Dual HER2-targeted approaches in HER2-positive breast cancer

Approximately 15–20% of all breast cancers are human epidermal growth factor receptor 2 (HER2) positive, with clinical studies having validated the HER2 receptor tyrosine kinase pathway as an important therapeutic target. Presently, two HER2-targeted therapies are approved by the Food and Drug Administration for treatment of HER2-positive breast cancer: the HER2-targeted humanized monoclonal antibody trastuzumab and the small-molecule tyrosine kinase inhibitor lapatinib. Despite use of these HER2-targeted agents, many patients still experience disease progression. For this reason, numerous new agents and therapeutic strategies are under investigation. Based on preclinical data suggesting synergistic effects from dual therapy targeting HER2, clinical trials that test the effects of combining anti-HER2 agents have been conducted and are ongoing. Here, we review recently presented data from several clinical trials, which indicate that the strategy of combining HER2 blockade therapies can offer greater clinical efficacy, with adverse effects of varying degrees. Specifically, we review new data reported at the 2010 San Antonio Breast Cancer Symposium (SABCS 2010), including the phase II NeoSphere and phase III NeoALTTO clinical trials, and data from three clinical trials reported at the 2011 American Society of Clinical Oncology (ASCO 2011) meeting. Together these trials elucidate the potential role of combining trastuzumab with lapatinib or pertuzumab. We also discuss additional ongoing studies that will help further define the role of dual HER2 blockade therapies and its impact on clinical practice.     

Trastuzumab-induced recruitment of Csk-homologous kinase (CHK) to ErbB2 receptor is associated with ErbB2-Y1248 phosphorylation and ErbB2 degradation to mediate cell growth inhibition

The inhibitory effect of trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of ErbB2, is associated with its ability to induce ErbB2-Y1248 phosphorylation, and the status of phosphorylated ErbB2-Y1248 (ErbB2-pY1248) may correlate with the sensitivity of breast cancers to trastuzumab. The mechanisms of which remain unclear. Here, we show that binding of trastuzumab to ErbB2 activates ErbB2 kinase activity and enhances ErbB2-Y1248 phosphorylation in trastuzumab-sensitive breast cancer cells. This in turn increases the interaction between ErbB2 and non-receptor Csk-homologous kinase (CHK), leading to growth inhibition of breast cancer cells. Overexpression of CHK mimics trastuzumab treatment to mediate ErbB2-Y1248 phosphorylation, Akt downregulation, and growth inhibition of trastuzumab-sensitive breast cancer cells. CHK overexpression combined with trastuzumab exerts an additive effect on cell growth inhibition. We further demonstrate that positive ErbB2-pY1248 staining in ErbB2-positive breast cancer biopsies correlates with the increased trastuzumab response in trastuzumab neoadjuvant settings. Collectively, this study highlights an important role for ErbB2-pY1248 in mediating trastuzumab-induced growth inhibition and trastuzumab-induced interactions between CHK and ErbB2-pY1248 is identified as a novel mechanism of action that mediates the growth inhibition of breast cancer cells. The novel mechanistic insights into trastuzumab action revealed by this study may impact the design of next generation of therapeutic monoclonal antibodies targeting receptor tyrosine kinases, as well as open new avenues to identify novel targets for the treatment of ErbB2-positive cancers.     

HER2过表达乳腺癌曲妥珠单抗耐药后靶向治疗策略

 曲妥珠单抗与化疗药物联用后提高了人类表皮生长因子受体2（HER2）过表达乳腺癌的反应率,但在开始治疗后数月易出现治疗性耐药,限制了患者的总生存期。曲妥珠单抗耐药机制主要与HER家族以外的蛋白酪氨酸激酶受体信号通路活化、PI3K-AKT通路扩增等有关。酪氨酸激酶抑制剂、磷脂酰肌醇3-激酶抑制剂等分子靶向药物作为曲妥珠单抗耐药者的替代治疗手段,单药疗效均不甚满意。多个大型临床试验证实,新型分子靶向药物联合应用能明显限制,甚或最终消除曲妥珠单抗初始治疗耐药性问题。     

Selective inhibition of ADAM metalloproteases blocks HER-2 extracellular domain (ECD) cleavage and potentiates the anti-tumor effects of trastuzumab

