Established Patient-derived Xenograft Models Research Articles

Integrative In Vivo Drug Testing Using Gene Expression Signature and Patient-Derived Xenografts from Treatment-Refractory HER2 Positive and Triple-Negative Subtypes of Breast Cancer.

Patient-derived xenografts (PDXs) are powerful tools for translational cancer research. Here, we established PDX models from different molecular subtypes of breast cancer for in vivo drug tests and compared the histopathologic features of PDX model tumors with those of patient tumors. Predictive biomarkers were identified by gene expression analysis of PDX samples using Nanostring nCount cancer panels. Validation of predictive biomarkers for treatment response was conducted in established PDX models by in vivo drug testing. Twenty breast cancer PDX models were generated from different molecular subtypes (overall success rate, 17.5%; 3.6% for HR+/HER2−, 21.4% for HR+/HER2+, 21.9% for HR−/HER2+ and 22.5% for triple-negative breast cancer (TNBC)). The histopathologic features of original tumors were retained in the PDX models. We detected upregulated HIF1A, RAF1, AKT2 and VEGFA in TNBC cases and demonstrated the efficacy of combined treatment with sorafenib and everolimus or docetaxel and bevacizumab in each TNBC model. Additionally, we identified upregulated HIF1A in two cases of trastuzumab-exposed HR−/HER2+ PDX models and validated the efficacy of the HIF1A inhibitor, PX-478, alone or in combination with neratinib. Our results demonstrate that PDX models can be used as effective tools for predicting therapeutic markers and evaluating personalized treatment strategies in breast cancer patients with resistance to standard chemotherapy regimens.

The significance of insulin receptor expression to predict the resistance to VEGFR-TKIs and induce PD-L1 expression in advanced clear cell renal cell carcinoma.

592 Background: Previously we have identified the prognostic gene set of ccRCC patients, where insulin receptor (INSR) expression was decreased in the patients with poor outcome (Proc Natl Acad Sci U S A., 2001). We examined the clinical significance of decreased INSR expression and sought to elucidate the underlying mechanisms. Methods: The INSR expression was immunohistochemically examined in the nephrectomy specimens of RCC patients (n = 33) who then received axitinib. We established patient derived Xenograft model (PDX) of ccRCC and examined the INSR expression in the axitinib resistant PDX tumors by Western blotting. As the INSR is expressed in the vascular endothelial cells, we co-cultured the RCC cell lines with the human renal glomerular endothelial cells (HGEC) treated with si-RNA of INSR (si-INSR) and the microarray experiment was conducted. Results: In RCC patients with axitinib, those with low INSR expression had poor outcome (median PFS 19.5 vs 2.3 months, p < 0.001; median OS 34.2 vs 5.6 months, p = 0.001). The INSR expression was the significantly independent predictor of PFS (p = 0.006). In the axitinib-resistant PDX tumors, the expression of INSR was decreased. In the co-culture experiments, the microarray experiments revealed that the decreased INSR expression in the HGECs may be involved with the important signaling pathway including interferon response in Caki-1 cells. Interferon- β was highly expressed in HGECs with si-INSR. The decreased INSR expression and the increased interferon-β expression in HGEC were confirmed when axitinib was administered. The Caki-1 cells that was co-cultured with HGECs treated with si-INSR demonstrated high expression of PD-L1. The PD-L1 expression was increased in a concentration-dependent manner of recombinant interferon-β and increased phosphorylation of STAT1 and STAT3 were observed. Conclusions: In conclusion, the decreased INSR expression could be a biomarker to predict the resistance to VEGFR-TKIs. The decreased INSR expression was correlated with the increased interferon-β expression in HGECs, which leads to the induction of PD-L1 through increased phosphorylation of STAT1 and STAT3.

Abstract P1-13-04: Efficacy of adjuvant 5-Fluorouracil in residual HER2-negative breast cancer following neoadjuvant chemotherapy

Abstract Background: Patients with residual disease following neoadjuvant chemotherapy have an increased risk of relapse. Recently the CREATE-X trial demonstrated that adjuvant capecitabine (oral prodrug of 5'deoxy-5-fluorouridine), prolonged disease-free survival and overall survival among patients with HER2-negative breast cancer who had residual invasive disease after neoadjuvant chemotherapy containing anthracycline, taxane, or both. Therefore, we sought to evaluate the antitumor efficacy of 5-fluorouracil (5-FU) in patient-derived xenografts (PDXs) from residual tumors resistant to neoadjuvant chemotherapy. Methods: Antitumor efficacy of 5-FU was assessed in vivo in three PDXs varying in hormone receptor status (0, 4% and 11% respectively), generated from residual tumors of primary breast cancer patients treated with neoadjuvant chemotherapy containing anthracycline, and taxanes. In addition, significance of timing of therapy was also assessed, comparing efficacy of initiating treatment upon implantation (immediate start cohort; mimicking treating residual disease with adjuvant therapy), with initiating treatment upon establishment of PDX (standard start cohort). Results: 5-FU was efficacious in established PDX models that are triple negative (0% ER; p< 0.0001), low ER positive (4% ER, p=0.0213) and ER-positive (11% ER; p= 0.0390), decreasing growth compared to the cohort. However, there was no statistically significant difference between the immediate start cohort and standard start cohort. Western blot analysis of the treatment-naïve derived mouse tumors recognized RB as a predictive biomarker for 5-FU response. Conclusion: 5-FU has anti-tumor activity in residual HER2-negative PDX models resistant to taxanes, and anthracyclines in the neoadjuvant setting. Citation Format: Owusu-Brackett N, Scott S, Yuca E, Evans KW, Tapia C, Meric-Bernstam F. Efficacy of adjuvant 5-Fluorouracil in residual HER2-negative breast cancer following neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-13-04.

Patient-derived bladder cancer xenograft models reveal VEGF and CDK4 enhancing tumor metastasis behavior.

