Keitaro Tsushita

Mutations of the p53 gene as a prognostic factor in aggressive B-cell lymphoma

BACKGROUND Mutations of the p53 gene are associated with a poor prognosis in several types of cancer. We investigated the prognostic importance of p53 mutations in patients with aggressive B-cell lymphoma. METHODS We examined the relation between the presence or absence of a detectable p53 mutation in lymphoma cells and the response to chemotherapy and overall survival in 102 previously untreated patients with aggressive B-cell lymphoma. Mutations of the p53 gene were identified by polymerase-chain-reaction-mediated analysis of single-strand conformation polymorphisms and by direct sequencing. RESULTS Of 102 cases of aggressive B-cell lymphoma, 22 (22 percent) involved p53 mutations. The rate of complete remission was significantly lower in patients with a tumor carrying a p53 mutation (6 of 22 patients, 27 percent) than in those with the wild-type p53 gene (61 of 80 patients, 76 percent) (P<0.001). Overall survival was significantly lower among patients with p53 mutations than among those with the wild-type p53 gene; the Kaplan-Meier estimates of survival at five years were 16 percent and 64 percent, respectively (P<0.001). Multivariate analysis incorporating prognostic factors from the international prognostic index demonstrated that p53 mutations had independent effects on the rates of complete remission and survival. When we categorized patients according to the international prognostic index, we found no effect of p53 mutations in patients in the groups at high-intermediate and high risk. However, these mutations were significantly associated (P< 0.001) with low rates of complete remission (33 percent vs. 91 percent) and survival (27 percent vs. 81 percent at five years) in the groups at low and low-intermediate risk. CONCLUSIONS Mutations of the p53 gene are associated with a poor prognosis in patients with aggressive B-cell lymphoma.

Combinations of cytogenetics and international scoring system can predict poor prognosis in multiple myeloma after high-dose chemotherapy and autologous stem cell transplantation

High‐dose chemotherapy followed by autologous stem cell transplantation (ASCT) is a standard therapy for newly diagnosed multiple myeloma. Combinations of recently proposed prognostic factors such as cytogenetics and international scoring system (ISS) may be useful to predict prognosis after ASCT. This study evaluated 60 consecutive patients who underwent ASCT in four institutes. The median age of patients was 57 years old. Cytogenetic analyses of bone marrow at diagnosis detected metaphase abnormalities in 9 of 51 patients and interphase abnormalities in six of 35 patients (17p13 deletion, t(4;14) and t(14;16)). Seventeen patients had ISS stage 3 at diagnosis. Twenty‐five patients who had any of these risk factors were defined as high risk. All patients were conditioned with high‐dose melphalan. With a median follow‐up of 3.4 years, overall survival and event‐free survival at 3 years were significantly worse in high‐risk patients (48% vs. 97%; P = 0.0005 and 16% vs. 37%; P = 0.038, respectively) despite the higher CR plus VGPR rate among high‐risk patients. In addition, survival at 1 year after progression was significantly worse in high‐risk patients despite salvage chemotherapy containing thalidomide (32% vs. 100%, P = 0.0001). Combinations of cytogenetics and ISS could readily predict prognosis. Quality of response is a poor surrogate marker for ultimate outcome. High‐risk patients may need more effective treatment. Am. J. Hematol. 2009.

Failure to Detect Epstein-Barr Virus (EBV) DNA in Plasma by Real-Time PCR in a Case of EBV-Associated Posttransplantation Lymphoproliferative Disorder Confined to the Central Nervous System

We report here a patient who developed multiple central nervous system (CNS) space-occupying lesions 6 months after bone marrow transplantation from an HLA-matched unrelated donor. He had extensive chronic graft-versus-host disease and severe thrombocytopenia. Posttransplantation lymphoproliferative disorder (PTLD) was diagnosed after biopsy of the lesion was facilitated by the transfusion of 40 units of platelets. Epstein-Barr virus (EBV) DNA was not initially detected in the peripheral blood by real-time polymerase chain reaction, and the blood became positive for EBV at a low level only after more than 6 weeks had passed since the initial identification of detectable intracranial lesions. The patient died of cerebral herniation while donor leukocyte infusion was being prepared, and an autopsy confirmed the diagnosis of EBV-associated PTLD restricted to the CNS.

