Skin Rash as the First Manifestation of Pediatric Chronic Neutrophilic Leukemia / 罕见病研究

A 6-year-old girl presented with recurrent skin rash at the initial stage, recent joint pain, and neutrophilia was found during a routine blood test. After a multidisciplinary case discussion, she was diagnosed with chronic neutrophil leukemia, and the symptoms were relieved after hydroxyurea and luxolitinib treatment. She received the allogeneic hematopoietic stem cell transplantation subsequently. At present, she is in stable condition and under follow-up. Chronic neutrophil leukemia is a rare disease, which rarely occurs in children. It is more difficult to diagnose in patients with skin rash as the first manifestation. The diagnosis and treatment of this case reflects the important role of multidisciplinary cooperation in the diagnosis and treatment of difficult and rare diseases.

Neutrophilic Leukemoid Reaction Associated with Malignancy Initially Suspected as Chronic Neutrophilic Leukemia

Although neutrophilia can manifest from various causes, it is important to be able to distinguish chronic neutrophilic leukemia (CNL) from neutrophilic leukemoid reactions (NLR). In this paper, we describe four cases of leukocytosis with neutrophilia, including one case of CNL with a T618I mutation in colony stimulating factor 3 receptor (CSF3R) and three cases of NLR associated with malignancy or sepsis, which were initially suspected as CNL. Of the three NLR cases, one was associated with ovarian cancer, one with monoclonal gammopathy of undetermined significance and one with multiple myeloma with sepsis. This study demonstrated that confirming the clonality of myeloid cells with CSF3R T618I could contribute to making an accurate differential diagnosis between CNL and NLR in patients with solid cancers or plasma cell neoplasms caused by paraneoplastic syndromes and/or infection.

Research on the Influence of new oncogenic CSF3R mutations in Chronic Neutrophilic Leukemia / Эрүүл мэндийн лаборатори

