HER2 Extracellular Domain Research Articles

Abstract 1227: Modulation of EGF-receptors signaling by small peptidomimetic molecules in breast cancer cells

Abstract Protein-protein interactions play a crucial role in regulating many biological processes as well as in disease pathology. Understanding the structural basis and modulating protein-protein interactions has a tremendous impact in developing new therapeutic strategies for many human diseases. Human epidermal growth factor receptor family of proteins (EGFR, HER2, 3 and 4) plays an important role in cell growth and proliferation. Aberrant interactions of EGFR family of proteins lead to tumor growth and metastasis. HER2, a member of EGFR family of proteins is overexpressed in approximately 25% of breast cancers. Deregulation of HER2 signaling pathways and overexpression of HER2 is known to occur in many cancers. HER2 is known to form heterodimers and is a major therapeutic target in breast cancer treatment. Formation of HER2 receptor heterodimerization with other EGFR family of proteins such as EGFR, HER3 is essential for intracellular kinase activation and is the basis for exploring direct inhibition of EGFR activation by blocking dimerization for therapeutic purposes. We have designed several small peptidomimetics to inhibit HER2-mediated signaling for cell growth. One of such peptidomimetics, Compound 5 (Arg-[3-amino-naphthyl-propionic acid]-Phe) designed, exhibited antiproliferative activity with IC50 values in the nM range against HER2 overexpressing breast cancer cell lines, SKBR-3 and BT-474. Binding studies using Florescence Microscopy, ELISA, Circular Dichroism (CD) and Surface Plasmon Resonance (SPR) confirmed that compound 5 binds specifically to HER2-extracellular domain. SPR studies revealed that compound 5 specifically binds to domain IV of HER2 extracellular region. Also, Compound 5 inhibited HER2-HER3 as well as HER2-EGFR heterodimerization evaluated by SPR. Pathhunter and proximity ligation assays confirmed the inhibition of HER2 heterodimerization with EGFR and HER3 by compound 5. Western blots revealed that compound 5 blocks the transphosphorylation of HER2 protein. Based on these observations small peptidomimetic inhibitor molecules can be potential therapeutic agents in disabling HER2 mediated heterodimerization. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1227. doi:1538-7445.AM2012-1227

Mechanism of resistance to trastuzumab and molecular sensitization via ADCC activation by exogenous expression of HER2-extracellular domain in human cancer cells.

Trastuzumab, a humanized antibody targeting HER2, exhibits remarkable therapeutic efficacy against HER2-positive breast and gastric cancers; however, acquired resistance presents a formidable obstacle to long-term tumor responses in the majority of patients. Here, we show the mechanism of resistance to trastuzumab in HER2-positive human cancer cells and explore the molecular sensitization by exogenous expression of HER2-extracellular domain (ECD) in HER2-negative or trastuzumab-resistant human cancer cells. We found that long-term exposure to trastuzumab induced resistance in HER2-positive cancer cells; HER2 expression was downregulated, and antibody-dependent cellular cytotoxicity (ADCC) activity was impaired. We next examined the hypothesis that trastuzumab-resistant cells could be re-sensitized by the transfer of non-functional HER2-ECD. Exogenous HER2-ECD expression induced by the stable transfection of a plasmid vector or infection with a replication-deficient adenovirus vector had no apparent effect on the signaling pathway, but strongly enhanced ADCC activity in low HER2-expressing or trastuzumab-resistant human cancer cells. Our data indicate that restoration of HER2-ECD expression sensitizes HER2-negative or HER2-downregulated human cancer cells to trastuzumab-mediated ADCC, an outcome that has important implications for the treatment of human cancers.

Amplification of the Oncogene HER2 in Breast Cancer: Molecular Basis and Therapeutic Significance

The epithelial cells of the breast are under the influence of a range of hormones and growth factors. The HER2, a member of the family of growth factor receptors with tyrosine kinase activity, is amplified in approximately 15-20% of breast tumors, defining a highly aggressive subgroup. The breast cancers that overexpress HER2 inaugurated the era of targeted therapy in breast cancer and became the focus of numerous studies to enable better understanding and development of more efficient therapeutic strategies. Trastuzumab, a humanized monoclonal antibody that binds to the extracellular domain of HER2, causes inhibition of cell growth and increased apoptosis. However, response rates to monotherapy with this monoclonal antibody vary from 12-30%, and most of the responders patients develop resistance within one year. In this mini-review we present the major signaling pathways involved with HER2, as well as major mechanisms of resistance to trastuzumab and new potential targets.

