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Optimal salvage chemotherapy has not been established for lymphoid malignancy, which is refractory to the conventional cyclophosphamide, doxorubicin, vincristine, and prednisone regimen. To explore an effective regimen, we conducted a phase I pilot study of combination chemotherapy with methotrexate, ifosfamide, l-asparaginase and dexamethasone (MILD), which are unaffected by MDR1-encoded P-glycoprotein. A total of 18 patients with lethal lymphoid malignancy were enrolled over a 2-yr period. The median age was 63 yr. Eleven patients had T/NK-cell malignancies, six had B-cell malignancies, and one was diagnosed with a blastic plasmacytoid dendritic cell neoplasm. Patients aged >/=60 and <60 yr were planned to receive a set of starting doses of methotrexate and ifosfamide, which should induce myelosuppression. Eleven patients completed two courses of MILD therapy. Treatment-related death because of systemic mucormycosis was observed in one patient. Major treatment-related adverse events were grade 3 or more hematologic toxicities, which included lymphopenia corresponding to dose-limiting toxicity. The most common grade 3 non-hematologic toxicity was febrile neutropenia. Of the 14 evaluated patients, three achieved a complete response, and four showed a partial response. The overall response rate was 57%. It was very interesting that all of seven responders had T/NK-cell malignancies. MILD therapy was feasible and presented acceptable toxicity in patients with refractory or lethal lymphoid malignancies. The efficacy for T/NK-cell malignancies should be further evaluated.

Splenic marginal zone lymphoma (SP-MZL) is a rare low-grade B-cell neoplasm that often shows leukemic manifestation. Less than 20% of cases of SP-MZL express CD5. We analyzed 11 cases of CD5-positive SP-MZL with leukemic manifestation. The clinical characteristics of these cases did not differ from those of CD5-negative SP-MZL. Flow cytometry revealed positive results as follows : CD3, 0/9 ; CD5, 11/11 ; CD10, 0/11 ; CD11c, 4/10, CD13, 5/11 ; CD19, 11/11 ; CD20, 10/11 ; CD21, 4/4 ; CD22, 7/7 ; CD23, 5/10 ; CD25, 8/11 ; FMC7, 5/7 ; κ type 6/9, and λ type 2/9. All 3 cases with monoclonal γ-globulinemia expressed CD13. Resected spleen exhibited a proliferation of neoplastic cells in white pulp in all 8 splenectomy patients and a marginal pattern was detected in 5 patients. Only 2 cases showed involvement of red pulp. Immunohistochemistry showed that the lymphoma cells were positive for CD5, CD20, and BCL-2 and negative for CD3, CD10, cyclin D1, BCL-6, and MUM-1 in all 11 cases. These results suggest that CD5-positive SP-MZL differs from B-cell chronic lymphocytic leukemia, that CD13 expression is found in about half of CD5-positive SP-MZL cases, and that CD5-positive SP-MZL may be related to memory B-cell neoplasm or plasma cell differentiation.

Based on the presence of the tumor-specific over-expression of Plk1 (polo-like kinases) in various malignancies, we examined Plk1 expression in nine cases of reactive follicular hyperplasia (RFH), 42 of diffuse large B cell lymphoma (DLBCL), 16 of follicular lymphoma (FL), and 10 of nasal NK/T lymphoma. There was no significant difference in the Plk1-positive cell percentage between RFH and DLBCL. The Plk1-positive cell percentage ranged from 6 to 20% with a median of 12.9% in DLBCL. In FL, Plk1-positivity was at most 7%. Plk1-positivity in nasal NK/T cell lymphoma (4.7-14.1% with a median of 9.2%) was significantly higher than that of FL and tended to be lower than DLBCL (p < 0.001, p = 0.05, respectively). Although a strong correlation between positive cell percentages for Plk1 and Ki-67 in these three lymphomas specified Plk1 as a proliferation marker (r = 0.83-0.91), the Plk1-positive cell percentage relative to the other proliferation markers tended to be particularly low in nasal NK/T cell lymphoma. In 41 cases of DLBCL, the positive cell percentages of Plk1 and Ki-67 were both correlated with overall survival. The 4-year overall survival rates by Kaplan-Meier analysis for Plk1-negative and positive patients were 80 and 38%, respectively (p = 0.02).

We present here clinical and hematological findings of 52 cases of granular lymphocyte-proliferative disorder (GLPD), which contained 35 indolent T-cell lineage granular lymphocyte-proliferative disorder (T-GLPD), two atypical T-GLPD, 12 chronic NK-cell lymphocytosis (CNKL), and three aggressive NK-cell leukemia (ANKL). The median period of follow up was 24 months. Hemoglobin level <8.0 g/dL was recognized in 21 cases of indolent T-GLPD (60%), among which 15 patients met the criteria of pure red cell aplasia. Neutrophil counts <500/microL occurred only in two cases of T-GLPD (6%). Although the median age and male-to-female distribution were similar, very frequent anemia and rare neutrocytopenia in indolent T-GLPD in the present study keenly contrasted with previous reports. CD56 was positive in three of 29 indolent T-GLPD cases with CD4-CD8+ phenotype, in three of four CD4+CD8-, and in none of two CD4-CD8- cases. Therefore, although two atypical T-GLPD cases were CD56-positive, CD56 should not be a specific marker for aggressive T-GLPD. All CNKL patients had a chronic course with a stable granular lymphocyte count. All three ANKL patients presented high fever and hepatosplenomegaly, barely responded to chemotherapies and died within 6 months. The present analysis of 52 cases of GLPD in Japan showed that Japanese and Western cases of indolent T-GLPD clearly differ in their hematological complications.

