Medulloblastoma is the most common malignant brain tumour of childhood. The identification of critical genes involved in its pathogenesis will be central to advances in our understanding of its molecular basis, and the development of improved therapeutic approaches.

Malignant glioblastoma (GBM) is a highly aggressive brain tumor with a dismal prognosis and limited therapeutic options. Genomic profiling of GBM samples has identified four molecular subtypes (Proneural, Neural, Classical and Mesenchymal), which may arise from different glioblastoma stem-like cell (GSC) populations. We previously showed that adherent cultures of GSCs grown on laminin-coated plates (Ad-GSCs) and spheroid cultures of GSCs (Sp-GSCs) had high expression of stem cell markers (CD133, Sox2 and Nestin), but low expression of differentiation markers (βIII-tubulin and glial fibrillary acid protein). In the present study, we characterized GBM tumors produced by subcutaneous and intracranial injection of Ad-GSCs and Sp-GSCs isolated from a patient-derived xenoline. Although they formed tumors with identical histological features, gene expression analysis revealed that xenografts of Sp-GSCs had a Classical molecular subtype similar to that of bulk tumor cells. In contrast xenografts of Ad-GSCs expressed a Mesenchymal gene signature. Adherent GSC-derived xenografts had high STAT3 and ANGPTL4 expression, and enrichment for stem cell markers, transcriptional networks and pro-angiogenic markers characteristic of the Mesenchymal subtype. Examination of clinical samples from GBM patients showed that STAT3 expression was directly correlated with ANGPTL4 expression, and that increased expression of these genes correlated with poor patient survival and performance. A pharmacological STAT3 inhibitor abrogated STAT3 binding to the ANGPTL4 promoter and exhibited anticancer activity in vivo. Therefore, Ad-GSCs and Sp-GSCs produced histologically identical tumors with different gene expression patterns, and a STAT3/ANGPTL4 pathway is identified in glioblastoma that may serve as a target for therapeutic intervention.

We report a new family with autosomal dominant inheritance of a late onset rapidly progressive leukodystrophy in which exome sequencing has revealed a novel mutation p.R782G in the Colony-Stimulating Factor 1 Receptor gene (CSF1R). Neuropathology of two affected family members showed cerebral white matter degeneration with axonal swellings and pigmented macrophages. The few recently reported families with CSF1R mutations had been previously labelled "hereditary diffuse leukencephalopathy with axonal spheroids" (HDLS) and "pigmentary orthochromatic leukodystrophy" (POLD), disorders which now appear to form a disease continuum. The term "adult-onset leukoencephalopathy with axonal spheroids and pigmented glia" (ALSP) has been proposed to encompass this spectrum. As CSF1R regulates microglia this mutation implies that dysregulation of microglia is the primary cause of the disease.

There is increasing evidence from genome-wide association studies for a strong inherited genetic basis of susceptibility to acute lymphoblastic leukaemia (ALL) in children, yet the effects of protein-coding variants on ALL risk have not been systematically evaluated. Here we show a missense variant in CDKN2A associated with the development of ALL at genome-wide significance (rs3731249, P=9.4 × 10(-23), odds ratio=2.23). Functional studies indicate that this hypomorphic variant results in reduced tumour suppressor function of p16(INK4A), increases the susceptibility to leukaemic transformation of haematopoietic progenitor cells, and is preferentially retained in ALL tumour cells. Resequencing the CDKN2A-CDKN2B locus in 2,407 childhood ALL cases reveals 19 additional putative functional germline variants. These results provide direct functional evidence for the influence of inherited genetic variation on ALL risk, highlighting the important and complex roles of CDKN2A-CDKN2B tumour suppressors in leukaemogenesis.

Infant acute lymphoblastic leukemia (ALL) with MLL rearrangements (MLL-R) represents a distinct leukemia with a poor prognosis. To define its mutational landscape, we performed whole-genome, exome, RNA and targeted DNA sequencing on 65 infants (47 MLL-R and 18 non-MLL-R cases) and 20 older children (MLL-R cases) with leukemia. Our data show that infant MLL-R ALL has one of the lowest frequencies of somatic mutations of any sequenced cancer, with the predominant leukemic clone carrying a mean of 1.3 non-silent mutations. Despite this paucity of mutations, we detected activating mutations in kinase-PI3K-RAS signaling pathway components in 47% of cases. Surprisingly, these mutations were often subclonal and were frequently lost at relapse. In contrast to infant cases, MLL-R leukemia in older children had more somatic mutations (mean of 6.5 mutations/case versus 1.3 mutations/case, P = 7.15 × 10(-5)) and had frequent mutations (45%) in epigenetic regulators, a category of genes that, with the exception of MLL, was rarely mutated in infant MLL-R ALL.

