- Email: [email protected]
Actionable mutations in cancers: Human vs. patient derived xenografts
Poster Session – Preclinical Models, Wednesday 29 November 2016 137 Poster (Board P108) Integrated pathway analysis of malignant rhabdoid tumour identifies key SMARCB1-pathways and therapeutic opportunities M.A. Finetti1 , M.P. Selby1 , A. Del Carpio Pons1 , J.P. Wong2 , M. Bashton1 , R.E. Cranston1 , J. Barker1 , S. Crosier1 , A. Smith1 , R.A. Ramli1 , Y. Grabovska1 , S. Bailey1 , P.H. Huang2 , S.C. Clifford1 , D. Williamson1 . 1 Newcastle University, NICR, Newcastle, United Kingdom; 2 The Institute of Cancer Research, Division of Cancer Biology, London, United Kingdom Background: Malignant Rhabdoid Tumours (MRTs) are aggressive paediatric tumour with very poor prognosis and life expectancy. These malignancies are characterized by biallelic inactivation of SMARCB1 a core subunit of the hSWI/SNF chromatin remodelling complex. SMARCB1 re-expression in MRT cells leads differentiation and growth arrest, deregulating hundreds of downstream genes/pathways. 450k array, RNA-seq, CHIP and CRISPR screening were used to identify SMARCB1 dependent downstream pathways and novel SMARCB1-dependent therapeutic targets that can improve the survival of MRT patients. Material and Methods: RNA-seq and 450k-methylation analyses in MRT human primary malignancies (n > 40) and in 4 MRT cell lines in which lentivirus was used to re-express SMARCB1 (G401, A204, CHLA-266, and STA-WT1) as well as 5’-Aza-cytidine demethylase treatment were used to investigate the downstream effects effects of SMARCB1 loss. Following SMARCB1 re-expression cells were expression and methylation profiled to identify genes/pathways regulated by SMARCB1. Cells with and without SMARCB1 re-expression were further screened for functional susceptibilities using the whole-genome GeCKO (Genome Scale CRISPR Knock-Out) library which contains 122441 guide constructs targeting 19,050 genes and 1,864 miRNA. Key pathways were identified by integrated bioinformatic analysis and validated functionally. Results: Integrated network-based pathway analysis identifies several key SMARCB1-dependent and potentially druggable genes/pathways deregulated at the transcriptional/methylation level in primary MRTs. Furthermore, CRISPR screening identifies gene targets which promote or inhibit growth/viability in a SMARCB1-dependent manner in our functional models. SMARCB1-dependent genes/pathway deregulation was verified and validated by targeted pathway manipulation and quantified by qRT-PCR and phenotypic changes. Key pathways/genes include but are not limited to E2F1, AURKA, BMI1, PLK1, SHH, Rho, TGF-b, PDGFRa, FGFR1, Wnt and STAT3/IL-6. PDGFRa, FGFR1, STAT3/IL-6, Wnt and TGFBeta mechanisms were further investigated in vitro using available inhibitors/drugs. Conclusions: Integrative analysis of -omics data using systems biology methods we have provided a catalogue of the key MRT tumourigenic genes/pathways changes in both primary and functional models. Rhabdoid cells showed sensitivity to pathway inhibition and cell death by dysregulation of several downstream SMARCB1-dependent oncogenes. We have isolated and validated critical genes/pathways which are targetable therapeutically and will lead to better treatments for what is currently one of the most lethal paediatric cancers known. No conflict of interest. 138 Poster (Board P109) Human HLA-A*02:01/CHM1+ allo-restricted T cell receptor-transgenic CD8+ T cells specifically inhibit Ewing sarcoma growth in vitro and in vivo U. Thiel1 , F. Blaeschke2 , G. Richter3 , S. Burdach3 . 1 Klinikum rechts der Isar, Pediatrics, Munich, Germany; 2 LMU, Pediatrics, Munich, Germany; 3 TUM, Pediatrics, Munich, Germany Background: The endochondral bone protein Chondromodulin-I (CHM1) provides oncogene addiction in Ewing sarcoma (ES). We pre-clinically tested the targetability of CHM1 by TCR transgenic, allo-restricted, peptide specific T cells to treat ES. We previously generated allo-restricted wildtype CD8+ T cells directed against the ES specific antigen CHM1319 causing specific responses against ES. However, utilization of these cells in current therapy protocols is hampered due to high complexity in production, relatively low cell numbers, and rapid T cell exhaustion. Material and Methods: In order to provide off-the-shelf products in the future, we aimed at generating HLA-A*02:01-restricted T cell receptor (TCR)-transgenic T cells directed against CHM1319 by retroviral transduction. These cells were tested in vitro and pre-clinically in Rag2−/− gc−/− mice. Results: After short-term expansion a 100% purified CHM1319TCR-transgenic T cell population expressed a CD62L+/CD45RO and CD62L+/CD45RA+ phenotype. These cells displayed specific in vitro IFNg and granzyme B release in co-culture with HLA-A*02:01+ ES cell lines expressing CHM1. When co-injected with ES cells in Rag2−/− gc−/− mice, CHM1-specific TCR-transgenic T cells significantly inhibited the formation of lung and liver metastases in contrast to control mice. Lungs and livers
