Eléonore Gravier

Frequent PTEN genomic alterations and activated phosphatidylinositol 3-kinase pathway in basal-like breast cancer cells

IntroductionBasal-like carcinomas (BLCs) and human epidermal growth factor receptor 2 overexpressing (HER2+) carcinomas are the subgroups of breast cancers that have the most aggressive clinical behaviour. In contrast to HER2+ carcinomas, no targeted therapy is currently available for the treatment of patients with BLCs. In order to discover potential therapeutic targets, we aimed to discover deregulated signalling pathways in human BLCs.MethodsIn this study, we focused on the oncogenic phosphatidylinositol 3-kinase (PI3K) pathway in 13 BLCs, and compared it with a control series of 11 hormonal receptor negative- and grade III-matched HER2+ carcinomas. The two tumour populations were first characterised by immunohistochemistry and gene expression. The PI3K pathway was then investigated by gene copy-number analysis, gene expression profiling and at a proteomic level using reverse-phase protein array technology and tissue microarray. The effects of the PI3K inhibition pathway on proliferation and apoptosis was further analysed in three human basal-like cell lines.ResultsThe PI3K pathway was found to be activated in BLCs and up-regulated compared with HER2+ tumours as shown by a significantly increased activation of the downstream targets Akt and mTOR (mammalian target of rapamycin). BLCs expressed significantly lower levels of the tumour suppressor PTEN and PTEN levels were significantly negatively correlated with Akt activity within that population. PTEN protein expression correlated significantly with PTEN DNA copy number and more importantly, reduced PTEN DNA copy numbers were observed specifically in BLCs. Similar to human samples, basal-like cell lines exhibited an activation of PI3K/Akt pathway and low/lack PTEN expression. Both PI3K and mTOR inhibitors led to basal-like cell growth arrest. However, apoptosis was specifically observed after PI3K inhibition.ConclusionsThese data provide insight into the molecular pathogenesis of BLCs and implicate the PTEN-dependent activated Akt signalling pathway as a potential therapeutic target for the management of patients with poor prognosis BLCs.

Polo-like Kinase 1: A Potential Therapeutic Option in Combination with Conventional Chemotherapy for the Management of Patients with Triple-Negative Breast Cancer

Breast cancers are composed of molecularly distinct subtypes with different clinical outcomes and responses to therapy. To discover potential therapeutic targets for the poor prognosis-associated triple-negative breast cancer (TNBC), gene expression profiling was carried out on a cohort of 130 breast cancer samples. Polo-like kinase 1 (PLK1) was found to be significantly overexpressed in TNBC compared with the other breast cancer subtypes. High PLK1 expression was confirmed by reverse phase protein and tissue microarrays. In triple-negative cell lines, RNAi-mediated PLK1 depletion or inhibition of PLK1 activity with a small molecule (BI-2536) induced an increase in phosphorylated H2AX, G(2)-M arrest, and apoptosis. A soft-agar colony assay showed that PLK1 silencing impaired clonogenic potential of TNBC cell lines. When cells were grown in extracellular matrix gels (Matrigel), and exposed to BI-2536, apoptosis was observed specifically in TNBC cancerous cells, and not in a normal cell line. When administrated as a single agent, the PLK1 inhibitor significantly impaired tumor growth in vivo in two xenografts models established from biopsies of patients with TNBC. Most importantly, the administration of BI-2536, in combination with doxorubicin + cyclophosphamide chemotherapy, led to a faster complete response compared with the chemotherapy treatment alone and prevented relapse, which is the major risk associated with TNBC. Altogether, our observations suggest PLK1 inhibition as an attractive therapeutic approach, in association with conventional chemotherapy, for the management of patients with TNBC.

TTK/hMPS1 is an attractive therapeutic target for triple-negative breast cancer.

