Eltrombopag Added to Immunosuppression in Severe Aplastic Anemia.

BACKGROUND: A single-group, phase 1-2 study indicated that eltrombopag improved the efficacy of standard immunosuppressive therapy that entailed horse antithymocyte globulin (ATG) plus cyclosporine in patients with severe aplastic anemia. METHODS: In this prospective, investigator-led, open-label, multicenter, randomized, phase 3 trial, we compared the efficacy and safety of horse ATG plus cyclosporine with or without eltrombopag as front-line therapy in previously untreated patients with severe aplastic anemia. The primary end point was a hematologic complete response at 3 months. RESULTS: Patients were assigned to receive immunosuppressive therapy (Group A, 101 patients) or immunosuppressive therapy plus eltrombopag (Group B, 96 patients). The percentage of patients who had a complete response at 3 months was 10% in Group A and 22% in Group B (odds ratio, 3.2; 95% confidence interval [CI], 1.3 to 7.8; P = 0.01). At 6 months, the overall response rate (the percentage of patients who had a complete or partial response) was 41% in Group A and 68% in Group B. The median times to the first response were 8.8 months (Group A) and 3.0 months (Group B). The incidence of severe adverse events was similar in the two groups. With a median follow-up of 24 months, a karyotypic abnormality that was classified as myelodysplastic syndrome developed in 1 patient (Group A) and 2 patients (Group B); event-free survival was 34% and 46%, respectively. Somatic mutations were detected in 29% (Group A) and 31% (Group Β) of the patients at baseline; these percentages increased to 66% and 55%, respectively, at 6 months, without affecting the hematologic response and 2-year outcome. CONCLUSIONS: The addition of eltrombopag to standard immunosuppressive therapy improved the rate, rapidity, and strength of hematologic response among previously untreated patients with severe aplastic anemia, without additional toxic effects. (Funded by Novartis and others; RACE ClinicalTrials.gov number, NCT02099747; EudraCT number, 2014-000363-40.).

Iron is a modifier of the phenotypes of JAK2-mutant myeloproliferative neoplasms.

JAK 2-V617F mutation causes myeloproliferative neoplasms (MPNs) that can manifest as polycythemia vera (PV), essential thrombocythemia (ET), or primary myelofibrosis. At diagnosis, patients with PV already exhibited iron deficiency, whereas patients with ET had normal iron stores. We examined the influence of iron availability on MPN phenotype in mice expressing JAK2-V617F and in mice expressing JAK2 with an N542-E543del mutation in exon 12 (E12). At baseline, on a control diet, all JAK2-mutant mouse models with a PV-like phenotype displayed iron deficiency, although E12 mice maintained more iron for augmented erythropoiesis than JAK2-V617F mutant mice. In contrast, JAK2-V617F mutant mice with an ET-like phenotype had normal iron stores comparable with that of wild-type (WT) mice. On a low-iron diet, JAK2-mutant mice and WT controls increased platelet production at the expense of erythrocytes. Mice with a PV phenotype responded to parenteral iron injections by decreasing platelet counts and further increasing hemoglobin and hematocrit, whereas no changes were observed in WT controls. Alterations of iron availability primarily affected the premegakaryocyte-erythrocyte progenitors, which constitute the iron-responsive stage of hematopoiesis in JAK2-mutant mice. The orally administered ferroportin inhibitor vamifeport and the minihepcidin PR73 normalized hematocrit and hemoglobin levels in JAK2-V617F and E12 mutant mouse models of PV, suggesting that ferroportin inhibitors and minihepcidins could be used in the treatment for patients with PV.

Absence of NKG2D ligands defines leukaemia stem cells and mediates their immune evasion.