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of representative mice displayed CD8+ T cell infiltration in the presence (control group treated with unspecific T cells) and in the absence (study group) of metastatic disease, respectively. Furthermore, mice receiving unspecific T cells showed signs of graft-versus-host disease in contrast to all mice receiving CHM1319-TCR-transgenic T cells. Conclusions: CHM1 specific TCR-transgenic T cells were successfully generated causing anti-ES responses in vitro and in vivo. In the future, CHM1319-TCR-transgenic T cells may control minimal residual disease rendering donor lymphocyte infusions more efficacious and less toxic. No conflict of interest. 139 Poster (Board P110) Identification of novel SEC61G-EGFR fusions in pediatric ependymoma T. Servidei1 , D. Meco1 , V. Muto2 , M. Tartaglia2 , R. Riccardi1 . 1 Catholic University "A. Gemelli" Hospital, Pediatric Oncology, Rome, Italy; 2 Ospedale Pediatrico Bambino Gesu, ` Genetics and Rare Disease Research Division, Rome, Italy Background: Ependymomas represent 10% of pediatric brain tumors, with 5-year survival rates at approximately 50%. Despite their histopathological similarities, ependymomas consist of heterogeneous molecular-genetic groups, arising from different glial stem cell (SC) populations. In cultures, SCs are forced into active proliferation by exogenous EGF/bFGF. Culture conditions might exert negative/positive selection pressure, leading to clonal expansion of genetically heterogenous tumor subpopulations. We addressed whether omission of EGF/bFGF might select mitogenindependent (MI) cells present in ependymomas and the underlying aberrant signaling. Material and Methods: The EPP-MI line was established by culturing the ependymoma SC line EPP in mitogen-free media. Biological and molecular features of lines (proliferation, tumorigenicity, expression of stemness markers, EGFR signaling and subcellular localization, sensitivity to the EGFR inhibitors gefitinib and AEE788) were assessed through standard laboratory assays. The sequences of SEC61G-EGFR fusions and presence in ependymomas were analyzed through RNA-seq, RT-PCR and Sanger sequencing. Results: Compared to EPP, EPP-MI cells displayed impaired proliferation in vitro, but much higher tumorigenicity, despite a lower expression of stemness markers. A constitutively activated, truncated EGFR was found in EPP-MI cells, that was associated with AKT and STAT3 phosphorylation and blocked by EGFR inhibitors. By transcriptome sequencing and validation, we discovered that EPP-MI cells harbored an intrachromosomal rearrangement that fused in frame EGFR downstream to Sec61G5’UTR. The SEC61G-EGFR fusions lacked the ligand-binding domain of EGFR, while retaining the transmembrane and the tyrosine kinase coding domains. Similarly to EGFR, the majority of SEC61G-EGFR protein was bound to the membranes. High levels of constitutively activated STAT3 was found in the nucleus of EPP-MI cells, whereas in EPP cells nuclear STAT3 was phosphorylated only in response to EGF. Compared to EPP, EPP-MI cells were three-fold more sensitive to the antiproliferative effects of EGFR inhibitors in vitro. In orthotopic models, AEE788 did not exert significant antitumoral activity on EPP xenografts, whereas it significantly prolonged survival of mice bearing EPP-MI xenografts (log-rank, P = 0.02). RT-PCR sequencing detected SEC61G-EGFR transcripts in one of 17 pediatric ependymomas. Conclusions: 1) In ependymoma SCs, SEC61G-EGFR fusions are associated with higher tumorigenicity, aberrant EGFR/STAT3 signaling, and sensitization to EGFR inhibitors both in vitro and in vivo. 2) SEC61G-EGFR fusions are expressed in pediatric ependymomas and might represent a drugable target. 3) Culture conditions might expand subpopulations present in tumors and unravel new driver mutations. (Supported by Fondazione per l’Oncologia Pediatrica). No conflict of interest.
