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Representative images of H&E, KIT and DOG1 staining performed in the xenograft models.

PDF file, 289KB, Western immunoblots and their densitometric analyses showing the pharmacodynamic response of GIST430 xenograft to 22 days of AT13387, imatinib or combination treatment.

PDF file, 56KB, Graphs showing the typical dose response of GIST882, GIST430 and GIST48 cell lines to AT13387 and imatinib.

Representative images of H&E, KIT and DOG1 staining performed in the xenograft models

The majority of gastrointestinal stromal tumors (GIST) are characterized by activating mutations of KIT, an HSP90 client protein. Further secondary resistance mutations within KIT limit clinical responses to tyrosine kinase inhibitors, such as imatinib. The dependence of KIT and its mutated forms on HSP90 suggests that HSP90 inhibition might be a valuable treatment option for GIST, which would be equally effective on imatinib-sensitive and -resistant clones. We investigated the activity of AT13387, a potent HSP90 inhibitor currently being evaluated in clinical trials, in both in vitro and in vivo GIST models. AT13387 inhibited the proliferation of imatinib-sensitive (GIST882, GIST-T1) and -resistant (GIST430, GIST48) cell lines, including those resistant to the geldanamycin analogue HSP90 inhibitor, 17-AAG. Treatment with AT13387 resulted in depletion of HSP90 client proteins, KIT and AKT, along with their phospho-forms in imatinib-sensitive and -resistant cell lines, irrespective of KIT mutation. KIT signaling was ablated, whereas HSP70, a marker of HSP90 inhibition, was induced. In vivo, antitumor activity of AT13387 was showed in both the imatinib-sensitive, GIST-PSW, xenograft model and a newly characterized imatinib-resistant, GIST430, xenograft model. Induction of HSP70, depletion of phospho-KIT and inhibition of KIT signaling were seen in tumors from both models after treatment with AT13387. A combination of imatinib and AT13387 treatment in the imatinib-resistant GIST430 model significantly enhanced tumor growth inhibition over either of the monotherapies. Importantly, the combination of AT13387 and imatinib was well tolerated. These results suggest AT13387 is an excellent candidate for clinical testing in GIST in combination with imatinib. Mol Cancer Ther; 11(8); 1799–808. ©2012 AACR.

Purpose: Oncogenic signaling in gastrointestinal stromal tumors (GIST) is sustained via PI3K/AKT pathway. We used a panel of six GIST xenograft models to assess efficacy of GDC-0941 as single agent or in combination with imatinib (IMA).Experimental Design: Nude mice (n = 136) were grafted bilaterally with human GIST carrying diverse KIT mutations. Mice were orally dosed over four weeks, grouped as follows: (A) control; (B) GDC-0941; (C) imatinib, and (D) GDC+IMA treatments. Xenografts regrowth after treatment discontinuation was assessed in groups C and D for an additional four weeks. Tumor response was assessed by volume measurements, micro-PET imaging, histopathology, and immunoblotting. Moreover, genomic alterations in PTEN/PI3K/AKT pathway were evaluated.Results: In all models, GDC-0941 caused tumor growth stabilization, inhibiting tumor cell proliferation, but did not induce apoptosis. Under GDC+IMA, profound tumor regression, superior to either treatment alone, was observed. This effect was associated with the best histologic response, a nearly complete proliferation arrest and increased apoptosis. Tumor regrowth assays confirmed superior activity of GDC+IMA over imatinib; in three of six models, tumor volume remained reduced and stable even after treatment discontinuation. A positive correlation between response to GDC+IMA and PTEN loss, both on gene and protein levels, was found.Conclusion: GDC+IMA has significant antitumor efficacy in GIST xenografts, inducing more substantial tumor regression, apoptosis, and durable effects than imatinib. Notably, after treatment withdrawal, tumor regression was sustained in tumors exposed to GDC+IMA, which was not observed under imatinib. Assessment of PTEN status may represent a useful predictive biomarker for patient selection. Clin Cancer Res; 19(3); 620–30. ©2012 AACR.

Introduction: The PI3K signaling pathway drives tumor cell proliferation and survival in gastrointestinal stromal tumor (GIST). We tested the in vivo efficacy of three PI3K inhibitors (PI3Ki) in patient-derived GIST xenograft models.Experimental Design: One hundred and sixty-eight nude mice were grafted with human GIST carrying diverse KIT genotypes and PTEN genomic status. Animals were dosed orally for two weeks as follows: control group (untreated); imatinib (IMA); PI3Ki (BKM120—buparlisib, BEZ235, or BYL719) or combinations of imatinib with a PI3Ki. Western blotting, histopathology, and tumor volume evolution were used for the assessment of treatment efficacy. Furthermore, tumor regrowth was evaluated for three weeks after treatment cessation.Results: PI3Ki monotherapy showed a significant antitumor effect, reflected in tumor volume reduction or stabilization, inhibitory effects on mitotic activity, and PI3K signaling inhibition. The IMA+PI3Ki combination remarkably improved the efficacy of either single-agent treatment with more pronounced tumor volume reduction and enhanced proapoptotic effects over either single agent. Response to IMA+PI3Ki was found to depend on the KIT genotype and specific model-related molecular characteristics.Conclusion: IMA+PI3Ki has significant antitumor efficacy in GIST xenografts as compared with single-agent treatment, resulting in more prominent tumor volume reduction and enhanced induction of apoptosis. Categorization of GIST based on KIT genotype and PI3K/PTEN genomic status combined with dose optimization is suggested for patient selection for clinical trials exploring such combinations. Clin Cancer Res; 20(23); 6071–82. ©2014 AACR.

