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.

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.

Genetic prediction of ICU hospitalization and mortality in COVID-19 patients using artificial neural networks.

There is an unmet need of models for early prediction of morbidity and mortality of Coronavirus disease-19 (COVID-19). We aimed to a) identify complement-related genetic variants associated with the clinical outcomes of ICU hospitalization and death, b) develop an artificial neural network (ANN) predicting these outcomes and c) validate whether complement-related variants are associated with an impaired complement phenotype. We prospectively recruited consecutive adult patients of Caucasian origin, hospitalized due to COVID-19. Through targeted next-generation sequencing, we identified variants in complement factor H/CFH, CFB, CFH-related, CFD, CD55, C3, C5, CFI, CD46, thrombomodulin/THBD, and A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS13). Among 381 variants in 133 patients, we identified 5 critical variants associated with severe COVID-19: rs2547438 (C3), rs2250656 (C3), rs1042580 (THBD), rs800292 (CFH) and rs414628 (CFHR1). Using age, gender and presence or absence of each variant, we developed an ANN predicting morbidity and mortality in 89.47% of the examined population. Furthermore, THBD and C3a levels were significantly increased in severe COVID-19 patients and those harbouring relevant variants. Thus, we reveal for the first time an ANN accurately predicting ICU hospitalization and death in COVID-19 patients, based on genetic variants in complement genes, age and gender. Importantly, we confirm that genetic dysregulation is associated with impaired complement phenotype.

Genetic justification of severe COVID-19 using a rigorous algorithm.

Recent studies suggest excessive complement activation in severe coronavirus disease-19 (COVID-19). The latter shares common characteristics with complement-mediated thrombotic microangiopathy (TMA). We hypothesized that genetic susceptibility would be evident in patients with severe COVID-19 (similar to TMA) and associated with disease severity. We analyzed genetic and clinical data from 97 patients hospitalized for COVID-19. Through targeted next-generation-sequencing we found an ADAMTS13 variant in 49 patients, along with two risk factor variants (C3, 21 patients; CFH,34 patients). 31 (32%) patients had a combination of these, which was independently associated with ICU hospitalization (p = 0.022). Analysis of almost infinite variant combinations showed that patients with rs1042580 in thrombomodulin and without rs800292 in complement factor H did not require ICU hospitalization. We also observed gender differences in ADAMTS13 and complement-related variants. In light of encouraging results by complement inhibitors, our study highlights a patient population that might benefit from early initiation of specific treatment.

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.

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.

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.

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.

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.

Targeted genotyping of COVID-19 patients reveals a signature of complement C3 and factor B coding SNPs associated with severe infection.

We have attempted to explore further the involvement of complement components in the host COVID-19 (Coronavirus disease-19) immune responses by targeted genotyping of COVID-19 adult patients and analysis for missense coding Single Nucleotide Polymorphisms (coding SNPs) of genes encoding Alternative pathway (AP) components. We have identified a small group of common coding SNPs in Survivors and Deceased individuals, present in either relatively similar frequencies (CFH and CFI SNPs) or with stark differences in their relative abundance (C3 and CFB SNPs). In addition, we have identified several sporadic, potentially protective, coding SNPs of C3, CFB, CFD, CFH, CFHR1 and CFI in Survivors. No coding SNPs were detected for CD46 and CD55. Our demographic analysis indicated that the C3 rs1047286 or rs2230199 coding SNPs were present in 60 % of all the Deceased patients (n = 25) (the rs2230199 in 67 % of all Deceased Males) and in 31 % of all the Survivors (n = 105, p = 0.012) (the rs2230199 in 25 % of all Survivor Males). When we analysed these two major study groups using the presence of the C3 rs1047286 or rs2230199 SNPs as potential biomarkers, we noticed the complete absence of the protective CFB rs12614 and rs641153 coding SNPs from Deceased Males compared to Females (p = 0.0023). We propose that in these individuals, C3 carrying the R102G and CFB lacking the R32W or the R32Q amino acid substitutions, may contribute to enhanced association dynamics of the C3bBb AP pre-convertase complex assembly, thus enabling the exploitation of the activation of the Complement Alternative pathway (AP) by SARS-CoV-2.

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.

Detection of two novel alleles, HLA-A*02:943 and -B*51:104:02, in Greek cord blood units.

The new alleles A*02:943 and B*51:104:02 were detected in Greek cord blood units.

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.

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.

Effect of chemotherapy on primary mucosa-associated lymphoid tissue lymphoma of the orbit.

PURPOSE: To present a treatment plan for localized mucosa-associated lymphoid tissue lymphoma of the orbit in order to preserve vital structures and function from the side effects of radiotherapy. MATERIALS AND METHODS: Study of 2 clinical cases by means of clinical and radiologic examination, with 2 and 3 years follow-up, respectively. RESULTS: Immediate remission of the disease after 6 cycles of chemotherapy, with no signs of recurrence after 2 and 3 years. Clinical examination of the oculomotor mechanism and visual activity gave excellent results. CONCLUSION: Although radiotherapy is preferable for localized lymphoproliferative lesions, chemotherapy should also be considered as an effective treatment that preserves the integrity and function of the ocular adnexa.