Triple-negative breast cancer (TNBC) represents a subgroup of breast cancers (BC) associated with the most aggressive clinical behavior. No targeted therapy is currently available for the treatment of patients with TNBC. In order to discover potential therapeutic targets, we searched for protein kinases that are overexpressed in human TNBC biopsies and whose silencing in TNBC cell lines causes cell death. A cohort including human BC biopsies obtained at Institut Curie as well as normal tissues has been analyzed at a gene-expression level. The data revealed that the human protein kinase monopolar spindle 1 (hMPS1), also known as TTK and involved in mitotic checkpoint, is specifically overexpressed in TNBC, compared to the other BC subgroups and healthy tissues. We confirmed by immunohistochemistry and reverse phase protein array that TNBC expressed higher levels of TTK protein compared to the other BC subgroups. We then determined the biological effects of TTK depletion by RNA interference, through analyses of tumorigenic capacity and cell viability in different human TNBC cell lines. We found that RNAi-mediated depletion of TTK in various TNBC cell lines severely compromised their viability and their ability to form colonies in an anchorage-independent manner. Moreover, we observed that TTK silencing led to an increase in H2AX phosphorylation, activation of caspases 3/7, sub-G1 cell population accumulation and high annexin V staining, as well as to a decrease in G1 phase cell population and an increased aneuploidy. Altogether, these data indicate that TTK depletion in TNBC cells induces apoptosis. These results point out TTK as a protein kinase overexpressed in TNBC that may represent an attractive therapeutic target specifically for this poor prognosis associated subgroup of breast cancer.

A prognostic DNA signature for T1T2 node-negative breast cancer patients†

Predicting evolution of small node‐negative breast carcinoma is a real challenge in clinical practice. The aim of this study was to search whether qualitative or quantitative DNA changes may help to predict metastasis of small node‐negative breast carcinoma. Small invasive ductal carcinomas without axillary lymph node involvement (T1T2N0) from 168 patients with either good (111 patients with no event at 5 years after diagnosis) or poor (57 patients with early metastasis) outcome were analyzed with comparative genomic hybridization (CGH) array. A CGH classifier, identifying low‐ and high‐risk groups of metastatic recurrence, was established in a training set of 78 patients, then validated, and compared with clinicopathological parameters in a distinct set of 90 patients. The genomic status of regions located on 2p22.2, 3p23, and 8q21‐24 and the number of segmental alterations were defined in the training set to classify tumors into low‐ or high‐risk groups. In the validation set, in addition to estrogen receptors and grade, this CGH classifier provided significant prognostic information in multivariate analysis (odds ratio, 3.34; 95% confidence interval 1.01–11.02; P = 4.78 × 10−2, Wald test). This study shows that tumor DNA contains important prognostic information that may help to predict metastasis in T1T2N0 tumors of the breast.

EMA - A R package for Easy Microarray data analysis

BackgroundThe increasing number of methodologies and tools currently available to analyse gene expression microarray data can be confusing for non specialist users.FindingsBased on the experience of biostatisticians of Institut Curie, we propose both a clear analysis strategy and a selection of tools to investigate microarray gene expression data. The most usual and relevant existing R functions were discussed, validated and gathered in an easy-to-use R package (EMA) devoted to gene expression microarray analysis. These functions were improved for ease of use, enhanced visualisation and better interpretation of results.ConclusionsStrategy and tools proposed in the EMA R package could provide a useful starting point for many microarrays users. EMA is part of Comprehensive R Archive Network and is freely available at http://bioinfo.curie.fr/projects/ema/.

Genomic Instability: A Stronger Prognostic Marker Than Proliferation for Early Stage Luminal Breast Carcinomas

Background The accurate prognosis definition to tailor treatment for early luminal invasive breast carcinoma patients remains challenging. Materials and Methods Two hundred fourteen early luminal breast carcinomas were genotyped with single nucleotide polymorphisms (SNPs) array to determine the number of chromosomal breakpoints as a marker of genomic instability. Proliferation was assessed by KI67 (immunohistochemistry) and genomic grade index (transcriptomic analysis). IHC3 (IHC4 score for HER2 negative tumors) was also determined. Results In the training set (109 cases), the optimal cut-off was 34 breakpoints with a specificity of 0.94 and a sensitivity of 0.57 (Area under the curve (AUC): 0.81[0.71; 0.91]). In the validation set (105 cases), the outcome of patients with > 34 breakpoints (11 events / 22 patients) was poorer (logrank test p < 0.001; Relative Risk (RR): 3.7 [1.73; 7.92]), than that of patients with < 34 breakpoints (19 events / 83 patients).Whereas genomic grade and KI67 had a significant prognostic value in univariate analysis in contrast to IHC3 that failed to have a statistical significant prognostic value in this series, the number of breakpoints remained the only significant parameter predictive of outcome (RR: 3.47, Confidence Interval (CI [1.29; 9.31], p = 0.014)) in multivariate analysis . Conclusion Genomic instability, defined herein as a high number of chromosomal breakpoints, in early stage luminal breast carcinoma is a stronger prognostic marker than proliferation.

