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

DNA Damage, Somatic Aneuploidy, and Malignant Sarcoma Susceptibility in Muscular Dystrophies

Albeit genetically highly heterogeneous, muscular dystrophies (MDs) share a convergent pathology leading to muscle wasting accompanied by proliferation of fibrous and fatty tissue, suggesting a common MD–pathomechanism. Here we show that mutations in muscular dystrophy genes (Dmd, Dysf, Capn3, Large) lead to the spontaneous formation of skeletal muscle-derived malignant tumors in mice, presenting as mixed rhabdomyo-, fibro-, and liposarcomas. Primary MD–gene defects and strain background strongly influence sarcoma incidence, latency, localization, and gender prevalence. Combined loss of dystrophin and dysferlin, as well as dystrophin and calpain-3, leads to accelerated tumor formation. Irrespective of the primary gene defects, all MD sarcomas share non-random genomic alterations including frequent losses of tumor suppressors (Cdkn2a, Nf1), amplification of oncogenes (Met, Jun), recurrent duplications of whole chromosomes 8 and 15, and DNA damage. Remarkably, these sarcoma-specific genetic lesions are already regularly present in skeletal muscles in aged MD mice even prior to sarcoma development. Accordingly, we show also that skeletal muscle from human muscular dystrophy patients is affected by gross genomic instability, represented by DNA double-strand breaks and age-related accumulation of aneusomies. These novel aspects of molecular pathologies common to muscular dystrophies and tumor biology will potentially influence the strategies to combat these diseases

Comparison of three different methods to detect bone marrow involvement in patients with neuroblastoma

Purpose!#!Neuroblastoma (NB) is the most frequent extracranial tumor in children. The detection of bone marrow (BM) involvement is crucial for correct staging and risk-adapted treatment. We compared three methods regarding the detection of NB involvement in BM.!##!Methods!#!Eighty-one patients with NB were included in this retrospective study. BM samples were obtained at designated time points at study entry and during treatment or follow-up. The diagnostic tools for BM analysis included cytomorphology (CM), flow cytometry (FCM) and automatic immunofluorescence plus fluorescence in situ hybridization (AIPF).!##!Results!#!We analyzed 369 aspirates in 81 patients in whom AIPF, CM, and FCM were simultaneously available. During the observation period, NB cells were detected in 86/369 (23.3%) cases, by CM in 32/369 (8.7%), by FCM in 52 (14.1%), and by AIPF in 72 (19.5%) samples. AIPF and/or FCM confirmed all positive results obtained in CM and detected 11 additional positive BM aspirates in 294 CM negative samples (p < 0,001). Survival of patients with BM involvement at study entry identified solely by FCM/AIPF was 17.4% versus 0% for patients in whom BM involvement was already identified by CM.!##!Conclusion!#!The combination of AIPF/FCM yielded the highest detection rate of NB cells in BM. AIPF was the single, most sensitive method in detecting these cells. Although CM did not provide any additional positive results, it is still a useful, readily available and cost-effective tool. The prognostic significance of FCM and AIPF should be confirmed in a prospective study with a larger number of patients

Tumor Touch Imprints as Source for Whole Genome Analysis of Neuroblastoma Tumors

<div><p>Introduction</p><p>Tumor touch imprints (TTIs) are routinely used for the molecular diagnosis of neuroblastomas by interphase fluorescence in-situ hybridization (I-FISH). However, in order to facilitate a comprehensive, up-to-date molecular diagnosis of neuroblastomas and to identify new markers to refine risk and therapy stratification methods, whole genome approaches are needed. We examined the applicability of an ultra-high density SNP array platform that identifies copy number changes of varying sizes down to a few exons for the detection of genomic changes in tumor DNA extracted from TTIs.</p><p>Material and Methods</p><p>DNAs were extracted from TTIs of 46 neuroblastoma and 4 other pediatric tumors. The DNAs were analyzed on the Cytoscan HD SNP array platform to evaluate numerical and structural genomic aberrations. The quality of the data obtained from TTIs was compared to that from randomly chosen fresh or fresh frozen solid tumors (n = 212) and I-FISH validation was performed.</p><p>Results</p><p>SNP array profiles were obtained from 48 (out of 50) TTI DNAs of which 47 showed genomic aberrations. The high marker density allowed for single gene analysis, e.g. loss of nine exons in the <i>ATRX</i> gene and the visualization of chromothripsis. Data quality was comparable to fresh or fresh frozen tumor SNP profiles. SNP array results were confirmed by I-FISH.</p><p>Conclusion</p><p>TTIs are an excellent source for SNP array processing with the advantage of simple handling, distribution and storage of tumor tissue on glass slides. The minimal amount of tumor tissue needed to analyze whole genomes makes TTIs an economic surrogate source in the molecular diagnostic work up of tumor samples.</p></div