Abstract
Ten-Eleven-Translocation 2 (TET2) encodes a critical dioxygenase that regulates DNA methylation and transcription. Somatic alterations in TET2 are observed in “age-related” clonal hematopoiesis (CH), with these alterations being prevalent along a spectrum of “pre-malignant” CH including clonal cytopenia(s) of undetermined significance (CCUS), and in hematological neoplasms such as chronic myelomonocytic leukemia (CMML). Single-cell flow based studies have reported that early loss of TET2 results in granulomonocytic differentiation and skewing of multipotent and common myeloid progenitor cells, as a result of early clonal dominance and enhanced fitness. CMML is a neoplasm characterized by sustained peripheral blood monocytosis, arising on a background of TET2 mutant CH (60% with TET2 mutations), commonly demonstrating tolerance to multiple truncating TET2 mutations. TET2 mutant CMML patients, especially in the absence of ASXL1 mutations, have lower risk stratifications, have better responses to hypomethylating agents, and have a distinct survival advantage. In addition, CMML patients have a proportional increase in the classical monocyte fraction (CD14 + /CD16- [M01 fraction] at > 94%) as assessed by flow cytometry, distinguishing CMML from reactive monocytosis and other myeloid neoplasms with monocytosis. Malignant monocytes often display an altered expression of CD13, CD33, CD117 and CD38, along with aberrant co-expression of CD7 and CD56 (not specific to neoplasia). However, individual cell-types associated with mutant TET2 in CH and the effects of mutant gene dosage, including TET2 somatic copy number alterations (SCNA) on clonal evolution, need further evaluation. We carried out this study using single cell-based multiomics, to capture TET2-specific cellular clonal surface marker expression compositions in patients with a spectrum of TET2-mutated CCUS and CMML.
Research was performed under the advisement of Mayo Clinic Institutional Review Board. All CMML cases were diagnosed using the 2016 World Health Organization defined criteria. Mononuclear cells were enriched from peripheral blood using gradient density centrifugation and submitted for testing using several applications (see Supplementary Methods for details). Briefly, we utilized the Tapestri platform (MissionBio, San Francisco, CA) which combines surface protein expression using 42 oligo-conjugated cell surface markers (Biolegend, San Diego, CA), and DNA based sequencing using a 45-gene panel specific for myeloid malignancies (see Supplementary Table 1 and Supplementary Fig. 1 for cell calling details). Of note, although SRSF2 mutations are frequent in CMML, given that the P95 hotspot mutation area is highly GC rich, single cell-based amplicon design was not feasible. All relevant mutations reported were found by clinical next generation sequencing (NGS) reporting.
