Tumor suppressor gene methylation on the short arm of chromosome 1 in chronic myelogenous leukemia.

OBJECTIVES: We previously reported loss of heterozygosity on 1p in chronic myelogenous leukemia (CML). We analyzed promoter methylation and mutation of tumor suppressor genes on 1p36 in CML. METHODS: We performed methylation-specific PCR (MS-PCR) analysis of the PRDM2, RUNX3, and TP73 genes in 61 patients with CML (43 chronic phase, CP; two accelerated phase; and 16 blast crisis, BC). Oxidative MS-PCR, PCR-single-strand conformation polymorphism, and real-time reverse transcriptase PCR were also analyzed. K-562 cells were grown in the presence of 5-Aza-dC and trichostatin A. RESULTS: Methylation of the PRDM2, RUNX3, and TP73 genes was detected in 24/60 (40%), 21/61 (34%), and 28/60 (47%) patients, respectively. Methylation of all three genes was detected in 19/59 (32%) patients. Methylation was more frequent in BC than in CP. Oxidative MS-PCR analysis detected 5-mC in the PRDM2, RUNX3, and TP73 genes in 10/22 (45%), 15/21 (71%), and 16/26 (62%) samples with methylation detected by MS-PCR, respectively. Decreased expression was observed in several samples with methylation, while no mutations were found in the genes. Treatment of K-562 cells induced growth suppression, demethylation, and reexpression of the PRDM2 and RUNX3 genes. CONCLUSION: Multiple tumor suppressor genes on 1p were inactivated in CML by methylation.

Peroxiredoxin 2 expression is increased in neutrophils of patients with refractory cytopenia with multilineage dysplasia.

Myelodysplastic syndromes (MDS) are heterogeneous clonal disorders characterized by cytopenias that arise due to ineffective haematopoiesis and morphological dysplasia and carry an increased risk of incident acute myeloid leukaemia. The pathogenesis of marrow dysfunction in MDS is multifactorial and consistent with a multistep model and may lead to heterogeneity of MDS. We investigated the proteome profile of circulating neutrophils purified from patients with refractory cytopenia with multilineage dysplasia (RCMD) to identify proteins that have a role in the pathogenesis. Using 2-dimensional difference gel electrophoresis and protein identification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we found that peroxiredoxin 2 (PRDX2), a member of the peroxiredoxin family that regulates reactive oxygen species, was markedly upregulated in neutrophils of RCMD patients compared to healthy donors. Increased PRDX2 expression in the neutrophils of RCMD patients was confirmed using quantitative reverse transcription polymerase chain reaction, immunoblotting and immunocytochemical analysis. In addition, white blood cell and neutrophil counts in RCMD patients correlated inversely with the PRDX2 expression of. Oxidative stress is a known factor involved in the pathogenesis of MDS, and PRDX2 is associated with tumourigenesis of several solid tumours. Accordingly, our results suggest that PRDX2 may perform an important function in the pathogeneis of RCMD.

[Analysis of molecular mechanism involved in development of acute myeloid leukemia].

We examined the role of molecules related to drug resistance, such as P-glycoprotein (P-gp) and telomerase (TERT), signaling molecules of STATs and FLT3 in leukemia pathogenesis in de novo acute myeloid leukemia (AML), and myelodysplastic syndrome in the phase of overt leukemia (MDS-OL). Subjects were 18 patients with de novo AML, in which expression of P-gp, TERT, STAT3, STAT5, and FLT3 was observed in 11, 14, 16, 18, and 14 of patients, respectively. Phosphorylation of STAT3, STAT5, and FLT3 in patients with de novo AML was observed in 10 out of 14, 14 out of 18, and 10 out of 14 patients, respectively. Phosphorylation of STAT5 was associated with expression of both P-gp and TERT, suggesting that STAT5 is one of the transcription factors for these genes. On the other hand, P-gp, TERT, STAT3, STAT5, and FLT3 were expressed in 3, 1, 1, 6, and 1 of the 7 patients with MDS-OL, respectively. While phosphorylation of STAT5 was observed in 4 out of 7 patients, phosphorylation of STAT3 or FLT3 was not detected in all cases examined. Telomere length varied from 2.7 kb to 6.0 kb in de novo AML, accompanied by an increased level of telomerase activity in 4 of 5 patients with de novo AML. In contrast, all MDS-OL cases showed a similar telomere length of 4-5 kb. These results indicate that consideration should be given to the differences of molecular mechanisms in the pathogenesis of de novo AML and MDS-OL for the treatment strategy of AML.

