Search

Background: The majority of GIST are driven by constitutively activated KIT/PDGFRA kinases and are susceptible to treatment with tyrosine kinase inhibitors. During treatment, most of these tumors will develop secondary mutations in KIT or PDGFRA inducing drug resistance, so there is an unmet need for novel therapies. We tested the efficacy of IDRX-42, a novel selective KIT inhibitor with high activity towards the most relevant KIT mutations, in 4 GIST xenograft models. Methods: NMRI nu/nu mice were transplanted with patient-derived GIST xenograft models UZLX-GIST9 (KIT:p.P577del;W557LfsX5;D820G), UZLX-GIST2B (KIT:p.A502_Y503dup), UZLX-GIST25 (KIT:p.K642E) and the cell-line derived model GIST882 (KIT:p.K642E). Mice were treated daily with vehicle (control), imatinib (100mg/kg), sunitinib (20mg/kg), avapritinib (5mg/kg), or IDRX-42 (10mg/kg, 25mg/kg). Efficacy was assessed by tumor volume evolution, histopathology, grading of histological response and immunohistochemistry. The Kruskal-Wallis and Wilcoxon Matched Pairs tests were used for statistical analysis, with p

Detailed characterization of established STS PDX models.

Given the very limited efficacy of doxorubicin (doxo) in soft tissue sarcoma, there is a clear need for more active and less toxic treatments for this family of diseases. However, due to the rarity of these malignancies and lack of reliable preclinical models, development of new therapies has lagged behind. We evaluated the efficacy of PhAc-ALGP-doxorubicin (ALGP-doxo), a prodrug metabolized to doxo by peptidases present in tumor cells and/or tumor microenvironment, in a synovial sarcoma (SynSa) and two dedifferentiated liposarcoma (DDLPS) patient-derived xenograft models. Sixty-eight mice were engrafted bilaterally with human DDLPS or SynSa and randomized to control, doxo, or ALGP-doxo treatment, which were administered using an intraperitoneal minipump. Tumor volume measurement, histopathology, and Western blotting were used to assess treatment efficacy. Tumor regrowth was evaluated in a subset of mice over a period of 2 weeks after treatment cessation. Although tumor volume in the control and doxo groups increased steadily, ALGP-doxo caused tumor volume stabilization in the DDLPS xenografts and significant tumor shrinkage in the SynSa model, continuing after treatment cessation. A significant decrease in proliferation and increase in apoptosis compared with control and doxo was observed during and after treatment with ALGP-doxo in all models. In conclusion, ALGP-doxo shows considerably higher antitumoral efficacy compared with doxo in all patient-derived xenograft models tested. Administration of a 30- to 40-fold higher dose of ALGP-doxo than doxo is tolerated without significant adverse events. These results warrant further testing of this prodrug in anthracycline-sensitive and -resistant models of soft tissue sarcoma. Mol Cancer Ther; 16(8); 1566–75. ©2017 AACR.

PDF file - 271K

Details on the synthetic method to make PhAc-ALGP-doxorubicin

Evolution of relative tumor volume of each of the six tumors entered in the UZLX-STS7 regrowth experiment.

Genomic profile, describing copy number gains and losses, obtained using low coverage whole genome sequencing of subsequent passages of STS PDX models, revealing a stable copy number profile throughout passaging. Copy number profile of (A) the MPNST model UZLX-STS39 and (B) the mesenchymal chondrosarcoma model UZLX-STS41.

The activity of the receptor tyrosine kinase KIT is crucial for gastrointestinal stromal tumor (GIST) growth and survival. Imatinib and sunitinib are very effective in advanced GIST, but have no curative potential. The observation that heat shock protein 90 (HSP90) inhibition results in KIT degradation prompted us to assess the efficacy of the HSP90 inhibitor retaspimycin hydrochloride (IPI-504) alone or in combination with imatinib or sunitinib in two GIST xenografts with distinctive KIT mutations. Nude mice were grafted with human GIST carrying KIT exon 13 (GIST-882; n = 59) or exon 11 (GIST-PSW; n = 44) mutations and dosed with imatinib (50 mg/kg twice daily), sunitinib (40 mg/kg once daily), IPI-504 (100 mg/kg 3 times per week), IPI-504 + imatinib, or IPI-504 + sunitinib. We evaluated tumor volume, proliferation and apoptosis, KIT expression and activation, as well as adverse events during treatment. Treatment with IPI-504 alone resulted in tumor regression, proliferation arrest, and induction of tumor necrosis. We documented downregulation of KIT and its signaling cascade in IPI-504–treated animals. Treatment effects were enhanced by combining IPI-504 with imatinib or sunitinib. On histologic examination, liver damage was frequently observed in animals exposed to combination treatments. In conclusion, IPI-504 shows consistent antitumor activity and induces KIT downregulation in GIST, as a single agent, and is more potent in combination with imatinib or sunitinib. The sequence of drug administration in the combination arms warrants further studies. Mol Cancer Ther; 10(10); 1897–908. ©2011 AACR.