It is currently accepted that activity of the integrin alpha(IIb)beta(3) is modulated by direct interaction between its cytoplasmic tails (CTs) and association of cytoplasmic proteins with them, and disruption of the close linkage between CTs leads to activation of alpha(IIb)beta(3) (inside-out signaling). We previously reported that ancient ubiquitous protein (Aup1) binds to the membrane-proximal sequence of integrin alphaCTs that plays a pivotal role in the inside-out signaling. To explore biological function of Aup1, we examined in this study interaction of Aup1 with Src and Syk that are quickly activated in platelets before fibrinogen binding following thrombin stimulation. By immunoprecipitation assay with resting platelets, we first found that alpha(IIb)beta(3), Src, Syk, and Aup1 are constitutively complexed. In vitro binding study with recombinant Syk and glutathione (GSH) S-transferase (GST) - Src, -Aup1, and -alpha(IIb) and - beta(3) CTs that are immobilized to GSH- beads revealed direct binding of Syk to Aup1 as well as the beta(3) CT. Dot blot analysis with synthetic peptides for alpha(IIb) and beta(3) CTs, and GST-Aup1 and -Src immobilized to PVDF membrane exhibited concordant result with the GST pull-down assay. Immunoprecipitation of platelet lysates 10 seconds after thrombin stimulation, when activity and tyrosine phosphorylation of Syk are maimum, exhibited that active Syk does not coprecipitate with Aup1. In vitro kinase assay with GST-Syk and -Aup1 proteins at the presence or absence of active Src, a potent activator of Syk, revealed that Aup1 does not directly influence activation of Syk by autophosphorylation or tyrosine phosphorylation by Src. These results indicate that Aup1 is an adaptor recruiting Syk to the alpha(IIb) CT, and suggest that the alpha(IIb) -Aup1- Syk- beta(3) complex formation links alpha(IIb) and beta(3) CTs to sustain alpha(IIb)beta(3) in an inactive state and Syk dissociates from Aup1 after activation.

Objectives: Nasal natural killer (NK)/T-cell lymphoma is characterized by chemo-resistance, angiodestruction, and aggressive tumor progression. Few studies exist on molecular characteristics of this disease entity., Methods: Expression levels of major apoptosis-related proteins Bcl-2, Bcl-x, Mcl-1, Bax, and a proliferative marker Ki-67 were analyzed in 11 nasal NK/T-cell lymphoma cases by immunohistochemical methods. Nine cases were of NK-cell lineage and two cases were of T-cell lineage. For comparison, 12 follicular lymphoma (FL) cases and 16 diffuse large B-cell lymphoma (DLBCL) cases were also studied., Results and Conclusions: Bax expression was low in all nasal NK-cell lymphoma cases, which constitute the major population of nasal NK/T-cell lymphoma. Bax expression in nasal NK-cell lymphoma was similar to FL and significantly lower compared with DLBCL. Bcl-2 expression was significantly lower in nasal NK/T-cell lymphoma compared with that of FL and DLBCL. Bcl-x expression was high in all three lymphomas. Two distinct Mcl-1 expression groups existed for nasal NK/T-cell lymphoma (6.2 +/- 5.2% and 59.1 +/- 12.3%, 95% CI). Ki-67 expression was high in nasal NK/T-cell lymphoma, and worse prognostic groups tended to express higher levels of Ki-67. The results suggest a combination of impaired apoptosis and aggressive proliferation in nasal NK/T-cell lymphoma, and may provide explanations for its poor prognosis.

Background/aims: Secondary hemophagocytic syndrome (hemophagocytic lymphohistiocytosis, HLH) follows viral infection, malignant disorders, and autoimmune disease. Criteria for HLH diagnosis, which were proposed in 2004, include hypertriglyceridemia. However, some studies reported the absence of hypertriglyceridemia in patients with secondary HLH, differing from those with primary HLH., Subjects and Methods: In this study, we investigated the presence or absence of hypertriglyceridemia in 28 patients who were diagnosed with secondary HLH between 1997 and 2007 retrospectively. There were no patients undergoing treatment for those with a history of hyperlipidemia., Results: The subjects consisted of 14 patients with lymphoma-associated HLH, 11 with virus-associated HLH, 2 with autoimmune disease-associated HLH, and 1 with post transplantation HLH. In 19 patients (68%), hypertriglyceridemia was noted on diagnosis or during the disease period (mean: 242 mg/dL). Furthermore, the triglyceride (TG) level decreased with the treatment-related amelioration of HLH (mean level before and after treatment: 297 and 136 mg/dL, respectively, p=0.0001)., Conclusion: These results suggest that the TG level is useful for diagnosing HLH and evaluating the treatment response. TG measurement is simple and inexpensive; therefore, this parameter can be determined several times to evaluate the treatment response.

Extranodal natural killer (NK)/T cell lymphoma, nasal type, is a recently recognized distinct entity and the most common type of non-B cell extranodal lymphoma in Asia. This retrospective analysis studied the potential survival benefits of hematopoeitic stem cell transplantation (HSCT) compared with a historical control group. A total of 47 patients from 3 previously published series of HSCT were matched according to NK/T cell lymphoma International Prognostic Index (NKIPI) risk groups and disease status at transplantation with 107 patients from a historical control group for analysis. After a median follow-up of 116.5 months, the median survival time was not determined for the HSCT group, but it was 43.5 months for the control group (95% confidence interval [CI] = 6.7 to 80.3 months; P = .127, log-rank test). In patients who were in complete remission (CR) at the time of HSCT or at surveillance after remission, disease-specific survival rates were significantly higher in the HSCT group compared with the control group (disease-specific 5-year survival rate, 87.3% for HSCT vs 67.8% for non-HSCT; P = .027). In contrast, in subgroup analysis on non-CR patients at the time of HSCT or non-HSCT treatment, disease-specific survival rates were not significantly prolonged in the HSCT group compared with the control group (1-year survival rate, 66.7% for HSCT vs 28.6% for non-HSCT; P = .141). The impact of HSCT on the survival of all patients was significantly retained at the multivariate level with a 2.1-fold (95% CI =1.2- to 3.7-fold) reduced risk of death (P = .006). HSCT seems to confer a survival benefit in patients who attained CR on postremission consolidation therapy. These findings suggest that, in particular, patients in CR with high NKIPI risk scores at diagnosis should receive full consideration for HSCT.