Adjuvant systemic therapy for early breast cancer

Breast cancer-specific mortality has declined over the last two decades as a result of the introduction of screening programmes and advances in adjuvant systemic therapy. The most notable progress has been achieved in the case of Her2-positive disease, following the introduction of trastuzumab, a humanized monoclonal antibody targeting the extracellular domain ofHer2. Indeed, addition of trastuzumab to chemotherapy for a total duration of 1 year increases progression-free survival (PFS) as well as overall survival (OS) compared with chemotherapy alone. In hormone receptor-positive disease, treatment with aromatase inhibitors (AIs) prolongs PFS compared with tamoxifen, and in some clinical trials they have been found to increase OS also. Chemotherapy remains the mainstay of treatment for patients with triple-negative disease and also plays a role in Her2-positive and high-risk hormone receptor-positive tumours. Although, overall, only limited progress has been achieved in this field, most trials suggest that the introduction of taxanes has resulted in improved outcomes. Dose-dense chemotherapy is superior to older adjuvant regimens comprising 3-weekly administration of paclitaxel, while high-dose chemotherapy with autologous stem cell support currently has no place in the treatment of breast cancer. Neoadjuvant chemotherapy increases the rate of breast-conserving surgeries; importantly, the pathological complete remission (pCR) rate has been identified as a surrogate for improved OS in hormone receptor-negative patients. Thus, clinical trials have aimed to increase pCR rates by addition of further cytotoxic substances or biologicals such as trastuzumab, lapatinib or bevacizumab. However, the optimum treatment strategy for hormone receptor-negative patients without pCR remains elusive. This review discusses recent developments and open questions in the field of adjuvant systemic therapy for early breast cancer.

Targeting of the non-mutated tumor antigen HER2/neu to mature dendritic cells induces an integrated immune response that protects against breast cancer in mice

IntroductionGiven their relative simplicity of manufacture and ability to be injected repeatedly, vaccines in a protein format are attractive for breast and other cancers. However, soluble human epidermal growth factor receptor (HER2)/neu protein as a vaccine has not been immunogenic. When protein is directly targeted to antigen uptake receptors, such as DEC205 (DEC), efficient processing and presentation of antigen take place. The aim of this study was to determine the immunogenicity of a HER2 protein vaccine that directly targets to DEC+ dendritic cells (DCs) in a mouse breast cancer model.MethodsWe genetically engineered the HER2 extracellular domain into a monoclonal antibody specific for DEC (DEC-HER2). Mice of various genetic backgrounds were immunized with DEC-HER2 in combination with DC maturation stimuli (poly IC ± CD40 Ab). Vaccine-induced T cell immunity was determined by analyzing the ability of CD4+/CD8+ T cell to produce interferon (IFN)-gamma and proliferate upon antigen rechallenge. Sera were assessed for the presence of antigen specific antibody (Ab). For vaccine efficacy, FVB/N mice were immunized with DEC-HER2 in combination with poly IC and protection against neu-expressing mammary tumors was assessed. Protection mechanisms and tumor-specific T cell responses were also evaluated.ResultsWe demonstrate that DEC-HER2 fusion mAb, but not Ctrl Ig-HER2, elicits strong, broad and multifunctional CD4+ T cell immunity, CD8+ T cell responses, and humoral immunity specific for HER2 antigen. Cross-reactivity to rat neu protein was also observed. Importantly, mice xeno-primed with DEC-HER2 were protected from a neu-expressing mammary tumor challenge. Both CD4+ and CD8+ T cells mediated the tumor protection. Robust anti-tumor T cell immunity was detected in tumor protected mice.ConclusionsImmunization of mice with HER2 protein vaccine targeting DEC+ DCs in vivo induced high levels of T- and B-cell immunity. Non-targeted HER2 protein was poorly immunogenic for CD4+ and CD8+ T cells. This vaccination approach provided long-term survival benefit for mice challenged with neu-expressing tumor following as little as 2.7 μg of HER2 protein incorporated in the vaccine. Vaccine-induced CD4+ and CD8+ T cells were both essential for tumor protection. This immunization strategy demonstrates great potential towards the development of vaccines for breast cancer patients.

Treatment of HER2 positive breast cancer patients

Breast cancer (BC) is the most common cancer in women. It is known already for years that BC is a heterogeneous disease. This has now been confirmed by BC gene signature by which at least four types of BC subtypes can be distinguished. Approximately 15 % of patients with BC have a »HER2 like« tumor. A distinctive feature of this subtype is an overexpression of HER2 protein and/or amplification of the HER2 gene. HER2 protein is a transmembrane receptor protein, which promotes tumor growth, replication, invasion and dissemination. Consequently, when diagnosed, HER2 positive tumors are usually larger, less-differentiated, and are more often accompanied by metastatic involvement of the axillary lymph nodes, as compared to HER2 negative tumors. Women with HER2 positive tumors are therefore at higher risk of cancer recurrence and death. With the introduction of targeted anti- HER2 therapy, the prognosis of HER2 disease has significantly improved. There are currently two antiHER2 drugs registered for use in general oncology practice. Trastuzumab is a monoclonal antibody directed toward the extracellular domain of HER2 protein. It is used for the treatment of metastatic BC and in adjuvant setting. It is more effective when combined with chemotherapy compared to monotherapy. Combined with chemotherapy, it prolongs time to progression and overall survival of patients with metastatic BC. A year of adjuvant treatment with trastuzumab reduces the risk of recurrence and death from BC by 50 % and 30 %, respectively. Lapatinib is a small molecule that binds to the intracellular domain of HER2 receptor. The drug is registered for the treatment of metastatic HER2 positive BC after failure to trastuzuamb therapy. Combined with chemotherapy or hormonal therapy, lapatinib prolongs time to progression in patients with metastatic BC. Several other antiHER2 drugs are currently under evaluation in clinical trials.