Patients with acute myeloid leukaemia (AML) often achieve remission after therapy, but subsequently die of relapse1 that is driven by chemotherapy-resistant leukaemic stem cells (LSCs)2,3. LSCs are defined by their capacity to initiate leukaemia in immunocompromised mice4. However, this precludes analyses of their interaction with lymphocytes as components of anti-tumour immunity5, which LSCs must escape to induce cancer. Here we demonstrate that stemness and immune evasion are closely intertwined in AML. Using xenografts of human AML as well as syngeneic mouse models of leukaemia, we show that ligands of the danger detector NKG2D-a critical mediator of anti-tumour immunity by cytotoxic lymphocytes, such as NK cells6-9-are generally expressed on bulk AML cells but not on LSCs. AML cells with LSC properties can be isolated by their lack of expression of NKG2D ligands (NKG2DLs) in both CD34-expressing and non-CD34-expressing cases of AML. AML cells that express NKG2DLs are cleared by NK cells, whereas NKG2DL-negative leukaemic cells isolated from the same individual escape cell killing by NK cells. These NKG2DL-negative AML cells show an immature morphology, display molecular and functional stemness characteristics, and can initiate serially re-transplantable leukaemia and survive chemotherapy in patient-derived xenotransplant models. Mechanistically, poly-ADP-ribose polymerase 1 (PARP1) represses expression of NKG2DLs. Genetic or pharmacologic inhibition of PARP1 induces NKG2DLs on the LSC surface but not on healthy or pre-leukaemic cells. Treatment with PARP1 inhibitors, followed by transfer of polyclonal NK cells, suppresses leukaemogenesis in patient-derived xenotransplant models. In summary, our data link the LSC concept to immune escape and provide a strong rationale for targeting therapy-resistant LSCs by PARP1 inhibition, which renders them amenable to control by NK cells in vivo.

Publisher Correction: Absence of NKG2D ligands defines leukaemia stem cells and mediates their immune evasion.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Continuous and differential improvement in worldwide access to hematopoietic cell transplantation: activity has doubled in a decade with a notable increase in unrelated and non-identical related donors.

Promoting access to and excellence in hematopoietic cell transplantation (HCT) by collecting and disseminating data on global HCT activities is one of the principal activities of the Worldwide Network for Blood and Marrow Transplantation, a non-Governmental organization in working relations with the World Health Organization. HCT activities are recorded annually by member societies, national registries and individual centers including indication, donor type (allogeneic/autologous), donor match and stem cell source (bone marrow/peripheral blood stem cells/cord blood). In 2018, 1,768 HCT teams in 89 countries (six WHO regions) reported 93,105 (48,680 autologous and 44,425 allogeneic) HCT. Major indications were plasma cell disorders and lymphoma for autologous, and acute leukemias and MDS/MPN for allogeneic HCT. HCT number increased from 48,709 in 2007. Notable increases were seen for autoimmune diseases in autologous and hemoglobinopathies in allogeneic HCT. The number of allogeneic HCT more than doubled with significant changes in donor match. While HCT from HLA identical siblings has seen only limited growth, HCT from non-identical related donors showed significant increase worldwide. Strongest correlation between economic growth indicator of gross national income/capita and HCT activity/ten million population was observed for autologous HCT (r=0.79). HCT from unrelated donors showed strong correlation (r=0.68), but only moderate correlation (r=0.51) was detected from related donors. The use of HCT doubled in about a decade worldwide at different speed and with significant changes regarding donor match as a sign of improved access to HCT worldwide. Although narrowing, significant gaps remain between developing and non-developing countries.

Hematopoietic cell transplantation and cellular therapies in Switzerland. Evolution over 25 years. A report from the stem cell transplantation and cellular therapies working groups of the SBST 1997-2021.

The Swiss Blood Stem Cell Transplantation and Cellular Therapy Group (SBST) leads a mandatory national registry for all hematopoietic stem cell transplants (HCT) and cellular therapies. After 25 years, information was available for 11,226 patients receiving an HCT (4031 allogeneic and 7195 autologous), including 925 pediatric patients. We compared patient characteristics and outcome by quinquennia 1997-2001, 2002-2006, 2007-2011, 2012-2016, and 2017-2021. There were numerous changes over time. Allogeneic transplant recipients became older (median age 33.7 vs. 54.3) and had more frequently unrelated donors and reduced intensity conditioning in later quinquennia. Similarly, age increased for recipients of autologous HCT (median 48.3 vs. 59.9). We did not see a significant drop in transplant activity during the SARS-CoV-2 pandemic. Analysis of outcome showed overall survival (relative risk (RR) of death 0.664 (0.529-0.832) and progression free survival (RR 0.708 (0.577-0.870) being improved over time comparing the latest to the first quinquennium adjusting for risk factors. Non-relapse mortality decreased in recipients of allogeneic HCT (RR: 0.371 (0.270-0.509)) over time but relapse risks did not. Outcome of autologous HCT improved as well across quinquennia, this improvement was mainly due to decreased relapse risks (RR 0.681 (0.597-0.777)), possibly related to maintenance treatment or rescue treatment for relapse mainly in myeloma patients. Cellular therapies other than allogeneic or autologous HCT, particularly chimeric antigen receptor T-cells (CAR-T) treatment have started to increase after 2019, year of approval of the first commercial CAR-T product in Switzerland. Data on chimeric antigen receptor T-cell treatment are too early for comparative analyses. Detailed analyses of changes over time are presented. This study includes all HCTs, and cellular therapies, data useful for quality assurance programs, health care cost estimation and benchmarking. Between 50% and 60% of patients are long-term survivors after both types of HCT, indicating growing populations of surviving patients requiring long-term care.

