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Abstract

  1. Top of page
  2. Abstract
  3. Pathophysiology of the HER Family
  4. HER2 in Breast Cancer Pathogenesis
  5. Mechanisms of Action of Trastuzumab
  6. Mechanisms of Resistance to Trastuzumab
  7. FCGR3A Polymorphism
  8. Potential Therapeutic Strategies for Overcoming Trastuzumab Resistance
  9. Future Direction
  10. Conclusions
  11. References

Approximately 20% of breast cancers are characterized by overexpression of human epidermal growth factor receptor 2 (HER2) protein and associated gene amplification, and the receptor tyrosine kinase is believed to play a critical role in the pathogenesis of these tumors. The development and implementation of trastuzumab, a humanized monoclonal antibody against the extracellular domain of HER2 protein, has significantly improved treatment outcomes in patients with HER2-overexpressing breast cancer. However, despite this clinical usefulness, unmet needs for better prediction of trastuzumab’s response and overcoming primary and acquired resistance remain. In this review, we discuss several potential mechanisms of resistance to trastuzumab that have been closely studied over the last decade. Briefly, these mechanisms include: impaired access of trastuzumab to HER2 by expression of extracellular domain-truncated HER2 (p95 HER2) or overexpression of MUC4; alternative signaling from insulin-like growth factor-1 receptor, other epidermal growth factor receptor family members, or MET; aberrant downstream signaling caused by loss of phosphatase and tensin homologs deleted from chromosome 10 (PTEN), PIK3CA mutation, or downregulation of p27; or FCGR3A polymorphisms. In addition, we discuss potential strategies for overcoming resistance to trastuzumab. Specifically, the epidermal growth factor receptor/HER2 tyrosine kinase inhibitor lapatinib partially overcame trastuzumab resistance in a clinical setting, so its efficacy results and limited data regarding potential mechanisms of resistance to the drug are also discussed. (Cancer Sci 2011; 102: 1–8)

Breast cancer is the leading cause of cancer death among women worldwide, with approximately 1 000 000 new cases reported each year.(1,2) Approximately 20% of breast cancer tumors show overexpression of human epidermal growth factor receptor 2 (HER2) protein, and HER2 overexpression has been repeatedly identified as a factor indicating poor prognosis.(3,4) Trastuzumab is a humanized monoclonal antibody that targets the extracellular domain of the HER2 protein, and clinical studies have intensively investigated the potential therapeutic roles of this compound since the late 1990s.

In particular, studies have shown that a combination of trastuzumab and conventional chemotherapy is significantly more effective at treating HER2-overexpressing metastatic breast cancers than chemotherapy alone.(5) In light of this finding, trastuzumab has come to be used in adjuvant or neo-adjuvant settings for operable HER2-overexpressing breast cancers.(6–8) However, despite trastuzumab’s promising usefulness in clinical settings, only a relatively small percentage of patients are reported to benefit from trastuzumab therapy alone, with response rates to trastuzumab as a single agent of approximately 20%.(9) In addition, even when trastuzumab therapy leads to temporary tumor shrinkage, clinical relapse is observed in virtually all metastatic patients. More effective treatment of HER2-overexpressing breast cancer requires a deeper understanding of the mechanisms of resistance to trastuzumab.

In this review, we discuss several proposed mechanisms of resistance to trastuzumab and potential ways to overcome this resistance. Given that lapatinib, a dual inhibitor of epidermal growth factor receptor (EGFR)/HER2 tyrosine kinase, has been clinically proven to overcome at least some resistance to trastuzumab,(10) mechanisms of resistance to lapatinib will also be discussed. Although a number of potential biomarkers to predict response to trastuzumab or lapatinib have been suggested in clinical studies, these will not be discussed here unless a cell-based experimental model exists.

Pathophysiology of the HER Family

  1. Top of page
  2. Abstract
  3. Pathophysiology of the HER Family
  4. HER2 in Breast Cancer Pathogenesis
  5. Mechanisms of Action of Trastuzumab
  6. Mechanisms of Resistance to Trastuzumab
  7. FCGR3A Polymorphism
  8. Potential Therapeutic Strategies for Overcoming Trastuzumab Resistance
  9. Future Direction
  10. Conclusions
  11. References

With the exception of HER2, each member of the HER family, which consists of human epidermal growth factor receptors 1–4, has cognate-identified ligands (Fig. 1). Ligand binding to extracellular domains induces conformational changes in the receptor, which subsequently promotes homo- or heterodimerization and activation as receptor tyrosine kinase (RTK). Each dimer can then trigger various intracellular signaling pathways, including those of PI3K/Akt, Ras/Raf/MEK/ERK, and STATs, which all play important roles in cellular oncogenic processes such as proliferation, survival, motility, and angiogenesis.

image

Figure 1.  Ligand binding and dimerization of epidermal growth factor receptor (EGFR) family members. AR, amphiregulin; BTC, betacellulin; EGF, epidermal growth factor; EPRG, epiregulin; HB-EGF, heparin-binding EGF; HER2, human epidermal growth factor receptor 2; NRG, neuregulin; TGF-α, transforming growth factor-α.

