Too many white cells-TAM, JMML, or something else?

Leukocytosis is a common finding in pediatric patients, and the differential diagnosis can be broad, including benign reactive leukocytosis and malignant myeloproliferative disorders. Transient abnormal myelopoiesis is a myeloproliferative disorder that occurs in young infants with constitutional trisomy 21 and somatic GATA1 mutations. Most patients are observed, but outcomes span the spectrum from spontaneous resolution to life-threatening complications. Juvenile myelomonocytic leukemia is a highly aggressive myeloproliferative disorder associated with altered RAS-pathway signaling that occurs in infants and young children. Treatment typically involves hematopoietic stem cell transplantation, but certain patients can be observed. Early recognition of these and other myeloproliferative disorders is important and requires a clinician to be aware of these diagnoses and have a clear understanding of their presentations. This paper discusses the presentation and evaluation of leukocytosis when myeloproliferative disorders are part of the differential and reviews different concepts regarding treatment strategies.

Chronic Myeloid Leukaemia In A 14-Year-Old Child: An Unusual Case Report.

Chronic myeloid leukaemia is a myeloproliferative stem cell disorder characterized by dysregulated production and proliferation of myeloid cells. Hallmark of the disease is the reciprocal translocation between chromosome 9 and 22 giving rise to an abnormal chromosome known as Philadelphia chromosome. Approximately 15% of all leukaemias are chronic myeloid leukaemia with a slight male predominance. This is a case of a 14-year-old boy with no premorbid presented with 4 months history of fatigue and shortness of breath on exertion. On examination patient was vitally stable. On general physical examination there was pallor and sternal tenderness. On abdominal examination spleen was palpable with a size of about 8cm. Respiratory, cardiovascular and musculoskeletal examination was unremarkable. Complete blood picture showed leukocytosis, low haemoglobin and normal platelets. Erythrocyte sedimentation rate was 65 mm/hr. Liver function tests, Renal function tests, Serum electrolytes, Urine routine examination and c-reactive protein were normal. Chest x-ray and Electrocardiography was normal. Peripheral blood smear showed neutrophils 56% with 3% lymphocytes, 1% blasts and retic count of 0.5%. Bone marrow biopsy was suggestive of chronic myeloid leukaemia which was confirmed by FISH and Cytogenetic studies.

A homozygous stop-gain variant in ARHGAP42 is associated with childhood interstitial lung disease, systemic hypertension, and immunological findings.

ARHGAP42 encodes Rho GTPase activating protein 42 that belongs to a member of the GTPase Regulator Associated with Focal Adhesion Kinase (GRAF) family. ARHGAP42 is involved in blood pressure control by regulating vascular tone. Despite these findings, disorders of human variants in the coding part of ARHGAP42 have not been reported. Here, we describe an 8-year-old girl with childhood interstitial lung disease (chILD), systemic hypertension, and immunological findings who carries a homozygous stop-gain variant (c.469G>T, p.(Glu157Ter)) in the ARHGAP42 gene. The family history is notable for both parents with hypertension. Histopathological examination of the proband lung biopsy showed increased mural smooth muscle in small airways and alveolar septa, and concentric medial hypertrophy in pulmonary arteries. ARHGAP42 stop-gain variant in the proband leads to exon 5 skipping, and reduced ARHGAP42 levels, which was associated with enhanced RhoA and Cdc42 expression. This is the first report linking a homozygous stop-gain variant in ARHGAP42 with a chILD disorder, systemic hypertension, and immunological findings in human patient. Evidence of smooth muscle hypertrophy on lung biopsy and an increase in RhoA/ROCK signaling in patient cells suggests the potential mechanistic link between ARHGAP42 deficiency and the development of chILD disorder.

Inherited deficiency of stress granule ZNFX1 in patients with monocytosis and mycobacterial disease.

