Neuroblastoma is a malignant neoplasm of the developing sympathetic nervous system that is notable for its phenotypic diversity. High-risk patients typically have widely disseminated disease at ...diagnosis and a poor survival probability, but low-risk patients frequently have localized tumors that are almost always cured with little or no chemotherapy. Our genome-wide association study (GWAS) has identified common variants within FLJ22536, BARD1, and LMO1 as significantly associated with neuroblastoma and more robustly associated with high-risk disease. Here we show that a GWAS focused on low-risk cases identified SNPs within DUSP12 at 1q23.3 (P = 2.07 × 10⁻⁶), DDX4 and IL31RA both at 5q11.2 (P = 2.94 × 10⁻⁶ and 6.54 × 10⁻⁷ respectively), and HSD17B12 at 11p11.2 (P = 4.20 × 10⁻⁷) as being associated with the less aggressive form of the disease. These data demonstrate the importance of robust phenotypic data in GWAS analyses and identify additional susceptibility variants for neuroblastoma.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Neuroblastoma cell lines are an important and cost-effective model used to study oncogenic drivers of the disease. While many of these cell lines have been previously characterized with SNP, ...methylation, and/or mRNA expression microarrays, there has not been an effort to comprehensively sequence these cell lines. Here, we present raw whole transcriptome data generated by RNA sequencing of 39 commonly-used neuroblastoma cell lines. These data can be used to perform differential expression analysis based on a genetic aberration or phenotype in neuroblastoma (e.g., MYCN amplification status, ALK mutation status, chromosome arm 1p, 11q and/or 17q status, sensitivity to pharmacologic perturbation). Additionally, we designed this experiment to enable structural variant and/or long-noncoding RNA analysis across these cell lines. Finally, as more DNase/ATAC and histone/transcription factor ChIP sequencing is performed in these cell lines, our RNA-Seq data will be an important complement to inform transcriptional targets as well as regulatory (enhancer or repressor) elements in neuroblastoma.
MYCN amplification and 11q deletion are two inversely correlated prognostic factors of poor outcome in neuroblastoma. Here we identify common variants at 11q22.2 within MMP20 that associate with ...neuroblastoma cases harboring 11q deletion (rs10895322), using GWAS in 113 European-American cases and 5109 ancestry-matched controls. The association is replicated in 44 independent cases and 1902 controls. Our study yields novel insights into the genetic underpinnings of neuroblastoma, demonstrating that the inherited common variants reported contribute to the origin of intra-tumor genetic heterogeneity in neuroblastoma.Chromosomal abnormalities such as 11q deletion are associated with poor prognosis in neuroblastoma. Here, the authors perform a genome-wide association study and identify an association between a variant within a Matrix metalloproteinase (MMP) gene member, MMP20, and 11q-deletion subtype neuroblastoma.
Neuroblastoma is an often fatal pediatric cancer more frequent in European-American than African-American children. African-American children, however, are at higher risk for the more severe form of ...neuroblastoma and have worse overall survival than European-American children. Genome-wide association studies (GWAS) have identified several single-nucleotide polymorphisms (SNP) associated to neuroblastoma in children of European descent. Knowledge of their association to neuroblastoma in African-American children is still lacking.
We genotyped and imputed SNPs located in three gene regions reported to be associated to neuroblastoma in children of European descent, and tested them for association in 390 African-American patients with neuroblastoma compared with 2,500 healthy, ethnically matched controls.
SNPs in the BARD1 gene region show a similar pattern of association to neuroblastoma in African-American and European-American children. The more restricted extent of linkage disequilibrium in the African-American population suggests a smaller candidate region for the putative causal variants than previously reported. Limited association was observed at the other two gene regions tested, including LMO1 in 11p15 and FLJ22536 in 6p22.
Common BARD1 SNPs affect risk of neuroblastoma in African-Americans. The role of other SNPs associated to neuroblastoma in children of European descent could not be confirmed, possibly due to different patterns of linkage disequilibrium or limited statistical power to detect association to variants with small effect on disease risk. Extension of GWAS to populations of African descent is important to confirm their results and validity beyond the European populations and can help to refine the location of the putative causal variants.