New strategies to treat advanced bladder cancer are urgently required. A patient-derived xenograft (PDX) model has become a useful tool to evaluate chemotherapeutics and investigate personalized cancer treatment options. In this study, fresh human bladder cancer specimens (C09303 and C21391) were transplanted subcutaneously into nude mice to establish PDX models. These models well retained pathological characteristics and molecular markers of the original tumor. Primary cells derived from C09303 were cultured and perfused into the bladders of nude mice to establish orthotopic xenograft models. Evident signals of lung and kidney metastases were detected by whole-body optical imaging. Gemcitabine displayed a significant therapeutic effect on the subcutaneous or orthotopic xenograft tumors of C09303. In vitro, matrigel invasion assays showed that C09303 primary culture cells are remarkably invasive. To study the metastatic properties of C09303, we sequenced the genomes of C09303 and C21391, and found that the expression of VEGF and CDK4 was significantly upregulated, and VEGF-knockdown or CDK4-knockdown C09303 cells showed attenuated invasiveness and migration. This PDX model revealed that VEGF and CDK4 enhanced tumor metastasis behavior, suggesting them as novel molecular therapeutic targets. This framework provides a tool for research on metastasis mechanism and individualized treatment for bladder cancer.

Abstract 1040: Patient derived xenografts of hematologic malignancies for translational research

Abstract Acute leukemias and lymphomas represent a very heterogeneous group of hematologic malignancies and pose an important challenge in the clinical routine. They frequently develop resistance to the treatment with standard-of-care (SoC) drugs and have a high incidence of disease recurrence. Recent progress in molecular profiling has helped to identify new potential drivers for the different leukemia and lymphoma subtypes, with some of them being potential therapeutic targets. Further target validation and drug development projects are highly dependent on corresponding preclinical models representing the different clinical subtypes. Therefore, we started to establish and characterize new patient derived xenografts (PDX) of AML, ALL and lymphomas for drug development and translational research. AML- and ALL-PDX were derived from bone marrow aspirates or peripheral blood samples, from primary or relapsed acute leukemia patients. Purified cells were transplanted either intravenously (i.v.) and/or subcutaneously (s.c.) into immunodeficient mice. Some mice developed a systemic AML, which was monitored by flow cytometric analysis of blood samples. Non-Hodgkin- or Hodgkin lymphoma-PDX were derived from peripheral blood, lymph node extirpations or core needle biopsies, and were usually transplanted subcutaneously into immunodeficient mice. For further characterization, established PDX models were treated with SoC and investigational drugs. In parallel, gene expression profiles as well as mutations were analyzed within the first in vivo passages. More than 20 new PDX models from AML, ALL, NHL and HL have been successfully established and characterized. Highly individual response to the treatments were observed, correlation analyses with mutations and gene expression are ongoing. Our newly and extensively characterized PDX models from hematologic malignancies are suitable tools for preclinical drug development. They provide an exceptional platform for the identification and validation of new targets and allow the preclinical screening of new compounds and combinations for translational research projects. Citation Format: Antje Siegert, Bernadette Brzezicha, Stephan Fuhrmann, Leonid Karawajew, Martin Janz, Clemens Schmitt, Iduna Fichtner, Jens Hoffmann. Patient derived xenografts of hematologic malignancies for translational research [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 1040.

Abstract 3433: Genomic analysis of patient-derived xenograft (PDX) model from metastatic breast cacner

Abstract Background: Patient-derived xenograft (PDX) models have been shown to be predictive of clinical outcomes and are being used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Molecular subtyping of metastatic breast cancer is specifically important for clinical decision for targeted therapy, endocrine therapy, and chemotherapy. The PDX model, reflecting the same molecular characteristics of a patient's tumor, can provide valuable information prior to treatment. We explored the possibility of generating PDX from metasttic breast cancer and examined whether the primary or metastatic cancer of the patient and the tumor implanted in the mouse have the same genetic characteristics. Method: We established PDX models using small biopsy samples of tumors from primary breast or metastatic cancer using NSG mouse. Clinicopathologic characteristics and outcome were collected from the electronic medical records. Whole-exome sequencing was performed using Illumina platform. Single-nucleotide polymorphisms (SNPs) and small insertions and deletions (Indels) were called using muTect(1.1.7) and IndelGenotyper provided by GATK (3.6.0). We also investigated whether copy number variations (CNVs) are maintained in the PDX models. Conifer was used for counting the read framents. The read counts of normal and tumors were normalized by a log2 scale. Results: We established 24 PDX models from 34 biopsy samples (11 primary breast cancer and 23 metastatic cancer). 11 samples were hormone receptor-positive luminal type, 9 samples were HER2-positive type and 13 samples were triple-negative subtype. Our results indicate that the most frequent mutated genes are TP53, ARAF, GNAQ, ATRX, and PIK3CA in total samples. TP53 was the most frequently altered gene in all subtypes. Other mutated genes were slightly different for each subtype, followed by GNAQ and GATA3 in hormone receptor-positive subtype. ARAF and ATRX were in TNBC subtype. In HER2+ subtype, GNAQ, BRCA2, PIK3CA and ARID1B were same number of mutations. EIF3E and IKZF3 were the most frequently amplified genes and USP6 and SSX4 were the most frequently deleted genes. In addition, we compared the results in 5 cases that had all of the patient's tumor sample and the xenografted tumor sample; the same mutation was found and reflected the molecular features of the patient. Conclusion: In this study, it was possible to establish PDX from biopsy tissues and PDX models maintain the genomic characteristics of the patient tumor. We believe that by using PDX models and bioinformatics analysis, it is possible to find new druggable targets and eventually the proper drug for personalized medicine. Citation Format: Dongjin Shin, Seock-Ah Im, Seongyeong Kim, Minjung Kim, Yu Jin Kim, Jinjoo Kang, Ahrum Min, Jieun Lee, Giyong Jang, Deukchae Na, Kyung-Hun Lee, Jongil Kim. Genomic analysis of patient-derived xenograft (PDX) model from metastatic breast cacner [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 3433.

