Prospects and Potential for Chimerism Analysis after Allogeneic Hematopoietic Stem Cell Transplantation.

Chimerism analysis after allogeneic hematopoietic stem cell transplantation serves to confirm engraftment, indicate relapse of hematologic malignancy, and attribute graft failure to either immune rejection or poor graft function. Short tandem repeat PCR (STR-PCR) is the prevailing method, followed by quantitative real-time PCR (qPCR), with detection limits of 1-5% and 0.1%, respectively. Chimerism assays using digital PCR or next-generation sequencing, both of which are more sensitive than STR-PCR, are increasingly used. Stable mixed chimerism is usually not associated with poor outcomes in non-malignant diseases, but recipient chimerism may foretell relapse of hematologic malignancies, so higher detection sensitivity may be beneficial in such cases. Thus, the need for and the type of intervention, e.g., immunosuppression regimen, donor lymphocyte infusion, and/or salvage second transplantation, should be guided by donor chimerism in the context of the feature and/or residual malignant cells of the disease to be treated.

Impaired Repopulating Ability of Uhrf2-/- Hematopoietic Progenitor Cells in Mice.

UHRF proteins catalyze the ubiquitination of target proteins and are involved in regulating gene expression. Some studies reported a reduced expression of UHRF2 in acute leukemia cells, but the role of UHRF2 in hematopoiesis remains unknown. Here, we generated Uhrf2-/- mice to clarify the role of UHRF2 deletion in hematopoiesis. Compared to Uhrf2+/+ mice, Uhrf2-/- mice showed no differences in complete blood counts, as well as bone marrow (BM) findings and spleen weights. Proportions of cells in progenitor fractions in BM were comparable between Uhrf2+/+ mice and Uhrf2-/- mice. However, in competitive repopulation assays with BM transplants (BMT), the proportions of Uhrf2-/- cells were decreased relative to Uhrf2+/+ cells in all lineages. After the second BMT, Uhrf2-/- neutrophils were few, while 20-30% of Uhrf2-/- T cells and B cells were still detected. RNA sequencing showed downregulation of some genes associated with stem-cell function in Uhrf2-/- hematopoietic stem/progenitor cells (HSPCs). Interestingly, trimethylated histone H3 lysine 9 was increased in Uhrf2-/- HSPCs in a cleavage under targets and tagmentation assay. While UHRF2 deletion did not cause hematologic malignancy or confer a growth advantage of HSPCs, our results suggest that UHRF2 may play a role in the regulation of hematopoiesis.

Evaluation of a quantitative PCR-based method for chimerism analysis of Japanese donor/recipient pairs.

Chimerism analysis is a surrogate indicator of graft rejection or relapse after allogeneic hematopoietic stem cell transplantation (HSCT). Although short tandem repeat PCR (STR-PCR) is the usual method, limited sensitivity and technical variability are matters of concern. Quantitative PCR-based methods to detect single nucleotide polymorphisms (SNP-qPCR) are more sensitive, but their informativity and quantitative accuracy are highly variable. For accurate and sensitive chimerism analysis, a set of KMR kits (GenDx, Utrecht, Netherlands), based on detection of insertions/deletions (indels) by qPCR, have been developed. Here, we investigated informativity and validated the accuracy of KMR kits in Japanese donor/recipient pairs and virtual samples of DNA mixtures representative of Japanese genetic diversity. We found that at least one recipient-specific marker among 39 KMR-kit markers was informative in all of 65 Japanese donor/recipient pairs. Moreover, the percentage of recipient chimerism estimated by KMRtrack correlated well with ratios of mixed DNA in virtual samples and with the percentage of chimerism in HSCT recipients estimated by STR-PCR/in-house SNP-qPCR. Moreover, KMRtrack showed better sensitivity with high specificity when compared to STR-PCR to detect recipient chimerism. Chimerism analysis with KMR kits can be a standardized, sensitive, and highly informative method to evaluate the graft status of HSCT recipients.