Human inborn errors of IFN-γ underlie mycobacterial disease, due to insufficient IFN-γ production by lymphoid cells, impaired myeloid cell responses to this cytokine, or both. We report four patients from two unrelated kindreds with intermittent monocytosis and mycobacterial disease, including bacillus Calmette-Guérin-osis and disseminated tuberculosis, and without any known inborn error of IFN-γ. The patients are homozygous for ZNFX1 variants (p.S959* and p.E1606Rfs*10) predicted to be loss of function (pLOF). There are no subjects homozygous for pLOF variants in public databases. ZNFX1 is a conserved and broadly expressed helicase, but its biology remains largely unknown. It is thought to act as a viral double-stranded RNA sensor in mice, but these patients do not suffer from severe viral illnesses. We analyze its subcellular localization upon overexpression in A549 and HeLa cell lines and upon stimulation of THP1 and fibroblastic cell lines. We find that this cytoplasmic protein can be recruited to or even induce stress granules. The endogenous ZNFX1 protein in cell lines of the patient homozygous for the p.E1606Rfs*10 variant is truncated, whereas ZNFX1 expression is abolished in cell lines from the patients with the p.S959* variant. Lymphocyte subsets are present at normal frequencies in these patients and produce IFN-γ normally. The hematopoietic and nonhematopoietic cells of the patients tested respond normally to IFN-γ. Our results indicate that human ZNFX1 is associated with stress granules and essential for both monocyte homeostasis and protective immunity to mycobacteria.

Abnormal DNA methylation patterns in patients with infection­caused leukocytopenia based on methylation microarrays.

The present study aimed to investigate the association between gene methylation and leukocytopenia from the perspective of gene regulation. A total of 30 patients confirmed as having post­infection leukocytopenia at People's Hospital of Xinjiang Uygur Autonomous Region between January 2016 and June 2017 were successively recruited as the leukocytopenia group; 30 patients with post­infection leukocytosis were enrolled as the leukocytosis group. In addition, 30 healthy volunteers who received a health examination at the hospital during the same period were included as the normal control group. In each group, four individuals were randomly selected for whole genome methylation screening. After selection of key methylation sites, the remaining samples in each group were used for verification using matrix­assisted laser desorption/ionization­time of flight mass spectrometry. The levels of serum complement factors C3 and C5 in the leukocytopenia group were significantly lower than those in the other two groups (P<0.05). According to whole­genome DNA methylation detection, 66 and 27 methylation loci may be associated with leukocytopenia and leukocytosis, respectively. Most of these abnormal loci are located on chromosomes 2, 6, 7, 1, 17 and 11. The rates of WW domain containing E3 ubiquitin protein ligase 2 gene methylation at cytosine­phosphate­guanine (CpG)_1, CpG_5/6 and CpG_7 in the leukocytopenia group were higher than in the other two groups (P<0.05); the rate of AKT2 CpG_1 methylation was higher in the leukocytopenia group than in the other two groups (P<0.05); the rate of calcium­binding atopy­related autoantigen 1 gene CpG_2 methylation was higher in the leukocytosis group than in the normal control group (P<0.05); and the rate of NADPH oxidase 5 gene CpG_3 methylation was higher in the leukocytosis group than in the normal control group (P<0.05). Chemotactic factor secretion and cell migration abnormalities, ubiquitination modification disorders and reduced oxidative burst may participate in infection­complicated leukocytopenia. The results of this study shed new light on the molecular biological mechanisms of infection­complicated leukocytopenia and provide novel avenues for diagnosis and treatment.

Clinical and Genetic Spectrum of a Large Cohort of Patients With Leukocyte Adhesion Deficiency Type 1 and 3: A Multicentric Study From India.

