ErbB2-positive Breast Cancer Research Articles

Management of ErbB2-positive Breast Cancer: Insights from Preclinical and Clinical Studies with Lapatinib

The management of human epidermal growth factor receptor 2-positive (ErbB2+) breast cancer is challenging; patients with ErbB2+ breast tumors have more aggressive disease and a poor prognosis. The increasing incidence of breast cancer in Asia and the limitations of existing treatments pose additional challenges. In this review, we summarize the preclinical and clinical evidence that indicates how lapatinib, a novel inhibitor that targets the human epidermal growth factor receptor (ErbB1) and ErbB2 may help clinicians address four particularly challenging issues in the management of ErbB2+ breast cancer. These issues are: (i) trastuzumab therapy failure, (ii) development of central nervous system metastases, (iii) minimizing toxicity and (iv) selecting the most appropriate partners (chemotherapy and non-chemotherapy) for combination therapy with lapatinib. Lapatinib, in combination with chemotherapeutic agents, such as capecitabine, provides clinical benefits to patients with ErbB2+ breast cancer, including patients who develop progressive disease on trastuzumab. Lapatinib, in combination with non-chemotherapeutic agents, such as letrozole, may also provide a chemotherapy-free treatment option for postmenopausal patients with estrogen receptor-positive/ErbB2+ metastatic breast cancer. Encouraging results have also emerged regarding the synergistic effects of lapatinib in combination with other agents for the treatment of ErbB2+ breast cancer. Promising findings have also been reported for the use of lapatinib to prevent and treat central nervous system metastases. Collectively, these results indicate that the judicious use of lapatinib, an effective oral therapy with a manageable toxicity profile, can enhance the management of patients with ErbB2+ breast cancer.

P130Cas is an essential transducer element in ErbB2 transformation

The ErbB2 oncogene is often overexpressed in breast tumors and associated with poor clinical outcome. p130Cas represents a nodal scaffold protein regulating cell survival, migration, and proliferation in normal and pathological cells. The functional role of p130Cas in ErbB2-dependent breast tumorigenesis was assessed by its silencing in breast cancer cells derived from mouse mammary tumors overexpressing ErbB2 (N202-1A cells), and by its reexpression in ErbB2-transformed p130Cas-null mouse embryonic fibroblasts. We demonstrate that p130Cas is necessary for ErbB2-dependent foci formation, anchorage-independent growth, and in vivo growth of orthotopic N202-1A tumors. Moreover, intranipple injection of p130Cas-stabilized siRNAs in the mammary gland of Balbc-NeuT mice decreases the growth of spontaneous tumors. In ErbB2-transformed cells, p130Cas is a crucial component of a functional molecular complex consisting of ErbB2, c-Src, and Fak. In human mammary cells, MCF10A.B2, the concomitant activation of ErbB2, and p130Cas overexpression sustain and strengthen signaling, leading to Rac1 activation and MMP9 secretion, thus providing invasive properties. Consistently, p130Cas drives N202-1A cell in vivo lung metastases colonization. These results demonstrate that p130Cas is an essential transducer in ErbB2 transformation and highlight its potential use as a novel therapeutic target in ErbB2 positive human breast cancers.

Taiwanese patients with breast cancer with brain metastasis (BM) enrolled in the Lapatinib Expanded Access Program (LEAP).

1111 Background: LEAP is an expanded access study of lapatinib in combination with capecitabine in 42 countries. The primary objective of LEAP was to provide patients with pre-approval access to lapatinib based on positive clinical data from a pivotal phase III trial. Methods: LEAP enrolled patients with ErbB2-positive, locally advanced and metastatic breast cancer (MBC) showing progressive disease following prior therapies with anthracycline-, taxane-, and trastuzumab-containing regimens. Patients received lapatinib (1,250 mg/day) and capecitabine (2,000 mg/m2/day, days 1-14, every 21 days). In this study we analyzed Taiwanese LEAP patients with BM at baseline. Brain and systemic response were assessed using RECIST criteria. Independent radiologist assessed response of BM. Results: From March 2007 to September 2008, there were 238 patients enrolled in Taiwan. We retrospectively analyzed data from 194 patients with accessible medical charts, which included 52 patients with baseline BM. Among these 52 patients, 26 (50.0%) had baseline and follow up brain computed tomography (CT). With protocol treatment 1 (3.8%) achieved complete response of brain lesions, 8 ( 30.8%) had partial response, 10 ( 38.5%) had stable disease, and 7 ( 26.9%) had progressive disease. The overall brain response rate was numerically better than the overall systemic response rate in the whole 194 subjects (34.6% vs. 22.4%, p=0.2171). The median progression-free survival (PFS) and median time to progression (TTP) of BM are significantly longer than in 194 subjects (PFS: 8.47 mon vs. 5.23 months, p=0.0054. TTP: 9.60 months vs. 5.53 months, p=0.0376). Within these 26 subjects, the tendency of better brain response than systemic response rate (34.6% vs. 25.0%, p=0.5448), longer PFS (8.47 months vs. 4.77 months, p=0.3461) and longer TTP (9.60 months vs. 5.53 months, p=0.3103) were observed. Furthermore, only 8 subjects developed BM during LEAP treatment in total 194 subjects. Conclusions: Brain is a common metastatic site for ErbB2-positive MBC patients and usually implies poor prognosis and short survival. Based on the Taiwanese LEAP experience, lapatinib plus capecitabine treatment demonstrated a significant benefit in this group of patients. No significant financial relationships to disclose.

