MtDNA haplogroups and elite Korean athlete status.

Mitochondrial DNA (mtDNA) variation has recently been suggested to have an association with athletic performance or physical endurance. Since mtDNA is haploid and lacks recombination, specific mutations in the mtDNA genome associated with human exercise tolerance or intolerance arise and remain in particular genetic backgrounds referred to as haplogroups. To assess the possible contribution of mtDNA haplogroup-specific variants to differences in elite athletic performance, we performed a population-based study of 152 Korean elite athletes [77 sprint/power athletes (SPA) and 75 endurance/middle-power athletes (EMA)] and 265 non-athletic controls (CON). The overall haplogroup distribution of EMA differed significantly from CON (p<0.01), but that of SPA did not. The EMA have an excess of haplogroups M* (OR 4.38, 95% CI 1.63-11.79, p=0.003) and N9 (OR 2.32, 95% CI 0.92-5.81, p=0.042), but a dearth of haplogroup B (OR 0.26, 95% CI 0.09-0.75, p=0.003) compared with the CON. Thus, our data imply that specific mtDNA lineages may provide a significant effect on elite Korean endurance status, although functional studies with larger sample sizes are necessary to further substantiate these findings.

Adoptive immunotherapy using human peripheral blood lymphocytes transferred with RNA encoding Her-2/neu-specific chimeric immune receptor in ovarian cancer xenograft model.

The current gene transfer technology for single chain (scFv)-based chimeric immune receptor (CIR) has relied on retrovirus and lentivirus vectors which require a long time to obtain sufficient number of transduced cells and stably incorporate into genome. To ameliorate these limitations, we applied RNA electroporation to human peripheral blood lymphocytes (PBLs) activated with anti-CD3 antibody and interleukin-2 (IL-2) for 3 days and assessed that PBL transiently expressing anti-Her-2/neu CIR (CIR-PBL) containing signaling portion of CD28 and CD3zeta could elicit strong cytotoxicity in vitro and antitumor responses in vivo. The CIR-PBL expressed high level of CIR in CD4+, CD8+ and CD56+ cells. Her-2/neu-specific stimulation induced secretion of type-I cytokines including interferon-gamma (IFN-gamma), IL-8 and granulocyte-macrophage colony-stimulating factor, and IFN-gamma secretion was mainly mediated by CD8+ T cells. CIR-PBL specifically killed SKOV3 cell line expressing Her-2/neu. Adoptive transfer of CIR-PBL in SKOV3 xenograft model led to significant inhibition of tumor growth compared with transfer of mock-transduced PBL and showed higher inhibition than those with Herceptin, humanized monoclonal antibody specific for Her-2/neu. These results provided evidence that electroporation of CIR RNA to human PBLs could be used for rapid generation and high number of therapeutic antigen-specific T cells for adoptive immunotherapy.

Comparison of four hematology analyzers, CELL-DYN Sapphire, ADVIA 120, Coulter LH 750, and Sysmex XE-2100, in terms of clinical usefulness.

We evaluated the clinical usefulness (leukocyte distribution classification, morphologic classification, and morphologic flags) of the following four hematology analyzers: CELL-DYN Sapphire (CD-Sapphire) (Abbott Diagnostics, Santa Clara, CA, USA), ADVIA 120 (Bayer Diagnostics, Tarrytown, NY, USA), Beckman Coulter LH 750 (Beckman Coulter, Miami, FL, USA), and Sysmex XE-2100 (TOA Medical Electronics Co., Kobe, Japan). Four hundred thirty samples from patients and 100 samples from healthy individuals were analyzed. For distributional classification, the sensitivity rates of CD-Sapphire, ADVIA 120, LH 750, and XE-2100 were 93.1, 95.9, 94.9, and 94.9%, respectively, and the efficiency rates were 80.7, 81.6, 84.1, and 84.2%, respectively. For morphologic classification, the sensitivity rates of CD-Sapphire, ADVIA 120, LH 750, and XE-2100 were 88.6, 93.2, 77.3, and 94.3%, respectively, and the efficiency rates were 80.9, 73.0, 79.5, and 74.2%, respectively. Comparing the findings in different morphologic flags, XE-2100 showed the highest sensitivity for Blasts flag (90.9%); CD-Sapphire showed the highest sensitivity for Immature granulocytes and/or Left-shift flag (85.5%); ADVIA 120 showed the highest sensitivity for Atypical lymphocytes flag (60.0%); and LH 750 showed the highest sensitivity for Nucleated RBC flag (75.0%). Our results demonstrate that the four analyzers are comparable in overall performance.

