Donor Chimerism Research Articles

Low Incidence of Relapse with a Moderate Conditioning Regimen of Fludarabine, Busulfan, and Melphalan for Patients with Myeloid Malignancies: A Single-Center Analysis of 100 Patients

Relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT) with standard myeloablative conditioning regimens such as Flu-Bu (fludarabine and busulfan) remains a major concern in patients with myeloid malignancies. A low relapse rate was reported when thiotepa or melphalan (Mel) were added to Flu-Bu, but this might increase the non-relapse mortality (NRM). Here, we evaluated the outcomes of 100 patients (70 acute myeloid leukemia, 23 myelodysplastic syndrome, 4 chronic myelomonocytic leukemia, and 3 granulocytic sarcoma) who received their first allo-HSCT after moderate-dose FBM conditioning regimen (fludarabine 150 mg/m2, busulfan 6.4 mg/kg, and melphalan 140 mg/m2, n=69; or lower-dose melphalan at 100 mg/m2 for patients over 55 years old and/or those with HCT-CI ≥3, n=31). The donors were HLA-matched siblings (n=19), unrelated donors (n=4), and haplo-identical donors (n=77). A total of 88% patients had an intermediate or high disease risk index (DRI). Out of 96 evaluable patients, 94 achieved neutrophil engraftment and had full donor chimerism on day 30. After a median follow-up of 468 days (range, 55-1039 days), only four patients relapsed with 2-year cumulative incidence of relapse (CIR) of 5.3%±3.6%. The 100-day and 2-year NRM were 6.8%±4.4% and 12.3%±3.6%, respectively. At last follow-up, the 2-year disease-free survival (DFS) and overall survival (OS) were 82.4%±4.2% and 80.3%±6.0%, respectively. Moreover, when comparing the transplantation outcomes between patients with melphalan 100 mg/m2 and 140 mg/m2, the NRM and CIR were not significantly different and the 2-year DFS and OS were similar in both groups, although the Mel 100 group was associated with a higher median age (58 vs 42 years, p<0.001) and a higher percentage of patients with HCT-CI ≥3 (p=0.005). For the whole group, the only independent factor associated with transplantation outcomes was HCT-CI ≥3, which led to higher NRM and inferior DFS and OS. Our study suggested that moderate-intensity FBM conditioning was feasible for patients with myeloid malignancies with a low relapse rate without increased NRM. A lower dose of melphalan at 100 mg/m2 maintained the low relapse risk without excess NRM for older adults. However, the FBM regimen should be used with caution in patients with high-risk HCT-CI (≥3).

Maternal dendritic cells influence fetal allograft response following murine in-utero hematopoietic stem cell transplantation

BackgroundIntrauterine hematopoietic stem cell transplantation (IUT), potentially curative in congenital haematological disease, is often inhibited by deleterious immune responses to donor cells resulting in subtherapeutic donor cell chimerism (DCC). Microchimerism of maternal immune cells (MMc) trafficked into transplanted recipients across the placenta may directly influence donor-specific alloresponsiveness, limiting DCC. We hypothesized that dendritic cells (DC) among trafficked MMc influence the development of tolerogenic or immunogenic responses towards donor cells, and investigated if maternal DC-depletion reduced recipient alloresponsiveness and enhanced DCC.MethodsUsing transgenic CD11c.DTR (C57BL/6) female mice enabled transient maternal DC-depletion with a single dose of diphtheria toxin (DT). CD11c.DTR females and BALB/c males were cross-mated, producing hybrid pups. IUT was performed at E14 following maternal DT administration 24 h prior. Bone marrow-derived mononuclear cells were transplanted, obtained from semi-allogenic BALB/c (paternal-derived; pIUT), C57BL/6 (maternal-derived; mIUT), or fully allogenic (aIUT) C3H donor mice. Recipient F1 pups were analyzed for DCC, while maternal and IUT-recipient immune cell profile and reactivity were examined via mixed lymphocyte reactivity functional assays. T- and B-cell receptor repertoire diversity in maternal and recipient cells were examined following donor cell exposure.ResultsDCC was highest and MMc was lowest following pIUT. In contrast, aIUT recipients had the lowest DCC and the highest MMc. In groups that were not DC-depleted, maternal cells trafficked post-IUT displayed reduced TCR & BCR clonotype diversity, while clonotype diversity was restored when dams were DC-depleted. Additionally, recipients displayed increased expression of regulatory T-cells and immune-inhibitory proteins, with reduced proinflammatory cytokine and donor-specific antibody production. DC-depletion did not impact initial donor chimerism. Postnatal transplantation without immunosuppression of paternal donor cells did not increase DCC in pIUT recipients; however there were no donor-specific antibody production or immune cell changes.ConclusionsThough maternal DC depletion did not improve DCC, we show for the first time that MMc influences donor-specific alloresponsiveness, possibly by expanding alloreactive clonotypes, and depleting maternal DC promotes and maintains acquired tolerance to donor cells independent of DCC, presenting a novel approach to enhancing donor cell tolerance following IUT. This may have value when planning repeat HSC transplantations to treat haemoglobinopathies.