Leukocyte adhesion deficiency (LAD) syndrome is a group of inborn errors of immunity characterized by a defect in the cascade of the activation and adhesion leading to the failure of leukocyte to migrate to the site of tissue injury. Three different types of LAD have been described. The most common subtype is LAD type 1 (LAD1) caused due to defects in the ITGß2 gene. LAD type 2 (LAD2) is caused by mutations in the SLC35C1 gene leading to a generalized loss of expression of fucosylated glycans on the cell surface and LAD type 3 (LAD3) is caused by mutations in the FERMT3 gene resulting in platelet function defects along with immunodeficiency. There is a paucity of data available from India on LAD syndromes. The present study is a retrospective analysis of patients with LAD collated from 28 different centers across India. For LAD1, the diagnosis was based on clinical features and flow cytometric expression of CD18 on peripheral blood leukocytes and molecular confirmation by Sanger sequencing. For patients with LAD3 diagnosis was largely based on clinical manifestations and identification of the pathogenic mutation in the FERMT3 gene by next-generation Sequencing. Of the total 132 cases diagnosed with LAD, 127 were LAD1 and 5 were LAD3. The majority of our patients (83%) had CD18 expression less than 2% on neutrophils (LAD1°) and presented within the first three months of life with omphalitis, skin and soft tissue infections, delayed umbilical cord detachment, otitis media, and sepsis. The patients with CD18 expression of more than 30% (LAD1+) presented later in life with skin ulcers being the commonest manifestation. Bleeding manifestations were common in patients with LAD3. Persistent neutrophilic leukocytosis was the characteristic finding in all patients. 35 novel mutations were detected in the ITGß2 gene, and 4 novel mutations were detected in the FERMT3 gene. The study thus presents one of the largest cohorts of patients from India with LAD, focusing on clinical features, immunological characteristics, and molecular spectrum.

Myelodysplastic/myeloproliferative neoplasm with ring sideroblasts and thrombocytosis (MDS/MPN with RS-T) complicated by hyperleukocytosis and gene analysis in relation to leukocytosis.

Myelodysplastic/myeloproliferative neoplasm (MDS/MPN) with ring sideroblasts and thrombocytosis (MDS/MPN with RS-T), which exhibits both an increased number of marrow ring sideroblasts and thrombocytosis, is a rare disorder classified as one of the newly established forms of MDS/MPN in the WHO 2016 classification. A 77-year-old female with marked thrombocytosis of 1,024×109/L was tentatively diagnosed with essential thrombocythemia in 2011, and the thrombocytosis was controlled using hydroxycarbamide and low-dose busulfan. In 2016, the leukocyte count increased to a peak value of 68.8×109/L (86.6% mature neutrophils) during platelet-reduction therapy. Bone marrow aspirate exhibited hypercellularity with ring sideroblasts comprising 41.5% erythroblasts without excess myeloblasts. Cytogenetic examination demonstrated the JAK2 V617F mutation and chromosomal abnormality of 46,XX,del(20)(q1?). Furthermore, dysplastic features of erythroid and granuloid precursors, as well as many large atypical megakaryocytes, were observed. Further genetic examinations revealed the SF3B1 K700E mutation, but not amplification of the JAK2 gene or pathogenic mutations in the 13 other genes examined. A diagnosis of MDS/MPN with RS-T was established and hyperleukocytosis was controlled using a higher dose of hydroxycarbamide. Although the patient maintained a stable disease state, she became RBC transfusion-dependent. Hyperleukocytosis, regardless of chemotherapy, is rare and may be novel in this disorder.

GATA2 hypomorphism induces chronic myelomonocytic leukemia in mice.

The transcription factor GATA2 regulates normal hematopoiesis, particularly in- stem cell maintenance and myeloid differentiation. Various heteroallelic GATA2 gene mutations are associated with a variety of hematological neoplasms, including myelodysplastic syndromes and leukemias. Here, we report that impaired GATA2 expression induces myelodysplastic and myeloproliferative neoplasm development in elderly animals, and this neoplasm resembles chronic myelomonocytic leukemia in humans. GATA2 hypomorphic mutant (G2f GN / fGN ) mice that were generated by the germline insertion of a neocassette into the Gata2 gene locus avoided the early embryonic lethality observed in Gata2-null mice. However, adult G2f GN / fGN mice suffered from exacerbated leukocytosis concomitant with progressive anemia and thrombocytopenia and eventually developed massive granulomonocytosis accompanied by trilineage dysplasia. The reconstitution activity of G2f GN / fGN mouse stem cells was impaired. Furthermore, G2f GN / fGN progenitors showed myeloid lineage-biased proliferation and differentiation. Myeloid progenitor accumulation started at a younger age in G2f GN / fGN mice and appeared to worsen with age. G2f GN / fGN mice showed increased expression of transcripts encoding cytokine receptors, such as macrophage colony-stimulating factor receptor and interleukin-6 receptor, in granulocyte-monocyte progenitors. This increased expression could be correlated with the hypersensitive granulomonocytic proliferation reaction when the mice were exposed to lipopolysaccharide. Taken together, these observations indicate that GATA2 hypomorphism leads to a hyperreactive defense response to infections, and this reaction is attributed to a unique intrinsic cell defect in the regulation of myeloid expansion that increases the risk of hematological neoplasm transformation.