Preclinical Models 140 Poster (Board P111) Actionable mutations in cancers: Human vs. patient derived xenografts L.J. Xue1 , W. Qian1 , S. Guo1 , J. Cai1 , H. Li1 . 1 crown bioscience, translational oncology, Santa Clara, USA Background: Precision cancer medicine targets specific genetic abnormality, holding great promise. It is at present largely built on the proposed “actionable” somatic mutations found in patients, but their validation in various patient populations remains to be the major challenges. PDX is
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Poster abstracts
Poster Session – Preclinical Models, Wednesday 29 November 2016
a well-accepted predictive experimental model mimicking original patient’s in histo- and molecular pathology, per accumulated anecdote data, and broadly used to assess drug efficacy, discover biomarker, guide clinical development/use of cancer therapy, and thus, potentially validate actionable mutations provided the presence of comparable actionable mutation profiles in patient and PDXs. Methods: 8147 patients in TCGA datasets for a set of 50 genes with frequent actionable somatic mutations were identified and compared to those from a cohort of 749 PDXs per RNA-seq data of CRC, NSCLC, PAN, GA, H&N, ES, OV, HCC, where only mutations in both datasets were used and those in dbSNP removed. Frequencies of mutated genes were compared with and without weighing sample number per disease types. Results: 513 PDXs (68.5%) have at least one potentially actionable mutation, comparable to TCGA patients (61.3%). In PDXs, TP53 has the highest mutation rate (47.4% un-weighted, 35.2% weighted respectively), followed by APC (15.6%, 16.7%), ATM (5.07%, 6.09%), FBXW7 (4.01%, 5.08%), PIK3CA (3.07%, 4.07%), CDKN2A (2.40%, −2.88%) and other genes. Similar results were observed in TCGA patients with TP53 having the highest mutation rate (26.4%, 43.8%), followed by APC (6.53%, 12.7%), PTEN (6.21%, 3.86%), PIK3CA (5.45%, 5.0%), ATM (5.11%, 6.96%). The difference between un-weighted and weight reflects its uneven occurrence among cancers. In PDXs, 22.3% models have >1 gene with potentially actionable mutations, also comparable to TCGA patient data (33.1%). Furthermore, 22.0% of PDXs have mutations that are likely to affect protein function, based on SIFT and Polyphen-2 prediction, which is higher than patients (13.8%). We have also validated some of the actionable mutations in PDXs. Conclusions: The profile of putative actionable mutations in patient hints potential for target therapy, whereas comparable profile of PDX suggests being an ideal experimental system to test validity of precision medicine. No conflict of interest. 141 Poster (Board P112) Identifying targeted therapies to eliminate chemo-resistant LSCs and improve disease-free survival in AML disseminated models C. Zhang1 , Z. Yan2 , Q. Zong3 . 1 Pfizer Inc, ORU, San Diego, USA; 2 Pfizer, ORU, San Diego, USA; 3 Pfizer, DSRD, San Diego, USA Background: Leukemic stem cell is one of the leading causes for disease recurrence in acute myeloid leukemia (AML) patients as it lacks sensitivity to conventional therapy. In this report, we aimed to understand whether leukemic stem cell (LSC) plays a key role for developing chemoresistance after treatment of high dose induction therapy in AML models, and subsequently to identify targeted agents that can eliminate LSC population and enhance chemotherapy. Material and methods: AML models were developed by introvenously injecting MV4−11 or PDX cells in NSG (NOD/SCID IL2RG−/− ) mice. Disease progression in AML disseminated models was tracked either by in vivo bioluminescence imaging or via ex vivo flow cytometry analysis of AML in peripheral blood and bone marrow. Immunophenotyping of the bone marrow residual disease were performed. Results: In the tested AML