Granulocyte transfusion (GTX) is a therapeutic option for patients with prolonged neutropenia suffering from severe infections. Efficient granulocyte collection by apheresis from donors requires ...clear separation of granulocytes from red blood cells (RBCs), and infusion of high-molecular-weight (MW) hydroxyethyl starch (HES) facilitates RBC sedimentation. Recent research has shown that apheresis with medium-MW HES may prevent adverse effects of high-MW HES on donors, but the rationale for collection with medium-MW HES has yet to be evaluated. To validate the use of medium-MW HES, we first performed experiments with whole blood samples to determine how efficiently high-, medium- and low-MW HES separated granulocytes from RBCs, and found that medium-MW HES was just as efficient as high-MW HES. We also reviewed clinical data of granulocyte apheresis at our institution to evaluate granulocyte yields. Retrospective analysis of granulocyte collection revealed that apheresis with medium-MW HES yielded sufficient granulocytes for GTX and that donor anemia reduced collection efficiency. These results collectively may help us to establish a safer method for apheresis targeting polymorphonuclear granulocytes as an alternative to high-MW HES.
To establish effective therapeutic strategies for eosinophil-related disorders, it is critical to understand the developmental pathway of human eosinophils. In mouse hematopoiesis, eosinophils ...originate from the eosinophil lineage-committed progenitor (EoP) that has been purified downstream of the granulocyte/macrophage progenitor (GMP). We show that the EoP is also isolatable in human adult bone marrow. The previously defined human common myeloid progenitor (hCMP) population (Manz, M.G., T. Miyamoto, K. Akashi, and I.L. Weissman. 2002. Proc. Natl. Acad. Sci. USA. 99:11872-11877) was composed of the interleukin 5 receptor alpha chain(+) (IL-5Ralpha(+)) and IL-5Ralpha(-) fractions, and the former was the hEoP. The IL-5Ralpha(+)CD34(+)CD38(+)IL-3Ralpha(+)CD45RA(-) hEoPs gave rise exclusively to pure eosinophil colonies but never differentiated into basophils or neutrophils. The IL-5Ralpha(-) hCMP generated the hEoP together with the hGMP or the human megakaryocyte/erythrocyte progenitor (hMEP), whereas hGMPs or hMEPs never differentiated into eosinophils. Importantly, the number of hEoPs increased up to 20% of the conventional hCMP population in the bone marrow of patients with eosinophilia, suggesting that the hEoP stage is involved in eosinophil differentiation and expansion in vivo. Accordingly, the phenotypic definition of hCMP should be revised to exclude the hEoP; an "IL-5Ralpha-negative" criterion should be added to define more homogenous hCMP. The newly identified hEoP is a powerful tool in studying pathogenesis of eosinophilia and could be a therapeutic target for a variety of eosinophil-related disorders.
Current mouse lines efficient for human cell xenotransplantation are backcrossed into NOD mice to introduce its multiple immunodeficient phenotypes. Our positional genetic study has located the ...NOD-specific polymorphic Sirpa as a molecule responsible for its high xenograft efficiency: it recognizes human CD47 and the resultant signaling may cause NOD macrophages not to engulf human grafts. In the present study, we established C57BL/6.Rag2nullIl2rgnull mice harboring NOD-Sirpa (BRGS). BRGS mice engrafted human hematopoiesis with an efficiency that was equal to or even better than that of the NOD.Rag1nullIl2rgnull strain, one of the best xenograft models. Consequently, BRGS mice are free from other NOD-related abnormalities; for example, they have normalized C5 function that enables the evaluation of complement-dependent cytotoxicity of antibodies against human grafts in the humanized mouse model. Our data show that efficient human cell engraftment found in NOD-based models is mounted solely by their polymorphic Sirpa. The simplified BRGS line should be very useful in future studies of human stem cell biology.
•NOD-specific Sirpa polymorphism is the genetic determinant of highly efficient xenograft activity in NOD-based immunodeficient mouse models.
Relapse of acute myeloid leukemia (AML) is a critical problem in clinics. Particularly, the prognosis of patients who relapsed after allogeneic stem cell transplantation (allo-SCT) is still poor. ...Self-renewing leukemic stem cells (LSCs), which are mainly enriched within CD34+CD38- fraction, cause re-growth of leukemia and eventually lead to recurrence. Therefore, evaluation of residual LSCs is crucial to estimate the efficacy of therapies including allo-SCT. As we previously described, T-cell immunoglobulin mucin-3 (TIM-3) is a useful marker for discrimination of normal and malignant HSCs. We, therefore, prospectively evaluated the frequencies of LSCs at multiple time points and assessed the validity of TIM-3 as a minimal residual disease (MRD) marker in the setting of allo-SCT.
