Molecular and biochemical evaluation of oxidative effects of cord blood CD34+ MPs on hematopoietic cells.

A graft source for allogeneic hematopoietic stem cell transplantation is umbilical cord blood, which contains umbilical cord blood mononuclear cells (MNCs and mesenchymal stem cells, both an excellent source of extracellular microparticles (MPs). MPs act as cell communication mediators, which are implicated in reactive oxygen species formation or detoxification depending on their origin. Oxidative stress plays a crucial role in both the development of cancer and its treatment by triggering apoptotic mechanisms, in which CD34+ cells are implicated. The aim of this work is to investigate the oxidative stress status and the apoptosis of HL-60 and mononuclear cells isolated from umbilical cord blood (UCB) following a 24- and 48-hour exposure to CD34 + microparticles (CD34 + MPs). The activity of superoxide dismutase, glutathione reductase, and glutathione S-transferase, as well as lipid peroxidation in the cells, were employed as oxidative stress markers. A 24- and 48-hour exposure of leukemic and mononuclear cells to CD34 + -MPs resulted in a statistically significant increase in the antioxidant activity and lipid peroxidation in both cells types. Moreover, CD34 + MPs affect the expression of BCL2 and FAS and related proteins and downregulate the hematopoietic differentiation program in both HL-60 and mononuclear cells. Our results indicate that MPs through activation of antioxidant enzymes in both homozygous and nonhomozygous cells might serve as a means for graft optimization and enhancement.

Perspectives for the Use of Umbilical Cord Blood in Transplantation and Beyond: Initiatives for an Advanced and Sustainable Public Banking Program in Greece.

The umbilical cord blood (UCB) donated in public UCB banks is a source of hematopoietic stem cells (HSC) alternative to bone marrow for allogeneic HSC transplantation (HSCT). However, the high rejection rate of the donated units due to the strict acceptance criteria and the wide application of the haploidentical HSCT have resulted in significant limitation of the use of UCB and difficulties in the economic sustainability of the public UCB banks. There is an ongoing effort within the UCB community to optimize the use of UCB in the field of HSCT and a parallel interest in exploring the use of UCB for applications beyond HSCT i.e., in the fields of cell therapy, regenerative medicine and specialized transfusion medicine. In this report, we describe the mode of operation of the three public UCB banks in Greece as an example of an orchestrated effort to develop a viable UCB banking system by (a) prioritizing the enrichment of the national inventory by high-quality UCB units from populations with rare human leukocyte antigens (HLA), and (b) deploying novel sustainable applications of UCB beyond HSCT, through national and international collaborations. The Greek paradigm of the public UCB network may become an example for countries, particularly with high HLA heterogeneity, with public UCB banks facing sustainability difficulties and adds value to the international efforts aiming to sustainably expand the public UCB banking system.

Ruxolitinib in patients with polycythemia vera resistant and/or intolerant to hydroxyurea: European observational study.

BACKGROUND: Hydroxyurea (HU) is a commonly used first-line treatment in patients with polycythemia vera (PV). However, approximately 15%-24% of PV patients report intolerance and resistance to HU. METHODS: This phase IV, European, real-world, observational study assessed the efficacy and safety of ruxolitinib in PV patients who were resistant and/or intolerant to HU, with a 24-month follow-up. The primary objective was to describe the profile and disease burden of PV patients. RESULTS: In the 350 enrolled patients, 70% were >60 years old. Most patients (59.4%) had received ≥1 phlebotomy in the 12 months prior to the first dose of ruxolitinib. Overall, 68.2% of patients achieved hematocrit control with 92.3% patients having hematocrit <45% and 35.4% achieved hematologic remission at month 24. 85.1% of patients had no phlebotomies during the study. Treatment-related adverse events were reported in 54.3% of patients and the most common event was anemia (22.6%). Of the 10 reported deaths, two were suspected to be study drug-related. CONCLUSION: This study demonstrates that ruxolitinib treatment in PV maintains durable hematocrit control with a decrease in the number of phlebotomies in the majority of patients and was generally well tolerated.

