CD20 gene deletion causes a CD20-negative relapse in diffuse large B-cell lymphoma.

In diffuse large B-cell lymphoma (DLBCL), a CD20-negative relapse is clinically significant because it is associated with chemo-refractory phenotypes and loss of a therapeutic target. The alteration of the CD20 gene is reported as infrequent in CD20-negative relapse in B-cell lymphoma. We established a DLBCL cell line with loss of CD20 expression (SD07) from a patient at CD20-negative relapse. She was initially diagnosed with CD20-positive DLBCL and received repeated immuno-chemotherapy that included rituximab. SD07, which has an immunoglobulin κ rearrangement identical to that of lymphoma cells at CD20-negative relapse, showed homozygous deletion of the CD20 gene with loss of the copy number of 11q12. SD07 is the first case in which it is proven that the loss of CD20 expression in relapsed DLBCL is the result of deletion of the CD20 gene. Deletion of the CD20 gene is a molecular mechanism of CD20-negative relapse in a subset of DLBCL.

Clinical efficacy and safety of biapenem for febrile neutropenia in patients with underlying hematopoietic diseases: a multi-institutional study.

A multi-institutional study was conducted to assess efficacy and safety of biapenem (BIPM), a carbapenem antibiotic, as an initial-stage therapeutic agent for febrile neutropenia (FN) in patients with hematopoietic diseases. A total of 216 patients from 25 medical institutions were enrolled in this study; of these, 204 were included in the safety analysis and 178 in the efficacy analysis. The combined (excellent and good) response rate was 67.9%, and antipyretic effect (subsidence + tendency to subsidence) was achieved within 3 and 5 days of treatment in 67.3 and 75.9% of patients, respectively. Thus, the clinical responses were gratifying. A response rate of 61.7% (37/60) was observed even in high-risk FN patients in whom neutrophil counts prior to and at 72 h after the start of BIPM were ≤100/µl. BIPM is considered to be a highly promising drug, with prompt onset of clinical benefit, as an initial-stage therapeutic agent for the treatment of FN in patients with hematopoietic diseases.

[Bilateral renal infiltration of acute lymphoblastic leukemia cells at relapse after allogeneic stem cell transplantation].

A 44-year-old male patient was diagnosed with acute lymphoblastic leukemia (CD10+, CD19+, CD20 weak) and underwent unrelated bone marrow transplantation (uBMT) with a conditioning regimen of cyclophosphamide plus total body irradiation during first complete remission (CR). Twenty months post-uBMT, the serum creatinine level (Cre) increased gradually, up to ≥ 1.5 mg/dl at 23 months. Since the increase in Cre was observed continuously, imaging examinations were performed and showed significant bilateral enlargement of the kidneys. Renal biopsy showed diffuse invasion of TdT, CD10 and CD19 positive lymphoid cells in the tubulo-interstitial region. Since leukemia cells were observed in the bone marrow, it was diagnosed as relapse in the bone marrow and kidney. Following reinduction chemotherapy, both kidneys returned to normal size. The patient entered into a second CR, but relapse occurred 6 months thereafter. The patient underwent uBMT again with a reduced-intensity conditioning regimen and CR has been maintained up to 5 months post-second uBMT. Although it is considered rare for relapse to occur with diffuse enlargement of both kidneys, as shown in this case, it is important to confirm the state of the kidney by performing blood tests and image diagnosis during the early phase, when renal dysfunction of an uncertain cause occurs after transplantation.

Over-expression of CCL3 MIP-1alpha in a blastoid mantle cell lymphoma with hypercalcemia.

