Merel Gijsen

HER2 Phosphorylation Is Maintained by a PKB Negative Feedback Loop in Response to Anti-HER2 Herceptin in Breast Cancer

Herceptin (trastuzumab) is used in patients with breast cancer who have HER2 (ErbB2)-positive tumours. However, its mechanisms of action and how acquired resistance to Herceptin occurs are still poorly understood. It was previously thought that the anti-HER2 monoclonal antibody Herceptin inhibits HER2 signalling, but recent studies have shown that Herceptin does not decrease HER2 phosphorylation. Its failure to abolish HER2 phosphorylation may be a key to why acquired resistance inevitably occurs for all responders if Herceptin is given as monotherapy. To date, no studies have explained why Herceptin does not abolish HER2 phosphorylation. The objective of this study was to investigate why Herceptin did not decrease HER2 phosphorylation despite being an anti-HER2 monoclonal antibody. We also investigated the effects of acute and chronic Herceptin treatment on HER3 and PKB phosphorylation in HER2-positive breast cancer cells. Using both Förster resonance energy transfer (FRET) methodology and conventional Western blot, we have found the molecular mechanisms whereby Herceptin fails to abolish HER2 phosphorylation. HER2 phosphorylation is maintained by ligand-mediated activation of EGFR, HER3, and HER4 receptors, resulting in their dimerisation with HER2. The release of HER ligands was mediated by ADAM17 through a PKB negative feedback loop. The feedback loop was activated because of the inhibition of PKB by Herceptin treatment since up-regulation of HER ligands and ADAM17 also occurred when PKB phosphorylation was inhibited by a PKB inhibitor (Akt inhibitor VIII, Akti-1/2). The combination of Herceptin with ADAM17 inhibitors or the panHER inhibitor JNJ-26483327 was able to abrogate the feedback loop and decrease HER2 phosphorylation. Furthermore, the combination of Herceptin with JNJ-26483327 was synergistic in tumour inhibition in a BT474 xenograft model. We have determined that a PKB negative feedback loop links ADAM17 and HER ligands in maintaining HER2 phosphorylation during Herceptin treatment. The activation of other HER receptors via ADAM17 may mediate acquired resistance to Herceptin in HER2-overexpressing breast cancer. This finding offers treatment opportunities for overcoming resistance in these patients. We propose that Herceptin should be combined with a panHER inhibitor or an ADAM inhibitor to overcome the acquired drug resistance for patients with HER2-positive breast cancer. Our results may also have implications for resistance to other therapies targeting HER receptors.

Potential of PET to Predict the Response to Trastuzumab Treatment in an ErbB2-Positive Human Xenograft Tumor Model

Currently, an alteration in the gross volume of a tumor is used to assess its response to trastuzumab; however, this approach provides only a late indication of response. Tissue-sample ex vivo assays are potentially valuable, but their procurement through biopsies is invasive and might be biased by tumor heterogeneity. We studied the feasibility of using PET to quantify changes in ErbB2 (HER2/neu) expression and to predict the response to trastuzumab in BT474 breast cancer xenografts with N-[2-(4-18F-fluorobenzamido)ethyl]maleimide (18F-FBEM)-HER2:342 Affibody. Methods: Mice bearing BT474 tumors were given trastuzumab (50 mg/kg loading dose, 25 mg/kg maintenance dose, administered intraperitoneally twice a week) or saline (control) for a total of 5 doses. Tumor size was monitored twice a week. Animals were scanned before the treatment, at 48 h, and 2 wk after the beginning of therapy. After the final scan, PET results were correlated with tumor response and immunohistochemical assessment of ErbB2 level, as well as with vasculature in the treated tumors. Results: Analysis of PET images indicated that tracer uptake was significantly reduced after 1 dose of trastuzumab, compared with baseline, suggesting applicability as an early indicator of changes in ErbB2 expression. After 5 doses of trastuzumab, the overall decrease in 18F-FBEM-HER2:342 Affibody uptake also correlated with tumor response and downregulation of ErbB2 expression by immunohistochemical assessment. However, individual animals had different responses. There was a correlation between bigger PET changes and a higher vessel count in the tumors, suggesting that an increased number of vessels could lead to better trastuzumab delivery. We confirmed that the difference in average vessel count in the tumors was not related to the size of the tumors and therefore was not due to the selection of more vascular tumors. This finding is consistent with previous findings demonstrating that the number of vessels in a tumor could be a useful prognostic marker for treatment response. Conclusion: Our data suggest that Affibody-based PET can noninvasively provide specific information on changes in receptor expression and could be a valuable strategy for predicting tumor response to trastuzumab.