models, daunorubicin/cytarabine (DA) chemotherapy showed early antileukemic efficacy through apoptosis induction and antiproliferation. Unfortunately, treatment showed minimal survival benefits despite early antileukemic effects. In the DA residual diseases of the BM2407 and BM0407 AML PDX models, flow cytometry analyses showed enriched fractions of CD33+/CD34+ and CLL1+/CD117− subpopulations, respectively. Subsequently, we performed self-renewal functional test by in vivo limiting dilution assay using sorted CD33+/CD34+ and CLL1+/CD117− cells. These chemoresistant CD33+/CD34+ or CLL1+/CD117− cells were much more tumorigenic than their counterpart CD33+/CD34− and CLL1+/CD117+ cells, respectively. By combing chemotherapy with targeted therapeutic agents including palbociclib and CD33-ADC, we observed significant improved survival of the AML disease mice compared to the single agent treatment. In the mechanistic studies, the chemoresistant LSCs highly express CD33 thus can be eliminated by CD33-ADC. Palbociclib promotes the premature entry of quiescent LSCs into S phase therefore enhanced the cytotoxicity by chemotherapy. Conclusion: These study results revealed that the anti-LSC effect of these targeted agents played a key role for the combinatorial benefits with chemotherapy. Conflict of interest: Ownership: Pfizer Inc. Other Substantive Relationships: Authors are Pfizer employees.
142 Poster (Board P113) New preclinical models for neuro-oncology: Orthotopic patientderived glioblastoma xenografts in mice 3 A. Orthmann1 , J. Hayback ¨ 2 , A. Jodicke ¨ , S. Kuhn4 , M. Linnebacher5 , I. Fichtner1 , J. Hoffmann1 . 1 EPO GmbH, Experimental Pharmacology & Oncology, Berlin, Germany; 2 Institute of Pathology, Medical University of Graz, Neuropathology, Graz, Austria; 3 Vivantes medical centre Berlin, Clinic for Neurosurgery, Berlin, Germany; 4 Medical centre Ernst von Bergmann Potsdam, Clinic for Neurosurgery, Potsdam, Germany; 5 Medical University of Rostock, Molecular Oncology and Immunotherapy, Rostock, Germany
Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults. These tumors frequently show a very heterogeneous, diffuse infiltrative and aggressive growth with a mean survival time between 8 and 18 months. The current standard care for glioma patients is of limited benefit. To support basic and translational research initiated the development of new preclinical glioma models from patient tumors. We started with subcutaneous (s.c.) transplantation of 85 glioma tissue samples to immunodeficient mice and were able to establish and characterize 21 novel patient-derived xenograft (PDX) models (engraftment rate 25%). To better mimic the crucial interaction of human gliomas with host microenvironment (endothelial, stromal and immune cells, extracellular matrix) we utilized orthotopic transplantation, since tumor-host interactions are frequently organ specific. Up to now, 8 of the established PDX models were also transplanted intracranially (orthotopic) by an injection of 50.000 cells in the right hemisphere. After successful engraftment, both the s.c. and the orthotopically transplanted glioma PDX were screened for sensitivity towards a broad drug panel (everolimus, sorafenib, bevacizumab, irinotecan, salinomycin, thalidomide, regorafenib or temozolomide) in monotherapy or combinations. In the s.c. model system, the strongest response was induced by bevacizumab (7/21), irinotecan (15/21), and temozolomide (16/21), whereas the other drugs had no activity. From the evaluated combination therapies, we learned that combinations of temozolomide with other drugs did not provide any additive effects. Interestingly first data showed differences in sensitivity levels to treatments between orthotopic and s.c. transplantation of the same PDX model. We found for example drugs bevacizumab, irinotecan