We analyzed a case with the blastoid variant of mantle cell lymphoma (MCL-BV), a rare subtype of B-cell lymphoma, presenting with marked hypercalcemia at diagnosis. Enzyme-linked immunosorbent assay (ELISA) showed elevated serum levels of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-1alpha (MIP-1alpha), and type I collagen telopeptide, but not parathyroid hormone, calcitriol or parathyroid hormone-related peptide at diagnosis, suggesting local osteoclastic hypercalcemia in this case. By reverse transcription polymerase chain reaction (RT-PCR) analysis, we found predominant expression of mRNA for MIP-1alpha in addition to those for receptor-activator of nuclear-factor kappa B ligand (RANKL), TNF-alpha, and IL-6 in lymphoma cells obtained from the patient. Furthermore, recombinant MIP-1alpha significantly stimulated (3)H-thymidine uptake by isolated MCL cells in vitro. Treatment with intravenous fluids, bisphosphonate, and methylprednisolone followed by combination chemotherapy promptly corrects the hypercalcemia and successfully induced complete remission, which was accompanied by a decrease of these cytokines in the serum, including MIP-1alpha. In the present case, MIP-1alpha, an osteoclast-activating factor produced by mantle lymphoma cells, may contribute to the development of hypercalcemia. It likely acts through RANKL expression in tumor cells and/or stroma cells, as indicated in multiple myeloma (MM) and adult T-cell leukemia/lymphoma (ATLL). Furthermore, MIP-1alpha is also involved in the development of an aggressive phenotype on MCL by stimulating proliferation of these lymphoma cells. In summary, the present study demonstrated that MIP-1alpha is an important factor in the development of both hypercalcemia and an aggressive phenotype in some types of B-cell lymphoma.

Megakaryopoiesis and platelet function in polycythemia vera and essential thrombocythemia patients with JAK2 V617F mutation.

Patients with Ph chromosome negative myeloproliferative disease (Ph-MPD) have an increased risk of vascular complications. It remains controversial whether patients with the JAK2 V617F mutation (V617F) exhibit increased risk, while recent growing evidence has shown a critical role for V617F in clonal erythropoiesis in Ph-MPD. We studied 53 patients with Ph-MPD especially in relation to megakaryopoiesis, the thrombotic complications and the presence of V617F. Using novel mutation-specific PCR which is a highly sensitive PCR-based assay for detection of JAK2 mutated allele(s), we identified V617F in 38 Ph-MPD, which include 13 polycythemia vera (PV), 23 essential thrombocythemia (ET) and 2 chronic idiopatic myelofibrosis. The numbers of megakaryocytes were significantly increased in PV and ET patients with V617F, but the platelet counts were slightly lower. Although statistically not significant, the incidence of thrombotic events was higher in the group with V617F compared to in those without the mutation. Agonist-induced in vitro platelet aggregation and platelet adhesion were not affected by the presence of this mutation. Nonetheless, we found a hypercoagulable state in Ph-CMPD with V617F by employing whole blood thromboelastography. It suggests pre-thrombotic tendencies in CMPD are complex and JAK2 V617F mutation might have a role in vivo blood coagulation by altering not only the number, but function(s) of all three myeloid cells, including red blood cells, white blood cells and platelets in Ph-CMPD.

Histopathology of bone marrow reconstitution after umbilical cord blood transplantation for hematological diseases.

To study hematopoietic reconstitution in umbilical cord blood transplantation (CBT), bone marrow (BM) histology was investigated in 35 biopsies after bone marrow transplantation (BMT) and in 40 biopsies after CBT. BM biopsies were obtained at different times after transplantation and were evaluated for cellularity, number of megakaryocytes and CD34-positive cells, and fibrosis. In biopsies up to 29 days after BMT, cellularity was increased and megakaryocytes were observed, but at 29 days after CBT, biopsies showed severe cellular depletion and almost no megakaryocytes. In addition, fewer CD34-positive cells were observed after CBT compared to after BMT. After day 30 after CBT, hematopoietic recovery of the BM was gradually observed and after day 100 after transplantation, no essential differences were observed between BMT and CBT. Hematopoietic recovery of the BM after CBT was delayed compared to that after BMT, but engraftment of donor cells after CBT was also observed in histopathologically. To the best of the authors' knowledge this is the first histopathological description of BM reconstitution after CBT.

[Splenic irradiation as a successful treatment for an elderly patient with B-cell prolymphocytic leukemia].