Abstract 1437: De novo fatty acid synthesis-driven sphingolipid metabolism promotes metastatic potential of colorectal cancer

Abstract Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the USA. Development of metastasis is the most common cause of death in these patients. Fatty acid synthase (FASN) and Sphingosine Kinases 1 and 2 (SPHK1 and 2) are overexpressed in many cancers, including CRC. However, the contribution of FASN-mediated upregulation of sphingolipid metabolism to CRC metastasis and potential of these pathways as targets for therapeutic intervention remains unknown. The purpose of this study was to determine (i) expression of FASN, SPHK1 and SPHK2 in human CRC tissues, (ii) the effect of upregulation of FASN on sphingolipid metabolism and (iii) functional significance of the FASN/SPHK axis in advanced CRC. Methods: Expression of FASN, SPHK1 and SPHK2 was assessed in a CRC tumor microarray (matched normal colon and tumor; 56 cases) by immunohistochemistry. Sphingolipids were measured by mass spectrometry. Primary CRC cells were established from CRC patient-derived xenograft (PDX) tumors and treated with TVB-3664, a novel FASN inhibitor, or FTY-720, an S1P mimetic that inhibits SPHKs and S1P receptors. Cellular proliferation was measured using a cell counter. Migration capabilities of cells were assessed by live cell imaging using Nikon BioStation. Invadopodia and focal adhesions were assessed by total internal reflection fluorescence microscopy. Tumor tissues were implanted into 6 to 8-week-old NOD scid gamma mice to establish PDX models. Expression of proteins involved in adhesion, migration and invasion were assessed by western blot. Results: SPHK1 and SPHK2 were overexpressed in CRC as compared to normal mucosa and expression of FASN correlates with expression of SPHK2 (Spearman's r=0.27894, p=0.0374). Furthermore, FASN and SPHKs co-localized within invadopodia of primary CRC cells. Moreover, FASN inhibition decreased expression of SPHK2 and the level of Sphingosine-1-phosphate (S1P) in primary and established CRC cells. Inhibition of de novo lipogenesis using TVB-3664 or FTY-720 significantly inhibited proliferation, migration, focal adhesion formation and gelatin degradation ability of primary CRC cells. Inhibition of the FASN/SPHK/S1P axis was accompanied by a decrease in activation of p-MET, p-FAK, and p-Paxilin in vitro and in vivo. S1P treatment rescued FASN-mediated inhibition of these proteins suggesting that FASN promotes metastatic properties of CRC cells, in part, through an increase in sphingolipid metabolism. Conclusion: Upregulation of the FASN/SPHK/S1P axis promotes CRC progression by enhancing cellular proliferation, adhesion and migration. Therefore, this study provides a strong rationale for further investigation of the interconnection of de novo lipogenesis and sphingolipid metabolism that would potentially lead to identification of new therapeutic targets and strategies for CRC. Citation Format: Naser Jafari, James Drury, Andrew J. Morris, Fredrick O. Onono, Payton D. Stevens, Tianyan Gao, Eun Y. Lee, Heidi L. Weiss, B Mark Evers, Yekaterina Zaytseva. De novo fatty acid synthesis-driven sphingolipid metabolism promotes metastatic potential of colorectal cancer [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 1437.

Establishment of lung cancer patient-derived xenograft models and primary cell lines for lung cancer study

BackgroundThe overall 5-year survival rate of lung cancer is about 15% even with therapeutic drugs like tyrosine kinase inhibitors. Ideal models are urgently needed for exploring mechanisms and finding new drugs. Patient-derived xenografts (PDX) models and primary cells are both used to screen therapeutic regimens for cancer. However, PDX models and primary cells from the same patient are difficult to establish. Their consistency to the original tumor tissue is not well studied.Methods31 lung cancer patient tissues were procured to establish the lung cancer PDX models and primary cell lines. Tumor growth measurements, histological and immunohistochemistry analysis, Western blotting, EGFR and K-RAS mutation detection and gefitinib sensitive assay were performed to evaluate the characteristic of established PDX models. Immunofluorescence analysis, anchorage-independent cell growth, Western blotting and gefitinib sensitive assay were performed to assay the characteristic of established primary cell lines. The whole-exome sequencing was used to compare the characteristic of the patient’s tumor tissue, established PDX and primary cell line.ResultsTwenty-one lung cancer PDX models (67.74%, 21/31) and ten primary cell lines (32.25%, 10/31) were established from patients’ tumor tissues. The histology and pathological immunohistochemistry of PDX xenografts are consistent with the patients’ tumor samples. Various signal pathways were activated in different PDX models (n = 5) and primary cell lines (n = 2). EGFR mutation PDX model and primary cell line (LG1) were sensitive to gefitinib treatment. The expression of CK8/18, TTF1 and NapsinA in LG1 and LG50 primary cells were also positive. And the activated signal pathways were activated in LG1 and LG50 primary cell lines. Furthermore, the gene mutation in PDX tumor tissues and primary cell line (LG50) was consistent with the mutation in LG50 patient’s tumor tissues.ConclusionThese data suggested that established lung cancer PDX models and primary cell lines reserved mostly molecular characteristics of primary lung cancer and could provide a new tool to further understand the mechanisms and explore new therapeutic strategies.

