Osamu Imataki

Antithymocyte globulin affects the occurrence of acute and chronic graft-versus-host disease after a reduced-intensity conditioning regimen by modulating mixed chimerism induction and immune reconstitution

Background. There have been no detailed analyses of the induction of donor cell–type chimerism, the onset and incidence of acute and chronic graft-versus-host disease (GVHD), and the immune recovery kinetics after reduced-intensity stem cell transplantation (RIST). Methods. To address these, with particular emphasis on the impact of the use of antithymocyte globulin (ATG) in RIST, we compared 39 consecutively registered patients who underwent RIST from an HLA-matched related donor and 33 patients who underwent conventional marrow-ablative transplantation. Results. The incidences of grades II to IV acute and chronic GVHD tended to be less in RIST with ATG than in either RIST without ATG or conventional marrow-ablative transplantation. In a multivariate analysis, the predictive factors for acute and chronic GVHD included, respectively, ATG and grades II to IV acute GVHD. In a chimerism analysis, the achievement of complete donor chimera in T-cell lineage was delayed in RIST without ATG compared with RIST with ATG (P =0.038), which might explain the observed delayed onset of acute GVHD in RIST with ATG compared with the other two regimens. The ratio of type 1 and 2 dendritic cells did not affect the development of GVHD, whereas the number of naive CD4+ T cells did. No difference was observed in the incidence of clinically definitive infection, including cytomegalovirus, among the three cohorts, regardless of the use of ATG. Conclusions. We suggest that the conditioning regimen and immunosuppressive strategy after RIST should be carefully balanced against the risk of GVHD and of relapse of the basic disorder caused by the lack of a graft-versus-leukemia benefit.

TRALI after the infusion of marrow cells in a patient with acute lymphoblastic leukemia

BACKGROUND:  TRALI is one of the most serious, life‐threatening complications after blood transfusion. Antibodies against neutrophils or HLA molecules from the donor are thought to be the primary causative agents. Rarely, antibodies in the recipient may react with transfused neutrophils and initiate the same events, which raises the possibility that TRALI may also occur in an allogeneic PBPC transplantation setting.

A prospective trial to evaluate the safety and efficacy of pravastatin for the treatment of refractory chronic graft-versus-host disease.

This prospective study evaluates the safety and efficacy of pravastatin for the treatment of chronic graft-versus-host disease (GVHD). We included 18 patients with refractory chronic GVHD. Oral pravastatin was started at 10 mg/day, and the dose was increased up to 40 mg/day in 4 weeks. This maximum dose was administered over 8 weeks. There were no severe adverse events caused by pravastatin. A clinical response was observed in the skin score in two patients, mouth score in five patients, eye score in two patients, liver score in three patients, platelet count score in one patient, and weight loss in two patients. The overall response rate was 28%. Immunophenotypic analyses showed that T-helper (Th)1 cells were dominant in all but one patient before treatment and that the Th1/Th2 ratio tended to be lower in the responders than in the nonresponders. A randomized controlled trial is warranted to evaluate the efficacy of pravastatin against chronic GVHD.

Multivariate analysis of factors influencing oral mucositis in allogeneic hematopoietic stem cell transplantation

Little information is available regarding the effect of oral intervention on the outcome of hematopoietic stem cell transplantation (HSCT). We retrospectively analyzed the incidence of oral mucositis after allogeneic HSCT with or without oral intervention among 96 consecutive patients in our hospital between January 1988 and March 2006. We combined two oral intervention strategies: cryotherapy and oral health care. The former was applied beginning in 2003 for patients being treated with melphalan, and the latter, which was the study’s main strategy, was applied to all HSCT recipients beginning in 2004. Oral mucositis was evaluated according to NCI CTCAE v3.0. The incidence of oral mucositis was 30.9% (17/55) in reduced-intensity stem cell transplantation (RIST), which was significantly lower than the 90.2% (37/41) in conventional stem cell transplantation (CST; P < 0.001). Among these 96 patients, severe oral mucositis was observed in 19 (46.3%) CST cases and in 6 (10.9%) RIST cases (P < 0.001). The occurrence of oral mucositis apparently decreased after oral health care instructions were given. Multiple logistic analysis revealed that the conditioning regimen and oral health care were independent risk factors for the incidence of oral mucositis. The cryotherapy did not exert enough potency to prevent oral mucositis in patients who had undergone CST or RIST. We concluded that oral health care improved tissue damage due to an overall upgrade in oral hygiene during chemotherapy.

