Graft-versus-host Disease Grade Research Articles

Treosulfan is a safe and effective alternative to busulfan for conditioning in adult allogeneic HSCT patients: Data from a single center.

Type of conditioning regimen impacts the outcome of patients who undergo allogeneic HSCT since graft versus host disease (GVHD), infections, regimen related toxicities (RRT) are important causes of post-transplant mortality. Despite the RRT profile of busulfan, it is frequently used worldwide. Treosulfan has advantages in terms of dose of administration, lower incidence of sinusoidal obstruction syndrome and lower neurotoxicity. We retrospectively investigated outcomes of patients who underwent allogeneic HSCT with treosulfan or busulfan based conditioning regimens in our institution. Treosulfan was administered to 94 patients while 85 patients received busulfan. Our outcomes were RRT, chronic and acute GVHD, relapse related mortality (RRM), non-relapse mortality, and fungal infection. The clinical follow up data, regarding the primary and secondary endpoints of our study, of the patients who received treosulfan or busulfan based conditioning regimens were statistically analyzed. The median follow-up was 14 months for the treosulfan group while it was 11 months for the busulfan group (p = 0.16). RRT was 11.7% and 7.1% for treosulfan and busulfan respectively. The incidence of extensive chronic GVHD was less frequent in the treosulfan group compared to the busulfan group (15.7% vs. 32.1%) (p < 0.001). The incidence of acute GVHD (Grade 3 or higher) was 32.2% in the treosulfan group while it was 31.6% in the busulfan group. The RRM was 17% in the treosulfan group while it was 34% in the busulfan group. The non-relapse mortality was 35.5% and 29.4% in the treosulfan group and in the busulfan group respectively (p = 0.962). Treosulfan, with a lower RRM, lower chronic GVHD incidence and with a similar RRT profile appears to be a safe alternative to busulfan.

Treatment responsive acute GVHD after posttransplant cyclophosphamide-based prophylaxis: incidence and clinical outcomes

BackgroundPost-transplant cyclophosphamide (PTCy) following hematopoietic cell transplantation (HCT) has emerged as standard of care for graft-versus-host disease (GVHD) prevention in adult patients without increasing malignant relapse. We previously defined acute GVHD (aGVHD) treatment response categories as either corticosteroid-sensitive (SS), dependent (SD), or resistant (SR) based on response to first-line corticosteroids and reported their clinical outcomes following non-PTCy based prophylaxis. Over one-third of patients developed aGVHD requiring systemic therapy. Cases were predominantly SR with a 14% overall incidence of SR aGVHD. The incidence and clinical outcomes of these three distinct aGVHD treatment response groups following PTCy-based prophylaxis is not well described. ObjectiveThe objective of this retrospective, single-institution, cohort study was to assess the incidence and clinical outcomes of SS, SD, and SR aGVHD following HCT with PTCy-based prophylaxis using a prophylactic regimen of PTCy, tacrolimus, and mycophenolate mofetil (MMF). Study designWe included 196 consecutive (2017-2021) adult and pediatric patients receiving allogeneic HCT for malignant and non-malignant disorders at the University of Minnesota. Patients received PTCy on days +3 and +4 plus tacrolimus and mycophenolate mofetil prophylaxis. Bone marrow (BM) and peripheral blood stem cell (PBSC) graft sources and related and unrelated donors were included. Recipients received myeloablative (MAC) or reduced-intensity conditioning (RIC) regimens. ResultsIn 196 allografts, 54 (28%) developed aGVHD before day +180 with median time to onset of 50 days (IQR 34-71 days). Of those, 32 patients (16% overall) developed maximum grade II-III aGVHD requiring systemic corticosteroids with the following response: 13 (41%) SS, 10 (31%) SD, and 9 (28%) SR. Overall incidence of SR aGVHD was 4.6%. Only 12 patients (6%) developed maximum grade III aGVHD while none had grade IV aGVHD. 2-year overall survival analyzed from 80 days after initiation of systemic treatment was similar in the SS and SD groups (77 and 75%, respectively), comparable to those without aGVHD (81%), but was lowest in the SR group (20%) with GVHD being the primary cause of death. Non-relapse mortality (NRM) was highest in the SR group. MN high risk and higher GVHD grade at onset were risk factors for developing SR aGVHD. ConclusionsOverall, we report a low incidence (16%) of aGVHD requiring systemic corticosteroids with PTCy-based prophylaxis. Acute GVHD cases were predominantly SS aGVHD with a lower incidence of SD and SR aGVHD. Our findings suggest that PTCy-based prophylaxis reduces rates of treatment resistant aGVHD. Patients with SR aGVHD had the worst clinical outcomes and poorest survival. Those with SS and SD aGVHD had similar clinical outcomes, both better than SR aGVHD.

Post-transplant cyclophosphamide, calcineurin inhibitor, and mycophenolate mofetil compared to anti-thymocyte globulin, calcineurin inhibitor, and methotrexate combinations as graft-versus-host disease prophylaxis post allogeneic stem cell transplantation from sibling and unrelated donors in patients with acute myeloid leukemia: a study on behalf of the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation.

Post-transplant cyclophosphamide plus calcineurin inhibitor (CNI)(tacrolimus or cyclosporine A) plus mycophenolate mofetil (PTCy/TAC or CSA/MMF) and anti-thymocyte globulin plus CNI (tacrolimus or cyclosporine A) plus methotrexate (ATG/TAC or CSA/MTX) are common graft-versus-host disease (GVHD) prophylaxis regimens. We compared the two regimens in patients with acute myeloid leukemia (AML) undergoing allogeneic transplantation from matched siblings or unrelated donors. 402 received PTCy/TAC or CSA/MMF and 5648 received ATG/TAC or CSA/MTX. Patients in the PTCy-based group were younger (48.7 vs. 51.5 years, p = 0.024) and there was a higher frequency of patient cytomegalovirus seropositivity and female donor to male patient combination in this group (77.8% vs. 71.8%, p = 0.009 and 18.4% vs. 14.4%, p = 0.029, respectively). More patients in the PTCy-based group received reduced-intensity conditioning (51.5% vs. 41%, p < 0.0001). No differences were observed in the incidence of acute GVHD grade II-IV and III-IV (21.2% vs. 20.4%, p = 0.92 and 8.1% vs. 6%, p = 0.1) or 2-year total and extensive chronic GVHD (33.7% vs. 30%, p = 0.09 and 10.7% vs. 11.2%, p = 0.81) between the groups. In the multivariate analysis, all transplant outcomes did not differ between the groups. PTCy/CNI/MMF and ATG/CNI/MTX are alternative regimens for GVHD prophylaxis in AML patients.

Comparable outcomes of allogeneic peripheral blood versus bone marrow hematopoietic stem cell transplantation from a sibling donor for pediatric patients.

