András Sablauer

Targeting the DNA Repair Pathway in Ewing Sarcoma

Ewing sarcoma (EWS) is a tumor of the bone and soft tissue that primarily affects adolescents and young adults. With current therapies, 70% of patients with localized disease survive, but patients with metastatic or recurrent disease have a poor outcome. We found that EWS cell lines are defective in DNA break repair and are sensitive to PARP inhibitors (PARPis). PARPi-induced cytotoxicity in EWS cells was 10- to 1,000-fold higher after administration of the DNA-damaging agents irinotecan or temozolomide. We developed an orthotopic EWS mouse model and performed pharmacokinetic and pharmacodynamic studies using three different PARPis that are in clinical development for pediatric cancer. Irinotecan administered on a low-dose, protracted schedule previously optimized for pediatric patients was an effective DNA-damaging agent when combined with PARPis; it was also better tolerated than combinations with temozolomide. Combining PARPis with irinotecan and temozolomide gave complete and durable responses in more than 80% of the mice.

The Dynamic Epigenetic Landscape of the Retina During Development, Reprogramming, and Tumorigenesis

SUMMARY In the developing retina, multipotent neural progenitors undergo unidirectional differentiation in a precise spatiotemporal order. Here we profile the epigenetic and transcriptional changes that occur during retinogenesis in mice and humans. Although some progenitor genes and cell cycle genes were epigenetically silenced during retinogenesis, the most dramatic change was derepression of cell type–specific differentiation programs. We identified developmental stage–specific super-enhancers and showed that most epigenetic changes are conserved in humans and mice. To determine how the epigenome changes during tumorigenesis and reprogramming, we performed integrated epigenetic analysis of murine and human retinoblastomas and induced pluripotent stem cells (iPSCs) derived from murine rod photoreceptors. The retinoblastoma epigenome mapped to the developmental stage when retinal progenitors switch from neurogenic to a terminal patterns of cell division. The epigenome of retinoblastomas was more similar to that of normal retina than was that of retina-derived iPSCs, and we identified retina-specific epigenetic memory.

The Childhood Solid Tumor Network: A new resource for the developmental biology and oncology research communities

Significant advances have been made over the past 25 years in our understanding of the most common adult solid tumors such as breast, colon, lung and prostate cancer. Much less is known about childhood solid tumors because they are rare and because they originate in developing organs during fetal development, childhood and adolescence. It can be very difficult to study the cellular origins of pediatric solid tumors in developing organs characterized by rapid proliferative expansion, growth factor signaling, developmental angiogenesis, programmed cell death, tissue reorganization and cell migration. Not only has the etiology of pediatric cancer remained elusive because of their developmental origins, but it also makes it more difficult to treat. Molecular targeted therapeutics that alter developmental pathway signaling may have devastating effects on normal organ development. Therefore, basic research focused on the mechanisms of development provides an essential foundation for pediatric solid tumor translational research. In this article, we describe new resources available for the developmental biology and oncology research communities. In a companion paper, we present the detailed characterization of an orthotopic xenograft of a pediatric solid tumor derived from sympathoadrenal lineage during development.

Alert dwell time: introduction of a measure to evaluate interruptive clinical decision support alerts

Metrics for evaluating interruptive prescribing alerts have many limitations. Additional methods are needed to identify opportunities to improve alerting systems and prevent alert fatigue. In this study, the authors determined whether alert dwell time-the time elapsed from when an interruptive alert is generated to when it is dismissed-could be calculated by using historical alert data from log files. Drug-drug interaction (DDI) alerts from 3 years of electronic health record data were queried. Alert dwell time was calculated for 25,965 alerts, including 777 unique DDIs. The median alert dwell time was 8 s (range, 1-4913 s). Resident physicians had longer median alert dwell times than other prescribers (P < 001). The 10 most frequent DDI alerts (n = 8759 alerts) had shorter median dwell times than alerts that only occurred once (P < 001). This metric can be used in future research to evaluate the effectiveness and efficiency of interruptive prescribing alerts.

Development and characterization of a human orthotopic neuroblastoma xenograft

Neuroblastoma is a pediatric cancer of the developing sympathoadrenal lineage. The tumors are known to develop from the adrenal gland or paraspinal ganglia and have molecular and cellular features of sympathetic neurons such as dense core vesicles and catecholamine production. Here we present the detailed molecular, cellular, genetic and epigenetic characterization of an orthotopic xenograft derived from a high-risk stage 4 neuroblastoma patient. Overall, the xenografted tumor retained the high risk features of the primary tumor and showed aggressive growth and metastasis in the mouse. Also, the genome was preserved with no additional copy number variations, structural variations or aneuploidy. There were 13 missense mutations identified in the xenograft that were not present in the patient’s primary tumor and there were no new nonsense mutations. None of the missense mutations acquired in the xenograft were in known cancer genes. We also demonstrate the feasibility of using the orthotopic neuroblastoma xenograft to test standard of care chemotherapy and molecular targeted therapeutics. Finally, we optimized a new approach to produce primary cultures of the neuroblastoma xenografts for high-throughput drug screening which can be used to test new combinations of therapeutic agents for neuroblastoma.

