Identification of putative noncanonical driver mutations in patients with essential thrombocythemia.

Essential thrombocythemia (ET) cases without canonical JAK2, CALR, or MPL mutations, that is, triple-negative (TN) ET, have been found in 10%-20% of ET cases. Owing to the limited number of TN ET cases, its clinical significance remains unclear. This study evaluated TN ET's clinical characteristics and identified novel driver mutations. Among 119 patients with ET, 20 (16.8%) had no canonical JAK2/CALR/MPL mutations. Patients with TN ET tended to be younger and had lower white blood cell counts and lactate dehydrogenase values. We identified putative driver mutations in 7 (35%): MPL S204P, MPL L265F, JAK2 R683G, and JAK2 T875N were previously reported as candidate driver mutations in ET. Moreover, we identified a THPO splicing site mutation, MPL*636Wext*12, and MPL E237K. Four of the seven identified driver mutations were germline. Functional studies on MPL*636Wext*12 and MPL E237K revealed that they are gain-of-function mutants that increase MPL signaling and confer thrombopoietin hypersensitivity with very low efficiency. Patients with TN ET tended to be younger, although this was thought to be due to the inclusion of germline mutations, hereditary thrombocytosis. Accumulating the genetic and clinical characteristics of noncanonical mutations may help future clinical interventions in TN ET and hereditary thrombocytosis.

Allogeneic hematopoietic stem cell transplantation from a 2-HLA-haplotype-mismatched family donor for posttransplant relapse: a prospective phase I/II study.

HLA haploidentical hematopoietic stem cell transplantation (HSCT), i.e., HSCT from a 1-HLA-haplotype-mismatched family donor, has been successfully performed even as a second transplantation for posttransplant relapse. Is the haploidentical the limit of HLA mismatches in HSCT? In order to explore the possibility of HLA-mismatched HSCT from family donors beyond haploidentical relatives, we conducted a prospective phase I/II study of 2-HLA-haplotype-mismatched HSCT (2-haplo-mismatch HSCT). We enrolled 30 patients with posttransplant relapse (acute myeloid leukemia: 18, acute lymphoblastic leukemia: 11, non-Hodgkin lymphoma: 1). 2-haplo-mismatch HSCT was performed as the second to sixth transplantations. The donors were siblings (n = 12), cousins (n = 16), and second cousins (n = 2). The conditioning regimen consisted of fludarabine, cytarabine, melphalan, low-dose anti-thymocyte globulin, and 3 Gy of total body irradiation. Graft-versus-host disease (GVHD) prophylaxis consisted of tacrolimus, methylprednisolone, and mycophenolate mofetil. All patients achieved neutrophil engraftment, except for a case of early death. The cumulative incidences of grades II-IV and III-IV acute GVHD were 36.7% and 16.7%, respectively. The overall survival at 1 year, relapse, and non-relapse mortality rates was 30.1%, 38.9%, and 44.3%, respectively. Considering the poor prognosis of posttransplant relapse, 2-haplo-mismatch HSCT can be an alternative option in a second or third transplantation.

Toxoplasmic Encephalitis Followed by Primary EBV-Associated Post-Transplant Lymphoproliferative Disorder of the Central Nervous System in a Patient Undergoing Allogeneic Hematopoietic Stem Cell Transplant: A Case Report.

Toxoplasmic encephalitis (TE) and post-transplant lymphoproliferative disorder of the central nervous system (CNS-PTLD) are major complications after allogeneic hematopoietic stem cell transplant (allo-SCT); both are fatal without timely diagnosis and disease-specific treatment. Differential diagnosis of TE and CNS-PTLD can be challenging because brain biopsy, a gold standard for diagnosis, is sometimes not possible, owing to poor patient condition after allo-SCT. Here, we describe a case of isolated CNS-PTLD arising during the therapeutic course of TE. A 51-year-old man was admitted with mental abnormalities and fever on Day 106 after allo-SCT to treat myelodysplastic syndrome. Magnetic resonance imaging (MRI) revealed multiple nodular and ring-enhanced lesions in the brain, and the result of polymerase chain reaction (PCR) for Toxoplasma gondii in cerebrospinal fluid was positive; therefore, he was diagnosed with TE. Anti-Toxoplasma therapy led to clinical improvement, and the result of subsequent PCR was negative. However, he developed left-sided hemiplegia on Day 306. Head MRI revealed a new lesion and a growing lesion, presenting as ring-enhanced nodules. Brain biopsy was performed, and a pathologic diagnosis of Epstein-Barr virus-associated CNS-PTLD was made. There was no evidence of TE. He was treated successfully by reducing immunosuppressants, followed by rituximab administration and a donor lymphocyte infusion, resulting in complete remission. While T.gondii-specific PCR has great value for diagnosis of TE, CNS-PTLD can be diagnosed only by brain biopsy; hence, brain biopsy may be warranted in cases of suspected PTLD.

