Side scatter ratio of the CD105-positive and CD105-negative red blood cell fractions is useful for the detection of low-grade myelodysplastic neoplasms by flow cytometry.

OBJECTIVES: We assessed the utility of red blood cell (RBC) CD105 and side scatter (SSC) parameters by flow cytometry for the detection of low-grade myelodysplastic neoplasms (MDS) in bone marrow specimens. METHODS: Ten RBC parameters incorporating CD105 or SSC combined with the Meyerson-Alayed scoring system (MASS) metrics were retrospectively evaluated by flow cytometry for utility in detecting low-grade MDS (n = 56) compared with cytopenic controls (n = 86). RESULTS: Myelodysplastic neoplasms were associated with 7 of the RBC parameters in univariate analysis. Multivariate analysis using cutoff values based on optimal and 95% specificity levels of the RBC metrics and the MASS parameters revealed the SSC ratio of CD105-positive and CD105-negative RBC fractions (CD105+/- SSC); the percentage and coefficient of variation of the CD105-positive fraction of RBCs (CD105%, CD105+CV) emerged as significant RBC variables. Two simple scoring schemes using these RBC values along with MASS parameters were identified: 1 using CD105+/- SSC, CD105%, and CD105+CV combined with the percentage of CD177-positive granulocytes (CD177%), myeloblast percentage (CD34%), and granulocyte SSC (GranSSC), and the other incorporating CD105+/- SSC, CD105+CV, CD177%, CD34%, GranSSC, and B-cell progenitor percentage. Both demonstrated a sensitivity of approximately 80%, with a specificity of roughly 90% for the detection of MDS compared with cytopenic controls. CONCLUSIONS: The red blood cell parameter, CD105+/- SSC, appears to be beneficial in the evaluation of low-grade MDS by flow cytometry.

CD5 B-Cell Predominant Primary Immunodeficiency: Part of the Spectrum of MAGT1 Deficiency.

Background: Selective anti-polysaccharide antibody deficiency (SPAD) with CD5 B-cell predominance and autoimmune phenomena was identified in a male cohort first reported by Antall et al in 1999. The phenotypically likewise and genotypically identical X-linked immunodeficiency with magnesium defect, Epstein-Barr Virus infection, and neoplasia (XMEN) disease was defined as a novel primary immunodeficiency (PID) in 2011. Recent studies of the magnesium transporter 1 (MAGT1) gene mutation reveal glycosylation defects contributing to more phenotypic variance than the "XMEN" title pathologies. The updated title, "X-linked MAGT1 deficiency with increased susceptibility to EBV-infection and N-linked glycosylation defect," was proposed in 2020. Objectives: To reflect the patient population more accurately, a prospective classification update may consider MAGT1 glycobiological errors contributing to phenotypic variance but also pre-genetic testing era reports with CD5 B-cell predominance. Methods: Patient 1 from Antall et al presented at 28 years of age for further immunological evaluation of his CD5/CD19 B-cell predominance diagnosed at 5 years old. Design: Immune re-evaluation done through flow cytometry and next-generation sequencing. Results: Flow cytometry B-cell phenotyping revealed persistent CD5+CD19+ (93%). Flow cytometric histogram quantified reduced activator CD16+CD56+ natural killer and CD8+ T-cell receptor, Group 2, Member D (NKG2D) glycoprotein expression. A c.923-1_934 deletion loss of function mutation was identified in the MAGT1 gene. Conclusion: We suggest the novel PID XMEN, based on its CD5 B-cell predominance, had been discovered and reported over a decade earlier as CD5+ PID based on the MAGT1 mutation found in the same. We encourage consideration of combining these labels and recent findings to offer the most accurate classification of this disease.

Flow Cytometric Findings in Clonal Cytopenia of Undetermined Significance.