Feasibility of electronic patient-reported outcome monitoring and self-management program in aplastic anemia and paroxysmal nocturnal hemoglobinuria-a pilot study (ePRO-AA-PNH).

INTRODUCTION: Electronic patient-reported outcomes (ePRO) are increasingly recognized in health care, as they have been demonstrated to improve patient outcomes in cancer, but have been less studied in rare hematological diseases. The aim of this study was to develop and test the feasibility of an ePRO system specifically customized for aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH). METHODS: After performing a user-centered design evaluation an ePRO system for AA and PNH patients could be customized and the application was tested by patients and their medical teams for 6 months. Symptom-reporting triggered self-management advice for patients and prompts them to contact clinicians in case of severe symptoms, while the medical team received alerts of severe symptoms for patient care. RESULTS: All nine included patients showed a high adherence rate to the weekly symptom-reporting (72%) and reported high satisfaction. The system was rated high for usage, comprehensibility, and integration into daily life. Most patients (78%) would continue and all would recommend the application to other AA/PNH patients. Technical performance was rarely a barrier and healthcare providers saw ePRO-AA-PNH as a useful supplement, but the lacking integration into the hospital information system was identified as a major barrier to usage. CONCLUSION: An ePRO system customized for AA and PNH was feasible in terms of adherence, satisfaction, and performance, showing a high potential for these rare conditions in terms of data collection and patient guidance. However, the integration into clinical workflows is crucial for further routine use. TRIAL REGISTRATION: ClinicalTrials.gov NCT04128943.

Antibody response to mRNA SARS-CoV-2 vaccination in 182 patients after allogeneic hematopoietic cell transplantation.

INTRODUCTION: Patients after allogeneic stem cell transplantation are at high risk for infection-related complications, and vaccination efficacy might be impaired depending on the immune reconstitution. In this study, we evaluate their response to mRNA vaccines against SARS-CoV-2. METHODS: During routine follow-up visits, patients were asked about their vaccination status and if they had a previous infection with SARS-CoV-2. In fully vaccinated patients, the antibody titer was measured using the Roche Elecsys Anti-SARS-CoV-2 S test. A titer of <1 U/L was considered as negative, titers of ≥250 U/ml as a high antibody titer, and a titer of 50-249 U/ml as a low antibody titer. Patient characteristics were evaluated by chart review to identify risk factors for poor vaccination response. RESULTS: The majority of patients developed a high antibody titer (138 out 182 patients, 75.8%). Risk factors for a low antibody titer were immunosuppressive therapy, a lymphocyte count <0.9 G/L, ongoing treatment for the underlying malignancy, and active graft-versus-host disease (GvHD). Donor type, underlying disease, a previous SARS-CoV-2 infection, and sex did not significantly influence the response to the vaccination. DISCUSSION: While patients undergoing allogeneic stem cell transplantation have been excluded from the initial registration trials, our real-world experience with a large patient cohort confirms the data of previous studies, showing that most patients do have a good response to mRNA vaccines against SARS-CoV-2. Nevertheless, a significant proportion of patients shows an inadequate vaccination, which can be improved after a third vaccination in most cases despite immunosuppressive therapy.

Frequency, reactivity and evolution of human leukocyte antigen and human platelet antigen antibodies in the setting of hematopoietic cell transplantation.

