XIST loss impairs mammary stem cell differentiation and increases tumorigenicity through Mediator hyperactivation.

X inactivation (XCI) is triggered by upregulation of XIST, which coats the chromosome in cis, promoting formation of a heterochromatic domain (Xi). XIST role beyond initiation of XCI is only beginning to be elucidated. Here, we demonstrate that XIST loss impairs differentiation of human mammary stem cells (MaSCs) and promotes emergence of highly tumorigenic and metastatic carcinomas. On the Xi, XIST deficiency triggers epigenetic changes and reactivation of genes overlapping Polycomb domains, including Mediator subunit MED14. MED14 overdosage results in increased Mediator levels and hyperactivation of the MaSC enhancer landscape and transcriptional program, making differentiation less favorable. We further demonstrate that loss of XIST and Xi transcriptional instability is common among human breast tumors of poor prognosis. We conclude that XIST is a gatekeeper of human mammary epithelium homeostasis, thus unveiling a paradigm in the control of somatic cell identity with potential consequences for our understanding of gender-specific malignancies.

MiR-662 is associated with metastatic relapse in early-stage breast cancer and promotes metastasis by stimulating cancer cell stemness.

BACKGROUND: Breast cancer (BC) metastasis, which often occurs in bone, contributes substantially to mortality. MicroRNAs play a fundamental role in BC metastasis, although microRNA-regulated mechanisms driving metastasis progression remain poorly understood. METHODS: MiRome analysis in serum from BC patients was performed by TaqMan™ low-density array. MiR-662 was overexpressed following MIMIC-transfection or lentivirus transduction. Animal models were used to investigate the role of miR-662 in BC (bone) metastasis. The effect of miR-662-overexpressing BC cell conditioned medium on osteoclastogenesis was investigated. ALDEFLUOR assays were performed to study BC stemness. RNA-sequencing transcriptomic analysis of miR-662-overexpressing BC cells was performed to evaluate gene expression changes. RESULTS: High levels of hsa-miR-662 (miR-662) in serum from BC patients, at baseline (time of surgery), were associated with future recurrence in bone. At an early-stage of the metastatic disease, miR-662 could mask the presence of BC metastases in bone by inhibiting the differentiation of bone-resorbing osteoclasts. Nonetheless, metastatic miR-662-overexpressing BC cells then progressed as overt osteolytic metastases thanks to increased stem cell-like traits. CONCLUSIONS: MiR-662 is involved in BC metastasis progression, suggesting it may be used as a prognostic marker to identify BC patients at high risk of metastasis.

CD95/Fas and metastatic disease: What does not kill you makes you stronger.

CD95 (also known as Fas) is the prototype of death receptors; however, evidence suggests that this receptor mainly implements non-apoptotic signaling pathways such as NF-κB, MAPK, and PI3K that are involved in cell migration, differentiation, survival, and cytokine secretion. At least two different forms of CD95 L exist. The multi-aggregated transmembrane ligand (m-CD95 L) is cleaved by metalloproteases to release a homotrimeric soluble ligand (s-CD95 L). Unlike m-CD95 L, the interaction between s-CD95 L and its receptor CD95 fails to trigger apoptosis, but instead promotes calcium-dependent cell migration, which contributes to the accumulation of inflammatory Th17 cells in damaged organs of lupus patients and favors cancer cell invasiveness. Novel inhibitors targeting the pro-inflammatory roles of CD95/CD95 L may provide attractive therapeutic options for patients with chronic inflammatory disorders or cancer. This review discusses the roles of the CD95/CD95 L pair in cell migration and metastasis.

Publisher Correction: PH-domain-binding inhibitors of nucleotide exchange factor BRAG2 disrupt Arf GTPase signaling.

In the version of this article originally published, several co-authors had incorrect affiliation footnote numbers listed in the author list. Tatiana Cañeque and Angelica Mariani should each have affiliation numbers 3, 4 and 5, and Emmanuelle Charafe-Jauffret should have number 6. Additionally, there was an extra space in the name of co-author Robert P. St.Onge. These errors have been corrected in the HTML and PDF versions of the paper and the Supplementary Information PDF.

PH-domain-binding inhibitors of nucleotide exchange factor BRAG2 disrupt Arf GTPase signaling.

