Toll-like receptor 3: implications for proinflammatory microenvironment in human breast cancer

Under many circumstances, the host constituents that are found in the tumor microenvironment support a malignancy network and provide the cancer cells with advantages in proliferation, invasiveness and metastasis establishment at remote organs. It is known that Toll like receptors (TLRs) are expressed not only on immune cells but also on cancer cells and it has suggested a deleterious role for TLR3 in inflammatory disease. Hypothesizing that altered IFN gamma signaling may be a key mechanism of immune dysfunction common to cancer as well CXCR4 is overexpressed among breast cancer patients, the mRNA expression of TLR3, CXCR4 and IFN gamma in breast cancer tumor tissues was investigated. No statistically significant differences in the expression of CXCR4 mRNA, IFN gamma and TLR3 between healthy and tumor tissues was observed, however, it was verified a positive correlation between mRNA relative expression of TLR3 and CXCR4 (p < 0.001), and mRNA relative expression of TLR3 was significantly increased in breast cancer tumor tissue when compared to healthy mammary gland tissue among patients expressing high IFN gamma (p = 0.001). Since the tumor microenvironment plays important roles in cancer initiation, growth, progression, invasion and metastasis, it is possible to propose that an overexpression of IFN gamma mRNA due to the pro-inflammatory microenvironment can lead to an up-regulation of CXCR4 mRNA and consequently to an increased TLR3 mRNA expression even among nodal negative patients. In the future, a comprehensive study of TLR3, CXCR4 and IFN gamma axis in primary breast tumors and corresponding healthy tissues will be crucial to further understanding of the cancer network.

Efficacy of the dietary histone deacetylase inhibitor butyrate alone or in combination with vitamin A against proliferation of MCF-7 human breast cancer cells

The combined treatment with histone deacetylase inhibitors (HDACi) and retinoids has been suggested as a potential epigenetic strategy for the control of cancer. In the present study, we investigated the effects of treatment with butyrate, a dietary HDACi, combined with vitamin A on MCF-7 human breast cancer cells. Cell proliferation was evaluated by the crystal violet staining method. MCF-7 cells were plated at 5 x 10(4) cells/mL and treated with butyrate (1 mM) alone or combined with vitamin A (10 µM) for 24 to 120 h. Cell proliferation inhibition was 34, 10 and 46% following treatment with butyrate, vitamin A and their combination, respectively, suggesting that vitamin A potentiated the inhibitory activities of butyrate. Furthermore, exposure to this short-chain fatty acid increased the level of histone H3K9 acetylation by 9.5-fold (Western blot), but not of H4K16, and increased the expression levels of p21WAF1 by 2.7-fold (Western blot) and of RARβ by 2.0-fold (quantitative real-time PCR). Our data show that RARβ may represent a molecular target for butyrate in breast cancer cells. Due to its effectiveness as a dietary HDACi, butyrate should be considered for use in combinatorial strategies with more active retinoids, especially in breast cancers in which RARβ is epigenetically altered.

Bone mineral density in postmenopausal women with and without breast cancer

OBJECTIVE: The values of bone mineral density (BMD) were compared in postmenopausal women with and without breast cancer. METHODS: A cross-sectional study was conducted, including 51 breast cancer survivors (BCS) and 71 women without breast cancer, who were non-users of hormone therapy, tamoxifen, or aromatase inhibitors. BMD T-scores and measurements in grams per centimeter squared (g/cm²) were obtained at the femoral neck, trochanter, Ward's triangle, and lumbar spine. Osteopenia and osteoporosis were grouped and categorized as abnormal BMD. Unconditional logistic regression analysis was used to estimate the odds ratios (OR) of abnormal BMD values as measures of association, with 95% confidence intervals (CIs), adjusting for age, years since menopause, parity, and body mass index (BMI). RESULTS: The mean age of the women with and without breast cancer was 54.7 ± 5.8 years and 58.2 ± 4.8 years (p < 0.01), respectively. After adjusting for age, parity and BMI, abnormal BMD at the femoral neck (adjusted OR: 4.8; 95% CI: 1.5-15.4), trochanter (adjusted OR: 4.6; 95% CI: 1.4-15.5), and Ward's triangle (adjusted OR: 4.5; 95% CI: 1.5-12.9) were significantly more frequent in postmenopausal BCS than in women without breast cancer. Postmenopausal BCS had a significantly lower mean BMD at the trochanter (0.719 vs. 0.809 g/cm², p < 0.01) and at the Ward's triangle (0.751 vs. 0.805 g/cm², p = 0.03). CONCLUSION: The prevalence of abnormal BMD was higher in postmenopausal BCS than in postmenopausal women without breast cancer. Bone health requires special vigilance and the adoption of interventions should be instituted early to minimize bone loss in BCS.