and everolimus less active in the orthotopic model. Bioinformatic analysis did not reveal a correlation between common “oncomutations” (Illumina TrueSeq Cancer panel) and drug sensitivity. The most frequent mutations in our glioma PDX were KDR, FGFR3, PIK3CA, PTEN, P53, NOTCH1 and KIT. Histopathological analyses demonstrate that our glioma PDX models have retained the heterogeneity of the disease. Particularly the orthotopically transplanted glioma PDX are showing the original tumor histology and have better take rates in comparison to the s.c. engrafted tumors. The available panel of the broadly characterized 21 glioma PDX models provides an exceptional platform for translational oncology research projects, the identification and validation of new targets and biomarker candidates, screening of new drugs and combinations, as well as for personalizing patient treatment. No conflict of interest. 143 Poster (Board P114) Characterization of breast cancer preclinical models reveals a specific macrophage polarization D. Vallerand1 , G. Massonnet1 , F. Kebir2 , D. Gentien1 , Z. Maciorowski2 , P. De la Grange3 , S. Humbert4 , F. Assayag1 , L. De Plater1 , A. Nicolas2 , E. Marangoni1 , S. Weigand5 , S. Roman-Roman1 , A. Savina6 , D. Decaudin7 . 1 Institut Curie, Translational Research, Paris, France; 2 Institut Curie, Tumor Biology, Paris, France; 3 Genosplice, Genosplice technology, Paris, France; 4 Institut Curie, Research Department, Paris, France; 5 Roche, Oncology, Penzberg, Germany; 6 Institut Roche, Oncology, Boulogne Billancourt, France; 7 Institut Curie, Translatioinal Research, Paris, France Background: Tumor microenvironment has been the target of drug discovery effort in the last years. However few studies have addressed the characterization of the stroma component in patient-derived xenografts (PDXs) and genetically engineered models (GEMs). The aim of this study was to characterize the stroma displayed by different models of breast cancer tumors in mice. Materials and Methods: For this purpose, transcriptomic and flow cytometry analyses on murine populations were performed in a series of 25 PDXs and 2 most commonly used GEMs (MMTV-PyMT and MMTV-erBb2). Specifically, macrophages from 5 models were sorted and profiled by gene expression and phenotypically characterized by flow cytometry (FC).
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of representative mice displayed CD8+ T cell infiltration in the presence (control group treated with unspecific T cells) and in the absence (study group) of metastatic disease, respectively. Furthermore, mice receiving unspecific T cells showed signs of graft-versus-host disease in contrast to all mice receiving CHM1319-TCR-transgenic T cells. Conclusions: CHM1 specific TCR-transgenic T cells were successfully generated causing anti-ES responses in vitro and in vivo. In the future, CHM1319-TCR-transgenic T cells may control minimal residual disease rendering donor lymphocyte infusions more efficacious and less toxic. No conflict of interest. 139 Poster (Board P110) Identification of novel SEC61G-EGFR fusions in pediatric ependymoma T. Servidei1 , D. Meco1 , V. Muto2 , M. Tartaglia2 , R. Riccardi1 . 1 Catholic University "A. Gemelli" Hospital, Pediatric Oncology, Rome, Italy; 2 Ospedale Pediatrico Bambino Gesu, ` Genetics and Rare Disease Research Division, Rome, Italy Background: Ependymomas represent 10% of pediatric brain tumors, with 5-year survival rates at approximately 50%. Despite their histopathological similarities, ependymomas consist of heterogeneous molecular-genetic groups, arising from different glial stem cell (SC) populations. In cultures, SCs are forced into active proliferation by exogenous EGF/bFGF. Culture conditions might exert negative/positive selection pressure, leading to clonal expansion of genetically