Relationship between serum HER2 level and histologic HER2 status in patients with advanced/recurrent gastric cancer.

24 Background: The HER2 extracellular domain can be quantitatively measured in terms of the serum HER2 level by using a CLIA. Increased serum HER2 levels have been reported to be found in 20-25% of metastatic breast cancer patients and correlated with HER2 protein overexpression, increased tumor volume, and worse prognosis. However, the prevalence of HER2 expression in the serum of gastric cancer (GC) patients is not known. Methods: This study retrospectively analyzed the tumor tissue and serum samples of 100 patients who had histologically confirmed advanced/recurrent stomach adenocarcinoma, had started chemotherapy at National Cancer Center Hospital East during 2007–2011, did not have any other active malignancies, and whose serum and tumor tissue samples had been collected and stored at the baseline. HER2 protein expression was determined by immunohistochemistry (IHC; I-VIEW Pathway 4B5 kit) with the GC scoring system (Hofmann, 2008), and gene amplification was determined by fluorescence in situ hybridization (FISH; PathVysion HER2 DNA probe kit), with HER2:CEP17 ≥ 2.0 seen in tumor tissue. Serum HER2 levels were measured using a CLIA (ADIVA Centaur XP). HER2 positivity was defined as IHC 2+ with FISH positive or IHC 3+. Results: The baseline characteristics were as follows: men, 66; median age, 66 years (range, 29–85); advanced/recurrent, 96/4; and intestinal/diffuse/mixed, 38/41/21. An IHC score of 0/1+/2+/3+ was noted in 41/32/6/21 patients, respectively. Gene amplification in FISH was noted in 28 cases. The number of HER2 positive patients was 21. The median serum HER2 level was 9.3 ng/mL (range, 5.0–332.4). With a cutoff level of 15 ng/mL (mean + 2SD of 241 healthy control women), the sensitivity of serum HER2 levels with respect to HER2 positivity was 52.4%; specificity, 93.7%; positive predictive value, 68.8%; negative predictive value, 84.1%; and accuracy, 85.0%. The area under the receiver operating characteristic curve was 0.894. Conclusions: Increased serum HER2 levels were noted in GC patients and were associated with HER2 overexpression in tumor tissue. Further investigations of serum HER2 in terms of the clinical significance are warranted.

Pertuzumab

The humanized monoclonal antibody pertuzumab is the first in a new class of drugs, the human epidermal growth factor receptor (HER) dimerization inhibitors. Given that pertuzumab binds to a different epitope of the HER2 extracellular domain than trastuzumab, combination therapy with pertuzumab plus trastuzumab may result in more comprehensive blockade of HER2 signalling than can be achieved with trastuzumab alone. The efficacy of adding pertuzumab to trastuzumab plus docetaxel for the first-line treatment of HER2-positive metastatic breast cancer was demonstrated in the randomized, double-blind, multinational, phase III CLEOPATRA trial. Both independently assessed progression-free survival (primary endpoint) and investigator-assessed progression-free survival were significantly improved in patients receiving pertuzumab plus trastuzumab and docetaxel compared with those receiving placebo plus trastuzumab and docetaxel. The prespecified interim analysis of survival revealed a strong trend towards a survival benefit associated with pertuzumab, although this was not considered statistically significant. The objective response rate was higher with pertuzumab than with placebo. Intravenous pertuzumab had an acceptable tolerability profile when added to trastuzumab and docetaxel in the CLEOPATRA trial.

Clinical pharmacology of trastuzumab emtansine (T-DM1): an antibody–drug conjugate in development for the treatment of HER2-positive cancer