Risk Factors, Prevalence, and Outcomes of Invasive Fungal Disease Post Hematopoietic Cell Transplantation and Cellular Therapies: A Retrospective Monocenter Real-Life Analysis.

(1) Background: Autologous, allogeneic hematopoietic cell transplantation (HCT) and other cellular therapies, including CAR T cell and gene therapy, constitute a cornerstone in the management of various benign and malignant hematological disorders. Invasive fungal infections (IFD) remain a significant cause of morbidity and mortality in HCT recipients. Therefore, we investigated the prevalence and risk factors of IFD following HCT and other cellular therapies in an era of novel antifungal prophylaxis. (2) Methods: In this study, we retrospectively enrolled adult HCT recipients who were treated at our JACIE-accredited center according to standard operating procedures over the last decade (2013-2022). (3) Results: 950 patients who received cellular therapies were studied. None of the 19 CAR T cell and neither of the two gene therapy recipients developed IFD whereas 3/456 autologous HCT recipients who suffered from primary refractory/relapsed lymphomas presented with probable IFD. Overall, 11 patients who received allogeneic HCT experienced probable IFD, possible IFD was found in 31/473, and IFD was proven in 10/473. A second IFD episode was present in three patients. Four-year OS was significantly lower in proven compared to probable IFD (p = 0.041) and was independently associated with HCT-CI (p = 0.040) and chronic GVHD (p = 0.045). (4) Conclusions: In this real-world cohort, the prevalence of proven and probable IFD in an era of novel antifungal prophylaxis was found to be relatively low. However, IFDs were associated with poor outcomes for patients who received allogeneic HCT.

Adenoviral Infections in Bone Marrow Transplanted Adult Patients: A Review of the 44 Cases Reported in the Last 25 Years.

Adenoviral infections in immunocompromised individuals may be life-threatening conditions. The aim of this review is to document all the reported cases of adenoviral infection is patients having undergone bone marrow transplantation (BMT). A comprehensive literature search of the databases Pubmed, Science Direct, and Google Scholar was conducted to identify all the case reports of adenoviral infections in BMT patients. A total of 30 articles with 44 patients were included. The most common underlying condition was acute lymphocytic leukemia (23%) followed by acute myeloid leukemia (18%). The most common site of infection was disseminated (50%), followed by liver infection (8%) and hemorrhagic cystitis (8%). Cidofovir was administered in 40.9% of the cases, and death was reported in 34.4% of them. Ribavirin was administered as monotherapy in 15.9% of patients, with a mortality rate of 57.1%. We found that the antiviral drug option had no statistically significant effect on the mortality rate (p=0.242). Also, the absence of graft-versus-host disease (GVHD) was not associated with an improved outcome (p=0.523). There was, however, a statistically significant difference in the outcome based on the site of infection (p=0.005), with a higher rate of mortality in the disseminated and gastrointestinal cases. To the best of our knowledge, this is the first review documenting all the cases of adenoviral infections in BMT patients. Future randomized studies are needed to validate the results of the present study.

Solitary extramedullary plasmacytoma of the parotid gland in a patient with mixed connective tissue disease.

Solitary extramedullary plasmacytomas of the head and neck region are rare entities. Very few have been described in the parotid gland. They can clinically and radiologically mimic the rather common benign tumors of the parotid gland (pleomorphic adenoma and Wharthin's tumor), and subsequently the need careful consideration by the involving surgeon and pathologist is needed for a proper diagnosis. In the present study, a case of solitary extramedullary plasmacytoma in the left parotid gland of a 55-year-old patient with mixed connective tissue disease is reported, along with the relevant clinical, imaging, operative, and histopathological findings. Postoperative hematological investigation to confirm the singularity of the lesion was performed. Complementary treatment with radiotherapy was followed. Disease-free, 1 year follow up is also presented.

Non-transplantable cord blood units as a source for adoptive immunotherapy of leukaemia and a paradigm of circular economy in medicine.

