Prophylactic fresh frozen plasma may prevent development of hepatic VOD after stem cell transplantation via ADAMTS13-mediated restoration of von Willebrand factor plasma levels.

We initially conducted a multicenter, randomized trial (n=43), and subsequently a questionnaire study (n=209) of participating hospitals, to evaluate whether infused fresh frozen plasma (FFP) could prevent the occurrence of hepatic veno-occlusive disease (VOD) after stem cell transplantation (SCT). Forty-three patients were divided into two groups: 23 receiving FFP infusions and 20 not receiving it. VOD developed in three patients not receiving FFP. Plasma von Willebrand factor (VWF) antigen levels were lower at days 0, 7 and 28 after SCT in patients receiving FFP than in those not receiving it, whereas plasma ADAMTS13 activity (ADAMTS13:AC) did not differ between them. Plasma VWF multimer (VWFM) was demonstrated to be defective in the high approximately intermediate VWFM during the early post-SCT phase, but there was a significant increase in high VWFM just before VOD onset. This suggests that a relative enzyme-to-substrate (ADAMTS13/high-VWFM) imbalance is involved in the pathogenesis of VOD. To strengthen this hypothesis, the incidence of VOD was apparently lower in patients receiving FFP infusions than in those not receiving it (0/23 vs 3/20) in the randomized trial. Further, the results combined with the subsequent questionnaire study (0/36 vs 11/173) clearly showed the incidence to be statistically significant (0/59 vs 14/193, P=0.033).

Identification of novel MUNC13-4 mutations in familial haemophagocytic lymphohistiocytosis and functional analysis of MUNC13-4-deficient cytotoxic T lymphocytes.

BACKGROUND: Familial haemophagocytic lymphohistiocytosis (FHL) has an autosomal recessive mode of inheritance and consists of at least three subtypes. FHL2 subtype with perforin (PRF1) mutation accounts for 30% of all FHL cases, while FHL with MUNC13-4 mutation was recently identified and designated as FHL3 subtype. OBJECTIVE: To examine MUNC13-4 mutations and the cytotoxic function of MUNC13-4 deficient T lymphocytes in Japanese FHL patients METHODS: Mutations of MUNC13-4 and the cytotoxicity of MUNC13-4-deficient cytotoxic T lymphocytes (CTL) were analysed in 16 Japanese families with non-FHL2 subtype. RESULTS: Five new mutations of the MUNC13-4 gene were identified in six families. The mutations were in the introns 4, 9, and 18, and exons 8 and 19. Two families had homozygous mutations, while the remaining four had compound heterozygous mutations. Cytotoxicity of MUNC13-4 deficient CTL was low compared with control CTL, but was still present. Clinically, the onset of disease tended to occur late; moreover, natural killer cell activity was not deficient in some FHL3 patients. CONCLUSIONS: MUNC13-4 mutations play a role in the development of FHL3 through a defective cytotoxic pathway.

High frequency of QPY allele and linkage disequilibrium of granzyme-B in Epstein-Barr-virus-associated hemophagocytic lymphohistiocytosis.

Mediation of Epstein-Barr virus (EBV)-specific cytotoxicity in T lymphocyte via the perforin/granzyme pathway has been demonstrated; therefore, a study involving cytolytic molecules was essential for the clarification of hemophagocytic lymphohistiocytosis (HLH) pathogenesis. This investigation, which analysed the frequency of three allelic mutations of granzyme-B (55Q/R, 95P/A and 247Y/H) in patients with EBV-HLH and infectious mononucleosis, identified the high prevalence of the QPY haplotype in EBV-HLH patients in comparison with healthy controls. A > G polymorphism was also detected in intron 5; furthermore, nearly complete linkage disequilibrium was observed among these polymorphisms. The recessive role of the QPY haplotype of granzyme-B might be responsible for the pathogenesis of EBV-HLH. Cytotoxicity and DNA fragmentation of cytotoxic T lymphocytes did not differ among patients characterized by the QPY/QPY, RAH/RAH and QPY/RAH genotypes. This finding suggested that DNA fragmentation in target cells is mediated not only by granzyme-B but also by other molecules, including other granzymes or Fas.