PurposeTrastuzumab emtansine (T-DM1) is an antibody–drug conjugate comprising trastuzumab and DM1, a microtubule polymerization inhibitor, covalently bound via a stable thioether linker. To characterize the pharmacokinetics (PK) of T-DM1 in patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer, data from four studies (TDM3569g, TDM4258g, TDM4374g, and TDM4688g) of single-agent T-DM1 administered at 3.6 mg/kg every 3 weeks (q3w) were assessed in aggregate.MethodsMultiple analytes—T-DM1, total trastuzumab (TT), DM1, and key metabolites—were quantified using enzyme-linked immunosorbent assays or liquid chromatography tandem mass spectrometry. PK parameters of T-DM1, TT, and DM1 exposure were calculated using standard noncompartmental approaches and correlated to efficacy (objective response rate) and safety (platelet counts, hepatic transaminase concentrations). Immunogenicity was evaluated by measuring anti-therapeutic antibodies (ATA) to T-DM1 after repeated dosing using validated bridging antibody electrochemiluminescence or enzyme-linked immunosorbent assays.ResultsPK parameters for T-DM1, TT, and DM1 were consistent across studies at cycle 1 and steady state. T-DM1 PK was not affected by residual trastuzumab from prior therapy or circulating extracellular domain of HER2. No significant correlations were observed between T-DM1 exposure and efficacy, thrombocytopenia, or increased concentrations of transaminases. Across the studies, ATA formation was detected in 4.5% (13/286) of evaluable patients receiving T-DM1 q3w.ConclusionsThe PK profile of single-agent T-DM1 (3.6 mg/kg q3w) is predictable, well characterized, and unaffected by circulating levels of HER2 extracellular domain or residual trastuzumab. T-DM1 exposure does not correlate with clinical responses or key adverse events.

OPCML, a novel systems regulator of tyrosine kinase signaling in ovarian and other cancers

OPCML, a GPI anchored tumor suppressor gene is inactivated by somatic methylation in multiple cancers. We previously identified this gene by LOH mapping and demonstrated that it was inactivated by somatic methylation in 80% of ovarian cancers. Restoring OPCML expression by stable transfection suppressed in-vitro growth and in-vivo tumorigenicity. We hypothesized that OPCML might abrogate growth signaling pathways. In SKOV-3 and PEO1, ovarian cancer cell lines with no expression of OPCML, we demonstrated that OPCML negatively regulates a specific repertoire of receptor tyrosine kinases (RTKs) EPHA2, FGFR1, FGFR3, HER2 and HER4, and reciprocally, OPCML siRNA and shRNA knockdown in normal ovarian surface epithelial cells up-regulates these same RTKs, with no effect on RTKs EPHA10, FGFR2, FGFR4, EGFR, HER3, VEGFR1 and VEGFR3. shRNA knockdown shows that loss of OPCML accelerates the growth of normal ovarian surface epithelial cells in vitro. Example immunoprecipitation experiments revealed that OPCML binds to EphA2, FGFR1 and HER2 extracellular domains with no such interaction to EGFR, thus OPCML binds directly to RTKS that it negatively regulates. Cotransfection of ECD less and ECD containing Her2/Neu shows that the functional tumor suppressor phenotype of OPCML is mediated via interaction with the ECD. We demonstrate that OPCML is located exclusively in the raft membrane fraction and sequesters RTKs that it binds to the raft fraction, leading to polyubiquitination and proteosomal degradation via a cav-1 endosomal mechanism resulting in systems depletion of this specific RTK repertoire, that does not occur with RTKs that OPCML does not bind. Pulse-chase experiments confirm rapid loss of HER2 in OPCML expressing cells and not in OPCML deficient cells. We demonstrate that OPCML abrogates EGF mediated phosphorylation of FGFR1, HER2 and EGFR and the downstream phosphosignaling of pErk and pAKT. A recombinant modified OPCML-like protein without a GPI anchor, signal peptide or glycosylation was constructed and expressed in E. coli. This rOPCML tumor suppressor protein therapeutic caused growth inhibition by apoptosis in 6/7 ovarian cancer cell lines tested with no effect on OPCML expressing normal ovarian surface epithelium by an identical mechanism to the transfected normal protein. pERk and pAKT inhibition was seen with functional growth inhibition and increased apoptosis in rOPCML treated cells. rOPCML was then injected intraperitoneally twice weekly in two murine intraperitoneal models of ovarian cancer (nude mouse A2780 and SKOV3) and demonstrated profound inhibition of tumour weight, ascites volume and peritoneal dissemination compared with BSA control. Western analysis from rescued tumors confirmed that the same mechanism of specific RTK downregulation was also evident in vivo. In Summary, the OPCML tumour suppressor mediates its suppressor function by systems level negative regulation of at least 5 RTKs and a recombinant modified derivative is a potent tumor suppressor protein therapeutic in-vitro and in-vivo that recapitulates the in-vitro mechanism.

Monitoring soluble HER2 extracellular domain in the serum of breast cancer patients using a refined ELISA assay

Monitoring soluble HER2 extracellular domain in the serum of breast cancer patients using a refined ELISA assay

S5-1: Neoadjuvant Pertuzumab (P) and Trastuzumab (H): Biomarker Analyses of a 4-Arm Randomized Phase II Study (NeoSphere) in Patients (pts) with HER2−Positive Breast Cancer (BC).