PDF file - 566K, Xenograft tumor volume assessment, combined for all models. Tumor volume is expressed in percentage, as ratio between time point x baseline. Data are presented for all models (A), based on PTEN protein expression (B), and PTEN status by FISH (C). In GIST-PSW GDC-0941 treatment as single agent lasted 19 days (*). The dotted line shows the time of treatment withdrawal.

Table S2. Comparison in tumour volumes, proliferative and apoptotic activity between two weeks (end of treatment) and 5 weeks (end of regrowth experiment).

Supplemental Figures Legend

PDF file - 1222K, PTEN protein expression. Strong expression of PTEN was observed in GIST-DFR (A), GIST-BOE (B), GIST882 (C) and GIST48 (D). In GIST-PSW (E) and GIST882Ly (F) the majority of the tumor cells were PTEN negative, only scattered cells showed weak cytoplasmatic staining. The endothelium of blood vessels was used as internal positive control.

PDF file - 58K, Xenografts used in GDC-0941 efficacy experiments. All mice were subcutaneously transplanted with tumor fragment on both sides.

PDF file - 4757K, Western blotting showing KIT oncogenic signaling cascade. Three different tumor lysates were analyzed per treatment group.

Figure S1. Xenograft tumour volume assessment for the efficacy experiment. Tumour volume is displayed as percentage and normalized for baseline values and presented for UZLX-GIST3 (A), UZLX-GIST4 (B), UZLX-GIST2 (C), and GIST48 (D).

PDF file - 1683K, Representative micro-PET images from GIST-PSW (A) and GIST-DFR (B) mice treated with IMA or GDC+IMA are shown. Scans were perfromed at baseline, week four (end of the treatment) and weeks six and eight - during treatment discontinuation.

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PDF file - 2344K, Representative images of control tumors from GIST882Ly (A-E) and GIST-DFR (F-J). The H&E staining was shown to illustrate tumor morphology (A, F). Expression of KIT (B, G), ANO1 (C, H), ETV1 (D, I) and CD44 (E, J) were studied to further characterize GIST xenograft models.

Purpose: KIT activity is crucial for gastrointestinal stromal tumors (GIST). Imatinib (IMA) and sunitinib (SUN) are very effective KIT-inhibitors in patients with advanced GIST but have no curative potential. We evaluated the efficacy of the novel HSP90 inhibitor IPI-493 alone, or in combination with IMA or SUN in GIST xenografts with KIT mutations.Experimental Design: Nude mice (n = 98) were grafted bilaterally with human GIST carrying KIT exon 11 (GIST-PSW), KIT exon 9 (GIST-BOE), or double, KIT imatinib-sensitive exon 11 and imatinib-resistant exon 17 mutations (GIST-48). Mice were divided into six treatment groups and dosed orally for 15 days as follows: (i) control group, sterile water; (ii) IMA alone; (iii) SUN alone; (iv) IPI-493 alone; (v) IPI-493+IMA; and (vi) IPI-493+SUN.Results: Treatment with IPI-493 resulted in tumor growth stabilization, variable proliferation arrest, induction of apoptosis and necrosis, and downregulation of KIT and its signaling cascade, especially in the GIST-BOE model. Significant reduction of vessel density was observed with IPI-493 treatment, and was equal to SUN treatment in GIST-PSW and GIST-BOE xenografts. IPI-493 treatment effects were enhanced in combination with TKIs, especially with IPI-493+SUN. In our hands, IPI-493 showed dose-dependent liver damages.Conclusions: When administered as a single agent in a xenograft model, the HSP90 inhibitor IPI-493 has consistent antitumor activity and induces KIT downregulation in GISTs with heterogeneous KIT mutations. IPI-493 synergizes with TKIs that are commonly used for the treatment of advanced or IMA-resistant GIST. The antitumor response of IPI-493 is particularly enhanced in combination with SUN. Clin Cancer Res; 17(17); 5604–14. ©2011 AACR.

PDF file - 84K, Xenograft models characterization.

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The study objective was to characterize the excretion and metabolic profile of the respiratory syncytial virus fusion protein inhibitor, JNJ-53718678. Prior animal and in vitro studies suggested three main elimination pathways: N-glucuronidation to M8; CYP(3A4) metabolism leading to circulating metabolites M5, M12, M19 and M37; and JNJ-53718678 biliary excretion. To gain insight into the relative contribution of JNJ-53718678 and M8 biliary excretion, duodenal fluid sampling was incorporated into this mass balance study. A single oral dose of 500 mg 14C-JNJ-53718678 was administered to six healthy male subjects. Four hours after study drug intake, gallbladder contraction was stimulated and duodenal fluid samples were collected. JNJ-53718678, its key circulating metabolites and total radioactivity (TR) were quantified in plasma, feces, urine and duodenal fluid. Safety was monitored throughout. JNJ-53718678 and M12 represented 47.4% and 17.8%, respectively, of TR area under the curve (AUC)∞ in plasma. M37 (9.6%), M19 (5.2%), M5 (4.3%) and M8 (1.4%) were minor metabolites; 70.6% of TR was recovered in feces and 19.9% in urine. Duodenal fluid concentrations (% of TR) were highest for JNJ-53718678 (11.6%) followed by M8 (10.4%), M5 (5.9%) and M12 (1.1%). In feces, 10–16% of TR was JNJ-53718678, 5–8% M5