OBJECTIVES: To examine flow cytometric (FCM) findings in clonal cytopenia of undetermined significance (CCUS) in relation to variant allele fraction (VAF) and mutation risk. METHODS: Nine FCM parameters, including 5 FCM metrics (Meyerson-Alayed scoring scheme [MASS] parameters) we previously used to identify myelodysplastic syndromes (MDS), were compared among 96 CCUS samples, 100 low-grade MDS samples and 100 samples from patients without somatic alterations (controls). RESULTS: FCM findings did not differ between CCUS samples with less than 20% VAF and controls. CCUS samples with more than 20% VAF (CCUS >20% VAF) demonstrated more than 1 abnormal FCM parameter at a frequency between MDS and controls. Abnormalities in CCUS with high-risk alterations (CCUS(hi)) were similar to MDS, with no statistical difference in the percentage of cases with more than 1 FCM abnormality or a positive MASS score. The positive predictive value (PPV) for clinically significant myeloid processes; MDS, CCUS(hi), and CCUS >20% VAF compared with other CCUS samples and controls was 94.8%, with 96.5% specificity and 61% sensitivity using a modified MASS score. A subset of MDS (43%) was distinguished from CCUS(hi) and CCUS >20% VAF using 3 parameters, with a 93.5% PPV and 83.3% specificity. CONCLUSIONS: FCM abnormalities can distinguish high-risk CCUS based on VAF or alteration type from low-risk CCUS and MDS in many cases. The findings are of potential utility in the evaluation of patients with cytopenias.

Decreased CD177pos neutrophils in myeloid neoplasms is associated with NPM1, RUNX1, TET2, and U2AF1 S34F mutations.

A decreased percentage of CD177pos neutrophils is frequently present in MDS and AML and is a useful flow cytometry (FCM) marker for the identification of MDS. The underlying mechanism leading to the low percentage of CD177pos neutrophils in MDS has not been explained. The aim of this study was to identify whether specific somatic mutations in myeloid neoplasms are associated with the low percentage of CD177pos neutrophils. 507 myeloid neoplasms with one or more pathogenic molecular abnormality identified by NGS and in which CD177 expression was assessed were evaluated. Correlation with CD177 expression was determined for 39 variables (including genes mutated, diagnostic groups and gender) using a 40 % cutoff level for low CD177 expression. In multivariate analysis mutations involving NPM1 (OD 0.26), RUNX1 (OD 0.39), TET2 (OD 0.58), and U2AF1 S34F (OD 0.25) were associated with low percentage of CD177pos neutrophils when all cases were evaluated. JAK2 (OD 2.5) alteration was associated with increased percentage of CD177pos neutrophils. Differences were noted between diagnostic subgroups with no single mutation associated with decreased CD177pos neutrophils in MDS and CCUS. The findings demonstrate an association between the percentage of CD177pos neutrophils and somatically acquired mutations involving several genes.

Initial assessment of α-synuclein structure in platelets.

Over the last few years data from our group have indicated that α-synuclein is important in development of immune cells as well as potentially erythrocytes and platelets. The latter is important since this protein may work as negative regulator of granule release. Thus, we sought to begin to understand the structure of this protein in platelets. Flow cytometric analysis of this protein using region-specific (N-terminus, central region and C-terminus) monoclonal antibodies was performed. Antibody to the central region gave the strongest shift among all three antibodies, with the C-terminus having intermediate shift and N-terminus minimal shift. Western blotting using the same antibodies showed similar binding of all antibodies to α-synuclein. These results suggest a similar arrangement of this protein in platelets as seen in neurons. Future studies ought to look at the role that each protein region plays in platelets.

Clinical Utility of Targeted Next-Generation Sequencing in the Evaluation of Low-Grade Lymphoproliferative Disorders.

OBJECTIVES: We investigated the usefulness of a custom-designed 31-gene next-generation sequencing (NGS) panel implemented on a routine basis for the evaluation of low-grade lymphoproliferative disorders (LPDs). METHODS: In total, 147 blood, bone marrow, and tissue specimens were sequenced, including 81% B-cell, 15% T-cell, and 3% natural killer (NK)-cell neoplasms. RESULTS: Of the cases, 92 (63%) of 147 displayed at least one pathogenic variant while 41 (28%) of 147 had two or more. Low mutation rates were noted in monoclonal B-cell lymphocytoses and samples with small T- and NK-cell clones of uncertain significance. Pathogenic molecular variants were described in specific disorders and classified according to their diagnostic, prognostic, and potential therapeutic value. Diagnostically, in addition to confirming the diagnosis of 15 of 15 lymphoplasmacytic lymphomas, 10 of 12 T large granular lymphocytic leukemias, and 2 of 2 hairy cell leukemias (HCLs), the panel helped resolve the diagnosis of 10 (62.5%) of 16 challenging cases lacking a specified diagnosis based on standard morphology, phenotype, and genetic analysis. CONCLUSIONS: Overall, implementation of this targeted lymphoid NGS panel as part of regular hematopathology practice was found to be a beneficial adjunct in the evaluation of low-grade LPDs.

