Studies of paediatric cancers have shown a high frequency of mutation across epigenetic regulators. Here we sequence 633 genes, encoding the majority of known epigenetic regulatory proteins, in over ...1,000 paediatric tumours to define the landscape of somatic mutations in epigenetic regulators in paediatric cancer. Our results demonstrate a marked variation in the frequency of gene mutations across 21 different paediatric cancer subtypes, with the highest frequency of mutations detected in high-grade gliomas, T-lineage acute lymphoblastic leukaemia and medulloblastoma, and a paucity of mutations in low-grade glioma and retinoblastoma. The most frequently mutated genes are H3F3A, PHF6, ATRX, KDM6A, SMARCA4, ASXL2, CREBBP, EZH2, MLL2, USP7, ASXL1, NSD2, SETD2, SMC1A and ZMYM3. We identify novel loss-of-function mutations in the ubiquitin-specific processing protease 7 (USP7) in paediatric leukaemia, which result in decreased deubiquitination activity. Collectively, our results help to define the landscape of mutations in epigenetic regulatory genes in paediatric cancer and yield a valuable new database for investigating the role of epigenetic dysregulations in cancer.
Heterozygous de novo variants in the eukaryotic elongation factor EEF1A2 have previously been described in association with intellectual disability and epilepsy but never functionally validated. Here ...we report 14 new individuals with heterozygous EEF1A2 variants. We functionally validate multiple variants as protein‐damaging using heterologous expression and complementation analysis. Our findings allow us to confirm multiple variants as pathogenic and broaden the phenotypic spectrum to include dystonia/choreoathetosis, and in some cases a degenerative course with cerebral and cerebellar atrophy. Pathogenic variants appear to act via a haploinsufficiency mechanism, disrupting both the protein synthesis and integrated stress response functions of EEF1A2. Our studies provide evidence that EEF1A2 is highly intolerant to variation and that de novo pathogenic variants lead to an epileptic‐dyskinetic encephalopathy with both neurodevelopmental and neurodegenerative features. Developmental features may be driven by impaired synaptic protein synthesis during early brain development while progressive symptoms may be linked to an impaired ability to handle cytotoxic stressors.
Heterozygous de novo variants in the eukaryotic elongation factor EEF1A2 have previously been described in association with intellectual disability and epilepsy but never functionally validated. Here ...we report 14 new individuals with heterozygous EEF1A2 variants. We functionally validate multiple variants as protein-damaging using heterologous expression and complementation analysis. Our findings allow us to confirm multiple variants as pathogenic and broaden the phenotypic spectrum to include dystonia/choreoathetosis, and in some cases a degenerative course with cerebral and cerebellar atrophy. Pathogenic variants appear to act via a haploinsufficiency mechanism, disrupting both the protein synthesis and integrated stress response functions of EEF1A2. Our studies provide evidence that EEF1A2 is highly intolerant to variation and that de novo pathogenic variants lead to an epileptic-dyskinetic encephalopathy with both neurodevelopmental and neurodegenerative features. Developmental features may be driven by impaired synaptic protein synthesis during early brain development while progressive symptoms may be linked to an impaired ability to handle cytotoxic stressors.
We performed a retrospective analysis of longitudinal real-world data (RWD) from patients with breast cancer to replicate results from clinical studies and demonstrate the feasibility of generating ...real-world evidence. We also assessed the value of transcriptome profiling as a complementary tool for determining molecular subtypes.
De-identified, longitudinal data were analyzed after abstraction from records of patients with breast cancer in the United States (US) structured and stored in the Tempus database. Demographics, clinical characteristics, molecular subtype, treatment history, and survival outcomes were assessed according to strict qualitative criteria. RNA sequencing and clinical data were used to predict molecular subtypes and signaling pathway enrichment.