Imatinib at a daily dose of 400 mg is the standard treatment for chronic myelogenous leukemia in the chronic phase. However, the feasibility of this dose for small Japanese adults has not been clarified. We prospectively investigated the toxicity and efficacy of this dose in adult Japanese patients. Among the 89 evaluable patients with a median body weight of 62.8 kg, imatinib therapy was held in 40 subjects (45%), due to Grade 3-4 toxicities in 30 patients (75%) and Grade 2 toxicities at the discretion of the attending physician in 10 patients (25%). However, treatment was resumed and the dose was gradually increased until 62 of the 89 patients tolerated a maintenance dose of 400 mg. Older age and lower body weight were significant independent risk factors for discontinuation of imatinib. After a median follow-up period of 31 months, 84 patients were alive without progression. The complete cytogenetic response rate was 60 and 90% at 6 months and 1 year after starting imatinib, respectively. Older patients and those with a lower body weight were less likely to achieve a complete cytogenetic response. These findings suggest that the body weight has a significant influence on the toxicity and efficacy of imatinib in patients with a small body size, although dose reduction in proportion to weight may result in an inadequate response to imatinib., (Copyright 2008 Wiley-Liss, Inc.)

Checkpoint protein Chk1 has been identified as an Hsp90 client. Treatment with 100 nM geldanamycin (GM) for 24 h markedly reduced the Chk1 amount in Jurkat and ML-1 leukemia cell lines. Because Chk1 plays a central role in G2 checkpoint, we added GM to G2-arrested Jurkat and HL-60 cells pretreated with 50 nM doxorubicin for 24 h. GM slowly released both cell lines from doxorubicin-induced G2 arrest into G1 phase. GM also abrogated ICRF-193-induced decatenation G2 checkpoint in Jurkat and HL-60 cells. Western blot analysis showed that addition of GM attenuates doxorubicin- and ICRF-193-induced Chk1 phosphorylation at Ser345. GM, however, failed to abrogate G2 arrest in p53-positive ML-1 cells maybe due to the p21 induction. GM released HeLa cells from doxorubicin-induced G2 arrest but trapped them at M phase. Flow cytometric analysis showed that addition of GM converted doxorubicin-induced necrosis into apoptosis in Jurkat cells. Colony assay indicated that although GM has a weak cytotoxic effect as a single agent, it dramatically intensifies the cytotoxicity of doxorubicin and ICRF-193 in Jurkat and HL-60 cells. These results suggest that abrogation of G2 checkpoint by GM may play a central role in sensitizing p53-negative tumor cells to DNA-damaging and decatenation-inhibiting agents.

A 63-year-old man who was incidentally found to have thrombocytopenia at a periodic physical examination visited our hospital. The spleen was palpable 3 finger-breadths below the navel level, and the liver was palpable 1 finger-breadth below the right costal margin. Peripheral blood examination showed WBC 2,900/microl, Hb 13.4 g/dl, and platelets 54 X 10(3)/ microl. Gaucher cells were recognized in the bone marrow by aspiration, and serum levels of total acid phosphatase and angiotensin converting enzyme were increased. Glucocerebrosidase activity was lower than the control level in bone marrow stroma cells, and modification of glucocerebrosidase genotype N188S was shown, which had been identified in the past. Furthermore, neurological examination was normal. Based on these results, we diagnosed the patient with Gaucher disease type I, and started enzyme replacement therapy. Gaucher disease is rare in Japanese, approximately 100 cases having been reported; diagnosis at older age is also relatively rare and, as far as we know, the oldest age reported in Japanese was 57 years old. Gaucher disease should be considered a differential diagnosis when thrombocytopenia and splenomegaly are found in elderly patients, although it is relatively rare.

Extranodal natural killer (NK)/T-cell lymphoma, nasal type, and aggressive NK-cell leukemia are rare, and their standard therapy has not been established. They are Epstein-Barr virus-associated lymphoid malignancies, and tumor cells express P-glycoprotein leading to multidrug resistance of the disease. Patients with stage IV, relapsed or refractory diseases have a dismal prognosis, with survival measured in months only. To develop an efficacious chemotherapeutic regimen, we conducted a dose-escalation feasibility study of a new chemotherapeutic regimen, SMILE, comprising the steroid dexamethasone, methotrexate, ifosfamide, L-asparaginase, and etoposide. The components of SMILE are multidrug resistance-unrelated agents and etoposide. Etoposide shows both in vitro and in vivo efficacy for Epstein-Barr virus-associated lymphoproliferative disorders. Eligible patients had newly diagnosed stage IV, relapsed or refractory diseases after first-line chemotherapy, were 15-69 years of age, and had satisfactory performance scores (0-2). Four dose levels of methotrexate and etoposide were originally planned to be evaluated. At level 1, six patients with extranodal NK/T-cell lymphoma, nasal type, were enrolled. Their disease status was newly diagnosed stage IV (n = 3), first relapse (n = 2), and primary refractory (n = 1). All of the first three patients developed dose-limiting toxicities, and one of them died of sepsis with grade 4 neutropenia. A protocol revision stipulating early granulocyte colony-stimulating factor administration was made. Two out of three additional patients developed dose-limiting toxicities that were all manageable and transient. For the six enrolled patients, the overall response rate was 67% and the complete response rate was 50%. Although its safety and efficacy require further evaluation, we recommend a SMILE chemotherapy dose level of 1 for further clinical studies.

