Malformation of the endoplasmic reticulum system evolving into giant inclusions and Auer bodies in acute promyelocytic leukemia: an ultrastructural study of 6 cases.

The endoplasmic reticulum（ER）is the largest membranous network serving as a region for protein, lipid and steroid synthesis, transport and storage. Detailed information about ER-cisternae, ER-tubules and rough endoplasmic reticulum (rER) is scarce in human blood cells. This study describes a series of giant inclusions and Auer bodies in promyeloblasts in six patients with acute promyelocytic leukemia (APL), by light microscopy, transmission electron microscopy (TEM) and cytochemical stains. TEM revealed that giant inclusions and pro-Auer bodies were associated with rER and surrounded by tubular structures composed of degenerated or redundant membrane in promyeloblasts, which corresponded with elements of the ER system. This paper reveals that in the promyeloblasts of APL, ER is the source of and transforms progressively into giant inclusions and Auer bodies.

Progression in Myeloid Neoplasms: Beyond the Myeloblast.

Disease progression in myelodysplastic syndromes (MDS), myelodysplastic-myeloproliferative neoplasms (MDS/MPN), and myeloproliferative neoplasms (MPN), altogether referred to as myeloid neoplasms (MN), is a major source of mortality. Apart from transformation to acute myeloid leukemia, the clinical progression of MN is mostly due to the overgrowth of pre-existing hematopoiesis by the MN without an additional transforming event. Still, MN may evolve along other recurrent yet less well-known scenarios: (1) acquisition of MPN features in MDS or (2) MDS features in MPN, (3) progressive myelofibrosis (MF), (4) acquisition of chronic myelomonocytic leukemia (CMML)-like characteristics in MPN or MDS, (5) development of myeloid sarcoma (MS), (6) lymphoblastic (LB) transformation, (7) histiocytic/dendritic outgrowths. These MN-transformation types exhibit a propensity for extramedullary sites (e.g., skin, lymph nodes, liver), highlighting the importance of lesional biopsies in diagnosis. Gain of distinct mutations/mutational patterns seems to be causative or at least accompanying several of the above-mentioned scenarios. MDS developing MPN features often acquire MPN driver mutations (usually JAK2), and MF. Conversely, MPN gaining MDS features develop, e.g., ASXL1, IDH1/2, SF3B1, and/or SRSF2 mutations. Mutations of RAS-genes are often detected in CMML-like MPN progression. MS ex MN is characterized by complex karyotypes, FLT3 and/or NPM1 mutations, and often monoblastic phenotype. MN with LB transformation is associated with secondary genetic events linked to lineage reprogramming leading to the deregulation of ETV6, IKZF1, PAX5, PU.1, and RUNX1. Finally, the acquisition of MAPK-pathway gene mutations may shape MN toward histiocytic differentiation. Awareness of all these less well-known MN-progression types is important to guide optimal individual patient management.

Molecular Heterogeneity of Pediatric AML with Atypical Promyelocytes Accumulation in Children-A Single Center Experience.

Acute promyelocytic leukemia (APL) pathogenesis is based on RARA gene translocations, which are of high importance in the diagnosis of and proper therapy selection for APL. However, in some cases acute myeloid leukemia (AML) demonstrates APL-like morphological features such as atypical promyelocytes accumulation. This type of AML is characterized by the involvement of other RAR family members or completely different genes. In the present study, we used conventional karyotyping, FISH and high-throughput sequencing in a group of 271 de novo AML with atypical promyelocytes accumulation. Of those, 255 cases were shown to carry a typical chromosomal translocation t(15;17)(q24;q21) with PML::RARA chimeric gene formation (94.1%). Other RARA-positive cases exhibited cryptic PML::RARA fusion without t(15;17)(q24;q21) (1.8%, n = 5) and variant t(5;17)(q35;q21) translocation with NPM1::RARA chimeric gene formation (1.5%, n = 4). However, 7 RARA-negative AMLs with atypical promyelocytes accumulation were also discovered. These cases exhibited TBL1XR1::RARB and KMT2A::SEPT6 fusions as well as mutations, e.g., NPM1 insertion and non-recurrent chromosomal aberrations. Our findings demonstrate the genetic diversity of AML with APL-like morphological features, which is of high importance for successful therapy implementation.

A Pathogenic NRAS c.38G>A (p.G13D) Mutation in RARA Translocation-negative Acute Promyelocytic-like Leukemia with Concomitant Myelodysplastic Syndrome.

