Haploidentical Hematopoietic Stem Cell Transplantation Research Articles

Humanized Anti-CD25 Monoclonal Antibody Replacing MTX for Graft-Versus-Host Disease Prophylaxis in Haploidentical Hematopoietic Stem Cell Transplantation

Introduction Currently, a short course of methotrexate (MTX) remains the classical regimen for the prevention of graft-versus-host disease (GVHD) in haploidentical hematopoietic stem cell transplantation (haplo-HSCT). Although MTX was widely applied for GVHD prophylaxis, it has been reported to be associated with significant toxicity and considerable side effects. We conducted a prospective, single-center, non-randomized controlled study to evaluate whether MTX can be safely removed from GVHD prophylaxis regimen after transplantation. Methods We prospectively reported and analyzed the data of 149 patients with hematologic malignancies who received haplo-HSCT from May 2022 to January 2023, and followed up until July 10, 2023. Patients were voluntarily enrolled in two groups according to different prevention programs of GVHD. A total of 82 patients were administrated with a combination of cyclosporine (CsA) or tacrolimus, mycophenolate mofetil (MMF), anti-human thymocyte immunoglobulin (ATG), MTX (15mg, +1d, 10mg, +3d, 10mg, +6d), and humanized anti-CD25 monoclonal antibody (Xenopax, 25mg, +0d, +4d) (single-dose anti-CD25 with MTX group). A total of 67 patients were given with CsA or tacrolimus, MMF, ATG and humanized anti-CD25 monoclonal antibody (Xenopax, 50mg, +0d, +4d) (double-dose anti-CD25 group). This study is registered with the Chinese Clinical Trial Registry (chictr.org.cn: ChiCTR2200060184). Results Compared with the single-dose anti-CD25 with MTX group, the median time to achieve neutrophil and platelet engraftment was shorten in double-dose anti-CD25 group (neutrophil: 9 days (7-20) vs 11 days (7-39), P=0.000; platelet: 9 days (7-30) vs 11 days (7-46), P=0.001). The incidence of total oral mucositis (OM) and grade 2-4 OM were significantly reduced in double-dose anti-CD25 group (total OM: 40.29% vs 63.41%, P=0.003; grade 2-4: 7.46% vs 32.05%, P=0.000). The median duration of OM was remarkably shorter in double-dose anti-CD25 group (5 days (2-18) vs 11 days (3-30), P=0.000). Lower cumulative incidence (CI) of grade II-IV aGVHD was observed in double-dose anti-CD25 group than in single-dose CD25 with MTX group (16.7 ± 4.6% vs 28.4 ± 5.0%, P=0.063). There was no statistical difference in two groups of the 100-day CI of grade III-IV aGVHD (6.1 ± 3.0% vs 13.6 ± 3.8%, P=0.123). Lower CI of cGVHD were occurred in double-dose anti-CD25 group (8.4 ± 4.1% vs 20.3 ± 5.1%, P=0.073). The CI of GVHD/relapse-free survival in double-dose anti-CD25 group was remarkably improved than that in single-dose CD25 with MTX group (73.9 ± 5.5% vs 55.6 ± 5.7%, P=0.044). No significant difference occurred in two groups of the overall CIs of CMV viremia, EBV viremia, overall survival, disease-free survival, relapse and treatment-related mortality. Conclusions Based on the efficacy and safety of the clinical trial results at our transplant center, replacing MTX with a single dose of humanized anti-CD25 monoclonal antibody is a promising GVHD prophylaxis for haplo-HSCT.

Memory T Cell Donor Lymphocyte Infusion As a Treatment for Viral Reactivations after Pediatric Haploidentical Hematopoietic Stem Cell Transplant

Introduction Viral reactivations, including cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus (ADV), human herpesvirus 6 (HHV6), and BK virus (BKV), constitute significant infectious complications following pediatric hematopoietic stem cell transplant (HSCT). abT cell depletion of haploidentical graft prevents risk of graft-versus-host disease (GVHD) but leads to delayed immune recovery and increased risk of viral reactivations. Donor lymphocyte infusion (DLI) with CD45RA-depleted memory T cells has been reported as an effective method to rapidly recover T cell immunity post HSCT. We hereby report the experience of CD45RA-depleted memory T cell infusion to treat viral reactivation following pediatric abT cell depleted haploidentical HSCT in our center over the last three years. Methods We retrospectively reviewed pediatric patients with abT cell depleted haploidentical transplant from related donors in our center from 2021-2023, and those who received memory T cell DLI for one or more viral reactivations. We routinely infuse CD45RA depleted memory T cells (dose at 1x10 6 CD3 T cells/kg) on the day of stem cell infusion prophylactically. Surplus of memory T cells harvested was cryopreserved for future use. Viral reactivations were monitored regularly after transplant by blood PCR assays (twice weekly for CMV; once weekly for EBV, ADV, and HHV-6). BK virus of urine and blood were monitored if patients became symptomatic for hemorrhagic cystitis. Once virus reactivation is identified, we start antiviral medications and infuse cryopreserved donor memory T cells for treatment. If subsequent virus PCR tests remain positive, donor memory T cells are infused every 1-2 weeks until the test is negative. Results A total of 52 memory T cell infusions were administered to 22 patients for viral reactivation after HSCT. Half of the patients (n=11) required only one infusion for viral clearance, whereas the other half received memory T cell infusion for 2-9 times. 19 patients (86.4%) are alive at time of data collection and analysis (July 2023), and 18 patients (81.8%) are in complete remission. The characteristics of patients and transplants are summarized in Table 1. The viral reactivations, which are intended to treat, include CMV (57.7%), EBV (7.7%), ADV (23.1%), BKV (15.4%) and HHV6 (9.6%), and some patients suffered from multiple viral reactivations at the time of memory T cell infusion (so the total percentage is more than 100%). The median day of memory T cell infusion post-transplant was day 46 (range 4-183). The median and most commonly used cell dose was 2 (0.025 - 20.5) x 10 6/kg CD3+ memory T cells (CD45RA depleted). The median duration to achieve negative PCR test for each virus are 13 days (CMV), 24 days (EBV),72 days (ADV), 15 days (HHV-6) and 88 days (BKV) (Figure 1). Thus most of the patients were CMV PCR negative within 15 days after DLI. All DLI were well tolerated. One patient (4.5%) was reported to suffer from stage 2 skin GVHD post donor memory T cell infusion. Conclusions CD45RA-depleted donor memory T cell infusion is a safe, feasible and effective treatment for viral reactivation following pediatric haploidentical stem cell transplant.

Clinical Implications of Germline Predisposition Gene Variants in Patients with Refractory or Relapsed B Acute Lymphoblastic Leukemia