heterogenous tumor subpopulations. We addressed whether omission of EGF/bFGF might select mitogenindependent (MI) cells present in ependymomas and the underlying aberrant signaling. Material and Methods: The EPP-MI line was established by culturing the ependymoma SC line EPP in mitogen-free media. Biological and molecular features of lines (proliferation, tumorigenicity, expression of stemness markers, EGFR signaling and subcellular localization, sensitivity to the EGFR inhibitors gefitinib and AEE788) were assessed through standard laboratory assays. The sequences of SEC61G-EGFR fusions and presence in ependymomas were analyzed through RNA-seq, RT-PCR and Sanger sequencing. Results: Compared to EPP, EPP-MI cells displayed impaired proliferation in vitro, but much higher tumorigenicity, despite a lower expression of stemness markers. A constitutively activated, truncated EGFR was found in EPP-MI cells, that was associated with AKT and STAT3 phosphorylation and blocked by EGFR inhibitors. By transcriptome sequencing and validation, we discovered that EPP-MI cells harbored an intrachromosomal rearrangement that fused in frame EGFR downstream to Sec61G5’UTR. The SEC61G-EGFR fusions lacked the ligand-binding domain of EGFR, while retaining the transmembrane and the tyrosine kinase coding domains. Similarly to EGFR, the majority of SEC61G-EGFR protein was bound to the membranes. High levels of constitutively activated STAT3 was found in the nucleus of EPP-MI cells, whereas in EPP cells nuclear STAT3 was phosphorylated only in response to EGF. Compared to EPP, EPP-MI cells were three-fold more sensitive to the antiproliferative effects of EGFR inhibitors in vitro. In orthotopic models, AEE788 did not exert significant antitumoral activity on EPP xenografts, whereas it significantly prolonged survival of mice bearing EPP-MI xenografts (log-rank, P = 0.02). RT-PCR sequencing detected SEC61G-EGFR transcripts in one of 17 pediatric ependymomas. Conclusions: 1) In ependymoma SCs, SEC61G-EGFR fusions are associated with higher tumorigenicity, aberrant EGFR/STAT3 signaling, and sensitization to EGFR inhibitors both in vitro and in vivo. 2) SEC61G-EGFR fusions are expressed in pediatric ependymomas and might represent a drugable target. 3) Culture conditions might expand subpopulations present in tumors and unravel new driver mutations. (Supported by Fondazione per l’Oncologia Pediatrica). No conflict of interest.
Preclinical Models 140 Poster (Board P111) Actionable mutations in cancers: Human vs. patient derived xenografts L.J. Xue1 , W. Qian1 , S. Guo1 , J. Cai1 , H. Li1 . 1 crown bioscience, translational oncology, Santa Clara, USA Background: Precision cancer medicine targets specific genetic abnormality, holding great promise. It is at present largely built on the proposed “actionable” somatic mutations found in patients, but their validation in various patient populations remains to be the major challenges. PDX is
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Poster abstracts
Poster Session – Preclinical Models, Wednesday 29 November 2016
a well-accepted predictive experimental model mimicking original patient’s in histo- and molecular pathology, per accumulated anecdote data, and broadly used to assess drug efficacy, discover biomarker, guide clinical development/use of cancer therapy, and thus, potentially validate actionable mutations provided the presence of comparable actionable mutation profiles in patient and PDXs. Methods: 8147 patients in TCGA datasets for a set of 50 genes with frequent actionable somatic mutations were identified and compared to those from a cohort of 749 PDXs per RNA-seq data of CRC, NSCLC, PAN, GA, H&N, ES, OV, HCC, where only mutations in both datasets were used and those in dbSNP removed. Frequencies of mutated genes were compared with and