The clinical abstraction cohort (n = 4000) mirrored the demographics and clinical characteristics of patients with breast cancer in the US, indicating feasibility for RWE generation. Among patients who were human epidermal growth factor receptor 2-positive (HER2+), 74.2% received anti-HER2 therapy, with ∼70% starting within 3 months of a positive test result. Most non-treated patients were early stage. In this RWD set, 31.7% of patients with HER2+ immunohistochemistry (IHC) had discordant fluorescence in situ hybridization results recorded. Among patients with multiple HER2 IHC results at diagnosis, 18.6% exhibited intra-test discordance. Through development of a whole-transcriptome model to predict IHC receptor status in the molecular sequenced cohort (n = 400), molecular subtypes were resolved for all patients (n = 36) with equivocal HER2 statuses from abstracted test results. Receptor-related signaling pathways were differentially enriched between clinical molecular subtypes.
RWD in the Tempus database mirrors the overall population of patients with breast cancer in the US. These results suggest that real-time, RWD analyses are feasible in a large, highly heterogeneous database. Furthermore, molecular data may aid deficiencies and discrepancies observed from breast cancer RWD.
Longitudinal real-world data (RWD) from a large cohort of patients with breast cancer (n = 4000) were analyzed to test whether results were consistent with previous clinical studies and demonstrate real-world evidence validity. Then, whole-transcriptome sequencing was evaluated as a complementary diagnostic tool (n = 400). The cohort mirrored the general population with breast cancer in the United States, indicating real-world evidence feasibility, whereas transcriptome profiling supported and augmented standard diagnostic tests.
BACKGROUND: The nuclear factor-kB (NF-kB) family of transcriptional regulators are central mediators of the cellular inflammatory response. Although constitutive NF-kB signaling is present in most ...human tumours, mutations in pathway members are rare, complicating efforts to understand and block aberrant NF-kB activity in cancer. METHODS: To identify additional genetic alterations that drive ependymoma, we sequenced the whole genomes (WGS) of 41 tumours and matched normal blood, and the transcriptomes (RNAseq) of 77 tumours. The transforming significance of alterations were tested in mouse NSCs that we showed previously to be cells of origin of ependymoma. RESULTS: Here, we show that more than two thirds of supratentorial ependymomas contain oncogenic fusions between RELA, the principal effector of canonical NF-kB signalling, and an uncharacterized gene, C11orf95. In each case, C11orf95-RELA fusions resulted from chromothripsis involving chromosome 11q13.1. C11orf95-RELA fusion proteins translocated spontaneously to the nucleus to activate NF-kB target genes, and rapidly transformed neural stem cells-the cell of origin of ependymoma-to form these tumours in mice. CONCLUSIONS: Our data identify the first highly recurrent genetic alteration of RELA in human cancer, and the C11orf95-RELA fusion protein as a potential therapeutic target in supratentorial ependymoma. SECONDARY CATEGORY: Neuropathology & Tumor Biomarkers.
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Pathologic germ line mutations that predispose patients to cancer are estimated to occur in 4-30% of all pediatric oncology cases. In addition to leukemia specific familial predisposition ...syndromes, children with rare constitutional syndromes, heterogeneous dysmorphic syndromes, and multiple-cancer hereditary predisposition syndromes are all at an increased risk for hematologic malignancies. However, to date no genome-wide analysis has been done to define the range of germ line mutations that occur in pediatric patients with hematological malignancies.
To determine the frequency of pediatric cancer patients that have germ line variants of pathological significance in genes that predisposed to cancer, we analyzed the germ line and tumor DNA from 1120 pediatric cancer patients that were enrolled in the St. Jude – Washington University Pediatric Cancer Genome Project (PCGP). Samples were analyzed by whole-genome sequencing (n = 595), whole-exome sequencing (n = 456), or both (n = 69). Single nucleotide variants (SNVs), insertions/deletions (indels), structural variations (SV) and copy number alterations (CNAs) were detected using our analytical pipeline and all single nucleotide polymorphisms (SNPs) previously identified in non-cancer populations were filtered out. Our analysis then focused on the 23 cancer predisposition genes recently recommended for germ line analysis by the American College of Genetics and Genomics, along with an additional 8 genes that have been previously shown to predispose to pediatric cancer at a high penetrance. All variants in these 31 genes were classified as pathologic, likely pathologic, uncertain significance, likely benign, and benign based on literature review and in-silico predictions on the effect of novel mutations. An expanded analysis including a total of 565 genes known to play a role in oncogenesis was also evaluated.