To clarify the clinical features of adult patients with acute leukemia (AL) with 11q23 abnormalities, we performed a retrospective analysis of data from 58 adult Japanese patients: 51 with acute myeloid leukemia (AML), and 7 with acute lymphoblastic leukemia (ALL). The incidences according to fusion partners in AML were: t(9;11), 31.3%; t(11;19), 27.4%; t(6;11), 21.5%. The incidence of patients with t(11;19) was higher than those in the US and Europe, and the incidence of t(4;11) was lower than that in childhood. The results indicated the poor prognosis of AML with 11q23 abnormalities regardless of the fusion partners. AML patients with 11q23 aged <60 years in the first CR who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) showed a more favorable outcome than those treated without allo-HSCT, although the differences were not statistically significant (P = 0.322 for DFS, P = 0.138 for OS). This result suggests that treatment strategies including allo-HSCT may be considered in the first CR in cases of AML with 11q23 abnormalities. However, further studies involving a large number of cases are required to assess the effect of allo-HSCT on adult AL with 11q23 abnormalities.

The mitotic spindle checkpoint prevents the onset of anaphase and subsequent cell division until chromosomes are properly aligned on a bipolar spindle. Thus, it regulates the cell division cycle by keeping cells with defective spindles from leaving mitosis. The budding uninhibited by benzimidazole (Bub1) is a key component of mitotic checkpoint. Bub1 encodes a serine/threonine kinase required for mitotic spindle checkpoint function. The regulation of cell morphology in eukaryotic cells is a complex process involving major components of the cytoskeleton including actin microfilaments, microtubules, and intermediate filaments (IFs). Here we show that Bub1 directly affects the structural integrity of IFs. Constitutive expression of Bub1 caused disappearance of filamentous vimentin, a type III IF, and consequently changed cell morphology. Expression of kinase domain-deleted Bub1 induced neither morphological change nor disappearance of vimentin. These observations suggest that Bub1 not only regulates the cell cycle, but also may be involved in the cytoskeletal control in interphase cells.

A 72-year-old Japanese man presented with 43.1 x 10(9)/l hairy cells and apparent splenomegaly. The leukemia cells had unevenly distributed microvilli and round nuclei with dense chromatin and one or two clear nucleoli, lacked CD25 expression and were negative for tartrate-resistant acid phosphatase. The case was diagnosed as hairy cell leukemia variant (HCLv) and proved refractory to various chemotherapies, including cladribine, pentostatin, interferon-alpha, CHOP and rituximab. Because of the CD52 expression, we treated the patient with alemtuzumab. Pretreatment with 22.5 Gy to the spleen reduced the spleen size from 12 to 4 cm below the left costal margin, and the number of circulating leukemic cells decreased from 229.0 to 63.6 x 10(9)/l. Subsequent administration of 24.0 mg of alemtuzumab eliminated leukemic cells in the peripheral blood on day 12, and the spleen was not palpable after the administration of 54.0 mg of alemtuzumab. In vitro treatment with alemtuzumab confirmed the cytotoxic effect against the patient's leukemic cells in the presence of complement. This is the first report showing clinical effectiveness of splenic irradiation and alemtuzumab against refractory HCLv., (2008 S. Karger AG, Basel)

To study the clinical characteristics of hypereosionophilic syndrome and chronic eosinophilic leukemia (HES/CEL) in Japan, the clinical data of 29 HES/CEL patients throughout the country were surveyed. Moreover, the involvement of the FIP1L1-alphaPDGFR fusion gene resulting from a cryptic del (4)(q12q12) was examined in 24 cases. The FIP1L1-alphaPDGFR messenger RNA (mRNA) was detected in three patients (13% of patients fulfilled WHO criteria and 17% of Chusid criteria). One had a novel fusion transcript, which skipped the exon 12 of alphaPDGFR. The transcript appears to be generated by a splicing mechanism that is different from the previously reported splicing patterns. In silico analysis, the exon skipping was not related to a disruption of the exonic splicing enhancers within the exon but strongly associated with the loss of the vast majority of the FIP1L intron 8a where intronic splicing enhancers were accumulated. Unexpectedly, pseudo-chimera DNA fragments with some shared characteristic features were occasionally generated from healthy control samples by reverse transcriptase polymerase chain reaction (RT-PCR). Considering the relatively low incidence of the FIP1L1-alphaPDGFR transcript positive case, extreme care must therefore be taken when making a diagnosis using RT-PCR before imatinib therapy.

The World Health Organization classification of haematolymphoid tumours recognizes three categories of natural killer (NK)-cell neoplasms: blastic NK-cell lymphoma, aggressive NK-cell leukaemia, and extranodal NK/T-cell lymphoma, nasal-type. Recent studies indicate that CD4+CD56+ blastic NK-cell lymphoma is of plasmacytoid dendritic cell origin, and true tumours of precursor NK-cell origin may be present mainly in the CD4-CD56+ subset. Myeloid/NK-cell precursor acute leukaemia may also develop from precursor NK cells. However, because the developmental pathway of normal NK cells is not well understood, tumours of precursor NK-cell origin are not clearly identified. Among mature NK-cell tumours, extranodal NK/T-cell lymphoma is relatively common in Asia and Latin America. In localized disease, chemoradiotherapy seems to be promising, and in advanced disease, new combination chemotherapies are under active investigation. Aggressive NK-cell leukaemia is rare and has a poor prognosis. Because NK-cell neoplasms are rare and difficult to manage, rigorous studies are required for their understanding and management.

