Poor graft function after haploidentical stem cell transplantation with post-transplant cyclophosphamide.

This is a retrospective cohort study of consecutive adult patients who received a haploidentical-SCT (haplo-SCT) with post-transplant cyclophosphamide (PT-Cy) in a single centre. Poor graft function (PGF) was defined as the occurrence of either persistent neutropenia (ANC < 0.5 × 109/µL) with poor response to granulocyte colony-stimulating factors (G-CSF) and/or thrombocytopenia (platelets < 20 × 109/L) with transfusion dependence, with complete donor chimerism and without concurrent severe GVHD or underlying disease relapse, during the first 12 months after transplantation. Forty-four (27.5%) out of 161 patients were diagnosed with PGF. Previous CMV reactivation was significantly more frequent in patients with PGF (88.6% versus 73.5%, p = 0.04) and the number of reactivations was also higher in these patients. Besides, early CMV reactivations in the first 6 months post-SCT were also significantly more frequent among patients with PGF (88.6% versus 71.8% p = 0.025). Thirty-two percent of patients with PGF were treated with increasing doses of thrombopoietin-receptor agonists (TRA) and 7 patients were treated with a donor CD34 + selected boost. In total, 93.2% of patients reached adequate peripheral blood counts in a median time of 101 days (range 11-475) after diagnosis. PGF is a frequent complication after haplo-SCT with PT-Cy. CMV reactivation might be the most relevant factor associated to its development. Even when most patients recover peripheral counts with support therapy, there is a group of patients with persistent cytopenias who can effectively be treated with TRA and/or a boost of CD34 + selective cells.

COVID-19 in transplant recipients: The Spanish experience.

We report the nationwide experience with solid organ transplant (SOT) and hematopoietic stem cell transplant (HSCT) recipients diagnosed with coronavirus disease 2019 (COVID-19) in Spain until 13 July 2020. We compiled information for 778 (423 kidney, 113 HSCT, 110 liver, 69 heart, 54 lung, 8 pancreas, 1 multivisceral) recipients. Median age at diagnosis was 61 years (interquartile range [IQR]: 52-70), and 66% were male. The incidence of COVID-19 in SOT recipients was two-fold higher compared to the Spanish general population. The median interval from transplantation was 59 months (IQR: 18-131). Infection was hospital-acquired in 13% of cases. No donor-derived COVID-19 was suspected. Most patients (89%) were admitted to the hospital. Therapies included hydroxychloroquine (84%), azithromycin (53%), protease inhibitors (37%), and interferon-ß (5%), whereas immunomodulation was based on corticosteroids (41%) and tocilizumab (21%). Adjustment of immunosuppression was performed in 85% of patients. At the time of analysis, complete follow-up was available from 652 patients. Acute respiratory distress syndrome occurred in 35% of patients. Ultimately, 174 (27%) patients died. In univariate analysis, risk factors for death were lung transplantation (odds ratio [OR]: 2.5; 95% CI: 1.4-4.6), age >60 years (OR: 3.7; 95% CI: 2.5-5.5), and hospital-acquired COVID-19 (OR: 3.0; 95% CI: 1.9-4.9).

Clinical applications of donor lymphocyte infusion from an HLA-haploidentical donor: consensus recommendations from the Acute Leukemia Working Party of the EBMT.

Donor lymphocyte infusion has been used in the management of relapsed hematologic malignancies after allogeneic hematopoietic cell transplantation. It can eradicate minimal residual disease or be used to rescue a hematologic relapse, being able to induce durable remissions in a subset of patients. With the increased use of haploidentical hematopoietic cell transplantation, there is renewed interest in the use of donor lymphocytes to either treat or prevent disease relapse post transplant. Published retrospective and small prospective studies have shown encouraging results with therapeutic donor lymphocyte infusion in different haploidentical transplantation platforms. In this consensus paper, finalized on behalf of the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation, we summarize the available evidence on the use of donor lymphocyte infusion from haploidentical donor, and provide recommendations on its therapeutic, pre-emptive and prophylactic use in clinical practice.

Bendamustine as part of conditioning of autologous stem cell transplantation in patients with aggressive lymphoma: a phase 2 study from the GELTAMO group.