Introduction: The PI3K signaling pathway drives tumor cell proliferation and survival in gastrointestinal stromal tumor (GIST). We tested the in vivo efficacy of three PI3K inhibitors (PI3Ki) in patient-derived GIST xenograft models. Experimental Design: One hundred and sixty-eight nude mice were grafted with human GIST carrying diverse KIT genotypes and PTEN genomic status. Animals were dosed orally for two weeks as follows: control group (untreated); imatinib (IMA); PI3Ki (BKM120—buparlisib, BEZ235, or BYL719) or combinations of imatinib with a PI3Ki. Western blotting, histopathology, and tumor volume evolution were used for the assessment of treatment efficacy. Furthermore, tumor regrowth was evaluated for three weeks after treatment cessation. Results: PI3Ki monotherapy showed a significant antitumor effect, reflected in tumor volume reduction or stabilization, inhibitory effects on mitotic activity, and PI3K signaling inhibition. The IMA+PI3Ki combination remarkably improved the efficacy of either single-agent treatment with more pronounced tumor volume reduction and enhanced proapoptotic effects over either single agent. Response to IMA+PI3Ki was found to depend on the KIT genotype and specific model-related molecular characteristics. Conclusion: IMA+PI3Ki has significant antitumor efficacy in GIST xenografts as compared with single-agent treatment, resulting in more prominent tumor volume reduction and enhanced induction of apoptosis. Categorization of GIST based on KIT genotype and PI3K/PTEN genomic status combined with dose optimization is suggested for patient selection for clinical trials exploring such combinations. Clin Cancer Res; 20(23); 6071–82. ©2014 AACR.

Insufficiency of phosphatase and tensin homolog (PTEN) occurs in numerous tumor types and has been implicated as a resistance mechanism to receptor tyrosine kinase-targeted therapies in human cancer. In this study, we have performed a comprehensive molecular and immunohistochemical characterization of PTEN in 58 imatinib-naive and 54 imatinib-treated gastrointestinal stromal tumors (GISTs). The findings were correlated with clinicopathological data. At the genomic level, PTEN was affected mainly by mono-allelic loss, which was significantly less frequent in imatinib-naive vs imatinib-resistant tumors (9% vs 39%, P

Purpose: KIT activity is crucial for gastrointestinal stromal tumors (GIST). Imatinib (IMA) and sunitinib (SUN) are very effective KIT-inhibitors in patients with advanced GIST but have no curative potential. We evaluated the efficacy of the novel HSP90 inhibitor IPI-493 alone, or in combination with IMA or SUN in GIST xenografts with KIT mutations. Experimental Design: Nude mice (n = 98) were grafted bilaterally with human GIST carrying KIT exon 11 (GIST-PSW), KIT exon 9 (GIST-BOE), or double, KIT imatinib-sensitive exon 11 and imatinib-resistant exon 17 mutations (GIST-48). Mice were divided into six treatment groups and dosed orally for 15 days as follows: (i) control group, sterile water; (ii) IMA alone; (iii) SUN alone; (iv) IPI-493 alone; (v) IPI-493+IMA; and (vi) IPI-493+SUN. Results: Treatment with IPI-493 resulted in tumor growth stabilization, variable proliferation arrest, induction of apoptosis and necrosis, and downregulation of KIT and its signaling cascade, especially in the GIST-BOE model. Significant reduction of vessel density was observed with IPI-493 treatment, and was equal to SUN treatment in GIST-PSW and GIST-BOE xenografts. IPI-493 treatment effects were enhanced in combination with TKIs, especially with IPI-493+SUN. In our hands, IPI-493 showed dose-dependent liver damages. Conclusions: When administered as a single agent in a xenograft model, the HSP90 inhibitor IPI-493 has consistent antitumor activity and induces KIT downregulation in GISTs with heterogeneous KIT mutations. IPI-493 synergizes with TKIs that are commonly used for the treatment of advanced or IMA-resistant GIST. The antitumor response of IPI-493 is particularly enhanced in combination with SUN. Clin Cancer Res; 17(17); 5604–14. ©2011 AACR.

INTRODUCTION: The rarity of dedifferentiated liposarcoma (DDLPS) and the lack of experimental DDLPS models limit the development of novel therapeutic strategies. Pazopanib (PAZ) is a tyrosine kinase inhibitor that is approved for the treatment of non-adipocytic advanced soft tissue sarcoma. The activity of this agent has not yet been properly explored in preclinical liposarcoma models nor in a randomized phase Ш clinical trial in this entity. The aim of the present study was to investigate whether PAZ had antitumor activity in DDLPS models in vivo. MATERIAL AND METHODS: We established two patient-derived DDLPS xenograft models (UZLX-STS3 and UZLX-STS5) through implantation of tumor material from sarcoma patients in athymic nude NMRI mice. An animal model of the SW872 liposarcoma cell line was also used. To investigate the efficacy of PAZ in vivo, mice bearing tumors were treated for 2 weeks with sterile water, doxorubicin (1.2 mg/kg, intraperitoneally, twice per week), PAZ [40 mg/kg, orally (p.o.), twice per day], or PAZ plus doxorubicin (same schedules as for single treatments). RESULTS: Patient-derived xenografts retained the histologic and molecular features of DDLPS. PAZ significantly delayed tumor growth by decreasing proliferation and inhibited angiogenesis in all models tested. Combining the angiogenesis inhibitor with an anthracycline did not show superior efficacy. CONCLUSION: These results suggest that PAZ has potential antitumor activity in DDLPS primarily through antiangiogenic effects and therefore should be explored in clinical trials.