Upregulation of ADAM proteases and HER ligands through a feedback loop mediates acquired resistance to trastuzumab in HER2-amplified breast cancer

The response rarely sustains long among the responders for Herceptin (trastuzumab) monotherapy treatment. It is still poorly understood how Herceptin exerts its mechanism of action and how the acquired resistance to this drug occurs.

Reply: PET Prediction of Response to Trastuzumab in ErbB2-Positive Human Xenograft Model

REPLY: The work that we have recently described tested the feasibility of the use of Affibody molecules (Affibody AB) and PET to predict tumor response to ErbB2-targeted therapy. It is clear that additional studies will be needed to dissect the mechanistic events underlying the observed changes. Differences among tumor cell lines could affect responses as well. Clinical studies show that the assessment of ErbB2 level by immunohistochemistry produces variable results among laboratories. This variation may be due to differences in immunohistochemistry staining techniques and scoring criteria (1). For antigen-retrieval processes, the solution used (e.g., citrate buffer or ethylenediaminetetraacetic acid and their pH), the duration of heating, and antigen retrieval may all affect detection of the ErbB2 antigen by immunohistochemistry (2). Different anti-ErbB2 antibodies used for immunohistochemistry staining have also been shown to produce different degrees of ErbB2 staining in tumors, even in the presence of gene amplification (3), although applying calibration may help in minimizing those differences (4). The HercepTest (Dako) using Dako antibody was proposed as the standardized immunohistochemistry method to overcome the problem of interlaboratory variations. The scoring system uses the intensity of ErbB2 staining as its basis, and an ErbB2-positive tumor is defined as a tumor with greater than 10% of cells stained 31 (5). Despite use of the HercepTest, there still was a high discrepancy between local and central ErbB2 testing in the N9831 Intergroup Adjuvant Trial, with a concordance of only 81.6% for a diagnostic test for the presence of the ErbB2 protein (6). The American Society of Clinical Oncology and College of American Pathologists recommended an algorithm defining positive, equivocal, and negative values for both ErbB2 protein expression and gene amplification. A positive ErbB2 result from immunohistochemistry staining is defined by uniform, intense membrane staining of more than 30% of invasive tumor cells instead of the original 10% (5). However, despite this new algorithm and definition, not all laboratories have adopted this new guideline and there still are variable results in ErbB2 testing among laboratories. Furthermore, both the old and the new “ErbB2 counting” definitions have problems in detecting subtle ErbB2 changes induced by the treatment. For example, if trastuzumab decreases ErbB2 staining from 100% of the cells to 40%, both the old and the new ErbB2 definition will score preand post-treatment samples as “positive” and may fail to detect ErbB2 changes after trastuzumab treatment. After trastuzumab treatment, we found in human breast carcinoma BT474 xenografts a significant reduction of tracer uptake related to ErbB2 decrease rather than tumor size reduction (7). The observable reduction in PET signals could be due to partial-volume effect, but this possibility is rather unlikely since the images were acquired with high contrast and in the absence of background activity. When large enough regions are drawn around the tumor, the partial-volume effect does not cause any loss of signal, and the signal that is measured indicates the actual activity distribution. Moreover, for PET quantification, we deliberately chose the value related to maximum counts per pixel within the tumor that is least affected by partial-volume effect. Importantly, we have also shown that tumor ErbB2 membrane staining and PET changes correlated with tumor volume after 5 doses of trastuzumab treatment (7). There was a correlation between PET and immunohistochemistry, and the radionuclide concentrations measured with PET agreed with the radioactivity concentrations obtained by g-counting (data were not presented). Although there was a large overlap in ErbB2 staining between the trastuzumab-treated and control groups, we found a significant reduction of ErbB2 downregulation after 5 doses of trastuzumab. This finding is consistent with several cell line experiments from different groups finding that trastuzumab downregulates ErbB2 receptors (8–10). After a single dose of trastuzumab, we could clearly see the differences in ErbB2 membrane staining between control and trastuzumab-treated samples, but the intensity percentage scoring failed to detect these changes (7). The dose and duration of trastuzumab will clearly affect the amount of ErbB2 downregulation and the detection of ErbB2 changes by immunohistochemistry. In our paper, the dose of trastuzumab was deliberately high (50 mg/kg loading dose followed by 4 more doses of 25 mg/kg each) to ensure that changes in receptor expression ErbB2 would be possible (7). Reddy et al. (11) treated BT474 xenografts with a lower dose of 10 mg/kg for only 6 d. They found a decrease in PET tracer using C6.5 diabody but could not detect any ErbB2 changes by immunohistochemistry. They concluded that “The exact mechanism by which trastuzumab treatment inhibits C6.5db binding is not yet understood.” On the other hand, McLarty et al. (12) reported that trastuzumab reduced ErbB2 membrane staining in SKBR3 cells and in MDAMB361 and MDAMB361 xenograft models. In this case, mice were treated only with 4 mg of trastuzumab per kilogram for 3 d or 3 wk. At 3 d, the authors did not see ErbB2 membrane changes, but 3 wk later immunohistochemistry analysis of tumor tissues indicated significant ErbB2 downregulation, associated with almost complete eradication of viable tumor cells. This finding is consistent with our study as we did not see a difference in intensity percentage scoring after a single dose of trastuzumab (7); we observed differences in ErbB2 membrane staining after 5 doses of the drug (7). We believe that the differences seen in ErbB2 staining between Reddy et al. (11), McLarty et al. (12), and our study (7) may be related to the dose and duration of trastuzumab used. However, the differences may also be related to the ErbB2 testing methods and the scoring criteria, which could not detect subtle ErbB2 changes after trastuzumab treatment. Trastuzumab was used as monotherapy before surgery in patients with primary operable ErbB2-positive breast tumors in a pilot study by Gennari et al. (13). They observed no change in ErbB2-positive staining using monoclonal antibody CB11 in the trastuzumab-treated samples. However, they provided figures from only 1 patient, as shown in their Figures 2A and 2B (13). Although these figures suggest some changes between preand posttreatment samples, the authors found no variations in the ErbB2 status (13). Furthermore, the use of a different anti-ErbB2 antibody and different scoring criteria may also have contributed