Esophageal actinomycosis after allogeneic peripheral blood stem cell transplantation for extranodal natural killer/T cell lymphoma, nasal type.

We report a 19-year-old man with extranodal natural killer (NK)/T cell lymphoma, nasal type treated by allogeneic peripheral blood stem cell transplantation (allo-PBSCT). His lymphoma was chemoresistant, and disseminated during local radiotherapy. The patient received allo-PBSCT from his HLA-1 locus mismatched sister using busulfan (BU), cyclophosphamide (CY) and VP-16 as the conditioning regimen. His course was complicated by esophageal actinomycosis 9 months after transplantation, which resulted in the rupture of the right common carotid artery. These observations suggest that actinomycosis should be monitored carefully after transplantation in patients who have received local radiation therapy before the procedure.

Unrelated bone marrow transplantation for Epstein-Barr virus-associated T/NK-cell lymphoproliferative disease.

Epstein-Barr virus (EBV)-associated T/NK-cell lymphoproliferative disease (LPD) has been linked to several different disorders, including chronic active EBV infection, EBV-associated hemophagocytic syndrome, hypersensitivity to mosquito bites, hydroa vacciniforme, aggressive NK-cell leukemia, and nasal/nasal-type NK-cell lymphoma. In most instances, these disorders are refractory to conventional treatments and have a poor prognosis. Here, we report a new treatment strategy for EBV-associated T/NK-cell LPD, consisting of immunochemotherapy, intensive combination chemotherapy, and stem cell transplantation. The five patients studied, two with T-cell and three with NK-cell LPD, lacked a human leukocyte antigen-matched, related donor, and therefore received bone marrow grafts from HLA-matched, unrelated donors. The preconditioning regimen consisted of total-body irradiation (12 Gy), etoposide (900 mg/m(2)), and cyclophosphamide (120 mg/kg) or melphalan (210 mg/m(2)). All patients had residual LPD by a quantitative PCR technique prior to transplantation. After unrelated bone marrow transplantation (UBMT), four of the five patients remain in continuous complete remission at a median of 19 months, without detectable EBV-DNA in peripheral blood. Thus, UBMT appears to be a reasonable option for the treatment of patients with EBV-associated T/NK-cell LPD. Detection of EBV-DNA by PCR offers an important tool for assessing minimal residual disease in patients with EBV-associated T/NK-cell LPD.

Allogeneic hematopoietic stem cell transplantation for 27 children with juvenile myelomonocytic leukemia diagnosed based on the criteria of the International JMML Working Group.

Prognostic factors of juvenile myelomonocytic leukemia (JMML) have not been clarified because of its very low incidence and inaccuracy in the diagnosis. The purpose of this study was to evaluate children with JMML given an allogeneic hematopoietic stem cell transplantation (SCT) and the role of different variables potentially influencing outcome in a nationwide survey in Japan based on the newly proposed criteria by the International JMML Working Group. The study patients were 27 children who underwent SCT among 55 JMML patients retrospectively collected in the survey. The source of grafts was HLA-identical siblings in 12 cases, HLA-matched unrelated individuals in 10 and others in five. Total body irradiation was used in 18 cases. Event-free and overall survival (OS) at 4 years after SCT were 54.2 +/- 11.2% (s.e.) and 57.9 +/- 11.0% (s.e.), respectively. Six patients died of relapse and three of complications. Patients with abnormal karyotypes showed a significantly lower OS than those with normal karyotypes (P < 0.001). Patients below 1 year of age showed a significantly higher OS than those of 1 year of age or more (P = 0.02). Patients with grade 0-1 acute graft-versus-host disease (GVHD) or chronic GVHD had a more favorable OS than those without them, although they were not statistically significant (P > 0.05). Other variables studied were not associated with OS. A multivariate analysis of these factors yielded the abnormal karyotype as the only significant risk factor for lower OS (risk ratio: 11.0; 95% CI: 2.7-45.1).

Intensification of chemotherapy using block therapies as consolidation and reinduction therapies for acute lymphoblastic leukemia during childhood.

