Acute myeloid leukemia (AML) is characterized by clonal heterogeneity, leading to frequent relapses and drug resistance despite intensive clinical therapy. Although AML's clonal architecture has been addressed in many studies, practical monitoring of dynamic changes in those subclones during relapse and treatment is still understudied.
Fifteen longitudinal bone marrow (BM) samples were collected from three relapsed and refractory (R/R) AML patients. Using droplet digital polymerase chain reaction (ddPCR), the frequencies of patient's leukemic variants were assessed in seven cell populations that were isolated from each BM sample based on cellular phenotypes. By quantifying mutant clones at the diagnosis, remission, and relapse stages, the distribution of AML subclones was sequentially monitored.
Minimal residual (MR) leukemic subclones exhibit heterogeneous distribution among BM cell populations, including mature leukocyte populations. During AML progression, these subclones undergo active phenotypic transitions and repopulate into distinct cell population regardless of normal hematopoiesis hierarchic order. Of these, MR subclones in progenitor populations of patient BM predominantly carry MR leukemic properties, leading to more robust expansion and stubborn persistence than those in mature populations. Moreover, a minor subset of MR leukemic subclones could be sustained at an extremely low frequency without clonal expansion during relapse.
In this study, we observed treatment persistent MR leukemic subclones and their phenotypic changes during the treatment process of R/R AML patients. This underscores the importance of preemptive inhibition of clonal promiscuity in R/R AML, proposing a practical method for monitoring AML MR subclones.
© 2024 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Down syndrome (DS) is characterized by the occurrence of three copies of human chromosome 21 (HSA21). HSA21 contains a cluster of four interferon receptor (IFN-R) genes: IFNAR1, IFNAR2, IFNGR2, and IL10RB. DS patients often develop mucocutaneous infections and autoimmune diseases, mimicking patients with heterozygous gain-of-function (GOF) STAT1 mutations, which enhance cellular responses to three types of interferon (IFN). A gene dosage effect at these four loci may contribute to the infectious and autoimmune manifestations observed in individuals with DS. We report high levels of IFN-αR1, IFN-αR2, and IFN-γR2 expression on the surface of monocytes and EBV-transformed-B (EBV-B) cells from studying 45 DS patients. Total and phosphorylated STAT1 (STAT1 and pSTAT1) levels were constitutively high in unstimulated and IFN-α- and IFN-γ-stimulated monocytes from DS patients but lower than those in patients with GOF STAT1 mutations. Following stimulation with IFN-α or -γ, but not with IL-6 or IL-21, pSTAT1 and IFN-γ activation factor (GAF) DNA-binding activities were significantly higher in the EBV-B cells of DS patients than in controls. These responses resemble the dysregulated responses observed in patients with STAT1 GOF mutations. Concentrations of plasma type I IFNs were high in 12% of the DS patients tested (1.8% in the healthy controls). Levels of type I IFNs, IFN-Rs, and STAT1 were similar in DS patients with and without recurrent skin infections. We performed a genome-wide transcriptomic analysis based on principal component analysis and interferon modules on circulating monocytes. We found that DS monocytes had levels of both IFN-α- and IFN-γ-inducible ISGs intermediate to those of monocytes from healthy controls and from patients with GOF STAT1 mutations. Unlike patients with GOF STAT1 mutations, patients with DS had normal circulating Th17 counts and a high proportion of terminally differentiated CD8+ T cells with low levels of STAT1 expression. We conclude a mild interferonopathy in Down syndrome leads to an incomplete penetrance at both cellular and clinical level, which is not correlate with recurrent skin bacterial or fungal infections. The constitutive upregulation of type I and type II IFN-R, at least in monocytes of DS patients, may contribute to the autoimmune diseases observed in these individuals.