Most influenza pandemics have been caused by H1N1 viruses of purely or partially avian origin. Here, using Cox proportional hazard model, we attempt to identify the genetic variations in the whole genome of wild-type North American avian H1N1 influenza A viruses that are associated with their virulence in mice by residue variations, host origins of virus (Anseriformes-ducks or Charadriiformes-shorebirds), and host-residue interactions. In addition, through structural modeling, we predicted that several polymorphic sites associated with pathogenicity were located in structurally important sites, especially in the polymerase complex and NS genes. Our study introduces a new approach to identify pathogenic variations in wild-type viruses circulating in the natural reservoirs and ultimately to understand their infectious risks to humans as part of risk assessment efforts towards the emergence of future pandemic strains.

Repetitive transcranial magnetic stimulation (rTMS) has increasingly been studied over the past decade to determine whether it has a therapeutic benefit on focal cerebral ischemia. However, the underlying mechanism of rTMS in this process remains unclear. In the current study, we investigated the effects of rTMS on the proliferation of adult neural stem cells (NSCs) and explored microRNAs (miRNAs) that were affected by rTMS. Our data showed that 10 Hz rTMS significantly increased the proliferation of adult NSCs after focal cerebral ischemia in the subventricular zone (SVZ), and the expression of miR-25 was obviously up-regulated in the ischemic cortex after rTMS. p57, an identified miR-25 target gene that regulates factors linked to NSC proliferation, was also evaluated, and it exhibited down-regulation. To further verify the role of miR-25, rats were injected with a single dose of antagomir-25 and were subjected to focal cerebral ischemia followed by rTMS treatment. The results confirmed that miR-25 could be repressed specifically and could drive the up-regulation of its target gene (p57), which resulted in the inhibition of adult NSC proliferation in the SVZ after rTMS. Thus, our studies strongly indicated that 10 Hz rTMS can promote the proliferation of adult NSCs in the SVZ after focal cerebral ischemia by regulating the miR-25/p57 pathway.

To define the mutation spectrum in non-Down syndrome acute megakaryoblastic leukemia (non-DS-AMKL), we performed transcriptome sequencing on diagnostic blasts from 14 pediatric patients and validated our findings in a recurrency/validation cohort consisting of 34 pediatric and 28 adult AMKL samples. Our analysis identified a cryptic chromosome 16 inversion (inv(16)(p13.3q24.3)) in 27% of pediatric cases, which encodes a CBFA2T3-GLIS2 fusion protein. Expression of CBFA2T3-GLIS2 in Drosophila and murine hematopoietic cells induced bone morphogenic protein (BMP) signaling and resulted in a marked increase in the self-renewal capacity of hematopoietic progenitors. These data suggest that expression of CBFA2T3-GLIS2 directly contributes to leukemogenesis.

Genomic landscapes of 92 adult and 111 pediatric patients with B-cell acute lymphoblastic leukemia (B-ALL) were investigated using next-generation sequencing and copy number alteration analysis. Recurrent gene mutations and fusions were tested in an additional 87 adult and 93 pediatric patients. Among the 29 newly identified in-frame gene fusions, those involving MEF2D and ZNF384 were clinically relevant and were demonstrated to perturb B-cell differentiation, with EP300-ZNF384 inducing leukemia in mice. Eight gene expression subgroups associated with characteristic genetic abnormalities were identified, including leukemia with MEF2D and ZNF384 fusions in two distinct clusters. In subgroup G4 which was characterized by ERG deletion, DUX4-IGH fusion was detected in most cases. This comprehensive dataset allowed us to compare the features of molecular pathogenesis between adult and pediatric B-ALL and to identify signatures possibly related to the inferior outcome of adults to that of children. We found that, besides the known discrepancies in frequencies of prognostic markers, adult patients had more cooperative mutations and greater enrichment for alterations of epigenetic modifiers and genes linked to B-cell development, suggesting difference in the target cells of transformation between adult and pediatric patients and may explain in part the disparity in their responses to treatment.

Posterior fossa ependymoma comprises two distinct molecular variants termed EPN_PFA and EPN_PFB that have a distinct biology and natural history. The therapeutic value of cytoreductive surgery and radiation therapy for posterior fossa ependymoma after accounting for molecular subgroup is not known.

Telomeres are the protective arrays of tandem TTAGGG sequence and associated proteins at the termini of chromosomes. Telomeres shorten at each cell division due to the end-replication problem and are maintained above a critical threshold in malignant cancer cells to prevent cellular senescence or apoptosis. With the recent advances in massive parallel sequencing, assessing telomere content in the context of other cancer genomic aberrations becomes an attractive possibility. We present the first comprehensive analysis of telomeric DNA content change in tumors using whole-genome sequencing data from 235 pediatric cancers.