Abstract 1737: Overcoming trastuzumab resistance with the irreversible Pan-HER inhibitor neratinib

Overexpression of HER2 is seen in 15-20% of breast cancer and is a predictor of poor prognosis. Trastuzumab, a monoclonal antibody to the ECD of the HER2 receptor, has been shown to increase survival in patients especially in combination with chemotherapy. However, a significant number of patients do not respond to trastuzumab treatment and resistance develops in those given trastuzumab as a monotherapy. The purpose of this study was to investigate whether the small molecule irreversible tyrosine kinase inhibitor neratinib (HKI-272) could be used to overcome resistance in trastuzumab resistant BT474 and SKBR3 cell lines and to look at the effects of neratinib on trastuzumab naive cell lines. The effects of acute trastuzumab, neratinib, and trastuzumab and neratinib combination treatments were investigated in SKBR3 and BT474 cells. It has already been established that trastuzumab does not abolish pHER2 expression and this has been implicated in trastuzumab resistance. Western blotting showed an increase in pHER2 and pERK with acute trastuzumab treatment. Conversely, acute neratinib treatment alone and in combination with trastuzumab reduced pERK and pHER2 expression. Furthermore, although acute treatment with trastuzumab decreased the expression of pHER3 and pAKT, the combination of neratinib with trastuzumab gave a much greater decrease in expression. Fluorescence microscopy was also performed on both the resistant and native SKBR3 cells after acute trastuzumab, neratinib and combination treatments. Trastuzumab treatment increased pHER2 membrane localisation in both native and resistant cells in a dose dependent manner, whilst neratinib treatment or its combination with trastuzumab reduced membrane pHER2. Cell viability experiments on BT474 and SKBR3 cells showed that neratinib treatment was much more effective at reducing cell viability than trastuzumab(p=0.0078, p=0.0499) and that combined trastuzumab and neratinib treatment was more effective at reducing cell viability than neratinib alone (p=0.0028, p=0.0148). In the trastuzumab resistant SKBR3 and BT474 cells, withdrawal of trastuzumab lead to an increase in cell viability. However, neratinib treatment was able to reverse trastuzumab resistance and significantly reduce cell viability (p=0.0022, p=0.0087). Furthermore, combined treatment with trastuzumab and neratinib was more effective at reducing cell viability than neratinib alone(p=0.0087, p=0.0260) suggesting that trastuzumab treatment should be continued even when resistance develops. These results showed that the combined treatment with neratinib and trastuzumab was more effective in the inhibition of pHER2, pHER3, pAkt and pERK, and led to a greater decrease in cell viability than trastuzumab treatment alone. We would therefore recommend neratinib to be used in combination with trastuzumab in both trastuzumab naive and resistant HER2 over-expressing breast cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1737. doi:10.1158/1538-7445.AM2011-1737