Between April 1994 and March 1997, 143 children (age range, 1-15 years) with newly diagnosed acute lymphoblastic leukemia (ALL), except for those patients with t(9;22), were treated according to protocol-94 of the Osaka Childhood Leukemia Study Group. In this trial, the intensity of chemotherapy was enforced in the consolidation and reinduction phases by introducing AML-type block therapies consisting of concentrated administration of 4 to 6 drugs during 5 or 6 days. For patients in the higher risk groups, rotational combination chemotherapy was introduced following the early phase. A total of 124 children with B-cell precursor ALL (B-pre ALL) were classified into 3 groups, the ultrahigh-risk group (UHRG) (15 patients), the high-risk group (HRG) (61 patients), or the standard-risk group (SRG) (48 patients), based on age. leukocyte count, immunophenotype, central nervous system leukemia, response to treatment, and selected chromosomal abnormalities. The complete remission rate was 93%, and the 6-year event-free survival (EFS) rate was 79%+/-4%. EFS rates for the UHRG, HRG, and SRG groups were 67%+/-12%, 80%+/-6%, and 81%+/-6%, respectively. Nineteen patients with T-cell ALL were treated with the protocol for the UHRG. Thirteen patients (68%) attained complete remission, and the 6-year EFS rate was 55%+/-12%. Thus, intensification of chemotherapy improved the EFS rate and AML-type block therapies appeared to be effective as the consolidation and reinduction therapies for B-pre ALL.

t(10;11)-acute leukemias with MLL-AF10 and MLL-ABI1 chimeric transcripts: specific expression patterns of ABI1 gene in leukemia and solid tumor cell lines.

The recurrent translocation t(10;11) is associated with acute myeloid leukemia (AML). The AF10 gene on chromosome 10 at band p12 and MLL at 11q23 fuse in the t(10;11)(p12;q23). Recently, we have identified ABI1 as a new partner gene for MLL in an AML patient with a t(10;11)(p11.2;q23). The ABI1 is a human homologue of the mouse Abl-interactor 1 (Abi1), encoding an Abl-binding protein. The ABI1 protein exhibits sequence similarity to homeotic genes, and contains several polyproline stretches and a src homology 3 (SH3) domain. To clarify the clinical features of t(10;11)-leukemias, we investigated 6 samples from acute leukemia patients with t(10;11) and MLL rearrangement and detected MLL-AF10 chimeric transcripts in 5 samples and MLL-ABI1 in one. The patient with MLL-ABI1 chimeric transcript is the second case described, thus confirming that the fusion of the MLL and ABI1 genes is a recurring abnormality. Both of the patients with MLL-ABI1 chimeric transcript are surviving, suggesting that these patients have a better prognosis than the patients with MLL-AF10. To investigate the roles of AF10 and ABI1 further, we examined the expression of these genes in various cell lines and fresh tumor samples using the reverse transcriptase-polymerase chain reaction method. Although AF10 was expressed in almost all cell lines similarly, the expression patterns of ABI1 were different between leukemia and solid tumor cell lines, suggesting the distinctive role of each isoform of ABI1 in these cell lines. We also determined the complete mouse Abi1 sequence and found that the sequence matched with human ABI1 better than the originally reported Abi1 sequence. Further functional analysis of the MLL-AF10 and MLL-ABI1 fusion proteins will provide new insights into the leukemogenesis of t(10;11)-AML.

Effect of chemotherapy and stem cell transplantation on T lymphocyte clones in familial haemophagocytic lymphohistiocytosis.

