Donor Lymphocyte Infusion Research Articles

Chidamide-Based Pre-Emptive Treatment for High-Risk AML Patients after Hematopoietic Stem Cell Transplantation: A Real-World Experience from Chinese Single-Center

Background: The treatment of relapsed or refractory acute myeloid leukemia ( AML) remains challenging. Measurable residual disease (MRD) persistence at transplantation or post-transplantation has been identified as the strongest risk factor for relapse and poor outcomes for patients with hematological malignancies (HMs). Chidamide, a novel oral histone deacetylase inhibitor, has shown potential therapeutic effects in various types of HMs by inducing cell apoptosis and regulating immunity. This study aimed to evaluate the efficacy and safety of Chidamide-based prophylactic or pre-emptive treatment for patients who are MRD positive before or who have sustained MRD positive after transplantation. Aim: The rate of 6 months of conversion to MRD- and relapse-free survival of high-risk AML after Chidamide-based treatment. Methods: From December 2020 to July 2023, 50 patients with high-risk HMs received chidamide-based therapy after allo-HSCT. 39 patients were diagnosed with AML ( 4 secondary AML), 2 with myelodysplastic syndromes, 6 with acute lymphoid leukemia, and 3 with mixed-phenotype acute leukemia. 46 patients (92%) with relapsed/refractory status or detectable MRD (MRD+) at transplantation or after allo-HSCT received chidamide as pre-emptive, and the remaining 4 high-risk patients with MRD− conducted as prophylactic intervention. Patients with detectable MRD before transplantation and those who remained positive for MRD after transplantation were defined as very high-risk AML (VHR-AML). Chidamide was administered at a dose of 5 mg per day orally, 6 times per week, lasting at least 1 year after engraftment. Additional agents included hypomethylating agents (HMAs), donor lymphocyte infusion (DLI) or tyrosine kinase inhibitor (TKI) in patients with Philadelphia chromosome-positive. In this study, 28 patients received chidamide monotherapy, 13 received chidamide combined with HMAs, and 9 cases with other therapy. Results: The median age of all patients was 38 years old (range 17-67), comprising 24 males and 26 females. Up to 1st July 2023, with a median follow-up period of 376 days (25-984d), the overall rate of MRD negative showed 64%. The rate of MRD- survival, Relapse-free survival (RFS) and overall survival (OS) were 56%, 66% and 76%, respectively. The 6 months MRD negative rate, RFS and OS rate were 86%, 82%, 86% and 88%, which maintained at 82%, 82% and 86% with 12 months follow-up, respectively. In 46 cases of the pre-emptive intervention arm, the overall MRD- rate was 61.7%. Among them, 83.3% of patients (20/24) who received chidamide monotherapy exhibited a much higher conversion to MRD− than those combined with HMAs (7/13, 53.8%) or other drugs (2/9, 22.2%) (P=0.004). In 4 cases who received chidamide monotherapy as prophylactic intervention, 3 patients sustained MRD negativity up to the last follow-up, except for one patient diagnosed with MPAL(B+M) relapsed after 1 year of HSCT with taking chidamide for 6 months. A total of 30 patients (including 25 AML) with VHR diseases remained in a status of MRD+ or non-remission after transplantation. Eighteen patients (60%) achieved MRD− and sustained for a median of 251 days (range 31-531 d). VHR-AML patients account for 94.4% (17/18) of MRD- responders(Figure 1A). Similarly, the results showed a higher rate of conversion from MRD+ to MRD− in chidamide monotherapy than the combination groups (Chidamide monotherapy, 81.8%; Chidamide + HMA, 63.6% and Chidamide + others, 25%, P=0.042,Figure 1B). It highlighted that treatment with chidamide resulted in increased MRD− response, especially for AML. On the contrary, of 7 patients with B-cell malignancy, including 5 B-ALL and 2 MPAL (B + M) patients, only 2 (28.6%) patients converted to MRD−. Reversible grade 3/4 neutropenia occurred in 38% of patients, and thrombocytopenia was observed in 24% during the first four treatment cycles. Five patients experienced grade 1/2 non-hematological adverse events, such as fatigue, gastrointestinal reaction, pneumonia and infection. No new-onset GvHD was reported in patients without a prior history of GvHD during chidamide treatment. Conclusion: The chidamide-based regimen is safe and effective in pre-emptive treatment for patients with high-risk relapsed AML after transplantation. Whether as a monotherapy or combined with HMA, chidamide shows significant efficacy in eliminating MRD, warranting further clinical research.

Sensitive Molecular Detection of JAK2 V617F Is a Predictive Marker of Relapse in Patients with Myelofibrosis after Allogeneic Stem Cell Transplantation

Background: Myelofibrosis (MF) is a BCR-ABL1-negative myeloproliferative neoplasm (MPN) associated with JAK2 V617F mutation in approximately 50% of patients. Allogeneic stem cell transplantation (alloSCT) is the only curative treatment option for MF but a significant proportion of patients undergoing alloSCT experience relapse. Although measurable residual disease (MRD) is an established predictor of relapse in other hematological malignancies, the dynamics of JAK2 V617F variant allele frequency (VAF) in MF post-alloSCT and its prognostic impact are yet to be clearly delineated. In this study, we assessed the utility of sensitive molecular detection of JAK2 V617F as a marker of post-alloSCT outcomes in MF particularly with respect to the optimal threshold and time points for predicting early relapse. Methods: A retrospective analysis was conducted of consecutive patients with JAK2 V617F-mutated MF receiving alloSCT at our institution between 2016 and 2022. Data on clinicopathological features, alloSCT characteristics, treatment response assessments, graft-versus-host disease (GVHD) and outcomes were collected. JAK2 V617F was enumerated by droplet digital polymerase chain reaction (ddPCR) using the Bio-Rad QX200 ddPCR platform on archived bone marrow or peripheral blood samples at defined time points pre- and post-alloSCT. JAK2 V617F VAF was defined as the percentage of JAK2 V617F mutant copies amongst total JAK2 (mutant plus wild-type) copies, with a routine assay sensitivity of 0.05%. Results: 31 patients with JAK2 V617F-mutated MF underwent alloSCT during the study period (Table 1). Five patients experienced relapse at a median of 14.2 (range: 3.2-22) months post-alloSCT. 13 patients died of infection (n = 5), overt relapse (n = 4) or GVHD (n = 4). After a median duration of follow-up of 17.5 (range: 0.9-77.9) months, the 5-year overall survival (OS) and relapse-free survival (RFS) were 52.8% and 49.6%, respectively. The 2-year cumulative incidences of relapse (CIR) and non-relapse mortality (NRM) were 26.6% and 35.5%, respectively. On univariate analysis, superior OS and RFS were only significantly associated with chronic GVHD ( P = 0.017 and 0.013, respectively). A total of 174 samples were obtained within 1 month of alloSCT (n = 31) and on days +30 (n = 31), +60 (n = 30), +100 (n = 29), +180 (n = 23) and +360 (n = 21) after alloSCT. All patients had detectable JAK2 at the time of alloSCT with a median VAF of 43.8% (range: 0.2-95%). The incidence of JAK2 V617F detection was 45.2% on day +30, 43.3% on day +60, 44.8% on day +100, 34.8% on day +180 and 23.8% on day +360 post-alloSCT. Of 6 patients who received ruxolitinib for GVHD treatment post-alloSCT, one eventually relapsed while the other 5 had undetectable JAK2 prior to ruxolitinib commencement. JAK2 VAF was higher in patients who eventually relapsed than in non-relapsed patients on days +60, +100, +180 and +360 ( P = 0.008, 0.016, 0.001 and <0.001, respectively) (Figure 1). However, JAK2 positivity at each of these time points was not significantly associated with survival outcomes (OS, RFS, NRM or GVHD-free relapse-free survival [GRFS]). Receiver operating characteristic (ROC) analysis indicated that JAK2 VAF on days +100, +180 and +360 post-alloSCT were significant predictors of eventual relapse ( P = 0.022, 0.019 and 0.01, respectively). The optimal JAK2 VAF thresholds at each of these time points were 0.2% (sensitivity 80%; specificity 83.3%; area under curve [AUC] 0.788) at day +100, 7.1% (sensitivity 75%; specificity 94.7%; AUC 0.842) at day +180, and 11.7% (sensitivity 75%; specificity 94.1%; AUC 0.86) at day +360. Comparisons of JAK2 VAF at each time point to the immediately preceding VAF showed that ≥2-fold increases at days +100 (from day +60) and +180 (from day +100) were significantly associated with relapse ( P = 0.002 and 0.001 respectively). Conclusion: Although persistence of JAK2 is common for more than a year post-alloSCT for JAK2 V617F-mutated MF, serial sensitive molecular monitoring of JAK2 at defined time points appears to be a predictive marker of relapse. In this study, doubling of JAK2 VAF at days +100 and +180 post-alloSCT were associated with relapse. These data support the notion that sensitive molecular detection of JAK2 may have a role akin to MRD in informing the pre-emptive use of immunotherapeutic intervention in this setting, including immunosuppression withdrawal and/or donor lymphocyte infusions.