Expression of neural cell-adhesion molecule CD56 is a rare event in B-cell lymphoma. We described four cases of CD56-positive B-cell lymphoma, including follicular lymphoma and diffuse large B-cell lymphoma. These lymphoma cells expressed CD10 and bcl-6, which suggests germinal center-stage phenotype. Immunohistochemistry showed that CD56-positive cells aggregated and displayed a cohesive growth pattern, indicating that homotypic adhesion through the molecules might affect the manner of tumor growth and expansion. Although CD56 expression level varies among the cases, this molecule might play some roles in the manner of growth and expansion of CD56-positive B-cell lymphomas.

Hemophagocytic lymphohistiocytosis (HLH), a disorder of the mononuclear phagocyte system, can be classified into two distinct forms: primary HLH (FHL) and secondary HLH. To clarify the epidemiology and clinical outcome for each HLH subtype, we conducted a nationwide survey of HLH in Japan. Since 799 patients were diagnosed in 292 institutions of Japan between 2001 and 2005, the annual incidence of HLH was estimated as 1 in 800,000 per year. Among them, 567 cases were actually analyzed in this study. The most frequent subtype was Epstein-Barr virus (EBV)-associated HLH, followed by other infection- or lymphoma-associated HLH. Age distribution showed a peak of autoimmune disease- and infection-associated HLH in children, while FHL and lymphoma-associated HLH occurred almost exclusively in infants and the elderly, respectively. The 5-year overall survival rate exceeded 80% for patients with EBV- or other infection-associated HLH, was intermediate for those with FHL or B-cell lymphoma-associated HLH, and poor for those with T/NK cell lymphoma-associated HLH (<15%). Although this nationwide survey establishes the heterogeneous characteristics of HLH, the results should be useful in planning prospective studies to identify the most effective therapy for each HLH subtype.

Extensive bone marrow necrosis and symptomatic hypercalcemia have been described independently as rare complications of chronic myeloid leukemia. Here we report a 66-year-old man who developed B cell blastic transformation 10 years after diagnosis of CML in the chronic phase. Extensive bone marrow necrosis and symptomatic hypercalcemia concurrently developed after transformation, with development of disseminated intravascular coagulation and multifocal osteolysis. Most necrotic cells were readily identifiable as blasts. Mediators related to hypercalcemia, including prostaglandin E2, transforming growth factor-alpha and transforming growth factor-beta, were significantly elevated in the serum. As far as we know, this is the first case report of chronic myeloid leukemia concurrently developing bone marrow necrosis and hypercalcemia; this association was not reported in other types of leukemia or bone marrow malignancies., (2007 S. Karger AG, Basel)

Bortezomib is a proteasome inhibitor that can be effective in the treatment of refractory and relapsed multiple myeloma. Recently, severe pulmonary complications associated with bortezomib therapy have been reported in Japan. Because bortezomib has not yet been approved for general use in Japan and is imported by attending physicians on the request of patients, The Japanese Society of Hematology and The Japanese Society of Clinical Hematology sent urgent questionnaires to the councilors of both societies to explore the situation and the details of pulmonary complications associated with bortezomib therapy. Clinical details were available for 46 patients who had been treated with personally imported bortezomib in Japan. Seven patients (15.2%), including 3 who died from respiratory failure, showed complications definitely or probably caused by bortezomib. Of the 7 patients, 6 had a prior history of stem cell transplantation (SCT), whereas only 14 of 39 patients without lung injury had received SCT treatment (P = .033, Fisher exact test). Multivariate analysis revealed that the concomitant use of corticosteroids might reduce the risk of lung injury (P = .024; odds ratio, 0.055) and that a previous SCT might increase the risk (P = .042; odds ratio, 13.140). We summarized these data from questionnaires for a limited Japanese cohort and therefore do not know the precise incidence of lung injury linked to fatal progression. Thus, future verification concerning this matter is warranted after the approval of bortezomib for use in Japan. Clinicians should be aware of the possibility of severe pulmonary complications associated with bortezomib therapy.

The feasibility and effects of combination chemotherapy for very elderly patients with acute myeloid leukemia was examined in 65 patients (including previous myelodysplastic syndrome) aged 76 years or morewith a performance status of 0 - 3. Induction chemotherapy was performed with 30 mg/m2 daunorubicin on days 1 - 3, 150 mg/m2 behenoyl cytosine arabinoside on days 1 - 7, and 70 mg/m2 6-mercaptopurine with 300 mg allopurinol taken orally on days 1 - 7 (BHAC-DM). The complete remission (CR) rate was 38.5%, whereas overall survival at 2 and 5 years was 22.0% and 4.7%, respectively. Two- and 5-year survival of CR patients was 41.8% and 11.2%, respectively. The relapse rate of the 25 CR patients was 64.0% and disease-free survival at 2 and 5 years was 21.0% and 11.2%, respectively. The therapy-related mortality rate at induction was 13.8%. BHAC-DM is feasible and effective for selected very elderly acute myeloid leukemia patients.

We report a male patient who was diagnosed as having hyper-IgM syndrome at the age of 28 years old. The patient had a history of recurrent infectious diseases since childhood such as bronchitis and otitis media, with decreased and increased levels of serum IgG and IgM, respectively. Genomic analysis of the CD40 ligand gene revealed deletion of six nucleotides from 475 to 480 followed by a T to A change at 481. These findings were compatible with the diagnosis of type 1 hyper-IgM syndrome. Bearing in mind the fact that only 20% of such patients survive over the age of 25, this patient is considered to be a rare case who was not actually diagnosed as having this disease until 28 years old.

