HER3 p95HER2 and HER2 protein expression levels

Authors: Allan Lipton Laurie Goodman Kim Leitzel Jennifer Cook Jeff Sperinde Mojgan Haddad Wolfgang J Köstler Weidong Huang Jodi M Weidler Suhail Ali Alicia Newton EvaMarie Fuchs Agnes Paquet Christian F Singer Reinhard Horvat Xueguang Jin Joyee Banerjee Ali Mukherjee Yuping Tan Yining Shi Ahmed Chenna Jeff Larson Yolanda Lie Thomas Sherwood Christos J Petropoulos Stephen Williams John Winslow Gordon Parry Michael Bates

Publish Date: 2013/08/20

Volume: 141, Issue: 1, Pages: 43-53

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Abstract

Trastuzumab is effective in the treatment of HER2/neu overexpressing breast cancer but not all patients benefit from it In vitro data suggest a role for HER3 in the initiation of signaling activity involving the AKT–mTOR pathway leading to trastuzumab insensitivity We sought to investigate the potential of HER3 alone and in the context of p95HER2 p95 a trastuzumab resistance marker as biomarkers of trastuzumab escape Using the VeraTag® assay platform we developed a dual antibody proximitybased assay for the precise quantitation of HER3 total protein H3T from formalinfixed paraffinembedded FFPE breast tumors We then measured H3T in 89 patients with metastatic breast cancer treated with trastuzumabbased therapy and correlated the results with progressionfree survival and overall survival using Kaplan–Meier and decision tree analyses that also included HER2 total H2T and p95 expression levels Within the subpopulation of patients that overexpressed HER2 high levels of HER3 and/or p95 protein expression were significantly associated with poor clinical outcomes on trastuzumabbased therapy Based on quantitative H3T p95 and H2T measurements multiple subtypes of HER2positive breast cancer were identified that differ in their outcome following trastuzumab therapy These data suggest that HER3 and p95 are informative biomarkers of clinical outcomes on trastuzumab therapy and that multiple subtypes of HER2positive breast cancer may be defined by quantitative measurements of H3T p95 and H2TTrastuzumab Herceptin® Genentech a humanized monoclonal antibody that targets the extracellular domain of the ErbB2 HER2 receptor is an effective treatment of both early and metastatic HER2positive breast cancer MBC particularly when used in combination with chemotherapy 1 2 3 4 5 6 However many patients treated with trastuzumab do not respond or experience disease recurrence despite disease classification as HER2positive by immunohistochemistry IHC or fluorescence in situ hybridization FISHMuch attention has been focused on improving the prediction of clinical benefit as well as overcoming the technical shortcomings of current diagnostic methods 7 8 9 10 It is plausible that clinical outcomes can be improved simply through better selection of patient candidates for trastuzumab by increasing the accuracy of HER2 assessments if the increased accuracy extends the treatment indication to cases which were otherwise excluded However assuming HER2 is the sole indicator of trastuzumab responsiveness ignores the complex biological context of the cell signaling system in which HER2 operatesHER2 functions as a partner with other HER receptor family members in the formation of heterodimers which initiate signaling through several key pathways that are known to drive proliferation and survival in epithelial cancers 11 12 Given the overlap and redundancy of cell signaling pathways the measurement of HER2 alone is likely insufficient to accurately characterize trastuzumab sensitivity for any given tumor While overexpression of HER2 protein may be necessary for trastuzumab activity in vivo the concomitant expression of other HERfamily receptors may provide opportunities for escape from trastuzumab antagonism via their role as dimerization partners of HER2 Unless methods are developed to measure additional biomarkers of trastuzumab response it will remain difficult to distinguish HER2positive tumors that are driven primarily by HER2 overexpression and thus may be highly susceptible to trastuzumab from HER2positive tumors that are driven by HER2 heterodimers eg HER2HER3 and may not be easily antagonized by trastuzumab 13Although studies suggest that ligandindependent HER2HER3 heterodimers represent a trastuzumabsensitive oncogenic driver in HER2positive breast cancer 14 15 one can hypothesize that the concomitant high expression of HER3 or its ligands in HER2positive tumors may confer escape from trastuzumab inhibition Recent data suggests that upregulation of a set of receptor tyrosine kinases including HER3 is pivotal in attenuating the antitumor effects of a number of inhibitors of the PI3K/AKT pathway 16 including those that act directly on HER2 17 18 Furthermore the downregulation of HER3 mRNA has been postulated as a surrogate for activation of HER2 dimerization 19 and HER3 mRNA levels have been correlated with clinical benefit in ovarian tumors treated with the HER2HER3 dimerizationinhibiting antibody pertuzumab 20 Lastly in vitro data suggest that heregulininduced HER2HER3 heterodimers mediate trastuzumab insensitivity by activating signaling through the AKT–mTOR pathway and that trastuzumab is ineffective in abrogating this signal 13 Recent clinical data demonstrating the enhanced efficacy in both early and metastatic HER2positive breast cancer with the addition of the heterodimerblocking HER2 antibody pertuzumab to trastuzumab is consistent with ligandactivated and possibly ligandindependent HER2HER3 signaling in HER2positive breast cancer 21 22In this study we sought to explore the association between HER3 protein expression and clinical outcome following trastuzumab therapy in a cohort of MBC patients with HER2 positive tumors We hypothesized that elevated HER3 protein expression in the setting of HER2 overexpression is a reasonable surrogate for the presence of HER2HER3 heterodimers given a correlation between the two measurements 23 24 and therefore may correlate with suboptimal response to trastuzumab HER3 is expressed at relatively low levels and is difficult to quantify by traditional IHC procedures in FFPE sections therefore we used the VeraTag platform 25 26 to develop a proximitybased assay for the precise quantitation of HER3 protein expression in FFPE specimens We then measured HER3 expression in a cohort of trastuzumabtreated MBC patients and performed statistical analyses to explore the relationship between HER3 expression and clinical outcomes following trastuzumabbased treatmentFinally having previously found a dependence of outcome on quantitative HER2 27 and p95 truncated HER2 levels 28 in the current cohort we further examined the relationship of HER3 expression with clinical outcome in the context of HER2 protein expression and p95 expression using regression tree analysis

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