Search Results (25)

Background: Extracorporeal photopheresis (ECP) consists of the collection of a patient’s peripheral blood mononuclear cells (MNCs) that, after incubation with a photosensitive molecule, are exposed to ultraviolet-A (UVA) and then reinfused into the patient. There are two methods for performing the ECP procedure: the “in-line” methods and the “off-line” methods. In the “in-line” method, all the phases of ECP (leukapheresis, photoactivation, and reinfusion) are achieved sequentially in extracorporeal circulation using a single instrument and a single sterile disposable kit without disconnection from the patient’s blood circulation. In this paper, we report our real-life experience with a recently licensed in-line ECP system proposed by Fresenius-Kabi. Methods: The ECP procedures (n = 211) were performed using an Amicus cell separator and a Phelix UV irradiator with Amicus software 4.51 and Phelix software 1.0. A targeted 2000 mL of whole blood (WB) was processed, and 1.5 J/cm² of UVA light was delivered to the collected mononuclear cells (MNCs). Results: From May 2023 to April 2024, we performed 211 ECP procedures in 11 patients with graft-versus-host disease (GvHD). The processed blood volume was between 1992 and 2000 mL, and the blood flow speed during the procedures was highly variable (from 30 to 50 mL/min), so the total duration of the procedure was quite variable (from 92 to 118 min). The collection efficiency (CE2) for mononuclear cells was always satisfactory (from 55% to 73%), with a minimal presence of RBCs and PLTs. Conclusions: In our experience, the Amicus system-based ECP procedure is safe and well tolerated as we observed only one side effect. The duration of the procedure was always under two hours. The collection efficiency (CE2) for MNCs was satisfactory, with minimal platelet and RBC product contamination. Full article

Autologous dendritic cell (DC)-based immunotherapy is a cell-based advanced therapy medicinal product (ATMP) that was first introduced more than three decades ago. In the current study, our objective was to establish a harmonized protocol using two varied antigenic sources and a good manufacturing practice (GMP)-compliant, manual method for generating clinical-grade DCs at a limited-resource academic setting. After obtaining ethical committee-approved informed consent, the recruited patients underwent leukapheresis, and single-batch DC production was carried out. Using responder-independent flow cytometric assays as quality control (QC) criteria, we propose a differentiation and maturation index (DI and MI, respectively), calculated with the QC cut-off and actual scores of each batch for comparison. Changes during cryopreservation and personnel variation were assessed periodically for up to two to three years. Using our harmonized batch production protocol, the average DI was 1.39 and MI was 1.25. Allogenic responder proliferation was observed in all patients, while IFN-gamma secretion, evaluated using flow cytometry, was detected in 10/36 patients and significantly correlated with CD8+ T cell proliferation (p value-0.0002). Tracking the viability and phenotype of cryopreserved MDCs showed a >90% viability for up to three years, while a mature DC phenotype was retained for up to one year. Our results confirm that the manual/semi-automated protocol was simple, consistent, and cost-effective, without the requirement for expensive equipment and without compromising on the quality of the final product. Full article

Purpose: We aimed to identify subsets of patients who benefit from emergency LA and to establish a therapeutic algorithm for AML patients with hyperleukocytosis. Methods: In this single-center retrospective cohort study, a total of 20 consecutive patients underwent LA because of their clinical symptoms. Overall survival (OS) analysis was conducted using the Kaplan–Meier plot method. Univariate and multivariate analyses (using multiple logistic regression) were performed. At the time of diagnosis, all patients received a standard diagnostic workup for AML including FLT3-ITD mutational analysis. Results: FLT3-ITD mut AML patients receiving LA had a median OS of 437 days (range 5–2379 days) with a corresponding 14-day survival of 92.3%, while FLT3 wt AML patients displayed a significantly lower median OS of only 5 days (range 1–203 days) with a corresponding 14-day survival of 14.3% (p = 0.0001). Conclusions: Among patients with clinical symptoms of leukostasis, the subset of FLT3-ITD mut AML patients showed a better outcome with lower early mortality after emergency LA. Based on these observations, we established a therapeutic algorithm for AML patients with hyperleukocytosis. Full article

