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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.

For adult granulosa cell tumors (aGCTs), the preferred treatment modality is surgery. Chemotherapy and anti-hormonal therapy are also frequently used in patients with recurrent aGCT. We aimed to review the existing literature on the response to chemotherapy and anti-hormonal therapy in patients with aGCT. Embase and MEDLINE were searched from inception to November 2021 for eligible studies. Objective response rate (ORR) was calculated as the total number of cases with a complete response (CR) or a partial response (PR). Disease control rate (DCR) was defined as the sum of cases with CR, PR or stable disease (SD). A total of 10 studies were included that reported on chemotherapy and 13 studies were included that reported on anti-hormonal therapy. The response rates of the 56 chemotherapy regimens that could be evaluated resulted in an ORR of 30% and DCR of 58%. For anti-hormonal therapy, the results of 73 regimens led to an ORR of 11% and a DCR of 66%. Evidence on systemic therapy in aGCT only is limited. For both chemotherapy and anti-hormonal therapy, the ORR is limited, but the response is considerably higher when patients achieving SD are included. New approaches are needed to provide more evidence and standardize treatment in aGCT.

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.

Background Subsets of high grade endometrial cancer (EnCa) over-express HER2 (ERBB2), yet clinical trials have failed to demonstrate any anti-tumor activity utilizing trastuzumab, an approved platform for HER2 positive breast cancer (BrCa). A truncated p95HER2 variant lacking the trastuzumab binding site may confer resistance. The objective of this investigation was to characterize the expression of the p95HER2 truncated variant in EnCa. Materials and methods With institutional approval, 86 high grade EnCa tumors were identified with tumor specimens from surgeries performed between 2000 and 2011. Clinical data were collected and all specimens underwent tumor genotyping, HER2 immunohistochemistry (IHC, HercepTest®), HER2 fluorescent in situ hybridization (FISH), along with total HER2 (H2T) and p95HER2 assessment with VeraTag® testing. Regression models were used to compare a cohort of 86 breast tumors selected for equivalent HER2 protein expression. Results We identified 44 high grade endometrioid and 42 uterine serous carcinomas (USC). IHC identified high HER2 expression (2+ or 3+) in 59% of the tumors. HER2 gene amplification was observed in 16 tumors (12 USC, 4 endometrioid). Both HER2 gene amplification and protein expression correlated with H2T values. High p95HER2 expression above 2.8RF/mm 2 was observed in 53% ( n =54) with significant correlation with H2T levels. When matched to a cohort of 107 breast tumors based on HercepTest HER2 expression, high grade EnCa presented with higher p95 levels ( p Conclusions These data demonstrate that compared to BrCa, high grade EnCa expresses higher levels of p95HER2 possibly providing rationale for the trastuzumab resistance observed in EnCa.

Uterine serous carcinoma (USC) is an aggressive subtype of endometrial cancer that commonly harbors HER2 gene amplification. Clinical trial has demonstrated that USC is impervious to trastuzumab therapy, though the mechanism is poorly understood. Since HER3 mediated signaling has been implicated in trastuzumab resistance in breast cancer, we sought to understand the relevance of HER3 activation in USC xenografts derived from the HER2 gene amplified (HER2:Chr 17 > 15) non-immortalized USC cell line ARK2. Cohorts of mice harboring xenografts derived from ARK2 were treated with either vehicle, trastuzumab (10 mg/kg IP BIW), lapatinib (150 mg/kg QD oral gavage) or the combination of trastuzumab and lapatinib for 21 days. Acute and chronic post treatment tumor samples were assessed for downstream signaling alterations. Single agent trastuzumab had no impact on xenograft growth compared to vehicle. Lapatinib alone resulted in significant tumorstatic effects (p < 0.01) and dual therapy with trastuzumab and lapatinib induced synergistic activity that significantly decreased tumor volume compared to all other arms (p < 0.01). Single agent trastuzumab was associated with rapid elevation in pHER3 levels with unchanged pAKT and pERK expression compared to vehicle. These elevated pHER3 levels were similarly elevated after the 21 day treatment. Anti-tumor activity observed in the lapatinib and dual lapatinib/trastuzumab arm was not associated with any alterations in pHER3 levels following 21 day treatment, though acute elevations in pHER3 were noted in the dual HER2 blockade arm at 24 hours after treatment. In conclusion, trastuzumab alone failed to impact USC xenograft growth and this innate resistance was associated with an elevation in pHER3 levels that was still evident in xenografts at the end of the treatment period. Unlike single agent trastuzumab, treatment arms that utilized lapatinib demonstrated significant anti-tumor activity with no increase in pHER3 above vehicle over the course of treatment. These data highlight HER3 activation as a possible trastuzumab resistance mechanism in USC that can be abrogated through the addition of lapatinib. Citation Format: Silvia F. Hernandez, Celeste DiGloria, Jolijn Groeneweg, Darrell Borger, Rosemary Foster, Bo Rueda, Whitfield Growdon. Phosphorylated HER3 levels associated with trastuzumab resistance in HER2 gene amplified uterine serous carcinoma xenograft tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 658. doi:10.1158/1538-7445.AM2015-658