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

PDF file - 78K

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

PDF file - 604K

Soft-tissue sarcomas (STS) represent a heterogeneous group of rare, malignant tumors of mesenchymal origin. Reliable in vivo sarcoma research models are scarce. We aimed to establish and characterize histologically and molecularly stable patient-derived xenograft (PDX) models from a broad variety of STS subtypes. A total of 188 fresh tumor samples from consenting patients with localized or advanced STS were transplanted subcutaneously in NMRI-nu/nu–immunodeficient mice. Once tumor growth was observed, the material was passaged to a next generation of mice. A patient-derived tumor sample was considered “successfully engrafted” whenever the sample was transplanted to passage 1. A PDX model was considered “established” when observing stable morphologic and molecular features for at least two passages. With every passage, histologic and molecular analyses were performed. Specific genomic alterations and copy-number profile were assessed by FISH and low coverage whole-genome sequencing. The tumor engraftment rate was 32% (61/188) and 188 patient samples generated a total of 32 PDX models, including seven models of myxofibrosarcoma, five dedifferentiated liposarcoma, five leiomyosarcoma, three undifferentiated pleomorphic sarcoma, two malignant peripheral nerve sheet tumor models, and single models of synovial sarcoma and some other (ultra)rare subtypes. Seventeen additional models are in early stages of engraftment (passage 1–2). Histopathologic and molecular features were compared with the original donor tumor and were stable throughout passaging. The platform is used for studies on sarcoma biology and suited for in vivo preclinical drug testing as illustrated by a number of completed and ongoing laboratory studies.

Detailed description of the number of mice/tumors and the doses of drugs in all in vivo experiments.

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.

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

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).

Purpose:Gastrointestinal stromal tumors (GIST) are commonly treated with tyrosine kinase inhibitors (TKI). The majority of patients with advanced GIST ultimately become resistant to TKI due to acquisition of secondary KIT mutations, whereas primary resistance is mainly caused by PDGFRA p.D842V mutation. We tested the activity of avapritinib, a potent and highly selective inhibitor of mutated KIT and PDGFRA, in three patient-derived xenograft (PDX) GIST models carrying different KIT mutations, with differential sensitivity to standard TKI.Experimental Design: NMRI nu/nu mice (n = 93) were transplanted with human GIST xenografts with KIT exon 11+17 (UZLX-GIST9KIT 11+17), exon 11 (UZLX-GIST3KIT 11), or exon 9 (UZLX-GIST2BKIT9) mutations, respectively. We compared avapritinib (10 and 30 mg/kg/once daily) versus vehicle, imatinib (50 mg/kg/bid) or regorafenib (30 mg/kg/once daily; UZLX-GIST9KIT11+17); avapritinib (10, 30, 100 mg/kg/once daily) versus vehicle or imatinib [UZLX-GIST3KIT11]; and avapritinib (10, 30, 60 mg/kg/once daily) versus vehicle, imatinib (50, 100 mg/kg/twice daily), or sunitinib (40 mg/kg/once daily; UZLX-GIST2BKIT9).Results:In all models, avapritinib resulted in reduction of tumor volume, significant inhibition of proliferation, and reduced KIT signaling. In two models, avapritinib led to remarkable histologic responses, increase in apoptosis, and inhibition of MAPK-phosphorylation. Avapritinib showed superior (UZLX-GIST9KIT 11+17 and -GIST2BKIT 9) or equal (UZLX-GIST3KIT 11) antitumor activity to the standard dose of imatinib. In UZLX-GIST9KIT 11+17, the antitumor effects of avapritinib were significantly better than with imatinib or regorafenib.Conclusions:Avapritinib has significant antitumor activity in GIST PDX models characterized by different KIT mutations and sensitivity to established TKI. These data provide strong support for the ongoing clinical trials with avapritinib in patients with GIST (NCT02508532, NCT03465722).

Mouse body weight during the course of treatment is presented as percent of the baseline value in the three xenografts.

Grading scheme for pMAPK immunostaining. Staining was graded based on the intensity of the staining and the percent of tumor area showing pMAPK positivity.