Cardiotoxic effects, or lack thereof, of anti-ErbB2 immunoagents.

e11054 Background: Herceptin (H), an anti-ErbB2 humanized antibody prescribed for treatment of ErbB2-positive breast cancer, has proved to be an essential tool in immunotherapy. However, large-scale clinical studies with H have shown that it engenders cardiomyopathy, particularly in patients treated either concurrently or previously with anthracyclines. Two novel human antitumor immunoconjugates were engineered in our laboratory by fusion of a human anti-ErbB2 scFv, Erbicin, with either a human RNase or the Fc region of a human IgG1, and thus called Erb- hRNase and Erb-hcAb (human anti-ErbB2-compact antibody), respectively. Both immunoagents are selectively cytotoxic for ErbB2-positive cancer cells in vitro and vivo. The Erbicin-derived immunoagents (EDIA) target on breast cancer cells an ErbB2 epitope different from that of H. We report that EDIA did not show in vitro cardiotoxic effects on rat and human cardiomyocytes, whereas H was strongly toxic. Methods: Methods for measuring cardiac function in M-mode with shortening fraction (FA) and in B-mode with ejection fraction are not very sensitive in detecting early myocardial damage. In this study we evaluated myocardial strain by speckle tracking technique (ST) by Color Doppler echocardiography (Visual sonic Vevo 2100) to identify early left ventricular dysfunction in mice treated with EDIA, doxorubicin (D), H, their associations and in a sham group. We measured ST and FA by assessing M-mode short axis projection in C57BL/6 mice at time 0, 2 and 6 days of daily administration. Results: EDIA did not impair cardiac function in vivo in a mouse model whereas H significantly reduced radial strain at 3 days of treatment and fractional shortening (% FS) at 6 days of treatment compared to the sham group in a fashion similar to D. Similarly, cardiac fibrosis, an index of collagen accumulation following cardiac muscle deterioration, was significantly attenuated in mice treated with either Erb-hcAb or Erb-hRNase as compared to those treated with H or D. Conclusions: These results suggest that the Erb-hcAb and the immuno RNase are immunoagents which can fulfil the therapeutic need of patients ineligible to H treatment due to cardiac dysfunction. No significant financial relationships to disclose.

Abstract 614: ErbB-2 Suppresses Notch-1 Signaling by Regulating Jagged-1 Localization in ErbB-2 positive breast cancer

Abstract ErbB-2 is amplified and overexpressed in 20-30% of breast tumors. Today, ErbB-2 positive breast cancers are treated with trastuzumab plus chemotherapy. In spite of dramatic improvements in survival for women with ErbB-2+ breast tumors, resistance remains a major problem in metastatic disease. We have recently published that ErbB-2 suppresses Notch-1 activity and this is reversed by trastuzumab or a dual EGFR/ErbB-2 tyrosine kinase inhibitor similar to lapatinb. Furthermore, we demonstrated that trastuzumab resistance is reversed with a Notch-1 siRNA or a gamma-secretase inhibitor (GSI). Coincidentally, co-overexpression of Notch-1 and its ligand, Jagged-1, in breast cancer have been associated with the poorest overall survival. We know that activated Notch-1 plays an important role in ErbB-2 positive breast cancer and trastuzumab resistance; however, the precise mechanism by which Notch-1 is activated is not yet known. Thus, we hypothesized that overexpression of ErbB-2 inhibited expression and/or cell surface localization of Jagged-1 to suppress Notch-1 activity. This would be reversed in response to anti-ErbB-2 treatment. The results demonstrated that trastuzumab or lapatinib treatment of SKBr3 cells increased protein expression of Jagged-1 by Flow Cytometry. Notch-1 and Jagged-1 co-localized with early endosome antigen-1 (EEA-1) to submembraneous endosomes in ErbB-2 positive breast cancer cells as measured by confocal immunofluorescence. However upon trastuzumab treatment, Notch-1 and Jagged-1 exited EEA-1 positive vesicles and Notch-1 accumulated in the nucleus while Jagged-1 localized to the cell membrane. Cell surface biotinylation assays showed that Jagged-1 was enriched on the cell surface in response to trastuzumab treatment and this was mimicked by expression of K44A Dynamin. Cell surface accumulation of Jagged-1 was associated with an increase in Notch-1 transcriptional activity as measured by an increase in Deltex1 mRNA by real-time PCR. Conversely, knockdown of Jagged-1 by siRNA in SKbr3 and BT474 cells prevented the trastuzumab-induced increase in Deltex1 mRNA. Co-culture studies confirmed that culturing SKBr3 cells with fibroblast-expressing Jagged-1 or DLL4 increased Notch activity as measured by a CBF-1/Notch reporter assay. Taken together, these results suggest that ErbB-2 suppresses Notch-1 activity by retention of Jagged-1 or possibly other Notch ligands within early endosomes, which may be reversed by trastuzumab or lapatinib treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 614.