Comparison of cryoprotectants in hematopoietic cell infusion-related adverse events.

BACKGROUND: The standard cryoprotectant for human cellular products is dimethyl sulfoxide (DMSO), which is associated with hematopoietic cell infusion-related adverse events (HCI-AEs) in hematopoietic stem cell transplantation including peripheral blood stem cell (PBSC) transplantation (PBSCT). DMSO is often used with hydroxyethyl starch (HES), which reduces DMSO concentration while maintaining the postthaw cell recovery. The cryoprotectant medium CP-1 (Kyokuto Pharmaceutical Industrial) is widely used in Japan. After mixture of a product with CP-1, DMSO and HES concentrations are 5% and 6%, respectively. However, the safety profile of CP-1 in association with HCI-AEs has not been investigated. STUDY DESIGN AND METHODS: To compare CP-1 with other cryoprotectants, we conducted a subgroup analysis of PBSCT recipients in a prospective surveillance study for HCI-AEs. Moreover, we validated the toxicity of CP-1 in 90 rats following various dose administration. RESULTS: The PBSC products cryopreserved with CP-1 (CP-1 group) and those with other cryoprotectants, mainly 10% DMSO (non-CP-1 group), were infused into 418 and 58 recipients, respectively. The rate of ≥grade 2 HCI-AEs was higher in the CP-1 group, but that of overall or ≥grade 3 HCI-AEs was not significantly different, compared to the non-CP-1 group. Similarly, after propensity score matching, ≥grade 2 HCI-AEs were more frequent in the CP-1 group, but the ≥grade 3 HCI-AE rate did not differ significantly between the groups. No significant toxicity was detected regardless of the CP-1 dose in the 90 rats. CONCLUSIONS: Infusion of a CP-1-containing PBSC product is feasible with the respect of HCI-AEs.

Clonal hematopoiesis with JAK2V617F promotes pulmonary hypertension with ALK1 upregulation in lung neutrophils.

Pulmonary hypertension (PH) is a progressive cardiopulmonary disease characterized by pulmonary arterial remodeling. Clonal somatic mutations including JAK2V617F, the most frequent driver mutation among myeloproliferative neoplasms, have recently been identified in healthy individuals without hematological disorders. Here, we reveal that clonal hematopoiesis with JAK2V617F exacerbates PH and pulmonary arterial remodeling in mice. JAK2V617F-expressing neutrophils specifically accumulate in pulmonary arterial regions, accompanied by increases in neutrophil-derived elastase activity and chemokines in chronic hypoxia-exposed JAK2V617F transgenic (JAK2V617F) mice, as well as recipient mice transplanted with JAK2V617F bone marrow cells. JAK2V617F progressively upregulates Acvrl1 (encoding ALK1) during the differentiation from bone marrow stem/progenitor cells peripherally into mature neutrophils of pulmonary arterial regions. JAK2V617F-mediated STAT3 phosphorylation upregulates ALK1-Smad1/5/8 signaling. ALK1/2 inhibition completely prevents the development of PH in JAK2V617F mice. Finally, our prospective clinical study identified JAK2V617F-positive clonal hematopoiesis is more common in PH patients than in healthy subjects. These findings indicate that clonal hematopoiesis with JAK2V617F causally leads to PH development associated with ALK1 upregulation.

Myeloproliferative neoplasm-driving Calr frameshift promotes the development of pulmonary hypertension in mice.