Retinal Cell Type DNA Methylation and Histone Modifications Predict Reprogramming Efficiency and Retinogenesis in 3D Organoid Cultures

SUMMARY Diverse cell types can be reprogrammed into pluripotent stem cells by ectopic expression of Oct4 (Pou5f1), Klf4, Sox3, and Myc. Many of these induced pluripotent stem cells (iPSCs) retain memory, in terms of DNA methylation and histone modifications (epigenetic memory), of their cellular origins, and this may bias subsequent differentiation. Neurons are difficult to reprogram, and there has not been a systematic side-by-side characterization of reprogramming efficiency or epigenetic memory across different neuronal subtypes. Here, we compare reprogramming efficiency of five different retinal cell types at two different stages of development. Retinal differentiation from each iPSC line was measured using a quantitative standardized scoring system called STEM-RET and compared to the epigenetic memory. Neurons with the lowest reprogramming efficiency produced iPSC lines with the best retinal differentiation and were more likely to retain epigenetic memory of their cellular origins. In addition, we identified biomarkers of iPSCs that are predictive of retinal differentiation.

Detection of Phenotypic Alterations Using High-Content Analysis of Whole-Slide Images

Tumors exhibit spatial heterogeneity, as manifested in immunohistochemistry (IHC) staining patterns. Current IHC quantification methods lose information by reducing this heterogeneity in each whole-slide image (WSI) or in selective fields of view to a single staining index. The aim of this study was to investigate the sensitivity of an IHC quantification method that uses this heterogeneity to reliably compare IHC staining patterns. We virtually partitioned WSIs by a grid of square tiles, and computed the staining index distributions to quantify heterogeneities. We used samples from these distributions as inputs to non-parametric statistical comparisons. We applied our grid method to fixed tumor samples from 26 tumors obtained from a double-blind preclinical study of a patient-derived orthotopic xenograft model of pediatric neuroblastoma in CD1 nude mice. We compared the results of our grid method to the results based on whole-slide indices, the current practice. We show that our grid method reliably detects phenotypic alterations that other tests based on whole-slide indices fail to detect. Based on robustness and increased sensitivity of statistical inference, we conclude that our method of whole-slide grid quantification is superior to existing whole-slide quantification techniques.

Abstract 2708: Development of an individualized 3D transport model of topotecan for a patient-derived orthotopic xenograft model of pediatric neuroblastoma

Resistance to chemotherapeutics and targeted therapies in pediatric solid tumors including neuroblastoma is a common cause of poor clinical outcome. These failures in part stem from shortcomings in understanding inter- and intra-tumor heterogeneities of drug penetration due to heterogeneities in blood perfusion. Herein we propose to develop an individualized 3D transport model of topotecan (TPT) for a patient-derived orthotopic xenograft model of pediatric NB5 neuroblastoma to account for inter- and intra-tumor heterogeneities in blood perfusion. The transport model uses a 3D reaction-diffusion equation to simulate diffusion of TPT from blood vessels into the tumor tissue and its flux in and out of intracellular space. Our transport model takes three types of inputs to predict TPT exposure maps defined over the volume of an individual tumor: a) plasma concentration-time profiles from an individualized physiologically-based pharmacokinetic (PBPK) model of TPT (separate cohort), b) 3D blood perfusion map of the individual tumor from contrast enhanced ultrasound (CEUS) using VisualSonics VEVO 2100 imaging system, and c) in vitro TPT cellular uptake and efflux kinetics from two-photon imaging. We use in vitro pharmacodynamics (PD) experiments with NB5 cells exposed to TPT to derive probabilistic PD-rules for drug effects (e.g., γ-H2AX response). Based on these rules and the exposure maps, we then compute probabilities of effects for the entire tumor volume. We will validate the predicted drug effect maps by comparing them to the observed effects measured by immunohistochemistry marker for γ-H2AX from the same tumor (location matched) using spatial correlation techniques. Citation Format: Abbas Shirinifard, Suresh Thiagarajan, Yogesh T. Patel, Abigail D. Davis, Megan O. Jacus, Stacy L. Throm, Jessica Roberts, Vinay Daryani, Clinton F. Stewart, Andras Sablauer. Development of an individualized 3D transport model of topotecan for a patient-derived orthotopic xenograft model of pediatric neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2708.

