The genetic landscape of high-risk neuroblastoma

PMID: 23334666 · DOI: 10.1038/ng.2529 · Journal: Nature Genetics (2013)

TL;DR

This study characterized the somatic mutational landscape of 240 high-risk, metastatic (stage 4) neuroblastomas using whole-exome, whole-genome, and transcriptome sequencing as part of the TARGET initiative. The authors found a remarkably low median exonic mutation frequency (0.60 per megabase) and very few recurrently mutated genes: ALK (9.2%), PTPN11 (2.9%), ATRX (2.5% mutations plus 7.1% focal deletions), MYCN (1.7%, a recurrent p.Pro44Leu alteration), and NRAS (0.83%). Pathway analysis implicated RAS/MAPK signaling. Rare germline pathogenic variants were enriched in ALK, CHEK2, PINK1, and BARD1, suggesting a larger role for germline predisposition than previously suspected.

Cohort & data

• 240 matched tumor/normal pairs from high-risk stage 4 NBL patients (>18 months at diagnosis, metastatic disease)
• Dataset: nbl_broad_2013
• Whole-exome sequencing (221 cases, ~33 Mb targeted, 124X mean coverage)
• Whole-genome sequencing (19 cases: 10 Illumina at 29.7X, 10 Complete Genomics at 59.9X)
• RNA-seq (10 Illumina WGS cases, >10 Gbp per case)
• Significance testing with MutSig
• Males 62%, median age 3.4 years, MYCN amplification in 32%
• Reference genome: hg19/GRCh37

Key findings

• Median exonic mutation frequency of 0.60 per megabase (0.48 non-silent), consistent with other pediatric cancers and far below adult solid tumors.
• Only 5 genes with both statistical significance and biological rationale for neuroblastoma involvement: ALK (9.2%), PTPN11 (2.9%), ATRX (2.5% mutations + 7.1% deletions = 9.6% total), MYCN (1.7%), and NRAS (0.83%).
• ALK mutation was the only significantly mutated gene associated with decreased overall survival (p=0.0103).
• ATRX alterations were mutually exclusive with MYCN amplification and enriched in older children (p=0.0021).
• MYCN harbored a recurrent p.Phe44Leu activating mutation in 4 cases without MYCN amplification, scored as functional by multiple prediction tools.
• Gene set analysis: 12 of 857 gene sets enriched for somatic mutation (q<0.1), with 8 implicating RAS/MAPK signaling.
• Two hyper-mutated tumors (7.27 and 4.29 mutations/Mb) harbored MLH1 alterations.
• Structural analysis of 19 WGS cases: median 41.5 breakpoints per case; no recurrent fusion transcripts; NBAS most commonly rearranged gene.
• Germline analysis identified enrichment of pathogenic variants in ALK, CHEK2, PINK1, BARD1, and TP53.

Genes & alterations

Gene	Alteration	Frequency	Notes
ALK	Kinase domain missense mutations	9.2% (22/240)	All restricted to kinase domain; germline p.Arg1275Gln also found; associated with worse OS (p=0.0103)
PTPN11	Activating missense mutations	2.9% (7/240)	All previously reported in COSMIC
ATRX	Loss-of-function mutations + multi-exon deletions	9.6% (6 mutations + 17 deletions)	Mutually exclusive with MYCN amplification; enriched in older children
MYCN	p.Phe44Leu point mutation	1.7% (4/240)	Recurrent in non-amplified cases; highly conserved residue; predicted functional
NRAS	Activating missense mutations	0.83% (2/240)	Significant only in COSMIC-restricted analysis
CHEK2	Germline destabilizing variants	3 patients	Cancer predisposition alleles not previously described in NBL
BARD1	Germline loss-of-function (p.Arg112, p.Arg641)	2 patients	Known NBL susceptibility locus; rare variants at common-variant susceptibility loci
PINK1	Germline variants (p.Leu437Pro, p.Arg279His)	2 patients	Transcriptionally regulated by MYC proteins
TP53	Germline p.Pro219Ser	1 patient	Associated with Li-Fraumeni syndrome
PALB2	Germline splice-site variant + somatic missense	2 patients	BRCA complex member
ARID1A	Somatic missense (p.G1139V, p.G1942D)	2/240	Chromatin remodeling
ARID1B	Focal deletions	2/240	Chromatin remodeling
MLH1	Nonsense mutation + deletion	1 patient	Associated with hyper-mutated phenotype

Clinical implications

• ALK kinase domain mutations represent an actionable therapeutic target (crizotinib and next-generation ALK inhibitors), but only 9.2% of high-risk cases carry these mutations.
• The low overall somatic mutation burden challenges the concept that druggable targets can be defined in each patient by DNA sequencing alone.
• Germline predisposition variants in ALK, CHEK2, BARD1, PINK1, and TP53 suggest that genetic testing may have broader utility in neuroblastoma families than previously suspected.
• The data suggest that the majority of high-risk neuroblastomas may be driven by copy number alterations, epigenetic modifications, and rare germline variants rather than recurrent somatic point mutations.

Limitations & open questions

• Cohort restricted to high-risk stage 4 disease (>18 months, metastatic); findings may not generalize to lower-risk or infant neuroblastoma.
• Whole-genome sequencing limited to 19 cases; structural rearrangements and non-coding mutations likely under-ascertained.
• Phi29-based whole-genome amplification used for 80/222 WES cases; oxoG artifacts required computational correction.
• No recurrent fusion transcripts identified, but RNA-seq available for only 10 cases.
• Germline analysis compared to European American controls only (ESP); not corrected for multiple testing.
• Epigenetic drivers (methylation, chromatin state) not directly profiled.
• The relative paucity of druggable targets raises the question of whether functional genomics, epigenomics, or combinatorial approaches are needed for therapeutic development in neuroblastoma.

Citations from this paper used in the wiki

• “We studied 240 cases using a combination of whole exome, genome and transcriptome sequencing […] Here we report a low median exonic mutation frequency of 0.60 per megabase (0.48 non-silent), and remarkably few recurrently mutated genes in these tumors.” (Abstract)
• “Genes with significant somatic mutation frequencies included ALK (9.2% of cases), PTPN11 (2.9%), ATRX (2.5%, an additional 7.1% had focal deletions), MYCN (1.7%, a recurrent p.Pro44Leu alteration), and NRAS (0.83%).” (Abstract)
• “Rare, potentially pathogenic germline variants were significantly enriched in ALK, CHEK2, PINK1, and BARD1.” (Abstract)
• “ALK was the only significantly mutated gene with an association with clinical outcome, as mutation positive cases had a decreased overall survival probability (p=0.0103)” (Results)
• “The relative paucity of recurrent somatic mutations in neuroblastoma challenges current therapeutic strategies reliant upon frequently altered oncogenic drivers.” (Abstract)

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