Natural Course of Human T-Cell Leukemia Virus Type 1 Proviral DNA Levels in Carriers During Pregnancy.

The measurement of human T-cell leukemia virus type 1 (HTLV-1) proviral DNA levels by using polymerase chain reaction has been beneficial for confirming HTLV-1 infection during pregnancy. However, the influence of pregnancy on HTLV-1 infection and proviral DNA levels among pregnant women with HTLV-1 has not been clarified. We prospectively gathered blood samples from 36 pregnant women in whom HTLV-1 carriage was previously diagnosed and sequentially measured their proviral DNA levels. The HTLV-1 proviral DNA levels remained at a plateau during pregnancy but were elevated after delivery. Moreover, flow cytometry and serological analyses revealed that the regulatory T-cell population and soluble interleukin 2 receptor levels were similarly elevated after birth in comparison with those in control pregnant women. This study is the first to provide data on sequential changes in HTLV-1 proviral DNA levels during and after pregnancy. These findings will guide the establishment of a better program to prevent mother-to-child transmission of HTLV-1.

Detection of de novo single nucleotide variants in offspring of atomic-bomb survivors close to the hypocenter by whole-genome sequencing.

Ionizing radiation released by the atomic bombs at Hiroshima and Nagasaki, Japan, in 1945 caused many long-term illnesses, including increased risks of malignancies such as leukemia and solid tumours. Radiation has demonstrated genetic effects in animal models, leading to concerns over the potential hereditary effects of atomic bomb-related radiation. However, no direct analyses of whole DNA have yet been reported. We therefore investigated de novo variants in offspring of atomic-bomb survivors by whole-genome sequencing (WGS). We collected peripheral blood from three trios, each comprising a father (atomic-bomb survivor with acute radiation symptoms), a non-exposed mother, and their child, none of whom had any past history of haematological disorders. One trio of non-exposed individuals was included as a control. DNA was extracted and the numbers of de novo single nucleotide variants in the children were counted by WGS with sequencing confirmation. Gross structural variants were also analysed. Written informed consent was obtained from all participants prior to the study. There were 62, 81, and 42 de novo single nucleotide variants in the children of atomic-bomb survivors, compared with 48 in the control trio. There were no gross structural variants in any trio. These findings are in accord with previously published results that also showed no significant genetic effects of atomic-bomb radiation on second-generation survivors.

Induction of apoptosis by HBI-8000 in adult T-cell leukemia/lymphoma is associated with activation of Bim and NLRP3.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T-cell malignancy caused by human T-cell lymphotropic virus 1. Treatment options for acute ATL patients include chemotherapy, stem cell transplantation, and recently the anti-chemokine (C-C motif) receptor 4 antibody, although most patients still have a poor prognosis and there is a clear need for additional options. HBI-8000 is a novel oral histone deacetylase inhibitor with proven efficacy for treatment of T-cell lymphomas that recently received approval in China. In the present study, we evaluated the effects of HBI-8000 on ATL-derived cell lines and primary cells obtained from Japanese ATL patients. In most cases HBI-8000 induced apoptosis in both primary ATL cells and cell lines. In addition, findings obtained with DNA microarray suggested Bim activation and, interestingly, the contribution of the NLR family, pyrin domain containing 3 (NLRP3) inflammasome pathway in HBI-8000-induced ATL cell death. Further investigations using siRNAs confirmed that Bim contributes to HBI-8000-induced apoptosis. Our results provide a rationale for a clinical investigation of the efficacy of HBI-8000 in patients with ATL. Although the role of NLRP3 inflammasome activation in ATL cell death remains to be verified, HBI-8000 may be part of a novel therapeutic strategy for cancer based on the NLRP3 pathway.

Relevance of molecular tests for HTLV-1 infection as confirmatory tests after the first sero-screening.