Establishment and Characterization of an Acute Lymphocytic Leukemia Cell Line Expressing CD13 and CD33 with a Complex Philadelphia Translocation.

Objective To investigate the pathogenesis of Philadelphia (Ph)-positive acute lymphocytic leukemia (ALL), we established a lymphoblastoid cell line. Methods Bone marrow cells from a patient with Ph-positive ALL were enriched by Ficoll-Hypaque centrifugation and cultured in medium with fetal calf serum. Materials The mononuclear cells of bone marrow aspirate were obtained from an adult man with ALL after he experienced relapse following induction therapy including imatinib mesylate. Results The cell line termed TNA-M was established, carrying a three-way Ph translocation involving two chromosome 9s and one chromosome 22 as a sole karyotypic abnormality. Furthermore, the cells were positive for CD13 and CD33 in addition to CD19, CD22 and CD79a antigens. Conclusion This unique cell line is expected to be a valuable tool for understanding the pathogenesis of Ph-positive ALL.

Discontinuation of imatinib in Japanese patients with chronic myeloid leukemia.

It was recently recognized that some chronic myeloid leukemia patients with a complete molecular response could sustain that response after discontinuation of imatinib. To characterize the clinical outcomes and profiles of chronic phase chronic myeloid leukemia patients who could discontinue imatinib, we conducted a nationwide survey in Japan. Among 3,242 imatinib-treated chronic myeloid leukemia patients, we identified 50 who had discontinued imatinib for at least six months; of these we analyzed 43. Molecular recurrence was detected in 19 patients, and a complete molecular response rate was estimated to be 47% following imatinib discontinuation. Based on multivariate regression analysis, imatinib dose intensity and prior interferon-α administration were independently predictive of molecular recurrence within 12 months. The depth of the molecular response should be a factor influencing long-term sustained complete molecular response after discontinuation of imatinib. Additionally, an immunological mechanism modified by interferon-α might control chronic myeloid leukemia stem cells.

Acquired hemophilia A in a patient with essential thrombocythemia.

A 69-year-old woman with essential thrombocythemia (ET) developed giant ecchymosis, and she was admitted to hospital. Marked anemia (Hb 8.1 g/dl) accompanied by a prolonged activated partial thromboplastin time (89.6 s) was observed, and she received red blood cells (RBC) and fresh frozen plasma (FFP). On day 2 after admission, consciousness disturbance suddenly occurred, whereas computed tomography of the brain showed no evidence of bleeding. As the ecchymosis progressed, she developed shock. Although RBC and FFP transfusions were administered, she developed multi-organ failure and died 48 h after admission. Low factor VIII activity (<1%) accompanied by factor VIII inhibitor (17 Bethesda units) was found after her death. An autopsy revealed cerebral infarction without cerebral herniation. To date, acquired hemophilia A accompanying ET has been described in only one other patient. Although acquired factor VIII inhibitor is a rare disease, it should be tested for in ET patients with marked hemorrhagic tendency.

Risk factor analysis of vasovagal reaction from blood donation.

BACKGROUND: Vasovagal reaction (VVR) is the most frequent side effect at blood collection sites. AIMS: To protect donors, factors contributing to VVR were analysed. MATERIALS AND METHODS: Complications following whole blood and apheresis donations have been recorded and accumulated by the Japanese Red Cross Tokyo Blood Centre. A dataset of 43,948 donors who had no complications was prepared as a control by randomly selecting days in each season in the 2006 and 2007 fiscal years. Factors contributing to 4924 VVR incidents in the 2006 and 2007 fiscal years were analysed by univariate and multivariate logistic regression. RESULTS: The age, weight, body mass index (BMI), predonation systolic and diastolic pressure, and circulating blood volume were lower, and the pulse was higher, for the VVR group compared to the control group (p<0.0001). The VVR group had more female donors, less sleep, and more time since a meal than the control. In multivariate analysis, significant risk factors for 400 ml whole blood donors, which are the majority of donors, were an age <50 years, being female, a BMI <25, pulse ≥90/min, sleep duration <8 h, the time after eating ≥4 h, a first time donation and circulating blood volume of <4.3 l. Sleep duration of <6 h was shown to be a VVR risk as much as a first time donation. CONCLUSION: From our analysis, the amount of sleep obtained the previous night should be considered at the reception of donors.

[Long-term remission after nonmyeloablative allogeneic bone marrow transplantation in a relapsed Hodgkin lymphoma patient with CNS involvement].

