Medicinal Research Reviews 2012, 32(1), 166–215 ErbB family receptor inhibitors as therapeutic agents in breast cancer: Current status and future clinical perspective Ruchi Saxena and Anila Dwivedi* Division of Endocrinology Central Drug Research Institute, CSIR, Lucknow-26001, (U.P.), India *Corresponding author: A. Dwivedi, e-mail: [email protected]; Phone: 091- 0522- 2612411-18, Extn 4438 Fax: +91-091-(522)-2623405 / 2623938 / 2629504 ABSTRACT Breast cancer is the most common cancer diagnosed in women and the second most common cause of female cancer related deaths with more than one million new cases diagnosed per year throughout the world. With the recent advances in the knowledge of cellular processes and signaling pathways involved in the pathogenesis of breast cancer, the current focus of researchers and clinicians is to develop novel treatment strategies that can be included in the armamentarium against breast cancer. With the failure of endocrine- targeted therapy and the development of resistance to existing chemotherapy, the most explored pathway as next generation target for breast cancer therapy has been the EGFR (ErbB-1)/HER-2 (ErbB-2) pathway. The present review focuses on the rationale for targeting members of ErbB receptor family and numerous agents that are in use for inhibiting the pathway. The mechanism of action, preclinical and clinical trial data of the agents that are in use for targeting the EGFR/HER-2 pathway and the current status thereof have been discussed in detail. In addition, the future clinical promises these agents hold either as monotherapy or as combination therapy with conventional agents or with other antisignaling agents have been pondered, so as to provide better and more efficacious treatment strategies for breast cancer patients. KEYWORDS: ErbB; EGFR; HER-2; breast cancer; tyrosine kinase 1. INTRODUCTION Breast cancer is the most common female cancer and the second most common cause of female cancer related deaths with more than one million new cases diagnosed per year throughout the world.1 Despite advances in the early detection of breast cancer and the advent of novel targeted therapies, breast cancer still remains a significant public health problem due to the involvement of multiple aberrant and redundant signaling pathways in the tumorigenesis and the development of resistance to the existing therapeutic agents. Initially, various cytotoxic agents were employed for the treatment of breast cancer. These agents were non-selective and included alkylating agents, anthracyclines, anti-metabolites and tumor antibiotics that kill neoplastic cells by causing DNA damage, interference with DNA repair mechanisms and disturbance of metabolic pathways.2 Soon after combination strategy was formulated as the simultaneous 3-4 combination of two or more agents provided better results. In 1980s clinical trials in breast cancer patients gave many new agents including the taxanes – paclitaxel and docetaxel.5 Though these agents caused tumor regression but cellular toxicity caused by these agents was a serious problem. Subsequently, the endocrine therapy was introduced for early stage breast cancer as large number of tumors were found to be estrogen dependent 6. With the increasing understanding of cellular processes involved in breast cancer at molecular level and the signaling pathways involved, focus Medicinal Research Reviews 2012, 32(1), 166–215 was later, directed towards the development of targeted therapies for more efficacious treatment of breast cancer. Besides, for the formulation of correct therapy for an individual, it became essential to identify the class of the tumor based on the phenotypic markers 7 and a newer classification, was proposed: (i) Estrogen receptor (ER) - positive or luminal type: These low grade tumors are characterized by gene expression patterns similar to normal cells that line the breast ducts and glands and show positive immunohistochemical staining for luminal cytokeratins 8/18.These are further categorized into two types –(a) Luminal A type that express higher levels of estrogen receptor and have better prognosis, (b) Luminal B type that more often express EGFR or HER-2 and have poor prognosis.8,9 The arrival of estrogen antagonist tamoxifen and more recently aromatase inhibitors have significantly prolonged the survival of patients with ER positive luminal A type disease.6, 10-11 (ii) HER-2 type: These are characterized by extra copies of the HER-2 (ErbB-2) gene and are usually of high grade. These cancers grow faster and show poor patient outcome. However, targeted therapies such as trastuzumab and lapatinib are proving to be useful in the treatment of such cancers.12-13 (iii) Basal type: The gene expression pattern of such cancers is similar to those of basal cells that line the outer basal layer of mammary duct, including expression of keratin 5, keratin 6, keratin 17 and four kallikrein genes (klk5-klk8). There tumors show normal expression of HER-2 (ErbB-2) and lower expression of ER.13-14 The “triple negative subgroup” of the basal type lacks expression of estrogen receptor and progesterone receptors, have normal expression of HER-2 and are p53 mutated.15-16 These are high grade cancers, grow faster and have a lower ‘recurrence –free’ and overall survival, regardless of disease stage at diagnosis. This type of cancer is more common among women with BRCA1 mutations.17-18 These cancers do not respond to hormonal therapy and anti-HER-2 targeted therapy. The poly (ADP-ribose) polymerase (PARP) inhibitors appear to be among the most promising treatments under investigation for BRCA-associated cancers and sporadic triple-negative disease.19-22 However, recent findings report that most (>60 %) basal like tumors are EGFR-positive and EGFR-inhibitors are being evaluated in clinical trials in pre-selected patients with basal like tumors.23-24 This modern classification of breast cancer based on molecular features, has directed the efforts of scientists towards the identification and development of agents that would target specific cellular processes/ receptor type with a view to benefit a particular group of patients. In this review, the targeted therapies for breast cancer patients currently undergoing clinical trials have been discussed with special focus on ErbB family receptor inhibitors. 2. TARGETED THERAPIES FOR BREAST CANCER The first selective and targeted therapy came when it was found that majority of cases of breast cancer express higher levels of estrogen receptor-α (ERα).25-28 With the evolving understanding of the biology of ERα pathway, selective estrogen receptor modulators (SERMs) were developed that represented the first targeted endocrine therapy.29-30 Tamoxifen is currently the first line endocrine agent for the treatment of ERα-positive primary and advanced breast cancers.28,30-33 As an adjuvant therapy in early breast cancer, tamoxifen improves overall survival (OS) and its widespread use has led to a reduction of about 26% in breast cancer mortality for both pre- and post-menopausal patients.6,32-34 In previously untreated metastatic breast cancer, more than 50% of ERα- positive tumors achieve an objective response or tumor stabilization on tamoxifen treatment.35-39 Further, tamoxifen shows clinical application as a preventive agent for hormone dependent breast cancer and it also appears to maintain bone density.40-42 Despite the obvious advantages and benefits associated with the use of tamoxifen, there are some negative side-effects that include increased incidences of endometrial cancer in post menopausal women,43-44 increased occurrences of hot flashes and other menopausal symptoms,43 increased blood clots and increased cataracts.45 Another problem with the use of tamoxifen is that all patients with metastatic disease and about 40% patients on adjuvant Medicinal Research Reviews 2012, 32(1), 166–215 tamoxifen therapy eventually relapse and ultimately die from this disease and many ERα-positive patients do not respond to tamoxifen therapy at all.46-47 Several mechanisms of resistance to tamoxifen therapy have been proposed.48-52 These include: (1) decrease in or loss of expression of ERα;49 (2) increased expression of coactivator proteins like Amplified in breast and ovarian cancer-1 (AIB-1) gene amplification51 (AIB-1 associates with other coactivators and the ER transcription machinery to form large complexes capable of synergistically activating ER mediated transcription); (3) decreased expression of corepressors like Nuclear receptor Corepressor (NCoR)52 (NCoR associates with the transcription assembly and influence transcription by recruiting the histone deacetylase complex which leads to chromatin-condensation and decreased rates of transcription); (4) activated growth factor pathways that lead to phosphorylation of ER-α and ligand independent growth signals and cross-talk between ER-α and EGFR/HER-2 pathways which is discussed in detail in Section 3.2.53-55 Fig 1: Potential sites for therapeutic intervention in growth factor mediated pathway. Therapeutic agents specifically targeted at the inhibition of growth factor receptors and events within the signal transduction pathway include antireceptor antibodies, receptor tyrosine kinase inhibitor, farnesyl transferase inhibitor, bcl-2 antisense nucleotide, topoisomerase II inhibitors, and inhibitors of several other signaling intermediates like PKC inhibitor, Ras/Raf/MEK pathway and mTOR/PI3K/Akt pathway inhibitors. In many clinical cases, the lack of response to endocrine therapy together with increased metastasis was found to be associated with overexpression of EGFR/HER-2 and increased crosstalk of this pathway with ERα.56-58 Thus, targeting the growth factor - mediated signaling has become an important therapeutic option for breast cancer treatment. Modes of blocking growth factor signaling include (i) ligand antagonists, (ii) anti-receptor antibodies, (iii) small molecule tyrosine kinase inhibitors, (iv) farnesyl transferase inhibitors and (v) antisense oligonucleotides. These inhibitors can be employed at various steps to ultimately effect gene transcription via growth factor signaling (Fig.1). Therapeutic agents specifically targeted at the inhibition of growth factor receptors and several downstream targets including PKC, MAPK pathway, mTOR/PI3K/Akt pathway, inhibitors of bcl-2, topoisomerase II and ubiquitin proteasome pathway are being developed.59-65 These inhibitors have met various degrees of success and Medicinal Research Reviews 2012, 32(1), 166–215 are under different phases of clinical trials. A summary and overview of the targeted therapies for breast cancer is given in Table - I. Table-Ι: A bird’s eye view of targeted therapies for breast cancer S.No. Pathway targeted /type of therapy Agent Phase of clinical trial for breast cancer 1. Endocrine Therapy 1. Selective Estrogen Receptor Modulators (SERMs) Tamoxifen Raloxifene FDA approved FDA approved for BC risk reduction 2. Aromatase Inhibitors Anastrazole FDA approved for adjuvant therapy of ER+ve BC FDA approved for adjuvant therapy of ER+ve BC FDA approved for adjuvant therapy of ER+ve BC FDA approved for refractory ER+ve MBC Letrozole Exemestane 3. Pure antiestrogen 2. 3. 4. 5. 6. 7. ICI 182,780 Epidermal growth factor receptor 1. Monoclonal antibody Trastuzumab Pertuzumab Cetuximab Approved for HER2 overexpressing BC Phase II Phase II 2. Small molecule inhibitors Gefitinib Erlotinib Lapatinib Canertinib HKI-272 Phase II Phase II FDA approved for MBC Phase II Phase II 1. Monoclonal antibody Bevacizumab FDA approved for ER-ve BC 2. Small molecule inhibitors Pazopanib Sunitinib Axitinib Sorafenib Phase II Phase II Phase II Phase II 1. Farnesyl Transferase Inhibitor 2. Raf inhibitor Antisense oligonucleotide Small molecule inhibitor 3. MEK inhibitor Mammalian Target of Rapamycin and the PI3K/Akt pathway Topoisomerase II R115777 Phase II ISIS 5132 Bay 43-9006 CI-1040 CCI-779 RAD001 C1311 Phase II Phase I Phase II Phase II Phase II Phase II Hsp90 (ubiquitin proteasome pathway) Tanespimycin Phase II Vascular endothelial Growth factor receptor Ras/Raf/MEK/ERK pathway The following sections will briefly review the current status of ErbB receptor inhibitors particularly the EGFR/HER-2 receptor inhibitors developed / being developed so far, for breast cancer therapy and their future clinical implications will be discussed. Medicinal Research Reviews 2012, 32(1), 166–215 3. ErbB RECEPTORS AS A TARGET FOR BREAST CANCER THERAPY The epidermal growth factor receptor family i.e. ErbB family serves as an excellent candidate for therapeutic intervention based on studies of tumor formation, which is defined by aberrant cell proliferation.66-67 To date, four members of ErbB receptor family have been identified: (i) EGFR (HER1/ErbB-1), (ii) HER-2 (ErbB-2/neu), (iii) HER-3 (ErbB-3) and (iv) HER-4 (ErbB-4). These ErbB receptor family members promote tumor cell proliferation as well as survival in a variety of malignancies including breast, lung, prostate, head and neck, stomach, kidney, brain and pancreas.68-75 EGFR is overexpressed in 16-48% of the human breast cancers and an association has been reported between EGFR expression and poor prognosis.76-77 EGFR is also found to be overexpressed in ‘triple-negative’ breast cancers which are characterized by their unique molecular profile, aggressive behavior and distinct patterns of metastasis.1315 Hence EGFR could also serve as a target for therapeutic intervention in subgroup of triple negative breast cancer patients that overexpress EGFR. HER-2 is overexpressed in 25-30% of all human breast carcinomas and a significant correlation between overexpression and reduced survival of breast cancer patients has been found.73-78 Further, overexpression of these ErbB receptor family members may mediate endocrine resistance, due to crosstalk with the ER signal transduction pathways, 56-58,68 as already mentioned in the previous section . Fig 2: Structure of a typical ErbB family receptor showing (1) The extracellular domain containing cysteine rich ligand binding domains I, II, III and IV (2) Transmembrane domain and (3) The intracellular domains i.e. juxtamembrane domain; tyrosine kinase domain and regulatory region domain, including autophosphorylated tyrosine. 