BACKGROUND AND OBJECTIVES: Antibodies (Ab) against HLA and HPA antigens play an important role in HCT. In this prospective study we evaluated prevalence and kinetics of HLA- and HPA-Ab after HCT, including a possible donor-recipient transfer and their clinical relevance in respect to platelet transfusion refractoriness (PTR). MATERIALS AND METHODS: Patients were consecutively recruited. Ab were determined by microbead assay technique and a mean fluorescence intensity cut-off of 1,000. RESULTS: At baseline, 21 donors (42 %) and 27 patients (54 %) had HLA-Ab with a mean panel reactivity (cPRA) of 34.9 ± 29.4 % and 46.1 ± 36.5 %, respectively. We observed a significant higher number of HLA-Ab specificities in female donors and patients and a predominance of HLA-class I Ab. At day 0 we detected an increase of HLA-Ab (from 526 to 673) and cPRA (55.2 ± 31.9 %). Thirty-six patients (72 %) developed new HLA-Ab, mainly 3 weeks after HCT. In 7 patients an HLA-Ab with the same specificity as detected in the corresponding donor emerged, suggesting a possible transfer from the donor to the recipient. Overall, MFI showed a high variation. Type and number of transfusions were not associated with number and intensity of HLA-Ab (ρ: -0.05 - 0.02). Number of HLA-Ab, cPRA and intensity were not associated with PTR, which occurred in 9 patients (18 %) and none had bleeding WHO > 2. CONCLUSIONS: Although a considerable number of patients have and develop HLA-Ab before and early after HCT, we found no association with PTR and bleeding and management should be individualized.

Molecular Minimal Residual Disease in Acute Myeloid Leukemia.

BACKGROUND: Patients with acute myeloid leukemia (AML) often reach complete remission, but relapse rates remain high. Next-generation sequencing enables the detection of molecular minimal residual disease in virtually every patient, but its clinical value for the prediction of relapse has yet to be established. METHODS: We conducted a study involving patients 18 to 65 years of age who had newly diagnosed AML. Targeted next-generation sequencing was carried out at diagnosis and after induction therapy (during complete remission). End points were 4-year rates of relapse, relapse-free survival, and overall survival. RESULTS: At least one mutation was detected in 430 out of 482 patients (89.2%). Mutations persisted in 51.4% of those patients during complete remission and were present at various allele frequencies (range, 0.02 to 47%). The detection of persistent DTA mutations (i.e., mutations in DNMT3A, TET2, and ASXL1), which are often present in persons with age-related clonal hematopoiesis, was not correlated with an increased relapse rate. After the exclusion of persistent DTA mutations, the detection of molecular minimal residual disease was associated with a significantly higher relapse rate than no detection (55.4% vs. 31.9%; hazard ratio, 2.14; P<0.001), as well as with lower rates of relapse-free survival (36.6% vs. 58.1%; hazard ratio for relapse or death, 1.92; P<0.001) and overall survival (41.9% vs. 66.1%; hazard ratio for death, 2.06; P<0.001). Multivariate analysis confirmed that the persistence of non-DTA mutations during complete remission conferred significant independent prognostic value with respect to the rates of relapse (hazard ratio, 1.89; P<0.001), relapse-free survival (hazard ratio for relapse or death, 1.64; P=0.001), and overall survival (hazard ratio for death, 1.64; P=0.003). A comparison of sequencing with flow cytometry for the detection of residual disease showed that sequencing had significant additive prognostic value. CONCLUSIONS: Among patients with AML, the detection of molecular minimal residual disease during complete remission had significant independent prognostic value with respect to relapse and survival rates, but the detection of persistent mutations that are associated with clonal hematopoiesis did not have such prognostic value within a 4-year time frame. (Funded by the Queen Wilhelmina Fund Foundation of the Dutch Cancer Society and others.).

Posttransplant cyclophosphamide vs antithymocyte globulin in HLA-mismatched unrelated donor transplantation.