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As it lacks a cognate ligand, HER2 must dimerize with other HER family members under physiological conditions. However, under conditions of overexpression, HER2 can be constitutively active and transform NIH3T3 cells in the absence of a ligand.(11) HER2 can theoretically form four different types of dimers (with HER1, HER2, HER3, or HER4), but the HER2/HER3 heterodimer is thought to be the most mitogenic and transforming.(12–15) Two peculiar characteristics distinguish HER3 from other HER family members: it lacks tyrosine kinase activity on its own; and it contains at least six docking domains for p85, the regulatory unit of Class I PI3K. These properties allow HER3 to function as a scaffold protein to efficiently trigger the PI3K pathway. Indeed, a study has suggested that breast cancer cell lines expressing both HER2 and HER3 appear to have a higher degree of Akt phosphorylation.(16)

HER2 in Breast Cancer Pathogenesis

  1. Top of page
  2. Abstract
  3. Pathophysiology of the HER Family
  4. HER2 in Breast Cancer Pathogenesis
  5. Mechanisms of Action of Trastuzumab
  6. Mechanisms of Resistance to Trastuzumab
  7. FCGR3A Polymorphism
  8. Potential Therapeutic Strategies for Overcoming Trastuzumab Resistance
  9. Future Direction
  10. Conclusions
  11. References

The HER2 protein is overexpressed in 20–25% of breast cancers and has been shown to be associated with poor rates of disease-free survival and increased resistance to some chemotherapeutic drugs.(3,4) Gene amplification is considered the main mechanism of HER2 protein overexpression. HER2 expression is known to contribute to breast cancer carcinogenesis; indeed, a series of experimental studies have indicated that transfection of HER2 into mammary epithelial cells induces oncogenic transformation.(17) However, HER2 has been reported to require HER3 to drive breast cancer cell proliferation, emphasizing the importance of the HER2/HER3 heterodimer complex mentioned above.(18)

Mechanisms of Action of Trastuzumab

  1. Top of page
  2. Abstract
  3. Pathophysiology of the HER Family
  4. HER2 in Breast Cancer Pathogenesis
  5. Mechanisms of Action of Trastuzumab
  6. Mechanisms of Resistance to Trastuzumab
  7. FCGR3A Polymorphism
  8. Potential Therapeutic Strategies for Overcoming Trastuzumab Resistance
  9. Future Direction
  10. Conclusions
  11. References

The mechanisms of action for trastuzumab can be roughly divided into two components: inhibition of intracellular signaling; and induction of an immune system-mediated antitumor response. HER2 is known to trigger multiple signaling pathways, and thus inhibition of HER2 should theoretically result in inactivation of those pathways. Although trastuzumab’s method of inhibiting HER2 activity is not fully understood, some studies have suggested that the drug might promote internalization and degradation of HER2.(19,20) Other recent studies have suggested that the HER2/HER3/PI3K complex and subsequent PI3K/Akt signaling pathway play central roles in cell proliferation in HER2-overexpressing cells, and thus disruption of this complex may be the key molecular mechanism of action of trastuzumab (Fig. 2).(21,22)

image

Figure 2.  Proposed mechanism of action of trast-uzumab. When trastuzumab binds to overexpressing human epidermal growth factor receptor 2 (HER2), the HER2/HER3/PI3K complex is disrupted, followed by decreased degradation of p27. CDK, cyclin-dependent kinase; PTEN, phosphatase and tensin homologs deleted from chromosome 10.

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When trastuzumab is given, one important cellular response is cell cycle arrest at the G1/S boundary, which is often accompanied by an increase in p27 level and decreases in cyclin D1 and cyclin-dependent kinase 2 activity.(23,24) When used as a single agent in vitro, trastuzumab induces little apoptosis, if any at all.(25,26) However, a series of in vitro studies showed the synergistic effects of trastuzumab when combined with chemotherapeutic agents, thus forming a basis for clinical studies combining trastuzumab with chemotherapy.(27) This synergistic effect may be explained by inhibition of the PI3K/Akt signaling pathway, which normally promotes cell survival.