Frameshifts in the Calreticulin (CALR) exon 9 provide a recurrent driver mutation of essential thrombocythemia (ET) and primary myelofibrosis among myeloproliferative neoplasms (MPNs). Here, we generated knock-in mice with murine Calr exon 9 mimicking the human CALR mutations, using the CRISPR-Cas9 method. Knock-in mice with del10 [Calrdel10/WT (wild-type) mice] exhibited an ET phenotype with increases of peripheral blood (PB) platelets and leukocytes, and accumulation of megakaryocytes in bone marrow (BM), while those with ins2 (Calrins2/WT mice) showed a slight splenic enlargement. Phosphorylated STAT3 (pSTAT3) was upregulated in BM cells of both knock-in mice. In BM transplantation (BMT) recipients from Calrdel10/WT mice, although PB cell counts were not different from those in BMT recipients from CalrWT/WT mice, Calrdel10/WT BM-derived macrophages exhibited elevations of pSTAT3 and Endothelin-1 levels. Strikingly, BMT recipients from Calrdel10/WT mice developed more severe pulmonary hypertension (PH)-which often arises as a comorbidity in patients with MPNs-than BMT recipients from CalrWT/WT mice, with pulmonary arterial remodeling accompanied by an accumulation of donor-derived macrophages in response to chronic hypoxia. In conclusion, our murine model with the frameshifted murine Calr presented an ET phenotype analogous to human MPNs in molecular mechanisms and cardiovascular complications such as PH.

Crucial role of hematopoietic JAK2 V617F in the development of aortic aneurysms.

JAK2V617F is the most frequent driver mutation in myeloproliferative neoplasms (MPNs) and is associated with vascular complications. However, the impact of hematopoietic JAK2V617F on the aortic aneurysms (AAs) remains unknown. Our cross-sectional study indicated that 9 (23%) out of 39 MPN patients with JAK2V617F exhibited the presence of AAs. Next, to clarify whether the hematopoietic JAK2V617F contributes to the AAs, we applied a bone marrow transplantation (BMT) with the donor cells from Jak2V617F transgenic (JAK2V617F) mice or control wild-type (WT) mice into lethally irradiated apolipoprotein E-deficient mice. Five weeks after BMT, the JAK2V617F-BMT mice and WT-BMT mice were subjected to continuous angiotensin II infusion to induce AA formation. Four weeks after angiotensin II infusion, the abdominal aorta diameter in JAK2V617F-BMT mice was significantly enlarged compared to that in the WT-BMT mice. Additionally, the abdominal AA-free survival rate was significantly lower in the JAK2V617F-BMT mice. Hematopoietic JAK2V617F accelerated aortic elastic lamina degradation as well as activation of matrix metalloproteinase (MMP)-2 and MMP-9 in the abdominal aorta. The numbers of infiltrated macrophages were significantly upregulated in the abdominal aorta of the JAK2V617F-BMT mice accompanied by STAT3 phosphorylation. The accumulation of BM-derived hematopoietic cells carrying JAK2V617F in the abdominal aorta was confirmed by use of reporter GFP-transgene. BM-derived macrophages carrying JAK2V617F showed increases in mRNA expression levels of Mmp2, Mmp9, and Mmp13. Ruxolitinib decreased the abdominal aorta diameter and the incidence of abdominal AA in the JAK2V617F-BMT mice. Our findings provide a novel feature of vascular complications of AAs in MPNs with JAK2V617F.

Hematopoietic cell infusion-related adverse events in pediatric/small recipients in a prospective/multicenter study.

BACKGROUND: Hematopoietic cell infusion-related adverse events (HCI-AEs) in hematopoietic stem cell transplantations (HSCTs) have been largely attributed to toxicity of dimethyl sulfoxide (DMSO) for cryopreservation, but HSC products also contain various cells and plasma components. Our recent prospective study of 1125 HSCT recipients revealed the highest overall HCI-AE rate in bone marrow transplantation (BMT) using fresh/noncryopreserved products, although products of peripheral blood stem cell transplantation and cord blood transplantation (CBT) are generally cryopreserved with DMSO containing smaller plasma volumes. We aimed to clarify if product volume and component effects are more substantial in small recipients including children. STUDY DESIGN AND METHODS: We performed subgroup analysis on 219 recipients of 45 kg or less body weight (whole small recipients), including 90 children (pediatric recipients), from the original cohort (general recipients). RESULTS: Whereas overall HCI-AE rates did not differ among hematopoietic stem cell sources in the general recipients, bradycardia most often occurred after CBT in whole small recipients. Conversely, whole small and general recipients shared the same trend of having the highest rate of hypertension in BMT. The overall HCI-AE rate was higher in allogeneic HSCT compared with autologous HSCT. Notably, pediatric recipients showed a 10-fold higher incidence of nausea and vomiting in allogeneic HSCT compared with autologous HSCT, suggesting a possible role of allogeneic antigens. Multivariate analysis identified a relatively large infusion volume per body weight as a significant factor correlating with HCI-AE in whole small recipients. CONCLUSIONS: We should be aware of product volume and specific HCI-AEs such as nausea and vomiting in small patients including children.