The role of plerixafor in conditioning regimens before unmanipulated bone marrow transplantation in patients with Wiscott–Aldrich syndrome

Our previous experience of using plerixafor and granulocyte colony stimulating factor (G-CSF) in addition to treosulfan-based conditioning in patients with Wiscott–Aldrich syndrome (WAS) demonstrated efficacy and a decreased risk of severe graft failure after hematopoietic stem cell transplantation (HSCT) with TCRab + /CD19 + graft depletion. Because of the remaining risk of graft failure in WAS patients following HSCT with unmanipulated grafts reported in a number of large-scale retrospective studies, we used plerixafor and G-CSF in conditioning regimens in 6 WAS patients who received native bone marrow as a graft source. None of the patients developed severe organ toxicity in the early post-transplantation period. All of them had long-term full donor chimerism in whole blood and the CD3 + line and a good graft function. At the last follow-up, 5 patients are alive at 3.7 to 74.7 months after HSCT (median follow-up time: 24.0 months). One patient died of chronic lung graft-versus-host-disease at 18 months after the transplantation. Despite limited experience, we believe that additional hematopoietic stem cell mobilization with plerixafor and G-CSF in treosulfan-based conditioning regimens may be effective in WAS patients undergoing HSCT with an unmanipulated graft. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. Key words: plerixafor, conditioning regimen, hematopoietic stem cell transplantation, Wiscott–Aldrich syndrome, unmanipulated graft

Interferon gamma (IFN-g) continuous infusion affects engraftment in a murine transplant model

IntroductionEffects of Interferon gamma (IFN-g) on hematopoiesis have been reported as both stimulatory and suppressive. In humans, IFN-g appears to be deleterious in conditions such as chronic neutropenia, anemia of chronic disease, and aplastic anemia, suggesting some level of bone marrow suppression. Inborn errors of immunity often have an inflammatory component as part of their phenotype, and this inflammation may impact on their transplant outcomes leading to increased graft failure. We thus developed a murine model using implanted pumps to assess the impact of exogenous IFN-g administered by continuous infusion during transplantation. MethodsMurine model and IFN-g administration: Bone marrow nucleated cells (BMNCs) were collected from femurs and tibias of 8- to 10-week-old B6. SJL-Ptprca-Pep3b-/BoyJ donors (CD45.1+) and 5 million cells were injected via tail vein into 10- to 12-week-old recipient C57BL6 mice (CD45.2+) after 400cGy irradiation. Micro-osmotic pumps (Alzet®1007D/2001) were surgically implanted 2 days before transplant to provide continuous infusion of recombinant mouse IFN-g at 5ug/day for 7 days. Donor chimerism%CD45.1 + engraftment inT, B, NK, and myeloid cells was determined in peripheral blood every 2 weeks (from 4 to 16 weeks) post-transplant using cell specific monoclonal antibodies. 16 weeks after transplant, mice were euthanized, and %CD45.1+ engraftment was measured in spleen and bone marrow. ResultsAlthough the absolute peripheral blood numbers were similar (Fig.1A), the infusion of IFN-g in this non myeloablative transplant resulted in lower donor engraftment as compared to control transplants (Fig.1B). Myeloid engraftment appeared to be most affected, with a statistically significant reduction of engraftment (p < 0.005) in monocytes and granulocytes. The quantitative reduction was higher than 70% as compared to control transplants worsening over time, suggesting that the long-term repopulating cells were most affected (Fig.1C). Bone marrow was affected in a similar pattern (Fig.1D). [Display omitted] ConclusionsWe established a mixed-chimera model to assess the impact of exogenous IFN-g on engraftment. Our results provide evidence that continuously elevated IFN-g levels are deleterious for engraftment and may help explain the clinical phenomena of graft loss in this patients. Further analysis of other inflammatory pathways and IFN-g blockers are ongoing.