Differential expression of CD90 and CD14 stem cell markers in malignant breast cancer cell lines

The recently emerged concept of cancer stem cell (CSC) has led to a new hypothesis on the basis for tumor progression. Basically, the CSC theory hypothesizes the presence of a hierarchically organized and relatively rare cell population, which is responsible for tumor initiation, self-renewal, and maintenance, in addition to accumulation of mutation and resistance to chemotherapy. CSCs have recently been described in breast cancer. Different genetic markers have been used to isolate breast CSCs, none of which have been correlated with the tumorigenicity or metastatic potential of the cells, limiting their precise characterization and clinical application in the development of therapeutic protocols. Here, we sought for subpopulations of CSCs by analyzing 10 judiciously chosen stem cell markers in a normal breast cell line (MCF10-A) and in four human breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-435, and Hs578-T) displaying different degrees of metastatic and invasiveness potential. We were able to identify two markers, which are differentially expressed in nontumorigenic versus tumor cells. The CD90 marker was highly expressed in the malignant cell lines. Interestingly, the CD14 molecule displayed higher expression levels in the nontumorigenic cell line. Therefore, we demonstrated that these two markers, which are more commonly used to isolate and characterize stem cells, are differentially expressed in breast tumor cells, when compared with nontumorigenic breast cells. (C) 2012 International Society for Advancement of Cytometry

TGF-beta 1 modulates the homeostasis between MMPs and MMP inhibitors through p38 MAPK and ERK1/2 in highly invasive breast cancer cells

Background: Metastasis is the main factor responsible for death in breast cancer patients. Matrix metalloproteinases (MMPs) and their inhibitors, known as tissue inhibitors of MMPs (TIMPs), and the membrane-associated MMP inhibitor (RECK), are essential for the metastatic process. We have previously shown a positive correlation between MMPs and their inhibitors expression during breast cancer progression; however, the molecular mechanisms underlying this coordinate regulation remain unknown. In this report, we investigated whether TGF-beta 1 could be a common regulator for MMPs, TIMPs and RECK in human breast cancer cell models. Methods: The mRNA expression levels of TGF-beta isoforms and their receptors were analyzed by qRT-PCR in a panel of five human breast cancer cell lines displaying different degrees of invasiveness and metastatic potential. The highly invasive MDA-MB-231 cell line was treated with different concentrations of recombinant TGF-beta 1 and also with pharmacological inhibitors of p38 MAPK and ERK1/2. The migratory and invasive potential of these treated cells were examined in vitro by transwell assays. Results: In general, TGF-beta 2, T beta RI and T beta RII are over-expressed in more aggressive cells, except for T beta RI, which was also highly expressed in ZR-75-1 cells. In addition, TGF-beta 1-treated MDA-MB-231 cells presented significantly increased mRNA expression of MMP-2, MMP-9, MMP-14, TIMP-2 and RECK. TGF-beta 1 also increased TIMP-2, MMP-2 and MMP-9 protein levels but downregulated RECK expression. Furthermore, we analyzed the involvement of p38 MAPK and ERK1/2, representing two well established Smad-independent pathways, in the proposed mechanism. Inhibition of p38MAPK blocked TGF-beta 1-increased mRNA expression of all MMPs and MMP inhibitors analyzed, and prevented TGF-beta 1 upregulation of TIMP-2 and MMP-2 proteins. Moreover, ERK1/2 inhibition increased RECK and prevented the TGF-beta 1 induction of pro-MMP-9 and TIMP-2 proteins. TGF-beta 1-enhanced migration and invasion capacities were blocked by p38MAPK, ERK1/2 and MMP inhibitors. Conclusion: Altogether, our results support that TGF-beta 1 modulates the mRNA and protein levels of MMPs (MMP-2 and MMP-9) as much as their inhibitors (TIMP-2 and RECK). Therefore, this cytokine plays a crucial role in breast cancer progression by modulating key elements of ECM homeostasis control. Thus, although the complexity of this signaling network, TGF-beta 1 still remains a promising target for breast cancer treatment.