Traditionally, bone marrow (BM) has been preferred as a source of stem cells (SCs) in pediatric hematopoietic SC transplantation (HSCT); however, the use of peripheral blood SCs (PBSC) has recently increased. With advancing graft-versus-host disease (GVHD) prophylaxis, whether the BM is still a better SC source than PB in sibling donor HSCT remains controversial. Here, we compared the results of BM transplantation (BMT) and PBSC transplantation (PBSCT) in pediatric patients with malignant or non-malignant diseases receiving sibling HSCT using a total of 7.5mg/kg of anti-thymocyte globulin (ATG). We retrospectively reviewed children who received HSCT from a sibling donor between 2005 and 2020 at Seoul National University Children's Hospital. Of the 86 patients, 40 underwent BMT, and 46 underwent PBSCT. Fifty- six patients had malignant diseases, whereas thirty patients had non-malignant diseases. All conditioning regimens comprised ATG. Busulfan-based myeloablative conditioning regimens were administered to patients with malignant diseases and approximately half of those with non-malignant diseases. The remaining half of the patients with non-malignant diseases were administered cyclophosphamide-based reduced- intensity conditioning regimens. According to studies conducted at our center, all BM donors received G-CSF before harvest to achieve early engraftment. In all 86 patients (47 males and 39 females), the median age at the time of HSCT was 11.4 (range, 0.7 - 24.6) years. The median follow-up period was 57.9 (range, 0.9-228.6) months, and the corresponding values for those with BM and PBSC were 77 (range, 2.4-228.6) months and 48.7 (range, 0.9-213.2) months, respectively. Engraftment failure occurred in one patient with BM and no patient with PBSC. The cumulative incidence of acute GVHD with grades II-IV was higher in PBSC (BM 2.5%, PBSC 26.1%, p = 0.002), but there was no significant difference in those with grades III-IV acute GVHD (BM 0%, PBSC 6.5%, p = 0.3703) and extensive chronic GVHD (BM 2.5%, PBSC 11.6%, p = 0.1004). There were no significant differences in treatment-related mortality (TRM) (BM 14.2%, PBSC 6.8%, p = 0.453), 5-year event-free survival (EFS) (BM 71.5%, PBSC 76.2%, p = 0.874), and overall survival (OS) rates (BM 80.8%, PBSC 80.3%, p = 0.867) between BM and PBSC in the univariate analysis. In the multivariate analysis, which included all factors with p < 0.50 in the univariate analysis, there was no significant prognostic factor for EFS or OS. There was no significant difference in the relapse incidence between BM and PBSC among patients with malignant diseases (BM 14.2%, PBSC 6.8%, p = 0.453). Additionally, there were no significant differences in the TRM, 5-year EFS, and OS rates between malignant and non-malignant diseases nor between the busulfan-based myeloablative regimen and reduced-intensity chemotherapy using cyclophosphamide. In this study, we showed no significant differences in EFS, OS, TRM, and GVHD, except for acute GVHD grades II-IV, between BMT and PBSCT from sibling donors, using ATG (a total of 7.5mg/kg). Therefore, PB collection, which is less invasive for donors and less labor-intensive for doctors, could also be considered an acceptable SC source for sibling donor HSCT in children.

Graft-versus-host disease and impact on relapse in myelofibrosis undergoing hematopoietic stem cell transplantation

Allogeneic hematopoietic stem cell transplantation (alloHSCT) remains the only curative treatment for myelofibrosis (MF). Relapse occurs in 10–30% and remains a major factor for dismal outcomes. Previous work suggested that graft-versus-host disease (GVHD) might be associated with risk of relapse. This study included 341 patients undergoing their first (n = 308) or second (n = 33) alloHSCT. Anti-T-lymphocyte or antithymocyte globulin was used for GVHD prophylaxis in almost all patients. Median time to neutrophile and platelet engraftment was 13 days and 19 days, respectively. The cumulative incidence of acute GVHD grade II-IV was 41% (median, 31 days; range, 7–112). Grade III-IV acute GVHD was observed in 22%. The cumulative incidence of chronic GVHD was 61%. Liver was affected in 23% of acute GVHD cases and 46% of chronic GVHD cases. Severe acute GVHD was associated with high non-relapse mortality. The development of acute GVHD grade II and moderate GVHD was an independent factor for reduced risk for relapse after transplantation without increased risk for non-relapse mortality, while especially acute GVHD grade IV was associated with high non-relapse mortality. Last, we identified that ongoing response to ruxolitinib, accelerated-phase MF at time of transplantation and splenectomy prior to transplantation were independent predictors for relapse.

A Day 14 Endpoint for Acute GVHD Clinical Trials

The overall response rate (ORR) 28 days after treatment has been adopted as the primary endpoint for clinical trials of acute graft versus host disease (GVHD). However, physicians often need to modify immunosuppression earlier than day (D) 28, and non-relapse mortality (NRM) does not always correlate with ORR at D28. We studied 1144 patients that received systemic treatment for GVHD in the Mount Sinai Acute GVHD International Consortium (MAGIC) and divided them into a training set (n=764) and a validation set (n=380). We used a recursive partitioning algorithm to create a Mount Sinai model that classifies patients into favorable or unfavorable groups that predicted 12 month NRM according to overall GVHD grade at both onset and D14. In the Mount Sinai model grade II GVHD at D14 was unfavorable for grade III/IV GVHD at onset and predicted NRM as well as the D28 standard response model. The MAGIC algorithm probability (MAP) is a validated score that combines the serum concentrations of suppression of tumorigenicity 2 (ST2) and regenerating islet-derived 3-alpha (REG3α) to predict NRM. Inclusion of the D14 MAP biomarker score with the D14 Mount Sinai model created three distinct groups (good, intermediate, poor) with strikingly different NRM (8%, 35%, 76% respectively). This D14 MAGIC model displayed better AUC, sensitivity, positive and negative predictive value, and net benefit in decision curve analysis compared to the D28 standard response model. We conclude that this D14 MAGIC model could be useful in therapeutic decisions and may offer an improved endpoint for clinical trials of acute GVHD treatment.

Acute graft versus host disease 1976-2020: reduced incidence and predictive factors.

We studied the incidence of acute graft versus host disease (GvHD) and its outcome in three consecutive time frames (year <2000; 2000-2010; >2010), in 3,120 patients allografted in two transplant Centers between 1976 and 2020. The median age increased over the three periods from 32 to 42 to 54 years (p < 0.00001). The median day of onset of GvHD in the three periods was day +14, day +16, and day +30, respectively (p < 0.0001). The cumulative incidence (CI) of GvHD grades II-IV in the three periods was 47, 24, and 16%, respectively (p < 0.00001). The CI of GvHD grades III-IV was 13, 5, and 4% (p < 0.001). In multivariate analysis, significant predictive factors for GvHD II-IV, on top of year of transplant, were anti-thymocyte globulin (ATG) (RR 0.67, p > 0.001); post-transplant cyclophosphamide (PTCY) (RR 0.41, p < 0.001), a family mismatched donor (RR 1.31, p = 0.03) a matched unrelated donor (RR 2.1, p < 0.001), an unrelated mismatched donor (RR1.8, p = 0.001), donor age above 40 years (RR 1.27, p < 0.001), hematological malignancy-as compared to aplastic anemia (RR 2.3, p < 0.001). When selecting only GvHD grade II, in a multivariate analysis, there was a significant reduction of transplant-related mortality (TRM) for patients grafted in 2001-2010 (RR 0.62, p < 0.0001) and for patients grafted in 2011-2020 (RR 0.35, p < 0.0001) as compared to grafts before the year 2000. A similar reduction in time was seen for patients with GvHD grades III-IV. The overall TRM in the three periods was 30, 22, and 16% (p < 0.0001) and survival was 47, 51, and 58% (p < 0.0001). Relapse risk was unchanged. In conclusion, we showed improved prevention of acute GvHD with time, together with a significant delay in the onset of the disease. Treatment of GvHD has also improved over time, as suggested by both reduced TRM and improved survival in more recent transplant periods.