Type-1 regulatory T cells are critical for curative immunotherapy outcomes

Abstract IL-10 producing CD4 +type-1 regulatory T cells (Tr1) mediate immune tolerance during chronic infection, autoimmunity, and transplantation. We previously demonstrated that eomesodermin (Eomes) promotes Tr1 cell (Eomes +IL-10 +) development after allogenic bone marrow transplantation (alloBMT). Here we define the differentiation trajectory of Tr1 cells and the functional states therein. Eomes −IL-10 −, Eomes +IL-10 −and Eomes +IL-10 +subsets (defined by mCherry and GFP expression respectively) were FACS sorted after alloBMT and transferred into secondary recipients. Eomes +IL-10 +T cells were stable in phenotype after transfer while the Eomes +IL-10 −T cell subset differentiated into Eomes +IL-10 +Tr1 cells. Tr1 differentiation was associated with progressive enrichment for cytotoxicity (Perforin, Nkg7, Granzyme K, Tnfrsf9), immune regulatory (IR) (Tim-3, Lag3, Tox, Nr4a2) and chemokine (Ccl3, Ccl4, Ccl5) RNA expression. Equivalent IR, cytotoxic and cytokine/chemokine expression was confirmed at a protein level. The conditional deletion of Eomes in CD4 T cells prevented Tr1 differentiation and resulted in impaired cytotoxicity in vivo, the accumulation of donor CD11c +dendritic cells, mixed donor chimerism and leukemia relapse, consistent with a critical role for this lineage in tolerance, engraftment and graft-versus-leukemia effects. The deletion of Eomes from CD19-targeted chimeric antigen receptor (CAR) T cells, reduced cytotoxic molecule expression (perforin, granzyme A/B) in CD4 CAR T cells and resulted in immune escape due to CD19 antigen loss in leukemia cells. In sum, Tr1 cells mediate critical Eomes-dependent cytotoxic and regulatory function that are critical for curative immunotherapy outcomes.

The Use of SARS-CoV-2-Positive Donors in Hematopoietic Stem Cell Transplantation

Since 2019, the SARS-CoV-2 pandemic has become a global issue due to its high fatality rate. Over time, the characteristics of the virus have evolved and led to the creation of an omicron strain with higher infectivity but a significantly decreased fatality rate. For patients in urgent need of hematopoietic stem cell transplantation (HSCT), whether the SARS-CoV-2 infection status of donors has a significant impact on HSCT recipients should be clarified. To estimate the transplantation risk of SARS-CoV-2-positive donors, 24 patients who underwent HSCT from December 1, 2022 to January 30, 2023 were retrospectively included. The ratio of the observation group (SARS-CoV-2-positive donors, n=12) to the control group (SARS-CoV-2-negative donors, n=12) was 1:1. We observed the time of hematopoietic reconstruction, donor chimerism, severe infection, acute graft vs host disease, and hepatic vein occlusion disease during hematopoietic reconstruction. In the observation group, the average time of myeloid hematopoietic reconstruction was 11.58 days, and in the control group, it was 12.17 days (P=.3563 [>.05]). On average, all patients achieved a 90% donor chimerism rate of +13.58 (±4.5) days (P=.5121 [>.05]). The average percentage of patients that achieved successful hematopoietic reconstruction was 96.75% in the observation group and 96.31% in the control group (P=.7819 [>.05]). A total of 6 adverse events occurred during this study: 3 in the observation group and 3 in the control group. Our preliminary results showed favorable short-term outcomes in recipients of SARS-CoV-2-positive HCST donors.

Cord blood transplantation for nonmalignant disorders: early functional immunity and high survival.