Risk of Hormone Escape in a Human Prostate Cancer Model Depends on Therapy Modalities and Can Be Reduced by Tyrosine Kinase Inhibitors

Almost all prostate cancers respond to androgen deprivation treatment but many recur. We postulated that risk of hormone escape -frequency and delay- are influenced by hormone therapy modalities. More, hormone therapies induce crucial biological changes involving androgen receptors; some might be targets for escape prevention. We investigated the relationship between the androgen deprivation treatment and the risk of recurrence using nude mice bearing the high grade, hormone-dependent human prostate cancer xenograft PAC120. Tumor-bearing mice were treated by Luteinizing-Hormone Releasing Hormone (LHRH) antagonist alone, continuous or intermittent regimen, or combined with androgen receptor (AR) antagonists (bicalutamide or flutamide). Tumor growth was monitored. Biological changes were studied as for genomic alterations, AR mutations and protein expression in a large series of recurrent tumors according to hormone therapy modalities. Therapies targeting Her-2 or AKT were tested in combination with castration. All statistical tests were two-sided. Tumor growth was inhibited by continuous administration of the LH-RH antagonist degarelix (castration), but 40% of tumors recurred. Intermittent castration or complete blockade induced by degarelix and antiandrogens combination, inhibited tumor growth but increased the risk of recurrence (RR) as compared to continuous castration (RRintermittent: 14.5, RRcomplete blockade: 6.5 and 1.35). All recurrent tumors displayed new quantitative genetic alterations and AR mutations, whatever the treatment modalities. AR amplification was found after complete blockade. Increased expression of Her-2/neu with frequent ERK/AKT activation was detected in all variants. Combination of castration with a Her-2/neu inhibitor decreased recurrence risk (0.17) and combination with an mTOR inhibitor prevented it. Anti-hormone treatments influence risk of recurrence although tumor growth inhibition was initially similar. Recurrent tumors displayed genetic instability, AR mutations, and alterations of phosphorylation pathways. We postulated that Her-2/AKT pathways allowed salvage of tumor cells under castration and we demonstrated that their inhibition prevented tumor recurrence in our model.

Predictive gene signature of response to the anti-TweakR mAb PDL192 in patient-derived breast cancer xenografts.

Purpose (1) To determine TweakR expression in human breast cancers (BC), (2) evaluate the antitumor effect of the anti-TweakR antibody PDL192, used alone or after chemotherapy-induced complete remission (CR), on patient-derived BC xenografts (PDX) and (3) define predictive markers of response. Experimental Design TweakR expression was analyzed by IHC on patients and PDXs BC samples. In vivo antitumor effect of PDL192 was evaluated on eight TweakR-positive BC PDXs alone or after complete remission induced by a combination of doxorubicin and cyclophosphamide. Using both responding and resistant PDX tumors after PDL192 administration, RT-QPCR were performed on a wide list of selected candidate genes to identify predictive markers of response. Results TweakR protein was expressed in about half of human BC samples. In vivo PDL192 treatment had significantly anti-tumor activity in 4 of 8 TweakR-positive BC PDXs, but no correlation between the expression level of the Tweak receptor and response to therapy was observed. PDL192 also significantly delayed tumor relapse after CR. Finally, an 8 gene signature was defined from sensitive and resistant PDXs. Conclusions PDL192 was highly efficient in some BC PDXs. We found 8 genes that were differentially expressed in responding and resistant tumors and could constitute a gene expression signature which would need to be extended to other xenograft models for confirmation. These data confirm the therapeutic potential of TweakR targeting in BC and the possibility of prospectively selecting patients who might benefit from therapy.

P3-05-03: Transcriptomic Validation of Molecular Classification of Invasive Ductal Carcinoma Based on Immunohistochemical Markers and Grade.