The optimal detection strategies for effective convalescent immunity after SARS-CoV-2 infection and vaccination remain unclear. The objective of this study was to characterize convalescent immunity targeting the SARS-CoV-2 spike protein using a multiparametric approach. At the beginning of the pandemic, we recruited 30 unvaccinated convalescent donors who had previously been infected with COVID-19 and 7 unexposed asymptomatic controls. Peripheral blood mononuclear cells (PBMCs) were obtained from leukapheresis cones. The humoral immune response was assessed by measuring serum anti-SARS-CoV-2 spike S1 subunit IgG via semiquantitative ELISA, and T-cell immunity against S1 and S2 subunits were studied via IFN-γ enzyme-linked immunosorbent spot (ELISpot) and flow cytometric (FC) activation-induced marker (AIM) assays and the assessment of cytotoxic CD8⁺ T-cell function (in the subset of HLA-A2-positive patients). No single immunoassay was sufficient in identifying anti-spike convalescent immunity among all patients. There was no consistent correlation between adaptive humoral and cellular anti-spike responses. Our data indicate that the magnitude of anti-spike convalescent humoral and cellular immunity is highly heterogeneous and highlights the need for using multiple assays to comprehensively measure SARS-CoV-2 convalescent immunity. These observations might have implications for COVID-19 surveillance, and the determination of optimal vaccination strategies for emerging variants. Further studies are needed to determine the optimal assessment of adaptive humoral and cellular immunity following SARS-CoV-2 infection, especially in the context of emerging variants and unclear vaccination schedules. Full article

Circulating tumor cells (CTCs) serve as crucial metastatic precursor cells, but their study in animal models has been hindered by their low numbers. To address this challenge, we present DanioCTC, an innovative xenograft workflow that overcomes the scarcity of patient-derived CTCs in animal models. By combining diagnostic leukapheresis (DLA), the Parsortix microfluidic system, flow cytometry, and the CellCelector setup, DanioCTC effectively enriches and isolates CTCs from metastatic breast cancer (MBC) patients for injection into zebrafish embryos. Validation experiments confirmed that MDA-MB-231 cells, transplanted following the standard protocol, localized frequently in the head and blood-forming regions of the zebrafish host. Notably, when MDA-MB-231 cells spiked (i.e., supplemented) into DLA aliquots were processed using DanioCTC, the cell dissemination patterns remained consistent. Successful xenografting of CTCs from a MBC patient revealed their primary localization in the head and trunk regions of zebrafish embryos. DanioCTC represents a major step forward in the endeavors to study the dissemination of individual and rare patient-derived CTCs, thereby enhancing our understanding of metastatic breast cancer biology and facilitating the development of targeted interventions in MBC. Summary statement: DanioCTC is a novel workflow to inject patient-derived CTCs into zebrafish, enabling studies of the capacity of these rare tumor cells to induce metastases. Full article

Background: Regulatory T cell (Treg) therapy is considered an alternative approach to induce tolerance in transplantation. If successful, this therapy may have implications on immunosuppression minimization/withdrawal to reduce drug-induced toxicity in patients. The aim of this study was to assess the efficacy of the mTORC1/C2 inhibitor, AZD8055, in the manufacturing of clinically competent Treg cells and compare the effects with those induced by rapamycin (RAPA), another mTOR inhibitor commonly used in Treg expansion protocols. Methods: Primary human Treg cells were isolated from leukapheresis product. Cell viability, expansion rates, suppressive function, autophagy, mitochondrial unfolded protein response (mitoUPR), and cell metabolic profile were assessed. Results: We observed a stronger inhibition of the mTORC2 signaling pathway and downstream events triggered by Interleukin 2 (IL2)-receptor in AZD8055-treated cells compared with those treated with RAPA. AZD8055 induced progressive metabolic changes in mitochondrial respiration and glycolytic pathways that disrupted the long-term expansion and suppressive function of Tregs. Unlike RAPA, AZD8055 treatment impaired autophagy and enhanced the mitoUPR cell stress response pathway. Conclusions: A distinct pattern of mTOR inhibition by AZD, compared with RAPA, induced mitochondrial stress response and dysfunction, impaired autophagy, and disrupted cellular bioenergetics, resulting in the loss of proliferative potential and suppressive function of Treg cells. Full article