Objectives:Gene variants are important factors in prognosis of the patients with hematological malignancies. In current study, our team investigate the relationship between blood and immune system germline predisposition variants and refractory/relapsed acute B lymphoblastic leukemia in patients. Methods:The study included 75 patients with refractory/recurrent acute B-lymphoblastic leukemia between January 2020 and December 2022. The first group of 41 patients, median age 13.5 years (range: 2-66 years), had not had a bone marrow transplant. The second group of 34 patients with a median age of 28.5 (range: 4-51 years old, once had a bone marrow transplant. More than 700 genetic susceptibility genes related to blood and immune system diseases were detected in all patient samples using high-throughput DNA sequencing technology to detect the type and number of Class I and Class II gene variants. The average sequencing depth was 150×. Class I refers to the gene variants that are clearly significant in guidelines or authoritative research literature. Class II are genetic variants that software analysis suggests may be harmful. Results:In the first group, 41 patients had an average number of Class I genetic susceptibility gene variants of 3.8 and Class II genetic susceptibility gene variants of 14.5 per patient. A total of 94 Class I genetic susceptibility gene variants were found, Thirty-two of them were recurrent more than 3 times that including HIF1A、SERPINE1、CFTR、CHEK2、ELANE、IFIH1、IRF7、JAK3、NCF2、PRF1、TGFBR2、TNFRSF13B、ATM、CYP3A4、ITGA2、NUDT15、THBD、BTLA、DNASE1、MPEG1、ADAMTS13、EP300、FANCA、KIT、BRCA2、F7、MLH1、STK11、TP53、YARS2、CYP2C19、TNFAIP3. The functions of these genes involve in combined immunodeficiency, autoinflammatory disease, complement deficiency, immune deficiency, T-cell dysfunction and antibody deficiency. After remission by CART cell therapy, twenty-three of the 41 patients underwent haploidentical hematopoietic stem cell transplantation, among which 5 patients relapsed after transplantation, three of the five donors of relapsed patients were also found HIF1A and SERPINE1 gene variants. In the second group of 34 patients, the average number of Class I genetic susceptibility gene variants in each patient was 3, and the average number of class 2 genetic susceptibility gene variants was 14. A total of 53 class 1 genetic susceptibility gene variants were found, Eleven of them were recurrent more than 3 times that Including BTLA、MPEG1 、KIT、SERPINE1、STK11、TP53、UGT1A1、EP300、FANCA、YARS2、F7,and four donors also carried the above gene variants, which were EP300. MPEG1, SERPINE1, YARS2 . After 34 patients achieved remission by CART cell therapy, 9 patients underwent the second hematopoietic stem cell transplantation (related half-matched), and 3 of them relapsed after the second transplantation (average 2.3 months). Two of the 3 donors of relapsed patients after second hematopoietic stem cell transplantation were also found BTLA and MPEG1 gene variants. Conclusions:Our results have shown that genetic susceptibility gene variants related to blood and immune system diseases are associated with refractory or relapsed acute B lymphoblastic leukemia. The number of genetic susceptibility gene variants may be related to the relapse of leukemia. 32 Class I genetic susceptibility gene variants may be related to the relapse of leukemia during chemotherapy, and 11 class I genetic susceptibility gene variants may be associated with relapse after bone marrow transplantation. Donor carrying genetic susceptibility gene variants (HIF1A and SERPINE1, EP300, MPEG1, YARS2, BTLA) may be associated with relapse after transplantation. Genetic susceptibility gene variants may also be helpful in the selection of bone marrow transplant donors. Keywords Acute lymphoblastic leukemia, germline predisposition gene, hematopoietic stem cell transplant, , relapse, hematological malignancy

Oligoclonal Bands in CSF May be Associated with Chronic Graft Versus Host Disease after Haploidentical Hematopoietic Stem Cell Transplantation

Introduction Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is a straightforward and effective therapy for hematology patients, and chronic graft-versus-host disease (cGVHD) is recognized as a major and important complication of haplo-HSCT. cGVHD can affect a number of organs, such as the skin, eyes, oral mucosa, liver, and lung.In addition to classical manifestations, cGVHD can imitate almost any autoimmune disease, and its central nervous system (CNS) involvement is exceedingly rare. In our studies, IgG synthesis and anti-myelin oligodendrocyte glycoprotein antibody were risk factors for CNS demyelination ( Annals of Hematology, 2018), aGVHD and hypertension were associated with posterior reversible encephalopathy syndrome ( Bone Marrow Transplantation, 2020), and aGVHD and cGVHD were identified as factors for idiopathic inflammatory demyelinating disease ( Am J Hematol, 2021). However, there are limited data available on cGVHD in the CNS. Therefore, we performed a thorough assessment of cGVHD, including examining the CSF of patients with cGVHD. Methods We performed a retrospective nested case‒control study in patients receiving haplo-HSCT between January 2013 and June 2022 in our center. In total, 86 patients suffering from cGVHD who also received CSF examinations were analyzed, of whom 42 patients showed evidence of CNS-cGVHD. Written informed consent was, and ethical approval was obtained before sample collection by the Ethics Committee of Peking University People's Hospital. The CSF samples were collected by lumbar puncture (LP). At least 1 ml of CSF was obtained from all patients by nontraumatic LP. All CSF samples were routinely screened for infection with bacterial, fungal, and virological pathogens, examined by histopathology and analyzed by fluorescence-activated cell sorting to identify tumor cells. Immune biomarkers were also evaluated. Results The median time from transplantation to cGVHD was 236 days. In the 86 cGVHD patients, aGVHD (HR 0.431, 95% CI 0.229-0.812, P=0.009), CSF protein (HR 0.634, 95% CI 0.484-0.784, P=0.)and oligoclonal bands (OCB) (HR 0.527, 95% CI 0.312-0.890, P=0.017) were identified as risk factors for the onset of cGVHD after haplo-HSCT. Moreover, we combined the variables to establish a nomogram for the cumulative incidence of cGVHD. The test showed that the fitness of the multivariate logistic regression model was suitable (P = 0.9516). The area under the ROC curve was 0.741. There were no differences in overall survival (OS), disease-free survival (DFS), or the cumulative incidence of relapse (CIR) between the cGVHD group and the control group without cGVHD. Nonetheless, there was a significant difference in nonrelapse mortality (NRM) (P=0.009). The 2-year cumulative NRM was 48.2% (95% CI 45.8%-50.6%) versus 12.6% (95% CI 12.2%-13.0%) between the cGVHD and without cGVHD groups, respectively. Then, we performed multivariate analysis for data from 42 patients who were diagnosed with CNS-cGVHD. The underlying diseases (HR 1.334, 95% CI 1.027-1.733, P=0.030) and CSF proteins (HR 0.313, 95% CI 0.126-0.776, P=0.012) were independently associated with the occurrence of CNS-cGVHD. Conclusions In conclusion, CSF immunity may be associated with the onset of cGVHD after haplo-HSCT, especially OCB, and the identification of cGVHD increases NRM after haplo-HSCT. Further multicenter prospective studies are needed to confirm the results and establish the standard diagnostic criteria and therapy for CNS-cGVHD.

Adoptive Transfer of CMV-Specific TCR-T Cells for the Treatment of CMV Infection after Haploidentical Hematopoietic Stem Cell Transplantation

Background: CMV reactivation after unmanipulated haploidentical stem cell transplantation (SCT) frequently occurs, causing life-threatening morbidities and transplantation failure. Preemptive therapy upon the detection of CMV viremia using antiviral agents is currently the standard of care but its efficacy is quite limited and associated with significant toxicity. The CMV antigen-specific cytotoxic T lymphocytes (CTLs) therapy was limited by the time-consuming manufacture process and relatively low success rate. More effective and safer approaches for the treatment of CMV reactivation after haploidentical SCT are in urgent need. Methods: A single-arm, open-label Phase I clinical trial evaluating the safety and efficacy of CMV-targeting TCR-T (CMV-TCR-T) cell therapy as the first-line preemptive therapy for patients with CMV reactivation post haploidentical PBSCT was conducted in the Chinese PLA General Hospital (ClinicalTrials.gov Identifier: NCT05140187). Six patients with CMV reactivation after haploidentical SCT were adoptively transferred by 1 to 3 doses of SCT donors-derived CMV-TCR-T cells. This trial was a dose-escalation study with doses ranging from 1×10 3 CMV-TCR-T cells/kg body weight per dose to 5×10 5 CMV-TCR-T cells/kg per dose Results: Except for the Grade 1 CRS observed in one patient and mild fever in 2 patients, no other adverse events were observed. Four patients had complete response within a month after CMV-TCR-T cells infusion without the administration of any antiviral agents. The other two patients who initially didn't completely respond to CMV-TCR-T cell therapy had salvage ganciclovir and foscarnet administration and then had rapid CMV clearance. The CMV-TCR-T cells displayed overall robust expansion and persistence in the peripheral blood after infusion. The CMV-TCR-T cells were firstly detected in the peripheral blood of these patients 3 to 7 days after the 1 st dose of CMV-TCR-T infusion, rapidly expanded and persisted for at least 1~4 months, providing long-term protections against CMV reactivation. In one patient, the CMV-TCR-T cells started to expand even when the anti-GvHD reagents were still being used , further indicating the proliferation potential of CMV-TCR-T cells. Conclusions: Our study firstly showed CMV-targeting TCR-T cell as a highly feasible, safe and effective first-line preemptive treatment for CMV reactivation after haploidentical PBSCT.