We conducted a phase 2 trial to evaluate the safety and efficacy of bendamustine instead of BCNU (carmustine) in the BEAM (BCNU, etoposide, cytarabine and melphalan) regimen (BendaEAM) as conditioning for autologous stem-cell transplantation (ASCT) in patients with aggressive lymphomas. The primary endpoint was 3-year progression-free survival (PFS). Sixty patients (median age 55 [28-71] years) were included. All patients (except one who died early) engrafted after a median of 11 (9-72) and 14 (4-53) days to achieve neutrophil and platelet counts of >0.5 × 109 /l and >20 × 109 /l, respectively. Non-relapse mortality at 100 days and 1 year were 3.3% and 6.7%, respectively. With a median follow-up of 67 (40-77) months, the estimated 3-year PFS and overall survival (OS) were 58% and 75%, respectively. Patients in partial response at study entry had significantly worse PFS and OS than patients who underwent ASCT in complete metabolic remission, and this was the only prognostic factor associated with both PFS (Relative risk [RR], 0.27 [95% confidence interval {CI} [0.12-0.56]) and OS (RR, 0.40 [95% CI 0.17-0.97]) in the multivariate analysis. BendaEAM conditioning is therefore a feasible and effective regimen in patients with aggressive lymphomas. However, patients not in complete metabolic remission at the time of transplant had poorer survival and so should be considered for alternative treatment strategies.

Factors predicting peripheral blood progenitor cell mobilization in healthy donors in the era of related alternative donors: Experience from a single center.

BACKGROUND: Poor mobilization results are unexpected after G-CSF-induced peripheral blood stem cell collection in healthy donors. However, 2%-5% of the donors are poor mobilizers. Factors predicting CD34+-cell yield after mobilization in related alternative donors are still poorly known. PATIENTS AND METHODS: Baseline characteristics and efficacy results of G-CSF induced mobilization of 159 adult healthy donors in our institution from 2008 to 2016 were retrospectively analyzed. All donors received 10 µg/kg of G-CSF once a day subcutaneously for 4 days. Leukapheresis started on the 5th day of G-CSF treatment. Donors were classified as poor mobilizers if they had less than 20 000 CD34 + cell/mL peripheral blood count in the 5th day of G-CSF treatment or if they needed three or more leukapheresis for graft collection. RESULTS: Age, weight, and platelet count before and after mobilization were significantly different between poor and good mobilizers. Poor mobilizers (n = 16) were older (50.6 vs 41.7 years, P = 0.002), weight lower (64 vs 75 kg, P = 0.00) and showed a lower platelet count before (199.5 vs 219.0 × 109 /L, P = 0.03) and after (192.5 vs 206 × 109 /L, P = 0.019) mobilization. In the multivariate analysis only the 30% of the variability of mobilization was explained by the model (sensitivity 80%, specificity 70%). CONCLUSION: In this cohort of healthy donors in a single institution, older age, less weight, and lower platelet count was associated with poorer mobilization. With clinical and analytic factors it is not possible to predict more than 30% of the variability. Further studies are needed to investigate new variables.

Mononuclear cell collection for extracorporeal photopheresis by using the "off-line" system: A comparative study between COBE Spectra and Spectra Optia devices.

