Defective monocyte plasticity and altered cAMP pathway characterize USB1-mutated poikiloderma with neutropenia Clericuzio type.

Poikiloderma with neutropenia (PN) Clericuzio type (OMIM #604173) is a rare disease with areas of skin hyper- and hypopigmentation caused by biallelic USB1 variants. The current study was spurred by poor healing of a perianal tear wound in one affected child homozygous for c.266-1G>A (p.E90Sfster8) mutation, from a family reported previously. Treatment with G-CSF/CSF3 or GM-CSF/CSF2 transiently increased neutrophil/monocytes count with no effect on wound healing. Analysis of peripheral blood revealed a lack of non-classical (CD14+/- CD16+ ) monocytes, associated with a systemic inflammatory cytokine profile, in the two affected brothers. Importantly, despite normal expression of cognate receptors, monocytes from PN patients did not respond to M-CSF or IL-34 in vitro, as determined by cytokine secretion or CD16 expression. RNAseq of monocytes showed 293 differentially expressed genes, including significant downregulation of GATA2, AKAP6 and PDE4DIP that are associated with leucocyte differentiation and cyclic adenosine monophosphate (cAMP) signalling. Notably, the plasma cAMP was significantly low in the PN patients. Our study revealed a novel association of PN with a lack of non-classical monocyte population. The defects in monocyte plasticity may contribute to disease manifestations in PN and a defective cAMP signalling may be the primary effect of the splicing errors caused by USB1 mutation.

T-large granular lymphocyte frequencies and correlates in disease states detected by multiparameter flow cytometry in pediatric and young adult population.

T-large granular lymphocytes (T-LGL) characterized by dim CD5 staining, although not completely understood, have unique roles in the immune system. Expansion of peripheral blood (PB) clonal T-LGL populations is associated with various entities in adults. We have previously demonstrated clonal T-LGL proliferations in pediatric immune dysregulation/inflammatory/proliferative conditions. However, T-LGL populations have not been studied in broader spectrum pathologies. In this study we evaluated sizes and correlates of T-LGL populations in the pediatric and young adult populations with various disease states. Lymphocytes including T-LGL were investigated retrospectively by reviewing PB multiparameter flow cytometric data with various indications over a 4-year period. Associations with clinical, laboratory findings, and T-LGL population sizes were sought. Among 520 cases reviewed, 240 were females and 280 males with a mean age of 9 years (0-33 years); mean T-LGL population constituted 14% (1-67%) in PB T cells. There were significant differences between T-LGL and CD5-bright, regular T cells. T-LGL correlated with CD8 + /DR + (R = 0.570; P < 0.01) and CD8 + /CD11b + (R = 0.597; P < 0.01) expression, indicating activated cytotoxic phenotype. The highest average T-LGL were seen in bone marrow transplant recipients (23.7%), Evans syndrome (23.7%), lymphoma (20.6%), and acute EBV infection (20.4%) cases, all with underlying immune dysregulation pathologies. In pediatric and young adult patients with different clinical conditions, PB T-LGL constitute an average of 14% of the T cells and have a predominantly activated cytotoxic T cell phenotype. Higher relative presence was seen in cases with an immune dysregulation background. These results may serve as a reference for T-LGL research efforts.

Aberrant myelomonocytic CD56 expression in Down syndrome is frequent and not associated with leukemogenesis.

Children with Down syndrome (DS) are at an increased risk of developing transient abnormal myelopoiesis (TAM) and acute leukemia. Aberrant expression of CD56 has been observed on myeloid leukemic blasts in DS patients. In general, CD56 expression in acute myeloid leukemia (AML) is considered a promoter of leukemogenesis. We did a retrospective flow cytometric study to investigate mature myelomonocytic cell CD56 expression patterns in TAM, non-TAM, and leukemia cases with DS. Flow cytometric analysis showed that granulocyte and monocyte aberrant/dysplastic CD56 expression is an inherent characteristic of most DS patients irrespective of the presence of TAM or leukemia. Increased CD56 expression in monocyte and granulocyte populations in DS could be multifactorial; greater expression of RUNX1 secondary to the gene dose effect of trisomy 21 along with the maturational state of the cells are the potential contributors. Unlike AML seen in non-DS patients, CD56 overexpression in DS AML cases does not appear to play a role in leukemogenesis.

Distinctive phenotypes in two children with novel germline RUNX1 mutations - one with myeloid malignancy and increased fetal hemoglobin.

