Lapatinib Sensitivity Research Articles

Abstract A6: Sustained MAPK signaling contributes to reduced sensitivity to lapatinib

Abstract Background: Approximately 20% to 30% of metastatic breast cancers overexpress the HER2 receptor tyrosine kinase. The standard front-line therapy for HER2-overexpressing breast cancer is the HER2 monoclonal antibody trastuzumab. However, for patients whose disease progresses on trastuzumab-based therapy, the dual EGFR/HER2 small-molecule tyrosine kinase inhibitor lapatinib is available. Unfortunately, the majority of metastatic breast cancer patients who do not respond to trastuzumab also show poor response to lapatinib. The mechanisms underlying lapatinib resistance are poorly understood. Elucidating these mechanisms may ultimately allow new therapeutic strategies to be developed for patients with HER2-overexpressing breast cancer. We have found that breast cancer cells with reduced sensitivity to lapatinib exhibit sustained mTORc1 and MAPK signaling. We previously showed that genetic knockdown or pharmacologic inhibition of PI3K/mTORc1 signaling improves response to lapatinib. In our current work, we examined the role of MAPK signaling in lapatinib sensitivity. Materials and Methods: Lapatinib-sensitive BT474 and HCC1419 cells, and lapatinib-resistant MDA361 cells were purchased from American Type Culture Collection, Manassas, VA. Lapatinib-resistant JIMT1 cells were purchased from DSMZ, Germany. MAPK signaling was blocked pharmacologically and genetically, and lapatinib response was then evaluated using trypan blue exclusion and anchorage-independent growth assays. Combined inhibition of MEK and lapatinib was analyzed statistically by CalcuSyn software to generate drug combination indices. Western blotting was performed to assess the effects of combination lapatinib and MEK inhibitor on HER2 signaling. Results and Discussion: MDA361 and JIMT1 cells, which have reduced sensitivity to lapatinib, exhibited sustained MAPK signaling after lapatinib treatment relative to lapatinib-sensitive BT474 and HCC1419 cells. Pharmacologic MEK inhibition increased sensitivity to lapatinib in MDA361 and JIMT1 cells, as measured by trypan blue cell viability and anchorage-independent three-dimensional assays. The combination of MEK inhibitor plus lapatinib showed pharmacologic synergy in JIMT1 and MDA361 cells, as indicated by combination indices below 1.0 using CalcuSyn software. At the molecular level, combined inhibition of MEK plus lapatinib resulted in greater reduction in phosphorylated MAPK versus either agent alone. In addition, our initial experiments suggested that combination MEK inhibitor plus lapatinib results in induction of Foxo1a and Foxo3a and reduced expression of FoxM1. Ongoing work will confirm the regulation of these forkhead transcription factors in lapatinib-resistant cells treated with MEK inhibitor plus lapatinib using Western blots, PCR, and immunofluorescence to examine nuclear translocation. Genetic knockdown of MEK will also be tested for the ability to increase lapatinib-mediated cell cycle arrest or apoptosis in JIMT1 and MDA361 cells. Finally, future xenograft studies will determine if pharmacologic inhibition of MEK increases lapatinib sensitivity in HER2-overexpressing breast cancers that are resistant to trastuzumab and lapatinib. Conclusions: In addition to our previous data showing that sustained mTORc1 signaling contributes to lapatinib resistance, we now show that resistant cells also exhibit sustained MAPK signaling. Our data supports further study of pharmacologic inhibition of mTORc1 and MAPK to improve response to lapatinib in HER2-overexpressing breast cancer.

Pharmacologic Inhibition of mTOR Improves Lapatinib Sensitivity in HER2-Overexpressing Breast Cancer Cells with Primary Trastuzumab Resistance

