Hyperactivation of the PI3K-AKT Pathway Commonly Underlies Resistance to Trastuzumab in HER2 Amplified Breast Cancer.
Authors: Chandarlapaty S. , King T. , Sakr R. , Barbashina V. , Arroyo N. , Morrogh M. , Wynveen C. , Shanu M. , Norton L. , Rosen N. , Hudis C. 
Published: December 2010

Abstract
BACKGROUND: Targeted therapy of HER2 amplified breast cancers with Trastuzumab has resulted in a survival benefit for patients with this disease. However, de novo and acquired resistance to Trastuzumab is commonly encountered. Many laboratory models of Trastuzumab resistant cancer have been developed and suggest that hyperactivation of the HER3-PI3K-AKT pathway downstream of HER2 may enable resistance. It has also been suggested that changes to HER2 itself, such as through loss of HER2 amplification or induction of p95-HER2 expression, may mediate resistance. A comprehensive in vivo assessment of multiple mechanisms of resistance has yet to be reported. Moreover, it is not known if these lesions are always present in pretreatment tumor samples or can be acquired over time. The aim of this study was to identify which mechanisms of resistance are present in human tumor samples after progression of disease on a Trastuzumab containing regimen. METHODS: Patients with HER2 amplified breast cancer with progression of disease by RECIST criteria on a Trastuzumab containing regimen were enrolled on an IRB approved protocol to obtain biopsies of progressive tumors. One tumor biopsy was flash frozen for nucleic acid studies and one biopsy preserved in formalin. For the pre-Trastuzumab comparison, archival formalin-fixed paraffin embedded (FFPE) blocks were obtained. Manual microdissection was performed on the frozen blocks to isolate tumor-containing sections for DNA isolation. PIK3CA hotspot mutations were identified using the Sequenom MassARRAY system. Following antibody optimization, IHC staining for HER3, PTEN and P-AKT (S473) was performed on freshly cut sections. Stained sections were scored by two, independent reviewing pathologists. HER3, PTEN and P-AKT expression were scored relative to staining in adjacent normal ductal epithelium. Staining and scoring for ER, PR, and HER2 was performed in CLIA-certified clinical laboratory. RESULTS: 19 patients have been enrolled and completed biopsy on protocol. Changes in HER2 overexpression status from the pretreatment biopsy to the post-progression biopsy were noted in 2/15 evaluated samples, with the remaining cases demonstrating stability. PTEN expression was diminished or absent in four cases. Activating mutations in hotspot regions of PIK3CA were identified in 3/10 cases. Enhanced expression of the HER3 receptor was identified in 4/11 cases with 2 of the cases showing an increase in expression compared to the pretreatment sample. CONCLUSIONS: HER2 overexpression status infrequently changed from the pre-Trastuzumab to post-Trastuzumab biopsy. Abnormalities in HER3, PI3K, and PTEN were not coexistent (e.g. PTEN loss and PI3K mutation were not seen in the same samples) in most cases. In some instances, the pretreatment tumor sample did not demonstrate lesions that were found on the post-progression sample, suggesting the possibility that molecular lesions may arise in response to therapy. Finally, in most cases, an abnormality in HER3, PI3K, or PTEN was identified implying a critical role for this pathway in mediating resistance to Trastuzumab.
Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 709.