Optimizing Outcomes in HER2-Positive Breast Cancer: The Molecular Rationale

The epidermal growth factor (EGF) receptor HER2 is a transmembrane receptor tyrosine kinase that plays a crucial role in the regulation of cell proliferation and survival. The overexpression of HER2 correlates strongly with prognosis in breast cancer. The targeted blockade of HER2 activity with monoclonal antibodies (eg, trastuzumab [Herceptin]) and small-molecule tyrosine kinase inhibitors (eg, lapatinib) results in the inhibition of tumor growth in HER2-positive cancers. Anti-HER2 therapies have also shown efficacy in combination with chemotherapy in clinical trials in patients with HER2- positive breast cancer. Their efficacy may, however, be limited by molecular mechanisms that compensate for HER2 suppression (eg, activity of EGF receptor) or mechanisms of resistance (eg, loss of PTEN). HER2 continues, however, to be overexpressed by the cancer cells, and the continued suppression of HER2 may be required for maximum antitumor effect. It should be noted that in the absence of definitive data from randomized trials showing an absence or presence of benefit, the use of anti-HER2 agents such as trastuzumab in multiple sequential regimens has become the standard of care. Combining HER2 blockers with agents that overcome the compensatory or resistance mechanisms may increase the efficacy of anti-HER2 therapies. In addition, anti-HER2 therapies can have synergy with common chemotherapy regimens and remain effective through multiple lines of therapy. Optimizing the use of therapies that target HER2 signaling will lead to further advances in the treatment of breast cancer.

The epidermal growth factor (EGF) receptor family of tyrosine kinases regulates a complex signaling cascade that controls the proliferation, survival, adhesion, migration, and differentiation of cells.[1,2] The dysregulation of EGF receptor signaling by mechanisms such as receptor or ligand overexpression and constitutive activation of receptors can lead to greater cell proliferation and other tumor-promoting activities.[ 3-5] This pathway is therefore tightly regulated in normal cells. The EGF receptor family consists of four distinct receptors: EGFR (ErbB-1), HER2 (HER2/neu, ErbB- 2), HER3 (ErbB-3), and HER4 (ErbB- 4),[2] and their abnormal activation is associated with human cancers of various origins.[6,7] The HER2 gene was cloned in the early 1980s by investigators at Massachusetts Institute of Technology[8,9] and the National Institutes of Health[10] and in Japan.[11] Overexpression of HER2, which has been reported in approximately a third of breast cancers, was found to correlate with tumor resistance to chemotherapy and poor prognosis.[12-15] A meta-analysis that related the expression of HER2 with outcome found that HER2 gene amplification or overexpression of HER2 protein predicted outcome in breast cancer on either univariate or multivariate analysis in 92% of the studies evaluated.[16] Three cellular mechanisms underlie the poor prognosis in patients with HER2-overexpressing tumors.[16-18] First, HER2 overexpression increases the metastatic properties of cancer cells, such as invasion, angiogenesis, and greater survival. Second, HER2 overexpression also confers greater resistance to therapeutic agents (eg, chemotherapy and hormone therapy), which can result in a poor response to treatment. This may correlate with the absence of steroid hormone receptors on HER2-positive cells. Third, HER2 overexpression confers a strong proliferative advantage to tumor cells that are characterized by a high percentage of S-phase cells. In addition, HER2 overexpression has been correlated with larger tumor size and aneuploidy.[ 16,18]