Transplant Outcomes for Acute Myeloid Leukemia or Myelodysplastic Syndromes Secondary to Acquired Aplastic Anemia or Paroxysmal Nocturnal Hemoglobinuria: A Report from the EBMT Severe Aplastic Anemia Working Party

Introduction Clonal evolution into myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) is the most concerning late event among non-transplanted acquired aplastic anemia (AA) or paroxysmal nocturnal hemoglobinuria (PNH) patients, occurring in nearly 10% of cases. MDS/AML secondary to AA differ from primary MDS/AML, presenting with higher risk cytogenetics and poorer prognosis, usually involving abnormalities in chromosome 7 (Gurnari C. et al., JCO 2023). The survival is poor, and the only curative treatment is allogeneic hematopoietic stem cell transplantation (HSCT). We present the HSCT outcomes of secondary MDS/AML following AA/PNH. Methods We have extracted data from the EBMT database for patients with AA or PNH diagnosed after 2010, who later presented a clonal evolution into AML, MDS, or isolated cytogenetic abnormality insufficient for MDS diagnosis according to WHO 2016 classification, who have undergone HSCT after the clonal evolution diagnosis. We have excluded AA/PNH patients with HSCT before clonal evolution, inherited bone marrow failure syndromes, and non-transplanted patients. Our primary objective was to describe the overall survival (OS). The secondary aims were transplant outcomes according to clonal evolution category and conditioning regimen intensity. Results: We have studied 278 patients from 114 different EBMT centers, 250 patients diagnosed with AA, and 28 with classical PNH. The median age at AA/PNH diagnosis was 34.7 (20.5-51.7) years. At first evolution, 153 (56.5%) patients had a diagnosis of MDS, 45 (16.6%) AML, and 73 (26.9%) isolated abnormal cytogenetics insufficient for a diagnosis of MDS (missing data for 7 patients). The median interval from AA/PNH diagnosis to clonal evolution was 30.1 (8.1-67) and 39.8 (21.5-82.6) months for MDS and AML, respectively. The interval from clonal evolution to HSCT was 5 (3-10.2) and 3.4 (2.3-5.1) months for MDS and AML, respectively. We present transplant characteristics in Table 1. With a median follow-up of 5 (4.3 - 5.9) years, the 5-year overall survival (OS) was 65% for the entire cohort. A subgroup analysis did not find a significant 5-year OS difference among groups: 73, 62, and 60% for isolated cytogenetic abnormality, MDS, and AML, respectively (p=0.3, Figure 1). The main causes of death were transplant-related toxicities (66.7% and 75% for isolated cytogenetic abnormality and MDS, respectively) and relapse or progression for AML (43.8%). AML patients relapsed more frequently than MDS patients by 5 years [cumulative incidences of 22 (9-35) vs. 10% (4-17) for AML and MDS (p=0.046), respectively]. The 5-year non-relapse mortality (NRM) was 19 (7-31) and 28% (19-36) for AML and MDS patients, respectively (p=0.3). When stratifying by conditioning regimen intensity, the 5-year NRM was 34% (19-40) for myeloablative (MAC) vs. 18% (9-26) for reduced-intensity (RIC, p=0.025), with similar incidence of relapse in both groups (4 vs 10% for MAC and RIC, respectively, p=0.2). AML patients were more prone to have presented acute graft-versus-host disease (GvHD) by day+100 than MDS or isolated cytogenetic abnormality patients (cumulative incidences of 36, 26, and 14% respectively, p=0.022). Likewise, AML patients had more chronic GvHD (43, 28, and 12% respectively, p=0.008). Graft-failure, GvHD, relapse-free survival (GGRFS) was 71 (61-82), 60 (51-68), and 45% (29-60) for isolated abnormality, MDS, and AML, respectively (p=0.026). Discussion This study is the largest cohort of transplanted patients with secondary MDS/AML following AA/PNH. The major limitations of our study are its retrospective nature, non-adjusted analyses, and missing data on complete cytogenetics. We found a 5-year OS higher than 60% for MDS/AML compared to 45% in the previously published EBMT study (Hussein A. et al., BBMT 2014) and did not find a survival difference between AML and MDS. Interestingly, the main cause of death for MDS patients was HSCT complications. Furthermore, MAC was also associated with a higher NRM. Clinicians should indicate HSCT with discretion for post-AA/PNH MDS or isolated cytogenetic abnormality, considering the transplant toxicity, especially with MAC, and the risk of primary disease progression. For patients with AML and a high risk of relapse, early tapering of immunosuppressive agents and post-transplant maintenance, such as hypomethylating agents and donor lymphocyte infusion, should be considered.

Practice Patterns of Transplant Centers Regarding Maintenance Treatment Post Allogeneic Hematopoietic Cell Transplantation in Acute Myeloid Leukemia: A Survey on Behalf of the EBMT Acute Leukemia Working Party

Introduction: Disease recurrence in acute myeloid leukemia (AML) remains the major reason of allogeneic hematopoietic cell transplantation (allo-HCT) failure. Strategies to reduce the risk of relapse such as prophylactic pharmacologic interventions post allo-HCT are more frequently applied by some transplant physicians, especially after the growing evidence of its efficacy and tolerability. Others are still being skeptical regarding its implementation due to difficulty proving efficacy by large randomized trials, or concerns of long-term toxicities. The aim of this study was to conduct a survey among the European Society for Blood and Marrow Transplantation (EBMT)-affiliated transplant centers to identify clinical practice patterns regarding maintenance treatment post-allo-HCT in AML. Methods: After the approval of the board of the Acute Leukemia Working Party (ALWP), EBMT-affiliated centers received by email a questionnaire-based survey between January and June 2023. The questionnaire consisted of 13 questions assessing clinical practices regarding maintenance treatment post allo-HCT in AML, specifically the indications of maintenance, the type, the drug used, and the duration of the treatment. Results: A total of 115 (out of 424) EBMT-affiliated centers from 31 countries responded to the survey with a response rate of 27.12%. Overall, 102 (88.8%) centers implement maintenance strategy whether pharmacological intervention or cellular therapy post allo-HCT in AML, most of the centers (N=67, 58.3%) use it based on a personalized decision, 17 (14.8%) use it routinely, and 18 (15.7%) use it exceptionally. Regarding the indication, the majority of the centers recommend maintenance treatment for AML patients in second remission or beyond (N=64, 62.7%) or in AML patients in first remission with high risk of relapse based on cytogenetic or molecular data (N=61, 59.8%), whereas 34 (33.3%) centers recommend it only for FLT3-mutant disease, and 11 (10.8%) centers in all patients regardless of the risk of disease. The majority of centers (N=77, 75.5%) use a combination of pharmacologic and cellular therapies, and 20 (19.6%) centers use only pharmacologic therapies. Among centers using pharmacologic therapies, 62 (63.9%) use hypomethylating agents (HMA) (azacitidine N=51 (82.3%), decitabine N=6 (9.6%), HMA with venetoclax N=23 (37%), HMA with donor lymphocyte infusion (DLI) N=38 (61%), or a combination of modalities in 42 (67.7%)). In general, most centers (N=35; 87.5%) do it pre-emptively either minimal residual disease (MRD) driven or chimerism driven, whereas 22 (55%) centers do it prophylactically. The majority of centers continue HMA for one-year duration (N=37, 59.7%), 15 (24.2%) for two years and only 10 (16.1%) continue until disease recurrence or toxicity. Out of 97 responding centers regarding the use of FLT3 inhibitors as maintenance, 93 (95.9%) are implementing this strategy. The most common choice was sorafenib in 82/93 (88.2%) centers, midostaurin in 28 (30%), and gilteritinib in 30 (32.2%). Among these, 56 (83.5%) centers use FLT3 inhibitors as prophylaxis whereas 28 (41.7%) use a pre-emptive approach. The preferred maintenance duration was for two years in 51 (54.8%) centers, followed by one-year duration in 30 (32.3%) centers, continuous until disease relapse or toxicity in 9 (9.7%) centers, and for five years in only 3 (3.2%) centers. Novel agents are used in 71 (70.2%) centers as maintenance either alone or in combination with HMA, of them 52 (73.2%) use venetoclax, and 13 (13.2%) use enasidenib. Finally, regarding practice patterns, 32 (29.6%) centers claim that maintenance treatment is part of their routine practice, 46 (42.6%) only for FLT3 mutant AML. Others are not routinely implementing maintenance because they are still skeptical and need more evidence by randomized clinical trials (N=8), whereas 5 centers are concerned regarding tolerability, and 16 (14.8%) cannot implement because of regulatory restrictions or lack of access. Conclusion: In this survey, the majority of responding EBMT-affiliated transplant centers is implementing post-transplant AML maintenance treatment, predominantly HMA and FLT3 inhibitors. Further studies are needed to clarify the appropriate strategy and duration of maintenance therapy. The study will help the EBMT to incorporate post-transplant strategies as a routine registry capture parameter.