We report a case of B-cell prolymphocytic leukemia (B-PLL) that was treated successfully with splenic irradiation (SI). An 86-year-old man underwent a medical examination for lumbago and general fatigue at another hospital in June 2007. A compressed lumbar fracture and splenomegaly were found using computed tomography (CT). Thereafter, the patient consulted our hospital because of leukocytosis. Peripheral blood showed hemoglobin level 9.8 g/dl and white blood cell count 38.1x10(9)/l with 91% atypical cells. Surface marker analysis demonstrated that atypical cells were positive for CD20, CD22, FMC7, surface IgM, surface IgD and kappa, but were negative for CD5, TdT and lambda. The morphology of these cells was compatible with prolymphocytes with prominent nucleoli and condensed nuclear chromatin. A diagnosis of B-PLL was made. SI (total dose 20 Gy) was chosen for the treatment and a single course of SI was very effective without causing any significant adverse events. This case demonstrates that SI may remain valuable for the treatment of B-PLL in an elderly patient.

[Successful treatment with prednisolone for autoimmune myelofibrosis accompanied with Sjögren syndrome].

A 66-year-old woman presented with anemia in January 2006. She was admitted to our Department in February, after laboratory data showed pancytopenia and bone marrow biopsy reticulin fibrosis. The results of the diagnostic work-up, which included the anti-SS-A antibody, anti-SS-B antibody positivity and salivary gland scintigraphy, Schirmer test and Rose Bengal test, supported the classification criteria of Sjögren syndrome. Due to secondary myelofibrosis accompanied by Sjögren syndrome, she was started on prednisolone (PSL) and recovered completely from the anemia and thrombocytopenia. After the PSL was tapered, a recent follow-up indicated that the peripheral blood had normalized with the PSL therapy. As a causal disease of autoimmune myelofibrosis in collagen disease, systemic lupus erythematosus occurs frequently. This patient is considered to be a rare case in whom secondary myelofibrosis was accompanied by Sjögren syndrome.

Hypermethylation of CpG islands in p16 as a prognostic factor for diffuse large B-cell lymphoma in a high-risk group.

PURPOSE: The aim of the study was to analyze the methylation status of the promoter regions of p15 and p16 and to assess the prognostic significance of promoter hypermethylation in diffuse large B-cell lymphoma (DLBCL). EXPERIMENTAL DESIGN: DLBCL was diagnosed by morphology and immunohistochemical analysis according to the World Health Organization (WHO) classification. The methylation status of CpG islands in the p15 and p16 promoters was analyzed by methylation-specific polymerase chain reaction in 49 DLBCLs. RESULTS: Hypermethylation of the p15 and p16 promoters was detected in 20 (41%) and 22 (45%) of the 49 DLBCLs, respectively. Among all patients with DLBCL, there was no significant difference in the overall survival between those with hypermethylated and unmethylated p15 (P=0.442) or between those with hypermethylated and unmethylated p16 (P=0.468). Therefore, methylation was analyzed in combination with evaluation of clinical features using the international prognostic index (IPI). In the high-intermediate-risk and high-risk groups, patients with hypermethylated p16 had significantly lower survival rates than those of patients in the same risk group with unmethylated p16 (P=0.010). CONCLUSIONS: Our results suggest that hypermethylation of the p16 promoter indicates a poor prognosis in high-intermediate-risk and high-risk DLBCL patients, and may be a useful marker for selection of appropriate treatment when used in conjunction with the IPI.

Cotylenin A-induced differentiation is independent of the transforming growth factor-beta signaling system in human myeloid leukemia HL-60 cells.

Cotylenin A, which has been isolated as a plant growth regulator, potently induces the differentiation of human myeloid leukemia cells. Treatment of HL-60 cells with a combination of transforming growth factor (TGF)-beta and 1alpha, 25-dihydroxyvitamin D(3) (VD3) resulted in increased differentiation compared to separate treatments, but TGF-beta did not affect the cotylenin A-induced differentiation of HL-60 cells. It is possible that the signal transduction pathway used by cotylenin A for inducing the differentiation of leukemia cells is the same as that used by TGF-beta. However, cotylenin A did not affect the expression of TGF superfamily or Smad genes in HL-60 cells. Treatment with neutralizing anti-TGF-beta antibody or an inhibitor of TGF-beta signaling did not inhibit cotylenin A-induced differentiation, although VD3-induced differentiation was significantly suppressed by these treatments. The subcellular distribution of Smad3 was also unaffected by cotylenin A. These results suggest that the cotylenin A-induced differentiation of leukemia cells is independent of the TGF-beta signaling system, although TGF-beta acts as an autocrine mediator of the growth arrest and differentiation of leukemia cells induced by VD3 and other inducers.