An acute promyelocytic leukemia (APL) patient not demonstrating the retinoic acid receptor α (RARA) translocation is rare. A 76-year-old man was diagnosed with myelodysplastic syndrome (MDS). After a year, abnormal promyelocytes were detected with pancytopenia and disseminated intravascular coagulopathy. Morphologically, the patient was diagnosed with APL; however, a genetic examination failed to detect RARA translocation. Thereafter, whole-genome sequencing revealed an NRAS missense mutation [c.38G>A (p.G13D)]. This mutation was not detected in posttreatment bone marrow aspirate, despite residual MDS. Few reports are available on similar cases. Furthermore, the NRAS c.38G>A mutation may be a novel pathogenic variant exacerbating RARA translocation-negative acute promyelocytic-like leukemia.

Asxl1 deletion disrupts MYC and RNA polymerase II function in granulocyte progenitors.

Mutations in the gene Additional Sex-Combs Like 1 (ASXL1) are recurrent in myeloid malignancies as well as the pre-malignant condition clonal hematopoiesis, where they are universally associated with poor prognosis. However, the role of ASXL1 in myeloid lineage maturation is incompletely described. To define the role of ASXL1 in myelopoiesis, we employed single cell RNA sequencing and a murine model of hematopoietic-specific Asxl1 deletion. In granulocyte progenitors, Asxl1 deletion leads to hyperactivation of MYC and a quantitative decrease in neutrophil production. This loss of granulocyte production was not accompanied by significant changes in the landscape of covalent histone modifications. However, Asxl1 deletion results in a decrease in RNAPII promoter-proximal pausing in granulocyte progenitors, indicative of a global increase in productive transcription. These results suggest that ASXL1 inhibits productive transcription in granulocyte progenitors, identifying a new role for this epigenetic regulator in myeloid development.

Nano-chemically Modified Tetracycline-3 (nCMT-3) Attenuates Acute Lung Injury via Blocking sTREM-1 Release and NLRP3 Inflammasome Activation.

BACKGROUND: Intratracheal (IT) lipopolysaccharide (LPS) causes severe acute lung injury (ALI) and systemic inflammation. CMT-3 has pleiotropic anti-inflammatory effects including matrix metalloproteinase (MMP) inhibition, attenuation of neutrophil (PMN) activation, and elastase release. CMT-3's poor water solubility limits its bioavailability when administered orally for treating ALI. We developed a nano-formulation of CMT-3 (nCMT-3) to test the hypothesis that the pleiotropic anti-inflammatory activities of IT nCMT-3 can attenuate LPS-induced ALI. METHODS: C57BL/6 mice were treated with aerosolized IT nCMT-3 or saline, then had IT LPS or saline administered 2 h later. Tissues were harvested at 24 h. The effects of LPS and nCMT-3 on ALI were assessed by lung histology, MMP level/activity (zymography), NLRP3 protein, and activated caspase-1 levels. Blood and bronchoalveolar lavage fluid (BALF) cell counts, PMN elastase, and soluble triggering receptor expressed on myelocytes-1 (sTREM-1) levels, TNF-α, IL-1ß, IL-6, IL-18, and BALF protein levels were also measured. RESULTS: LPS-induced ALI was characterized by histologic lung injury (PMN infiltration, alveolar thickening, edema, and consolidation) elevated proMMP-2, -9 levels and activity, increased NLRP-3 protein and activated caspase-1 levels in lung tissue. LPS-induced increases in plasma and BALF levels of sTREM-1, TNF-α, IL-1ß, IL-6, IL-18, PMN elastase and BALF protein levels demonstrate significant lung/systemic inflammation and capillary leak. nCMT-3 significantly ameliorated all of these LPS-induced inflammatory markers to control levels, and decreased the incidence of ALI. CONCLUSIONS: Pre-treatment with nCMT3 significantly attenuates LPS-induced lung injury/inflammation by multiple mechanisms including: MMP activation, PMN elastase, sTREM-1 release, and NLRP3 inflammasome/caspase-1 activation.

CSF3R T618I, SETBP1 G870S, SRSF2 P95H, and ASXL1 Q780* tetramutation co-contribute to myeloblast transformation in a chronic neutrophilic leukemia.