Lapatinib, a dual EGFR/HER2 kinase inhibitor, is approved for use in patients with trastuzumab-refractory HER2- overexpressing breast cancer. Increased PI3K signaling has been associated with resistance to trastuzumab, although its role in lapatinib resistance remains unclear. The purpose of the current study was to determine if PI3K/mTOR activity affects lapatinib sensitivity. Reduced sensitivity to lapatinib was associated with an inability of lapatinib to inhibit Akt and p70S6K phosphorylation. Transfection of constitutively active Akt reduced lapatinib sensitivity, while kinase-dead Akt increased sensitivity. Knockdown of 4EBP1 also increased lapatinib sensitivity, in contrast to p70S6K knockdown, which did not affect response to lapatinib. Pharmacologic inhibition of mTOR using rapamycin or ridaforolimus increased lapatinib sensitivity and reduced phospho-Akt levels in cells that showed poor response to single-agent lapatinib, including those transfected with hyperactive Akt. Finally, combination mTOR inhibition plus lapatinib resulted in synergistic inhibition of proliferation, reduced anchorage-independent growth, and reduced in vivo tumor growth of HER2- overexpressing breast cancer cells that have primary trastuzumab resistance. Our data suggest that PI3K/mTOR inhibition is critical for achieving optimal response to lapatinib. Collectively, these experiments support evaluation of lapatinib in combination with pharmacologic mTOR inhibition as a potential strategy for inhibiting growth of HER2-overexpressing breast cancers that show resistance to trastuzumab and poor response to lapatinib.

Neuregulin-1-Mediated Autocrine Signaling Underlies Sensitivity to HER2 Kinase Inhibitors in a Subset of Human Cancers

HER2 kinase inhibitors, such as lapatinib, have demonstrated clinical efficacy in HER2-amplified breast cancers. By profiling nearly 700 human cancer cell lines, we identified a subset of non-HER2 amplified cancer cells with striking sensitivity to HER2 kinase inhibition-particularly from head and neck tumors. These cells were found to depend on a neuregulin-1 (NRG1)-mediated autocrine loop driving HER3 activation, which can be disrupted by lapatinib. Elevated NRG1 expression and activated HER3 are strongly associated with lapatinib sensitivity invitro, and these biomarkers were enriched in a subset of primary head and neck cancer samples. The findings suggest that patients with NRG1-driven tumors lacking HER2 amplification may derive significant clinical benefit from HER2:HER3-directed therapies.

Phosphoproteomic mass spectrometry profiling links Src family kinases to escape from HER2 tyrosine kinase inhibition.

Despite the initial effectiveness of the tyrosine kinase inhibitor lapatinib against HER2 gene-amplified breast cancers, most patients eventually relapse after treatment, implying that tumors acquire mechanisms of drug resistance. To discover these mechanisms, we generated six lapatinib-resistant HER2-overexpressing human breast cancer cell lines. In cells that grew in the presence of lapatinib, HER2 autophosphorylation was undetectable whereas active PI3K-Akt and MAPK were maintained. To identify networks maintaining these signaling pathways, we profiled the tyrosine phosphoproteome of sensitive and resistant cells using an immunoaffinity-enriched mass spectrometry method. We found increased phosphorylation of Src family kinases (SFK) and putative Src substrates in several resistant cell lines. Treatment of these resistant cells with Src kinase inhibitors partially blocked PI3K-Akt signaling and restored lapatinib sensitivity. Further, SFK mRNA expression was upregulated in primary HER2+ tumors treated with lapatinib. Finally, the combination of lapatinib and the Src inhibitor AZD0530 was more effective than lapatinib alone at inhibiting pAkt and growth of established HER2-positive BT-474 xenografts in athymic mice. These data suggest that increased Src kinase activity is a mechanism of lapatinib resistance and support the combination of HER2 antagonists with Src inhibitors early in the treatment of HER2+ breast cancers in order to prevent or overcome resistance to HER2 inhibitors.

Abstract 708: Integrated analysis of drug resistance networks in breast cancer

Abstract Targeted inhibition of HER2 tyrosine kinase receptor presents a promising strategy in the treatment of HER2-positive breast cancer patients. However, despite favorable response during the acute phase, majority of patients develop resistance to anti-HER2 drugs during the chronic phase of treatment. Here, we developed a cell culture model of chronic lapatinib (a dual inhibitor of EGFR and ERBB2 receptor tyrosine kinases) treatment by continuous passaging of HER2-overexpressing SKBR3 cells in increasing doses of lapatinib over a year. The resultant cell line, SKBR3-R, is almost 100-fold more resistant to lapatinib-induced growth inhibition when compared to the parental line. We have verified that this difference in lapatinib sensitivity is not due to altered expression or signaling at the level of HER2 or HER3 receptors in SKBR3-R cells. By integrating genome-wide gene expression profiling with our novel network-based genomic data analysis approach, we show that 1) cell toxicity mediated by acute lapatinib treatment in HER2-positive cells is associated with nutrient starvation and energy stress, and 2) chronic lapatinib treatment leads to the activation of compensatory cellular networks involved in the response to nutrient starvation, which confers resistance to lapatinib-induced cell death. Accordingly, while imposing a glucose starvation response phenotype on SKBR3 cells protects from lapatinib-mediated cell death, pharmacological inhibition of the nutrient starvation response in SKBR3-R cells reverses lapatinib resistance. In order to see if these networks play a role in breast cancer pathogenicity within clinical settings, we carried out a detailed network-based correlations of tumor gene expression profiles of breast cancer patients with their clinical data. Importantly, we found that the metabolic networks associated with lapatinib resistance are also associated with poor outcome within clinical settings. This study implicates nutrient deprivation response as a major factor in the acquired resistance of breast cancer cells to lapatinib, and suggests novel targets for combinatorial therapy of HER2-positive breast cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 708. doi:10.1158/1538-7445.AM2011-708