The HER-2 receptor tyrosine kinase is an important regulator of cell proliferation and survival, and it is a clinically validated target of therapeutic intervention for HER-2 positive breast cancer patients. Its extracellular domain (ECD) is frequently cleaved by protease(s) in HER-2 overexpressing breast cancer patients, rendering the remaining membrane-bound portion (p95) a constitutively activated kinase. The presence of both serum ECD and cellular p95 protein has been linked to poor clinical outcome as well as reduced effectiveness of some therapeutic treatments. We have identified a series of potent, selective small molecule inhibitors of ADAM proteases, exemplified here by INCB003619, and demonstrate that these inhibitors effectively block HER-2 cleavage in HER-2 overexpressing human breast cancer cell lines. Intriguingly, when used in combination, INCB003619 dramatically enhances the antiproliferative activity of suboptimal doses of the anti-HER-2 antibody, trastuzumab, in HER-2 overexpressing/shedding breast cancer cell lines, accompanied by reduced ERK and AKT phosphorylation. Furthermore, INCB003619, in combination with trastuzumab, augments the pro-apoptotic and antiproliferative effects of the chemotherapeutic agent paclitaxel. Consistent with these in vitro data, INCB003619 reduces serum ECD levels and enhances the antitumor effect of trastuzumab in a xenograft tumor model derived from the HER-2 overexpressing BT-474 breast cancer cell line. Collectively, these findings suggest that blocking HER-2 cleavage with selective ADAM inhibitors may represent a novel therapeutic approach for treating HER-2 overexpressing breast cancer patients.     

Landscape of combination therapy trials in breast cancer brain metastasis

Combination therapy has become a cornerstone in cancer treatment to potentiate therapeutic effectiveness and overcome drug resistance and metastasis. In this work, we explore combination trials in breast cancer brain metastasis (BCBM), highlighting deficiencies in trial design and underlining promising combination strategies. On October 31, 2019, we examined ClinicalTrials.gov for interventional and therapeutic clinical trials involving combination therapy for BCBM, without limiting for date or location. Information on trial characteristics was collected. Combination therapies used in trials were analyzed and explored in line with evidence from the medical literature. Sixty-five combination therapy trials were selected (n = 65), constituting less than 0.7% of all breast cancer trials. Most trials (62%) combined ≥2 chemotherapeutic agents. Chemotherapy with radiation was main-stay in 23% of trials. Trastuzumab was mostly used in combination (31%), followed by lapatinib (20%) and capecitabine (15%). Common strategies involved combining tyrosine kinase inhibitors with thymidylate synthase inhibitors (6 trials), dual HER-dimerization inhibitors (3 trials), microtubule inhibitors and tyrosine kinase inhibitors (3 trials), and HER-dimerization inhibitors and tyrosine kinase inhibitors (3 trials). The combination of tucatinib and capecitabine yielded the highest objective response rate (83%) in early phase trials. The triple combination of trastuzumab, tucatinib and capecitabine lowered the risk of disease progression or death by 52% in patients with HER2-positive BCBM. Combining therapeutic agents based on biological mechanisms is necessary to increase the effectiveness of available anti-cancer regimens. Significant survival benefit has yet to be achieved in future combination therapy trials. Enhancing drug delivery through blood–brain barrier permeable agents may potentiate the overall therapeutic outcomes.     

Trastuzumab in breast cancer

Trastuzumab (Herceptin) is a therapeutic monoclonal antibody specific for the human epidermal growth factor receptor type 2 (HER2), a cell-surface tyrosine kinase receptor overexpressed by 25% to 30% of breast cancers. The drug is now regarded as one option for standard therapy in HER2-overexpressing metastatic breast cancers. It is associated with a moderate response rate as a single agent, and in combination with standard chemotherapy, can produce greater response rates and prolong the survival of women with advanced breast cancer. Its activity in metastatic breast cancer has led to active clinical trials examining its potential role in the neoadjuvant and adjuvant settings. The successful clinical development of trastuzumab provides further proof of principle that biologically targeted therapies can have a profound impact on the management of breast cancer. Here we review the clinical development of this novel agent, emphasizing the potential for therapeutic synergy when trastuzumab is combined with both standard chemotherapy and innovative molecularly targeted and biologic agents.     