Background:Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative neoplasm (MPN), since the first description of CNL in 1920, more than 150 cases have reported in the literature. The World Health Organization (WHO) recognizes CNL, as a MPN and, for the frst time, provides recognized criteria to permit the operational classifcation of this poorly defned disease. Until recently, the molecular pathogenesis of CNL was unknown and the diagnosis was based on morphological aspects, clinical criteria and exclusion of known genetic entities like the Philadelphia translocation indicative of CML, or JAK2 mutations indicative of MPNs. Recent discovery of highfrequency granulocyte-colony stimulating factor receptor (CSF3R) mutations in CNL identifes a new major diagnostic criterion, and lend more specificity to the WHO diagnostic criteria for CNL, which are variably applied in routine clinical practice. In 2013 Maxson et al., and Pardanani and colleagues identified granulocyte-colony stimulating factor 3 receptor (CSF3R) mutations in 8 of 9 (89%), and in 13 of 13 (100%) patients with CNL, respectively. CSF3R mutations fall into 2 classes: nonsense or frameshift mutations that lead to premature truncation of the cytoplasmic tail of the receptor (truncation mutations) and point mutations in the extracellular domain of CSF3R (membrane proximal mutations). These nonsense or frameshift mutations truncate the cytoplasmic tail of CSF3R, impair its internalization,and alter its interactions with proteins such as SHP-1/2 and SOCS family members. These structural and functional alterations are thought to perturb the capacity of CSF3R to regulate granulocyte differentiation and to increase granulocytic proliferative capacity. Thetwo types of CSF3R mutations may have differential susceptibility to classes of tyrosine kinase inhibitors,with CSF3R truncation mutations showing activation of SRC family–TNK2 kinase signaling and sensitivity to dasatinib, and CSF3Rmembrane proximal mutations strongly activate the JAK/signal transducer and activator of transcription pathway and are sensitive to JAK kinase inhibitors such as ruxolitinib.The most common CSF3R mutation in CNL is themembrane proximal mutation: T618I. On September 2012 we got a case (a 67-years-old Chinese man), which had fulflled the WHO diagnostic criteria for CNL with a novel mutation site of colony stimulation factor 3 receptor (CSF3R). In our case was identifed a membrane proximal mutations CSF3RT618I and also a unreported novel mutation site of CSF3R-H54A in the CD34+ and CD15+ cell fractionsby sorting bone marrow samples (BD FACSAria™ III; BD Biosciences) using a PCR-based DNA pyrosequencing method. Thus, we sought to determine CSF3R-FL, CSF3R-T618I, CSF3R-H54A mutations have some correlation with molecular pathogenesis of CNL.Objective:To determine CSF3R-FL, CSF3R-T618I, CSF3RH54A mutations that have some signifcance on the molecular pathogenesis of CNL.Materials/Methods:1. Plasmid construction. Plasmids were constructed by PCR amplifcation of the insert, restriction digestion and ligation using standard molecular biology methods, briefly: the host vectors pLV-EF1α-EYFP-N, pLP-2, pVSV-G, pLP-1 gag pol were purifed with Omegabiotek maxi prep kit and digested by restriction enzymes (ECO RI, NOTI). The linearized vectors were purified from agarose gel using a AxyPrep TM DNA Gel Extraction Kit and the concentration of the samples was estimated on an agarose gel stained with ethidium bromide.The inserts (CSF3R-FL, CSF3R-T618I, CSF3R- H54A) were generated by PCR, the sequences of the primer-pairs used and the conditions of the PCR reactions. The amplifed DNA fragments were purifed from agarose gels using AxyPrep TM DNA Gel Extraction kits and digested by ECO RI, NOTI was used to linearize the acceptor vector. Enzymatic reactions in the case of the i) insert: 100–3000 ng purifed PCR product was digested by appropriate amount of enzyme and 4 µl of 10x reaction buffer in 40 µl of fnal volume for overnight at the appropriate temperature; ii) vector: 2000–4000 ng plasmid DNA was digested by appropriate amount of enzyme and 2 µl of 10x reaction buffer in 20 µl of fnal volume for 4 hours at the appropriate temperature. When necessary Research on the Influence of new oncogenic CSF3R mutations in Chronic Neutrophilic Leukemia Otgonbat Altangerel1, Ming Feng Zhao2, Wu Ri Mao21Department of Internal Medicine, Division of Hematology, Mongolian National University of Medical Sciences, Mongolia 2Department of Hematology, Tianjin First Central Hospital, First Central Clinical College of Tianjin Medical University,P.R. China11 the digested fragments were purifed again and the concentrations of the inserts were estimated on agarose gels, as described above. A 1:3 vector: insert molar ratio was used for the ligation reactions. Chemically competent DH5α Escherichia coli bacteria were transformed with the products of the ligation reactions and were grown on Luria Bertrani (LB) agar plates containing the required antibiotic, such as ampicillin (Sigma). A day later single colonies were picked from the plate, inoculated into and grown overnight in LB medium containing with ampicillin. Plasmids were purifed from the overnight cultures as above and tested by restriction mapping for the presence of the insert. Selected clones were sequenced by Sanger sequencing.2. Lentiviral packaging system we used 3 main components, such as the lentiviral expression vector(Plasmid DNA of CSF3R-FL, CSF3R-T618I), the lentiviral packaging plasmids (pLP-1, pLP-2 plasmids that encode for gag, pol, and rev from the HIV or FIV genome and pVSV-G), 293TNN producer cells. We seed 1X105 293TNN cells per 10 cm2 culture plate in 2-3 ml of culture medium containing DMEM medium supplemented with 4 mM L-glutamine, 4.5 g/l glucose, and fetal bovine serum (10%) without antibiotics. Grow for 18-24 hours at 37 °C with 5% CO 2 so that the cell density reaches ~60 - 80% confluency at the time of transfection. We used a GFP as a positive control, to confrm transfection experiment was successful. Then we collected the cell culture supernatant, which is containing infectious pseudoviral particles.3. Transduction of Pseudotyped Viral Particles into the primary cell of mouse. In the fnal step we have used the Mouse Colony Forming Unit Assay using MethoCultTM to assess the effects of CNL-associated oncogenes on the morphology and number of primary murine cells derived bone marrow. For this purpose cells are transduced with either control, which is without viral construct or a construct expressing the oncogene of interest (CSF3R-FL, CSF3R-T618I).Results:1. On the Plasmid construction step we successfully extracted and purifcated of recombinant plasmids ofCSF3R-FL and CSF3R-T618I cloning, but we still didexperiment to obtain the recombinant plasmid CSF3R- H54A cloning.2. After 24 hours of transfection 293TNN Cells with Packaging Plasmids and the Expression Construct, cells we visualized with green fluorescence protein under the fluorescence microscope.3. The both two of CSF3Rcloning were capable of transforming murine colony forming cells. After transforming, CFU-GM colonies were counted manually by light microscopy seven days after plating. We found that the membrane proximal mutation (T618I) transformed CFU-GM colonies number was more than the full length non-mutants (CSF3RFL), which indicates that T618 mutation of CSF3R conferred the clonal advantage of CNL leukemia cells.Conclusions:1. The establishment of the plasmid reconstruction, lentiviral packaging system and Mouse Colony Forming Unit Assay were done successfully.2. We confrmed the transformation capacity of the CSF3R mutations, especially CSF3R-T618I was higher than CSF3R-FL. This result demonstrates that T618 mutation of CSF3R conferred the clonal advantage of CNL leukemia cells.3. Further studies will be continued and are needed to prove the effects of the novel mutation site CSF3RH54A on the transduced murine bone marrow progenitor cells by using CFC assay

Evaluation of a father and son with atypical chronic myeloid leukemia with SETBP1 mutations and a review of the literature

We report the case of a father and son diagnosed with atypical chronic myeloid leukemia (aCML). Both patients harbored SETBP1 mutations, which are present in 24.3% of aCML patients. Moreover, both shared the variant encoding p.Pro737His, but the aCML severity was greater in the son because of the presence of two other missense mutations causing p.Asp868Asn and p.Ser885Arg alterations. SETBP1 mutations may be associated with an adverse prognosis, so their detection would help in the diagnosis of aCML and the determination of a patient's prognosis.