Clinical, Cytogenetic and Immunophenotypic Spectra of Post-Transplant Relapse Following Allogeneic Hematopoietic Stem Cell Transplantation for Myelodysplastic Syndrome

Introduction Allogeneic hematopoietic stem cell transplantation (HCT) is a curative therapy used to treat myelodysplastic syndrome (MDS). Relapse post-HCT confers poor outcomes and subsequent therapy is not standardized. Like acute myeloid leukemia (AML), several mechanisms are known to be involved in post-HCT relapse including clonal evolution in favor of chemotherapy-resistant clones and tumor immune escape. Systemic reviews are scarce in the MDS post-HCT relapse population. Accordingly, the present study aimed to investigate clinical, cytogenetic, and immunophenotypic (IP) spectra for MDS post-HCT relapse patients (pts), along with subsequent therapies, and clinical outcomes following post-HCT relapse. Patients and methods A retrospective study was conducted and found that 231 MDS pts received HCT between April 2010 and September 2022 at the Princess Margaret Cancer Centre (PMCC). The primary endpoint was overall survival (OS) following post-HCT relapse. Secondary endpoints explored time to relapse (TTR), relapse presentation (i.e., MDS or AML), clonal evolution (i.e., changes in cytogenetics or IP profiles), and therapeutic strategies delivered after relapse. Results Of the 231 MDS pts treated with HCT, 52 (23%) relapsed in a median of 4.5 months (range 0.9-59.8 months). Twenty-five (48%) pts relapsed with MDS (i.e., blast <20%), while 27 (52%) relapsed with AML (i.e., blast ≥20%) in the marrow. Fifteen (65%) pts showed cytogenetic changes at the time of post-HCT relapse. Eleven (48%) of which presented with more aggressive findings (i.e., additional structural abnormalities and monosomal karyotype). Ten of 17 (59%) pts presented with changes in their IP profile at post-HCT relapse, 7 (70%) of which relapsed with AML. Combined these findings give evidence of clonal evolution in favor of more aggressive malignant clones that are often responsible for post-HCT relapse. Treatment with hypomethylating agents (HMA) alone or in combination after post-HCT relapse increased OS to 5.6 months in comparison to those not receiving HMA of whom OS was 1.4 months (p=0.0003). Stratified by disease presentation cytogenetic clonal evolution was seen in 6/12 (50%) of the post-HCT MDS relapses, and 9/11 (82%) of the post-HCT AML relapses. Cytogenetic abnormalities at diagnosis and post-HCT relapse, respectively included in descending order: structural abnormalities (n=14/23, 61%; n=20/23, 87%), complex karyotype (n=12/23, 52%; 17/23, 74%), -5/del(5q) (n=11/23, 48%; n=10/23, 43%), -7/del(7q) (n=8/23, 35%; n=11/23, 48%), monosomy karyotype (n=8/23, 35%; n=11/23, 48%), -17 (n=3/23, 13%; n=5/23, 22%), der(5)/t(5;17) (n=2/23, 9%; n=5/23, 22%), -20 (n=2/23, 9%; n=3/23, 13%), and inv(3) (n=1/23, 4%, n=2/23, 9%). Seventeen pts had paired IP profiles available both prior to HCT and at post-HCT relapse, of which 10 (59%) had profile changes of at least 1 antigen. Seven of 10 (70%) pts that relapsed with AML post-HCT had IP expression changes, compared to 3/7 (42.9%) pts that relapsed with MDS post-HCT. Summarized IP findings include reduced expression of CD7 (n=2/6, 33%), altered expression of CD34 (n=3/15, 20%), altered expression of CD117 (n=3/14, 21%), and reduced expression of HLA DR (n=4/15, 27%). The presence of IP expression changes at post-HCT relapse suggests evidence of clonal evolution or immune escape as the mechanism behind the relapsed disease. Overall survival duration was 3.97 months; the 12-month OS rate was 20.0% (10.0-32.5%). Post-HCT relapse treatments included hypomethylating agents (HMA; n=13/46, 28%), HMA with donor lymphocyte infusion (DLI; n= 7/46, 15%), HMA with Venetoclax (VEN; n=7/46, 15%), chemotherapy (n=9/46, 19%), best supportive care (8/46, 17%) and second HCT (n=2/46, 4%). Patients that received HMA with or without DLI or VEN had a longer survival duration of 5.6 months (95% CI [3.4-13.2 months]) compared to those not receiving HMA 1.4 months (95% CI [0.6-0.39 months, p=0.0003]). The 12-month survival rate was also significantly in favor of pts that received HMA following post-HCT relapse (35.3% [95% CI 17.2-54.0%] vs 4.3% [95% CI 0.3-18.0%]). Conclusion Patients with MDS undergoing HCT may relapse with either MDS or AML. The relapsed disease often shows evidence of clonal evolution in its immunophenotype and/or cytogenetic profile toward a more aggressive disease. Approaches to post-HCT relapse remain heterogenous but HMA is beneficial.

A Phase I Study of Venetoclax in Combination with Inotuzumab Ozogamicin for Relapsed or Refractory ALL in Adults

Introduction Adults with acute lymphoblastic leukemia (ALL), especially those who are older and/or have unfavorable disease biology frequently relapse. Inotuzumab ozogamicin (INO), an anti-CD22 monoclonal antibody covalently linked to calicheamicin, is approved for treatment of relapsed or refractory (R/R) CD22+ ALL. Although INO induces measurable residual disease negative (MRD-neg) complete remission (CR) in >75% of patients (pts), the duration of response (DOR) is limited (<6 months). Pre-clinical work demonstrates that malignant lymphoblasts are BCL2-dependent (Del Gaizo Moore Blood 2008). Initial results of the BCL2 inhibitor venetoclax (VEN) combination therapies for ALL are promising (Jain ASH 2019, Pullarkat Cancer Discov 2021). Due to distinct mechanisms of activity, non-overlapping toxicity, and possible synergy (Kirchhoff Blood 2021), we hypothesized that INO and VEN can be safely and effectively combined. Study Design and Eligibility This is an investigator-sponsored phase I study (NCT05016947) with a 3+3 dose escalation design. The primary objective is to determine the maximum tolerated dose (MTD) of VEN plus INO at the FDA-approved doses in adults with R/R CD22+ ALL. Secondary objectives assess efficacy (hematologic response, MRD by multi-parametric flow cytometry [MFC] threshold <0.01%, and DOR). A 14 pt expansion cohort is planned. Dose-limiting toxicity (DLT) is defined as treatment-related CTCAE v5 non-heme toxicity grade 3+, sinusoidal obstructive syndrome (SOS) any grade, or liver toxicity during Induction (Ind) Cycle (C) 1 and/or failure of hematologic recovery (ANC ≥500, Plts ≥25 K/mL) by day 42 of Ind C1 in responding pts. Eligibility: ≥18 years (yrs); ALL (≥5% blasts) or LBL (marrow involvement not required); CD22 expression on ≥20% of blasts; R/R to one or more cycles of CC or in the case of Philadelphia-chromosome (Ph)-positive ALL, failed by two or more TKIs, or refractory to/ineligible for ponatinib; ≥60 days from bone marrow transplant (BMT), and off immune suppression ≥2 weeks; adequate liver and kidney function; no history of liver disease including SOS; blast count ≤25 K/mL prior to treatment; no symptomatic central nervous system (CNS) disease. Pts with prior INO or VEN treatment for R/R ALL are excluded. Regimen Pts are treated with a VEN Ramp-Up over 3 days (DL1: 100/200/200; DL2: 100/200/400) followed by Induction C1: IV INO days 1, 8, 15 (0.8/0.5/0.5 mg/m2) and VEN daily per DL days 1-21. DEX 10 mg/m2 IV/PO is administered during Ramp-Up and Ind C1 days 1-4 (7 days total). Pts with >5% blasts after C1 receive Ind C2 without DEX. Pts with <5% blasts after Ind (1 or 2 cycles) continue with up to four 28-day consolidation cycles (INO 0.5 mg/m2 days 1, 8, 15 plus VEN per DL days 1-21). Pts receive CNS prophylaxis with intrathecal chemotherapy. Results The 2-level dose-escalation phase has been completed; we enrolled 9 pts (5 female/4 male) with Ph-negative (n=7) or Ph-positive (n=2) ALL. The median age was 45 yrs (range 25 -74). Pts had received 1 (n=5) or 2 (n=4) prior lines of therapy. DL1 enrolled 3 pts without DLT. All DL1 pts achieved an MRD-neg CR and bridged to consolidation: 1 pt XRT to involved disease site (ankle) after 3 cycles and 2 pts to BMT after 2 cycles (Table). Three pts enrolled at DL2; no DLTs were observed. All achieved an MRD-neg CR. Two pts bridged to consolidation (donor lymphocyte infusion after 3 cycles, blinatumomab after 5 cycles) while 1 pt with KMT2A-r ALL experienced early relapse with lineage switch during second cycle. A confirmatory cohort of 3 pts enrolled at DL2 without DLT. All achieved a CR (n=2, MRD-neg; n=1, MRD-pos). The pt with MRD-pos CR as best response proceeded to blinatumomab as bridge to BMT after 2 cycles with 2 pts continuing treatment (currently cycle 2). Overall, CR was achieved in 100% (9/9) of pts with 7/8 (87%) achieving MRD-negativity. With median follow-up of 195 days (95% CI 80 - NR), 2 pts have relapsed 48 (on treatment) and 177 (after BMT) days from registration while other pts remain alive in CR. Non-heme grade 3-4 AEs include febrile neutropenia (2), DIC (1), tumor lysis syndrome (1), anxiety (1), hypertension (1), and hypotension (1). There have been no SOS. There were no delays in count recovery during C1, with median time from C1 to C2 being 30 days (range 27-42). Conclusion VEN 400mg x 21 days in combination with INO at standard doses was well tolerated and declared the recommended phase 2 dose (RP2D) with a CR rate of 100%.