Abstract LB-307: Overexpression of AXL as a novel acquired resistance mechanism in EGFR-mutated non-small cell lung cancer cells

Abstract Activating mutations of EGFR have been characterized as important mechanisms for carcinogenesis in a subset of EGFR-dependent non-small cell lung cancers (NSCLC). EGFR tyrosine kinase inhibitors (TKI), such as erlotinib and gefitinib, have dramatic clinical effects on EGFR-addicted lung cancers and are used as first-line therapy for EGFR-mutant tumors. However, this therapeutic sensitivity is invariably temporary and eventually all tumors acquire secondary resistance to the drugs and progress. Erlotinib-resistant EGFR mutations such as EGFR T790M and MET activation have been reported in drug-resistant tumors, but the mechanism of resistance remains unclear in a significant fraction of cases. Here we report a novel molecular mechanism leading to secondary resistance to erlotinib. HCC827, an EGFR-mutant and highly erlotinib-sensitive NSCLC cell line was exposed to increasing doses of erlotinib for 5 months and 2 subclonal cell lines were established, both of which grow well in the presence of high-dose erlotinib (15µM). In contrast to parental cells, the two erlotinib-resistant cell lines demonstrated sustained activation of Ras/Mek/Erk and Akt signaling pathways in the presence of erlotinib despite complete inhibition of EGFR phosphorylation. Sequencing of the entire EGFR gene sequence revealed no secondary changes, and expression of EGFR and MET, as well as MET activation did not differ from parental cells suggesting the presence of an alternative, novel mechanism of resistance. Gene expression profiling revealed a limited number of genes with altered expression levels which were verified by quantitative rt-PCR. We focused on the cell surface receptor tyrosine kinase, AXL (10-fold overexpressed in one resistant clone) given its known role in cell proliferation, angiogenesis and recent findings indicating its involvement in lapatinib-resistance in ErbB2-positive breast cancer. Prominent AXL overexpression was further confirmed in this clonal isolate on the protein level. siRNA-mediated downregulation of AXL reconstituted erlotinib-sensitivity, leading to cell death and was accompanied by inhibition of AKT activation. However, AXL knockdown in the absence of erlotinib did not have growth inhibitory effects on the cells. These results suggest that AXL signaling can serve as a backup control of proliferative pathways in our resistant cell models in the presence of EGFR inhibition serving as a novel paradigm of oncogenic switch. Mechanisms underlying overexpression of AXL and its restoration of TKI sensitivity are under investigation. Our results suggest that an oncogenic switch from EGFR-dependent to EGFR/AXL-codependent signaling can lead to secondary EGFR-TKI resistance in NSCLC, indicating that dual blockade of EGFR and AXL may serve as a novel therapeutic strategy for the treatment of EGFR-mutant lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-307.

Abstract 5064: The non-receptor tyrosine kinase Fer is required for activated ErbB2-mediated tumorigenesis

Abstract The fer proto-onogene encodes a non-receptor protein-tyrosine kinase (PTK) which together with the parologous Fps/Fes kinase makes up a distinct F-BAR domain containing subclass of PTKs. F-BARs have recently been shown to mediate membrane binding and bending properties that confer roles in receptor endocytosis and vesicle trafficking. Among known substrates of the Fer PTK are cortactin and F-BAR/SH3 containing adaptor proteins that regulate cytoskeletal remodeling and membrane dynamics important to cellular functions such as polarity determination, migration and mitogenesis. Mice deficient in Fer, Fps/Fes or both kinases are overtly healthy; although they do display enhanced responses to endotoxin challenges. Biochemical studies have implicated Fer in growth factor signaling, cadherin-dependent cell-cell adhesion, integrin-mediated adhesion and redox signaling. Cell biological studies have suggested a role for Fer in potentiating proliferation and migration. Recent results in our laboratory have provided compelling biological proof-in-principle that Fer is required for mammary tumorigenesis. RNAi-based knockdown of Fer reduced the tumorigenesis of the metastatic MDA-MB-231 breast cancer cells in mouse xenografting studies. In addition, tumor development in a transgenic mouse model of Her2/Neu/ErbB2-mediated breast tumorigenesis was significantly delayed in Fer-deficient mice by virtue of a targeted knock-in kinase-inactivating mutation in the mouse fer locus. Biochemical internalization assays suggested that EGFR endocytosis was enhanced in Fer-deficient tumor-derived epithelial cell cultures. These data point to a tumor promoting function for Fer in mammary tumorigenesis through a mechanism involving prolongation of ErbB2 signaling. Taken together, these studies suggest that pharmacological intervention of Fer may have therapeutic benefit in human ErbB2 positive breast cancer and potentially, in other epithelial cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5064.