Abstract Background: NeoSphere showed that P with H and docetaxel (T) had a significantly higher pCR rate (45.8%) compared with TH (29%) or other P combinations (HP, TP) (Gianni, SABCS 2010). HER2+/ER+ and HER2+/ER− BC have different patterns of gene expression, and treatment response seems to be driven by different biologic pathways (Bianchini, ASCO 2011). This effect was also seen in NeoSphere. A broad panel of biomarkers (BMs) was assessed in tumor specimens and in sera to characterize the molecular profile of the ITT population and to explore BMs with different treatments and in different subsets of pts. Methods: Core biopsies and sera from 387/417 pts collected at baseline were available for BM analyses. HER2, all its truncated forms, HER3, IGF1R, PTEN, and pAKT were assessed by IHC and a modified H-score was derived per each marker. HER1, HER2, HER3, amphiregulin (AREG) and betacellulin mRNA expression in tumor tissue was assessed by qRT-PCR. C-myc was assessed by FISH. Mutational analyses of 8 hot spots within PIK3CA on tumor DNA was done via TaqMan-PCR. ELISA was used to detect HER2 extracellular domain (ECD), AREG, EGF, and TGFα in serum. Treatment interaction test was performed to test for association between BMs and pCR. In the analysis, median values were used as cutoffs, except for PTEN, PIK3CA mutation, and c-myc amplification, where cutoffs were applied following a biologic rationale. Results: In the overall population, high HER2 membrane protein expression measured by modified H-score was associated with a significantly higher pCR rate in the THP arm (p=0.001), with a large benefit from combination of P with TH (OR=3.74, CI 80%, 2.21−6.34; p=0.0009). The cutoff derived from NeoSphere has no immediate clinical applicability. A decreased likelihood of pCR was detected for pts with PI3K mutations in all 4 arms in a preliminary analysis limited to about 70% of cases. A final analysis will be presented. A significantly different distribution of BM expression in the ER+ vs ER− subgroups was found for 10 of 16 BMs (FDR<0.05). Low levels of IGF1R were found to be associated with higher pCR with combination of P with TH in the ER− (OR=4.40, CI 80%, 2.17−8.9; p=0.006) but not ER+ subgroup. High HER2 mRNA expression was associated with higher pCR rate in the HP arm in ER− (p=0.021) but not ER+ (p=0.74) tumors. Data on the full BM analysis including those on the ratio between HER2 ECD and intracellular domain (p95) will be presented. The levels measured for AREG and betacellulin were generally very low (less than the limits of variability for these assays), hence further analysis was not felt meaningful. Summary: The level of HER2 membrane protein expression was associated with increased benefit from combination of P with TH. The PI3K mutational status appears to have a relevant role in defining the likelihood of response with all treatments. The different treatment effects according to ER status are reflected in a different molecular BM profile in ER+ vs ER− tumors, with the IGF1R membrane score being correlated with ER− tumors. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S5-1.

P2-01-14: High Level of CD44 Expression and Soluble CD44 Secretion Mediate Trastuzumab Resistance in HER2 Positive Breast Cancer.

Abstract Background: Overexpression of HER2 occurs in 15–25% of invasive breast cancer. Trastuzumab, a humanized monoclonal antibody targeting the extracellular domain of HER2, markedly improves disease-free and overall survival in patients with HER2−positive breast cancer. However, resistance to trastuzumab develops in most patients with HER2−overexpressing metastatic breast cancer. Breast cancer stem cells have been shown to express HER2, although its role in the development or maintenance of stem cells is not well characterized. CD44 is one of the proposed markers for breast cancer stem cells. We investigated the role of CD44 in acquired trastuzumab resistance in HER2 positive breast cancer. Methods: CD44 mRNA expression was measured by quantitative real-time PCR. The proportion of CD44 positive cells was measured by fluorescence-activated cell sorting (FACS). Soluble CD44 level was detected by enzyme-linked immunosorbent assay (ELISA). In vitro tumorigenicity was evaluated by soft agar colony formation assay. Cell invasion was tested by using a matrigel invasion assay. In vivo tumor growth was evaluated using a mammary fat pad xenograft mouse model. Results: CD44 mRNA expression, CD44 positive cell population by FACS, and CD44 secretion were significantly increased in both SKBR3 derived trastuzumab resistant SKBR3 clone 3 and BT474 derived trastuzumab resistant HR20 cells compared to parental, trastuzumab sensitive cells. Consistent with the increase in CD44, trastuzumab resistant cells showed an increase in soft agar colony formation and cell invasion, which were blocked by siRNA-induced downregulation of CD44 expression. Tumor growth and incidence were enhanced in mice injected with trastuzumab resistant HR20 cells compared to the mice injected with trastuzumab sensitive parental BT474 cells. Knock-down of CD44 expression by the transfection with CD44-specific siRNA suppressed in vivo tumor growth in the mice injected with trastuzumab resistant HR20 cells. Finally, we found high serum CD44 levels correlated with advanced tumor progression in HER2 positive breast cancer. Conclusion: Our findings indicate that high levels of CD44 mediate trastuzumab resistance in HER2 positive breast cancer cells as well as tumor progression in mice. Circulating CD44 is prognostic in patients with HER2 positive breast cancer. CD44 may be a therapeutic target for HER2 positive breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-14.