MP72-06 CORRELATION OF THE INSULIN RECEPTOR EXPRESSION AND THE RESISTANCE TO VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR TYROSINE KINASE INHIBITORS IN ADVANCED CLEAR CELL RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology II1 Apr 2018MP72-06 CORRELATION OF THE INSULIN RECEPTOR EXPRESSION AND THE RESISTANCE TO VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR TYROSINE KINASE INHIBITORS IN ADVANCED CLEAR CELL RENAL CELL CARCINOMA Masayuki Takahashi, Kei Daizumoto, Yayoi Fukuhara, Keisuke Ozaki, Megumi Tsuda, Yoshito Kusuhara, Hidehisa Mori, Tomoya Fukawa, Yasuyo Yamamoto, Kunihisa Yamaguchi, Tomoharu Fukumori, and Hiroomi Kanayama Masayuki TakahashiMasayuki Takahashi More articles by this author , Kei DaizumotoKei Daizumoto More articles by this author , Yayoi FukuharaYayoi Fukuhara More articles by this author , Keisuke OzakiKeisuke Ozaki More articles by this author , Megumi TsudaMegumi Tsuda More articles by this author , Yoshito KusuharaYoshito Kusuhara More articles by this author , Hidehisa MoriHidehisa Mori More articles by this author , Tomoya FukawaTomoya Fukawa More articles by this author , Yasuyo YamamotoYasuyo Yamamoto More articles by this author , Kunihisa YamaguchiKunihisa Yamaguchi More articles by this author , Tomoharu FukumoriTomoharu Fukumori More articles by this author , and Hiroomi KanayamaHiroomi Kanayama More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2290AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Vascular endothelial growth factor receptor tyrosine kinase inhibitors (VEGFR-TKIs) demonstrate the significant efficacy for advanced clear cell renal cell carcinoma (ccRCC), however, it eventually becomes resistant to VEGFR-TKIs during the treatment. So far, the mechanisms for the resistance to VEGFR-TKIs remain to be fully elucidated. Previously we have identified the gene set which could predict poor prognosis of ccRCC patients (Takahashi M et.al., Proc Natl Acad Sci U S A., 98: 9754, 2001). We examined whether the insulin receptor (INSR) expression in the gene set may correlate with the resistance to VEGFR-TKIs. METHODS The INSR expression was examined immunohistochemically in the nephrectomy specimens of the RCC patients (n=33) who then received axitinib as the VEGFR-TKI and correlated with their survival outcome. We compared the INSR expression of the nephrectomy or metastasectomy specimens before and after the administration of VEGFR-TKIs in 5 cases. The INSR expression of the human renal glomerular endothelial cells (HGEC) was compared before and after the administration of axitinib by Western blotting. In addition, we established patient derived Xenograft model (PDX) of ccRCC. Tumors of PDX were resected when it regrew and showed the resistance for axitinib and the INSR expression was compared before and after the treatment by Western blotting. RESULTS The INSR was expressed at the vessels surrounding tumor cells. Progression-free survival (PFS) was significantly shorter in the INSR-negative group than in the INSR-positive group. Multivariate analysis revealed that the INSR expression was the significantly independent predictor of PFS. In the specimens resected after VEGFR-TKI, the INSR expression was more frequently decreased. As the concentration of axitinib increased, the INSR expression in the HGEC was decreased. The tumors of PDX that were resected after demonstrating the resistance to axitinib had the decreased INSR expression. CONCLUSIONS The decreased INSR expression could be correlated with the resistance to VEGFR-TKI and its expression may be useful in selecting appropriate drugs for advanced ccRCC patients. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e954 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Masayuki Takahashi More articles by this author Kei Daizumoto More articles by this author Yayoi Fukuhara More articles by this author Keisuke Ozaki More articles by this author Megumi Tsuda More articles by this author Yoshito Kusuhara More articles by this author Hidehisa Mori More articles by this author Tomoya Fukawa More articles by this author Yasuyo Yamamoto More articles by this author Kunihisa Yamaguchi More articles by this author Tomoharu Fukumori More articles by this author Hiroomi Kanayama More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract P5-05-01: A molecularly annotated collection of breast cancer patient-derived xenograft models aligned with ongoing clinical trials built from fine needle aspiration samples throughout neoadjuvant treatment

Abstract BACKGROUND: Patient-derived xenograft (PDX) models of breast cancer replicate the diverse histologic and molecular features of patient tumors and provide a renewable source of human tumor tissue. However, collection of tissue by core needle biopsy is problematic due to patient discomfort, bleeding risk and the limited number of passes a patient can tolerate. Several studies have catalogued the maintenance of molecular features of patient tumors in PDX models of breast cancer. METHODS: To support the neoadjuvant molecular diagnostic and drug development program in triple negative breast cancer (TNBC), a pilot study was conducted to determine if fine needle aspiration (FNA) could be used for building PDX models. Subsequently, PDX models are being established in alignment with ongoing clinical trials at MDACC. The molecular evolution of patient's tumors, matched with PDXs engrafted from their tumors, is under study throughout the neoadjuvant treatment of TNBC using RNA sequencing, whole-exome sequencing, deep sequencing of cancer genes, and histologic analyses. RESULTS: To date, 20 established PDX models have been developed and stable PDX models continue to be generated at a rate of 2-3 per month. Several of these models are derived from serial FNAs derived from patients throughout neoadjuvant treatment. These models retain histologic and molecular features of the original patient tumors. Serial patient biopsies, matched with PDX models, have enabled measurement of the mutational and transcriptomic evolution in vivo of TNBC undergoing neoadjuvant treatment. We have standardized the use of FNAs to generate PDX models both pre- and post-neoadjuvant therapy in the following ongoing neoadjuvant clinical trials: 1. MDACC 2014-0185 (PI Stacy Moulder, 360 patients), 'ARTEMIS: A Randomized TNBC-Enrolling trial to confirm Molecular profiling Improves Survival' 2. MDACC 2014-0045 (PI Jennifer Litton, 20+ patients), 'A pilot study of BMN673 as a neoadjuvant study in patients with a diagnosis of invasive breast cancer and a deleterious BRCA mutation' CONCLUSION: We demonstrated that PDX models from tissue collected by FNA recapitulate the biology and clinical course of the patient's tumor. Sequencing analyses revealed that neoadjuvant chemotherapy and PDX engraftment enrich for cancer gene mutations. We observe association of the rate of successful PDX engraftment with clinical parameters such as the patient's residual cancer burden (RCB) status at the time of surgery (upon completion of neoadjuvant treatment). In addition, we observe that PDX models derived from serial patient biopsies throughout treatment are more resistant to chemotherapy treatment. These models recapitulate the variety of chemotherapy responses observed in patients with TNBC and serve as powerful tools for preclinical biomarker and discovery studies. Citation Format: Echeverria GV, Cai S, Tu Y, McCoy A, Lau R, Redwood A, Rauch G, Adrada B, Candelaria R, Santiago L, Thompson A, Litton J, Moulder S, Symmans F, Chang JT, Piwnica-Worms H. A molecularly annotated collection of breast cancer patient-derived xenograft models aligned with ongoing clinical trials built from fine needle aspiration samples throughout neoadjuvant treatment [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-05-01.

Effects of siRNA Silencing of TUG1 and LCAL6 Long Non-coding RNAs on Patient-derived Xenograft of Non-small Cell Lung Cancer.

