Prognostic factors for mature natural killer (NK) cell neoplasms: aggressive NK cell leukemia and extranodal NK cell lymphoma, nasal type.

BACKGROUND: Patients with natural killer (NK) cell neoplasms, aggressive NK cell leukemia (ANKL) and extranodal NK cell lymphoma, nasal type (ENKL), have poor outcome. Both diseases show a spectrum and the boundary of them remains unclear. The purpose of this study is to draw a prognostic model of total NK cell neoplasms. PATIENTS AND METHODS: We retrospectively analyzed 172 patients (22 with ANKL and 150 with ENKL). The ENKLs consisted of 123 nasal and 27 extranasal (16 cutaneous, 9 hepatosplenic, 1 intestinal and 1 nodal) lymphomas. RESULTS: Complete remission rate for ENKL was 73% in stage I, but 15% in stage IV, which was consistent with that for ANKL (18%). The prognosis of ENKL was better than that of ANKL (median survival 10 versus 1.9 months, P < 0.0001) but was comparable when restricted to stage IV cases (4.0 months, P = 0.16). Multivariate analysis showed that four factors (non-nasal type, stage, performance status and numbers of extranodal involvement) were significant prognostic factors. Using these four variables, an NK prognostic index was successfully constructed. Four-year overall survival of patients with zero, one, two and three or four adverse factors were 55%, 33%, 15% and 6%, respectively. CONCLUSION: The current prognostic model successfully stratified patients with NK cell neoplasms with different outcomes.

Direct association of adenosine deaminase with a T cell activation antigen, CD26.

CD26, the T cell activation molecule dipeptidyl peptidase IV (DPPIV), associates with a 43-kilodalton protein. Amino acid sequence analysis and immunoprecipitation studies demonstrated that this 43-kilodalton protein was adenosine deaminase (ADA). ADA was coexpressed with CD26 on the Jurkat T cell lines, and an in vitro binding assay showed that the binding was through the extracellular domain of CD26. ADA deficiency causes severe combined immunodeficiency disease (SCID) in humans. Thus, ADA and CD26 (DPPIV) interact on the T cell surface, and this interaction may provide a clue to the pathophysiology of SCID caused by ADA deficiency.

The fabrication and testing of electrospun silica nanofiber membranes for the detection of proteins.

In this study, we fabricated electrospun silica nanofiber membranes and investigated their use in biomolecular sensing. The diameter, porosity and surface-to-volume ratio of nanofiber membranes were investigated under different fabrication conditions. Using this type of nanofiber membrane, enzyme-linked immunosorbent assay (ELISA) was performed, and the results were compared with those obtained with conventional ELISA using polystyrene well plates. The minimum detectable concentration was determined as 0.19 ng ml(-1) (1.6 pM), which is 32 times lower than that of conventional ELISA. In addition, the detection time for all processes for the nanofiber membrane was reduced to 1 h, compared with 1 day for conventional ELISA. The increased sensitivity, faster reaction time, and affordability of the nanofiber membrane make it well suited for bio-chip use.

Myeloablative cord blood transplantation for adults with hematological malignancies using tacrolimus and short-term methotrexate for graft-versus-host disease prophylaxis: single-institution analysis.

We studied clinical outcomes of 25 adult patients with hematological malignancies who underwent cord blood transplantation (CBT) after a myeloablative conditioning regimen, including high-dose cytosine arabinoside (CA) (8 g/m(2)), cyclophosphamide (CY) (120 mg/kg), and total-body irradiation (TBI) (12 Gy). For graft-versus-host disease (GVHD) prophylaxis, all patients received a combination of tacrolimus and short-term methotrexate (sMTX). Neutrophil engraftment was achieved in 20 of 25 patients. Of the 22 evaluable patients, 2 and 7 had grades I and II acute GVHD, respectively, and only 1 developed grade III acute GVHD after discontinuation of tacrolimus due to encephalopathy. Chronic GVHD developed in 13 of 19 evaluable patients, including 4 with the extensive type. However, the Karnofsky scores of survivors at 1 year after CBT were 90% or 100%. Eight of 25 patients died of nonrelapse causes (n = 4) and relapse/progressive disease (n = 4); 17 patients are currently alive with 15 free of disease at the present time (median follow-up, 24 months). The probability of disease-free survival at 2 years among patients with standard risk was 89% and that of high-risk patients was 30%. Transplantation-related mortality within 100 days was 12%. These results suggested that the CA/CY/TBI combination is a promising conditioning regimen for myeloablative CBT. Furthermore, tacrolimus and sMTX seemed to have suppressed severe acute GVHD and chronic GVHD, which may also contribute to the favorable results.