P2-18-06: Conventional Trastuzumab Is an Antagonist of Natural Killer Cells: Making the Case for Fucose-Depleted Trastuzumab.

Abstract The discovery of the HER2 receptor and later the development of trastuzumab (an anti-HER2 antibody) were major advances leading toward the treatment of a significant proportion of human breast cancers. Trastuzumab (Herceptin®) is a humanized monoclonal IgG1 whose discrete antitumor effects are exerted through the 2 main components of the antibody. Trastuzumab's FAB portion binds to the HER2 extracellular domain, interrupting HER2 signaling. The FC portion then engages the activating receptor (FcγRIIIa) on an effector cell, usually a natural killer (NK) cell and instigates a lytic attack via antibody-dependent cellular cytotoxicity (ADCC). The value of trastuzumab in breast cancer therapy is well established, but, there are limitations to the drug's effectiveness. These include: (1) unresponsive tumors with low or equivocal HER2 expression; (2) de novo (primary) resistance to therapy in over 50% of patients with HER2−positive tumors; (3) frequent emergence of secondary resistance often after short exposure periods; (4) competition of plasma IgG proteins with antibodies for binding to the activating FC receptors on NK cells; and (5) the high cost of trastuzumab treatment, raising concerns for the economic sustainability of this form of therapy. Trastuzumab and other therapeutic antibodies share a structural feature that is critical to their effectiveness: a specific oligosaccharide which is covalently linked to the FC region at asparagine 297 (Asn297) in each of the 2 heavy chains. This oligosaccharide, a mannosyl-chitobiose core, is “core-fucosylated” with a single α-L-fucose (fucose) sugar attached via an α(1,6) linkage. In 2002, Shields et al. developed a novel form of trastuzumab which lacked core fucose in the Fc region [J Biol Chem 2002; 277: 26733–40]. When compared to conventional trastuzumab, the fucose-depleted trastuzumab was 43-fold more potent in provoking ADCC against HER2−expressing human breast cancer cells in vitro. Subsequent studies with several monoclonal antibodies have elucidated the role of core fucose in inhibiting ADCC, by reducing the affinity of trastuzumab's Fc portion for the activating FC receptors located on effector cells. The superior ADCC effect of fucose-depleted trastuzumab has now been demonstrated in vivo [Junttila TT et al., Cancer Res 2010; 70: 4481–4489]. We review both in vitro and in vivo evidence from multiple laboratories (working with trastuzumab, rituximab and other therapeutic antibodies) to argue that fucose-depleted trastuzumab offers several potential advantages over conventional trastuzumab. Such advantages in clinical use, we postulate, include (1) treatment of patients with trastuzumab-resistant tumors, (2) treatment of patients with equivocal or low expression of HER2, and (3) decreased cost of treatment. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-18-06.

Targeting HER2 in breast cancer: beyond trastuzumab

Trastuzumab has altered the natural history of HER2+ breast cancer. In the metastatic setting, it has improved progression-free and overall survival. In patients with operable breast cancer, adjuvant trastuzumab, when added to chemotherapy, has improved disease-free and overall survival. Unfortunately, virtually all patients with metastatic breast cancer develop disease that is at least partially resistant to trastuzumab. In these patients, there is still value in continuing trastuzumab in combination with other treatments, but trastuzumab alone is unable to fully suppress tumor growth. Multiple mechanisms of resistance to trastuzumab have been suggested including activation of other growth factor receptors, preferential finding of HER2 to HER3, loss of the extracellular domain of HER2, and activation of the PI3 kinase pathway as a result of PTEN loss or a PIK3CA mutation. It is unknown to what extent these mechanisms are relevant in individual patients, but it is probable that many different mechanisms of resistance are clinically important. Over the past decade, a number of treatments have been developed for patients with trastuzumab-resistant disease. At present, only lapatinib, a small-molecule inhibitor of HER1 and HER2, is commercially available. It is active when administered with either chemotherapy or trastuzumab. A variety of other therapies are under investigation in phase III clinical trials. Pertuzumab, a monoclonal antibody that inhibits HER2-HER3 heterodimers, appears to be effective when combined with trastuzumab ± chemotherapy. T-DM1, an antibody-drug conjugate, has also displayed remarkable activity in the setting of refractory disease and has limited toxicity. It is presently under investigation in multiple randomized trials. Neratinib is an oral irreversible tyrosine kinase inhibitor that targets HER1, HER2, and HER4. As a single agent, it appears to be more active than lapatinib, but is associated with more significant toxicity. It, too, is under evaluation in phase III trials in the adjuvant and metastatic settings. A variety of other agents are under active study including the mTOR inhibitors, the PI3kinase inhibitors, angiogenesis inhibitors, and IGFR antagonists. It is likely that a number of new agents will be available for the treatment of HER2+ breast cancer in the next several years, and outcomes for this group of patients will continue to improve.