The aim of the present study was to establish a patient-derived xenograft (PDX) mouse model of non-small cell lung cancer (NSCLC) and investigate the anti-tumor efficacy of silencing of TUG1 and LCAL6 long non-coding RNA in the PDX model. PDXs were established by subcutaneously implanting NSCLC surgical tumor fragments into immunodeficient mice. PDX characterization was performed by histopathological, immunohistochemical and real-time polymerase chain reaction (RT-PCR) analyses for NSCLC subtype-specific markers and expression of LCAL6 and TUG1. Anti-tumor efficacy of siRNA silencing of TUG1 and LCAL6 was also investigated in the PDX model. The effect of TUG1 and LCAL6 silencing on protein expression of proliferation marker Ki67 and HOX-gene family HOXB7 in the tumors was assessed by immunohistochemical staining and Western blotting. Establishment of NSCLC PDX models resulted in 9 of 26 cases (34.6%). Lung squamous cell carcinomas (SCC) had a higher engraftment rate (58.3%) than lung adenocarcinomas (ADC) (18.2%) (p<0.05). Comparative analysis indicated these established PDX models of NSCLC closely resembled the original tumors with regard to NSCLC subtype-specific markers TTF-1, napsin A, p63 and expression of LCAL6 and TUG1. The tumor volume and weight were significantly reduced in the TUG1-silenced group as compared to the control group (p<0.05). However, no significant tumor growth inhibition was found in the LCAL6-silenced group (p>0.05). Expression of both TUG1and LCAL6 was reduced by siRNA treatment. Expression of Ki67 and HOXB7 was significantly suppressed in both the TUG1- and LCAL6-silenced groups compared to the control group (p<0.01). The TUG1-silenced group showed more reduced Ki67 expression than the LCAL6-silenced group (p<0.05). PDX NSCLC models were established with a high degree of similarity with the original tumor with regard to histological, immunohistochemical features and RNA expression of TUG1 and LCAL6. Silencing of TUG1 inhibited both tumor growth and expression of the proliferation marker Ki67 and HOX-gene family HOXB7 in the PDX model of NSCLC.

Establishment of a human primary pancreatic cancer mouse model to examine and investigate gemcitabine resistance.

Pancreatic cancer is one of the most fatal types of cancer and is associated with a dismal prognosis. Gemcitabine-based chemotherapy is clinically used for the treatment of advanced pancreatic cancer. However, many forms of pancreatic cancer have acquired resistance to gemcitabine. In order to prevent patients from suffering from the side effects of chemotherapy and to have the chance to receive more effective intervention, assessment of whether the patient pancreatic cancer cells are resistant to gemcitabine before clinical practice is crucial. Recently, patient-derived xenograft (PDX) models have been regarded as a practical approach for preclinical drug resistance test. In the present study, we harvested tumor specimens from 28 pancreatic cancer patients to establish PDX models. The tumor formation rate of the xenografts was 100%, several of which could be re-implanted in nude mice for more than 10 passages. Primary cells were further obtained from the PDX xenografts to determine their morphological features and evaluate their proliferation rate, migration capacity and angiopoietic ability. In addition, the sensitivities of the primary cells and PDX xenografts to gemcitabine were correlated with each other. When compared to the gemcitabine-sensitive cells, the gemcitabine-resistant cells had a higher level of MCF2L expression, suggesting that MCF2L plays an important role in gemcitabine resistance.

Investigation of factors affecting the efficacy of 3C23K, a human monoclonal antibody targeting MISIIR.

MISIIR is a potential target for ovarian cancer (OC) therapy due to its tissue-specific pattern of expression. 3C23K is a novel therapeutic monoclonal anti-MISIIR antibody designed to recruit effector cells and promote cell death through ADCC (antibody dependent cell-mediated cytotoxicity). Our objective was to determine the tolerability and efficacy of 3C23K in OC patient-derived xenografts (PDX) and to identify factors affecting efficacy. Quantitative RT-PCR, immunohistochemistry (IHC), and flow cytometry were used to categorize MISIIR expression in established PDX models derived from primary OC patients. We selected two high expressing models and two low expressing models for in vivo testing. One xenograft model using an MISIIR over-expressing SKOV3ip cell line (Z3) was a positive control. The primary endpoint was change in tumor size. The secondary endpoint was final tumor mass. We observed no statistically significant differences between control and treated animals. The lack of response could be secondary to a number of variables including the lack of known biomarkers of response, the low membrane expression of MISIIR, and a limited ability of 3C23K to induce ADCC in PDX models. Further study is needed to determine the magnitude of ovarian cancer response to 3C23K and also if there is a threshold surface expression to predict response.

Abstract 3885: Establishment, characterization and drug response of primary and metastatic prostate organoids