This article discusses the molecular mechanisms of the oncogenicity of HER2 overexpression, reviews approaches that use molecular targeting for treating HER2-positive cancers, and describes strategies for optimizing outcomes by using agents that target HER2. Molecular Mechanism: Oncogenicity of HER2 Overexpression All members of the EGF receptor family share a similar structure: they consist of an extracellular ligand-binding domain, a single membrane-spanning region, and an intracellular domain with tyrosine kinase activity. On ligand binding to the extracellular domain, EGF receptors form heterodimers or homodimers, resulting in the activation of intracellular tyrosine kinase and autophosphorylation of specific tyrosine residues.[ 2,19] The phosphotyrosine residues in turn recruit adaptor proteins or enzymes, which initiate signaling cascades to produce a physiologic outcome.[2] Receptor signaling is terminated primarily by endocytosis of the receptor-ligand complex, followed by its recycling to the cell surface or degradation. In normal cells, HER2 does not bind to any known ligand with high affinity, but can signal only by recruiting another activated EGF receptor.[20] The subsequent transactivation and autophosphorylation of HER2 generates intracellular signals that are significantly stronger and of substantially longer duration than signals that emanate from other receptor pairs.[21] There are several molecular reasons for the strength of the signal generated by HER2-containing heterodimers (Table 1). First, HER2 is the preferred heterodimerization partner of all other EGF receptors in normal cells, as well as tumor cells.[22] Overexpression of HER2 in tumor cells may further drive its dimerization by increasing its availability to pair with ligand-activated receptors.[23] HER2 does not bind to any known ligand with high affinity, but on dimerization it increases the affinity of ligands to their receptors by decreasing the rate at which ligands dissociate from the active dimers. In addition, HER2 makes its dimerization partner more promiscuous, allowing it to bind to a broader spectrum of EGF-like ligands.[24] As a result, HER2- containing heterodimers can respond to more ligands with a prolonged and stronger signal. Cancers do not necessarily result from an increased rate of cell proliferation; a disruption of the balance between cell division and cell survival is also a crucial factor. Another basis for the oncogenicity of HER2 stems from the potent activation of both the cell-proliferative Ras-MAPK pathway and the cell-survival pathway that is mediated by PI3K/Akt (Figure 1). Because it does not bind any ligand, HER2 cannot signal directly through these pathways, but it can gain control of the pathways by dimerizing with EGFR and HER3. Epidermal growth factor receptor signaling is terminated by the internalization of cell surface receptors, followed by their degradation. HER2 evades this process of signal attenuation by two mechanisms, resulting in its prolonged signaling.[25] Heterodimers that contain HER2 are internalized more slowly than other heterodimers, resulting in impaired signal attenuation. In addition, HER2- containing heterodimers are not targeted to a degradative pathway; instead, they are recycled to the cell surface. By defective internalization and greater recycling, HER2 heterodimers remain at the cell surface longer, increasing the strength and duration of the intracellular signal. It should be noted that the overexpression of HER2 has been associated with its homodimerization and ligand-independent activation (Figure 1).[26-28] Signaling through the MAPK pathway is also significantly enhanced and prolonged in cells that overexpress HER2 when compared with cells that express low levels.[24] This constitutive activity may play a crucial role in the transformation and proliferation of HER2-positive breast cancer cells.

Targeting HER2 in Breast Cancer Because the overexpression of HER2 correlates with the pathogenesis of and prognosis in breast cancer, it is an important therapeutic target. Anti-HER2 therapies (Table 2) reduce the proliferation and survival of tumors that overexpress HER2. Immunologic Therapies
Trastuzumab (Herceptin), a recombinant humanized monoclonal antibody to the extracellular domain of HER2, is the only anti-HER2 agent that has been approved by the US Food and Drug Administration to treat patients with metastatic breast cancer whose tumors overexpress HER2. Trastuzumab has been shown to have both cytostatic and cytotoxic effects in vitro (Table 3). Trastuzumab disrupts receptor signaling through the downstream proapoptotic PI3K/Akt cell-survival pathway (Figure 2).[29-31] Trastuzumab has also been shown to activate the phosphatase activity of the tumor suppressor PTEN, which reverses the activation of PI3K and Akt.