Ji-Hun Lim

Establishment of a reference interval for natural killer cell activity through flow cytometry and its clinical application in the diagnosis of hemophagocytic lymphohistiocytosis

Recently, the Histiocyte Society revised the diagnostic criteria for hemophagocytic lymphohistiocytosis (HLH) to include low or absent natural killer (NK) cell activity, according to local laboratory reference. The aim of this study was to establish reference interval for functional NK‐cell activity in 63 healthy Korean individuals using a flow‐cytometric assay. We used peripheral blood mononuclear cells (PBMCs) as effector cells and Fluorescein isothiocyanate‐labeled K562 cells as target cells. NK‐cell activity was calculated using the following equation: NK‐cell activity (%) = (test lysis − spontaneous lysis) × 100/(maximum lysis − spontaneous lysis). NK‐cell activity was analyzed in 13 known HLH patients and 16 suspected non‐HLH patients using a flow‐cytometric assay. The mean (±SD) cytotoxicity of PBMCs from healthy individuals was 20.9 ± 5.3% and the reference interval was 11.8–31.9%. The mean NK‐cell activity of HLH patients (8.3 ± 8.9%) was significantly lower (P = 0.001) than that of non‐HLH patients (20.1 ± 7.8%). The sequential changes in NK‐cell activity in the HLH group corresponded to clinical and laboratory findings following treatment. We successfully developed a functional NK‐cell activity test for use in the clinical laboratory and obtained a reference interval of NK‐cell activity from healthy donors. This assay, and associated reference interval, was used to analyze 30 clinically relevant specimens and the results were shown to be well correlated.

RANBP2-ALK fusion combined with monosomy 7 in acute myelomonocytic leukemia.

Anaplastic lymphoma receptor tyrosine kinase (ALK) is located on chromosome 2p23; the chromosomal rearrangements of this gene are common genetic alterations, resulting in the creation of multiple fusion genes involved in tumorigenesis. However, the presence of an ALK fusion in myeloid malignancies is extremely rare. We report a case of acute myelomonocytic leukemia in a 31-year-old woman with an unusual rearrangement between RAN-binding protein 2 (RANBP2) and ALK and a karyotype of 45,XX,inv(2)(p23q21),-7[20]. We detected an ALK rearrangement using fluorescence in situ hybridization, identified the ALK fusion partner by using RNA transcriptome sequencing, and demonstrated the RANBP2-ALK fusion transcript by reverse transcriptase--PCR and Sanger sequencing. Immunohistochemistry for ALK showed strong staining of the nuclear membrane in leukemic cells. The patient had an unfavorable clinical course. Our results, together with a literature review, suggest the RANBP2-ALK fusion combined with monosomy 7 may be related to a unique clonal hematologic disorder of childhood and adolescence, characterized by myelomonocytic leukemia and a poor prognosis.

VLA-4 and CXCR4 expression levels show contrasting prognostic impact (favorable and unfavorable, respectively) in acute myeloid leukemia

Very late antigen-4 (VLA-4) and CXC chemokine receptor 4 (CXCR4) perform critical roles in the adhesion of hematopoietic and leukemic stem cells to marrow stromal cells. This mechanism is associated with chemoresistance in patients with acute myeloid leukemia (AML). Here, we measured VLA-4 and CXCR4 expressions in leukemic myeloblasts to determine their prognostic implications. Using multicolor flow cytometry, positive VLA-4 and CXCR4 expressions were measured in leukemic myeloblasts in bone marrow aspirates that were obtained from newly diagnosed adult AML patients (n = 98). VLA-4 expression was higher in patients at favorable or intermediate cytogenetic risk than in patients at poor risk (p < 0.001 and p = 0.002, respectively), but CXCR4 expression was not significantly different. Among the 72 non-promyelocytic leukemia patients analyzed who received cytarabine + anthracycline-based induction chemotherapy, high VLA-4 expression was independently associated with a high probability of complete remission (p = 0.019) and superior relapse-free survival (RFS) (p < 0.001). However, high CXCR4 expression independently increased the probability of relapse (p = 0.002) and was associated with a shorter RFS (p = 0.006). When categorizing patients into three groups according to VLA-4 and CXCR4 expression levels, the group of high VLA-4 and low CXCR4 showed longer RFS (p = 0.001) and overall survival (OS) (p = 0.011) than the group of low VLA-4 or high CXCR4.