[Adult T-cell leukemia-lymphoma with severe hepatic damage and fluid retention successfully treated with mogamulizumab].

Adult T-cell leukemia-lymphoma (ATL) is a peripheral T-cell malignancy caused by the human T-cell lymphotropic virus, type I and it has an extremely poor prognosis. A 66-year-old man with severe hepatic damage, massive pleural effusion and ATL cell infiltration-induced ascites was referred to our department. Reduced-intensity cytotoxic chemotherapy was attempted, but could not continue due to persistent hyperbilirubinemia. Laboratory results also showed elevated lactate dehydrogenase (LDH) and serum albumin levels were profoundly decreased. A humanized monoclonal antibody against chemokine receptor type 4 (CCR4), mogamulizumab (Moga), was thereby challenged and it successfully resolved the hepatic damage. Finally, a standard dose of chemotherapy could be administered, and it induced a complete remission. The patient is still in remission more than three years after the final dosage of standard chemotherapy. These results indicate that Moga, whose pharmacokinetics are not significantly influenced by hepatic function or serum albumin, could be a promising treatment option for patients with ATL complicated by severe hepatic damage due to infiltration of ATL cells.

Loss of HLA-DR expression is related to tumor microenvironment and predicts adverse outcome in diffuse large B-cell lymphoma.

The interaction between tumor cells and the tumor microenvironment is essential in the development and progression of diffuse large B-cell lymphoma (DLBCL). Loss of human leukocyte antigen DR (HLA-DR) in DLBCL is a robust adverse prognostic marker. We evaluated the immunohitochemical expression of HLA-DR in lymphoma and the biologic implications of the loss of HLA-DR. The loss of HLA-DR correlated with clinical stage (p < 0.05), International Prognosis Index (p < 0.05), soluble interleukin-2 receptor (p < 0.05) and poor outcome in patients with DLBCL, especially among elderly patients. Flow cytometry analysis of the infiltrating T-cells showed that the mean CD4 + CD25 +/CD8 ratio of the infiltrating T-cells was higher in the HLA-DR positive group than in the HLA-DR negative group (p < 0.05). These data suggest that loss of HLA-DR expression in DLBCL decreases the ratio of helper T-cell within the T-cell population in the tumor microenvironment and might contribute to escape from immunosurveillance.

A new ATL xenograft model and evaluation of pyrrolidine dithiocarbamate as a potential ATL therapeutic agent.

Adult T-cell leukemia/lymphoma (ATL) is caused by human T-lymphotrophic virus type 1 infection and is one of the most refractory malignant T-cell lymphomas. Improvement of ATL therapy options requires the establishment of appropriate ATL animal models. In this study, we successfully generated an ATL mouse model by xenotransplantation of primary peripheral blood mononuclear cells (PBMCs) isolated from ATL patients (ATL cells) into nonobese diabetic/severe combined immunodeficiency/Jak3-null mice (NOJ mice). To generate the model, the ATL S1T cell line was subcutaneously injected into mice. Primary ATL cells were then transplanted subcutaneously, intraperitoneally, or intravenously. ATL cells infiltrated multiple organs, and elevated human soluble interleukin 2 receptor (IL-2R) levels were detected in peripheral blood. Injection of one million primary ATL cells was needed for successful engraftment into host mice. Thawed cells, frozen long-term in liquid nitrogen, could also be transplanted; however, more cells were required to achieve similar results. The median mouse survival time was proportional to the number of cells injected. Successful secondary transplantation of ATL cells from one NOJ mouse into another was achieved and confirmed by T-cell receptor analysis. Finally, we examined the effects of the antioxide pyrrolidine dithiocarbamate (PDTC) as an antitumor agent in vivo. PDTC administration inhibited the increase of soluble IL-2R and improved mouse survival, suggesting that this compound has potential as an anti-ATL agent. We demonstrated that ATL cells could be stably xenotransplanted into NOJ mice using primary cells. This model will be useful in the establishment of novel therapies to treat ATL.

A retrospective analysis of treatment outcomes in adult T cell leukemia/lymphoma patients with aggressive disease treated with or without allogeneic stem cell transplantation: A single-center experience.