Hematopoietic stem cell transplantation for natural killer-cell lineage neoplasms.

Neoplasms of natural killer (NK)-lineage are rare. Their prognosis is generally poor except for cases of solitary nasal NK-cell lymphoma. The NK-cell Tumor Study Group performed a survey in Japan on patients diagnosed between 1994 and 1998. Of 228 patients selected for analysis, 40 underwent HSCT (15 allografts and 25 autografts). The underlying diseases were myeloid/NK cell precursor acute leukemia (n = 4), blastic NK-cell lymphoma (n = 11), aggressive NK-cell leukemia (n = 3), and nasal-type extranodal NK-cell lymphoma (n = 22). At the time of HSCT, 22 patients were in complete remission (CR), 11 were in relapse, and seven were primary refractory. All patients received myeloablative conditioning regimens including total-body irradiation. Sixteen died of disease progression, and six of treatment-related causes. Overall, 4-year survival was 39% with a median follow-up of 50 months; this was significantly better than that of patients who did not undergo HSCT (21%, P = 0.0003). For patients transplanted in CR, the 4-year overall survival was 68%, which was significantly better than that of patients who went into CR but did not undergo HSCT (P = 0.03). These findings suggest that the HSCT is a promising treatment strategy for NK-cell lineage.

Overexpression of AML1 renders a T hybridoma resistant to T cell receptor-mediated apoptosis.

The AML1 gene, which encodes the DNA binding subunit of the heterodimeric transcription factor, PEBP2/CBF, is involved in several types of chromosomal translocations associated with human acute myeloid leukemia, and has been shown by gene targeting to be essential for the development of definitive hematopoiesis in the murine fetal liver. In addition, the gene is expressed abundantly in T lymphocytes and has been implicated in T cell specific gene expression. In the present study we examined the function of AML1 in T cell receptor (TCR)-mediated, Fas/Fas-ligand dependent apoptosis of a T hybridoma line, DO11.10. Several independent cell clones overexpressing the AML1 protein were isolated by transfecting AML1 cDNA into these cells. These clones possessed an increased level of PEBP2/CBF DNA binding activity and were found to be resistant to apoptosis induced by anti-CD3 antibody treatment. Northern blot analysis revealed that induction of the Fas-ligand transcript was markedly suppressed in the anti-CD3 treated clones. Instead, expression of IL-2 receptor alpha subunit (IL-2R alpha), which is a manifestation of proliferative TCR signaling, was induced. This was in contrast to the parental, anti-CD3 treated DO11.10 cells where induction of Fas-ligand but not of IL-2R alpha was observed. Resistance of the AML1 overexpressing cell clones to TCR-mediated apoptosis is most likely attributable to the lack of Fas-ligand induction, since simultaneous treatment with anti-CD3 and anti-Fas antibodies caused apoptosis of the clones. The overall results suggest that the AML1 protein may play a pivotal role in switching TCR signaling between apoptosis and cell proliferation in T lymphocytes.

A cutaneous agranular CD2- CD4+ CD56+ "lymphoma": report of two cases and review of the literature.