Pathologic or likely pathologic germ line variants in one of the 31 genes were detected in 8% (90/1120) of patients, including: 16% (46/287) of patients with solid tumors, 8.6% (21/245) with brain tumors, and 3.9% (23/588) with leukemia. Expanding this analysis to 565 cancer gene resulted in only a slight increase, with a pathologic or likely pathologic variant being detected in 8.6% (97/1120) of patients. The most frequently effected genes included TP53 (n=48), APC (n=7) and BRCA2(n=6). Importantly, in >50% of these patients, analysis of their tumor DNA revealed the absence of a wild type allele for the cancer predisposition gene that was altered in the germ line.
The 588 pediatric patients with leukemia included 116 acute myeloid leukemias (AMLs: FAB M7 n=20; Core Binding Factor leukemias n=86; MLL-R n=10) and 472 acute lymphoblastic leukemias (ALLs: E2A-PBX1 n=53; ERG-R n=39; TEL-AML1 n=53; Hyperdiploid n=69; Hypodiploid n=47; BCR-ABL1 n=40; T-ALL n=32; MLL-R n=40; BCR-ABL-like n=31; and Other n=68). Across this cohort, 3.9% (23/588) of leukemia patients harbored a pathologic germ line mutations in one of the 31 cancer pre-disposing genes. This number increased to 4.6% (27/588; 28 mutations) when the expanded gene list was evaluated. TP53 (n=10) was the most frequently altered germ line gene in pediatric leukemia patients and was found predominantly in low-hypodiploid ALL, as previously reported. Germ line pathologic variants were also identified in KRAS, RUNX1, APC, BRCA2, and RET (2 cases each), and NRAS, SH2B3, BRCA1, MUTYH, PTCH1, SDHA,VHL, and NF2 (1 case each). Although germ line mutations in RUNX1 and SH2B3are typically associated with myeloid neoplasms, we identified these lesions in 3 cases of B lineage ALL suggesting an association with a wider spectrum of leukemia.
In conclusion, a small but significant proportion of pediatric patients with leukemia carry a germ line variant of pathologic significance in a cancer predisposition gene. These results suggest that these germ line lesions likely play a direct role in the pathogenesis of the patient’s presenting leukemia. Moreover, our results suggest that these patients would benefit from future clinical surveillance for the development of a second cancer. Lastly, these data demonstrate the power of comprehensive next generation DNA/RNA sequencing for the identification of pediatric patients who carry a germ line pathologic variant in a cancer predisposition gene.
No relevant conflicts of interest to declare.
Members of the nuclear factor-κB (NF-κB) family of transcriptional regulators are central mediators of the cellular inflammatory response. Although constitutive NF-κB signalling is present in most ...human tumours, mutations in pathway members are rare, complicating efforts to understand and block aberrant NF-κB activity in cancer. Here we show that more than two-thirds of supratentorial ependymomas contain oncogenic fusions between RELA, the principal effector of canonical NF-κB signalling, and an uncharacterized gene, C11orf95. In each case, C11orf95-RELA fusions resulted from chromothripsis involving chromosome 11q13.1. C11orf95-RELA fusion proteins translocated spontaneously to the nucleus to activate NF-κB target genes, and rapidly transformed neural stem cells-the cell of origin of ependymoma-to form these tumours in mice. Our data identify a highly recurrent genetic alteration of RELA in human cancer, and the C11orf95-RELA fusion protein as a potential therapeutic target in supratentorial ependymoma. PUBLICATION ABSTRACT
Presents the recommendations of the Human Genetic Education Subcommittee of the American Society of Human Genetics (ASHG) on the development of introductory biology courses for non-science majors ...addressing the list of concepts including evolution, transmission, gene expression, gene regulation, and genetics and society. Used an online survey to determine the recommended concepts. (YDS)
Abstract 68
Early T-cell precursor acute lymphoblastic leukemia (ETP ALL) is characterized by an immature T-lineage immunophenotype (cCD3+, CD1a-, CD8- and CD5dim) aberrant expression of myeloid and ...stem cell markers, a distinct gene expression profile and very poor outcome. The underlying genetic basis of this form of leukemia is unknown.
