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P. Valent et al.
Mutation profiles in CMML: current standards and lim- itations
Somatic mutations are detectable in the vast majority of patients with CMML.8,11,103-106 The clonal architecture, clone sizes and clonal evolution patterns vary from patient to patient.106-108 In some cases, initially small clones expand over time. It is, therefore, standard to apply next-genera- tion sequencing assays with sufficient sensitivity to iden- tify bona fide somatic mutations associated with CMML. The most frequently detected somatic mutations in CMML are mutations in TET2 (60%), SRSF2 (50%), and ASXL1 (40%) (Table 6).31,103-110 The presence of a SRSF2 mutation, particularly in combination with mutated TET2, correlates strongly with a CMML phenotype.31,109,110 It is also worth noting that two of these mutations (TET2, ASXL1) are also known as CHIP/ARCH-related muta- tions. However, only mutated ASXL1 has been associated with a poor prognosis in CMML.104,109 An overview of somatic mutations recurrently detected in CMML is pro- vided in Table 6. Somatic mutations with independent prognostic impact include several RAS-pathway muta- tions as well as mutations in ASXL1, RUNX1 and SETBP1 (Table 6).31,103-111 RAS-pathway mutations trigger cell signal- ing and proliferation and have been associated with cytokine-independent growth of CMML progenitor cells, the proliferative variant of CMML, AML transformation and poor survival.10,22,23,112-116 Other driver mutations involved in cell signaling, such as JAK2 V617F or KIT D816V, are also major triggers of cellular differentiation (Online Supplementary Table S7). These drivers alone can- not induce transformation, but they may act together with other (e.g., ‘RAS pathway’) mutations to cause disease progression. Whereas JAK2 V617F is a strong indicator of MPN-like differentiation, the presence of KIT D816V is almost always associated with concomitant mast cell dif- ferentiation and mastocytosis (SM-CMML).6-8,32-36,39,42,43 The other mutations found in CMML act as modulators of epi- genetic events and transcription (e.g., ASXL1) or DNA methylation (e.g., TET2), as regulators of the spliceosome machinery (e.g., SRSF2), or as modulators of the DNA damage response, such as TP53 (Table 6). During progres- sion of CMML to secondary AML and especially during therapy, the mutational landscape(s) and clonal architec- ture(s) may change.109-113 For example initially small clones may expand and may be selected because of resistance- mediating molecular features. It is worth noting that sev- eral mutated gene products also serve as potential targets of therapy (Table 6).
Our faculty recommends that next-generation sequenc- ing studies should be regarded as a standard approach in all patients with suspected or known CMML as well as in patients with idiopathic monocytosis of unknown signifi-
cance and in those with persistent reactive monocytosis (in order to exclude an additional clonal component). When a CMML-related mutation is found in an individual with idiopathic monocytosis of unknown significance or reactive monocytosis, the diagnosis may change to clonal monocytosis of unknown significance or oligomonocytic CMML, depending on additional findings.
Our faculty also recommends that the next-generation sequencing assay should have sufficient sensitivity (to detect 2-5% clonal cells) and should cover all relevant lesions shown in Table 6. In the context of CHIP/ARCH, a cutoff variant allele frequency of 2% is considered diag- nostic,69 whereas in the context of CMML, we propose 10% as the variant allele frequency diagnostic cut off and thus marker to count as a co-criterion of CMML when, for example, no diagnostic morphological dysplasia can be documented (Tables 1 and 3), similar to the definition in MDS.71,73 Determining the variant allele frequency is also useful for documenting the clinical impact of certain driver lesions in special CMML variants (e.g., with JAK2 V617F or KIT D816V) and clone expansion during follow- up. Therefore, our faculty recommends that molecular studies in CMML should report variant allele frequencies with sufficient precision and sufficient sensitivity – in the same way as in MDS.71,73 Finally, our faculty recommends that molecular markers should increasingly be used to optimize prognostic scoring systems in CMML.117-120
Flow cytometry in CMML: standards and limitations
Flow cytometry studies are an essential diagnostic tool in patients with (suspected) classical CMML, pre-CMML conditions and special CMML variants.121-132 Therefore, our faculty is of the opinion that it is standard practice to perform multi-color flow cytometry (MFC) in the PB and BM in all cases with suspected or known CMML or a sus- pected pre-CMML condition. MFC studies are helpful to confirm the monocyte and blast cell counts in these patients and to exclude AML. In addition, MFC is useful to confirm the presence of distinct monocyte popula- tions. Monocytes are defined as CD14+ cells in these analyses. Based on the expression of CD14 and CD16, monocytes are further divided into classical (MO1) monocytes (CD14bright/CD16−), intermediate (MO2) monocytes (CD14bright/CD16+) and non-classical (MO3) monocytes (CD14dim/CD16+) (Table 7).127,128,132 Compared to age-matched healthy donors133 and patients with reac- tive monocytosis, but also myeloid neoplasms other than CMML (even MDS), the percentages of MO1 monocytes in the PB are higher and the percentage of MO3 mono- cytes is lower in patients with CMML.127,131,132 When the absolute monocyte count is increased in the PB, a cutoff value of >94% MO1 monocytes, based on their
Table 7. Phenotypic classification of monocytes and distribution of monocyte subsets in patients with chronic myelomonocytic leukemia and in controls.*
Monocyte-subset
Classical (MO1)
Intermediate (MO2)
Non-classical (MO3)
Defining phenotype
CD14bright/CD16−
CD14bright/CD16+
CD14dim/CD16+
Typical relative frequency in: CMML MDS or MPN
≥94% 70-97% <20% 5-20% <5% 5-10%
Reactive BM
<94%
5-15%
5-20%
*Data refer to published results presented in references #125 through #132. CMML: chronic myelomonocytic leukemia; MDS: myelodysplastic syndrome; MPN: myeloprolifera- tive neoplasm; BM: bone marrow.
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haematologica | 2019; 104(10)