Intracranial involvement of relapsed Hodgkin lymphoma (HL) is quite rare and its prognosis is very poor. We report a patient with relapsed HL with central nervous system (CNS) involvement after autologous stem cell transplantation successfully treated with allogeneic bone marrow transplantation with reduced intensity conditioning regimen. A standard therapy for relapsed CNS HL has not yet established. To the best of our knowledge, this is the first case report describing allogeneic stem cell transplantation for relapsed CNS HL in an elderly patient. Our results in this case suggest that allogeneic stem cell transplantation could be a useful therapeutic option in relapsed CNS HL patients, if their CNS lesions are controlled before stem cell transplantation.

[Usefulness of (18)F-FDG PET/CT for detecting lesions in patients with POEMS syndrome].

Many patients with POEMS syndrome have osteosclerotic plasmacytoma. Radiation therapy is useful for patients who have localized lesions, although chemotherapy is necessary for patients who have widespread lesions. Thus, evaluation of these lesions is important to determine the therapeutic strategy. We evaluated the activities of lesions in two patients with POEMS syndrome by (18)F-FDG positron emission tomography (PET)/computed tomography (CT) scan. In the first patient, PET/CT scan revealed osteosclerotic lesions, which were not detected by Ga-scintigraphy or plain X-ray. It also detected residual disease activity and relapse. In the second patient, lymph node involvement was suggested by (18)F-FDG uptake, and plasmacytoma was confirmed by subsequent biopsy. In the extramedullary lesions of this case, FDG uptake was as marked as in myeloma, whereas bone lesion was only detectable by CT scan. In POEMS syndrome, the PET and CT are complementary, and the combined PET/CT scan is considered to be very useful for evaluation of involved lesions.

Cladribine combined with rituximab (R-2-CdA) therapy is an effective salvage therapy in relapsed or refractory indolent B-cell non-Hodgkin lymphoma.

Although cladribine has been reported to be an active purine analog against indolent B-cell non-Hodgkin lymphoma (B-NHL), there are few reports of combination use of cladribine and rituximab. This multicenter phase II study evaluated the efficacy and toxicity of cladribine with rituximab (R-2-CdA) therapy in relapsed or refractory indolent B-NHL. Twenty patients with the median age of 58.5 yrs (range, 42-72) were enrolled and received R-2-CdA therapy from April 2005 to July 2007. The median number of prior regimens was 2 (range, 1-3), and fifteen patients (75%) were previously treated with rituximab-containing regimens. Disease histology included follicular lymphoma in 16 patients, MALT lymphoma in two patients, nodal marginal B-cell lymphoma in one patient, and lymphoplasmacytic lymphoma in one patient. The overall response rate (ORR) was 90%, with a complete response rate (CRR) of 70%. Estimated median progression-free survival (PFS) time was 22.4 months (95%CI, 10.9-32.6 months) at a median follow-up time of 27 months (range, 12-43). Two-year PFS and 2-yr overall survival (OS) were 52.6% (95%CI, 31.0-73.2%) and 89.5% (95%CI, 66.1-97.3%), respectively. Grade 3 or grade 4 toxicities were neutropenia in 74% and thrombocytopenia in 11%. R-2-CdA therapy was demonstrated to have a high activity with durable PFS and acceptable toxicity in relapsed or refractory indolent B-NHL mostly pretreated with rituximab-containing therapy. Although a large-scale trial is needed for confirmation, R-2-CdA therapy could be a good salvage therapy option in relapsed or refractory indolent B-NHL.

Absence of mutations on the SNF5 gene in hematological neoplasms with chromosome 22 abnormalities.

BACKGROUND: The relation with SNF5 mutation and chromosome 22 abnormalities is not clear in hematological neoplasms. METHODS: To elucidate the relevance of the SNF5 gene on 22q11.2, karyotypes were reviewed in 283 hematological neoplasms. Loss of heterozygosity (LOH) on 22q was analyzed in 21 plasma cell myelomas without chromosome 22 abnormalities. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) on the SNF5 gene was analyzed in 8 hematological neoplasms with 22q- or -22, and 8 chronic myelogenous leukemias (CMLs) in blast crisis. Fluorescence in situ hybridization (FISH) was performed in 1 myelodysplastic syndrome (MDS) case with -22,del(22)(q11.2 q13). RESULTS: 22q- or -22 was observed in 36 patients. LOH on 22q was detected in 1 of the 21 myelomas. Mobility shifts were found by PCR-SSCP analysis in 2 CMLs, whereas sequence analysis showed polymorphisms. FISH analysis revealed the SNF5 gene was not deleted in the MDS case. CONCLUSION: These results suggest that alterations of the SNF5 gene are rare in hematological neoplasms with chromosome 22 abnormalities. Haploinsufficiency may contribute to the development of these neoplasms.