Abstract 2235: FRET assay to assess EGFR/HER2 dimerization in cancer cell lines

Expression level of the HER family is unreliable as a predictive marker for the outcome of targeted therapies in cancer. Only a third of HER2 positive breast cancer patients respond to trastuzumab monotherapy. Thus there is significant need for the development of new biomarkers and the role of dimerization may prove a more effective predictor than expression itself. It has been shown that specific HER dimerization pairs are important. However, there has been no protocol proposed which can accurately assess dimerization to facilitate the development of an assay viable for use in a clinical setting. Previous attempts have either been based upon transfection of proteins whose interactions can be monitored which would not seem to be directly applicable to in-situ monitoring or do not explicitly separate true dimerization from mere colocalization. Our approach also allows intracellular localization to be visualized, this is significant as nuclear translocation of EGFR can lead to a number of signaling events. Utilizing measurement of Foerster Resonance Energy Transfer (FRET) to assess distances of the order 7 nm between receptors we can quantify dimerization. Our secondary conjugation technique uses anti-mouse and anti-rabbit primary antibodies and species specific dye-conjugated secondary antibodies, with Alexa488 as the donor and Alexa546 as the acceptor dye. An increase in dimerization results in increased occurrence of FRET which can be measured as a reduction in donor fluorescence lifetime. We have validated our assay for dimerization of EGFR and HER2 in 3 cell lines, BT474, SKBr3 and A431, and furthermore in paraffin embedded cell pellets. Comparison of preparations with donor antibodies alone with those containing donor and acceptor we demonstrate basal EGFR/HER2 dimerization levels are present (p We then applied the assay to monitor response to targeted therapies, the HER2 specific monoclonal antibodies, trastuzumab and pertuzumab. Trastuzumab is seen to decrease lifetime while dimerization inhibitor pertuzumab produces an increase. This is consistent with findings using biochemical methods. Finally, we applied and validated our assay in paraffin embedded cell pellets. These data demonstrate the robustness of our assay and the potential clinical application to paraffin-embedded tissue microarrays in order to assess the contribution of HER signaling in-situ. The assay represents a proof of principle, from here this same technique could be applied to other dimerization pairs. This will allow the prognostic potential of dimerization to be assessed and used to inform treatment regimes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2235. doi:10.1158/1538-7445.AM2011-2235

Abstract 531: HER4 cleavage and nuclear localization mediate Trastuzumab resistance in HER2-positive breast cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background. Trastuzumab is used in HER2 positive breast cancer patients and has been shown to increase survival in these patients. However, drug resistance imposes a crucial limitation to the successful treatment of HER2 positive breast cancer. Multiple mechanisms have been proposed but the role of HER4 in relation to Trastuzumab treatment and resistance remains unclear. This study aims to investigate the role of HER4 in relation to Trastuzumab treatment and resistance in HER2 positive breast cancer. Methods. The effects of Trastuzumab on the protein and mRNA levels of HER4 were studied in both naive and Trastuzumab resistant cells using western blot and RT-PCR. HER4 localisation after Trastuzumab treatment was determined by confocal microscopy and nuclear fractionation. A gamma-secretase inhibitor (GSI) and a pan-HER kinase inhibitor, Neratinib, were used to inhibit HER4 cleavage during Trastuzumab treatment. The role of HER4 was further validated using siRNA experiments. HER4 level and its localisation were also assessed by IHC in BT474 xenograft samples treated with Trastuzumab or control. To understand the prognostic role of HER4, TMAs of a cohort of HER2 positive breast cancer patients were stained for HER4 expression using IHC and the levels were correlated with patients’ outcome. Results. The basal level of HER4 in HER2 positive SKBR3 and BT474 is comparable to HER2 negative MCF7 cells. In SKBR3 and BT474, Trastuzumab treatment upregulated HER4 mRNA and protein expression in a dose responsive manner. Moreover, HER480kDa was also increased and an enhanced nuclear localisation was observed, indicating that Trastuzumab promotes HER4 cleavage. Resistant cells appear to have more nuclear HER4 and Trastuzumab withdrawal causes a decrease in nuclear intensity. HER4 downregulation via siRNA reduces cell viability in Trastuzumab sensitive and resistant cells. In SKBR3, GSI decreased HER4 nuclear staining and decreased Trastuzumab induced HER4 nuclear translocation. In addition, GSI also enhanced response to Trastuzumab in decreasing cell viability. Neratinib also blocks HER480kDa formation induced by Trastuzumab, and combination treatment of Trastuzumab and Neratinib had an additive inhibition effect on cell viability. There was a significant upregulation of nuclear HER4 in a Trastuzumab treated BT474 xenograft model compared to the control. Nuclear HER4 was also prognostic in a cohort of HER2 positive breast cancer patients. Conclusion. This study shows that Trastuzumab upregulates HER4 in vitro and in vivo. Nuclear HER4 seems to mediate Trastuzumab resistance in HER2 positive breast cancer cells. HER4 knockdown as well as drug treatments that decrease nuclear HER4 may enhance sensitivity to Trastuzumab. We believe that the prognostic and predictive values of nuclear HER4 in relation to Trastuzumab resistance need to be further validated in HER2 positive breast cancer patients. Citation Format: Siti Norasikin Mohd Nafi, Merel Gijsen, Ioannis Roxanis, Gabriela Kramer-Marek, Jacek Capala, Anthony Kong. HER4 cleavage and nuclear localization mediate Trastuzumab resistance in HER2-positive breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 531. doi:10.1158/1538-7445.AM2013-531