Synergistic Effects of Foretinib with HER-Targeted Agents in MET and HER1- or HER2-Coactivated Tumor Cells

The HER and MET receptor tyrosine kinases (RTK) are coactivated in a subset of human tumors. This study characterizes MET and HER expression and signaling in a panel of human tumor cell lines and the differential susceptibility of these cell lines to single agents or combinations of foretinib, a multikinase MET inhibitor, with HER-targeted agents, erlotinib or lapatinib. Most MET-amplified tumor lines without HER1 or HER2 amplification are sensitive to foretinib, whereas MET-amplified lines with HER1 or HER2 amplification are more sensitive to the combination of foretinib with lapatinib or erlotinib. Interestingly, MET-overexpressing tumor cell lines with HER1 or HER2 amplification also exhibited reduced sensitivity to lapatinib or erlotinib in the presence of hepatocyte growth factor (HGF), indicating MET activation can decrease the effectiveness of HER1/2 inhibitors in some cell lines. Consistent with this observation, the effect of HGF on lapatinib or erlotinib sensitivity in these cells was reversed by foretinib, other MET inhibitors, or siRNA to MET. Western blot analyses showed that combining foretinib with erlotinib or lapatinib effectively decreased the phosphorylation of MET, HER1, HER2, HER3, AKT, and ERK in these cells. Furthermore, HER2-positive advanced or metastatic breast cancer patients treated with lapatinib who had higher tumor MET expression showed shorter progression-free survival (19.29 weeks in MET-high patients vs. 28.14 weeks in MET-low patients, P < 0.0225). These data suggest that combination therapy with foretinib and HER-targeted agents should be tested as a treatment option for HER1- or HER2-positive patients with MET-amplified or -overexpressing tumors.

Abstract P3-14-14: Activation of SRC and MAPK Mediates Resistance to Lapatinib in ER-/ERBB2+ Breast Cancer

Abstract Background Resistance to lapatinib represents a significant problem in the treatment of ERBB2+ breast cancer, especially after failure of prior trastuzumab therapy. A number of resistance mechanisms have been identified in recent years that limit the anti-tumor effects of lapatinib. These mechanisms include the activation of ERα signaling, and sustained activation of AKT either through coexpression of receptor tyrosine kinases (IGF-IR, AXL, MET, ERBB3), loss of PTEN or activating mutations in PIK3CA. However, these mechanisms have been shown to confer resistance primarily in ER+/ERBB2+ breast cancer cells; the mechanisms of resistance to lapatinib in ER-/ERBB2+ breast cancer (∼ 50% of ERBB2+ breast cancers) remain poorly understood. We hypothesized that resistance to lapatinib in ER-/ERBB2+ breast cancer cells can be mediated by activation of alternative signaling pathways which bypass the PI3K/AKT signaling axis. Materials and Methods To investigate the mechanisms of acquired resistance to lapatinib we generated ER-/ERBB2+ and ER+/ERBB2+ breast cancer cells resistant to lapatinib at the clinically relevant concentration of 2.6 uM (SK-lapR and BT-lapR). Resistance to lapatinib was achieved by exposure of SKBR3 and BT474 cells to increasing concentrations of the drug, and resistant cells were selected through serial passage. The activation status of multiple signaling pathways in lapatinib sensitive and resistant cells was determined by western blot analysis, and the role of individual signaling pathways in promoting resistance to lapatinib was investigated in cell proliferation assays using selective small molecule inhibitors. Results and Discussion Consistent with previous studies, we show that inhibition of ERα signaling using 100 nM fulvestrant in combination with lapatinib restored lapatinib sensitivity and effectively leads to growth arrest and cell death in ER+/ERBB2+ BT-lapR cells (P&amp;lt;0.001). However, none of the previously proposed mechanisms of resistance to lapatinib were apparent in our SK-lapR cells (ER-/ERBB2+). Strikingly, these cells which are stably resistant to lapatinib show no activation of AKT even in the absence of lapatinib. Rather, they exhibit increased phosphorylation of SRC kinase (3.9 fold, analyzed with ImageJ) and no inhibition of ERK1/2 after treatment with lapatinib compared to sensitive SKBR3 cells. Inhibition of SRC activity with 1uM AZD0530 in combination with lapatinib significantly reduced growth of SK-lapR cells compared to treatment with lapatinib alone (P&amp;lt;0.05). These data reveal potential novel mechanisms for lapatinib resistance in ER-/ERBB2+ breast cancer, and may indicate therapeutic strategies to prevent or overcome lapatinib resistance. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-14-14.