3.1. ErbB RECEPTOR SIGNALING ErbB family receptors are transmembrane receptor tyrosine kinases, composed of an extracellular ligandbinding domain;79-81 a trans-membrane domain that anchors the receptor to the membrane; a juxtamembrane domain which is believed to regulate various functional aspects of ErbB receptor including control of the tyrosine kinase activity (Fig. 2), receptor downregulation, ligand internalization, Medicinal Research Reviews 2012, 32(1), 166–215 and receptor sorting. The domain also has binding motifs that mediate its interaction with second messengers like calmodulin82-83; and an intracellular tyrosine kinase domain with enzymatic activity.84 The cytoplasmic domain also consists of a carboxy-terminal tail containing tyrosine autophosphorylation sites which link these receptors to proteins containing Src homology 2 (SH2) and phosphotyrosine-binding (PTB) domain motifs. In the absence of a ligand, the receptors exist as inactive monomers. When the ligands like epidermal growth factor (EGF) and transforming growth factor-α (TGF- α) bind to receptors causing some conformational changes, it results in homo- or hetero- dimerization of the receptors.85 Dimerization of receptor brings the intracellular C-terminal tyrosine kinase domains in close proximity to each other, leading to autophosphorylation. This in turn allows for docking of second messenger proteins like Src homology domain consensus protein (Shc) and growth factor receptor-bound protein (Grb-2) containing SH2 or PTB domains on these phosphorylated tyrosine residues on the receptor,86 activating multiple downstream pathways involved in tumor progression and metastatic disease. Major pathways associated with ErbB signaling include the Ras/mitogen activated protein kinase (MAPK) pathway, the phosphatidyl inositol 3-kinase (PI3K)/Akt pathway, the Janus kinase(JAK)/signal transducers and activators of transcription (STAT) pathway, and the phospholipase Cγ (PLCγ) pathway (Fig. 3). These signaling pathways ultimately affect cell proliferation, survival, motility, and adhesion.87-92 Fig 3: The ErbB signaling pathway. The ErbB family receptors are activated by binding to ligands, including EGF, TGF-heparin-binding EGF-like growth factor, amphiregulin, betacellulin, and epiregulin.Ligand binding induces formation of functionally active dimers (details given in Fig. 4). Dimerization induces the activation of the intracellular tyrosine kinase domain, which leads to autophosphorylation of the receptor on multiple tyrosine residues. This in turn leads to recruitment of adaptor proteins like Shc, Grb-2 and activates a series of intracellular signaling cascades to effect gene transcription resulting in cancer cell proliferation, invasion, metastasis and also stimulates tumor induced angiogenesis. The extracellular portion i.e. the N-terminus of ErbB receptors is the ligand binding domain and binds a variety of ligands.93 The ligands of ErbB family receptors can be divided into three groups based on their affinities for various receptors: (i) epidermal growth factor (EGF), transforming growth factor α (TGF- α), and amphiregulin bind to EGFR (HER-1 i.e. ErbB-1);94-96 (ii) betacellulin, heparin-binding growth factors, and epiregulin can interact with both EGFR (HER-1/ ErbB-1) and HER-4 (ErbB-4);97-100 and (iii) tomoregulins and heregulins/neuregulins (NRG-1, NRG-2, NRG-3, NRG-4) bind to HER-4 (ErbB-4). NRG-1 and NRG-2 also bind to HER-3 (ErbB-4).101-102 There is no known ligand for HER-2 Medicinal Research Reviews 2012, 32(1), 166–215 (ErbB-2), but it is the preferred hetero-dimerization partner for other members of the ErbB receptor family (Fig. 4).93 A List of ErbB family receptors and their cognate ligands is given in Table - II. Fig 4: ErbB family receptor dimerization and downstream signaling. Ligand binding to the receptor induces formation of homo- or hetero-dimers. Upon dimerization the intracellular tyrosine kinase domains of the receptors get phosphorylated and thus get activated. The active dimers subsequently initiate downstream signaling. (details given in Fig. 3). 1, EGFR (HER-1); 2,- HER-2; 3, HER-3; 4, HER-4. Table-ІІ: ErbB family receptors and their ligands Receptor Ligands EGFR (ErbB-1/HER-1) Epidermal growth factor (EGF) Transforming growth factor-α (TGFα) Epiregulin (EP) Amphiregulin (AR) Betacellulin (BTC) Heparin-binding EGF-like growth factor (HB-EGF) HER-2 (ErbB-2) Unknown HER-3 (ErbB-3) Neuregulin(NRG-1)/heregulin(HRG) isoforms NRG-2α and β HER-4 (ErbB-4) NRG-1/HRG isoforms NRG-2α and β NRG-3 NRG-4 Tomoregulin HB-EGF BTC EP The ErbB receptor family regulates cell proliferation, differentiation, apoptosis, invasion, and angiogenesis and thus plays an important role in normal organogenesis by mediating morphogenesis and Medicinal Research Reviews 2012, 32(1), 166–215 differentiation in normal conditions.103-105 In normal cells, the EGFR/HER-2 signaling pathway is under tight regulation by different regulatory mechanisms but this tight control is often lost in case of tumor cells due to which they gain the advantage to proliferate under adverse conditions, metastasize to surrounding tissues, and increase angiogenesis.66-67 Under normal conditions, when a ligand binds to ErbB family receptor, the receptor gets activated which triggers the downstream signaling but in tumor cells, ligand independent activation of the receptor can occur.85-90 Several mechanisms have been proposed for this ligand -independent activation : (i) Overexpression of the wild type EGFR/HER-2 in some cases like cancers of breast, lung, glioblastoma, head and neck cancer, bladder carcinoma, ovarian carcinoma, and prostate cancer, may lead to ligand independent receptor dimerization and subsequent up regulation of EGFR/HER-2 signalling.95,106-107 (ii) Gene amplification is not a commonly reported phenomenon in tumors, with the exception of glioblastoma108 and 20-25% of ER-positive breast cancers that overexpess HER-2.78 (iii) Presence of mutant EGFR causing it to be inappropriately activated. These mutations include point mutations or deletions in the tyrosine kinase activation domain,109-110 seen in about 81% non-small cell lung cancer (NSCLC), and deletion in the extracellular domain (EGFRviii variant), seen in about 2% NSCLC,109-111 25-50% cases of glioblastomas112-113 and in 42% of head and neck cancers.110 However, there are no known tyrosine kinase domain mutations in breast cancer. (iv) Dysregulation of the EGFR pathway in some cancers could be a result of overexpression of TGF-α, an EGFR ligand, leading to the development of an autocrine loop.114 3.2. ROLE OF EGFR/HER-2 IN ENDOCRINE RESISTANCE : CROSS TALK MECHANISM The ER-induced signaling mechanism coupled with the fact that over two thirds of breast cancers exhibit high expression of ER, have provided the rationale for preventing and treating breast cancer by estrogen antagonism, highlighted by the discovery of tamoxifen. However, a serious problem emerging with the use of tamoxifen is intrinsic or acquired resistance to endocrine agents.46-47 Cumulative clinical data suggest that patients with HER-2 and EGFR overexpressing tumors have a poorer outcome when treated with tamoxifen.115-117 Many patients present with primary (de novo) resistance to endocrine therapy, despite high tumor levels of ER, and all patients with advanced disease eventually acquire resistance.48-50 Although as mentioned in earlier Section 2, a number of probable explanations for the development of resistance to endocrine therapy have been given, the activation of ER and the cross-talk between ER-α and EGFR/HER-2 pathways appears to be one of the major players. For example, the membrane bound ERs play key role in the mechanism of cross-talk between ERs and growth factor receptors.118-120 In addition to the activation of gene transcription via the genomic pathway, ER regulates its nongenomic functions via the membrane estrogen receptor mediated signaling.121-123 The membrane estrogen receptors functionally mimic the growth factor ligands and increase the levels of second messengers such as cyclic-adenosine monophosphate (c-AMP) within minutes124-126 which in turn activate various tyrosine kinase receptors such as Insulin-like growth factor-1 receptor (IGF-1R) , EGFR, and HER-2.127-128 ER can also interact with the growth factor receptors either by associating with adaptor molecules or by direct phosphorylation of EGFR/HER-2. Finally, ER-induced signaling pathway also induce EGFR ligands such as TGF-and cause downregulation of EGFR and HER-2.129-131 The crosstalk between the ER and the growth factor signaling pathway is bidirectional. The ER can be phosphorylated at serine-118 within its activation function-1 (AF-1) domain by the MAPKs (Erk1/2) and Akt which are downstream components of EGFR/HER-2 pathway, leading to ligandindependent ER activation.132-133Also, serine-167 in AF-1 is phosphorylated by the ribosomal S6 kinase (RSK) which is itself activated by Erk1 and Erk2.134-136 In addition, phosphorylation of ER coregulatory 137-138 proteins by growth factor kinases modulate the ER signaling pathway. For example, phosphorylation of ER coactivator AIB1 by MAPK increases ER-dependent transcription,139-140 and overexpression of AIB1 converts tamoxifen-bound ER into an estrogen agonist rather than an Medicinal Research Reviews 2012, 32(1), 166–215 antagonist.124 These finding suggests that the crosstalk between the ER and EGFR/HER-2 signaling pathways has an important role in tamoxifen resistance. Delineation of the interplay between the estrogens, ER, and ER cross-talk with receptors like EGFR and HER-2 should be an important diagnostic and prognostic objective in anti-EGFR/ HER-2 therapy. 4. EGFR/HER-2 INHIBITORS IN BREAST CANCER THERAPY Overexpression of at least two of the members (EGFR and HER-2) of the ErbB receptor family has been associated with a more aggressive clinical behavior. Thus, ErbB receptor family was identified early as an important target for drug development. Till date, a number of therapeutic strategies have been developed which specifically target either intracellular or extracellular domains of the EGFR and its family members.74-76,129-130 The extracellular portion of the receptor can be targeted either by the use of ligand antagonists or by monoclonal antibodies against the receptor. Attempts to make small molecules that could compete with EGFR/HER-2 ligands for the ligand binding domain of the receptor have not met success and this strategy is discontinued.141 Monoclonal antibodies bind to the extracellular domain of the receptor preventing its activation by the ligand. A summary of monoclonal antibodies is given in TableIII. The intracellular portion of the receptor can be targeted by using the small molecule tyrosine kinase inhibitors that block the adenosine triphosphate (ATP) binding site of the tyrosine kinase domain as summarized in Table- IV. Table-ΙΙI: Summary of the anti-EGFR/HER-2 monoclonal antibodies for breast cancer treatment Agent Class of EGFR/HER-2 inhibitor Phase of clinical developm -ent FDA approved Adverse effects Clinical activity in Source breast cancer Refer ences Trastuzumab Anti HER-2 antibody Cardiac dysfunction, cardiotoxicity As monotherapy in HER2 overexpressing breast cancer, c/w TKIs and Tam Roche 155160 Pertuzumab Anti HER-2 antibody Phase II Diarrhea, pain, c/w Trastuzumab nausea, Vomiting, mucositis Roche 216221 Cetuximab Anti-EGFR antibody Phase II Rash, diarrhea, c/w carboplatin nausea, triple negative BC vomiting c/w Ironotecan 234237 Agent Gefitinib Erlotinib Lapatinib Canertinib Neratinib S.No. 1. 2. 3. 4. 