Telomerase Is a Prognostic Marker of Poor Outcome and a Therapeutic Target in Neuroblastoma.

PURPOSE: Telomere maintenance is a hallmark of high-risk neuroblastoma; however, the contribution of telomerase and alternative lengthening of telomeres (ALT) to clinical phenotypes has remained unclear. We aimed to determine the clinical relevance of telomerase activation versus ALT as biomarkers in pretreatment neuroblastoma and to assess the potential value of telomerase as a therapeutic target. MATERIALS AND METHODS: The genomic status of TERT and MYCN was assessed in 457 pretreatment neuroblastomas by fluorescence in situ hybridization. ALT was examined in 273 of 457 tumors by detection of ALT-associated promyelocytic leukemia nuclear bodies, and TERT expression was determined by microarrays in 223 of these. Cytotoxic effects of telomerase-interacting compounds were analyzed in neuroblastoma cell lines in vitro and in vivo. RESULTS: We detected TERT rearrangements in 46 of 457 cases (10.1%), MYCN amplification in 93 of 457 cases (20.4%), and elevated TERT expression in tumors lacking TERT or MYCN alterations in 10 of 223 cases (4.5%). ALT activation was found in 49 of 273 cases (17.9%). All these alterations occurred almost mutually exclusively and were associated with unfavorable prognostic variables and adverse outcome. The presence of activated telomerase (ie, TERT rearrangements, MYCN amplification, or high TERT expression without these alterations) was associated with poorest overall survival and was an independent prognostic marker in multivariable analyses. We also found that the telomerase-interacting compound 6-thio-2'-deoxyguanosine effectively inhibited viability and proliferation of neuroblastoma cells bearing activated telomerase. Similarly, tumor growth was strongly impaired upon 6-thio-2'-deoxyguanosine treatment in telomerase-positive neuroblastoma xenografts in mice. CONCLUSION: Our data suggest telomerase activation and ALT define distinct neuroblastoma subgroups with adverse outcome and that telomerase may represent a promising therapeutic target in many high-risk neuroblastomas.

A mechanistic classification of clinical phenotypes in neuroblastoma.

Neuroblastoma is a pediatric tumor of the sympathetic nervous system. Its clinical course ranges from spontaneous tumor regression to fatal progression. To investigate the molecular features of the divergent tumor subtypes, we performed genome sequencing on 416 pretreatment neuroblastomas and assessed telomere maintenance mechanisms in 208 of these tumors. We found that patients whose tumors lacked telomere maintenance mechanisms had an excellent prognosis, whereas the prognosis of patients whose tumors harbored telomere maintenance mechanisms was substantially worse. Survival rates were lowest for neuroblastoma patients whose tumors harbored telomere maintenance mechanisms in combination with RAS and/or p53 pathway mutations. Spontaneous tumor regression occurred both in the presence and absence of these mutations in patients with telomere maintenance-negative tumors. On the basis of these data, we propose a mechanistic classification of neuroblastoma that may benefit the clinical management of patients.

Telomerase activation by genomic rearrangements in high-risk neuroblastoma.

Neuroblastoma is a malignant paediatric tumour of the sympathetic nervous system. Roughly half of these tumours regress spontaneously or are cured by limited therapy. By contrast, high-risk neuroblastomas have an unfavourable clinical course despite intensive multimodal treatment, and their molecular basis has remained largely elusive. Here we have performed whole-genome sequencing of 56 neuroblastomas (high-risk, n = 39; low-risk, n = 17) and discovered recurrent genomic rearrangements affecting a chromosomal region at 5p15.33 proximal of the telomerase reverse transcriptase gene (TERT). These rearrangements occurred only in high-risk neuroblastomas (12/39, 31%) in a mutually exclusive fashion with MYCN amplifications and ATRX mutations, which are known genetic events in this tumour type. In an extended case series (n = 217), TERT rearrangements defined a subgroup of high-risk tumours with particularly poor outcome. Despite a large structural diversity of these rearrangements, they all induced massive transcriptional upregulation of TERT. In the remaining high-risk tumours, TERT expression was also elevated in MYCN-amplified tumours, whereas alternative lengthening of telomeres was present in neuroblastomas without TERT or MYCN alterations, suggesting that telomere lengthening represents a central mechanism defining this subtype. The 5p15.33 rearrangements juxtapose the TERT coding sequence to strong enhancer elements, resulting in massive chromatin remodelling and DNA methylation of the affected region. Supporting a functional role of TERT, neuroblastoma cell lines bearing rearrangements or amplified MYCN exhibited both upregulated TERT expression and enzymatic telomerase activity. In summary, our findings show that remodelling of the genomic context abrogates transcriptional silencing of TERT in high-risk neuroblastoma and places telomerase activation in the centre of transformation in a large fraction of these tumours.