Prognostic value of platelet indices as determined by ADVIA 120 in patients suspected of having disseminated intravascular coagulation.

Recent technological advances have made it possible to record a variety of platelet indices using automated hematology analyzers. Disseminated intravascular coagulation (DIC) is associated with the dramatic hemostasis activation, with evidence of fibrin formation and platelet consumption. We investigated the prognostic significance of platelet indices as measured by ADVIA in 222 patients suspected of having DIC. The presence of overt DIC was defined using the scoring system of the International Society of Thrombosis and Haemostasis Subcommittee. Twenty-eight day hospital mortality was used as a clinical prognosis parameter. Median platelet count and platelet-crit (PCT) levels markedly decreased in nonsurvivors, whereas mean platelet volume (MPV), platelet component distribution width (PCDW) and platelet dry mass distribution width (PMDW) were significantly increased in nonsurvivors. In terms of ROC analysis, which was conducted to predict 28-day mortality, areas under the receiver operating characteristic curve (AUC) were; 0.73 platelet count, 0.72 for PCT, 0.69 for PCDW, 0.65 for PMDW and 0.61 for MPV. The odds ratio of a reduced platelet count for the relative risk of 28-day mortality was 5.249 (95% CI: 2.399-11.486), and the odds ratio for PCDW was 3.240 and for PMDW 3.262. Among these indices, platelet count, PCDW and PMDW were found to be more predictive of 28-day hospital mortality. Our results suggest that these indices may provide prognostic information on hospital mortality in the patients suspected of having DIC.

Therapeutic use of granulocyte-colony stimulating factor could conceal residual malignant cells in patients with AML1/ETO+ acute myelogenous leukemia.

We have experienced a number of cases of AML1/ETO+ acute myelogenous leukemia that showed remission based on bone marrow (BM) morphological criteria, but that revealed clonal abnormalities in most cells by fluorescence in situ hybridization (FISH). Interestingly, most of these cases had AML with AML1/ETO rearrangement. The malignant cells were differentiated and considered mature cells after granulocyte-colony stimulating factor (G-CSF) treatment. To clarify the possible mechanisms underlying this phenomenon, we investigated the expression levels of G-CSFR in AML cells with AML1/ETO rearrangement by flow cytometry and real-time polymerase chain reaction (PCR). The number of AML1/ETO+ cells expressing G-CSFR at baseline was significantly higher than that of AML1/ETO- AML cells (2673 vs 522). In addition, the G-CSFR gene was more highly expressed in AML1/ETO+ cells than in AML1/ETO- cells by real-time PCR. This study reveals that cases showing remission after treatment with G-CSF mostly had leukemia with AML1/ETO rearrangement. This finding might be explained by the higher expression of G-CSF receptor in AML1/ETO+ cells than in AML1/ETO- cells. We recommend that remission should be confirmed by FISH, because malignant clones can be differentiated and masked in morphological examination or chromosome test, especially for AML with AML1/ETO rearrangement.

Methylenetetrahydrofolate reductase A1298C genotypes are associated with the risks of acute lymphoblastic leukaemia and chronic myelogenous leukaemia in the Korean population.

