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Supplementary Figure 4. Ki-67 staining of non-HER2 gene amplified USC xenografts. Unlike the HER2 amplified models, no changes in proliferation were seen upon treatment with lapatinib and/or trastuzumab in SPEC2 (A) and EnCa2 (B) xenografts. Tumors harvested at the completion of each in vivo treatment study were subjected to Ki-67 immunohistochemical analysis. Scale bars: 100 µm.

Purpose: Uterine serous carcinoma (USC) is an aggressive subtype of endometrial cancer that commonly harbors HER2 gene amplification. We investigated the effectiveness of HER2 inhibition using lapatinib and trastuzumab in vitro and in xenografts derived from USC cell lines and USC patient-derived xenografts.Experimental Design: Immunohistochemistry and FISH were performed to assess HER2 expression in 42 primary USC specimens. ARK1, ARK2, and SPEC2 cell lines were treated with trastuzumab or lapatinib. Cohorts of mice harboring xenografts derived from ARK2 and SPEC2 cell lines and EnCa1 and EnCa2 primary human USC samples were treated with either vehicle, trastuzumab, lapatinib, or the combination of trastuzumab and lapatinib. Acute and chronic posttreatment tumor samples were assessed for downstream signaling alterations and examined for apoptosis and proliferation.Results:HER2 gene amplification (24%) correlated significantly with HER2 protein overexpression (55%). All models were impervious to single-agent trastuzumab treatment. Lapatinib decreased in vitro proliferation of all cell lines and in vivo growth of HER2-amplified xenografts (ARK2, EnCa1). In addition, dual therapy with trastuzumab and lapatinib resulted in significant antitumor activity only in ARK2 and EnCa1 tumors. Dual HER2 therapy induced on target alteration of downstream MAPK and PI3K pathway mediators only in HER2-amplified models, and was associated with increased apoptosis and decreased proliferation.Conclusions: Although trastuzumab alone did not impact USC growth, dual anti-HER2 therapy with lapatinib led to improved inhibition of tumor growth in HER2-amplified USC and may be a promising avenue for future investigation. Clin Cancer Res; 20(24); 6517–28. ©2014 AACR.

Supplementary Table 1. Clinical characteristics of the 42 USC patients whose tissue specimens were utilized for HER2 immunohistochemical and FISH analyses.

Supplementary Figure 3. Mouse weight changes over the course of each four-arm treatment study. In all experiments, lapatinib and trastuzumab treatments did not significantly affect mouse weights. Mice were weighed weekly, and the percent changes in weight compared to baseline weights (100%) are shown. Error bars represent the standard error of the mean.

Supplementary Figure 2. Anti-tumor activity of single agent lapatinib treatment was observed in HER2 amplified ARK2 xenografts (p = 0.02), while no response was seen in non-HER2 amplified SPEC2 xenografts. Mice harboring tumors derived from ARK2 or SPEC2 cell lines were treated with either vehicle or lapatinib. Tumors were measured twice weekly, and the percent change in tumor volume compared to baseline volume (100%) is shown. Error bars represent the standard error of the mean.

Supplementary Figure Legends. The supplementary figure legends are in this document as requested.