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.
Highlights
Neuroblastoma, an embryonal tumor of neuroectodermal origin, accounts for 11% of all cancerrelated deaths in children [1]
We assessed droplet digital PCR (ddPCR) sensitivity in detecting MYCN amplification in a mixture of genomic DNA isolated from two neuroblastoma cell lines
We validated our protocols in a panel of 15 neuroblastoma cell lines and 2 MYC-amplified non-neuroblastoma cell lines to control for assay specificity, and reveal evidence for different MYCN or ALK status than previously reported for 7 cell lines commonly used in research
Summary
Neuroblastoma, an embryonal tumor of neuroectodermal origin, accounts for 11% of all cancerrelated deaths in children [1]. Treatment scenarios range between observation only and multimodal concepts including high-dose chemotherapy with autologous stem cell rescue, surgery, radiotherapy and immunotherapy [1, 4]. Two major remaining obstacles are managing resistance to induction therapy, which causes progression and early death, and managing chemotherapy-resistant relapses due to minimal residual disease, which can occur years after initial diagnosis. MYCN amplifications [5, 6] and activating ALK mutations or amplifications [7,8,9,10] define, among other molecular aberrations, patient subgroups with highly aggressive and frequently therapy-resistant neuroblastomas. One of the first targeted treatment options to become available for chemoresistant neuroblastomas is targeting activating ALK mutations or amplifications by blocking ALK tyrosine kinase activity [11,12,13,14,15]. Promising preclinical strategies include binding or enzymatic inhibition of epigenetically acting proteins such as the BRD4 bromodomain protein [16, 17], the EZH2 [18] or DNMT1 [19] methyltransferases or the histone deacetylases [20, 21], and disturbing mechanisms maintaining MYCN protein stability via the inhibition of aurora kinase A (AURKA) [22]
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