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This figure: (A) Exo-Spin SEC columns were used to isolate exosomes from control and patient plasma. SEC fractions were evaluated for CD63 by ELISA and protein by BCA. There was minimal protein contamination. (B) Exosome-containing fractions (8-12) were pooled and RNA was isolated using Qiagen columns; GAPDH levels were assessed by qPCR (Ct values are presented; error bars represent 2 PCR replicates). This confirms our ability to detect mRNA cargo from isolated exosomes. (C) Exosomes were isolated from the conditioned media of the A673 Ewing sarcoma cell line. RNA was isolated from exosomes and from A673 whole cell lysates (WCL).GUSB and EWS-FLI1 levels were determined in the A673 WCL, and in undiluted and 1:10 diluted exosomal samples. Two replicates were performed per sample. The positive EWSR1-FLI1 fusion transcript confirms the presence of tumor-derived exosomes in the media of the A673 EWS cell line.
As a component of my Pediatric Hematology, Oncology, Neuro-Oncology, and Stem Cell Transplant Fellowship at Lurie Children’s, I worked with Dr. Elizabeth Perlman, investigating mutations in microRNA processing genes and SIX1/2 homeodomains in pre-therapy Favorable Histology Wilms Tumors through the National Cancer Institute’s “Therapeutically Applicable Research to Generate Effective Treatments” (TARGET) Initiative. Through this project, we identified recurrent mutations in the microRNA processing genes DGCR8 and DROSHA as well as in the early developmental genes SIX1 and SIX2.
This was followed by my close participation in two additional TARGET initiative manuscripts. This experience was crucial as it forms the nidus of my experience as a clinician-investigator. It also taught me the importance of developing therapies targeted at the mutations and pathways impacted by an individual patient’s tumor.
As a result, I chose to participate in the Developmental Therapeutics Fellowship through the Northwestern Medicine Developmental Therapeutics Program, which allowed me to develop a deeper understanding of the intricacies in the design, implementation, and conduction of early phase clinical trials. During this time, I completed a review of the potential role of Glycogen Synthase Kinase-3β in the treatment of cancer.
As a result of my investigative and clinical accomplishments, I began my career as an attending physician on the solid tumor team at Lurie Children’s in July 2016. My overall goal is to develop a career in Pediatric Oncology, with a focus on translational research and the development of new biomarkers in pediatric solid tumors.
With the new Precision Medicine initiative at Lurie Children’s, I was excited to secure the Hyundai Hope on Wheels Impact Grant to begin to investigate the use of exosomes as biomarkers to support earlier diagnosis of pediatric solid tumors, track response to treatment, and monitor for the development of progressive or metastatic disease. In addition, I was also appointed to the Children’s Oncology Group’s Renal Tumor Steering Committee in 2018, where I serve as the Developmental Therapeutics lead and participate in clinical trial and biomarker research development.
Therefore, I am uniquely positioned to move my exosome research forward into the clinical realm once able to establish reliable and reproducible methods for the isolation and analysis of exosomes from the peripheral blood of solid tumor patients.
Funded through the Department of Health and Human Services R21 Grant, PAR-16-277
This project seeks to develop reliable and reproducible methods for the isolation and analysis of exosomes from the peripheral blood of pediatric patients with solid tumor containing known fusion transcripts. Exosomes will be isolated from peripheral blood samples and purified to obtain a population of tumor-specific exosomes. These tumor-specific exosomes will then be analyzed for the known fusion transcript identified in the primary tumor sample.
Funded thorough the Children’s Oncology Group Solid Malignancy Integrated Translational Science Center Pilot Grant
This project seeks to develop reliable and reproducible methods for the isolation of exosomes from the peripheral blood of pediatric patients with solid tumor containing known fusion transcripts. Exosomes will be isolated from peripheral blood samples and purified to obtain a population of tumor-specific exosomes. These tumor-specific exosomes will then be analyzed for the known fusion transcript identified in the primary tumor sample.
Amy L. Walz, MD, Principal Investigator
Elizabeth J. Perlman, MD, Collaborator and Mentor
Donna Kersey, Lab Manager and Technician
Samantha Gadd, PhD, Research Scientist