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme involved in folate metabolism, DNA methylation and synthesis. We investigated the association between MTHFR polymorphisms and the risks of acute and chronic leukaemias. MTHFR C677T and A1298C were genotyped in 396 Korean individuals using multiplex polymerase chain reaction/restriction fragment-length polymorphism. They were acute lymphoblastic leukaemia (ALL, n = 89), acute myeloid leukaemia (AML, n = 55), biphenotypic acute leukaemia (n = 12), chronic myelogenous leukaemia (CML, n = 40), and normal controls (n = 200). C677T genotypes were not associated with the risk of each disease. A1298C variants, however, significantly decreased the risks of ALL and CML compared with 1298AA. Odds ratios and 95% confidence intervals of 1298AC and 1298AC + CC were 0.53 (0.31-0.93) and 0.54 (0.31-0.93) in ALL, and 0.34 (0.14-0.80) and 0.40 (0.18-0.89) in CML, respectively, compared with 1298AA. These findings demonstrate that the development of ALL and CML is more dependent on folate status, and more susceptible to DNA instability than that of AML. In addition, A1298C rather than C677T may be a more important genetic risk modifier in leukaemogenesis at least in the Korean population.

Solanum nigrum produces nitric oxide via nuclear factor-kappaB activation in mouse peritoneal macrophages.

Nitric oxide (NO) is an antitumour molecule produced in activated macrophages and Solanum nigrum is a plant used in oriental medicine to treat tumours. In this study using mouse peritoneal macrophages, we have examined the mechanism by which Solanum nigrum regulates NO production. When Solanum nigrum was used in combination with 20 U/ml of recombinant interferon-gamma (rIFN-gamma), there was a marked cooperative induction of NO production. The increase in NO synthesis was reflected as an increased amount of inducible NO synthase (iNOS) protein. The production of NO from rIFN-gamma plus Solanum nigrum-stimulated peritoneal macrophages was decreased by treatment with N-monomethyl-L-arginine or N-tosyl-Phe chloromethyl ketone, an iNOS inhibitor. Additionally, the increased production of NO from rIFN-gamma plus Solanum nigrum-stimulated cells was almost completely inhibited by pretreatment with 100 micromol/l of pyrrolidine dithiocarbamate, an inhibitor of nuclear factor kappaB (NF-kappaB). Furthermore, Solanum nigrum increased activation of NF-kappaB. These findings suggest that Solanum nigrum increases the production of NO by rIFN-gamma-primed macrophages and NF-kappaB plays a critical role in mediating these effects.

Pseudobasophilia as an erroneous white blood cell differential count with a discrepancy between automated cell counters: report of two cases.

We report two cases that showed erroneous white blood cell differential counts by automated cell counters. Each case showed an interesting discrepancy of differential count between cell counters, and marked pseudobasophilia was observed by one of the two counters. The first patient was a 44-year-old female who suffered from multiple myeloma for more than one and a half years. Increased myeloma cells (43%) in peripheral blood were counted as basophils by the ADVIA 120, and as monocytes by SE-9000, respectively. The second patient was a 72-year-old female diagnosed as having chronic myelomonocytic leukemia. Dysgranulopoietic neutrophils (50%) and monocytes (31%) were increased in the peripheral blood. Dysgranulopoietic neutrophils were counted as basophils by STKS. In contrast, about half of the increased monocytes were counted as neutrophils by the ADVIA 120. These interesting findings highlight the importance of microscopic examination of the blood film in routine laboratory practice, and automated cell counters, especially for the hematologic patients, cannot completely substitute for it. These results also imply that at least some subpopulations with different membrane or cytoplasmic properties may exist even in the similarly classified cells.

Protein 4.1 deficiency and deletion of chromosome 20q are associated with acquired elliptocytosis in myelodysplastic syndrome.

We report a case of myelodysplastic syndrome (MDS), associated with prominent elliptocytosis. A 66-year-old male presented with peripheral pancytopenia, and was diagnosed with MDS [refractory anaemia (RA)]. Apart from marked elliptocytosis, dyshaematopoietic features were not evident in his peripheral blood or hypercellular bone marrow. After 18 months, he had progressed to RA with excess blasts in transformation. Analysis of red blood cell membrane proteins by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a reduced quantity of protein 4.1 (30% of control). Deletion of chromosome 20q was identified by conventional cytogenetic analysis and fluorescence in situ hybridization. Marked elliptocytosis, persistent for more than 17 months, decreased strikingly after chemotherapy with idarubicin and Ara-C. These findings suggest that acquired elliptocytosis occurred as an unusual morphological feature of MDS, associated with abnormalities of protein 4.1 and chromosome 20q.