Role of MmTRA1b/phospholipid scramblase1 gene expression in the induction of differentiation of human myeloid leukemia cells into granulocytes.

OBJECTIVE: We previously cloned a human normal counterpart (MmTRA1b/phospholipid scramblase 1) of the mouse leukemogenesis-associated gene MmTRA1a. MmTRA1b gene expression was increased during differentiation of human monoblastic leukemia U937 cells using some differentiation inducers but not 1alpha,25-dihydroxyvitamin D(3) (a typical monocytic differentiation inducer). To further elucidate the role of human MmTRA1b gene expression in the differentiation of myelogenous leukemia cells, we measured MmTRA1b gene expression in several myeloid leukemia cell lines and primary leukemia cells. MATERIALS AND METHODS: The expression of MmTRA1b mRNA was determined by semiquantitative reverse transcriptase polymerase chain reaction. RESULTS: Expression of the MmTRA1b gene was markedly induced during granulocytic differentiation of promyelocytic leukemia NB4 and HT93 cells induced by all-trans retinoic acid (ATRA). The level of MmTRA1b mRNA was significantly increased during differentiation toward granulocytes, but not monocytes/macrophages, in bipotential myeloid leukemia HL-60 cells. The level of MmTRA1 mRNA was not increased during erythroid differentiation induced by hemin in erythroid leukemia K562 and HEL cells or during megakaryocytic differentiation induced by 12-O-tetradecanoylphorbol-13-acetate in K562 cells. Expression of the MmTRA1b gene also was not induced when apoptosis of NB4 cells was induced by antileukemic drugs. ATRA-induced differentiation of antisense MmTRA1b-transfected NB4 cells was significantly suppressed. On the other hand, ATRA induced the differentiation of MmTRA1b-transfected NB4 cells more efficiently than that of mock-transfected cells. MmTRA1b mRNA also was clearly induced in ATRA-treated primary acute promyelocytic leukemia cells during granulocytic differentiation. CONCLUSION: MmTRA1b mRNA was specifically induced during granulocytic differentiation of acute promyelocytic leukemia cells and was associated with induction of their differentiation.

MmTRA1b/phospholipid scramblase 1 gene expression is a new prognostic factor for acute myelogenous leukemia.

We previously found that expression of the Mm-1 cell-derived transplantability-associated gene 1b (MmTRA1b)/phospholipid scramblase 1 gene was markedly induced during the granulocytic differentiation of human myeloid leukemia cells. To evaluate the role of MmTRA1b expression in human myeloid leukemia, we investigated the relative levels of MmTRA1b transcripts in 81 patients with acute myelogenous leukemia (AML) by the reverse transcriptase polymerase chain reaction. The expression of MmTRA1b in AML-M1, -M5a and -M5b was significantly lower than that in normal bone marrow cells. The levels of MmTRA1b expression in AML-M2 and -M4 varied among patients. Higher MmTRA1b mRNA levels were associated with significantly longer overall survival in AML, especially in AML-M4 patients, independent of chromosomal aberrations such as t(8;21) and inv(16). The present results suggest that the MmTRA1b mRNA level is a new prognostic factor for AML, especially the AML-M4 subtype.

Acute respiratory distress syndrome during the third infusion of rituximab in a patient with follicular lymphoma.

We present the case of a 66-year-old man with follicular lymphoma who developed acute respiratory distress syndrome (ARDS) during a third infusion of rituximab. High fever, tachypnea, and progressive hypoxemia accompanied by massive bilateral pleural effusions appeared suddenly approximately 3 hours after the third infusion was started, although the 2 prior infusions of rituximab had produced only mild adverse effects. The patient was treated successfully with high-dose methylprednisolone and 3 days of mechanical ventilatory support. No evidence was obtained to indicate that the ARDS had been caused by either cytokine release or tumor lysis, and serum human antichimeric antibody was not detected. Although the cause of ARDS was not confirmed, our experience in this case suggested that an anaphylactic reaction induced by repeated infusion of rituximab was involved in the onset of pulmonary disease. Although ARDS is rarely seen with rituximab infusion, careful management is required for safe administration of the newly developed rituximab therapy. This management includes monitoring biological reactions not only during the initial infusion but also during subsequent infusions of the antibody.

