To study clonal evolution in chronic myeloid leukemia (CML), we searched for BCR-ABL-independent gene mutations in both Philadelphia chromosome (Ph)-negative and Ph-positive clones in 29 chronic-phase CML patients by targeted deep sequencing of 25 genes frequently mutated in myeloid disorders. Ph-negative clones were analyzed in 14 patients who developed clonal cytogenetic abnormalities in Ph-negative cells during treatment with tyrosine kinase inhibitors (TKI). Mutations were detected in 6/14 patients (43%) affecting the genes DNMT3A, EZH2, RUNX1, TET2, TP53, U2AF1 and ZRSR2. In two patients, the mutations were also found in corresponding Ph-positive diagnostic samples. To further investigate Ph-positive clones, 15 randomly selected CML patients at diagnosis were analyzed. Somatic mutations additional to BCR-ABL were found in 5/15 patients (33%) affecting ASXL1, DNMT3A, RUNX1 and TET2. Analysis of individual hematopoietic colonies at diagnosis revealed that most mutations were part of the Ph-positive clone. In contrast, deep sequencing of subsequent samples during TKI treatment revealed one DNMT3A mutation in Ph-negative cells that was also present in Ph-positive cells at diagnosis, implying that the mutation preceded the BCR-ABL rearrangement. In summary, BCR-ABL-independent gene mutations were frequently found in Ph-negative and Ph-positive clones of CML patients and may be considered as important cofactors in the clonal evolution of CML.

Our understanding of the significance of epigenetic dysregulation in the pathogenesis of myeloid malignancies has greatly advanced in the past decade. Enhancer of Zeste Homolog 2 (EZH2) is the catalytic core component of the Polycomb Repressive Complex 2 (PRC2), which is responsible for gene silencing through trimethylation of H3K27. EZH2 dysregulation is highly tumorigenic and has been observed in various cancers, with EZH2 acting as an oncogene or a tumor-suppressor depending on cellular context. While loss-of-function mutations of EZH2 frequently affect patients with myelodysplastic/myeloproliferative neoplasms, myelodysplastic syndrome and myelofibrosis, cases of chronic myeloid leukemia (CML) seem to be largely characterized by EZH2 overexpression. A variety of other factors frequently aberrant in myeloid leukemia can affect PRC2 function and disease pathogenesis, including Additional Sex Combs Like 1 (ASXL1) and splicing gene mutations. As the genetic background of myeloid malignancies is largely heterogeneous, it is not surprising that EZH2 mutations act in conjunction with other aberrations. Since EZH2 mutations are considered to be early events in disease pathogenesis, they are of therapeutic interest to researchers, though targeting of EZH2 loss-of-function does present unique challenges. Preliminary research indicates that combined tyrosine kinase inhibitor (TKI) and EZH2 inhibitor therapy may provide a strategy to eliminate the residual disease burden in CML to allow patients to remain in treatment-free remission.

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Clonal hematopoiesis is frequently observed in elderly people. To investigate the prevalence and dynamics of genetic alterations among healthy elderly individuals, a cohort of 50 people >80 years was genotyped for commonly mutated leukemia-associated genes by targeted deep next-generation sequencing. A total of 16 somatic mutations were identified in 13/50 (26%) individuals. Mutations occurred at low variant allele frequencies (median 11.7%) and remained virtually stable over 3 years without development of hematologic malignancies in affected individuals. With DNMT3A mutations most frequently detected, another cohort of 160 healthy people spanning all age groups was sequenced specifically for DNMT3A revealing an overall mutation rate of 6.2% (13/210) and an age-dependent increase of mutation prevalence. A significant difference (p = 0.017) in the DNMT3A expression pattern was detected between younger and healthy elderly people as determined by qRT-PCR. To evaluate the selection of clonal hematopoietic stem cells (HSCs), bone marrow of two healthy individuals with mutant DNMT3A was transplanted in a humanized mouse model. Xenografts displayed stable kinetics of DNMT3A mutations over 8 months. These findings indicate that the appearance of low-level clones with leukemia-associated mutations is a common age-associated phenomenon, but insufficient to initiate clonal selection and expansion without the additional influence of other factors.