VLA-4 Agonist Enhances the Capacity of HSC Engraftment and Immune Reconstitution

Poor donor cell implantation and delayed hematopoietic reconstitution are important factors affecting the prognosis of patients after allogeneic hematopoietic stem cell transplantation (Allo-HSCT). However, our understanding of the critical endogenous signals that reconstitute hematopoiesis in transplant patients remains limited，which is essential for achieving targeted interventions and preventing donor-recipient chimeric states after transplantation. Here, we revealed the reconstitution dynamics of human allogeneic hematopoietic stem and progenitor cells (HSPCs) and immune cell subpopulations in the post-transplantation mixed donor chimerism (MDC) to fully donor chimerism (FDC) state at single-cell resolution. The hematopoietic stem cell (HSC) implantation process involves extensive interactions with immune cells mainly through surface Kcnq5, Itga4 (encoding a subunit of VLA-4), Tfrc, CD44 receptor, and ligands for Rps19, B2m, etc. Further identification of VLA-4(integrin α4β1) as a potential regulatory target for HSC implantation by single-cell sequencing of a stable allogeneic bone marrow transplantation (Allo-BMT) MDC-FDC mouse model. Grafts Itgα4 and Itgβ1 deletion impair donor implantation and further diminish the efficacy of donor lymphocyte infusion (DLI) following Allo-BMT, rather than directly affecting DLI. Critically, we identified a VLA-4 agonist that promotes donor implantation in the mixed chimeric mouse model of Allo-BMT in a time-dependent manner, with the most pronounced promotion of early T cell implantation in particular. Moreover, it was found to enhance the self-renewal capacity of HSC and lymphoid reconstitution in competitive transplantation and human umbilical cord blood transplantation xenograft experiments. Fortunately, VLA-4 agonist does not exacerbate the acute graft-versus-host disease(aGVHD) process while maintaining the graft-versus-leukemia (GVL) effect. VLA-4 agonist not only upregulates of both Itgα4 and Itgβ1 expression, but also promotes the ERK/2 phosphorylation pathway, which affects HSC biological functions, and this process can be inhibited by ERK kinase inhibitors. These results provide insights into the regenerative process of transplanted HSPCs in human patients，and demonstrate a previously unknown positive regulation of HSC engraftment and immune reconstitution by VLA-4, and allow for a new and simple translational strategy to enhance HSC transplantation.

Olutasidenib for the Treatment of mIDH1 Acute Myeloid Leukemia in Patients Relapsed or Refractory to Hematopoietic Stem Cell Transplant, Prior mIDH1 Inhibitor, or Venetoclax

Introduction. Olutasidenib is a small molecule, oral, mutated-IDH1 (mIDH1) inhibitor approved for the treatment of relapsed/refractory (R/R) acute myeloid leukemia (AML). In the pivotal cohort of the registrational phase 1/2 trial, 51/147 (35%) patients achieved a complete remission (CR) or CR with partial hematologic recovery (CRh) to olutasidenib, with a duration of response of 25.9 months and an overall response rate (ORR) of 48%. The phase 1/2 study included 10 cohorts for a total of 335 patients, and there were patient subsets that were R/R to previous hematopoietic stem cell transplant (HSCT), ivosidenib (IVO) or venetoclax (VEN). We conducted post hoc analyses to better understand the response to olutasidenib in these poor-prognosis subgroups of mIDH1 AML. Methods. This Phase 1/2, open-label, multi-center study enrolled patients with confirmed AML or intermediate, high-, or very high-risk myelodysplastic syndromes/neoplasms (MDS). Patients received olutasidenib 100mg QD, 150mg QD or 150mg BID monotherapy or the combination of olutasidenib 150mg BID plus azacitidine. Patients were grouped by prior therapy. Baseline demographics and disease characteristics are shown in Table 1. The study was not powered to statistically evaluate subpopulations; descriptive statistics are provided. Data cutoff date is June 2021. Results. The post-HSCT group (n=31) had a relatively high median baseline bone marrow blast count (58%) and a median of 4 prior treatment regimens ( Table 1). Six (19%) of these patients had a CR (including the 1 patient with MDS), and 3 (10%) patients had a CR with incomplete count recovery (CRi) resulting in a 29% composite complete remission (CRc) rate. One patient had a morphologic leukemia-free status, resulting in a 32% ORR. Half the responders received monotherapy, and half received a combination of olutasidenib and azacitidine. Two patients were enrolled in the monotherapy maintenance cohort while in CR to prior HSCT; one of these patients maintained a CR for 13 months before progressing, and the other patient progressed and discontinued due to a new central nervous system disease ( Table 2). For the 10 patients with responses (ORR), the median duration of response was 7.1 months (range 1-23.4+). At data cutoff, 2 patients were ongoing responders, 3 patients proceeded to a second HSCT, and 3 to donor lymphocyte infusion (DLI). The post-IVO group (n=9) had a high median baseline bone marrow blast count (83%) and a median of 4 prior treatment regimens ( Table 1). Two patients (22%) achieved a CR; both CRc and ORR were 22% ( Table 2). Both responders had received olutasidenib in combination with azacitidine. One responder proceeded to HSCT after 3.1 months, and one responded for 5.6 months and then had disease progression. Response rates in the post-VEN group (n=20) included CR in 6 patients (30%), CRh in 1 (5%), and CRi in 2 (10%) resulting in a CRc of 9 (45%) and an ORR of 45% ( Tables 1 and 2). One responder received combination olutasidenib and azacitidine. Two patients were enrolled in the monotherapy maintenance cohort while in CRi to prior treatment; both improved their response to a CR. Median duration of response was not reached and ongoing at 28.5 months. At data cutoff, 5 patients continued treatment, and 15 had discontinued. Conclusions. This descriptive analysis suggests that olutasidenib alone or in combination with azacitidine may induce complete remissions in patients with mIDH1 AML or MDS that is R/R to VEN, IVO or even HSCT. Further investigation of these difficult-to-treat subpopulations is needed. Although only a small number of patients receiving maintenance therapy were included in this analysis, the data show that maintenance of a CR and even improvement of response from CRi to CR is possible with olutasidenib.

Explorations of Post-DLI Low-Dose Cyclophosphamide for Preventing Severe aGVHD

Background Relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a serious life-threatening complication. The granulocyte colony-stimulated factor mobilized donor lymphocyte infusions (gDLI) combined with chemotherapy is currently a commonly used treatment method. Nevertheless, gDLI may cause so severe acute graft-versus-host disease (aGVHD) as to impact prognosis. Posttransplant cyclophosphamide (PTCy) has been the backbone for GVHD prophylaxis by inducing tolerance to minor histocompatibility antigens in recipients, while the application of post-gDLI low-dose cyclophosphamide (PDCy) for GVHD prophylaxis has not yet been attempted. Methods To explore this possibility, a retrospective study was conducted. 20 patients relapsing after HSCT were administered 20 mg/kg/d cyclophosphamide（Cy）on day 3 (for matched related transplantation) or on days 3 and 4 (for haplo-identical or unrelated transplantation) after gDLI to prevent aGVHD (the PDCy group). Furthermore, through propensity score matching, 58 matched controls received other (for HID and URD) or no (for MSD) immunosuppressive therapy for GVHD prophylaxis (the Non-Cy group). Patients from the PDCy and the Non-Cy group were matched by transplantation type, CD3+ T cell counts, and age and gender of recipients and donors. An MRD complete response was undetectable MRD lower than 0.01% with qPCR, and a reduction of at least one-log fold in MRD load post gDLI was defined as an MRD response. Results The PDCy group comprised 9 and 11 men and women, respectively, with a median age of 38 (34-52, IQR) years. The Non-Cy group comprised 34 and 24 men and women, respectively, with a median age of 40 (29-47, IQR) years. The infused CD3+T cell dose in the PDCy and Non-Cy group was 3.0 *10^7/kg and 2.9 *10^7/kg, respectively (p=1.0). The interval between HSCT and gDLI was longer in the PDCy group when compared to the Non-Cy group (30.8 vs 6.5 months, p<0.01). And first recurrence accounted for a higher percentage in the Non-Cy group (93% vs 45%, p<0.01). Other transplant- and DLI-specific data did not exhibit any significant difference between the groups. With a median follow-up of 4.8 (0-37.1) months, the PDCy group had lower cumulative incidence of severe aGVHD (III-IV, 5% vs 31%, p=0.02; II-IV, 25% vs 52%, p=0.04, figure 1A, B), but no significant differences existed in 4-month OS (64% vs 59%, p=0.51, figure 1E) and NRM (20% vs 19%, p=0.5, figure 1D). Among 49 patients undergoing salvage treatment (PDCy group, n=16; Non-Cy group, n=33), there was no statistical difference in 4-month CIR (20% vs 47%, p=0.11, figure 1C) and rates of objective response (PR+CR, 68.8% vs 54.5%, p=0.6). Among 29 patients undergoing preemptive treatment (PDCy group, n=4; Non-Cy group, n=25), there was no statistical difference in the rates of MRD complete response (25% vs 42%, p=1) and MRD response (25% vs 50%, p=0.6) between the PDCy group and the Non-Cy group. The PDCy regimen didn't increase the incidence of severe bacterial infection (45% vs 33%, p=0.3), bacteremia (25% vs 16%, p=0.11), fungus infection (20% vs 29.3%, p=0.4), virus infection (5% vs 19%, p=0.1), hemorrhagic cystitis (n=1 vs n=1), and cardiac events (n=0 vs n=0). Conclusions In conclusion, on the premise of safety, the PDCy regimen could effectively protest against severe aGVHD after gDLI while preserving therapeutic response rates. However, the research results still require verification through longer follow-up and large prospective randomized studies.