Purpose: We investigated whether vitreous cytology and the measurement of intravitreous cytokine were useful for the diagnosis of intraocular malignant lymphoma., Subjects and Methods: 8 eyes of 5 patients with suspected intraocular malignant lymphoma during the past 15 months. 3 vitreous samples were collected from 3 patients at the time of pars plana vitrectomy. Polymerase chain reaction(PCR) amplification and flowcytometric analysis(FACS) of the vitreous samples were performed. Interleukin (IL)-10 and IL-6 concentrations were measured., Results: Vitreous cytology showed increased atypical B lymphocytes. The vitreous IL-10/IL-6 ratio was higher than 1 in all cases. Monoclonal rearrangement of the immunoglobulin heavy chain gene and the light chain restriction of immunoglobulin were detected., Conclusion: The detection of the monoclonality of infiltrated cells into the vitreous by PCR amplification and FACS, and the measurements of IL-10 and IL-6 concentrations in the vitreous fluid may be useful in the diagnosis of intraocular malignant lymphoma.

Bub1 is a component of the mitotic spindle checkpoint apparatus. Abnormality of this apparatus is known to cause multinuclei formation, a hallmark of chromosomal instability (CIN). A549, aneuploid cell line, aberrantly passed through the mitotic phase and became multinuclei morphology in the presence of nocodazole. Time-lapse videomicroscopy showed unreported bizarre morphology, which we named 'mitotic lobulation' in A549 cells just before the exit from mitosis and multinuclei formation. External expression of wild-type Bub1-EGFP clearly suppressed the multinuclei formation by retaining A549 cells at the mitotic phase during 48 h of time-lapse observation. This suppressive effect on mitotic aberrancy should not be mere restoration of normal Bub1 function, because A549 cells express proper amount of Bub1, which distributed cytoplasm during interphase and concentrated at kinetochore in metaphase. Furthermore, external expression of wild-type Bub1-EGFP suppressed multinuclei formation induced by Tax both in A549 and HeLa cells. Tax is known to induce mitotic abnormality by binding and inactivating Mad1. These observations, therefore, suggest functional redundancy between Bub1 and other mitotic checkpoint protein(s) and a possibility of correction of mitotic aberrancy by external Bub1 expression.

Bortezomib is a novel proteasome inhibitor with significant antimyeloma activity. Its frequent adverse effects are manageable, including gastrointestinal symptoms, peripheral neuropathy, and thrombocytopenia. Severe lung toxicity has not previously been reported. Between June 2004 and September 2005, 13 Japanese patients with multiple myeloma were treated with bortezomib in Toranomon Hospital, Juntendo University School of Medicine, and Jichi Medical School. Four of them developed severe pulmonary complications, and 2 died of respiratory failure without progression of underlying disease. To our knowledge, this is the first report on life-threatening pulmonary adverse effects after bortezomib therapy. Previous clinical studies on bortezomib, mostly in the United States and Europe, have shown low incidences of pulmonary adverse effects. Our study suggests that bortezomib can cause serious lung injury, and that its incidence might vary among different ethnicities. Clinicians need to be alert to the possibility.

In 2001, a 45-year-old man with severe aplastic anemia received a bone marrow transplantation from his HLA-identical sister, 2.5 years after which he developed severe liver dysfunction together with abdominal pain, disturbance of consciousness and generalized vesicles. Disseminated varicella-zoster virus infection was diagnosed by the detection of VZV DNA. Acyclovir and plasma exchange rapidly controlled his VZV-related symptoms and signs. Disseminated VZV infection is often fatal, but acyclovir and plasma exchange may be useful for such infection by reducing the circulating viral load.

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.

Background: Blastic natural killer (NK) cell lymphoma/leukemia (BNKL) is an immature CD56-positive neoplasm, which was recognized recently and characterized by systemic proliferation of tumor cells including skin, lymph node, and bone marrow., Methods: The current study analyzed 47 patients with BNKL (27 had leukemias and 20 had lymphomas). Patient data were collected for the survey of the NK-Cell Tumor Study Group., Results: There were 33 males and 14 females, with a median age of 53 years (range, 3 months to 89 years). There were few clinicopathologic differences between the leukemia and lymphoma types. Cutaneous involvement was noted at diagnosis in 28 patients, who presented a tendency for older age of onset (median: 56 vs. 46 years, P = 0.11) than patients with noncutaneous BNKL. Cutaneous BNKL showed less frequent mediastinal involvement (4% vs. 53%, P = 0.0002) and less severe thrombocytopenia (P =0 .03). Phenotypic characteristics were also different, with cutaneous BNKL favoring CD4 and HLA-DR expression, and noncutaneous BNKL favoring CD16 and CD34 expression. Both groups responded well to chemotherapy for lymphoid malignancies, but disease recurrence was frequent. The prognosis of patients with noncutaneous BNKL was significantly poorer than that of patients with cutaneous BNKL (median survival: 15 vs. 25 months, P = 0.02). Multivariate analysis confirmed that cutaneous involvement was a significant and independent prognostic factor for BNKL, as were age of onset and leukocyte count., Conclusions: These findings suggested that BNKL is a heterogeneous disease and contains at least two subtypes. Although further investigations are needed to settle a marker for distinction, the presence of cutaneous involvement is a useful prognostic factor.