[32] Early studies have shown that trastuzumab induces HER2 internalization and degradation in HER2- overexpressing cells[29,33]; however, recent findings suggest that this may not be true.[34] Cells treated with trastuzumab also undergo growth arrest in the G₁ phase, accompanied by the induction of the cyclin-dependent kinase inhibitor p27.[33,35,36] Trastuzumab has been shown to suppress angiogenesis in vivo by inducing antiangiogenic factors, such as thrombospondin 1, and suppressing proangiogenic factors, such as vascular endothelial growth factor, transforming growth factor, angiopoietin 1, and plasminogen activator inhibitor 1.[37,38] In addition, trastuzumab can block the process of metalloproteinase-mediated HER2 ectodomain shedding, which has been shown to cause constitutive HER2 signaling.[39]

By virtue of being an antibody, trastuzumab can harness immune-mediated responses to cause tumor cell toxicity. For example, trastuzumab has been shown to initiate antibodydependent cell-mediated cytotoxicity (ADCC).[40] Trastuzumab has also been shown to potentiate the effects of chemotherapy by multiple mechanisms of action in vitro, as well as in vivo.[41] Pietras and colleagues have shown that treatment with trastuzumab can prevent DNA repair after treatment with DNA-damaging agents.[42] Another molecular explanation for this synergy may be the suppression of the Akt-mediated survival pathway; trastuzumab can therefore induce apoptosis. In fact, a recent trial of trastuzumab in the neoadjuvant setting in primary breast cancers showed that trastuzumab induced apoptosis, confirming that the antibody exerts a cytotoxic effect in vivo.[43] Pertuzumab, another humanized monoclonal antibody to HER2, is currently in phase III trials in patients with breast cancer. In contrast to trastuzumab, pertuzumab binds HER2 near the center of the dimerization arm[44] and can prevent the formation of ligand-induced HER2-containing dimers.[45] As a dimerization inhibitor, pertuzumab diminishes ligandactivated HER2 signaling, including HER2 phosphorylation and activation of MAPK and Akt.[45,46] Pertuzumab would also be expected to recruit effector cells such as macrophages and monocytes to the tumor through the binding of the antibody constant Fc domain to specific receptors on those immune cells. Other antibody-based strategies to attenuate HER2 signaling are in various stages of development. These strategies involve the use of intracellular single-chain Fv antibody fragments as well as armed antibodies. Examples of the latter are toxinlabeled antibodies to HER2[47,48] and antibodies to HER2 labeled with radionuclides such as yttrium-90 and iodine-131.[49,50] Small-Molecule Tyrosine Kinase Inhibitors
The inhibition of tyrosine kinase activity is another strategy for targeting EGF receptor pathways in the treatment of cancer. Small-molecule tyrosine kinase inhibitors (TKIs) have a range of activity, with some specific for a single receptor kinase and others equally active against several receptors.[ 51] Tyrosine kinase inhibitors that are specific for EGFR, eg, gefitinib (Iressa) and erlotinib (Tarceva), have shown only limited efficacy as monotherapies for breast cancer in the preclinical and clinical settings, suggesting that EGFR does not drive tumor growth.[52-54] Dual-kinase inhibitors are a new generation of TKIs that can block signal transduction through both EGFR and HER2 (Table 4). These TKIs inhibit the growth and survival of tumor cells by reducing both MAPK and PI3K signaling (Figure 2).[55,56] Lapatinib, a member of this class of TKIs, has shown promising activity in preclinical and early clinical investigations. In clinical trials lapatinib induced apoptosis and caused growth arrest of tumors that overexpressed HER2 or EGFR.[57] Lapatinib is a reversible inhibitor, and EKB-569 is another dual-kinase inhibitor that irreversibly inhibits the kinase activity of EGFR and HER2; however, the clinical significance of irreversible inhibition has not yet been determined.[51] The potential advantages of dualkinase inhibitors are that they inhibit both ligand-dependent and ligand-independent signaling, they can potentially overcome resistance to trastuzumab in tumors that develop compensatory mechanisms, and they appear to have synergy with chemotherapy.