A Phase I, Single-Arm, Open Label, Dose Escalation, Multicenter Study of Off-the-Shelf Natural Killer (NK) Cells (SAR445419) in Patients with Relapsed/Refractory Acute Myeloid Leukemia (R/R AML)

Background and Significance: SAR445419 (SAR'419) is an allogeneic, off-the-shelf, donor-derived, natural killer (NK) cell investigational immunotherapy. NK cells in SAR'419 are expanded ex vivo using PM21 particles expressing membrane-bound interleukin-21 and 4-1BB ligand from the plasma membranes of feeder cells (Ciurea et al, Leukemia, 2022). Ex vivo, PM21 expansion allows for large numbers of hyperactive NK cells to be generated and administered, thereby increasing therapeutic potential to patients. It is hypothesized that mature, fully functional NK cells expanded ex vivo will have superior anti-tumor activity, favorable safety profile, and can induce remissions, enabling patients to receive curative allogeneic hematopoietic stem cell transplantation (HSCT), thereby improving outcomes in patients with R/R AML. Two phase 1/2 dose-escalation and expansion studies (NCT01787474, NCT02809092) of ex vivo expanded haploidentical donor NK cells infused as 6 doses over a course of 2 weeks, after fludarabine and cytarabine chemotherapy, demonstrated this approach to be feasible with favorable safety profile in patients with R/R AML. This study aims to determine the candidate dose(s), evaluate safety, tolerability, and preliminary anti-tumor activity of SAR'419 administered after fludarabine and cytarabine chemotherapy for adult patients with R/R AML. Study Design and Methods: This is a phase 1, single-arm, dose escalation study (NCT05712278). The primary objective is to determine the candidate dose(s) based on the evaluation of dose-limiting toxicities (DLTs). The DLT evaluation period is from start of chemotherapy (Day -6) until 28 days after the first administration of SAR'419, starting at the lowest dose. Eligible patients will be administered fludarabine, 30 mg/m 2/day and cytarabine, 2 g/m 2/day (Day -6 to Day -2) for 5 days followed by 6 doses of SAR'419 (intravenous) given thrice weekly over 2 weeks beginning on Day 1. Approximately 12 DLT-evaluable patients will be enrolled. The treatment period will start from the first administration of chemotherapy (Day -6) until end of treatment on Day 56, and subsequently patients will have follow-up visits every 2 months. Dose escalation will be guided by the modified toxicity probability interval 2 design. Key secondary objectives include characterization of overall safety, tolerability, HSCT rate, impact of SAR'419 on infection, and preliminary anti-leukemic activity of SAR'419 (as per international working group criteria). Biomarkers in whole blood (including serum and plasma) and bone marrow (aspirates-liquid, and biopsy-solid) will be evaluated to assess the impact of SAR'419 treatment. Key inclusion criteria are adult participants with confirmed diagnosis of R/R AML according to World Health Organization classification, including participants with relapsed AML in the bone marrow after allogeneic HSCT (including those who have received donor lymphocyte infusions); with or without concomitant or with isolated central nervous system or extramedullary disease who have received at least 1 prior line of therapy for AML. Key exclusion criteria are participants with a second primary malignancy requiring active therapy (adjuvant hormonal therapy is allowed); with known acquired immunodeficiency syndrome or human immunodeficiency virus disease requiring antiretroviral treatment or having active hepatitis B or C infection, or symptomatic severe acute respiratory syndrome coronavirus 2 infection; and who are receiving >10 mg/day of oral prednisone or equivalent. In the safety analyses, all DLT modeling will be performed on the DLT-evaluable population or those who experienced a DLT. All other safety analyses will be performed on the safety population using descriptive statistics. This study is actively recruiting at this time and is being conducted in the United States.

Opsoclonus-Myoclonus Syndrome and Acquired Von Willebrand Syndrome As Manifestations of Graft-Versus-Host Disease

Background: Opsoclonus-myoclonus syndrome (OMS), known as dancing eye syndrome, is a rare neurological disorder characterised by a triad of opsoclonus (rapid, involuntary eye movements), myoclonus (uncontrolled muscle twitches) and ataxia. The syndrome is usually seen in children, often associated with neuroblastoma, and is extremely rare in adults. The pathogenesis is thought to be immune in nature and aetiology is variable although most cases are associated with solid tumors or after an infection. Treatment involves a combination of managing the underlying condition and immunosuppression. Acquired von Willebrand disease is a rare acquired bleeding disorder with a range of different aetiologies including lymphoproliferative, myeloproliferative, cardiovascular and autoimmune disorders. The pathogenic mechanism, in most cases is due to increased plasma clearance of von Willebrand factor (vWF) caused by such mechanisms as antibodies, cell adsorption, shear stress or increased proteolysis. Therapeutic approaches focus on prevention of bleeding through supportive treatment, and management of the underlying cause. To the best of our knowledge, this is the first case of opsoclonus-myoclonus syndrome with concurrent acquired von Willebrand disease as manifestations of graft versus host disease. Case: Here we present the case of a 47-year-old man presenting with opsoclonus myoclonus syndrome and concurrent acquired von Willebrand disease as manifestations of graft versus host disease. The patient had a background of relapsed myeloid sarcoma after haploidentical allogeneic haematopoietic stem cell transplant with subsequent chemotherapy to achieve full remission. He received donor lymphocyte infusion as consolidation therapy to enhance the graft-versus leukaemia effect. Six weeks following donor T cell infusion of 1 x 10^6 CD3 cells/kg, he presented with initial features suggestive of graft versus host disease (GVHD) of the digestive tract and subsequently developed neurological manifestations in the form of rapid, repetitive, involuntary and multidirectional eye movements; tremors and ataxia. Further tests prior to planned invasive testing revealed an additional new diagnosis of acquired von Willebrand disease, requiring supportive von Willebrand factor concentrate infusion. Multiple extensive investigations including cerebral MRI imaging, cerebrospinal fluid analysis and paraneoplastic antibodies were all negative for associated malignancy, infection, other autoimmune disorder or disease relapse. Skin, bone marrow examination and cross-sectional imaging confirmed complete remission with complete donor chimerism in all blood cell lineages. The patient commenced steroid immunosuppression for treatment of gastrointestinal GVHD with complete resolution of all symptoms and clinical parameters. Conclusion: We highlight a rare case of central nervous system involvement with graft versus host disease following cellular therapy, manifesting as the rare opsoclonus-myoclonus syndrome with associated acquired von Willebrand disease. The proposed pathological mechanism is the activation of host tissue antigen-specific T and B cells, resulting in autoantibody and cytokine-mediated effects upon neuronal tissue and coagulation proteins. Use of glucocorticoids in this case to suppress auto-antibody formation, block transcription of pro-inflammatory genes and induce lymphocyte depletion was rapidly effective and provided durable remission of symptoms, without recurrence on cessation of immunosuppression. This case highlights the need for close clinical and biochemical monitoring of patients during the risk period following allogeneic cellular therapy, for serious allo-immune manifestations not fitting classical descriptions of GVHD.

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.