The use of anti-thymocyte globulin (ATG) has represented the standard of care in graft-versus-host disease (GVHD) prophylaxis in patients undergoing a mismatched unrelated donor (MMUD) transplant. The safety and feasibility of posttransplant cyclophosphamide (PTCY) in this setting have been reported recently, but no study has compared the outcomes of PTCY vs ATG in 9/10 MMUD transplants. Using the registry data of the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation, we performed a matched-pair analysis comparing those 2 strategies in a 9/10 MMUD setting. Ninety-three patients receiving PTCY were matched with 179 patients receiving ATG. A significantly lower incidence of severe acute GVHD was observed with PTCY compared with ATG. Recipients of the former also showed higher leukemia-free survival and GVHD/relapse-free survival (GRFS). When performing a subgroup analysis including patients receiving peripheral blood stem cells, being in complete remission, or receiving the same associated immunosuppressive agents, superiority of PTCY over ATG was confirmed. Similar to the haploidentical setting, use of PTCY is an effective anti-GVHD prophylaxis in the 9/10 MMUD transplant. Use of PTCY may also provide better outcomes in long-term disease control. These results need confirmation in large prospective randomized trials.

Cellular immunotherapy with multiple infusions of in vitro-expanded haploidentical natural killer cells after autologous transplantation for patients with plasma cell myeloma.

BACKGROUND AIMS: To investigate the feasibility and safety of haploidentical natural killer (NK) cell infusions as consolidation immunotherapy after autologous stem cell transplant (ASCT) in patients with plasma cell myeloma. METHODS: Ten patients (median age, 59 years) received induction treatment followed by high-dose melphalan (200 mg/m2) at day -1, ASCT at day 0 and increasing NK cell doses (1.5 × 106, 1.5 × 107 and multiple doses of 1.0 × 108 cells/kg body weight) from day +1 to day +30 after ASCT. NK cells were harvested and purified from peripheral blood of haploidentical donors and expanded for 19 days with interleukin (IL)-2 and IL-15 under Good Manufacturing Practice conditions. RESULTS: NK cell numbers increased 56.0-fold (37.4- to 75.5-fold). Patients received a median of 3.8 × 108 (0.9-5.7 × 108) NK cells/kg body weight in six (three to eight) infusions. Multiparametric mass cytometry analysis demonstrated an altered surface receptor repertoire of expanded NK cells with increased degranulation and cytokine production activities but diminished expression of perforin. Donor NK cells were detectable in the peripheral blood, peaking 1 h after each dose (up to 90% donor NK cells). The treatment was safe and well tolerated, without evidence of graft-versus-host disease. Comparison with a control patient population receiving ASCT without NK cell infusions showed no significant difference in relapse, progression-free survival and overall survival. CONCLUSIONS: This study demonstrates reliable manufacturing of high numbers of activated NK cells for multiple-dose infusions and safe administration of these cellular products. The trial was registered at ClinicalTrials.gov (identifier no. NCT01040026).

Busulfan-cyclophosphamide versus cyclophosphamide-busulfan as conditioning regimen before allogeneic hematopoietic cell transplantation: a prospective randomized trial.

Busulfan and cyclophosphamide (BuCy) is a frequently used myeloablative conditioning regimen for allogeneic hematopoietic cell transplantation (allo-HCT). Theoretical considerations and pharmacological data indicate that application of busulfan prior to subsequent cyclophosphamide (BuCy) may trigger liver toxicity. Reversing the order of application to cyclophosphamide-busulfan (CyBu) might be preferable, a hypothesis supported by animal data and retrospective studies. We performed a prospective randomized trial to determine impact of order of application of Bu and Cy before allo-HCT in 70 patients with hematological malignancy, 33 patients received BuCy and 37 CyBu for conditioning. In the short term, there were minimal differences in liver toxicity favoring CyBu over BuCy, significant only for alanine amino transferase at day 30 (p = 0.03). With longer follow-up at 4 years, non-relapse mortality (6% versus 27%, p = 0.05) was lower and survival (63% versus 43%, p = 0.06) was higher with CyBu compared to BuCy. Other outcomes, such as engraftment (p = 0.21), acute and chronic graft-versus-host disease (p = 0.40; 0.36), and relapse (p = 0.79), were similar in both groups. We prospectively show evidence that the order of application of Cy and Bu in myeloablative conditioning in allo-HCT patients has impact on outcome.

The impact of cytogenetic risk on the outcomes of allogeneic hematopoietic cell transplantation in patients with relapsed/refractory acute myeloid leukemia: On behalf of the acute leukemia working party (ALWP) of the European group for blood and marrow transplantation (EBMT).

