Abstract A28: Clinical next-generation sequencing (NGS) reveals genomic alterations (GAs) to guide targeted therapy in advanced neuroblastoma patients
Authors: Ali Siraj M. , Hawryluk Matthew J. , Wang Kai , Chmielecki Juliann , Palmer Gary A. , Garcia Lazaro , White Emily , Yelensky Roman , Stephens Philip J. , Ross Jeffrey S. , Maris John M. , Miller Vince A. 
Published: May 2015

Abstract
Introduction: High-risk neuroblastoma patients have a survival rate below 50% despite dose-intensive chemoradiotherapy. Treatment using molecularly targeted therapy could more effectively manage patients with less toxicity, but would be best deployed through identification of GAs that suggest responsiveness to such therapies. Recent work from the TARGET initiative (Pugh et al., Nature Genetics 2013) demonstrates a near 10% frequency of ALK GAs in high-risk neuroblastoma patients; such patients could benefit from crizotinib or similar agents targeting the ALK kinase. However, the majority of high-risk neuroblastoma patients still lack identifiable options for targeted therapy. We reviewed the GAs in 17 advanced, high-risk neuroblastoma patients who underwent prospective genomic profiling by clinical NGS to identify actionable alterations that might allow successful trials of targeted therapy.
Methods: Diagnostic genomic profiling was performed to characterize all classes of GAs (base substitutions, small insertions/deletions, copy number alterations, and rearrangements) on primary tumors or metastatic specimens, either pre- or post-chemotherapy, of 17 advanced neuroblastoma patients. For each specimen, 3,320 exons of 182 cancer-related genes and selected introns of 14 frequently rearranged genes or 3769 exons of 236 cancer-related genes and selected introns of 19 frequently rearranged genes (earlier and current version of assay, respectively) were sequenced to a minimum coverage depth of 250x in a CLIA-certified, CAP-accredited lab (Foundation Medicine, Cambridge, MA). Actionable GAs were defined as those for which there were FDA-approved agents and/or agents being evaluated in clinical trials.
Results: The patient population had a median age of 4.6 yrs (range 2 - 18 yrs), 13 were males, and 16 had Stage 4 disease (one Stage 3). Specimens were sequenced to an average depth of 861x, and GAs were present in 100% (17/17) of cases. These 17 cases harbored 27 GAs (1.6 alterations per tumor; range 1 to 5). Ninety-four percent of cases harbored at least one actionable GA, with a mean of 1.6 actionable GAs per tumor (range 1 to 5). Previously described GAs in neuroblastoma were present in this series, such as GAs in ALK (5 cases; 29%), MYCN (5 cases; 29%), and ATRX (2 cases; 12%), which occurred in a largely mutually exclusive fashion. Alterations in ALK were predominantly base substitutions (80%) and included F1174L and R1275Q at frequencies of 40% and 20%, respectively. One ALK alteration was a novel fusion gene predicted to be active in vivo and potentially responsive to crizotinib. Another recurrent actionable alteration is potentially targetable by FGFR inhibitors, as two neuroblastoma cases contained the activating base substitution N546K in FGFR1.
Conclusions: Profiling the tumor genomes of 17 high-risk neuroblastoma patients led to the identification of actionable GAs in a high proportion of patients including alterations previously unseen in neuroblastoma. Specifically, novel fusions of ALK and activating alterations in FGFR1 would not be detected by current molecular diagnostic assays employed in neuroblastoma but offer possible immediate benefit from targeted treatment. Such findings suggest the value of rebiopsying progressive or recurrent high-risk neuroblastoma to identify unforeseen avenues for treatment with targeted therapy.
Citation Format: Siraj M. Ali, Matthew J. Hawryluk, Kai Wang, Juliann Chmielecki, Gary A. Palmer, Lazaro Garcia, Emily White, Roman Yelensky, Philip J. Stephens, Jeffrey S. Ross, John M. Maris, Vince A. Miller. Clinical next-generation sequencing (NGS) reveals genomic alterations (GAs) to guide targeted therapy in advanced neuroblastoma patients. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A28.