Shuichi Shirane

Activation of the thrombopoietin receptor by mutant calreticulin in CALR-mutant myeloproliferative neoplasms.

Recurrent somatic mutations of calreticulin (CALR) have been identified in patients harboring myeloproliferative neoplasms; however, their role in tumorigenesis remains elusive. Here, we found that the expression of mutant but not wild-type CALR induces the thrombopoietin (TPO)-independent growth of UT-7/TPO cells. We demonstrated that c-MPL, the TPO receptor, is required for this cytokine-independent growth of UT-7/TPO cells. Mutant CALR preferentially associates with c-MPL that is bound to Janus kinase 2 (JAK2) over the wild-type protein. Furthermore, we demonstrated that the mutant-specific carboxyl terminus portion of CALR interferes with the P-domain of CALR to allow the N-domain to interact with c-MPL, providing an explanation for the gain-of-function property of mutant CALR. We showed that mutant CALR induces the phosphorylation of JAK2 and its downstream signaling molecules in UT-7/TPO cells and that this induction was blocked by JAK2 inhibitor treatment. Finally, we demonstrated that c-MPL is required for TPO-independent megakaryopoiesis in induced pluripotent stem cell-derived hematopoietic stem cells harboring the CALR mutation. These findings imply that mutant CALR activates the JAK2 downstream pathway via its association with c-MPL. Considering these results, we propose that mutant CALR promotes myeloproliferative neoplasm development by activating c-MPL and its downstream pathway.

JAK2 , CALR , and MPL mutation spectrum in Japanese patients with myeloproliferative neoplasms

Recurrent somatic mutations in the JAK2 , MPL , and CALR genes have been described in patients diagnosed with Philadelphia-negative myeloproliferative neoplasms (MPN), including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). These mutations are generally

JAK2V617F mutation status and allele burden in classical Ph-negative myeloproliferative neoplasms in Japan

JAK2V617F, a gain-of-function mutation in the tyrosine kinase JAK2, is frequently detected in classical myeloproliferative neoplasms (MPNs). In the present study, we determined the JAK2V617F allele burden in Japanese MPN patients using alternately binding probe competitive-polymerase chain reaction, a highly quantitative method recently developed by our group. Although we observed strong similarities in terms of epidemiological parameters associated with the JAK2V617F allele burden between our cohort and others, we found a higher JAK2V617F allele burden in Japanese polycythemia vera (PV) patients and lower frequencies of thrombosis in Japanese MPN patients compared with previous reports. In addition, despite the presence of high red blood cell counts, some patients bearing the JAK2V617F mutation were not diagnosed as PV, as their hemoglobin values were lower than the WHO PV criterion. In these patients, the JAK2V617F allele burden was strikingly similar to that in PV patients fulfilling the 2008 WHO criteria, suggesting that these patients can be classified as PV. Although isotopic measurement of red cell mass (RCM) is required for definitive diagnosis of PV, our data suggest that precise measurement of the JAK2V617F allele burden may improve the diagnosis of PV when RCM has not been determined.

The 2014 BCSH criteria and the 2016 WHO criteria for essential thrombocythemia: A comparison in a large-scale cohort

There are currently 2 representative diagnostic criteria for essential thrombocythemia (ET), the 2014 British Committee for Standards in Hematology Guidelines (BCSH) criteria and the 2016 World Health Organization (WHO) criteria. We compare and discuss the advantages and disadvantages of the 2 criteria.

The 2016 WHO diagnostic criteria for polycythemia vera renders an accurate diagnosis to a broader range of patients including masked polycythemia vera: comparison with the 2008 WHO diagnostic criteria

Tomasz Sacha, Joanna G ora-Tybor, Monika Szarejko, Gra_ zyna Bober, Olga Grzybowska-Izydorczyk, Joanna NiesiobeRdzka-KreR _ zel, Marek Dudzi nski, Ewa Wasilewska, Krzysztof My sliwiec, Justyna Gil, Michał Gniot, Iwona Pietkun, Ewa MeRdra s, Jadwiga Hołojda, Joanna Wącław, Krzysztof Giannopoulos Chair and Department of Hematology, Jagiellonian University Medical College, Krak ow, Poland Department of Hematology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland Department of Hematology and Transplantology, Medical University of Gda nsk, Gda nsk, Poland Hematology and Bone Marrow Transplantation Department, Medical Silesian University, Katowice, Poland Department of Hematology, Medical University of Lodz, Copernicus Memorial Hospital, Lodz, Poland Hematology, Oncology, and Internal Medicine Department, Medical University of Warsaw, Warsaw, Poland Hematology Department, Teaching Hospital No 1 in Rzesz ow, Rzesz ow, Poland Hematology Department, Medical University of Białystok, Białystok, Poland Hematology Department, Clinical Hospital in Zielona G ora, Zielona G ora, Poland Department of Hematology, Markiewicz Memorial Oncology Center Brzozow, Brzozow, Poland Hematology Department, Medical University in Pozna n, Pozna n, Poland Hematology and Internal Medicine Department, Rydygier Specialistic Hospital, Krak ow, Poland Department of Hematology, Neoplastic Blood Disorders and Bone Marrow Transplantation in Wrocław, Poland Department of Hematology, Specialized Hospital in Legnica, Poland Experimental Hematooncology Department, University of Lublin, Lublin, Poland