Background Acquired resistance to tyrosine kinase inhibitors (TKIs) in gastrointestinal stromal tumours (GISTs) is most commonly caused by secondary KIT or PDGFRA mutations. In this study we characterize a newly established GIST xenograft model, UZLX-GIST9, and evaluate the in vivo response of the model to standard TKIs (imatinib, sunitinib, and regorafenib). Methods Tumour fragments from a metastatic lesion of a GIST patient clinically progressing after treatment with imatinib, sunitinib and regorafenib were engrafted in a nude, immunodeficient mouse. Upon sequential passaging from mouse to mouse, tumour fragments were collected for histopathological and molecular characterization. The sensitivity of the model to treatment with TKIs was evaluated in 28 mice [passage 2 (n = 8), passage 4 (n = 20), 41 tumours]. Mice were grouped as follows: control (untreated), imatinib (50 mg/kg/BID), imatinib (100 mg/kg/BID), sunitinib (40 mg/kg/QD), and regorafenib (30 mg/kg/QD). After three weeks of oral treatment, tumours were collected for subsequent analysis. The efficacy of treatment was assessed by tumour volume, histopathology and Western immunoblotting. Results UZLX-GIST9 maintains the same typical morphological features and immunohistochemical characteristics as the original patient biopsy and expresses CD117 and DOG1. The KIT mutational profile (p.P577del + W557LfsX5+ D820G) remains the same as the original tissue sample originating from an intraspinal metastatic site. Three week treatment with different TKIs showed that the model is resistant to imatinib. Sunitinib induces tumour growth delay and regorafenib reduces the tumour burden by 30% as compared to control animals. While none of the TKIs had a significant effect on cell proliferation or cell survival, a remarkable increase of necrosis and significant reduction of microvessel density was observed under sunitinib and regorafenib. Western immunoblotting showed a mild reduction in KIT and AKT activation only in regorafenib treated tumours. Conclusions We established a novel human GIST xenograft, UZLX-GIST9, harbouring KIT exon 11 and 17 mutations and maintaining the pheno-and genotype of the original tumour. UZLX-GIST9 shows different levels of response to standard TKIs. This model will help to study TKI resistance and to explore novel treatment approaches for patients with TKI-resistant GIST.

Background: STS constitutes a rare and very heterogeneous family of mesenchymal tumors. The limited treatment options available for advanced STS underline the need for reliable preclinical models to test novel therapeutic strategies. Methods: A panel of patient-derived xenografts (PDX) was established by subcutaneous implantation of fresh, surgically resected tumor specimens in immunodeficient athymic nude NMRI mice. Once tumor growth was observed, pieces of tumor were re-transplanted to next generations of mice. At each passage tumor fragments were collected for histolopathological and molecular characterization. A model was considered established after observing stable histological and molecular features for at least two passages. To evaluate the stability of the genomic profile we performed low-coverage whole-genome sequencing on DNA isolated from tumors obtained from at least two passages. In addition RNA-seq was used to better characterize the mutational profile of the ex-mouse tumors. Results: Until now 139 STS samples from consenting patients treated at the University Hospitals, Leuven, Belgium, have been transplanted. Twenty six well-characterized, stable STS PDX models have been established, maintaining the histopathological and molecular features of the original tumor. Seventeen models, analyzed with low-coverage DNA sequencing, showed a stable genomic profile in at least two different tumor passages. At this point the panel includes models of gastrointestinal stromal tumor (6 models), myxofibrosarcoma (5), dedifferentiated liposarcoma (3), malignant peripheral nerve sheath tumor (3), synovial sarcoma (2), leiomyosarcoma (3) epithelioid haemangioendothelioma (1), mesenchymal chondrosarcoma (1) and undifferentiated high grade sarcoma (2). Some of these models have already been successfully used for in vivo testing of novel agents, including both targeted and cytotoxic (pro-)drugs, and results served as a rationale for at least four prospective clinical trials. In addition 22 other xenografts are still in early stages of engraftment, not yet fulfilling our criteria of an “established model”. Conclusion: Our panel of mesenchymal PDX models is characterized by stable histological and molecular features. These clinically well-annotated models can contribute to reliable preclinical studies for new anticancer treatments for STS. The availability of unique, rare STS xenografts also allows to study the biology of these diseases. The platform is made available to collaborators from academia and industry. Citation Format: Agnieszka Wozniak, Jasmien Cornillie, Yemarshet K. Gebreyohannes, Bram Boeckx, Haifu Li, Thomas Van Looy, Giuseppe Floris, Jasmien Wellens, Lise Vreys, Daphne Hompes, Marguerite Stas, Friedl Sinnaeve, Diether Lambrechts, Maria Debiec-Rychter, Raf Sciot, Patrick Schoffski. Patient-derived xenograft (PDX) models of soft tissue sarcoma (STS): a preclinical platform for early drug testing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5197.

Purpose: Oncogenic signaling in gastrointestinal stromal tumors (GIST) is sustained via PI3K/AKT pathway. We used a panel of six GIST xenograft models to assess efficacy of GDC-0941 as single agent or in combination with imatinib (IMA). Experimental Design: Nude mice (n = 136) were grafted bilaterally with human GIST carrying diverse KIT mutations. Mice were orally dosed over four weeks, grouped as follows: (A) control; (B) GDC-0941; (C) imatinib, and (D) GDC+IMA treatments. Xenografts regrowth after treatment discontinuation was assessed in groups C and D for an additional four weeks. Tumor response was assessed by volume measurements, micro-PET imaging, histopathology, and immunoblotting. Moreover, genomic alterations in PTEN/PI3K/AKT pathway were evaluated. Results: In all models, GDC-0941 caused tumor growth stabilization, inhibiting tumor cell proliferation, but did not induce apoptosis. Under GDC+IMA, profound tumor regression, superior to either treatment alone, was observed. This effect was associated with the best histologic response, a nearly complete proliferation arrest and increased apoptosis. Tumor regrowth assays confirmed superior activity of GDC+IMA over imatinib; in three of six models, tumor volume remained reduced and stable even after treatment discontinuation. A positive correlation between response to GDC+IMA and PTEN loss, both on gene and protein levels, was found. Conclusion: GDC+IMA has significant antitumor efficacy in GIST xenografts, inducing more substantial tumor regression, apoptosis, and durable effects than imatinib. Notably, after treatment withdrawal, tumor regression was sustained in tumors exposed to GDC+IMA, which was not observed under imatinib. Assessment of PTEN status may represent a useful predictive biomarker for patient selection. Clin Cancer Res; 19(3); 620–30. ©2012 AACR.