Familial haemophagocytic lymphohistiocytosis (FHL) is a rare disorder in infancy, curative only by an allogeneic stem cell transplantation (SCT). We recently confirmed the clonal evidence of T cells in FHL. To confirm the effect of chemotherapy and SCT in FHL, the change of T-cell clones was analysed in two patients using inverse reverse transcription-polymerase chain reaction (RT-PCR) of the T-cell receptor variable region (TCR V) gene, followed by PCR for the junctional region (Jbeta-PCR), a single-strand conformation polymorphism (SSCP) and sequencing analysis at diagnosis, after chemotherapy and after SCT. A high frequency (> 15%) of alphabeta T-cell clones and a predominant bias (Jbeta1:Jbeta2, 85:15) for the Jbeta1 subgroup were observed in the two patients at diagnosis. In one patient, however, an inverted predominant bias (Jbeta1:Jbeta2, 9:91) for the Jbeta2 subgroup and oligoclonal expansion were observed at relapse after chemotherapy. In the other patient, correction of both restricted Jbeta cluster usage and variation of TCR were observed after chemotherapy and SCT. Using sequence analysis, the clonal T cells detected at diagnosis were found to be substituted at low frequency (< 0.75%) by several new clones after chemotherapy and SCT. These results indicate that any genetic defect could influence the regulation of the T-cell network, and normalization of both the variation in each Vbeta repertoire and the Jbeta1/Jbeta2 ratio is needed to achieve remission, and might support the rationale that the only acceptable curative therapy of FHL is allogeneic SCT.

Requirement for etoposide in the treatment of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis.

PURPOSE: We sought to identify the clinical variables most critical to successful treatment of Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (HLH). PATIENTS AND METHODS: Among the factors tested were age at diagnosis (< 2 years or > or = 2 years), time from diagnosis to initiation of treatment with or without etoposide-containing regimens, timing of cyclosporin A (CSA) administration during induction therapy, and the presence or absence of etoposide. RESULTS: By Kaplan-Meier analysis, the overall survival rate for the entire cohort of 47 patients, most of whom had moderately severe to severe disease, was 78.3% +/- 6.7% (SE) at 4 years. The probability of long-term survival was significantly higher when etoposide treatment was begun less than 4 weeks from diagnosis (90.2% +/- 6.9% v 56.5% +/- 12.6% for patients receiving this agent later or not at all; P <.01, log-rank test). Multivariate analysis with the Cox proportional hazards model demonstrated the independent prognostic significance of a short interval from EBV-HLH diagnosis to etoposide administration (relative risk of death for patients lacking this feature, 14.1; 95% confidence interval, 1.16 to 166.7; P =.04). None of the competing variables analyzed had significant predictive strength in the Cox model. However, concomitant use of CSA with etoposide in a subset of patients appears to have prevented serious complications from neutropenia during the first year of treatment. CONCLUSION: We conclude that early administration of etoposide, preferably with CSA, is the treatment of choice for patients with EBV-HLH.

Separation of N2-ethyl-2'-deoxyguanosine-5'-monophosphate and four native deoxyribonucleoside monophosphates using capillary zone electrophoresis with polyethylene glycol as buffer additive.

We investigated the separation of five deoxyribonucleoside monophosphates: 2'-deoxyguanosine-5'-monophosphate (dGMP), 2'-deoxyadenosine-5'-monophosphate (dAMP), 2'-deoxycytosine-5'-monophosphate (dCMP), 2'-deoxythymidine-5'-monophosphate (dTMP) and a dGMP adduct possessing N2-ethyl-guanine, which has been noted in relation to mutagenesis of alcohol, using capillary zone electrophoresis (CZE). The concentration of polyethylene glycol (PEG) as a modifier and the pH of the running solutions can efficiently control the observed separation. Interaction of PEG with analytes was quantitatively evaluated. PEG worked effectively as a hydrophobic selector in these separations. The values of pKa of the acidic-NH-groups in the base moieties of dGMP, dTMP, and the dGMP adduct are close to that of boric acid used as buffer of the running solutions. The control of their charge was facilitated, enabling improved separations. A more sufficient and fast separation was achieved by both optimization of pH of the running solutions and PEG concentration compared with that obtained by pH control alone. On-line concentration using a stacking method followed by the PEG-assisted CZE was briefly studied.

Synthesis and applications of [1-(15)N]-labeled 4,6-dimethyl-4H-[1,2,5]oxadiazolo[3,4-d]pyrimidine-5,7-dione 1-oxide as a useful tool for mechanistic investigations.