Soft tissue sarcoma (STS) is a heterogeneous family of rare mesenchymal tumors, characterized by histopathological and molecular diversity. Tissue microarray (TMA) is a tool that allows performing research in orphan diseases in a more efficient and cost-effective way. TMAs are paraffin blocks consisting of multiple small representative tissue cores from biological samples, for example, from multiple donors, diverse sites of disease, or multiple different diseases. In 2015, we began constructing TMAs using archival tumor material from STS patients. Specimens were well annotated in terms of histopathological diagnosis, treatment, and clinical follow-up of the tissue donors. Each TMA block contains duplicate or triplicate 1.0–1.5 mm tissue cores from representative tumor areas selected by sarcoma pathologists. The construction of TMAs was performed with TMA Grand Master (3DHistech). So far, we have established disease-specific TMAs from 7 STS subtypes: gastrointestinal stromal tumor (72 cases included in the array), alveolar soft part sarcoma (n = 12 + 47), clear cell sarcoma (n = 22 + 32), leiomyosarcoma (n = 55), liposarcoma (n = 42), inflammatory myofibroblastic tumor (n = 12 + 21), and alveolar rhabdomyosarcoma (n = 24). We also constructed a multisarcoma TMA covering a representative number of important histopathological subtypes on arrays for screening purposes, namely, angiosarcoma, dedifferentiated liposarcoma, pleomorphic liposarcoma, and myxoid liposarcoma, leiomyosarcoma, malignant peripheral nerve sheath tumor, myxofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, and undifferentiated pleomorphic sarcoma, with 7–11 individual cases per subtype. We are currently expanding the list of TMAs with additional sarcoma entities, considering the heterogeneity of this family of tumors. Our extensive STS TMA platform is suitable for rapid and cost-effective morphological, immunohistochemical, and molecular characterization of the tumor as well as for the identification of potential novel diagnostic markers and drug targets. It is readily available for collaborative projects with research partners.

Soft-tissue sarcomas (STS) represent a heterogeneous group of rare, malignant tumors of mesenchymal origin. Reliable in vivo sarcoma research models are scarce. We aimed to establish and characterize histologically and molecularly stable patient-derived xenograft (PDX) models from a broad variety of STS subtypes. A total of 188 fresh tumor samples from consenting patients with localized or advanced STS were transplanted subcutaneously in NMRI-nu/nu-immunodeficient mice. Once tumor growth was observed, the material was passaged to a next generation of mice. A patient-derived tumor sample was considered "successfully engrafted" whenever the sample was transplanted to passage 1. A PDX model was considered "established" when observing stable morphologic and molecular features for at least two passages. With every passage, histologic and molecular analyses were performed. Specific genomic alterations and copy-number profile were assessed by FISH and low coverage whole-genome sequencing. The tumor engraftment rate was 32% (61/188) and 188 patient samples generated a total of 32 PDX models, including seven models of myxofibrosarcoma, five dedifferentiated liposarcoma, five leiomyosarcoma, three undifferentiated pleomorphic sarcoma, two malignant peripheral nerve sheet tumor models, and single models of synovial sarcoma and some other (ultra)rare subtypes. Seventeen additional models are in early stages of engraftment (passage 1-2). Histopathologic and molecular features were compared with the original donor tumor and were stable throughout passaging. The platform is used for studies on sarcoma biology and suited for in vivo preclinical drug testing as illustrated by a number of completed and ongoing laboratory studies. ispartof: MOLECULAR CANCER THERAPEUTICS vol:18 issue:6 pages:1168-1178 ispartof: location:United States status: published

