Generation Of Patient-derived Xenografts Research Articles

Abstract 6900: Patient-derived xenografts from circulating cancer stem cells and their potential utility in personalized medicine of pancreatic cancer

Abstract Background: Pancreatic cancer carries a poor prognosis in the majority of patients, with an overall 5-year survival rate of less than 10%. Therefore, the development of new therapies and preclinical models is crucial and urgently needed. Circulating cancer stem cells (cCSCs) are a subset of circulating cancer cells that have stem cell-like properties and play a significant role in tumor progression, metastasis, and treatment resistance. Patient-derived xenografts (PDX) reflect the genotypic and phenotypic landscape of patients' cancers and are useful for drug screening, biomarker development, and preclinical evaluation of personalized medicine strategies. In this study, we describe a fast, simple, and cost effective method to generate PDX on Chorioallantoic Membrane (CAM) from circulating cancer stem cells. Methods: In this study, primary cultures from circulating cancer stem cells were established using sphere-forming assays from 30 patients with newly diagnosed pancreatic cancer. Subsequently, tumorspheres were transplanted onto the CAM membrane of fertilized chicken eggs to form patient- derived xenografts. Results: For the identification of cCSCs from peripheral blood of pancreatic cancer patients, an innovative in vitro sphere-forming assay was established. The number of tumorspheres was correlated with clinicopathological parameters like tumor size, lymph node involvement and grading status of the primary tumor. Implantation of tumorspheres onto the CAM was successful in ~90 % of the applied samples. The histological analysis suggests that the PDX tumors were morphologically and pathologically identical to primary tumor tissue. Conclusions: The number of tumorspheres was associated with clinicopathological parameters. Further follow-up is needed to evaluate the prognostic significance of tumorsphere detection in pancreatic cancer. Generation of PDX from cCSCs on CAM has a high success rate and may become an integral part of the drug development arena, including drug screening and biomarker development. Citation Format: Monika Pizon, Dorothea Schott, Andreas Ettner-Sittner, Benedikt J. Wagner, Katharina Pachmann, Thiha Aung, Christina Hackl, Silke Haerteis. Patient-derived xenografts from circulating cancer stem cells and their potential utility in personalized medicine of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6900.

The Power and Promise of Patient-Derived Xenografts of Human Breast Cancer.

In 2016, a group of researchers engaged in the development of patient-derived xenografts (PDXs) of human breast cancer provided a comprehensive review of the state of the field. In that review, they summarized the clinical problem that PDXs might address, the technical approaches to their generation (including a discussion of host animals and transplant conditions tested), and presented transplantation success (take) rates across groups and across transplantation conditions. At the time, there were just over 500 unique PDX models created by these investigators representing all three clinically defined subtypes (ER+, HER2+, and TNBC). Today, many of these PDX resources have at least doubled in size, and several more PDX development groups now exist, such that there may be well upward of 1000 PDX models of human breast cancer in existence worldwide. They also presented a series of open questions for the field. Many of these questions have been addressed. However, several remain open, or only partially addressed. Herein, we revisit these questions, and recount the progress that has been made in a number of areas with respect to generation, characterization, and use of PDXs in translational research, and re-present questions that remain open. These open questions, and others, are now being addressed not only by individual investigators, but also large, well-funded consortia including the PDXNet program of the National Cancer Institute in the United States, and the EuroPDX Consortium, an organization of PDX developers across Europe. Finally, we discuss the new opportunities in PDX-based research.

Chronic lymphocytic leukemia patient-derived xenografts recapitulate clonal evolution to Richter transformation

Chronic lymphocytic leukemia (CLL) is a B-cell neoplasm with a heterogeneous clinical behavior. In 5–10% of patients the disease transforms into a diffuse large-B cell lymphoma known as Richter transformation (RT), which is associated with dismal prognosis. Here, we aimed to establish patient-derived xenograft (PDX) models to study the molecular features and evolution of CLL and RT. We generated two PDXs by injecting CLL (PDX12) and RT (PDX19) cells into immunocompromised NSG mice. Both PDXs were morphologically and phenotypically similar to RT. Whole-genome sequencing analysis at different time points of the PDX evolution revealed a genomic landscape similar to RT tumors from both patients and uncovered an unprecedented RT subclonal heterogeneity and clonal evolution during PDX generation. In PDX12, the transformed cells expanded from a very small subclone already present at the CLL stage. Transcriptomic analysis of PDXs showed a high oxidative phosphorylation (OXPHOS) and low B-cell receptor (BCR) signaling similar to the RT in the patients. IACS-010759, an OXPHOS inhibitor, reduced proliferation, and circumvented resistance to venetoclax. In summary, we have generated new RT-PDX models, one of them from CLL cells that mimicked the evolution of CLL to RT uncovering intrinsic features of RT cells of therapeutical value.

Establishment of experimental salivary gland cancer models using organoid culture and patient-derived xenografting

PurposeDepending on its histological subtype, salivary gland carcinoma (SGC) may have a poor prognosis. Due to the scarcity of preclinical experimental models, its molecular biology has so far remained largely unknown, hampering the development of new treatment modalities for patients with these malignancies. The aim of this study was to generate experimental human SGC models of multiple histological subtypes using patient-derived xenograft (PDX) and organoid culture techniques.MethodsTumor specimens from surgically resected SGCs were processed for the preparation of PDXs and patient-derived organoids (PDOs). Specimens from SGC PDXs were also processed for PDX-derived organoid (PDXO) generation. In vivo tumorigenicity was assessed using orthotopic transplantation of SGC organoids. The pathological characteristics of each model were compared to those of the original tumors using immunohistochemistry. RNA-seq was used to analyze the genetic traits of our models.ResultsThree series of PDOs, PDXs and PDXOs of salivary duct carcinomas, one series of PDOs, PDXs and PDXOs of mucoepidermoid carcinomas and PDXs of myoepithelial carcinomas were successfully generated. We found that PDXs and orthotopic transplants from PDOs/PDXOs showed similar histological features as the original tumors. Our models also retained their genetic traits, i.e., transcription profiles, genomic variants and fusion genes of the corresponding histological subtypes.ConclusionWe report the generation of SGC PDOs, PDXs and PDXOs of multiple histological subtypes, recapitulating the histological and genetical characteristics of the original tumors. These experimental SGC models may serve as a useful resource for the development of novel therapeutic strategies and for investigating the molecular mechanisms underlying the development of these malignancies.