Controlled internalization of Her-2/ neu receptors by cross-linking for targeted delivery

Receptor mediated internalization is a crucial step for targeted intracellular delivery of therapeutic and imaging agents. It was recently demonstrated that trastuzumab, an FDA approved humanized monoclonal antibody against Her-2/ neu tyrosine kinase receptor, did not induce endocytosis of the internalization resistant Her-2/ neu receptor. Here we report that accelerated internalization of trastuzumab can be induced by cross-linking the cell membrane bound antibody-receptor complex with an avidin/streptavidin-biotin system. We demonstrated that internalization was achieved both in vitro and in vivo in Her-2/ neu expressing human breast cancer cell lines (BT-474, SK-BR-3 and AU-565) and that repetitive labeling cycles further amplified the loading of cargo molecules within the targeted cells. No trastuzumab binding and internalization was observed in Her-2/ neu negative MDA-MB-231 cells, whereas weak membrane binding and negligible internalization were detected in MCF-7 cells with low expression level of Her-2/ neu receptor. The method was used to noninvasively image Her-2/ neu receptors in isolated cells and in a preclinical breast cancer model with MRI. The controlled internalization of Her-2/ neu receptors can potentially enhance intracellular delivery of drugs and imaging probes, and improve imaging sensitivity and selectivity as well as therapeutic efficacy, through antibody-directed binding and internalization using a pretargeting approach.     

Trastuzumab Plus Pertuzumab Resistance Does Not Preclude Response to Lapatinib Plus Trastuzumab in HER2‐Amplified Colorectal Cancer

Human epidermal growth factor 2 (HER2) amplification represents a distinct molecular subgroup of colorectal cancers that is associated with anti‐epidermal growth factor receptor resistance and sensitivity to dual HER2 targeting. Although clinical trials have reported activity for trastuzumab/pertuzumab and trastuzumab/lapatinib combinations, there are no reports on lapatinib plus trastuzumab activity after resistance to trastuzumab plus pertuzumab. Presented are three cases of HER2 amplified colorectal cancer that developed acquired refractoriness to trastuzumab pertuzumab with subsequent clinical benefit to lapatinib plus trastuzumab, highlighting the potential for HER2 tyrosine kinase inhibition plus trastuzumab in overcoming trastuzumab/pertuzumab resistance.     

抗EGFR靶向治疗胃癌

EGFR家族在胃癌形成中具有重要的作用.针对EGFR的分子靶向药物主要包括胞外单克隆抗体和胞内酪氨酸激酶抑制剂,许多新药如曲妥珠单抗、西妥昔单抗、帕尼单抗、拉帕替尼,已进入胃癌治疗的Ⅲ期临床研究阶段,而ToGA研究结果更给晚期胃癌患者带来希望.     

Targeted Inhibition of Multiple Receptor Tyrosine Kinases in Mesothelioma

The receptor tyrosine kinases (RTKs) epidermal growth factor receptor (EGFR) and MET are activated in subsets of mesothelioma, suggesting that these kinases might represent novel therapeutic targets in this notoriously chemotherapy-resistant cancer. However, clinical trials have shown little activity for EGFR inhibitors in mesothelioma. Despite the evidence for RTK activation in mesothelioma pathogenesis, it is unclear whether transforming activity is dependent on an individual kinase oncoprotein or the coordinated activity of multiple kinases. Using phospho-RTK and immunoblot assays, we herein demonstrate activation of multiple RTKs (EGFR, MET, AXL, and ERBB3) in individual mesothelioma cell lines but not in normal mesothelioma cells. Inhibition of mesothelioma multi-RTK signaling was accomplished using combinations of RTK direct inhibitors or by inhibition of the RTK chaperone, heat shock protein 90 (HSP90). Multi-RTK inhibition by the HSP90 inhibitor 17-allyloamino-17-demethoxygeldanamycin (17-AAG) had a substantially greater effect on mesothelioma proliferation and survival compared with inhibition of individual activated RTKs. HSP90 inhibition also suppressed phosphorylation of downstream signaling intermediates (AKT, mitogen-activated protein kinase, and S6); upregulated the p53, p21, and p27 cell cycle checkpoints; induced G₂ phase arrest; induced caspase 3/7 activity; and led to an increase in the sub-G₁ apoptotic population. These compelling proapoptotic and antiproliferative responses indicate that HSP90 inhibition warrants clinical evaluation as a novel therapeutic strategy in mesothelioma.     

