Patient-Derived Xenograft Models Reveal Intratumor ... Translational Science Patient-Derived Xenograft

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  • Translational Science

    Patient-Derived Xenograft Models Reveal Intratumor Heterogeneity and Temporal Stability in Neuroblastoma No�emie Braekeveldt1, Kristoffer von Stedingk2,3, Susanne Fransson4, Angela Martinez-Monleon4, David Lindgren1, Ha

    � kan Axelson1, Fredrik Levander5,

    Jakob Willforss5, Karin Hansson5, Ingrid �ra2, Torbj€orn Backman6, Anna B€orjesson6, Siv Beckman1, Javanshir Esfandyari1, Ana P. Berbegall7, Rosa Noguera7, Jenny Karlsson8, Jan Koster3, Tommy Martinsson4, David Gisselsson8,9, Sven Pa

    � hlman1, and Daniel Bexell1,9

    Abstract

    Patient-derived xenografts (PDX) and the Avatar, a single PDX mirroring an individual patient, are emerging tools in preclinical cancer research. However, the consequences of intratumor heterogeneity for PDX modeling of biomarkers, target identification, and treatment decisions remain under- explored. In this study, we undertook serial passaging and comprehensive molecular analysis of neuroblastoma orthotopic PDXs, which revealed strong intrinsic genetic, transcriptional, and phenotypic stability for more than 2 years. The PDXs showed preserved neuroblastoma-associ- ated gene signatures that correlated with poor clinical out- come in a large cohort of patients with neuroblastoma. Furthermore, we captured spatial intratumor heterogeneity using ten PDXs from a single high-risk patient tumor. We

    observed diverse growth rates, transcriptional, proteomic, and phosphoproteomic profiles. PDX-derived transcription- al profiles were associated with diverse clinical character- istics in patients with high-risk neuroblastoma. These data suggest that high-risk neuroblastoma contains elements of both temporal stability and spatial intratumor heterogene- ity, the latter of which complicates clinical translation of personalized PDX–Avatar studies into preclinical cancer research.

    Significance: These findings underpin the complexity of PDX modeling as a means to advance translational applica- tions against neuroblastoma. Cancer Res; 78(20); 5958–69.�2018 AACR.

    Introduction Malignant tumors are often made up of distinct subclonal

    populations, which can evolve both spatially, that is, in different regions of the tumor, and temporally as a consequence of tumor growth or treatment. This evolving intratumor heterogeneity (ITH) has been implicated as fundamental in cancer progression, metastasis, treatment resistance, and cancer survival (1, 2), par- ticularly in adult cancers. Recent work suggests that pediatric tumors also exhibit genomic diversity.

    Neuroblastoma, a childhood cancer originating from the devel- oping sympathetic nervous system, is characterized by a diverse clinical phenotype (3). Patients with high-risk neuroblastomas often have a poor prognosis despite heavymultimodal treatment, and survivors can suffer from long-term, severe side-effects. Emerging data suggest that clonal evolution and genetic ITH also is a feature in neuroblastoma (4–11) and is involved in neuro- blastoma treatment response (8, 9, 11). We have previously shown that high genetic diversity is common in childhood cancer after chemotherapy and correlates to poor prognosis (4, 11). Less is known about transcriptional, proteomic, and functional ITH in neuroblastoma; yet, understanding this is important as it most likely influences biomarker discovery, target identification, treat- ment decisions, and treatment response.

    1Department of Laboratory Medicine, Division of Translational Cancer Research, Lund University, Lund, Sweden. 2Department of Clinical Sciences, Division of Pediatric Oncology, Lund University, University Hospital, Lund, Sweden. 3Department of Oncogenomics, Amsterdam UMC, University of Amsterdam, the Netherlands. 4Department of Pathology and Genetics, University of Gothen- burg, Gothenburg, Sweden. 5Department of Immunotechnology, Lund Univer- sity, Lund, Sweden. 6Division of Pediatric Surgery, Department of Clinical Sciences, Lund University, University Hospital, Lund, Sweden. 7Department of Pathology, Medical School, University of Valencia/INCLIVA/CIBERONC, Madrid, Spain. 8Division of Clinical Genetics, Department of Laboratory Medicine, Lund University, Lund, Sweden. 9Department of Pathology, Laboratory Medicine, Medical Services, University Hospital, Lund, Sweden.

    Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

    N. Braekeveldt and K. Stedingk contributed equally to this article.

    Corresponding Authors: Daniel Bexell, Lund University, Medicon Village 404: C3, SE-223 81 Lund, Sweden. Phone: 46-46-222-64-23, E-mail: daniel.bex ell@med.lu.se; and Kristoffer von Stedingk, Department of Oncogenomics, Academic UMC, University of Amsterdam, the Netherlands. E-mail: kristoffer.von_stedingk@med.lu.se

    doi: 10.1158/0008-5472.CAN-18-0527

    �2018 American Association for Cancer Research.

