Germline pathogenic variants in 786 neuroblastoma patients

Abstract

Importance: Neuroblastoma accounts for 12% of childhood cancer deaths. The genetic contribution of rare pathogenic germline variation in patients without a family history remains unclear.

Objective: To define the prevalence, spectrum, and clinical significance of pathogenic germline variation in cancer predisposition genes (CPGs) in neuroblastoma patients.

Design setting and participants: Germline DNA sequencing was performed on the peripheral blood from 786 neuroblastoma patients unselected for family history. Rare variants mapping to CPGs were evaluated for pathogenicity and the percentage of cases harboring pathogenic (P) or likely pathogenic (LP) variants was quantified. The frequency of CPG P-LP variants in neuroblastoma cases was compared to two distinct cancer-free control cohorts to assess enrichment. Matched tumor DNA sequencing was evaluated for "second hits" at CPGs and germline DNA array data from 5,585 neuroblastoma cases and 23,505 cancer-free control children was analyzed to identify rare germline copy number variants (CNVs) affecting genes with an excess burden of P-LP variants in neuroblastoma. Neuroblastoma patients with germline P-LP variants were compared to those without P-LP variants to test for association with clinical characteristics, tumor features, and patient survival.

Main outcomes and measures: Rare variant prevalence, pathogenicity, enrichment, and association with clinical characteristics, tumor features, and patient survival.

Results: We observed 116 P-LP variants in CPGs involving 13.9% (109/786) of patients, representing a significant excess burden of P-LP variants compared to controls (9.1%; P = 5.14 × 10^-5, Odds Ratio: 1.60, 95% confidence interval: 1.27-2.00). BARD1 harbored the most significant burden of P-LP variants compared to controls (1.0% vs. 0.03%; P = 8.18 × 10^-7; Odds Ratio: 32.30, 95% confidence interval: 6.44-310.35). Rare germline CNVs disrupting BARD1 were also identified in neuroblastoma patients (0.05%) but absent in controls (P = 7.08 × 10^-3; Odds Ratio: 29.47, 95% confidence interval: 1.52 - 570.70). Overall, P-LP variants in DNA repair genes in this study were enriched in cases compared to controls (8.1% vs. 5.7%; P = 0.01; Odds Ratio: 1.45, 95% confidence interval: 1.08-1.92). Neuroblastoma patients harboring a germline P-LP variant had a worse overall survival when compared to patients without P-LP variants (P = 8.6 × 10^-3), and this remained significant in a multivariate Cox proportional-hazards model (P = 0.01).

Conclusions and relevance: Neuroblastoma patients harboring germline P-LP variants in CPGs have worse overall survival and BARD1 is an important predisposition gene affected by both common and rare pathogenic variation. Germline sequencing should be performed for all neuroblastoma patients at diagnosis to inform genetic counseling and support future longitudinal and mechanistic studies. Patients with a germline P-LP variant should be closely monitored, regardless of risk group assignment.

Keywords: DNA repair; Neuroblastoma; genetic predisposition; germline; survival.

Figure 1.. Germline P-LP variants in cancer predisposition genes across 786 neuroblastoma cases.

Oncoprint of known cancer predisposition genes (CPGs) harboring rare germline variants classified as pathogenic (P) or likely pathogenic (LP). Samples without P-LP variants in these genes are not shown. Samples are ordered by COG risk group and annotated with patient clinical and tumor biologic features. Genes are color-coded according to mode of inheritance, when known. Bar chart to the right indicates the number of variants detected for each gene and whether pathogenicity was determined based on ClinVar or our modified InterVar automated assessment. All variants were manually reviewed for quality and evidence of pathogenicity.

Figure 2.. Neuroblastoma cases harbor an excess burden of rare P-LP variants in cancer predisposition genes.

A. Overall excess burden of P-LP variants (SNVs and indels) in neuroblastoma cases vs. PMBB and gnomAD v2.1 controls is shown for cancer predisposition genes and the subset of genes studied involved in DNA repair. B. Gene-based rare variant burden test results comparing the number of neuroblastoma subjects with P-LP variants to those detected in gnomAD v2.1 and PMBB. C. Lollipop figure depicting eight germline P-LP variants in BARD1. D. Rare P-LP variants in BARD1 are observed predominantly in patients diagnosed with high-risk neuroblastoma.

Figure 3.. Rare germline CNVs disrupting BARD1 in neuroblastoma.

A. BARD1 deletions were identified in 3 out of 5,585 neuroblastoma patients through copy number analysis of a large germline DNA array dataset (medium blue, top track). No deletions were observed in 23,505 chip-matched controls. The thick and thin bars represent minimum and maximum deletion coordinates, respectively. One deletion was validated and fine-mapped by WGS (light blue, middle track). No BARD1 protein coding deletions were observed in 10,847 individuals in the gnomAD v2.1 structural variant dataset (dark blue, bottom track). B-D. The three array-based CNV calls are shown in log R ratio (LRR) and B allele frequency (BAF) plots. Darker shading indicates the minimum deleted region, whereas lighter shading indicates the maximum region. E. WGS validation for patient PALXTB is shown as relative sequencing coverage for matched blood and tumor samples. F. Rare BARD1 deletion CNVs are enriched in neuroblastoma compared to cancer-free controls. G. Deletions disrupting BARD1 were observed exclusively in patients diagnosed with high-risk subset of neuroblastoma.

Figure 4.. Patient carriers of germline P-LP variants in CPGs have worse outcome.

Kaplan-Meier plots of overall survival probability in neuroblastoma patients with and without P/LP variants cancer predisposition genes. A. All patients B. Restricted to low- and intermediate risk (non-high-risk) groups. C. Restricted to high-risk group. Statistical significance in A-C assessed by log-rank test (P < 0.05). D. Forest plot of hazard ratios from Cox proportional-hazards model.