Peripheral membrane proteins orchestrate many physiological and pathological processes, making regulation of their activities by small molecules highly desirable. However, they are often refractory to classical competitive inhibition. Here, we demonstrate that potent and selective inhibition of peripheral membrane proteins can be achieved by small molecules that target protein-membrane interactions by a noncompetitive mechanism. We show that the small molecule Bragsin inhibits BRAG2-mediated Arf GTPase activation in vitro in a manner that requires a membrane. In cells, Bragsin affects the trans-Golgi network in a BRAG2- and Arf-dependent manner. The crystal structure of the BRAG2-Bragsin complex and structure-activity relationship analysis reveal that Bragsin binds at the interface between the PH domain of BRAG2 and the lipid bilayer to render BRAG2 unable to activate lipidated Arf. Finally, Bragsin affects tumorsphere formation in breast cancer cell lines. Bragsin thus pioneers a novel class of drugs that function by altering protein-membrane interactions without disruption.

[2021 update of the GEFPICS' recommendations for HER2 status assessment in invasive breast cancer in France]. / Mise à jour 2021 des recommandations du GEFPICS pour l'évaluation du statut HER2 dans les cancers infiltrants du sein en France.

The last international guidelines on HER2 determination in breast cancer have been updated in 2018 by the American Society of Clinical Oncology and College of American Pathologists, on the basis of a twenty-year practice and results of numerous clinical trials. Moreover, the emerging HER2-low concept for 1+ and 2+ non amplified breast cancers lead to refine French practices for HER2 status assessment. The GEFPICS group, composed of expert pathologists, herein presents the latest French recommendations for HER2 status evaluation in breast cancer, taking into account the ASCO/CAP guidelines and introducing the HER2-low concept. In the era of personalized medicine, HER2 status assessment remains one of the most important biomarkers in breast cancer and its quality guaranties the optimal patients' care. French pathologists' commitment in theranostic biomarker quality is more than ever required to provide the most efficient cares in oncology.

Transcriptomic Analysis of Breast Cancer Stem Cells and Development of a pALDH1A1:mNeptune Reporter System for Live Tracking.

Many solid cancers are hierarchically organized with a small number of cancer stem cells (CSCs) able to regrow a tumor, while their progeny lacks this feature. Breast CSC is known to contribute to therapy resistance. The study of those cells is usually based on their cell-surface markers like CD44high /CD24low/neg or their aldehyde dehydrogenase (ALDH) activity. However, these markers cannot be used to track the dynamics of CSC. Here, a transcriptomic analysis is performed to identify segregating gene expression in CSCs and non-CSCs, sorted by Aldefluor assay. It is observed that among ALDH-associated genes, only ALDH1A1 isoform is increased in CSCs. A CSC reporter system is then developed by using a far red-fluorescent protein (mNeptune) under the control of ALDH1A1 promoter. mNeptune-positive cells exhibit higher sphere-forming capacity, tumor formation, and increased resistance to anticancer therapies. These results indicate that the reporter identifies cells with stemness characteristics. Moreover, live tracking of cells in a microfluidic system reveals a higher extravasation potential of CSCs. Live tracking of non-CSCs under irradiation treatment show, for the first time, live reprogramming of non-CSCs into CSCs. Therefore, the reporter will allow for cell tracking to better understand the implication of CSCs in breast cancer development and recurrence.

[GEFPICS' guidelines for management of breast cancer tissue samples in the neoadjuvant setting]. / Recommandations du GEFPICS pour la prise en charge des prélèvements dans le cadre du traitement néoadjuvant du cancer du sein.

Neoadjuvant therapy is an increasing treatment option in the management of breast cancer. The tumor response to neoadjuvant therapy, especially the pathological complete response, is a validated endpoint frequently used in clinical trials. However, there is still a lack of standardization for the surgical specimen management in the neoadjuvant setting. This leads to heterogeneity in the specimen handling and might lead to significant bias for the prognostic assessment of patients or in clinical trials. The GEFPICS group, composed of expert breast cancer pathologists, herein presents guidelines for the management of breast and axillary specimen before treatment (management of biopsy, items of the pathological report) and after neoadjuvant therapy (specimen handling, histological assessment of response, items of the pathological report and response grading systems).

Circulating tumour cells from patients with colorectal cancer have cancer stem cell hallmarks in ex vivo culture.