Intravascular large B-cell lymphoma with FDG accumulation in the lung lacking CT/67gallium scintigraphy abnormality

Intravascular large B‐cell lymphoma (IVLBCL) is a rare lymphoma characterized by the presence of large tumour cells within the blood vessels. It has been considered that IVLBCL is a highly malignant disease with poor prognosis. However, it has been shown that a therapeutic effect resembling that of conventional B‐cell lymphomas may be obtained with the application of systemic chemotherapy at the early stage of this disease. Although involvement in the lung is often detected at autopsy, early diagnosis is quite difficult. In this report, we present a case of IVLBCL with pulmonary involvement where 18‐fluoro‐deoxyglucose positron emission tomography (FDG‐PET) was useful in the early diagnosis. Neither computed tomography (CT) nor 67gallium scintigraphy could reveal the presence of disease in the lung. Histological evidence of IVLBCL was obtained by TBLB after FDG uptake in the lung was confirmed by FDG‐PET. The patient exhibited a good response to the subsequent combination chemotherapy. We propose that FDG‐PET is a powerful tool for the early diagnosis of IVLBCL with pulmonary involvement, if the possibility of this disease presents in the patient with respiratory symptoms without abnormal findings by CT and 67gallium scintigraphy. Copyright

Detection of EML4-ALK fusion genes in a few cancer cells from transbronchial cytological specimens utilizing immediate cytology during bronchoscopy

The presence of fusion genes between the anaplastic lymphoma kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML4) genes is useful for determining appropriate molecular-targeted therapies in patients with non-small cell lung cancer (NSCLC). The diagnosis of NSCLC is often judged from transbronchial cytological specimens. The efficacy of RT-PCR for detection of EML4-ALK fusion genes in transbronchial cytological specimens has not been studied. Here, we evaluated the detection rate of EML4-ALK fusion genes in transbronchial cytological specimens positive for NSCLC by immediate cytology during bronchoscopic examination. Various numbers of H2228 cells carrying EML4-ALK variant 3 were combined with 1×10(6) wild-type WBCs. The RNA was extracted and the sensitivity of detection of the EML4-ALK fusion gene was determined using a nested RT-PCR. A total of 161 cell samples, from cases without available tissue samples, obtained by bronchoscopic examinations utilized for immediate cytology in patients with NSCLC were subsequently analyzed for EML4-ALK fusion genes using a nested multiplex RT-PCR. EML4-ALK variant 3 was detected in a small number of H2228 cells (10 cells), even in the presence of 1×10(6) WBCs (sensitivity: 0.001%). In the patient cytological samples, EML4-ALK fusion genes were detected in five of 161 NSCLCs (3.1%) and four of 88 adenocarcinomas (4.5%). Sequencing confirmed that these samples included three variant 1 genes, one variant 2 gene and one variant 3 gene. Using the same cytological samples, EGFR mutations were detected in 39 of 161 NSCLCs (24.2%) and 36 of 88 adenocarcinomas (40.9%). There was no case in which both EML4-ALK fusion and EGFR mutation were simultaneously detected. Rapid diagnosis during bronchoscopy utilizing immediate cytology contributed to the selection of the best samples for genetic analysis. EML4-ALK fusion genes as well as EGFR mutations were successfully detected in a small number of cancer cells from transbronchial cytological specimens using a nested multiplex RT-PCR. Our present strategy can be integrated into the clinical process without additional invasive examination of patients. In the era of molecular-targeted treatments for NSCLC, the combination of rapid diagnosis during bronchoscopic examination and stocking samples as cDNA could further correspond to genetic analyses of accumulating driver genes in NSCLC.

Micafungin does not influence the concentration of tacrolimus in patients after allogeneic hematopoietic stem cell transplantation.

BACKGROUND Tacrolimus is commonly used in stem cell transplant recipients for prophylaxis of graft-vs-host disease. Micafungin is widely used as a strong antifungal agent in empirical therapy in patients with febrile neutropenia. Both tacrolimus and micafungin are substrates of cytochrome P450 3A4 in vitro. Therefore, there is risk of drug interaction with concomitant administration of these drugs. OBJECTIVE To estimate the drug interaction of tacrolimus and micafungin by evaluating the pharmacokinetics in 6 patients who had undergone allogeneic stem cell transplantation. RESULTS The mean (SD) concentration-dose ratio of tacrolimus in all patients at 1, 4, 8, and 24 hours after concomitant administration of micafungin was 607 ± 306, 653 ± 328, 699 ± 340 and 671 ± 403 (ng/mL)/(mg/kg/d), respectively, and without micafungin was 756 ± 314 (ng/mL)/(mg/kg/d). The percentage of the concentration-dose ratio in patients treated with tacrolimus and micafungin vs patients treated with tacrolimus alone was 98%, 105%, 112%, and 108% at 1, 4, 8, and 24 hours, respectively. For both tacrolimus and micafungin, the 90% confidence intervals for the primary pharmacokinetic parameters (ie, the concentration-dose ratio at each point) ranged from 80% to 125%. CONCLUSION We conclude that there is no drug interaction between tacrolimus and concomitantly administered micafungin in stem cell transplantation recipients.