Data from several in vivo experiments have indicated that trastuzumab is capable of mediating the induction of immune responses such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity.(28) With ADCC, immno-effector cells expressing the Fcγ receptor recognize and bind to the Fc domain of the IgG1 antibody (trastuzumab) and subsequently lyse cells (in this case, tumor cells) attached to the antibody. Perhaps the most convincing evidence of the contribution of ADCC to trastuzumab-induced antitumor activity is in mice lacking the FC receptor (FcR−/−), in which trastuzumab treatment resulted in significantly lower rates of tumor regression than in FC receptor-expressing mice.(29) While directly proving the role of ADCC in trastuzumab’s activity is more difficult in breast cancer patients, Gennari et al.(30) noted an increase in infiltration by lymphoid cells in tumor samples collected following trastuzumab treatment compared to pretreatment tumor specimens.

Mechanisms of Resistance to Trastuzumab

  1. Top of page
  2. Abstract
  3. Pathophysiology of the HER Family
  4. HER2 in Breast Cancer Pathogenesis
  5. Mechanisms of Action of Trastuzumab
  6. Mechanisms of Resistance to Trastuzumab
  7. FCGR3A Polymorphism
  8. Potential Therapeutic Strategies for Overcoming Trastuzumab Resistance
  9. Future Direction
  10. Conclusions
  11. References

Impaired access of trastuzumab to HER2 protein.

Truncated HER2 (p95 HER2).  Several studies have identified a truncated version of HER2 that lacks the receptor’s extracellular domain, dubbing it “p95 HER2” after its molecular weight. Molina et al.(31) surveyed expression of p95 HER2 in surgically excised breast cancer samples and found that presence of the protein was more frequent in node-positive cases than in node-negative ones. Further, Xia et al.(32) showed that HER3 can be trans-phosphorylated by p95 HER2 in the presence of heregulin in BT-474 HER2-overexpressing breast cancer cell lines, and that this phosphorylation can be inhibited by lapatinib but not trastuzumab (Fig. 3).

image

Figure 3.  Proposed mechanisms of trastuzumab resistance. CDK, cyclin-dependent kinase; EGFR, epidermal growth factor receptor; HER2, human epidermal growth factor receptor 2; HGF, MET ligand; IGF-1R, insulin-like growth factor-1 receptor; PTEN, phosphatase and tensin homologs deleted from chromosome 10.

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Scaltriti et al.(33) transfected both complete HER2 and p95 HER2 into MCF-7 breast cancer cells and compared the cells with regard to in vivo sensitivity to trastuzumab and lapatinib. Results showed that while MCF-7/HER2 cells were sensitive to both trastuzumab and lapatinib, MCF-7/p95 HER2 cells were sensitive only to lapatinib.(33) These authors also retrospectively analyzed the presence of p95 HER2 in tumors from 46 patients who had been treated with trastuzumab-based regimens, and found that p95 HER2-positive patients (n = 9) were less likely to experience tumor shrinkage (p95 HER2-positive vs p95 HER2-negative  = 1/9 [11.1%] vs 19/37 [51.4%]).(33)

Masking with MUC4.  After establishing a cell line from a breast cancer patient whose tumor showed HER2 gene amplification and primary resistance to trastuzumab (JIMT-1), Nagy et al.(34) compared the line with other HER2-amplified and trastuzumab-sensitive breast cancer cell lines with respect to trastuzumab-binding to the cell surface. Results showed impaired binding of trastuzumab to JIMT-1 compared with trastuzumab-sensitive cells, which the authors hypothesized was due to potential masking of HER2 because permeabilization with surface active agents increased trastuzumab-binding to JIMT-1 cells.(34) Specifically, they suspected that MUC4, a membrane-associated mucin, was masking HER2 in the JIMT-1 line, as overexpression of rat Muc4 had been previously reported to result in reduced binding of antibodies against the extracellular region of HER2.(35) They then went on to show that MUC4 levels are increased in JIMT-1 compared with other trastuzumab-sensitive cells, and that knockdown of MUC4 by siRNA restored trastuzumab binding to cell surfaces in the JIMT-1 line (Fig. 3).(34)

Alternative signaling.