BACKGROUND: Extracorporeal photopheresis (ECP) is an efficient and established therapy to treat acute and chronic graft vs host disease (GVHD). Using an "off-line" method, the first step (mononuclear cell [MNC] collection) is decisive, as long as a high MNC yield and purity in the collected product is desirable. Two "off-line" devices were compared: the COBE Spectra and the Spectra Optia (Terumo BCT), using both continuous and intermittent protocols. PATIENTS AND METHODS: Twelve patients with GvHD (7 acute/5 chronic) were enrolled between June 2014 and May 2015 and were alternatively assigned for each procedure to either the COBE Spectra or the Spectra Optia cell separator. Patients characteristics and procedure/product parameters were analyzed. RESULTS: Two hundred procedures (100 per device) were included. The Spectra Optia system showed higher total nucleated cells and MNC collection efficiencies (18.6(10.2-29.7) vs 7.9(4.1-14.8)% and 43.6(20.3-59.5) vs 23.3(11.4-37.1)%, P < .001) and monocyte and lymphocyte collection efficiencies (55.2(17.7-83.2) vs 22.8(9-38.9)% and 38.3(26.7-53.4) vs 22.2(9-38.9)%, respectively, P < .001). Absolute platelet loss (PL) and PL per liter of blood processed were significantly lower in the Spectra Optia group (22.9(18.3-28.1) vs 33.6(26.5-41.1)%, P < .001 and 3.7(3.1-4.5) vs 4.3(3.5-4.2)%, P = .01, respectively). However, granulocyte contamination was higher (4.5(1.3-36) vs 1.2(0.4-5.7)%, P < .001) and a higher product haematocrit was obtained with the Spectra Optia (1(0.5-1.6) vs 0.3(0.2-0.5)%, P < .001), without an impact on irradiation time. CONCLUSIONS: In our study, Spectra Optia proved to be safe and effective in collecting MNC with high yield and purity for ECP in GvHD.

Inonotosis in Patient with Hematologic Malignancy.

We report a lung-invasive fungal disease with possible cutaneous needle tract seeding in a patient with a febrile neutropenia caused by the Basidiomycetes mold Inonotus spp. Although rare, Inonotus spp. should be added to the list of microorganisms causing invasive fungal disease in neutropenic patients with hematologic malignancies.

Donor Cell-Derived Hematologic Neoplasms after Hematopoietic Stem Cell Transplantation: A Systematic Review.

Development of de novo hematologic malignancies in donor cells after allogeneic stem cell transplantation (allo-SCT) provides a useful in vivo model to study the process of leukemogenesis. A systematic analysis of the cases reported in the literature was performed to identify risk factors and mechanisms involved in the pathogenesis of donor cell-derived hematologic neoplasms (DCHN) and leukemogenic transformation. Relevant data were extracted from 137 cases. Cases of DCHN show a wide heterogeneity with regard to recipient/donor age, sex mismatch, and conditioning regimen. Some characteristics, such as the type of primary disease, the type of hematologic malignancy of the DCHN, and the stem cell source used in the transplant procedure, differ from those expected. Mechanisms involved in the pathogenesis of DCHN are complex, and several hypotheses have been proposed, such as pre-existing hematologic neoplasms or premalignant clones in the donor, decreased immune surveillance, and damage to bone marrow microenvironment in the recipient. Most likely several if not all these mechanisms play a role in DCHN development. Novel approaches, such as next-generation sequencing to study consecutive samples after allo-SCT in these patients, appear to be promising to decipher the mechanisms of leukemogenesis.

Busulfan-based myeloablative conditioning regimens for haploidentical transplantation in high-risk acute leukemias and myelodysplastic syndromes.

BACKGROUND: High-risk acute leukemia (AL) and myelodysplastic syndrome (MDS) remain a therapeutic challenge. Unmanipulated haploidentical-related donor transplantation based on a myeloablative conditioning regimen (HAPLO-MAC) and post-transplant cyclophosphamide (PT-Cy) as prophylaxis against graft vs host disease (GvHD) is now a promising rescue strategy that could become universally available. OBJECTIVE: To evaluate the results of HAPLO-MAC with PT-Cy in patients with AL and MDS reported to the Haploidentical Transplantation Subcommittee of the Spanish Group for Hematopoietic Transplantation (GETH). PATIENTS AND METHODS: We report our multicenter experience using an IV busulfan-based HAPLO-MAC regimen and PT-Cy for treatment of 65 adults with high-risk AL and MDS. RESULTS: Engraftment was recorded in 64 patients (98.5%), with a median time to neutrophil and platelet recovery of 16 and 27 days, respectively. The cumulative incidence of grade II-IV acute GvHD and chronic GvHD was 28.6% and 27.5%, respectively. After a median follow-up of 31 months for survivors, the cumulative incidence of non-relapse mortality and relapse at 2 years was 18.8% and 25%, respectively. Estimated 30-month event-free survival and overall survival were 56% and 54.5%, respectively. CONCLUSION: HAPLO-MAC comprising an IV busulfan-based conditioning regimen enabled long-term disease control with acceptable toxicity in high-risk AL and MDS.