Full CD34 Donor Chimerism Following CD34 Chimerism Directed Donor Lymphocyte Infusion Results in Reduced Relapse and Superior Overall Survival Compared with Mixed Chimerism Status in Allogeneic Haemopoietic Stem Cell Transplant Recipients

Introduction Peripheral blood CD34 donor chimerism (DC) is a clinically valuable method for detecting relapse of myeloid malignancies following allogeneic hematopoietic stem cell transplantation (alloHSCT). Our institution has recently shown that CD34 DC ≤80% was superior to CD3 DC for predicting relapse (1). While pre-emptive DLI has demonstrated efficacy in preventing relapse in the setting of falling whole unfractionated and CD3 DC, little has been reported about its utility in falling CD34 DC. Methods We conducted a retrospective analysis of consecutive patients who received DLI at the Alfred Hospital in Melbourne, Australia between January 2012 and January 2023. Peripheral blood CD34 DC was monitored in alloHSCT patients transplanted for myeloid malignancies or acute lymphoblastic leukaemia (ALL). CD34+ cells were enriched using magnetic beads and DC measured using an STR-PCR method, which had a sensitivity of 1%. DLI was administered to patients with CD34 ≤80% that did not respond to immunosuppression taper. DLI doses were based on donor type and recipient weight, with subsequent doses administered incrementally according to a predefined protocol. Starting doses were 3x10 6/kg for sibling donors, 1x10 6/kg for unrelated donors and 0.5x10 6/kg for haploidentical donors. DLI was discontinued upon achieving full DC (FDC) defined as CD34 DC > 95%, clinically significant graft-versus-host disease (GVHD), disease progression, or lack of available donor cells. Results One hundred and six patients received DLI between 2012 and 2023. Twenty-five of these patients received DLI for CD34 DC ≤80% without morphologic relapse. Eleven of 25 (44%) patients received DLI alone, while 14 (56%) patients received it in combination with other therapies, primarily a hypomethylating agent. At the time of DLI, 5 (20%) of the 25 patients had concurrent decreased CD3 DC ≤ 80%. The median follow-up period was 1.84 years (range 0.58 to 8.43 years). Sixteen of 25 (66.7%) patients achieved CD34 FDC while 8 (33.3%) had persistent CD34 mixed DC (CD34 MDC). One patient could not be assessed due to death from infection. Patient characteristics and outcomes according to response to DLI are presented in Table 1. The cumulative incidence of relapse was significantly lower in the CD34 FDC group (12.5% vs 75% at 2 years, p<0.001). Patients achieving CD34 FDC had improved progression-free survival (PFS) and overall survival (OS) compared to the CD34 MDC group (PFS median years not reached [95% CI 1.94-ND] vs 0.29 years [95% CI 0.09-ND], p<0.001; OS median years not reached vs 0.68 years [95% CI 0.58-ND], p=0.003; Figure 1). Rates of grade II-IV acute graft-versus-host-disease (aGVHD) post-DLI were 12.5% in those achieving FDC and MDC (Table 1). A significant proportion of patients (37.5%) achieving FDC developed moderate-severe cGVHD while the MDC group did not survive long enough to develop cGVHD (Table 1). The non-relapse mortality observed in the CD34 FDC group was 12.5% at 3 years post-DLI. Conclusion Our centre experience demonstrated that CD34-directed DLI reduced relapse risk and improved overall survival when FDC is restored in alloHSCT patients, with an acceptable toxicity profile. Only 20% of patients with CD34 MDC had concurrent CD3 MDC, highlighting the additional value of CD34 monitoring in guiding salvage DLI administration. In patients with CD34 MDC ≤80%, the target of DLI should be to achieve FDC (>95%) given the clear association with improved outcomes in this group. References 1/ Das TP, North D, Fleming SA, Tan JLC, Ivey A, Cummings NJ, Spencer A, Patil SS, Widjaja JML, Swain MI, Bourke C, O'Brien ME, Kliman DS, Curtis DJ. Peripheral Blood CD34 Donor Chimerism has Greater Clinical Utility Than CD3 for Detecting Relapse after Allogeneic Stem Cell Transplantation for Acute Myeloid Leukemia or Myelodysplastic Syndrome. Transplant Cell Ther. 2023 Jul;29(7):454.e1-454.e8. doi: 10.1016/j.jtct.2023.03.025.

Clinical Characteristics, Management, and Allogeneic Hematopoietic Cell Transplantation of Patients with Toll-like Receptor 8 Gain-of-Function Mutations

Toll-like receptor 8 gain-of-function (TLR8 GOF) is a recently described inborn error of immunity due to germline or somatic mutations in the TLR8 gene and characterized by severe neutropenia, infections, lymphoproliferation, humoral immune defects, and in some cases, bone marrow failure. Treatment of TLR8 GOF has been challenging to date, and guidance for optimal therapy is lacking. We describe the clinical characteristics and management of 10 patients with TLR8 GOF variants, including the first female patient and further clinical outcome on the 6 patients initially reported with this disease with a focus on the role of allogeneic hematopoietic cell transplantation (HCT). All but one patient were male ranging from 0.5 months to 28 years at age of symptom onset. Eight patients had somatic mutations with variant allele frequencies of 7-26%; two patients had germline mutations. The 9 male patients all had severe neutropenia and varying degrees of anemia and/or thrombocytopenia. Oral ulcers, recurrent infections, hepatosplenomegaly, and hypogammaglobulinemia were common. Immune phenotype was variable, ranging from severe lymphopenia to marked T cell lymphoproliferation. Several patients had an inverted CD4:CD8 T cell ratio and skewing of T cell subsets to the terminal effector memory phenotype. Increased double-negative T cells were also observed. All patients had low class switched memory B cells. Bone marrows were hypo- to hypercellular with marked myeloid hypoplasia, and in most cases, with increased T cells, large granular lymphocytes, and/or lymphoid aggregates. Neutropenia was initially responsive to granulocyte colony stimulating factor (GCSF) +/- steroids in several patients but ultimately become refractory to therapy. Immunomodulatory agents and chemotherapy were unsuccessful. Six patients underwent allogeneic HCT; two patients died at 8 and 18 years without definitive therapy; and one patient was lost to follow-up. The female patient had germline disease and presented with pure red blood cell aplasia at 2 weeks of life and is scheduled to undergo HCT. Transplant data were available for 5 of 6 patients who underwent HCT. Age at transplant was 3 to 21 years. One patient who received a graft from a 7/8 HLA-matched unrelated donor developed severe veno-occlusive disease and secondary graft failure. The patient underwent a second transplant and ultimately died. Another patient received a matched sibling donor HCT while critically ill from Candida lusitaniae and mucormycosis infection with associated multisystem organ failure. The patient engrafted with 100% donor chimerism but died on day +15 post-HCT. The remaining 3 patients are alive and well 8 months to 2 years post-HCT. Two patients received grafts from HLA-matched donors (1 sibling and 1 unrelated) with reduced intensity conditioning and cyclophosphamide, mycophenolate mofetil and vorinostat for graft-versus-host disease (GVHD) prophylaxis. The third patient underwent a haploidentical HCT with myeloablative conditioning and post-transplant cyclophosphamide. Post-HCT complications included acute skin and/or gastrointestinal GVHD that was responsive to therapy in all patients, and severe lung GVHD in one patient. There were no major infectious complications. All three patients had full donor chimerism in myeloid and T cells with complete resolution of disease phenotype at last follow-up. The patient for whom detailed transplant data is not available received a haploidentical HCT. He developed mixed myeloid chimerism for which he received multiple donor lymphocyte infusions and was alive at last contact >3 years post-HCT. TLR8 GOF should be considered in male and female patients with severe, unexplained neutropenia refractory to GCSF, and particular care should be taken when investigating for mutations given the disease-causing variant allele frequencies as low as 7%. Neutropenia and immune dysregulation were refractory to all therapies in our cohort, and TLR8 GOF was fatal in two patients who did not receive definitive therapy. Allogeneic HCT is curative and should be considered without delay for medically suitable patients.

Allogeneic Bone Marrow Transplantation from HLA-Identical or Haploidentical Donors for Children and Adolescents with Sickle Cell Disease: A Feasible Curative Option