Haploidentical Hematopoietic Stem Cell Transplantation with Post-Transplant Cyclophosphamide in the Chilean National Health System. a Single Center Study

Haploidentical peripheral blood stem cell transplantation with post-transplant cyclophosphamide (Haplo-PTCy) is the most common transplantation modality in low-and-middle-income countries (LMIC). Registry data has shown comparable outcomes when using haploidentical donors versus Matched Unrelated Donors (MUD) in patients with acute leukemias. In the Chilean Public Health System Haplo-PTCy was incorporated in 2016 for adults up to 60 years old. We analyzed all adult patients who received an Haplo-PTCy at Hospital del Salvador, in a prospective registry study between 2016 and 2021. The primary outcome was overall survival (OS). Secondary outcomes were event-free survival (EFS), cumulative incidence of relapse, and incidence of grade II-IV acute graft-versus-host disease (aGVHD) at day +100. Overall survival and EFS were estimated using the Kaplan-Meier method. The cumulative incidence of relapse was calculated using relapse as the primary event and death without relapse as a competing event. Acute GVHD is shown as absolute incidence at day +100. All statistical analyses were performed with R. This study was done in compliance with the Helsinki Declaration and was approved by the Institutional Review Board. Eighty-five Haplo-PTCy were performed in 82 patients (Table 1). Three-second Haplo-PTCy were excluded from this analysis. The median age was 25 years (range, 15-51), and 65% were male. Ninety-four percent of patients had a neoplastic disease (77/82), and the most common diagnosis was acute lymphoblastic leukemia (57%). Forty-seven percent proceeded to transplant in the first complete response. Most of the patients (76%) presented a low hematopoietic cell transplantation (HCT)-specific comorbidity index. Conditioning was mostly myeloablative (96%). The median dose of CD34+ cells infused was 8.1 x 106/kg (range, 2.4-10.0). No patient needed a desensitization regimen. Cytokine release syndrome (CRS) was common (86%) but no grade 3 CRS was observed. Primary graft failure was observed in one patient (1.2%). Poor graft function was observed in 11 patients (13%). Five patients (6.1%) died before engraftment. The incidence of grade II-IV aGVHD at day +100 was 29% (grade IV was not observed). With a median follow-up of 33 months (range 0-84), the estimated 3-year OS and EFS were 68.3% (95% CI 59-79%) and 64.6% (95% CI 55-76%), respectively (Figure 1). In patients with neoplastic disease (n=77), the 3-year cumulative incidence of relapse was 23% (95% CI 15-33%). Haplo-PTCy is feasible in LMIC. This cohort of cases, mostly young patients with neoplastic disease, shows encouraging survival with an acceptable aGVHD and relapse incidence.

Allogeneic Hematopoietic Stem Cell Transplantation for Classical Paroxysmal Nocturnal Hemoglobinuria

Classical paroxysmal nocturnal hemoglobinuria (cPNH) is a rare acquired clonal hematopoietic cell disease. Eculizumab is effective in PNH patients, though, it must be administered indefinitely. As the technology of allogeneic hematopoietic stem cell transplantation (allo-HSCT) improves in recent years, what is the position of HSCT in treatment of cPNH? This study discusses the efficacy and safety of allo-HSCT in cPNH. The clinical data of 5 cPNH patients who underwent allo-HSCT from 2019 to 2022 were collected. All patients were diagnosed cPNH according to guidelines from International PNH Interest Group. Indications for HSCT were transfusion-dependent and nonavailability of eculizumab. The cohort was all male, with a median age of 26 years (range, 26 to 46 years) at the time of transplantation. All patients had documented hemolysis and no thrombosis during monitoring. The median time from diagnosis to allo-HSCT was 5.5 years (range, 3.6 to 18 years). The median PNH granulocyte clone size was 96.3% (range, 90% to 99.7%), and the median lactate dehydrogenase (LDH) level was 2224IU/L (8.896-fold of the upper limit of normal). All patients were detected bone marrow hyperplasia by trephine biopsy. The stem cell source came from 4 haploidentical donors and 1 HLA-identical sibling donor. Among the 5 patients, 3 patients underwent myeloablative conditioning (MAC) and 2 patients underwent reduced-intensity conditioning (RIC) treatment. None of the patients experienced primary implantation failure. Neutrophil implantation time was 15 (13-21) days, and platelet implantation time was 24 (13-60) days. 3 patients developed grade II acute graft-versus-host disease (aGVHD). No patients developed grade Ⅲ-Ⅳ aGVHD. 2 patients developed localized chronic graft-versus-host disease (cGVHD) involving lung and skin respectively, and no patients developed extensive cGVHD. PNH clones turned negative in all patients after 2 months (range, 1 to 3 months) of transplantation. At a median follow-up of 16 (6-34) months, 1 patient died of relapse and infection. The remaining 4 patients survived, of which 2 patients had discontinued all drugs. cPNH has its own unique clinical features. Allo-HSCT could cure cPNH patients who can withdrawal from treatment of all medication. Haploidentical donor HSCT is an acceptable choice for cPNH, and RIC constitutes a valid option.

Treosulfan Based Haploidentical Αß T Cell Depleted HSCT Represents a Curative Alternative in Pediatric and Adult Patients with Transfusion Dependent Thalassaemia

Background: Quality of life remains severely compromised in patients suffering from Transfusion Dependent Thalassemia (TDT) despite optimal supportive care. HSCT with a MSD, the current curative option, achieving a 92.1% 2y-OS in children and 84.4% in adults (EBMT registry). Unfortunately, availability of MDs is limited, so that haploidentical HSCT is increasingly explored. Methods: 13 TDT patients (median age: 11 years; range: 2-23) received either a CD3 +/CD19 + (n=4; all class II/III) or αß/CD19 + (n=10; 7 class II, II/III) T-haplo-HSCT and were compared with 8 TDT-patients (7 class II, II/III, III) receiving a BM graft from a MSD (median age: 11 years; range: 4-22). Indication for T-haplo HSCT patients was severe iron overload/ transfusion-associated complications. All patients received an identical conditioning regimen consisting of treosulfan, thiotepa, fludarabine (FTT) and ATG-Grafalon, with the only difference in the timing of ATG (upfront in T-haplo-HSCT, prior to d0 in MSD-HSCT). Immunosuppression (IST) was maintained for a minimum of 180 days consisting of calcineurin inhibitors (mainly tacrolimus) and MMF. Results: The OS and DFS for MSD and T-haplo-HSCT was 100%/100% and 100%/92%, respectively (Table 1). The median follow-up was 30 months for MSD (range 4-72) and 38 months for T-haplo-HSCT (range 4-86). Neutrophil engraftment was achieved after a median of 31 days for MSD patients and 17 days for T-haplo-HSCT with a median of 5 x 10 8 TNC/kg (range: 3.28-7.88) and 15 x 10 6 CD3 +/CD19 + or αβ/CD19 + depleted CD34 + cells/kg (range: 9.2-24.2), respectively. One patient received two T-haplo HSCT, due to primary graft failure partly due to a major AB0 incompatibility. A mixed chimerism was observed in 3/7 MSD-HSCT (median 92%; range 27.5-100%) and in T-haplo-HSCT in 2/13 patients (median 99%; range: 45.3 -100%). Transfusion independence was achieved in all MSD and in all but one T-haplo patient. In MSD and in T-haplo-HSCT, IST was terminated after a median of 173 days and 226 days (range: 112-347), respectively. MSD patients reached CD4 counts >50/µl on day +43 (median; range: 25-125) and T-haplo-HSCT patients on day +114 (median; range: 33-173). The treosulfan-based conditioning regimen was well tolerated with no VOD/SOS observed in this high-risk population. No case of acute or chronic Graft-versus-Host disease (GvHD) was observed in the MSD population. In T-haplo-HSCT, 3 patients experienced grade I acute GvHD (skin) which resolved with prednisolone and in 2 cases with additional extracorporeal photopheresis. Only the oldest patient experienced mild chronic skin GvHD, which resolved by day +580. No severe infectious complications occurred despite a prolonged chimerism-triggered weaning of the IST. Conclusions: Independent of donor availability, graft rejection and iron-overload related organ damage (VOD/SOS) are major pitfalls in transplanting advanced stage TDT patients. Treosulfan demonstrated to be an excellent alternative to busulfan in this setting reflected in the safety and efficacy data of this pilot series. Overall, outcomes of T-haplo-HSCT in TDT are very encouraging with a competitive OS, so that a transplant indication in pediatric and AYA TDT patients lacking a MD is feasible.