Development of a Pediatric AML Patient Derived Xenograft Program

Background: Survival rates for pediatric acute myeloid leukemia (AML) still lag far behind those for most other pediatric cancers. New agents are desperately needed, but new drug development has been hampered by a lack of clinically relevant pediatric AML patient-derived xenograft (PDX) models for research. Texas Children's Hospital (TCH) has the patient volume, a close collaboration between clinical groups and research laboratories, and the infrastructure to address this gap. Methods: TCH patients diagnosed with AML are offered participation in local research. Most samples from local patients are enriched for mononuclear cells (MNCs) and injected fresh on the day of collection. Additionally, cryopreserved bone marrow samples from patients who enrolled on Children's Oncology Group clinical trials and consented to bank tissue for research have been used for PDX generation. Leukemia cells are tail vein injected (2x10e5 cells per mouse) into immunodeficient mice without conditioning. Most models are generated in the NOD.scid.Il2Rγc-/-/SCFh/h/GM-CSFh/h/IL-3h/h (NSGS) strain. Mice are closely monitored for human CD45+/CD33+/CD3- (hAML) cells in peripheral blood by flow cytometry. Once moribund, the mice are humanely euthanized, and bone marrow, peripheral blood, and spleen are harvested and evaluated by flow cytometry for disease burden. Bone marrow cells are injected by tail vein into secondary recipients. Serially transplanting PDX models are validated and characterized by short tandem repeat (STR) fingerprinting and DNA/RNA sequencing. Results: Thus far, we have developed 24 serially transplanting pediatric AML PDX models representing a variety of clinical and cytomolecular categories. The patients from whom these models were derived ranged in age from 0.5-20 years of age at diagnosis (mean 9.4 years). Most are derived from samples collected at diagnosis. For AML005, we have a second serially transplanting model derived from relapse. Our collection includes 8 models with KMT2A fusions, 3 with NPM1 mutations, 4 with WT1 mutations, 3 with FLT3 internal tandem duplications or point mutations, 1 with a NUP98 rearrangement, and 1 CBFA2T3-GLIS2 model. Model identities are validated by comparing the PDX STRs, flow cytometry, and fluorescence in situ hybridization results with matched samples from the patient of origin. For 3 models we also have targeted DNA sequencing for 174 recurrent leukemia-associated mutations for both the patient and the corresponding PDX model. All mutations detected in the patient samples were also detected in the PDX models. The Pearson correlation coefficients comparing the variant allele frequencies for each patient-PDX pair range from 0.82 to 0.99, indicating that the models are faithful representations of the original disease even at the subclone level. A summary of the current collection of serially transplanting models is provided in Table 1. Over 15 additional samples have engrafted once and are currently in secondary recipients. Data on established and validated models are being made publicly available through the Baylor PDX Portal (https://pdxportal.research.bcm.edu), where investigators from Baylor and labs worldwide can access information about our collection and request viably frozen samples for their own research purposes. Our models have been distributed to labs around the world for preclinical studies of novel therapeutics. Conclusion: Through development and distribution of unique pediatric AML PDX models, we hope to create a renewable resource to speed preclinical research and expedite development of drugs for clinical testing for children with AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 704: Development of a patient-derived xenograft (PDX) modeling program to enable pediatric precision medicine