Patchy haemopoiesis in long-term remission of idiopathic aplastic anaemia

ABSTRACT: 13 patients with idiopathic aplastic anaemia in remission for more than 2 years were examined to define the haemopoietic status by means of bone marrow scintigraphy, ferrokinetics and bone marrow culture for haemopoietic progenitor cells. Haemoglobin levels reached the normal range in all these patients although mild neutropenia and thrombocytopenia were still observed in 5 patients. Bone marrow scintigrams using indium‐111 showed normal distribution in 2, diffuse low accumulation in 3, patchy distribution in 7, and expanded distribution with patchy uptake in 1 patient. The defective haemopoiesis was also confirmed by ferrokinetic and bone marrow culture studies. The patchy haemopoiesis appears to characterize the residual marrow damage in remission of idiopathic aplastic anaemia.

Tumor Necrosis Factor-p Gene Expression and Its Relationship to the Clinical Features and Histopathogenesis of Peripheral T-Cell Lymphomas

We investigated the levels of tumor necrosis factor-β (TNF-β) mRNA in the tumorous tissues of a series of 18 patients with peripheral T-cell lymphomas (PTCL), to assess the contribution of the expression of this gene to the features of the disease. Total RNA, extracted from diagnostic tissue specimens, was subjected to semiquantitative analysis by reverse transcription-coupled polymerase chain reaction (RT-PCR). The level of TNF-β mRNA was semiquantified against that in MT-2 cells, a line of human T cells infected with human T cell leukemia virus type I (HTLV-I). Expression of TNF-B in neoplastic T-cells was confirmed by immunohistochemistry. The extent of TNF-β gene expression was correlated with the histopathological features of neovascularization. There was also a relationship between the extent of TNF-β gene expression and the presence of B-symptoms. Results suggest that TNF-β produced by neoplastic T-cells influences clinical features and is involved in histopathogenesis of PTCL.

Phase III Study of Ranimustine, Cyclophosphamide, Vincristine, Melphalan, and Prednisolone (MCNU-COP/MP) versus Modified COP/MP in Multiple Myeloma: A Japan Clinical Oncology Group Study, JCOG 9301

To investigate whether combination chemotherapy with vincristine, cyclophosphamide, prednisolone, and melphalan (COP/ MP) with the addition of ranimustine (MCNU) (MCNU-COP/MP) is superior to the slightly modified COP/MP (mCOP/MP) regimen in multiple myeloma (MM), a multicenter randomized study was performed. Two hundred ten patients with newly diagnosed, overt MM not treated with chemotherapy were enrolled from 32 institutions of the Lymphoma Study Group of the Japan Clinical Oncology Group and were randomized to receive either MCNU-COP/MP or mCOP/MP. The response rate (RR) to mCOP/MP was 43.7% (95% confidence interval [CI], 33.9%–53.8%] and to MCNU-COP/MP was 56.1 % (95% CI, 46.1 %–65.7%) (P = .097). The progression-free survival (PFS) was significantly longer for patients treated with MCNU-COP/MP than for patients treated with mCOP/MP (median, 23.0 months [95% CI, 18.9–25.8] versus 15.8 months [95% CI, 14.1–19.4]) (P = .014). However, no significant difference in overall survival rate (OS) was observed between the groups (median, 49.9 months [95% CI, 40.4-59.1] versus 44.0 months [95% CI, 32.8–59.8]) ( P = .75). Grades 3 and 4 hematological toxicities were more frequently observed with MCNU-COP/MP than with mCOP/MP, but the incidence of grades 3 and 4 nonhematological toxicities was low in both groups. In conclusion, MCNU-COP/MP in comparison with mCOP/MP improved RR and PFS in overt MM; however, this outcome did not contribute to prolonging OS, indicating that addition of MCNU to mCOP/MP has no benefit on survival.

The dual expression of minor and major bcr/abl chimeric mRNA in blast crisis of chronic myelogenous leukemia.