Transcriptomic analyses identified four major groups among invasive ductal carcinomas, associated with different clinical outcomes. Their definitions in practice is still matter of debate. Our aims were 1) to validate definitions based on immunohistochemical markers and grade: luminal A= ER and or PR+ve, grade I HER2−ve, luminal B= ER+ve and/or PR+ve, HER2+ve or grade III, HER2 enriched carcinomas= HER2+ve and ER-ve, basal-like/triple negative carcinomas= grade III, ER-ve, PR-ve, HER2−ve and expression of at least one of the basal-like markers (CK5/6, CK14, EGFR); 2) to refine this immunohistochemical definition. 142 consecutive tumors were selected in our tumour bank (42 triple-negative and grade III; 31 HER2+ve and ER-ve; 35 luminal B ER or PR+ve and grade III (7 cases) or HER2+ve (28 cases); luminal A (34 ER+ve or PR+ve, grade I)). Transcriptomic analyses were performed using Affymetrix U133+2 arrays (good quality RNAs obtained for all frozen specimens). The molecular classes determined according to proposed definitions were compared to those obtained with unsupervised clustering analyses using datas after GC-RMA normalisation (with the intrinsic gene list genes and with the highest variance genes). Marker patterns of expression (CK 8/18, 5/6, 14, EGFR, BCL2 and Ki67) were analysed within each molecular class. Based on the phenotypical definition and grade, 10 and 9% of cases were misclassified respectively using unsupervised clustering either with the intrinsic gene list or the highest variance genes. The misclassified tumors were luminal B HER2+ve case with low ER level of expression, or HER2+ve and ER-ve cases classified among the triple-negative group. 39 out of 42 (93%) triple negative expressed at least one of the basal markers. RP expression pattern differed between luminal A and B carcinomas. All luminal A showed at least > 15% of positive cells and 65% of them harboured > 50% of positive cells. In contrast, luminal B showed 20% stained cells) in more than 90% of the luminal A cases. CK14 was positive (i.e. > 1% positive cell) in 65% of the triple negative cases, compared to CK5/6 positive in 58% of the cases. Ki67 was > 20% in 90% of the triple negative and in 55% of luminal B cases compared to less than 5% of the luminal A cases. 20 and 30% of HER2+ve ER-ve carcinomas expressed CK5/6, CK14 and EGFR associated in more than 90% of the cases to CK8/18 positivity (> 20% positive cells). Identification of molecular classes of breast carcinomas was accurately determined by immunohistochemistry and grade. Low level of RP expression and BCL2 negativity were part of the luminal B phenotype. CK14 was more sensitive in this population of triple negative carcinomas to identify basal-like carcinomas. KI67 was highly expressed in the vast majority if not all breast triple negative carcinomas. HER2+ve ER-ve carcinomas can express basal markers but in contrast to basal-like carcinomas, associated in the large majority of the cases to CK8/18 expression. The accurate determination of molecular groups of breast carcinomas should be a key parameter for development of targeted therapies within each group. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-05-03.

P4-09-01: Identification of Poor Prognosis T1T2N0 Luminal ERBB2-ve Breast Carcinomas.

Background: To identify ER+ve ERBB2-ve ductal T1T2N0 carcinomas associated with a poor prognosis remains challenging. We have previously demonstrated that the number of chromosomal breakpoints assessed by CGH could be a marker of worse outcome for breast carcinomas. Our aim was to validate the CGH based signature in a series of luminal ductal and T1T2 N0 carcinoma patients with long-term clinical follow-up. Patients and methods: We analyzed 214 patients treated for an invasive ductal ER+ve ERBB2-ve carcinomas, smaller than 30mm. The training set was composed of 109 patients (10.9 years of median follow-up; 30 cases associated with a metastatic event within less than 4 years/79 control cases with no metastastic event at 5 years) and the validation set of 105 patients (10.5 years of median follow-up; 30 relapses including contra-lateral breast carcinomas, loco-regional relapses and 8 metastatic events). None of the patient received adjuvant chemotherapy. 16 received an adjuvant hormonotherapy (10 in the training and 6 in the validation groups). We genotyped the sample set with the SNP6.0 affymetrix array. After RMA normalisation using Genotyping console, segmentation was performed according to the Zhang and Siegmund maximum method. In the training data set, the number of breakpoints was assessed, linked to outcome and the threshold optimising the sensitivity and specificity was determined (ROC curve). The threshold prognostic value was then tested on the validation series (Kaplan Meier analysis, log rank test, determination of relative risk and its confidence interval with a Cox model). Results: In the training set, median numbers of breakpoints were 7 in cases that experienced a metastatic event after more than 5 years and 40.5 in cases that experienced a metastatic event in less than 4 years. The threshold (Younden index ) was 34 breakpoints with a sensitivity of 0.57 and a specificity of 0.94 (AUC: 0.81[0.71;0.91]). In the validation set, the outcome of patients with more than 34 breakpoints was poorer than that of patients with less than 34 breakpoints ( 34 breakpoints: 11 events out of 22 patients with a median time to progression of 108 months; p 34 versus 34 breakpoints: 4 events out of 22 patients with a median time to progression of 108 months; p=0.009 (logrank test); RR: 5.29 [1.32; 21.26]). Conclusion: We demonstated that patients with T1T2 ( Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-09-01.