Karyotypic analysis at time of diagnosis has an important value in determining initial response to treatment, remission duration and overall survival (OS) in acute myeloid leukemia (AML). Less is known about its value before allogeneic hematopoietic cell transplantation (allo-HCT) in patients transplanted with active disease, either relapsed or primary refractory (Rel-Ref) AML. We explored the impact of cytogenetic risk (stratification according to MRC-UK) in 2089 patients with either Ref (n = 972) or Rel AML (n = 1117) transplanted during the period 2000-2017. Overall, 154 patients had a favorable risk, 1283 had an intermediate risk and 652 had an adverse cytogenetic risk. Median follow-up was 49 months. Compared to the favorable risk group, intermediate and adverse risk patients were associated with worse leukemia-free survival and OS and also with a higher incidence of relapse. In a subgroup analysis of patients in the intermediate risk group harboring Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD), this remained an important prognostic factor, being associated with worse outcomes. When analyzing patients according to the intensity of the conditioning regimen, no differences were observed for the main transplant outcomes. In conclusion, in patients diagnosed with AML and transplanted with active disease, karyotype remains an important prognostic factor, allowing splitting patients into different risk groups according to their cytogenetics. Similarly, FLT3-ITD mutation also remains a negative prognostic factor in this population.

Relapse of Aplastic Anemia with Majority Donor Chimerism (Donor-Type Aplasia) Occurring Late after Bone Marrow Transplantation.

There have been sporadic reports of the development of delayed disease recurrence after bone marrow transplantation for severe aplastic anemia despite sustained majority or full donor chimerism. This is termed "donor-type aplasia" (DTA). We describe the management and outcome of 11 pediatric patients from 8 institutions in Europe, the United States, and the Middle East who developed DTA at a mean of 35 months post-transplant. These patients were initially transplanted at a mean age of 10.0 years (range, 5.8 to 16.0 years), 9 from matched sibling donors and 2 from matched unrelated donors. Attempts to treat DTA with varying combinations of additional immunosuppression (including intravenous immunoglobulin, donor lymphocyte infusions, stem cell boosts, and other therapies) failed. Ten patients have received a conditioned second transplant, 9 from the same donor and 1 from a new matched unrelated donor. Aplasia has resolved in the remaining patient in response to ongoing eltrombopag therapy. All patients were alive at a mean of 92 months (range, 26 to 195) after a second transplant; 6 are in complete remission, but 4 suffered from second/recurrent DTA at 16 to 129 months after retransplant and required further transplant therapy.

Very long-term follow-up of aplastic anemia treated with immunosuppressive therapy or allogeneic hematopoietic cell transplantation.

INTRODUCTION: Since the 1970s outcome of aplastic anemia (AA) patients has improved significantly due to the introduction of immunosuppressive therapy (IST) and allogeneic hematopoietic transplantation (HCT). However, patients may suffer from persistent disease, relapse, clonal evolution, graft-versus-host disease and other late effects. Here, we analyse very long-term outcome of all AA patients at our institution comparing not only survival, but also response status and complications. METHODS: Patient charts of all 302 AA patients treated between 1973 and 2017 at the University Hospital Basel, Switzerland, were retrospectively analysed. RESULTS: First line treatment was IST in 226 (75%) and HCT in 76 (25%) patients. Overall survival at 30 years was similar in patients treated initially by HCT and IST (44% (±14%), and 40% (± 9%) respectively, with better results in more recent years. Partial and no response occurred more frequently after IST, relapse incidence after IST was 24 %, whereas non-engraftment and graft failure was documented in 15 patients (19 %) after HCT. Clonal evolution to myelodysplastic syndrome / acute myeloid leukemia was 16 % at 25 years in IST patients, 1.3 % in HCT patients, iron overload (18 versus 4 %, p = 0.002) and cardiovascular events (11 versus 1 %, p=0.011) occured significantly more often in IST than HCT treated patients. The majority of long-term survivors, 96% of those alive at 25 years, were in complete remission at last follow up, irrespective of the initial treatment modality. CONCLUSION: Very long term survivors after AA are those with stable hematopoietic recovery.

[Thrombocytopenia as an incidental finding]. / Thrombozytopenie als Zufallsbefund.

