Clinical and molecular characterization of patients with stage 4(M) neuroblastoma aged less than 18 months without MYCN amplification.

INTRODUCTION: The survival of children with stage 4(M) neuroblastoma without MYCN amplification and below the age of 18 months is considered better than the still dismal outcome of older high-risk neuroblastoma patients. This study analyzes the impact of clinical and molecular characteristics on the long-term outcome. PATIENTS AND METHODS: Clinical presentation, survival, and recurrence patterns of patients enrolled onto trials NB90, NB97, and NB2004 were retrospectively analyzed. Gene expression signatures based on RNA microarrays (TH10) were investigated if tumor material was available. RESULTS: Between 1990 and 2015, 177 patients with stage 4(M) MYCN nonamplified neuroblastoma aged less than 18 months at diagnosis were eligible. After a median follow-up of 9.7 years (IQR 5.0, 13.4), the proportions of 10-year event-free survival (EFS) and overall survival (OS) were 73% (95% confidence interval [CI] 67-79%) and 86% (95% CI 80-92%), respectively. Of the 27 neuroblastoma recurrences, 44% occurred in more than one site. Four additional patients presented histologically mature ganglioneuroma at recurrence. Six patients developed a secondary malignancy. The secondary 5-year EFS and OS of the 27 patients with neuroblastoma recurrence were 44% and 59%, respectively. TH10 gene expression signature was not prognostically predictive in the investigated subcohort. CONCLUSION: The outcome of patients with stage 4(M) neuroblastoma aged less than 18 months is favorable when treated with high-risk or otherwise intensive therapy. The development of secondary malignancies and the potential of maturation to ganglioneuroma call for a controlled stepwise reduction of treatment intensity.

TERT Expression and Clinical Outcome in Pulmonary Carcinoids.

PURPOSE: The clinical course of pulmonary carcinoids ranges from indolent to fatal disease, suggesting that specific molecular alterations drive progression toward the fully malignant state. A similar spectrum of clinical phenotypes occurs in pediatric neuroblastoma, in which activation of telomerase reverse transcriptase (TERT) is decisive in determining the course of disease. We therefore investigated whether TERT expression defines the clinical fate of patients with pulmonary carcinoid. METHODS: TERT expression was examined by RNA sequencing in a test cohort and a validation cohort of pulmonary carcinoids (n = 88 and n = 105, respectively). A natural TERT expression cutoff was determined in the test cohort on the basis of the distribution of TERT expression, and its prognostic value was assessed by Kaplan-Meier survival estimates and multivariable analyses. Telomerase activity was validated by telomere repeat amplification protocol assay. RESULTS: Similar to neuroblastoma, TERT expression exhibited a bimodal distribution in pulmonary carcinoids, separating tumors into TERT-high and TERT-low subgroups. A natural TERT cutoff discriminated unfavorable from favorable clinical courses with high accuracy both in the test cohort (5-year overall survival [OS], 0.547 ± 0.132 v 1.0; P < .001) and the validation cohort (5-year OS, 0.788 ± 0.063 v 0.913 ± 0.048; P < .001). In line with these findings, telomerase activity was largely absent in TERT-low tumors, whereas it was readily detectable in TERT-high carcinoids. In multivariable analysis considering TERT expression, histology (typical v atypical carcinoid), and stage (≤IIA v ≥IIB), high TERT expression was an independent prognostic marker for poor survival, with a hazard ratio of 5.243 (95% CI, 1.943 to 14.148; P = .001). CONCLUSION: Our data demonstrate that high TERT expression defines clinically aggressive pulmonary carcinoids with fatal outcome, similar to neuroblastoma, indicating that activation of TERT may be a defining feature of lethal cancers.

Reliable assessment of telomere maintenance mechanisms in neuroblastoma.

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.

Chromothripsis followed by circular recombination drives oncogene amplification in human cancer.

The mechanisms behind the evolution of complex genomic amplifications in cancer have remained largely unclear. Using whole-genome sequencing data of the pediatric tumor neuroblastoma, we here identified a type of amplification, termed 'seismic amplification', that is characterized by multiple rearrangements and discontinuous copy number levels. Overall, seismic amplifications occurred in 9.9% (274 of 2,756) of cases across 38 cancer types, and were associated with massively increased copy numbers and elevated oncogene expression. Reconstruction of the development of seismic amplification showed a stepwise evolution, starting with a chromothripsis event, followed by formation of circular extrachromosomal DNA that subsequently underwent repetitive rounds of circular recombination. The resulting amplicons persisted as extrachromosomal DNA circles or had reintegrated into the genome in overt tumors. Together, our data indicate that the sequential occurrence of chromothripsis and circular recombination drives oncogene amplification and overexpression in a substantial fraction of human malignancies.