The majority of gastrointestinal stromal tumors (GIST) are characterized by activating mutations of KIT, an HSP90 client protein. Further secondary resistance mutations within KIT limit clinical responses to tyrosine kinase inhibitors, such as imatinib. The dependence of KIT and its mutated forms on HSP90 suggests that HSP90 inhibition might be a valuable treatment option for GIST, which would be equally effective on imatinib-sensitive and -resistant clones. We investigated the activity of AT13387, a potent HSP90 inhibitor currently being evaluated in clinical trials, in both in vitro and in vivo GIST models. AT13387 inhibited the proliferation of imatinib-sensitive (GIST882, GIST-T1) and -resistant (GIST430, GIST48) cell lines, including those resistant to the geldanamycin analogue HSP90 inhibitor, 17-AAG. Treatment with AT13387 resulted in depletion of HSP90 client proteins, KIT and AKT, along with their phospho-forms in imatinib-sensitive and -resistant cell lines, irrespective of KIT mutation. KIT signaling was ablated, whereas HSP70, a marker of HSP90 inhibition, was induced. In vivo, antitumor activity of AT13387 was showed in both the imatinib-sensitive, GIST-PSW, xenograft model and a newly characterized imatinib-resistant, GIST430, xenograft model. Induction of HSP70, depletion of phospho-KIT and inhibition of KIT signaling were seen in tumors from both models after treatment with AT13387. A combination of imatinib and AT13387 treatment in the imatinib-resistant GIST430 model significantly enhanced tumor growth inhibition over either of the monotherapies. Importantly, the combination of AT13387 and imatinib was well tolerated. These results suggest AT13387 is an excellent candidate for clinical testing in GIST in combination with imatinib. Mol Cancer Ther; 11(8); 1799–808. ©2012 AACR.

Background and objective: Dedifferentiated liposarcoma (DDLPS) and synovial sarcoma (SynSa) are aggressive malignant tumors of mesenchymal origin. Prognosis of advanced disease is poor with low response rates to first-line single agent doxorubicin (doxo) treatment, which is regarded as standard of care in metastatic disease. We evaluated the efficacy of PhAc-ALGP-doxorubicin (ALGP-doxo), a prodrug which is metabolized to doxo by peptidases present in the tumor microenvironment and/or tumor cells, in one SynSa and two DDLPS patient-derived xenografts. Methods: NMRI mice (n = 24) were transplanted subcutaneously on both flanks with human DDLPS (models UZLX-STS3 and UZLX-STS5) or SynSa (UZLX-STS7). Mice were randomized to three treatment groups: control (vehicle), doxo (0.03 mmol/kg for UZLX-STS3; 0.04 mmol/kg for UZLX-STS5 and UZLX-STS7) or ALGP-doxo (1.2 mmol/kg). Treatments were administered using intraperitoneal osmotic pumps, continuously releasing drugs for 7 days. Afterwards, half of mice from each group were sacrificed and tumors were collected. Remaining mice were monitored for another 14 days and tumors were collected on day 21. Treatment efficacy was assessed by tumor volume, hematoxylin and eosin (H&E) staining, immunohistochemistry for proliferation [phospho-histone H3 (pHH3), Ki-67] and apoptosis [cleaved caspase 3 (CC3)] and by Western blotting (WB). Statistical analysis was performed with Wilcoxon and Mann-Withney U-test, defining p Results: While the tumor volume in the control and doxo group increased steadily, in UZLX-STS3 and UZLX-STS5 ALGP-doxo caused tumor volume stabilization. Moreover, in UZLX-STS7 the average relative tumor volume decreased to 60.1% on day 7 and to 4.6% on day 21 under ALGP-doxo treatment. Long-lasting therapeutic effects of ALGP-doxo even after treatment withdrawal were observed in all three models tested. Compared to the control and doxo group, ALGP-doxo induced a significant decrease in proliferation and a significant increase in apoptotic activity on day 7 in all three models. It was also observed on day 21 in UZLX-STS3 and UZLX-STS5, while in UZLX-STS7 tumor tissue was replaced by fibroblasts. WB with tumor samples collected on day 7 showed expression of cleaved PARP in ALGP-doxo treated samples of UZLX-STS7, but not in the control or doxo group, and decreased pHH3 levels in the ALGP-doxo treated tumors of UZLX-STS5 and UZLX-STS7. Conclusion: ALGP-doxo shows considerably higher anti-tumor activity than doxo in patient-derived DDLPS and SynSa xenografts. The delivery of a 30-40 fold higher dose of ALGP-doxo than doxo is tolerated without significant side effects, while comparable dose of doxo is lethal. The prodrug and its activation by the tumor or the microenvironment warrants further testing in anthracycline-sensitive and -resistant preclinical models of human malignancies. Citation Format: Jasmien Cornillie, Agnieszka Wozniak, Lise Vreys, Haifu Li, Thomas Van Looy, Jasmien Wellens, Ulla Vanleeuw, André Trouet, Peter Pokreisz, Daphne Hompes, Maria Debiec-Rychter, Raf Sciot, Patrick Schöffski. Efficacy of an innovative, enzyme-activated doxorubicin prodrug in patient-derived dedifferentiated liposarcoma and synovial sarcoma xenograft models. [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 5353. doi:10.1158/1538-7445.AM2015-5353