Thrombocytopenia as an incidental finding Abstract. Platelets play an important role in normal hemostasis and their number in the periphereal blood is normally very stable. Thrombocytopenia is frequently encountered in the clinic. As a first step, one has to exclude so called pseudothrombocytopenia due to preanalytic (EDTA as an anticoagulant) conditions. This can be done by determination of platelet counts in citrate or alternative (e. g. Thrombo-Exact) tubes or by performing a blood smear, where platelets are seen clumped. A blood smear is also the mainstay of further work-up as the differential diagnosis is very broad. In bleeding patients early therapeutic interventions have to be considered. Further work-up should be done stepwise and in collaboration with a hematologist. A definite diagnosis is essential in order to define specific therapeutic interventions, mainly in bleeding patients, in patients scheduled for surgery or patients taking antiaggregation and / or anticoagulation therapy.

The sympathomimetic agonist mirabegron did not lower JAK2-V617F allele burden, but restored nestin-positive cells and reduced reticulin fibrosis in patients with myeloproliferative neoplasms: results of phase II study SAKK 33/14.

The ß-3 sympathomimetic agonist BRL37344 restored nestin-positive cells within the stem cell niche, and thereby normalized blood counts and improved myelofibrosis in a mouse model of JAK2-V617F-positive myeloproliferative neoplasms. We therefore tested the effectiveness of mirabegron, a ß-3 sympathomimetic agonist, in a phase II trial including 39 JAK2-V617F-positive patients with myeloproliferative neoplasms and a mutant allele burden more than 20%. Treatment consisted of mirabegron 50 mg daily for 24 weeks. The primary end point was reduction of JAK2-V617F allele burden of 50% or over, but this was not reached in any of the patients. One patient achieved a 25% reduction in JAK2-V617F allele burden by 24 weeks. A small subgroup of patients showed hematologic improvement. As a side study, bone marrow biopsies were evaluated in 20 patients. We found an increase in the nestin+ cells from a median of 1.09 (interquartile range 0.38-3.27)/mm2 to 3.95 (interquartile range 1.98-8.79)/mm2 (P<0.0001) and a slight decrease of reticulin fibrosis from a median grade of 1.0 (interquartile range 0-3) to 0.5 (interquartile range 0-2) (P=0.01) between start and end of mirabegron treatment. Despite the fact that the primary end point of reducing JAK2-V617F allele burden was not reached, the observed effects on nestin+ mesenchymal stem cells and reticulin fibrosis is encouraging, and shows that mirabegron can modify the microenvironment where the JAK2-mutant stem cells are maintained. (Registered at clinicaltrials.gov identifier: 02311569).

The value of the post-thaw CD34+ count with and without DMSO removal in the setting of autologous stem cell transplantation.

BACKGROUND: CD34+ cell count correlates with engraftment potency after autologous stem cell transplantation. Assessment of CD34+ mainly occurs after apheresis and before cryopreservation with dimethyl sulfoxide (DMSO). The influence of postthaw CD34+ cell numbers over time to engraftment is not well studied, and determination of postthaw CD34+ cell counts is challenging for a variety of reasons. The aim of this retrospective study was to systematically assess the value of postthaw CD34+ cell counts in autologous grafts with and without DMSO removal. STUDY DESIGN AND METHODS: Between January 2008 and December 2015, 236 adult patients underwent a total of 292 autologous stem cell transplantations. Median age at transplantation was 56 years, and the main indication was multiple myeloma (60%). DMSO removal was done in 96 grafts (33%), either by centrifugation or by Sepax method. RESULTS: Patients receiving grafts containing DMSO showed a significantly faster platelet (p = 0.02) and RBC (p = 0.001) engraftment. DMSO removal was not associated with fewer infusion-related adverse events. We observed a good correlation between CD34+ cell count after apheresis and CD34+ cell count after thawing/washing (r = 0.931). Ninety grafts (31%) showed a significant loss of viable CD34+ cells, which translated into a delayed engraftment. CONCLUSION: DMSO removal was associated with delayed platelet and RBC engraftment without preventing adverse events. CD34+ cell enumeration after thawing remains difficult to perform, but grafts showing higher cell loss during cryopreservation and thawing are associated with slower engraftment. Prospective studies on the role of DMSO removal and postthaw CD34+ enumeration using defined protocols are needed.