TGF-β1 modulates the homeostasis between MMPs and MMP inhibitors through p38 MAPK and ERK1/2 in highly invasive breast cancer cells

Abstract Background Metastasis is the main factor responsible for death in breast cancer patients. Matrix metalloproteinases (MMPs) and their inhibitors, known as tissue inhibitors of MMPs (TIMPs), and the membrane-associated MMP inhibitor (RECK), are essential for the metastatic process. We have previously shown a positive correlation between MMPs and their inhibitors expression during breast cancer progression; however, the molecular mechanisms underlying this coordinate regulation remain unknown. In this report, we investigated whether TGF-β1 could be a common regulator for MMPs, TIMPs and RECK in human breast cancer cell models. Methods The mRNA expression levels of TGF-β isoforms and their receptors were analyzed by qRT-PCR in a panel of five human breast cancer cell lines displaying different degrees of invasiveness and metastatic potential. The highly invasive MDA-MB-231 cell line was treated with different concentrations of recombinant TGF-β1 and also with pharmacological inhibitors of p38 MAPK and ERK1/2. The migratory and invasive potential of these treated cells were examined in vitro by transwell assays. Results In general, TGF-β2, TβRI and TβRII are over-expressed in more aggressive cells, except for TβRI, which was also highly expressed in ZR-75-1 cells. In addition, TGF-β1-treated MDA-MB-231 cells presented significantly increased mRNA expression of MMP-2, MMP-9, MMP-14, TIMP-2 and RECK. TGF-β1 also increased TIMP-2, MMP-2 and MMP-9 protein levels but downregulated RECK expression. Furthermore, we analyzed the involvement of p38 MAPK and ERK1/2, representing two well established Smad-independent pathways, in the proposed mechanism. Inhibition of p38MAPK blocked TGF-β1-increased mRNA expression of all MMPs and MMP inhibitors analyzed, and prevented TGF-β1 upregulation of TIMP-2 and MMP-2 proteins. Moreover, ERK1/2 inhibition increased RECK and prevented the TGF-β1 induction of pro-MMP-9 and TIMP-2 proteins. TGF-β1-enhanced migration and invasion capacities were blocked by p38MAPK, ERK1/2 and MMP inhibitors. Conclusion Altogether, our results support that TGF-β1 modulates the mRNA and protein levels of MMPs (MMP-2 and MMP-9) as much as their inhibitors (TIMP-2 and RECK). Therefore, this cytokine plays a crucial role in breast cancer progression by modulating key elements of ECM homeostasis control. Thus, although the complexity of this signaling network, TGF-β1 still remains a promising target for breast cancer treatment.

HER-2, p53, p21 and hormonal receptors proteins expression as predictive factors of response and prognosis in locally advanced breast cancer treated with neoadjuvant docetaxel plus epirubicin combination

Abstract Background Neoadjuvant chemotherapy has been considered the standard care in locally advanced breast cancer. However, about 20% of the patients do not benefit from this clinical treatment and, predictive factors of response were not defined yet. This study was designed to evaluate the importance of biological markers to predict response and prognosis in stage II and III breast cancer patients treated with taxane and anthracycline combination as neoadjuvant setting. Methods Sixty patients received preoperative docetaxel (75 mg/m2) in combination with epirubicin (50 mg/m2) in i.v. infusion in D1 every 3 weeks after incisional biopsy. They received adjuvant chemotherapy with CMF or FEC, attaining axillary status following definitive breast surgery. Clinical and pathologic response rates were measured after preoperative therapy. We evaluated the response rate to neoadjuvant chemotherapy and the prognostic significance of clinicopathological and immunohistochemical parameters (ER, PR, p51, p21 and HER-2 protein expression). The median patient age was 50.5 years with a median follow up time 48 months after the time of diagnosis. Results Preoperative treatment achieved clinical response in 76.6% of patients and complete pathologic response in 5%. The clinical, pathological and immunohistochemical parameters were not able to predict response to therapy and, only HER2 protein overexpression was associated with a decrease in disease free and overall survival (P = 0.0007 and P = 0.003) as shown by multivariate analysis. Conclusion Immunohistochemical phenotypes were not able to predict response to neoadjuvant chemotherapy. Clinical response is inversely correlated with a risk of death in patients submitted to neoadjuvant chemotherapy and HER2 overexpression is the major prognostic factor in stage II and III breast cancer patients treated with a neoadjuvant docetaxel and epirubicin combination.