Advances in immunotherapy with T cells armed with chimeric antigen receptors (CAR-Ts), opened up new horizons for the treatment of B-cell lymphoid malignancies. However, the lack of appropriate targetable antigens on the malignant myeloid cell deprives patients with refractory acute myeloid leukaemia of effective CAR-T therapies. Although non-engineered T cells targeting multiple leukaemia-associated antigens [i.e. leukaemia-specific T cells (Leuk-STs)] represent an alternative approach, the prerequisite challenge to obtain high numbers of dendritic cells (DCs) for large-scale Leuk-ST generation, limits their clinical implementation. We explored the feasibility of generating bivalent-Leuk-STs directed against Wilms tumour 1 (WT1) and preferentially expressed antigen in melanoma (PRAME) from umbilical cord blood units (UCBUs) disqualified for allogeneic haematopoietic stem cell transplantation. By repurposing non-transplantable UCBUs and optimising culture conditions, we consistently produced at clinical scale, both cluster of differentiation (CD)34+ cell-derived myeloid DCs and subsequently polyclonal bivalent-Leuk-STs. Those bivalent-Leuk-STs contained CD8+ and CD4+ T cell subsets predominantly of effector memory phenotype and presented high specificity and cytotoxicity against both WT1 and PRAME. In the present study, we provide a paradigm of circular economy by repurposing unusable UCBUs and a platform for future banking of Leuk-STs, as a 'third-party', 'off-the-shelf' T-cell product for the treatment of acute leukaemias.

Risk Factors and Outcomes of Klebsiella pneumoniae Infection Before and After Allogeneic Hematopoietic Cell Transplantation.

Objectives: Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kp) emerge as a major healthcare concern worldwide. Despite the significance of infections before and after allogeneic hematopoietic cell transplantation (alloHCT), the burden of KP infections has not been extensively evaluated. Methods: We studied the incidence, risk factors, and outcomes of consecutive alloHCT recipients with Kp isolates before and after alloHCT. Results: Among 424 patients who underwent alloHCT in 2008-2018, we studied two groups: those with Kp isolates before (group 1, 52 patients) and those with Kp isolates after alloHCT (group 2, 66 patients). prE-transplant infections were associated with post-transplant infections (p = 0.010), despite secondary prophylaxis. KPC-Kp was isolated in 29% of group 1, and 80% of group 2. Both groups were characterized by a significant burden of moderate-severe acute graft- vs.-host disease (GVHD) [cumulative incidence (CI) of 44.5 and 61.9%, respectively] and severe chronic (CI of 56.7 and 61.9%). Kp infections and GVHD were independent predictive factors of treatment-related mortality (TRM) in both groups. Conclusions: Our study highlights the significant impact of Kp infections on TRM, with GVHD consisting an important underlying factor. As prophylactic measures did not improve rates of post-transplant infections, innovative interventions need to be further investigated to address this major healthcare concern.

Identification of miRNAs from stem cell derived microparticles in umbilical cord blood.

Umbilical cord blood CD34+ (UCB-CD34+) stem cells are clinically used in hematopoietic cell transplantation. However, there are limitations in the use of umbilical cord blood transplants because of the small number of cells and delayed engraftment. To gain a better understanding of functional components of UCB, we have detected and characterized CD34+ microparticles (CD34+MPs) from cord blood units. We collected cord blood units and assessed the numbers of CD34+MPs before and after red blood cell and plasma depletion by SEPAX processing using flow cytometry analysis. In parallel we identified MPs by electron microscopy. CD34+MPs and cells were isolated by MACs sorting. MicroRNAs (miR-106, miR-221, miR-517, miR-519, and miR-221) exhibited a characteristic microRNA profile that was further validated in isolated CD34+MPs. We found that in cord blood, there are CD34+MPs that carry microRNAs.

Isolated Extramedullary Relapse as a Poor Predictor of Survival after Allogeneic Hematopoietic Cell Transplantation for Acute Leukemia.