The diagnosis of human T-cell leukemia virus type-1 (HTLV-1) infection has been widely examined by serologics. In the first screening tests, serological false negative and positive samples have been reduced thanks to advances in assay techniques that apply new emission agents and sensors. On the other hand, western blot (WB) remains problematic. For example, WB analysis yields many samples equivalent to antibody positive ones. To reduce the need for WB, an alternative testing strategy is required to detect HTLV-1 infection. Polymerase chain reaction (PCR) for the HTLV-1 provirus has recently been recommended for a final diagnosis of infection. However, although PCR is thought to be one element, the validation of detection performance for HTLV-1 infection between serological and molecular testing is not always clear. Thus, this study aimed to evaluate the accuracy and test the validity of an improved methodology for serological detection of HTLV-infection, as well as that of PCR. In conclusion, the high values of kappa-statistics are expected to deliver high quality in chemiluminescent enzyme immunoassay (or chemiluminescent immunoassay), while the problems with WB assays remain to be elucidated. As an alternative to WB, a combination of real-time qPCR and nested PCR is proposed as a suitable confirmatory test.

[Classification and clinical findings of myelodysplastic syndromes].

Myelodysplastic syndromes (MDS) are a group of related disorders in which bone marrow stem cells malfunction, while the type is diagnosed based on the WHO classification revised in 2008. Although the diagnosis largely depends on the cytomorphology, it is difficult to diagnose MDS based on the morphology alone, particularly in patients with < 5% blasts in the bone marrow and a normal karyotype. In Japan, a grading system for the diagnostic accuracy of MDS was proposed in 2007, and evaluation of dysplasia (high, intermediate, low, minimal) is a characteristic part. Morphologic dysplastic changes are classified into highly specific category A (pseudo-Pelger-Huet anomaly, degranulation of neutrophils, micro-megakaryocytes, ringed sideroblasts) and less specific category B (dysplasia other than category A). With the use of this grading system, diagnostic problems should be reduced. Flow cytometry has also been proposed as a tool to improve the evaluation of marrow dysplasia, because immunophenotyping is an accurate method for quantitative and qualitative evaluations of hematopoietic cells, and MDS specimens have been found to exhibit abnormal expressions of several cellular antigens. In addition, the molecular classification of MDS has received marked attention in recent years. New molecular markers including RPS14, TET2, IDH1/2, SF3B1, ASXL1, RUNX1, TP53, EZH2, JAK2, and WT1 have been revealed to be important for the prognosis, as well as diagnosis and classification. In this report, we review MDS diagnostic approaches from the viewpoints of cytomorphology, immunophenotyping, and cytogenetics.

Pulmonary Nodular Lymphoid Hyperplasia Evaluated with Bronchoalveolar Lavage Fluid Findings: A Case Report and Review of the Literature on Japanese Patients.

Pulmonary nodular lymphoid hyperplasia (PNLH) is a very rare disease, and it is difficult to diagnose PNLH and distinguish it from mucosa-associated lymphoid tissue (MALT) lymphoma. In addition, information on bronchoalveolar lavage fluid (BALF) analyses is lacking. We herein report a 36-year-old Japanese woman diagnosed with PLNH by a surgical biopsy and analysis of BALF. The BALF showed an increase in B-cell marker-positive lymphocytes, normal patterns of B-cell clonality, mucosa-associated lymphoid tissue 1 gene, and immunoglobulin heavy chain at 14q32 translocations. We also reviewed Japanese cases of PNLH described in Japanese or English to explore the characteristics of such cases.

An instructive case suggesting warfarin resistance which is independent on the regulation of the CYP2C9 and VKORC1 genotype.

To improve the safety and effectiveness of warfarin (WF) therapy, the initial dose trends to be practically decided based on single nucleotide polymorphism (SNP) genotyping of two genes, cytochrome P450 (CYP) 2C9 and vitamin K epoxide reductase complex1 (VKORC1). We encountered a 43-year-old female who was hospitalized for investigation and treatment because of intermittent convulsive seizures. Right brain cortical vein thrombi were confirmed by magnetic resonance imaging (MRI) scan; therefore, a 3 mg dose of WF was empirically initiated. The prothrombin time (PT), expressed as the international normalized ratio (INR), did not change at all, even when WF was increased to a dose of 11 mg/day. Direct sequence analysis revealed *3 in CYP2C9 and 3673 GA, 6484 CT, 6853 GC and 9041 GA in VKORC1, indicating that the genotypic pattern of the two genes is the responsible SNP for the moderate phenotype on WF sensitivity. Conclusively, our case may present an unknown mechanism other than the concern mentioned above.

Oligosecretory Primary Plasma Cell Leukemia with Atypical Morphological Abnormality.

