Telomere Maintenance Mechanisms Define Clinical Outcome in High-Risk Neuroblastoma

Abstract

◥Neuroblastoma is a childhood cancer with heterogeneous clinical outcomes. To comprehensively assess the impact of telomere maintenance mechanism (TMM) on clinical outcomes in highrisk neuroblastoma, we integrated the C-circle assay [a marker for alternative lengthening of telomeres (ALT)], TERT mRNA expression by RNA-sequencing, whole-genome/exome sequencing, and clinical covariates in 134 neuroblastoma patient samples at diagnosis. In addition, we assessed TMM in neuroblastoma cell lines (n ¼ 104) and patient-derived xenografts (n ¼ 28). ALT was identified in 23.4% of high-risk neuroblastoma tumors and genomic alterations in ATRX were detected in 60% of ALT tumors; 40% of ALT tumors lacked genomic alterations in known ALT-associated genes. Patients with high-risk neuroblastoma were classified into three subgroups (TERT-high, ALT þ , and TERT-low/non-ALT) based on presence of C-circles and TERT mRNA expression (above or below median TERT expression). Event-free survival was similar among TERT-high, ALT þ , or TERT-low/non-ALT patients. However, overall survival (OS) for TERT-low/non-ALT patients was significantly higher relative to TERT-high or ALT patients (log-rank test; P < 0.01) independent of current clinical and molecular prognostic markers. Consistent with the observed higher OS in patients with TERT-low/non-ALT tumors, continuous shortening of telomeres and decreasing viability occurred in low TERT-expressing, non-ALT patient-derived high-risk neuroblastoma cell lines. These findings demonstrate that assaying TMM with TERT mRNA expression and C-circles provides precise stratification of high-risk neuroblastoma into three subgroups with substantially different OS: a previously undescribed TERT-low/non-ALT cohort with superior OS (even after relapse) and two cohorts of patients with poor survival that have distinct molecular therapeutic targets.Significance: These findings assess telomere maintenance mechanisms with TERT mRNA and the ALT DNA biomarker C-circles to stratify neuroblastoma into three groups, with distinct overall survival independent of currently used clinical risk classifiers.

Alternative lengthening of telomeres in childhood neuroblastoma from genome to proteome

“…ATRX mutations were identified in 55% of the ALT-positive neuroblastomas. In line with this observation, an ATRX mutation frequency of 60% was reported in a parallel study 46 . The most frequent type of ATRX mutations were large deletions.…”

ALT Neuroblastoma Chemoresistance due to ATM Activation by Telomere Dysfunction is Reversible with the ATM Inhibitor AZD0156

“…Previous studies, using less-than-specific means of identifying ALT, suggest that ALT neuroblastomas have distinct clinical behavior such as older age at diagnosis, protracted disease progression, and poor response to chemotherapy (30)(31)(32). Employing more specific ALT markers, especially the robust telomeric DNA C-circle assay, we showed that ALT activation occurs in ~25-30% of high-risk neuroblastomas with or without ATRX genomic alterations (29). Patients with ALT neuroblastoma have very poor long-term survival and are often non-salvageable following progression or relapse (29).…”

“…Neuroblastoma patients stratified as highrisk continue to have poor outcomes, with an overall survival rate of ~ 50% with current comprehensive therapy (24)(25)(26). Using next generation sequencing and molecular approaches, we and several other groups observed that most common genomic alterations in high-risk neuroblastoma (MYCN amplification, TERT rearrangements, and ATRX genomic alterations) converge on activating telomere maintenance mechanisms (20,(27)(28)(29). We have recently shown that telomere maintenance mechanisms (TMM) define clinical outcome in high-risk neuroblastoma, irrespective of currently employed risk factors, suggesting that TMM are pivotal for neuroblastoma pathogenesis and may potentially provide therapeutic targets (29).…”

“…Employing more specific ALT markers, especially the robust telomeric DNA C-circle assay, we showed that ALT activation occurs in ~25-30% of high-risk neuroblastomas with or without ATRX genomic alterations (29). Patients with ALT neuroblastoma have very poor long-term survival and are often non-salvageable following progression or relapse (29). Similar to ALT neuroblastoma, ALT pancreatic neuroendocrine tumors, gliomas, and soft tissue sarcomas are known to have distinct clinical behavior and outcome relative to non-ALT tumors (33)(34)(35)(36)(37), suggesting that ALT mediated telomere maintenance may impact cancer cell growth and the response to treatment.…”

Refining immunotherapeutic approaches to high‐risk neuroblastoma based on tumor genomic profiles

“…High-risk neuroblastoma is broadly subdivided into two major cohorts: younger patients (toddlers) whose tumors show MYCN amplification, 1p deletion and TERT overexpression; and older patients whose tumors harbor 11q deletion, occasionally along with other recurrent segmental chromosomal alterations (SCAs), maintain chromosome ends via the alternative lengthening of telomere (ALT) mechanism, and generally show a slightly more inflamed TME [8][9][10][11]. It remains unknown if these or other genomic alterations are associated with anti-GD2 efficacy or how they may coordinate immune escape.…”