5. Structure Irreversible pan-HER inhibitor Irreversible pan-HER inhibitor Irreversible Dual TKI for EGFR/ HER2 Reversible EGFR inhibitor Reversible EGFR inhibitor Class of EGFR inhibitor Phase I/II Phase II FDA approved for MBC Phase II Phase II Phase of clinical trial c/w trastuzumab; c/w paclitaxel monotherpy monotherapy in inflammatory BC; c/w trastuzumab; c/w bevacizumab c/w pertuzumab c/w anastrazole Clinical activity in breast cancer Pfizer Glaxo Smith Kline Genentech /Roche Astra Zeneca Source Covalently binds to a conserved cysteine residue located in the kinase domains of these proteins. covalently binds to the ATP binding site of the intracellular kinase domain inhibitor of the intracellular tyrosine kinase domains of both EGFR andHER2 receptors inhibits EGFR tyrosine kinase by binding to the ATPbinding site of the enzyme. inhibits EGFR tyrosine kinase by binding to the ATPbinding site of the enzyme. Mechanism of action Diarrhea, nausea Rash, nausea, vomiting, asthenia, diarrhea, mucosutis, hypersensitivity, thrombocytopenia Rash, diarrhea, nausea, vomiting Rash, diarrhea, nausea, fatigue, headache Rash, diarrhea, nausea, vomiting Adverse effects 302306 296297 269270, 288 264267 246247 Refer ences Medicinal Research Reviews 2012, 32(1), 166–215 Medicinal Research Reviews 2012, 32(1), 166–215 4.1. ANTI-EGFR/HER-2 MONOCLONAL ANTIBODIES 4.1.1. TRASTUZUMAB HER-2 overexpression is seen in 15-30% of breast cancers and is usually caused by gene amplification.142143 It is linked to higher grade and extensive forms of ductal carcinoma in situ144-145 and is also associated with adverse outcome in invasive lobular carcinoma.78,146 In order to select the patients that are likely to benefit from HER-2 targeted therapy, it is important to determine the status of HER-2 in breast cancer. Currently, two types of tests i.e. immunohistochemistry (IHC) that detects receptor overexpression and fluorescence in situ hybridization (FISH) that identifies HER-2 gene amplification, are used for the purpose.147-149 Though detection of HER-2 by FISH is more accurate but it requires special equipments that makes it more expensive. Recent reports indicate a considerable degree of concordance between the two methods of detection on same tumor specimens. While 100% concordance was observed in IHC 3+ readings when compared with FISH, cases with 2+ IHC score were not very reproducible.150 Thus, such patients must have a confirmatory FISH test before administration of trastuzumab therapy. When compared to IHC, FISH test is also found to be a better predictor of response to trastuzumab therapy and overall prognosis. Another test known as chromogenic in situ hybridization (CISH) uses small DNA probes to count the number of HER-2 genes in breast cancer cells.151-152 This has advantage over FISH of being less expensive as it measures color changes and doesn’t require a special microscope. Newer tests are being developed that could measure the amount of HER-2 protein in cancer cells more precisely and thus help in identification of patients who could respond to HER-2 targeted therapies such as trastuzumab. Trastuzumab is a humanized anti-HER-2 monoclonal antibody that has been approved for treatment of patients with breast cancers that overexpress HER-2 protein or that exhibit HER-2 gene amplification.153 It binds to the extracellular domain IV of HER-2, important in facilitating the overall conformational change induced by binding of the ligand to the receptor.154-155 In this way, trastuzumab exhibits its antitumor activity either by antibody-dependent cell mediated cytotoxicity, downregulation of signaling following antibody mediated receptor internalization, or inhibition of signaling through other 156-57 members of the ErbB receptor family by preventing the formation of heterodimer. This in turn will result in decreased angiogenesis, increased apoptosis and decreased proliferation. This antibody therapy was initially targeted specifically for patients with advanced relapsed breast cancer that overexpressed the HER-2 protein.158-159 Since its launch in 1998, trastuzumab has become an important therapeutic option for patients with HER-2/neu-positive breast cancer. It is widely used for its approved indication as a second line of treatment for advanced metastatic disease, and is also being studied in adjuvant treatment for earlier-stage disease and in neoadjuvant treatment protocols.160-163 188.8.131.52. TRASTUZUMAB AS FIRST/ SECOND LINE MONOTHERAPY IN MBC The efficacy and safety of trastuzumab as monotherapy has been assessed in various phase II trials. In one such trial, 164 trastuzumab was administered weekly to 46 patients with pretreated metastatic breast cancer whose tumors overexpressed HER-2. A loading dose of 250 mg trastuzumab was administered intravenously, followed by 10 weekly doses of 100 mg each. After 10 weeks, patients with no disease progression were given weekly maintenance dose of 100 mg. Toxicity was minimal, with no antibodies formed against trastuzumab. Objective responses were observed in 5 of the 43 evaluable patients, including 1 complete response (CR) and 4 partial responses (PR), for an overall response rate (ORR) of 11.6%. Responses were seen in mediastinum, lymph nodes, liver, and chest wall lesions. Minor responses (seen in 2 patients) and stable disease (14 patients) lasted for a median of 5.1 months. These results demonstrated that trastuzumab is well tolerated and Medicinal Research Reviews 2012, 32(1), 166–215 clinically active in patients with HER-2 overexpressing metastatic breast cancers (MBC) with extensive prior therapy. The efficacy of trastuzumab as a single agent was also assessed in a pivotal phase II study by Cobleigh et al. (1999), 165 on heavily pretreated patients whose tumor overexpressed HER-2 at the 2+ and 3+ levels by IHC. The ORR in this group was 15% with a median survival (MS) of 9.1 months in this population refractory to anthracyclines and taxoids treatment. Retrospective analysis of response rate (RR) and median survival (MS) restricted to the patients whose tumors overexpressed HER-2 at the highest levels (IHC 3+) showed a RR of 18% and MS of 16.4 months. Another Phase II study166 investigated the clinical efficacy and safety of trastuzumab monotherapy as first-line treatment given once every 3 weeks in woman with HER-2 positive MBC. In 105 patients receiving five cycles of therapy, the ORR was 19% and the clinical benefit rate (CBR) was 33%. Median time-to-progression (TTP) was 3.4 months. The monotherapy was well tolerated and no significant adverse events were reported. The most common treatment-related adverse events were only mild-tomoderate rigors, pyrexia, headaches, nausea, and fatigue. In a recently published study, 167 where trastuzumab was used as a single agent, the RR in 111 assessable patients with 3+ IHC staining was 35% and the RR for 2+ cases was 0%; the response rates in patients with and without HER-2 gene amplification detected by FISH were 34 and 7%, respectively. 103 It is now clear that 3+ expression by IHC method or gene amplification demonstrated by FISH is required for likely benefit from trastuzumab. The most common adverse effects of trastuzumab were mild to moderate infusion-related reactions, which are usually noted with the first infusion and decrease in frequency thereafter. The most clinically significant adverse event included symptomatic cardiac dysfunction, which occurred in 2% to 4.7% of patients on trastuzumab monotherapy. Thus trastuzumab was accepted to be effective and tolerable in first or second line treatment of HER-2 positive metastatic breast cancer as single agent. 184.108.40.206. TRASTUZUMAB IN COMBINATION THERAPY Many landmark phase II and phase III trials reported additive and synergistic activity of trastuzumab when used in combination with standard chemotherapy (either paclitaxel or anthracycline based). In the pivotal randomized phase III study by Slamon et al. 2001,168 patients were randomized to receive chemotherapy with or without trastuzumab. Patients were grouped according to whether or not adjuvant chemotherapy contained an anthracycline, such that the majority of patients who did not have adjuvant chemotherapy or adjuvant therapy not containing an anthracycline were randomized to doxorubicin and cyclophosphamide with or without trastuzumab. In the group, with adjuvant anthracycline, patients were randomized to paclitaxel with or without trastuzumab. TTP was significantly longer in each of the combination subgroups (cyclophosphamide vs. trastuzumab + cyclophosphamide, 6.1 months vs. 7.8 months; paclitaxel vs. trastuzumab + paclitaxel, 2.7 months vs.6.9 months). When both chemotherapy subsets were considered, a survival benefit attributable to trastuzumab with chemotherapy vs. chemotherapy alone was noted (MS, 25 months vs. 20 months). This observed survival difference was despite the fact that nearly three-quarters of patients treated initially with chemotherapy alone crossed over to trastuzumab as a single agent on progression of disease. The retrospective analysis showed that the difference was much greater in all the parameters in case of patients with HER-2 expression level of 3+ as determined by IHC. These two studies by Cobleigh et al.165 and Slamon et al.168 led to the licensing of trastuzumab as treatment for metastatic breast cancer. In the pivotal phase III trastuzumab combination trial, 169 trastuzumab was associated with class III or IV cardiac dysfunction in 27% of the anthracycline and cyclophosphamide plus trastuzumab-treated group compared with 8% of the group given an anthracycline and cyclophosphamide alone. Cardiac toxicity has remained a significant limiting factor for the use of trastuzumab since its FDA approval in late 1998. Trastuzumab trials since then have included cardiac eligibility criteria and prospective cardiac Medicinal Research Reviews 2012, 32(1), 166–215 monitoring. The incidence of congestive heart failure in a pooled analysis of six recent trials was 2.7%. Cardiotoxicity is usually reversible and manageable even with continued trastuzumab therapy. A randomized, multicenter trial, 170 compared first-line trastuzumab plus docetaxel vs. docetaxel alone in patients with HER-2 positive MBC. Trastuzumab plus docetaxel was significantly superior to docetaxel alone in terms of ORR (61% vs. 34%), Overall survival (OS) (median, 31.2 v 22.7 months; P < .0325), TTP (median, 11.7 vs. 6.1 months), time to treatment failure (median, 9.8 vs. 5.3 months), and duration of response (median, 11.7 v 5.7 months). There was little difference in the number and severity of adverse events between the arms. Another randomized Phase II study,171 evaluated the activity of weekly paclitaxel vs. its combination with trastuzumab for treatment of patients with advanced breast cancer overexpressing HER2. The combination group exhibited superior ORR (75% vs. 56.9%), particularly in the subset of IHC 3+ patients (84.5% vs. 47.5%). A statistically significant better median TTP was also seen in the subgroup with IHC 3+ (369 vs. 272 days) and visceral disease (301 vs. 183 days). Thus weekly paclitaxel plus trastuzumab was found to be highly active and safe and it is superior to paclitaxel alone in patients with IHC score of 3+. The combination of trastuzumab and navelbine has been tested in the phase II setting. 172 The ORR to the combination in patients with metastatic disease was 75%, and in patients whose tumors overexpressed HER-2 at the IHC 3+ level, it was 80%. The combination was well tolerated. Trastuzumab was also tested in combination with vinorelbine, 173 because vinorelbine therapy is not associated with cardiotoxicity, alopecia, or significant gastrointestinal side effects. In a phase II trial, 174 trastuzumab was administered weekly with vinorelbine on the same day. The ORR was 68%. Median time to treatment failure was 5.6 months; 38% of patients were progression free after one year. The data support the use of trastuzumab and vinorelbine as a safe, well-tolerated, and effective first-line treatment for women with HER-2 positive MBC. In a similar trial by Papaldo et al. (2006),175 that compared vinorelbine alone with vinorelbine plus weekly trastuzumab as combination therapy, the combination group showed higher ORR (51.4% vs.27.3%). The median duration of response was 8 months for women treated with vinorelbine and 10 months for those who received the combination. Patients in the combination arm also had a longer TTP (9 months vs. 6 months) and OS (27 months vs. 22 months) Toxicity was mild in both groups. Concerning cardiotoxicity in combination group, 20% patients had left ventricular systolic dysfunction. A study by De Maio et al. (2007), 176 tested the activity of the same combination, with trastuzumab given every 3 weeks. Activity of 3-weekly trastuzumab plus vinorelbine fell within the range of results reported with weekly schedules. Toxicity was prevalently manageable. This combination was found to be safe and active for metastatic breast cancer patients who received adjuvant taxanes with anthracyclines. Trastuzumab was also tested in combination with polychemotherpy. Burris et al. (2004),177 tested efficacy and toxicity of weekly paclitaxel/carboplatin with or without trastuzumab following initial treatment with trastuzumab. Patients were given trastuzumab (8 mg/kg followed by 4 mg/kg/wk) for 8 weeks. Out of 61 patients in the trial, 52 patients were assessable for response and all 61 patients were assessable for survival. Out of the 52 patients, 33% experienced a PR to trastuzumab monotherapy and given 8 additional weeks of trastuzumab, 29% had stable disease and proceeded to receive paclitaxel/carboplatin/trastuzumab. 