Chronic neutrophilic leukemia (CNL) is a rare but serious myeloid malignancy. In a review of reported cases for WHO-defined CNL, CSF3R mutation is found in about 90% cases and confirmed as the molecular basis of CNL. Concurrent mutations are observed in CSF3R-mutated CNL patients, including ASXL1, SETBP1, SRSF2, JAK2, CALR, TET2, NRAS, U2AF1, and CBL. Both ASXL1 and SETBP1 mutations in CNL have been associated with a poor prognosis, whereas, SRSF2 mutation was undetermined. Our patient was a 77-year-old man and had no significant past medical history and symptoms with leukocytosis. Bone marrow (BM) aspirate and biopsy revealed a markedly hypercellular marrow with prominent left-shifted granulopoiesis. Next-generation sequencing (NGS) of DNA from the BM aspirate of a panel of 28 genes, known to be pathogenic in MDS/MPN, detected mutations in CSF3R, SETBP1, and SRSF2, and a diagnosis of CNL was made. The patient did not use a JAK-STAT pathway inhibitor (ruxolitinib) but started on hydroxyurea and alpha-interferon and developed pruritus after 4 months of diagnosis and nasal hemorrhage 1 month later. Then, the patient was diagnosed with CNL with AML transformation and developed intracranial hemorrhage and died. We repeated NGS and found that three additional mutations were detected: ASXL1, PRKDC, MYOM2; variant allele frequency (VAF) of the prior mutations in CSF3R, SETBP1, and SRSF2 increased. The concurrence of CSF3RT618I, ASXL1, SETBP1, and SRSF2 mutation may be a mutationally detrimental combination and contribute to disease progression and AML transformation, as well as the nonspecific treatment of hydroxyurea and alpha-interferon, but the significance and role of PRKDC and MYOM2 mutations were not undetermined.

Pathogenic Mutations and Atypical Flow Cytometric Findings Characterize the Majority of Unclassifiable Myelodysplastic/Myeloproliferative Neoplasms.

OBJECTIVES: Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) are a group of rare and heterogeneous hematopoietic disorders that frequently present a diagnostic challenge. Here we present our institutional experience with next-generation sequencing (NGS), together with morphologic, flow cytometric, and cytogenetic evaluation, in the diagnosis of MDS/MPN, with particular emphasis on MDS/MPN unclassifiable (MPN-U). METHODS: We evaluated the morphologic, flow cytometric, cytogenetic, and molecular characteristics of all MDS/MPN cases that underwent NGS at our institution between April 2016 and February 2019. RESULTS: Thirty-seven cases of MDS/MPN were identified, including 14 cases of MDS/MPN-U. Ninety-seven percent harbored mutations and immunophenotypic aberrancies (36/37), while only 38% had cytogenetic abnormalities (12/32). The MDS/MPN-U group had the highest rate of myeloblast phenotypic abnormalities and had a high mutation rate of approximately 2.7 mutated genes per case, most commonly in JAK2, SRSF2, and ASXL1. CONCLUSIONS: No single ancillary study was abnormal in every case, but all cases had at least one abnormal finding, demonstrating the usefulness of a multiparameter approach to the diagnosis of MDS/MPN. Although a few specific mutations were found exclusively in MDS/MPN-U and JAK2 mutations were most prevalent, larger studies are needed to determine whether MDS/MPN-U has a mutational "fingerprint," which may aid in diagnosis and targeted therapy.

Synchronous detection of multiple myeloma and acute myeloid leukemia: A diagnostic and therapeutic challenge.

INTRODUCTION: Synchronous detection of multiple myeloma and acute myeloid leukemia in a single patient is a rare coincidence. Treatment of these patients is still unclear, mostly based on acute myeloid leukemia strategies combined with bortezomib. CASE REPORT: A 72-year-old male with no medical history was investigated for pancytopenia. On medical examination, he was complicated with a wide and severe skin infection on arm. On examination of bone marrow aspirate, 25% myeloblasts infiltration and additional 10% plasma cells were seen. Acute myeloid leukemia was diagnosed and plasma cell proliferation was attributed to reactive plasmacytosis due to skin infection. However, flowcytometric studies and immunohistochemical examination revealed two different cell populations with 30-40% atypical plasma cells and >20% myeloblasts. Serum M-protein detected by serum electrophoresis test and immunofixation test revealed a monoclonal IgG lambda band. He was diagnosed with concurrent acute myeloid leukemia and multiple myeloma without history of chemotherapy.Management and outcome: The patient was initially treated with bortezomib and dexamethasone for the myeloma. Subsequently, azacitidine was administered subcutaneously for the acute myeloid leukemia treatment. The tru-cut biopsy of the lesion on his arm revealed suppurative inflammatory findings and no malign cells detected. Antibiotherapy was started according to susceptibility. He expired after three months of survival. DISCUSSION: The synchronous occurrence of these two different clonal hematological malignancies is rare in hematology practice. Patient-based prospective studies and case series are needed to guide diagnosis and treatment strategies. Furthermore, this report highlights the importance of ruling out reactive plasmacytosis in patients with hematological malignancy who developed severe infections.