Efficacy of Triple Prophylaxis for Prevention of Graft-Versus-Host Disease in Matched Sibling Allogeneic Peripheral Blood Hematopoietic Stem Cell Transplantation in Pediatric Patients

BACKGROUND: Graft-versus-host disease (GVHD) continues to compromise the overall success of allogenic hematopoietic stem cell transplantation. It is the most important cause of morbidity and non-relapse mortality after allogeneic hematopoietic stem cell transplantation for malignant disease. In adults, post-transplant cyclophosphamide (Cy-PT) has been shown to be a feasible, economically accessible, and effective strategy to reduce the incidence of GVHD in matched sibling hematopoietic stem cell transplantation in combination with a calcineurin inhibitor plus mycophenolate mofetil (MMF). AIM: Demonstrate the clinical benefit of Cy-PT plus a calcineurin inhibitor combined with MMF for GVHD prophylaxis in HLA-matched, related peripheral blood stem cell transplants in pediatric patients with malignant hematologic neoplasms compared to standard therapy in historical controls. METHODS: A retrospective study was conducted on 22 pediatric patients with malignant hematologic neoplasms who underwent HLA-matched related peripheral blood allogenic stem cell transplantation (alloSCT) between July 2012 and December 2022. A comparison was made between two groups, one with triple prophylaxis using Cy-PT, cyclosporine (CsA), and MMF, and a historical cohort that received standard GVHD prophylaxis based on CsA and methotrexate (MTX). Patients were identified from a hospital registry. Descriptive and inferential statistics will be reported, using SPSS version 25 for analysis. RESULTS: Twenty-two patients received HLA-matched alloSCT from first-degree relatives genotypically identical in HLA-A, HLA-B, and HLA-DRB1 alleles. The demographic characteristics of both groups are summarized in the table. No patient experienced primary graft failure, sinusoidal obstructive syndrome, or hemorrhagic cystitis. Acute GVHD grade II-IV did not develop in patients who received Cy-PT, whereas in the CsA/MTX prophylaxis group, 1 (9%) grade IV patient was observed who died due to this cause before day 100. Two patients (18%) developed Moderate/Severe GVHD in the CsA/MTX group, and 1 (9%) had moderate GVHD in the Cy-PT group. Two-year overall survival was similar between the two groups (Cy-PT 53% and CsA/MTX 67%), as was event-free survival (PTCy 48% vs. CsA/MTX 61%). CONCLUSION: Peripheral blood represents a feasible option in our setting due to rapid graft engraftment, short hospital stays, and low incidence of primary graft failure, but with a higher incidence of GVHD as reported in the literature. We explored the efficacy and safety of triple prophylaxis for HLA-matched alloSCTs from peripheral blood using Cy-PT + CsA + MMF in a small group of patients, with outcomes slightly better than standard prophylaxis. These preliminary results motivate us to continue with this new regimen.

Hematopoietic stem cell transplantation for B-thalassemia major with alemtuzumab

While matched related donor (MRD) allogeneic hematopoietic stem cell transplantation (HSCT) is a curative option for transfusion-dependent beta-thalassemia (TDT), the use of alternative sources has increased, resulting in the exploration of novel transplant-conditioning regimens to reduce the contribution of graft-versus-host disease (GVHD) and graft failure (GF) to transplant-related morbidity and mortality. Alemtuzumab is a CD52 monoclonal antibody that has been successfully incorporated into myeloablative conditioning regimens for other hematologic conditions, yet there have been limited studies regarding the use of alemtuzumab in HSCT for TDT. The purpose of this study was to evaluate engraftment, incidence of GVHD, and transplant related morbidity and mortality in patients with TDT who received alemtuzumab in addition to standard busulfan-based conditioning. The primary endpoint was severe GVHD-free, event-free survival (GEFS). Our cohort included 24 patients with a median age of 6.8 years (range 1.5-14.9). Eleven patients received a 10/10 MRD HSCT, eleven 10/10 unrelated donor (UD), and two mismatched UD. All patients achieved primary engraftment. For all patients, 5-year GEFS was 77.4% and 5-year overall survival (OS) was 91%. The 5-year cumulative incidence of GF (attributed to poor graft function) without loss of donor chimerism was 13.8% (95% CI: 4.5, 35.3). We report low rates of significant acute GVHD grade II–IV (12.5%) and chronic GVHD (4.4%). Younger age and MRD were associated with significantly improved GEFS, OS and EFS. Our results show that the use of alemtuzumab promotes stable engraftment, may reduce rates of severe GVHD, and results in acceptable GEFS, OS, and EFS.

The MAGIC Algorithm Probability Predicts Long-Term Outcomes and Treatment Response to Second-Line Therapy for Acute Gvhd

Introduction: Risk stratification approaches using clinical criteria or biomarkers in acute graft-versus-host disease (GVHD) have traditionally been implemented at diagnosis or initiation systemic treatment with corticosteroids. For patients who require second-line treatment, the significance of biomarkers at salvage therapy and the relationship between biomarker risk and choice of therapy are unknown. Methods: We studied adult patients who received second-line treatment for acute GVHD between January 2016 and December 2021 in the Mount Sinai Acute GVHD Consortium (MAGIC) database, which collects clinical data and longitudinal serum samples from allogeneic HCT recipients using a rigorous PRoBE study design. Exclusion criteria included relapse of underlying malignant disease prior to second-line therapy, missing response data, or missing serum sample. Serum samples collected from 7 days prior to 3 days after initiation of second-line therapy met criteria for biomarker analysis. The MAGIC algorithm probability (MAP) was calculated as a single value based on concentrations of ST2 and REG3α and was stratified at a threshold of 0.290 to define high MAP and low MAP groups. The primary objective evaluated the association between MAP and survival for patients receiving second-line therapy for acute GVHD. The key secondary objective was to investigate the MAP and choice of second-line systemic agent on Day 28 overall response rate (ORR) and survival. Results: In total, 316 adult patients received second-line treatment for acute GVHD; 149 patients (47%) were excluded for disease relapse (n=27), missing response data (n=48), or missing sample (n=74).Of the 167 evaluable patients, most had steroid-refractory acute GVHD (n=146, 87%), rather than steroid-dependent (n=8, 5%) or steroid-sensitive (n=14, 8%) disease. 62 patients received ruxolitinib while 102 received other systemic agents (best available therapy: BAT). Baseline clinical characteristics of the study population according to treatment choice were similar, except for donor type (Rux: MRD 15%, MUD 74%, haplo 11%; BAT: MRD 30%, MUD 67%, haplo 3%; P=0.014) and median days from corticosteroids to second-line treatment (Rux: 18, BAT: 12; P=0.02).In total, 81 patients had high MAPs and 86 patients had low MAPs, with similar distribution in each treatment cohort (P=0.53). For the entire cohort, the median follow-up for survivors was 22 months (range, 1.5-27).The MAP separated patients into 2 groups (high vs low) with significantly different mortality (NRM at 2 years, 76% vs 29%, P&amp;lt;.0001) and survival (OS at 2 years, 12% vs 63%, P&amp;lt;.0001). Patients who received ruxolitinib as second-line therapy had lower mortality (NRM at 2 years, 35% vs 60%, P&amp;lt;.002) and improved survival (OS at 2 years, 51% vs 32%, P&amp;lt;.008), compared to those who received other systemic agents. However, the outcomes of patients with high MAP were poor, regardless of second-line treatment choice ( Figure 1A). The MAP (low vs high), time from corticosteroids to second-line treatment (≥14 days vs &amp;lt; 14 days), GVHD grade at second-line (0-2 vs 3-4), and choice of second-line therapy (Rux vs BAT) were all significant (P&amp;lt;0.05) predictors of lower NRM and higher OS in multivariate analysis. The MAP also separated patients into 2 groups with significantly different D28 ORR (low: 49%, high: 30%, P&amp;lt;.0001). In agreement with prospective trials, ruxolitinib resulted in higher D28 response rates compared to other systemic therapies (ORR 54.9% vs 30.5%, P=0.005), which was observed in both low MAP (ORR, 64.5% vs 38.5%, P=0.017) and high MAP cohorts (ORR, 42.9% vs 22.6%, P=0.06) ( Figure 1B). The MAP (low vs high), GVHD grade at second line (0-2 vs 3-4), and choice of second-line therapy (Rux vs BAT) were significant predictors (P&amp;lt;0.05) of higher D28 ORR in multivariate analysis. Conclusion: The MAP measured at the initiation of second-line systemic treatment for acute GVHD predicts NRM and OS, as well as treatment response. The outcomes of patients with high MAP are poor, regardless of second-line treatment choice, and the higher CR rate in low MAP patients may drive higher survival rates. Incorporation of MAP into clinical trials in this setting warrants investigation. The results also support the current use of ruxolitinib as second-line treatment for acute GVHD, particularly in patients with low MAP.