Disappearance of CD21-positive follicular dendritic cells preceding the transformation of follicular lymphoma: immunohistological study of the transformation using CD21, p53, Ki-67, and P-glycoprotein.

Some follicular lymphomas histologically transform into diffuse aggressive lymphomas, the prognosis of which is poor. There are, however, no reliable histological criteria for predicting which cases will later undergo such transformation. In low-grade B-cell lymphomas, follicular dendritic cells form dense mesh-like networks that contain accumulating neoplastic B-cells. These are rare in high-grade lymphomas. We immunohistochemically analyzed CD21-positive follicular dendritic cells in 32 follicular lymphomas, including 3 transformed lymphomas, in addition to immunohistological study using P-glycoprotein, p53, and Ki-67. We found that the mesh-like networks in follicles are more clearly defined in low-grade lymphomas than in high-grade lymphomas (p = 0.015). Neoplastic follicles in 2 transformed lymphomas lost the networks of follicular dendritic cells before transformation despite the existence of morphologically clear follicles. This differed from the non-transformed cases of the same cytological grades. Prognosis was statistically better for patients with low-grade tumor than for those with high-grade tumor (p = 0.026), and there was a trend toward poorer survival among CD21-negative cases (p = 0.186). P-glycoprotein, p53, and Ki-67 expressions did not provide sufficient information to predict the transformation of follicular lymphoma. The presence of CD21-positive follicular dendritic cells in neoplastic follicles might help predict the potential of follicular lymphoma to transform to diffuse large B-cell lymphoma.

[Primary subcutaneous mantle cell lymphoma treated successfully with THP-COP therapy].

A 73-year-old man noticed a subcutaneous tumor on the left upper palpebra from April 1998, but did not seek therapy for it. Facial subcutaneous tumors appeared from November 1999, and multiple tumors appeared on the skin of the chest and both upper arms from January 2000. Tumor biopsy revealed that these tumors were non-Hodgkin lymphoma showing CD19 (+), CD20 (+), CD5 (+), CD10 (-), smIgM (+), sm lambda (+) and cyclin D1 (+). The karyotype was t(11;14) (q13;q32), but bcl-1 gene rearrangement was not detected. On the basis of these data, primary mantle cell lymphoma (MCL) of the subcutis was diagnosed. The patient underwent eight courses of THP-COP therapy, and complete remission was achieved. Primary subcutaneous B-cell lymphoma, especially MCL, is rare. MCL is aggressive and difficult to cure; the median survival of patients is 3 to 5 years, and the 5-year survival is 30%. However, the present patient showed a good response to chemotherapy, and complete remission has continued for 17 months since the MCL was first diagnosed.

[Hepatitis-associated aplastic anemia preceded by a hemophagocytic syndrome-like state].

A 21-year-old man was admitted to our hospital for acute hepatitis of unknown cause. His liver function improved with rest, but worsened 2 months later. He developed a high fever and pancytopenia. The serum level of cytokines including TNF-alpha, IFN-gamma, IL-6, and M-CSF was elevated, and hemophagocytes were seen in bone marrow. These findings suggested a hemophagocytic syndrome-like state. With prednisolone, gamma-globulin, and G-CSF, the high fever disappeared and the patient's liver function gradually recovered. However, the severe pancytopenia persisted. The bone marrow became acellular with a small number of hemophagocytes, and hepatitis-associated aplastic anemia was diagnosed. After immunosuppressive therapy with ATG, CyA and G-CSF was started, and the patient showed hematopoietic reconstitution. The bone marrow CD4+/CD8+ lymphocyte ratio recovered to within the normal range, and the serum cytokines including TNF-alpha and IFN-gamma decreased. The increase in serum cytokines, particularly TNF-alpha and INF-gamma, as well as the presence of activated T cells associated with the preceding hemophagocytic syndrome-like state may have predisposed this patient to aplastic anemia.