Isolated extramedullary relapse in the pleural fluid of a patient with acute myeloid leukemia following allogeneic BMT.

We report an unusual case of AML, in which the patient showed extramedullary relapse in the pleural fluid and the skin without bone marrow recurrence even 3 years after allogeneic BMT. On examination of the pleural effusion and the skin, which relapsed 31 months and 40 months, respectively, after BMT, we found that most of cells were as the XY-type recipient by quantitative X/Y FISH (fluorescence in situ hybridization). However, 100% of the bone marrow cells remained XX-type donor cells. In the present case, we believe that the graft-versus-leukemia (GVL) response in the extramedullary site was not so effective as that in the bone marrow, where it remains effective.

Mutation analysis of the PIG-A gene in Korean patients with paroxysmal nocturnal haemoglobinuria.

AIM: Paroxysmal nocturnal haemoglobinuria (PNH) is caused by deficient biosynthesis of the glycosylphosphatidylinositol (GPI) anchor in haemopoietic stem cells. Mutation of the phosphatidylinositol glycan class A (PIG-A) gene, an X linked gene that participates in the first step of GPI anchor biosynthesis, is responsible for PNH. The characteristics of somatic mutation of the PIG-A gene in Korean patients with PNH were studied. METHODS: Twenty four patients with PNH were selected. Ham tests and sucrose haemolysis tests were carried out on all patients. The expression of CD59 in erythrocytes and granulocytes was investigated in 14 and five patients, respectively, to confirm the diagnosis. Dideoxy fingerprinting (ddF) was used to screen mutations, and direct sequencing of DNA was performed to characterise the mutations. RESULTS: Gene mutation was detected in 12 of the 24 patients. The other 12 patients were negative in ddF screening. Ten new mutations and two known mutations were detected. The mutations consisted of five deletions, six substitutions, and one insertion. These mutations resulted in six premature terminations, three abnormal splicings, one missense mutation in exon 2, and two nonsense mutations. Two patients with venous thrombosis showed mutations in exon 3 only. Substitution mutations were seen in six patients and frameshift mutations in the other six. CONCLUSIONS: There were 10 new mutations among the 12 mutations in the Korean patients with PNH and the characteristics of the mutations varied, with no significant hot spots in sites or types.

Lymphoid preponderance and the absence of basophilia and splenomegaly are frequent in m-bcr-positive chronic myelogenous leukemia.

Chronic myelogenous leukemia (CML) with a minor bcr-abl transcript is a rare entity. We describe a 66-year-old female who was diagnosed with CML in the chronic phase. Molecular analysis of her Philadelphia chromosome using the reverse transcriptase polymerase chain reaction and subsequent sequencing revealed a minor bcr-abl transcript. Monocytosis resembling chronic myelomonocytic leukemia was observed without splenomegaly and basophilia. Her clinical course was indolent and maintained the chronic phase of CML for nearly 3 years under hydroxyurea treatment. A review of the 23 cases of m-bcr CML including this case showed the presence of monocytosis and the absence of basophilia and splenomegaly in 55.0%, 55.0%, and 70.0% of patients, respectively. The absence of basophilia was a significant finding in patients without monocytosis ( P=0.01). Although the hematological features or clinical outcomes were variable in m-bcr CML cases, all three cases at the onset of the blastic phase showed lymphoid crisis, implying an increased lymphoid leukemogenicity of minor bcr-abl transcripts.

Granulocytic sarcoma in MLL-positive infant acute myelogenous leukemia: fluorescence in situ hybridization study of childhood acute myelogenous leukemia for detecting MLL rearrangement.