OBJECTIVE: Although counting of circulating tumour cells (CTC) has attracted a broad interest as potential markers of tumour progression and treatment response, the lack of functional characterisation of these cells had become a bottleneck in taking these observations to the clinic. Our objective was to culture these cells in order to understand them and exploit their therapeutic potential to the full. DESIGN: Here, hypothesising that some CTC potentially have cancer stem cell (CSC) phenotype, we generated several CTC lines from the blood of patients with advanced metastatic colorectal cancer (CRC) based on their self-renewal abilities. Multiple standard tests were then employed to characterise these cells. RESULTS: Our CTC lines self-renew, express CSC markers and have multilineage differentiation ability, both in vitro and in vivo. Patient-derived CTC lines are tumorigenic in subcutaneous xenografts and are also able to colonise the liver after intrasplenic injection. RNA sequencing analyses strikingly demonstrate that drug metabolising pathways represent the most upregulated feature among CTC lines in comparison with primary CRC cells grown under similar conditions. This result is corroborated by the high resistance of the CTC lines to conventional cytotoxic compounds. CONCLUSIONS: Taken together, our results directly demonstrate the existence of patient-derived colorectal CTCs that bear all the functional attributes of CSCs. The CTC culture model described here is simple and takes <1 month from blood collection to drug testing, therefore, routine clinical application could facilitate access to personalised medicine. CLINICAL TRIAL REGISTRATION: ClinicalTrial.gov NCT01577511.

Stromal Expression of MARCKS Protein in Ovarian Carcinomas Has Unfavorable Prognostic Value.

Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer. Identification of new therapeutic targets is crucial. MARCKS, myristoylated alanine-rich C-kinase substrate, has been implicated in aggressiveness of several cancers and MARCKS inhibitors are in development. Using immunohistochemistry (IHC), we retrospectively assessed MARCKS expression in epithelial and stromal cells of 118 pre-chemotherapy EOC samples and 40 normal ovarian samples from patients treated at Salah Azaiez Institute. We compared MARCKS expression in normal versus cancer samples, and searched for correlations with clinicopathological features, including overall survival (OS). Seventy-five percent of normal samples showed positive epithelial MARCKS staining versus 50% of tumor samples (p = 6.02 × 10-3). By contrast, stromal MARCKS expression was more frequent in tumor samples (77%) than in normal samples (22%; p = 1.41 × 10-9). There was no correlation between epithelial and stromal IHC MARCKS statutes and prognostic clinicopathological features. Stromal MARCKS expression was correlated with shorter poor OS in uni- and multivariate analyses. Stromal MARCKS overexpression in tumors might contribute to cancer-associated fibroblasts activation and to the poor prognosis of EOC, suggesting a potential therapeutic interest of MARCKS inhibition for targeting the cooperative tumor stroma.

Bevacizumab plus neoadjuvant chemotherapy in patients with HER2-negative inflammatory breast cancer (BEVERLY-1): a multicentre, single-arm, phase 2 study.

BACKGROUND: Addition of bevacizumab to standard chemotherapy in the neoadjuvant setting in patients with HER2-negative metastatic breast cancer improves progression-free survival and the proportion of patients achieving pathological complete response. In the BEVERLY-1 (UCBG-0802) trial we aimed to assess the addition of bevacizumab to neoadjuvant and adjuvant chemotherapy in the treatment of patients with HER2-negative inflammatory breast cancer. METHODS: We did this phase 2, single-arm trial at 20 hospitals in France. We enrolled women aged 18 years or older who had non-metastatic HER2-negative inflammatory breast cancer. Patients underwent 3-week treatment cycles, receiving neoadjuvant intravenous fluorouracil (500 mg/m(2)), epirubicin (100 mg/m(2)), cyclophosphamide (500 mg/m(2)), and bevacizumab (15 mg/kg) during cycles 1-4, then docetaxel (100 mg/m(2)) and bevacizumab during cycles 5-8. 2-4 weeks after surgery, patients received adjuvant radiotherapy, hormone therapy (if they had a hormone receptor-positive tumour), and adjuvant intravenous bevacizumab. The primary endpoint was pathological complete response in breast and axillary lymph nodes after neoadjuvant treatment, determined after centralised review in accordance with Sataloff classification and assessed in the intention-to-treat population. Our analysis of toxic effects included all patients who received at least one dose of bevacizumab. The trial is complete and follow-up is ongoing. This study is registered with ClinicalTrials.gov, number NCT00820547. FINDINGS: Between Jan 16, 2009, and Sept 8, 2010, we enrolled 101 patients, one of whom withdrew consent before treatment, leaving 100 patients in the primary endpoint analysis. After neoadjuvant therapy, 19 (19% [95% CI 12-28]; p=0·16) of 100 patients achieved a pathological complete response according to centralised review. The most frequent grade 3-4 events during the neoadjuvant phase were neutropenia (89 [89%] of 100 patients), febrile neutropenia (37 [37%]), and mucositis (23 [23%]) and during the adjuvant phase the most frequent grade 3-4 adverse event was proteinuria (5 [7%] of 75 patients). One (1%) patient died of thrombotic microangiopathy after cycle 1, which was thought to be related to bevacizumab. Two patients (3%) developed transitory heart failure. 48 (48%) patients had serious adverse events, the most frequent of which was febrile neutropenia (28 [28%]). INTERPRETATION: Our results suggest that the addition of bevacizumab to neoadjuvant and adjuvant chemotherapy does not provide clinical benefit to patients with non-metastatic HER2-negative inflammatory breast cancer. Longer follow-up and correlative studies to identify patients who might benefit from bevacizumab are needed. FUNDING: Roche, La Ligue Nationale contre le Cancer, UNICANCER, and Chugai Pharma.