Disseminated cryptococcosis in a non-Hodgkin's lymphoma patient with late-onset neutropenia following rituximab-CHOP chemotherapy: a case report and literature review.

Rituximab-related late-onset neutropenia (R-LON) is an adverse event associated with rituximab. A 65-year-old woman presented with diffuse large B-cell lymphoma of the kidney without bone marrow involvement. She was treated with 4 cycles of CHOP chemotherapy consisting of doxorubicin, cyclophosphamide, vincristine, and prednisolone at 4-week intervals. Rituximab was also administrated of the second, third, fourth CHOP cycles. She developed a high fever of 38°C, nausea, and severe neutropenia following the four cycles of R-CHOP chemotherapy. Her leukocyte count was 160/µl without neutrophils. Initially, a blood and pleural fluid and cerebrospinal fluid cultures were positive for Cryptococcus neoformans. Once she became asymptomatic following treatment with fluconazole and neutropenia was recovered with lenograstim, she had neck stiffness and admitted soon. Cerebro-spinal fluid (CSF) culture was positive for Cryptococcus neoformans. Treatment with amphotericin B(AMPH-B) and flucytosine(5-FC) was initiated as diagnosis of cryptococcus meningitis. Lenograstim was administrated for 9 months, and amount of dose was 9,750 µg. Cryptococcosis with malignant lymphoma is rare disease, and previously 17 cases were reported. Of note, mortality of disseminated cryptococcosis with malignant lymphoma is 54%. The more and more rituximab is widely used; the cases of severe infection in R-LON may increase.

[Angioimmunoblastic T-cell lymphoma with marked polyclonal plasmacytosis in peripheral blood and bone marrow mimicking plasma cell leukemia].

A 70-year-old man was admitted to our hospital with fever, generalized lymphadenopathy and hypoxia in October 2009. Blood examination demonstrated leukocytosis, anemia, thrombocytopenia and hyper γ-globulinemia. Peripheral blood and bone marrow smear showed marked plasma cell proliferation mimicking plasma cell leukemia. However, flow cytometric analysis showed that plasma cells were of polyclonal origin and M-protein was not detected by immunofixation of serum protein. Elevations of soluble interleukin 2 receptor and serum IL-6 were observed. A heavy Epstein-Barr viral load was detected in serum by real-time PCR. Biopsy was obtained from the right inguinal lymph node. The pathological diagnosis was angioimmunoblastic T-cell lymphoma (AITL) and rearrangement of the T-cell receptor Cß1 gene was detected. The patient was treated with CHOP therapy, and all clinical manifestations, including fever, lymphadenopathy, anemia, thrombocytopenia, hyper γ-globulinemia, plasmacytosis and hypoxia, were improved. Only a few reported cases have demonstrated AITL with marked polyclonal plasmacytosis. Although pathological mechanisms of plasmacytosis in AITL patients have not been fully elucidated, it is suggested that IL-6 and IL-10 were involved in its pathogenesis in the present case.

[Factors affecting the response of thalidomide therapy for patients with multiple myeloma].

Factors that affect the response of multiple myeloma patients to thalidomide were evaluated in 40 patients who were not eligible for chemotherapy (untreated: 14, relapse/refractory: 26). The complete response (CR) rate was 2.5%; partial response (PR) 50.0%; minimal response (MR) 25.0%; no change (NC) 12.5%; and progressive disease 10.0%. The response to thalidomide could be evaluated after four weeks of treatment. Significantly higher responses were associated with untreated patients, patients with combined use of thalidomide plus dexamethasone, and patients with kappa light chain. Patients who responded well to thalidomide showed a significantly higher progression-free survival (PFS) rate. In patients with kappa light chain, PFS and overall survival rates were significantly higher than those with lambda light chain. Frequent adverse reactions were numbness (47.5%), constipation (32.5%), and eruption (30.0%). In patients previously treated with vincristine, numbness occurred in a significantly higher percentage of patients.

Expression analysis of genes located within the common deleted region of del(20q) in patients with myelodysplastic syndromes.