Transgenic mice with cardiac-specific over-expression of tumor necrosis factor-α (TNF1.6) progress to dilated heart failure. A significant inflammatory response precedes functional deterioration, and ...may contribute to cardiac damage in this model. To evaluate the underlying molecular mechanisms, we assessed the gene expression in six groups of mouse hearts defined by age, gender, and phenotype (
n = 3/group) using Affymetrix microarray analysis. Phenotype was defined as compensated (in young TNF1.6) or decompensated (in older TNF1.6) via echocardiogram. Of the >1000 transcripts altered in the compensated hearts (fold change > 2,
P < 0.05 vs. wild-type (WT)), 102 were identified as immune response genes, 20 of which function in antigen presentation and processing. When comparing the compensated and decompensated hearts, >50 genes were differentially regulated, including seven immunoglobulin genes. Real-time reverse transcriptase-polymerase chain reaction and cDNA microarray confirmed the Affymetrix data. Mac3+ macrophages, CD4+ T and CD45/B220+ B-cells were identified in both compensated and decompensated hearts. However, a large amount of IgG was found deposited in areas devoid of B-lymphocytes in the myocardium of decompensated TNF1.6 mice; no such accumulation was seen in the compensated or age-matched controls. Furthermore, nuclei density analyses showed a two-fold increase in the myocardium of both compensated and decompensated TNF1.6 mice (vs. WT). This study suggests that TNF-α over-expression activates not only the inflammatory response, but also humoral immune responses within the transgenic hearts. The autoimmune response occurs concomitantly with cardiac decompensation and may participate in triggering the transition to failure in TNF1.6 mice.
Abstract
We recently showed that relapsed neuroblastoma (NB) tumors harbor an increased somatic mutational burden, enriched for ALK or RAS-MAPK activating lesions, nominating targeted therapeutic ...strategies for a condition that is largely incurable. We also showed that these mutations can be present in subclonal tumor cell populations at diagnosis, suggesting that they may contribute to acquired chemoradiotherapy resistance. We hypothesize that subclonal mutations in NB oncogenes are common at diagnosis, are biomarkers for treatment failure, and can be targeted in conjunction with standard chemotherapy to improve patient survival. Here, we focus on the first aspect of this broad hypothesis by presenting the subclonal landscape of diagnostic, high-risk neuroblastomas. A meta-analysis of our previously published primary/relapse (N=23), whole exome (N=232) and whole genome (N=159) diagnostic data, and recent sequencing study of 78 diagnostic and 67 relapse cases were used to inform design of a custom amplicon panel for ultra-deep sequencing. The 28-gene panel covers 57 mutations in frequently mutated genes. Here, 250 high-risk primary tumors were sequenced to an average depth of 50,000X. More subclonal mutations were called (mean=36) compared to clonal alterations (mean=3), demonstrating the complex subclonal architecture in these tumors. We validated five clonal ALK mutations we originally discovered and identified 15 additional pathogenic ALK mutations with mutant allele frequencies (MAFs) ranging from 0.03-23%. Known ALK activating mutations were found at codons 1170, 1174, 1196, 1245, 1275 in 15 of 38 diagnostic primary tumors. This 39.5% frequency is much higher than the 14% frequency defined by us using Sanger or other sequencing methods. Improvement of subclonal mutation detection of actionable, relapse-specific driver mutations demonstrates the clinical utility of utilizing this sequencing methodology at diagnosis to enable timely improvement of outcomes for children with high-risk refractory NB.
Citation Format: Jo Lynne Harenza, Maura A. Diamond, Derek A. Oldridge, Olivia Padovan-Merher, Pichai Raman, Yael P. Mosse, John M. Maris. Defining the subclonal landscape of high-risk neuroblastoma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3000. doi:10.1158/1538-7445.AM2017-3000
Abstract
BACKGROUND: A genome-wide association study (GWAS) in neuroblastoma identified 8 regions where common polymorphisms are associated with neuroblastoma susceptibility. However, the biological ...mechanisms and causal SNPs are largely unknown. Rare variants, potentially in linkage with common polymorphisms but with a higher effect size, may contribute to cancer susceptibility at GWAS-defined loci and provide biological insight into mechanism.