Alternative signaling from insulin-like growth factor-1 receptor (IGF-1R).  IGF-1R is an RTK characterized as a major trigger of the PI3K pathway. Lu et al.(36) reported that cells overexpressing both HER2 and IGF-1R (MCF-7/HER2-18) showed resistance to trastuzumab (Fig. 3), and that this resistance was able to be overcome using co-treatment with a blocking antibody against IGF-1R, such as α-IR3, or insulin-like growth factor binding protein-3 and trastuzumab. Lu et al.(37) also later reported that IGF-1 signaling increases expression levels of SKP2, a ubiquitin ligase for p27, and enhances SKP2′s association with p27. As a result, the level of p27 is reduced and G1/S cell cycle arrest does not occur, even in the presence of trastuzumab.(37)

Another group further reported that IGF-1R is overexpressed in a trastuzumab-resistant clone selected from a trastzumab-sensitive SKBR-3 cell line.(38) These authors also showed that IGF-1R shares a mechanical connection with HER2 in the cells and that the PI3K/Akt and RAS/MAPK pathway can be activated more rapidly by IGF-1 stimulation than in parental SKBR-3 cells.(38) Of particular importance, the trastzumab resistance attributable to IGF-1R signaling was able be overcome using co-treatment with trastuzumab and α-IR3.(38)

However, apart from these above-mentioned preclinical data, results obtained in clinical studies evaluating the roles of IGF-1R expression in trastuzumab studies are inconsistent.(39,40)

Alternative signaling from EGFR family members.  Ritter et al. developed a trastuzumab-resistant cell line in vivo by exposing BT-474 cells transplanted into nude mice to trastuzumab, then reharvesting and plating the residual tumor cells. The resistant cells were found to express higher levels of phosphorylated EGFR and EGFR/HER2 heterodimers than parental BT-474 (Fig. 3), and phosphorylation of HER2 and the basal association of p85 with HER3 in the cells were found to be inhibited by EGFR tyrosine kinase inhibitors (TKIs).(41) Of particular interest, the authors showed that EGFR TKIs and lapatinib induced apoptosis and inhibited in vivo growth in these resistant cells.(41)

In an examination of the role of transforming growth factor-α (TGF-α), a ligand for EGFR, in trastuzumab resistance, Valabrega et al. found that cells gene-manipulated to overexpress TGF-α became resistant to trastuzumab. These authors also obtained tumor samples from three breast cancer patients before starting trastuzumab-based therapy and after experiencing progression, and comparatively analyzed the samples for levels of TGF-α expression. In each case, they noted no TGF-α expression before the therapy but high levels during periods of regrowth.

Potentially supporting those preclinical findings, a positive correlation was observed between HER2/HER2 homodimer expression quantified on tumor specimens and an elevated response rate and longer survival with trastuzumab-based treatment in HER2-positive metastatic breast cancer patients.(42,43)

Alternative signaling from MET.  In their analysis of expression levels of MET, another RTK, in HER2-overexpressing breast cancer tumors and cell lines, Shattuck et al.(44) found that tumor samples from patients who showed clinical resistance to combination therapy with vinorelbine and trastuzumab had higher levels of MET and its ligand HGF than did patients who responded to the treatment. In addition, in an in vitro study, these authors showed that exogenous HGF inhibited trastuzumab-induced growth inhibition and upregulation of p27 (Fig. 3),(44) and that combination of trastuzumab and MET kinase inhibitors or MET knockdown produced a synergistic growth inhibitory effect. They further showed that 48-h exposure of HER2-overexpressing breast cancer cell lines to trastuzumab resulted in increased expression of MET protein.(44) This finding is extremely interesting given that MET-amplification and certain alternative signals from MET to HER3 have been found capable of inducing acquired resistance to the EGFR TKIs gefitinib and erlotinib in non-small-cell lung cancer.(45) Involvement of MET may therefore be a common mechanism of acquired resistance to anti-EGFR family drugs.

Aberrant activation of downstream signaling.

