-based regimen with a taxane-containing regimen in patients with large operable or locally advanced breast cancers. Fresh frozen tumour biopsies were mandatory before starting chemotherapy. Frozen sections were examined centrally and the p53 test was done if the invasive tumour cell content was above 20%. RNA extract was used to assess p53 status: p53 wild type tumours were defined as tumours whose cDNA transfected in yeast gave less than 20% red colonies (background); p53 mutated tumours gave 20% or more red colonies. Results: The trial accrual was completed in November 2006 after inclusion of 1856 patients. There was no frozen sample available in 70 patients. In 276 patients the % of tumour cells was <20. The p53 test failed in 62 patients, generally because the quality or quantity of RNA was too low for RT-PCR. P53 was successfully assessed in 1458 patients: 661 (45%) tumours are p53 mutated and 797 (55%) are p53 wt. p53 mutated and wild type tumours were grade III in 40% (265/661) and 19% (153/797), respectively, and estrogen receptor negative in 41% (270/661) and 14.5% (116/797), respectively. We did not see any correlation between p53 status and tumour size or nodal status at diagnosis. HER2 status data are being collected. Conclusion: P53 status has been successfully evaluated using a yeast assay in 78.5% (1458/1856) of cases in the context of a multicenter trial. In indirect comparisons the frequency of patients with functionally altered p53 status is higher than with sequenced based methods. Our results confirm an association between p53 mutation and high grade or estrogen receptor negative status, but not with age, tumour size or clinical nodal status. More data regarding the quality and quantity of RNA collected will be presented at the meeting.nnCitation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1067.

Abstract 2031: Antitumoral synergy of iron chelators and chemotherapies in triple-negative breast cancer cell lines and patient-derived xenograft

Introduction: Tumor cells present an iron metabolic disorder with high proliferation rate, increased iron storage (ferritin and Labile Iron Pool - LIP) and high sensibility to iron deprivation, which could be a therapeutic target. Anticancer effect of iron chelators deferoxamine (DFO) and deferasirox (DFX) has been revealed in several types of cancers. In breast cancer (BC), the development of new therapeutic approaches is urgently needed for triple negative (TN) subtype which presents poor prognosis and lacks targeting therapy. We investigated the therapeutic potential of iron chelators combined with chemotherapeutic agents in TNBC cell lines and patient-derived xenografts (PDX). Methods: Anticancer effects of iron chelators combined with chemotherapeutic agents (doxorubicin, cisplatin or carboplatin) were evaluated in vitro in 4 TNBC cell lines by MTT assay, annexin V/PI staining and assessment of caspase 3/7 activity. Assessment of LIP, transferrin receptor 1 (TfR1) expression level, Reactive Oxygen Species (ROS) production and mitochondrial membrane potential variations were performed by flow cytometry, and ELISA assay for ferritin level. The activity of DFX alone or combined with doxorubicin/cyclophosphamide (AC) was tested in the HBCx-10 TNBC PDX selected because of its relapse to AC. Iron homeostasis, hypoxia and PI3K pathway were analyzed by immunohistochemistry (IHC) and Western-blot in both cell lines and PDX tumors. In DFX+/-AC treated and untreated tumors, induction of apoptosis was performed by TUNEL assay and a transcriptome analysis is ongoing. Results: Iron chelators acted in synergy effect with three chemotherapies in all cell lines which were tested to inhibit cell proliferation and induce apoptosis. Chelators increased cytotoxicity until 60% compared to chemotherapies alone. In all cell lines, chelators treatment increased TfR1 expression level and decreased LIP and ferritin. Furthermore, down-regulation of PI3K pathway, hypoxia, mitochondrial membrane potential, and the production increasing of ROS were observed. In HBCx-10 PDX, a trend for antitumoral activity of DFX alone was observed (p=0.09) at the end of the experiment (day 81). A significant difference of Relative Tumor Volume (RTV) was observed since day 18 between the AC group and the DFX+AC group (tumor growth inhibition: 37 to 61%, tumor growth delay: 10 to 14 days, 0.005 Conclusions: Iron chelators may increase the effectiveness of conventional chemotherapies for TNBC treatments. This antitumoral synergy involves PI3K pathway downregulation, ROS production and decrease mitochondrial membrane potential. Citation Format: Sandrine Tury, Sophie Vacher, Veronique Becette, Franck Assayag, Sophie Chateau-Joubert, Jean-Luc Servely, Elisabetta Marangoni, Ivan Bieche, Celine Callens. Antitumoral synergy of iron chelators and chemotherapies in triple-negative breast cancer cell lines and patient-derived xenograft [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2031. doi:10.1158/1538-7445.AM2017-2031