Heat Shock Protein 90 Inhibitors
Another class of small-molecule inhibitors influences EGF signaling by increasing receptor degradation. These inhibitors (eg, geldanamycin) block heat shock protein 90 (HSP90), a chaperone protein that is crucial in maintaining EGF receptors in a signaling- competent form.[58,59] As a consequence, HSP90 inhibitors prevent the stabilization of EGF receptors at the membrane and target these receptors for degradation.[60,61] Geldanamycin, in particular, binds to members of the HSP90 family, blocking the assembly of HSP90 heterocomplexes and destabilizing existing heterocomplexes.[62] As a result, geldanamycin downregulates surface HER2 through greater degradative sorting in endosomes.[34] Treatment with geldanamycin has been shown to result in decreased Akt activity and is correlated with a loss of Akt phosphorylation in breast cancer cells that overexpress HER2.[63] The major drawback of HSP90 inhibitors, however, is their relatively low specificity for EGF receptors; their use can affect the function of many other cellular proteins that require HSP90 for structural stability. Their specificity may be increased by using them in combination with specific anti-EGF receptor TKIs. Other Strategies
Gene therapy strategies for targeting EGF receptor activity aim at blocking the transcription, translation, and maturation of members of EGF receptor transcripts or proteins.[2] Among the agents in development are the adenovirus type 5 early region 1A gene product,[64] triplex-forming oligonucleotides, antisense oligonucleotides, and ribozymes.[65-67] Further experience with these agents may lead to novel strategies that significantly reduce HER2 signaling. Determinants of Clinical Response to Anti-HER2 Agents The clinical benefits of anti-HER2 agents may not be observed in all HER2-positive patients. For example, trastuzumab monotherapy produces an objective response in about a third of patients with HER2-positive disease and clinical benefits in almost half the patients who overexpress HER2.[68] These responses are higher when trastuzumab is used in combination with chemotherapy; objective responses were observed in 50% of patients. Recent studies suggest that there may be a correlation between the decrease in serum concentration of HER2 between 2 and 4 weeks after the start of trastuzumab- based treatment and progression- free survival.[69] The predictive value of serum HER2 levels should be investigated further. It is important to understand the molecular mechanisms that confer resistance to trastuzumab so that patients with disease that will not respond to the therapy are not exposed to its potential adverse effects.[32] Some HER2-positive tumors may have intrinsic resistance to trastuzumab and other anti-HER2 agents, and others may have acquired resistance, ie, they may have developed compensatory mechanisms (discussed in greater detail in the next section). Ongoing research indicates that the intrinsic resistance of tumor cells to trastuzumab may have a number of causes. An in vitro study showed that primary resistance to trastuzumab could stem from a masking of membrane proteins by a membraneassociated mucin.[70] This masking is thought to result in decreased accessibility to and lack of activation of HER2.[70] Evaluation of a HER2- overexpressing breast cancer cell line after exposure to trastuzumab showed an association between downregulation of p27kip1 levels and secondary resistance.[71] Loss or mutation of the tumor suppressor gene PTEN is another important cause of tumor-cell resistance to trastuzumab.[32,72] A retrospective analysis of breast carcinomas found that the responses to trastuzumab-based therapy were significantly poorer in patients with PTEN-deficient tumors than in those with normal PTEN expression.[32] A recent study has elucidated the role of PTEN in increasing sensitivity to HER2 blockers such as trastuzumab. On binding to HER2, trastuzumab stabilizes and activates the PTEN tumor suppressor, thereby downregulating the proapoptotic PI3K/Akt signaling pathway. Thus, PTEN sensitizes tumor cells to trastuzumab, and in the absence of PTEN the antitumor effects of trastuzumab are impaired.[ 32] These findings suggest that drugs that augment PTEN activity may sensitize tumors to trastuzumab. Combinations of HER2 blockers and PI3K inhibitors (once these are commercially available) may have greater efficacy. The mammalian target of rapamycin (mTOR) kinase, which is an important downstream mediator of the PI3K/Akt pathway, is another potential target for overcoming resistance to trastuzumab. The greater proapoptotic activity of PI3K in the absence of PTEN can be attenuated by inhibiting the downstream mediator, mTOR. The addition of mTOR inhibitors, such as sirolimus (rapamycin [Rapamune]) and temsirolimus, may help overcome resistance to trastuzumab in tumors that lack PTEN.[73]Optimizing Outcomes in Breast Cancer: Molecular Rationale The use of agents that target HER2 has greatly increased survival in patients with HER2-positive breast cancer. The use of anti-HER2 agents is now considered the standard of care for patients with HER2-positive disease, but many questions about the appropriate use of these agents remain to be resolved in clinical trials:

• How should anti-HER2 therapy be used in the adjuvant setting?