Study of electron densities of normal and neoplastic human breast tissues by Compton scattering using synchrotron radiation

Electron densities of 33 samples of normal (adipose and fibroglangular) and neoplastic (benign and malignant) human breast tissues were determined through Compton scattering data using a mono-chromatic synchrotron radiation source and an energy dispersive detector. The area of Compton peaks was used to determine the electron densities of the samples. Adipose tissue exhibits the lowest values of electron density whereas malignant tissue the highest. The relationship with their histology was discussed. Comparison with previous results showed differences smaller than 4%. (C) 2012 Elsevier Ltd. All rights reserved.

Germline DNA copy number variation in familial and early-onset breast cancer

Introduction: Genetic factors predisposing individuals to cancer remain elusive in the majority of patients with a familial or clinical history suggestive of hereditary breast cancer. Germline DNA copy number variation (CNV) has recently been implicated in predisposition to cancers such as neuroblastomas as well as prostate and colorectal cancer. We evaluated the role of germline CNVs in breast cancer susceptibility, in particular those with low population frequencies (rare CNVs), which are more likely to cause disease." Methods: Using whole-genome comparative genomic hybridization on microarrays, we screened a cohort of women fulfilling criteria for hereditary breast cancer who did not carry BRCA1/BRCA2 mutations. Results: The median numbers of total and rare CNVs per genome were not different between controls and patients. A total of 26 rare germline CNVs were identified in 68 cancer patients, however, a proportion that was significantly different (P = 0.0311) from the control group (23 rare CNVs in 100 individuals). Several of the genes affected by CNV in patients and controls had already been implicated in cancer. Conclusions: This study is the first to explore the contribution of germline CNVs to BRCA1/2-negative familial and early-onset breast cancer. The data suggest that rare CNVs may contribute to cancer predisposition in this small cohort of patients, and this trend needs to be confirmed in larger population samples.

Angiotensin II Facilitates Breast Cancer Cell Migration and Metastasis

Breast cancer metastasis is a leading cause of death by malignancy in women worldwide. Efforts are being made to further characterize the rate-limiting steps of cancer metastasis, i.e. extravasation of circulating tumor cells and colonization of secondary organs. In this study, we investigated whether angiotensin II, a major vasoactive peptide both produced locally and released in the bloodstream, may trigger activating signals that contribute to cancer cell extravasation and metastasis. We used an experimental in vivo model of cancer metastasis in which bioluminescent breast tumor cells (D3H2LN) were injected intra-cardiacally into nude mice in order to recapitulate the late and essential steps of metastatic dissemination. Real-time intravital imaging studies revealed that angiotensin II accelerates the formation of metastatic foci at secondary sites. Pre-treatment of cancer cells with the peptide increases the number of mice with metastases, as well as the number and size of metastases per mouse. In vitro, angiotensin II contributes to each sequential step of cancer metastasis by promoting cancer cell adhesion to endothelial cells, trans-endothelial migration and tumor cell migration across extracellular matrix. At the molecular level, a total of 102 genes differentially expressed following angiotensin II pretreatment were identified by comparative DNA microarray. Angiotensin II regulates two groups of connected genes related to its precursor angiotensinogen. Among those, up-regulated MMP2/MMP9 and ICAM1 stand at the crossroad of a network of genes involved in cell adhesion, migration and invasion. Our data suggest that targeting angiotensin II production or action may represent a valuable therapeutic option to prevent metastatic progression of invasive breast tumors.

Analysis of the concordance rates between core needle biopsy and surgical excision in patients with breast cancer