50 AML patients who had been detected CD34+CD38-TIM-3+ LSCs in BM sample at least once before SCT were analyzed. All 50 patients underwent allo-SCT at Fukuoka Blood and Marrow Transplantation Group (FBMTG)-related hospitals from July 2015 to May 2018. 43 out of 50 patients achieved complete donor chimerism confirmed by short tandem repeat PCR (STR-PCR) at the time of engraftment (day 15-36), and these 43 patients was prospectively evaluated the frequencies of residual CD34+CD38-TIM-3+ LSCs using multi-color flow cytometry. Median age of the evaluated 43 AML patients at SCT was 44.8 years (28-67). 26 patients underwent SCT on disease and 17 patients at hematological CR. Relapse-free survival (RFS) was calculated from the time of SCT until last follow-up or documentation of relapse.
In each timing (at diagnosis, relapse, engraftment, etc.), we tracked the frequencies of TIM-3+ cells within CD34+CD38- fraction in BM. Of note, the proportion of CD34+CD38- fraction was 0.016 0.0056 - 0.0267 % of BMMNC at engraftment. Then, according to the frequency of TIM-3+ cells within CD34+CD38- fraction, we classified patients into 3 groups; ‘high’ (> 90 % of CD34+CD38- cells were positive for TIM-3, n=5), ‘intermediate’ (61-90 % of CD34+CD38- cells were positive for TIM-3, n=11), and ‘low’ (< 60 % of CD34+CD38- cells were positive for TIM-3, n=27). This classification revealed that the recurrence rate within the observation period (median 334.4 days) was 100 % (5 of 5) in ‘high’ group, 45.4 % (5 of 11) in ‘intermediate’ group and 14.8 % (4 of 27) in ‘low’ group. Relapse-free survival was 83 (72-92) days in ‘high’ group, 368 (56-869) days in ‘intermediate’ group and 482.6 (45-1029) days in ‘low’ group, respectively (p<0.01) (Figure 1). Of note, all of 5 patients in ‘high’ group exhibited hematological relapse within 100 days. Thus, our new classification using TIM-3 as LSC-specific marker could isolate the patients at high risk for early relapse in allo-SCT settings even if they were considered as hematological CR and maintained complete donor chimerism assessed by clinically available methods (three-color FACS, STR-PCR, FLT3-ITD status and fusion gene specific qPCR) at the same timepoint. It suggests that monitoring of LSCs using TIM-3 should be a more sensitive and versatile strategy to predict early relapse of AML than aberrant surface markers monitoring by multi-color FACS because conventional FACS did not detect MRD population at engraftment while our strategy detected (we could detect LSCs (CD34+CD38-TIM-3+ fraction) within BMMNC in 0.0053 0 - 0.012 %), and, the cases whose aberrant markers could be traced were only 28 cases of 43 in this study.
We also confirmed that the identical driver mutations were detected at both the initial diagnosis and relapse using whole exome sequencing (WES) of purified LSCs. As a typical example, WES detected CEBPA mutation (c.11dupG, 72.7 % and 50.0 %) and WT1 mutation (c.1091_1092insTTGTACGGTC, 41.9 % and 39.5 %) in a single patient. Additionally, we also validated that purified TIM-3+ LSCs at engraftment harbored the identical mutations by amplicon sequencing. It indicates that CD34+CD38-TIM-3+ cells should represent LSCs throughout the clinical course, from diagnosis to relapse.
In summary, TIM-3 expression represents the clones of functional LSCs are involved in relapse. Evaluation of TIM-3+ LSCs by multi-color FACS should be a highly sensitive strategy to predict relapse in clinical allo-SCT settings, and might enable us to appropriately intervene to overcome the poor clinical outcomes of AML.
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Akashi:Taiho Pharmaceutical: Research Funding; Novartis pharma: Research Funding; Pfizer: Research Funding; Kyowa Hakko Kirin: Research Funding, Speakers Bureau; Eisai: Research Funding; Celgene: Research Funding, Speakers Bureau; Astellas Pharma: Research Funding; sanofi: Research Funding; MSD: Research Funding; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Chugai Pharma: Research Funding; Eli Lilly Japan: Research Funding; Ono Pharmaceutical: Research Funding; Otsuka Pharmaceutical: Research Funding; Asahi-kasei: Research Funding.
We stratified patients with newly diagnosed acute promyelocytic leukemia (APL) according to a white blood cell (WBC) count of ≥ 3 × 10
9
/L (high risk) or < 3 × 10
9
/L (low risk) before ...administering risk-adapted chemotherapy in combination with all-
trans
retinoic acid (ATRA). In total, 27 low-risk and 23 high-risk patients were assigned to receive induction and three courses of consolidation with ATRA and anthracycline, followed by 2-year maintenance regimen. High-risk group additionally received cytarabine during 1st consolidation and another one-shot idarubicin treatment during 3rd consolidation. We prospectively monitored measurable residual disease (MRD) after induction and each consolidation. In the low-risk and high-risk groups, 5-year disease-free survival (DFS) rates were 86.5% and 81.2% (
p
=
0.862
), and 5-year overall survival rates were 100% and 84.8% (
p
=
0.062
), respectively. In the MRD-negative and MRD-positive groups, 5-year DFS rates were 91.7% and 78.4% (
p
=
0.402
) and 84.7% and 60.0% (
p
=
0.102
) after induction and 1st consolidation, respectively. Relapse rates were 8.3% and 13.3% (
p
=
0.570
) and 9.0% and 40.0% (
p
=
0.076
) after induction and 1st consolidation, respectively. Achieving MRD-negativity after 1st consolidation, rather than after induction, was a potential predictor of relapse and DFS in patients with APL treated with ATRA + chemotherapy.