Intermediate to highly suspicious calcification in breast lesions: a radio-pathologic correlation

Targeted therapies against the human epidermal growth factor receptor HER2, have led to revolutionary strides in breast cancer research and treatment. Clinical therapeutic decisions in the treatment of patients with HER2 positive metastatic breast cancer are based on appropriate patient selection, the clinical situation, and data related to the available therapeutic agents trastuzumab and lapatinib. Trastuzumab was the first agent tested and approved in 1996 as single agent for patients with chemotherapy-refractory disease, and in combination with paclitaxel as first-line treatment. This intravenous humanized monoclonal antibody is directed against the extracellular domain of the HER2 protein. Lapatinib, an oral small molecule tyrosine kinase inhibitor has more recently become available (in 2007), approved for used in combination with capecitabine for patients with HER2 positive metastatic disease that has progressed on trastuzumab. Important questions and controversies still remain and are reviewed, including patient selection for anti-HER2 treatment of metastatic disease based on HER2 testing, dose scheduling of trastuzumab, duration of therapy, tolerability, role of lapatinib and clinical significance of trastuzumab resistance and efficacy. Ongoing trials designed to maximize the therapeutic ratio of these agents, are also discussed.     

Monoclonal antibodies, small molecules, and vaccines in the treatment of breast cancer

The human epidermal growth factor receptor (HER, ErbB) family of receptors is considered an important therapeutic target, and various types of molecularly based small molecules, including monoclonal antibodies, protein tyrosine kinase inhibitors, and therapeutic vaccines, are in development as potential therapies for metastatic breast cancer. Trastuzumab (Herceptin; Genentech, Inc.; South San Francisco, CA), the first approved monoclonal antibody for HER-2 (ErbB-2)-overexpressing metastatic breast cancer, provided the proof of principle that targeting specific receptors results in clinical benefit. Other monoclonal antibodies and the small molecule dual protein tyrosine kinase inhibitors show great promise as treatments for metastatic breast cancer but require evaluation in clinical trials to assess their benefits. Therapeutic vaccines may have a role, particularly in early-stage disease, but they are associated with greater limitations and study design issues that make their evaluation difficult. Optimum combination therapy regimens with a variety of novel approaches that incorporate small molecule targeted therapies need to be developed, and the population most likely to benefit from targeted therapies needs to be identified.     

Combining lapatinib (GW572016), a small molecule inhibitor of ErbB1 and ErbB2 tyrosine kinases, with therapeutic anti-ErbB2 antibodies enhances apoptosis of ErbB2-overexpressing breast cancer cells

Antibodies and small molecule tyrosine kinase inhibitors targeting ErbB2 exhibit distinct, noncross resistant mechanisms of action. Here, apoptosis of ErbB2-overexpressing breast cancer cells was enhanced by combining lapatinib, an inhibitor of ErbB1 and ErbB2 tyrosine kinases, with anti-ErbB2 antibodies, including (i) trastuzumab, a humanized monoclonal antibody, and (ii) pAb, rabbit polyclonal antisera generated by vaccination with a human ErbB2 fusion protein. Treating ErbB2-overexpressing breast cancer cell lines with a relatively low concentration of lapatinib alone resulted in a minimal increase in tumor cell apoptosis with an associated decrease in steady-state protein levels of p-ErbB2, p-Akt, p-Erk1/2, and notably survivin, compared to baseline. Exposure to pAb alone reduced total ErbB2 protein, disrupting ErbB3 transactivation, leading to a marked inhibition of p-Akt; however, survivin protein levels remained unchanged and apoptosis only increased slightly. Treatment with trastuzumab alone had relatively little effect on survivin and apoptosis was unaffected. Combining lapatinib with either pAb or trastuzumab markedly downregulated survivin protein and enhanced tumor cell apoptosis. The association between the inhibition of survivin and enhanced apoptosis following the combination of ErbB2-targeted therapies provides a biological effect in order to identify therapeutic strategies that promote tumor cell apoptosis and might improve clinical response.     