Purpose: Gastrointestinal stromal tumors (GIST) are commonly treated with tyrosine kinase inhibitors (TKI). The majority of patients with advanced GIST ultimately become resistant to TKI due to acquisition of secondary KIT mutations, whereas primary resistance is mainly caused by PDGFRA p.D842V mutation. We tested the activity of avapritinib, a potent and highly selective inhibitor of mutated KIT and PDGFRA, in three patient-derived xenograft (PDX) GIST models carrying different KIT mutations, with differential sensitivity to standard TKI. Experimental Design: NMRI nu/nu mice (n = 93) were transplanted with human GIST xenografts with KIT exon 11+17 (UZLX-GIST9KIT 11+17), exon 11 (UZLX-GIST3KIT 11), or exon 9 (UZLX-GIST2BKIT9) mutations, respectively. We compared avapritinib (10 and 30 mg/kg/once daily) versus vehicle, imatinib (50 mg/kg/bid) or regorafenib (30 mg/kg/once daily; UZLX-GIST9KIT11+17); avapritinib (10, 30, 100 mg/kg/once daily) versus vehicle or imatinib [UZLX-GIST3KIT11]; and avapritinib (10, 30, 60 mg/kg/once daily) versus vehicle, imatinib (50, 100 mg/kg/twice daily), or sunitinib (40 mg/kg/once daily; UZLX-GIST2BKIT9). Results: In all models, avapritinib resulted in reduction of tumor volume, significant inhibition of proliferation, and reduced KIT signaling. In two models, avapritinib led to remarkable histologic responses, increase in apoptosis, and inhibition of MAPK-phosphorylation. Avapritinib showed superior (UZLX-GIST9KIT 11+17 and -GIST2BKIT 9) or equal (UZLX-GIST3KIT 11) antitumor activity to the standard dose of imatinib. In UZLX-GIST9KIT 11+17, the antitumor effects of avapritinib were significantly better than with imatinib or regorafenib. Conclusions: Avapritinib has significant antitumor activity in GIST PDX models characterized by different KIT mutations and sensitivity to established TKI. These data provide strong support for the ongoing clinical trials with avapritinib in patients with GIST (NCT02508532, NCT03465722).

Background: The majority of GISTs are driven by constitutively activated KIT/PDGFRA kinases and susceptible to treatment with tyrosine kinase inhibitors such as imatinib, sunitinib and regorafenib. During treatment most tumors will develop heterogeneous secondary mutations in KIT or PDGFRA inducing drug resistance, so there is an unmet need for novel therapies. We tested the efficacy of M4205, a novel specific KIT inhibitor with high activity towards the most relevant KIT mutations, in patient-derived GIST xenograft models. Methods: NMRI nu/nu mice (n=146) were transplanted with patient-derived GIST xenograft models UZLX-GIST9 (KIT:p.P577del;W557LfsX5;D820G) known to be resistant to both imatinib and sunitinib, with the dose-dependent imatinib-sensitive and sunitinib-sensitive models UZLX-GIST2B (KIT:p.A502_Y503dup), UZLX-GIST25 (KIT: p.K642E) and the cell-line derived model GIST882 (KIT: p.K642E)*. Mice were treated daily with vehicle (control), imatinib (100mg/kg), avapritinib (5mg/kg), sunitinib (20mg/kg), or M4205 (10mg/kg, 25mg/kg). Efficacy was assessed by tumor volume evolution, histopathology [hematoxilin & eosine staining (H&E)] and immunohistochemistry [Ki-67, phospho-Histone H3 (pHH3), cleaved PARP]. Histologic response (HR) was graded as previously described°. Mann Whitney U and Wilcoxon Matched Pairs tests were used for statistical analysis, with p Results: M4205 (25mg/kg) caused tumor volume shrinkage in UZLX-GIST2B, -GIST25 and GIST882 with relative decrease to 45.6%, 35.1% and 57.3% on the last day as compared to baseline. In UZLX-GIST9 tumor growth to 132.4% was observed in M4205 (25mg/kg)-treated tumors as compared to baseline. In all models we observed significant smaller tumor volumes in M4205 (25mg/kg)-treated tumors compared to control, and this antitumor activity was superior to imatinib in UZLX-GIST9, -GIST2B and GIST882, and to sunitinib in -GIST25. Compared to controls, M4205 (25mg/kg) induced a significant decrease in mitosis (H&E) in all models, confirmed on pHH3 and Ki-67 immunostainings in three models. In -GIST25 and GIST882 grade 2-4 HR with myxoid degeneration was observed in all tumors. All treatments were well tolerated. Conclusion: M4205 has significant antitumor activity in patient- and cell line-derived GIST xenograft models. The novel kinase inhibitor induces volumetric responses, decreases mitotic activity, has antiproliferative effects and in models with KIT exon 13 mutation leads to characteristic myxoid degeneration. * Cornillie et al. Mol Cancer Ther 2019; 18(6):1168-1178 ° Agaram et al. Clin Cancer Res 2007; 13:170-81 Citation Format: Luna De Sutter, Agnieszka Wozniak, Jasper Verreet, Ulla Vanleeuw, Lore De Cock, Nina Linde, Christine Drechsler, Christina Esdar, Raf Sciot, Patrick Schöffski. Anti-tumor effects of the novel KIT mutant inhibitor M4205 in patient-derived gastrointestinal stromal tumor (GIST) xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2666.