A Day 14 Endpoint for Acute Gvhd Clinical Trials

The overall response rate (ORR) 28 days (D28) after treatment has been adopted as the primary endpoint for clinical trials of acute graft versus host disease (GVHD). ORR combines complete response (CR) and partial response (PR) because CR and PR have very similar non relapse mortality (NRM). D28 ORR has some disadvantages: first, physicians usually decide to modify immunosuppression much earlier than D28, and second, NRM does not always correlate well with ORR at D28, particularly for patients who present with intermediate risk or low risk disease. MAGIC serum biomarkers at day 14 (D14) of GVHD treatment have been shown to correlate better with NRM than clinical symptom response (Srinagesh et al, Blood Advances, 2019). We therefore hypothesized that a combination of clinical symptom severity and biomarkers at D14 of treatment would predict NRM at least as well and possibly better than clinical response at D28. We studied 1144 patients from 23 MAGIC sites who were systemically treated with at least 0.25 mg/kg/day of systemic steroids (prednisone equivalents) and had clinical data and serum samples available in the MAGIC database and biorepository. We divided the patients into a training set (n=764) and a validation set (n=380). We used 12-month NRM from the time of acute GVHD onset as the primary outcome of interest. We first trained a recursive partitioning algorithm in the training set to create a response model (MAGIC D14 clinical response) according to overall GVHD grade at both onset and D14 that predicted 12-month NRM and then applied it to the validation set. Patients that either progressed to grade III/IV GVHD, continued having grade III/IV GVHD, or only improved to grade II at D14 from grade III/IV at D0 had high NRM and were classified as non-responders. Patients with grade I/II GVHD at day 0 who had grade 0-II GVHD at day 14 and patients who improved to grade 0/I at day 14 from grade III/IV at day 0 had low NRM and were classified as responders. Although counterintuitive, responders included patients whose GVHD remained at grade II because the 12 month NRM of this group was only 20%. The new clinical model predicted NRM as well as the D28 standard response model in the validation set. When we integrated the D14 MAP biomarker score (low: ≤ 0.290 vs high: &amp;gt;0.290) with the new D14 clinical response (D14 MAGIC integrated response), we observed three response groups of integrated response with strikingly different 12 month NRM (8%, 35%, 76%, p&amp;lt;0.001), in contrast to the D28 standard response (Figure 1A, 1B). D14 complete response required MAGIC clinical response with low MAP; D14 partial response required either MAGIC clinical response with high MAP or MAGIC clinical non-response with low MAP; and D14 non-response required MAGIC clinical non-response with high MAP. The D14 response groups also displayed significant differences in 12 month OS (82%, 58%, 14% respectively, p&amp;lt;0.001) We then compared the D14 integrated response with the D28 standard response for 12 month NRM prediction. This D14 integrated response model was superior for time-dependent AUC (0.78 vs 0.71, P=0.03), sensitivity (0.71 vs 0.54), positive predictive value (0.52 vs 0.48) and negative predictive value (0.92 vs 0.89), with minimal loss in specificity (0.85 vs 0.87) in the prediction of 12-month NRM compared to the D28 standard response model. Using decision curve analysis, the D14 integrated response displayed higher net benefit (correct identification of patients that will experience NRM by 12 months) over the whole range of threshold probabilities/preferences for changing immunosuppression when 12-month NRM was used as the outcome. We conclude first, that the definition of response that uses onset and D14 grades is as accurate as D28 ORR model; and second, that a model using clinical grades and biomarkers on D14 more accurately predicts long term NRM than the standard D28 definition and may be useful as a clinical trial endpoint.

Outcomes of Treosulfan Vs. Busulfan Based Conditioning Regimens in Adult Patients Undergoing Allogeneic HSCT: Real Life Data from a Single Center

Introduction The type of conditioning regimen has a great impact on the outcome of patients who undergo allogeneic HSCT since graft versus host disease (GVHD), infections, regimen related organ and tissue toxicities are among the most important causes of post-transplant mortality. Despite the regimen related toxicity profile of busulfan, supported by information from the results of clinical trials and real-world data, it is still commonly used worldwide. Treosulfan has advantages in terms of dose of administration, lower incidence of sinusoidal obstruction syndrome and lower neurotoxicity. We aimed to retrospectively investigate outcomes and compare results of patients who underwent matched and haploidentical allogeneic HSCT with treosulfan based or busulfan based reduced intensity conditioning, non-myeloablative conditioning and myeloablative conditioning regimens in our institution. Methods From September 2016 to November 2022, treosulfan was administered to 94 patients while 85 patients received busulfan. We analyzed outcomes as regimen related-toxicity, chronic and acute GVHD, overall survival, non-relapse mortality, relapse related mortality and fungal infection. The myeloablative dose of busulfan was 12.8 mg/kg while it was 9.6 mg/kg for the non myeloablative regimen which was combined with fludarabine. The myeloablative dose of treosulfan was 42 mg/m2 while a dose range of 30-36 mg/m2 was administered for the reduced intensity conditioning. Results The median follow up was 14 months for the treosulfan group while it was 11 months for the busulfan group (p = 0.16). Regimen related toxicity, which was monitored during the hospital stay and 3 months after discharge were 11.7% and 7.1% for treosulfan and busulfan respectively and were similar between two groups. Extensive chronic GVHD was encountered in 15.7% of the patients in the treosulfan group compared to 32.1% in the busulfan group (p &amp;lt; 0.001). Similarly, chronic skin GVHD was observed in 3.6% of the patients who received treosulfan while it was seen in 22.6% of the busulfan group (p &amp;lt; 0.001). Acute GVHD (grade 3 or higher) was encountered in 32.2% of the patients within the treosulfan group while it was encountered in 31.6% of the patients in the busulfan group. Overall survival was 47.5% and 36.5 % in the treosulfan group and busulfan groups respectively. The relapse-related mortality was 17 % in the treosulfan group while it was 34% in the busulfan group. Fungal infection was observed in 23.4% and 36.5% of the patients in treosulfan and busulfan groups respectively. Conclusion Treosulfan, with a lower chronic GVHD incidence and similar regimen related toxicity profile appears to be a safe alternative to busulfan and prospective randomized controlled trials are needed to confirm the results in our study. Keywords: Busulfan; treosulfan; conditioning regimen; stem cell transplantation

Umbilical Cord Blood Stem Cells As Third-Party Adjuvant Infusion in Human Leukocyte Antigen Antibody-Positive Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplantation

Introduction: The presence of human leukocyte antigen (HLA) antibodies, even if not donor-specific antibodies (DSA), is an important risk factor for graft failure (GF) or poor graft function (PGF) in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, there is no ideal strategy for eradicating or descending HLA antibodies in this population. In the present study, we investigated the efficacy and safety of unrelated umbilical cord blood stem cells (CBCs) as a third-party adjuvant infusion in patients with HLA antibodies undergoing allo-HSCT. Methods:Patients undergoing allo-HSCT from July 2017 to June 2021 were enrolled in this study and HLA antibodies were routinely tested before transplantation. Patients were divided into three groups: Group A (patients with positive/weak positive HLA antibodies and with CBCs infusion), Group B (patients with HLA antibodies positive/weak positive without CBCs infusion), and Group C (patients without HLA antibody nor CBCs infusion). Single unrelated CBCs (HLA4/6 or more compatible ) were infused four hours before donor stem cells. The clinical characteristics of the patients and the paired donors were collected. The occurrence of granulocyte and platelet engraftment time, graft-versus-host disease (GVHD), cytomegalovirus (CMV), Epstein-Barr virus (EBV) reactivation, one-year and two-year relapse-free survival (RFS), transplantation-related mortality (TRM), and overall survival (OS) for all three groups of patients were also analyzed in this study. Results:A total of 90 patients were included in this study (17 in group A, 36 in group B, and 37 in group C). The baseline clinical characteristics of the patients were presented in Table 1. No obvious adverse effects were observed in the process of CBCs infusion. All the patients achieved 100% donor chimerism without CBCs engraftment. There were no significant differences in granulocyte and platelet engraftment time between the three groups. Two patients in Group A and C, and 3 patients in Group B developed PGF, respectively. There were no significant differences in the incidence and grades of acute or chronic GVHD among the three groups ( Figure A, B). In the first 3 months after allo-HSCT, the incidence of EBV reactivation was higher in patients in Group B than that in Group A and C, respectively ( Figure C). There were no significant differences in the CMV reactivation between the three groups in the first 3 months after transplantation ( Figure D). Patients in Group A and B both had lower 1-year RFS and higher 1-year TRM, compared with Group C, respectively ( Figure E, F). However, there were no significant statistical differences in 2-year RFS and TRM between the three groups. Although patients in Group A had longer 2-year OS than that in Group B, there were no significant differences between the two groups: Group A vs Group B (76.5% vs 55.6%, P = 0.194), Group B vs Group C (55.6% vs 85.5%, P &amp;lt; 0.05)( Figure G). A multifactorial Cox regression risk model was established to explore the risk factors affecting OS and suggested that patients' pre-transplantation status (with active malignant disease or minimal residual disease positive) and HLA antibodies pre-transplant were the most independent risk factors for OS (HR = 4.380, 95% CI = 1.544-12.427, P&amp;lt;0.05; HR = 0.196, 95% CI = 0.065-0.587, P&amp;lt;0.05). Conclusions: Patients with positive/weak positive HLA antibodies receiving CBCs as a third-party adjunctive infusion were safe, and there was a benefit in the incidence of EBV reactivation compared to such a population without CBCs infusion. The patients with negative HLA antibodies had higher 1-year RFS and lower 1-year TRM than those with positive/weakly positive HLA antibodies regardless CBCs infusion. Due to the small number of included cases, we will expand the sample number to further validate our results in the future.

Inflammatory Change of FoxP3 + Regulatory T Cells in Human Acute Graft-Versus-Host Disease: Implication in Immunopathogenesis

FoxP3 + regulatory T (Treg) cells are a subset of CD4 + T cells that are requisite for control of immune responses in various settings, thereby preventing excessive inflammation and maintaining homeostasis of the immune system. Paradoxically, in several autoimmune disorders, it has been suggested that Treg cells undergo inflammatory conversion to produce effector cytokines promoting tissue inflammation. Although Treg cells are known to play an important role in the pathogenesis of acute graft-versus-host disease (GvHD) following allogeneic stem cell transplantation (SCT), such inflammatory changes have not been focused on so far especially in the setting of human acute GvHD. Herein, in a prospective cohort of patients with hematologic malignancies who were treated with allogeneic SCT, we investigated the quantitative and qualitative changes of Treg cells in subjects with acute GvHD, including production of inflammatory cytokines from Treg cells. We also studied a cellular mechanism for production of effector cytokines by Treg cells and clinical significance of inflammatory changes of Treg cells. We first determined the frequency of Treg cells in the peripheral blood of patients with acute GvHD and compared it with that of patients who did not develop acute GvHD. Percentage of CD25 hiCD127 loFoxP3 + Treg cells among CD4 + T cells was significantly lower in patients with acute GvHD. When we analyzed for the composition of Treg cell subsets in more detail, we observed that the proportion of CD45RA -FoxP3 hi (actively suppressive) Treg cells was decreased and the proportion of CD45RA -FoxP3 lo (cytokine-producing non-suppressive) Treg cells was increased in patients with acute GvHD. Next, we assessed the production of inflammatory cytokines from Treg cells following T cell receptor (TCR) stimulation of peripheral blood lymphocytes with anti-CD3/CD28 antibody, using intracellular cytokine staining. As a result, we found that a remarkable proportion of CD4 +CD25 hiFoxP3 + Treg cells of acute GvHD patients produced TNF-α upon TCR stimulation. Interestingly, a significant proportion of TNF-α-producing Treg cells also secreted IL-17A simultaneously. These findings are consistent with the increased CD45RA -FoxP3 lo fraction among the Treg cell subsets. To explore a potential mechanism underlying inflammatory change of Treg cells, we quantified a variety of soluble factors in systemic circulation before and after onset of acute GvHD. The concentration of IL-6 and IL-21 was significantly higher in the serum drawn after onset of acute GvHD compared to baseline, suggesting that these Th17-polarizing cytokines might be responsible for the inflammatory conversion of Treg cells during acute GvHD. We further checked the suppressive activity of TNF-α-producing CD4 +CD25 hiFoxP3 + Treg cells of acute GvHD patients. TNF-α + Treg cells of acute GvHD patients expressed lower level of FoxP3 than the TNF-α - counterpart. Similarly, the percentage of CD39 + cells was lower in TNF-α + Treg cells than in the TNF-α - counterpart. In fact, Treg cell suppressive activity during acute GvHD was significantly reduced compared with that of pre-SCT control when non-Treg CD4 + T cells or CD8 + T cells were used as responder cells. Finally, we examined the clinical significance of TNF-α-producing CD4 +CD25 hiFoxP3 + Treg cells in acute GvHD patients. The frequency of TNF-α + cells among circulating Treg cells significantly correlated with the severity of acute GvHD, with higher proportion of TNF-α + Treg cells in patients with the higher grade of acute GvHD. This result indicates that the pathologic conversion of Treg cells to produce TNF-α contributes to immune-mediated tissue injury during acute GvHD. Taken together, during human acute GvHD, Treg cells undergo functional alteration to produce proinflammatory cytokines including TNF-α and IL-17A and to exhibit the reduced suppressive activity. TNF-α production by Treg cells is closely associated with the clinical severity of acute GvHD.

Evaluation of Artificial Intelligence (AI) for Acute Graft-Versus-Host Disease(GVHD) Classification

Introduction Graft-Versus-Host Disease(GVHD) following hematopoietic stem cell transplant is a significant complication, causing a 10-30% mortality rate. Staging and grading are often performed by physicians in training, such as residents and fellows, and the assessments can be inconsistent between different physicians and clinical centers. This inconsistency may lead to confusion for both the medical team and the patients. This study evaluates the performance of an artificial intelligence (AI) tool in determining the grade of acute GVHD, exploring its potential role in the classification of GVHD. Method We conducted a performance assessment of Chat-GPT-3.5 and Chat-GPT-4 in grading acute GVHD. We constructed a dataset comprising simulated patients with acute GVHD grades 1-4. We then posed a question to both Chat-GPT-3.5 and Chat-GPT-4.0, asking for the severity of GVHD in each patient. The physicians' assessment was based on the modified Glucksberg criteria. Result Both Chat-GPT tools use the Glucksberg criteria as a reference source. Overall, Chat-GPT-4.0 demonstrated 75% concordance with physician assessment, whereas Chat-GPT-3.5 showed 62.5% concordance. When provided with numerical data, both tools successfully graded acute GVHD with 100% accuracy. However, when given subjective data, Chat-GPT-4.0 exhibited 50% concordance with physician assessment, whereas Chat-GPT-3.5 showed 25%. Although Chat-GPT was able to provide staging for each organ involvement, it struggled to estimate the volume of diarrhea based on the number of episodes. For instance, when asked about the grading of a patient experiencing more than 20 episodes of diarrhea per day, the system concluded that the patient had acute GVHD grade 4. This suggests that Chat-GPT-4.0 has difficulty accurately assessing the amount of diarrhea unless it's exceeding significant amount. Chat-GPT-3.5 exhibited challenges in accurately quantifying the Body Surface Area (BSA). It was unable to assess the BSA if it was less than 25%. Moreover, if the rash involved more than two areas of the body, even when described with alternative language (e.g., ‘torso’, ‘entire torso‘), it would disproportionately inflated the BSA. Conclusion The above results demonstrate that both tools are insufficient for accurately determining the grading of acute GVHD when only subjective data is provided, primarily due to the incapacity of Chat-GPT-3.5 to assess the Body Surface Area (BSA) and Chat-GPT-4.0 to evaluate the volume of diarrhea. However, both tools displayed 100% accuracy when objective data was provided, with Chat-GPT-4.0 notably capable of accurately indicating the stages of organ involvement. Healthcare professionals should not solely rely on Chat-GPT for assessment, and it is advisable to instruct patients against utilizing Chat-GPT to evaluate their own acute GVHD.