Adult T cell leukemia/lymphoma (ATL) is an aggressive peripheral T cell neoplasm with very poor prognosis. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been reported as a curative treatment modality for ATL. However, there are no reports comparing chemotherapy alone with allo-HSCT in ATL. In this report, we retrospectively analyzed data for patients treated with (n = 29, median age 55 years) or without allo-HSCT (n = 37, median age 58 years) for ATL in Kagoshima University Hospital, located in one of the most endemic areas of human T cell lymphotropic leukemia virus type 1 infection. Forty patients (61%) started coordination for allo-HSCT. Ten patients (34.4%) received allo-HSCT while in complete remission (CR), whereas the others were not in CR. Twenty-five patients (86.2%) received reduced-intensity conditioning, and the others received myeloablative conditioning. With a median follow-up period for survivors of 41 months (range, 5 to 125 months), the 3-year overall survival (OS) rate from first chemotherapy for all patients (with or without allo-HSCT) was 35.2%. The 3-year OS from first chemotherapy for patients who received allo-HSCT or only chemotherapy was 44.9% and 27.7%, respectively. Univariate analyses revealed that high serum soluble IL-2 receptor (sIL-2R) levels (≥ 2000 U/mL) just before the conditioning regimen and progressive disease (PD) status at HSCT (according to Japan Clinical Oncology Group Study 0907 criteria) were significant risk factors for OS in the allo-HSCT group. Multivariate analyses revealed that PD status was a significant risk factor for OS in the allo-HSCT group. In the chemotherapy-only group, the 3-year OS rate was 61.5% (95% CI, 30.8% to 81.8%) in patients with serum sIL-2R levels < 2000 U/mL for > 3 months. In contrast, the 3-year OS rate was 5.7% (95% CI, .4% to 22.4%) in patients who did not achieve serum sIL-2R levels < 2000 U/mL for >3 months. Our single-center cohort experience indicates that chemosensitivity is the most important prognostic factor for OS in ATL patients and the use of allo-HSCT is limited in chemorefractory patients with aggressive ATL disease. In the chemosensitive patients, allo-HSCT demonstrated a tendency toward better OS. Further clinical studies are warranted to determine optimal treatments for patients who are less sensitive to conventional chemotherapy.

In vitro evaluation of dry powder inhaler devices of corticosteroid preparations.

BACKGROUND: Although investigations of the drug aerosols generated from inhaled corticosteroid (ICS) preparations and combined drug preparations provide basic information about inhalation therapy, many clinicians have one-sided data about the precision of drug aerosols from the manufacturer. The present study was conducted to analyze and compare the performances of dry powder inhaler (DPI) devices of ICS and combined drug preparations. METHODS: The particle size of individual aerosols was measured according to the time-of-flight principle in terms of their aerodynamic diameter by using the aerodynamic particle sizer spectrometer Model 3321. Percent aerosolization was measured using only stage #0 and backup filters of the Andersen non-viable sampler model AN-200. RESULTS: The particle size distribution of aerosols generated from a Turbuhaler™ and Twisthaler™ showed a mono-modal distribution of less than 5 µm. In contrast, Diskus™ showed a polydisperse distribution, ranging from 0.5 to 20 µm. The percentages of DPI preparations converted into aerosols with a particle size less than 11 µm at a suction flow rate of 28.3 L/min were 5.7-6.2% for Diskus, 37.5-47.0% for Turbuhaler, and 19.8% for Twisthaler. At a suction flow rate of 60 L/min, the conversion percentages for DPI preparations into aerosols with a particle size less than 7.6 µm were 5.9-7.5%, 78.2-86.7%, and 43.5%, respectively. CONCLUSIONS: Because in vitro differences in the aerosolization among different DPI devices containing ICS and combined drug preparations were observed, prescribers of these preparations should consider whether the patients will benefit more from the treatment of the central airways versus the peripheral airways.

[A study of drug aerosols generated by various devices for inhaled steroids].

PURPOSE: To evaluate the performances of various devices for inhaled steroid. METHODS: Aerosol particle diameters were determined using the Aerodynamic Particle Sizer Spectrometer. Conversion ratios to fine particle aerosol were determined using an Andersen non-bubble sampler. RESULTS: The mass median aerodynamic diameter was between 1 and 4 microm for all devices examined, tending to decrease with the increase in a suction flow rate. The percent of drug aerosolized less than 11 microm in diameter at a suction flow rate of 28.3 L/min was 100% for BDP-HFA and CIC-HFA, 87.5% for FP-HFA, 47.0% for BUD-DPI, 6.2% for FP-DPI, and 4.9% for SFC-DPI. The percent of drug aerosolized less than 7.6 microm in diameter at a suction flow rate of 60 L/min was 78.2% for BUD-DPI, 5.9% for FP-DPI, and 6.5% for SFC-DPI. DISCUSSION: For MDI preparations, devices permitting easy synchronization of drug atomization and inspiration exhibited higher inhalation efficiency. For DPI preparations, the BUD-DPI device exhibited higher inhalation efficiency, which was enhanced by increasing the suction flow rate.