RUNX1 associated familial platelet disorder (FPD) is a rare autosomal dominant hematologic disorder characterized by thrombocytopenia and/or altered platelet function. There is an increased propensity to develop myeloid malignancy (MM) - acute myeloid leukemia, myeloproliferative neoplasms or myelodysplastic syndrome often in association with secondary somatic variants in other genes. To date, 23 FPD-MM pediatric cases have been reported worldwide. Here, we present two new kindreds with novel RUNX1 pathogenic variants in which children are probands. The first family is a daughter/mother diad, sharing a heterozygous frameshift variant in RUNX1 gene (c.501delT p.Ser167Argfs*9). The daughter, age 13 years, presented with features resembling juvenile myelomonocytic leukemia - severe anemia, thrombocytopenia, high white cell count with blast cells, monocytosis, increased nucleated red cells and had somatic mutations with high allele burden in CUX1, PHF6, and SH2B3 genes. She also had increased fetal hemoglobin and increased LIN28B expression. The mother, who had a long history of hypoplastic anemia, had different somatic mutations- a non-coding mutation in CUX1 but none in PHF6 or SH2B3. Her fetal hemoglobin and LIN28B expression were normal. In the second kindred, the proband, now 4 years old with thrombocytopenia alone, was investigated at 3 months of age for persistent neonatal thrombocytopenia with large platelets. Molecular testing identified a heterozygous intragenic deletion in RUNX1 encompassing exon 5. His father is known to have increased bruising for several years but is unavailable for testing. These two cases illustrate the significance of secondary mutations in the development and progression of RUNX1-FPD to MM.

CD14/16 monocyte profiling in juvenile myelomonocytic leukemia.

Monocyte subset analysis by flow cytometry has been shown to be a useful diagnostic tool in chronic myelomonocytic leukemia in adults. An increase in the classical monocyte fraction (CD14++/CD16-) greater than 94.0% of total monocytes is considered highly sensitive and specific in distinguishing chronic myelomonocytic leukemia from other myeloproliferative disorders. In a pilot study of juvenile myelomonocytic leukemia cases, we noted that CD14++/CD16- monocyte fraction was >95% in de novo juvenile myelomonocytic leukemia (JMML) with somatic PTPN11 mutations but normal in those with monosomy 7 or Noonan syndrome. Monocyte subgroup profiling by itself is not diagnostic of JMML but may distinguish molecular subgroups within JMML.

Clonal T-cell large granular lymphocyte proliferations in childhood and young adult immune dysregulation conditions.

BACKGROUND: Proliferation of large granular lymphocytes (LGL) and T-cell LGL (T-LGL) in peripheral blood along with demonstration of clonality are the hallmarks of a heterogeneous group of disorders, including T-LGL leukemia or T-LGL lymphocytosis. They are often associated with neutropenia and responsive to immunosuppression. The true nature of this entity is not well understood. Some cases are reported as reactive phenomena with very limited experience in pediatric population. METHODS: Hematology/Oncology Flow Cytometry Laboratory database has been reviewed retrospectively. Patients with identifiable distinct CD5-dim T-cell population and positive clonal T-cell receptor rearrangement were included in the analysis. Clinical and laboratory data were then reviewed. RESULTS: Sixteen cases of children and young adults with increased peripheral blood clonal T-LGL population characterized by dim CD5 expression with wide range of underlying immune dysregulation/stimulation disorders were reviewed. Extended follow up with repeat testing suggested the reactive nature of persistent clonal T-LGL proliferations in this group. CONCLUSIONS: Our observations indicate that clonal T-LGL proliferations in children and young adults are reactive in nature and some can be persistent with an indolent course with unknown consequentiality. Clonal T-LGL cells could be targeting the most prominent immunogenic stressor(s) involved as a control mechanism.

Mild erythrocytosis as a presenting manifestation of PIEZO1 associated erythrocyte volume disorders.