There is no consensus on the best donor for children with nonmalignant disorders and immune deficiencies in the absence of a matched related donor (MRD). We evaluated the 2-year overall survival (OS) after umbilical cord blood transplantation (UCBT) in patients with nonmalignant disorders from 2009 to 2020 enrolled in a prospective clinical trial using either 5/6 or 6/6 UCB as the cell source. Patients receive a fully ablative busulfan, cyclophosphamide, and fludarabine without serotherapy. Fifty-five children were enrolled, median age 5 months (range, 1-111 months); primary immune deficiency (45), metabolic (5), hemophagocytic lymphohistiocytosis (1), and hematologic disorders (4). Twenty-six patients had persistent infections before transplant. Nineteen of them (34%) were 6/6 matched, and 36 (66%) were 5/6 human leukocyte antigen-matched. The OS at 2 years was 91% (95% cumulative incidence, 79-96), with a median follow-up of 4.3 years. The median time to neutrophil and platelet recovery were 17 days (range, 5-39 days) and 37 days (range, 20-92 days), respectively. All but one evaluable patient achieved full donor chimerism. The cumulative incidence of acute GVHD grades 2-4 on day 100 was 16% (n= 9). All patients with viral infections at the time of transplant cleared the infection at a median time of 54days (range, 44-91 days). All evaluable patients underwent correction of their immune or metabolic defects. We conclude that in the absence of MRD, UCBT following myeloablative conditioning without serotherapy is an excellent curative option in young children with nonmalignant disorders. This trial has been registered at www.clinicaltrials.gov as NCT00950846.

Total Marrow Irradiation for Second Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Advanced Acute Leukemia

Second allogeneic hematopoietic stem cell transplantation (HSCT) is a treatment option for patients with acute leukemia who relapse after a first HSCT. Although myeloablative conditioning (MAC) regimens before the first HSCT are considered superior to reduced- intensity conditioning (RIC) in terms of disease control in acute leukemia patients, the optimal conditioning regimen for the second allogeneic HSCT remains controversial. The most important prognostic factors are the remission disease phase at the time of the second HSCT and an interval >12 months from the first HSCT to the second HSCT. Total marrow irradiation (TMI) is an advanced high-precision radiation treatment that delivers therapeutic doses over extensively selected targets while substantially reducing radiation to vital organs compared to conventional total body irradiation (TBI). Here we report the results of a retrospective analysis of second allogeneic HSCT treated with TMI as an MAC regimen with the intent of limiting toxicity. We investigated the efficacy of high dose per fraction TMI in combination with thiotepa, fludarabine, and melphalan in 13 consecutive patients with acute leukemia who had relapsed after a first allogeneic HSCT treated between March 2018 and November 2021. Donor type was haploidentical in 10 patients, unrelated in 2 patients, and HLA-identical sibling in 1 patient. The conditioning regimen consisted of 8 Gy TMI in 5 patients on days -8 and -7 and 12 Gy TMI in 8 patients on days -9 to -7, plus thiotepa 5 mg/kg on day -6, fludarabine 50 mg/day on days -5 to -3, and melphalan 140 mg/day on day -2. The TMI was delivered at the dosage og 4 GY for 2 consecutive days (total = 8 GY) or for 3 consecutive days (total = 12 GY). The median patient age was 45 years (range, 19 to 70 years); 7 patients were in remission, and 6 had active disease at the time of their second allogeneic HSCT. The median time to a neutrophil count of >.5×109/L was 16 days (range, 13 to 22 days), and the median time to a platelet count of >20×109/L was 20 days (range, 14 to 34 days). All patients showed complete donor chimerism on day +30 post-transplantation. The cumulative incidence of grade I-II acute graft-versus-host disease (GVHD) was 43%, and that of chronic GVHD was 30%. The median duration of follow-up was 1121 days (range, 200 to 1540 days). Day +30 and +100 transplantation-related mortality (TRM) was 0. The overall cumulative incidence of TRM, relapse rate, and disease free-survival were 27%, 7%, and 67%, respectively. This retrospective study demonstrates the safety and efficacy of a hypofractionated TMI conditioning regimen in patients with acute leukemia undergoing second HSCT with encouraging outcomes in terms of engraftment, early toxicity, GVHD, and relapse.