Abstract Background: Recapitulation of the full spectrum of genomic changes driving patient tumors have resulted in increased use of patient-derived xenograft (PDX) models in studies of basic cancer biology and preclinical drug development. Given the translational potential of PDXs and limited availability of pediatric cancer models, we established a PDX program to expand the existing collection of pediatric PDXs in the community and enable pre- and post-clinical studies. Methods: PDX generation requests were integrated into clinical workflows to maximize identification of eligible patients for informed consent and tissue collection at Memorial Sloan Kettering Cancer Center. Methodologies for tissue procurement and cryopreservation were optimized to facilitate implantation into host immunodeficient mice and enable multi-institutional tissue exchange for model building. A bioinformatics pipeline was established to allow molecular validation of engrafted PDXs using a next-generation targeted gene panel (MSK-IMPACT) evaluating concordance based on acquired mutations, copy number alterations and clonal structure. Results: Between November 2016 - October 2021, 379 PDX models were developed (265 distinct models) representing 69 discrete diagnoses. Sarcoma represents the most common model type (50 discrete osteosarcoma, 20 desmoplastic small round cell tumor, 14 Ewing sarcoma, 24 rhabdomyosarcoma, 2 CIC/DUX4 and 2 BCOR-rearranged sarcoma) followed by neuroblastoma (n=35), leukemia (n=44), and Wilms tumor (n=15). While the majority of PDXs were established from recurrent or metastatic tissue, 7 paired diagnostic/pre-therapy and post-therapy or relapse models were generated. Genomic characterization of PDXs demonstrate excellent concordance and recapitulation of single nucleotide variants (90%), structural (88%) and copy number variants (94%) between patient tumor and matched PDX. Discrepancies between matched patient/PDX pairs are due to sub-clonal heterogeneity in source tumors with clonal outgrowth in the PDX. Analysis of serial PDX passages also demonstrate stable recapitulation of the genomic profile. Establishment of a diverse PDX collection allowed preclinical evaluation of 10 targeted agents across a spectrum of pediatric tumors and provided the preclinical rationale for 3 investigator-initiated pediatric clinical trials. Conclusions: Investment in the development of a phenotypically diverse and biologically faithful collection of pediatric PDX models enables the goals of precision medicine. Optimization of PDX workflows and methods has also enabled the development of a pediatric PDX consortium (PROXC - Pediatric Research in Oncology Xenografting Consortium) to further support the development of pre- and post-clinical studies for pediatric cancer. Citation Format: Filemon S. Dela Cruz, Joseph G. McCarter, Daoqi You, Nancy Bouvier, Xinyi Wang, Kristina C. Guillan, Armaan H. Siddiquee, Katie B. Souto, Hongyan Li, Teng Gao, Dominik Glodzik, Daniel Diolaiti, Neerav N. Shukla, Joachim Silber, Umeshkumar K. Bhanot, Faruk Erdem Kombak, Diego F. Coutinho, Shanita Li, Juan E. Arango Ossa, Juan S. Medina-Martinez, Michael V. Ortiz, Emily K. Slotkin, Michael D. Kinnaman, Sameer F. Sait, Tara J. O'Donohue, Marissa Mattar, Maximiliano Meneses, Michael P. LaQuaglia, Todd E. Heaton, Justin T. Gerstle, Nicola Fabbri, Chelsey M. Burke, Irene M. Rodriquez-Sanchez, Christine A. Iacobuzio-Donahue, Julia L. Glade Bender, Ryan D. Roberts, Jason T. Yustein, Nino C. Rainusso, Brian D. Crompton, Elizabeth Stewart, Alejandro Sweet-Cordero, Leanne C. Sayles, Andrika D. Thomas, Michael H. Roehrl, Elisa de Stanchina, Elli Papaemmanuil, Andrew L. Kung. Development of a patient-derived xenograft (PDX) modeling program to enable pediatric precision medicine [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 704.

Lenvatinib inhibits the growth of gastric cancer patient-derived xenografts generated from a heterogeneous population

BackgroundLenvatinib is a multitargeted tyrosine kinase inhibitor that is being tested in combination with immune checkpoint inhibitors to treat advanced gastric cancer; however, little data exists regarding the efficacy of lenvatinib monotherapy. Patient-derived xenografts (PDX) are established by engrafting human tumors into immunodeficient mice. The generation of PDXs may be hampered by growth of lymphomas. In this study, we compared the use of mice with different degrees of immunodeficiency to establish PDXs from a diverse cohort of Western gastric cancer patients. We then tested the efficacy of lenvatinib in this system.MethodsPDXs were established by implanting gastric cancer tissue into NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) or Foxn1nu (nude) mice. Tumors from multiple passages from each PDX line were compared histologically and transcriptomically. PDX-bearing mice were randomized to receive the drug delivery vehicle or lenvatinib. After 21 days, the percent tumor volume change (%Δvtumor) was calculated.Results23 PDX models were established from Black, non-Hispanic White, Hispanic, and Asian gastric cancer patients. The engraftment rate was 17% (23/139). Tumors implanted into NSG (16%; 18/115) and nude (21%; 5/24) mice had a similar engraftment rate. The rate of lymphoma formation in nude mice (0%; 0/24) was lower than in NSG mice (20%; 23/115; p < 0.05). PDXs derived using both strains maintained histologic and gene expression profiles across passages. Lenvatinib treatment (mean %Δvtumor: -33%) significantly reduced tumor growth as compared to vehicle treatment (mean %Δvtumor: 190%; p < 0.0001).ConclusionsNude mice are a superior platform than NSG mice for generating PDXs from gastric cancer patients. Lenvatinib showed promising antitumor activity in PDXs established from a diverse Western patient population and warrants further investigation in gastric cancer.

Abstract PD7-01: Towards personalized medicine - patient-derived breast tumor grafts as predictors of relapse and response to therapy. Preliminary results