Brief reports: A distinct DNA methylation signature defines breast cancer stem cells and predicts cancer outcome.

Self-renewal and differentiation are two epigenetic programs that regulate stem cells fate. Dysregulation of these two programs leads to the development of cancer stem cells (CSCs). Recent evidence suggests that CSCs are relatively resistant to conventional therapies and responsible for metastasis formation. Deciphering these processes will help understand oncogenesis and allow the development of new targeted therapies. Here, we have used a whole genome promoter microarray to establish the DNA methylation portraits of breast cancer stem cells (bCSCs) and non-bCSCs. A total of 68 differentially methylated regions (DMRs) were more hypomethylated in bCSCs than in non-bCSCs. Using a differentiation assay we demonstrated that DMRs are rapidly hypermethylated within the first 6 hours following induction of CSC differentiation whereas the cells reached the steady-state within 6 days, suggesting that these DMRs are linked to early CSC epigenetic regulation. These DMRs were significantly enriched in genes coding for TGF-ß signaling-related proteins. Interestingly, DMRs hypomethylation was correlated to an overexpression of TGF-ß signaling genes in a series of 109 breast tumors. Moreover, patients with tumors harboring the bCSC DMRs signature had a worse prognosis than those with non-bCSC DMRs signature. Our results show that bCSCs have a distinct DNA methylation landscape with TGF-ß signaling as a key epigenetic regulator of bCSCs differentiation.

MicroRNA93 regulates proliferation and differentiation of normal and malignant breast stem cells.

MicroRNAs (miRNAs) play important roles in normal cellular differentiation and oncogenesis. microRNA93 (mir-93), a member of the mir106b-25 cluster, located in intron 13 of the MCM7 gene, although frequently overexpressed in human malignancies may also function as a tumor suppressor gene. Using a series of breast cancer cell lines representing different stages of differentiation and mouse xenograft models, we demonstrate that mir-93 modulates the fate of breast cancer stem cells (BCSCs) by regulating their proliferation and differentiation states. In "claudin(low)" SUM159 cells, expression of mir-93 induces Mesenchymal-Epithelial Transition (MET) associated with downregulation of TGFß signaling and downregulates multiple stem cell regulatory genes, including JAK1, STAT3, AKT3, SOX4, EZH1, and HMGA2, resulting in cancer stem cell (CSC) depletion. Enforced expression of mir-93 completely blocks tumor development in mammary fat pads and development of metastases following intracardiac injection in mouse xenografts. The effect of mir-93 on the CSC population is dependent on the cellular differentiation state, with mir-93 expression increasing the CSC population in MCF7 cells that display a more differentiated "luminal" phenotype. mir-93 also regulates the proliferation and differentiation of normal breast stem cells isolated from reduction mammoplasties. These studies demonstrate that miRNAs can regulate the states and fates of normal and malignant mammary stem cells, findings which have important biological and clinical implications.