Abstract Introduction &amp; objectives: Prostate cancer (PCa)-associated mortality results from metastasis to bone and resistance to androgen deprivation or cytotoxic therapy. Despite early detection of primary PCa, advanced castration resistant prostate cancer (CRPC) and bone metastases (BM) are detected in 10% of patients already at the time of initial diagnosis. The majority of recurrences might be due to cancer cells with stem cell-like properties (cancer stem cell-like, CSC-like). These could be therapy-resistant in a dormant state at the primary site or have metastasize prior to diagnosis of the primary tumor. CSC-like cells are the most tumorigenic and metastatic, however, current treatments target the differentiated tumor bulk cells. Understanding the mechanisms of PCa tumor initiation and metastasis by CSC-like cells is crucial for proper prognosis of high risk patient groups. Materials &amp; methods: To model CSC-like cells we generated organoids from patient-derived tissues (here termed as “canceroids”) and established patient-derived xenografts (PDXs). Canceroids were derived from bulk tumor tissues; primary PCa, bone metastasis from PCa tissue (BM-PCa), established PDX models LAPC9 and BM18 and lymph node (LN) metastasis from PCa (LAPC4) PDX models. Cytotoxic compounds and androgen inhibitors are tested on the canceroids from different BM or LN tissues using viability assays (CellTiter Glo assay) and 3D imaging by confocal and light sheet microscopy. Results: We have generated several canceroid lines from human primary PCa and from established PDX models that maintain key features of the CSC-like cells, previously characterized on the tumor itself (AR, PSA, Cytokeratins). The luminal phenotype of BM18 is evidently maintained in the BM18 canceroids, based on positive cytokeratin (CK)18 and absent CK5 expression. LAPC4 organoids contain CK5 and CK18 cells, in line with the mixed basal and luminal phenotype. Imaging of PSA and cytokeratin distribution confirms the luminal phenotype of the canceroids. BM18 and LAPC9 canceroids are maintained both in presence and absence of dihydrotestosterone indicating that androgen independent growth properties of the tumor are conserved in the canceroids. Viability assays indicate that canceroids respond to chemotherapeutics (cabacitaxel, docetaxel) and partially to hormone inhibitors (abiraterone, enzalutamide). Conclusions: Identification of the oncogenic properties of metastatic CSC-like (sub)populations has both prognostic and therapeutic applications. Establishment of CSC-derived organoids is the first step towards routine derivation from metastasis or primary PCa tissues as a potential platform for personalized drug compound evaluation. Citation Format: Sofia Karkampouna, Federico la Manna, Eugenio Zoni, Lijkele Beimers, Peter Kloen, Antoinette Wetterwald, Joël Grosjean, Irena Klima, Marco Giovanni Cecchini, Martin Spahn, George Niklaus Thalmann, Marianna Kruithof- de Julio. Establishment, characterization and drug response of primary and metastatic prostate organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3885. doi:10.1158/1538-7445.AM2017-3885

Abstract 1935: Molecular characterization of orthotopic patient-derived xenograft models of pediatric brain tumors

Abstract [Introduction: Solid tumors of the nervous system are the most common childhood cancers after leukemias. Even though we might be able to cure more and more patients, survivors still severely suffer long-term from the intensive treatments. Therefore, new treatment strategies are urgently needed. Orthotopic patient-derived xenograft (PDX) models are an excellent platform for biomarker and preclinical drug development. However, the rarity of pediatric brain tumors and the multitude of different sub entities hinder the generation of large collection of PDX models of specific entities within single institutions. In order to generate an overview about existing PDX models in the community, we started collecting established PDX models from various centers all over the world and performed extensive molecular characterization to precisely determine the distinct molecular subgroup and constellation of genetic alterations for each PDX model, and thus identify its targetable oncogenic drivers. Material and Methods: PDX models were established and maintained by dissociating tumor material into a single cell suspension and then orthotopically injecting it into the brain of immunodeficient animals. All PDX models and matching primary tumors (if available) have been analyzed by whole-exome and low-coverage whole-genome sequencing, as well as DNA methylation and gene expression profiling at the German Cancer Research Center (DKFZ). Results and Discussion: Thus far, we have collected and characterized 70 established PDX models from 6 ATRTs, 8 ependymomas, 16 high-grade gliomas, 38 medulloblastomas, and 2 CNS-PNETs. PDX models always retain their molecular subtype and in the vast majority of cases also the mutations and copy number alterations when compared to their primary tumors. Only in rare cases do we observe additional aberrations, which most likely represent outgrowths of subclones from the primary tumor. Analysis of our entire cohort identified an overrepresentation of the most aggressive tumor subtypes, but also subtypes which have not been available for preclinical testing before due to lack of genetically engineered mouse models or suitable cell lines, such as Group 4 medulloblastoma. Based on our current analysis, the PDX models within the community are not yet covering the entire heterogeneity within the patient population. As a follow up, we aim to make these models and data accessible in a user-friendly manner so that the community can use them for preclinical research. Conclusion: PDX models of pediatric brain tumors are very rare. Our molecular characterization allows researchers all over the world to find the right models for their specific scientific question. Therefore, this work will provide an unprecedented resource to study tumor biology and pave the way for improving treatment strategies for children with malignant brain tumors. Citation Format: Sebastian Brabetz, Susanne N. Gröbner, Huriye Seker-Cin, Florian Selt, Till Milde, David T. Jones, Madison T. Wise, Jessica M. Rusert, Kyle Pedro, Andy Strand, Olaf Witt, Sarah E. Leary, Xiao-Nan Li, Robert J. Wechsler-Reya, James M. Olson, Stefan M. Pfister, Marcel Kool. Molecular characterization of orthotopic patient-derived xenograft models of pediatric brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1935. doi:10.1158/1538-7445.AM2017-1935

Abstract 5015: Innovative and predictive models against breast cancer

Abstract Many reports support that xenografts from patient-derived xenografts (PDXs) in mice or rats well recapitulate the molecular diversity, cellular heterogeneity, and histology seen in patient tumors. However, shortcomings including limited clinical diversity of the PDXs, absence of influence of human microbiome, absence of drug human metabolism and reduced immune system restrain the predictive values of these PDX models. To set-up a holistic integration of these criticisms, we have associated efforts from public hospitals, academic groups, biotechs and private pharmaceutical companies with the financial support of the French Ministry of Industry. Patient surgical specimens from different breast tumor subtypes, among 9 other cancer pathologies, are collected to establish large collections of PDXs in mice. In addition, in vitro primary cultures of cells from these samples are conducted to establish a collection of cell lines from the stromal and the tumoral compartments. The 40 established breast PDX models represent the 4 major molecular clinical subtypes among which 37% are triple negative, 33% are Luminal B HER2-, 22% are Luminal B Her2+ and 8% Her2+. The models are evaluated for ex vivo and in vivo sensitivities to 4 clinically relevant anticancer drugs, for histology, immune infiltrates, and molecular characteristics including the analysis of gene polymorphism, RNAseq gene expression and NGS exome sequencing of 112 genes. The metagenomic sequencing of the gut microbiota from stools collected in both patients and tumor-bearing mice before and after chemotherapeutic treatments improves our knowledge on the role of microbiota in cancer progression and treatment. Secondly, to increase the predictability of the models, we are generating mice with humanized liver showing distinct drug pharmacokinetic profiles as compared with parental mice. Finally, the humanization of the immune system in mice is developed by several approaches including the use of induced pluripotent stem cells from cancer patients. These tumor collection and model characterization were performed under harmonized procedures within the consortium, allowing high quality material and results. All model characteristics are being compiled in a web-based database for efficient features search and data mining. We will present the first characterized breast cancer models and will discuss their usefulness and chance to bring benefit to patients via this holistic strategy developed within the IMODI initiative. Citation Format: Olivier Duchamp, Severine Tabone-Eglinger, Caroline Mignard, Isabelle Goddard, Alain Bruno, Maia Chanrion, Loreley Calvet, Olivier Degoul, Kevin Dhondt, Liliane Goetsch, Julie Miralves, Christophe Lautrette, Cindy Pensec, Françoise Le Vacon, Isabelle Treilleux, Laurent Arnould, Philippe Vaglio, Grégoire Prevost, Olivier Tredan. Innovative and predictive models against breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5015. doi:10.1158/1538-7445.AM2017-5015