Phase II Study of Yttrium-90-Ibritumomab Tiuxetan as Part of Reduced-Intensity Conditioning (with Melphalan, Fludarabine ± Thiotepa) for Allogeneic Transplantation in Relapsed or Refractory Aggressive B Cell Lymphoma: A GELTAMO Trial.

We designed a phase II clinical trial including Y-90 ibritumomab-tiuxetan as part of a reduced-intensity conditioning (RIC) allogeneic stem cell transplantation (AlloSCT) in high-risk non-Hodgkin lymphoma (Clinical Trials Identifier: NCT00644371). Eligible patients had high-risk relapsed/refractory aggressive lymphoma. The conditioning regimen consisted of rituximab 250 mg (days -21 and -14), Y-90 ibritumomab IV (.4 m Ci/kg, day -14), fludarabine 30 mg/m2 i.v. (days -3 and -2) plus melphalan 70 mg/m2 i.v. (days -3 and -2) or 1 dose of melphalan and thiotepa 5 mg/kg (day -8). Donors were related. Eighteen patients were evaluable. At the time of transplantation, responses were complete remission (CR) (n = 7, 39%), partial remission (n = 6, 33%) or refractory disease (n = 4, 28%). Y-90-ibritumomab infusions were well tolerated, with no adverse reactions. Nonrelapse mortality at 1 year was 28%. Median follow-up was 46 (range, 39 to 55) months. Estimated 1-year progression-free survival (PFS) was 50%, and 4-year overall survival (OS) and PFS were both 44.4%. CR at the moment of AlloSCT had significant impact on PFS (71% versus 27%, P = .046) and OS (71% versus 27%, P = .047). Our results show that Y-90-ibritumomab-tiuxetan as a component of RIC for AlloSCT is feasible in patients with high-risk B cell lymphoma. Development of phase III clinical trials is needed to clarify the contribution of radioimmunotherapy to RIC AlloSCT.

Transient hemolysis due to anti-D and anti-A1 produced by engrafted donor's lymphocytes after allogeneic unmanipulated haploidentical hematopoietic stem cell transplantation.

BACKGROUND: Development of de novo alloantibodies against recipient's red blood cell (RBC) antigens by engrafted donor's lymphocytes is a known phenomenon in the setting of allogeneic hematopoietic stem cell transplantation (HSCT). This situation is usually clinically insignificant. We report a case of early clinically relevant hemolytic anemia in a blood group A1 D+ patient, due to a limited production of anti-D and anti-A1 produced by nonpreviously sensitized newly engrafted donor's immune system. CASE REPORT: A 31-year-old Caucasian woman, blood group A1 , D+, with Hodgkin's lymphoma, received an unmanipulated haploidentical allogeneic peripheral blood HSCT after a nonmyeloablative conditioning regimen. Donor blood group was A2 B, D-. The patient had an uneventful course until Day +34, when she developed clinically significant hemolytic anemia with a positive direct antiglobulin test. Anti-D and anti-A1 produced by the donor-engrafted lymphocytes were detected both in serum and in eluate. The hemolysis produced an accelerated group change, turning the patient's ABO group into A2 B 2 weeks after the detection of the alloantibodies. As the residual patient's RBCs progressively disappeared, anti-D and anti-A1 production decreased and were not detected in serum by Day +41. CONCLUSION: This case illustrates that de novo alloantibody production against ABO and D antigens by the newly engrafted donor's lymphocytes can occasionally cause clinically significant anemia. To our knowledge, this is the first case reported of clinically significant hemolytic anemia due to a transient anti-D anti-A1 alloimmunization after T-cell-repleted haploidentical HSCT.

Umbilical cord blood transplantation in adults with advanced hodgkin's disease: high incidence of post-transplant lymphoproliferative disease.