STS constitutes a family of rare mesenchymal tumors with more than 70 subtypes described. The limited treatment options available for advanced STS patients underline the need for reliable preclinical models to test new therapeutic approaches. We established a panel of PDX models (XenoSarc) by subcutaneous implantation of fresh tumor specimens in athymic mice (nu/nu NRMI). Once tumor growth was observed, pieces of tumor were re-transplanted to next generations of animals. At each passage tumor fragments were collected for histopathological and molecular characterization. In an ongoing effort 493 STS samples from 414 consenting patients treated at the University Hospitals, Leuven (Belgium) have been transplanted. A total of 67 PDX models from 20 STS subtypes have been established, meaning they have stable morphological, immunohistochemical and genetic characteristics over at least 2 passages. The PDX platform includes more common STS subtypes such as myxofibrosarcoma (n=12 models), gastrointestinal stromal tumors (9), dedifferentiated liposarcoma (10), and leiomyosarcoma (8), as well as models from ultra-rare subtypes, e.g. pulmonary intimal sarcoma, extraskeletal osteosarcoma, mesenchymal chondrosarcoma, myxoinflammatory fibroblastic sarcoma and others. All relevant details about the donor patient and tumor characteristics, including sensitivity to the standard treatments, are known for every model. The models are well-characterized, with availability of molecular information on genomic profile (by low-coverage whole genome sequencing), and expression profile (by RNA sequencing). Xenografts are accompanied by ready to use tissue microarrays (TMA) from the models, which can be exploited for target identification and model selection for preclinical studies. Ex-mouse material can also be used to establish primary cell cultures and 3D organoids for in vitro screening purposes. The XenoSarc platform offers opportunities for studying the biology of various sarcoma subtypes including ultra-rare entities and was found to be a very reliable tool for early drug screening in STS in preparation of clinical testing of novel compounds. The platform is available for collaborative preclinical projects with academic and industrial partners. Citation Format: Agnieszka Wozniak, Luna De Sutter, Lore De Cock, Britt Van Renterghem, Che-Jui Lee, Yannick Wang, Ulla Vanleeuw, Kimberly Verbeeck, Daphne Hompes, Friedl Sinnaeve, Hazem Wafa, Baki Topal, Joris Jaekers, Dirk Van Raemdonck, Maria Debiec-Rychter, Raf Sciot, Patrick Schöffski. XenoSarc: patient-derived xenograft (PDX) models of soft tissue sarcoma (STS) and their histopathological and molecular characterization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3100.

Background: STS constitutes a rare family of mesenchymal tumors with more than 70 subtypes classified by WHO (2013). The limited treatment options available for advanced STS underline the need for reliable preclinical models, especially from ultra-rare subtypes, to test novel therapeutic strategies. Methods: A panel of PDX models was established by subcutaneous implantation of fresh 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 histopathological and molecular characterization. A model was considered “established” after observing stable histological and molecular features for at least two passages. Furthermore, ex-mouse tumor tissue samples were stored, further characterized by immunocytology and flow cytometry and cultured to be used for in vitro drug testing. Results: Between September 2011 and November 2019, 375 STS samples from 325 consenting patients treated at the University Hospitals, Leuven, Belgium have been transplanted. A total of 56 PDX models were established, maintaining the histopathological and molecular features of the original tumor. Detailed clinical information about the donor patient and tumor characteristics (including sensitivity to standard and experimental agents), is known for every model. At present the XenoSarc platform includes ready to use models of dedifferentiated liposarcoma (11 models), gastrointestinal stromal tumor (8), myxofibrosarcoma (8), leiomyosarcoma (7), malignant peripheral nerve sheath tumor (3), synovial sarcoma (2), pulmonary artery intimal sarcoma (2), epithelioid hemangioendothelioma (1), mesenchymal chondrosarcoma (1), pleomorphic rhabdomyosarcoma (1), CIC-rearranged round cell sarcoma (1), myxoinflammatory fibroblastic sarcoma (1), rhabdomyosarcoma not otherwise specified (NOS) (1), telangiectatic extraskeletal osteosarcoma (1), extraskeletal osteosarcoma (1) and high-grade undifferentiated pleomorphic sarcoma (7). These models are well-characterized, including molecular information on copy number changes (by low-coverage whole genome sequencing), gene expression profile (by RNA-Seq) and targeted sequencing to detect mutations in genes involved in tumorigenesis. In addition, we have constructed tissue microarrays (TMA) from the xenografts which are used for target identification and model selection for preclinical studies. We are using the xenografts for in vivo testing of novel agents, including targeted and cytotoxic (pro-)drugs, and results already served as rationale for a number of prospective clinical trials. A total of 27 other xenografts are still in early stages of engraftment, not yet fulfilling our criteria of an “established” model. Conclusion: The XenoSarc platform offers a lot of opportunities for studying the biology of a variety of important sarcoma subtypes including ultra-rare entities, and has proven efficiency for early drug screening in STS in preparation of clinical testing of novel compounds. The platform is well maintained and continuously expanded, and available to collaborators from academia and industry. Citation Format: Agnieszka Wozniak, Britt Van Renterghem, Jasmien Cornillie, Yannick Wang, Che-Jui Lee, Jasmien Wellens, Ulla Vanleeuw, Madita Nysen, Daphne Hompes, Friedl Sinnaeve, Hazem Wafa, Baki Topal, Tom Verbelen, Maria Debiec-Rychter, Raf Sciot, Patrick Schöffski. XenoSarc: Patient-derived xenograft (PDX) models of soft tissue sarcoma (STS) and their histopathological and molecular characterization [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1117.