Deletion of the long arm of chromosome 20 (del(20q)) is observed in 5-10% of patients with myelodysplastic syndromes (MDS). We examined the expression of 28 genes within the common deleted region (CDR) of del(20q), which we previously determined by a CGH array using clinical samples, in 48 MDS patients with (n = 28) or without (n = 20) chromosome 20 abnormalities and control subjects (n = 10). The expression level of 8 of 28 genes was significantly reduced in MDS patients with chromosome 20 abnormalities compared to that of control subjects. In addition, the expression of BCAS4, ADA, and YWHAB genes was significantly reduced in MDS patients without chromosome 20 abnormalities, which suggests that these three genes were commonly involved in the molecular pathogenesis of MDS. To evaluate the clinical significance, we analyzed the impact of the expression level of each gene on overall survival (OS). According to the Cox proportional hazard model, multivariate analysis indicated that reduced BCAS4 expression was associated with inferior OS, but the difference was not significant (HR, 3.77; 95% CI, 0.995-17.17; P = 0.0509). Functional analyses are needed to understand the biological significance of reduced expression of these genes in the pathogenesis of MDS.

JAK2 V617F mutation is rare in idiopathic erythrocytosis: a difference from polycythemia vera.

A single mutation 1849G>T in the JAK2 gene (V617F) has recently been described in classical myeloproliferative disorders (MPD). To investigate the incidence and clinical significance of the JAK2 mutation, we performed allele-specific polymerase chain reaction (PCR) and enzyme-based assessment in 11 idiopathic erythrocytosis (IE) and 15 polycythemia vera (PV) patients. Aberrant bands indicating the V617F mutation were detected in only one of 11 patients with IE, whereas all of the 15 patients with PV showed the JAK2 mutation. Sequence analysis was subsequently performed in the IE patient showing aberrant bands on allele-specific PCR, and a nucleotide change corresponding to the V617F mutation was detected in four of 29 clones tested. This patient might have progressed to PV according to the new WHO diagnostic criteria proposed in 2007, since a gradual increase in platelet counts was observed 4 years after the time of diagnosis. A further longitudinal study monitoring V617F positive-cells will clarify the process of progression from IE to PV in such a patient.

Isolated granulocytic sarcoma of the small intestine successfully treated with chemotherapy and bone marrow transplantation.

Isolated primary granulocytic sarcoma is a rare disease that presents as an extramedullary tumor of myeloid lineage cells. Most patients subsequently develop acute myelogenous leukemia (AML) within a short period, and their prognosis is poor. Herein, we report the case of a 33-year-old woman with a primary isolated granulocytic sarcoma which originated in the small intestine. After she recovered from surgery, she received intensive chemotherapy equivalent to that for AML, followed by allogeneic bone marrow transplantation from an HLA-matched, unrelated donor. Four years after the transplantation, she remains in complete remission without graft-versus-host disease or any other symptoms. This case illustrates the effectiveness of our therapeutic strategy for isolated granulocytic sarcoma, not only with surgical resection of the tumor and intensive chemotherapy equivalent to that for AML, but also with allogeneic bone marrow transplantation, performed while no sign of AML is observed.

Knockdown of the Wnt receptor Frizzled-1 (FZD1) reduces MDR1/P-glycoprotein expression in multidrug resistant leukemic cells and inhibits leukemic cell proliferation.

Multidrug resistance (MDR) is a major obstacle to leukemia treatment. The Frizzled-1 (FZD1) Wnt receptor is involved in MDR in some solid cancers, but has rarely been reported to act in acute myeloid leukemia (AML). We investigated whether the knockdown of FZD1 affects MDR1 expression and P-glycoprotein (P-gp) function in multidrug resistant leukemic cell lines, as well as FZD1 and MDR1/P-gp expression in leukemic cells taken from patients with AML (n = 112). FZD1 knockdown significantly reduced MDR1 expression through the Wnt/ß-catenin pathway, disrupted the P-gp efflux function, induced the recovery of sensitivity to chemotherapeutic agents, and hindered cell proliferation in cell lines. FZD1 expression in leukemic cells was significantly higher in patients experiencing relapse (n = 34) than in those with no relapse (n = 44, P = .003). Leukemic cells unable to achieve complete response (CR) showed an increased expression of MDR1 and P-gp, compared to patients who achieved CR. Obtaining CR in patients with higher FZD1 expression at diagnosis is difficult. Moreover, they tend to present instances of relapse, suggesting that AML cells with increased FZD1 expression are resistant to chemotherapy. We conclude that the activated FZD1 observed in leukemic cells likely confers acquired drug resistance, whereas FZD1 silencing may be more effective in reversing MDR.