We report the outcome of 30 consecutive patients with Hodgkin disease (HD) who underwent single-unit UCBT. Most (90%) patients had failed previous autologous hematopoietic stem cell transplantation. The conditioning regimens were based on combinations of thiotepa, busulfan, cyclophosphamide or fludarabine, and antithymocyte globulin. The cumulative incidence (CI) of myeloid engraftment was 90% [95% confidence interval (C.I.), 74-98%] with a median of 18 d (range, 10-48). CI of acute graft-versus-host disease (GvHD) grades II-IV was 30% (95% C.I., 17-44%), while the incidence of chronic GVHD was 42% (95% C.I., 23-77%). The non-relapse mortality (NRM) at 100 d and 4 yr was 30% (95% C.I., 13-46%) and 47% (95% C.I., 29-65%), respectively. EBV-related post-transplant lymphoproliferative disease (EBV-PTLD) accounted for more than one-third of transplant-related death, with an estimate incidence of 26% (95% C.I., 9-44). The incidence of relapse at 4 yr was 25% (95% C.I., 9-42%). Four-year event-free survival (EFS) and overall survival (OS) were 28% and 30%, respectively. Despite a high NRM and an unexpected high incidence of EBV-PTLD, UCBT in heavily pretreated HD patients is an option for patients lacking a suitable adult donor, provided the disease is not in refractory relapse.

Inhibitory killer cell immunoglobulin-like receptor (iKIR) mismatches improve survival after T-cell-repleted haploidentical transplantation.

Alloreactivity triggered by interaction between killer cell immunoglobulin-like receptors (KIRs) and natural killer (NK) cells plays a role in the graft-versus-tumor effect after hematopoietic stem cell transplantation (SCT). Our aim in this study was to evaluate this role in the setting of T-cell-repleted haploidentical SCT with postinfusion high-dose cyclophosphamide (PT-Cy). We included 33 patients. Among patient-donor pairs with at least 1 inhibitory KIR (iKIR) gene mismatch, event-free survival (EFS) and cumulative incidence of relapse 1 year after transplant were significantly better (85% vs. 37% [P = 0.008] and 18% vs. 46% [P = 0.041], respectively). A subanalysis in 12 patients with Hodgkin's lymphoma (HL) showed an improvement in EFS 1 year after transplant in those patients with KIR ligand mismatch (100% vs. 25%, P = 0.012), although overall survival (OS) was not affected (85% vs. 80%, P = 0.2). Eight of 12 patient-donors pairs presented iKIR mismatches. Of note, this outcome was better in the small subgroup, both for EFS (100% vs. 25%, P = 0.012) and for OS (100% vs. 37%, P = 0.004). Our data suggest that in the setting of T-cell-repleted haploidentical SCT with PT-Cy, iKIR mismatch is associated with improved survival, with particularly good results for both iKIR and KIR ligand mismatches in patients with HL.

Haplo-cord transplantation using CD34+ cells from a third-party donor to speed engraftment in high-risk patients with hematologic disorders.

Among the strategies to optimize engraftment of cord blood (CB) stem cell transplantation (SCT), single CB with the coinfusion of CD34(+) stem cells from an HLA-mismatched auxiliary donor (haplo-cord) provides a valid alternative for adult patients without a suitable donor. A total of 132 high-risk adult patients with hematological malignancies from 3 Spanish institutions underwent myeloablative haplo-cord SCT. The median age was 37 years and median weight was 70 kg; 37% had active disease. The median number of postprocessing CB total nucleated and CD34(+) cells was 2.4 × 10(7)/kg (interquartile range [IQR], 1.8 to 2.9) and 1.4 × 10(5)/kg (IQR, .9 to 2), respectively. Neutrophil engraftment occurred in a median of 11.5 days (IQR, 10.5 to 16.5) and platelet engraftment at 36 days (IQR, 25.5 to 77). Graft failure was 2% overall and only 9% for CB. Cumulative incidence of acute graft-versus-host disease (GHVD) grades II to IV was 21% and cumulative incidence of chronic GVHD was 21%. Median follow-up was 60 months (range, 3.5 to 163). Overall survival was 43.5%, event-free survival was 38.3%, nonrelapse mortality was 35%, and relapse was 20% at 5 years. Myeloablative haplo-cord SCT results in fast engraftment of neutrophils and platelets, low incidences of acute and chronic GVHD, and favorable long-term outcomes using single CB units with relatively low cell content. Moreover, CB cell dose had no impact on CB engraftment and survival in this study. Therefore, haplo-cord SCT expands donor availability while reducing CB cell dose requirements.