Plasma cell leukemia (PCL) is a rare variant of multiple myeloma. The detection of plasma cells in the peripheral blood and monoclonal protein in the serum or urine is important for the diagnosis of PCL. However, it is sometimes difficult to diagnose PCL in patients with atypical plasma cell morphology and/or those without detectable monoclonal protein. We herein report a case of oligosecretory PCL showing atypical morphology in leukemic cells with a convoluted nucleus and basophilic cytoplasm but without detectable monoclonal protein, except for serum free light chain. A flow cytometric analysis and pathological analysis were useful for the early diagnosis of PCL.

High IL-21 receptor expression and apoptosis induction by IL-21 in follicular lymphoma.

We surveyed IL-21 receptor (IL-21R) in leukemia and lymphoma and found that follicular lymphoma cells showed exceptionally high IL-21R expression. Notably, IL-21 showed divergent effects depending on the cell origin: growth stimulation in Burkitt lymphoma cell lines and adult T cell leukemia/lymphoma cell lines but induction of apoptosis in B lymphoma cell lines with t(14;18)(q32;q21), a marker karyotype of follicular lymphoma. IL-21 activated caspase-8 and -3 and reduced mitochondrial membrane potential. More importantly, IL-21 decreased Bcl-2 expression but increased Bax expression. These results support a new therapeutic approach using the IL-21/IL-21R system in follicular lymphoma.

Impact of p53 aberration on the progression of Adult T-cell Leukemia/Lymphoma.

Based on statistical analysis of its age-dependent occurrence, a multi-step carcinogenesis model has been proposed for Adult T-cell Leukemia/Lymphoma (ATLL). We have previously reported that the deletion of the p16 gene is a key event in ATLL progression. In the current study, we report for the first time that the aberrations of p16 and p53 are mutually exclusive in ATLL and either of the two events is sufficient for the ATLL progression. More than half of the patients had one of the two aberrations, and both aberrations emerged as significant markers for a poor prognosis.

Survey of chemokine receptor expression reveals frequent co-expression of skin-homing CCR4 and CCR10 in adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATLL) is a malignancy of mature T-cell origin with multi-organ involvement. Because the chemokine receptors play crucial roles in tissue-specific homing of mature lymphocytes, particular chemokine receptors expressed on ATLL cells may be involved in their tissue infiltration. We thus performed a comprehensive survey on the chemokine receptor expression in ATLL. ATLL cells expressed transcripts of CCR1, CCR4, CCR7, CCR8, CCR10 and CXCR4 but hardly expressed those of CCR2, CCR3, CCR5, CCR6, CCR9, CXCR1, CXCR2, CXCR3 and CXCR5. These results were confirmed at the protein level by flow cytometric analysis. Notably, patients who have skin lesions showed significantly higher levels of CCR10 mRNA expression than patients without skin lesions. ATLL cells migrated efficiently to the CCR4 ligand, CCL22, and moderately to the CCR10 ligands, CCL27 and CCL28. Moreover, ATLL skin lesions consistently contained transcripts of CCR10 and its ligands CCL27 and CCL28 besides those of CCR4 and its ligands CCL17 and CCL22 that have been reported previously. Collectively, the frequent co-expression of CCR4 and CCR10, the known pair of skin-homing chemokine receptors, may play an important role in ATLL invasion into the skin.

Association between circulating CD34-positive cells and serum alkaline phosphatase in relation to body mass index for elderly Japanese men.

BACKGROUND: Recent studies have confirmed an association between bone metabolism and vascular homeostasis. However, no study has examined the relationship between serum alkaline phosphatase (ALP) (a marker of bone metabolism) and circulating immature cell such as CD34-positive cells (a marker of vascular homeostasis). METHODS: We conducted a cross-sectional study of this association in 272 elderly Japanese men (60-79 years). Because low body mass index (BMI) status is a known characteristic of Japanese with a high incidence rate of stroke, we used a stratified analysis based on BMI. RESULTS: Multivariable linear regression analysis adjusted for confounding factors showed a significant correlation between serum ALP and the number of circulating CD34-positive cells, especially for participants with low BMI (<23 kg/m(2)). The parameter estimates (ß) and 95% confidence intervals (CI) for one standard deviation increments in serum ALP levels (62 IU/L) for the circulating CD34-positive cell count were ß = 0.25 (0.04, 0.45) for total subjects, ß = 0.45 (0.16, 0.75) for participants with low BMI (<23 kg/m(2)), and ß = 0.04 (-0.25, 0.34) for participants with high BMI (≥23 kg/m(2)). CONCLUSION: Serum ALP correlates positively with circulating CD34-positive cells among a general population of elderly Japanese men, especially those with low BMI (<23 kg/m(2)). These findings suggest that serum ALP levels may constitute an efficient tool for estimating the risk of insufficient vascular homeostasis, especially for participants with relatively few classical cardiovascular risk factors.