A Parallel Comparison of the Safety and Efficacy of Allogeneic Hematopoietic Stem Cell Transplantation for Refractory and Relapsed T-ALL/Lbl Patients Who Achieved Complete Remission with CD7 CAR-T Versus Patients Who Achieved First Complete Remission with Chemotherapy before Transplantation

Introduction We have developed a fratricide-resistant CD7 CAR-T cells from bulk T cells transduced with CD7 CAR without gene editing or additional gene transfer, named naturally selected CD7 CAR-T (NS7CAR-T). NS7 CAR-T therapy has shown a robust complete remission (CR) rate in patients with refractory or relapsed (R/R) T-cell acute lymphoblastic leukemia and T-cell lymphoblastic lymphoma (T-ALL/LBL). Nonetheless, the risk of relapse remains a challenge post-CD7 CAR-T therapy alone, suggesting the potential benefits of consolidation allogeneic hematopoietic stem cell transplantation (allo-HSCT). The question remains whether CD7 CAR-T therapy could increase the incidence of post-transplant complications. Here we conduct a parallel study to compare the safety and efficacy of allo-HSCT in R/R T-ALL/LBL patients who achieved CR with NS7 CAR-T therapy vs those who reached first CR (CR1) using chemotherapy prior to transplantation. Methods Between November 2020 and January 2023, a total of 223 consecutive patients with T-ALL/LBL who underwent allo-HSCT at our center were retrospectively analyzed. Patients who met the following criteria were included in this parallel study: 1) underwent the first allo-HSCT in our hospital, 2) achieved CR before transplantation either from NS7CAR-T therapy or CR1 from chemotherapy (Fig. 1). A total of 139 patients met the criteria for analysis and underwent allo-HSCT after achieving CR either from NS7CAR-T therapy (n=43) or chemotherapy (CR1=96). The median patient age at the time of transplantation was 15 years (range: 1-51 years). The median follow-up time was 370 days (2-941 days). CD7 CAR-T patients were from two clinical trials registered at www.clinicaltrials.gov NCT04572308, NCT04916860. Transplant donor types included sibling identical (n=12), haploidentical (n=110), and unrelated donors (n=17). We used total body irradiation (TBI)-based conditioning regimens (n=128) or Busulfan/Melphalan-based regimens (n=11). To prevent graft-versus-host disease (GVHD), we used ATG along with a short-term regimen combining methotrexate, cyclosporine/tacrolimus, and mycophenolate mofetil. Results The characteristics of patients from both groups are detailed in Table 1. The median interval between CD7 CAR-T treatment and transplantation was 59 (39-131) days. Within the CAR-T group, 16 patients had primary refractory disease, 18 had relapsed disease and 9 in minimal residual disease (MRD)-positive status in bone marrow before CD7 CAR-T therapy. Prior to CAR-T therapy, 55.8% (24 patients) had extramedullary lesions. In comparing the CAR-T and chemotherapy CR1 groups, we observed no significant differences in long-term efficacy post-allo-HSCT. The 2-year leukemia-free survival (LFS) rates were 64.9 (95% CI, 49.1-79.2)% and 69.8 (95% CI, 59.0-79.6)% (p=0.340); the 2-year overall survival (OS) rates were 64.1 (95%CI, 48.0-78.6)% and 71.8 (95%CI, 60.5-81.9)% (p=0.107); the 2-year relapse incidence (RI) rates were 9.3 (95%CI, 3.7-23.7)% and 11.5 (95%CI, 6.4-20.7)% (p=0.873); and the 2-year non-relapsed mortality (NRM) rates were 28.3 (95% CI, 16.9-47.4)% and 21.6 (95% CI, 13.9-33.4)% (p=0.244), for the CD7 CAR-T and non-CAR-T groups, respectively. The incidence of all grades of acute GVHD (aGVHD) 37.2 (95%CI, 25.2-54.9)% vs. 42.1 (95%CI, 33.3-53.3)% did not significantly differ between the CAR-T and non-CAR-T groups. A trend toward an increased rate but no P significance was observed in Grade 3-4 aGVHD incidence of 20.93 (95%CI, 11.7-37.4)% vs. 9.5 (95%CI, 5.1-17.6)% (p=0.139). One-year incidences of moderate to severe chronic GVHD (cGVHD) were 12.2 (95%CI, 5.4-28.0)% and 18.0 (95%CI, 11.5-28.0)% (p=0.357) in the CD7CAR-T and non-CAR-T groups, respectively (Table 2). No statistically significant differences were detected in the occurrences of transplant-associated thrombotic microangiopathy (TA-TMA), CMV, EBV, and HHV-6 infections between the two groups. Upon analyzing, we found that r/r patients who received allo-HSCT after achieving remission with NS7 CAR-T had a similar OS to CR1 patients (Fig. 2). Conclusions Our parallel study showed that T-ALL/LBL patients, who achieved CR via CD7 CAR-T therapy followed by a consolidation allo-HSCT, had similar favorable OS and LFS compared to patients who underwent allo-HSCT in the CR1 state induced by chemotherapy. This was achieved without increasing the incidence of GVHD, infections, or TA-TMA.

Outcome of Allogeneic Stem Cell Transplantation in FLT3-TKD-Mutated AML - a Study on Behalf of the Acute Leukemia Working Party of the EBMT