Background: Upregulation of receptor tyrosine kinase signaling pathways, including vascular endothelial growth factor receptor (VEGFR) and MET (also known as hepatocyte growth factor receptor), is found in different subtypes of soft tissue sarcoma. Cabozantinib (CABO) is a multi-target inhibitor of receptor tyrosine kinases including VEGFR, MET, RET, and KIT. We aimed to evaluate the anti-angiogenic and anti-tumor effect of CABO alone and in combination with the standard-of-care cytotoxic agent doxorubicin (DOX) in a patient-derived xenograft model of dedifferentiated liposarcoma (DDLPS). Methods: NMRI nu/nu mice were bilaterally transplanted with human UZLX-STS3 DDLPS tumors, a model originating from a patient with DDLPS. Twenty eight tumor-bearing mice were assigned to 4 groups and treated for three weeks with either vehicle (control), DOX (i.p., 1.2mg/kg/twice per week), CABO (p.o., 30mg/kg/QD) or CABO+DOX (same dose/schedule as for the single agents). Tumor volume was measured three times a week. Mitotic and apoptotic activity were assessed on hematoxylin and eosin (H&E) staining and with phospho-histone H3 (pHH3) and cleaved-caspase 3 (CC3) immunostainings. Microvascular density (MVD) was evaluated by counting CD34 positive vessels. Statistics were calculated using the Mann-Whitney U pairs tests. p < 0.05 was considered as statistically significant. Results: A significant delay in tumor growth was observed in CABO and CABO+DOX treated groups as compared to controls (p Conclusions: CABO has anti-tumor activity in the UZLX-STS3 DDLPS xenograft model by inhibiting proliferation, inducing apoptosis and suppressing angiogenesis. These in vivo results support ongoing and planned clinical trials with cabozantinib in mesenchymal malignancies. Citation Format: Haifu Li, Agnieszka Wozniak, Karel Van Den Bossche, Thomas Van Looy, Jasmien Wellens, Daphne Hompes, Dana Aftab, Maria Debiec-Rychter, Raf Sciot, Patrick Schöffski. Cabozantinib, a multi-target receptor tyrosine kinase inhibitor, decreases tumor growth and angiogenesis in a patient-derived dedifferentiated liposarcoma xenograft. [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 774. doi:10.1158/1538-7445.AM2015-774

Background: STS constitutes a rare and very heterogeneous family of tumors of mesenchymal origin. The limited treatment options available for advanced STS underline the need for reliable preclinical models to test novel therapeutic strategies. Methods: A panel of patient-derived xenografts was established and is currently expanded in nu/nu NMRI mice by subcutaneous implantation of fresh, surgically resected tumor specimens from consenting patients with STS, treated at the University Hospitals Leuven, Belgium. We mainly focused on STS subtypes which harbor specific genetic alterations (e.g. translocations, gene amplifications). Once tumor growth was observed, pieces of tissue were re-transplanted to the next generation of mice. At each passage tumor fragments were collected for histological and molecular characterization. Fluorescence in situ hybridization (FISH) was performed to confirm specific genomic alterations in selected xenografts. A STS xenograft model was considered established after observing maintained histological and molecular features for at least two passages. Results: Until now a total of 78 STS patient samples have been transplanted. Fifteen well-characterized, stable STS models have already been established, maintaining the features of the original tumor: myxofibrosarcoma (4 models), dedifferentiated liposarcoma (2), synovial sarcoma (2), epithelioid haemangioendothelioma (1), malignant peripheral nerve sheath tumor (2), mesenchymal chondrosarcoma (1), leiomyosarcoma (2) and sarcoma not otherwise specified (1). Some of these models have already been successfully used for in vivo testing of novel agents, including tyrosine kinase inhibitors or cytotoxic (pro-)drugs. Twelve other xenografts are still in early stages of engraftment, not yet fulfilling our criteria of “established model”. Conclusion: This newly established, representative panel of STS xenografts is characterized by stable histological and molecular features reflecting the pheno- and genotype of the original patient samples. This panel is thus well suited for in vivo drug testing of innovative agents. The unique availability of models of some ultra-rare entities will also allow us to study the biology of these diseases. The platform is available for collaboration with academic and commercial partners (contact patrick.schoffski@uzleuven.be). Citation Format: Haifu Li, Jasmien Cornillie, Agnieszka Wozniak, Thomas Van Looy, Yemarshet Gebreyohannes, Jasmien Wellens, Ulla Vanleeuw, Daphne Hompes, Marguerite Stas, Friedl Sinnaeve, Maria Debiec-Rychter, Raf Sciot, Patrick Schöffski. Establishment and characterization of a panel of patient-derived soft tissue sarcoma (STS) xenograft models for in vivo testing of novel therapeutic approaches. [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 1460. doi:10.1158/1538-7445.AM2015-1460