METHODS: To identify rare variants at neuroblastoma GWAS loci we performed ultradeep targeted resequencing of BARD1 and LMO1 on peripheral blood DNA using RainDance microdroplet RDT1000 PCR enrichment, and HiSeq2000 single end 100 bp reads. We aggregated existing whole-genome and exome sequence data from constitutional DNA of neuroblastoma cases (Molenaar et al, Nature 2012; NCI TARGET) on all replicated neuroblastoma GWAS regions.
RESULTS: Exon sequence data were available for 517 cases for BARD1, and 330 cases for LMO1, LINC00340, LIN28B-HACE1, LMO1, DUSP12, DDX4-IL3RA, and HSB17B12 (all genes with replicated associations to NB). The highest frequency of rare potentially damaging variants occurred in BARD1 with 7/517 cases (1.4%) containing novel (n=4) or rare (n=3) variants as defined by ESP6500, 1000 genomes project, and dbSNP135. Rare variants at BARD1 were associated with homozygosity or heterozygosity for the protective alleles. Using SIFT, PolyPhen-2 or MutationTaster, 6/7 variants were predicted to be deleterious, including a nonsense mutation in the domain mediating BARD1-BRCA1 heterodimerization and a nonsense mutation in a BRCT domain implicated in homology directed repair. Sanger sequencing of tumor DNA and RNA confirmed both nonsense mutations were present in their respective primary tumors. HEK293 cells transfected with BARD1 nonsense variants showed decreased stabilization of BRCA1.
CONCLUSION: Novel and potentially damaging mutations in BARD1 exons are present in the constitutional DNA of ∼1% of patients with neuroblastoma and may contribute to disease susceptibility. However, BARD1 rare variants were not tagged by the common risk alleles providing an alternative mechanism to prime neuroblasts towards transformation. Further functional work, expansion of the targeted resequencing discovery cohort as a prelude to a larger case-control comparison, and incorporation of ENCODE data into analysis of non-coding SNPs may provide critical insights into neuroblastoma pathogenesis.
Citation Format: Andrew C. Wood, Trevor J. Pugh, Olena Morozova, Jan Koster, Jan J. Molenaar, Vanessa Pineros, Kristopher R. Bosse, Juan C. Perin, Sharon Diskin, Maura A. Diamond, Marco Marra, Matthew Meyerson, Rogier Versteeg, John M. Maris. Rare DNA variants are enriched at the BARD1 locus and likely influence neuroblastoma susceptibility. abstract. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3804. doi:10.1158/1538-7445.AM2013-3804
Scientific Data 4:170033 doi: 10.1038/sdata.2017.33 (2017); Published 28 March 2017; Updated: 5 December 2017. The growth media for the cell lines CHP-212 and IMR-32 listed in Table 1 of this Data ...Descriptor were incorrectly stated. The cell line labelled as ‘NB-16’ in Fig. 2, Table 1, and Table 2 was incorrectly recorded.
Neuroblastoma is a malignant neoplasm of the developing sympathetic nervous system that is notable for its phenotypic diversity. High-risk patients typically have widely disseminated disease at ...diagnosis and a poor survival probability, but low-risk patients frequently have localized tumors that are almost always cured with little or no chemotherapy. Our genome-wide association study (GWAS) has identified common variants within FLJ22536, BARD1, and LMO1 as significantly associated with neuroblastoma and more robustly associated with high-risk disease. Here we show that a GWAS focused on low-risk cases identified SNPs within DUSP12 at 1q23.3 (P = 2.07×10-6), DDX4 and IL31RA both at 5q11.2 (P = 2.94×10-6 and 6.54×10-7 respectively), and HSD17B12 at 11p11.2 (P = 4.20×10-7) as being associated with the less aggressive form of the disease. These data demonstrate the importance of robust phenotypic data in GWAS analyses and identify additional susceptibility variants for neuroblastoma.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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