We examined the effectiveness of various anti-tumour agents to natural killer (NK)-cell tumour cell lines and samples, which are generally resistant to chemotherapy, using flow cytometric terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labelling (TUNEL) assay. Although NK-YS and NK-92 were highly resistant to various anti-tumour agents, l-asparaginase induced apoptosis in these two NK-cell lines. NK-cell leukaemia/lymphoma and acute lymphoblastic leukaemia (ALL) samples were selectively sensitive to l-asparaginase and to doxorubicin (DXR) respectively. Samples of chronic NK lymphocytosis, an NK-cell disorder with an indolent clinical course, were resistant to both drugs. Our study clearly separated two major categories of NK-cell disorders and ALL according to the sensitivity to DXR and l-asparaginase. We examined asparagine synthetase levels by real-time quantitative polymerase chain reaction (RQ-PCR) and immunostaining in these samples. At least in nasal-type NK-cell lymphoma, there was a good correlation among asparagine synthetase expression, in vitro sensitivity and clinical response to l-asparaginase. In aggressive NK-cell leukaemia, although asparagine synthetase expression was high at both mRNA and protein levels, l-asparaginase induced considerable apoptosis. Furthermore, samples of each disease entity occupied a distinct area in two-dimensional plotting with asparagine synthetase mRNA level (RQ-PCR) and in vitrol-asparaginase sensitivity (TUNEL assay). We confirmed rather specific anti-tumour activity of l-asparaginase against NK-cell tumours in vitro, which provides an experimental background to the clinical use of l-asparaginase for NK-cell tumours.

The efficacy of allogeneic haematopoietic stem-cell transplantation (allo-HSCT) for natural killer (NK)-cell neoplasms is unknown. We investigated the results of allo-HSCT for NK-cell neoplasms between 1990 and 2003 through questionnaires. After reclassification by a haematopathologist, of 345 patients who underwent allo-HSCT for malignant lymphoma, 28 had NK-cell neoplasms (World Health Organization classification): extranodal NK/T-cell lymphoma (n=22), blastic NK-cell lymphoma (n=3), and aggressive NK-cell leukaemia (n=3). Twelve were chemosensitive and 16 chemorefractory. Twenty-two had matched-related donors. Stem-cell source was bone marrow in eight and mobilised peripheral blood in 20. Conditioning regimens were myeloablative (n=23) and non-myeloablative (n=5). Grade 2-4 acute graft-versus-host disease (GVHD) and chronic GVHD developed in 12 and 8 respectively. Eight died of disease progression, three of infection, two of acute GVHD, one of veno-occlusive disease, one of interstitial pneumonitis, and one of thrombotic microangiopathy. Two-year progression-free and overall survivals were 34% and 40% respectively (median follow-up, 34 months). All patients who did not relapse/progress within 10 months achieved progression-free survival (PFS) during the follow-up. In multivariate analysis, stem cell source (BM versus peripheral blood; relative risk 3.03), age (>or=40 years vs. <40 years; relative risk 2.85), and diagnoses (extranodal NK/T-cell lymphoma versus others; relative risk 3.94) significantly affected PFS. Allo-HSCT is a promising treatment for NK-cell neoplasms.

Aggressive natural killer cell leukemia (ANKL) is an intractable malignancy that is characterized by the outgrowth of NK cells. To identify transforming genes in ANKL, we constructed a retroviral cDNA expression library from an ANKL cell line KHYG-1. Infection of 3T3 cells with recombinant retroviruses yielded 33 transformed foci. Nucleotide sequencing of the DNA inserts recovered from these foci revealed that 31 of them encoded KRAS2 with a glycine-to-alanine mutation at codon 12. Mutation-specific PCR analysis indicated that the KRAS mutation was present only in KHYG-1 cells, not in another ANKL cell line or in clinical specimens (n=8).

Dlk1 (Pref-1) is a transmembrane and secreted protein, which is a member of the epidermal growth factor-like family, homologous to Notch/Delta/Serrate. We have found by real-time RT-PCR that Dlk1 mRNA levels were high in CD34(+) cells in 10 of 12 MDS samples compared with CD34(+) cells from 11 normals. Also, Dlk1 mRNA was elevated in mononuclear, low density bone marrow cells from 11/38 MDS patients, 5/11 AML M6 and 2/4 AML M7 samples. Furthermore, 5/6 erythroleukemia and 2/2 megakaryocytic leukemia cell lines highly expressed Dlk1 mRNA. Levels of Dlk1 mRNA markedly increased during megakaryocytic differentiation of both CMK megakaryoblasts as well as normal CD34(+) hematopoietic stem cells. High serum levels of Dlk1 occurred in RA (4/10) and essential thrombocythemia (2/10) patients. Functional studies showed that forced expression of Dlk1 enhanced proliferation of K562 cells growing in 1% fetal bovine serum. Analysis of hematopoiesis of Dlk1 knockout mice suggested that Dlk1 contributed to granulocyte, megakaryocyte and B-cell clonogenic growth and was needed for generation of splenic B-cells. In summary, Dlk1 is overexpressed in selected samples of MDS (especially RA and RAEB) and AML (particularly M6, M7), and it appears to be associated with normal development of megakaryocytes and B cells., (Leukemia (2005) 19, 1404-1410.)

A 59-year-old woman with goiter complained of nausea, vomiting and weight loss in April 2000. She underwent an endoscopic examination and was admitted to our hospital because gastric biopsy specimens revealed that she had diffuse large B-cell lymphoma. A thyroid biopsy also detected the diffuse infiltration of lymphoma cells, which were positive for CD19, CD20, CD38 and HLA-DR. Although the cells expressed surface immunoglobulin a chain, they lacked expressions of the kappa and lambda light chains. Chromosomal analysis of the thyroid cells showed 47, XX, t(2 ; 3)(q31 ; q13), + 3, t(8 ; 22)(q24 ; q11). After five courses of biweekly CHOP chemotherapy, she received autologous peripheral blood stem cell transplantation in October 2000. Currently, she has maintained complete remission for more than 4 years.