31 patients with measurable disease were assessable for response after initial single-agent trastuzumab followed by paclitaxel/carboplatin/trastuzumab. An ORR of 84%, median TTP of 14.2 months, and median OS of 32.2 months was reported with the triplet combination. In the patients treated with paclitaxel/carboplatin alone after disease progression on initial trastuzumab, an ORR of 69%, median TTP of 8.3 months, and median OS of 22.2 months was reported. Median TTP for all 61 patients is 10 months and the median OS is 26.7 months. This trial confirmed the activity and tolerability of weekly paclitaxel/carboplatin alone or in combination with trastuzumab in women with HER-2 overexpressing MBC. Robert et al. (2006), 178 conducted a phase III multicenter trial to evaluate the safety and efficacy of trastuzumab and paclitaxel with or without carboplatin in HER-2 overexpresing MBC. Patients were randomized to recieve six cycles of either trastuzumab 4 mg/kg loading dose plus 2 mg/kg weekly thereafter with paclitaxel 175 mg/m2 every 3 weeks , or trastuzumab 4 mg/kg loading dose plus 2 Medicinal Research Reviews 2012, 32(1), 166–215 mg/kg weekly thereafter with paclitaxel 175 mg/m2 and carboplatin area under the time-concentration curve = 6 every 3 weeks followed by weekly trastuzumab alone. The ORR was 52% for the triplet combination versus 36% for combination of trastuzumab and paclitaxel, median progression free survival (PFS) was 10.7 months and 7.1 months respectively. The improved clinical response with triple combination was more evident in HER-2 3+ patients. Both regimens were well tolerated, and febrile neutropenia and neurotoxicity occurred infrequently; Grade 4 neutropenia occurred more frequently with triple combination. 220.127.116.11. TRASTUZUMAB AFTER DISEASE PROGRESSION Trastuzumab has clearly revolutionized treatment for HER-2 positive patients; however in majority of the cases disease progression occurs within 1 year of treatment. It is also uncertain whether further trastuzumab either as monotherapy or in combination with chemotherapy is of any benefit. It is also unknown if retreatment on relapse is useful for patients on adjuvant trastuzumab. Preclinical studies by Fujimoto-Ouchi et al. (2005)179 suggested that trastuzumab in combination with chemotherapy can slow down tumor growth in the presence of disease progression on trastuzumab monotherapy. Clinical evidences for the use of trasuzumab after disease progression are largely derived from the retrospective studies.180-183 The retrospective analyses suggest that continuation of trastuzumab beyond disease progression in patients with HER-2 overexpressing metastatic breast cancer appears to be of value, producing responses and clinical benefit, and is well tolerated without significant cardiac toxicity. The feasibility of this approach warranted examination in prospective trials. However a similar analysis by Montemurru et al.184 gave contrasting results. A phase II trial by Bartsch et al. 185 evaluated the efficacy and safety of gemcitabine and trastuzumab after earlier exposure to anthracyclines, docetaxel and/or vinorelbine, and trastuzumab. Patients received gemcitabine at a dose of 1,250 mg/m² on day one and eight, every 21 days. Trastuzumab was administered in three-week cycles. Earlier therapies consisted of trastuzumab (100%), anthracyclines (100%), vinorelbine (96.6%), docetaxel (72.4%), and capecitabine (72.4%). Nineteen point two percent patients experienced PR, and stable disease ≥ 6 months was observed in a further 26.9%, resulting in a clinical benefit rate of 46.2%. TTP was median 3 months, and OS 17 months. Neutropenia (20.7%), thrombocytopenia (13.8%), and nausea (3.4%) were the only treatment-related adverse effects that occurred with grade 3 or 4 intensity. Four patients (13.8%) developed brain metstasis (BM) while on therapy. Together with the favourable toxicity profile, this regimen appeared to be a safe and potentially effective salvage therapy option in a heavily pre-treated population. A phase III trial By O’Shaughnessy et al. 186 comparing trastuzumab and lapatinib with lapatinib alone did not give significant evidence in favor of using trastuzumab after disease progression. Another phase III randomized trial, 187 GBG26/TBP, investigated the efficacy of continuing trastuzumab plus capecitabine compared to capecitabine alone in patients progressing on any prior trastuzumab treatment. The study required 482 patients, with the first interim analysis to be done after 150 events. However, it was closed early on after recruiting 156 patients. The final analysis reported statistically significant advantage for the combination arm with increase in RR (48% vs. 27%) and mean TPP (8.2 vs. 5.6 months) with no significant increase in OS. In a recent trial, the effect of Ttrastuzumab on survival after BM was analyzed in 78 HER-2 positive breast cancer patients.188 Patients were grouped according to trastuzumab therapy; no treatment and treatment before and after BM were diagnosed. The OS after the diagnosis of BM as well as TTP of intracranial tumors was prolonged in patients who received trastuzumab after BM was diagnosed. Conversely, BM occurred much later in patients who received trastuzumab before BM. In the multivariate Cox regression model, age at BM <50 years, disease-free interval of about 24 months, TTP of intracranial tumor of about 4.8 months, and trastuzumab treatment after BM were signiﬁcantly associated with longer survival after the onset of BM. Thus trastuzumab therapy after the onset of BM in HER-2 positive breast Early-stage invasive BC, node positive or high-risk node negative Early-stage invasive BC, node positive or high-risk node negative Early stage, node positive, invasive BC Node positive or high risk node negative Early stage, node positive or node negative BC NCCTG N9831 NSABP B-31 BCIRG 006 FINHER Tumor characteristics HERA Study 232 3222 3969 3401 Patient no. 3 A: doxorubicin+cyclophosphamide→ docetaxel B: doxorubicin+cyclophosphamide→docetaxel+ trastuzumab A: docetaxel or vinorelbine→cyclophosphamide+ fluorouracil+epirubicin B: docetaxel or vinorelbine +trastuzumab→ cyclophosphamide +fluorouracil+epirubicin 3 3 A: doxorubicin+cyclophosphamide→paclitaxel B: doxorubicin+cyclophosphamide →paclitaxel+ trastuzumab C: docetaxel+carbopltin+trastuzumab 3 2 Median follow up A: doxorubicin+cyclophosphamide→paclitaxel B: doxorubicin+cyclophosphamide →paclitaxel+ trastuzumab C: doxorubicin+cyclophosphamide →paclitaxel →trastuzumab A: chemotherpy→observation B: chemotherapy→trastuzumab for 1 yr C: chemotherapy→trastuzumab for 2 yrs Arms Table V: Summary of the randomized phase III trials of Trastuzumab in adjuvant therapy 88% at 3 yrs 95% at 3 yrs 0.41(0.16-1.08) 89% at 3 yrs 0.42(0.21-0.83) 90% at 4 yrs 0.66(0.47-0.93) 86% at 4 yrs 92% at 4 yrs 0.59(0.40-0.85) 89% at yrs 93% at 4 yrs 0.63(0.49-0.81) 92% at 3 yrs 90% at 3 yrs 0.66 (0.47-0.91) OS HR(95%CI) 78% at 3 yrs 82% at 4 yrs 0.67(0.54-0.83) 77% at 4 yrs 83% at 4 yrs 0.61(0.37-0.65) 73% at 4 yrs 86% at 4 yrs 0.49(0.41-0.58) 4 % at 3 yrs 83% at 3 yrs 0.64(0.54-0.76) DFS HR(95%CI) Medicinal Research Reviews 2012, 32(1), 166–215 Medicinal Research Reviews 2012, 32(1), 166–215 cancer patients is associated with a signiﬁcant survival beneﬁt after BM diagnosis compared with patients who never received or completed trastuzumab before the BM diagnosis. Thus, trastuzumab appears be useful in preventing BM, but there are no definitive evidences to support the continuation of trastuzumab after disease progression. Also, because trastuzumab does not appear to cross the blood brain barrier efficiently, it seems to be of limited use in preventing metastasis. 18.104.22.168. TRASTUZUMAB IN ADJUVANT SETTING Some large international randomized clinical trial have been conducted to test the efficacy of trastuzumab in the adjuvant settings and have given encouraging outcomes except for the PACS04 trial.189, 277 Characteristics of the included trials for trastuzumab are summarized in Table-V. HER-2 positivity was assessed by IHC and FISH in the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-31, North Central Cancer Treatment Group (NCCTG) 9831, and Herceptin Adjuvant (HERA) trials, by IHC and CISH in the Finland Herceptin2 (FinHer) trial, and by FISH only in the Breast Cancer International Group (BCIRG) 006 trial. Patients at high risk for recurrence were enrolled in all studies, as demonstrated by the high prevalence of hormone receptor negative disease and node-positive disease in the populations accrued. Three trials (the NASBP B-31, NCCTG N9831, and BCIRG 006 trials) evaluated the combination of doxorubicin and cyclophosphamide followed by an anthracycline, with or without trastuzumab. The HERA, BCIRG 006, and NCCTG N9831 trials were three-arm studies, while the FinHer trial was a 2 x 2 study (randomizing all patients to vinorelbine or docetaxel and then randomizing the HER-2 positive subgroup to trastuzumab or observation).190-191 Similar sudy design of the (NSABP) B-31 and (NCCTG) 9831 led to the joint analysis of these trials192. Both these trials included treatment with the standard adjuvant chemotherapy regimen of doxorubicin plus cyclophosphamide followed by paclitaxel and 1 year with or without concurrent trastuzumab therapy in women with operable HER-2 positive breast cancer. The combined analysis of data from these two treatment arms was approved by the National Cancer Institute (NCI). A third arm included in the N9831 study, in which patients received sequential trastuzumab after administration of doxorubicin plus cyclophosphamide and paclitaxel, was not included in the combined analysis, but the interim analysis by Perez et al. (2006),193 compared this sequential arm with the control and concurrent trastuzumab–paclitaxel arms. The absolute difference in disease-free survival (DFS) between the trastuzumab group and the control group was 12% at three years. Trastuzumab therapy was associated with a 33% reduction in the risk of death. The three-year cumulative incidence of class III or IV congestive heart failure or death from cardiac causes in the trastuzumab group was 4.1% in trial B-31 and 2.9% in trial N9831. Both the studies suggest that trastuzumab combined with paclitaxel after doxorubicin and cyclophosphamide improves outcomes among women with surgically removed HER-2 positive breast cancer. The HERA trial,190,194 was a multicenter, randomized, three-arm trial in patients with HER-2 positive early stage invasive BC who have completed at least four cycles of (neo)adjuvant chemotherapy, with or without radiotherapy. Patients were randomized to observation only, 1 year of trastuzumab, or 2 years of trastuzumab, given on a 3-weekly schedule. Efficacy results for the standard approach and the 1year trastuzumab therapy treatment arms have been reported. At a 1-year median follow-up, patients treated with trastuzumab in the HERA trial experienced a 46% lower risk of a first event than patients under observation. This corresponded to an absolute benefit of 8.4% in DFS favoring trastuzumab at 2 years. Overall survival in the two groups was not significantly different (29 deaths with trastuzumab vs. 37 with observation). Severe cardiotoxicity developed in 0.5% of the women who were treated with trastuzumab. The HERA trial thus concluded that one year of treatment with trastuzumab after adjuvant chemotherapy significantly improves DFS among women with HER-2 positive breast cancer. The Breast Cancer International Research Group (BCIRG) 006 trial, 195 was designed to assess the role of trastuzumab in a docetaxel-containing chemotherapy regimen with or without doxorubicin. Patients were randomized to one of three regimens: doxorubicin plus cyclophosphamide followed by docetaxel, doxorubicin plus cyclophosphamide followed by docetaxel with concurrent trastuzumab, or combined Medicinal Research Reviews 2012, 32(1), 166–215 docetaxel, carboplatin, and trastuzumab that did not contain an anthracycline. In an interim analysis presented at the 2006 San Antonio Breast Cancer Symposium (SABCS), investigators confirmed that, at a median follow-up of 3 years, the addition of trastuzumab to chemotherapy resulted in a significantly longer survival time than with standard adjuvant chemotherapy alone for patients