Downregulation of Notch Pathway by a γ-Secretase Inhibitor Attenuates AKT/Mammalian Target of Rapamycin Signaling and Glucose Uptake in an ERBB2 Transgenic Breast Cancer Model

ERBB2/neu and Notch signaling are known to be deregulated in many human cancers. However, pathway cross-talk and dependencies are not well understood. In this study, we use an ERBB2-transgenic mouse model of breast cancer (neuT) to show that Notch signaling plays a critical role in tumor maintenance. Inhibition of the Notch pathway with a gamma-secretase inhibitor (GSI) decreased both the Notch and the mammalian target of rapamycin/AKT pathways. Antitumor activity resulting from GSI treatment was associated with decreased cell proliferation as measured by Ki67 and decreased expression of glucose transporter Glut1. Positron emission tomography (PET) imaging showed that the functional consequences of decreased Glut1 translated to reduced glucose uptake and correlated with antitumor effects as measured by micro-computed tomography imaging. The decrease of Glut1 in neuT tumors was also observed in several human breast cancer cell lines following GSI treatment. We provide evidence that approximately 27% of ERBB2-positive human breast cancer specimens display high expression of HES1, phospho-S6RP, and GLUT1. Together, these results suggest that pathways downstream of Notch signaling are, at least in part, responsible for promoting tumor growth in neuT and also active in both neuT and a subset of human breast cancers. These findings suggest that GSI may provide therapeutic benefit to a subset of ERBB2-positive breast cancers and that [(18)F]FDG-PET imaging may be useful in monitoring clinical response.

An RNA Interference Screen Identifies Metabolic Regulators NR1D1 and PBP as Novel Survival Factors for Breast Cancer Cells with the ERBB2 Signature

Overexpression of the adverse prognostic marker ERBB2 occurs in 30% of breast cancers; however, therapies targeting this gene have not proved to be as effective as was initially hoped. Transcriptional profiling meta-analyses have shown that there are approximately 150 genes co-overexpressed with ERBB2, suggesting that these genes may represent alternative factors influencing ERBB2-positive tumors. Here we describe an RNA interference-based analysis of these genes that identifies transcriptional regulators of fat synthesis and storage as being critical for the survival of these cells. These transcription factors, nuclear receptor subfamily 1, group D, member 1 (NR1D1) and peroxisome proliferator activated receptor gamma binding protein (PBP), both reside on ERBB2-containing 17q12-21 amplicons and are part of the ERBB2 expression signature. We show that NR1D1 and PBP act through a common pathway in upregulating several genes in the de novo fatty acid synthesis network, which is highly active in ERBB2-positive breast cancer cells. Malate dehydrogenase 1 and malic enzyme 1, enzymes that link glycolysis and fatty acid synthesis, are also regulated by NR1D1. The resulting high-level fat production from increased expression of these genes likely contributes to an abnormal cellular energy metabolism based on aerobic glycolysis. Together, these results show that the cells of this aggressive form of breast cancer are genetically preprogrammed to depend on NR1D1 and PBP for the energy production necessary for survival.

Neratinib, an Irreversible ErbB Receptor Tyrosine Kinase Inhibitor, in Patients With Advanced ErbB2-Positive Breast Cancer

Neratinib is an oral, irreversible pan-ErbB receptor tyrosine kinase inhibitor. The efficacy and safety of neratinib were evaluated in two cohorts of patients with advanced ErbB2-positive breast cancer-those with and those without prior trastuzumab treatment-in an open-label, multicenter, phase II trial. Patients in the two cohorts (prior trastuzumab, n = 66; no prior trastuzumab, n = 70) received oral neratinib 240 mg once daily. The primary end point was the 16-week progression-free survival (PFS) rate for the evaluable population (prior trastuzumab, n = 63; no prior trastuzumab, n = 64), as assessed by independent review. The 16-week PFS rates were 59% for patients with prior trastuzumab treatment and 78% for patients with no prior trastuzumab treatment. Median PFS was 22.3 and 39.6 weeks, respectively. Objective response rates were 24% among patients with prior trastuzumab treatment and 56% in the trastuzumab-naïve cohort. The most common adverse events were diarrhea, nausea, vomiting, and fatigue. Diarrhea was the most frequent grades 3 to 4 adverse event, occurring in 30% of patients with prior trastuzumab treatment and in 13% of patients with no prior trastuzumab treatment, which prompted dose reductions in 29% and 4% of patients, respectively, but treatment discontinuation in only one patient. No neratinib-related, grades 3 or 4 cardiotoxicity was reported. Oral neratinib showed substantial clinical activity and was reasonably well tolerated among both heavily pretreated and trastuzumab-naïve patients who had advanced, ErbB2-positive breast cancer. Diarrhea was the most common adverse effect but was manageable with antidiarrheal agents and dose modification.