Clonal cytopenia of undetermined significance (CCUS) with dysplasia is enriched for MDS-type molecular findings compared to CCUS without dysplasia.

OBJECTIVES: Although morphologic dysplasia is not typically considered a feature of CCUS, we have consistently observed low-level bone marrow (BM) dysplasia among CCUS patients. We sought to determine whether sub-diagnostic BM dysplasia in CCUS patients is associated with other clinico-pathologic findings of myelodysplastic syndrome (MDS). METHODS: We identified 49 CCUS patients, 25 with sub-diagnostic dysplasia (CCUS-D), and 24 having no dysplasia (CCUS-ND). We compared the clinical, histologic, and laboratory findings of CCUS-D and CCUS-ND patients to 49 MDS patients, including blood cell counts, BM morphology, flow cytometry, cytogenetics, and results of next-generation sequencing. RESULTS: No statistically significant differences were observed between CCUS-D and CCUS-ND patients in the degree of cytopenias, BM cellularity, myeloid-to-erythroid ratio, or the presence of flow cytometric abnormalities. However, compared to CCUS-ND, CCUS-D patients exhibited increased mutations in myeloid malignancy-associated genes, including non-TET2/DNMT3A/ASXL1 variants, spliceosome (SF3B1, SRSF2, ZRSR2, or U2AF1) variants, and IDH2/RUNX1/CBL variants. CCUS-D patients were also enriched for higher variant allele frequencies and co-mutation of TET2/DNMT3A/ASXL1 with other genes. CONCLUSIONS: CCUS-D patients exhibit a molecular (but not clinical) profile more similar to MDS patients than CCUS-ND, suggesting CCUS-D may represent a more immediate precursor to MDS and may warrant closer clinical follow-up.

CD177 Enhances the Detection of Myelodysplastic Syndrome by Flow Cytometry.

OBJECTIVES: Previously we demonstrated that a decreased percentage of CD177-positive granulocytes detected by flow cytometry (FCM) was associated with myelodysplastic syndrome (MDS). Here we expand on those findings to more rigorously evaluate the utility of CD177 for the detection of MDS. METHODS: Two hundred patient samples (100 MDS and 100 controls) were evaluated for granulocyte expression of CD177 and 11 other flow cytometric parameters known to be associated with MDS. RESULTS: We show that CD177, as a single analyte, is highly correlated with MDS with a receiver operating characteristic area under curve value of 0.8. CD177 expression below 30% demonstrated a sensitivity of 51% and a specificity of 94% for detecting MDS with a positive predictive value of 89.5%. In multivariate analysis of 12 MDS-associated FCM metrics, CD177 and the Ogata parameters were significant indicators of MDS, and CD177 increased sensitivity of the Ogata score by 16% (63%-79%) for predicting MDS. Finally, diagnostic criteria incorporating these parameters with a 1% blast cutoff level and CD177 resulted in a sensitivity of 90% and specificity of 91% for detecting MDS. CONCLUSIONS: The findings indicate CD177 is a useful FCM marker for MDS.

Flow Cytometry Identifies a Spectrum of Maturation in Myeloid Neoplasms Having Plasmacytoid Dendritic Cell Differentiation.