Abstract 921: HER3 reactivation: A compensatory shift between Akt-mediated positive and negative feedback loops in HER2-overexpressed human breast cancer cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The activated serine/threonine protein kinase Akt (or Protein Kinase B, PKB) regulates cell survival and metabolism by phosphorylation of numerous adaptor proteins and kinases. Akt is known as a key downstream target of HER3 signaling axis. It is also suggested to be an important modulator for the HER3 reactivation via a compensatory shift in the HER3 phosphorylation-dephosphorylation equilibrium during TKI-induced drug resistance. However, the mechanism of Akt-mediated HER3 reactivation during TKI treatment is still unclear. Interestingly, HER3 was rapidly dephosphorylated when an Akt inhibitor (Akt inhibitor VIII, Akti) was applied to mimic the inhibitory effect of TKI on the PI3K/Akt axis. The same phenomenon was also found when PI3K inhibitor was utilized instead. This positive feedback within the HER3 signaling axis was shown to be mediated by PTPN9, a phosphatase that is known to negatively regulate the EGFR activation state. We showed that PTPN9 was upregulated once Akt was inhibited. This lead to EGFR deactivation and eventually caused the dephosphorylation of HER3, which was reversed when PTPN9 was locked down. Furthermore, reactivation of HER3 was also found in the Akti-treated cells at 24h. This was shown to be due to a negative feedback loop via the ADAM17-mediated heregulin release, which could be suppressed by ADAM17 inhibitor INCB4298. Our results are crucial in advancing our knowledge in HER3/PI3K/Akt axis, where the positive loop revealed the mechanism of Akt suppression in regulating HER3 phosphorylation; and the negative loop is implicated in resistance mechanisms to various targeted therapies that affect Akt activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 921. doi:10.1158/1538-7445.AM2011-921

Abstract 5362: The role of adam10 in trastuzumab resistance of her2 positive breast cancer cell lines