Neoplasms putatively originating from precursor and mature natural killer (NK) cells are rare, and their clinical features are unclear. A nationwide survey was performed in Japan to clarify the clinical features of these neoplasms diagnosed between 1994 and 1998, and data for 237 patients who met the criteria for putative NK cell-lineage neoplasms were analyzed. Among them, 11 had myeloid/NK-cell precursor acute leukemia, 15 blastic NK-cell lymphoma, 21 precursor NK-cell acute lymphoblastic leukemia, 22 aggressive NK-cell leukemia/lymphoma, 149 nasal-type NK-cell lymphoma (123 nasal and 26 extranasal) and 19 chronic NK lymphocytosis. The median overall survival time of patients with aggressive NK-cell leukemia/lymphoma was 2 months, which for chronic NK lymphocytosis was more than 8 years, and that for the other types of NK-cell neoplasms was between 6 and 22 months. Nasal NK-cell lymphoma and extranasal NK-cell lymphoma share the same histology. The age of affliction was the same, but the sex was different with males predominantly having nasal NK-cell lymphoma and females extranasal NK-cell lymphoma. Patients with extranasal NK-cell lymphoma had the tendency to exhibit a more advanced state of disease, with significantly higher International Prognostic Index and LDH levels, and significantly lower hemoglobin and platelet levels. The overall survival, however, did not differ significantly. Precursor NK-cell acute lymphoblastic leukemia and blastic NK-cell lymphoma were arbitrarily defined by the presence or absence of 30% or more of blastic cells in the bone marrow or peripheral blood, but there were no significant differences for affected age, gender, involved sites or prognosis. Aggressive NK-cell leukemia/lymphoma and extranasal NK-cell lymphoma were arbitrarily defined by the presence or absence of 30% or more of large granular lymphocytes in the bone marrow or peripheral blood and it is possible that aggressive NK-cell leukemia/lymphoma is a leukemic phase of extranasal NK-cell lymphoma. The incidence of skin involvement, however, was significantly higher for extranasal NK-cell lymphoma, suggesting that the two diseases are different. In nasal NK-cell lymphoma, Epstein-Barr virus in tumor cells was detected in all patients tested, suggesting its causative role.

Natural killer (NK) cell disorders are rare diseases. Genetic abnormalities of the several tumor suppressor genes, including p15INK4B, p16INK4A/p14ARF, p53, p73, and Rb genes have been reported. Deletions and point mutations of these genes are frequently detected in these diseases. It has been reported that tumor suppressor genes are inactivated by DNA methylation of the promoter region and/or first exon of the genes in a variety of human cancers. In this study we analyze the methylation status of the genes associated with cell cycle regulation, including p16INK4A, p15INK4B, p21/Waf1/Cip1, p27/Kip1, p73, and p14ARF, by methylation specific (MS) PCR and/or bisulfite sequencing. We examined 29 cases of NK cell disorders (five aggressive NK cell leukemia/lymphoma, three blastic NK cell lymphoma/leukemia, five nasal NK cell lymphoma, three myeloid/NK cell precursor acute leukemia, 13 chronic NK lymphocytosis). We found methylation of the first exon of the p16INK4A gene in two cases (one aggressive, one blastic), and methylation of the p14ARF gene in one aggressive NK cell leukemia. Bisulfite sequencing revealed that methylation of the p15 and p27 genes was rare in these disorders. MS-PCR suggested that the p73 and p21 genes were methylated in seven cases, respectively (p73: one blastic, one nasal, five chronic; p21: one myeloid/NK, one aggressive, one nasal, and four chronic); bisulfite sequencing confirmed that methylated alleles of these genes were dominant in the samples except three cases (one myeloid/NK, one aggressive, and one chronic) in which methylated alleles of the p21 genes were less than 34% of all alleles. These results suggested that inactivation of the cell cycle regulatory genes by DNA methylation could be associated with tumorigenesis in NK cell disorders, not only aggressive subtypes but also chronic subtype.

Mxi1 protein is a basic helix-loop-helix, leucine zipper (bHLHZIP) transcriptional factor, which dimerizes with the Max protein. This heterodimer binds a specific DNA sequence (E-box) and suppresses transcription of target genes. On the other hand, c-Myc protein is also a bHLHZIP protein that dimerizes with Max, binds the identical E-box sequence but activates transcription of the target genes. We report that hematopoietic cells have three novel Mxi1 transcripts: Mxi-D lacks exon 3, which encodes the basic region; Mxi-ND lacks the N-terminal mSin3 binding region and the DNA binding region; and Mxi-NF lacks the N-terminal mSin3 binding region. In normal bone marrow and hematological cell lines, the dominantly expressed isoforms are Mxi-D and full-length Mxi1 (Mxi-F). In GST-pull down assays, the Mxi-D protein binds the Max protein and the PHA2 regions of mSin3 proteins. Whereas the Mxi-F/Max heterodimer binds the E-box sequence, Mxi-D/Max heterodimer can not bind this sequence in electrophoretic mobility shift assays. In reporter gene assay, Mxi-D suppresses transcription as strongly as Mxi-F. In colony formation assay using Rat1 fibroblast cells, Mxi-D cannot suppress clonal growth stimulated by c-Myc as strongly as Mxi-F. In summary, Mxi-D may play an important role in the c-Myc family protein network acting as a dominant negative isoform of Mxi-F since: i) Mxi-D can dimerize with Max in vitro; ii) Mxi-D/Max heterodimers cannot bind E-box in vitro, iii) Mxi-D cannot suppress clonal growth stimulated by c-Myc.