    Cancer Research

    Cancer Res; 78(20) October 15, 20185958

    on June 11, 2020. © 2018 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

    Published OnlineFirst August 28, 2018; DOI: 10.1158/0008-5472.CAN-18-0527

    http://crossmark.crossref.org/dialog/?doi=10.1158/0008-5472.CAN-18-0527&domain=pdf&date_stamp=2018-9-29 daniel.bexell@med.lu.se daniel.bexell@med.lu.se http://cancerres.aacrjournals.org/

  • Patient-derived xenografts (PDX) have emerged as promising preclinical cancer models because PDXs can retain many of the molecular and functional features of their ancestral tumors in patients (12). Thousands of PDX models have thus been estab- lished in major academic and industrial preclinical drug testing programs. The PDX–Avatar and the coclinical trial concepts depend on the establishment of a single PDX model mirroring a patient tumor. By simultaneously establishing and treating a PDX–Avatar of a patient enrolled in a clinical trial with a new agent, underlyingmechanisms canbe studied, novel combination strategies can be evaluated, and potential biomarkers identified. The use of well-characterized PDX models thus holds promise to improve the transition of preclinical drug evaluation data to the clinics. However, the functional consequences of ITH for PDX– Avatar and coclinical trial studies have not been addressed. This could be crucial because clonal evolution and ITH are essential factors in the response to anticancer drugs and the development of treatment resistance (2).

    We have previously established neuroblastoma patient-derived orthotopic xenografts (PDOX), which retain the histopathologic, stromal, and metastatic features of aggressive patient tumors (13, 14). We prefer to use PDOXs instead of subcutaneous PDXs, because orthotopic tumors have more relevant biology (15) and retain spontaneous metastatic capacity (16). In this study, up to eight in vivo generations of neuroblastoma PDOXs were estab- lished through serial passaging in NSG mice. PDOXs were trans- planted as undissociated tumor fragments to avoid genetic aber- rations and clonal selections associated to culturing procedures (17). Comprehensive molecular analyses of multiple in vivo passages from each PDOX model were performed to examine temporal evolution of human high-risk neuroblastoma.We dem- onstrate high genetic, transcriptional and phenotypic stability over time, and identify PDOX-derived gene signatures, which correlate with neuroblastoma-associated processes and with clin- ical characteristics including patient prognosis. These gene signa- tures are preserved for more than 2 years of in vivo serial passaging in mice. However, using multiple PDOXs derived from a single high-risk patient tumor, we uncovered diverse transcriptional, proteomic, and phosphoproteomic profiles, suggesting a signif- icant spatial ITH of the patient tumor. Our findings highlight functional and molecular spatial ITH as an element of high-risk neuroblastoma. This complicates the interpretation of PDX– Avatar studies (1 mouse, 1 patient) for biomarker discovery as well as target identification and treatment-guiding decisions in personalized oncology.

    Materials and Methods Animal procedures

    Animal procedures were performed as described previously (13, 14). Briefly, 4-to-6-week-old female or male NSG mice were housed under pathogen-free conditions and received autoclaved

    water and food. We have previously established neuroblastoma PDOXs through the implantation of undissociated patient tumor fragments into the paraadrenal space of immunodeficient NSG mice (13, 14). Established PDOXs underwent serial orthotopic transplantation into next generation NSG mice to establish up to eight in vivo generations for each PDOX model. The study was approved by the Regional Ethics Board of Southern Sweden (289- 2011). The Malm€o–Lund Ethical Committee for the use of lab- oratory animals approved all animal experiments (M146-13). See SI Appendix for further information on animal procedures.

    IHC and microscopy Xenograft tumors andmice organswere formalin-fixed, embed-

    ded in paraffin, and 4 mm tissue sections were cut and analyzed. See SI Appendix for further information.

    SNP analysis PDOXs were snap frozen and stored at �80�C for SNP array

    analysis. Briefly, DNA was extracted from the tissues using the DNeasy Blood and Tissue Kit (Qiagen) according to the manu- facturer's instructions. The Affymetrix CytoScan HD platformwas used for SNP array analysis as described previously (13).

    Exome sequencing DNAwas extracted from frozen tumors or constitutional blood

    fromneuroblastomapatients and from thePDOXsusing standard procedures prior to fluorometric quantitation and DNA integrity assessment on Agilent Tapestation (Agilent). Exome sequencing was performed on tumors fro