A Case of Acute Myeloid Leukemia Transformed from JAK2 V617F-Positive Chronic Neutrophilic Leukemia

Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative neoplasm (MPN) characterized by persistent peripheral blood neutorphilia, bone marrow hypercellularity of neutrophilic granulocyte proliferation and hepatosplenomegaly. Acquired somatic mutation, JAK2 V617F, is the only molecular marker known to have association with classic BCR-ABL1 negative MPNs. However, JAK2 V617F has been detected occasionally in other MPNs such as CNL or other disease entities. We experienced a case of CNL with JAK2 V617F mutation. The patient was diagnosed according to the 2008 WHO classification criteria, and developed AML 9 months after the diagnosis of CNL. The JAK2 V617F was detected in the bone marrow throughout the clinical course. More cases are needed to establish the role of JAK2 V617F in the pathogenesis, prognosis and disease course of CNL.

A Case of Chronic Neutrophilic Leukemia with Multiple Myeloma and an Increased Function of Her Neutrophils / 대한혈액학회지

We describe here a 64-year-old woman who simultaneously presented with chronic neutrophilic leukemia (CNL) and multiple myeloma (MM). The patient presented with mature neutrophilic leukocytosis, hepatosplenomegaly, the absence of Philadelphia chromosome and the BCR-ABL fusion gene, along with IgG kappa type monoclonal gammopathy in her serum and urine. The bone marrow aspirates showed hypercellularity with marked granulocytic hyperplasia and an increase in immature plasma cells. The neutrophil function tests showed increased phagocytosis, chemotaxis and respiratory burst activity, but there was normal microbial killing activity. The patient was treated with dexamethasone and pamidronate for MM and with hydroxyurea for CNL, and she was discharged from the hospital in an improved condition.

A Case of Chronic Neutrophilic Leukemia Associated with Increased Respiratory Burst Activity of Neutrophils / 대한혈액학회지

Chronic neutrophilic leukemia is a rare myeloproliferative disorder. We have experienced a typical case of chronic neutrophilic leukemia in a 76-year-old man who complained abdominal distension due to hepatosplenomegaly. White blood cell count of peripheral blood was 50,500/nL with 90% segmented neutrophils. The underlying disease for a leukemoid reaction had not been detected. Leukocyte alkaline phosphatase score and the serum levels of vitamin B12 and uric acid were elevated. Chromosome study showed a normal karyotype without Philadelphia chromosome or bcr/abl rearrangement. Phorbol myristate acetate-activated respiratory burst activity of neutrophils measured with chemiluminescence showed increased activity.

A Case of Chronic Neutrophilic Leukemia with Multiple Myeloma / 대한혈액학회지

Chronic neutrophilic leukemia(CNL) is a rare disorder characterized by persistent neutrophilia of mature cell type without evidence of infection, hepatosplenomegaly, elevated neutrophil alkaline phosphatase score and the absence of Philadelphia chromosome We report a typical case of CNL with multiple myeloma(IgG-lambda type) in 68 years old female. Many neutrophils had toxic granule and vacuole. The peripheral blood smear showed neutrophlic leukocytosis(50,940/microliter, neutrophil 94%) with rare immature form. The cytogenetic study showed normal karyotype without Philadelphia chromosome. She was diagnosed as multiple myeloma with paraproteinemia, lytic bone lesion of skull, and immature plasma cells in bone marrow.

Pure red cell aplasia associated with chronic neutrophilic leukemia: A case report.

A 79-yesr-old woman was admitted to the hospital with evidence of pure red cell aplasia and chronic neutrophilic leukemia. Bone marrow aspiration showed almost absence of erythropoiesis, marked hyperplastic granulopoiesis with normal maturation and normal megakaryocytes. Serum vitamin B12, unsaturated vitamin B12 binding capacity, transcobalamin I and transcobamin III increased considerably. Chronic myelocytic leukaemia could be ruled out by the blood and bone marrow examination and a high leucocyte alkaline phosphatase score. This case of pure red cell aplasia accompanied by proliferative feature in other cell lines is an example of a link between erythrodysplastic and myeloproliferative disorders.