[1-(15)N]-Labeled 4,6-dimethyl-4H-[1,2,5]oxadiazolo[3,4-d]pyrimidine-5,7-dione 1-oxide (1-(15)N1) was easily prepared by nitration of commercially available 6-amino-1,3-dimethyl-1H-pyrimidine-2,4-dione using 15N-enriched nitric acid followed by an intramolecular oxidative cyclization with iodosylbenzene diacetate under mild conditions. On the basis of the experimental results using 1-(15)N1, the formation of 8-phenyltheophylline (3), the 1,3-dimethylalloxazines (4: n = 0, 1), and 1,3,7,9-tetramethyl-1H,9H-pyrimido[5,4-g]pteridine-2,4,6,8-tetraone++ + (5) in the thermal reaction of the N-oxide 1 with benzylamine, aniline, or piperidine, and the generation of NO or NO-related species in the reaction with N-acetylcysteamine were reasonably explained by considering the initial attack of the employed nucleophiles on the 3a-position of 1.

Genetic defect in human X-linked agammaglobulinemia impedes a maturational evolution of pro-B cells into a later stage of pre-B cells in the B-cell differentiation pathway.

Surrogate light chains (lambda 5/VpreB) are selectively expressed in early precursors of B cells. B-cell defects in X-linked agammaglobulinemia (XLA) are caused by mutations in the gene for Bruton's tyrosine kinase. To elucidate the nature of early B-lineage cells in bone marrow (BM), samples from 13 XLA patients and 24 healthy controls of different ages were comparatively analyzed using an antihuman VpreB monoclonal antibody. Expression of surrogate light (SL) and mu-heavy chains were examined after cell membrane permeabilization because they are mainly expressed in the cytoplasm of early B-lineage cells. A flow cytometric analysis of normal BM identified 5 discrete cell types of B cells: mu(-)SL(++) (pro-B [B-cell progenitor]), mu(low)SL(++) (pre-B1a), mu(low)SL(+) (pre-B1b), mu(low)SL(- )(pre-B2), and mu(high)SL(- )(B). The large cells, presumably in cycling states, were enriched in pre-B1a cells. The frequencies of B-lineage cells in BM were higher in young children, and declined with advancing age. In contrast, XLA showed a profound reduction in BM B-lineage cells. In XLA BM, an expansion of pro-B cells with some small pre-B1a cells was marked, but other cells were negligible. These observations illustrate a B-cell maturation defect in XLA as well as a normal human B-cell differentiation pathway. The results suggest that the genetic defect in XLA may impede the evolution of pro-B cells beyond the earlier pre-B stage into the later stage of pre-B cells in B-cell development. (Blood. 2000;96:610-617)

An effective chemotherapeutic regimen for acute myeloid leukemia and myelodysplastic syndrome in children with Down's syndrome.

In recent pediatric collaborative studies of acute myeloid leukemia (AML), patients with Down's syndrome (DS) have better outcome than other patients when they were treated according to their intensive AML protocols. This may be attributed to enhanced sensitivity of DS AML cells to selected chemotherapeutic agents. We evaluated a less intensive chemotherapeutic regimen which was specifically designed for children with AML-DS. Remission induction chemotherapy consisted of daunorubicin (25 mg/m2/day for 2 days), cytosine arabinoside (100 mg/m2/day for 7 days), and etoposide (150 mg/m2/day for 3 days). Patients received one to seven courses of consolidation therapy of the same regimen. Thirty-three patients were enrolled on the study and their clinical, hematologic and immunophenotypic features were analyzed. Of the 33 patients, all were younger than 4 years and diagnosed as having acute megakaryoblastic leukemia or myelodysplastic syndrome. All patients achieved a complete remission and estimated 8 year event-free survival rate was 80+/-7%. Three patients relapsed and two died due to cardiac toxicity and one due to septic shock. The results of our study showed that patients with AML-DS constitute a unique biologic subgroup and should be treated according to a less intensive protocol designed for AML-DS.

Splenectomy in haemophagocytic lymphohistiocytosis: report of histopathological changes with CD19+ B-cell depletion and therapeutic results.