Objective: To evaluate whether immunohistochemical marker studies performed on core needle biopsy (CNB) specimens accurately reflect the marker status of the tumor obtained from final surgical specimen. Methods: This was a retrospective study that used the database of the Division of Mastology of the Hospital das Clinicas, Sao Paulo, Brazil. Sixty-nine patients submitted to ultrasound-guided CNB diagnosed with breast cancer were retrospectively analyzed. Immunohistochemistry (IHC) on core biopsy specimens was compared to that of excisional biopsy regarding estrogen receptor (ER), progesterone receptor (PR), human epidermal gowth factor receptor 2 gene (HER2), p53, and Ki67. The analysis of the concordance between CNB and surgical biopsy was performed using the kappa (k) coefficient (95% CI). Results: A perfect concordance between the labeling in the surgical specimens and the preoperative biopsies in p53 (k = 1.0; 95% CI: 0.76-1.0) was identified. There was an almost perfect concordance for ER (k = 0.89; 95% CI: 0.65-1.0) and a substantial concordance for PR (k = 0.70; 95% CI: 0.46-0.93). HER2 (k = 0.61; 95% CI: 0.38-0.84) and Ki-67 (k = 0.74; 95% CI: 0.58-0.98) obtained a substantial concordance this analysis. Conclusion: The results of this study indicate that the immunohistochemical analysis of ER, PR, Ki-67, and p53 from core biopsy specimens provided results that accurately reflect the marker status of the tumor. The concordance rate of HER2 was less consistent; although it produced substantial concordance, values were very close to moderate concordance.

Expression of BAG-1 and PARP-1 in Precursor Lesions and Invasive Cervical Cancer Associated with Human Papillomavirus (HPV)

Cervical cancer remains persistently the second most common malignancies among women worldwide, responsible for 500,000 new cases annually. Only in Brazil, the estimate is for 18,430 new cases in 2011. Several types of molecular markers have been studied in carcinogenesis including proteins associated with apoptosis such as BAG-1 and PARP-1. This study aims to demonstrate the expression of BAG-1 and PARP-1 in patients with low-grade squamous intraepithelial lesions (LSILs), high-grade squamous intraepithelial lesions (HSILs) and invasive squamous cell carcinomas (SCCs) of the uterine cervix and to verify a possible association with HPV infection. Fifty samples of LSILs, 50 samples of HSILs and 50 samples of invasive SCCs of the uterine cervix were analyzed by immunohistochemistry for BAG-1 and PARP-1 expression. PCR was performed to detect and type HPV DNA. BAG-1 expression levels were significantly different between LSILs and HSILs (p = 0,014) and between LSILs and SCCs (p = 0,014). In regards to PARP-1 expression, we found significant differences between the expression levels in HSILs and SCCs (p = 0,022). No association was found between BAG-1 expression and the presence of HPV. However, a significant association was found between PARP-1 expression and HPV positivity in the HSILs group (p = 0,021). In conclusion our research suggests that BAG-1 expression could contribute to the differentiation between LSIL and HSIL/SCC whereas PARP-1 could be useful to the differentiation between HSIL HPV-related and SCC. Further studies are needed to clarify the molecular aspects of the relationship between PARP-1 expression and HPV infection, with potential applications for cervical cancer prediction.

Decreased expression of ADAMTS-1 in human breast tumors stimulates migration and invasion

Abstract Background ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motifs) is a member of the ADAMTS family of metalloproteases. Here, we investigated mRNA and protein levels of ADAMTS-1 in normal and neoplastic tissues using qPCR, immunohistochemistry and immunoblot analyses, and we addressed the role of ADAMTS-1 in regulating migration, invasion and invadopodia formation in breast tumor cell lines. Results In a series of primary breast tumors, we observed variable levels of ADAMTS-1 mRNA expression but lower levels of ADAMTS-1 protein expression in human breast cancers as compared to normal tissue, with a striking decrease observed in high-malignancy cases (triple-negative for estrogen, progesterone and Her-2). This result prompted us to analyze the effect of ADAMTS-1 knockdown in breast cancer cells in vitro. MDA-MB-231 cells with depleted ADAMTS-1 expression demonstrated increased migration, invasion and invadopodia formation. The regulatory mechanisms underlying the effects of ADAMTS-1 may be related to VEGF, a growth factor involved in migration and invasion. MDA-MB-231 cells with depleted ADAMTS-1 showed increased VEGF concentrations in conditioned medium capable of inducing human endothelial cells (HUVEC) tubulogenesis. Furthermore, expression of the VEGF receptor (VEGFR2) was increased in MDA-MB-231 cells as compared to MCF7 cells. To further determine the relationship between ADAMTS-1 and VEGF regulating breast cancer cells, MDA-MB-231 cells with reduced expression of ADAMTS-1 were pretreated with a function-blocking antibody against VEGF and then tested in migration and invasion assays; both were partially rescued to control levels. Conclusions ADAMTS-1 expression was decreased in human breast tumors, and ADAMTS-1 knockdown stimulated migration, invasion and invadopodia formation in breast cancer cells in vitro. Therefore, this series of experiments suggests that VEGF is involved in the effects mediated by ADAMTS-1 in breast cancer cells.