Different Sources of Hematopoietic Stem Cell Transplantation in Patients with Thalassemia Major: Relationship between Immune Reconstitution, Graft-Versus-Host Disease, and Viral Infections

Objective: To compare the relationship among immune reconstitution, graft-versus-host disease (GVHD), and viral infections in three kinds of hematopoietic stem cell transplantation(HSCT) using different host sources for thalassemia major(TM) patients: matched unrelated donor (MUD) HSCT, haploidentical (Haplo) HSCT with post-transplant cyclophosphamide (PTCy)-based prophylactic therapy, and TCRαβ-T cell-depleted HSCT (TDH). Methods: A total of 191 TM patients in our center between October 2018 and June 2022 were included in this study, comprising three transplant groups: MUD group (n=56), PTCy group (n=45), and TDH group (n=90). Four-color flow cytometry was used to detect the proportions and absolute counts of lymphocyte subsets at the 12th month after transplantation for the three different donor types. The immune reconstitution was analyzed and compared with the occurrence of GVHD and virus infections. Results: 1. Immune Reconstitution: (1) The ratio of helper cells (CD3 +CD4 +)/cytotoxic T cells (CD3 +CD8 +) at the 12 months after transplantation was compared among the three groups. There were statistically significant differences between the TDH and PTCy groups, as well as between the TDH and MUD groups(P=022 and 0.011, respectively). No statistical difference was found between the PTCy and MUD groups (P=0.932). The CD4 +/CD8 + ratio in the TDH group recovered the fastest and had already returned to the normal reference range (0.7-2.7). (2) The comparison of B cells (CD3 -CD19 +) at the 12 months after transplantation showed statistically significant differences between the TDH and PTCy groups, as well as between the MUD and PTCy groups (P=0.049 and 0.034, respectively). No statistical difference was found between the TDH and MUD groups (P=0.447). The recovery of B cells was faster in the TDH and MUD groups, while it was slowest in the PTCy group. (3) The comparison of natural killer cells (NK cells) (CD3 -CD56 +) at the 12 months after transplantation showed statistically significant differences between the TDH and PTCy groups, as well as between the TDH and MUD groups (P=0.048 and 0.000, respectively). No statistical difference was found between the PTCy and MUD groups (P=0.366). The TDH group exhibited the fastest recovery of NK cells. 2. Chronic GVHD (cGVHD): (1) 7/90 cases (7.78%) of cGVHD appeared in the TDH group, 19/45 cases (42.22%) in the PTCy group, and 2/56 cases (3.57%) n the MUD group. (2) No statistical difference showed in the incidence of cGVHD between the TDH and MUD groups (P=0.500). There was a statistical difference in the incidence of cGVHD between the TDH and PTCy groups (P=0.000), as well as between the MUD and PTCy groups (P=0.000). 3. Viral Infection: (1) 22/90 cases (24.44%) of viral infection occurred in the TDH group, 7/45 cases (15.56%) in the PTCy group, and 6/56 cases (10.71%) in the MUD group. (2) Among the 90 cases of TDH group, the CD4/CD8 ratio at the 6 months after transplantation was 0.41±0.24 in the 22 cases with viral infection and 0.69±0.40 in the 68 cases without that. There was a statistical difference in the mean CD4/CD8 ratio between the two divisions in the TDH group (P=0.013). Conclusion:The TDH group exhibited faster immune reconstitution compared to the other two groups, especially in terms of CD4/CD8 ratio, B cells, and NK cells. This may be related to two special factors for patients underwent TDH in our center: a rather high average infusion of CD34 cells (30×10 6/kg) , and the preservation of γδ cells, B cells, and NK cells with the removing of TCRαβ-T cells in the TDH treatment protocol. The TDH and MUD groups showed a lower incidence of cGVHD, while the PTCy group had a higher incidence. The occurrence of viral infection in the TDH group was 24.44%. Also, the lower a CD4/CD8 ratio at the 6 months after transplantation, the higher the incidence of viral infection. Table 1. The Absolute Counts of Lymphocyte Subsets at the 12th Month after Transplantation

Early Hematopoietic Reconstitution Status after Haploidentical Transplantation in AML Patients Is Indicative of Early Complications

Objective To investigate the early hematopoietic reconstruction process of haploidentical allogeneic hematopoietic stem cell transplantation (haplo-allo-HSCT) in patients with acute myeloid leukemia, and to explore its correlation with the occurrence of early complications after transplantation. Methods A retrospective analysis was performed on the clinical information of 187 AML patients who underwent haploidentical allo-HSCT at the Hematology Hospital of the Chinese Academy of Medical Sciences from January 2017 to December 2022, and all of these patients achieved complete remission before transplantation. The results of pre-transplantation-7d-after-tranplantation+14d blood routine were analyzed. The occurrence of aGVHD within 100 days after transplantation was the primary outcome event, and CMV infection within 60 days was the secondary outcome event. The diagnosis and grading of aGVHD is described in Glucksberg and MAGIC. R-4.2.0 statistical software was used for statistical analysis. Results A total of 187 AML patients who underwent haploid transplantation after achieving complete remission were included, with a median age of 35 (27-55) years, including 105 male patients (56.31%) and 82 female patients (43.69%). All patients were grouped into aGVHD patients according to different risk levels: (1) There were differences between the I~IV°aGVHD group and the patients without aGHVD (100 versus 87 patients) in the early post-transplantation plt and WBC absolute levels, and the I~IV°aGVHD group showed a lower plt trend in 1d and 2d, and a higher WBC level (0.08×10 9/L vs 0.07×10 9/L, p=0.036) at 10 days. (2) Compared with patients without aGHVD (70 versus 87 cases), the former showed lower plt levels (62.5×10 9/L vs 80×10 9/L, p=0.029) in 1d and higher WBC levels (0.03×10 9/L vs 0.02×10 9/L, p=0.047; 0.11×10 9/L vs 0.07×10 9/L, p=0.033) in 9d~10d), the Hb level was lower at 1d~3d (91g/L vs 99g/L, p=0.047; 91g/L vs 97.5g×109/L, p=0.039; 89.5g/L vs 95.5g/L, p=0.013). (3) Compared with patients without aGHVD (33 versus 87 patients), the III~IV°aGVHD group showed lower plt levels (60×10 9/L vs 74×10 9/L, p=0.006; 48×10 9/L vs 52×10 9/L, p=0.018; 26×10 9/L vs 36.5×10 9/L, p=0.003) in 1~3d, and high WBC level (0.04×10 9/L vs 0.02×10 9/L, p=0.025; 0.14×10 9/L vs 0.07×10 9/L, p=0.008) showed in 10 days, and lower Hb level in 1d~3d (89g/L vs 99g/L, p=0.047; 89g/L vs 97.5g/L, p=0.039; 83g/L vs 95.5g/L, p=0.013). Based on the maintenance of platelets at 20×109/L for 3 days after transplantation, the cumulative incidence of 100 days III~IV°aGVHD in the ≤3d group was higher (63% vs 30%, p=0.015). Univariate analysis showed that the absolute value of 1~3d plt after transplantation was low, and the PLT maintained at 20×10 9/L after HSCT was short, which was a risk factor for the occurrence of aGVHD. Group analysis of CMV occurring within 60 days showed that in the early post-transplantation period, patients in the non-CMV group had higher absolute platelet values (63.50×10 9/L vs 47.00×10 9/L, p=0.03) than patients in the CMV group at 0 days. Age, pretreatment regimen, transplant type, aGVHD prevention regimen, time to first PLT infusion after transplantation, absolute value of 0 d platelets, and platelet transfusion efficiency were included in univariate and multivariate analysis, and multivariate analysis showed that pretreatment regimen (p=0.017, 1.002 [0.606-0.953]) and transplant type (p<0.001, 0.264 [0.147-0.476]) were independent risk factors affecting the occurrence of CMV. No correlation between platelet transfusion efficiency and PLT infusion frequency and CMV was found. Conclusion Early hematopoietic reconstruction after transplantation in AML-CR patients who received haploid transplantation has certain indicative significance for the occurrence of early complications, and its indicative significance becomes more significant with the increase of aGHVD degree.