Generation of lymphocytes potentiated against leukemic lymphoblasts by stimulation using leukemic cell lysate-pulsed dendritic cells in patients with acute lymphoblastic leukemia and measurement of in vitro anti-leukemic cytotoxicity

Abstract Using granulocyte-macrophage colony stimulating factor, interleukin4 and tumor necrosis factor α, we generated dendritic cells (DCs) from mononuclear cells isolated from the peripheral blood (PB) of eight patients with acute lymphoblastic leukemia (ALL), who were in complete remission (CR), and pulsed these DCs with leukemic cell lysates. Specific cytotoxicity assays were performed by incubation of effector cells (lymphocytes generated from cryopreserved mononuclear cells isolated in CR state of ALL) and targets (cryopreserved leukemic cells at diagnosis). Patients showing decreased cytotoxicity had poorer clinical courses. When we measured lymphocyte subsets, we found positive correlations between cytotoxicity levels and the proportions of T lymphocytes and CD8+ T lymphocytes, but negative correlations between cytotoxicity levels and the proportions of NK cells and regulatory T lymphocytes. In conclusion, we show here that leukemia-specific autologous DCs can be generated from the PB of ALL patients in CR, that the incubation of these DCs with leukemic cell lysates can generate lymphocytes potentiated against leukemic cells, and that relationships are evident among all of cytotoxicity, lymphocyte subsets, and patient prognosis.

Description of Mycobacterium chelonae subsp. bovis subsp. nov., isolated from cattle (Bos taurus coreanae), emended description of Mycobacterium chelonae and creation of Mycobacterium chelonae subsp. chelonae subsp. nov.

Three rapidly growing mycobacterial strains, QIA-37T, QIA-40 and QIA-41, were isolated from the lymph nodes of three separate Korean native cattle, Hanwoo (Bos taurus coreanae). These strains were previously shown to be phylogenetically distinct but closely related to Mycobacterium chelonae ATCC 35752T by taxonomic approaches targeting three genes (16S rRNA, hsp6 and rpoB) and were further characterized using a polyphasic approach in this study. The 16S rRNA gene sequences of all three strains showed 99.7 % sequence similarity with that of the M. chelonae type strain. A multilocus sequence typing analysis targeting 10 housekeeping genes, including hsp65 and rpoB, revealed a phylogenetic cluster of these strains with M. chelonae. DNA-DNA hybridization values of 78.2 % between QIA-37T and M. chelonae indicated that it belongs to M. chelonae but is a novel subspecies distinct from M. chelonae. Phylogenetic analysis based on whole-genome sequences revealed a 95.44±0.06 % average nucleotide identity (ANI) value with M. chelonae, slightly higher than the 95.0 % ANI criterion for determining a novel species. In addition, distinct phenotypic characteristics such as positive growth at 37 °C, at which temperature M. chelonae does not grow, further support the taxonomic status of these strains as representatives of a novel subspecies of M. chelonae. Therefore, we propose an emended description of Mycobacterium chelonae, and descriptions of M. chelonae subsp. chelonae subsp. nov. and M. chelonae subsp. bovis subsp. nov. are presented; strains ATCC 35752T(=CCUG 47445T=CIP 104535T=DSM 43804T=JCM 6388T=NCTC 946T) and QIA-37T (=KCTC 39630T=JCM 30986T) are the type strains of the two novel subspecies.

FISH analysis of MLL gene rearrangements: detection of the concurrent loss or gain of the 3' signal and its prognostic significance.

The rearrangement of the mixed‐lineage leukemia (MLL) gene occurs through translocations and insertions involving a variety of partner chromosome genes. However, there are few studies on aberrant MLL signal patterns such as concurrent 3ʹ MLL deletion.

Severe Fever with Thrombocytopenia Syndrome Presenting with Hemophagocytic Lymphohistiocytosis

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease caused by the newly discovered SFTS Bunyavirus, and there have been no case reports of SFTS patients presenting with hemophagocytic lymphohistiocytosis (HLH) in the English literature. We report a case of SFTS presenting with HLH in a 73-year-old immunocompetent male farmer. Although the patient had poor prognostic factors for SFTS, such as old age and central nervous system symptoms, he recovered fully with supportive care.