with HER-2 positive breast cancer. A 39% longer DFS duration for patients in the doxorubicin plus cyclophosphamide followed by docetaxel with concurrent trastuzumab arm and a 41% longer OS time were found compared with the doxorubicin plus cyclophosphamide followed by docetaxel control arm. Similarly, a 33% longer DFS time for combined docetaxel, carboplatin, and trastuzumab and a 34% longer OS time were found, compared with the the doxorubicin plus cyclophosphamide followed by docetaxel control arm. The difference in DFS between the two investigational arms was not considered to be statistically significant. The FinHer trial, 196 compared docetaxel with vinorelbine for the adjuvant treatment of early breast cancer. The patients were randomized to three cycles of docetaxel or vinorelbine followed by three cycles of fluorouracil, epirubicin, and cyclophosphamide. The patients of HER-2 positive subgroup were further randomized to either receive or not receive trastuzumab for 9 weeks along with the first three cycles of docetaxel or vinorelbine. DFS at three years with docetaxel was 91% vs. 86% with vinorelbine but OS did not differ between the groups. Within the subgroup of patients who had HER-2 positive cancer, those who received trastuzumab had a DFS of 89 % vs. 78 % for the subgroup without trastuzumab. The trial led to the conclusion that docetaxel was associated with more adverse events than vinorelbine. Trastuzumab was not associated with decreased left ventricular ejection fraction or cardiac failure. Adjuvant treatment with docetaxel, as compared with vinorelbine, improves DFS in women with early breast cancer. A short course of trastuzumab administered concomitantly with docetaxel or vinorelbine is effective in women with breast cancer who have an amplified HER-2 gene. Though trastuzumab seems to be promising in the adjuvant settings, the question that remains unanswered is the treatment duration and regimen and if it should be given simultaneously or sequentially after chemotherapy. Comparison of trials B-31 and N9831 and the HERA trial suggests that cardiotoxicity is lower after sequential administration. However, we cannot rule out the possibility that simultaneous administration of chemotherapy and trastuzumab is more effective than sequential administration. Simutaneous administration gives cytotoxic effects while sequential administration is cytostatic. Another problem associated with the combination of chemotherapy with adjuvant trastuzumab is cardiotoxicity which limits the benefits of the therapy. Further follow-up of the adjuvant trials will add to the knowledge of the nature and reversibility of cardiac events associated with trastuzumab use and will help in formulating an optimal combination for an individual patient. Presently, trastuzumab appears to be the best treatment available for HER-2 positive breast cancer patients, but there are some issues associated with its use. Firstly, selection of patients who could benefit from trastuzumab therapy should be more accurate. The cardiotoxicity associated with trastuzumab is another serious problem which could be combated with the formulation of optimum treatment strategy in relation to dose, duration and combination with other agents. The emerging problem with trastuzumab therapy is development of resistance to the agent. The majority of HER-2 overexpressing tumors demonstrated primary (de novo or intrinsic) resistance to single-agent trastuzumab. In fact, the rate of primary resistance to single-agent trastuzumab for HER-2 overexpressing MBC is 66% to 88%.165-168 The majority of patients who achieve an initial response to trastuzumab in combination with chemotherapy develop resistance within one year.169-171 In the adjuvant setting, administration of trastuzumab in combination with or following chemotherapy improves the DFS and OS rates in patients with early stage breast cancer.192-195 However, approximately 15% of these women still develop metastatic disease despite trastuzumab-based adjuvant chemotherapy. 22.214.171.124. MECHANISM OF DEVELOPMENT OF TRASTUZUMAB RESISTANCE The majority of HER-2 overexpressing breast cancers either develops resistance or do not respond to trastuzumab therapy alone. This could be due to the one of the following reasons: Medicinal Research Reviews 2012, 32(1), 166–215 (1) Overexpression of MUC4 sterically hinders trastuzumab from binding HER-2 surface receptor and may mediate cross-talk to activate HER-2 leading to disrupted interaction between HER-2 and trastuzumab.197-198 (2) Expression of redundant survival signaling pathways like the insulin-like growth factor (IGF) receptor; growth factor ligands of EGFR, HER-3, or HER-4 (EGF, betacellulin, heregulin) reduce growth inhibitory effect of trastuzumab by 57, 84, and 90 percent, respectively.199-200,202 (3) Deficient expression of the PTEN tumor suppressor gene and enhanced PI3K signaling.203 (4) Expression of p95 truncated form of HER-2 that lacks the extracellular domain, which is the recognition site for trastuzumab.204 In HER-2 overexpressing cancer cells, the extracellular domain (ECD) of HER-2 is cleaved by the sheddases like ADAM (a disintegrin and metalloproteinase) family of zincdependent, membrane-associated metalloprotease.204-205 The ectodomain shed of HER-2 renders its remaining transmembrane portion, p95, a constitutively active, phosphorylated tyrosine kinase. 206-208 In vitro studies indicate that the p95 fragment of HER-2 is 10–100 times more oncogenic than the full length receptor.195 In the clinic, the presence of the ECD in the serum of cancer patients has been linked to a poor prognosis,208-211 with decreases in serum ECD levels during treatment being a predictor of response to trastuzumab therapy.212-214 Accordingly, inhibition of the sheddases responsible for ECD shedding and p95 production may have potential therapeutic benefit in HER-2 positive patients. (5) Downregulation of the cyclin-dependent kinase inhibitor p27kip1.215-217 However, these mechanisms of trastuzumab resistance do not appear to preclude the antitumor activity of small molecule inhibitors of ErbB family receptors. 4.1.2. PERTUZUMAB / 2C4 Pertuzumab is a recombinant humanized monoclonal antibody (2C4) that binds to the extracellular domain II of the HER-2 receptor.218-219 Through its binding, it blocks the ability of dimerization between HER-2 receptor with other ErbB family receptors. Pertuzumab is referred to as a HER dimerization inhibitor, or HDI. Pertuzumab binds at different positions on the receptor from trastuzumab, blocking the pathway through different mechanisms and inhibiting cellular proliferation.201,220 Phase I clinical trial221-222 with pertuzumab as monotherapy has given promising results. Initial phase II studies223 of pertuzumab in MBC patients showed that pertuzumab was safe and well tolerated but had limited efficacy in this group of patients. Phase I and II trials have both demonstrated synergy between pertuzumab and trastuzumab, with the addition of pertuzumab to trastuzumab providing responses among women refractory to trastuzumab therapy. The first Phase II study224 evaluating pertuzumab plus trastuzumab included 66 patients with HER-2 positive, metastatic breast cancer who had progressed on trastuzumab therapy. Overall responses were achieved in 24% of patients with 7.6% CR and 16.7% partial response PR. Disease stabilization for at least six months was achieved in nearly 26% of patients. The adverse effects of this combination included diarrhea, pain, nausea, vomiting and mucositis. There is a hope that the combination of trastuzumab and pertuzumab used with chemotherapy will be even more effective if used to treat women newly diagnosed with advanced cancer. The combination is being evaluated in first-line MBC patients in another study; CLEOPATRA (CLinical Evaluation Of Pertuzumab and TRAstuzumab) conducted by Roche.225 This phase III study began recruiting patients in January 2008 and is underway in 18 countries worldwide. If this study is successful, this combination of trastuzumab plus pertuzumab and chemotherapy has the potential to become a new standard of care in HER-2 positive MBC patients. 4.1.3. CETUXIMAB / C225 Cetuximab is a human-mouse chimeric monoclonal antibody derived from the murine anti-EGFR monoclonal antibody M225.226-227 It has shown growth inhibitory effects in EGFR overexpressing cell lines and tumor xenografts 226,228 and so was considered for the treatment of EGFR overexpressing breast cancer patients including the triple negative cases that overexpress EGFR.15,22-23 It competitively binds to the accessible extracellular domain III of EGFR with high affinity preventing EGFR ligand binding and Medicinal Research Reviews 2012, 32(1), 166–215 inhibiting receptor dimerization.229-230 Irreversible binding of EGFR by Cetuximab facilitates receptor internalization and subsequent degradation. Receptor downmodulation induces cell-cycle arrest, upregulation of p27Kip1 and subsequently inhibits tumor growth and metastasis.228, 231 Cetuximab may also work by mediating complement fixation and ADCC (antibody dependent- cell mediated cytotoxicity). More importantly, this antibody is able to block the activation of the tyrosine kinase domain of the EGFR following stimulation with a specific ligand.232-233 In addition, blockade of the EGFR with monoclonal antibodies results in a significant inhibition of neoangiogenesis. This phenomenon seems to be related to the reduction in the synthesis of angiogenic factors such as interleukin-8 (IL-8), vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in tumor cells following treatment with anti-EGFR monoclonal antibodies.234-235 Although cell lines treated with cetuximab show only a 20–40% antitumor response in vitro, tumors in athymic mice are growth inhibited by more than 75% on cetuximab treatment. Improved antitumor responses in vivo could be due to downmodulation of angiogenic factors.236-237 A supra-additive antitumor effect was observed in preclinical studies when cetuximab was combined with paclitaxel in breast cancer models. However, this combination was not found to be promising in a phase I trial due to disappointing preliminary efficacy.238 A phase II study on 163 MBC patients was done to evaluate the combination of cetuximab with irinotecan and carboplatin, the standard chemotherapeutic agents used for breast cancer therapy.239 The patients were randomized to receive either irinotecan (90 mg/m2) followed by carboplatin on days 1 and 8 of each 21-day cycle or the same treatment except with cetuximab at a dose of 400 mg/m2 i.v. for dose 1 then 250 mg/m2 weekly thereafter. The ORR was significantly improved with the addition of cetuximab than with irinotecan and carboplatin alone (39% vs. 19%) but it is associated with greater toxicity. In another phase II multi-center randomized clinical trial240 on 102 patients with metastatic triple negative (basal like) breast cancer, patients were randomly given either carboplatin in combination with weekly 2 cetuximab (250 mg/m ) or carboplatin alone. In the carboplatin monotherapy cohort 6% achieved a PR, 4% achieved stable disease, and the CBR was 10%. In the combination arm, the ORR was 18%, 9% of patients had stable disease and the CBR was 27%. Although, due to the progressive nature of the disease, most patients progressed rapidly, the combination of carboplatin and cetuximab show significantly improved anti-tumor activity in comparison to carboplatin alone. In another phase II trial cetuximab was combined with Irinotecan in patients with MBC pre-treated with an anthracycline or a taxane-based therapy but the results were not very promising in pretreated patients.241 In this study, 19 patients were treated with cetuximab 250 mg/m2 weekly and Irinotecan 80 mg/m2, the ORR was 11% with one patient achieving a PR and 1 patient achieving a CR. One patient had stable disease for 11 cycles. The combination was well tolerated with dermatologic toxicities in some cases. Thus cetuximab seems to a promising agent and requires further clinical trials to be used for the treatment of breast cancer. As it targets specifically EGFR, it also holds promise for the subgroup of patients with aggressive triple negative phenotype that overexpress EGFR. But for this patient selection is an important criterion. The majority of the studies with anti- EGFR agents merely required EGFR to be present in the tumor or have not preselected at all for patient selection. Currently, there are no accurate diagnostic methods of determining the level of EGFR expression of a tumor and hence, the clinical benefits from anti EGFR therapies are limited. More work is required to be done for the precise indication of EGFR status and predictive value of currently used preclinical models should a lso be reassessed so as to improve the clinical outcome of EGFR targeting agents. 