Granulocytic sarcoma is considered to be rare and its frequent occurrence is associated with specific genetic changes such as t(8;21). To investigate an association between MLL (mixed lineage leukemia or myeloid-lymphoid leukemia) rearrangement and granulocytic sarcoma, we applied fluorescence in situ hybridization for detection of the 11q23/MLL rearrangements on the bone marrow cells of 40 patients with childhood acute myelogenous leukemia (AML). Nine (22.5%) of 40 patients exhibited MLL rearrangements. Three (33.3%) of these nine patients had granulocytic sarcoma and were younger than 12 months of age. Of these three patients one presented as granulocytic sarcoma of both testes with cerebrospinal fluid involvement, the second case presented in the form of an abdominal mass, and the third as a periorbital granulocytic sarcoma. On the other hand, no granulocytic sarcomas were found among MLL-negative patients. It is likely that MLL-positive infant AML may predispose granulocytic sarcoma. Regarding the findings of our study and those of other reports, we would guess that the incidence of granulocytic sarcoma in pediatric MLL-positive AML may be equal to or greater than the 18 to 24% described in AML with t(8;21). Further investigations designed to identify 11q23/MLL abnormalities of leukemic cells or extramedullary tumor may be helpful for the precise diagnosis of granulocytic sarcoma.

Immunophenotypic and cytogenetic changes in acute leukaemia at relapse.

To gain more insight into the immunophenotypic and cytogenetic changes in acute leukaemia at relapse, 99 Korean patients treated at a single institution were studied: acute myelogenous leukaemia (AML, n=46), acute lymphoblastic leukaemia (ALL, n=44) and biphenotypic and mixed leukaemia (n=9). Immunophenotypic changes at relapse were observed in 51 of 99 patients (51.5%) with almost even distribution in AML and ALL. Overall expression of aberrant markers on leukaemic cells was more frequent at relapse than at initial diagnosis (P < 0.05), and this finding was most prominent in B lineage ALL (41.4% versus 10.3%, P=0.007). Gain of aberrant CD13 or CD33 at relapse of B lineage ALL was most frequently observed. Cytogenetic changes at relapse were observed in 28 of 46 patients (60.8%). The initially abnormal karyotypes were more frequently associated with clonal changes at relapse compared to initially normal karyotypes (78.3% versus 43.5%, P=0.016). Cytogenetic changes were more frequent in B lineage ALL than in AML (90% versus 47.8%, P=0.05). In ALL, patients showing cytogenetic changes at relapse were significantly younger than those showing no changes (mean age of 15.0 versus 38.8, P=0.002), whereas in AML there was no significant difference between the two groups. In conclusion, the gain of aberrant markers and cytogenetic changes at relapse, which are suggestive of clonal instability, are more prevalent in B lineage ALL compared to AML, and lymphoid leukaemic cells of younger patients are more susceptible to clonal changes at relapse.

Generation of cytotoxic T lymphocytes specific for human cytomegalovirus using dendritic cells in vitro.

For the adoptive immunotherapy in immunodeficient bone marrow transplant recipients to prevent and treat human cytomegalovirus (HCMV)-associated diseases, HCMV-pulsed dendritic cells (DCs) were used as antigen-presenting cells for the induction of cytotoxic T lymphocytes (CTLs) specific to HCMV antigens in vitro. The antiviral CTL responses induced by HCMV-pulsed DCs were as highly efficient as those induced by HCMV-infected dermal fibroblasts, and endogenous viral gene expression was not required to induce virus-specific T-cell lines. The strong cytotoxic activity against HCMV-pp65, known as HCMV major antigen, was identified using autologous B lymphoblastoid cell line expressing pp65 antigen. The cytotoxic activity toward HCMV-infected target cells was found to be mediated primarily by CD8+ T cells, although both CD8+ cells and CD4+ cells were able to lyse autologous virus-infected target cells. The CTLs contained a mixture of effector cells that recognized virus peptides in the context of major histocompatibility complex. This system may be useful for defining the cellular immune response to HCMV and for the treatment of HCMV infection in immunocompromised patients.

Congenital monoblastic leukemia with 9;11 translocation in monozygotic twins : a case report.

We report an autopsy case of congenital monoblastic leukemia that developed in monozygotic twins. The twin presented with progressive hepatosplenomegaly at 4 weeks after birth. One twin died of massive bleeding and hypovolemic shock before the treatment started. At autopsy, the liver was diffusely enlarged and showed a diffuse whitish discoloration except for the subcapsular and perivenular areas. Microscopic examination disclosed infiltration of histiocyte-like atypical cells along the sinusoids and portal areas of the liver. Spleen, lymph nodes and choroid plexus were also infiltrated by the tumor cells. However, bone marrow involvement of the tumor was minimal although multifocal. On immunohistochemical staining, these atypical cells were reactive for CD68 (PGM-1) and lysozyme, suggesting that the tumor cells might have been derived from mono- histiocyte. Cytogenetic study revealed 9;11 translocation, which is frequently associated with acute monoblastic leukemia. To the best of our knowledge, this is the first report of congenital monoblastic leukemia of monozygotic twins in Korea.