Tumors of the same histologic type often comprise clinically and molecularly distinct subgroups; however, the etiology of these subgroups is unknown. Here, we report that histologically identical, but genetically distinct, ependymomas exhibit patterns of gene expression that recapitulate those of radial glia cells in the corresponding region of the central nervous system. Cancer stem cells isolated from ependymomas displayed a radial glia phenotype and formed tumors when orthotopically transplanted in mice. These findings identify restricted populations of radial glia cells as candidate stem cells of the different subgroups of ependymoma, and they support a general hypothesis that subgroups of the same histologic tumor type are generated by different populations of progenitor cells in the tissues of origin.

Animal models provide an in vivo system to study gene function by transgenic and knockout approaches. Targeted knockout approaches have been very successful in mice, but are currently not feasible in zebrafish due to the inability to grow embryonic stem cells. As an alternative, a reverse genetic approach that utilizes screening by resequencing and/or TILLING (Targeting Induced Local Lesions INGenomes) of mutagenized genomes has recently gained popularity in the zebrafish field. Spermatogonia of healthy males are mutagenized using ENU (N-ethyl-N-nitrosourea) and F1 progeny is collected by breeding treated males with healthy wild type females. Sperm and DNA banks are generated from F1 males. DNA is screened for ENU-induced mutations by sequencing or TILLING. These mutations can then be studied by in vitro fertilization (IVF) from the cryopreserved sperm of the corresponding F1 male followed by breeding to homozygosity. A high-throughput method of screening for rare heterozygotes and efficient recovery of mutant lines are important in identification of a large number of mutations using this approach. This article provides optimized protocols for resequencing and TILLING based on our experiences. We performed a pilot screen on 1235 F1 males by resequencing 54 exons from 17 genes and analyzed the sequencing data using multiple programs to maximize the mutation detection with minimal false positive detection. As an alternative to sequencing, we developed the protocols for TILLING by capillary electrophoresis using an ABI Genetic analyzer 3100 platform followed by fragment analysis using GeneScan and Genotyper softwares. PCR products generated by fluorescently labeled universal primers and tailed exon-specific primers were pooled 4-fold prior to heteroduplex formation. Overall, our pilot screen shows that a combination of TILLING and sequencing is optimal for achieving cost-effective, high-throughput screening of a large number of samples. Amplicons with fewer common SNPs are ideal for TILLING whereas amplicons with multiple SNPs and in/del polymorphisms are best suited for sequencing followed by analysis with SNPdetector.

A novel candidate metastasis modifier, ribosomal RNA processing 1 homolog B (Rrp1b), was identified through two independent approaches. First, yeast two-hybrid, immunoprecipitation, and functional assays demonstrated a physical and functional interaction between Rrp1b and the previous identified metastasis modifier Sipa1. In parallel, using mouse and human metastasis gene expression data it was observed that extracellular matrix (ECM) genes are common components of metastasis predictive signatures, suggesting that ECM genes are either important markers or causal factors in metastasis. To investigate the relationship between ECM genes and poor prognosis in breast cancer, expression quantitative trait locus analysis of polyoma middle-T transgene-induced mammary tumor was performed. ECM gene expression was found to be consistently associated with Rrp1b expression. In vitro expression of Rrp1b significantly altered ECM gene expression, tumor growth, and dissemination in metastasis assays. Furthermore, a gene signature induced by ectopic expression of Rrp1b in tumor cells predicted survival in a human breast cancer gene expression dataset. Finally, constitutional polymorphism within RRP1B was found to be significantly associated with tumor progression in two independent breast cancer cohorts. These data suggest that RRP1B may be a novel susceptibility gene for breast cancer progression and metastasis.

Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging-with substantial inter-observer variability in the histopathological diagnosis of many tumour types. Here we present a comprehensive approach for the DNA methylation-based classification of central nervous system tumours across all entities and age groups, and demonstrate its application in a routine diagnostic setting. We show that the availability of this method may have a substantial impact on diagnostic precision compared to standard methods, resulting in a change of diagnosis in up to 12% of prospective cases. For broader accessibility, we have designed a free online classifier tool, the use of which does not require any additional onsite data processing. Our results provide a blueprint for the generation of machine-learning-based tumour classifiers across other cancer entities, with the potential to fundamentally transform tumour pathology.

