Whole-Exome Sequencing Reveals Novel Candidate Driver Mutations and Potential Druggable Mutations in Patients with High-Risk Neuroblastoma

Abstract

Neuroblastoma is the most prevalent solid tumor in early childhood, with a 5-year overall survival rate of 40-60% in high-risk cases. Therefore, the identification of novel biomarkers for the diagnosis, prognosis, and therapy of neuroblastoma is crucial for improving the clinical outcomes of these patients. In this study, we conducted the whole-exome sequencing of 48 freshly frozen tumor samples obtained from the Biobank. Somatic variants were identified and selected using a bioinformatics analysis pipeline. The mutational signatures were determined using the Mutalisk online tool. Cancer driver genes and druggable mutations were predicted using the Cancer Genome Interpreter. The most common mutational signature was single base substitution 5. MUC4, MUC16, and FLG were identified as the most frequently mutated genes. Using the Cancer Genome Interpreter, we identified five recurrent cancer driver mutations spanning MUC16, MUC4, ALK, and CTNND1, with the latter being novel and containing a missense mutation, R439C. We also identified 11 putative actionable mutations including NF1 Q1798*, Q2616*, and S636X, ALK F1174L and R1275Q, SETD2 P10L and Q1829E, BRCA1 R612S, NOTCH1 D1670V, ATR S1372L, and FGFR1 N577K. Our findings provide a comprehensive overview of the novel information relevant to the underlying molecular pathogenesis and therapeutic targets of neuroblastoma.

Keywords: cancer driver gene; druggable mutations; mutational signatures; neuroblastoma; somatic variants; tumor mutational burden; tumor-only; whole-exome sequencing.

Figure 1

Landscape of somatic variants in patients with NB. (A) The mutation summary in NB displays the distribution of variant classification, variant type distribution, base substitution type, the number of somatic variants in each sample, a summary of variant classification, and the top 10 genes with the highest number of mutations. (B) Oncoplot of the top 20 mutated genes in 48 NB cases. (C) Co-occurring and mutually exclusive interactions among the top 20 frequently mutated genes. A tendency toward co-occurrence and mutual exclusivity is represented with bluish-green and brown, respectively. The statistical significance for each pair of genes is determined using Fisher’s exact test.

Figure 2

The lollipop plot of the top six most commonly mutated genes. (A) MUC4. (B) MUC16. (C) FLG. (D) OBSCN. (E) RNF213. (F) DMD. The x-axis represents the mutation position relative to a schematic representation of a gene and the y-axis represents the total number of mutations at each alteration.

Figure 3

Correlation between somatic mutational status and overall survival (OS) of patients with NB. (A) Wild-type and mutated MUC4. (B) Wild-type and mutated MUC16. (C) Wild-type and mutated FLG. (D) Wild-type and mutated OBSCN. (E) Wild-type and mutated RNF213. (F) Wild-type and mutated DMD. Censored subjects are denoted with tick marks on the curves. The log-rank test was used to compare differences in OS between patients with wild-type genes and mutations.

Figure 4

Correlation between mutated MUC4 LRRN and OS of patients with NB. Censored subjects are denoted with tick marks on the curves. The log-rank test was used to compare differences in OS between patients with wild-type genes and mutations.

Figure 5

Tumor mutational burden (TMB), its prognostic value and association with clinicopathological characteristics, and comparison of TMB. (A) TMB scores of each patient in the NB cohort. (B–D) Correlation analysis between TMB and clinicopathological factors, including age, gender, and primary site. (E) Survival analysis of patients with high and low TMB.

Figure 6

Mutational signatures enriched in the NB cohort. Columns represent the COSMIC signatures and rows indicate the samples. The numbers in the matrix represent the percentage of the relative contribution of signatures, with darker brown indicating a higher percentage contribution of the COSMIC signature.

Figure 7

Enriched oncogenic signaling pathways in NB. (A) Oncogenic pathways based on the number of mutated genes in the pathway and samples. (B–D) Somatic variants in genes of the RTK-RAS, NOTCH, and Hippo pathways across different samples. Blue text represents oncogenes, and red text represents tumor suppressor genes.

Figure 8

Enrichment analysis of mutated genes in NB. (A–C) Top 10 significantly enriched Gene Ontology terms in biological process, cellular component, and molecular function. (D) Top 10 significantly enriched KEGG pathways. (E) Top 10 hub genes. The color gradient from yellow to red indicates the degree of hub importance—yellow for less important hub genes and red for the most important hub genes.

Figure 9

An oncoplot of somatic mutations associated with chemotherapy resistance in patients from both chemosensitivity and chemoresistance groups.