Gastrointestinal and Cardiovascular Safety Profiles of Neratinib Monotherapy in Patients with Advanced ErbB2-Positive Breast Cancer.

Abstract Background: In a phase 2 study, oral neratinib was administered to patients (pts) with advanced breast cancer in 2 cohorts, those with prior trastuzumab treatment (prior T, n=66) and those with no prior trastuzumab treatment (no prior T, n=70). Neratinib demonstrated robust antitumor activity with objective response rates of 26% and 51%, respectively, and was generally tolerable (Burstein et al. Cancer Res 2009;69:72S). Diarrhea, all grades, occurred in 89% of total pts. Characteristics of the diarrhea and left ventricular ejection fraction (LVEF) measurements are described. Methods: Pts were required to have ErbB2 gene amplification in tumor tissue as measured by fluorescence in situ hybridization by independent assessment. Serial LVEF measurements were made with a multigated acquisition scan or echocardiogram. Pts were ineligible if baseline LVEF was <50%. Adverse events were graded based on the NCI Common Terminology Criteria, v 3.0. Pts received oral neratinib 240 mg daily. Results: Data collected by 18 Mar 2009 are reported; the median (range) duration of neratinib treatment was 4.5 (0.2-23.5) months for pts with prior T and 7.8 (0.5-24.2) months for pts with no prior T. Diarrhea, all grades occurred in 93% of the total population (prior T: 97%, no prior T: 89%); grade 3-4 diarrhea occurred in 21% of the total population (prior T: 30%, no prior T: 13%). Median times of onset were 2 and 3 days after first dose of neratinib, respectively, and median durations of diarrhea were 7 and 5 days. In study week 1, 86% and 73% of pts with prior T and no prior T, respectively, had diarrhea; this decreased to 12-14% in months 3 and 4 (Table). Diarrhea was the cause of dose interruptions in 36% of pts with prior T and 11% of pts with no prior T and of dose reductions in 30% of pts with prior T and 5% of pts with no prior T; only 1 pt (prior T) discontinued treatment due to diarrhea. Pts used anti-diarrheal medications for supportive therapy (prior T: 91%, no prior T: 67%). For LVEF measurements, there was little variation from baseline in most pts during the study. Four pts had at least 1 LVEF measurement <50% (2 of these pts had measurements within the institutional range of normal). None had congestive heart failure. One of these pts had grade 3 atrioventricular block and grade 3 bradycardia, which were considered unrelated to neratinib. Discussion: Neratinib treatment was not associated with clinically significant cardiotoxicity. Pts who developed diarrhea with neratinib monotherapy had early onset, but frequency and severity decreased with time on study. Despite the high frequency of diarrhea with neratinib treatment, it was readily managed with supportive therapy and dose interruptions and/or reductions.Time Course of Diarrhea in ErbB2-Positive Breast Cancer Patients Treated With NeratinibStudy PeriodPrior T: No. on StudyPrior T: % With Diarrhea, All Grades*Prior T: % With Diarrhea, Gr 3-4*No Prior T: No. on StudyNo Prior T: % With Diarrhea, All Grades*No Prior T: % With Diarrhea, Gr 3-4*Week 166861770737Week 2-466611170436Month 260331066152Month 34713060120Month 44312058140*For pts with multiple toxicity grades in a period, the maximum grade was reported. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5096.

A Phase I, Dose Escalation Study of Lapatinib in Combination with Carboplatin, Paclitaxel, with or without Trastuzumab in Patients with Metastatic Breast Cancer: An Update.