Choroid plexus carcinoma (CPC) is a rare and aggressive tumor of infancy without a clear treatment strategy. This study describes the outcomes of children with CPC treated on the multi-institutional phase 2 SJYC07 trial and reports on the significance of clinical and molecular characteristics.

Clear cell meningioma represents an uncommon variant of meningioma that typically affects children and young adults. Although an enrichment of loss-of-function mutations in the SMARCE1 gene has been reported for this subtype, comprehensive molecular investigations are lacking. Here we describe a molecularly distinct subset of tumors (n = 31), initially identified through genome-wide DNA methylation screening among a cohort of 3093 meningiomas, of which most were diagnosed histologically as clear cell meningioma. This cohort was further supplemented by an additional 11 histologically diagnosed clear cell meningiomas for analysis (n = 42). Targeted DNA sequencing revealed SMARCE1 mutations in 33/34 analyzed samples, accompanied by a nuclear loss of expression determined via immunohistochemistry and a decreased SMARCE1 transcript expression in the tumor cells. Analysis of time to progression or recurrence of patients within the clear cell meningioma group (n = 14) in comparison to those with meningioma WHO grade 2 (n = 220) revealed a similar outcome and support the assignment of WHO grade 2 to these tumors. Our findings indicate the existence of a highly distinct epigenetic signature of clear cell meningiomas, separate from all other variants of meningiomas, with recurrent mutations in the SMARCE1 gene. This suggests that these tumors may arise from a different precursor cell population than the broad spectrum of the other meningioma subtypes.

Colorectal cancer is a common malignancy occurring in the digestive system, which is the third common cause of cancer mortality in developed countries. Shikonin, a naphthoquinone compound extracted from the root of Lithospermum erythrorhizon, is extensively reported to exert antitumor activity against various types of cancer. However, the systematic effect of shikonin in colon cancer remains poorly understood. In the present study, we evaluated the antitumor activity of shikonin in human colon cancer cells and the therapeutic effect on a xenograft mouse model. Transcriptomics and metabolomics were further integrated to provide a systematic perspective of the shikonin-induced antitumor mechanism. The results demonstrated that shikonin had a remarkable antitumor potency both in vitro and in vivo. Moreover, metabolic pathways, including the purine metabolism, amino acid metabolism, and glycerophospholipid metabolism, were perturbed and subsequently led to cell cycle arrest in the G2/M phase. In particular, the disturbance of purine metabolism may account for the major mechanism resulting from shikonin antitumor activity.

Aggressive cancers often have activating mutations in growth-controlling oncogenes and inactivating mutations in tumor-suppressor genes. In neuroblastoma, amplification of the MYCN oncogene and inactivation of the ATRX tumor-suppressor gene correlate with high-risk disease and poor prognosis. Here we show that ATRX mutations and MYCN amplification are mutually exclusive across all ages and stages in neuroblastoma. Using human cell lines and mouse models, we found that elevated MYCN expression and ATRX mutations are incompatible. Elevated MYCN levels promote metabolic reprogramming, mitochondrial dysfunction, reactive-oxygen species generation, and DNA-replicative stress. The combination of replicative stress caused by defects in the ATRX-histone chaperone complex, and that induced by MYCN-mediated metabolic reprogramming, leads to synthetic lethality. Therefore, ATRX and MYCN represent an unusual example, where inactivation of a tumor-suppressor gene and activation of an oncogene are incompatible. This synthetic lethality may eventually be exploited to improve outcomes for patients with high-risk neuroblastoma.

Organic anion transporter 3 (OAT3) plays a vital role in removing a broad variety of anionic drugs from kidney, thus avoiding their possible toxicity in the body. In the current study, we investigated the role of insulin-like growth factor 1 (IGF-1) in the regulation of OAT3. We showed that IGF-1 induced a dose- and time-dependent increase in OAT3 transport activity, which correlated well with an increase in OAT3 expression. The IGF-1-induced increase in OAT3 expression was blocked by protein kinase A (PKA) inhibitor H89. Moreover, IGF-1 induced an increase in OAT3 phosphorylation, which was also blocked by H89. These data suggest that the IGF-1 modulation of OAT3 occurred through PKA signaling pathway. To further confirm the involvement of PKA, we treated OAT3-expressing cells with PKA activator Bt2-cAMP, followed by examining OAT activity and phosphorylation. We showed that OAT3 activity and phosphorylation were much enhanced in Bt2-cAMP-treated cells as compared to that in control cells. Finally, linsitinib, an anticancer drug that blocks the IGF-1 receptor, abrogated IGF-1-stimulated OAT3 transport activity. In conclusion, our study demonstrated that IGF-1 regulates OAT3 expression and transport activity through PKA signaling pathway, possibly by phosphorylating the transporter.