Score to Guide Donor Choice in Haploidentical Stem Cell Transplant Using Post-Transplant Cyclophosphamide for Patients with Acute Myeloid Leukemia: A Study from the Acute Leukemia Working Party of the EBMT

Introduction: There is paucity of information to guide selection of the most appropriate stem cell donor for haploidentical (Haplo) hematopoietic stem cell transplant (HSCT). Patients and Methods: We retrospectively analyzed the characteristics of haploidentical family donors that may affect transplant outcomes in patients with acute myeloid leukemia (AML) who received graft-versus-host disease (GVHD) prophylaxis with postransplant cyclophosphamide (PTCy) registered in the EBMT database. The primary endpoint was GVHD and relapse-free survival (GRFS). Results: Overall, 2200 patients were included with a median age of 56 years (range, 18-75); 1742 (79%) were in complete remission and 1246 (56.6) received reduced intensity conditioning. Regarding donors, the median age was 37 years (range, 8-71), 820 (37%) were females of which 458 (21%) were used for male recipients, 1252 (57%) had positive CMV serostatus, 1631 (74%) donated peripheral blood (PB) and 1638 (75%) had ≥ 4/8 HLA mismatch with the recipient. 100-day acute GVHD grade II-IV and III-IV and 2-year chronic and chronic extensive GVHD were 28% (95% CI 26-30), 11% (95% CI 10-12), 33% (95% CI 31-35) and 14% (95% CI 12-15), respectively. After median follow-up of 24 months, the cumulative incidence of relapse and non-relapse mortality (NRM) and the probability of leukemia-free survival (LFS), overall survival (OS) and GRFS were 26% (95% CI 24-28), 22% (95% CI 20-24), 52% (95% CI 50-55), 57% (95% CI 55-60) and 41% (95% CI 39-43), respectively. In multivariable analysis, donor-related risk factors with a negative impact on GRFS were older age (HR 1.1; 95% CI 1.04-1.15), use of PB (HR 1.19; 95% CI 1.04-1.37), and female donors to male recipients (HR 1.25; 95% CI 1.09-1.43). We further developed a score that distinguished 4 groups with 0 (241), 1 (913), 2 (863) or 3 (183) donor-related risk factors. GRFS was 57% (95% CI 50-63), 43% (95% CI 39-46), 37% (95% CI 33-40), and 30% (95% CI 23-38), respectively (P < 0.001) (Figure 1). The score was also able to stratify risk groups for acute and chronic GVHD, NRM, LFS and OS. Conclusion: Donor variables have an important impact on AML patient's outcome after Haplo-HSCT using PTCy. With three simple donor characteristics, age, gender and stem cell source, we were able to generate a score that may help to select the most appropriate donor in clinical practice.

A Prospective, Multi-Center, Randomized, Controlled Clinical Trial to Explore the Role of Mesenchymal Stem Cells in Preventing Graft Versus Host Disease

Background: There are few studies currently available on the prevention of GVHD by mesenchymal stem cells (MSCs) that use a systematic approach to prevent both aGVHD and cGVHD simultaneously. The clinical investigation of MSCs preventing either aGVHD or cGVHD independently is the main focus of the earlier studies. Aim:Using the strategy of co-transplantation of MSCs and HSCs and phased application of MSCs after transplantation (MSCs 100-day scheme), we seek to develop a secure and dependable system that can avoid both aGVHD and cGVHD based on our preliminary research. Method:Patients undergoing haploidentical adult hematopoietic stem cell transplantation will receive unaltered, fourth-generation UC-MSCs(Umbilical Cord). In addition to the usual GVHD preventive measures, UC-MSCs are given four hours before the transplantation of peripheral blood stem cells on day 0. UC-MSCs are administered once weekly for 1-4 weeks, once every 2-8 weeks, and once every 4-12 weeks after transplantation. The dose needed for each transfusion is 1 10 6/kg. Our research will look at how well UC-MSCs prevent GVHD following haplo-HSCT, as well as recurrence rates after transplantation, transplant-related issues, and the state of immunological reconstruction. the frequency, severity, 1- and 2-year survival rates, cumulative death rates, and the occurrence of aGVHD and cGVHD.Real-time tracking will be performed to monitor the occurrence of aGVHD and cGVHD, as well as their severity, frequency, 1-year and 2-year survival rates, and cumulative mortality rates. The analysis of the differences between the control group and the experiment will take into account the pre-transplant ferritin levels, CMV serological status of the donor-recipient, blood type matching situation, HLA locus matching situation, disease remission status, residual disease conditions, recipient age, donor-recipient gender matching situation, stem cell quantities (nucleated cells and CD34+ cell counts), and stem cell numbers (nucleated cells and CD34+ cell counts). Results: 96 patients from the MSCs group and 96 patients from the control group are now accessible for statistical purposes. Demographic factors for age and gender distribution do not differ considerably. Acute myeloid leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome-EB2, and acute mixed cell leukemia,which were all classified as having the same level of risk. The cumulative incidence of aGVHD grades II to IV is 19.8% in the MSCs group, while grades III to IV is 0%. Comparable indications for the control group are 43.8% and 9.3%, respectively. The overall incidence of aGVHD differs between the two groups in a statistically significant way (P=0.0047). The rates of moderate and severe cGVHD in the control group were 10.4% and 9.4%, respectively, whereas they were 13.5% and 4.2% in the MSCs group. Over the course of 1 year, the cumulative incidence of cGVHD for the MSCs group was 36.5%, compared to 39.6% for the control group. Regarding the overall two-year OS, there is no discernible difference between the MSCs group and the control group (P=0.43).While the MSCs group outperformed the control group in terms of GRFS (GVHD free and relapse free survival). Conclusion: The strategy of MSCs 100-day scheme infusion can successfully lower the incidence of acute and chronic GVHD and is beneficial for enhancing patient GRFS.

Post-Transplant Cyclophosphamide-Based Graft-Versus-Host Disease Prophylaxis in HLA-Matched and Haploidentical Donor Transplants for Patients with Hodgkin disease: A Comparative Study of the LWP EBMT