Background: The majority of GISTs are driven by KIT/PDGFRA gain of function mutations. Advanced GIST is routinely treated with tyrosine kinase inhibitors (TKI; e.g imatinib, sunitinib and regorafenib). However, with time, heterogeneous resistance to these agents occurs, mainly due to acquired mutations in KIT. We tested the efficacy of dovitinib, which acts against VEGFR, FGFR, FLT3, PDGFRB and KIT, using patient-derived GIST xenograft models. Methods: NMRI nu/nu mice (n = 47) were bilaterally transplanted with the human GIST xenografts: a dose-dependent-imatinib-sensitive model UZLX-GIST2 (KIT: p.A502_Y503dup) or a model resistant to both imatinib and sunitinib, UZLX-GIST9 (KIT: p.P577del+p.W557LfsX5+p.D820G). Mice were divided to four treatment groups: control (untreated), imatinib (50 mg/kg/bid p.o.), imatinib (100 mg/kg/bid p.o.) and dovitinib (30 mg/kg/qd p.o.). Efficacy was assessed by tumor volume measurement (3x/week), histopathology [hematoxylin & eosin staining (H&E)], immunohistochemistry [Ki67, phospho-Histone H3 (pHH3), cleaved PARP] and Western blotting analysis of KIT signaling. Histologic response (HR) was graded as previously described1. Microvascular density (MVD) was assessed by counting CD31 positive vessels. Mann Whitney U test was used for statistical analysis. Results: After three weeks of treatment, dovitinib caused tumor volume reduction (to 37%) in UZLX-GIST2 and disease stabilization in UZLX-GIST9. Only minimal HR was observed in imatinib-treated cohorts, whereas dovitinib induced grade 2-3 HR in >50% of tumors in both models. Compared to control, dovitinib reduced mitotic activity by 22.6 fold (p Conclusion: The multi-kinase inhibitor dovitinib showed anti-tumor efficacy in GIST xenograft models, though the effect was more pronounced in KIT exon 9 mutant (UZLX-GIST2). The decrease in MVD in both models suggested that the anti-tumor effect in these models was at least partially mediated by an anti-angiogenic effect of dovitinib. 1 Antonescu et al. Clin Cancer Res. 2005; 11:4182-90 Citation Format: Yemarshet Kelemework Gebreyohannes, Thomas Van Looy, Agnieszka Wozniak, Jasmien Wellens, Haifu Li, Jasmien Cornillie, Ulla Vanleeuw, Lise Vreys, Matthew Squires, Ana-Maria Rodringuez, Maria Debiec-Rychter, Raf Sciot, Patrick Schöffski. Anti-tumor effects of dovitinib, a multi-target kinase inhibitor, in patient-derived gastrointestinal stromal tumor (GIST) xenograft models. [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 775. doi:10.1158/1538-7445.AM2015-775

Objective: Most GIST are dependent on the KIT/PDGFRA signaling pathway and therefore can be treated with specific tyrosine kinase inhibitors (TKI). With time GIST develop resistance to TKI. We need reliable models which can be used for preclinical drug testing. Methods: Patient-derived xenografts were established in nu/nu NMRI mice by subcutaneous implantation of fresh, surgically resected tumor specimens from consenting patients with GIST treated at the University Hospitals Leuven. Once tumor growth was observed, pieces of tumor were re-transplanted to next generations of mice. At each passage tumor fragments were collected for histological and molecular characterization. A model was considered to be established after observing stable histological and molecular features for at least two passages. Results: At present we have access to five successfully established GIST models, with different KIT mutations and different sensitivity to standard TKI (Table). Untreated tumors from subsequent passages show morphological and immunohistochemical features resembling the original patient biopsy, verifying the stability of GIST xenografts growth. KIT mutational status has also been confirmed in samples obtained from all models. These models are used for pre-clinical testing of new therapeutic approaches, and current results served as a rationale for at least four prospective clinical trials. If needed, we parallel our patient-derived models with in vitro or in vivo studies using one of the few established GIST cell lines. Conclusion: Our established GIST xenografts maintain the histological and molecular characteristics of the original patient sample and are a very useful and reliable preclinical platform for testing the new targeted approaches in GIST. The platform is available for collaboration with academic and commercial partners (contact patrick.schoffski@uzleuven.be). The overview of patients-derived GIST xenograftsModel*KIT mutationIn vivo efficacy [% of relative tumor volume after three weeks]PrimarySecondaryImatinib (50mg/kg, BID)Sunitinib (40mg/kg, QD)UZLX-GIST1p.V560Dn/a2020UZLX-GIST2p.A502_Y503dupn/a17080UZLX-GIST3p.W557_V559delinsFn/a2525UZLX-GIST4p.K558_G565delinsRn/a4520UZLX-GIST9p.P577delp.W557LfsX5, p.D820G250170*models which showed tumor growth for at least two passages; n/a-not applicable Citation Format: Agnieszka Wozniak, Thomas Van Looy, Giuseppe Floris, Yemarshet K. Gebreyohannes, Jasmien Wellens, Haifu Li, Jasmien Cornillie, Ulla Vanleeuw, Daphne Hompes, Marguerite Stas, Maria Debiec-Rychter, Raf Sciot, Patrick Schoffski. A panel of patient derived gastrointestinal stromal tumors (GIST) xenograft models for in vivo preclinical drug testing. [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 1461. doi:10.1158/1538-7445.AM2015-1461

10532 Background: Advanced GIST is treated with the tyrosine kinase inhibitors imatinib, sunitinib and regorafenib, but the majority of patients develop heterogeneous resistance to these agents. In...