Introduction The overall prognostic impact of point mutations in the tyrosine-kinase domain 1 of FLT3 (FLT3-TKD mut) in patients (pts) with acute myelogenous leukemia (AML) remains controversial. Furthermore, co-occurrence of FLT3-TKD mut and mutated nucleophosmin-1 (NPM1 mut) was shown to have a favorable prognostic impact on AML pts receiving conventional chemotherapy. However, little is known about the outcome of AML pts with FLT3-TKD mut with or without co-occurring NPM1 mut treated with allogeneic stem cell transplantation (alloSCT). Patients and Methods This is a retrospective study of the acute leukemia working party (ALWP) of the European Society for Blood and Marrow transplantation (EBMT) investigating the outcome of adult AML pts with FLT3-TKD mut with or without NPM1 mut after first alloSCT using matched sibling (MSD), unrelated (UD) or haploidentical (haplo) donors between 2005 and 2022. All patients were in first or second complete remission (CR1 of CR2). Information on cytogenetic risk was necessary for inclusion. No ex-vivo T-cell depletion (TCD) was allowed. The primary endpoint was leukemia-free survival (LFS) at two years after alloSCT. Secondary endpoints were overall survival (OS), cumulative incidence of relapse (RI), non-relapse mortality (NRM), acute and chronic graft-versus-host disease (GVHD) and GVHD-free, relapse free survival (GRFS). One hundred and eighty-two adult AML pts with FLT3-TKD mut were identified, of which 74 (40.7%) had a co-occurring mutation in NPM1. Median age was 55.1 years (range 18.6-79.1) and 46.7% were male. Median follow-up was 21.4 months [IQR 13.6-28.1]. 78% of the pts were in CR1. Cytogentic risk was favorable, intermediate and adverse in 12.6%, 75.3% and 12.1% of pts, respectively. Additional FLT3 internal tandem duplication (FLT3-ITD) was present in 25.8% of pts. Information on measurable residual disease (MRD) was available for 68.7% of pts, being positive in 48% of these. Karnofsky performance score was &amp;lt; 90% in 21.1%, and hematopoietic cell transplantation-specific comorbidity index (HCT-CI) was &amp;gt;=3 in 28.1% of pts. 69.6% of pts were cytomegalovirus (CMV) positive. Conditioning was myeloablative in 52.8% and reduced intensity in 47.2% of pts. Stem cell donors were MSD, UD and haplo in 25.8%, 55.5% and 18.7% of cases. Stem cell source was bone marrow in 10.4%, peripheral blood in 87.4% and both in 2.2%. In-vivo TCD was performed in 59.2% of pts. GVHD prophylaxis was calcineurin inhibitor-based in 92.2% of pts. Results Engraftment was achieved in 99.4% of pts with a day-60 absolute neutrophil count (ANC) &amp;gt;0.5 x 10 9/L of 97.7% [95% CI: 93.7-99.2]. Day-180 rates of acute GVHD (aGVHD) grade II-IV and grade III-IV were 26.2% (95% CI:19.9-32.9) and 8.1% (95% CI:4.6-12.8), respectively. At two years, rates of total chronic GVHD (cGVHD) and extensive cGVHD were 30% (95% CI: 22.5-38) and 16.6% (95% CI:10.6-23.7), respectively. Two-year LFS and OS were 66.1% (95% CI: 57.6-73.2) and 74% (95% CI: 65.4-80.8), respectively. Two-year RI, NRM and GRFS were 22.5% (95% CI:16-29.8), 11.4% (95% CI: 7-17), and 51.1% (95% CI: 42.4-59.1), respectively. Cause of death was original disease in 47.5%, infection in 27.5%, GVHD in 22.5% and non-SCT related in 2.5% of cases. In univariate analysis the only factor with significant impact on LFS was donor type (haplo [n=34] vs. others, p=0.006). For GRFS, patient CMV negativity (p=0.014) and in-vivo TCD (p=0.01) were significant. Interestingly, co-occurring mutations in FLT3 (FLT3-ITD) or NPM1 were not identified as having a significant impact on LFS or GRFS. Moreover, MRD status before alloSCT was also not shown to have a significant impact on these outcomes. In multivariate analysis of LFS and GRFS, only donor type (haplo [n=34] vs. others) was shown to be significant for both endpoints, hazard ratio (HR) 2.54; 95% CI: 1.4-4.6; p=0.002, and HR 2.01; 95% CI: 1.18-3.41; p=0.01, respectively. Conclusion Acknowledging the limitations of our retrospective study in this rare entity results are encouraging given the high-risk population (median age 55.1 years, CR2 in 22%, co-occurring FLT3-ITD in 25.8%, positive MRD in 48%, HCT CI &amp;gt;=3 in 28.1%). LFS, OS and GRFS rates were 66.1%, 74% and 51.1%, respectively, and both RI and NRM were comparably low with rates of 22.5% and 11.4%, respectively. No positive prognostic impact of co-occurring mutations in FLT3-TKD and NPM1 was detected, unlike what was found for conventional chemotherapy.

Characteristics of Alloreactive T-Cell Clones That Predict Early Development of Acute GvHD Following Allogeneic HCT

Acute Graft-versus-Host Disease (GvHD) is mediated by T-cells recognizing alloantigens. Despite advances in prophylactic regimens that broadly deplete T-cells, incidence of acute GvHD remains high, suggesting that there is a need to further characterize alloreactive T-cells specifically. Using mixed-lymphocytes reactions (MLR) and high-throughput T cell receptor (TCR) sequencing of third complementarity-determining region (CDR3) of the TCRβ chain, we identified and characterized alloreactive T-cell clones in donor-recipient pairs of allogeneic hematopoietic cell transplantation. We sequenced and identified a total of 34,903 unique donor-derived CDR3β functional sequences (FS) from 20 donor-recipient in vitro MLRs using peripheral blood mononuclear cells (PBMC) collected from donors and patients pre-transplant. We also sequenced the respective unstimulated donor PBMC samples (Donor Type: 13 HLA-Matched/7 Mismatched, GvHD prophylaxis: 11 Tacrolimus (Tac)-Methotrexate/8 Tac-MMF/1 Sirolimus, PTCy: 12 Yes/8 No). On average, our identified alloreactive FS make up a small fraction of the sampled donor repertoire in comparison to nonalloreactive FS. (Mean fraction: 0.004 vs. 0.996, p &amp;lt; 0.0001) Surprisingly, we found that alloreactive FS have significantly shorter CDR3β length compared to nonalloreactive FS (mean nt 43.50 vs. 44.40, p &amp;lt; 10E-15). We also found that alloreactive FS which can be located within the unstimulated donor sample (n= 4522) are shorter in comparison to all derived alloreactive FS (mean nt 42.43 vs. 43.50, p &amp;lt; 10E-15) and to nonalloreactive FS (mean nt 42.43 vs. 44.40, p &amp;lt; 10E-15) (Figure 1A). Publicly shared FS have, on average, shorter CDR3β length and are overrepresented among FS associated with autoreactivity and cross-reactivity (Khosravi-Maharlooei et al., J Clin Invest, 2019). Thus, we hypothesized that alloreactive FS may be also publicly shared. We derived a list of shared FS from our donor samples, where if a FS is found in more than one donor, it is considered shared. Alloreactive clones were significantly more likely to be shared compared to nonalloreactive (OR 1.94; CI 95%: 1.88 to 2.01). In addition, using a publicly available set of cross-reactive FS (https://vdjdb.cdr3.net/search), we found that alloreactive sequences are also highly cross-reactive compared to nonalloreactive sequences (OR 6.6; CI 95%: 5.18 to 8.42). FS with greater levels of sharedness appear more frequently within a repertoire (Madi et al., Genome Res, 2014). As alloreactive sequences are highly shared, we hypothesized that donor repertoires with greater proportion of shared and alloreactive FS will increase the odds of developing clinically significant GvHD (Grade 2-4) in the recipient. Indeed, within donor repertoires, we found a higher fraction of alloreactive sequences amongst the highly expressed FS (The top 50% of FS in repertoire frequency) which correlated with development of GvHD in recipients post-transplant (mean fraction: GvHD+ group 0.013 vs GvHD- group 0.004, p &amp;lt; 10E-7). Due to the higher portion of shared and alloreactive FS in the top half of the donor T-cell repertoire, we used R50, an index that estimates the diversity of the top 50% of a donor repertoire, to predict the development of GvHD. Utilizing an initial validation cohort of 12 donors, we found that donors whose corresponding recipients developed GvHD within the first 3 months post-transplant had significantly lower R50 compared to those that did not develop GvHD (R50 of 0.096 vs 0.345 p &amp;lt; 0.0001). We also conducted a time-to-event analysis regarding GvHD development in the first 90 days post-transplant. Using the average R50 of the validation cohort as the cutoff for low and high R50 (Average R50: 0.20), we found, in an expanded bi-institutional cohort (n=57), that those with lower R50 values had a significantly greater risk of GvHD compared to those with higher R50 (Figure 1B). In conclusion, our data suggests that functional alloreactive T lymphocytes are characterized by shorter TCRβ CDR3 length. These FS are highly shared and cross-reactive, suggesting a common mechanism underlying the selection of alloreactive and autoreactive clones. From there, we were able to develop and validate a diversity metric that predicts GvHD development pre-transplant based on the donor T-cell repertoire. These findings have implications for donor selection and need to be examined prospectively.