Optimal timing of apheresis for the efficient mobilization of peripheral blood progenitor cells recruited by high-dose granulocyte colony-stimulating factor in healthy donors.

Predictors of peripheral blood stem cell (PBSC) yield can potentially improve the comfort, safety, and efficacy of CD34+ cell collection from donors treated with recombinant human granulocyte colony-stimulating factor (G-CSF). We investigated 181 apheresis procedures on 109 healthy allogeneic donors to identify factors correlating with efficient PBSC collection. Apheresis started on Day 4 or 5 and continued up to Day 6 of G-CSF administration. CD34+ cell　yields on Days 4 and 5 were comparable, and significantly higher than on Day 6. This suggests that starting apheresis on Day 4 rather than Day 5 may be preferable, to reduce G-CSF exposure and optimize yield, even if multi-day collection is required. More CD34+ cells were collected from male and cytomegalovirus (CMV)-seronegative donors than from female and CMV-seropositive donors, respectively. The yields of CD34+ cells were similarly high in both male and female donors aged 20-29 years; yields decreased in female donors in their thirties, and were comparably low in both male and female donors in their forties and thereafter. These findings should guide decision-making about when to begin apheresis, and encourage careful consideration of donor factors such as gender, age, and CMV serostatus when collecting PBSCs.

Adverse Events Associated With Infusion of Hematopoietic Stem Cell Products: A Prospective and Multicenter Surveillance Study.

Adverse events (AEs) associated with blood transfusions, including component-specific red cell, platelet, and plasma products, have been extensively surveyed. In contrast, surveillance of AEs associated with hematopoietic stem cell (HSC) products in HSC transplantation (HSCT) has been less rigorous, even though HSC products include a diversity of immature and mature hematopoietic cells, substantial plasma, and dimethyl sulfoxide (DMSO) in the case of cryopreserved HSC products. HSC infusion-related AEs have been attributed to DMSO toxicity, but AEs associated with the infusion of noncryopreserved HSC products are not uncommon. To quantify the frequencies, types, and risk factors of HSC infusion-related AEs, we implemented national surveillance for AEs observed within 24 hours after infusion. Herein we report on 1125 HSCTs, including 570 peripheral blood stem cell transplantations (PBSCTs) (290 autologous [auto-] and 280 allogeneic [allo-]), 332 allo-bone marrow transplantations (allo-BMTs) and 223 allo-cord blood transplantations (allo-CBTs). Unexpectedly, incidences of grade ≥ 2 AEs were most frequent in allo-BMTs (37.7%) with no DMSO in any product compared with auto-/allo-PBSCTs (20.9%, P < .001) and allo-CBTs (19.3%, P < .001) typically cryopreserved with DMSO. Hypertension was most often noted in BMTs, whereas nausea/vomiting, fever, and allergic reactions were most frequent in allo-PBSCTs. In a multivariate analysis, a history of transfusion reactions was a risk factor for overall AEs in all HSCTs (odds ratio [OR] = 1.459, P = .045). For grade ≥ 2 AEs in allo-HSCTs, a history of transfusion reactions (OR = 1.551, P = .044) for overall AEs, and high infusion volume (OR = 7.544, P = .005) and allo-PBSCTs (versus BMTs, OR = 9.948, P = .002) for allergic reactions were identified as risk factors. These findings suggest that some factors unrelated to DMSO, such as allo-antigens, contribute to HSC infusion-related AEs. As severe AEs, a total of 117 grade ≥ 3 AEs were reported in 1125 HSCTs, including life-threatening complications in 3 (0.3%) HSCTs: 1 allo-CBT (anaphylaxis) and 2 allo-PBSCTs (hypoxia, kidney injury) with cryopreserved product. Our data show that HSC infusion risks vary by product, can be severe, and should be monitored with the same rigor as modern transfusion hemovigilance programs.