Abstract Background: Predicting the risk of relapse in patients with non-metastatic breast cancer is important for medical decisions and patient counseling. For patients who receive neoadjuvant chemotherapy, pathologic response and especially pathologic complete response (pCR) has been associated with risk of relapse; however this association is imperfect. Our prior work in patient-derived xenograft (PDX) models indicated that tumor engraftment in mice correlated with risk of recurrence. To understand further the prognostic utility of PDX, we designed a prospective clinical trial to determine the correlation between PDX generation with residual disease following neoadjuvant chemotherapy and relapse. Preliminary data are presented. Methods: Women with newly diagnosed non-metastatic hormone receptor low-positive (HR-low, ER and/or PR ≤ 10%) or negative breast cancer planned to receive systemic chemotherapy prior to definitive surgery were eligible. Tumor tissue at diagnosis was orthotopically implanted in NOD/SCID mice. The primary objective of the study is to correlate the ability of a tumor to engraft in mice with pathologic responses and clinical outcomes. Results: Between 12/2016 and 2/2020, 58 patients enrolled (triple negative breast cancer (TNBC), n=37; HR-low or negative/Her2+, n=11; or HR-low/Her2-, n=8; mixed, n=1). PDXs were successfully established from 16 patients. Patients uniformly received intensive preoperative chemotherapy per standard of care. Among the 12 patients whose tumors engrafted in mice (PDX(+)) and underwent surgery, the pCR rate was 41.7%. In the subgroup of patients with postoperative follow up &amp;gt;6 months (n=9), 3 patients had achieved a pCR, 1 of whom recurred; and 6 patients had residual disease, 3 of whom recurred (overall relapse rate 44.4%). All patients who relapsed (TNBC n=3; HR-low/Her2- n=1), experienced a very early relapse (&amp;lt;12 months) after their definitive surgery. Disease progressed to metastatic during preoperative chemotherapy in one patient and 2 patients with residual disease relapsed while on adjuvant capecitabine. One patient experienced an early relapse despite having achieved a pCR. Patients who relapsed, died of breast cancer ≤1 year after diagnosis; the only surviving patient has not reached this landmark yet. Among the 38 patients whose tumors did not engraft (PDX(-)) and underwent surgery, the overall pCR rate was 60.6%. In the subgroup of patients with postoperative follow up &amp;gt;6 months (n=30), 18 patients had achieved a pCR of whom none recurred, and 12 had residual disease of whom 1 patient (with TNBC) had locoregional recurrence 35.1 months after her definitive surgery (relapse rate 3.3%). She underwent repeat surgery followed by radiation therapy and 5.5 months after her recurrence, she remains disease-free. Achievement of pCR was not statistically different between the PDX(+) and PDX(-) group. In the entire cohort, the presence of residual disease was associated with high risk of relapse (22.2%) as compared to the achievement of pCR (4.8%, p = 0.16). However, successful tumor engraftment was associated not only with a higher risk of relapse (44.4% vs 3.3%, p = 0.0068, odds ratio 20.45), but also an early and exceptionally aggressive relapse. Conclusion. Our functional studies not only identify a patient population with a high risk of relapse with greater precision than the achievement of pCR, but also identify patients whose relapse has a particularly aggressive natural history. We established models that recapitulate this aggressive disease and in-depth genomic studies are underway. These studies will lead us to better patient stratification on the basis of risk of relapse and novel therapeutic strategies focused on patients with exceptionally aggressive breast cancers that our current diagnostic methods cannot identify. Citation Format: Christos Vaklavas, Zhengtao Chu, Rachel E Factor, Alana L Welm, Bryan E Welm, Cindy B Matsen. Towards personalized medicine - patient-derived breast tumor grafts as predictors of relapse and response to therapy. Preliminary results [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD7-01.

Estimating molecular variability of patient-derived xenografts of esophageal cancer.

e13649 Background: The survival of patients with esophageal squamous cell carcinoma depends not only on clinical signs (TNM, tumor site), but also on the molecular genetic subtype of the tumor. Identification of the molecular genetic subtype and the presence/absence of clinically significant mutations is an important step towards finding new effective drugs and choosing the most appropriate therapeutic strategies. A patient-derived xenograft (PDX) model is a valuable resource to solving the problem, provided that the model accurately reproduces the basic clinical and molecular genetic characteristics of a human tumor. Our purpose was to create a PDX model of a human tumor and to study 7 polymorphisms of the xenograft tissue and tissues of a donor tumor. Methods: PDX models of esophageal cancer were produced by transplanting a tumor fragment from a patient with esophageal squamous cell carcinoma to the BALB/c Nude athymic mice (n = 10 for one PDX generation). 5 PDX were generated. 7 polymorphisms (NFE2L2 (c.85G &gt; A), NOTCH1 (c.1379C &gt; T), NOTCH1 (c.1451G &gt; T), ZNF750 (c.414C &gt; A), ZNF750 (c.1621G &gt; A), SMARCA4 (c.2272C &gt; T), KMT2D (c.15508C &gt; T)) were determined by the HRM analysis in tissues of each PDX generation and in the donor tumor. Results: All examined samples demonstrated the absence of ZNF750 (c.1621G &gt; A) and NOTCH1 (c.1379C &gt; T) polymorphisms and the presence of ZNF750 (c.414C &gt; A), SMARCA4 (c.2272C &gt; T) and KMT2D (c.15508C &gt; T) polymorphisms. Polymorphisms in the NFE2L2 (c.85G &gt; A) gene and in the NOTCH1 (c.1451G &gt; T) gene were found in the F3, F4 and F5 PDX generations, but were absent in the donor tumor and the F1 and F2 generations. Conclusions: Molecular and genetic characteristics of the donor tumor change through several PDX generations. Early PDX generations are recommended for the studies as they better reproduce molecular and genetic characteristics of the original tumors.

Comparison of the tumor cells proliferative activity in donor tissue and samples of xenograft generations.

e15619 Background: Esophageal cancer is a common tumor of the mucous layer of the esophagus and the sixth most common cause of cancer deaths worldwide. Studying the mechanisms of its occurrence and development and the search for new approaches to its prevention and treatment are relevant. Such studies require a reliable experimental basis with such an important component as the use of animal models of cancer. In vivo models are an important tool both for testing new drugs and for studying the mechanisms of disease development. The purpose of the study was to create a patient-derived xenograft (PDX) model of esophageal cancer and to evaluate the proliferative activity of cells in the donor tumor and in xenograft samples in a series of successive generations. Methods: A PDX model of esophageal cancer was created by transplanting a tumor fragment from a patient with esophageal squamous cell carcinoma to the distal esophagus of BALB/c Nude athymic mice. The tumor histotype was confirmed histologically (hematoxylin and eosin staining). The proliferative activity of cells was evaluated by immunohistochemistry (IHC) with anti-Ki-67 antibodies. Results: Histological identity of xenograft samples and the donor tumor was confirmed. IHC showed an increase in the proliferative activity index in samples of transplanted tumors compared to the donor material: 12.4% in the primary tumor and 58%, 65.2%, 54.2% and 60.7% in generations 1, 3, 4 and 5, respectively. Conclusions: Histological characteristics of the donor tumor are preserved in all PDX models. The study demonstrated an increase of the proliferative activity in the first PDX generation compared to the primary tumor which persisted in the subsequent models.