We report 2 cases of agranular CD2- CD4+ CD56+ non-Hodgkin lymphoma in which skin seemed to be the primary site. A 21-year-old woman's initial symptom was a skin nodule on the right cheek. She also had tumors in the nasopharynx, and the bone marrow subsequently became involved. No lymphadenopathy was present. She experienced complete remission after dose-intensified therapy with cyclophosphamide, hydroxydaunomycin, vincristine [Oncovin], and prednisone (CHOP), but the disease relapsed in the central nervous system 6 months later. An 81-year-old man experienced an 11-month history of skin nodules in the left forearm. On admission, he had a bone marrow infiltration of lymphoma cells. He died of pneumonia during chemotherapy. The malignant cells of the 2 patients had similar morphologic features, with a monocytoid nucleus and no cytoplasmic granules. The cells in both cases showed a unique phenotype: CD2-, CD3-, CD4+, CD8-, CD13-, CD14-, CD34-, CD16-, CD56+, CD57-, HLA-DR-positive. Staining for peroxidase and alpha-naphthyl butyrate esterase was negative. The T-cell receptor beta, gamma, delta, IgH, kappa, lambda genes were of germ line configurations. The DNA of Epstein-Barr virus was not detected from the bone marrow cells by polymerase chain reaction. Only 3 other cases with similar phenotypes have been reported; all had skin lesions. Although the origin of these cells remains unknown, we propose that this is a distinct clinicopathologic entity.

A novel translocation involving chromosomes 2, 9, 14, and 22 in chronic myeloid leukemia.

A 46-year-old man with chronic myelogenous leukemia was found to have a new complex translocation. In chronic phase, all of the bone marrow cells had a rearrangement of a t(2;9;14;22) (p21;q34;q32;q11). Southern blot analysis of leukocyte DNA revealed rearrangement of the breakpoint cluster region (bcr) within the 5.8-Kb bcr. The patient eventually died in blast crisis 28 months later. The cytogenetic findings of bone marrow cells showed a 46,XY,t(2;9;14;22)(p21;q34;q32;q11),add(1p),del(3q) karyotype in blast crisis.

Clonal evolution from trisomy into tetrasomy of chromosome 8 associated with the development of acute myeloid leukemia from myelodysplastic syndrome.

Tetrasomy 8, though rare, is usually associated with trisomy 8, a far more common chromosomal abnormality in acute myeloid leukemia (AML). Yet the clonal relationship between trisomy 8 and tetrasomy 8 in the cases with these chromosomal abnormalities has been unclear. Here, we report a case of a 17-year-old male, diagnosed as having a myelodysplastic syndrome (MDS). Chromosome analysis showed the presence of trisomy 8. Five years later, he developed overt AML exhibiting tetrasomy 8 only. After chemotherapy, the blast cells in the bone marrow decreased to 3.4%, and the karyotype showed trisomy 8 alone. Fluorescence in situ hybridization using a probe specific for chromosome 8 showed that the percentages of cells exhibiting 2/ 3 /4 signals were 7.8/89.2/2.0 at the MDS stage, 20.5/36.1/41.0 when overt AML developed and 24.0/72.1/2.4 after chemotherapy. These results suggested that tetrasomy 8 is derived from the AML clone, possibly evolved from the MDS clone with trisomy 8. To our knowledge, this is the first detailed case report of clonal evolution from trisomy 8 into tetrasomy 8 associated with the development of AML from MDS.

Reassessment of non-hodgkins's lymphoma with a "nodular" growth variant: a clinicopathologic study of follicular, mantle cell and marginal zone lymphomas prospectively diagnosed with multiparameter analyses.