Given the very limited efficacy of doxorubicin (doxo) in soft tissue sarcoma, there is a clear need for more active and less toxic treatments for this family of diseases. However, due to the rarity of these malignancies and lack of reliable preclinical models, development of new therapies has lagged behind. We evaluated the efficacy of PhAc-ALGP-doxorubicin (ALGP-doxo), a prodrug metabolized to doxo by peptidases present in tumor cells and/or tumor microenvironment, in a synovial sarcoma (SynSa) and two dedifferentiated liposarcoma (DDLPS) patient-derived xenograft models. Sixty-eight mice were engrafted bilaterally with human DDLPS or SynSa and randomized to control, doxo or ALGP-doxo treatment, which were administered using an intraperitoneal minipump. Tumor volume measurement, histopathology and Western blotting were used to assess treatment efficacy. Tumor regrowth was evaluated in a subset of mice over a period of two weeks after treatment cessation. While tumor volume in the control and doxo groups increased steadily, ALGP-doxo caused tumor volume stabilization in the DDLPS xenografts and significant tumor shrinkage in the SynSa model, continuing after treatment cessation. A significant decrease in proliferation and increase in apoptosis compared to control and doxo was observed during and after treatment with ALGP-doxo in all models. In conclusion, ALGP-doxo shows considerably higher antitumoral efficacy compared to doxo in all patient-derived xenograft models tested. Administration of a 30-40 fold higher dose of ALGP-doxo than doxo is tolerated without significant adverse events. These results warrant further testing of this prodrug in anthracycline-sensitive and -resistant models of soft tissue sarcoma. ispartof: Molecular Cancer Therapeutics vol:16 issue:8 pages:1566-1575 ispartof: location:United States status: published

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.

The activity of the receptor tyrosine kinase KIT is crucial for gastrointestinal stromal tumor (GIST) growth and survival. Imatinib and sunitinib are very effective in advanced GIST, but have no curative potential. The observation that heat shock protein 90 (HSP90) inhibition results in KIT degradation prompted us to assess the efficacy of the HSP90 inhibitor retaspimycin hydrochloride (IPI-504) alone or in combination with imatinib or sunitinib in two GIST xenografts with distinctive KIT mutations. Nude mice were grafted with human GIST carrying KIT exon 13 (GIST-882; n = 59) or exon 11 (GIST-PSW; n = 44) mutations and dosed with imatinib (50 mg/kg twice daily), sunitinib (40 mg/kg once daily), IPI-504 (100 mg/kg 3 times per week), IPI-504 + imatinib, or IPI-504 + sunitinib. We evaluated tumor volume, proliferation and apoptosis, KIT expression and activation, as well as adverse events during treatment. Treatment with IPI-504 alone resulted in tumor regression, proliferation arrest, and induction of tumor necrosis. We documented downregulation of KIT and its signaling cascade in IPI-504–treated animals. Treatment effects were enhanced by combining IPI-504 with imatinib or sunitinib. On histologic examination, liver damage was frequently observed in animals exposed to combination treatments. In conclusion, IPI-504 shows consistent antitumor activity and induces KIT downregulation in GIST, as a single agent, and is more potent in combination with imatinib or sunitinib. The sequence of drug administration in the combination arms warrants further studies. Mol Cancer Ther; 10(10); 1897–908. ©2011 AACR.