The only curative option widely available for sickle cell disease (SCD) is bone marrow transplantation (BMT). Since few patients have a healthy matched sibling donor (MSD), haploidentical (haplo) donors have expanded the donor pool to approximately 90% of the patients. The addition of thiotepa to the Johns Hopkins conditioning therapy backbone dor haplo BMT showed durable engraftment and excellent overall survival. However, graft rejection has remained an important obstacle. Our challenge in these patients has been to augment the conditioning therapy to provide adequate engraftment, while maintaining a low transplant-related mortality. Methods: Retrospective data of all 26 patients with SCD followed by our group after MSD (n=7) and haplo (n=19) BMT performed in four different institutions. With a MSD, conditioning was rATG 4.5mg/kg, Busulfan (Bu, AUC 4,500), Fludarabine (Flu, 120mg/m²) or rATG, Bu, Cyclophosphamide (Cy, 200mg/kg) and with haplo donors, rATG 4.5mg/kg, Thiotepa 10mg/kg, Flu 150mg/m², Cy 29 mg/kg and TBI 2Gy. Patients were prepared to BMT with intense hypertransfusion/bleeding or erythrocytapheresis to keep HbS<30 and Hb~10g/dL, reticulocytes <10% for 2-3 months prior to BMT, with intensive chelation to keep ferritin <1,000ng/mL. The presence of donor specific antibodies would exclude the patient from undergoing BMT. Due to two consecutive secondary graft failures (no detectable donor cells despite initial engraftment) and two falling mixed chimerism among the first 13 patients undergoing haplo, the doses of the conditioning therapy were increased to Cy 50 mg/kg and TBI 4Gy in a single fraction, as previously used in our patients with aplastic anemia undergoing BMT. Six consecutive patients received this augmented conditioning therapy. All patients had serial chimerism measured by STR. The graft-versus-host disease (GVHD) prophylaxis included Cyclosporine - Methotrexate in MSD and post-transplant Cy/mesna, mycophenolate mofetil and sirolimus in haplo. Results: Between September 2016 and April 2023, 26 patients underwent BMT, with a median age of 11 years (4-20), half of them females, 7 with a MSD and 19 an haplo (4 siblings, 6 fathers, and 9 mothers); 77% of the donors had sickle cell trait. The graft was bone marrow in all but one patient with Sb thal and Hodgkin's lymphoma. The median follow-up was 15.5 months. All patients engrafted. BMT with MSD had excellent outcomes and stable chimerism. Among the first 13 patients undergoing haplo, secondary graft failure occurred very suddenly in two patients (CD34 cell doses of 2 and 8x10 6 CD34/kg), both male teenagers; two additional patients had a significant drop in chimerism up to 30% donor cells but responded to serial donor leukocyte infusions 1-5 x10 6 CD3/kg (2 and 6) and developed very mild skin GVHD. The other patients maintained > 89-100% donor cells. Thereafter, with the augmented conditioning regimen, all six consecutive patients have stable 100% donor chimerism. Acute II-III GVHD was observed in 34% and chronic GVHD in 15% of the patients. Viral reactivations (CMV, HHV6, BKV, HHV7 and EBV) were observed in all patients. Overall survival is 100% and rejection-free survival is 83% at 1 year (Figures 1, 2). Conclusions: Allogeneic BMT with an HLA-identical or haploidentical family donor was feasible to treat SCD with an excellent overall survival. With a larger cohort and longer follow-up, the long-term effects of this strategy will be thoroughly studied.

Automatic generation of alloreactivity-reduced donor lymphocytes and hematopoietic stem cells from the same mobilized apheresis product

IntroductionIn vitro or in vivo depletion of alloreactive T cells can facilitate haplo-identical hematopoietic stem cell transplantation (HSCT). Very satisfactory transplant outcomes were thus reported for TCRαβ/CD19-depleted hematopoietic stem/progenitor cell (HSPC) grafts. The current semi-automatic manufacturing process on the CliniMACS Plus, although robust, still requires a significant amount of manual labor to be completed. Towards advancing and further facilitating large scale cell processing, a new TCRαβ/CD19 depletion module combined with the previously described CD45RA depletion module (to serve as allo-reactivity attenuated donor lymphocyte infusion) was established on the CliniMACS Prodigy.MethodsWe evaluated six apheresis products from G-CSF-mobilized volunteer donors which were split automatically by the Prodigy, one portion each depleted of CD45RA+ or of TCRαβ+ and CD19+ cells. We investigated critical quality attributes for both products. Products were assessed for recovery of HSPCs and mature subsets, as well as depletion efficiency of targeted cells using flow cytometry. Effects of apheresis and product age post 48 h storage at 2–6 °C as well as freeze-thawing on product viability and recovery of WBC and HPSCs were assessed by flow cytometry.ResultsTen sequential automatic processes were completed with minimal hands-on time beyond tubing set installation. Depletion efficiency of CD45RA+ resp. TCRαβ+ and CD19+ cells was equivalent to previous reports, achieving mean depletions of 4 log of targeted cells for both products. HSPC products retained TCRγδ+ and NK cells. 48 h storage of apheresis product was associated with the expected modest loss of HSPCs, but depletions remained efficient. Depleted products were stable until at least 72 h after apheresis with stem cell viabilities > 90%. Freeze-thawing resulted in loss of NK cells; post-thaw recovery of viable CD45+ and HSPCs was > 70% and in line with expectation.ConclusionThe closed, GMP-compatible process generates two separate medicinal products from the same mobilized apheresis product. The CD45RA-depleted products contained functional memory T cells, whereas the TCRαβ/CD19-depleted products included HSPCs, TCRγδ+ and NK cells. Both products are predicted to be effectively depleted of GVH-reactivity while providing immunological surveillance, in support of haplo-identical HSCT.Graphical

Blinatumomab Redirects Donor Lymphocytes against CD19+ Acute Lymphoblastic Leukemia without Relevant Bystander Alloreactivity after Haploidentical Hematopoietic Stem Cell Transplantation.

Blinatumomab alone or with donor leukocyte infusions (DLI) has been used after allogeneic hematopoietic stem cell transplantation (HSCT) as a salvage therapy in relapsing patients with CD19+ hematological malignancies. It was effective in a fraction of them, with low incidence of Graft-versus-Host Disease (GvHD). Immunosuppressive drugs used as GvHD prophylaxis hinder T cell function and reduce the efficacy of the treatment. Because T cell-depleted haploidentical HSCT with donor regulatory and conventional T cells (Treg/Tcon haploidentical HSCT) does not require post-transplant immunosuppression, it is an ideal platform for the concomitant use of blinatumomab and DLI. However, the risk of GvHD is high because the donor is haploidentical. We treated two patients with CD19+ acute lymphoblastic leukemia (ALL) who had relapsed after Treg/Tcon haploidentical HSCT with blinatumomab and DLI. Despite the mismatch for one HLA haplotype, they did not develop GvHD and achieved complete remission with negative minimal residual disease. Consistently, we found that blinatumomab did not enhance T cell alloreactivity in vitro. Eventually, the two patients relapsed again because of their high disease risk. This study suggests that treatment with blinatumomab and DLI can be feasible to treat relapse after haploidentical transplantation, and its pre-emptive use should be considered to improve efficacy.

Coordinated Immune Cell Networks in the Bone Marrow Microenvironment Define the Graft Versus Leukemia Response with Adoptive Cellular Therapy

Donor lymphocyte infusion (DLI) is an established therapy for relapsed acute myeloid leukemia (AML) after hematopoietic stem cell transplant (HSCT), but response rates are poor (~20%). Interactions between leukemia and immune cells within the leukemia microenvironment may determine responsiveness to adoptive cellular immunotherapies. We hypothesized that systematic characterization of the leukemic marrow microenvironment over treatment course with DLI would define leukemia-immune cell interactions critical to response and hence provide mechanistic understanding of the graft-versus-leukemia (GvL) effect. In particular, we anticipated that use of multi-modal single-cell sequencing across patients and time points before and after therapy accounting for temporal dependencies would facilitate disentangling of the complex microenvironment of leukemic marrow. To infer cell-cell interactions in a temporally resolved fashion from single cell RNA-sequencing (scRNA-seq) data, we developed DIISCO, a Bayesian model using a Gaussian process regression network. In total, we profiled 30 bone marrow aspirates (14 pre- and 16 post-DLI) from 9 patients (5 responder [R], 4 nonresponder [NR]) with post-HSCT relapsed AML treated with DLI by scRNA-seq, single cell TCR sequencing (scTCR-seq) and surface protein characterization (scCITE-seq). We obtained 51,371 high-quality transcriptomes from the marrow samples along with 43,918 cells from the DLI products of these patients (4 R, 3 NR) for a total of 95,289 cells. Cells were clustered with Phenograph resulting in 57 clusters, including 2 CD4+ T cell, 6 CD8+ T cell (C0, C5, C21, C25, C26, C40), 3 NK, 10 B cell, 3 AML leukemia, and several myelo-erythroid clusters. DIISCO revealed a cascading response post-DLI that centered around CD8 C0, CD8 C5, AML, and a B cell cluster in R, not observed in NR. Furthermore, C0 appeared to expand after DLI only in R. We observed a strong negative interaction from CD8 C0 to AML after DLI, suggesting an anti-leukemia immune response, supporting the notion that immune cell populations formed a coordinated interactome associated with effective GvL. Evaluation of inferred interacting pairs using DIISCO and with the tool CellPhoneDB pointed toward an activation circuit in R between CD226 on CD8 C0 and PVR/NECTIN2 on AML. Conversely, inhibitory interactions between the exhaustion marker TIGIT on CD8 C0 and AML were observed in NR. Because DIISCO pinpointed CD8 C0 as a central hub for response, we characterized the identity of this cluster and its post-DLI dynamics. scTCR-seq revealed that this cluster, but not other T cell clusters, underwent marked clonal expansion after DLI ( Figure 1A). scRNA-seq revealed high expression of activation/effector genes ( GZMB, IFNG, CX3CR1, PRF1) compared to other T cells. The clonally expanded cells of C0 also highly expressed ZNF683, a transcription factor associated with T cell cytotoxicity. scCITE-seq demonstrated expression of CD45RA and CD57 but not CD28, consistent with an effector memory re-expressing RA (TEMRA) phenotype. We validated post-DLI expansion of CD8 TEMRA cells (CD3+, CD8+, CD62L-, CD45RA+) in AML R by multi-parameter flow cytometry in an independent cohort of 53 AML patients (29 R, 24 NR) with post-HSCT relapse treated with DLI at our institution (fold change 2.56 pre to post for R, p<0.001; 1.3 pre to post for NR, p=0.474). Moreover, to examine whether CD8+ TEMRA cells were specifically activated after encounter with leukemia, we devised an in vitromodified mixed lymphocyte reaction. For 6 AML R, post-DLI donor-derived CD8+ TEMRA cells that were cultured with pre-DLI recipient leukemia cells consistently demonstrated greater CD137 (p=0.004) and IFN-γ secretion compared to co-culture of the leukemia cells with other T cell subsets (p=0.015) ( Figure 1B). CD8+ TEMRA cells cultured in the absence of leukemia cells did not show elevated expression of either CD137 or IFN-γ, indicating antigen-specific functional activation of CD8+ TEMRAs but not other cell types after encounter with autologous leukemia. Our results reveal clonal expansion and tumor-specific activation of CD8+ TEMRA cells, supporting an antigen specific functional role of these cells in mediating GvL in AML DLI responders. Further evaluation of this CD8 TEMRA population with the ability to recognize leukemia cells may translate to strategies to optimize adoptive cellular therapy or predict responsiveness to DLI.