Low incidence of TEL/AML1 fusion and TEL deletion in Korean childhood acute leukemia by extra-signal fluorescence in situ hybridization.

TEL/AML1 fusion in acute leukemia results from cryptic translocation of chromosome 12 and 21, the presence of which suggests a favorable prognosis. The incidence of TEL/AML1 fusion in B-lineage ALL is approximately 25%, but the incidence in Korea has not yet been reported. To investigate the incidence of TEL/AML1 fusion and TEL deletion, bone marrow specimens from 77 Korean children with newly diagnosed acute leukemia were analyzed by FISH. We applied extra-signal FISH to discriminate a true TEL/AML1 fusion from a false-positive fusion signal. To determine the cut-off value of the TEL/AML1 fusion signal, 20 normal bone marrow specimens and 28 normal peripheral blood specimens were also analyzed. The frequency of patients with TEL/AML1 fusion was 13.3% (4 cases) among 30 B-lineage ALL and 9.5% among 42 ALL. One TEL/AML1 fusion-positive patient was also found among 4 acute biphenotypic leukemias. TEL/AML1 fusion was not found in any samples from patients with T-lineage ALL or AML. The incidence of TEL deletion was 6.7% (2 cases) among 30 B-lineage ALL and 4.8% among 42 ALL. The incidences of TEL/AML1 fusion and TEL deletion in Korean children with acute leukemia appear to be lower than those in other countries, suggesting a racial difference.

Cytologic and histologic correlation of atypical glandular cells of undetermined significance.

To determine the cytologic and histologic correlation of atypical glandular cells of undetermined significance (AGUS) in Papanicolaou smears, a cytology file from January 1998 to May 1999 was reviewed. Surgical pathology files were searched to determine which patients received subsequent biopsies. One hundred thirty-two patients with AGUS were identified. Corresponding biopsies were available for 82 of these cases. AGUS has been sub-classified into 3 subtypes: 1) AGUS, favor reactive; 2) AGUS, not otherwise specified; and 3) AGUS, favor neoplasia. The pathologic findings for the respective Papanicolaou smears with the diagnosis of each subtype of AGUS through the follow-up period were as follows: benign lesions in 56.1%, 0%, and 1.2%; squamous intraepithelial lesions 2.4%, 0%, and 1.2%; glandular intraepithelial lesions 0%, 0%, and 17.1%; endometrial simple hyperplasia 1.2%, 0%, and 0%; and carcinoma 0%, 9.8%, and 11%, respectively. In conclusion, AGUS, on cervical cytologic screening, was correlated with significant pathologic findings in 41.5% of the patients (37.8% with preinvasive or invasive glandular lesions and 9.6% with combined squamous intraepithelial lesions). It is thought that intensive follow-up studies, including colposcopy, cervical biopsy, and curettage, should be recommended for complete evaluation of AGUS.

A Korean family with a dominantly inherited beta-thalassemia due to Hb Durham-N.C./Brescia.

We describe the molecular and the hematological characteristics of a Korean family with a dominantly inherited beta-thalassemia. Carriers were characterized by moderate anemia, hypochromia, microcytosis, elevated Hb A2 and Hb F levels, and splenomegaly. DNA analysis revealed a CTG (Leu) to CCG (Pro) substitution at codon 114 of the beta-globin gene, that leads to a highly unstable hemoglobin variant, Hb Durham-N.C./Brescia, and this was linked to the beta haplotype V, [+----+-], and framework 2. RNA analysis showed that the proband had comparable levels of mutant and normal beta-mRNA. Translation of the mutant mRNA would give rise to non-functional hyperunstable beta-globin chains, and their degradation would, by placing an additional burden on the proteolytic process of the red blood cell precursors, result in a more severe phenotype.