Functional epigenetic approach identifies frequently methylated genes in Ewing sarcoma

Using a candidate gene approach we recently identified frequent methylation of the RASSF2 gene associated with poor overall survival in Ewing sarcoma (ES). To identify effective biomarkers in ES on a genome-wide scale, we used a functionally proven epigenetic approach, in which gene expression was induced in ES cell lines by treatment with a demethylating agent followed by hybridization onto high density gene expression microarrays. After following a strict selection criterion, 34 genes were selected for expression and methylation analysis in ES cell lines and primary ES. Eight genes (CTHRC1, DNAJA4, ECHDC2, NEFH, NPTX2, PHF11, RARRES2, TSGA14) showed methylation frequencies of>20% in ES tumors (range 24-71%), these genes were expressed in human bone marrow derived mesenchymal stem cells (hBMSC) and hypermethylation was associated with transcriptional silencing. Methylation of NPTX2 or PHF11 was associated with poorer prognosis in ES. In addition, six of the above genes also showed methylation frequency of>20% (range 36-50%) in osteosarcomas. Identification of these genes may provide insights into bone cancer tumorigenesis and development of epigenetic biomarkers for prognosis and detection of these rare tumor types

Pediatric T-ALL type-1 and type-2 relapses develop along distinct pathways of clonal evolution

The mechanisms underlying T-ALL relapse remain essentially unknown. Multilevel-omics in 38 matched pairs of initial and relapsed T-ALL revealed 18 (47%) type-1 (defined by being derived from the major ancestral clone) and 20 (53%) type-2 relapses (derived from a minor ancestral clone). In both types of relapse, we observed known and novel drivers of multidrug resistance including MDR1 and MVP, NT5C2 and JAK-STAT activators. Patients with type-1 relapses were specifically characterized by IL7R upregulation. In remarkable contrast, type-2 relapses demonstrated (1) enrichment of constitutional cancer predisposition gene mutations, (2) divergent genetic and epigenetic remodeling, and (3) enrichment of somatic hypermutator phenotypes, related to BLM, BUB1B/PMS2 and TP53 mutations. T-ALLs that later progressed to type-2 relapses exhibited a complex subclonal architecture, unexpectedly, already at the time of initial diagnosis. Deconvolution analysis of ATAC-Seq profiles showed that T-ALLs later developing into type-1 relapses resembled a predominant immature thymic T-cell population, whereas T-ALLs developing into type-2 relapses resembled a mixture of normal T-cell precursors. In sum, our analyses revealed fundamentally different mechanisms driving either type-1 or type-2 T-ALL relapse and indicate that differential capacities of disease evolution are already inherent to the molecular setup of the initial leukemia

Frequent and sex-biased deletion of SLX4IP by illegitimate V(D)J-mediated recombination in childhood acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) accounts for ∼25% of pediatric malignancies. Of interest, the incidence of ALL is observed ∼20% higher in males relative to females. The mechanism behind the phenomenon of sex-specific differences is presently not understood. Employing genome-wide genetic aberration screening in 19 ALL samples, one of the most recurrent lesions identified was monoallelic deletion of the 5′ region of SLX4IP. We characterized this deletion by conventional molecular genetic techniques and analyzed its interrelationships with biological and clinical characteristics using specimens and data from 993 pediatric patients enrolled into trial AIEOP-BFM ALL 2000. Deletion of SLX4IP was detected in ∼30% of patients. Breakpoints within SLX4IP were defined to recurrent positions and revealed junctions with typical characteristics of illegitimate V(D)J-mediated recombination. In initial and validation analyses, SLX4IP deletions were significantly associated with male gender and ETV6/RUNX1-rearranged ALL (both overall P < 0.0001). For mechanistic validation, a second recurrent deletion affecting TAL1 and caused by the same molecular mechanism was analyzed in 1149 T-cell ALL patients. Validating a differential role by sex of illegitimate V(D)J-mediated recombination at the TAL1 locus, 128 out of 1149 T-cell ALL samples bore a deletion and males were significantly more often affected (P = 0.002). The repeatedly detected association of SLX4IP deletion with male sex and the extension of the sex bias to deletion of the TAL1 locus suggest that differential illegitimate V(D)J-mediated recombination events at specific loci may contribute to the consistent observation of higher incidence rates of childhood ALL in boys compared with girl

Genetic alterations of ALK in high-risk neuroblastoma patients: a SIOPEN study

Background: In neuroblastoma (NB), the ALK receptor tyrosine kinase can be constitutively activated either through genomic amplification or activating point mutations. We studied ALK genetic alterations in high-risk NB patients to determine their frequency and prognostic impact.N/

High frequency of subclonal ALK mutations in high risk neuroblastoma patients. A SIOPEN study

Introduction: In neuroblastoma (NB), activating ALK receptor tyrosine kinase point mutations are detected in 8–10% at diagnosis using conventional sequencing. To determine the potential occurrence and the prognostic impact of ALK mutations in a series of high risk NB patients we studied ALK variation frequencies using targeted deep sequencing in samples of patients enrolled in the SIOPEN HR-NBL01 stud

MiR-192 directly binds and regulates Dicer1 expression in neuroblastoma.

Neuroblastoma (NB) arises from the embryonic neural crest and is the most common extracranial solid tumor in children under 5 years of age. Reduced expression of Dicer1 has recently been shown to be in correlation with poor prognosis in NB patients. This study aimed to investigate the mechanisms that could lead to the down-regulation of Dicer1 in neuroblastoma. We used computational prediction to identify potential miRs down-regulating Dicer1 in neuroblastoma. One of the miRs that were predicted to target Dicer1 was miR-192. We measured the levels of miR-192 in 43 primary tumors using real time PCR. Following the silencing of miR-192, the levels of dicer1 cell viability, cell proliferation and migration capability were analyzed. Multivariate analysis identified miR-192 as an independent prognostic marker for relapse in neuroblastoma patients (p=0.04). We were able to show through a dual luciferase assay and side-directed mutational analysis that miR-192 directly binds the 3' UTR of Dicer1 on positions 1232-1238 and 2282-2288. An increase in cell viability, proliferation and migration rates were evident in NB cells transfected with miR-192-mimic. Yet, there was a significant decrease in proliferation when NB cells were transfected with an miR-192-inhibitor We suggest that miR-192 might be a key player in NB by regulating Dicer1 expression

Functional epigenetic approach identifies frequently methylated genes in Ewing sarcoma

Using a candidate gene approach we recently identified frequent methylation of the RASSF2 gene associated with poor overall survival in Ewing sarcoma (ES). To identify effective biomarkers in ES on a genome-wide scale, we used a functionally proven epigenetic approach, in which gene expression was induced in ES cell lines by treatment with a demethylating agent followed by hybridization onto high density gene expression microarrays. After following a strict selection criterion, 34 genes were selected for expression and methylation analysis in ES cell lines and primary ES. Eight genes (CTHRC1, DNAJA4, ECHDC2, NEFH, NPTX2, PHF11, RARRES2, TSGA14) showed methylation frequencies of>20% in ES tumors (range 24-71%), these genes were expressed in human bone marrow derived mesenchymal stem cells (hBMSC) and hypermethylation was associated with transcriptional silencing. Methylation of NPTX2 or PHF11 was associated with poorer prognosis in ES. In addition, six of the above genes also showed methylation frequency of>20% (range 36-50%) in osteosarcomas. Identification of these genes may provide insights into bone cancer tumorigenesis and development of epigenetic biomarkers for prognosis and detection of these rare tumor types