BACKGROUND: Neoplasms derived from plasmacytoid dendritic cells (PDCs) are currently divided into two broad categories: mature PDC proliferations associated with myeloid neoplasms (MPDMN) and blastic plasmacytoid dendritic cell neoplasm (BPDCN); only BPDCN is recognized in the WHO 2016 classification of hematopoietic neoplasms. We present seven patients with high grade myeloid neoplasms (MNs), mostly acute leukemias, having a spectrum of PDC differentiation and not fitting with MPDMN or BPDCN. METHODS: We analyzed seven MN cases having increased myeloblasts and prominent CD56-negative PDC proliferations comprising 5-26% of bone marrow or blood cellularity as measured by flow cytometry. The cases included five acute myeloid leukemia (three FAB M4 subtype, two unclassified), one mixed phenotype acute leukemia, and one case of unclassified MN. RESULTS: Six cases demonstrated immunophenotypic evidence of PDC differentiation from leukemic blasts, based on variable expression of CD34, CD45, CD123, and CD304 by the leukemic cells. Four cases had circulating PDC populations in blood. None of the cases met clinical or pathologic criteria for BPDCN. Morphologic review was available for four acute leukemia cases and demonstrated either nodular or interstitial infiltrates of PDCs. All cases had an aggressive clinical course, and three cases had FLT3 ITD mutation. CONCLUSIONS: These cases demonstrate that high grade MNs, in particular AML, can exhibit PDC differentiation, with or without monocytic differentiation, in a manner distinct from MPDMN or BPDCN. The existence of MNs with immature PDC proliferations suggests that there is a broader spectrum of PDC-associated neoplasms than currently recognized. © 2019 International Clinical Cytometry Society.

Factor XII and uPAR upregulate neutrophil functions to influence wound healing.

Coagulation factor XII (FXII) deficiency is associated with decreased neutrophil migration, but the mechanisms remain uncharacterized. Here, we examine how FXII contributes to the inflammatory response. In 2 models of sterile inflammation, FXII-deficient mice (F12-/-) had fewer neutrophils recruited than WT mice. We discovered that neutrophils produced a pool of FXII that is functionally distinct from hepatic-derived FXII and contributes to neutrophil trafficking at sites of inflammation. FXII signals in neutrophils through urokinase plasminogen activator receptor-mediated (uPAR-mediated) Akt2 phosphorylation at S474 (pAktS474). Downstream of pAkt2S474, FXII stimulation of neutrophils upregulated surface expression of αMß2 integrin, increased intracellular calcium, and promoted extracellular DNA release. The sum of these activities contributed to neutrophil cell adhesion, migration, and release of neutrophil extracellular traps in a process called NETosis. Decreased neutrophil signaling in F12-/- mice resulted in less inflammation and faster wound healing. Targeting hepatic F12 with siRNA did not affect neutrophil migration, whereas WT BM transplanted into F12-/- hosts was sufficient to correct the neutrophil migration defect in F12-/- mice and restore wound inflammation. Importantly, these activities were a zymogen FXII function and independent of FXIIa and contact activation, highlighting that FXII has a sophisticated role in vivo that has not been previously appreciated.

Compound Heterozygosity for Hb D-Ibadan (HBB: c.263C>A) and Hb C (HBB: c.19G>A).

We report an individual with a compound heterozygosity for Hb D-Ibadan (HBB: c.263C>A) and Hb C (HBB: c.19G>A), a hemoglobin (Hb) combination not previously identified. The compound hemoglobinopathy was detected in a young woman during routine prenatal screening. Variant Hbs were identified and confirmed by high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) followed by Sanger DNA sequencing. Hb D-Ibadan was present in significant excess over Hb C (70.3 to 24.4%). A complete blood count (CBC) revealed moderate microcytosis with slight anemia. The history suggests the Hb combination is clinically silent. The findings indicate the compound hemoglobinopathy demonstrates thalassemia minor-like red cell indices with an unequal distribution of the variant Hbs. Comparison with other Hb D-like heterozygous conditions is reviewed.

Gain-of-function mutations of Ptpn11 (Shp2) cause aberrant mitosis and increase susceptibility to DNA damage-induced malignancies.

Gain-of-function (GOF) mutations of protein tyrosine phosphatase nonreceptor type 11 Ptpn11 (Shp2), a protein tyrosine phosphatase implicated in multiple cell signaling pathways, are associated with childhood leukemias and solid tumors. The underlying mechanisms are not fully understood. Here, we report that Ptpn11 GOF mutations disturb mitosis and cytokinesis, causing chromosomal instability and greatly increased susceptibility to DNA damage-induced malignancies. We find that Shp2 is distributed to the kinetochore, centrosome, spindle midzone, and midbody, all of which are known to play critical roles in chromosome segregation and cytokinesis. Mouse embryonic fibroblasts with Ptpn11 GOF mutations show a compromised mitotic checkpoint. Centrosome amplification and aberrant mitosis with misaligned or lagging chromosomes are significantly increased in Ptpn11-mutated mouse and patient cells. Abnormal cytokinesis is also markedly increased in these cells. Further mechanistic analyses reveal that GOF mutant Shp2 hyperactivates the Polo-like kinase 1 (Plk1) kinase by enhancing c-Src kinase-mediated tyrosine phosphorylation of Plk1. This study provides novel insights into the tumorigenesis associated with Ptpn11 GOF mutations and cautions that DNA-damaging treatments in Noonan syndrome patients with germ-line Ptpn11 GOF mutations could increase the risk of therapy-induced malignancies.