[Adenomyoepithelioma with dominant myoepithelial contingent of the breast: A case report and literature review]. / Adénomyoépithéliome avec contingent myoépithélial prédominant du sein : à propos d'une observation et revue de la littérature.

We report a case of adenomyoepithelioma with predominant myoepithelial quota, a rare tumor of the breast due to proliferation of epithelial and myoepithelial cells in a patient of 71 years. This lesion, with difficult radiological and pathological diagnosis (biopsy) in the initial stage of the treatment, should benefit from surgical resection in healthy margin. In fact, this tumor is evolving in most cases on a benin mode, but cases of local or metastatic recurrences were reported. Histological and immunohistochemical arguments are important to reach the final diagnosis.

[Pre-analytical stage for biomarker assessment in breast cancer: 2014 update of the GEFPICS' guidelines in France]. / Recommandations du GEFPICS concernant la phase pré-analytique pour l'évaluation de HER2 et des récepteurs hormonaux dans le cancer du sein : mise à jour 2014.

Biomarker assessment of breast cancer tumor samples is part of the routine workflow of pathology laboratories. International guidelines have recently been updated, with special regards to the pre-analytical steps that are critical for the quality of immunohistochemical and in situ hybridization procedures, whatever the biomarker analyzed. Fixation and specimen handling protocols must be standardized, validated and carefully tracked. Cooperation and training of the personnel involved in the specimen workflow (e.g. radiologists, surgeons, nurses, technicians and pathologists) are of paramount importance. The GEFPICS' update of the recommendations herein details and comments the different steps of the pre-analytical process. Application of these guidelines and participation to quality insurance programs are mandatory to ensure the correct evaluation of oncotheranostic biomarkers.

[2014 update of the GEFPICS' recommendations for HER2 status determination in breast cancers in France]. / Mise à jour 2014 des recommandations du GEFPICS pour l'évaluation du statut HER2 dans les cancers du sein en France.

International guidelines on HER2 determination in breast cancer have just been updated by the American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP), on the basis of more than ten-year practice, results of clinical trials and concordance studies. The GEFPICS group, composed of expert pathologists in breast cancer, herein presents these recommendations, adapted to the French routine practice. These guidelines highlight the possible diagnosis difficulties with regards to HER2 status determination, such as intra-tumor heterogeneity, special histological subtypes and biomarker re-evaluation during metastatic relapse. Pre-analytical issues and updated scoring criteria (especially for equivocal cases) are detailed, in order to decrease the occurrence of false negative cases. In the era of personalized medicine, pathologists are more than ever involved in the quality of oncotheranostic biomarker evaluation.

A novel bioinformatic approach reveals cooperation between Cancer/Testis genes in basal-like breast tumors.

Breast cancer is the most prevalent type of cancer in women worldwide. Within breast tumors, the basal-like subtype has the worst prognosis, prompting the need for new tools to understand, detect, and treat these tumors. Certain germline-restricted genes show aberrant expression in tumors and are known as Cancer/Testis genes; their misexpression has diagnostic and therapeutic applications. Here we designed a new bioinformatic approach to examine Cancer/Testis gene misexpression in breast tumors. We identify several new markers in Luminal and HER-2 positive tumors, some of which predict response to chemotherapy. We then use machine learning to identify the two Cancer/Testis genes most associated with basal-like breast tumors: HORMAD1 and CT83. We show that these genes are expressed by tumor cells and not by the microenvironment, and that they are not expressed by normal breast progenitors; in other words, their activation occurs de novo. We find these genes are epigenetically repressed by DNA methylation, and that their activation upon DNA demethylation is irreversible, providing a memory of past epigenetic disturbances. Simultaneous expression of both genes in breast cells in vitro has a synergistic effect that increases stemness and activates a transcriptional profile also observed in double-positive tumors. Therefore, we reveal a functional cooperation between Cancer/Testis genes in basal breast tumors; these findings have consequences for the understanding, diagnosis, and therapy of the breast tumors with the worst outcomes.

Mevalonate metabolism regulates Basal breast cancer stem cells and is a potential therapeutic target.