4.2. SMALL MOLECULE TYROSINE KINASE INHIBITORS Numerous ErbB family receptor inhibitors are in clinical development but only lapatinib has received US Food and Drug Administration (FDA) approval for the treatment of breast cancer for MBC.280 These small molecules compete with ATP for binding to the kinase domain of the receptor.242 Tyrosine kinase inhibitors have several potential advantages over monoclonal antibodies. First, they are orally bioavailable Medicinal Research Reviews 2012, 32(1), 166–215 and generally well tolerated. Second, they appear active against truncated forms of HER2 receptors (p95) in vitro.243-244 Third, their small size may allow them to penetrate sanctuary sites, such as the central nervous system. Finally, by taking advantage of the homology between kinase domains of ErbB family receptors, tyrosine kinase inhibitors can be developed to target more than one member of the receptor family simultaneously.112 4.2.1. GEFITINIB Gefitinib a synthetic anilinoquinazoline is an oral, selective and reversible inhibitor of EGFR tyrosine kinase. It competes with ATP for EGFR ATP binding site within the tyrosine kinase domain of the receptor that ultimately blocks signal transduction pathway implicated in proliferation and survival of cancer cells.245 In vitro, gefitinib can bind to the related ErbB receptor family member , however, its affinity for HER-2 is 200-fold lower than that for EGFR.246 The efficacy of treatment with gefitinib improves with increasing levels of EGFR in the tumor.247 HER-2 overexpressing tumors are also susceptible to gefitinib treatment, theoretically by virtue of their heterodimerization with EGFR.248-250 While at low doses gefitinib is cytostatic in vitro, at higher doses it inhibits cellular proliferation, induces apoptosis and decreases in vitro colony formation. Several studies have demonstrated that gefitinib treatment results in a dose and time-dependent growth inhibition in solid tumors.249-251 Gefitinib treatment downmodulates levels of angiogenic factors including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) secreted by tumors in vivo, thereby lowering the degree of neoangiogenesis and microvessel production.252-253 In order to improve the antitumor efficacy of gefitinib it has been combined with chemotherapy or radiotherapy. Gefitinib administered with chemotherapeutic agents is reported to demonstrate supra additive tumor inhibition compared to either treatment alone. 253-254 Its use as monotherapy in advanced and refractory MBC has proven to be disappointing. A phase II multicenter trial was done to evaluate the antitumor activity and pharmacodynamic/biologic effect of geﬁtinib 500 mg/day monotherapy in patients with pre-treated, advanced breast cancer.255 The study showed clinically significant activity of gefitinib when used as monotherapy. However, a phase II trial by Von Minckwitz et al. (2005) evaluating gefitinib as monotherpy on patients with taxane and anthracycline 256 pretreated metastatic breast cancer did not gave encouraging results. Gefitinb monotherapy was found to be well tolerated and the side-eﬀect proﬁle was as expected from current knowledge of the drug. There was no correlation between EGFR expression and response in this study. However, its activity in combination therapy has shown more potential. Gefitinib has also been evaluated in combination therapy in two phase II studies for which it was combined with docetaxel as first line therapy. In one of these studies,257 41 patients were given oral gefitinib 250 mg per day along with docetaxel ( 75 mg/m2 or 100 mg/m2). The ORR was 54% with a CR and PR in 22 out of 41 patients. Toxicities included neutropenia, diarrhea, rash and anemia. The second study was a phase II multi-institutional trial258 to determine the efficacy and tolerability of gefitinib and docetaxel as first-line treatment in 33 patients with MBC.115 Patients received gefitinib 250 mg once daily and docetaxel 75 mg/m2 every 3 weeks, until tumor progression, toxicity or other reasons for discontinuation. The clinical benefit rate was 51.5% and the overall objective response rate was 39.4%. . The median duration of clinical benefit was 10.9 months. The most common reason for study discontinuation was disease progression (16 patients), followed by toxicity (10 patients). Toxicities were mainly attributable to docetaxel, including ≥ grade 3 neutropenia in 43% of patients rather than gefitinib. It was concluded that the combination of docetaxel and gefitinib was an active regimen in MBC, and the toxicities and efficacy were similar to those of docetaxel alone. Unfortunately, gefitinib's activity in MBC could not be elucidated in these two phase II studies as the trials did not include a docetaxel alone group for comparison. Gefitinib was also tested in combination with hormonal therapy that included anastrazole, an aromatase inhibitor approved for adjuvant therapy in the treatment of ER-positive breast cancer.259-260 In a double-blind, placebo-controlled randomized trial261 of 56 postmenopausal patients with ER-positive and EGFR-positive primary breast cancer, patients were randomly assigned to geﬁtinib (250 mg given orally Medicinal Research Reviews 2012, 32(1), 166–215 once a day) and the aromatase inhibitor, anastrazole (1 mg given orally once a day), and to geﬁtinib (250 mg given orally once a day) and placebo of identical appearance to anastrazole given orally once a day, all given for 4–6 weeks before surgery. The combination arm showed a significantly greater reduction of proliferation. Tumor size reduction of ≥30% was achieved in 14 out of 28 patients with the combination treatment and in 12 of 22 patients receiving gefitinib alone.Gefitinib was also evaluated in combination with anastrazole in a phase II multicenter, double blind, randomized trial to investigate its efficacy on reversing resistance to hormonal therapy in women with newly diagnosed hormone receptor positive MBC.262 The patients were randomized to receive anastrazole in combination with either gefitinib or placebo. The gefitinib group showed a superior PFS (14.5 months vs. 8.2 months) and CBR (49% vs. 34%) when compared with placebo. The treatment was well tolerated but adverse events were seen twice as often in the gefitinib arm when compared with the placebo arm. The results suggested that the combination of anastrazole plus gefitinib is well tolerated and shows increased anti-tumor activity when compared to anastrazole alone in MBC patients and further studies are required to evaluate its efficacy. 4.2.2. ERLOTINIB/ OSI-774 Erlotinib selectively, potently and reversibly inhibits EGFR tyrosine kinase activity of wild-type EGFR and also of the constitutively active mutant EGFRvIII.241, 263 Studies in human cancer cells found that it inhibits epidermal growth factor-dependent cell proliferation at nanomolar concentrations. It binds in a reversible fashion to the ATP binding site of the receptor, blocking the downstream pathway that leads to cellular proliferation. 132 Like gefitinib, erlotinib does not decrease the level of EGFR protein.264 Erlotinib shows activity against multiple breast cancer cell lines in vitro and in xenograft models.265 In a phase II dose escalation study of erlotinib in combination with the standard dose of trastuzumab, evidences of antineoplastic activity were observed with the recommended dose of 150 mg/day.265 In a phase II clinical trial erlotinib was given in combination with bevacizumab (a monoclonal antibody that inhibits VEGF pathway) to 13 pre-treated breast cancer patients, the response to therapy was observed in 1 out of 9 evaluable patients.266 The most commonly reported adverse events with erlotinib treatment are grade 1 or 2 rash, diarrhea, asthenia, nausea and vomiting. Erlotinib was also tried in combination with weekly docetaxel treatment as first line therapy; a RR of 55% was obtained in this trial.267 Both gefitinib and erlotinib did not show success in the treatment of breast cancer. One probable reason seems be the lack of tyrosine kinase domain mutations in cases of breast cancer. Another reason for the failure of these agents as breast cancer therapy could be inaccurate selection of patients. So there is need to develop techniques to assess the level of EGFR in tumors before the formulation of therapy for a patient. 4.2.3. LAPATINIB Lapatinib (lapatinib ditosylate), is an orally active dual inhibitor of the tyrosine kinase domain of both EGFR and HER-2. It binds to the ATP binding pocket of the tyrosine kinase domain of the receptor preventing autophosphorylation and thus inhibiting the growth signals.268 Lapatinib has shown activity in a number of diﬀerent metastatic and advanced tumor cell lines including breast cancer cell lines overexpressing either EGFR or HER-2,269-270 and has recently shown positive results in clinical testing as well. Lapatinib also binds with p95 (the truncated form of HER-2) and thus might be effective in trastuzumab resistant cases.271 This novel investigational agent has given enthusiastic results in patients with metastatic, treatment-refractory disease. It is the most clinically advanced of kinase inhibitors in breast cancer and has been approved in March 2007 for use in combination with capecitabine in patients with advanced, refractory MBC.285 Medicinal Research Reviews 2012, 32(1), 166–215 126.96.36.199. LAPATINIB AS MONOTHERAPY The efficacy of lapatinib as single agent second-line therapy in advanced/metastatic breast cancer has been studied in a number of trials. Initial suggestions of the clinical activity of lapatinib came from several phase I trials testing its safety, tolerability and pharmacokinetics. A phase I trial273 on 39 enrolled patients with solid tumors was conducted with dose-escalation from 175 to 1800 mg/day. Adverse events reported include grade 3 diarrhea (2 of 6 patients administered 900 mg lapatinib twice a day) and grade 1–2 skin rash, diarrhea, vomiting, constipation, fatigue and anorexia. Evidences of antitumor activity were observed in patients treated with 1200 mg/day of lapatinib. In another phase Ib dose ranging study by Dees et al (2004),274 in which 30 heavily pretreated breast cancer patients received lapatinib as monotherapy, 4 patients showed confirmed PR and 10 others had prolonged stable disease. All 10 had EGFR expression by IHC, and 8 of these 10 overexpressed HER-2.274 An open-label multicenter phase II study275 was done on 80 HER-2 positive MBC patients refractory to trastuzumab therapy, with an oral dose of 1500 mg daily of lapatinib. The ORR was 8%, 14% of patients achieved stable disease and 22% of patients achieved PFS. Adverse events with lapatinib were tolerable with anorexia, nausea, rash, vomiting, diarrhea, and weight loss being the most common. Cardiotoxicity was not significantly observed. Another phase II study by Gomez et al. (2005),276 assessed the efficacy of lapatinib as first line therapy in HER-2 positive MBC. The patients were randomized to receive either 1500 mg daily or 500 mg daily of lapatinib. The ORR achieved was about 24% with no significant difference between the two groups. These two phase II studies led the way for lapatinib to be investigated in further clinical trials. A phase II study (EGF20008) by Burstein et al. (2008),272 examined the safety and efficacy of lapatinib monotherapy in chemotherapy-refractory tumors. This study included 2 cohorts of patients, cohort A with HER-2 positive and cohort B with HER-2 negative patients. More than 95% of patients had stage IV disease, and nearly all patients had received 3 or more lines of anti-cancer therapy previously. 97% of HER-2 positive patients had received at least 12 weeks of prior trastuzumab therapy. Lapatinib 1500 mg daily was administered, with dose reduction to 1250 mg in the event of grade 3/4 toxicity. HER2 positive cohort showed an ORR of 1.4% versus 0.0% in the HER-2 negative cohort. The independent review reported that 5.7% of HER-2 positive patients received a clinical benefit, but there was no clinical benefit in the HER-2 negative group. Median OS was 29.4 weeks (cohort A) vs. 18.6 weeks (cohort B). These responses were modest, but this was a heavily pretreated cohort. Patients with HER-2 overexpressing breast cancer have been found to have a significantly higher risk of developing BM. Lapatinib is capable of penetrating the blood brain barrier and have been used in clinical trials for the treatment of BM. A phase II study (EGF20009)278 assessed the clinical activity and safety of lapatinib as a first line treatment in locally advanced or metastatic HER-2 positive breast cancer without prior targeted therapy. The ORR was 24% and did not differ significantly between the two dosage groups (1500 mg daily or 500 mg twice daily). The median duration of response was 28.4 weeks, and PFS was 63% at 4 months, and 43% at 6 months. This trial suggested a role for first-line lapatinib therapy in locally advanced or metastatic HER-2 positive breast cancer. Another phase II study (EGF105084) was done by Lin et al. 279 on 39 HER-2 positive patients with progressive BM. The primary end point was objective response in the CNS by Response Evaluation Criteria in Solid Tumors (RECIST). Secondary end points included objective response in non-CNS sites, time to progression, overall survival, and toxicity. One patient achieved a PR in the brain by RECIST (ORR 2.6%). 