Use of Optical Genome Mapping to Detect Structural Variants in Neuroblastoma.

BACKGROUND: Neuroblastoma is the most common extracranial solid tumour in children, accounting for 15% of paediatric cancer deaths. Multiple genetic abnormalities have been identified as prognostically significant in neuroblastoma patients. Optical genome mapping (OGM) is a novel cytogenetic technique used to detect structural variants, which has not previously been tested in neuroblastoma. We used OGM to identify copy number and structural variants (SVs) in neuroblastoma which may have been missed by standard cytogenetic techniques. METHODS: Five neuroblastoma cell lines (SH-SY5Y, NBLW, GI-ME-N, NB1691 and SK-N-BE2(C)) and two neuroblastoma tumours were analysed using OGM with the Bionano Saphyr® instrument. The results were analysed using Bionano Access software and compared to previous genetic analyses including G-band karyotyping, FISH (fluorescent in situ hybridisation), single-nucleotide polymorphism (SNP) array and RNA fusion panels for cell lines, and SNP arrays and whole genome sequencing (WGS) for tumours. RESULTS: OGM detected copy number abnormalities found using previous methods and provided estimates for absolute copy numbers of amplified genes. OGM identified novel SVs, including fusion genes in two cell lines of potential clinical significance. CONCLUSIONS: OGM can reliably detect clinically significant structural and copy number variations in a single test. OGM may prove to be more time- and cost-effective than current standard cytogenetic techniques for neuroblastoma.

Combination Therapies Targeting ALK-aberrant Neuroblastoma in Preclinical Models.

PURPOSE: ALK-activating mutations are identified in approximately 10% of newly diagnosed neuroblastomas and ALK amplifications in a further 1%-2% of cases. Lorlatinib, a third-generation anaplastic lymphoma kinase (ALK) inhibitor, will soon be given alongside induction chemotherapy for children with ALK-aberrant neuroblastoma. However, resistance to single-agent treatment has been reported and therapies that improve the response duration are urgently required. We studied the preclinical combination of lorlatinib with chemotherapy, or with the MDM2 inhibitor, idasanutlin, as recent data have suggested that ALK inhibitor resistance can be overcome through activation of the p53-MDM2 pathway. EXPERIMENTAL DESIGN: We compared different ALK inhibitors in preclinical models prior to evaluating lorlatinib in combination with chemotherapy or idasanutlin. We developed a triple chemotherapy (CAV: cyclophosphamide, doxorubicin, and vincristine) in vivo dosing schedule and applied this to both neuroblastoma genetically engineered mouse models (GEMM) and patient-derived xenografts (PDX). RESULTS: Lorlatinib in combination with chemotherapy was synergistic in immunocompetent neuroblastoma GEMM. Significant growth inhibition in response to lorlatinib was only observed in the ALK-amplified PDX model with high ALK expression. In this PDX, lorlatinib combined with idasanutlin resulted in complete tumor regression and significantly delayed tumor regrowth. CONCLUSIONS: In our preclinical neuroblastoma models, high ALK expression was associated with lorlatinib response alone or in combination with either chemotherapy or idasanutlin. The synergy between MDM2 and ALK inhibition warrants further evaluation of this combination as a potential clinical approach for children with neuroblastoma.

Loss of ALK hotspot mutations in relapsed neuroblastoma.

ALK is the most commonly mutated oncogene in neuroblastoma with increased mutation frequency reported at relapse. Here we report the loss of an ALK mutation in two patients at relapse and a paired neuroblastoma cell line at relapse. ALK detection methods including Sanger sequencing, targeted next-generation sequencing and a new ALK Agena MassARRAY technique were used to detect common hotspot ALK variants in tumors at diagnosis and relapse from two high-risk neuroblastoma patients. Copy number analysis including single nucleotide polymorphism array and array comparative genomic hybridization confirmed adequate tumor cell content in DNA used for mutation testing. Case 1 presented with an ALK F1174L mutation at diagnosis with a variant allele frequency (VAF) ranging between 23.5% and 28.5%, but the mutation was undetectable at relapse. Case 2 presented with an ALK R1257Q mutation at diagnosis (VAF = 39%-47.4%) which decreased to <0.01% at relapse. Segmental chromosomal aberrations were maintained between diagnosis and relapse confirming sufficient tumor cell content for mutation detection. The diagnostic SKNBE1n cell line harbors an ALK F1174S mutation, which was lost in the relapsed SKNBE2c cell line. To our knowledge, these are the first reported cases of loss of ALK mutations at relapse in neuroblastoma in the absence of ALK inhibitor therapy, reflecting intra-tumoral spatial and temporal heterogeneity. As ALK inhibitors are increasingly used in the treatment of refractory/relapsed neuroblastoma, our study highlights the importance of confirming whether an ALK mutation detected at diagnosis is still present in clones leading to relapse.