Comparison of Global versus Epidermal Growth Factor Receptor Pathway Profiling for Prediction of Lapatinib Sensitivity in Bladder Cancer

Chemotherapy for metastatic bladder cancer is rarely curative. The recently developed small molecule, lapatinib, a dual epidermal growth factor receptor (EGFR)/human epidermal growth factor receptor-2 receptor tyrosine kinase inhibitor, might improve this situation. Recent findings suggest that identifying which patients are likely to benefit from targeted therapies is beneficial, although controversy remains regarding what types of evaluation might yield optimal candidate biomarkers of sensitivity. Here, we address this issue by developing and comparing lapatinib sensitivity prediction models for human bladder cancer cells. After empirically determining in vitro sensitivities (drug concentration necessary to cause a 50% growth inhibition) of a panel of 39 such lines to lapatinib treatment, we developed prediction models based on profiling the baseline transcriptome, the phosphorylation status of EGFR pathway signaling targets, or a combination of both data sets. We observed that models derived from microarray gene expression data showed better prediction performance (93%-98% accuracy) compared with models derived from EGFR pathway profiling of 23 selected phosphoproteins known to be involved in EGFR-driven signaling (54%-61% accuracy) or from a subset of the microarray data for transcripts in the EGFR pathway (86% accuracy). Combining microarray data and phosphoprotein profiling provided a combination model with 98% accuracy. Our findings suggest that transcriptome-wide profiling for biomarkers of lapatinib sensitivity in cancer cells provides models with excellent predictive performance and may be effectively combined with EGFR pathway phosphoprotein profiling data. These results have significant implications for the use of such tools in personalizing the approach to cancers treated with EGFR-directed targeted therapies.

Novel Mechanism of Lapatinib Resistance in HER2-Positive Breast Tumor Cells: Activation of AXL

HER2-directed therapies, such as trastuzumab and lapatinib, are important treatments for breast cancer. However, some tumors do not respond or develop resistance to these agents. We isolated and characterized multiple lapatinib-resistant, HER2-positive, estrogen receptor (ER)-positive breast cancer clones derived from lapatinib-sensitive BT474 cells by chronic exposure to lapatinib. We show overexpression of AXL as a novel mechanism of acquired resistance to HER2-targeted agents in these models. GSK1363089 (foretinib), a multikinase inhibitor of AXL, MET, and vascular endothelial growth factor receptor currently in phase II clinical trials, restores lapatinib and trastuzumab sensitivity in these resistant cells that exhibit increased AXL expression. Furthermore, small interfering RNA to AXL, estrogen deprivation, or fulvestrant, an ER antagonist, decreases AXL expression and restores sensitivity to lapatinib in these cells. Taken together, these data provide scientific evidence to assess the expression of AXL in HER2-positive, ER-positive patients who have progressed on either lapatinib or trastuzumab and to test the combination of HER2-targeted agents and GSK1363089 in the clinic.

Phosphatidylinositol 3-kinase hyperactivation results in lapatinib resistance that is reversed by the mTOR/phosphatidylinositol 3-kinase inhibitor NVP-BEZ235.