Abstract 3861: Development of testicular cancer patient derived xenograft models to test combination therapies targeting PI3K/Akt and MDM2

Abstract Metastatic testicular cancer (TC) is highly sensitive to cisplatin-based chemotherapy. However, patients with advanced disease in the poor prognosis group only have a 50% 5-year survival resulting from chemoresistance. Previous data showed that both the PI3K/Akt pathway and the MDM2/p53 axis are involved in cisplatin resistance of TC cells. In this study we aim to establish and characterize TC patient-derived xenografts (PDX) in order to investigate PI3K/Akt and/or MDM2 inhibition as possible treatment options for TC. Excised and minced (8mm3) primary TC tissue was implanted subcutaneously into male NSG mice to obtain a first generation PDX model (F1). Animals were sacrificed when tumor volume exceeded 1500 mm3. The tumor was harvested and minced. Small pieces (8mm3) were implanted in a second generation (F2), as well as stored in FCS-5% DMSO to establish a biobank. In a panel of TC cell lines, combination treatment of Akt inhibition with or without cisplatin or Nutlin-3a was performed. Cleaved caspase-3 staining was assessed to measure apoptosis, and western blot was used to determine the effect of different treatments on sub-cellular localization and phosphorylation status of MDM2. We have established 3 TC PDX models. Histology of the primary tumors included the following subtypes: mixed germ cell tumors with embryonal- and yolk sac carcinoma and teratoma components. Engraftment efficacy was 100% and tumor growth initiated within 4-5 weeks after implantation. Interestingly, we successfully implanted a biopsy taken from a metastatic lesion of a patient presenting with progressive refractory disease after receiving standard chemotherapy. Histology of the different PDX generations was comparable to the patient material, although a loss of the teratoma component was observed. In our cell line panel, Akt inhibition using MK2206 sensitized the acquired cisplatin resistant cell line TeraCP towards cisplatin treatment, whereas an additive effect was observed in other cell lines. Combination of Akt and MDM2 inhibition was highly synergistic in apoptosis induction in all cell lines. No relation was observed between the synergistic effect of Akt combined with MDM2 inhibition and sub-cellular localization or phosphorylation levels of MDM2. Taken together, we have successfully established 3 TC PDX models, including a chemo-resistant model. The biobank is currently being expanded. Combined Akt and MDM2 inhibition resulted in synergistic induction of apoptosis, and these combinations or other novel therapies will be tested in established TC PDX models. Supported by Dutch Cancer Society grant RUG 2014-6691 and CONACYT grant 381543 Citation Format: Gerda de Vries, Ximena Rosas-Plaza, Marcel A. van Vugt, Albert J.H. Suurmeijer, Jourik A. Gietema, Steven de Jong. Development of testicular cancer patient derived xenograft models to test combination therapies targeting PI3K/Akt and MDM2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3861. doi:10.1158/1538-7445.AM2017-3861

Abstract 5674: Cancer immunogenomics for the development of personalized ovarian cancer vaccine

Abstract Ovarian cancer (OC) is the fifth leading cause of cancer death in the United States with approximately 20,000 women being diagnosed every year. Since OC development is mainly asymptomatic, patients are often diagnosed at late stage and present local and distal metastases. This offers a clinical challenge, as roughly 70% of the patients develop chemoresistant disease. With the advent of cancer immunogenomics it is now possible to identify tumor specific mutations, or neoantigens/neoepitopes, that can be exploited for the development of personalized T cell therapies via DC vaccines or adoptive cell therapy (ACT). We therefore hypothesize that tumor cells isolated from malignant ascites reflect the genomic landscape of the primary tumor, offering a unique opportunity to identify clinically actionable tumor neoantigens. We utilized primary tumor, tumor cells isolated from malignant ascites and metastatic samples to create patient-derived xenografts (PDX) mice and to interrogate the tumor mutational landscape. Somatic mutations were identified by whole exome sequencing (WES) and RNASeq analysis in tumor samples, using PBMCs as germline control. In parallel 20 PDX of ovarian cancer were established in NSG mice by injecting 2×106 cells/mouse subcutaneously (SQ, n=10) and intraperitoneally (IP, n=10). Tumor growth was monitored weekly via caliper measurement for mice injected SQ and via abdominal circumference for mice injected IP. Mutational analysis and prediction of high affinity peptides was done using bioinformatics approach. We have utilized different algorithms that are based on Artificial Neural Networks or published motifs for predicting binding of 8-11 mer peptides to number of Human MHC alleles (HLA) and out of 168 mutated peptides identified, 30 were found to have high affinity for different HLAs compared to wild type peptide. Mutated and wild type peptides were pulsed into dendritic cells and co-cultured with CD8+ T cells for 20 days. Validation of stimulated CD8+ T cells by these mutated peptides using ELISPOT is underway. TCR specific for neoantigen from CD8+T cells will be identified and cloned using retroviral vectors. Transfected CD8+ T cells with neoantigen specific TCR will be confirmed by co-culturing them with dendritic cells pulsed with wild type and mutated peptides. Further engineered CD8+ T cells specific for neoantigens will be tested for adoptive cell therapy in the established PDX models of ovarian cancer. Citation Format: Muzamil Y. Want, Takemasa Tsuji, Richard Koya, Sebastiano Battaglia. Cancer immunogenomics for the development of personalized ovarian cancer vaccine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5674. doi:10.1158/1538-7445.AM2017-5674