Imaging flow cytometry (IFC) produces up to 12 spectrally distinct, information-rich images of single cells at a throughput of 5,000 cells per second. Yet often, cell populations are still studied using manual gating, a technique that has several drawbacks, hence it would be advantageous to replace manual gating with an automated process. Ideally, this automated process would be based on stain-free measurements, as the currently used staining techniques are expensive and potentially confounding. These stain-free measurements originate from the brightfield and darkfield image channels, which capture transmitted and scattered light, respectively. To realize this automated, stain-free approach, advanced machine learning (ML) methods are required. Previous works have successfully tested this approach on cell cycle phase classification with both a classical ML approach based on manually engineered features, and a deep learning (DL) approach. In this work, we compare both approaches extensively on the problem of white blood cell classification. Four human whole blood samples were assayed on an ImageStream-X MK II imaging flow cytometer. Two samples were stained for the identification of eight white blood cell types, while two other sample sets were stained for the identification of resting and active eosinophils. For both data sets, four ML classifiers were evaluated on stain-free imagery with stratified 5-fold cross-validation. On the white blood cell data set, the best obtained results were 0.778 and 0.703 balanced accuracy for classical ML and DL, respectively. On the eosinophil data set, this was 0.871 and 0.856 balanced accuracy. We conclude that classifying cell types based on only stain-free images is possible with all four classifiers. Noteworthy, we also find that the DL approaches tested in this work do not outperform the approaches based on manually engineered features. © 2019 International Society for Advancement of Cytometry.
© 2019 International Society for Advancement of Cytometry.

The promyelocytic leukemia (PML)-retinoic acid receptor α (RARA) fusion is hypothesized to serve a vital role in the pathogenesis of acute promyelocytic leukemia (APL), which results from a reciprocal translocation between chromosomes 15 and 17, t(15;17)(q24;q21). A minority of APL cases lack the classical t(15;17) and have been identified to have cryptic or masked t(15;17) or complex translocations. The present study reports on a case of a 37-year-old male with APL harboring a complex three-way translocation t(1;17;15)(q21;q21;q24). This karyotypic interpretation was further confirmed by fluorescence in situ hybridization, and 98% of the bone marrow cells analyzed were positive for the PML-RARA fusion gene. After combined treatment with all-trans retinoic acid and arsenic trioxide, the patient achieved complete remission with no recurrence for 3 years to date. To the best of our knowledge, the present study is the first to report on the novel variant of t(15;17) involving the breakpoint 1q21.

Interleukin-21 (IL-21), a member of the common cytokine receptor γ chain (γc) family, is secreted by CD4+ T cells and natural killer T cells and induces effector function through interactions with the IL-21 receptor (IL-21R)/γc complex expressed on both immune and non-immune cells. Numerous studies suggest that IL-21 plays a significant role in autoimmune disorders. Therapeutic intervention to disrupt the IL-21/IL-21R/γc interaction and inhibit subsequent downstream signal transduction could offer a treatment paradigm for these diseases. Potent neutralizing antibodies reported in the literature were generated after extensive immunizations with human IL-21 alone and in combination with various adjuvants. To circumvent the laborious method of antibody generation while targeting a conserved functional epitope, we designed a novel alternating-antigen immunization strategy utilizing both human and cynomolgus monkey (cyno) IL-21. Despite the high degree of homology between human and cyno IL-21, our alternating-immunization strategy elicited higher antibody titers and more potent neutralizing hybridomas in mice than did the immunization with human IL-21 antigen alone. The lead hybridoma clone was humanized by grafting the murine complementarity-determining regions onto human germline framework templates, using a unique rational design. The final humanized and engineered antibody, MEDI7169, encodes only one murine residue at the variable heavy/light-chain interface, retains the sub-picomolar affinity for IL-21, specifically inhibits IL-21/IL-21R-mediated signaling events and is currently under clinical development as a potential therapeutic agent for autoimmune diseases. This study provides experimental evidence of the immune system's potential to recognize and respond to shared epitopes of antigens from distinct species, and presents a generally applicable, novel method for the rapid generation of exceptional therapeutic antibodies using the hybridoma platform.