The pathogenesis of haemophagocytic lymphohistiocytosis (HLH) in children without a known familial pattern of inheritance is often difficult to establish. Splenic enlargement, one of the main clinical findings in this disorder, has led to the use of splenectomy for uncontrollable coagulopathy, persistent cytopenia or both. This procedure is also thought to be a useful tool in making a differential diagnosis in cases of the immunochemotherapy-resistant HLH. We report here five cases of splenectomized childhood HLH, in which subsets of mononuclear spleen cells were analysed either by flow cytometry or immunohistochemistry, and the results were compared with those from cases of hereditary spherocytosis (controls). There was a statistically significant depletion of CD19+ B cells in the HLH cases (3.8 +/- 3.2% vs. 52.6 +/- 4.5%, P < 0. 0001) associated with an increase of T cells in three cases and of natural killer cells in another. The histopathological findings included atrophic white pulps, B-cell depletion with fibrosis and haemosiderosis in all five cases. Despite temporary therapeutic benefits, three of the HLH patients had a rapidly deteriorating post-splenectomy course and all three eventually died. These results demonstrate striking depletion of B cells in the enlarged spleens of children with HLH, which may be an intrinsic feature of HLH pathogenesis. Further study is needed to establish the therapeutic value of splenectomy in this disease.

Hemophagocytosis by leukemic blasts in 7 acute myeloid leukemia cases with t(16;21)(p11;q22): common morphologic characteristics for this type of leukemia.

BACKGROUND: In a previous study of a case of acute megakaryoblastic leukemia with t(16;21)(p11;q22), which displayed hemophagocytosis by leukemic blasts, the authors mentioned that the same type of morphology had been cited in the literature for 4 other cases of acute myeloid leukemia (AML) with the same translocation. This observation prompted the authors to examine more cases of AML with t(16;21)(p11;q22) for this morphology. METHODS: The authors reviewed bone marrow smears for the presence of hemophagocytosis in 7 patients with AML identified as having t(16;21)(p11;q22). RESULTS: The leukemias belonged to the FAB-M1/M7 (n = 5), M5b (n = 2), and contained phagocytic blasts in various percentages (< 0.2-36.7%). The blasts contained either single or multiple cytoplasmic vacuoles, in some of which the phagosomes were visible. The engulfed hemopoietic cells (red cells, erythroblasts, lymphocytes, and thrombocytes) were also noted in their cytoplasm. These observations confirmed that hemophagocytosis by leukemic blasts is a common and characteristic feature of this type of leukemia. CONCLUSIONS: The study of 12 cases (the 7 cases described here and the previous 5 cases) strongly supports the hypothesis that hemophagocytosis by leukemic blasts is common and characteristic in this type of leukemia, which may be related to the specific chromosome aberration of t(16;21)(p11;q22).

SN-1, a novel leukemic cell line with t(11;16)(q23;p13): myeloid characteristics and resistance to retinoids and vitamin D3.

The MLL gene is fused with the cAMP-responsive element binding protein-binding protein (CBP) gene in t(11;16)(q23;p13), which has been reported to be associated with therapy-related acute leukemia. We established a novel myeloid cell line, SN-1, from a patient with T-cell acute lymphoblastic leukemia with t(11;16)(q23;p13) having in-frame MLL-CBP fusion transcripts. The majority of the SN-1 cells were positive for myeloperoxidase when examined using an electron microscope and expressed CD13, CD33, CD56, and HLA-DR antigens, but not CD7, CD10, CD19, CD34, or CD41 antigens, suggesting that these cells are of myeloid origin. SN-1 cells underwent functional and morphological differentiation when treated with actinomycin D or sodium butyrate, but not with all-trans-retinoic acid (ATRA) or 1alpha,25-dihydroxyvitamin D3 (VD3). Exposure of SN-1 cells to ATRA hardly affected cell growth and differentiation, whereas the growth of HL-60 and NB4 cells treated with ATRA was effectively inhibited, and differentiation into mature granulocytes was induced. SN-1 cells were relatively insensitive to VD3 with respect to inhibiting the cell growth and inducing the ability to reduce nitroblue tetrazolium, lysozyme activity, and morphological differentiation, although the expression of CD11b was slightly induced by VD3. These results suggest that the cell line was impaired in the signal transduction systems of ATRA and VD3. This cell line should be useful for the study of the role of CBP as a transcriptional regulator in leukemia differentiation and for the functional analysis of the MLL-CBP fusion gene, which will provide new insights into leukemogenesis caused by 11q23 translocations.