Prognostic impact of minimal residual disease analysis by flow cytometry in patients with acute myeloid leukemia before and after allogeneic hemopoietic stem cell transplantation.

Allogeneic stem cell transplantation (allo-SCT) has become the treatment of choice in patients with intermediate-risk and high-risk acute myeloid leukemia (AML). The quality of response to treatment, assessed in terms of detection of minimal residual disease (MRD), has been consistently associated with prognosis and clinical outcome in patients with AML. The aim of the present study was to evaluate the prognostic impact of analyzing MRD in bone marrow using 4-color multiparametric flow cytometry (MFC) in 29 patients with AML before and after allo-SCT. Eighteen patients who were shown to be MRD-negative [≤0.1% leukemia-associated immunophenotypes (LAIPs)] by MFC at transplantation and underwent allo-SCT had lower rates of relapse (15% vs. 66%, P = 0.045), better overall 1-yr survival (83% vs. 52%, P = 0.021) and a lower cumulative incidence of relapse (P = 0.032) than patients who were MRD-positive (>0.1%). All post-transplant MRD-positive patients underwent a therapeutic intervention after transplant (tapering of immunosuppression, donor lymphocyte infusion, or re-transplant) with the intention of preventing relapse. Disease was controlled and MRD disappeared in five of these patients. Disease recurred in the other seven patients. We can conclude that follow-up with MFC for the detection of MRD in AML before and after SCT is useful for predicting relapse. In the post-transplant setting, monitoring of MRD by MFC could be a key preemptive intervention.

Digital PCR Improves Sensitivity and Quantification in Monitoring CAR-T Cells in B Cell Lymphoma Patients.

Chimeric antigen receptor T cells (CAR-T) has emerged as a promising therapy, over 60% of patients fail to sustain a long-term response. The underlying factors that leads to the effectiveness of this therapy are not completely understood, CAR-T cell persistence and monitoring seems to be pivotal for ensuring a successful response. Various monitoring methods such as multiparametric flow cytometry (MFC) or quantitative PCR (qPCR) have been applied. Our objective is to develop digital PCR (dPCR) assays for detection and quantification of CAR-T cells, comparing them with MFC and qPCR. Samples taken at different follow-up times from 45 patients treated with CAR-T therapy were analyzed to assess the correlation between the different methodologies. dPCR presented a high correlation with MFC and qPCR (r = 0.97 and r = 0.87, respectively), while offering a higher sensitivity (0.01%) compared to MFC (0.1%) and qPCR (1%). dPCR emerged as an alternative and highly sensitivity method for monitoring CAR-T cell dynamics. This technique is well-suited for implementation in clinical practice as a complementary technique to MFC.

Single cord blood combined with HLA-mismatched third party donor cells: comparable results to matched unrelated donor transplantation in high-risk patients with hematologic disorders.

Matched unrelated donor (MUD) transplantation is the first alternative in the absence of a matched sibling donor. For patients without a suitable adult donor, we have adopted the dual stem cell transplantation protocol consisting of cord blood (CB) in combination with CD34(+) cells from a third party HLA-mismatched donor. We analyzed the outcomes of patients undergoing both procedures in a single center. Starting in 2004, a total of 20 patients with high-risk disease underwent 22 dual transplants and 25 patients underwent myeloablative MUD transplantation. The 30-day cumulative incidence of neutrophil engraftment was similar in both groups (91% and 95%), with a median time to engraftment of 14 and 16 days, respectively. Grade II-IV acute graft-versus-host disease was more frequent in the MUD group (40% versus 5%). Except for a tendency toward a higher incidence of viral hemorrhagic cystitis in the dual transplantation group, posttransplantation infectious events were comparable in the 2 groups. The 3-year cumulative incidence rates of relapse (41% versus 44%) and nonrelapse mortality (30% versus 25%) were similar in the MUD and dual transplantation cohorts. Estimated 3-year overall survival and disease-free survival were 47% and 41%, respectively, with no survival advantage for either group. In our experience, dual transplantation offers survival rates comparable to those from myeloablative MUD transplantation with similar nonrelapse mortality rates.

Protection of haematopoietic progenitor cell donors: an updated overview of the European landscape.