Piezo1, encoded by the gene PIEZO1, is an erythrocytic cellular membrane mechanoactivated cation channel. Mutations have been implicated in erythrocyte volume disorders (EVDs)-especially hereditary xerocytosis (HX)/dehydrated stomatocytosis (DHS). We identified three patients, all with novel PIEZO1 mutations, but only one displaying the HX/DHS phenotype. Retrospective review of three cases. Osmotic gradient red cell deformability (Osmoscan) was assessed via the Technicon Ektacytometer. Red cell band 3 content was estimated using Eosin-5'-Maleimide staining. Patient 1 was evaluated for polycythemia. Osmoscans suggested mild spherocytosis; a novel PIEZO1 mutation (p.Thr1589Ile, exon 35) was identified, causing mild erythrocytosis without hemolytic anemia. Patient 2 was evaluated for macrocytosis/reticulocytosis, normal-to-high hemoglobin, and indirect hyperbilirubinemia. Osmoscans suggested increased cellular hydration; a second novel PIEZO1 mutation (p.Arg1728Cys, exon 37) was identified, resulting in overhydrated stomatocytosis with well-compensated hemolysis. Patient 3 was evaluated for indirect hyperbilirubinemia only. Osmoscans suggested dehydrated stomatocytosis (DHS, xerocytosis); a third novel PIEZO1 mutation (p.Arg2279Cys, exon 47) was identified. All three patients' blood smears demonstrated stomatocytes and spherocytes. EVDs may be underdiagnosed due to the lack of "expected" anemia in a hemolytic disorder; two of three patients had high hemoglobin and red cell counts and one had high normal values for both parameters and the presence of stomatocytes/dehydrated cells lead to identification of causative PIEZO1 mutations. PIEZO1-associated EVDs may be more common than previously suspected and should be included in the diagnostic algorithms for mild erythrocytosis/unexplained jaundice.

KLF1 E325K-associated Congenital Dyserythropoietic Anemia Type IV: Insights Into the Variable Clinical Severity.

We identified a child with KLF1-E325K congenital dyserythropoietic anemia type IV who experienced a severe clinical course, fetal anemia, hydrops fetalis, and postnatal transfusion dependence only partially responsive to splenectomy. The child also had complete sex reversal, the cause which remains undetermined. To gain insights into our patient's severe hematologic phenotype, detailed analyses were performed. Erythrocytes from the patient and parents demonstrated functional abnormalities of the erythrocyte membrane, attributed to variants in the α-spectrin gene. Hypomorphic alleles in SEC23B and YARS2 were also identified. We hypothesize that coinheritance of variants in relevant erythrocyte genes contribute to the clinical course in our patient and other E325K-linked congenital dyserythropoietic anemia IV patients with severe clinical phenotypes.

Evidence for Increased Response to Induced Endoplasmic Reticulum Stress in Myeloid Cells in Acquired Aplastic Anemia.

Autoimmune response targeting the hematopoietic stem cells highlights the current understanding of acquired aplastic anemia (AAA) pathogenesis. Upregulation of the unfolded protein response is the cell's rejoinder to a variety of stresses, which either result in restoring homeostasis or cell death by increased expression of the transcription factor C/EBP homologous protein. We hypothesized that there is an inherent increased sensitivity to various cellular stressors, including the ones that target endoplasmic reticulum (ER) in AAA leading to a decreased proliferation and potentially contributing to susceptibility to autologous cytotoxicity. Using archived bone marrow aspirate samples, we demonstrate that the culture-expanded AAA myeloid cells have an increased response to ER stress induced by tunicamycin leading to decreased cell proliferation. Within the AAA myeloid samples, we show that the disease status, active versus response to therapy at the time of sampling does not alter the ER stress response. This is the first report, which provides evidence for an inherent defective stress control in the myeloid cells as a possible mechanism of evolution of the disease process in AAA.

Flow Cytometry as the Tool to Define Peripheral Blood Leukocyte Signatures in Acute EBV Infection.

Primary Epstein-Barr virus (EBV) infection which can manifest as infectious mononucleosis (IM) is commonly acquired during childhood. EBV primarily invades B cells leading to a lytic reaction; the control of the infection is handled by natural killer and T cells in immunocompetent individuals. The infection has a wide spectrum of clinical findings and can lead to serious complications in patients with certain underlying immunological dysfunctions. We retrospectively investigated peripheral white blood cell populations' surface marker characteristics in IM using a comprehensive flow cytometry marker panel. Twenty-one cases of IM and seventeen EBV-seropositive cases without IM serving as controls were included. We observed novel alterations in lymphocyte, neutrophil, and monocyte populations. In addition to increased activated cytotoxic T cells and low B cells, we demonstrated high T-large granular lymphocyte (T-LGL) populations in IM cases. Furthermore, despite T cells' increased HLA-DR expression, another activation marker, CD11b, was lower in T-LGL populations. Monocytes showed increased CD16 expression; CD64 was higher in neutrophils. Our findings point to monocyte and neutrophil activation which may account for acute clinical features and may contribute to the understanding of IM immunobiology. Furthermore, they may serve as a useful tool in investigating inherited and post-transplant conditions characterized by deficiencies in controlling EBV infection.

Identification of Paroxysmal Nocturnal Hemoglobinuria-Type Bone Marrow Mast Cells.