Members of a disintegrin and metalloproteases (ADAMs) are responsible for shedding of a variety of ErbB ligands. One of them, ADAM10, has been shown to cleave several pro-ligands, including betacellulin and HB-EGF. ADAM10 further may play a role in trastuzumab resistance since it is also responsible for HER2 shedding, thereby removing the binding site for trastuzumab. It was reported that ADAM10 inhibitor INCB 7839 could reduce HER2 cleavage in patients with HER2 positive breast cancer. However, a direct correlation of trastuzumab treatment and ADAM10 and the precise role of ADAM10 in trastuzumab resistance have not been elucidated. The aim of this project is therefore to investigate the role of ADAM10 in trastuzumab treatment and resistance in HER2 positive breast cancer cell lines. Using western blot, we showed that one hour of trastuzumab treatment leads to an upregulation of ADAM10 in conditioned medium and cell lysates of BT474 cells, which was correlated with an elevated level of betacellulin. Additionally, the use of a specific ADAM10 inhibitor in turn leads to downregulation of betacellulin levels in culture medium. Whereas one hour of trastuzumab treatment could not decrease pHER2, its combination with ADAM10 inhibitor reduced pHER2 level. To further investigate the role of ADAM10 in relation to trastuzumab treatment, we used cell viability studies to assess the effect of a specific ADAM10 inhibitor with or without trastuzumab treatment. Although monotherapy with ADAM10 inhibitor only had minimal influence, combining it with trastuzumab significantly enhanced trastuzumab in reducing cell proliferation of BT474 cells. Interestingly, the addition of ADAM10 inhibitor to trastuzumab exerts an even greater inhibition of cell proliferation in the BT474 trastuzumab resistant cell line. Since the N-domain of ADAM10 was shown to be cleaved by ADAM9 in other cell lines, we also assessed ADAM9 level in response to trastuzumab treatment. In BT474 lysates, ADAM9 level was increased after one hour of treatment, correlating with increased ADAM10 level in the medium. ADAM9 level was also found to be increased in the trastuzumab-resistant HER2 positive cell line in comparison to the sensitive cell line. Thus, ADAM10 as well as ADAM9 may play a key role in trastuzumab resistance in HER2 positive breast cancer cells. Our results suggest that the increased level of ADAM10 results in HER ligand sheddase which in turn may lead to the activation of HER receptors and the maintenance of pHER2. We demonstrated that ADAM10 inhibitor in combination with trastuzumab is able to decrease pHER2 and potentiate the trastuzumab effect in HER2 positive breast cancer cells. The role of ADAM9 in its regulation of ADAM10 in relation to trastuzumab treatment is currently under investigation. We recommend that inhibition of ADAM10 should be considered as a potential strategy to improve trastuzumab response in HER2 positive breast cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5362. doi:10.1158/1538-7445.AM2011-5362

Abstract 1530: Trastuzumab treatment induces ADAM17 and HER ligands in vivo

Anti-HER2 monoclonal antibody trastuzumab is used as treatment in patients with HER2 positive breast cancer. However, resistance inevitably occurs for all responders if trastuzumab is given as monotherapy. Although trastuzumab was developed to block HER2 signaling, several studies have shown that trastuzumab does not decrease HER2 phosphorylation. In a recent study, we have shown that in BT474 and SKBR3 cells HER2 phosphorylation is maintained by activation of the other HER receptors via their dimerization with HER2. The activation of alternative HER receptors is due to an increase of HER ligand release, which is mediated by a negative feedback loop involving PKB and ADAM17. In this study, we investigated the effect of trastuzumab treatment in vivo. Our hypothesis was that the described PKB feedback loop could explain differences in response to trastuzumab in vivo. Resistant xenograft samples may have higher levels of ADAM17 and HER ligands compared to responding xenograft samples, and thus maintaining their HER2 signaling. To study this hypothesis, BT474 xenografts were treated with five doses of trastuzumab or were left to grow untreated. The xenograft samples were then paraffin-embedded and analysed by immunohistochemisty. We scored the samples for HER2 membrane and cytoplasmic levels using an Intensity Percentage Scoring (IPS) system. Although, membrane HER2 levels were significantly reduced after trastuzumab treatment, there was a lot of heterogeneity between samples. We hypothesized that differences in drug deliver could be an explanation for this heterogeneity, so we co-stained for HER2 and CD31 (a blood vessel marker). Ten different parts of one sections were then independently scored for HER2 levels and blood vessel count. A lower membrane HER2 staining was found to correlate with a higher number of blood vessels. This finding suggests that HER2 membrane levels are downregulated when trastuzumab is delivered to the tissue. Drug delivery is therefore very important for an effective response to trastuzumab treatment. In line with our previously published data, staining for ADAM17, HER ligand heregulin, phospho-EGFR and phospho-HER3 receptors was increased in trastuzumab treated xenografts compared to untreated samples. Interestingly, in our preliminary results not all of the trastuzumab treated samples showed this increase, indicating a possible difference in response to treatment. In future, comparing xenograft size to the staining intensity of these proteins should give us an understanding of the difference in response. These in vivo findings are in line with our in vitro findings and offer treatment opportunities for overcoming resistance in patients. We propose that trastuzumab should be combined with a panHER inhibitor or an ADAM inhibitor to overcome the acquired drug resistance for HER2 positive breast cancer patients. A similar strategy could help overcome resistance to other therapies targeting this pathway Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1530. doi:10.1158/1538-7445.AM2011-1530