INTRODUCTION Post-transplant cyclophosphamide (PTCy) has proven to be a highly effective strategy in preventing graft-versus-host disease (GVHD) in haploidentical (haplo) hematopoietic stem cell transplantation (HSCT), but it is being increasingly used in HLA-matched transplants. The outcomes of haplo HSCT with PTCY in various hematologic malignancies, particularly in relapsed/refractory Hodgkin disease (HD), have shown promising results, challenging those obtained in transplant patients from HLA-matched donors without PTCy. However, there is limited information on the impact of donor types on the outcomes of patients with HD undergoing allogenic HSCT when using homogeneous GVHD prophylaxis with PTCy. To address this knowledge gap, we have conducted an extensive study using the Lymphoma Working Party of the European Society for Blood and Marrow Transplantation (LWP-EBMT) registry. The aim of this study was to compare the outcomes of patients with HD undergoing HSCT from both HLA-matched donors, which include matched sibling donors (MSD) and matched unrelated donors (MUD), and haplo donors, using PTCy as GVHD prophylaxis approach in all cohorts. PATIENTS AND METHODS Inclusion criteria All adults (≥ 18 years) patients diagnosed with HD who underwent their first HSCT between January 2010 and December 2020 from either a MSD, MUD or haplo, and whose data were reported to the EBMT registry were included. For the purposes of this study, only transplants who received PTCy-based as GVHD prophylaxis were considered. Statistical Analysis The study utilized statistical methods such as Kaplan-Meier estimation, Log-Rank test, competing risks analysis, and Cox proportional hazards regression. The analyses included OS, PFS, NRM, aGVHD, cGVHD, and graft failure. The statistical software R and packages were used. RESULTS Patient and Transplantation Characteristics Patient, disease and transplant-related characteristics of the 860 patients are summarized in Table 1. Engraftment The cumulative incidence (Cum Inc) of neutrophil recovery at day 30 was 95% (95% CI, 93-96) in the HLA-matched group and 96% (95% CI, 91-98) in the haplo cohort (p=0.18). The 60-day Cum Inc of platelet recovery in similar order was 94% (95% CI, 88-97) and 86% (95% CI, 83-88) (p<0.001). GVHD The Cum Inc of acute GVHD grade II-IV at 100 days in the HLA-matched and the haplo cohorts was 24% (95% CI, 17-31) and 34% (95% CI, 30-37), respectively (p=0.01), whereas for grade III-IV it was 8% (95% CI, 4-13) and 10% (95% CI, 8-13), (p=0.44). In multivariable analysis (table 2), haplo was associated with an increased risk of acute GVHD grades II-IV, when compared with HLA-matched (HR 0.65; 95% CI, 0.46-0.93; p=0.01). Other variables are shown in table 2. The 2-year Cum Inc of chronic GVHD in the HLA-matched and haplo cohorts was 26% (95% CI, 19-33) and 27% (95% CI, 24-31), respectively (p=0.75). Other variables are shown in table 2. NRM and relapse The Cum Inc of NRM at 2 years was 10% (95% CI; 6-16) for HLA-matched and 18% (95% CI; 15-21) for haplo (p=0.02). In multivariable analysis (table 2), haplo was associated with an increased risk of NRM, when compared to HLA-matched (HR 0.5; 95% CI, 0.28-0.89; p=0.01). Other variables are shown in table 2. The Cum Inc of relapse at 2 years was 22% (95% CI; 15-29) for HLA-matched and 24% (95% CI; 20-27) for haplo (p=0.81). Other variables are shown in table 2. Survival The 2-year OS for the HLA-matched and haplo cohorts was 82% (95% CI; 75-88) and 70% (95% CI; 67-74), respectively (p=0.002). In multivariable analysis (table 2), compared to HLA-matched, haplo (HR, 0.51; 95% CI, 0.34-0.77; p=0.001) was associated with worse survival. Other variables are shown in table 2. The 2-year PFS was similar between HLA-matched and haplo (66%; 95% CI, 58-74; and 58%; 95% CI, 54-62, respectively; p=0.17). Other variables are shown in table 2. Comparison of outcomes for MSD and MUD With regards to the donor type, most posttransplant outcomes did not exhibit significant differences, except for a higher incidence of grades II-IV acute GVHD in the MUD group (33%; 95% CI, 32-44) compared to the MSD group (17%; 95% CI, 10-25) (p=0.01). CONCLUSION Our study shows that in patients with HD undergoing HSCT with PTCy for GVHD prophylaxis, no significant differences in 2-year PFS were observed between HLA-matched and haplo. A higher risk of acute GVHD and NRM is associated with haplo, leading to lower OS compared to HLA-matched transplantation.

TBI/Ratg-Based Conditioning for Haploidentical Allogeneic Hematopoietic Stem Cell Transplantation in 11 Cases of Chemotherapy-Resistant Advanced-Stage Peripheral T-Cell Lymphoma

Objectives: To evaluate the clinical outcomes and safety of haploidentical allogeneic hematopoietic stem cell transplantation(haplo-HSCT)with total body irradiation (TBI)/rabbit Anti-thymocyte globulin (rATG) based conditioning regimen in chemotherapy-resistant advanced-stage peripheral T-cell lymphoma (PTCL). Methods: From September 2019 to December 2022, eleven chemotherapy-resistant advanced-stage PTCL patients who underwent haplo-HSCT were enrolled in our study. All of them received TBI/rATG-based conditioning regimen in our centers. Results: ①Among 11 patients, there were 6 males and 5 females with a median age of 40(range：22~58) years old. Six cases of them are peripheral T-cell lymphoma not otherwise specified (PTCL NOS), 3 cases are angioimmunoblastic T-cell lymphoma (AITL), 1 case is large-cell transformation of mycosis fungoides (MF-LCT), and 1 case is T-cell large granular lymphocytic leukemia(T-LGLL). The Lugano stage was III or IV in all cases, and 8 patients had B symptoms. The median number of previous chemotherapy exposure before transplantation was 4 (range: 2~10) lines and the disease status before transplantation was progressive diseases (PD) in all cases. The median time from diagnosis to transplantation was 17(range：6~36) months.②The conditioning regimen consisted of TBI 2Gy d-8, 4Gy from day-7 to -6, rATG 2.5 mg/kg/d day-5 to -2, etoposide 15mg/kg/d day-5 to -4, cyclophosphamide (CTX) 50mg/kg/d, day-3 to -2. The patients with central nervous system involvement received a regimen of thiotepa 5 mg/kg/d， from day -5 to -4 instead of CTX and etoposide, and add 2.0 g/m2 of cytarabine twice a day, from day -3 to day -2 into the conditioning.③All patients were successfully engrafted. Only one patient developed Grade III-IV acute graft-vs-host disease (aGvHD). Among the 8 survivors, 4 cases developed chronic graft-vs-host disease (cGvHD).④After transplantation, CR was obtained in 9 patients. ⑤Regimen related toxicities: all were non-fatal. Hematopoietic suppression occurred in all patients after conditioning. Three patients had diarrhea and four had mucositis and three had elevated transaminase/bilirubin levels. Seven patients had infections.⑥The 1-year cumulative non-relapse mortality (NRM) and 1-year cumulative incidence of relapse (CIR) was 22.5±14.0% and 20.2±12.7%, respectively. The 1-year overall survival (OS) and 1-year disease-free survival (DFS) was 72.7±13.4% and 63.6±14.5%, respectively Conclusions: TBIand rATG based conditioning regimen is effective and safe for haplo-HSCT in chemotherapy-resistant advanced-stage PTCL.

Outcomes after Bone Marrow Versus Peripheral Blood Haploidentical Hematopoietic Stem Cell Transplantation Using Post-Transplant Cyclophosphamide-Based Gvhd Prophylaxis

Background: Patients who do not have a human leukocyte antigen (HLA)-matched donor can undergo hematopoietic stem cell transplantation (HSCT) by utilizing haploidentical (haplo) family donors and post-transplant cyclophosphamide (PTCy) for Graft-versus-host disease (GVHD) prophylaxis. Originally, Haplo transplants utilized a bone marrow (BM) source because of concern of higher incidence of GVHD with peripheral blood stem cells (PBSC) graft. Recent years have seen an increase in PBSC graft utilization. Some published studies have reported comparable survival between the two graft sources. These studies also reported lower rate of chronic GVHD and higher quality of life with BM and a lower relapse rate and faster engraftment with PBSCs. We aimed to investigate the outcomes following BM versus PBSC haplo HSCT using (PT-Cy)-based GVHD prophylaxis. Methods: In this single-center retrospective study, we included all (n=176) adult patients who underwent haplo HSCT from August 2016 to July 2021 and had at least one year of follow-up. Bivariate analyses, using the chi-square and t-test, were performed. Kaplan-Meier and regression analyses were conducted. Data were analyzed using SPSS version 28. Statistical significance was considered at p<0.05. Results: The study included 176 haplo transplants including 65% peripheral blood and 35% bone marrow grafts. The median age was 54 (18-74) years and 68% of recipients were males. Ethnic minorities comprised 30% of recipients. Hematologic diagnoses included myeloid disease (65%), lymphoid disease (29%), and others (6%). 31 % of the patients underwent a Myeloablative transplant. Hematopoietic cell transplantation comorbidity index of 3 or higher was noted in 53% of patients. With a Median follow-up 21 (0-73) months, Overall survival (OS) and disease-free survival (DFS) was not reached in either BM or PBSC haplo HSCT group. 1-year OS of 75% and 74% (p-0.898) and 1-year DFS of 63% and 63% (p-0.994) were noted in BM and PBSC group respectively. In PBSC versus BM haplo recipients, an earlier neutrophil engraftment (17 days vs 18 days, p=0.022) was seen. Similar incidences of grade II-IV acute GVHD (50% vs 51%, p=0.875), relapse (22% vs 26%, p=0.579), non-relapse mortality (NRM, 17% vs 20%, p=0.682), platelet engraftment (28 vs 31 days, p<0.092) and 1- year chronic GVHD (35% vs 38%, p=0.410) were noted. Conclusions: In this study, Haploidentical allogeneic hematopoietic stem cell transplant recipients demonstrated similar rates of overall and disease-free survival, relapse, non-relapse mortality, acute and chronic GVHD, and platelet engraftment regardless of graft source. Neutrophil engraftment was observed one day earlier in peripheral stem cell recipients. This supports the use of either PBSC or Bone marrow as the preferred source of Haploidentical transplants.