[Current problems in the diagnosis of Philadelphia-negative myeloproliferative neoplasms in Japan].

To investigate the current situation and issues regarding the diagnosis of Philadelphia-negative myeloproliferative neoplasms (MPN) in Japan, we retrospectively analyzed an accumulated cohort consisting of 1,081 patients with suspected MPN. Based on WHO2008 diagnostic criteria, we diagnosed 101 of these patients with polycythemia vera, 179 with essential thrombocythemia, 36 with primary myelofibrosis, 45 with unclassifiable MPN, and 4 with myelodysplastic syndromes. Out of 716 patients, 235 were not diagnosed with MPN despite the detection of a JAK2, CALR, or MPL mutation. Among 156 patients with undefined MPN receiving further follow-up, none underwent bone marrow examination and screening for BCR-ABL1 was not performed in 88 cases. Thus, diagnosis was not possible in these cases due to a lack of essential examinations. Since the prognosis and treatment strategy associated with MPN differ among disease types, in addition to mutation analysis, the importance of bone marrow examination and screening for BCR-ABL1 must be re-recognized.

Breakthrough Exophiala dermatitidis infection during prophylactic administration of micafungin during second umbilical cord blood transplantation after graft failure

Exophiala dermatitidis infections in patients with hematological malignancies are very rare. Our patient had a blood stream infection caused by E. dermatitidis following the second umbilical cord blood transplantation (UCBT) after graft failure during the first UCBT. To our knowledge, this is the first report describing a breakthrough fungal infection caused by E. dermatitidis during the prophylactic administration of micafungin (MCFG). Therefore, MCFG‐treated patients should be monitored for breakthrough E. dermatitidis infection during hematopoietic stem cell transplantation.

Outcome of patients with acute undifferentiated leukemia after allogeneic hematopoietic stem cell transplantation

The World Health Organization (WHO) has categorized acute undifferentiated leukemia (AUL) as a rare subtype of acute leukemias of ambiguous lineage (ALAL). The prognosis of AUL is considered poor and it expresses no known lineage-specific marker [1,2]. Although allogeneic hematopoietic stem cell transplantation (allo-HSCT) can potentially cure various hematological malignancies, little is known about the transplant modality and outcomes of patients with AUL. Most published reports to date have analyzed the clinical characteristics and prognosis of AUL together with those of mixed-phenotypic acute leukemia (MPAL) although they have quite different clinical characteristics [3–5]. Therefore, these studies might not be particularly informative for patients with AUL. Here, we analyzed the clinical outcomes of 10 patients with AUL after allo-HSCT at our institution. We retrospectively reviewed 911 patients with acute leukemia who were admitted to our institution between April 2005 and March 2017. Consensus diagnostic criteria for AUL have not yet been established. Therefore, we defined AUL based on the WHO classification [2] as leukemic cells that were not positive for any lineage-specific markers (myeloid lineage: myeloperoxidase [cytochemistry, immunohistochemistry, or flow cytometry]; B cell lineage: CD19, CD79a, or cytoplasmic CD22; T cell lineage: cytoplasmic CD3 or surface CD3). Our institutional committee on research ethics approved the study (approval number: 1973), which proceeded according to the Declaration of Helsinki. Transplant procedures have been described in detail elsewhere [6]. Generally, myeloablative conditioning mainly included a total body irradiation (TBI) regimen (12Gy) in combination with cyclophosphamide (CY) at 120mg/kg, or a non-TBI regimen that included intravenous busulfan at 12.8mg/kg, and CY at 120mg/kg. The preparative regimen for the reduced-intensity procedure consisted of fludarabine (30mg/m for 6 d), melphalan (40mg/m for 2 d), and TBI (4 Gy). The patients were given intravenous infusion of donor hematopoietic stem cells on day 0. All patients received acute graft-versus-host disease (GVHD) prophylaxis with cyclosporine or tacrolimus and short-term methotrexate. Tacrolimus was used in cases involving either unrelated or human leukocyte antigen (HLA) mismatched transplantation. Engraftment in alloHSCT is defined as the first of three consecutive days with an absolute neutrophil count of 0.5 10/l or greater. The probability of overall survival (OS) was estimated using Kaplan–Meier product limit method. We calculated OS from the date of allo-HSCT to the last assessment for survivors. The cumulative incidence of non-relapse mortality (NRM), relapse and acute GVHD were evaluated using Gray’s method. For each estimation of the cumulative incidence of an event, death without event was defined as a competing risk. All statistical analyses were performed with EZR, a graphical user interface for R software (The R Foundation for Statistical Computing, version 2.13.0; www.r-project.org). Among the 911 patients with acute leukemia, AUL was classified in only 12 (1.3%). Among those, one patient refused to undergo allo-HSCT and another died early. Table 1 shows the characteristics of 10 patients with AUL who underwent allo-HSCT. The median age at the time of transplantation was 45 (range: 22–63) years. Seven (70.0%) and three (30.0%) patients were male and female, respectively. Marrow fibrosis was found in two (20.0%) patients, but extramedullary disease was not present in any patient. Cytochemical findings were negative for myeloperoxidase in all patients. Immunophenotyping revealed the common expression of CD34 (90.0%), HLADR (80.0%), and CD13 (60.0%), but the lineage-specific markers were absent in all patients. Cytogenetic studies of nine patients revealed chromosomal abnormalities in five (55.6%) of them. In five patients (cases 2, 4, 5, 9, and 10) with available data, polymerase chain reaction (PCR)