Loss of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) function.  Recruitment of Class I PI3K by an RTK, including HER2/HER3 heterodimers, catalyzes the conversion of membrane-associated phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3) and triggers downstream signaling. As PTEN is a phosphatase that converts PIP3 back to PIP2, it functions as a negative regulator of Class I PI3K under physiological conditions. Point mutations, deletions (homo- and heterozygous), and epigenetic changes in PTEN that lead to functional loss have all been noted in a number of malignancies.(46) In breast cancer, for example, frequencies of loss of heterozygosity and PTEN mutations have been reported to be approximately 25% and 6%, respectively.(46)

Nagata et al.(47) reported that knocking down PTEN in HER2-overexpressing breast cancer cell lines induces trastuzumab resistance both in vitro and in vivo (Fig. 3), and that this resistance can be overcome by combined treatment with trastuzumab and the PI3K inhibitor LY294002. In addition, they immunohistochemically assessed PTEN protein expression levels in breast cancer tumor samples obtained from patients who had been treated with trastuzumab, with results showing that lower PTEN expression significantly correlated with lower response rates.(47)

PIK3CA-activating mutation.  Somatic mutations in PIK3CA were first identified in 2004 in several malignant tumors, including breast cancer.(48) Subsequent studies have reported that the E542K or E545K and H1047R hotspot mutations, found on exons 9 and 20, respectively, are the most frequent types of mutation, found in 8–40% of breast cancer tumors.(48–51) Exon 9 mutations are located in the helical domain of p110α and are considered to abrogate inhibitory intermolecular interaction between p85 and p110.(52,53) In contrast, exon 20 mutations are located near the activation loop and are considered to produce constitutive kinase activity.

Both of the above-mentioned hotspot mutations are gain-of-function mutations that transform normal mammary epithelial cells.(54,55) Berns et al.(56) investigated the role of gain-of-function mutations of the PIK3CA gene in trastuzumab resistance by transfecting wild-type and mutant (H1047R) forms of PIK3CA in SKBR-3 HER2-overexpressing breast cancer cells (Fig. 3). Results showed that both wild-type and mutant PIK3CA transfections imparted trastuzumab resistance absent in GFP controls. Further, analysis of PIK3CA genotypes in tumor samples obtained from breast cancer patients who had undergone trastuzumab-based therapy showed an association between the presence of PIK3CA hotspot mutations and reduced time to progression after therapy.(56) Consistent with these findings, our recent study using naturally derived HER2-amplified breast cancer cell lines showed that cell lines with PIK3CA hotspot mutations were significantly more resistant to trastuzumab than those without mutations.(57)

Downregulation of p27.  As described above, the primary cellular response to trastuzumab treatment is considered to be G1/S cell cycle arrest, which is induced by reduced degradation of p27 following inhibition of intracellular signaling pathways.(58) Nahta et al.(58) showed that expression of p27 is reduced in trastuzuamab-conditioned resistant cells derived from SKBR-3 and that sensitivity to trastuzumab can be restored through transfection of the p27 gene (Fig. 3). Although the mechanism remains to be clarified, p27 downregulation may be caused by alternative signaling pathways, as described above.

FCGR3A Polymorphism

  1. Top of page
  2. Abstract
  3. Pathophysiology of the HER Family
  4. HER2 in Breast Cancer Pathogenesis
  5. Mechanisms of Action of Trastuzumab
  6. Mechanisms of Resistance to Trastuzumab
  7. FCGR3A Polymorphism
  8. Potential Therapeutic Strategies for Overcoming Trastuzumab Resistance
  9. Future Direction
  10. Conclusions
  11. References

As mentioned above, ADCC has been suggested to play a role in the antitumor activity of trastuzumab. In ADCC, the antibody (trastuzumab) first binds to tumor cells, then is engaged by effector cells through their receptors for IgG. One group of IgG FcRs are expressed on leukocytes and are categorized into three distinct classes: FcRI; FcRII; and FcRIII. In humans, the latter two classes can be further divided into FcRIIa and FcRIIb, and FcRIIIa and FcRIIIb. Although FcRIIb and FcRIIIb do not trigger ADCC, FcRIIa and FcRIIIa are activating FcRs that are expressed on monocytes/macrophages and on monocytes/macrophages and natural killer cells, respectively.

A single nucleotide polymorphism corresponding to the phenotype expression of histidine (H)/arginine (R) is known to exist at position 131 on FcRIIa, and of valine (V) or phenylalanine (F) at position 158 on FcRIIIa. Further, FcRIIIa with the V allele is known to have greater affinity to human IgG1 than that with the F allele, and cells bearing the FcRIIIa V allele also mediate ADCC more effectively. In a previous study involving follicular lymphoma patients treated with rituximab, an anti-CD20 IgG1 mAb with the same FcRs as trastuzumab, patients with homozygous 158 V/V alleles of FcRIIIa and homozygous 131 H/H alleles of FcRIIa showed a higher rate of tumor response than did F carrier and R carrier, respectively.(59) Musolino et al.(60) analyzed FcRIIIa, FcRIIa, and FcRIIb polymorphisms and their correlations with clinical efficacy of trastuzumab-based therapy in patients with metastatic HER2-positive breast cancer. Their results showed that the FcRIIIa-158 V/V genotype was significantly correlated with objective response rate and progression-free survival, as was the FcRIIa 131 H/H genotype.(60) Further, the combination of these two favorable genotypes (V/V and/or H/H) was independently associated with better objective response rate and progression-free survival than in other combinations.(60)