Here we report results of whole genome sequencing (WGS) of tumor and normal DNA from 12 children with ETP ALL. Genomes were sequenced to 30-fold haploid coverage using the Illumina GAIIx platform, and all putative somatic sequence and structural variants were validated. The frequency of mutations in 43 genes was assessed in a recurrence cohort of 52 ETP and 42 non-ETP T-ALL samples from patients enrolled in St Jude, Children's Oncology Group and AEIOP trials. Transcriptomic resequencing was performed for two WGS cases, and whole exome sequencing for three ETP ALL cases in the recurrence cohort.
We identified 44 interchromosomal translocations (mean 4 per patient, range 0–12), 32 intrachromosomal translocations (mean 3, 0–7), 53 deletions (mean 4, 0–10) and 16 insertions (mean 1, 0–5). Three cases exhibited a pattern of complex rearrangements suggestive of a single cellular catastrophe (“chromothripsis”), two of which had mutations targeting mismatch and DNA repair (MLH3 and DCLRE1C). While no single chromosomal alteration was present in all cases, 10 of 12 ETP ALLs harbored chromosomal rearrangements, several of which involved complex multichromosomal translocations and resulted in the expression of chimeric in-frame novel fusion genes disrupting hematopoietic regulators, including ETV6-INO80D, NAP1L1-MLLT10, RUNX1-EVX1 and NUP214-SQSTM1, each occurring in a single case. An additional ETP case with the ETV6-INO80D fusion was identified in the recurrence cohort. Additionally, 51% of structural variants had breakpoints in genes, including those with roles in hematopoiesis and leukemogenesis, and genes also targeted by mutation in other cases (MLH3, SUZ12, RUNX1).
We identified a high frequency of activating mutations in genes regulating cytokine receptor and Ras signalling in ETP ALL (67.2% of ETP compared to 19% of non-ETP T-ALL) including NRAS (17%), FLT3 (14%), JAK3 (9%), SH2B3 (or LNK; 9%), IL7R (8%), JAK1 (8%), KRAS (3%), and BRAF (2%). Seven cases (5 ETP, 2 non-ETP) harbored in frame insertion mutations in the transmembrane domain of IL7R, which were transforming when expressed in the murine cell lines, and resulted in enhanced colony formation when expressed in primary murine hematopoietic cells. The IL7R mutations resulted in constitutive Jak-Stat activation in these cell lines and primary leukemic cells expressing these mutations.
Fifty-eight percent of ETP cases (compared to 17% of non-ETP cases) harbored mutations known or predicted to disrupt hematopoietic and lymphoid development, including ETV6 (33%), RUNX1 (16%), IKZF1 (14%), GATA3 (10%), EP300 (5%) and GATA2 (2%). GATA3 regulates early T cell development, and mutations in this gene were observed exclusively in ETP ALL. The mutations were commonly biallelic, and were clustered at R276, a residue critical for binding of GATA3 to DNA. Strikingly, mutations disrupting chromatin modifying genes were also highly enriched in ETP ALL. Genes encoding the the polycomb repressor complex 2 (EZH2, SUZ12 and EED), that mediates histone 3 lysine 27 (H3K27) trimethylation were deleted or mutated in 42% of ETP ALL compared to 12% of non-ETP T-ALL. In addition, alterations of the H3K36 trimethylase SETD2 were observed in 5 ETP cases, but not in non-ETP ALL. We also identified recurrent mutations in genes that have not previously been implicated in hematopoietic malignancies including RELN, DNM2, ECT2L, HNRNPA1 and HNRNPR.
Using gene set enrichment analysis we demonstrate that the gene expression profile of ETP ALL shares features not only with normal human hematopoietic stem cells, but also with leukemic initiating cells (LIC) purified from patients with acute myeloid leukemia (AML). These results indicate that mutations that drive proliferation, impair differentiation and disrupt histone modification cooperate to induce an aggressive leukemia with an aberrant immature phenotype. The similarity of the gene expression pattern with that observed in the LIC of AML raises the possibility that myeloid-directed therapies might improve the outcome of ETP ALL.
Evans:St. Jude Children’s research Hospital: Employment, Patents & Royalties; NIH & NCI: Research Funding; Aldagen: Membership on an entity’s Board of Directors or advisory committees.
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