• Should anti-HER2 therapy be continued after disease progression?
• Should anti-HER2 therapy be incorporated into treatment through multiple lines?

In the absence of data from prospective clinical trials, understanding the molecular basis of the disease and the mechanisms of action of anti- HER2 agents can provide insights into potential strategies to optimize outcomes in patients with breast cancer. Implications for the Neoadjuvant and Adjuvant Settings
The efficacy of trastuzumab in combination with chemotherapy in patients with HER2-positive metastatic breast cancers[74] has generated interest in evaluating this combination in the neoadjuvant and adjuvant settings. At the molecular level the suppression of HER2 should synergize with chemotherapy in these settings by the same mechanisms, namely, attenuation of the PI3K/Akt cell-survival pathway. The benefit to the patient, however, is likely to be much greater in earlier-stage disease because of the substantially smaller size and limited spread of the tumor. In fact, complete elimination of micrometastases with regimens that include targeted anti-HER2 therapeutics agents may be possible in the adjuvant setting. The preliminary results of clinical trials that address this question have recently become available. In the neoadjuvant setting single-agent trastuzumab has been shown to induce apoptosis in HER2-overexpressing breast cancers, even before a combination of trastuzumab and docetaxel (Taxotere) was given.[43] Furthermore, the cytotoxicity of trastuzumab translated to a clinical benefit, with tumor regression in 8 (23%) of 35 patients after only 3 weeks of trastuzumab; the median decrease in the size of the primary tumor in all patients in the study was 20% (range: 0% to 60%). Promising results have also been observed in a phase III trial of neoadjuvant trastuzumab with combination chemotherapy. This trial randomized patients with HER2-positive operable breast cancer to either four cycles of paclitaxel followed by four cycles of fluorouracil, epirubicin (Ellence), and cyclophosphamide or to the same chemotherapy combined with trastuzumab over 24 weeks.[75] After only 34 patients had completed therapy, it was determined that the addition of trastuzumab significantly increased the likelihood of a pathologic complete response (67% vs 25%; P = .02); the study was stopped because of the superiority of the trastuzumab-based combination. Four large trials are addressing the clinical benefit of combinations of trastuzumab with chemotherapy in the adjuvant setting. The preliminary results of two of these trials have recently been presented; trastuzumab with chemotherapy has shown significantly greater benefits in overall survival and risk of recurrence than chemotherapy alone in the adjuvant setting in patients with early-stage HER2-positive breast cancer.[76,77] Data from the North Central Cancer Treatment Group N9831 Intergroup trial combined with data from the National Surgical Adjuvant Breast and Bowel Project B-31 study showed a 52% reduction in risk of breast cancer recurrence as well as a 33% survival benefit with adjuvant trastuzumab therapy.[77] These results were supported by the HERA (HERceptin Adjuvant) trial of adjuvant trastuzumab, which found a 46% reduction in recurrence by adding trastuzumab to chemotherapy.[76] These results show great promise for patients with earlystage disease. The final results from these trials, as well as the two ongoing studies, are awaited. Consistent with these findings, the guidelines of the National Comprehensive Cancer Network (NCCN) recommend that trastuzumab be incorporated into the adjuvant therapy of patients with HER2-overexpressing node-positive breast cancers.[78] In addition, the guidelines suggest that trastuzumab be considered for patients with HER2- positive node-negative tumors. The guidelines also specify that trastuzumab should not be given concurrently with an anthracycline because of the greater risk of cardiac toxicity. Duration of Treatment With Targeted Therapies