A critical role for alpha-synuclein in development and function of T lymphocytes.

Alpha-synuclein is highly expressed in the central nervous system and plays an important role in pathogenesis of neurodegenerative disorders such as Parkinson's disease and Lewy body dementia. Previous studies have demonstrated the expression of α-synuclein in hematopoietic elements and peripheral blood mononuclear cells, although its roles in hematopoiesis and adaptive immunity are not studied. Using an α-synuclein knock out (KO) mouse model, we have recently shown that α-synuclein deficiency is associated with a mild defect in late stages of hematopoiesis. More importantly, we demonstrated a marked defect in B lymphocyte development and IgG, but not IgM production in these mice. Here we show a marked defect in development of T lymphocytes in α-synuclein KO mice demonstrated by a significant increase in the number of CD4 and CD8 double negative thymocytes and significant decreases in the number of CD4 single positive and CD8 single positive T cells. This resulted in markedly reduced peripheral T lymphocytes. Interestingly, splenic CD4(+) and CD8(+) T cells that developed in α-synuclein KO mice had a hyperactivated state with higher expression of early activation markers and increased IL-2 production. Moreover, splenic CD4(+) T cells from α-synuclein KO mice produced lower levels of IL-4 upon antigenic stimulation suggesting a defective Th2 differentiation. Our data demonstrate an important role for α-synuclein in development of T lymphocytes and regulation of their phenotype and function.

CD1c(+) myeloid dendritic cells in myeloid neoplasia.

We determined the normal level and phenotype of CD1c(+) myeloid dendritic cells (MDCs) in blood and bone marrow and evaluated the level of CD1c(+) MDCs in 295 myeloid neoplasms. CD1c(+) MDCs were increased above the mean level of non-neoplastic hospital controls in 18.0% (53/295) of myeloid malignancies, increased three standard deviations above the control mean in 14.2% (42/295) with a 10-fold or more increase compared to mean in 6.8% (20/295). Increased CD1c(+) MDCs were associated with chronic myelomonocytic leukemia (CMML) (12/24, 50%) and acute myeloid leukemia (AML) (31/140, 22%) with a strong association with AML with the inv(16) cytogenetic abnormality. The cells were not increased in chronic myelogenous leukemia (CML) and rarely increased in non-CML myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS). Immunohistochemical staining of cases with increased CD1c(+) MDCs did not reveal clustering of the cells unlike that observed with myeloid neoplasms associated with increased plasmacytoid dendritic cells. Our findings indicate CD1c(+) MDC elevations are not uncommon in myeloid leukemias and are associated with CMML and AML, particularly AML with inv(16). © 2015 International Clinical Cytometry Society.

Mycobacterium avium Complex Infection in a Patient with Sickle Cell Disease and Severe Iron Overload.

A 34-year-old female with sickle cell anemia (hemoglobin SS disease) and severe iron overload presented to our institution with the subacute presentation of recurrent pain crisis, fever of unknown origin, pancytopenia, and weight loss. A CT scan demonstrated both lung and liver nodules concerning for granulomatous disease. Subsequent biopsies of the liver and bone marrow confirmed the presence of noncaseating granulomas and blood cultures isolated Mycobacterium avium complex MAC. Disseminated MAC is considered an opportunistic infection typically diagnosed in the immunocompromised and rarely in immunocompetent patients. An appreciable number of mycobacterial infection cases have been reported in sickle cell disease patients without immune dysfunction. It has been reported that iron overload is known to increase the risk for mycobacterial infection in vitro and in vivo studies. While iron overload is primarily known to cause end organ dysfunction, the clinical relationship with sickle cell disease and disseminated MAC infection has not been reported. Clinical iron overload is a common condition diagnosed in the sub-Saharan African population. High dietary iron, genetic defects in iron trafficking, as well as hemoglobinopathy are believed to be the etiologies for iron overload in this region. Patients with iron overload in this region were 17-fold more likely to die from Mycobacterium tuberculosis. Both experimental and clinical evidence suggest a possible link to iron overload and mycobacterial infections; however larger observational studies are necessary to determine true causality.

