Methyl-CpG-binding domain sequencing reveals a prognostic methylation signature in neuroblastoma

Accurate assessment of neuroblastoma outcome prediction remains challenging. Therefore, this study aims at establishing novel prognostic tumor DNA methylation biomarkers. In total, 396 low- and high-risk primary tumors were analyzed, of which 87 were profiled using methyl-CpG-binding domain (MBD) sequencing for differential methylation analysis between prognostic patient groups. Subsequently, methylation-specific PCR (MSP) assays were developed for 78 top-ranking differentially methylated regions and tested on two independent cohorts of 132 and 177 samples, respectively. Further, a new statistical framework was used to identify a robust set of MSP assays of which the methylation score (i.e. the percentage of methylated assays) allows accurate outcome prediction. Survival analyses were performed on the individual target level, as well as on the combined multimarker signature. As a result of the differential DNA methylation assessment by MBD sequencing, 58 of the 78 MSP assays were designed in regions previously unexplored in neuroblastoma, and 36 are located in non-promoter or non-coding regions. In total, 5 individual MSP assays (located in CCDC177, NXPH1, lnc-MRPL3-2, lnc-TREX1-1 and one on a region from chromosome 8 with no further annotation) predict event-free survival and 4 additional assays (located in SPRED3, TNFAIP2, NPM2 and CYYR1) also predict overall survival. Furthermore, a robust 58-marker methylation signature predicting overall and event-free survival was established. In conclusion, this study encompasses the largest DNA methylation biomarker study in neuroblastoma so far. We identified and independently validated several novel prognostic biomarkers, as well as a prognostic 58-marker methylation signature

11q deletion or ALK activity curbs DLG2 expression to maintain an undifferentiated state in neuroblastoma

High-risk neuroblastomas typically display an undifferentiated or poorly differentiated morphology. It is therefore vital to understand molecular mechanisms that block the differentiation process. We identify an important role for oncogenic ALK-ERK1/2-SP1 signaling in the maintenance of undifferentiated neural crest-derived progenitors through the repression of DLG2, a candidate tumor suppressor gene in neuroblastoma. DLG2 is expressed in the murine "bridge signature'' that represents the transcriptional transition state when neural crest cells or Schwann cell precursors differentiate to chromaffin cells of the adrenal gland. We show that the restoration of DLG2 expression spontaneously drives neuroblastoma cell differentiation, high-lighting the importance of DLG2 in this process. These findings are supported by genetic analyses of high-risk 11q deletion neuroblastomas, which identified genetic lesions in the DLG2 gene. Our data also suggest that further exploration of other bridge genes may help elucidate the mechanisms underlying the differentiation of NC-derived progenitors and their contribution to neuroblastomas

1p36 deletion results in a decrease in glycosaminoglycans which is associated with aggressiveness in neuroblastic tumors

Despite our deep understanding of neuroblastic tumors, some patients still suffer treatment failure, so pre-treatment risk stratification still requires improvement and the search for new therapeutic targets must continue. Here we correlated prognostic clinical and biological features of neuroblastic tumors with the density of extracellular matrix glycosaminoglycans (the main components of the extracellular matrix ‘ground substance’), in nearly 400 primary samples. We also studied the relationship between the density of extracellular matrix glycosaminoglycans and the expression of B3GALT6, an enzyme required for their synthesis. We associated a decrease in glycosaminoglycans with neuroblastomas that were histopathologically poorly-differentiated or undifferentiated, as well as with metastatic disease, and 1p36 deleted tumors. This decrease in glycosaminoglycans was also related to abnormal nuclear B3GALT6 expression in neuroblastic cells. These findings point towards the importance of the ground substance in the aggressiveness of neuroblastic tumors, which should therefore be considered when developing novel therapies for treating neuroblastomas

Intra-Tumour Genetic Heterogeneity and Prognosis in High-Risk Neuroblastoma

Spatial ITH is defined by genomic and biological variations within a tumour acquired by tumour cell evolution under diverse microenvironments, and its role in NB patient prognosis is understudied. In this work, we applied pangenomic techniques to detect chromosomal aberrations in at least two different areas of each tumour and/or in simultaneously obtained solid and liquid biopsies, detecting ITH in the genomic profile of almost 40% of HR-NB. ITH was better detected when comparing one or more tumour pieces and liquid biopsy (50%) than between different tumour pieces (21%). Interestingly, we found that patients with ITH analysed by pangenomic techniques had a significantly better survival rate that those with non-heterogeneous tumours, especially in cases without MYCN amplification. Moreover, all patients in the studied cohort with high ITH (defined as 50% or more genomic aberration differences between areas of a tumour or simultaneously obtained samples) survived after 48 months. These results clearly support analysing at least two solid tumour areas (separately or mixed) and liquid samples to provide more accurate genomic diagnosis, prognosis and therapy options in HR-NB