Pre-Emptive Donor Lymphocyte Infusion As Optimal Treatment for Relapsed Acute Myeloid Leukemias and Myelodysplastic Syndromes Following Allogeneic Stem Cell Transplantation: Insights from a French-Italian Study on 134 Patients

Disease relapse after allogeneic stem cell transplantation (allo-SCT) is the main challenge in achieving a cure for acute myeloid leukemias (AMLs) and myelodysplastic syndromes (MDSs). The treatment of disease relapse can be offered in case of full hematological recurrence or in the presence of minimal residual disease (MRD) positivity and/or mixed chimerism. The latter approach is defined as pre-emptive therapy. Overall, treatment strategies may include chemotherapy, molecular-target drugs, hypomethylating agents (HMAs), donor lymphocyte infusions (DLIs) or even a second allo-SCT. We retrospectively analyzed a cohort of 553 AML and MDS adult patients consecutively allotransplanted between January 1, 2015, and December 31, 2021, at Saint-Antoine University Hospital, Paris (France) (n=420) and Spedali Civili di Brescia, Brescia (Italy) (n=133). 134/553 (24%) patients relapsed and were included in this study. 103/134 (77%) were subsequently treated. Among patients who received treatment, 40/103 (39%) were treated with a DLI-based regimen. In this group, 9 received DLIs alone, while the others also received HMAs (4 cases), HMAs+ venetoclax (12 cases), FLT3-inhibitors (3 cases), intensive chemotherapy (5 cases), a second allo-SCT (5 cases), or other therapies (2 cases). The remaining 63/103 (61%) patients were treated with regimens that did not include DLIs. Of these, 10 received HMAs, 27 HMAs+ venetoclax, 4 FLT3-inhibitors, 6 intensive chemotherapy, 9 a second allo-SCT, and 7 other therapies. With a median follow up of 1.6 years, the estimated 1-, 2- and 5-year OS rate of the 134 patients who relapsed after allo-SCT was 32%, 18 %, and 11 %, respectively. The OS of patients treated after allo-SCT relapse was 40 %, 20%, and 15% at 1, 2 and 5 years compared with 6 %, 3%, and 0 % for patients who did not receive therapy, respectively ( p<0.01).Moreover, the OS was significantly better in patients treated in a pre-emptive setting than in those treated for a morphological relapse (OS at 1, 2 and 5 years was 60%, 36% and 30%, respectively, for patients treated in a pre-emptive setting versus 26%, 12%, and 6%, respectively, for patients treated in hematological relapse ( p<0.01) (Figure 1A). Focusing on post-relapse treatment, patients who received DLI-based regimens had a 1-, 2- and 5-year OS of 55%, 32% and 32%, respectively, compared to 27%, 16% and 7% for patients treated with other therapies ( p<0.01). Among patients who received DLIs, 50% of patients were treated in the pre-emptive setting. Conversely, in the group of patients treated without DLI, only 22% were treated in the pre-emptive setting, reflecting the more advanced phase of relapse in this group. Finally, when patients were analyzed by the treatment strategy (pre-emptive therapy vs therapy in hematological relapse) and DLI administration (yes vs no), the best outcome was achieved when the relapse was promptly detected and pre-emptive therapy was started, particularly if a DLI was administered. Indeed, the 1-year OS of patients treated in the pre-emptive setting was 67% for those receiving a DLI and 54% for those treated without DLI, while for patients with hematologic relapse, the 1-year OS was 43% for those treated with the DLI-based regimen and 17% for those treated without DLI ( p<0.01) (Figure 1B). On multivariate analysis, treatment with DLI after relapse, and pre-emptive setting were independent factors associated with better OS ( p=0.03 and p<0.01), respectively. Our data showed that relapse treatment with pre-emptive therapies is associated with improved outcomes and this effect is more evident when combined with a DLI . These results confirm the primary role of the graft- versus leukemia effect in the treatment of relapse and underscore the importance of its early identification through MRD monitoring.

Fractionated Gemtuzumab Ozogamicin Followed By Non-Engraftment Donor Leukocyte Infusions for Relapsed/Refractory Acute Myeloid Leukemia

Background: Previous studies have shown durable AML patient responses following donor lymphocyte infusions (DLI) in the absence of engraftment in both the frontline and relapsed setting (Dey et al, BJH 2005, Colvin et al, BBMT 2009, Ai et al, Blood 2010). Herein we report results from the completed dose-escalation phase from our trial of fractionated dosing of gemtuzumab ozogamicin (GO) followed by non-engraftment mismatched allogeneic donor cells infusion in patients with relapsed/refractory (R/R) AML. In this trial, instead of engraftment, it is postulated that donor cells serve as a potent immune stimulator. Study Design and Methods: In this phase I/II study, GO 6-9mg/m 2 total dose was administered as a fractionated 2-3mg/m 2 dose (capped at 4.5mg) on days 1, 4, and 7, followed by infusion of HLA-mismatched (0-3/6 antigens) related donor cells on day 8 [NCT03374332]. The dose-escalation phase 1 cohort used a 3+3 design combining GO with donor leucocyte infusion (DLI) at dose 1-2x10 7 CD3+ cells/kg (Dose Level 1) or 1-2x10 8 CD3+ cells/kg (Dose Level 2). Patients enrolled in the phase II portion were treated at the maximum tolerated dose (MTD) level. Lab correlative studies were drawn pre and post DLI to assess immune effector cells and leukemic blast surface expression as well as cytokine release profiles. Patients 18 years of age and older with histologically confirmed R/R AML who had recurrence or progression after at least 1 prior standard treatment were eligible. Enrollees must have had no active systemic infections and adequate lung, liver, cardiac, and renal function with an ECOG PS 0-1. Patients in CR or CRi after initial treatment could receive up to 2 additional treatments with GO+DLI. Results: Eleven patients (7 men and 4 women) were enrolled into the study. The median age was 72 years (54-81 years) and patients had received 1-4 previous lines of treatment. All patients were refractory to hypomethylating agents and venetoclax. No patient was a candidate for allogeneic stem cell transplant at the time of study enrollment. Ten patients received 3/6 and one patient received 1/6 HLA mismatched donor cells from a first- or second-degree relative. The MTD was determined to be 2x10 8 CD3+ cells/kg. Three patients were enrolled on dose level 1 and eight patients were enrolled on dose level 2. Treatment was well tolerated with minimal Grade 3 or 4 non-hematologic adverse events attributable to the study ( Table 1). One patient at dose level 2 developed septic shock and died prior to DLI while another patient at dose level 2 developed cryptococcal fungemia and died within one week of DLI. No donor experienced a Grade 3 or 4 adverse event. All patients had fever post DLI that fit criteria for Grade 1 or 2 CRS while one patient developed Grade 3 CR. Two patients with Grade 2 CRS and one patient with Grade 3 CRS received tociluzimab. One patient attained a CR after one cycle of GO+DLI. All other patients had progressive disease. Ultimately the study was stopped early due to futility. Median IL-6 and IL-8 levels were higher in the first 3 patients at dose level 2 (605.5pg/mL and 218.5 pg/mL) compared to the three patients in dose level 1 (79pg/mL and 57.8 pg/mL) but the difference was not statistically significant. IL-10 levels were significantly higher in dose level 2 than dose level 1(125 vs 14.8 pg/mL, p=0.05) Conclusions: The MTD of DLI in combination with GO for relapsed/refractory AML patients is 2x10 8 CD3+ cells/kg. Treatment in an older relapsed/refractory population was well tolerated. Patients reliably developed fevers post DLI with Grade1 or 2 CRS. However, there was no evidence that fever development corresponded to a clinical benefit or that the addition of non-engraftment DLI augmented the effect of single agent gemtuzumab. Additional analysis of cytokine release profiles for the remaining patients as well as immune checkpoint inhibition on leukemic blasts for all patients will be presented at the meeting.