Blocking Lysosomal Two-Pore Channel 2 Function Inhibits Proliferation of Multidrug Resistant Leukemia Cells and Sensitizes Them to Vincristine Treatment

Introduction Two-pore channel 2 (TPC2) represents an exceptional ion channel due to its localization on lysosomes1. TPC2 is overexpressed in several cancer types, e.g. in prostate cancer, and its expression is associated with poor survival probability2. To date, a role of TPC2 in leukemia has not been investigated. Pharmacologic inhibition of TPC2 can be performed with tetrandrine (Tet), a bisbenzylisoquinoline alkaloid3. However, its use is restricted due to unfavorable toxicity in animal studies4. In our study, we aim to elucidate the role of TPC2 function in multidrug resistant leukemia and, concurrently, identify efficacious TPC2 inhibitors by screening a library of synthetic bisbenzylisoquinoline derivatives (BBIQDs). Results Firstly, via qPCR analysis, we detected a 2-fold increased TPC2 mRNA expression in a vincristine-resistant (VCR-R) CEM (VCR-R CEM, T-ALL) cell line, compared with the parental CCRF-CEM cell line. Secondly, we knocked out TPC2 in VCR-R CEM cells using the CRISPR-Cas9 system to investigate its involvement in cell growth and treatment response. By studying cell proliferation over a period of six days, we found that the doubling time of TPC2-deficient cells was increased to 30 h, compared with 24 h for the wildtype (wt) cell line. Additionally, treatment of TPC2 knockout (ko) cells with VCR resulted in increased sensitivity versus wt, as indicated by proliferation (IC50 wt: 3.3 µM, IC50 ko: 1.6 µM, 72 h) and apoptosis (EC50 wt: 3.0 µM, EC50 ko: 1.7 µM, 48 h) assays. Next, a library of BBIQDs was synthesized and screened for the ability to inhibit TPC2 function using the whole endolysosomal patch clamp method. Our small molecule hits SG-005 and SG-094 inhibited TPC2 current density by 50% and 70%, respectively, giving novel TPC2 inhibitors with either similar or even increased potency, compared to Tet (50%). Treatment of VCR-R CEM cells with Tet, SG-005 and 94 effectively inhibited proliferation (IC50: 5-15 µM, 48 h). Toxicity was assessed by propidium iodide exclusion assays using Peripheral Blood Mononuclear cells (PBMCs, n=3 healthy donors), indicating reduced toxicity of SG-094 (&lt;5% dead cells vs. 25% dead cells for Tet and SG-005, 20 µM, 48 h treatment). Additionally, SG-094 was well tolerated in vivo when applied to a mouse model (90 nmol/kg/d on three consecutive days). Remarkably, combination of VCR (0.01 and 0.1 µM) with Tet, SG-005 or 94 (1 and 5 µM) synergistically increased treatment response of VCR-R CEM cells (wt) and of B ALL patient-derived xenograft (PDX) cells from a relapse patient (Bliss values: 1.1-2.4). Methods CellTiter-Blue® cell proliferation assays, qPCR mRNA expression, propidium iodide exclusion and analysis of apoptosis were performed as described by the manufacturers. Whole endolysosomal patch clamp, analysis of specific apoptosis of PDX cells and generation of the TPC2 ko model using the CRISPR-Cas9 system were performed as described previously3, 5, 6. Analysis of doubling time was performed by counting cells daily using trypan blue staining and a Vi-CELL XR device as indicated by the manufacturer. Conclusion Taken together, we detected an upregulated mRNA expression of endolysosomal TPC2 in VCR-R CEM cells, indicating that it represents a potential target for therapeutic intervention for difficult-to-treat leukemia phenotypes. Furthermore, we could show that genetic ablation of the ion channel results in a reduced proliferation rate and an increased sensitivity towards VCR treatment. Finally, we successfully identified pharmacologic TPC2 inhibitors which show antiproliferative effects when applied as monotherapy and synergistically enhance treatment response of VCR-R CEM cells and PDX cells when combined with VCR. References S. Patel, B. S. Kilpatrick, Biochim Biophys Acta Mol Cell Res1865, 1678-1686 (2018). F. Li, J. P. Ji, Y. Xu, R. L. Liu, Clin Transl Oncol, (2019). O. N. Nguyen et al., Cancer Res77, 1427-1438 (2017). H. Jin et al., Chem Res Toxicol24, 2142-2152 (2011). F. Koczian et al., Haematologica104, 546-555 (2019). F. A. Ran et al., Nat Protoc8, 2281-2308 (2013). Disclosures No relevant conflicts of interest to declare.

Establishment of hepatocellular carcinoma patient-derived xenografts from image-guided percutaneous biopsies

While patient-derived xenograft (PDX) models of hepatocellular carcinoma (HCC) have been successfully generated from resected tissues, no reliable methods have been reported for the generation of PDXs from patients who are not candidates for resection and represent the vast majority of patients with HCC. Here we compare two methods for the creation of PDXs from HCC biopsies and find that implantation of whole biopsy samples without the addition of basement membrane matrix favors the formation of PDX tumors that resemble Epstein-Barr virus (EBV)-driven B-cell lymphomas rather than HCC tumors. In contrast, implantation with Matrigel supports growth of HCC cells and leads to a high rate of HCC tumor formation from these biopsies. We validate the resulting PDXs, confirm their fidelity to the patients’ disease and conclude that minimally invasive percutaneous liver biopsies can be used with relatively high efficiency to generate PDXs of HCC.