Exploring blast composition in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms: CD45RA and CD371 improve diagnostic value of flow cytometry through assessment of myeloblast heterogeneity and stem cell aberrancy.

BACKGROUND: Flow cytometry immunophenotyping (FCIP) can improve diagnosis of myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN), although its application is challenging due to difficulties in standardization, complexity of antibody panels and subjective interpretation of data. Since blasts are invariably affected in these disorders, we developed a FCIP approach for detailed and objective analysis of the blast population. METHODS: FCIP using a one-tube 10-color (13-marker) antibody panel was performed on bone marrow samples from 23 MDS and 8 MDS/MPN patients, 21 cytopenic patients non-diagnostic for MDS (Non-MDS), and 16 Control samples. RESULTS: MDS and MDS/MPN cases demonstrated one to several immunophenotypic abnormalities including: increased myeloblasts, decreased stage-1 hematogones, aberrant stem cells, abnormal myeloblast heterogeneity/divergence from normal, increased or decreased CD45 intensity, increased CD117 or CD123 intensity, decreased CD38 intensity, and aberrant expression of lineage markers (CD5, CD19, CD56). A Blast score was developed that showed sensitivity of 80.6% and specificity of 90.5% for immunophenotypic diagnosis of MDS and MDS/MPN. Expression levels of CD45RA and CD371 were used to evaluate abnormal myeloblast heterogeneity and stem cell aberrancy. Both these features were, for the first time, incorporated into a scoring system and resulted in 19% increase in the sensitivity of the assay for lower-risk MDS. CONCLUSION: Deep immunophenotypic analysis of the blast population is valuable for diagnosis of MDS and MDS/MPN and can potentially provide sensitivity and specificity figures comparable to those previously described using more comprehensive panels that assess maturing myelomonocytic and erythroid elements in addition to progenitor cells.

Microfluidic-Based Detection of AML-Specific Biomarkers Using the Example of Promyelocyte Leukemia.

A microfluidic assay for the detection of promyelocytic leukemia (PML)-retinoic acid receptor α (RARα) fusion protein was developed. This microfluidic-based system can be used for rapid personalized differential diagnosis of acute promyelocyte leukemia (APL) with the aim of early initiation of individualized therapy. The fusion protein PML-RARα occurs in 95% of acute promyelocytic leukemia cases and is considered as diagnostically relevant. The fusion protein is formed as a result of translocation t(15,17) and is detected in the laboratory by fluorescence in situ hybridization (FISH) or reverse transcriptase polymerase chain reaction (RT-PCR). Diagnostic methods require many laboratory steps with specialized staff. The developed microfluidic assay includes a sandwich enzyme-linked immunosorbent assay (ELISA) system for PML-RARα on surface of magnetic microparticles in a microfluidic chip. A rapid detection of PML-RARα in cell lysates is achieved in less than one hour. A biotinylated PML-antibody on the surface of magnetic streptavidin coated microparticles is used as capture antibody. The bound translocation product is detected by a RARα antibody conjugated with horseradish peroxidase and the substrate QuantaRed. The analysis is performed in microfluidic channels which involves automated liquid processing with stringent washing and short incubation times. The results of the developed assay show that cell lysates of PML-RARα-positive cells (NB-4) can be clearly distinguished from PML-RARα-negative cells (HL-60, MV4-11).

Expression of putative leukemia stem cell targets in genetically-defined acute myeloid leukemia subtypes.