A Multicenter Study of CAR-T Cell Therapy for Relapse of B-ALL Post Allogeneic Stem Cell Transplantation

BACKGROUND: Patients with acute B-cell lymphoblastic leukemia (B-ALL) relapsed after allogeneic stem cell transplantation (allo-HSCT) have very poor prognosis. Chimeric antigen receptor T (CAR-T) cells targeting CD19 have been developed as one of the effective immunotherapies for relapsed/refractory B-ALL. The safety and efficacy of CAR-T cells in patients relapsed after allo-HSCT remain unclear. METHODS: This study analyzed 60 B-ALL patients relapsed after allo-HSCT treated with CAR-T cells between October 2015 and February 2022 from four Chinese centers. Their pre-treatment patient characteristics, treatment regimens, efficacy, adverse effects, and follow-up data were comprehensively analyzed. RESULTS: One patient refused efficacy evaluation. 7 patients died before day 28 after CAR-T infusion. A total of 52 were assessed for efficacy within 28 days after CAR-T infusion.76.92% (40/52) patients achieved complete remission (CR) at Day 28 after CAR-T infusion, including 38 patients who achieved MRD-negative CR. 4 of the 40 patients relapsed at a median follow-up date of 12.1 months. The 1-year OS and EFS of all patients were 52.8% ± 7.0% and 51.5% ± 7.0%, respectively, and the 2-year OS and EFS were 40.1% ± 8.6% and 45.1% ± 8.6%, respectively. Multifactorial analysis showed that patients with severe CRS had worse OS and EFS, and other factors including age, gender, site of CAR-T tumor involvement, type of previous transplantation, previous transplantation type, CAR-T product and CRS grade, etc., did not affect the long-term survival of patients. The incidence of CRS was 80.00%, the incidence of grade ≥3 CRS was 20.83%, and the median time of CRS was 2 days after CAR-T cell infusion (range: 0-34 days). 3 patients (5.00%) died due to multiorgan failure caused by severe CRS, and the rest of the patients' CRS was controlled, and the grade of CRS was related to the proportion of blast cells in the bone marrow prior to CAR-T cell infusion., independent of the type of transplantation and the source of CAR-T cells. The incidence of ICANS was 5.00%. The incidence of aGvHD was 30.00%, and patients who had received prior haploidentical allogeneic HSCT were significantly more likely to develop aGvHD after CAR-T cell infusion than those who had received prior matched allogeneic HSCT. The occurrence of aGvHD after CAR-T treatment was not associated with the occurrence of CRS (p = 1), the presence of aGvHD after prior transplantation (p = 0.3484), the presence of cGvHD after prior transplantation (p = 0.3167), the presence of cGvHD at the time of CAR-T infusion (p = 0.5136), and the presence of prior donor lymphocyte infusion (p = 0.7404), and whether CAR-T cells were from autologous or donor (p = 0.4819). CONCLUSIONS: CAR-T cells are safe and effective in treating B-ALL patients with post-transplant relapse, and donor-derived CAR-T cells do not exacerbate CRS with aGVHD, and there is a high incidence of aGVHD after CAR-T therapy in patients with HLA-haplotype-matched transplantation.

Assessing Real-Time Sickling Kinetics in Individual Cells from Erythrocyte Populations of Subjects with Different HbA, HbS and HbF Percentages

INTRODUCTION: Polymerization of deoxygenated sickle hemoglobin (HbS) leads to erythrocyte sickling. Treatment in sickle cell disease (SCD) aims to reduce the risks of red blood cell (RBC) sickling and associated complications including, but not limited to vaso-occlusive crises (VOC). There is substantial promise in developing therapies targeting HbS polymerization that consequently decrease RBC sickling, a fundamental pathophysiological driver of SCD. Nonetheless, there are limited options available for reliable diagnostic tools for the assessment of disease status and therapeutic outcomes. A robust and easy-to-use system to assess population-scale sickling kinetics and to monitor responses to Hb-modifying therapies in clinical trials for the clinical management of patients may be beneficial. We have developed a dynamic sickling assay (DSA) that uses an enzymatic oxygen scavenging system to provide tight control over the rate and depth of induced-hypoxia. DSA is able to differentiate samples based on endogenous Hb expression and can be used to monitor sickling kinetics pre- and post HbS-modifying therapies, including hematopoietic stem cell transplantation (HSCT). METHODS: SCD and sickle cell trait (SCT) subjects were recruited under the RBC Health Initiative IRB protocol number FF-RBC-003. Four hematopoietic stem cell transplant (HSCT) SCD subjects were enrolled in NIH haploidentical HSCT protocol 17-H-0069. Diluted blood samples were supplemented with protocatechuic acid (PCA), followed by protocatechuate 3,4-dioxygenase (PCD) enzyme inducing hypoxia at a predetermined rate. The sample was then injected into a microfluidic chamber, and resulting RBC morphological changes were observed and captured with time-lapse photography. Image analysis identified and quantified RBC using an AI-trained SICKLE image analysis software. HbAS (n=3), HbSS with HbA (post-transfusion, n=3) and HbSS samples containing 3.1 to 34.5% HbF (n=9) were processed with DSA to assess the effects of hemoglobin distribution on sickling kinetics.Pre- and post-HSCT samples were run to ascertain the effect on sickling kinetics at baseline, 3M, 6M and 12M post-HSCT. Sickling profiles were constructed enabling a comprehensive assessment of RBC sickling and treatment outcomes. The reported parameters include: mPOS@5% (time to reach 5% induced sickling, min), mPOS@50% (time to reach 50% maximum induced sickling,min), rate of sickling (the maximum sickling rate, %/min), maximum induced sickling (the highest induced sickling percentage, %), AUC at 10min (area under the curve@10min of DSA, %∙minute) RESULTS: DSA inter-run CVs are 6.9% for mPOS@5%, 3.8% for mPOS@50%, 2.7% for max sickling, 4.2% for AUC and 10.8% for the rate of sickling. Statistically significant correlations are observed between %HbS and rate of sickling, max sickling, mPOS@5%, and AUC at 10min. As percentages of Hb increased in transfused SCD patient samples, there were noticeable correlations between the delay in mPOS@5% and decreased rate of sickling, maximum sickling, and AUC10. Similarly, as HbA percentages increased in SCT samples, a decrease in AUC10 and max sickling were observed. Transfused and SCT samples did not show statistical significance due to sample size limitations. However, there was a strong correlation in delay of mPOS@5% and mPOS@50%, as well as a decrease in the rate of sickling and AUC10 with increasing percentages of HbF in samples (Table 1). Comparing pre-HSCT (BL) sample sickling kinetics to 3, 6 and 12 month post-HSCT samples showed decreases in the rate of sickling (from 44.4 to 0.6%), maximum sickling (from 45-23%), decreases in AUC and delays in mPOS (Table 2). CONCLUSIONS: This study found significant linear relationships between %HbS and sickling parameters.As HbA percentage increases in transfused SCD and SCT samples, improvements in sickling parameters are observed. There is a strong correlation between increased HbF percentage and improved DSA parameters. Comparative data analysis from patients pre- and post-HSCT also show improvement in sickling parameters. Overall, DSA provides a simple, robust method for assessing responses to Hb differences and monitoring SCD therapies such as gene-editing.

