Abstract 5679: Using droplet digital PCR to analyze MYCN copy number in plasma from patients with high-risk neuroblastoma

Abstract

Abstract The invasive nature of surgical biopsies most often prevents their sequential application to monitor disease in patients with high-risk neuroblastoma. Even when available, single biopsies often fail to reflect neuroblastoma dynamics, intratumor heterogeneity and drug sensitivities likely to change during neuroblastoma evolution and treatment. Implementing molecular characterization of cell-free neuroblastoma-derived DNA (cfDNA) isolated from blood plasma would improve outcome prediction, patient monitoring and treatment selection for high-risk neuroblastoma patients, by providing a method to follow clonal evolution in tumor subpopulations and treatment response as well as capture the molecular landscape of all tumor clones. As a first step, we established a digital droplet PCR (ddPCR) protocol to analyze the MYCN copy number status. Analyzing the dilution series of a synthetic MYCN template consisting of 70 nucleotides demonstrated a strong correlation between theoretically calculated MYCN copy number and the MYCN copy number measured using our ddPCR protocol. We compared MYCN copy number status in a large panel of neuroblastoma cell lines with MYCN copy number determined from cfDNAs isolated from cell culture media from the corresponding cell lines. Our ddPCR protocol reliably detected MYCN status in the cell line using cfDNA analysis. Next, we turned to a subcutaneous MYCN-driven neuroblastoma xenograft model to compare MYCN copy number in the xenografted tumor to MYCN copy number determined from cfDNA isolated from the mouse blood plasma. MYCN copy numbers also positively correlated using this model. We then analyzed MYCN copy number status in primary neuroblastoma samples from 10 patients and compared these values to the MYCN copy number determined from cfDNA isolated from 200 µl of blood plasma collected from the corresponding patients. These final validation steps demonstrated that our ddPCR protocol for cfDNA from patient blood plasma reliably detects MYCN copy number status in the tumor. Our data justify the further development of molecular neuroblastoma characterization from cfDNA in patient blood plasma. An expanded molecular diagnostic monitoring palette will improve monitoring of disease progression including relapse and metastatic events as well as therapy success or failure in high-risk neuroblastoma patients. Citation Format: Marco Lodrini, Annika Sprüssel, Kathy Astrahantseff, Daniela Tiburtius, Robert Konschak, Holger Lode, Matthias Fischer, Johannes H. Schulte, Angelika Eggert, Hedwig Deubzer. Using droplet digital PCR to analyze MYCN copy number in plasma from patients with high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5679. doi:10.1158/1538-7445.AM2017-5679