without weighing sample number per disease types. Results: 513 PDXs (68.5%) have at least one potentially actionable mutation, comparable to TCGA patients (61.3%). In PDXs, TP53 has the highest mutation rate (47.4% un-weighted, 35.2% weighted respectively), followed by APC (15.6%, 16.7%), ATM (5.07%, 6.09%), FBXW7 (4.01%, 5.08%), PIK3CA (3.07%, 4.07%), CDKN2A (2.40%, −2.88%) and other genes. Similar results were observed in TCGA patients with TP53 having the highest mutation rate (26.4%, 43.8%), followed by APC (6.53%, 12.7%), PTEN (6.21%, 3.86%), PIK3CA (5.45%, 5.0%), ATM (5.11%, 6.96%). The difference between un-weighted and weight reflects its uneven occurrence among cancers. In PDXs, 22.3% models have >1 gene with potentially actionable mutations, also comparable to TCGA patient data (33.1%). Furthermore, 22.0% of PDXs have mutations that are likely to affect protein function, based on SIFT and Polyphen-2 prediction, which is higher than patients (13.8%). We have also validated some of the actionable mutations in PDXs. Conclusions: The profile of putative actionable mutations in patient hints potential for target therapy, whereas comparable profile of PDX suggests being an ideal experimental system to test validity of precision medicine. No conflict of interest. 141 Poster (Board P112) Identifying targeted therapies to eliminate chemo-resistant LSCs and improve disease-free survival in AML disseminated models C. Zhang1 , Z. Yan2 , Q. Zong3 . 1 Pfizer Inc, ORU, San Diego, USA; 2 Pfizer, ORU, San Diego, USA; 3 Pfizer, DSRD, San Diego, USA Background: Leukemic stem cell is one of the leading causes for disease recurrence in acute myeloid leukemia (AML) patients as it lacks sensitivity to conventional therapy. In this report, we aimed to understand whether leukemic stem cell (LSC) plays a key role for developing chemoresistance after treatment of high dose induction therapy in AML models, and subsequently to identify targeted agents that can eliminate LSC population and enhance chemotherapy. Material and methods: AML models were developed by introvenously injecting MV4−11 or PDX cells in NSG (NOD/SCID IL2RG−/− ) mice. Disease progression in AML disseminated models was tracked either by in vivo bioluminescence imaging or via ex vivo flow cytometry analysis of AML in peripheral blood and bone marrow. Immunophenotyping of the bone marrow residual disease were performed. Results: In the tested AML models, daunorubicin/cytarabine (DA) chemotherapy showed early antileukemic efficacy through apoptosis induction and antiproliferation. Unfortunately, treatment showed minimal survival benefits despite early antileukemic effects. In the DA residual diseases of the BM2407 and BM0407 AML PDX models, flow cytometry analyses showed enriched fractions of CD33+/CD34+ and CLL1+/CD117− subpopulations, respectively. Subsequently, we performed self-renewal functional test by in vivo limiting dilution assay using sorted CD33+/CD34+ and CLL1+/CD117− cells. These chemoresistant CD33+/CD34+ or CLL1+/CD117− cells were much more tumorigenic than their counterpart CD33+/CD34− and CLL1+/CD117+ cells, respectively. By combing chemotherapy with targeted therapeutic agents including palbociclib and CD33-ADC, we observed significant improved survival of the AML disease mice compared to the single agent treatment. In the mechanistic studies, the chemoresistant LSCs highly express CD33 thus can be eliminated by CD33-ADC. Palbociclib promotes the premature entry of quiescent LSCs into S phase therefore enhanced the cytotoxicity by chemotherapy. Conclusion: These study results revealed that the anti-LSC effect of these targeted agents played a key role for the combinatorial benefits with chemotherapy. Conflict of interest: Ownership: Pfizer Inc. Other Substantive Relationships: Authors are Pfizer employees.