The hypothesis that minor bcr/abl fusion mRNA is produced in blast crisis of chronic myelogenous leukemia (CML) is examined. The RNA transcripts encoding the minor and major bcr/abl fused protein were detected by polymerase chain reaction (PCR) using RNA from peripheral blood or bone marrow cells of eight patients with blast crisis or accelerated phase of CML. The mRNA encoding for major bcr/abl was detected in all eight cases. In four patients, however, transcripts encoding for minor bcr/abl mRNA were detected, as well as major bcr/abl mRNA. The presence of minor bcr/abl mRNA was verified with the hybridization with a junction-specific probe and DNA sequencing analysis of PCR products. The appearance of minor bcr/abl fusion mRNA was associated with the lymphoblastic immunophenotype of the blast cells. In two of these four patients, samples of initial diagnosis of chronic phase of CML were available, which did not show minor bcr/abl transcript. We conclude that the appearance of minor bcr/abl mRNA transcript is associated with the terminal evolution of CML in lymphoblastic crisis.

Increased T-cell responses to Epstein-Barr virus with high viral load in patients with Epstein-Barr virus-positive diffuse large B-cell lymphoma.

Abstract The immunological status of patients with Epstein–Barr virus-positive diffuse large B-cell lymphoma (EBV+ DLBCL) without obvious immunodeficiency has not been elucidated. A multicenter prospective study was conducted to assess pretreatment T-cell responses to EBV, EBV-DNA load and anti-EBV antibody in these patients. The proliferative and interferon (IFN)-γ-producing capacity of T-cells in response to autologous B-lymphoblastoid cell lines was determined using carboxyfluorescein diacetate succinimidyl ester (CFSE)-based assay. Frequencies of EBV-specific CD4+ T-cells in patients with EBV+ DLBCL (n = 13) were significantly higher than in healthy controls (HCs) (n = 16) after both ex vivo and in vitro stimulation. Frequencies of EBV-specific CD8+ T-cells in patients with EBV+ DLBCL tended to be higher than in HCs after in vitro stimulation. Patients with EBV+ DLBCL also showed increased immunoglobulin G (IgG) responses to lytic EBV-encoded antigens. Pretreatment plasma EBV-DNA level was significantly higher in patients with EBV+ DLBCL than in patients with EBV− DLBCL or HCs. In conclusion, EBV-specific T-cells showed increased reactivity, accompanied by higher levels of plasma virus DNA in patients with EBV+ DLBCL.

Successful Treatment of Hemosiderosis with Regular Phlebotomy and Recombinant Human Erythropoietin

A 55-year-old female patient with hemosiderosis induced by administration of excessive doses of parenteral iron was successfully treated with regular phlebotomy combined with recombinant human erythropoietin (rHuEPO). Ferrokinetic data before therapy showed 28.0 mumol/l of serum iron, 4.1 mumol/l of unsaturated iron-binding capacity, 4,060 ng/ml of serum ferritin, 148 min of plasma iron disappearance time, 45% of red cell iron utilization and 0.4 mg/kg/day of plasma iron turnover rate. She had 300-ml phlebotomies, first every other week then weekly, and subcutaneous injections of rHuEPO twice a week. Two years later, the total volume of phlebotomized blood reached 31 liters and her ferrokinetic data showed: serum iron 8.6 mumol/l, iron-binding capacity 39.6 mumol/l, serum ferritin 277 ng/ml, plasma iron disappearance time 52 min, red cell iron utilization 100% and plasma iron turnover rate 0.5 mg/kg/day. During the phlebotomy therapy, her hemoglobin levels were maintained above 12 g/dl. No adverse effect due to rHuEPO occurred. These findings provide evidence for the efficacy of rHuEPO in multiple phlebotomy therapy for hemosiderosis and may open new avenues for its clinical application.

Achievement of a complete cytogenetic response with hydroxyurea in a patient with chronic myelogenous leukemia

Hydroxyurea rarely produces a complete cytogenetic remission in patients with chronic myelogenous leukemia (CML). In this report, we describe one case of the CML patient who achieved complete cytogenetic remission (no Ph chromosome in 20-25 metaphase cells) by treatment with hydroxyurea alone. By the fluorescent in situ hybridization (FISH) methodology using bcr/abl specific translocation probe, sequential bone marrow specimens from the patient showed the characteristic 9;22 translocation at a higher rate (9-10%) than the normal control range (2.49-4.88%) at the time of complete cytogenetic remission. Thus, it is suggested that FISH is a more sensitive method to detect the bcr/abl fusion gene than conventional cytogenetic analysis for the detection of minimal residual disease in CML.