The central role of HER2 in the growth and survival of breast cancer raises the question of the optimal duration of use of anti-HER2 agents, which act as growth inhibitors, and whether their prolonged use is justified. At the molecular level, because overexpression of HER2 is a consequence of gene amplification, HER2 is likely to be expressed and to drive growth even in residual tumors. This hypothesis is supported by preclinical data, which show that HER2 continues to be expressed on tumors of mice that have been treated with trastuzumab[ 79]; however, the clinical relevance remains to be determined. Continued and prolonged suppression of HER2 may be expected to slow tumor growth to some degree by suppressing HER2 signaling mechanisms that could be reactivated by the withdrawal of a HER2 blocker, leading to accelerated tumor growth. A comparable situation is the indefinite continuation of androgen blockade in patients with metastatic prostate cancer even after disease progression.[80] It should be noted that the clinical value of this strategy for prostate cancer has also not been examined in randomized trials. Indeed, this question becomes especially relevant in the adjuvant setting, when patients are likely to be in remission for several years. The optimal duration of anti-HER2 treatment after chemotherapy should be investigated in clinical trials. In the absence of definitive clinical trial data, the NCCN guidelines recommend that in the adjuvant setting trastuzumab should be given for 1 year, with cardiac monitoring.[78] Treatment Through Multiple Lines: Progression After Treatment With Anti-HER2 Agents
An unresolved issue is the need to continue agents like trastuzumab after the failure of regimens that contained an anti-HER2 agent. Should HER2 suppression be incorporated in the subsequent regimen? As discussed earlier, HER2 continues to be expressed in tumors; there may, therefore, be a need to continuously suppress HER2 activity to avoid accelerated tumor regrowth. This hypothesis is supported by the results of a preclinical model of progressive disease in mice bearing a HER2-overexpressing human breast cancer.[79] Mice were treated with trastuzumab monotherapy until disease progression, at which time HER2 continued to be expressed in the tumor, consistent with its continued role in driving tumor growth. The mice were then treated with either paclitaxel or docetaxel alone or in combination with trastuzumab. It was shown that even after disease progression with trastuzumab monotherapy, the combination of trastuzumab and either of the taxanes was significantly more efficacious than taxane monotherapy. These results support the use of anti-HER2 therapy after disease progression. Clinical data suggest that the continued administration of trastuzumab beyond disease progression is safe and feasible.[81-83] Longer durations of therapy do not appear to increase the risk of cardiac dysfunction and may confer some benefit for tumor response and survival.[81,83] The value of continuing trastuzumab after there has been disease progression is also being addressed in ongoing clinical trials. One phase III trial is evaluating the efficacy and toxicity of vinorelbine (Navelbine) with or without trastuzumab in patients with metastatic breast cancer that had progressed after combination therapy with a taxane and trastuzumab.[84] The primary end point in this trial is progression-free survival. Although there are no definitive data to support the clinical benefit of continued trastuzumab following progression on first-line trastuzumabcontaining chemotherapy for metastatic breast cancer and the optimal duration of trastuzumab in patients with long-term control of disease is still unknown, the continued use of trastuzumab through multiple sequential lines of treatment is current practice.[ 78] The benefits of HER2 blockade despite disease progression after anti- HER2 therapy can have implications for several common clinical scenarios, which may not be mutually exclusive.

• Hypothesis 2: Tumor Growth by a Mechanism That Compensates for HER2 Suppression-This scenario describes a situation in which the growth of a HER2-positive tumor is incompletely suppressed despite treatment with a single anti-HER2 agent. Preclinical data support the upregulation of epidermal growth factor receptor HER1 (EGFR) as the primary mechanism of compensation (Figure 3).