Small molecule inhibitors of HER2 are clinically active in women with advanced HER2-positive breast cancer who have progressed on trastuzumab treatment. However, the effectiveness of this class of agents is limited by either primary resistance or acquired resistance. Using an unbiased genetic approach, we performed a genome wide loss-of-function short hairpin RNA screen to identify novel modulators of resistance to lapatinib, a recently approved anti-HER2 tyrosine kinase inhibitor. Here, we have identified the tumor suppressor PTEN as a modulator of lapatinib sensitivity in vitro and in vivo. In addition, we show that two dominant activating mutations in PIK3CA (E545K and H1047R), which are prevalent in breast cancer, also confer resistance to lapatinib. Furthermore, we show that phosphatidylinositol 3-kinase (PI3K)-induced lapatinib resistance can be abrogated through the use of NVP-BEZ235, a dual inhibitor of PI3K/mTOR. Our data show that deregulation of the PI3K pathway, either through loss-of-function mutations in PTEN or dominant activating mutations in PIK3CA, leads to lapatinib resistance, which can be effectively reversed by NVP-BEZ235.

Activity of lapatinib is independent of EGFR expression level in HER2-overexpressing breast cancer cells

Epidermal growth factor receptor (EGFR/ErbB1) and HER2 (ErbB2/neu), members of the ErbB receptor tyrosine kinase family, are frequently overexpressed in breast cancer and are known to drive tumor growth and progression, making them promising targets for cancer therapy. Lapatinib is a selective competitive inhibitor of both the HER2 and EGFR tyrosine kinases. Although lapatinib showed significant activity in patients with HER2-positive breast cancer, the role of EGFR in the response of breast cancer to lapatinib has not been defined. Here, we examined the role of EGFR expression levels in the sensitivity of HER2-overexpressing breast cancer cells to lapatinib. Depletion of EGFR by EGFR small-interfering RNA knockdown did not affect lapatinib sensitivity in these cells, whereas treated HER2 siRNA knockdown cells became more resistant to lapatinib. We conclude that the in vitro activity of lapatinib is not dependent on EGFR expression level in HER2-overexpressing breast cancer cells.

EXEL-7647 Inhibits Mutant Forms of ErbB2 Associated with Lapatinib Resistance and Neoplastic Transformation

Mutations associated with resistance to kinase inhibition are an important mechanism of intrinsic or acquired loss of clinical efficacy for kinase-targeted therapeutics. We report the prospective discovery of ErbB2 mutations that confer resistance to the small-molecule inhibitor lapatinib. We did in vitro screening using a randomly mutagenized ErbB2 expression library in Ba/F3 cells, which were dependent on ErbB2 activity for survival and growth. Lapatinib resistance screens identified mutations at 16 different ErbB2 amino acid residues, with 12 mutated amino acids mapping to the kinase domain. Mutations conferring the greatest lapatinib resistance cluster in the NH2-terminal kinase lobe and hinge region. Structural computer modeling studies suggest that lapatinib resistance is caused by multiple mechanisms; including direct steric interference and restriction of conformational flexibility (the inactive state required for lapatinib binding is energetically unfavorable). ErbB2 T798I imparts the strongest lapatinib resistance effect and is analogous to the epidermal growth factor receptor T790M, ABL T315I, and cKIT T670I gatekeeper mutations that are associated with clinical drug resistance. ErbB2 mutants associated with lapatinib resistance transformed NIH-3T3 cells, including L755S and T733I mutations known to occur in human breast and gastric carcinomas, supporting a direct mechanism for lapatinib resistance in ErbB2-driven human cancers. The epidermal growth factor receptor/ErbB2/vascular endothelial growth factor receptor inhibitor EXEL-7647 was found to inhibit almost all lapatinib resistance-associated mutations. Furthermore, no ErbB2 mutations were found to be associated with EXEL-7647 resistance and lapatinib sensitivity. Taken together, these data suggest potential target-based mechanisms of resistance to lapatinib and suggest that EXEL-7647 may be able to circumvent these effects.

Genome-wide DNA copy number predictors of lapatinib sensitivity in tumor-derived cell lines

A common aim of pharmacogenomic studies that use genome-wide assays on panels of cancers is the unbiased discovery of genomic alterations that are associated with clinical outcome and drug response. Previous studies of lapatinib, a selective dual-kinase inhibitor of epidermal growth factor receptor (EGFR) and HER2 tyrosine kinases, have shown predictable relationships between the activity of these target genes and response. Under the hypothesis that additional genes may play a role in drug sensitivity, a predictive model for lapatinib response was constructed from genome-wide DNA copy number data from 24 cancer cell lines. An optimal predictive model which consists of aberrations at nine distinct genetic loci, includes gains of HER2, EGFR, and loss of CDKN2A. This model achieved an area under the receiver operating characteristic curve of approximately 0.85 (80% confidence interval, 0.70-0.98; P < 0.01), and correctly classified the sensitivity status of 8 of 10 head and neck cancer cell lines. This study shows that biomarkers predictive for lapatinib sensitivity, including the previously described copy number gains of EGFR and HER2, can be discovered using novel genomic assays in an unbiased manner. Furthermore, these results show the utility of DNA copy number profiles in pharmacogenomic studies.