Simultaneous screening for JAK2 and calreticulin gene mutations in myeloproliferative neoplasms with high resolution melting.

INTRODUCTION: Recently, novel calreticulin (CALR) mutations were discovered in Janus kinase 2 (JAK2) non-mutated myelofibrosis (PMF) and essential thrombocythemia (ET) cases, with a frequency of 60-80%. We examined clinical correlations and CALR mutation frequency in our myeloproliferative neoplasms (MPN) cases, and introduce an effective test method for use in clinical practice. METHODS: We examined 177 samples previously investigated for the JAK2 mutation for differential diagnosis of MPN. JAK2 and CALR mutations were analyzed using melting curve analysis and microchip electrophoresis, respectively. Next, we constructed a test for simultaneous screening of the JAK2 and CALR mutations utilizing high resolution melting (HRM). RESULTS: Among 99 MPN cases, 60 possessed the JAK2 mutation alone. Of the 39 MPN cases without the JAK2 mutation, 14 were positive for the CALR mutation, all of which were ET. Using our novel screening test for the JAK2 and CALR mutations by HRM, the concordance rate of conventional analysis with HRM was 96% for the JAK2 mutation and 95% for the CALR mutation. CONCLUSION: Our novel simultaneous screening test for the JAK2 and CALR gene mutations with HRM is useful for diagnosis of MPN.

A novel plasmacytoid dendritic cell line, CAL-1, established from a patient with blastic natural killer cell lymphoma.

Blastic natural killer (NK) cell lymphoma corresponding to CD4+CD56+ malignancies is a novel disease entity, according to the results of clinical, morphologic, and immunologic studies. It is especially noteworthy that this disease likely arises from plasmacytoid dendritic cells (pDCs), described previously as plasmacytoid T-cells, which have an important role in innate and adaptive immunity. However, the exact relationship between the tumor cells and pDCs remains to be elucidated. We encountered a patient with typical blastic NK cell lymphoma, which later converted to leukemic manifestations, and tried to establish a cell line using the leukemic cells. We succeeded in establishment of a novel cell line, CAL-1, which originated from the primary malignant cells. The genetic and phenotypic features of CAL-1 cells bear a similarity to those of pDCs, namely, plasmacytoid morphology at light and electron microscopy; negative results for CD11c and lineage-associated markers of CD3, CD14, CD19, and CD16; positive results for HLA-DR, CD4, CD56, CD45RA, and CD123; and negative results for TCR and IgH gene rearrangements. An interesting finding was that CAL-1 cells change morphologically into the mature DC appearance with many long dendrites after short-term culture in the presence of granulocyte-macrophage colony-stimulating factor and interleukin 3. CAL-1 cells can secrete tumor necrosis factor alpha but not interferon alpha. Thus although they do not share in part phenotypic and functional features with their normal counterparts, CAL-1 cells mostly exhibit a striking pDC phenotype. We describe the first novel pDC cell line of CAL-1. This cell line should open the opportunity for study not only of CD4+CD56+ tumor cells but also of pDCs in vitro.

Aberrant processing of Fas transcripts in adult T-cell leukemia: a possible role in tumor cell survival.

In adult T-cell leukemia (ATL), tumor cells commonly express abundant membrane-bound Fas antigen. We reported a significant correlation between Fas expression status of ATL patients and their clinical outcome. In the current study, we analyzed the Fas cDNA sequence of the distinctive ATL cases that barely expressed mFas identified during the previous study. According to the results, changes in the Fas amino acid sequence were deduced in two of seven cases. Furthermore, we identified seven novel variants of Fas mRNA produced by alternative splicing. Our data indicates the diversity of Fas gene expression at a mRNA level in ATL.

Clinical and oncologic implications in epigenetic down-regulation of CD26/dipeptidyl peptidase IV in adult T-cell leukemia cells.