There is increasing evidence that breast tumors are organized in a hierarchy, with a subpopulation of tumorigenic cancer cells, the cancer stem cells (CSCs), which sustain tumor growth. The characterization of protein networks that govern CSC behavior is paramount to design new therapeutic strategies targeting this subpopulation of cells. We have sought to identify specific molecular pathways of CSCs isolated from 13 different breast cancer cell lines of luminal or basal/mesenchymal subtypes. We compared the gene expression profiling of cancer cells grown in adherent conditions to those of matched tumorsphere cultures. No specific pathway was identified to be commonly regulated in luminal tumorspheres, resulting from a minor CSC enrichment in tumorsphere passages from luminal cell lines. However, in basal/mesenchymal tumorspheres, the enzymes of the mevalonate metabolic pathway were overexpressed compared to those in cognate adherent cells. Inhibition of this pathway with hydroxy-3-methylglutaryl CoA reductase blockers resulted in a reduction of breast CSC independent of inhibition of cholesterol biosynthesis and of protein farnesylation. Further modulation of this metabolic pathway demonstrated that protein geranylgeranylation (GG) is critical to breast CSC maintenance. A small molecule inhibitor of the geranylgeranyl transferase I (GGTI) enzyme reduced the breast CSC subpopulation both in vitro and in primary breast cancer xenografts. We found that the GGTI effect on the CSC subpopulation is mediated by inactivation of Ras homolog family member A (RHOA) and increased accumulation of P27(kip1) in the nucleus. The identification of protein GG as a major contributor to CSC maintenance opens promising perspectives for CSC targeted therapy in basal breast cancer.

What drives breast cancer heterogeneity: oncogenic events or cell of origin?

Breast cancer is known to show considerable inter-tumoural heterogeneity. It is widely accepted that combinations of oncogenic events have a major role in determining tumour phenotype. However, accumulating evidence suggests that the identity of the cell that acquires the first oncogenic event, the so-called cell of origin, may define the molecular subtype of the resulting tumour. Recent work published in the Journal of Pathology by Natrajan and colleagues questions the origin of breast cancer heterogeneity. After studying BRCA1 tumours, they suggest that genomic alterations are not sufficient to determine tumour behaviour. These and other recent observations underscore the importance of defining what is causing tumour heterogeneity, so that appropriate therapy can be given.

Neoadjuvant bevacizumab, trastuzumab, and chemotherapy for primary inflammatory HER2-positive breast cancer (BEVERLY-2): an open-label, single-arm phase 2 study.

BACKGROUND: Bevacizumab and trastuzumab are efficacious for treatment of advanced or HER2-positive metastatic breast cancer; however, few data exist for this regimen in inflammatory breast cancer. In our phase 2 trial, we aimed to assess efficacy and safety of neoadjuvant bevacizumab combined with trastuzumab and chemotherapy in patients with primary HER2-positive inflammatory breast cancer. METHODS: In our phase 2, multicentre, open-label, single-arm, non-comparative trial, we enrolled women (aged ≥ 18 years) with histologically confirmed HER2-positive non-metastatic inflammatory breast cancer at private or public oncology centres in France. Before surgery, patients were treated with fluorouracil, epirubicin, cyclophosphamide, and bevacizumab (cycles 1-4) and docetaxel, bevacizumab, and trastuzumab (cycles 5-8) in 3-week cycles. After surgery, patients received adjuvant radiotherapy, trastuzumab, and bevacizumab. For the primary endpoint, we assessed the proportion of patients who achieved a pathological complete response (defined by central review of surgical specimens according to Sataloff classification, counting missing data as failure) and adverse events in all enrolled patients. This study is registered with ClinicalTrials.gov, number NCT00717405. FINDINGS: Between Oct 23, 2008, and Oct 28, 2009, we enrolled 52 patients at 21 centres. 42 (81%) of 52 patients received all eight cycles of neoadjuvant therapy and 49 (94%) underwent surgery. After neoadjuvant therapy, 33 of 52 patients had a pathological complete response according to central review (63·5%, 95% CI 49·4-77·5). The most common adverse events were asthenia and nausea (both occurred in 36 [69%] of 52 patients). 25 (48%) patients had grade 3-4 neutropenia, which was the most common grade 3-4 adverse event. Only one grade 3 or worse adverse event regarded as related to bevacizumab was reported (hypertension, one patient). Four patients (8%) had cardiac failure. INTERPRETATION: Neoadjuvant treatment with bevacizumab, trastuzumab, and chemotherapy was efficacious and well tolerated in patients with previously untreated primary inflammatory breast cancer. Further confirmation of use of bevacizumab in inflammatory breast cancer is needed. FUNDING: Roche (France).