e13528 Background: Analysis of primary clinical specimens of DDLPS revealed evidence of AKT activation in 27% of cases (Gutierrez A, et al. Aberrant AKT activation drives well-differentiated liposarcoma. PNAS 2011; 108(39):16386-91). We evaluated the efficacy of the PI3K inhibitor GDC-0941 (GDC) alone and in combination with doxorubicin (DOX) in patient- and cell-line derived DDLPS xenografts with proven PI3K/AKT pathway activation. Methods: NMRI nu/nu mice were either injected bilaterally with 1x107SW872 cells or transplanted with human DDLPS (UZLX-STS3). Animals were randomized to 4 groups (6 mice/group) and treated for two weeks: vehicle, DOX (i.p., 1.2mg/kg/biw), GDC (p.o., 75mg/kg/qd), GDC+DOX (same dose/schedule as single agents). Efficacy was assessed by tumor volume assessment, Western blotting and histological evaluation (H&E). Mitotic and apoptotic effects were confirmed using immunostainings for phospho-histone H3 (p-H3) (for proliferation), and cleaved caspase 3 (CC3) (for apoptosis). Results: GDC and GDC+DOX significantly delayed the growth of both xenografts (74% decrease under GDC and 77% under GDC+DOX in SW872, 67% under GDC and 56% under GDC+DOX in UZLX-STS3, vs. control). GDC and GDC+DOX treatment resulted in reduced mitotic activity and increase in apoptosis, as compared to untreated tumors in both models (Table). The activation of PI3K signaling was nearly completely suppressed in the GDC and GDC+DOX treated groups in SW872, whilea weak activation of the pathway was still observed in UZLX-STS3. Conclusions: GDC-0941 has anti-tumor activity, decreases tumor proliferation and induces apoptosis in DDLPS with PI3K/AKT activation. The GDC+DOX combination did not show additive effect in vivo. [Table: see text]

10030 Background: PI3K signaling is crucial for GIST proliferation and survival. We assessed the efficacy of the PI3K inhibitor NVP-BEZ235 (BEZ), alone or in combination with imatinib (IM), in GIST xenografts. Methods: Nude mice were grafted bilaterally with human GIST, carrying either KIT exon 9 (GIST-BOE, dose-dependent IM resistant) or exon 11 (GIST-DFR, IM sensitive) mutations. Animals, randomized into four groups (n=8/group) were dosed orally for 2 weeks with either vehicle, IM (50mg/kg/bid), BEZ (10mg/kg/qd), or IM+BEZ. Treatment efficacy was assessed by tumor volume, histopathology and Western immunoblotting. Moreover tumor regrowth was evaluated for 3 weeks after treatment cessation. Results: As a single agent IM and BEZ stabilized tumor growth of both GIST-BOE and DFR. Moderate to significant tumor regression was observed in GIST-BOE under BEZ (by 27%), and IM+BEZ (66%), and also in GIST-DFR under IM (75%) and IM+BEZ (75%). In GIST-BOE significant reduction in mitotic index was observed under BEZ (8.5 fold) and IM+BEZ (8.5 fold) as compared to control. In GIST-DFR mitotic activity was virtually absent under all regimens. Apoptotic activity increased significantly after treatment with IM (5.5 fold) and IM+BEZ (14.0 fold) in GIST-DFR, whereas it was almost unaffected by BEZ as single agent, as well as in all treatment groups in GIST-BOE. By Western, PI3K signaling was incompletely inhibited in all groups in GIST-DFR, and after BEZ in GIST-BOE. Complete inhibition of PI3K signaling was observed only after combination treatment. After treatment cessation long-lasting growth-inhibition was observed in IM+BEZ treated GIST-DFR. Moreover, mitotic index after BEZ and BEZ+IM in GIST-DFR was lower than in control even after treatment withdrawal. Conclusions: BEZ shows significant efficacy in GIST xenografts. Furthermore, combination with IM shows synergistic and long-lasting effects even after treatment withdrawal, which is not the case with drugs routinely used for GIST treatment.

Background: Activating mutations in the receptor tyrosine kinase, KIT, are found in the majority of gastrointestinal stromal tumors (GIST) and further secondary resistance mutations in KIT frequently arise upon treatment with tyrosine kinase inhibitors such as imatinib. KIT and its mutant forms are sensitive clients of HSP90 and it has been suggested that HSP90 inhibition could be an effective treatment for both imatinib-sensitive and imatinib-resistant GIST, which can occur simultaneously in different clones within the same patient. Methods: AT13387 is a fragment-derived, potent HSP90 inhibitor, which is currently being evaluated in clinical trials. To evaluate its anti-tumor activity against GIST, AT13387 was tested in both imatinib-sensitive (GIST882, GIST-PSW) and imatinib-resistant (GIST430, GIST48) in vitro and in vivo GIST models. Results: AT13387 inhibited the proliferation of GIST cell lines at sub-100 nM potencies, irrespective of their sensitivity to imatinib. Treatment of GIST882, GIST430 and GIST48 cells with AT13387 induced HSP70, a marker of HSP90 inhibition, whilst reducing the levels of the client proteins KIT, AKT and their phosphorylated forms. A concomitant decrease in the levels of phospho-ERK and phospho-S6 demonstrated that KIT signaling was being inhibited in all cell lines, whilst an increase in cleaved PARP indicated apoptosis. In contrast, treatment with imatinib inhibited KIT signaling only in the imatinib-sensitive line, GIST882. In vivo, the efficacy of AT13387 was tested in imatinib-sensitive (GIST-PSW) and imatinib-resistant (GIST430) xenograft models. AT13387, dosed once a week, inhibited the growth of both xenografts; depletion of phospho-KIT and inhibition of KIT signaling were again seen in these tumors. As expected, treatment with imatinib caused significant regression of the GIST-PSW tumors but not of GIST430. The combination of imatinib and AT13387 significantly enhanced tumor growth inhibition (T/C 21%) over either of the monotherapies (T/C 30% for AT13387, 46% for imatinib) in the GIST430 xenograft. Importantly, the combination was well tolerated. Conclusions: AT13387 is currently being evaluated in a Phase II GIST trial both as single agent and in combination with imatinib. These results support the potential efficacy of the compound in both imatinib-sensitive and imatinib-resistant GIST and its current testing in this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A217.