DNA methylation profiling for molecular classification of neuroblastoma

Neuroblastoma is a heterogeneous disease where patient stratification is critical for prognosis and treatment decisions and where it recently has been suggested that the presence of telomere maintenance mechanisms (TMM) should be considered in risk stratification. We investigated the utility of DNA methylation-based classification for neuroblastoma diagnostics by analysing 303 tumours samples from two cohorts. We show that of the total number of cases, an average of 90% of the samples classified as neuroblastoma, while 66% also achieved confident classification into the three NB subclasses: "MYCN-type", "ALT/TERT TMM positive" and "TMM negative". The tumours classified as MYCN-type showed genomic amplification of MYCN (MNA); however, some MYCN-type cases lacked evident MNA, suggesting that epigenetic states might be influenced by other factors such as activating ALK mutations. Survival analysis indicated similar poor survival probabilities for patients classified as TMM positive or MYCN type, distinct from the inferior survival of TMM-negative cases. All cases, with complementary genomic data available, associated with TMM positivity also presented features associated with telomere lengthening mechanisms, including TERT or ATRX alteration. However, some tumours positive for these features, especially TERT rearrangement, classified as MYCN type rather than TMM positive, indicating that MNA and other mechanisms introduce a methylation pattern that supersede or overlap with pattern imposed by TERT. Chromosomal copy number alterations (CNAs) characteristic of methylation subclasses were identified, including 1p deletion and 17q gain in MYCN type and combinations of 11q loss, 3p loss, 7q gain, and 17q gain in TMM-positive cases, highlighting the potential of the methylation arrays to replace SNP arrays for prognostic genomic assessments. Our study demonstrates that DNA methylation-based classification stratifies neuroblastoma into clinically relevant subgroups, aiding diagnostic and prognostic decisions, although discrepancy between genomic features and methylation classification does occur. The interplay between genomic alterations and methylation patterns could give clues into the discrepancy and underscores the complexity of neuroblastoma biology and the need for further research and validation of clinical outcomes of the patients in the respective subclasses.