Pediatric chronic myeloid leukemia (CML) is a very rare malignancy (age-related incidence 0.1/100,000) typically presenting with leucocyte counts >100,000/µL. However, clinical signs of leukostasis are observed at diagnosis in only approximately 10% of all cases and among these, priapism is infrequent. Here, we analyze data from pediatric CML registries on the occurrence of priapism heralding diagnosis of CML in 16/491 (3.2%) boys (median age 13.5 years, range 4–18) with pediatric CML. In the cohort investigated, duration of priapism resulting in a diagnosis of CML was not reported in 5 patients, and in the remaining 11 patients, occurred as stuttering priapism over 3 months (n = 1), over 6 weeks (n = 1), over 1–2 weeks (n = 2), over several days (n = 2), or 24 h (n = 1), while the remaining 4 boys reported continuous erection lasting over 11–12 h. All patients exhibited splenomegaly and massive leukocytosis (median WBC 470,000/µL, range 236,700–899,000). Interventions to treat priapism were unknown in 5 patients, and in the remaining cohort, comprised intravenous fluids ± heparin (n = 2), penile puncture (n = 5) ± injection of sympathomimetics (n = 4) ± intracavernous shunt operation (n = 1) paralleled by leukocyte-reductive measures. Management without penile puncture by leukapheresis or exchange transfusion was performed in 3 boys. In total, 7 out 15 (47%) long-term survivors (median age 20 years, range 19–25) responded to a questionnaire. All had maintained full erectile function; however, 5/7 had presented with stuttering priapism while in the remaining 2 patients priapism had lasted <12 h until intervention. At its extreme, low-flow priapism lasting for longer than 24 h may result in partial or total impotence by erectile dysfunction. This physical disability can exert a large psychological impact on patients’ lives. In a narrative review fashion, we analyzed the literature on priapism in boys with CML which is by categorization stuttering or persisting as mostly painful, ischemic (low-flow) priapism. Details on the pathophysiology are discussed on the background of the different blood rheology of hyperleukocytosis in acute and chronic leukemias. In addition to the data collected, instructive case vignettes demonstrate the diagnostic and treatment approaches and the outcome of boys presenting with priapism. An algorithm for management of priapism in a stepwise fashion is presented. All approaches must be performed in parallel with cytoreductive treatment of leukostasis in CML which comprises leukapheresis and exchange transfusions ± cytotoxic chemotherapy. Full article

Monoclonal antibody treatment initially heralded an era of molecularly targeted therapy in oncology and is now widely applied in modulating anti-cancer immunity by targeting programmed cell receptors (PD-1, PD-L1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and, more recently, lymphocyte-activation gene 3 (LAG3). Chimeric antigen receptor T-cell therapy (CAR-T) recently proved to be a valid approach to inducing anti-cancer immunity by directly modifying the host’s immune cells. However, such cell-based therapy requires extensive resources such as leukapheresis, ex vivo modification and expansion of cytotoxic T-cells and current Good Manufacturing Practice (cGMP) laboratories and presents significant logistical challenges. Bi-/trispecific antibody technology is a novel pharmaceutical approach to facilitate the engagement of effector immune cells to potentially multiple cancer epitopes, e.g., the recently approved blinatumomab. This opens the opportunity to develop ‘off-the-shelf’ anti-cancer agents that achieve similar and/or complementary anti-cancer effects as those of modified immune cell therapy. The majority of bi-/trispecific antibodies target the tumor-associated antigens (TAA) located on the extracellular surface of cancer cells. The extracellular antigens represent just a small percentage of known TAAs and are often associated with higher toxicities because some of them are expressed on normal cells (off-target toxicity). In contrast, the targeting of intracellular TAAs such as mutant RAS and TP53 may lead to fewer off-target toxicities while still achieving the desired antitumor efficacy (on-target toxicity). Here, we provide a comprehensive review on the emerging field of bi-/tri-specific T-cell engagers and potential therapeutic opportunities. Full article

Autologous hematopoietic stem cell transplantation (autoHSCT) is a standard of care for patients with hemato-oncologic diseases. This procedure is highly regulated, and a quality assurance system needs to be in place. Deviations from defined processes and outcomes are reported as adverse events (AEs: any untoward medical occurrence temporally associated with an intervention that may or may not have a causal relationship), including adverse reactions (ARs: a response to a medicinal product which is noxious and unintended). Only a few reports on AEs cover the procedure of autoHSCT from collection until infusion. Our aim was to investigate the occurrence and severity of AEs in a large data set of patients who were treated by autoHSCT. In this retrospective, observational, single-center study on 449 adult patients during the years 2016–2019, AEs occurred in 19.6% of the patients. However, only 6.0% of patients had ARs, which is a low rate compared to the percentages (13.5–56.9%) found in other studies; 25.8% of the AEs were serious and 57.5% were potentially serious. Larger leukapheresis volumes, lower numbers of collected CD34+ cells and larger transplant volumes significantly correlated with the occurrence and number of AEs. Importantly, we found more AEs in patients >60 years (see graphical abstract). By preventing potentially serious AEs of quality and procedural issues, AEs could be reduced by 36.7%. Our results provide a broad view on AEs and point out steps and parameters for the potential optimization of the autoHSCT procedure, especially in elderly patients. Full article