Cryopreservation of Allogeneic Hematopoietic Cell Products During COVID-19 Pandemic: Graft Characterization and Engraftment Outcomes

The COVID-19 pandemic triggered the deployment of unfamiliar measures to safeguard successful allogeneic hematopoietic cell transplantation (allo-HCT). Among these measures, cryopreservation offered logistical benefits that could outlast the pandemic, including graft availability and timely clinical service. The purpose of this study was to evaluate graft quality and hematopoietic reconstitution in patients transplanted with cryopreserved allogeneic stem cell products during the COVID-19 pandemic. We evaluated 44 patients who underwent allo-HCT using cryopreserved grafts consisting of hematopoietic progenitor cells (HPC) apheresis (A) and bone marrow (BM) products at Mount Sinai Hospital. Comparative analyses of 37 grafts infused fresh during the one-year period preceding the pandemic were performed. Assessment of cellular therapy products included total nucleated cell and CD34+ cell enumeration, viability, and post-thaw recovery. The primary clinical endpoint was the evaluation of engraftment (absolute neutrophil count [ANC] and platelet count) and donor chimerism (presence of CD33+ and CD3+ donor cells) at day +30 and +100 post-transplant. Adverse events related to cell infusion were also analyzed. Patient characteristics were comparable between the fresh and cryopreserved groups with 2 exceptions in the HPC-A cohort: the number of patients in the cryopreserved group that received haploidentical grafts was 6 times that in the fresh group, and the number of patients in the fresh group with a Karnofsky performance score >90 was double that in the cryopreserved group. The quality of HPC-A and HPC-BM products was not affected by cryopreservation, and all grafts met the release criteria for infusion. The pandemic did not affect the time between collection and cryopreservation (median, 24 hours) and time in storage (median, 15 days). Median time to ANC recovery was significantly delayed in recipients of cryopreserved HPC-A (15 vs 11 days, P=.0121), and there was a trend toward delayed platelet engraftment (24 vs 19 days, P=.0712). The delay in ANC and platelet recovery was not observed when only matched graft recipients were compared. Cryopreservation did not affect the ability of HPC-BM grafts to engraft and reconstitute hematopoiesis, and there was no difference in the rates of ANC and platelet recovery. Achievement of donor CD3/CD33 chimerism was not affected by cryopreservation of either HPC-A or HPC-BM products. Graft failure was observed in only 1 case, a recipient of cryopreserved HPC-BM. Three recipients of cryopreserved HPC-A grafts died before ANC engraftment from infectious complications. Remarkably, 22% of our studied population had myelofibrosis, and almost half received cryopreserved HPC-A grafts with no graft failure observed. Finally, patients receiving cryopreserved grafts were at a higher risk of infusion-related adverse events than those receiving fresh grafts. Cryopreservation of allogeneic grafts results in adequate product quality with minimal impact on short-term clinical outcomes, except for an increased risk of infusion-related adverse events. Cryopreservation is a safe option in terms of graft quality and hematopoietic reconstitution with logistical benefits, but additional data are needed to determine long-term outcomes and assess whether this is a suitable strategy for at-risk patients.

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

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

Allogeneic hematopoietic stem cell transplantation remains a feasible approach for elderly with acute myeloid leukemia: a 10-year experience

The incidence of AML increases with age. The implementation of reduced intensity conditioning and progress in supportive care enabled to perform allo-HSCT in elderly patients. The main objective of the study was to assess the safety and efficacy of allotransplantation in elderly AML.Forty nine patients (33 males) at median age of 68 years were identified. Data on patients’ and transplant’s related variables were retrieved from our local transplant registry. Most patients (65%) were transplanted from 10/10-HLA or 9/10-HLA matched unrelated donor, seven patients (14%) received stem cells from matched related donor and ten patients (20%) from haploidentical donor. All patients received reduced-intensity conditioning (RIC). Peripheral blood was a source of stem cells in all patients except one (98%). Acute GVHD developed in 22 patients (44%) with 5 individuals presenting grade III-IV. CMV reactivation was demonstrated in 19 patients (39%) till day + 100. In total, 22 patients (45%) have died. The main causes of death included infectious complications (n = 9), relapse with subsequent chemotherapy resistance (n = 7), steroid-resistant GvHD (n = 4) and other causes (n = 2). Twenty-seven patients (55%) were alive at the last contact, presented full donor chimerism and remained in the complete remission. The probability of OS and relapse-free survival (RFS) were 57% and 81% at 2 years, respectively. Older donor age showed negative impact on relapse. CMV reactivation, the severity of acute graft versus host disease and older donor age negatively influenced survival. Allo-HSCT remains a safe, feasible and effective procedure for elderly AML patients.