The Highly Metastatic Nature of Uterine Cervical/Endometrial Cancer Displaying Tumor-Related Leukocytosis: Clinical and Preclinical Investigations.

Purpose: The aim of this study was to investigate the metastatic potential of uterine cervical and endometrial cancer displaying tumor-related leukocytosis (TRL).Experimental Design: Clinical data on uterine cervical (N = 732) and endometrial cancer (N = 900) were collected, and the metastatic potential of TRL-positive cancer was evaluated in univariate and multivariate analyses. Tumor and blood samples obtained from patients with cervical cancer, cervical cancer cell lines, and a mouse model of cervical cancer were used to examine the mechanisms underlying the highly metastatic nature of TRL-positive cancer, focusing on tumor-derived G-CSF and the myeloid-derived suppressor cell (MDSC)-mediated premetastatic niche.Results: Pretreatment TRL was significantly associated with visceral organ metastasis in patients with uterine cervical or endometrial cancer. The patients with TRL-positive cervical cancer displayed upregulated tumor G-CSF expression, elevated G-CSF levels, and increased MDSC frequencies in the peripheral blood compared with the TRL-negative patients. In vitro and in vivo investigations revealed that MDSCs produced in response to tumor-derived G-CSF are involved in premetastatic niche formation, which promotes visceral organ metastasis of TRL-positive cancer. The depletion of MDSCs attenuated this premetastatic niche formation and effectively inhibited the visceral organ metastasis of TRL-positive cancer.Conclusions: Uterine cervical/endometrial cancer displaying TRL is a distinct clinical entity with high metastatic potential. Tumor-derived G-CSF and the MDSC-mediated premetastatic niche are responsible for the highly metastatic nature of this type of cancer. MDSC-targeting therapy might represent a potential strategy for combating metastasis derived from TRL-positive uterine cancer. Clin Cancer Res; 24(16); 4018-29. ©2018 AACR.

Hyperleukocytosis is associated with distinct genetic alterations and is an independent poor-risk factor in de novo acute myeloid leukemia patients.

OBJECTIVES: Acute myeloid leukemia (AML) with hyperleukocytosis (HL) is intuitively thought as a unique group with dismal prognosis. However, comprehensive studies regarding the genetic landscape and clinical outcome in this group of patients are limited. METHODS: A total of 693 newly diagnosed de novo non-M3 AML patients were consecutively enrolled. We compared relevant mutations in 20 genes between AML patients with or without HL and exposed their prognostic implications. RESULTS: Hyperleukocytosis, defined as initial white blood cell counts above 50 000/µL, occurred in 28.9% of AML patients. HL patients had higher incidences of FLT3-ITD, NPM1, DNMT3A, CEBPA, and TET2 mutations. Multivariate analysis demonstrated that HL was an independent poor prognostic factor for overall survival and disease-free survival in total patients, those with intermediate-risk cytogenetics and normal karyotype irrespective of genetic alterations. Intriguingly, HL predicted poor survival in CEBPA double mutated, NPM1 + /FLT3-ITD- and NPM1-/FLT3-ITD- patients. Further, HL patients who received allogeneic hematopoietic stem cell transplantation (allo-HSCT) in first complete remission (CR) had a significantly longer overall survival and disease-free survival than those without allo-HSCT. CONCLUSIONS: Hyperleukocytosis is an independent poor prognostic factor irrespective of cytogenetics and mutation status. Allo-HSCT in first CR seems to ameliorate the poor prognostic impact of HL.

Dexamethasone in hyperleukocytic acute myeloid leukemia.

