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Acquired resistance to lapatinib, an inhibitor of EGFR and HER2 kinases, is common. We found that reactivation of EGFR, HER2 and HER3 occurred within 24 hours of lapatinib treatment after their initial dephosphorylation. This was associated with increased expression of NRG1 in cells treated with lapatinib. Exogenous NRG1 partially rescued breast cancer cells from growth inhibition by lapatinib. In addition, both parental and lapatinib-resistant breast cancer cells were sensitive to SGP1, which inhibits binding of NRG1 and other HER3 ligands. Addition of pertuzumab to lapatinib further inhibited NRG1-induced signalling, which was not fully inhibited by either drug alone. In animal model, a combination of pertuzumab to lapatinib induced a greater tumor regression than either lapatinib or pertuzumab monotherapy. This novel combination treatment may provide a promising strategy in clinical HER2-targeted therapy and may inhibit a subset of lapatinib-resistant breast cancer, although the group of patients that will respond to this therapy requires further stratification.

Trastuzumab prolongs survival in HER2 positive breast cancer patients. However, resistance remains a challenge. We have previously shown that ADAM17 plays a key role in maintaining HER2 phosphorylation during trastuzumab treatment. Beside ADAM17, ADAM10 is the other well characterized ADAM protease responsible for HER ligand shedding. Therefore, we studied the role of ADAM10 in relation to trastuzumab treatment and resistance in HER2 positive breast cancer. ADAM10 expression was assessed in HER2 positive breast cancer cell lines and xenograft mice treated with trastuzumab. Trastuzumab treatment increased ADAM10 levels in HER2 positive breast cancer cells (p ≤ 0.001 in BT474; p ≤ 0.01 in SKBR3) and in vivo (p ≤ 0.0001) compared to control, correlating with a decrease in PKB phosphorylation. ADAM10 inhibition or knockdown enhanced trastuzumab response in naïve and trastuzumab resistant breast cancer cells. Trastuzumab monotherapy upregulated ADAM10 (p ≤ 0.05); and higher pre-treatment ADAM10 levels correlated with decreased clinical response (p ≤ 0.05) at day 21 in HER2 positive breast cancer patients undergoing a trastuzumab treatment window study. Higher ADAM10 levels correlated with poorer relapse-free survival (p ≤ 0.01) in a cohort of HER2 positive breast cancer patients. Our studies implicate a role of ADAM10 in acquired resistance to trastuzumab and establish ADAM10 as a therapeutic target and a potential biomarker for HER2 positive breast cancer patients.

Trastuzumab has been shown to improve the survival outcomes of HER2 positive breast cancer patients. However, a significant proportion of HER2-positive patients are either inherently resistant or develop resistance to trastuzumab. We assessed the effects of neratinib, an irreversible panHER inhibitor, in a panel of 36 breast cancer cell lines. We further assessed its effects with or without trastuzumab in several sensitive and resistant breast cancer cells as well as a BT474 xenograft model. We confirmed that neratinib was significantly more active in HER2-amplified than HER2 non-amplified cell lines. Neratinib decreased the activation of the 4 HER receptors and inhibited downstream pathways. However, HER3 and Akt were reactivated at 24 hours, which was prevented by the combination of trastuzumab and neratinib. Neratinib also decreased pHER2 and pHER3 in acquired trastuzumab resistant cells. Neratinib in combination with trastuzumab had a greater growth inhibitory effect than either drug alone in 4 HER2 positive cell lines. Furthermore, trastuzumab in combination with neratinib was growth inhibitory in SKBR3 and BT474 cells which had acquired resistance to trastuzumab as well as in a BT474 xenograft model. Innately trastuzumab resistant cell lines showed sensitivity to neratinib, but the combination did not enhance response compared to neratinib alone. Levels of HER2 and phospho-HER2 showed a direct correlation with sensitivity to neratinib. Our data indicate that neratinib is an effective anti-HER2 therapy and counteracted both innate and acquired trastuzumab resistance in HER2 positive breast cancer. Our results suggest that combined treatment with trastuzumab and neratinib is likely to be more effective than either treatment alone for both trastuzumab-sensitive breast cancer as well as HER2-positive tumors with acquired resistance to trastuzumab.

Unlabelled: 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-(18)F-fluorobenzamido)ethyl]maleimide ((18)F-FBEM)-HER(2:342) 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 (18)F-FBEM-HER(2:342) 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.