Using droplet digital PCR to analyze MYCN and ALK copy number in plasma from patients with neuroblastoma

// Marco Lodrini 1 , Annika Sprussel 1 , Kathy Astrahantseff 1 , Daniela Tiburtius 1 , Robert Konschak 2, 3 , Holger N. Lode 4 , Matthias Fischer 5, 6, 7 , Ulrich Keilholz 8 , Angelika Eggert 1, 3, 9 and Hedwig E. Deubzer 1, 9, 10 1 Department of Pediatric Hematology, Oncology and Stem Cell Transplantation, Charite–Universitatsmedizin Berlin, Berlin, Germany 2 Translational Radiation Oncology Research Laboratory, Department of Radiooncology and Radiotherapy, Charite–Universitatsmedizin Berlin, Berlin, Germany 3 German Cancer Consortium (DKTK), Partner Site Berlin, Berlin, Germany 4 Department of Pediatric Hematology and Oncology, University Medicine Greifswald, Ferdinand-Sauerbruch-Strase, Germany 5 Department of Pediatric Hematology and Oncology, University Hospital Cologne, Cologne, Germany 6 Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany 7 Max Planck Institute for Metabolism Research, Cologne, Germany 8 Charite Comprehensive Cancer Center, Charite – Universitatsmedizin Berlin, Berlin, Germany 9 Berlin Institute of Health (BIH), Berlin, Germany 10 Junior Neuroblastoma Research Group, Experimental and Clinical Research Center (ECRC), Berlin, Germany Correspondence to: Marco Lodrini, email: marco.lodrini@charite.de Keywords: intratumor heterogeneity, liquid biopsy, non-invasive biomarker, pediatric cancer, tumor dynamics Received: April 21, 2017      Accepted: June 20, 2017      Published: July 07, 2017 ABSTRACT The invasive nature of surgical biopsies deters sequential application, and single biopsies often fail to reflect tumor dynamics, intratumor heterogeneity and drug sensitivities likely to change during tumor evolution and treatment. Implementing molecular characterization of cell-free neuroblastoma-derived DNA isolated from blood plasma could improve disease assessment for treatment selection and monitoring of patients with high-risk neuroblastoma. We established droplet digital PCR (ddPCR) protocols for MYCN and ALK copy number status in plasma from neuroblastoma patients. Our ddPCR protocol accurately discriminated between MYCN and ALK amplification, gain and normal diploid status in a large panel of neuroblastoma cell lines, and discrepancies with reported MYCN and ALK status were detected, including a high-level MYCN amplification in NB-1, a MYCN gain in SH-SY5Y, a high-level ALK amplification in IMR-32 and ALK gains in BE(2)-C, Kelly, SH-SY5Y and LAN-6. MYCN and ALK status were also reliably determined from cell-free DNA derived from medium conditioned by the cell lines. MYCN and ALK copy numbers of subcutaneous neuroblastoma xenograft tumors were accurately determined from cell-free DNA in the mouse blood plasma. In a final validation step, we accurately distinguished MYCN and ALK copy numbers of the corresponding primary tumors using retrospectively collected blood plasma samples from 10 neuroblastoma patients. Our data justify the further development of molecular disease characterization using cell-free DNA in blood plasma from patients with neuroblastoma. This expanded molecular diagnostic palette may improve monitoring of disease progression including relapse and metastatic events as well as therapy success or failure in high-risk neuroblastoma patients.