Phase II Study of Predictive Biomarker Profiles for Response Targeting Human Epidermal Growth Factor Receptor 2 (HER-2) in Advanced Inflammatory Breast Cancer With Lapatinib Monotherapy

Inflammatory breast cancer (IBC) is one of the most aggressive forms of breast cancer. Lapatinib, an oral reversible inhibitor of epidermal growth factor receptor (EGFR) and human EGFR 2 (HER-2), demonstrated clinical activity in four of five IBC patients in phase I trials. We conducted a phase II trial to confirm the sensitivity of IBC to lapatinib, to determine whether response is HER-2 or EGFR dependent, and to elucidate a molecular signature predictive of lapatinib sensitivity. Our open-label multicenter phase II trial (EGF103009) assessed clinical activity and safety of lapatinib monotherapy in patients with recurrent or anthracycline-refractory IBC. Patients were assigned to cohorts A (HER-2-overexpressing [HER-2+]) or B(HER-2-/EGFR+) and fresh pretreatment tumor biopsies were collected. Forty-five patients (30 in cohort A; 15 in cohort B) received lapatinib 1,500 mg once daily continuously. Clinical presentation and biomarker analyses demonstrated a tumor molecular signature consistent with IBC. Lapatinib was generally well tolerated, with primarily grade 1/2 skin and GI toxicities. Fifteen patients (50%) in cohort A had clinical responses to lapatinib in skin and/or measurable disease (according to Response Evaluation Criteria in Solid Tumors) compared with one patient in cohort B. Within cohort A, phosphorylated (p) HER-3 and lack of p53 expression predicted for response to lapatinib (P < .05). Tumors coexpressing pHER-2 and pHER-3 were more likely to respond to lapatinib (nine of 10 v four of 14; P = .0045). Prior trastuzumab therapy and loss of phosphate and tensin homolog 10 (PTEN) did not preclude response to lapatinib. Lapatinib is well tolerated with clinical activity in heavily pretreated HER-2+, but not EGFR+/HER-2-, IBC. In this study, coexpression of pHER-2 and pHER-3 in tumors seems to predict for a favorable response to lapatinib. These findings warrant further investigation of lapatinib monotherapy or combination therapy in HER-2+ IBC.

The Role of Trastuzumab in Early Stage Breast Cancer: Current Data and Treatment Recommendations

Treatment of early stage breast cancer requires a multimodality approach in order to eradicate residual cancer and prevent recurrent disease. Targeting the pathways that promote or sustain cancer cell growth and invasion is critical to the effective treatment of breast and other cancers. Overexpression of the family of HER receptors have been associated with a variety of malignancies; the first and best studied is the association of overexpression of the HER2/neu receptor with a more aggressive breast cancer phenotype and poorer survival. A humanized antibody to HER2/neu, trastuzumab, is now FDA approved for the treatment of early stage, HER2/neu overexpressing breast cancer sequenced with chemotherapy including doxorubicin, cyclophosphamide, and paclitaxel. Additional international and national studies support the significant impact of trastuzumab on both disease free and overall survival in women with this aggressive form of breast cancer. Toxicity includes a low but clear risk of congestive heart failure, and the large phase III trials have helped to determine which patients are at higher risk for this complication. Non-anthracycline containing regimens are an alternative therapy associated with reduced cardiac toxicity. Trastuzumab therapy is now the standard of care for the treatment of early stage, HER2/neu positive breast cancer given in combination with one of several chemotherapy regimens. Ongoing questions include the appropriate duration of trastuzumab treatment as well as the optimal chemotherapy regimen and sequence. The next large phase III adjuvant trial for this subset of breast cancer is an international collaboration designed to evaluate the added or alternative benefit of an oral tyrosine kinase inhibitor targeting HER2/neu as well as the epidermal growth factor receptor (EGFR), lapatinib. Other trials are investigating differences in duration. Studies in the neoadjuvant setting should help to define markers of trastuzumab and lapatinib sensitivity and resistance. Preliminary data combining trastuzumab with the antiangiogenic antibody bevacizumab is encouraging; this combination will be tested in both early stage and late stage disease.