Clinical evaluation of integrated panel testing by next-generation sequencing for somatic mutations in neuroblastomas with MYCN unamplification

Abstract

Neuroblastomas (NBs) exhibit heterogeneity and show clinically significant prognosis classified by genetic alterations. Among prognostic genes or genome factors, MYCN amplification (MNA) is the most established genomic marker of poor prognosis in patients with NB. However, the prognostic classification of more than 60% of patients without MNA has yet to be clarified. In this study, the application of target next-generation sequencing (NGS) was extended on the basis of a comprehensive panel of regions where copy number variations (CNVs) or point mutations occurred to improve the prognostic evaluation of these patients and obtain the sequence of 33 patients without MNA. A mean coverage depth of 887× was determined in the target regions in all of the samples, and the mapped read percentage was more than 99%. Somatic mutations in patients without MNA could be precisely defined on the basis of these findings, and 17 unique somatic aberrations, including 14 genes, were identified in 11 patients. Among these variations, most were CNVs with a number of 13. The 3-year event-free survival (EFS) of CNV(-) patients was 60.0% compared with the EFS (16.7%) of CNV(+) patients (P = 0.015, HR = 0.1344, 95%, CI = 0.027 to 0.678). CNVs were also associated with unfavorable histological characteristics (P = 0.003) and likely to occur in stage 4 (P = 0.041). These results might further indicate the role of CNVs in NB chemotherapy resistance (P = 0.059) and show CNVs as a therapeutic target. In multivariate analysis, the presence of CNVs was a clinically negative prognostic marker that impaired the outcome of patients without MNA and associated with poor prognosis in this tumor subset. Comprehensive genetic/genomic profiling instead of focusing on single genetic marker should be performed through in-depth NGS that could reveal prognostic information, improve NB target therapy, and provide a basis for investigations on NB pathogenesis.

Keywords: DNA copy number variations; high-throughput nucleotide sequencing; neuroblastoma; prognosis; treatment.

Figure 1. 13 CNVs of 8 patients

(CN_amp: copy number amplification; CN_del: copy number deletion)

Figure 2. H&E staining of the patient with TIAM1 deletion

(A) HE 200 ×; (B) HE 400 ×

Figure 3. Four point mutations in four patients

(2 ALK: F1174L; BRCA2: K2392N; PHOX2B: F86fs)

Figure 4. Oncoprint outcome of 11 patients (detected somatic mutations were mostly CNVs, including CN amp and CN del)

CDK4, CCND1, and ALK variations appeared most frequently, and they were present in two patients)

Figure 5. Summary of three-year EFS rates and log-rank test comparisons by CNV outcome

Figure 6. Distribution of obtaining a VGPR after four cycles of chemotherapy between CNV(+) and CNV (−)