Abstract Background: Lapatinib is a selective and highly potent dual, competitive inhibitor of erbB1 and erbB2 tyrosine kinases with clinical activity in erbB2-positive metastatic breast cancer (MBC). The combination of carboplatin, paclitaxel, and trastuzumab has been shown to have significant clinical activity in MBC as well as in the adjuvant setting. Given the synergy of dual inhibition with trastuzumab and lapatinib observed in both the preclinical and clinical settings, the feasibility, safety, and early clinical activity of paclitaxel, carboplatin, lapatinib with or without trastuzumab in patients (pts) with MBC was assessed.Methods: MBC pts previously untreated with trastuzumab or cytotoxic chemotherapy for metastatic or locally recurrent disease were enrolled into either Group (Grp) A (erbB2 positive) or B (erbB2 positive or negative). Escalating doses of lapatinib (planned range of 750-1500 mg/d) were administered in combination with (Grp A) or without (Grp B) trastuzumab (4 mg/kg followed by weekly 2 mg/kg infusions). Paclitaxel (80 mg/m2) and carboplatin (AUC 2 mg/ml*min) were administered on days 1, 8, 15 with cycles repeated every 28 days. Starting doses of lapatinib were 750 mg in Grp A and 1000 mg in Grp B (Dose Level 0). A standard 3 + 3 Phase I design was used to determine the optimally tolerated regimen (OTR) dose level.Results: Twenty-seven pts (Grp A n=15, Grp B n=12) have been enrolled in the study (median age 45 yrs, range 27-74). A median of 5 cycles (range 1-21) in Grp A and 5.5 (range 2-15) in Grp B have been administered. Drug-related toxicities were reported in 14 pts (93%) in Grp A and 11 pts (92%) in Grp B. In Grp A, drug-related grade 3 toxicities ≥ 10% include neutropenia (47%), diarrhea (33%), and rash (20%). Two drug-related grade 4 toxicities were reported (urinary tract infection and hypocalcemia). In Grp B, drug-related grade 3 toxicities ≥ 10% include diarrhea (17%), fatigue (17%), and rash (17%). Drug-related grade 4 toxicity (neutropenia) occurred in 3 pts in Grp B. In Grp A, there were two patients with dose-limiting toxicity (DLT) (Gr 3 diarrhea, Gr 3 rash) at lapatinib 1000 mg (Dose Level +1). The OTR (Dose Level 0) was identified as lapatinib 750 mg, paclitaxel 80 mg/m2, carboplatin AUC 2, and trastuzumab 2 mg/kg. In Grp B, two patients had DLT (one with Gr 4 neutropenia, one with Gr 3 rash and hypokalemia) at Dose Level 0 (lapatinib 1000 mg). At Dose Level -1 (paclitaxel reduced to 70 mg/m2), one pt had a DLT (Gr 4 neutropenia) and this dose level will be expanded to 6 pts. Clinical activity to date in Grp A (12 pts) includes 2 CR, 8 PR, and 1 SD and in Grp B (11 pts) 5 PR and 5 SD. Two pts in Grp B were ErbB2+, both had PR. Response assessments were not available for 4 pts (3 pts were awaiting response assessment and 1 pt was removed from the study prior to response assessment due to toxicity (neutropenia, catheter infection)).Conclusions: Lapatinib may be administered safely in combination with carboplatin, paclitaxel, with or without trastuzumab at known effective doses. Clinical activity has been observed. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3097.

Notch-1 Confers Resistance to ErbB-2 Targeted Inhibition by Regulating PTEN, p27, and IGF-1R.

Abstract Background: Amplification, over-expression, and/or hyperactivity of ErbB-2 occur in 30% of breast tumors. Metastatic breast tumors that overexpress ErbB-2 are difficult to treat and generally resistant to trastuzumab or lapatinib. We have recently published that overexpression of ErbB-2 suppresses Notch-1 activity and this is reversed by inhibitors of ErbB-2, trastuzumab or a tyrosine kinase inhibitor (Osipo et al., 2008. Oncogene). Furthermore, we demonstrated that trastuzumab resistance is reversed when Notch-1 is downregulated by siRNA or when cells are treated with a gamma-secretase inhibitor. Overexpression of IGF-1R and/or downregulation of PTEN, and p27Kip1 have been shown to be very sensitive markers of trastuzumab resistance. Based on our results and what is known from the literature, we asked by what mechanism Notch-1 contributes to trastuzumab resistance and can a combination therapy prevent or reverse the resistant phenotype by using a pre-clinical in vivo model.Methods: Using a pcDNA3.1-NIC construct expressing activated Notch-1 and a Notch-1 siRNA, we either increased NIC levels in trastuzumab sensitive or downregulated Notch-1 in trastuzumab resistant BT474 cells, respectively and measured protein and mRNA expressions of PTEN, p27Kip1, and the IGF-1R. In addition, BT474 breast tumor xenografts were generated in athymic mice and randomized to vehicle, MRK-003 or Ly411,575 gamma-secretase inhibitor, trastuzumab, or trastuzumab plus a gamma-secretase inhibitor. Tumor size was measured up to 14 weeks with treatments and up to 12 weeks after stopping treatments.Results: The results showed that intracellular, active Notch-1 (ICN) increased mRNA and protein expression of the IGF-1R, while simultaneously decreased protein expressions of PTEN and p27Kip1 in ErbB-2 positive breast cancer cells sensitive to trastuzumab. Conversely, knockdown of Notch-1 by siRNA in resistant cells decreased IGF-1R, while increasing PTEN and p27Kip1. The xenograft studies demonstrated that combining a GSI with trastuzumab either completely prevented tumor recurrence (MRK-003 GSI) or significantly delayed tumor recurrence (Ly 411,575 GSI).Conclusions: Taken together, these results suggest that Notch-1 potentially contributes to trastuzumab resistance by upregulating the IGF-1R and downregulating PTEN and p27Kip1 in vitro. The benefit of inhibiting Notch activity using a GSI in combination with trastuzumab is prevention of resistance and tumor recurrence. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3123.

