Alternative Lengthening of Telomeres is not synonymous with mutations in ATRX/DAXX

The PCAWG Consortium has recently released an unprecedented set of tumor whole genome sequence (WGS) data from 2,658 cancer patients across 38 different primary tumor sites1. WGS is able to document the quantity and distribution of telomeric repeats2. In one of the papers analyzing the PCAWG dataset, Sieverling et al.3 confirmed previous data4 indicating that tumors with truncating ATRX or DAXX alterations, referred to as ATRX/DAXXtrunc, have an aberrant telomere variant repeat (TVR) distribution. By regarding these mutations, vs. TERT modifications (TERTmod; i.e. promoter mutations +/- amplifications +/- structural variations), as indicators of Alternative Lengthening of Telomeres (ALT) vs. telomerase, they built a random forest classifier for ALT-probability, and then associated genomic characteristics with the putative Telomere Maintenance Mechanism (TMM)3. However, we show here that equating ATRX/DAXXtrunc and TERTmod with ALT and telomerase, respectively, results in TMM predictions which correlate poorly with TMM assay data. ATRX/DAXXtrunc mutations are heterogeneously distributed in ALT-positive (ALT+) tumors of different types, as are TERTmod in telomerase-positive tumors4. Although these mutations are strongly associated with TMM, most tumors do not harbor them, making them an inadequate basis for building a classifier in a large-scale pan-cancer study4-7. Here, we provide a new analysis of the PCAWG data, based on C-circle assay (CCA)8 that is available for a subset of these tumors4,9,10. We show that the Sieverling et al. score overestimates the proportion of ALT associated with ATRX/DAXXtrunc and misclassifies ALT tumors when these mutations are absent. We also show some TVR correlate with ATRX/DAXXtrunc mutations, regardless of TMM. Finally, we propose a new classifier to identify ALT tumors in the PCAWG cohort.