Preclinical studies in cell cultures have shown that the overexpression of EGFR and activation of the Akt pathway may be responsible for insensitivity to trastuzumab.[87,88] These data implicate the HER3 pathway, which can activate PI3K and Akt, as well as the EGFR pathway as potential escape mechanisms from anti-HER2 therapy. The inhibition of both of these activities by the EGFRspecific inhibitor gefitinib suggests that EGFR may be the key player in the compensatory mechanism. This was confirmed in another preclinical study, which reported that the growthinhibitory effect of trastuzumab on HER2-overexpressing breast cancer cells was considerably modulated by coexpression of EGFR.[89] This finding is supported by the results of a microarray analysis of tissues from patients who were treated with combination chemotherapy and trastuzumab. An analysis of predictive biomarkers showed that the response to trastuzumab depended not only on the levels of HER2 expression but also on the expression of EGFR, expression of EGF receptor ligands, expression of other receptors, and phosphorylation of downstream proteins.[90] This scenario requires that signaling by both HER2 and EGFR must be blocked for tumor growth to be controlled. Based on the molecular mechanisms, several possible ways to overcome EGFR activity in HER2- positive tumors can be proposed. Strategy 1: Dual-kinase inhibitors. Dual-kinase TKIs, such as lapatinib, which inhibit the activity of both EGFR and HER2, may be effective in overcoming the compensatory mechanisms of EGFR upregulation.[91] Moreover, dual-kinase TKIs may inhibit both ligand-dependent and ligand-independent signaling.[41] This approach cannot, however, provide the additional immune effects, such as those provided by ADCC and antiangiogenesis. Preliminary results from a phase II trial with lapatinib support the value of this strategy; lapatinib was shown to have clinical efficacy in patients with HER2-overexpressing metastatic breast cancer that was refractory to trastuzumab-containing regimens.[92] The initial results also suggest that single- agent lapatinib has activity as a front-line therapy in patients with advanced or metastatic breast cancer with HER2 amplification,[93] but the advantage of lapatinib-based chemotherapy regimens is under investigation. Ongoing phase III trials are comparing the efficacy and safety of lapatinib plus capecitabine (Xeloda), paclitaxel, or letrozole (Femara) and those of chemotherapy alone in patients with metastatic breast cancer.[ 94-96] The findings of these trials should help clarify the role of lapatinib plus chemotherapy in the treatment of metastatic disease.[91] Data from ongoing phase II and III trials of lapatinib and from investigations of other dual-kinase TKIs will provide greater insight into the efficacy and safety of these therapies in metastatic breast cancer. Strategy 2: Trastuzumab plus TKIs. Another strategy to inhibit the growth of tumors that have overcome HER2 suppression by the upregulation of EGFR is to combine trastuzumab with an anti-EGFR TKI, eg, gefitinib or erlotinib. In addition to inhibiting signaling, the monoclonal antibody would add complementary mechanisms of action through the promotion of ADCC. This strategy is supported by preclinical data that show synergy with the two classes of agents; both trastuzumab and the TKI alone slowed the growth of tumor cells, but they completely inhibited cell growth in combination.[88,97] These combinations are being tested in clinical trials in patients with trastuzumab- refractory disease. Theoretically combinations of biologics would be expected to increase the rate and durations of response in patients with breast cancer. Combinations of trastuzumab and TKIs do have some activity in patients with breast cancer, but the clinical data have not yet shown a conclusive benefit with these agents (Table 5).[98] Trastuzumab can also be combined with a dual-kinase TKI following a similar reasoning. Combining lapatinib with trastuzumab resulted in synergistic tumor cell killing in several HER2- overexpressing breast cancer cell lines.[99] The preliminary results of an open-label phase I study show that lapatinib plus trastuzumab has substantial clinical activity in patients with heavily pretreated breast cancer in whom the disease had progressed during treatment with trastuzumab-containing regimens (Table 5).[100] This combination of agents, which may prove to be a viable strategy to treat acquired resistance to trastuzumab, should be investigated in randomized trials. Strategy 3: Trastuzumab plus pertuzumab. Another strategy to suppress HER2-mediated signaling is concurrent treatment with monoclonal antibodies with different specificities for HER2. For example, trastuzumab and pertuzumab target different regions of the HER2 tyrosine kinase receptor; trastuzumab binds the extracellular domain IV of HER2 and pertuzumab binds domain II, thereby blocking HER2 dimerization with other EGF receptors.[45] Synergy between trastuzumab and pertuzumab has been seen in a preclinical model, the combination synergistically decreasing survival in the HER2-overexpressing BT474 cancer cell line by increasing apoptosis.[101] Trastuzumab and pertuzumab appear to act together to reduce the levels of the HER2 protein and block receptor signaling through Akt; MAPK activity, however, was found not to be impaired after exposure to both antibodies. Other combinations of antibodies to HER2 have shown synergy in vivo in BT474 xenografts.[ 102,103] These data suggest that combining monoclonal antibodies to HER2 may be more effective in inhibiting the survival of cancer cells than using a single antibody.[101]