Bone marrow is an abundant source of both hematopoietic as well as non-hematopoietic stem cells. Embryonic, fetal and stem cells located in tissues (adipose tissue, skin, myocardium and dental pulp) express core transcription factors, including the SOX2, POU5F1 and NANOG gene responsible for regeneration, proliferation and differentiation into daughter cells. The aim of the study was to examine the expression of SOX2 and POU5F1 genes in CD34-positive peripheral blood stem cells (CD34+ PBSCs) and to analyze the influence of cell culture on the expression of SOX2 and POU5F1 genes. The study material consisted of bone marrow-derived stem cells isolated by using leukapheresis from 40 hematooncology patients. Cells obtained in this process were subject to cytometric analysis to determine the content of CD34+ cells. CD34-positive cell separation was conducted using MACS separation. Cell cultures were set, and RNA was isolated. Real-time PCR was conducted in order to evaluate the expression of SOX2 and POU5F1 genes and the obtained data were subject to statistical analysis. We identified the expression of SOX2 and POU5F1 genes in the examined cells and demonstrated a statistically significant (p < 0.05) change in their expression in cell cultures. Short-term cell cultures (<6 days) were associated with an increase in the expression of SOX2 and POU5F1 genes. Thus, short-term cultivation of transplanted stem cells could be used to induce pluripotency, leading to better therapeutic effects. Full article

The development of chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of hematological diseases. However, approximately 60% of patients relapse after CAR-T cell therapy, and no clear cause for this failure has been identified. The objective of the Bio-CAR-T BS study (ClinicalTrials.gov: NCT05366569) is to improve our understanding of the lymphocyte harvest to maximize the quality of the CAR-T cell product. Of the 14 patients enrolled, 11 were diagnosed with DLBCL, 2 with PMBCL, and 1 with ALL. Five of 11 DLBCL patients met the criteria for “pre-emptive” Lymphocytes-apheresis (being at high risk of second relapse), and 6 were included in the standard-of-care Lymphocytes-apheresis group. Previous autologous stem cell transplantation (ASCT) and age were significantly different between the two groups. At the time of Lymphocyte-apheresis, patients in the “pre-emptive” group had more “fit” lymphocytes (higher CD4+/CD8+ ratio; higher naïve T cells levels) compared with standard group, probably due to the impact of ASCT. At the same time, also being older than 60 years results in a more “exhausted” lymphocyte profile. Overall, “pre-emptive” Ly-apheresis in DLBCL patients at high risk of relapse appears to be feasible and may allow the timely collection of “fit” lymphocytes for CAR-T cell manufacturing. Full article

After a CellSearch-processed circulating tumor cell (CTC) sample is imaged, a segmentation algorithm selects nucleic acid positive (DAPI+), cytokeratin-phycoerythrin expressing (CK-PE+) events for further review by an operator. Failures in this segmentation can result in missed CTCs. The CellSearch segmentation algorithm was not designed to handle samples with high cell density, such as diagnostic leukapheresis (DLA) samples. Here, we evaluate deep-learning-based segmentation method StarDist as an alternative to the CellSearch segmentation. CellSearch image archives from 533 whole blood samples and 601 DLA samples were segmented using CellSearch and StarDist and inspected visually. In 442 blood samples from cancer patients, StarDist segmented 99.95% of CTC segmented by CellSearch, produced good outlines for 98.3% of these CTC, and segmented 10% more CTC than CellSearch. Visual inspection of the segmentations of DLA images showed that StarDist continues to perform well when the cell density is very high, whereas CellSearch failed and generated extremely large segmentations (up to 52% of the sample surface). Moreover, in a detailed examination of seven DLA samples, StarDist segmented 20% more CTC than CellSearch. Segmentation is a critical first step for CTC enumeration in dense samples and StarDist segmentation convincingly outperformed CellSearch segmentation. Full article

Hyperleukocytosis in pediatric acute leukemia is associated with increased morbidity and mortality and at present there is no consensus on the use of leukapheresis (LPH) for its management. Our aim was to review characteristics and outcomes of newly diagnosed leukemia patients with hyperleukocytosis (HL) comparing those who received LPH and those who did not. An IRB approved retrospective case control study reviewed data from a single institution over a 10 year period. At our institution, LPH was used in 8 of 62 (13%) patients with hyperleukocytosis with minimal complications. Mean leukocyte count in patients who received LPH versus those who did not was 498 k cells/mm³ and 237 k cells/mm³, respectively. Patients who had symptoms of neurologic (63 vs. 17%) or pulmonary leukostasis (75 vs. 17%) were more likely to have undergone leukapheresis. The time from presentation to the initiation of chemotherapy was not different between those who received LPH and those who did not (mean of 35 h vs. 34 h). There was one death in the LPH group, that was the result of neurologic sequelae of hyperleukocytosis and not LPH itself. The use of LPH in patients with hyperleukocytosis is safe, well tolerated and does not alter time to chemotherapy at our institution. Full article