Although most acute myeloid leukemia (AML) patients achieve complete remissions, the majority still eventually relapse and die of their disease. Rare primitive leukemia cells, so-called leukemia stem cells (LSCs), represent one potential type of resistant cell subpopulation responsible for this dissociation between response and cure. Several LSC targets have been described, but there is limited evidence about their relative utility or that targeting any can prevent relapse. LSCs not only appear to be biologically heterogeneous, but the classic immunocompromised mouse transplantation model also has serious shortcomings as an LSC assay. Out data suggest that the most immature cell phenotype that can be identified within a patient's leukemia may be clinically relevant and represent the de facto LSC. Moreover, although phenotypically heterogeneous, these putative LSCs show consistent phenotypes within individual genetically defined groups. Using this LSC definition, we studied several previously described putative LSC targets, CD25, CD26, CD47, CD96, CD123, and CLL-1, and all were expressed across heterogeneous LSC phenotypes. In addition, with the exception of CD47, there was at most low expression of these targets on normal hematopoietic stem cells (HSCs). CD123 and CLL-1 demonstrated the greatest expression differences between putative LSCs and normal HSCs. Importantly, CD123 monoclonal antibodies were cytotoxic in vitro to putative LSCs from all AML subtypes, while showing limited to no toxicity against normal HSCs and hematopoietic progenitors. Since minimal residual disease appears to be a more homogeneous population of cells responsible for relapse, targeting CD123 in this setting may be most effective.

ROS-producing immature neutrophils in giant cell arteritis are linked to vascular pathologies.

Giant cell arteritis (GCA) is a common form of primary systemic vasculitis in adults, with no reliable indicators of prognosis or treatment responses. We used single cell technologies to comprehensively map immune cell populations in the blood of patients with GCA and identified the CD66b+CD15+CD10lo/-CD64- band neutrophils and CD66bhiCD15+CD10lo/-CD64+/bright myelocytes/metamyelocytes to be unequivocally associated with both the clinical phenotype and response to treatment. Immature neutrophils were resistant to apoptosis, remained in the vasculature for a prolonged period of time, interacted with platelets, and extravasated into the tissue surrounding the temporal arteries of patients with GCA. We discovered that immature neutrophils generated high levels of extracellular reactive oxygen species, leading to enhanced protein oxidation and permeability of endothelial barrier in an in vitro coculture system. The same populations were also detected in other systemic vasculitides. These findings link functions of immature neutrophils to disease pathogenesis, establishing a clinical cellular signature of GCA and suggesting different therapeutic approaches in systemic vascular inflammation.

Mouse models of neutropenia reveal progenitor-stage-specific defects.

Advances in genetics and sequencing have identified a plethora of disease-associated and disease-causing genetic alterations. To determine causality between genetics and disease, accurate models for molecular dissection are required; however, the rapid expansion of transcriptional populations identified through single-cell analyses presents a major challenge for accurate comparisons between mutant and wild-type cells. Here we generate mouse models of human severe congenital neutropenia (SCN) using patient-derived mutations in the GFI1 transcription factor. To determine the effects of SCN mutations, we generated single-cell references for granulopoietic genomic states with linked epitopes1, aligned mutant cells to their wild-type equivalents and identified differentially expressed genes and epigenetic loci. We find that GFI1-target genes are altered sequentially, as cells go through successive states of differentiation. These insights facilitated the genetic rescue of granulocytic specification but not post-commitment defects in innate immune effector function, and underscore the importance of evaluating the effects of mutations and therapy within each relevant cell state.

Promyelocytic Differentiation in Infiltrates of Prurigo Pigmentosa: An Analogy to Histiocytoid Sweet Syndrome.

Prurigo pigmentosa (PP) is a rare inflammatory dermatosis of unknown etiology. Young women are affected most commonly. Clinically, heavily itchy papules erupt mainly on the trunk healing with residual reticulate pigmentation. Histopathologic descriptions of PP are somewhat controversial. First, PP was reported as lichenoid-interface dermatitis, and later, neutrophils were recognized as the characteristic feature, and the variation in histopathologic patterns was interpreted as a time-dependent phenomenon. Immunohistochemical studies on PP are rare. Biopsies of 5 patients with clinically typical PP were examined histopathologically, and infiltrates were characterized immunohistochemically: myeloperoxidase, CD11c, CD68, CD4, CD8, tryptase, and langerin. In all cases, myeloperoxidase-positive cells with band forms of nuclei and with histiocytoid cytomorphology were identified. They were seen in the epidermis (4/5) and in the dermal infiltrate (5/5). On staining with CD11c, myeloid dendritic cells could be demonstrated in the infiltrate (5/5). In conclusion, myeloid progenitor cells are part of the infiltrate in PP, and they may sometimes be more numerous than mature neutrophils, akin to the situation in histiocytoid Sweet syndrome. This supports the classification of PP as a "neutrophilic dermatosis." In biopsies of suspected PP in which mature neutrophils are sparse, the section should be searched for neutrophilic band forms and histiocytoid promyelocytic cells. Immunohistochemical staining with myeloperoxidase helps to identify such cells and may enable a diagnosis of PP even when mature neutrophils are few.