Successful generation of patient-derived xenografts and cell lines as preclinical models for sarcomas.

e22549 Background: The establishment of patient-derived preclinical models has been a challenge issue in sarcomas, because of the rarity of diseases. We report our successful experience in establishing patient-derived sarcoma xenografts and cell lines. Methods: Tumor tissue samples from sarcoma patients undergoing surgery were collected by a team of orthopedicians and pathologists. Small tumor fragments were subcutaneously transplanted to immunodeficient NOG mice. The other part was used to establish a cell line using serum-containing media. The omics profiling was performed to examine the preservation of molecular backgrounds during the model establishment. Effects of FDA-approved drugs were examined in the established models. Results: Since April 2014, tumor samples from 246 sarcoma cases have been processed. The sample characteristics are summarized in the table. Currently we have established PDXs and cell lines from 39 and 27 cases, respectively. Xenografts and cell lines were established from unique sarcomas such as CIC-DUX4 fusion positive sarcoma, extraskeletal osteosarcoma, primary leiomyosarcoma of bone, and alveolar soft part sarcoma. A take rate of xenografts and cell lines varied among the sarcoma types. Omics profiles considerably changed during the process of model establishment. Anti-cancer drugs with significant tumor suppressive effects were detected. Conclusions: Our patient-derived cancer models will facilitate translational sarcoma research. The original biological features preserved in the xenografts and cell lines should be clarified to optimize the utility of sarcoma models. Currently, our cell lines are available for the academic researchers upon a request.

SUN-337 Anti-tumorigenic Effects Of The Maternal Leucine Zipper Kinase (MELK) Inhibitor, OTSSP167, In Pre-clinical In Vivo Models Of Adrenocortical Carcinomas (ACC)

ACC is an aggressive endocrine cancer with limited therapeutic options and poor survival rates. In patients with progressive disease, standard EDP (etoposide, doxorubicin and cisplatin) chemotherapy is associated with only a modest response and progression free survival of 5 months. Mitotane, the only adjuvant FDA approved therapy for ACC is associated with 30% stable disease or partial response, as well as an association with toxicity and intolerability. The lack of pre-clinical models has also contributed to slow progress in the field. Previously, we developed CU-ACC1 and CU-ACC2 patient derived xenografts (PDX) and their corresponding ACC cell lines to study potential targets in ACC tumorigenesis. In this study we characterized a third ACC PDX, CU-ACC9. CU-ACC9 PDX was derived from a mild cortisol producing 15 cm primary ACC tumor involving adrenal, kidney, distal pancreas with an inferior vena cava (IVC) thrombus in a 55 year-old female patient. On analysis, the CU-ACC9 tumor had a T53 p.R248W mutation, intermediate microsatellite instability, and loss of MSH2 and MSH6 staining by immunohistochemistry, all suggestive of Lynch syndrome. Despite the initial poor prognosis associated with IVC thrombus, the patient underwent radical resection and subsequent therapy with anti-PD1 checkpoint inhibitor and mitotane, and survived for 2 years. STR profiling confirmed a 96% match between the CU-ACC9 PDX and the original tumor tissue. Immunohistochemistry of the PDX revealed positive expression of the adrenal markers, inhibin alpha and SF-1. Further gene expression studies using RNA seq revealed that CU-ACC9 PDX and CU-ACC1 PDX had high MELK expression, a kinase we previously identified as a potential therapeutic target in ACC. Our prior studies demonstrated a dose dependent induction of apoptosis by the MELK inhibitor, OTSSP167, in H295R, CU-ACC1 and CU-ACC2 cells which correlated with endogenous MELK expression. To test effects of the MELK inhibitor in vivo, we treated the F13 generation of CU-ACC1 and F7 generation of CU-ACC9 PDX with OTSSP167. Accounting for the variable tumor growth rate between the two PDXs, OTSSP167 treatment for 18 days and 28 days in CU-ACC1 and CU-ACC9 PDXs, respectively resulted in a 4 fold (p= 0.02) and 2.2-fold ( p<0.001) inhibition in tumor growth. The tumor inhibitory growth index (TGII) for both explants were <20 % suggesting a response to the single agent therapy with OTSSP167. There was no evidence of systemic toxicity as assessed by changes in body weight. These data establish the therapeutic potential of OTSSP167 in ACC and suggest that targeting MELK could have efficacy in a subset ACC patients. Future studies will focus on enhancing the therapeutic efficacy by combining OTSSP167 with other rational based therapies in our ACC PDX models, towards Phase I studies in patients with ACC. Funding: CLC grants, K08, Doris Duke CU-FRCS, VA Merit Review

Abstract A38: RNA-seq of diagnostic and relapse disseminated tumor cells (DTCs) in stage M neuroblastoma patients and first steps towards DTC-derived xenografts in zebrafish embryos