Clinical Implications This article outlines several hypotheses for the growth of HER2-positive tumors despite HER2 suppression. Based on the molecular rationale, we have proposed strategies to overcome the intrinsic resistance and compensatory mechanisms responsible for the limited response to HER2 suppression. An understanding of the molecular basis for tumor growth will allow tailored combinations of targeted therapies that can be used in combination with chemotherapy to potentially improve outcomes. Combining non- cross-resistant anti-HER2 drugs that act at different sites on the receptor may enhance the efficacy of both agents. For example, in the case of a patient presenting with metastatic breast cancer that is positive for both HER2 and EGFR, a HER2 blocker (eg, trastuzumab) and a dual-kinase TKI, such as lapatinib, may be effective. This combination can inhibit the signaling pathways mediated by both receptors by different mechanisms of action, resulting in substantial synergy. The toxicity profile of potential combinations should also be considered in making treatment decisions. For example, trastuzumab has been associated with cardiotoxicity when administered with anthracycline-based therapy.[104,105] Newer formulations of anthracyclines with reduced cardiotoxicity may provide additional treatment options for patients. The potential treatment strategies suggested in this article are theoretical and proposed based on preclinical data and the molecular mechanism of HER2- mediated signaling, as well as the mechanisms of action of potential therapeutic agents or combinations of agents. The clinical efficacy of these combinations is being tested in ongoing trials. Additional well-controlled trials should also be conducted to test the strategies proposed here. Data collected by registHER, the prospective, longitudinal study of women with HER2-positive metastatic disease, will also allow greater insight into the molecular and clinical characteristics of the tumor, as well as patient-specific factors, that are required for optimal outcomes.[ 106] In this article we suggest that EGFR or PTEN may play a role in the development of resistance to anti-HER2 agents. Other molecules, (eg, the insulin- like growth factor) or changes in cell cycle control (eg, the loss of p27- kip1) may also play a role in resistance to HER2 suppressors. A complete understanding of the molecular mechanisms of resistance will lead to the design of clinical trials of agents that target specific molecules. In the meantime it is common to continue the use of a HER2 suppressor and change the chemotherapy regimen in patients with metastatic breast cancer. Conclusions HER2 signaling plays a crucial role in the proliferation of epithelial cancers and is a significant target for pharmacologic intervention. Monoclonal antibodies and small-molecule TKIs to HER2 have shown antitumor activity as single agents and in combination regimens in patients with metastatic breast cancer. There is evidence, however, that HER2-positive tumors can grow despite HER2 inhibition. In the absence of data from clinical trials, understanding the molecular basis of the disease and the results of preclinical studies provides clues on strategies to optimize the outcomes in patients with breast cancer. In general, HER2-positive tumors continue to express HER2; the continued suppression of HER2 is therefore warranted even after disease progression. Tumor growth may result from compensatory mechanisms for HER2 suppression, and additional strategies to block these pathways should be considered. Clinical trials that address many of these questions are ongoing; the results of these trials will shed light on ways of improving patient outcomes with anti-HER2 therapies. Please visit our website at http:// www.curatiocme.com/OptimizingOutcomes/ to access the posttest and evaluation form, and to download a certificate of participation for this activity (monograph and CD-ROM). For CME questions, please contact karen.wetzel@curatiocme.com

Disclosures:

Dr. Esteva has served as a speaker for and consultant to Genentech, Inc, and as a consultant to GlaxoSmithKline.

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