Although three subtypes of non-Hodgkin's lymphoma (NHL), follicular lymphoma (FL), mantle cell lymphoma (MCL) and marginal zone lymphoma (MZL), are now well recognized as independent categories, their biological behavior has not been fully compared. One of the reasons for this may be that subclassification by histological examination alone is often difficult since they all have a common variant of a "nodular" growth pattern and occasionally show similar cytological morphology. Recently, we reviewed patients with FL, MCL and MZL, who were prospectively diagnosed, using multiparameter analyses with unfixed fresh biopsy materials. Of 407 NHL patients, 101 (24.8%) belonged to these three categories and 80 could be followed; FL (n=27), MCL (n=27) and MZL (n=26). Twenty eight cases with diffuse large B-cell (DL-B) lineage lymphoma were selected as control at random. The frequency of the MCL patients with performance status (PS) 2 to 4 (41%) was significantly higher than MZL patients (4%) [P< 0.001]. The 3 year survival rate with FL, MCL, MZL and DL-B was 71.5%, 57.4%, 93.3% and 53.1%, respectively. The survival rate for MZL was significantly better than both FL (p = 0.048) and MCL (p = 0.0085). Significant differences were also found in the overall survival rates among the four risk groups as defined by the International Index [I2](low, low-intermediate, high-intermediate and high; 97.4%, 79.6%, 39.4% and 18.2%, respectively). A multivariate analysis revealed that the International Index may be a significant predictor for short survival (p=0.0001) in the patients with FL, MCL or MZL. These results suggest that MZL shows an apparently better prognosis than FL and MCL and is found to be a prognostically independent category. In contrast, the clinical outcome in MCL is the worst among the three subtypes and was closer to that of DL-B. The International Index can be applied to a wide spectrum of NHL, including MCL, MZL and FL, to and can predict prognosis in these cases.

[Essential thrombocythemia transformed to acute myeloblastic leukemia].

A 48-year-old woman was referred to Tohoku University Hospital in November 1981 because of leukocytosis pointed out in a group examination. At that time white blood cell count was 26.8 x 10(3)/microliters with no blasts, platelet count 268.0 x 10(4)/microliters and hemoglobin 11.4 g/dl. Bone marrow aspirates showed marked increase of megakaryocytes (15,900/microliters). Bone marrow chromosome analysis revealed 46, XX, -18, +mar without Ph1 chromosome, and DNA analysis showed no bcr rearrangement. She was diagnosed as having essential thrombocythemia and was treated with busulfan. On November 1986, she developed remarkable leukocytosis with leukemic blasts. White blood cells reached 153 x 10(3)/microliters with 33% blasts. Her blasts were positive for peroxidase staining, but negative for platelet peroxidase on electron microscopic study and platelet specific glycoproteins. A diagnosis of acute myeloblastic leukemia (M2) was made. The patient received various combination chemotherapy, which was ineffective, and she died due to pneumonia on June, 1989. In Japan, there has been reported only 8 cases of essential thrombocythemia transformed to acute leukemia. The clinical pictures of these 9 cases were discussed.

Significance of colonoscopy in patients with intestinal graft-versus-host disease after hematopoietic stem cell transplantation.

BACKGROUND AND STUDY AIMS: Patients with intestinal graft-versus-host disease (GVHD) are generally in poor clinical condition. In this study we aimed to establish the clinical significance of endoscopic diagnosis of this condition, observing only the distal section of the large intestine. PATIENTS AND METHODS: Endoscopic and pathological findings at colonoscopy were evaluated retrospectively in 12 patients who were diagnosed with intestinal GVHD after undergoing hematopoietic stem cell transplantation. RESULTS: The main mucosal changes observed at endoscopy were granular change, edema, "spotty redness", and sloughing. These were clearly displayed after enhancement with Indigo carmine staining, and with insertion of the colonoscope only as far as the distal section of the large intestine. A histological diagnosis of intestinal GVHD was made in 50 % of the patients, whose intestinal epithelium specimens showed numerous apoptotic bodies. It was possible to perform total colonoscopy in two patients who were in relatively good condition clinically, but there were no remarkable differences in the endoscopic findings throughout the large intestine, from the terminal ileum to the rectum. In terms of clinical outcomes of the 12 patients, their prognosis was poor in that they all either went on to suffer from chronic GVHD or died. CONCLUSIONS: Endoscopic and histological findings on distal colonoscopy are clinically significant in the diagnosis of intestinal GVHD, and limiting this examination to the distal section of the large intestine avoids causing further clinical deterioration in patients who are already in very poor general condition and the possibility of causing endoscopy-related complications.

Bone marrow stromal cells selectively stimulate the rapid expansion of lineage-restricted myeloid progenitors.