7 patients (18%) were progression free in both CNS and non-CNS sites at 16 weeks. Exploratory analyses identified additional patients with some degree of volumetric reduction in brain tumor burden. The most common AEs were grade 3 diarrheas and fatigue. Medicinal Research Reviews 2012, 32(1), 166–215 Table VI: Summary of phase II trials of lapatinib as monotherapy Study (reference) Blackwell et al. 275 Johnston et al. 280 Burstein et al. 272 Gomez et al. 276 Lin et al. 279 Kaufman et al. 281 Tumor characteristics Patient no. Lapatinib dose Response rate HER-2+, T- refractory MBC 80 1500 mg od 8% Relapsed/refractory IBC;HER-2+(n=24), EGFR+/HER2-(n=12) 36 1500 mg od 62%(PR) HER2+ 8.3%(PR) EGFR-/ HER2+ Chemo-refractory ABC/MBC 229 1500 mg od 1.4% HER2+ 0.0% HER2- First-line HER-2+ LABC/MBC 138 Arm A:1500 mg od Arm B: 500 mg bid 24% 750 mg bid 2.6% 1500 mg bid 40%-HER2+ 1PR-EGFR+/ HER-2- ve T-refractory/relapsed HER-2+ with BM Chemo-/T-refractory/ relapsed IBC, HER-2+(n=126), EGFR+/HER2-(n=15) 39 141 Lapatinib monotherapy was evaluated in patients with HER-2 positive relapsed/refractory inflammatory breast cancer. In a phase II trial (EGF103009) by Spector et al. (2006), 280 of the 24 patients with HER-2 positive tumors, 62% achieved a PR, with additional 21% experiencing stabilization of the disease. In contrast, in EGFR positive/ HER-2 negative subgroup only 8.3% of the 12 patients achieved a PR. In another study, HER-2 positive patients with inflammatory breast cancer refractory to anthracyclines, taxanes, and trastuzumab were treated with continuous lapatinib monotherapy at 1500 mg daily.281 Preliminary data demonstrated an estimated ORR of 40%. The most frequent toxicities were diarrhea and skin rash. It was concluded that lapatinib monotherapy is active in the treatment of relapsed/refractory HER-2 positive inflammatory breast cancer where currently only a few effective therapies are available. 1 A summary of phase II trials of lapatinib monotherapy is given in Table-VI. 188.8.131.52. LAPATINIB IN COMBINATION WITH OTHER TARGETED THERAPIES Lapatinib is also giving promising results in combination with other targeted agents like inhibitors of the VEGFR signaling pathway that is involved in angigiogenesis and is believed to play an important role in metastsis of breast cancer. Lapatinib in combination with pazopanib, an investigational tyrosine kinase inhibitor and angiogenesis inhibitor,282 is in phase II trial (VEG20007) in patients who have not been treated for their progressive disease. Preliminary reports show a 44% versus 30% RR for the combination arm, with a 73% versus 43% reduction in target lesions at 12 weeks.283 Another phase II trial investigated the effects of combining lapatinib with the angiogenesis inhibitor bevacizumab.284 Bevacizumab is a monoclonal antibody that specifically inhibits VEGFR mediated signaling and has been approved by FDA for the treatment of ER-negative breast cancer.285-286 The combination therapy gave promising results in heavily pretreated patients conferring a 34.4% clinical benefit at Week 24 (CR/PR/ stable disease), while 62.5% of patients had PFS at Week 12 and the combination was well tolerated. A phase III trial (EGF104900) was conducted to assess the advantage of combining lapatinib with trastuzumab in heavily pretreated HER-2 positive MBC patients refractory to trastuzumab therapy.287 In the study, 296 patients heavily pretreated with trastuzumab, were randomized to either lapatinib 1000 mg Medicinal Research Reviews 2012, 32(1), 166–215 daily plus trastuzumab 2 mg/kg weekly or lapatinib 1500 mg daily alone. The combination group demonstrated signiﬁcantly improved PFS (3 months vs. 2.1 months), and CBR (25.2% vs. 13.2%). However the differences in OS and ORR were not statistically significant. Both treatments were well tolerated with similar side-effect profile and an asymptomatic decline in left ventricular ejection fraction occurring in 5% of the patients in the combination arm and in 2% of the patients in the lapatinib only arm. The data showed that combined targeting gave better clinical outcomes in comparison with lapatinib alone in the pretreated metastatic setting. Ongoing trials involving lapatinib and trastuzumab include a phase III trial comparing paclitaxel and trastuzumab with lapatinib or placebo in HER-2 positive metastatic breast cancer (EGF104383), and a phase I study combining lapatinib, trastuzumab, carboplatin and paclitaxel (EGF103892). 184.108.40.206. LAPATINIB IN COMBINATION WITH CHEMOTHERAPY Lapatinib has also been successfully combined with chemotherapy in breast cancer patients.The standard chemotherapeutic agents with which lapatinib was tested include capecitabine and taxanes and the combination groups exhibited better prognosis in comparison to chemotherapy alone. A randomized phase III trial (EGF100151) was conducted to compare lapatinib plus capecitabine with capecitabine alone in women with advanced, progressive HER-2 positive breast cancer who experienced disease progression after treatment with regimens that included an anthracycline, a taxane, and trastuzumab.288-289 The patients were randomly allocated to receive either combination of lapatinib 1250 mg daily plus capecitabine 2000 mg daily or capecitabine 2500 mg daily alone. The interim analysis showed that the addition of lapatinib to capecitabine was associated with a 51% reduction in the risk of disease progression. The median TTP was 8.4 months in the combination group as compared with 4.4 months in the capecitabine monotherapy group. The ORR was 22% vs. 14% in favor of combination group. This improvement was achieved without an increase in serious grade 3/4 events. Again, cardiotoxicity was not a significant event. This trial led to its first approval for use in MBC patients. Lapatinib was further tested in combination therapy when it was evaluated in conjunction with taxanes. A phase III randomized double-blind study (EGF3001),290 was carried out on 579 MBC patients which were either HER-2 negative or have never been tested, The patients were randomized to receive either lapatinib 1500 mg daily combined with paclitaxel 175 mg/m2 or with paclitaxel 175 mg/m2 alone as first-line treatment. The ORR was 35% vs. 25% in favor of the combination group. However, TTP and OS were not significantly different between the two arms except in a subgroup of patients with HER-2 positive advanced breast cancer. As expected, there was a significantly greater toxicity profile in the combination group over the paclitaxel monotherapy group with alopecia, nausea, vomiting, rash and diarrhea being the most common adverse events. 220.127.116.11. LAPATINIB IN COMBINATION WITH HORMONAL THERAPY The evidences of molecular crosstalk between ER- and EGFR/HER-2 pathway, and its association with the development of resistance to hormonal therapy provides the rationale for combining treatments targeting both the pathways. Lapatinib has been tested in preclinical and clinical studies in combination with anti-estrogens like letrozole (an aromatase inhibitor) and tamoxifen (a selective estrogen receptor modulator) that are already approved for the treatment of breast cancer. Preclinical studies by Xia et al. (2006),291 provided evidence that the combining lapatinib with antiestrogen might delay or prevent the development of resistance to lapatinib in HER-2 overexpressing, ER-positive cells. There is also evidence that lapatinib can overcome hormone resistance, caused by cross-talk between HER-2 and ER, in preclinical models.292 A phase III study by Chowdhary et al. (2007),293 to compare lapatinib in combination with letrozole versus letrozole alone in post-menopausal women with ER positive MBC is currently ongoing. Patients are randomized to letrozole with or without lapatinib regardless of HER-2 status to test if lapatinib treatment prevents the conversion of ER positive/ HER-2 negative to ER positive/ Medicinal Research Reviews 2012, 32(1), 166–215 HER-2 positive breast cancer, thus blocking the development of resistance to endocrine therapy. Two phase II trials in hormone resistant, ER positive MBC are currently examining lapatinib as single agent (NCT00225758) or in combination with tamoxifen (NCT00118157). 20 5.3 25 22.8 6.7 35 A: paclitaxel +laptinib B: paclitaxel +placebo 579 HER2-ve or untested MBC Di Leo et al. 2007 response not reached 8.4 4.4 22 14 A: Capecitabine B: Capecitabine +lapatinib Chemo-/Trastuzumab -refractory HER2+ MBC Geyer et al. 2006 324 12.9 9.7 3(PFS) 2.1(PFS) 10.3 6.9 A: lapatinib B: lapatinib +trastuzumab 296 T-refractory, HER2+ MBC O’Shoughnessy et al. 2008 TTP (months) RR (%) Arms Patient no. Tumor characteristics Study (reference) Table VII: Summary of the phase III trials of Lapatinib in combination therapy OS (months) Major phase III clinical trials of lapatinib have been summarized in table -VII. Based on the available data, lapatinib was found to be an active and well-tolerated oral dual tyrosine kinase inhibitor for the treatment of HER-2 overexpressing breast cancer. Lapatinib is also active in trastuzumab refractory MBC patients. Due to its small size it can efficiently cross blood brain barrier and hence has potential benefits in patients with BM. Lapatinib is also found to be useful in inflammatory Medicinal Research Reviews 2012, 32(1), 166–215 breast cancer patients. Lapatinib is associated with either a very low incidence of or no cardiotoxicity and also does not increase toxicity when combined with trastuzumab. Recently, lapatinib treatment in a neo-adjuvant setting has shown to decrease the number of breast cancer stem cells and in tumor biopsies as opposed to chemotherapy which led to an increase.294-295 Therefore, the agents like lapatinib which are less toxic and more efficacious could be of significant importance in the treatment of breast cancer. Hence, lapatinib holds the potential to become the mainstay of HER-2 positive breast cancer therapy in future. 4.2.4. CANERTINIB / CI-1033 Canertinib/CI-1033 is an irreversible pan-erbB inhibitor. It covalently binds to the ATP binding site of the intracellular kinase domain of the receptor thus blocking the downstream signaling pathway.1, 296-297 Canertinib is a non-selective inhibitor of the members of ErbB receptor family, and thus it has a broader range of antitumor activity. As it is irreversible it has prolonged clinical effect and needs less frequent dosing. It inhibits EGFR kinase activity at low nanomolar range and has antitumor activity in EGFR and HER-2 dependent preclinical models.299 It is also active against HER-3 and HER-4 but has no effect on other tyrosine kinases. Canertinib has been shown to be active in both in vitro and in vivo tumor xenograft models of breast cancer.296 In a phase I trials of 10 heavily pretreated patients, one patient achieved stable disease for more than 25 weeks, but objective responses have not yet been reported.299-301 The adverse events associated with canertinib include grade 1–2 diarrhea, rash, nausea, and vomiting.201 At higher doses hypersensitivity reactions have also been observed. In addition to the typical EGFR-related toxicity, there is a 28% incidence of thrombocytopenia associated with canertinib, which might complicate its combination with myelosuppressive cytotoxic agents. It is presently being evaluated in phase II clinical trials. Canertinib could be a potential therapeutic agent in breast cancer patients as it is a non-specific inhibitor of ErbB receptor family but does not inhibit other receptor tyrosine kinases. Hence it has broader range of action and needs further clinical evaluation before it can be used as a therapy for breast cancer. 4.2.5. NERATINIB/HKI-272 HKI-272 is an irreversible orally active pan-HER receptor tyrosine kinase inhibitor with potential antineoplastic activity. It is a dual EGFR and HER-2 inhibitor that is in phase II clinical trial for breast cancer.1 It forms a covalent bond with the conserved cysteine residue of the ATP-binding pocket within the kinase domain of the receptor which thus prevents autophosphorylation of the receptor. HKI-272 treatment of cells results in inhibition of downstream signal transduction events and cell cycle regulatory pathways that ultimately decreases tumor cell proliferation. 