Beyond trastuzumab: New treatment options for HER2-positive breast cancer

HER2-positive tumors comprise 15% to 20% of all breast cancers (BC) and are associated with worse clinical outcomes [Slamon et al., Science 1987;235:177-82]. Trastuzumab is a humanized monoclonal antibody designed to target the extracellular domain of the HER2 receptor, and is the foundation of care of women with early and advanced HER2-positive BC. However, a significant proportion of patients with this type of BC display either primary or secondary resistance to trastuzumab. Therefore, in an effort to overcome such resistance and further improve the outcome of patients with HER2-positive disease, several new anti-HER2 agents are currently being developed. These include small molecules that inhibit the HER2 tyrosine kinase activity (lapatinib, neratinib), monoclonal antibodies directed at other epitopes of the HER2 extracellular domain (pertuzumab), antibody-drug conjugates (trastuzumab-DMl), and heat shock protein 90 inhibitors (tanespimycin). A great deal of interest has been generated by recent data from the randomized neo-adjuvant studies NeoALTTO and NeoSphere, which have shown that dual blockade of the HER2 receptor with anti-HER2 agents is significantly superior to using one agent alone. If these results are validated in larger ongoing and planned phase III studies in early BC, they could lead to a paradigm shift in treatment strategy. Therefore, to avoid unnecessary toxicities and costs, it is critical to intensify the research for biomarkers that can identify those patients most likely to benefit from specific targeted therapies.

Her2 : un modèle de cible thérapeutique

Fifteen percent of breast cancers overexpress Her2, a tyrosin-kinase receptor. Because of its prognosis and its incidence, several efforts have been done in the last years to better understand mechanisms of Her2 action. This led to the development of targeted therapies such as trastuzumab, monoclonal antibody directed against the Her2 extra-cellular domain. Trastuzumab provides significant clinical benefit in women with Her2-positive breast cancers. However, many women will either not respond or progress despite this treatment. The aim of this article is to summarize mechanisms of action of Her2 and of trastuzumab, and to better understand pathways activated in resistant tumors in order to identify ways to overcome them.

HER2 splice variants and their relevance in breast cancer

<p class="MsoNormal" style="background: white; margin: 0cm 0cm 0pt; text-align: justify;"><span style="font-size: small;"><span lang="EN-US">The HER2 gene amplification occurs in 20-30% of breast cancer and is correlated with a poorer prognosis compared to HER2-negative disease due to increased proliferation and metastatic potential.<span style="color: #231f20;"> Two major types of receptor inhibitors have been developed for therapy and one for each categories is currently used in clinic: i) the humanized monoclonal antibody trastuzumab, directed against the HER2 extracellular domain; and ii) the EGFR/HER2 dual<span style="mso-spacerun: yes;"> </span></span>tyrosine kinase inhibitor lapatinib. </span><span lang="EN-US">However, patients may develop resistance to</span><span lang="EN-US"> drugs and show</span><span lang="EN-US"> disease progression</span><span lang="EN-US">. Several resistant mechanisms have been explored and are still under investigation. Here, </span><span lang="EN-GB">we focus our attention on the role played by the alternative splicing forms of HER2 in mediating HER2 oncogenic activity and in conditioning the response to HER2 therapies. Three HER2 splice variants have been described so far; the p100 and the herstatin gave raised to two secreted proteins of 100 kd and 68 kd, respectively that act as cell growth inhibitors. Herstatin has been described for its ability to interrupt the constitutive HER2 activation, but also for its capacity to hamper HER2 dimerization with the others HER receptors. Interestingly, herstatin, present as mRNA and protein in non cancerous tissue in areas adjacent to breast carcinoma, is absent as protein in 75% of mammary tumors, </span><span lang="EN-US">which indicates that cancer cells are protected by some intrinsic mechanism against the putative growth-inhibitory effects of this naturally occurring molecule. The third splice form of HER2 gene is the Δ16HER2, encoding for a receptor lacking exon16, whose absence determines a constitutive active dimers with transforming activity in vitro and in vivo. The Δ16HER2 binds to trastuzumab to a less extend, due to conformational changes of the extracellular domain.<span style="mso-spacerun: yes;"> </span>The Δ16HER2 accounts for almost 9% of the total HER2 transcripts in human breast cancers and, additionally, Δ16HER2 levels are supposed to increase proportionally at the increasing of the HER2 wild-type copy numbers in human primary breast cancers. The availability of a specific assay to determine and quantify the expression levels of this splicing form and the availability of Δ16HER2 transgenic mice models made this variant as the most promising for the development of biodrugs. </span></span></p><span style="font-size: 12pt;" lang="EN-US">Finally, HER2 carboxy-terminal fragments (CTFs), generated by alternative initiation of translation, were observed in breast cancer patients. In particular, 611-CTF was described to activate multiple signaling pathways since it is expressed as a constitutively active homodimer. Expression of 611-CTF led to development of aggressive and invasive mammary tumors and it was suggested to be a potent oncogene capable of promoting mammary tumor progression and metastasis.</span>