Late stages of hematopoiesis and B cell lymphopoiesis are regulated by α-synuclein, a key player in Parkinson's disease.

α-Synuclein plays a crucial role in Parkinson's disease and dementias defined as synucleinopathies. α-Synuclein is expressed in hematopoietic and immune cells, but its functions in hematopoiesis and immune responses are unknown. We utilized α-synuclein(-/-) (KO) mice to investigate its role in hematopoiesis and B cell lymphopoiesis. We demonstrated hematologic abnormalities including mild anemia, smaller platelets, lymphopenia but relatively normal early hematopoiesis in KO mice compared to wild-type (WT) as measured in hematopoietic stem cells and progenitors of the different cell lineages. However, the absolute number of B220(+)IgM(+) B cells in bone marrow was reduced by 4-fold in KO mice (WT: 104±23×10(5) vs. KO: 27±5×10(5)). B cells were also reduced in KO spleens associated with effacement of splenic and lymph node architecture. KO mice showed reduced total serum IgG but no abnormality in serum IgM was noted. When KO mice were challenged with a T cell-dependent antigen, production of antigen specific IgG1 and IgG2b was abolished, but antigen specific IgM was not different from WT mice. Our study shows hematologic abnormalities including anemia and smaller platelets, reduced B cell lymphopoiesis and defects in IgG production in the absence of α-synuclein. This is the first report to show an important role of α-synuclein late in hematopoiesis, B cell lymphopoiesis and adaptive immune response.

CD177 expression on neutrophils: in search of a clonal assay for myeloid neoplasia by flow cytometry.

OBJECTIVES: To determine whether the fraction of CD177+ neutrophils might be altered in clonal myeloid disorders, similar to the skewed κ/λ ratio for B-cell lymphomas, and could be used to identify myeloid neoplasms. METHODS: Blood and bone marrow samples were evaluated for the fraction of CD177+ neutrophils by flow cytometry. RESULTS: Skewed high neutrophil CD177(%) was not associated with neoplasia, but skewed low neutrophil CD177(%) was highly correlated with clonal myeloid disorders at values less than 40%. Specificity of low neutrophil CD177(%) for clonal myeloid disorders was 87% with a 40% cutoff and 95% with a 30% cutoff. Findings were most pronounced for myelodysplasia, with 52% (11/21) containing fewer than 40% CD177+ neutrophils. Specificity was also suggested by normalization of neutrophil CD177(%) in four patients who reached morphologic remission after therapy for myelodysplasia or acute leukemia. CONCLUSIONS: Skewed low neutrophil CD177(%) is highly associated with clonal myeloid disorders, particularly myelodysplasia, and may be useful for detecting clonal myeloid disorders.

Follicular center helper T-cell (TFH) marker positive mycosis fungoides/Sezary syndrome.

We identified 11 patients with CD10(+) cutaneous T-cell lymphoma by flow cytometry. All cases were CD4(+) and CD8(-). Three patients had extensive lymphadenopathy, systemic symptoms and an aggressive clinical course consistent with angioimmunoblastic T-cell lymphoma or peripheral T-cell lymphoma. However, 8 of the 11 patients had a prolonged disease course with gross morphology, histology and tumor cell phenotype indistinguishable from mycosis fungoides or Sezary syndrome. Immunohistochemical studies confirmed CD10 expression in seven of the eight cases and revealed the lymphoma cells were Bcl-6(+), PD-1(+), and EBV(-). Two had significant expression of CXCL-13(+). The findings indicate that lymphoma cells from mycosis fungoides or Sezary syndrome may express follicular center helper T-cell markers CD10, Bcl-6, and PD-1 and occasionally CXCL-13. The expression of these markers in some cases of mycosis fungoides/Sezary syndrome suggests follicular center helper T-cell differentiation and may lead to confusion in distinguishing mycosis fungoides/Sezary syndrome from other follicular center helper T-cell marker positive T-cell lymphomas with cutaneous manifestations.