Previous studies have predicted that reciprocal activation of GATA-1 and PU.1 regulates myelo-erythroid versus myelo-lymphoid lineage commitment in early hematopoiesis. Such PU.1-activating ...myelo-lymphoid progenitors exist within the lymphoid-primed multipotent progenitor (LMPP) population at the primitive Lineage(-) Sca-1(+) c-Kit(+) (LSK) stage. We here show that the counterpart of GATA-1-activating myelo-erythroid progenitor resides also at the LSK stage, expressing CD41 at a high level. Purified CD41(hi) LSK cells showed exceedingly strong and prolonged myelo-erythroid-restricted reconstitution, and primed myelo-erythroid gene expression with a more primitive molecular signature as compared to the original common myeloid progenitor (CMP). The CD41(hi) LSK cells more strongly contributed to emergent and malignant myelopoiesis than LMPPs, and produced the original CMP by downregulating Sca-1 and CD41, suggesting that they are the earliest CMPs. Thus, the hematopoietic developmental map should be revised by integrating the primary branchpoint comprised of the new, isolatable CD41(hi) CMP and the LMPP populations.
Donor-derived hematological malignancies have been recognized as rare but serious late complications in allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients. Most cases in the ...literature were diagnosed as myelodysplastic syndrome or acute leukemia, with very few malignant lymphoma reported. We herein present another case of donor-derived Burkitt lymphoma that occurred 9 years after allo-HSCT under continued administration of immunosuppressants for chronic graft-versus-host disease (GVHD). The patient achieved a partial response after rituximab-combined intensive chemotherapy. To reduce the risk of relapse and to avoid organ toxicities due to repeated chemotherapies, we performed upfront high-dose chemotherapy followed by stem cell rescue using donor-derived CD34
+
cells, called pseudo-autologous HSCT (pASCT), and adjusted immunosuppressants appropriately. The patient remained disease-free for 23 months after pASCT without exacerbation of cGVHD. Although the observation period has been relatively short and longer follow-up is needed, pASCT may be a feasible option for donor-derived lymphoma even in patients with active cGVHD.
FLT3/FLK2, a member of the receptor tyrosine kinase family, plays a critical role in maintenance of hematopoietic homeostasis, and the constitutively active form of the FLT3 mutation is one of the ...most common genetic abnormalities in acute myelogenous leukemia. In murine hematopoiesis, Flt3 is not expressed in self-renewing hematopoietic stem cells, but its expression is restricted to the multipotent and the lymphoid progenitor stages at which cells are incapable of self-renewal. We extensively analyzed the expression of Flt3 in human (h) hematopoiesis. Strikingly, in both the bone marrow and the cord blood, the human hematopoietic stem cell population capable of long-term reconstitution in xenogeneic hosts uniformly expressed Flt3. Furthermore, human Flt3 is expressed not only in early lymphoid progenitors, but also in progenitors continuously along the granulocyte/macrophage pathway, including the common myeloid progenitor and the granulocyte/macrophage progenitor. We further found that human Flt3 signaling prevents stem and progenitors from spontaneous apoptotic cell death at least through up-regulating Mcl-1, an indispensable survival factor for hematopoiesis. Thus, the distribution of Flt3 expression is considerably different in human and mouse hematopoiesis, and human FLT3 signaling might play an important role in cell survival, especially at stem and progenitor cells that are critical cellular targets for acute myelogenous leukemia transformation.
Hemophagocytic lymphohistiocytosis (HLH) is characterized by deregulated engulfment of hematopoietic stem cells (HSCs) by BM macrophages, which are activated presumably by systemic inflammatory ...hypercytokinemia. In the present study, we show that the pathogenesis of HLH involves impairment of the antiphagocytic system operated by an interaction between surface CD47 and signal regulatory protein α (SIRPA). In HLH patients, changes in expression levels and HLH-specific polymorphism of SIRPA were not found. In contrast, the expression of surface CD47 was down-regulated specifically in HSCs in association with exacerbation of HLH, but not in healthy subjects. The number of BM HSCs in HLH patients was reduced to approximately 207 of that of healthy controls and macrophages from normal donors aggressively engulfed HSCs purified from HLH patients, but not those from healthy controls in vitro. Furthermore, in response to inflammatory cytokines, normal HSCs, but not progenitors or mature blood cells, down-regulated CD47 sufficiently to be engulfed by macrophages. The expression of prophagocytic calreticulin was kept suppressed at the HSC stage in both HLH patients and healthy controls, even in the presence of inflammatory cytokines. These data suggest that the CD47-SIRPA antiphagocytic system plays a key role in the maintenance of HSCs and that its disruption by HSC-specific CD47 down-regulation might be critical for HLH development.
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