Genomic DNA was isolated (10 to 100 ng) and sequenced using the BigDye Terminator Cycle Sequencing Kit and an ABI 3730 automated capillary sequencer (Applied Biosystems, Foster City, CA). == Statistical Methods == Patient and tumor characteristics were summarized by descriptive statistics. Under low PTEN conditions, in vitro data indicate that lapatinib alone and in combination with trastuzumab was effective in decreasing p-MAPK and p-AKT levels, whereas trastuzumab was ineffective. In the clinical trials, we confirmed that low PTEN or activating mutation inPIK3CAconferred resistance to the trastuzumab regimen (P= .015), whereas low PTEN tumors were associated with a high pathologic complete response rate (P= .007). == Conclusion == Activation of PI3 kinase pathway is associated with trastuzumab resistance, whereas low PTEN predicted for response to lapatinib. These observations support clinical trials with the combination of both agents. == INTRODUCTION == The epidermal growth factor receptor (EGFR)/human epidermal growth factor receptor (HER) family of transmembrane type I receptor tyrosine kinases plays an important role in processes such as cell proliferation, differentiation, and survival. Conformational changes after receptor dimerization lead to autophosphorylation and initiation of divergent signal transduction cascades.1These type I receptors Diatrizoate sodium signal through the MAPK/ERK pathway, stimulating cell division.2Cell line evidence also suggests that these receptors also modulate cell survival through activation of the AKT/phosphoinositol-3 kinase (PI3K) pathway.3Aberrant HER1 and HER2 signaling contributes to cancer cell proliferation and survival. The HER2 monoclonal antibody, trastuzumab, has been approved as adjuvant treatment for patients with breast cancer with HER2 overexpression, reducing both the recurrence rate and mortality.4Lapatinib, a reversible dual kinase inhibitor against EGFR and HER2,5has activity in patients with HER2 overexpression when given either as first-line therapy or after treatment failure with trastuzumab68and has been approved in combination with capecitabine Diatrizoate sodium in patients with metastatic disease, with significantly improved progression-free survival.9Recent data have also shown that the addition of lapatinib to letrozole almost tripled progression-free survival rates in patients Diatrizoate sodium with breast cancer whose tumors coexpressed steroid receptors and HER2.10 The antitumor effects of HER2 inhibitors require the modulation of key signaling pathways and cell cycle/apoptosis regulatory molecules that mediate the transforming effects of HER2.1113Activation of the PI3K pathway, as a result of loss or low levels of the phosphatase and tensin homolog (PTEN), is associated with resistance to trastuzumab. Recent data further Diatrizoate sodium support the observation that activation of the PI3K pathway byPIK3CAmutation or loss of PTEN is associated with resistance to trastuzumab.14Mechanisms for lapatinib are less well established. Recent in vitro data suggest that, unlike trastuzumab, loss of PTEN function is not associated with lapatinib resistance.15Previously, we observed in repeat biopsies of human primary breast cancers that blocking activation of the PI3K/AKT survival pathway is the main mechanism of action of trastuzumab.16The objectives of this study were to expand on these earlier observations and, first, to define cellular mechanisms of action of trastuzumab and lapatinib in cell lines and in clinical human biopsy samples and, second, to define possible predictive markers of response, especially in the PI3K/AKT pathway. As such, we evaluated the effect of lapatinib or trastuzumab given alone or in combination in HER2-overexpressing cell lines. We confirmed our in vitro observations in two sequential neoadjuvant clinical trials in patients with HER2-overexpressing locally advanced breast cancer, for which repeat biopsies were analyzed for involvement of either the MAPK/ERK or PI3K/AKT pathway. The results of the first neoadjuvant trastuzumab study have been reported16and are reiterated in this current analysis for comparison with the results from the lapatinib trial. == METHODS == == In Vitro Cell Culture Transfections and Treatments == SKBR3 cells were grown in McCoy’s Media (Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (Cellgro, Manassas, VA) and 1 penicillin/streptomycin, and BT474 cells were grown in DMEM Glutamax Media (Invitrogen) with 10% fetal bovine serum and 1 penicillin/streptomycin. The cells were then transfected with 50 nmol/L of mock or PTEN short interfering Rabbit polyclonal to ACSS3 RNA (siRNA; Dharmacon, Lafayette, CO) for 24 hours using Dhramfect (Dharmacon), according to the manufacturer’s instruction, and treated in the following four groups: vehicle control; lapatinib.