Haematopoietic progenitor cell donation from bone marrow and mobilised peripheral blood obtained from related and unrelated donors is an established procedure. The donation process in general has proven to be safe, but in rare cases severe and even fatal events have been reported. The present study aimed at providing a description of the current situation of donor protection measures in Council of Europe member States. A specific questionnaire was developed to compile information on donation activities, graft sources, legal frameworks, donor protection measures, collection of donor outcome data, and long-term follow-up of paediatric and adult related and unrelated donors. The outcome of this survey served as a basis for elaborating the Recommendation CM/Rec(2020)6 of the Committee of Ministers to member States on establishing harmonised measures for the protection of haematopoietic progenitor cell donors.

Advantages of high cell concentration prior to cryopreservation of initial leukapheresis in CAR-T cell therapy.

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapy is increasingly used in patients affected by B-cell lymphoma and acute lymphoblastic leukemia. For logistical reasons, initial apheresis products may be cryopreserved for shipment to manufacturing centers. Due to the characteristics of these patients, cells are often collected in large volumes, meaning more bags must be cryopreserved. This requires increased storage, time and monetary costs. In this context, we aimed to evaluate a high cell concentration cryopreservation protocol by centrifugation to standardize the initial CAR-T manufacturing procedure. MATERIALS AND METHODS: Sixty-eight processes of leukapheresis of 57 patients affected by refractory/relapsed B cell lymphoma and 9 patients affected by acute lymphoblastic leukemia who were eligible for anti-CD19 CAR-T cell treatment performed between June 2019 and October 2022 were analyzed. Whole blood count, percentage and number of T cells were assessed on the apheresis final product. The apheresis product, which was alternatively stored overnight at 4°C, was centrifuged, adjusting the volume to approximately 40 mL. The product was immediately cryopreserved to achieve a final cell concentration of 50-200×106 cells/ml for cryopreservation. RESULTS: Leukapheresis volume was reduced by almost fivefold (median: 185 to 40 mL), resulting in a higher product concentration in one bag. In addition, the number of non-target cells (monocytes, platelets and erythrocytes) was also reduced during the development of CAR T cell therapy, thereby maintaining T lymphocyte levels and providing a purer starting material. DISCUSSION: The advantages of the protocol include reducing economic costs, saving storage space, simplifying the manufacturing process, and facilitating shipping logistics. In conclusion, we present a validated, simple, and cost-effective cell enrichment processing protocol that provides high-quality cryopreserved products as starting material for the CAR-T cell manufacturing process.

Evaluation of minimal residual disease by real-time quantitative PCR of Wilms' tumor 1 expression in patients with acute myelogenous leukemia after allogeneic stem cell transplantation: correlation with flow cytometry and chimerism.

Relapse remains the main cause of treatment failure in patients with acute myelogenous leukemia (AML) after allogeneic hemopoietic stem cell transplantation (SCT). The Wilms' tumor 1 gene (WT1) is reportedly overexpressed in >90% of patients with AML and thus can be useful for minimal residual disease (MRD) monitoring. The aim of this study was to evaluate the usefulness of WT1 expression as a relapse predictor marker in patients with AML after SCT and compare it with flow cytometry (FC) and chimerism studies. WT1 expression was assessed retrospectively using quantitative RT-PCR in bone marrow and peripheral blood from 21 patients. Patients were classified according to WT1 dynamics posttransplantation. Eleven of the 21 patients had low and stable WT1 levels. All of these 11 patients showed complete chimerism and negative MRD by FC and remained in complete remission with a median follow-up of 27 months (range, 18-98 months). In contrast, 10 of 21 patients showed WT1 overexpression after SCT, and 9 of these 10 patients relapsed. The incidence of relapse differed significantly between the 2 groups of patients according to WT1 expression post-SCT (P = .00003). Relapse in the 9 patients occurred at a median of 314 days (range, 50-560 days). Interestingly, in these patients, relapse was first predicted by WT1 (with negative FC and complete chimerism) in 7 patients. WT1 overexpression was correlated with disease burden in patients with AML before and after allogeneic SCT. In patients who relapsed, both medullary and extramedullary relapse were better anticipated by WT1 overexpression compared with FC and chimerism.