Abstract B28: Development of next-generation breast cancer PDX models by applying intra mammary fat pad and intraductal tumor transfer

Abstract Introduction: Worldwide, breast cancer is the second most common cancer after lung cancer and the leading cause of cancer death in women. These facts underline the urgent need for improved therapeutic options. However, with marked progress in the understanding of breast cancer biology, it has become obvious that breast cancer is not a single disease but a collection of very heterogeneous entities both between patients as well as within a tumor. This variability poses a major challenge for translational research and the development of new drugs. Most preclinical models fail to reproduce this inter- and intra-tumor heterogeneity which might at least partly contribute to the unacceptably high attrition rates in clinical testing. Although traditionally used in vivo models such as cell line xenografts have been of great value in the past, with the advent of personalized health care, improved models are required to properly reflect the complex physiology. Aim and experimental procedure: To this end, we aim at the development and evaluation of orthotopic patient-derived xenograft (PDX) models for breast cancer. Such PDX models have been shown repeatedly to preserve some tumor heterogeneity and a genetic profile similar to the original primary tumor. Specifically, two different approaches are tested to transfer primary tumor material to orthotopic locations in immunodeficient mice. On the one hand, small tumor fragments are transplanted into the mammary fat pad (i.mfp) and secondly, intraductal injection is applied for tumor cell transfer. We hypothesize that the intraductal implantation of primary tumor cells mimics breast carcinogenesis more closely than existing xenografts. Consequently, such a model would provide a unique system to study the whole spectrum of tumor progression to invasive and metastatic disease. Results: So far, five subcutaneously established PDX models, which were used for proof-of-concept studies, successfully engrafted using i.mfp transfer. As expected, histological analysis showed that tumor characteristics, such as expression of Her2, were faithfully retained in the grafted tumors. Intriguingly, two of the established PDX models tested were successfully injected intraductally and yielded invasive mammary carcinomas. These, to our knowledge, are the first breast cancer PDX models shown to grow invasively upon intraductal transfer. Furthermore, preliminary results showed an enhanced penetration of the tumor by lymphatic vessels after orthotopic transfer, especially after intraductal injection, compared to ectopic transplantation (subcutaneous), suggesting a more physiological growth and vessel supply. On top, we have observed tumor engraftment and invasive growth of seven primary samples (patient surgical specimen) upon i.mfp transfer, one of which also developed from intraductal injection, confirming that invasive tumors can form upon direct injection of patient material into milks ducts of mice. Conclusion and Outlook: Taken together, we want to create novel preclinical models for breast cancer which are able to provide a good phenocopy of several subtypes of human tumors and can consequently serve as models with improved predictivity. First results indicate that orthotopic transfer of breast PDX models displays a very physiological modeling of breast cancer and, importantly, that the most orthotopic placement of tumor cells (intra-ductal injection) allows invasive growth of breast tumor cells. In the future, we plan to include further models for intraductal injection to allow a thorough comparison between the two techniques and a reciprocal comparison to the original tumor. Additionally, application of the two implantation techniques in an immune-system humanized mouse background could provide very complex and physiological preclinical tools for cancer immunotherapeutic drug candidates. Citation Format: Lena Vockentanz, Adam Nopora. Development of next-generation breast cancer PDX models by applying intra mammary fat pad and intraductal tumor transfer. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B28.

Abstract A25: Establishment of orthotopic patient-derived xenograft models of pediatric brain tumors – the Heidelberg experience

Abstract Solid tumors of the nervous system are the most common childhood cancers after leukemias. Although brain tumors are the leading cause of cancer-related mortality in children, there are not enough adequate model systems to study their biology. We therefore started a pediatric preclinical testing program in Heidelberg to generate orthotopic patient-derived xenograft (PDX) models for a large variety of pediatric brain tumors. Freshly dissected primary material from multiple centers is being sent to us immediately after surgical resections. One part of the tumor is being reserved for pathological and molecular analysis and the other part is being dissociated into a single cell suspension and injected into the brain of immunodeficient mice. After successful engraftment and passaging, extensive molecular characterization of the PDX tumor and the matching primary tumor are being performed. Thus far, we have injected 95 tumors: 36 low-grade gliomas (LGG), 23 medulloblastomas (MB), 13 ependymomas (EPN), 7 high-grade gliomas (HGG), 6 atypical teratoid rhaboid tumors (AT/RT), 3 meningeal tumors (MT), 3 embryonal tumors with multilayered rosettes (ETMR), 2 gliomatosis cerebri (GC), 1 dysembryoplastic neuroepithelial tumor (DNT) and 1 primitive neuroectodermal tumor (PNET). No engraftment was observed for any of the low-grade tumors (LGG, MT, DNT). For high-grade tumors we established initial engraftments of MB (5/23, 22%), EPN (5/13, 38%), HGG (2/7, 29%), AT/RT (2/6, 33%), ETMR (1/3, 33%) and PNET (1/1, 100%). 11 out of 16 (69%) established PDX models were already passaged at least twice in mice and can be used for preclinical experiments. We conclude that it is possible to generate preclinical models for most malignant pediatric brain tumor entities, but not for low-grade tumors using our current protocol. However, even for the malignant entities there seems to be a selection for only the most aggressive subtypes that successfully engraft. Therefore, due to the low engraftment rate of some tumor types, the rarity of pediatric brain tumors and the multitude of different subtypes, international collaborations are absolutely necessary in this field in order to generate and characterize a broad repertoire of PDX models for all pediatric brain tumor subtypes for preclinical testing. Citation Format: Norman L. Mack, Sebastian Brabetz, Florian Selt, Xanthopolous Christina, David Sumerauer, Heidi Bächli, Arnulf Pekrun, Martin U. Schuhmann, Stefan M. Pfister, Olaf Witt, Till Milde, Marcel Kool. Establishment of orthotopic patient-derived xenograft models of pediatric brain tumors – the Heidelberg experience. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A25.