Prognostic Factors Impacting Post-Transplant Outcomes in Adult T-Cell Acute Lymphoblastic Leukemia: A Registry-Based Study By the EBMT Acute Leukemia Working Party

Background: T-cell acute lymphoblastic leukemia (T-ALL) predominantly affects individuals in late childhood and young adulthood and historically had high mortality rates. The outcome of Pediatric inspired chemo protocols in the T-ALL have improved in the past years due to the advancement in treatment and supportive care but the data is less mature, although adult patients still face challenges. Limited studies have directly explored the impact of patient- and transplant-related factors on post-transplant outcomes in adult T-ALL patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods: A retrospective registry-based analysis was conducted across multiple centers using data from the European Society for Blood and Marrow Transplantation (EBMT) registry. Inclusion criteria were adult T-ALL patients (&amp;gt;18 years old) who underwent their first allo-HSCT in first complete remission from matched sibling donors (MSD), unrelated donors (UD) (10/10 or 9/10) or haploidentical donors between 2010 and 2021. Cord bloods were excluded. Multivariable Cox regression analysis was performed to examine the associations between patient/transplant characteristics and outcomes. Results: A total of 1907 patients were included in the study. The median age at transplant was 33.4 years (18.1-75). The median follow up was 2.9 years (2.6-3.1), and the median year of HSCT was 2016 (2010-2021). Patients were 70 % male with 67% being CMV-positive, at the time of transplant. Donors were predominantly male (64%), and (23%) of transplants involved female donors to male patients. The median period between diagnosis and HSCT was 5.9 months (0 -23.8). Most transplants were from matched sibling donors (MSD) (45%) followed by UD (43%) and 12% were from Haploidentical transplants. Most patients underwent myeloablative conditioning with total body irradiation (TBI)-based regimens (69%) and most patients received peripheral blood stem cells (84%). Cyclosporine with methotrexate was the most common graft-versus-host-disease (GVHD) prophylaxis (54 %). In vivo T cell depletion was used in 44%. The 2-year overall survival (OS) was 69.4%, and leukemia -free survival (LFS) was 62.1%. The cumulative incidence of acute GVHD (aGVHD) grades II-IV at 100 days was 32.6%, and grades III-IV was 10%. The cumulative incidence of chronic GVHD (cGVHD) at 2 years was 37.3%, and extensive cGVHD was 16.8%. Multivariate analysis yielded significant associations between patient/transplant characteristics and outcomes. Advanced age at transplant was associated with poorer outcomes, including LFS (HR=1.11, p=0.004), GVHD-free, relapse-free survival (GRFS) (HR=1.06, p=0.04), OS (HR=1.12, p=0.002), and higher non-relapse mortality (NRM) (HR=1.23, p=0.001); HR for 10y increment. A later year of HSCT was associated with improved GFRS (HR=0.89, p=0.001), OS (HR=0.9, p=0.02), and decreased NRM (HR=0.82, p=0.008), aGVHD II-IV (HR=0.83, p=0.001), and cGVHD (HR=0.8, p=0.001); HR for 3y increment. TBI was specifically superior with improved LFS (HR=0.79, p=0.02), GRFS (HR=0.83, p=0.04), and lower relapse incidence (RI) (HR=0.65, p=0.001). Female-to-male transplant combination had a negative impact on GFRS (HR=1.21, p=0.02), OS (HR=1.23, p=0.048), and on the risk of cGVHD in general (HR=1.39, p=0.002) and specifically extensive cGVHD (HR=1.47, p=0.01). The use of in vivo T-cell depletion had significant benefits in terms of GFRS, aGVHD grade III-IV, cGVHD, and extensive cGVHD. Conclusion: This large study identified prognostic factors, such as age at transplant, donor type, and conditioning regimen, in influencing key outcomes including OS, LFS, GVHD incidence, and NRM in adult T-ALL patients undergoing allo-HSCT. Importantly, a significant improvement over time in post-transplant outcomes was noted. These findings hold great promise for new adapted treatment strategies and can serve as a benchmark for future studies in that setting.

Monocyte Subpopulations and Their Expression of HLA-DR and CD300e after Allogeneic Stem Cell Transplantation in Patients with or without GvHD

Introduction: Graft versus host disease (GvHD) is a complex immune process that can affect up to 50% of patients undergoing allogeneic stem cell transplantation (allo-SCT). Even though prophylactic immunosuppressive regimens have been established over the last decades, GvHD is still one of the main causes of morbidity and mortality among these patients. Monocytes are cells of the innate immune system, that represent, as professional antigen-presenting cells, a connection to the adaptive immune system, and play a role in several autoimmune disorders. They are classified according to the expression of CD14 and CD16 into classical (CD14++/CD16-), intermediate (CD14++/CD16+), and non-classical (CD14+/CD16++) monocytes. Previous research has shown higher levels of intermediate monocytes in pediatric patients with GvHD. Furthermore, non-classical monocytes in allogenic bone marrow grafts have also been correlated with the development of GvHD. The expression of other surface markers such as HLA-DR and CD300e (IREM-2) are related to monocyte function and engagement in inflammatory processes. Nevertheless, the role of CD300e in GvHD remains unclear. Here, we present a description of monocyte subgroups and their expression of HLA-DR and CD300e in allo-SCT patients with or without GvHD as well as in healthy subjects. Methods: Transversal observational study with patients undergoing allo-SCT. Peripheral blood of patients was collected once after engraftment. Both acute and chronic GvHD patients were enrolled. Five healthy volunteers served as controls. Flow cytometry was performed to determine the immunophenotype of monocytes according to their expression of CD14 and CD16 stratifying into classical, intermediate, and non-classical subgroups as described above. In addition, the expression of HLA-DR and CD300e was analyzed for all monocytes and individual subgroups. Clinical characteristics are described in the table below. Statistical analysis was performed using GraphPad Prism 9. Comparisons between groups were made using non-parametrical tests and tests for multiple comparisons (Mann-Whitney and Kruskal-Wallis respectively). Results: 29 patients were enrolled. Two patients with GvHD were excluded due to neutropenia. 19 patients presented GvHD grade II or higher. Of those, ten had acute GvHD, five had prolonged acute GvHD, two had late onset acute GvHD, and two had chronic GvHD. Eight patients did not show GvHD or had only mild skin disease (grade one). The mean age was 60 years for all patients, 62.5 and 58.5 years among GvHD and non-GvDH patients, respectively. The image summarizes patient characteristics and comparisons. We compared transplant recipients with and without GvHD which each other and with healthy controls. In single comparisons, patients with GvHD showed significantly lower percentages of total monocytes as well as CD14+/HLA-DR+, total HLA-DR+, and intermediate monocytes compared to patients without GvHD. In addition, we observed a trend toward lower levels of CD16+/HLA-DR+ and CD300e-expressing monocytes in GvHD patients. When compared to healthy controls, allogeneic recipients without GvHD showed significantly higher levels of total monocytes, intermediate monocytes, and CD16+/HLA-DR+ monocytes and lower levels of classical and non-classical monocytes. Compared to controls, patients with GvHD showed significantly lower levels of total monocytes expressing CD300e (also true in all subgroups) as well as lower levels of classical monocytes, but higher levels of intermediate and non-classical monocytes. P values are shown in Table 1. Means and ranges for all groups are represented in Image 1. Conclusions: In our cohort, patients with GvHD showed higher levels of intermediate and non-classical monocytes compared to controls, however, these populations were lower when compared to allogeneic recipients without GvHD. This could indicate a consumption process similar to that observed in COVID-19 patients and a direct involvement of these monocytes in the GvHD process. In addition, we report for the first time the involvement of CD300e in GvHD, which was significantly lower expressed in the GvHD patients, compared to controls. However, there was only a strong trend compared to transplanted non-GvHD patients. This indicates a possible contribution of CD300e in the pathogenesis of GvHD. Further studies are needed in order to confirm these findings.