Bone marrow stromal cells serve hematopoietic microenvironments where different blood cells are controlled in their growth and differentiation. To characterize functions of stromal cells, 33 bone marrow stromal cells including preadipocytes, endothelial cells, and fibroblasts were established from transgenic mice harboring temperature-sensitive SV40 T-antigen gene and their selective stimulatory abilities to support large colony formation of lineage-specific hematopoietic progenitor cells (erythroid, monocyte/macrophage, granulocyte, and monocyte-granulocyte) were examined. Among established stromal cells, 27 clones showed erythropoietic stimulatory activity in the presence of erythropoietin. On myeloid progenitors, the stromal cells showed lineage-restricted stimulatory activity and a reciprocal relationship was observed between granulocyte formation and macrophage formation, but these activities were not dependent on the amount of produced colony-stimulating factors (CSFs). Our present study with many stromal cells established from bone marrow indicated that each stromal cell in the bone marrow may provide the preferable microenvironment for a rapid expansion of the lineage-restricted progenitor cells in combination with CSFs.

Edwardsiella tarda septicemia complicating acute leukemia.

We describe here an unusual case of Edwardsiella tarda septicemia preceded by acute gastroenteritis in a patient with acute leukemia in complete remission receiving maintenance chemotherapy. She also had ulcerated rectal cancer with bleeding, which was thought to serve as the portal of entry into the blood stream. She was successfully treated with cefmetazole and gentamicin in combination. However, the concomitant resolution of chemotherapy-induced granulocytopenia may have had a favorable effect as well.

A polymeric microfluidic chip for CE/MS determination of small molecules.

A polymeric microfluidic chip made of Zeonor 1020 was fabricated using conventional embossing techniques to perform capillary electrophoresis for selected ion monitoring and selected reaction monitoring mass spectrometric detection of small molecules. A silicon master was microfabricated using photolithographic and dry etching processes. The microfluidic channel was embossed in the plastic from a silicon master. The embossed chip was thermally bonded with a Zeonor 1020 cover to form an enclosed channel. This channel (60-microm width, 20-microm depth, 2.0- and 3.5-cm length) provided capillary electrophoresis (CE) separation of polar small molecules without surface treatment of the polymer. A microsprayer coupled via a microliquid junction provided direct electrospray mass spectrometric detection of CE-separated components. An electric field of 0.5-2 kV/cm applied between the microsprayer and a separation buffer reservoir produced a separation of carnitine, acylcarnitine, and butylcarnitine with separation efficiencies ranging from 1,650 to 18,000 plates. Injection quantities of 0.2 nmol of these compounds produced a separation of the targeted polar small molecules without surface treatment of the polymer-abundant ion current signals and baseline separation of these compounds in less than 10 s. These results suggest the feasibility of polymeric chip-based devices for ion spray CE/MS applications.

Cross-linking of CD26 by antibody induces tyrosine phosphorylation and activation of mitogen-activated protein kinase.

CD26, a T-cell activation antigen that has dipeptidyl peptidase IV activity in its extracellular domain and has also been shown to play an important role in T-cell activation. The earliest biochemical events seen in stimulated T lymphocytes activated through the engagement of the T-cell receptor (TCR) is the tyrosine phosphorylation of a panel of cellular proteins. In this study we demonstrate that antibody-induced cross-linking of CD26-in CD26-transfected Jurkat cells induced tyrosine phosphorylation of several intracellular proteins with a similar pattern to that seen after TCR/CD3 stimulation. Herbimycin A, an inhibitor of the src family protein tyrosine kinases dramatically inhibited this CD26-mediated effect on tyrosine phosphorylation. Major tyrosine phosphorylated proteins were identified by immunoblotting, and included p56lck, p59fyn, zeta associated protein-tyrosine kinase of 70,000 MW (ZAP-70), mitogen-activated protein (MAP) kinase, c-Cb1, and phospholipase C gamma. CD26-induced tyrosine phosphorylation of MAP kinase correlated with increased MAP kinase activity. In addition, CD26 was costimulatory to CD3 signal transduction since co-cross-linking of CD26 and CD3 antigens induced prolonged and increased tyrosine phosphorylation in comparison with CD3 activation alone. We therefore conclude that CD26 is a true costimulatory entity that can up-regulate the signal transducing properties of the TCR.