Her-2/neu-triggered intracellular tyrosine kinase activation: in vivo relevance of ligand-independent activation mechanisms and impact upon the efficacy of trastuzumab-based treatment

Proteolytic cleavage of the Her-2/neu extracellular domain (ECD) has been shown to initiate receptor phosphorylation representing Her-2/neu activation in vitro. The present investigation was performed to evaluate the clinical relevance of ECD cleavage for Her-2/neu activation and the consequences of active intracellular Her-2/neu signalling reflected by tyrosine kinase phosphorylation in patients treated with the anti-Her-2/neu antibody trastuzumab. Sera from 62 patients receiving trastuzumab-based treatment for Her-2/neu overexpressing metastatic breast cancer were assessed for pretreatment ECD levels using an enzyme-linked immunosorbent assay. In parallel, Her-2/neu activation status of tumour specimens was assessed by immunohistochemistry using a Her-2/neu phosphorylation state specific antibody (PN2A) and correlated with the patients' ECD levels and clinical course of disease. Serum ECD levels were significantly higher in 15 (24%) patients with tumours exhibiting activated Her-2/neu as compared to those without detectable Her-2/neu phosphorylation (median 148.2 vs 28.5 ng ml(-1), P=0.010). Whereas response rate only showed a trend to be higher in patients with Her-2/neu-phosphorylated breast cancer (47 vs 34%, P=0.197), both uni- and multivariate analyses revealed that the median progression-free survival under trastuzumab-based treatment was significantly longer in patients with Her-2/neu-phosphorylated breast cancer-11.7 (95% CI 5.2-18.3) months-when compared to the progression-free survival of 4.5 (95% CI 3.4-5.6) months observed in patients with tumours lacking phosphorylated Her-2/neu (P=0.001). Proteolytic cleavage of the ECD represents a biologically relevant ligand-independent mechanism of Her-2/neu activation in vivo. The influence of Her-2/neu activation status upon the outcome of trastuzumab-based therapies merits further investigation in larger prospective trials.     

Current status of antibody therapy for breast cancer

Antibody therapy with trastuzumab has greatly impacted breast cancer treatment. Combination treatment with trastuzumab is regarded currently as a first-line therapy for metastatic breast cancers that overexpress Her-2. It has become routine practice to examine the status of Her-2 expression in primary tumors. The impact of this therapy might be as great as that of endocrine therapy from a historical point of view. A number of new approaches using trastuzumab for seeking individualized treatment are being tested in current clinical trials. We reviewed recent advances in trastuzumab treatment and discuss the future of antibody therapy for breast cancer.     

Recent advances in systemic therapy: Advances in systemic therapy for HER2-positive metastatic breast cancer

Human epidermal growth factor receptor (HER)2 over-expression is associated with a shortened disease-free interval and poor survival. Although the addition of trastuzumab to chemotherapy in the first-line setting has improved response rates, progression-free survival, and overall survival, response rates declined when trastuzumab was used beyond the first-line setting because of multiple mechanisms of resistance. Studies have demonstrated the clinical utility of continuing trastuzumab beyond progression, and further trials to explore this concept are ongoing. New tyrosine kinase inhibitors, monoclonal antibodies, PTEN (phosphatase and tensin homolog) pathway regulators, HER2 antibody-drug conjugates, and inhibitors of heat shock protein-90 are being evaluated to determine whether they may have a role to play in treating trastuzumab-resistant metastatic breast cancer.     

Carcinoma of an unknown primary: are EGF receptor,Her-2/neu,and c-Kit tyrosine kinases potential targets for therapy?

Carcinomas of an unknown primary site (CUP) are heterogeneous tumours with a median survival of only 8 months. Tyrosine kinase inhibitors are promising new drugs. The aim of this study was to determine the expression of EGF-receptor, Her-2/neu, and c-Kit tyrosine kinases in CUP. Paraffin-embedded specimens were obtained from 54 patients with a CUP who were included in the GEFCAPI 01 randomised phase II trial. Immunohistochemistry was performed using the Dako autostainer with antibodies directed against HER-2/neu protein, EGFR protein, and c-Kit protein (CD117). EGFR expression was found in 36 out of 54 samples (66%). In contrast, Her-2/neu overexpression and c-Kit positivity were only detected in 4 and 10% of patients, respectively. No significant association was found between the expression of the tyrosine kinase receptors and prognosis. EGFR expression was significantly associated with response to cisplatin-based chemotherapy: the response rates were 50 and 22% in patients with EGFR-positive tumours and EGFR-negative tumours, respectively (P<0.05). This study shows that EGFR is frequently expressed in CUP. This finding may prompt clinical trials investigating EGFR inhibitors in this setting. In contrast, c-Kit expression and Her-2/neu overexpression occur infrequently in CUP. EGFR expression was correlated to tumour chemosensitivity.     