Sorafenib in Combination With Capecitabine: An Oral Regimen for Patients With HER2-Negative Locally Advanced or Metastatic Breast Cancer

Purpose Sorafenib is a multikinase inhibitor with antiangiogenic/antiproliferative activity. A randomized, double-blind, placebo-controlled phase IIB trial assessed sorafenib with capecitabine for locally advanced or metastatic human epidermal growth factor receptor 2 (HER2) -negative breast cancer. Patients and Methods Patients were randomly assigned to first-or second-line capecitabine 1,000 mg/m(2) orally twice a day for days 1 to 14 of every 21-day cycle with sorafenib 400 mg orally twice a day or placebo. The primary end point was progression-free survival (PFS). Results In total, 229 patients were enrolled. The addition of sorafenib to capecitabine resulted in a significant improvement in PFS versus placebo (median, 6.4 v 4.1 months; hazard ratio [HR], 0.58; 95% CI, 0.41 to 0.81; P = .001) with sorafenib favored across subgroups, including first-line (HR, 0.50; 95% CI, 0.30 to 0.82) and second-line (HR, 0.65; 95% CI, 0.41 to 1.04) treatment. There was no significant improvement for overall survival (median, 22.2 v 20.9 months; HR, 0.86; 95% CI, 0.61 to 1.23; P = .42) and overall response (38% v 31%; P = .25). Toxicities (sorafenib v placebo) of any grade included rash (22% v 8%), diarrhea (58% v 30%), mucosal inflammation (33% v 21%), neutropenia (13% v 4%), hypertension (18% v 12%), and hand-foot skin reaction/hand-foot syndrome (HFSR/HFS; 90% v 66%); grade 3 to 4 toxicities were comparable between treatment arms except HFSR/HFS (44% v 14%). Reasons for discontinuation in the sorafenib and placebo arms included disease progression (63% v 82%, respectively), adverse events (20% v 9%, respectively), and death (0% v 1%, respectively). Conclusion Addition of sorafenib to capecitabine improved PFS in patients with HER2-negative advanced breast cancer. The dose of sorafenib used in this trial resulted in unacceptable toxicity for many patients. A phase III confirmatory trial has been initiated with a reduced sorafenib dose.

Pertuzumab plus Trastuzumab plus Docetaxel for Metastatic Breast Cancer

BACKGROUND The anti-human epidermal growth factor receptor 2 (HER2) humanized monoclonal antibody trastuzumab improves the outcome in patients with HER2-positive metastatic breast cancer. However, most cases of advanced disease eventually progress. Pertuzumab, an anti-HER2 humanized monoclonal antibody that inhibits receptor dimerization, has a mechanism of action that is complementary to that of trastuzumab, and combination therapy with the two antibodies has shown promising activity and an acceptable safety profile in phase 2 studies involving patients with HER2-positive breast cancer. METHODS We randomly assigned 808 patients with HER2-positive metastatic breast cancer to receive placebo plus trastuzumab plus docetaxel (control group) or pertuzumab plus trastuzumab plus docetaxel (pertuzumab group) as first-line treatment until the time of disease progression or the development of toxic effects that could not be effectively managed. The primary end point was independently assessed progression-free survival. Secondary end points included overall survival, progression-free survival as assessed by the investigator, the objective response rate, and safety. RESULTS The median progression-free survival was 12.4 months in the control group, as compared with 18.5 months in the pertuzumab group (hazard ratio for progression or death, 0.62; 95% confidence interval, 0.51 to 0.75; P<0.001). The interim analysis of overall survival showed a strong trend in favor of pertuzumab plus trastuzumab plus docetaxel. The safety profile was generally similar in the two groups, with no increase in left ventricular systolic dysfunction; the rates of febrile neutropenia and diarrhea of grade 3 or above were higher in the pertuzumab group than in the control group. CONCLUSIONS The combination of pertuzumab plus trastuzumab plus docetaxel, as compared with placebo plus trastuzumab plus docetaxel, when used as first-line treatment for HER2-positive metastatic breast cancer, significantly prolonged progression-free survival, with no increase in cardiac toxic effects. (Funded by F. Hoffmann-La Roche/Genentech; ClinicalTrials.gov number, NCT00567190.)