Efficacy of D- red blood cell transfusion and rituximab therapy in autoimmune hemolytic anemia with anti-D and panreactive autoantibodies arising after hematopoietic stem cell transplant.

BACKGROUND: Autoimmune hemolytic anemia (AIHA) is caused by autoantibodies to red blood cells (RBCs), which can be panreactive and/or specific to Rh/other blood group antigens. We report a severe case of AIHA after bone marrow transplantation (BMT) due to autoanti-D triggered by reactivation of Epstein-Barr virus (EBV) infection. A combined strategy of D- RBC transfusion and administration of anti-CD20 monoclonal antibody (MoAb) resolved the hemolysis. CASE REPORT: A 33-year-old male underwent allogeneic BMT from an ABO-identical and HLA-matched unrelated male donor. Five months later, while having mild chronic graft-versus-host disease, he manifested AIHA, with a hemoglobin (Hb) level of 5.1 g/dL on AIHA Day 2 (Posttransplant Day 156) and was refractory to D+ RBCs, with a Hb level of 2.4 g/dL on AIHA Day 6. Anti-D-like autoantibodies (titer 1280, subclass immunoglobulin G1 , monocyte monolayer assay 28.7%) and panreactive (titer 40) were identified. Changing the RBC transfusion strategy to D- increased his Hb level to 6.7 g/dL on Day 10. Administration of anti-CD20 MoAb mitigated EBV-related B-cell proliferation and reduced anti-D autoantibody titer to 320 by Day 16 with normalized Hb concentration after 6 months. CONCLUSION: In severe AIHA, when standard treatment and regular RBC transfusions are ineffective, transfusion of RBCs lacking the target antigen(s) of autoantibodies and administration of anti-CD20 MoAb should be considered.

Prospective randomized and crossover comparison of two apheresis machines for peripheral blood stem cell collection: a multicenter study.

BACKGROUND: Improving apheresis technology may lead to an efficient and safe peripheral blood stem cell (PBSC) collection. Recently, the Spectra Optia (Optia, Terumo BCT) was introduced as an automated apheresis instrument, but comparisons with other instruments have been few. This is the first randomized multicenter and crossover comparison of the Optia with the automated program of the established apheresis instrument, the Spectra (Spectra-Auto, Terumo BCT). STUDY DESIGN AND METHODS: A total of 233 apheresis procedures performed in 46 autologous patients and 108 allogeneic donors were investigated. Apheresis performed in the first day for all subjects using the Spectra-Auto (n = 79) and the Optia (n = 75) were evaluated as first-day analysis. Seventy-nine subjects, who required another session on the second day, underwent apheresis using the other instrument than the first-day instrument and were compared with each other in a paired crossover analysis. RESULTS: The two instruments processed similar volumes with comparable run times and volumes of acid-citrate-dextrose used. The volumes of collected products were greater in the Optia. Yields of mononuclear cells and CD34+ cells were not different, but collection efficiencies were higher in the Optia (p = 0.008 in CE1 of crossover analysis). Spectra-Auto-collected products contained more contaminating red blood cells (RBCs), whereas there was a trend of more contaminating platelets (PLTs) in the Optia-collected products. Slight reductions were noted in the RBC or PLT counts of subjects who underwent apheresis with the Spectra-Auto or the Optia, respectively. CONCLUSION: The Optia is safe and more efficient in the PBSC collection compared with the Spectra-Auto.