HER2 splice variants and their relevance in breast cancer

The HER2 gene amplification occurs in 20-30% of breast cancer and is correlated with a poorer prognosis compared to HER2-negative disease due to increased proliferation and metastatic potential. Two major types of receptor inhibitors have been developed for therapy and one for each categories is currently used in clinic: i) the humanized monoclonal antibody trastuzumab, directed against the HER2 extracellular domain; and ii) the EGFR/HER2 dual tyrosine kinase inhibitor lapatinib. However, patients may develop resistance to drugs and show disease progression. Several resistant mechanisms have been explored and are still under investigation. Here, we focus our attention on the role played by the alternative splicing forms of HER2 in mediating HER2 oncogenic activity and in conditioning the response to HER2 therapies. Three HER2 splice variants have been described so far; the p100 and the herstatin gave raised to two secreted proteins of 100 kd and 68 kd, respectively that act as cell growth inhibitors. Herstatin has been described for its ability to interrupt the constitutive HER2 activation, but also for its capacity to hamper HER2 dimerization with the others HER receptors. Interestingly, herstatin, present as mRNA and protein in non cancerous tissue in areas adjacent to breast carcinoma, is absent as protein in 75% of mammary tumors, which indicates that cancer cells are protected by some intrinsic mechanism against the putative growth-inhibitory effects of this naturally occurring molecule. The third splice form of HER2 gene is the Δ16HER2, encoding for a receptor lacking exon16, whose absence determines a constitutive active dimers with transforming activity in vitro and in vivo. The Δ16HER2 binds to trastuzumab to a less extend, due to conformational changes of the extracellular domain. The Δ16HER2 accounts for almost 9% of the total HER2 transcripts in human breast cancers and, additionally, Δ16HER2 levels are supposed to increase proportionally at the increasing of the HER2 wild-type copy numbers in human primary breast cancers. The availability of a specific assay to determine and quantify the expression levels of this splicing form and the availability of Δ16HER2 transgenic mice models made this variant as the most promising for the development of biodrugs. Finally, HER2 carboxy-terminal fragments (CTFs), generated by alternative initiation of translation, were observed in breast cancer patients. In particular, 611-CTF was described to activate multiple signaling pathways since it is expressed as a constitutively active homodimer. Expression of 611-CTF led to development of aggressive and invasive mammary tumors and it was suggested to be a potent oncogene capable of promoting mammary tumor progression and metastasis.

HER2 splice variants and their relevance in breast cancer

The HER2 gene amplification occurs in 20-30% of breast cancer and is correlated with a poorer prognosis compared to HER2-negative disease due to increased proliferation and metastatic potential. Two major types of receptor inhibitors have been developed for therapy and one for each categories is currently used in clinic: i) the humanized monoclonal antibody trastuzumab, directed against the HER2 extracellular domain; and ii) the EGFR/HER2 dual tyrosine kinase inhibitor lapatinib. However, patients may develop resistance to drugs and show disease progression. Several resistant mechanisms have been explored and are still under investigation. Here, we focus our attention on the role played by the alternative splicing forms of HER2 in mediating HER2 oncogenic activity and in conditioning the response to HER2 therapies. Three HER2 splice variants have been described so far; the p100 and the herstatin gave raised to two secreted proteins of 100 kd and 68 kd, respectively that act as cell growth inhibitors. Herstatin has been described for its ability to interrupt the constitutive HER2 activation, but also for its capacity to hamper HER2 dimerization with the others HER receptors. Interestingly, herstatin, present as mRNA and protein in non cancerous tissue in areas adjacent to breast carcinoma, is absent as protein in 75% of mammary tumors, which indicates that cancer cells are protected by some intrinsic mechanism against the putative growth-inhibitory effects of this naturally occurring molecule. The third splice form of HER2 gene is the Δ16HER2, encoding for a receptor lacking exon16, whose absence determines a constitutive active dimers with transforming activity in vitro and in vivo. The Δ16HER2 binds to trastuzumab to a less extend, due to conformational changes of the extracellular domain. The Δ16HER2 accounts for almost 9% of the total HER2 transcripts in human breast cancers and, additionally, Δ16HER2 levels are supposed to increase proportionally at the increasing of the HER2 wild-type copy numbers in human primary breast cancers. The availability of a specific assay to determine and quantify the expression levels of this splicing form and the availability of Δ16HER2 transgenic mice models made this variant as the most promising for the development of biodrugs. Finally, HER2 carboxy-terminal fragments (CTFs), generated by alternative initiation of translation, were observed in breast cancer patients. In particular, 611-CTF was described to activate multiple signaling pathways since it is expressed as a constitutively active homodimer. Expression of 611-CTF led to development of aggressive and invasive mammary tumors and it was suggested to be a potent oncogene capable of promoting mammary tumor progression and metastasis.