CD26/dipeptidyl peptidase IV (DPPIV), a T-cell-activation antigen, is a 110-kD type II surface glycoprotein expressed on various types of normal cells. CD26/DPPIV is considered a multifunction housekeeping protein. Malignant cells often show altered CD26/DPPIV expression or no CD26/DPPIV expression, thus suggesting a useful marker for assessing some T-cell malignancies. In this study, cell surface protein and messenger RNA expression profiles for CD26/DPPIV were examined in 49 patients with adult T-cell leukemia (ATL), 10 carriers of human T-lymphotropic virus I (HTLV-I), and 4 HTLV-I-infected cell lines to assess the utility of CD26/DPPIV expression as a useful molecular marker for ATL pathology. In contrast to normal lymphocytes, ATL cells and HTLV-I-infected cell lines apparently down-regulated or completely lost the CD26/DPPIV antigen. Furthermore, the positive rate and antigen density for CD26/DPPIV in ATL cells gradually declined along with the advancement of ATL stage. Analysis of genomic DNA and the CD26/DPPIV transcript showed that CD26- ATL cells possessed faintly detected transcripts of the gene that were aberrantly methylated at the CpG islands within the promoter region in parallel with the advancement of ATL, a finding supported by a rescue experiment for transcript reexpression using 5-azacytidine as demethylation agent. Moreover, there was no relationship between loss of CD26/DPPIV and HTLV-I tax expression. These results indicate that ATL cells down-regulate CD26 antigens by means of epigenetic machinery and that this antigen abnormality is a useful molecular marker for the pathology of ATL.

Automation of bone marrow aspirate examination using the XE-2100 automated hematology analyzer.

BACKGROUND: Attempts to analyze bone marrow aspirates have been reported with the use of several automated blood cell counters, but sufficient accuracy in examination is not acquired yet. Major problems have included difficulties in correctly differentiating various immature cells and interference by lipid in bone marrow aspirates. The goal of this study was to solve these problems to attain more accurate assessment of bone marrow aspirates with automated blood cell counters. METHODS: We modified the XE-2100 Automated Hematology Analyzer (Sysmex Corporation, Kobe, Japan) to fit it for bone marrow aspirate measurement and evaluated its performance. Measurements were performed with the modified XE-2100 on 81 patient samples of bone marrow aspirates; as a reference, the manual visual method was used and flow cytometric analysis were carried out. RESULTS: Good correlations between results with the modified XE-2100 and the manual visual method were obtained for total nucleated cell count (TNCC; r = 0.99), erythroblast/TNCC ratio (r = 0.93), and myeloid cell/TNCC ratio (r = 0.75). CONCLUSIONS: When this device is used, bone marrow aspirate differentials can be determined quickly and easily. This device will be useful for preliminary examination to obtain a summary of various blood cell ratios in bone marrow aspirates before performance of microscopic examination.

Relationships of diverse apoptotic death process patterns to mitochondrial membrane potential (Δψ(m)) evaluated by three-parameter flow cytometric analysis.

Recently, it has been proposed that novel methodologies are needed to re-evaluate apoptotic cell death, as studies of apoptosis have shown it to be a complex process. Since mitochondria are key regulators in cell death pathways, we developed a simultaneous 3-parameter flow cytometric analysis that incorporates the change in mitochondrial membrane potential (Δψ(m)) in an Annexin-V [for phosphatidyl-serine (PS)] and propidium iodide (PI) assay system (3 parameters with 4 colours), and evaluated the apoptotic process using various haematological malignant cell lines and death triggers. The present method enabled visualization of cell composition during apoptosis and captured complicated molecular events. For example, apoptotic cells that lost Δψ(m) did not always externalize PS, while some late apoptotic cells had polarized Δψ(m). The findings of unchanged PS-externalization and aberrant cell death suggest that there is no relationship of PS externalization and apoptosis with an unknown apoptotic mechanism. Based on PS-externalization, sensitivity to staurosporine, and the combination of cell lines and triggers, the apoptotic process was classified into 2 types. Importantly, most of our findings could not be observed by PS-PI and Δψ(m) assays when independently performed. Our method may be useful for examining mitochondrial-related apoptosis and death signalling pathways, as well as screening novel apoptosis-inducing cancer drugs.