Limited and conflicting data exist on outcomes of patients with extramedullary relapses (EMRs) after allogeneic hematopoietic cell transplantation (allo-HCT) for acute leukemias. We retrospectively reviewed charts of consecutive allo-HCT recipients who underwent transplantation in our center with the indication of acute leukemia (July 1990 to July 2018). Incidences of isolated EMR (iEMR) and bone marrow relapse (BMR) were calculated using cumulative incidence (CI) analysis, with each and treatment-related mortality considered a competing risk. We studied 554 allo-HCT recipients for 1.8 years (range, .04 to 27.75). Ten-year CI of 10.5% for iEMR was associated only with advanced disease phase at transplantation, whereas 10-year CI of 34.8% for BMR was independently associated with pretransplant disease phase, lines of treatment, and fungal infections. Most iEMR and BMR patients (75% and 81%, respectively) received systemic treatment combined with local radiation for iEMR (26%) and donor lymphocyte infusions (16% and 28%, respectively) when feasible. Extensive chronic graft-versus-host disease (GVHD) was recorded in 47% of iEMR and 48% of BMR patients. Outcomes were poor both in iEMR (10-year overall survival [OS], 18.3%) and BMR (10-year OS, 19.1%). Independent predictors of OS were disease phase, type of donor, acute and chronic GVHD, fungal infections, iEMR, and BMR. In a large population with long-term follow-up, incidence of iEMR was relatively high, developed at the late post-transplant period, and was associated only with disease phase at transplantation. Furthermore, iEMR and BMR conferred similarly poor outcomes despite systemic treatment or extensive chronic GVHD.

Long-term outcomes of total body irradiation plus cyclophosphamide versus busulfan plus cyclophosphamide as conditioning regimen for acute lymphoblastic leukemia: a comparative study.

The role of total body irradiation (TBI) in allogeneic hematopoietic stem cell transplantation (HCT) for adult acute lymphoblastic leukemia (ALL) remains controversial. Therefore, we investigated long-term treatment outcomes of transplanted ALL patients aiming to identify prognostic factors and the impact of conditioning. We enrolled consecutive ALL patients transplanted from 1990 to 2016, following TBI- or busulfan (Bu)-based conditioning regimen. We studied 151 ALL patients transplanted in first complete remission (CR) (60), other CR (33), or relapsed/refractory disease (58) from sibling (87), and HLA-matched (42) or mismatched (17) unrelated and alternative donors (5). High-dose fractionated TBI-based conditioning was administered in 84. No differences were observed in baseline characteristics, except for disease stage at transplant, donor type, and graft source. With a follow-up of 19.0 (0.5-170.5) in TBI and 14.5 (1.2-319.1) months in non-TBI patients, there was no difference in acute (grades II-IV) or chronic GVHD, thrombotic microangiopathy, and bacterial or fungal infections. Only viral infections were significantly increased in the non-TBI group. There was no significant difference in the cumulative incidence (CI) of treatment-related or relapse mortality and disease-free or overall survival (OS). In the multivariate analysis, unfavorable pre-transplant predictors of OS were age (p = 0.024), advanced disease stage (p = 0.007), and female-to-male donor (p = 0.006). Interestingly, TBI patients younger than 40 years had significantly higher OS (55.1%, p = 0.023) and DFS (48.6%, p = 0.020). In conclusion, high-dose TBI is feasible in younger patients providing better survival. The choice between TBI- or Bu-conditioning regimens remains challenging.

The Role of Low-dose Anti-thymocyte Globulin as Standard Prophylaxis in Mismatched and Matched Unrelated Hematopoietic Peripheral Stem Cell Transplantation for Hematologic Malignancies.