Abstract Neuroblastoma (NB) is the most common tumor diagnosed in the first year of life. More than 90% of stage M NB patients present with disseminated tumor cells (DTCs) in the bone marrow (BM). Although these cells represent a major obstacle to treatment, their global gene expression has, so far, been only poorly analyzed. GD2POS DTCs of stage M NB patients were separated from the remaining BM-derived nontumor mononuclear cells (MNCs) by applying magnetic associated cell sorting. mRNA of diagnostic DTCs (n=22) and MNCs (n=14), primary tumors (n=16), relapse DTCs (n=20) and MNCs (n=14) was isolated and used for RNA-seq (50bp, single-end) in order to characterize the biology of DTCs. Furthermore, four DTC-derived neuroblastoma cell lines were established and used to generate a robust patient-derived zebrafish xenograft model. Annexin V staining (apoptosis), EdU proliferation assays, and transwell migration assays of the four cell lines were performed in vitro at temperatures between 34°C and 37°C. 322 differently expressed genes (DEG) were identified comparing primary tumors and DTCs (q &amp;lt; 0.001, |log2FC|&amp;gt;2). Especially, genes encoded by mitochondrial DNA (mtDNA) were upregulated in DTCs. Furthermore, we found the gene expression signature of relapse DTCs largely resembling those of diagnostic DTCs, with only 113 DEG under relaxed cut-offs (q &amp;lt; 0.01, |log2FC|&amp;gt;0.5). Notably, relapse DTCs showed a positional enrichment of 31 downregulated genes on chromosome 19, including five tumor-suppressor genes: SIRT6, BBC3/PUMA, STK11, CADM4, and GLTSCR2. Genomic analysis of 34 DTC samples revealed clonal deletions affecting a portion of the 31 downregulated genes encoded by chromosome 19. Although these deletions occurred at a higher frequency in relapse patients (9 out of 17 samples) as compared to the diagnostic samples (4 out of 17 samples), the positional distribution of downregulated genes did not coincide with the deleted ones. While chromosomal deletions in relapse samples were mainly found on 19q (8 out of 20 samples), most of the downregulated genes were encoded by 19p, which rarely contained deletions (3 out of 20 samples). The downregulation of PUMA (p=0.002) and CADM4 (p=0.02) was associated with a worse event-free survival in stage M patients (survival analysis performed in R2 with the Oberthuer dataset). Furthermore, four DTC-derived cell lines were tested for suitability of injecting into zebrafish embryos and subsequent functional testing. In vitro analysis revealed that in two cell lines (STA-NB-8 and STA-NB-12) proliferation and apoptosis were not affected by lower temperatures (34°C), at which zebrafish embryos still can develop properly, while in the remaining two cell lines both were impaired. Fewer STA-NB-15 and STA-NB-19 cells were in the S phase at 34°C (18.6% of STA-NB-15 and 24.2% of STA-NB-19 cells) as compared to 37°C (37.8% of STA-NB-15 and 45.1% of STA-NB-19 cells). Additionally, more of the STA-NB-15 cells were undergoing apoptosis at 34°C (50.3% Annexin V positive cells) as compared to 37°C (33.9% Annexin V positive cells). This first RNA-seq analysis of neuroblastoma DTCs revealed their unique expression profile in contrast to tumors, but showed less pronounced differences between diagnostic and relapse DTCs. The latter preferentially affected the downregulation of genes encoded by chromosome 19, which might be associated with treatment failure and disease progression. The establishment of xenografts in zebrafish embryos with DTC-derived cells will enable in vivo functional studies and optimize the generation of patient-derived xenografts in zebrafish embryos, which will allow drug screening and functional studies for personalized treatment approaches. Citation Format: Fikret Rifatbegovic, Sabine Taschner-Mandl, Martin Distel, M. Reza Abbasi, Christian Frech, Andrea Ziegler, Inge M. Ambros, Peter F. Ambros. RNA-seq of diagnostic and relapse disseminated tumor cells (DTCs) in stage M neuroblastoma patients and first steps towards DTC-derived xenografts in zebrafish embryos [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A38.

Abstract IA21: NCI's Patient-Derived Models Repository: Generating models from racial and ethnic minorities

Abstract The National Cancer Institute (NCI) has developed a Patient-Derived Models Repository (PDMR) comprising quality-controlled, early-passage, clinically annotated patient-derived xenografts (PDXs) to serve as a resource for public-private partnerships and for academic drug discovery efforts. The first 100 models were released in May 2017, are clinically annotated with pathologic and molecular information available in a publicly accessible database, and are available to the extramural community for research use (pdmr.cancer.gov). The PDMR was established by NCI at the Frederick National Laboratory for Cancer Research (FNLCR) in direct response to discussions with academia and industry; the oncology community's highest-priority need was identified as better preclinical models that more faithfully reflect the patient's tumor and are associated with the patient's treatment history. NCI has focused on collecting specimens from patients with cancers that are under-represented in many other PDX collections such as head and neck, pancreatic, bladder and gynecologic cancers, melanomas, and sarcomas. Recently, NCI has also increased its focus on generation of PDXs from racial and ethnic minorities through new funding opportunities. The initial goal is to have 20-25% of the models in the PDMR from racial and ethnic minorities in diagnoses that are most pertinent to cancer health disparities. The overall goal of NCI is to create a long-term home for at least 1,000 models such that sufficient biologic, clinical, and population diversity is represented to allow researchers to ask questions such as: what is the impact of tumor heterogeneity on target qualification or clinical response; do PDXs more faithfully represent the human tumor for pharmacodynamic assay and predictive marker development; or can an adequately powered preclinical PDX clinical trial lead to better evaluation of therapies for future clinical use? Grant Support: This project has been funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. government. Citation Format: Yvonne A. Evrard. NCI's Patient-Derived Models Repository: Generating models from racial and ethnic minorities [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr IA21.

Genomic and Functional Fidelity of Small Cell Lung Cancer Patient-Derived Xenografts.

