Telomere maintenance pathway activity analysis enables tissue- and gene-level inferences

Nersisyan, Lilit; Simonyan, Arman; Binder, Hans; Arakelyan, Arsen

doi:10.1101/2021.02.01.429081

Cited by 1 publication

(1 citation statement)

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“…Telomerase-mediated TMM in neuroblastoma can be determined by detecting the respective genomic alterations, i.e., amplification of MYCN (MNA) or rearrangements of TERT (TERT RA) [10], or by examining TERT expression levels [6,9,10] or telomerase activity [6,12]. Assessment of ALT has remained more challenging, as ALT-related molecular markers may lack sensitivity or specificity [9,12,[16][17][18][19][20]. Both analysis of ALT-associated promyelocytic leukemia nuclear bodies (APB) and detection of extrachromosomal circular partially double-stranded telomeric DNA (C-circles) have been used to assess ALT in neuroblastoma, however, the accuracy and comparability of these two methods have not been systematically determined yet.…”

Section: Introductionmentioning

confidence: 99%

Reliable assessment of telomere maintenance mechanisms in neuroblastoma

et al. 2022

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Background Telomere maintenance mechanisms (TMM) are a hallmark of high-risk neuroblastoma, and are conferred by activation of telomerase or alternative lengthening of telomeres (ALT). However, detection of TMM is not yet part of the clinical routine, and consensus on TMM detection, especially on ALT assessment, remains to be achieved. Methods Whole genome sequencing (WGS) data of 68 primary neuroblastoma samples were analyzed. Telomere length was calculated from WGS data or by telomere restriction fragment analysis (n = 39). ALT was assessed by C-circle assay (CCA, n = 67) and detection of ALT-associated PML nuclear bodies (APB) by combined fluorescence in situ hybridization and immunofluorescence staining (n = 68). RNA sequencing was performed (n = 64) to determine expression of TERT and telomeric long non-coding RNA (TERRA). Telomerase activity was examined by telomerase repeat amplification protocol (TRAP, n = 15). Results Tumors were considered as telomerase-positive if they harbored a TERT rearrangement, MYCN amplification or high TERT expression (45.6%, 31/68), and ALT-positive if they were positive for APB and CCA (19.1%, 13/68). If all these markers were absent, tumors were considered TMM-negative (25.0%, 17/68). According to these criteria, the majority of samples were classified unambiguously (89.7%, 61/68). Assessment of additional ALT-associated parameters clarified the TMM status of the remaining seven cases with high likelihood: ALT-positive tumors had higher TERRA expression, longer telomeres, more telomere insertions, a characteristic pattern of telomere variant repeats, and were associated with ATRX mutations. Conclusions We here propose a workflow to reliably detect TMM in neuroblastoma. We show that unambiguous classification is feasible following a stepwise approach that determines both, activation of telomerase and ALT. The workflow proposed in this study can be used in clinical routine and provides a framework to systematically and reliably determine telomere maintenance mechanisms for risk stratification and treatment allocation of neuroblastoma patients.

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