INTRODUCTION: Anti-thymocyte globulin (ATG)-based immunosuppressive therapy is often used in allogeneic hematopoietic cell transplantation to reduce incidence and severity of graft-versus-host disease (GVHD). PATIENTS AND METHODS: In our observational study, ATG (rabbit, Thymoglobulin; Sanofi, 5 mg/kg) was administered as a standardized part of the conditioning in 97 patients with a median age of 34 years (range, 14-58 years), allotransplanted for hematologic malignancies from matched (8/8; n = 52) and allele or antigen mismatched (7/8; n = 43 and 6/8; n = 2) unrelated donors. RESULTS: Five-year overall survival (OS) was 46.9%, disease-free survival was 46.5%, and treatment-related mortality was 20.6%, with a median follow-up of 29 months (range, 1-145 months). Acute GVHD (grade ≥ II) cumulative incidence was 26.9% in matched versus 50.5% in mismatched patients (P = .060), whereas extensive chronic GVHD was 45.6% versus 50%, respectively (P = .310). Five-year OS was 62.2% for the matched patients and 27.9% for the mismatched patients (P = .003) owing to a higher treatment-related mortality rate in mismatched patients (P = .032). In multivariate analysis including age, gender, time until transplantation, disease phase, mismatched donor, ABO mismatch, number of CD34+ infused, acute and chronic GVHD, the significant unfavorable factors for OS were HLA mismatch and advanced disease phase. CONCLUSION: A relatively low-dose ATG is effective in acute GVHD prophylaxis, leading to promising survival rates in matched transplants. Further comparative studies with adjusted ATG dose depending on Human leukocyte antigen disparity or alternative donors are warranted.

Survival Advantage and Comparable Toxicity in Reduced-Toxicity Treosulfan-Based versus Reduced-Intensity Busulfan-Based Conditioning Regimen in Myelodysplastic Syndrome and Acute Myeloid Leukemia Patients after Allogeneic Hematopoietic Cell Transplantation.

Treosulfan has been incorporated in conditioning regimens for sustained remission without substantial toxicity and treatment-related mortality (TRM). We aimed to analyze the safety and efficacy of a fludarabine 150 mg/m2 and treosulfan 42 g/m2 (FluTreo) conditioning regimen in medically infirm patients. Outcomes were compared with those of a similar historical group treated with fludarabine 150 mg/m2 to 180 mg/m2, busulfan 6.4 mg/kg, and antithymocyte globulin (ATG) 5 mg/kg to 7.5 mg/kg (FluBuATG). Thirty-one consecutive patients with acute myeloid leukemia (AML; n = 21), myelodysplastic syndrome (MDS; n = 6), or treatment-related AML (n = 4) received FluTreo conditioning. The historical group consisted of 26 consecutive patients treated with FluBuATG. In the FluTreo group, engraftment was prompt in all patients and 74% achieved >99% donor chimerism by day +30. No grades III or IV organ toxicities were noted. One-year cumulative incidences (CI) of acute and chronic graft-versus-host disease (GVHD) were 19.4% and 58.4%. The groups were similar for age, disease risk, lines of treatment, hematopoietic cell transplantation-specific comorbidity index, and acute or chronic GVHD incidence, except that there were more matched unrelated donor recipients in the FluTreo group (P < .001). With 20 (range, 2 to 36) months follow-up for FluTreo and 14 (range, 2 to 136) for FluBuATG, the 1-year cumulative overall survival (OS) probability was 76% versus 57%, respectively (P = .026); 1-year disease-free survival (DFS) was 79% versus 38% (P < .001). In multivariate analysis, the only significantly favorable factor for OS and DFS was FluTreo (P = .010 and P = .012). The CI of relapse mortality was markedly decreased in FluTreo versus FluBuATG (7.4% versus 42.3%, P < .001). In conclusion, the treosulfan-based regimen resulted in favorable OS and DFS with acceptable toxicity and low relapse rates compared with busulfan-based conditioning.

In vitro and in vivo study on the effect of antifungal agents on hematopoietic cells in mice.

