Telomere Maintenance Mechanisms in a Cohort of High-Risk Neuroblastoma Tumors and Its Relation to Genomic Variants in the TERT and ATRX Genes

Abstract

Tumor cells are hallmarked by their capacity to undergo unlimited cell divisions, commonly accomplished either by mechanisms that activate TERT or through the alternative lengthening of telomeres pathway. Neuroblastoma is a heterogeneous pediatric cancer, and the aim of this study was to characterize telomere maintenance mechanisms in a high-risk neuroblastoma cohort. All tumor samples were profiled with SNP microarrays and, when material was available, subjected to whole genome sequencing (WGS). Telomere length was estimated from WGS data, samples were assayed for the ALT biomarker c-circles, and selected samples were subjected to methylation array analysis. Samples with ATRX aberration in this study were positive for c-circles, whereas samples with either MYCN amplification or TERT re-arrangement were negative for c-circles. Both ATRX aberrations and TERT re-arrangement were enriched in 11q-deleted samples. An association between older age at diagnosis and 1q-deletion was found in the ALT-positive group. TERT was frequently placed in juxtaposition to a previously established gene in neuroblastoma tumorigenesis or cancer in general. Given the importance of high-risk neuroblastoma, means for mitigating active telomere maintenance must be therapeutically explored.

Keywords: ALT; ATRX; TERT; TMM; neuroblastoma; pediatric cancer; telomeres.

Figure 1

TERT-associated alterations. (A) An overview of the location of breakpoints affecting the TERT locus together with corresponding gene regions associated with the translocation. (B) Detailed view showing the juxtaposition of TERT in relation to super enhancers for six different cases. Super enhancers are specified by blue boxes with positions as indicated by previous studies by van Groningen et al. 2017 [29] and Gartlgruber et al. 2021 [30].

Figure 2

ATRX-associated alterations. Visualization of position and size of detected ATRX deletions together with annotation of genomic subgroup.

Figure 3

C-circle assay. C-circle assay slot blots are shown for all samples subdivided in groups according to presence of genomic alterations in ATRX, TERT, or MNA as well as a reference group consisting of NB samples lacking these aberrations. Cases in the ATRX group with other alterations than multi-exon deletions are indicated by asterisks.

Figure 4

Common cases and cases of particular interest. Genomic tumor profiles generated by SNP microarray are shown as genome-wide chromatograms and aberrations are indicated with blue arrows for loss, red arrows for gain and black arrows for amplification. C-circle assay results by slot blot are shown to the right of respective chromatogram together with scores and neuroblastoma subclass indicated from methylation-based classification. (A) NB cases representative of different genomic subtypes. (B) NB cases with indefinite features.

Figure 5

Telomere length estimation and segmental aberrations at chromosome 1. (A) Normalized tumor telomere length as calculated from WGS data using TelSeq software v.1.0 are shown as blue bars. Genomic subgroup as indicated on the X axis; presence of alteration in ATRX, TERT, MNA or 11q-deletion (11q-del), other segmental alterations (OS), 17q-gain (17q) and numerical only (Num). Different tumor materials stemming from same individual are marked by dotted line. (B) The samples with chromosome 1 aberrations are grouped into four categories according to alterations in ATRX, TERT, MNA and 11q-deleted (non- ATRX, TERT, MNA). Blue bars represent deletions and red bars represent gains. Genes of interest are highlighted with a dashed line.

Figure 6

Overview of TMM in neuroblastoma. Immortalization is a hallmark of tumor progression and can be achieved through different molecular mechanisms commonly connected to an ATRX associated ALT phenotype or telomerase activation.