Angioimmunoblastic T-Cell Lymphoma in a Patient with Klinefelter Syndrome

Patient: Male, 61 Final Diagnosis: AITL in Klinefelter syndrome Symptoms: — Medication: — Clinical Procedure: Chemotherapy Specialty: Hematology Objective: Rare disease Background: Although patients with Klinefelter syndrome have elevated risk and incidence rates for several solid cancers, reports on the incidence of hematological malignancies have been equivocal. Case Report: We report a patient diagnosed with angioimmunoblastic T-cell lymphoma in whom Klinefelter syndrome was newly detected. Moreover, we discuss the development of a variety of lymphomas in patients with Klinefelter syndrome. Conclusions: This is the first case describing angioimmunoblastic T-cell lymphoma in a patient with Klinefelter syndrome who was treated with chemotherapy.

A description of Mycobacterium chelonae subsp. gwanakae subsp. nov., a rapidly growing mycobacterium with a smooth colony phenotype due to glycopeptidolipids

Three rapidly growing mycobacterial strains, MOTTH4W, MOTT36WT and MOTT68W, were isolated from the sputa of three independent Korean patients co-infected with Mycobacterium yongonense Type II strains. The 16S rRNA gene sequences of all three strains were unique, which were closest to that of Mycobacterium chelonae subsp. bovis KCTC 39630T (99.9 % similarity). Multilocus sequence typing analysis targeting 10 housekeeping genes including hsp65 and rpoB revealed the distinct phylogenetic location of these strains, which were clustered with M. chelonae subsp. chelonae ATCC 35752T and M. chelonae subsp. bovis KCTC 39630T. Phylogenetic analysis based on whole genome sequences revealed a 95.89 % average nucleotide identity (ANI) value with M. chelonae subsp. chelonae, slightly higher than the 95.0 % ANI criterion for determining a novel species. In addition, phenotypic characteristics such as a smooth colony morphology and growth inhibition at 37 °C, distinct MALDI-TOF MS profiles of extracted total lipids due to surface glycopeptidolipids, and distinct drug susceptibility profiles further supported the taxonomic characterization of these strains as representing a novel subspecies of Mycobacterium chelonae. Mycobacterium chelonae subsp. gwanakae subsp. nov. is proposed and the type strain is MOTT36WT (=KCTC 29127T=JCM 32454T).

Investigation of a nosocomial outbreak of fungemia caused by Candida pelliculosa (Pichia anomala) in a Korean tertiary care center

BACKGROUND Candida pelliculosa is a rare pathogen of fungemia. There have been a few nosocomial outbreaks of C. pelliculosa fungemia in nurseries and pediatric intensive care units (ICU), hematologic units, and surgical ICU. We describe an epidemiologic outbreak investigation, including case findings of C. pelliculosa fungemia in South Korea. METHODS This outbreak investigation conducted in a 940-bed, tertiary referral center, Ulsan, South Korea and included active microbial surveillance and a case-control study. RESULTS A patient in the trauma intensive care unit (ICU) with multiple trauma developed C. pelliculosa fungemia, and 10 patients in the trauma ICU, medical ICU, and 2 general wards subsequently contracted C. pelliculosa fungemia during the next 24 days (November 16 and December 9, 2015). The 16s rRNA sequencing of 4 isolates showed that C. pelliculosa was verified with 99-100% similarity (GenBank accession number: KF317892.1), and these isolates were identical in the randomly amplified polymorphic DNA (RAPD) assay. A case-control study showed that medical staff and staying in the interventional radiology procedure room were risk factor for development of C. pelliculosa fungemia. After intervention including strict hand washing, disinfecting medical equipment, and contact precautions, there have been no new C. pelliculosa infections since December 10, 2015. CONCLUSIONS This is the first report of a nosocomial outbreak involving 11 patients in 2 ICUs and 2 general wards caused by C. pelliculosa in South Korea. Infection control measures are important for decreasing transmission of C. pelliculosa in the hospital.