142 Poster (Board P113) New preclinical models for neuro-oncology: Orthotopic patientderived glioblastoma xenografts in mice 3 A. Orthmann1 , J. Hayback ¨ 2 , A. Jodicke ¨ , S. Kuhn4 , M. Linnebacher5 , I. Fichtner1 , J. Hoffmann1 . 1 EPO GmbH, Experimental Pharmacology & Oncology, Berlin, Germany; 2 Institute of Pathology, Medical University of Graz, Neuropathology, Graz, Austria; 3 Vivantes medical centre Berlin, Clinic for Neurosurgery, Berlin, Germany; 4 Medical centre Ernst von Bergmann Potsdam, Clinic for Neurosurgery, Potsdam, Germany; 5 Medical University of Rostock, Molecular Oncology and Immunotherapy, Rostock, Germany
Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults. These tumors frequently show a very heterogeneous, diffuse infiltrative and aggressive growth with a mean survival time between 8 and 18 months. The current standard care for glioma patients is of limited benefit. To support basic and translational research initiated the development of new preclinical glioma models from patient tumors. We started with subcutaneous (s.c.) transplantation of 85 glioma tissue samples to immunodeficient mice and were able to establish and characterize 21 novel patient-derived xenograft (PDX) models (engraftment rate 25%). To better mimic the crucial interaction of human gliomas with host microenvironment (endothelial, stromal and immune cells, extracellular matrix) we utilized orthotopic transplantation, since tumor-host interactions are frequently organ specific. Up to now, 8 of the established PDX models were also transplanted intracranially (orthotopic) by an injection of 50.000 cells in the right hemisphere. After successful engraftment, both the s.c. and the orthotopically transplanted glioma PDX were screened for sensitivity towards a broad drug panel (everolimus, sorafenib, bevacizumab, irinotecan, salinomycin, thalidomide, regorafenib or temozolomide) in monotherapy or combinations. In the s.c. model system, the strongest response was induced by bevacizumab (7/21), irinotecan (15/21), and temozolomide (16/21), whereas the other drugs had no activity. From the evaluated combination therapies, we learned that combinations of temozolomide with other drugs did not provide any additive effects. Interestingly first data showed differences in sensitivity levels to treatments between orthotopic and s.c. transplantation of the same PDX model. We found for example drugs bevacizumab, irinotecan and everolimus less active in the orthotopic model. Bioinformatic analysis did not reveal a correlation between common “oncomutations” (Illumina TrueSeq Cancer panel) and drug sensitivity. The most frequent mutations in our glioma PDX were KDR, FGFR3, PIK3CA, PTEN, P53, NOTCH1 and KIT. Histopathological analyses demonstrate that our glioma PDX models have retained the heterogeneity of the disease. Particularly the orthotopically transplanted glioma PDX are showing the original tumor histology and have better take rates in comparison to the s.c. engrafted tumors. The available panel of the broadly characterized 21 glioma PDX models provides an exceptional platform for translational oncology research projects, the identification and validation of new targets and biomarker candidates, screening of new drugs and combinations, as well as for personalizing patient treatment. No conflict of interest. 143 Poster (Board P114) Characterization of breast cancer preclinical models reveals a specific macrophage polarization D. Vallerand1 , G. Massonnet1 , F. Kebir2 , D. Gentien1 , Z. Maciorowski2 , P. De la Grange3 , S. Humbert4 , F. Assayag1 , L. De Plater1 , A. Nicolas2 , E. Marangoni1 , S. Weigand5 , S. Roman-Roman1 , A. Savina6 , D. Decaudin7 . 1 Institut Curie, Translational Research, Paris, France; 2 Institut Curie, Tumor Biology, Paris, France; 3 Genosplice, Genosplice technology, Paris, France; 4 Institut Curie, Research Department, Paris, France; 5 Roche, Oncology, Penzberg, Germany; 6 Institut Roche, Oncology, Boulogne Billancourt, France; 7 Institut Curie, Translatioinal Research, Paris, France Background: Tumor microenvironment has been the target of drug discovery effort in the last years. However few studies have addressed the characterization of the stroma component in patient-derived xenografts (PDXs) and genetically engineered models (GEMs). The aim of this study was to characterize the stroma displayed by different models of breast cancer tumors in mice. Materials and Methods: For this purpose, transcriptomic and flow cytometry analyses on murine populations were performed in a series of 25 PDXs and 2 most commonly used GEMs (MMTV-PyMT and MMTV-erBb2). Specifically, macrophages from 5 models were sorted and profiled by gene expression and phenotypically characterized by flow cytometry (FC).