Liposomal amphotericin B, voriconazole, and caspofungin are currently used for systemic and severe fungal infections. Patients with malignant diseases are treated with granulocyte-colony stimulating factor (G-CSF) for the recovery of granulocytes after chemotherapy or hematopoietic cell (HC) transplantation. Since they have a high incidence of fungal infections, they inevitably receive antifungal drugs for treatment and prophylaxis. Despite their proven less toxicity for various cell types comparatively with amphotericin B and the decrease in the number of leukocytes that has been reported as a possible complication in clinical studies, the effect of liposomal amphotericin B, voriconazole, and caspofungin on HCs has not been clarified. The present study aimed to examine the in vitro and in vivo effect of these three modern antifungals on HCs. Colony-forming unit (CFU) assays of murine bone marrow cells were performed in methylcellulose medium with or without cytokines and in the presence or absence of various concentrations of liposomal amphotericin B, voriconazole, and caspofungin. In the in vivo experiments, the absolute number of granulocytes was determined during leukocyte recovery in sublethally irradiated mice receiving each antifungal agent separately, with or without G-CSF. In vitro, all three antifungal drugs were nontoxic and, interestingly, they significantly increased the number of CFU-granulocyte-macrophage colonies in the presence of cytokines, at all concentrations tested. This was contrary to the concentration-dependent toxicity and the significant decrease caused by conventional amphotericin B. In vivo, the number of granulocytes was significantly higher with caspofungin plus G-CSF treatment, higher and to a lesser extent higher, but not statistically significantly, with voriconazole plus G-CSF and liposomal amphotericin B plus G-CSF treatments, respectively, as compared with G-CSF alone. These data indicate a potential synergistic effect of these antifungals with the cytokines, in vitro and in vivo, with subsequent positive effect on hematopoiesis.

Carmustine, etoposide, cytarabine and melphalan versus a newly designed intravenous busulfan-based Busulfex, etoposide and melphalan conditioning regimen for autologous hematopoietic cell transplant: a retrospective matched-pair analysis in advanced Hodgkin and non-Hodgkin lymphomas.

Optimal conditioning remains a challenge in lymphomas. We designed a regimen consisting of Busulfex, etoposide and melphalan (BuEM). We retrospectively analyzed the outcome of patients conditioned with carmustine, etoposide, cytarabine and melphalan (BEAM) or BuEM in matched-pair analysis on a planned 2:1 ratio. Eighty-seven patients treated with BEAM who fulfilled the matching criteria were randomly selected. Two-year progression-free survival/overall survival (PFS/OS) were 63.2%/76.7% for BEAM vs. 65.6%/79.8% for BuEM after 64.7 and 42.7 months, respectively. Furthermore, marginally better PFS and OS were noted in Hodgkin lymphoma (HL) after BuEM. In multivariate analysis, PFS was superior in HL, chemosensitive disease and complete remission post-transplant. BEAM correlated with faster engraftment, reduced infections, less mucositis and liver toxicity, and BuEM with less need for blood cell and platelet transfusions and granulocyte colony-stimulating factor administration. In conclusion, BuEM was well tolerated and equally highly efficacious as BEAM for non-Hodgkin lymphoma and offered marginally significantly improved PFS and OS in HL with acceptable toxicity and zero mortality.

An outbreak of Burkholderia cepacia bacteremia in hospitalized hematology patients selectively affecting those with acute myeloid leukemia.

BACKGROUND: Burkholderia cepacia complex (Bcc) is a group of common environmental bacteria that preferentially colonize and infect patients with cystic fibrosis but are also emerging as nosocomial pathogens, possibly due to their resistance to disinfectants and antimicrobials. METHODS: We investigated a 3-month outbreak of Bcc bacteremia among hospitalized hematology patients. Environmental investigation and infection control measures were implemented. A retrospective, cross-sectional study was conducted to identify risk factors. RESULTS: Bcc was repeatedly isolated from the blood of 9 patients without central venous catheter who did not easily respond to targeted antibiotic treatment and 3 died of the infection. A point source was not identified and horizontal spread was suspected. Strict infection control measures terminated the outbreak. Interestingly, diagnosis of acute myeloid leukemia but not neutropenia or prior chemotherapy was a risk factor for infection acquisition. Neutropenia was positively correlated with infection duration. CONCLUSIONS: Bcc is not only a serious threat among immunocompromized hematology patients, but is also transmissible in clinical settings. Acute myeloid leukemia appears to confer additional risk for infection acquisition.