Hematopoietic stem cells (HSCs) give rise to mast cells (MCs), and a relative increase in bone marrow (BM) MC is common in various BM failure (BMF) conditions. Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal HSC disorder, frequently associated with BMF, characterized by decreased expression of glycosylphosphatidylinositol membrane anchor for complement regulatory proteins. Eculizumab, a monoclonal antibody that blocks complement factor 5, successfully controls PNH symptoms. In this study, we, for the first time, demonstrated PNH-type culture-grown BM MC (c-BMMC) using flow cytometry in two BMF patients and monitored population size during eculizumab therapy. Further research may unravel the properties of PNH-type c-BMMC.

Lymphocyte HLA-DR/CD-38 co-expression correlates with Hodgkin lymphoma cell cytotoxicity in vitro independent of PD-1/PD1-L pathway.

The interactions between Hodgkin and Reed Sternberg cells and tumor microenvironment, the changes that occur with therapy and, in particular, checkpoint inhibition are not fully understood. Understanding these is key to optimizing outcomes for patients with Hodgkin lymphoma (HL). We evaluated the immunophenotypic characteristics of cytotoxic, helper T and NK lymphocytes upon in vitro stimulation, cell-mediated cytotoxicity against HL cells, HDLM-2 and KM-H2, and the association with effector cell activation state, as well as changes in cytotoxicity following PD-1 or PDL-1 blockade. Higher HLA-DR/CD38 expression on effector cells was associated with increased cytotoxicity against HL cells. All effector cell types were cytotoxic of HL cells, though achieved maximum activation and cytotoxicity at variable timepoints. HLA-DR/CD38 co-expression correlated with cytotoxicity, but PD-1 expression did not. There was no significant change in cell-mediated cytotoxicity following PD-1/PDL-1 blockade. The mechanism of action of checkpoint inhibitors may not be limited to direct PD-1/PDL-1 blockade.

Differing reflections of paediatric classical Hodgkin's lymphoma on local and distant immunological microenvironments: a flow cytometric study.

AIMS: To compare immunological microenvironments in local and distant lymphoid tissues in Hodgkin's lymphoma (HL) in children. METHODS: We have analysed diagnostic bone marrow (BM) samples in 22 and corresponding involved lymph node (LN) in eight and peripheral blood (PB) in eight cases of HL by flow cytometry and sought correlations with clinical features retrospectively. RESULTS: While there were significant differences in lymphocyte compositions of BM and LN tissues, the distribution of lymphocyte subsets mimicked each other in BM and PB. CD8-positive cytotoxic T cells predominate the bone marrow in contrast to CD4-positive helper T cells in LN tissue with corresponding CD4/CD8 ratios (0.85 and 5.3, respectively; p=0.002). Additionally, T-large granular lymphocytes population was much higher in BM in comparison to LN tissue (10.5% vs 4.5%; p=0.036). CONCLUSIONS: Local immunological microenvironment appears to be highly influenced by HL tumour cells and distant site lymphocyte composition reflects immune response to control the neoplastic process.

Clinical Diagnosis of Red Cell Membrane Disorders: Comparison of Osmotic Gradient Ektacytometry and Eosin Maleimide (EMA) Fluorescence Test for Red Cell Band 3 (AE1, SLC4A1) Content for Clinical Diagnosis.

The measurement of band 3 (AE1, SLC4A1, CD233) content of red cells by eosin-5- maleimide (EMA) staining is swiftly replacing conventional osmotic fragility (OF) test as a tool for laboratory confirmation of hereditary spherocytosis across the globe. Our group has systematically evaluated the EMA test as a method to screen for a variety of anemias in the last 10 years, and compared these results to those obtained with the osmotic gradient ektacytometry (osmoscans) which we have used over three decades. Our overall experience allowed us to characterize the distinctive patterns with the two tests in several congenital erythrocyte membrane disorders, such as hereditary spherocytosis (HS), hereditary elliptocytosis (HE), Southeast Asian Ovalocytosis (SAO), hereditary pyropoikilocytosis (HPP) variants, erythrocyte volume disorders, various red cell enzymopathies, and hemoglobinopathies. A crucial difference between the two methodologies is that osmoscans measure red blood cell deformability of the entire sample of RBCs, while the EMA test examines the band 3 content of individual RBCs. EMA content is influenced by cell size as smaller red cells have lower amount of total membrane than larger cells. The SAO mutation alters the EMA binding site resulting in a lower EMA MCF even as the band 3 content itself is unchanged. Thus, EMA scan results should be interpreted with caution and both the histograms and dot plots should be analyzed in the context of the clinical picture and morphology.