Potential Therapeutic Strategies for Overcoming Trastuzumab Resistance

  1. Top of page
  2. Abstract
  3. Pathophysiology of the HER Family
  4. HER2 in Breast Cancer Pathogenesis
  5. Mechanisms of Action of Trastuzumab
  6. Mechanisms of Resistance to Trastuzumab
  7. FCGR3A Polymorphism
  8. Potential Therapeutic Strategies for Overcoming Trastuzumab Resistance
  9. Future Direction
  10. Conclusions
  11. References

Lapatinib.

Mechanism of action.  Lapatinib is a dual EGFR/HER2 TKI (Table 1)(61) which, unlike many other TKIs, has been found to be highly selective to EGFR and HER2.(62) In preclinical models of trastuzumab resistance, lapatinib was able to inhibit phosphorylation of HER2 and overall growth in HER2-overexpressing breast cancer cell lines specifically selected for their in vitro resistance to trastuzumab.(63) Xia et al.(64) reported that lapatinib combined with trastuzumab downregulated survivin to a greater degree and induced more apoptosis than either agent alone.

Table 1.   Potential strategies for trastuzumab-resistant breast cancer
AgentType of agent and targetClinical phase of development
  1. FDA, Food and Drug Administration (USA); EGFR, epidermal growth factor receptor; HER2, human epidermal growth factor receptor 2; IGF-1R, insulin-like growth factor-1 receptor; TKI, tyrosine kinase inhibitor; VEGFRs, vascular endothelial growth factor receptors.

LapatinibTKI; EGFR, HER2FDA-approved
PertuzumabmAb; HER2II
ErtumaxomabBispecific antibody against HER2 and Fcγ RI/IIIII
Trastuzumab–DM1mAb–toxin; HER2II–III
CP-751,871mAb; IGF-1RIII
Foretinib (GSK1363089)TKI; MET, VEGFRs, RON, AXLII
BEZ235mTOR/PI3KI–II
PerifostineAktI–II
TemsirolimusmTORFDA-approved for advanced renal cell cancer
EverolimusmTORFDA-approved for soft-tissue and bone saromas
HER2 vaccinesHER2 peptide-based vaccineII
Defucosylated trastuzumabmAb; HER2Preclinical

Clinical achievement of lapatinib.  Geyer et al. carried out a randomized phase III trial in 399 patients with breast cancer under recurrent/metastatic conditions comparing capecitabine in combination with lapatinib to capecitabine alone. All patients had previously received therapy with the anthracycline, taxane, and trastuzumab. The median time to progression was longer in the lapatinib-receiving arm (27.1 vs 18.6 weeks; < 0.001),(65) and the overall response rate was also higher in this arm (23.7%vs 13.9%; P = 0.017).(65) Overall survival also tended to be longer among patients in the combination group than those in the capecitabine-only group.(65)

A significant number of HER2-positive metastatic breast cancer patients who are treated with trastuzumab experience symptomatic central nervous system (CNS) metastasis, potentially as a consequence of relatively good control of visceral diseases by trastuzumab. Unlike trastuzumab, lapatinib has been suggested to cross the blood–brain barrier, providing rationale for testing lapatinib in patients with CNS metastases.(66) In a phase II trial of lapatinib in 39 patients with HER2-positive breast cancer and brain metastases, two patients experienced a partial response based on the Response Evaluation Criteria in Solid Tumors, and five additional patients experienced at least a 30% shrinkage of CNS lesions.(67)

Consistent with the preclinical data introduced above,(64) in a recent phase III clinical trial comparing lapatinib and trastuzumab in combination to lapatinib alone in HER2-positive metastatic breast cancer patients who progressed on trastuzumab-based regimens, combined treatment was found to be superior to lapatinib alone in progression-free survival as the primary endpoint (hazard ratio = 0.73; 95% confidence interval, 0.57 to 0.93; P = 0.008).(68)

The potential role of lapatinib alone or combined with trastuzumab for use in adjuvant or neo-adjuvant treatments is presently being evaluated in large randomized trials.