Small cell lung cancer (SCLC) patient-derived xenografts (PDX) can be generated from biopsies or circulating tumor cells (CTC), though scarcity of tissue and low efficiency of tumor growth have previously limited these approaches. Applying an established clinical-translational pipeline for tissue collection and an automated microfluidic platform for CTC enrichment, we generated 17 biopsy-derived PDXs and 17 CTC-derived PDXs in a 2-year timeframe, at 89% and 38% efficiency, respectively. Whole-exome sequencing showed that somatic alterations are stably maintained between patient tumors and PDXs. Early-passage PDXs maintain the genomic and transcriptional profiles of the founder PDX. In vivo treatment with etoposide and platinum (EP) in 30 PDX models demonstrated greater sensitivity in PDXs from EP-naïve patients, and resistance to EP corresponded to increased expression of a MYC gene signature. Finally, serial CTC-derived PDXs generated from an individual patient at multiple time points accurately recapitulated the evolving drug sensitivities of that patient's disease. Collectively, this work highlights the translational potential of this strategy.Significance: Effective translational research utilizing SCLC PDX models requires both efficient generation of models from patients and fidelity of those models in representing patient tumor characteristics. We present approaches for efficient generation of PDXs from both biopsies and CTCs, and demonstrate that these models capture the mutational landscape and functional features of the donor tumors. Cancer Discov; 8(5); 600-15. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 517.

TMOD-10. INTRACRANIAL PATIENT-DERIVED XENOGRAFT FROM A PEDIATRIC GLIOBLASTOMA RETAINS MOLECULAR CHARACTERISTICS AND GROWTH PATHWAYS

BACKGROUND: Pediatric glioblastomas (pGBM) are characterized by overlapping and unique molecular driver mutations compared to their adult counterparts. Primary cell cultures and patient-derived xenograft (PDX) models of pGBM are rare which may contribute to difficulty in advancing patient outcomes. We have established an orthotopic PDX mouse model from a pGBM resected from a 12 yo male with recurrent disease. The patient received focal radiation and several chemotherapy regimens prior to the recurrence leading to this tumor resection. METHODS: The surgical tumor specimen was dissociated and directly implanted into the brains of immunocompromised (NSG) mice. DNA and immunohistochemical analyses of the patient tumor and PDX were performed. Multiple in vitro culture conditions were tested on the dissociated primary tumor cells and PDX tumor cells. RESULTS: Implantation of the patient tumor cells in mice resulted in tumor growth after 120–250 days. Tumors were serially transplanted successfully. Attempts to establish in vitro cell cultures from primary tumor and from established PDX tumors failed. DNA sequencing of the patient tumor sample and tumors from each PDX generation revealed the H3F3A G34R mutation characteristic of cortical pGBM, and mutations in the TP53, PTEN and PIK3CA genes, all of which were conserved in PDX tumors. Interestingly, the patient tumor showed evidence of telomerase activity and alternative lengthening of telomeres (ALT) as did the PDX tumors. The combined PTEN and PIK3CA mutations in this tumor resulted in activation of the PI3K-mTOR pathway. Treatment of tumor-bearing mice with an mTOR or PI3K inhibitor resulted in suppression of the pathway and reduced proliferation. CONCLUSION: This pGBM PDX model conserved key driver mutations and pathways from the patient tumor. In vitro culture of pGBM can be extremely challenging to establish and more difficult methods such as orthotopic PDX may be required to study these tumors.

Generation of patient-derived xenografts from fine needle aspirates or core needle biopsy

Patient-derived xenografts have recently become a powerful tool for cancer research and may be used to guide personalized therapy. Thus far, patient-derived xenografts have been grown from tumor tissue obtained after operative resection; however, many cancer patients never undergo operative intervention for a variety of reasons. We hypothesized that xenograft tumors could be grown from smaller volumes of patient tissue, such as those obtained during diagnostic biopsies. Surgical specimens were obtained after resection of primary or metastatic lesions of the following cancers: pancreatic carcinoma, non-small cell lung cancer, bladder (urothelial) carcinoma, and melanoma. At least 10 cases of each cancer were included in this study. To mimic clinical biopsies, small fragments of the surgical specimens were biopsied with a 22-gauge needle, and the needle contents were injected subcutaneously in immunocompromised mice. The tumor fragment from which the biopsy was taken was also implanted subcutaneously in the contralateral side of the same mouse as a control. Success rates of the traditional method of xenograft implantation ranged from 27.3%-70%. Success rates of the fine needle aspirate technique ranged from 0%-36.4%. An attempt to engraft a percutaneous core needle liver biopsy of a metastatic pancreatic adenocarcinoma also was successful. We have found that it is possible to engraft fine needle aspirates and core biopsies of solid tumors in order to generate patient-derived xenografts. This may open up xenografting to a wider cancer patient population than previously possible.

Patient-derived xenograft (PDX) models in basic and translational breast cancer research.

Patient-derived xenograft (PDX) models of a growing spectrum of cancers are rapidly supplanting long-established traditional cell lines as preferred models for conducting basic and translational preclinical research. In breast cancer, to complement the now curated collection of approximately 45 long-established human breast cancer cell lines, a newly formed consortium of academic laboratories, currently from Europe, Australia, and North America, herein summarizes data on over 500 stably transplantable PDX models representing all three clinical subtypes of breast cancer (ER+, HER2+, and "Triple-negative" (TNBC)). Many of these models are well-characterized with respect to genomic, transcriptomic, and proteomic features, metastatic behavior, and treatment response to a variety of standard-of-care and experimental therapeutics. These stably transplantable PDX lines are generally available for dissemination to laboratories conducting translational research, and contact information for each collection is provided. This review summarizes current experiences related to PDX generation across participating groups, efforts to develop data standards for annotation and dissemination of patient clinical information that does not compromise patient privacy, efforts to develop complementary data standards for annotation of PDX characteristics and biology, and progress toward "credentialing" of PDX models as surrogates to represent individual patients for use in preclinical and co-clinical translational research. In addition, this review highlights important unresolved questions, as well as current limitations, that have hampered more efficient generation of PDX lines and more rapid adoption of PDX use in translational breast cancer research.