Background: STS constitutes a rare family of mesenchymal tumors with more than 70 subtypes described. The limited treatment options available for advanced STS underline the need for reliable preclinical models to test novel therapeutic strategies. Methods: Panel of PDX models was established by subcutaneous implantation of fresh tumor specimens in immunodeficient, athymic nude NMRI mice. Once tumor growth was observed, pieces of tumor were re-transplanted to next generations of animals. At each passage tumor fragments were collected for detailed characterization. A model was considered established after observing stable histological and molecular features for at least two passages. Results: Between 09/2011 and 11/2017, 228 STS samples from 203 consenting patients treated at University Hospitals Leuven, Belgium, have been transplanted. Thirty-three stable PDX models have been established, maintaining the histopathological and molecular features of the original tumor. Detailed clinical information about a donor patient, including sensitivity to given therapy, is linked to every model. Higher engraftment rate was observed in samples collected from patients who developed metastasis throughout the course of disease (38% vs. 23%, p Conclusion: Our XenoSarc platform contains a number of well-annotated models, characterized by stable histological and molecular features. This platform is a reliable tool for the evaluation of new anticancer treatments for STS and for studying the biology of these rare diseases. The platform is made available to collaborators from academia and industry. Citation Format: Agnieszka Wozniak, Jasmien Cornillie, Yemarshet K. Gebreyohannes, Yannick Wang, Jasmien Wellens, Ulla Vanleeuw, Daphne Hompes, Marguerite Stas, Friedl Sinnaeve, Hazem Wafa, Maria Debiec-Rychter, Raf Sciot, Patrick Schöffski. XenoSarc: Patient-derived xenograft (PDX) models of soft tissue sarcoma (STS)—an update on a preclinical platform for early drug testing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1031.

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: Elevated levels of matrix metalloproteinases have been found to associate with poor prognosis in various carcinomas. This study aimed at evaluating plasma levels of MMP1, MMP8 and MMP13 as diagnostic and prognostic markers of breast cancer. METHODS: A total of 208 breast cancer patients, of which 21 with inflammatory breast cancer, and 42 healthy controls were included. Plasma MMP1, MMP8 and MMP13 levels were measured using ELISA and correlated with clinicopathological characteristics. RESULTS: Median plasma MMP1 levels were higher in controls than in breast cancer patients (3.45 vs. 2.01 ng/ml), while no difference was found for MMP8 (10.74 vs. 10.49 ng/ml). ROC analysis for MMP1 revealed an AUC of 0.67, sensitivity of 80% and specificity of 24% at a cut-off value of 4.24 ng/ml. Plasma MMP13 expression could not be detected. No correlation was found between MMP1 and MMP8 levels. We found a trend of lower MMP1 levels with increasing tumour size (p = 0.07); and higher MMP8 levels with premenopausal status (p = 0.06) and NPI (p = 0.04). The median plasma MMP1 (p = 0.02) and MMP8 (p = 0.007) levels in the non-inflammatory breast cancer patients were almost twice as high as those found in the inflammatory breast cancer patients. Intriguingly, plasma MMP8 levels were positively associated with lymph node involvement but showed a negative correlation with the risk of distant metastasis. Both controls and lymph node negative patients (pN0) had lower MMP8 levels than patients with moderate lymph node involvement (pN1, pN2) (p = 0.001); and showed a trend for higher MMP8 levels compared to patients with extensive lymph node involvement (pN3) and a strong predisposition to distant metastasis (p = 0.11). Based on the hypothesis that blood and tissue protein levels are in reverse association, these results suggest that MMP8 in the tumour may have a protective effect against lymph node metastasis. CONCLUSION: In summary, we observed differences in MMP1 and MMP8 plasma levels between healthy controls and breast cancer patients as well as between breast cancer patients. Interestingly, our results suggest that MMP8 may affect the metastatic behaviour of breast cancer cells through protection against lymph node metastasis, underlining the importance of anti-target identification in drug development. ispartof: BMC Cancer vol:8 issue:1 pages:77-85 ispartof: location:England status: published

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: 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...

