Mutated Cancer-Related Genes Detected at Diagnosis of CML and a Novel Class of Variant Associated with the Philadelphia Translocation Are Both Independent Predictors of Inferior Outcomes
Authors: Shanmuganathan Naranie , Wadham Carol , Shahrin Nur Hezrin , Thomson Daniel , Feng Jinghua , Saunders Verity A , Lin Ming , Kenyon Rosalie , King Rob , Wang Paul , Ross David M , Yeung David T , Schreiber Andreas W , Hughes Timothy P. , Branford Susan 
Published: February 2021

Background
We previously reported a high incidence of mutated cancer-related genes at CML diagnosis in selected chronic phase patients with a poor outcome compared to those with a good outcome. We also found a novel class of variant associated with the formation of the Ph chromosome comprising fusions and/or rearrangement of genes on the translocated chromosomes, with evidence of fragmentation, inversion, and imperfect sequence reassembly. These were termed 'Ph-associated events' and were more frequent in patients with poor outcome. However, the risk attributable to these mutational events at diagnosis has not been defined in unselected cohorts.
Aim
To assess the impact of genomic events in a cohort of consecutively treated patients at diagnosis of chronic phase CML.
Methods
A hybridization capture sequencing method targeting genes implicated in myeloid and lymphoid malignancies was applied to diagnostic RNA of patients enrolled in the TIDEL II trial. Patients were treated with upfront imatinib with active intervention, dose escalation or nilotinib switch, primarily for lack of time-dependent molecular milestones. Single base variants, small insertions/deletions, splice variants, gene fusions, and focal gene deletions were assessed with pre-defined criteria for pathogenicity. These were further classified as pathogenic mutations in cancer-related genes or Ph-associated events. Univariate and multivariate analyses were performed to evaluate the influence of mutational events and other key clinical and demographic variables on outcome at 4 years. Failure events were as defined by the ELN 2020 recommendations.
Results
160/210 TIDEL II patients have so far been sequenced. 33 relevant mutations with variant allele frequencies ≥5% were identified in 9 genes in 25 patients (16%). ASXL1 was most frequently mutated (10% of all patients) and other recurrently mutated genes at diagnosis were RUNX1, BCORL1, IKZF1 and DNMT3A. Ph-associated events occurred in 25 patients (16%). Most of these (14/25 patients) involved fusions between genes on chromosomes 9 and 22 consistent with deletions adjacent to BCR and ABL1, or fusions between BCR or ABL1 and genes/regions on chromosomes other than 9 or 22. These were consistent with variant translocation and some were cytogenetically cryptic. Among these and other Ph-associated events were complex rearrangements involving inversions and large duplications. These were detected in 14/25 patients. Five patients had both cancer-related gene mutations and Ph-associated events, totalling 45 patients (28%) with at least 1 genomic event.
Cancer-related mutations at diagnosis were associated with inferior progression-free survival (PFS) 82% vs 91% P=.03, and failure-free survival (FFS) 55% vs 83% P&lt;.001. Molecular outcomes were also inferior: MMR 71% vs 89% P=.02; and MR4 27% vs 71%; P=.01. Similarly, Ph-associated events predicted inferior outcomes: FFS 63% vs 81% P=.01; MMR 70% vs 88% P=.01; and MR4 47% vs 68% P=.01. Importantly, patients with either or both of these mutational events had an increased likelihood of progression to accelerated phase or blast crisis or development of a BCR-ABL1 kinase domain mutation (Figure).
Independent predictors of all survival and molecular outcomes were assessed with univariate and multivariate modelling (Table). Candidate prognostic variables were age at diagnosis, sex, transcript, Sokal and ELTS scores and the genomic variables. The only independent predictor of PFS was mutations in cancer-related genes. Cancer-related gene mutations, Ph-associated events and the ELTS score were independent predictors of FFS, MMR and MR4.
We evaluated whether genomic data could be additive to the ELTS risk score. Low risk ELTS patients with any mutational event had inferior outcomes: FFS 76% vs 86%, P=.07; MMR 75% vs 92%, P=.02; MR4 41% vs 75%, P=0.006. Similar findings were observed in Intermediate risk ELTS patients: FFS 22% vs 91%, P&lt;.001; MMR 81% vs 84%, P=.06; MR4 9% vs 72%, P=0.047. The number of high risk ELTS patients were inadequate to perform this analysis.
Conclusion
Despite a proactive strategy for TKI switch and a higher imatinib starting dose, the presence of cancer-related gene mutations or Ph-associated events conferred inferior outcomes. Combining the ELTS score with any mutational event further differentiated patient outcomes, demonstrating the power of integrating genomic data with current risk stratification.

Disclosures
Shanmuganathan: Janssen: Other: travel expenses; Novartis: Honoraria, Other: Travel expenses; Gilead: Other: Travel expenses; Amgen: Other: travel expenses. Hughes:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Branford:Bristol Myers Squibb: Honoraria; Cepheid: Honoraria, Membership on an entity's Board of Directors or advisory committees; Qiagen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.