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G. Nteliopoulos et al.
responders, somatic variants impacted clinical outcome only in those treated with imatinib. This mirrors clinical experience in which the 2G-TKI can result in deep and durable responses in patients who were resistant to ima- tinib, and induce these responses more rapidly and in a larger proportion of patients when used as first-line thera- py. One possible explanation is that the increased potency of the 2G-TKI results in the rapid eradication of the mutat- ed clones and thus overcome the adverse prognostic impact. This hypothesis is supported by the fact that 90% of 2G-TKI-treated subjects with somatic variants achieved MR3 compared with <50% of subjects treated with ima- tinib. Data from in vitro liquid cultures corroborated our original findings, since at least cells containing some vari- ants were eradicated on treatment with dasatinib but per- sisted or were eradicated more slowly on treatment with imatinib. We also have additional indirect evidence that our findings may predict response to imatinib. First, we were able to show distinct methylation patterns between imatinib-treated subjects with and without variants, and second, the PPI network indicates close interactions between p210BCRABL1 and proteins affected by somatic vari- ation.
Somatic variants had better predictive power for out- comes than other widely-used predictive variables such as the Sokal score4,14,47 and BCRABL1 transcript type48,49 but less compared with the newly defined ELTS score.10 Previous clinical risk scores identified individuals at high risk of early progression but were less successful in pre- dicting poor-risk subjects in the low/intermediate cohorts. Combining a clinical risk score with somatic variants is a potentially promising approach, and may be particularly valuable in those with low Sokal /ELTS scores, who are heavily influenced by age, such that a young patient with inherently poor prognosis may be inappropriately classi- fied as non-high risk.
By using paired leukemia and control DNA, we found most somatic variants were part of a Ph+ clone. Therapy with imatinib eradicated the Ph+ clones with somatic vari- ants in responders achieving MR3 but not in non-respon- ders who progressed. These persistent somatic variants may be implicated in disease evolution or may be passen- ger mutations and require confirmation in larger cohorts.
The presence of pre-leukemia variants was implicated in three subjects. DNMT3A, ASXL1 variants were found in Ph+ and, albeit at lower levels, in Ph– cells. This suggests that these variants preceded the acquisition of BCRABL1.
Variants in DNMT3A, ASXL1 and TET2 are thought to be latent initiating mutations31 and are described in CML.27,28 However, ASXL1 mutations have also been found in chil- dren and young adults with CML.26 Rare germline variants had no impact on clinical outcomes in either of the ima- tinib or 2G-TKI cohorts, contrary to other reports.43 Variants detected only in Ph– cells from subjects aged >60 years in molecular remission may have developed during therapy, or have been present at diagnosis and unmasked in remission.
In summary, we showed potentially pathogenic somatic variants of epigenetic modifiers are common in CML-CP at diagnosis, and when combined with other risk factors may be promising predictive biomarkers determining which is the best TKI for each individual.
Our study has some limitations, the most important of which are small sample size (although it is the largest study to date assessing the effect of genetic variants on survival), the potential exaggeration of the effect size due to the selection of the extreme responders / non-respon- ders, the limited number of target genes, and the retro- spective nature of our observations. Furthermore, we were unable to assess the impact of additional chromoso- mal abnormalities (ACA)50 due to the absence of cytoge- netic data at diagnosis. The intriguing question remains as to whether any of the variants identified has more or less impact on prognosis, but because of the small numbers of each variant we were unable to explore this in more detail. We now wish to see our panel enriched by the addition of genes found to be altered in CML-CP by targeted, exome or WGS, and validated in larger, unselected, consecutive cohorts of individuals with CML. Our findings, if con- firmed in a prospective study, could assist in distinguish- ing individuals who would benefit starting therapy with a more potent 2G-TKI rather than imatinib.
Funding
JFA is a NIHR Senior Investigator and we are grateful for the support from the Imperial College NIHR Biomedical Research Centre.
Acknowledgments
We would also like to thank the Molecular Pathology Laboratory and the John Goldman Centre for Cellular Therapy for providing us with guanidinium thiocyanate (GTC) lysates and cells from CML and normal subjects’ samples. We are grate- ful to individuals with CML for participating in this study.
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