Current and emerging targeted therapies for metastatic breast cancer

The success of endocrine therapies for hormone receptor-positive breast cancer and trastuzumab and lapatinib for targeting human epidermal growth factor receptor 2 (HER2)-positive tumors has paved the way for the clinical development of several other metastatic breast cancer (MBC)-targeted therapies. Although the benefit of the anti-VEGF (vascular endothelial growth factor) monoclonal antibody bevacizumab in the MBC setting has become a topic of debate, clinical trial results are accumulating, and phase 3 evaluations are ongoing for newer HER2-targeted agents (pertuzumab and trastuzumab-maytansine immunoconjugate) and VEGF-targeted agents (aflibercept), as well as dual, epidermal growth factor receptor/HER2-targeted agents (afatinib [BIBW 2992] and neratinib), multitargeted tyrosine kinase inhibitors (sunitinib and pazopanib), and mammalian target of rapamycin (everolimus) and poly (ADP-ribose) polymerase 1 inhibitors (iniparib, olaparib). These agents as well as other novel classes of anticancer agents are being tested in clinical trials with the potential of addressing unmet therapeutic needs in the MBC patient population.     

Monitoring of FLT3 phosphorylation status and its response to drugs by flow cytometry in AML blast cells

FLT3 mutation and overexpression in most acute myeloid leukaemia (AML) patients make this tyrosine kinase receptor an attractive therapeutic target. FLT3 kinase inhibitors are actually in clinical trials, thus it is critical to develop a reproducible and standardized method for screening of FLT3 activation and for monitoring its inhibition in response to drug in AML patients. We developed a flow cytometry method to analyse phosphorylated FLT3 (P-FLT3) in samples with <10(5) cells. The method was first validated in FLT3 wild-type (HL60/WT) and mutant (MV4-11/ITD(+)) as well as FLT3 negative (K562) cell lines. The method also proved to be reproducible in AML patient samples. Analysis was performed after exposure to drugs (CEP-701 and SU11657), in vitro and in vivo. In response to increasing drug concentrations, there was a linear reduction in P-FLT3. Intracellular flow cytometry analysis correlated with Western blot and XTT assays; flow cytometry data also correlated with FLT3 mutational status. The results highlight a rapid method to detect P-FLT3 protein at the single cell level by flow cytometry which enables an accurate assessment of FLT3 kinase activity in blast cells in response to novel tyrosine kinase inhibitors.     

Genomic Alterations in Advanced Esophageal Cancer May Lead to Subtype‐Specific Therapies

The development of targeted agents for metastatic esophageal or gastroesophageal junction (GEJ) tumors has been limited when compared with that for other common tumors. To date, the anti‐human epidermal growth factor receptor‐2 (HER‐2) antibody, trastuzumab, in combination with chemotherapy, is the only approved novel agent for these cancers, and its use is limited to the small population of patients whose tumors overexpress HER‐2. Despite recent progress in the field, median overall survival remains only 8–12 months for patients with stage IV esophageal or GEJ cancer. In this article, we examine the molecular aberrations thought to drive the development and spread of esophageal cancer and identify promising targets for specific tumor inhibition. Data from clinical studies of targeted agents are reviewed, including epidermal growth factor receptor antibodies, tyrosine kinase inhibitors, HER‐2, and vascular endothelial growth factor‐directed therapy. Current and future targets include MET, fibroblast growth factor receptor, and immune‐based therapies. Evidence from trials to date suggests that molecularly unselected patient cohorts derive minimal benefit from most target‐specific agents, suggesting that future collaborative investigation should focus on preselected molecular subgroups of patients with this challenging heterogeneous disease.