Aim: STS constitutes a rare, heterogeneous family of tumours of mesenchymal origin. The limited treatment options available for advanced STS underline the need for reliable preclinical models to test novel therapeutic strategies. Methods: Patient-derived xenografts were established in nu/nu NMRI mice by subcutaneous implantation of fresh, surgically resected tumour specimens from consenting patients with STS treated at University Hospitals Leuven. We mainly focused on STS subtypes which harbour specific genetic alterations (e.g. translocations, gene amplifications). Once tumour growth was observed, pieces of tumour were re-transplanted to a next generation of mice. At each passage tumour fragments were collected for histological and molecular characterization. Specific genomic alterations were confirmed in subsequent passages by fluorescence in situ hybridization. A STS xenograft model was considered to be established after observing stable histological and molecular features for at least two passages. Results: Until now we have transplanted a total of 65 patient-derived STS samples. We were able to successfully establish 12 well-characterized STS models maintaining the features of the original tumour: myxofibrosarcoma (3 models); dedifferentiated liposarcoma (2); synovial sarcoma (2); epithelioid haemangioendothelioma (1); malignant peripheral nerve sheath tumour (1); mesenchymal chondrosarcoma (1); leiomyosarcoma (1) 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 prodrugs. In addition, 12 other xenografts are still in early stages of engraftment, not yet fulfilling our criteria of “established model”. Conclusions: Our newly established STS xenografts maintain the histological and molecular characteristics of the original patient sample, therefore offering a promising preclinical platform for testing anti-cancer drugs. The availability of models of some ultra-rare entities would also allow us to study the biology of these diseases. Disclosure: All authors have declared no conflicts of interest.

Aim: Most GIST are dependent on the KIT/PDGFRA signalling pathway and therefore can be treated with specific tyrosine kinase inhibitors (TKI). With time GIST develop resistance to the 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 tumour specimens from consenting patients with GIST treated at the University Hospitals Leuven. Once tumour growth was observed, pieces of tumour were re-transplanted to next generations of mice. At each passage tumour 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 TKIs (Table). Established GIST xenograft models and their sensitivity to standard treatment. In vivo efficacy was assessed by % of relative tumour volume after 3 weeks under either imatinib (50mg/kg BID) or sunitinib (40mg/kg QD) Model* KIT mutation In vivo efficacy Imatinib Sunitinib UZLX-GIST1 p.V560D 20 20 UZLX-GIST2 p.A502_Y503dup 170 80 UZLX-GIST3 p.W557_V559delinsF 25 25 UZLX-GIST4 p.K558_G565delinsR 45 20 UZLX-GIST9 p.P577del; p.W557LfsX5; p.D820G 250 170 *models which showed tumor growth for at least two passages Untreated tumours from subsequent passages show morphological and immunohistochemical features resembling the original patient biopsy, verifying the stability GIST xenografts growth. KIT mutational status has also been confirmed in samples obtained from all models. These models are used for subsequent testing of new therapeutic approaches and current results have had an impact on 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. Conclusions: 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. Disclosure: All authors have declared no conflicts of interest.

Elevated levels of matrix metalloproteinases (MMPs) have been found to associate with poor prognosis in various carcinomas. This study aimed at evaluating plasma levels of the collagenases MMP1, MMP8 and MMP13 as diagnostic and prognostic markers of breast cancer. Using ELISA, plasma levels of MMP1, MMP8 and MMP13 were measured in 42 control individuals and in 208 patients – of which 21 were inflammatory breast cancer patients – and were correlated with standard clinicopathological data. Plasma MMP1 levels were higher in breast cancer patients than in control individuals, while the opposite was true for MMP8. Plasma MMP13 levels could not be detected. We found a negative correlation of plasma MMP1 with tumour size (P = 0.07); and a positive association of MMP8 with the premenopausal status (P = 0.06), Nottingham Prognostic Index (P = 0.06) and Her2 expression (P = 0.07). Further, a twofold decrease in MMP1 (P = 0.025) and MMP8 (P = 0.007) levels was observed in inflammatory breast cancer patients, a very rare and not well understood aggressive disease. Most interestingly, we observed a peculiar relation between plasma MMP8 levels and lymph node metastasis. We found that both control individuals and patients without lymph node involvement (pN0) have lower plasma MMP8 levels than patients with moderate lymph node involvement (pN1, pN2) (P = 0.001); and that they show a trend for higher MMP8 levels as compared with patients with extensive lymph node metastasis (pN3) and a strong predisposition to distant metastasis. In summary, we observed differences in MMP1 and MMP8 plasma levels between distinct breast cancer patient groups. As it is not clear to date whether MMPs in blood and body fluids have a physiological function per se, we hypothesize that altered levels in blood reflect local changes in the extracellular microenvironment. As such, a positive association of blood MMP levels with clinical characteristics and tumour features reflects a negative association with tissue MMP levels and vice versa. Therefore, our results suggest that both MMP1 and MMP8 in the tumour may contribute to the aggressive phenotype of inflammatory breast carcinomas. Interestingly, our results suggest that tumour MMP8 expression may affect the metastatic behaviour of breast cancer cells with a greater protective effect against lymph node metastasis than against distant metastasis.