Abstract Background Differential leukocyte counts are usually measured based on cellular morphology or surface marker expression. It has recently been shown that leukocyte counts can also be determined by cell-type–specific DNA methylation (DNAm). Such epigenetic leukocyte counting is applicable to small blood volumes and even frozen material, but for clinical translation, the method needs to be further refined and validated. Methods We further optimized and validated targeted DNAm assays for leukocyte deconvolution using 332 venous and 122 capillary blood samples from healthy donors. In addition, we tested 36 samples from ring trials and venous blood from 266 patients diagnosed with different hematological diseases. Deconvolution of cell types was determined with various models using DNAm values obtained by pyrosequencing or digital droplet PCR (ddPCR). Results Relative leukocyte quantification correlated with conventional blood counts for granulocytes, lymphocytes, B cells, T cells (CD4 or CD8), natural killer cells, and monocytes with pyrosequencing (r = 0.84; r = 0.82; r = 0.58; r = 0.50; r = 0.70; r = 0.61; and r = 0.59, respectively) and ddPCR measurements (r = 0.65; r = 0.79; r = 0.56; r = 0.57; r = 0.75; r = 0.49; and r = 0.46, respectively). In some patients, particularly with hematopoietic malignancies, we observed outliers in epigenetic leukocyte counts, which could be discerned if relative proportions of leukocyte subsets did not sum up to 100%. Furthermore, absolute quantification was obtained by spiking blood samples with a reference plasmid of known copy number. Conclusions Targeted DNAm analysis by pyrosequencing or ddPCR is a valid alternative to quantify leukocyte subsets, but some assays require further optimization.
Bosutinib (SKI-606) is an orally available, once-daily, dual Src and Abl kinase inhibitor with promising clinical potential in first-, second-, and third-line treatment of chronic myeloid leukemia (CML). Bosutinib effectively inhibits wild-type BCR-ABL and most imatinib-resistant BCR-ABL mutations except for V299L and T315I. Low hematologic toxicity is a remarkable characteristic of this novel second-generation tyrosine kinase inhibitor, and this has been ascribed to its minimal activity against the platelet-derived growth factor receptor and KIT. Low-grade, typically self-limiting diarrhea, which usually appears within the first few weeks after treatment initiation, represents the predominant toxicity of bosutinib. Other treatment-associated adverse events are mostly mild to moderate. Bosutinib has been approved by the US Food and Drug Administration for the treatment of chronic, accelerated, or blast phase Philadelphia chromosome-positive CML in adult patients with resistance or intolerance to prior therapy. This review summarizes the main properties of bosutinib and the currently available data on its clinical potential in the treatment of CML.
A better understanding of events triggering chronic myeloid leukemia progression is critical for optimized clinical management of chronic myeloid leukemia (CML). We sought to validate that increased expression of Musashi 2 (MSI2), a post-transcription regulator, is associated with progression and prognosis. Screening of 152 patients with CML showed that MSI2 was significantly decreased among patients with CML in chronic phase (CP) at diagnosis (p < 0.0001), but found no significant difference between the normal control group and treated patients with CML in CP. Moreover MSI2 was significantly increased (p < 0.0001) in patients with advance disease (AD) CML. Furthermore, our human hematopoietic cell line data imply that MSI2 and BCR-ABL1 mRNA expression are correlated. However, these data cast a doubt on earlier reports that MSI2 effects HES1 expression via NUMB-NOTCH signaling.
Abstract Background: Interferon alpha (IFNa) is approved for the therapy of patients (pts) with polycythemia vera (PV), a subtype of myeloproliferative neoplasms (MPN), achieving high hematologic response (HR) rates. A fraction of pts also achieve molecular responses (MR), but clonal factors sensitizing for MR remain elusive. Methods: We integrated colony formation and differentiation assays with single-cell RNA sequencing (scRNAseq) and cell genotyping in PV patient-derived cells vs. healthy controls (HC) to dissect how IFNa targets diseased clones during erythroid differentiation. Results: IFNa significantly decreased colony growth in MPN and HC. Differences in MR were observed, with a higher degree of MR in pts with high variant allele frequency (VAF). While lower STAT1 but higher IFIT2 inducibility of PV vs. HC was observed, responses in individual colonies were much more variable. scRNAseq of colonies demonstrated more mature erythroid PV-derived colonies compared with HC and showed differential activation of JAK-STAT and hypoxia signaling. JAK2V617F-mutant cells showed significantly upregulated STAT5A-, heme- and G2M checkpoint-associated pathways relative to JAK2WT cells from the same pts. IFNa induced viral response genes in mutated cells from PV pts and antagonized the upregulation of metabolism genes. Subgroup analysis revealed that IFNa significantly decreased immature erythrocytic cells in PV (basophilic erythroblasts p <0.05; polychromatic erythroblasts p <0.05) but not in HC. CD71neg‑CD235a+ cells from HC ( p <0.05) but not from PV pts were inhibited by IFNa, and the number of reticulocytes was less affected in PV than in HC. Robust IFNa responses persisted throughout differentiation, with PV showing more pronounced effects than HC. Apoptosis was significantly upregulated by IFNa in PV but not HC, and the most apoptotic cells were characterized by downregulation of ribosomal genes. This link between apoptosis and ribosomal genes was corroborated through analysis of mitochondrial variants, demonstrating IFNa-induced eradication of specific clones, which harbored significantly elevated expression of ribosomal genes. Conclusion: Our findings indicate that PV-derived clones either undergo apoptosis or pass through their typical differentiation cycle, contributing to the long-term exhaustion of mutant cells. The pivotal roles of ribosomal genes and clonal prerequisites in the therapeutic mechanism of IFNa are underscored by our study.