IPSS-M Predicts Survival Outcomes Significantly Better Than IPSS-R in Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplantation for Myelodysplastic Neoplasms

Background The newly published molecular International Prognostic Scoring System (IPSS-M) represents a powerful risk stratification tool for treatment decision-making in myelodysplastic neoplasms (MDS); however, its utility in the context of allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains to be fully explored. Methods We retrospectively analyzed a large multicenter cohort of 347 MDS patients who underwent allo-HSCT between January 2017 and October 2022. The prediction accuracy of IPSS-M at diagnosis for 3-year survival outcomes was assessed and compared to that of the conventional revised International Prognostic Scoring System (IPSS-R). Results Among the 347 patients, median age at transplant was 48.3 years and 41.2% of patients were female. According to IPSS-M, patients were clustered as very low risk (1.2%), low risk (11.2%), moderately low risk (11.5%), moderately high risk (18.2%), high risk (33.4%), and very high risk (24.5%), resulting in a restratification of 49.3% of the entire cohort when compared with IPSS-R. Of these reclassified patients, 52.6% patients were upstaged and 47.4% were downstaged. Median follow-up time among the survivors was 28.6 (range, 4.7 to 76.6) months. With the IPSS-M model, overall survival (OS) and leukemia-free survival (LFS) discrimination was refined relative to the IPSS-R as evidenced by a 7.0 percentage- and 5.7 percentage-point increase in the concordance index (C-index), which was further supported by the lower Akaike Information Criterion and higher C-indexes in multivariate analyses. Among patients undergoing haploidentical HSCT, IPSS-M model also demonstrated significantly improved prognostic performance for LFS versus IPSS-R (C-index, 0.707 vs 0.604) which was validated by multivariate analyses. When restricting our analyses to younger patients (<49 years) and patients carrying detectable mutations, IPSS-M retained greater prognostic value with respect to OS and LFS; while it failed to stratify individual probability of OS and LFS in their counterparts. Conclusions IPSS-M was confirmed to increase prognostic discrimination at the individual level and is applicable to transplant-specific settings, which also had an advantage for subjects carrying mutations and younger patients.

Risk Factors for Positive Posttransplantation Measurable Residual Disease in Patients with Acute Lymphoblastic Leukemia

Background: The level of MRD before and after transplantation is related to inferior transplant outcomes, and post-hematopoietic stem cell transplantation measurable residual disease (post-HSCT MRD) has higher prognostic value in determining risk than pre-HSCT MRD. However, no work has been devoted to the risk factors for positive post-HSCT MRD in patients with ALL. This study evaluated the risk factors for post-HSCT MRD positivity in patients with acute lymphoblastic leukemia (ALL) who underwent allogeneic HSCT (allo-HSCT). Methods: A total of 1683 ALL patients were enrolled. Cox proportional hazard regression models were built for time-to-event outcomes. Multivariate analysis was performed to determine independent influencing factors from the univariate analysis. Results: Both in total patients and in T-ALL or B-ALL, pediatric or adult, HLA-matched sibling donor transplantation or haploidentical HSCT subgroups, positive pre-HSCT MRD was a risk factor for post-HSCT MRD positivity ( P < 0.001 for all). Disease status was also a risk factor for post-HSCT MRD positivity in all patients and in the B-ALL, pediatric, or haploidentical SCT subgroups ( P =0.003; P=0.035; P =0.003, respectively). A risk score for post-HSCT MRD positivity was developed using the variables pre-HSCT MRD and disease status. The cumulative incidence of post-HSCT MRD positivity was 12.3%, 25.1%, and 38.8% for subjects with scores of 0, 1, and 2-3, respectively ( P<0.001). Multivariate analysis confirmed the association of the risk score with the cumulative incidence of post-HSCT MRD positivity and relapse as well as LFS and OS. Conclusion: Our results indicated that positive pre-MRD and disease status were two independent risk factors for post-HSCT MRD positivity in patients with ALL who underwent allogeneic HSCT.

Impact of TP53 Allelic State on Outcomes of Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplantation for Myelodysplastic Neoplasm

Background : Tumor protein p53 ( TP53) mutations are associated with dismal outcomes in myelodysplastic neoplasms (MDS). However, the impact of TP53 allelic state in the context of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for MDS has not been fully explored. Method: We conducted a multi-center study in 347 MDS patients undergoing allo-HSCT between January 2017 and October 2022, with the median follow-up time among survivors of 28.6 (range, 4.7 - 76.6) months, and 45 had TP53 mutations among them. Results: Twenty-one patients were detected with TP53 monoallelic mutations ( TP531mut), while 24 had multiple hits ( TP53multi). Complex karyotype was more common in patients with mutated TP53 (52.4% of patients with TP531mutand 75.0% of patients with TP53multi), whereas this proportion was only 10.6% in patients with wild-type TP53 ( TP53wt). The prognosis varied according to different TP53 state, showing the 2-year overall survival (OS) rates of 51.4% (95% confidence interval [CI]: 33.7% - 78.6%) for TP531mut group versus 15.8% (95% CI: 3.3% - 76.4%) for TP53multi group versus 73.5% (95% CI: 68.5% - 78.9%) for TP53wt group ( P<0.001), and the 2-year cumulative incidence of relapse (CIR) rates of 28.6% (95% CI: 11.2% - 48.8%) for TP531mut group versus 47.3% (95% CI: 18.3% - 71.9%) for TP53multi group versus 10.8% (95% CI: 7.5% - 14.9%) for TP53wt group ( P<0.001). Similarly, the leukemia-free survival (LFS) and graft-versus-host disease-free/relapse-free survival (GRFS) of patients with TP531mut or TP53multi were obviously lower than those with TP53wt (both P<0.001). However, there was no significant difference on non-relapse mortality among these three groups ( P=0.424). The adverse effects of TP53 mutationson OS, LFS, GRFS and CIR were also found in patients undergoing haploidentical HSCT or those with molecular International Prognostic Scoring System high- and very high-risk categories (all P<0.05). In the subgroup with complex karyotype ( n = 32 with non-mutated TP53 and n = 29 with mutated TP53), significantly inferior OS, LFS and GFRS and higher CIR were observed in patients with mutated TP53 while in the subgroup without complex karyotype ( n = 270 with non-mutated TP53 and n = 16 with mutated TP53), there were no differences in these prognostic indicators (all P>0.05). Notably, among patients who received conventional conditioning regime without hypomethylating agents (HMA) ( n = 264 with non-mutated TP53 and n = 38 with mutated TP53), the inferior 2-year OS, LFS and GRFS rates, and the higher 2-year CIR rate of patients still presented in TP53 mutant patients(all P<0.05), but among those receiving conditioning regime containing HMA, similar outcomes were found between mutated ( n = 7) and non-mutated ( n = 38) TP53 groups (all P>0.05). Multivariable cox proportional hazards models demonstrated that monoallelic TP53 state was an independent risk factor for survival and relapse (HR OS=2.859, 95% CI: 1.372 - 5.957, P=0.005; HR LFS=2.566, 95% CI: 1.342 - 4.905, P=0.004; HR GRFS=2.146, 95% CI: 1.106 - 4.163, P=0.024; HR CIR=2.507, 95% CI: 1.033 - 6.086, P=0.042), and multi-hit TP53 state also predicted worse outcomes (HR OS=3.038, 95% CI: 1.453 - 6.356, P=0.003; HR LFS=2.716, 95% CI: 1.383 - 5.336, P=0.004; HR GRFS=1.882, 95% CI: 1.002 - 3.535, P=0.049). C onclusions: Monoallelic and multi-hit TP53 state identify a high-risk group for poor outcomes in MDS patients undergoing allo-HSCT, complex karyotype could amplify the adverse prognostic effect of TP53, and these patients might benefit from receiving conditioning regime containing HMA.