302-303 It is highly active against HER-2 overexpressing human breast cancer cell lines, inhibits the EGFR kinase and proliferation of EGFRdependent cells in culture and also in xenograft models. HKI-272 also inhibits the growth of cultured cells that contain sensitizing and resistance-associated EGFR mutations.304 HKI-272 has the particular advantage of having inhibitory activity in tumors that have mutated and become resistant to reversible inhibitors like erlotinib and gefitinib.285 In a preliminary phase I trial on 51 patients, 23 with advanced stage breast cancer, resulted in two confirmed and two unconfirmed PR in breast cancer.305 The encouraging response rate in this phase I trial, led to the initiation of a phase II clinical trial of HKI-272 in patients with advanced stage breast cancer. A phase II study was carried out involving 49 advanced HER-2 positive breast cancer patients who were divided into two treatment arms. The first arm included HER-2 positive breast cancer patients pre-treated with trastuzumab and the second arm included HER-2 patients with no prior trastuzumab treatment. Tumor response and PFS data continues to be gathered, but out of 32 evaluable patients, 6 patients achieved a confirmed PR with additional patients achieving an unconfirmed PR. Diarrhea and nausea were the major adverse effects noted in the study. In another Phase II open label study306 on 102 patients with stage IIIB, IIIC, or IV breast cancer, daily oral doses of 240 mg HKI-272 were generally well tolerated Medicinal Research Reviews 2012, 32(1), 166–215 with diarrhea as the predominant adverse event.1 The primary end point was the rate of PFS defined at 16 weeks. Patients not pretreated with trastuzumab had a PFS rate of 75% while patients with prior trastuzumab treatment had a 16-week PFS of 51%. These data show HKI-272 to be useful in HER-2 positive advanced breast cancer. It is also being evaluated in combination therapy with paclitaxel and with vinorelbine in patients with advanced breast cancer.1 These clinical investigations appear promising and may lead to further treatment options for patients with advanced HER-2 positive breast cancers and also for patients refractory to agents like erlotinib and gefitinib. 5. SUMMARY AND FUTURE IMPLICATIONS In recent years, there has been significant advancement in the treatment of breast cancer, and the number of mortalities due to the disease has substantially reduced for the past few years. These accomplishments have been led by the emergence of targeted therapies that include hormonal inhibitors, growth factor receptor tyrosine kinase inhibitors,59 the farnesyl transferase inhibitors,60 the bcl-2 antisense oligonucleotides61-62 and several other signaling intermediates like mTOR/PI3K/Akt pathway inhibitors63 and inhibitors of ubiquitin proteasome pathway.64-65 Many of these agents are now integrated in the first line therapy for several groups of MBC patients and continue to be the basis of successful treatment. With increasing incidence of tumor resistance to chemotherapy in breast cancer, it is of crucial importance to apply novel therapeutic agents alongwith current treatment standards and to explore newer agents that are under investigation in clinic. The epidermal growth factor receptor family i.e. ErbB family serves as an excellent example for therapeutic intervention based on studies of tumor formation, which is defined by aberrant cell proliferation. The ErbB receptor family consists of four members viz. EGFR, HER-2, HER-3 and HER-4 and promotes tumor cell proliferation in a variety of malignancies including breast cancer Two members of the family EGFR and HER-2 are frequently overexpressed in breast cancer. EGFR is overexpressed in 16-48% of the human breast cancers and an association has been reported between EGFR expression and poor prognosis. HER-2 is over expressed in 25-30% of all human breast carcinomas and a significant correlation between overexpression and reduced survival of breast cancer patients has been found. Trastuzumab has revolutionized the HER-2 overexpressing breast cancer treatment by improving survival in metastatic breast cancers when endocrine therapy failed. However, the majority of HER-2 overexpressing breast cancers do not respond to trastuzumab therapy alone. Several reasons have been proposed for this resistance including expression of redundant survival signaling pathways like IGFR, deficient expression of tumor suppressor gene PTEN, expression of p95HER2, a truncated form of HER-2 lacking extracellular domain recognized by trastuzumab; and downregulation of cyclin dependent kinase inhibitor p27kip. Cetuximab is another monoclonal antibody that is being evaluated in clinical trials and giving promising results. Since it targets EGFR, it also holds promise for subgroup of breast cancer patients with triple negative phenotype that overexpress EGFR. Small molecule tyrosine kinase inhibitors (TKIs) have shown promise in cases that are resistant to trastuzumab therapy as these compounds compete with ATP for binding to the EGFR/HER-2 catalytic kinase domain of the receptor to block the signaling pathway. These agents are also associated with lower risk of cardio-toxicity. Though research in this field has been going on for a long time and numerous EGFR inhibitors are under development, but to date only lapatinib has been approved by FDA for MBC. Lapatinib when combined with capecitabine has shown clinical efficacy in heavily pre-treated HER-2 overexpressing breast cancer patients. A number of other small molecule inhibitors like canertinib and HKI-272 are under development and numerous clinical trials are being conducted to assess the efficiency of these agents but unfortunately only limited gains have been achieved when these agents are used as monotherapy. Among the available therapies, TKIs appear to be better as compared to antibodies targeting EGFRs, in terms of side effects, cost-effectiveness and efficient delivery to tumor sites. However, a big challenge still lies ahead for medicinal chemists and pharmacologists for want of improved strategies, synthesis and identification of novel chemical entities targeting EGFR/HER-2. Medicinal Research Reviews 2012, 32(1), 166–215 The major obstacles in development of successful ErbB targeted therapy include the redundancy of cellular pathways, the concomitant aberrations and involvement of multiple cross talk mechanisms in cancer cells which eventually lead to the development of resistance. Therefore, it is likely that multiple targets need to be addressed for maximal clinical effects and to minimize development of resistance. There are evidences to show that EGFR/HER-2 inhibitors hold greater promise when used in combination with radiation or chemotherapy compared to either treatment alone. So the combination therapy may involve use of EGFR inhibitors with HER-2 inhibitors like trastuzumab with lapatinib that has shown promising results in clinic. The crosstalk between estrogen receptor α and EGFR/HER-2 provides a rationale for combining antiestrogens with HER-2 targeting therapies. Till date only tamoxifen and fulvestrant have been used for ER positive tumors.307 There is also a need for introducing newer antiestrogens with improved profile e.g. toremefine, raloxifene, into combination therapy. Similarly, IGFR1 inhibitors can also be used in combination with EGFR/HER-2 inhibitors.308 Crosstalk between VEGFR and EGFR/HER-2 pathways provides rationale for combining anti-VEGF antibodies or VEGFR tyrosine kinase inhibitors with ErbB inhibitors. Clinical trials are underway to investigate the efficacy of these combination therapies in breast cancer. EGFR/HER-2 inhibitors can also be used with hsp90 antagonists that leads to proteolysis of EGFR/HER-2 or with the inhibitors of PI3K/Akt/mTOR pathway.64-65 Future strategies may also be designed towards the combination of ErbB inhibitors with integrin antagonists to be utilized for enhanced outcome in MBC patients.309 In case of triple negative cancers having EGFR positive status, the PARP inhibitors combined with EGFR inhibitors, may be another ray of hope. Currently, the challenge remains ahead to formulate an appropriate combination strategy to maximize the treatment outcome. On the other hand, it is highly desirable to explore various resistance mechanisms as well. Another recent approach to combat development of resistance involves targeting the breast cancer stem cells.310 This subset of breast cancer cells has the unique property to proliferate and develop new tumors that might be resistant to the ongoing therapy. So it would be advantageous to target these stem cells in contrast to merely treating the symptoms of the disease. Lapatinib treatment in a neo-adjuvant setting has shown to decrease the number of breast cancer stem cells and in tumor biopsies as opposed to chemotherapy which led to an increase.294-295 Therefore, the agents like lapatinib which are less toxic and more efficacious could be of significant importance in the treatment of breast cancer. It is also important to identify the genes that trigger molecular pathways involved in chemoresistance, tumor formation and malignant cell self - renewal associated with the cancer stem cells. New therapies to inhibit breast cancer stem cells would be of vital importance and if these are not targeted, any targeted therapy would be of limited efficacy. Important work also needs to be done to identify patients who will benefit from targeting the ErbB family receptors. This requires the molecular characterization of the tumor of every individual patient and also the identification of biological markers predictive of response or development of resistance to the targeted agents. Development of more precise and accurate methods for the evaluation of expression levels of EGFR/HER-2 is highly mandatory. The selection of appropriate dose and schedule with new agents entering the clinic and implementation of the appropriate combination therapy with conventional therapies as well as with other anti-signaling agents, will aid to the success of upcoming candidate drugs. With the advancement of techniques in genomics and proteomics analysis, it is hoped that future research will also be directed towards the identification of signature genes associated with activation of specific signaling pathway.295 This would help in the identification of the major pathway involved in the tumorigenesis in any case which would lead to the formulation of more specific and appropriate individualized targeted therapy. ACKNOWLODGEMENT CDRI Communication No. 7932 Medicinal Research Reviews 2012, 32(1), 166–215 ABBREVIATIONS AIB1 amplified in breast and ovarian cancer-1 BM brain metastasis CISH chromogenic in situ hybridisation CR complete response c/w combination with DFS disease-free survival EGF epidermal growth factor EGFR epidermal growth factor receptor ERα estrogen receptor α FasL fas ligand FISH fluorescent in situ hybridization FKHR forkhead in rhabdomyosarcoma Grb growth factor receptor-bound protein GSK glycogen synthase kinase HER-2 herceptin-2 IGFR insulin –like growth factor receptor IHC immunohistochemistry JAK janus kinase LBD ligand binding domain MAPK mitogen activated protein kinase MBC metastatic breast cancer MS median survival mTOR mammalian target of rapamycin NRG neuregulin NFκB nuclear factor kappa B ORR overall response rate OS overall survival PFS progression free survival PI3K phosphotidyl inositol 3 kinase PKC Protein Kinase C PLCγ phospho lipase C γ PR partial response PTEN phosphatase and tensin homolog deleted on chromosome 10 RR response rate SOS sf sevenless guanine nucleotide exchange factor STAT signal transducers and activators of transcription TTP time to progression VEGFR vascular endothelial growth factor receptor REFERENCES 1. 2. 3. 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Silverman AP, Levin AM, Lahti JL and Cochran JR. Engineered Cystine-Knot Peptides that Bind αvβ3 Integrin with Antibody-Like Affinities. J Mol Biol 2009; 385:1064–1075. 310. Zhang M, Rosen JM. Stem cells in the etiology and treatment of cancer. Curr Opin Genet Dev 2006;16(1):60-64. Ms. Ruchi Saxena has done Masters in Biochemistry from the Department of Biochemistry, University of Lucknow, India and is presently pursuing Ph.D. under the supervision of Dr. Anila Dwivedi at the Division of Endocrinology at Central Drug Research Institute. She has qualified national level Entrance and Fellowship exams of Council of Scientific and Industrial Research and Indian Council of Medical Research. She is working as a Junior Research Fellow in a project on identification and development of novel therapeutic agent(s) for breast cancer. Dr Anila Dwivedi is a senior research scientist in the Division of Endocrinology at Central Drug Research Institute, Lucknow. She did her Masters in Biochemistry from Lucknow University and obtained Ph.D. in the area of Reproductive Biochemistry and Endocrinology from CDRI in 1986. Currently, she is pursuing research projects on Endocrine Related Cancers and Reproductive Biology. Her research projects are supported by Ministry of Health and Family Welfare, Govt of India and Indian Council of Medical Research, New Delhi, India. She has more than 50 peer- reviewed publications and three national / international patents.
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