Neuroblastoma after Childhood: Prognostic Relevance of Segmental Chromosome Aberrations, ATRX Protein Status, and Immune Cell Infiltration

Neuroblastoma (NB) is a common malignancy in children but rarely occurs during adolescence or adulthood. This subgroup is characterized by an indolent disease course, almost uniformly fatal, yet little is known about the biologic characteristics. The aim of this study was to identify differential features regarding DNA copy number alterations, α-thalassemia/mental retardation syndrome X-linked (ATRX) protein expression, and the presence of tumor-associated inflammatory cells. Thirty-one NB patients older than 10 years who were included in the Spanish NB Registry were considered for the current study; seven young and middle-aged adult patients (range 18-60 years) formed part of the cohort. We performed single nucleotide polymorphism arrays, immunohistochemistry for immune markers (CD4, CD8, CD20, CD11b, CD11c, and CD68), and ATRX protein expression. Assorted genetic profiles were found with a predominant presence of a segmental chromosome aberration (SCA) profile. Preadolescent and adolescent NB tumors showed a higher number of SCA, including 17q gain and 11q deletion. There was also a marked infiltration of immune cells, mainly high and heterogeneous, in young and middle-aged adult tumors. ATRX negative expression was present in the tumors. The characteristics of preadolescent, adolescent, young adult, and middle-aged adult NB tumors are different, not only from childhood NB tumors but also from each other. Similar examinations of a larger number of such tumor tissues from cooperative groups should lead to a better older age–dependent tumor pattern and to innovative, individual risk-adapted therapeutic approaches for these patients

Vascular patterns provide therapeutic targets in aggressive neuroblastic tumors.

Angiogenesis is essential for tumor growth and metastasis, nevertheless, in NB, results between different studies on angiogenesis have yielded contradictory results. An image analysis tool was developed to characterize the density, size and shape of total blood vessels and vascular segments in 458 primary neuroblastic tumors contained in tissue microarrays. The results were correlated with clinical and biological features of known prognostic value and with risk of progression to establish histological vascular patterns associated with different degrees of malignancy. Total blood vessels were larger, more abundant and more irregularly-shaped in tumors of patients with associated poor prognostic factors than in the favorable cohort. Tumor capillaries were less abundant and sinusoids more abundant in the patient cohort with unfavorable prognostic factors. Additionally, size of post-capillaries & metarterioles as well as higher sinusoid density can be included as predictive factors for survival. These patterns may therefore help to provide more accurate pre-treatment risk stratification, and could provide candidate targets for novel therapies

Genetic Instability and Intratumoral Heterogeneity in Neuroblastoma with <em>MYCN</em> Amplification Plus 11q Deletion

<div><h3>Background/Aim</h3><p>Genetic analysis in neuroblastoma has identified the profound influence of <em>MYCN</em> amplification and 11q deletion in patients’ prognosis. These two features of high-risk neuroblastoma usually occur as mutually exclusive genetic markers, although in rare cases both are present in the same tumor. The purpose of this study was to characterize the genetic profile of these uncommon neuroblastomas harboring both these high-risk features.</p> <h3>Methods</h3><p>We selected 18 neuroblastomas with MNA plus 11q loss detected by FISH. Chromosomal aberrations were analyzed using Multiplex Ligation-dependent Probe Amplification and Single Nucleotide Polymorphism array techniques.</p> <h3>Results and Conclusion</h3><p>This group of tumors has approximately the same high frequency of aberrations as found earlier for 11q deleted tumors. In some cases, DNA instability generates genetic heterogeneity, and must be taken into account in routine genetic diagnosis.</p> </div

Graphic representation of chromosomes 11, 1 and 17.

<p>Bars illustrate the deleted/gain region. The positions of the breakpoints are indicated in megabases. (A) Deletions of chromosome 11q. SRO: 1117.7 to qter (22.8 Mb). (B) Deletions of chromosome 1p. SRO: 3.5 to 8.6 Mb (5.1 Mb). (C) Gain of chromosome 17q. SRO: 41.7 to qter (37.1 Mb). The cases have been listed according to chromosome 2 aberrations.</p

Schematic representation of the amplicons in chromosome 2.

<p>All data are illustrated according to the NCBI build Hg19. (A) Genes included in the <i>MYCN</i> amplicon. The crossed lines in cases 1, 7, 9 and 10 indicate that the breakpoint mapped more distal in chromosome 2 than the area represented. (B) Boxes and arrows indicate position of the different amplicons in the cases with complex amplification. The cases have been listed according to chromosome 2 aberrations.</p