Abstract 3133A: Circulating tumor DNA as “liquid biopsy” in pediatric osteosarcoma
Authors: Fremed Michael , Piperdi Sajida , Zhang Wendong , Maqbool Shahina , Calder Brent , Castellanos Raquel , Gill Jonathan , Roth Michael , Hoang Bang , Geller David , Gorlick Richard , Weiser Daniel 
Published: July 2016

Abstract
BACKGROUND: Osteosarcoma (OS) is the most common primary malignant bone tumor and children and adolescents represent half of new cases diagnosed each year. During and after treatment there is no non-invasive test to assess disease response and early relapse, respectively. Monitoring of circulating tumor material as a “liquid biopsy” has potential to provide this critical information in OS, as it has proven useful in other solid tumors. We hypothesized that circulating tumor DNA (ctDNA) can be extracted from mouse plasma and identified using next generation sequencing. This could ultimately be used to assess tumor burden, evaluate response to treatment, and monitor for recurrence in OS. We overcome the challenge of identifying tumor DNA in a background of host DNA by first using primary tumor material to identify genomic aberrations in a targeted gene region known to be altered in &gt;95% of patients with OS. We then use next generation sequencing to identify in circulation the rare tumor DNA based on known aberrations expected to be present when there is a tumor burden.
MATERIALS AND METHODS: Human osteosarcoma cell lines 143b and M17 were grown in culture and introduced into SCID mice via tail vein, subcutaneous flank, and tibial plateau injections. Terminal bleeds were performed 30 minutes after tail vein injection and at max tumor growth respectively. DNA was extracted from primary tumor cell lines as well as from the plasma of injected mice. We then performed next generation sequencing (NGS) with the Illumina HiSeq 2000 using custom designed probes capturing genes commonly altered in osteosarcoma including TP53, RB1, ATRX, DLG2, MET, PTEN, and SLC19A1. DNA extracted from the plasma of a SCID mouse that did not receive tumor cell injection was used as a negative control.
RESULTS: Gene coverage of approximately 80% was obtained for targeted genes to a depth ranging from 250x to 2000x coverage for tumor cell lines. Cell free circulating tumor DNA was identified in plasma of mice injected with 143b mice via tail vein and flank injections but not from tumor cell free plasma negative control. Over 1000 mutations were identified, most notably c.467G&gt;C p.R156P, a well documented osteosarcoma mutation, which was present in the 143b cell line as well as the DNA extracted from the plasma of tail vein and flank injected mice.
CONCLUSIONS: Circulating cell free tumor DNA can be successfully extracted from SCID mouse plasma and identified using next generation sequencing of target genes. Based on these mouse findings, we anticipate that this will serve as a non-invasive biomarker of disease burden and response to therapy, as well as a biomarker to assess recurrence. The pilot work provides rationale to expand these findings into clinical trials that can prospectively validate our methods and findings. Our approach has the potential to improve outcomes for a childhood cancer frequently associated with poor survival.
Citation Format: Michael Fremed, Sajida Piperdi, Wendong Zhang, Shahina Maqbool, Brent Calder, Raquel Castellanos, Jonathan Gill, Michael Roth, Bang Hoang, David Geller, Richard Gorlick, Daniel Weiser. Circulating tumor DNA as “liquid biopsy” in pediatric osteosarcoma. [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 3133A.