Mechanisms of resistance to lapatinib.  Given that lapatinib was developed almost a decade after trastuzumab, far less research has been dedicated to the mechanisms of resistance to lapatinib than for trastuzumab. Activating mutations of PIK3CA have previously been suggested as causes of trastuzumab resistance,(56,57) but evidence has also been found suggesting that these mutations may contribute to resistance to lapatinib and other anti-HER2 small molecule inhibitors as well.(57,69) However, in contrast to the relatively strong supporting data for trastuzumab, loss of PTEN expression has not been found to be associated with lapatinib resistance in any cell lines or clinical specimens.(70) Xia et al.(71) developed an acquired resistance model for lapatinib by chronically exposing HER2-overexpressing cells to the drug, finding that resistant cells were more dependent on estrogen receptor signaling in terms of cell survival than parent cells. Importantly, they also noted increased estrogen receptor signaling in tumor biopsies from patients who had undergone lapatinib therapy.(71)

Pertuzumab.  Pertuzumab is another monoclonal antibody against the extracellular domain of HER2 protein (Table 1), but it attaches to a different epitope of HER2 from trastuzumab. Pertuzumab is believed to inhibit heterodimer formation between HER2 and EGFR or HER3.(72) Although the HER2/HER3 heterodimer is considered extremely important in HER2-driven cell signaling, as previously mentioned, Junttila et al.(21) reported that the heregulin-dependent HER2/HER3 heterodimer is not disrupted by trastuzumab, but is disrupted by pertuzumab. In a phase II clinical trial involving combination treatment with pertuzumab and trastuzumab in HER2-positive breast cancer patients, treatment produced a response rate and disease control rate of 24.2% and 50%, respectively.(73)

Trastuzumab-DM1.  Trastuzumab-DM1 is comprised of trastuzumab, DM1, an inhibitor of tubulin polymerization derived from maytansine, and the stable MCC linker that conjugates DM1 and trastuzumab (Table 1). The compound is designed to efficiently deliver DM1 to HER2-overexpressing cancer cells. Preclinical studies have indicated the growth-inhibitory effect of trastuzumab-DM1 in HER2-overexpressing and trastuzumab-resistant cells.(74) In a phase II clinical trial involving HER2-positive metastatic breast cancer patients with disease progression despite trastuzumab-based therapy (n = 112), trastuzumab-DM1 yielded an independently reviewed response rate and progression-free survival of 26.9% and 4.6 months, respectively.(75) Importantly, trastuzumab-DM1 had similar antitumor activity and an independently reviewed response rate of 24.2% even in patients previously treated with lapatinib and trastuzumab (= 66).(75)

PI3K pathway inhibitors.  As mentioned above, HER2-overexpressing breast cancer cells are believed to be dependent on the PI3K signaling pathway, and a number of genetic or epigenetic alterations in PI3K signaling molecules have been implicated as causes of resistance to trastuzumab or small-molecule HER2 kinase inhibitors. A previous study showed that HER2-overexpression and PIK3CA mutations frequently occur simultaneously in breast cancer cells,(76) and cell lines with either HER2 amplification or PIK3CA mutation are shown to be equally Akt-dependent.(57,77) PI3K pathway inhibitors may therefore be useful in overcoming resistance to anti-HER2 agents. Indeed, PI3K/mTOR dual inhibitor and Akt inhibitor were shown to effectively inhibit cellular growth in trastuzumab- and lapatinib-resistant cells.(69,77) At present, many classes of PI3K pathway inhibitors are in clinical development (Table 1), and their roles in overcoming trastuzumab resistance will be tested in the future.

Inhibitors of alternative signaling molecules.  As mentioned above, evidence has suggested that alternative signaling from IGF-1R or MET may cause trastuzumab resistance. Small-molecule IGF-1R or MET receptor tyrosine kinase, anti-IGF-1 antibody, and anti-HGF antibody are all in clinical development at present (Table 1). Monotherapy or combination therapy with these agents and trastuzumab may therefore be an attractive therapeutic strategy.