Abstract ED02-01: Molecular targets of cancer preventive agents

Abstract ED02-01: Molecular targets of cancer preventive agents

Neratinib, an irreversible pan erB receptor tyrosine kinase inhibitor active for advanced HER2+breast cancer

Neratinib (HKI-272) is a dual inhibitor of the tyrosine kinase receptors, erbB1 (EGFR) and erbB2 (HER2). In a phase I study, neratinib was tolerable and demonstrated antitumor activity in patients with solid tumors, including 8 of 25 evaluable patients with erbB2-positive advanced breast cancer. In an open-label, two-arm phase II study, patients with stage IIIB, IIIC or IV erbB2-positive advanced breast cancer were evaluated to further characterize the safety and efficacy of neratinib. The maximum tolerated dose of once-daily oral neratinib is 320 mg. The most common neratinib-related toxicity was diarrhea. Antitumor activity was observed in patients with breast cancer who had previous treatment with trastuzumab, anthracyclines, and taxanes, and tumors with a baseline ErbB-2 IHC staining intensity of 2+ or 3+. Neratinib has demonstrated a potent activity against trastuzumab-resistant and trastuzumab-naive HER2-positive breast cancer in a phase II study that evaluated the safety and efficacy of a daily 240 mg oral dose on 136 women with locally advanced or metastatic breast cancer (stage IIIB, IIIC or IV). The 16-week progression-free survival (PFS) rate was the primary end point. Secondary end points included safety, objective response rate and clinical benefit rate. Patients were assigned to one of two study arms based on prior treatment with trastuzumab. The efficacy analysis included 127 evaluable patients, 61 in arm A and 66 in arm B. In patients who were previously treated with trastuzumab (arm A), the 16-week PFS rate was 60%, and the median PFS was 23 weeks. The objective response rate was 26%, and the clinical benefit rate was 36%. At the present time, several phase II studies of neratinib in combination with chemotherapy (capacitabine, vinorelbine, paclitaxel and doxorubicin) are ongoing, some of them already finished. The tolerance and efficacy will be presented. A large worldwide randomized phase III study of the combination of trastuzumab versus neratinib plus paclitaxel in chemonaive advanced HER2-positive breast cancer patients will start very soon in order to determine the efficacy of neratinib compared with standard treatment.

Rac1 contributes to trastuzumab resistance of breast cancer cells: Rac1 as a potential therapeutic target for the treatment of trastuzumab-resistant breast cancer

Although treatment with trastuzumab improves outcomes for women with ErbB2-positive breast cancer, many patients who achieve an initial response to trastuzumab subsequently acquire resistance within 1 year. Rac1, a Ras-like small GTPase, has been implicated in the control of cell growth and morphology and is believed to be associated with breast cancer progression and metastasis. Here, we show that when parental SKBR3 cells become resistant to trastuzumab, Rac1 activity is increased, leading to altered cell morphology, which is accompanied by significant cytoskeleton disorganization. Furthermore, both trastuzumab-mediated down-regulation of ErbB2 and epidermal growth factor-induced down-regulation of epidermal growth factor receptor are impaired in the trastuzumab-resistant SKBR3 cells, indicating that the endocytic down-regulation of ErbB receptors is compromised in the resistant cells. This results in an aberrant accumulation of ErbB2 on the cell surface and enhanced ErbB2 and extracellular signal-regulated kinase activity in trastuzumab-resistant SKBR3 cells. Additionally, overexpression of constitutively active Rac1G12V in parental SKBR3 cells reduces sensitivity to trastuzumab. After reduction of Rac1 activity by NSC23766, a specific Rac1 inhibitor, trastuzumab-resistant SKBR3 cells display a cellular morphology similar to parental SKBR3 cells. Moreover, we show that NSC23766 restores trastuzumab-mediated endocytic down-regulation of ErbB2 and reduces extracellular signal-regulated kinase activity in resistant SKBR3 cells. Our findings highlight an important role for Rac1 in trastuzumab resistance of human breast cancer cells and identify the impaired trastuzumab-mediated endocytic down-regulation of ErbB2 as a novel mechanism of trastuzumab resistance. The significant effects of NSC23766 on trastuzumab-resistant SKBR3 cells warrant further study of NSC23766 as a potential treatment of trastuzumab-resistant breast cancers.