Abstract 1495: Quantification of blood perfusion in rhabdomyosarcoma xenograft in 3D using contrast-enhanced ultrasound imaging

This study aims to estimate individual 3D perfusion maps using nonlinear contrast-enhanced ultrasound imaging (CEUS) over the volume of individual rhabdomyosarcoma xenograft tumors in CD1 nude mice. We implanted the tumors subcutaneously close to the right kidney. We administered MicroMarker® microbubbles via tail vein catheter and acquired images using VisualSonics VEVO 2100 imaging system. We used the burst-replenishment technique to image tumor perfusion. To maintain a steady concentration of microbubbles, we used a programmable pump to inject a small bolus followed by constant infusion, achieving a steady state in less than 1 min, significantly shorter than the commonly used constant infusion without an initial bolus. Our preliminary analysis showed that the nonlinear CEUS signal intensities of kidney cortex have less than 20% variation between mice. We used a custom program to acquire the CEUS perfusion images over a 3D volume that included the tumor and the right kidney. We used the kidney as a reference organ to normalize whole tumor perfusion data. We applied temporal clustering methods to identify regions with similar perfusion profiles. We fitted three perfusion models to each region: a) mono-exponential, b) lognormal, c) beam-specific. We compared the performance of the models based on their overall quality of fit (across all the regions) and their error in estimated pre-burst signal level. Our preliminary analysis showed that log-normal and multi-vessel beam-specific models provide better performance than the mono-exponential model. We estimated individual 3D perfusion maps based on the model fits. These perfusion maps over the entire tumor volume represent tumor perfusion more accurately than the commonly used methods based on a single 2D plane over a region of interest and without including a reference organ. Our approach quantifies intratumoral blood perfusion heterogeneity and enables comparison of perfusion across individuals. Our method is extensible to other animal models and cancer types provided they can be imaged using ultrasound and a reference tissue can be imaged reliably in tandem on a subset of planes containing the tumor. Citation Format: Abbas Shirinifard, Suresh Thiagarajan, Andras Sablauer. Quantification of blood perfusion in rhabdomyosarcoma xenograft in 3D using contrast-enhanced ultrasound imaging. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1495. doi:10.1158/1538-7445.AM2015-1495

Abstract 1496: Quantification of tumor blood perfusion of an orthotopic mouse model of neuroblastoma using nonlinear contrast-enhanced ultrasound imaging

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA This study quantifies tumor perfusion in individual tumors to estimate blood flow and blood volume parameters of an individualized tumor compartment of a comprehensive physiologically-based pharmacokinetic model of topotecan using an orthotopic xenograft model of pediatric neuroblastoma. We non-invasively imaged perfusion in orthotopic neuroblastoma (NB5) xenograft tumors (n = 3 CD1 nude mice/time point) using nonlinear contrast enhanced ultrasound technique (CEUS). Tumor tissue and organs from the mice were harvested at predefined time-points. We used a programmable syringe pump to inject MicroMarker® microbubbles via tail vein catheter and acquired images using VisualSonics VEVO 2100 imaging system. We used the burst-replenishment technique to image tumor perfusion, which requires a constant concentration of microbubbles in blood during acquisition. To maintain a steady concentration of microbubbles, we programmed the pump to inject a small bolus followed by constant infusion. Our preliminary analysis showed that healthy kidneys rapidly reach a steady state in less than 1 min, significantly shorter than the commonly used constant infusion without an initial bolus. The nonlinear CEUS signal intensities of kidney cortex showed less than 20% variation between mice. We used a custom program to acquire the CEUS perfusion images over a 3D volume that included the tumor and a kidney. We used the kidney as a reference organ to normalize whole tumor perfusion data. We fitted the log-normal perfusion model to estimate perfusion parameters for individual tumors. Our perfusion quantification over the entire tumor volume represents tumor perfusion more accurately than the commonly used methods based on a single 2D plane without a reference organ. Our approach provides population estimates of blood perfusion based on properly normalized estimates of individual blood perfusion parameters. Citation Format: Suresh Thiagarajan, Abbas Shirinifard, Megan O. Jacus, Abigail D. Davis, Yogesh T. Patel, Stacy L. Throm, Vinay Daryani, Clinton F. Stewart, Andras Sablauer. Quantification of tumor blood perfusion of an orthotopic mouse model of neuroblastoma using nonlinear contrast-enhanced ultrasound imaging. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1496. doi:10.1158/1538-7445.AM2015-1496