HER2 vaccines.  Vaccines and adoptive immunotherapy targeting the HER2 extracellular domain have been tested in clinical trials, with results showing that significant levels of durable T-cell HER2 immunity can be generated with active immunization without significant consequences with regard to autoimmunity against normal tissues.(78) Early data from clinical trials testing the potential use of HER2-specific vaccines in adjuvant therapy for high-risk breast cancer patients have shown promising results.(79)

Ertumaxomab.  Ertumaxomab is an intact bispecific antibody targeting HER2 and CD3 on T cells with preferential binding to activating Fcγ type I/III receptors and redirecting T cells, macrophages, dendritic cells, and natural killer cells to HER2-expressing tumor sites (Table 1).(80) In a phase I trial (n = 17), ertumaxomab treatment was associated with one complete response, two partial responses, and two stable diseases in patients with metastatic breast cancer who had received extensive prior treatment.(81) At present, the effects of ertumaxomab are being evaluated in phase II studies.

Defucosylated trastuzumab.  Removal of fucose from antibody oligosaccharides attached to the heavy chain of Asn297 (defucosylation) has been shown to significantly enhance ADCC compared to the activity of regular antibodies. In addition, defucosylation of trastuzumab was also found to enhance ADCC in an in vitro assay as compared to regular trastuzumab.(82) Juntilla et al.(83) also recently reported that defucosylated trastuzumab more than doubled the median progression-free survival compared with conventional trastuzumab in preclinical models of HER2-amplified breast cancer.

Future Direction

  1. Top of page
  2. Abstract
  3. Pathophysiology of the HER Family
  4. HER2 in Breast Cancer Pathogenesis
  5. Mechanisms of Action of Trastuzumab
  6. Mechanisms of Resistance to Trastuzumab
  7. FCGR3A Polymorphism
  8. Potential Therapeutic Strategies for Overcoming Trastuzumab Resistance
  9. Future Direction
  10. Conclusions
  11. References

In this review, we discussed several proposed mechanisms of resistance to trastuzumab. This particular theme has been in the focus of a significant amount of research in the field of translational science over the past decade. A number of potential mechanisms have been proposed, perhaps more than can be properly explored. However, it should be noted that not all proposed mechanisms have been found to be clinically relevant, and virtually all clinical data available have been obtained by retrospectively analyzing tumor specimens collected from patients, most of whom were treated with trastuzumab combined with certain cytotoxic agents. This “contamination” of samples hampers certain determination that any detected biomarkers predict trastuzumab sensitivity. To determine the clinical significance of these mechanisms, continuous bedside and bench collaboration will be required. In particular, preoperative treatment with trastuzumab will provide an optimal platform for this kind of research, as investigators can easily compare pre- and post-trastuzumab specimens, and patients’ backgrounds vary to a lesser degree than in a metastatic setting.

The clinical usefulness of trastuzumab was recently proven in metastatic HER2-overexpressing gastric cancer in a large phase III trial, opening the door to molecular-based, rather than organ-based, treatment choice in treating solid tumors. However, despite the wealth of preclinical and clinical data regarding mechanisms of resistance to trastuzumab in breast cancer, the question of whether or not these mechanisms are common in other types of HER2-overexpressing tumors, such as gastric cancer, remains unanswered. Given that gastric cancer specimens are generally obtainable by endoscopy, serial biopsies before, during, and after trastuzumab therapy may provide precious information in the future.

Conclusions

  1. Top of page
  2. Abstract
  3. Pathophysiology of the HER Family
  4. HER2 in Breast Cancer Pathogenesis
  5. Mechanisms of Action of Trastuzumab
  6. Mechanisms of Resistance to Trastuzumab
  7. FCGR3A Polymorphism
  8. Potential Therapeutic Strategies for Overcoming Trastuzumab Resistance
  9. Future Direction
  10. Conclusions
  11. References

Mechanisms of resistance to trastuzumab have been studied in depth, and potential strategies for overcoming resistance are constantly being developed. However, well-designed cooperative and prospective clinical studies will be needed to determine the significance of these mechanisms and treatments.

Abbreviations
FDA

Food and Drug Administration (USA)

EGFR

epidermal growth factor receptor

HER2

human epidermal growth factor receptor 2

IGF-1R

insulin-like growth factor-1 receptor

TKI

tyrosine kinase inhibitor

VEGFRs

vascular endothelial growth factor receptors

References

  1. Top of page
  2. Abstract
  3. Pathophysiology of the HER Family
  4. HER2 in Breast Cancer Pathogenesis
  5. Mechanisms of Action of Trastuzumab
  6. Mechanisms of Resistance to Trastuzumab
  7. FCGR3A Polymorphism
  8. Potential Therapeutic Strategies for Overcoming Trastuzumab Resistance
  9. Future Direction
  10. Conclusions
  11. References
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