Breast cancer ErbB2 status in Asian women: A review of local literature

e22164 Background: Breast cancer incidence is increasing throughout Asia. However, the characteristics of Asian breast cancer are not always well understood due to the limited availability of local data and their inadequate inclusion in global literature databases. Effective breast cancer treatment in this region requires a better understanding of Asian breast cancer biology, including ErbB2 status. Methods: We performed a literature search in seven Asian countries on breast cancer studies in which tumour ErbB2 overexpression was assessed. The keywords erbB2 OR HER2 OR ErbB-2 OR HER-2 AND breast cancer AND (country) were used to search PubMed, international and local conference abstracts and local-language journals from the year 2000 onwards. Where available, we selected up to five representative studies from each country on the basis of population size, multi-institution patient populations, institution reputation and journal impact factor. We excluded studies containing only mRNA, serum, or cell line data or those conducted on biased populations or in western countries on Asian populations. Results: ErbB2 data from 26 Asian breast cancer studies are summarized in the table . The mean or median age of the study populations ranged from 46–56 years, with one exception. In most studies that evaluated tumour ErbB2 and hormone status, ErbB2 over-expression correlated negatively with ER positivity. Conclusions: The larger studies in particular confirm that the proportion of ErbB2-positive breast cancer in Asia is generally similar to the 20–30% reported for western women. Definitions of ErbB2 positivity using IHC vary between institutions and FISH is not routinely performed in several Asian countries. This may contribute to the fact that reliable, representative data on breast tumour ErbB2 status is scarce in Malaysia, the Philippines, India and Thailand. The increased availability of accurate ErbB2 testing and data would aid improved treatment of ErbB2-positive breast cancer in these countries. [Table: see text] [Table: see text]

No cure in sight for the world's drug problem

If you are a woman with ERBB2-positive advanced breast cancer that has failed to respond to therapy, lapatinib treatment could give you the chance to control your disease. Unless, that is, you are a patient of the UK National Health Service (NHS). Because on March 4, 2009, the UK National Institute of Clinical Excellence (NICE) ruled that the NHS should not prescribe lapatinib treatment as it is “not a cost-effective use of NHS resources”.

Peroxisome proliferator-activated receptor-γ protects ERBB2-positive breast cancer cells from palmitate toxicity

IntroductionAccumulation of fatty acids and neutral lipids in nonadipose tissues is cytotoxic. We recently showed that ERBB2-positive breast cancer cells produce significantly high amounts of fats, because of overexpression of the peroxisome proliferator-activated receptor (PPAR)γ-binding protein and the nuclear receptor NR1D1 (nuclear receptor subfamily 1, group D, member 1; Rev-erbα). These genes upregulate de novo fatty acid synthesis, which is a critical pathway for the energy production and survival of these cells. NR1D1 and PPARγ-binding protein are functionally related to PPARγ, a well established positive regulator of adipogenesis and lipid storage.MethodsThe effects of GW9662 and exogenously added palmitate on breast cells (BT474, MDA-MB-361, MCF-7, and human mammary epithelial cells) in monolayer culture were assessed. Mass spectrometric quantitation of fatty acids and fluorescence-based high content microscopy assays of cell growth, apoptosis, triglyceride storage and reactive oxygen species production were used.ResultsERBB2-positive breast cancer cells are more sensitive to inhibition of PPARγ activity by the antagonist GW9662. PPARγ inhibition results in increased levels of total fats in the cells, mostly because of increased amounts of palmitic and stearic unsaturated acids. Administration of exogenous palmitate is lethal to ERBB2-positive but not to ERBB2-negative cells. GW9662 exacerbates the effects of palmitate addition on BT474 and MDA-MB-361 cells, but it has no significant effect on MCF-7 and human mammary epithelial cells. Palmitate administration results in a fivefold to tenfold greater increase in fat stores in ERBB2-negative cells compared with ERBB2-positive cells, which suggests that the ERBB2-positive cells have maximized their ability to store fats and that additional palmitate is toxic to these cells. Both PPARγ inhibition and palmitate administration result in increased reactive oxygen species production in BT474 cells. The cell death that results from this treatment can be counteracted by the antioxidant N-acetyl cysteine.ConclusionsOur findings indicate that PPARγ activity enables ERBB2-positive breast cancer cells, which produce high levels of fat, to convert fatty acids to triglycerides, allowing these cells to avert the cell death that results from lipotoxicity. Endogenous palmitate toxicity represents a genetically based property of ERBB2-positive breast cancer that can be exploited for therapeutic intervention.

Expression of Epidermal Growth Factor Receptor or ErbB3 Facilitates Geldanamycin-Induced Down-Regulation of ErbB2

Overexpression of the epidermal growth factor receptor (EGFR), ErbB2, and ErbB3 promotes growth and antiapoptotic signaling. Overexpression of ErbB2 in breast cancer is associated with poor clinical outcome, and ways of down-regulating ErbB2 are important as therapeutic approaches. In contrast to EGFR, ErbB2 has been shown to be endocytosis deficient. However, down-regulation of ErbB2 can be induced by incubation of cells with geldanamycin and geldanamycin derivatives, counteracting the stabilizing function of heat shock protein 90 on ErbB2. In the present study, we have made use of stably transfected isogenic cell lines expressing ErbB2 only or ErbB2 together with EGFR and/or ErbB3. We now show that whereas ErbB2 can be down-regulated by incubation with geldanamycin in cells expressing ErbB2 only, the rate of geldanamycin-induced down-regulation increases significantly when the cells additionally express EGFR and/or ErbB3. This increase does, however, not correlate with activation/phosphorylation of ErbB2. The potential of heterodimer formation in ErbB2-positive breast cancer cells could thus turn out to be prognostically predictive with respect to outcome of treatment with geldanamycin derivatives.