Lineage switch from precursor B cell acute lymphoblastic leukemia to acute monocytic leukemia at relapse

A lineage switch in leukemia, in which the leukemic cell lineage at onset converts to another lineage at a later time, is an uncommon type of hybrid (mixed) leukemia regarded as a variation of bilineage leukemia. We present a case of a 60-year-old female diagnosed with precursor B cell acute lymphoblastic leukemia (ALL), whose markers in flow cytometry shifted from their original status of CD19+, 22+, 79a+, 13+, HLA-DR+, and TdT+. Although her bone marrow achieved remission after induction therapy, there was a small residual population of leukemic cells in the liver. Residual disease was proved by biopsy and pathologically shown to have an immature phenotype of CD5+, CD10−, CD20−, CD79a− and myeloperoxidase negativity. Two weeks after liver biopsy, blast cells progressively appeared in the peripheral blood; these cells had a monocytoid morphology and phenotype (CD13, 14) but were accompanied by myeloid (CD33) and lymphoid (CD2, 4, 20) cells. Markers CD7, 10 and 19 were negative by flow cytometry. This phenotypical conversion from B-ALL to hybrid leukemia featuring monocytoid characteristics is known as a lineage switch. This case suggests that leukemic subclones tend to carry out de-differentiation, occasionally in extramedullary sites, which serve as a hotbed for the selection of resistant clones.

Potentiated activation of VLA-4 and VLA-5 accelerates proplatelet-like formation

Fibronectin (FN) plays important roles in the proliferation, differentiation, and maintenance of megakaryocytic-lineage cells through FN receptors. However, substantial role of FN receptors and their functional assignment in proplatelet-like formation (PPF) of megakaryocytes are not yet fully understood. Herein, we investigated the effects of FN receptors on PPF using the CHRF-288 human megakaryoblastic cell line, which expresses VLA-4 and VLA-5 as FN receptors. FN and phorbol 12-myristate 13-acetate (PMA) were essential for inducing PPF in CHRF-288 cells. Blocking experiments using anti-β1-integrin monoclonal antibodies indicated that the adhesive interaction with FN via VLA-4 and VLA-5 were required for PPF. PPF induced by FN plus PMA was accelerated when CHRF-288 cells were enforced adhering to FN by TNIIIA2, a peptide derived from tenascin-C, which we recently found to induce β1-integrin activation. Adhesion to FN enhanced PMA-stimulated activation of extracellular signal-regulated protein kinase 1 (ERK1)/2 and enforced adhesion to FN via VLA-4 and VLA-5 by TNIIIA2-accelerated activation of ERK1/2 with FN plus PMA. However, c-Jun amino-terminal kinase 1 (JNK1), p38, and phosphoinositide-3 kinase (PI3K)/Akt were not stimulated by FN plus PMA, even with TNIIIA2. Thus, the enhanced activation of ERK1/2 by FN, PMA plus TNIIIA2 was responsible for acceleration of PPF with FN plus PMA.

Expansion of α-galactosylceramide-stimulated Vα24+ NKT cells cultured in the absence of animal materials

Vα24+ NKT is an innate lymphocyte with potential antitumor activity. Clinical applications of Vα24+ natural killer (NK) T cells, which are innate lymphocytes with potential antitumor activity, require their in vitro expansion. To avoid the potential dangers posed to patients by fetal bovine serum (FBS), the authors evaluated non-FBS culture conditions for the selective and efficient expansion of human Vα24+ NKT cells. Mononuclear cells (MNCs) and plasma from the peripheral blood of normal healthy donors were used before and after G-CSF mobilization. MNCs and plasma separated from apheresis products were also used. MNCs were cultured for 12 days in AIM-V medium containing α-galactosylceramide (α-GalCer) (100 ng/mL) and IL-2 (100 U/mL) supplemented with FBS, autologous plasma, or autologous serum. The cultured cells were collected and their surface markers, intracellular cytokines, and cytotoxicity were evaluated. The highest expansion ratio for Vα24+ NKT cells was obtained from G-CSF-mobilized MNCs cultured in medium containing 5% autologous plasma. Cultures containing MNCs and autologous plasma obtained before and after G-CSF mobilization had approximately 350-fold and 2,000-fold expansion ratios, respectively. These results suggest that G-CSF mobilization conferred a proliferative advantage to Vα24+ NKT cells by modifying the biology of cells and plasma factors. Expanded Vα24+ NKT cells retained their surface antigen expression and production of IFN-γ and exhibited CD1d-independent cytotoxicity against tumor cells. Vα24+ NKT cells can be efficiently expanded from G-CSF-mobilized peripheral blood MNCs in non-FBS culture conditions with α-GalCer and IL-2.