Neuroblastoma Molecular Risk-Stratification of DNA Copy Number and ALK Genotyping via Cell-Free Circulating Tumor DNA Profiling

Abstract

Background: MYCN amplification (MNA), segmental chromosomal aberrations (SCA) and ALK activating mutations are biomarkers for risk-group stratification and for targeted therapeutics for neuroblastoma, both of which are currently assessed on tissue biopsy. Increase in demand for tumor genetic testing for neuroblastoma diagnosis is posing a challenge to current practice, as the small size of the core needle biopsies obtained are required for multiple molecular tests. We evaluated the utility of detecting these biomarkers in the circulation. Methods: Various pre-analytical conditions tested to optimize circulating-tumor DNA (ctDNA) copy number changes evaluations. Plasma samples from 10 patients diagnosed with neuroblastoma assessed for SCA and MNA using single nucleotide polymorphism (SNP) array approach currently used for neuroblastoma diagnosis, with MNA status assessed independently using digital-droplet PCR (ddPCR). Three patients (one in common with the previous 10) tested for ALK activating mutations p.F1174L and p.F1245I using ddPCR. Results: Copy number detection is highly affected by physical perturbations of the blood sample (mimicking suboptimal sample shipment), which could be overcome using specialized preservative collection tubes. Pre-analytical DNA repair procedures on ctDNA before SNP chromosome microarray processing improved the lower limit of detection for SCA and MNA, defined as 20% and 10%, respectively. We detected SCA in 10/10 (100%) patients using SNP array, 7 of which also presented MNA. Circulating-free DNA (cfDNA) and matched tumor DNA profiles were generally identical. MNA was detected using ddPCR in 7/7 (100%) of MNA and 0/12 (0%) non-MNA cases. MNA and ALK mutation dynamic change was assessed in longitudinal samples from 4 and 3 patients (one patient with both), respectively, accurately reflected response to treatment in 6/6 (100%) and disease recurrence in 5/6 (83%) of cases. Samples taken prior to targeted treatment with the ALK inhibitor Lorlatinib and 6–8 weeks on treatment showed reduction/increase in ALK variants according to response to treatment. Conclusions: These results demonstrate the feasibility of ctDNA profiling for molecular risk-stratification, and treatment monitoring in a clinically relevant time frame and the potential to reduce fresh tissue requirements currently embedded in the management of neuroblastoma.

Circulating Tumor DNA in Pediatric Cancer

“…CtDNA could also be applied in other solid tumor treatment assessments where poor response is usually observed, such as end of induction therapy for high-risk neuroblastoma. In children with ALK mutant or ALK amplified high-risk neuroblastoma, ctDNA monitoring of this marker could identify patients who may benefit from alternative treatment strategies such as the ALK inhibitor lorlatinib, as demonstrated by Kahana-Edwin et al ( Kahana-Edwin et al, 2021b ). There is strong evidence for the importance of profiling tumors at relapse, including RAS-MAPK pathway mutations known to occur in relapsed/refractory disease ( Eleveld et al, 2015 ).…”

“…While the available evidence points to the importance of ctDNA in therapeutic decision-making and response evaluation, this is yet to be conclusive. For risk stratification, a prospective study of 13 patients revealed that ctDNA was a successful surrogate marker for molecular diagnosis of segmental chromosomal aberrations, MYCN amplifications and ALK variants, thereby presenting a relevant model for ctDNA analysis ( Kahana-Edwin et al, 2021b ).…”

Neuroblastoma Heterogeneity, Plasticity, and Emerging Therapies

“…For neuroblastoma with ALK mutations, the TITAN study (transatlantic integration targeting ALK in neuroblastoma) will investigate the frontline addition of lorlatinib, an ALK inhibitor with activity against a wider range of ALK mutations and penetrance to CNS [ 93 – 95 ]. In this context, liquid biopsies for ALK mutations might prove useful to monitor lorlatinib treatment response [ 96 ].…”

Liquid biopsies in pediatric oncology: opportunities and obstacles

“…Taken together, pediatric assays should capture a proportional or higher ratio of ctDNA:cfDNA because of relative tumor burden in similarly staged diseases [98]. Indeed, pediatric studies in neuroblastoma [65,99], EWS [66], and hepatoblastoma [80] have demonstrated the feasibility of sensitive detection from less than 1 ml of plasma, and studies of unilateral intraocular retinoblastoma, characterized by exceptionally small tumor volumes, detect ctDNA in plasma [83,100].…”

“…Intermediate-risk neuroblastoma patients without MYCN amplification or specific SCA maintain excellent outcomes with treatment reduction [60]; therefore, genomic misclassification would lead to inappropriate de-escalation of treatment. Multiple studies demonstrate sensitive and tissue concordant molecular characterization of these genes and SCA in neuroblastoma using liquid biopsies [61][62][63][64][65]. The promise of liquid biopsy for risk stratification is further illustrated in pediatric sarcomas.…”