Background: The treatment of relapsed or refractory leukemia remains a major problem. Among the new therapeutic approaches, the use of modified T lymphocytes, called chimeric antigen receptor T cells (CAR-T cells), seems promising. The first step of their preparation is leukapheresis, which involves the collection of mononuclear cells from the patient. This medical procedure requires numerous medical devices (MDs) made of plasticized polyvinylchloride (PVC). These compounds can leach out of the devices during contact with the patient’s blood. The aim of our study was to evaluate the migration of the plasticizers contained in the MD during a simulated pre-CAR-T cell leukapheresis procedure, and to measure the patient’s and their lymphocytes’ exposure to them. Methods: The qualitative and quantitative composition of the MD used for pre-CAR-T cell apheresis was determined by gas chromatography–mass spectrometry (GC–MS). Then, an ex vivo leukapheresis model using an ethanol/water simulant was performed to evaluate the plasticizers’ migration under simulated clinical conditions of pre-CAR-T cells’ cytapheresis. The plasticizers released into the simulant were quantified by GC–MS. Results: Diethylhexylphthalate (DEHP) was found in the apheresis kit, with amounts ranging from 25% to 59% (g/100 g of PVC). Bis(2-ethylhexyl) adipate was detected at trace levels. A total of 98.90 ± 11.42 mg of DEHP was released into the simulant, corresponding to an exposure dose of 1.4 mg/kg for a 70 kg patient. Conclusions: Patients undergoing a pre-CAR-T cell apheresis are mainly exposed to DEHP, which can impact their health because of its endocrine disruption effect, but could also lead to a decrease in CAR-T cells’ efficiency/quality. Full article

Cystinosis is an autosomal recessive metabolic disease that belongs to the family of lysosomal storage disorders. The gene involved is the CTNS gene that encodes cystinosin, a seven-transmembrane domain lysosomal protein, which is a proton-driven cystine transporter. Cystinosis is characterized by the lysosomal accumulation of cystine, a dimer of cysteine, in all the cells of the body leading to multi-organ failure, including the failure of the kidney, eye, thyroid, muscle, and pancreas, and eventually causing premature death in early adulthood. The current treatment is the drug cysteamine, which is onerous and expensive, and only delays the progression of the disease. Employing the mouse model of cystinosis, using Ctns^−/− mice, we first showed that the transplantation of syngeneic wild-type murine hematopoietic stem and progenitor cells (HSPCs) led to abundant tissue integration of bone marrow-derived cells, a significant decrease in tissue cystine accumulation, and long-term kidney, eye and thyroid preservation. To translate this result to a potential human therapeutic treatment, given the risks of mortality and morbidity associated with allogeneic HSPC transplantation, we developed an autologous transplantation approach of HSPCs modified ex vivo using a self-inactivated lentiviral vector to introduce a functional version of the CTNS cDNA, pCCL-CTNS, and showed its efficacy in Ctns^−/− mice. Based on these promising results, we held a pre-IND meeting with the Food and Drug Administration (FDA) to carry out the FDA agreed-upon pharmacological and toxicological studies for our therapeutic candidate, manufacturing development, production of the GMP lentiviral vector, design Phase 1/2 of the clinical trial, and filing of an IND application. Our IND was cleared by the FDA on 19 December 2018, to proceed to the clinical trial using CD34⁺ HSPCs from the G-CSF/plerixafor-mobilized peripheral blood stem cells of patients with cystinosis, modified by ex vivo transduction using the pCCL-CTNS vector (investigational product name: CTNS-RD-04). The clinical trial evaluated the safety and efficacy of CTNS-RD-04 and takes place at the University of California, San Diego (UCSD) and will include up to six patients affected with cystinosis. Following leukapheresis and cell manufacturing, the subjects undergo myeloablation before HSPC infusion. Patients also undergo comprehensive assessments before and after treatment to evaluate the impact of CTNS-RD-04 on the clinical outcomes and cystine and cystine crystal levels in the blood and tissues for 2 years. If successful, this treatment could be a one-time therapy that may eliminate or reduce renal deterioration as well as the long-term complications associated with cystinosis. In this review, we will describe the long path from bench-to-bedside for autologous HSPC gene therapy used to treat cystinosis. Full article