Mutational subtypes of JAK2 and CALR correlate with different clinical features in Japanese patients with myeloproliferative neoplasms

The majority of patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) harbor JAK2, CALR, or MPL mutations. We compared clinical manifestations of different subtypes of JAK2 and CALR mutations in Japanese patients with MPNs. Within our cohort, we diagnosed 166 patients as polycythemia vera (PV), 212 patients as essential thrombocythemia (ET), 23 patients as pre-primary myelofibrosis (PMF), 65 patients as overt PMF, and 27 patients as secondary myelofibrosis following the 2016 WHO criteria. Compared to patients with JAK2V617F-mutated PV, JAK2 exon 12-mutated PV patients were younger, showed lower white blood cell (WBC) counts, lower platelet counts, higher red blood cell counts, and higher frequency of thrombotic events. Compared to JAK2-mutated ET patients, CALR-mutated ET patients were younger, showed lower WBC counts, lower hemoglobin levels, higher platelet counts, and fewer thrombotic events. CALR type 1-like mutation was the dominant subtype in CALR-mutated overt PMF patients. Compared with JAK2V617F-mutated ET patients, JAK2V617F-mutated pre-PMF patients showed higher LDH levels, lower hemoglobin levels, higher JAK2V617F allele burden, and higher frequency of splenomegaly. In conclusion, Japanese patients with MPNs grouped by different mutation subtypes exhibit characteristics similar to those of their Western counterparts. In addition, ET and pre-PMF patients show different characteristics, even when restricted to JAK2V617F-mutated patients.

Dasatinib-induced anti-leukemia cellular immunity through a novel subset of CD57 positive helper/cytotoxic CD4 T cells in chronic myelogenous leukemia patients

Dasatinib induces lymphocytosis of large granular lymphocytes (LGLs) in a proportion of patients with chronic myelogenous leukemia (CML), and is associated with better clinical outcomes. LGLs consist of cytotoxic T lymphocytes and natural killer cells; however, the context and phenotypic/functional features of each type of LGL are unknown. To better define features of these LGLs, we investigated lymphocytosis in CML patients treated with dasatinib. D57-positive and CD4-positive type I T-helper (Th) cells (CD57+ Th cells) rarely occur in CML patients without lymphocytosis and in healthy individuals; however, a substantial increase in the proportion of CD57+ Th cells was observed in CML patients treated with dasatinib. In addition, these cells showed appreciable levels of cytocidal activity via cytotoxic degranulation. Analysis of T-cell receptor α and β sequences showed a skewed T-cell repertoire in the CD57+ Th cells. Furthermore, patients with LGLs and CD57+ Th lymphocytosis achieved stronger molecular responses than did those without lymphocytosis. While further studies are warranted, our observations suggest that dasatinib induces the expansion of CD57+ Th-LGLs, which may play a crucial role in the dasatinib-induced response against Philadelphia chromosome-positive leukemia.