Patients with acute myeloid leukemia and a high white blood cell count are at increased risk of early death and relapse. Because mediators of inflammation contribute to leukostasis and chemoresistance, dexamethasone added to chemotherapy could improve outcomes. This retrospective study evaluated the impact of adding or not adding dexamethasone to chemotherapy in a cohort of 160 patients with at least 50×109 white blood cells. In silico studies, primary samples, leukemic cell lines, and xenograft mouse models were used to explore the antileukemic activity of dexamethasone. There was no difference with respect to induction death rate, response, and infections between the 60 patients in the dexamethasone group and the 100 patients in the no dexamethasone group. Multivariate analysis showed that dexamethasone was significantly associated with improved relapse incidence (adjusted sub-HR: 0.30; 95% CI: 0.14-0.62; P=0.001), disease-free survival (adjusted HR: 0.50; 95% CI: 0.29-0.84; P=0.010), event-free survival (adjusted HR: 0.35; 95% CI: 0.21-0.58; P<0.001), and overall survival (adjusted HR: 0.41; 95% CI: 0.22-0.79; P=0.007). In a co-culture system, dexamethasone reduced the frequency of leukemic long-term culture initiating cells by 38% and enhanced the cytotoxicity of doxorubicin and cytarabine. In a patient-derived xenograft model treated with cytarabine, chemoresistant cells were enriched in genes of the inflammatory response modulated by dexamethasone. Dexamethasone also demonstrated antileukemic activity in NPM1-mutated samples. Dexamethasone may improve the outcome of acute myeloid leukemia patients receiving intensive chemotherapy. This effect could be due to the modulation of inflammatory chemoresistance pathways and to a specific activity in acute myeloid leukemia with NPM1 mutation.

ASXL1 mutations in AML are associated with specific clinical and cytogenetic characteristics.

Mutations of ASXL1 are early events in acute myeloid leukemia (AML) leukemogenesis and have been associated with unfavorable prognosis. In this study, we investigated the type and frequency of ASXL1 mutations in a large cohort of patients with de novo or secondary AML (s-AML) and looked for correlations with cytogenetic findings and disease features. ASXL1 mutations were associated with older age, s-AML and higher peripheral leukocytosis. We observed more frequent co-occurrence of ASXL1 mutations with trisomy 8 and chromosome 11 aberrations but a negative correlation with myelodysplastic syndromes (MDS)-related cytogenetic abnormalities, especially -5/del(5q) and -7/del(7q). ASXL1 mutations were also found in other genetically defined AML subgroups such as those with t(9;22), inv(3)/t(3;3), t(8;21) or t(15;17); however, none of our inv(16) cases carried ASXL1 mutations. We detected two previously unreported ASXL1 mutations, p.IIe593Val and p.Cys688Tyr. Our findings suggest that ASXL1 mutations tend to cluster with specific clinical and cytogenetic profiles of AML patients.

Deletion of MCP-1 Impedes Pathogenesis of Acid Ceramidase Deficiency.

Farber Disease (FD) is an ultra-rare Lysosomal Storage Disorder caused by deficient acid ceramidase (ACDase) activity. Patients with ACDase deficiency manifest a spectrum of symptoms including formation of nodules, painful joints, and a hoarse voice. Classic FD patients will develop histiocytes in organs and die in childhood. Monocyte chemotactic protein (MCP-1; CCL2) is significantly elevated in both FD patients and a mouse model we previously generated. Here, to further study MCP-1 in FD, we created an ACDase;MCP-1 double mutant mouse. We show that deletion of MCP-1 reduced leukocytosis, delayed weight loss, and improved lifespan. Reduced inflammation and fibrosis were observed in livers from double mutant animals. Bronchial alveolar lavage fluid analyses revealed a reduction in cellular infiltrates and protein accumulation. Furthermore, reduced sphingolipid accumulation was observed in the lung and liver but not in the brain. The neurological and hematopoietic defects observed in FD mice were maintained. A compensatory cytokine response was found in the double mutants, however, that may contribute to continued signs of inflammation and injury. Taken together, targeting a reduction of MCP-1 opens the door to a better understanding of the mechanistic consequences of ceramide accumulation and may even delay the progression of FD in some organ systems.