Optimizing Donor Chimerism Threshold for Next-Generation Sequencing Monitoring of Measurable Residual Disease Post-Allogeneic Stem Cell Transplantation for Myeloid Neoplasms

Next-generation sequencing (NGS) is used to monitor genetically measurable residual disease (gMRD) following allogeneic stem cell transplantation (aSCT). It is unknown whether an upper limit of chimerism exists such that gMRD NGS testing can be safely forgone. We reviewed 61 patients with acute myeloid leukemia and 24 patients with myelodysplastic syndrome who had at least 1 NGS panel before and after aSCT between 2016 and 2020. Donor chimerism was quantified. Logistic regression characterized which factors predicted gMRD. Receiver operating characteristic (ROC) curves were used to determine the optimal chimerism threshold for which gMRD would not be detected. Data from an additional 22 patients with follow-up NGS testing in 2022 were also analyzed to validate our proposed threshold. A: s expected, donor chimerism was a significant predictor of gMRD (odds ratio, .38; 95% confidence interval, .10 to .62; P=.02). Age, sex, conditioning regimen, presence of a related donor, and diagnosis were not associated with gMRD. A chimerism threshold of 92.5% optimized sensitivity (97.7%) and specificity (95.4%) such that values >92.5% strongly predicted the absence of gMRD (area under the ROC curve [AUC], .986). The validation cohort demonstrated similarly strong predictive capability (AUC, .974) with appropriate sensitivity (100%) and specificity (90.9%). NGS monitoring of gMRD is redundant at chimerism values greater than a more conservative threshold of 92.5% after aSCT.

CD34 Stem Cell Boost in Pediatric Allogeneic Stem Cell Transplant Recipients: A Case Series and Review of Literature

Patients with poor graft function (PGF) or declining donor chimerism (DC) post allogeneic hematopoietic cell transplantation (HCT) may benefit from a CD34-selected stem cell boost (SCB). We retrospectively studied outcomes of fourteen pediatric patients (PGF: 12 and declining DC: 2), with a median age of 12.8 (range 0.08–20.6) years at HCT, who received a SCB. Primary and secondary endpoints included resolution of PGF or improvement in DC (≥ 15% increase), overall survival (OS) and transplant-related mortality (TRM), respectively. The median CD34 dose infused was 7.47 × 106/kg (range 3.51 × 106–3.39 × 107/kg). Among patients with PGF who survived ≥ 3 months post-SCB (n = 8), we observed a non-significant decrease in the cumulative median number of red cell transfusions, platelet transfusions, and GCSF but not intravenous immunoglobulin doses in the 3 months before and after SCB. Overall response rate (ORR) was 50%, with 29% complete and 21% partial responses. ORR was better in recipients who received lymphodepletion (LD) pre-SCB versus none (75% versus 40%; p = 0.56). The incidence of acute and chronic graft-versus-host-disease was 7% and 14%, respectively. The 1-year OS was 50% (95% CI 23–72%) and TRM was 29% (95% CI 8–58%). SCB was effective in half of our cohort with possible benefit of LD pre-SCB.

Bone Turnover Marker for the Evaluation of Skeletal Remodelling in Autosomal Recessive Osteopetrosis after Haematopoietic Stem Cell Transplantation: A Case Report

Background: Autosomal recessive osteopetrosis (ARO) is a rare genetic disorder of bone metabolism, primarily affecting the remodelling function of osteoclasts. Haematopoietic stem cell transplantation (HSCT) is the first-line treatment for ARO. Traditional tools for the assessment of therapeutic response, such as measuring donor chimerism, do not provide information on bone remodelling. The use of bone turnover markers (BTMs) might be ideal. Here, we report a case of a paediatric ARO patient undergoing successful HSCT. Methods: For the evaluation of donor-derived osteoclast activity and skeletal remodelling throughout the transplantation, the bone resorption marker β-CTX (β-C-terminal telopeptide) was used. Results: The low baseline level of β-CTX markedly increased after transplantation and remained in the elevated range even after 3 months. Donor-derived osteoclast activity reached its new baseline level around the 50th percentile range after 5 months and proved to be stable during the 15-month follow-up time. The apparent increase of the baseline osteoclast activity after HSCT was in consonance with the radiographic improvement of the disease phenotype and the correction of bone metabolic parameters. Despite the successful donor-derived osteoclast recovery, craniosynostosis developed, and reconstructive surgery had to be performed. Conclusions: The use of β-CTX may be of aid in assessing osteoclast activity throughout the transplantation. Further studies could help to establish the extended BTM profile of ARO patients using the available osteoclast- and osteoblast-specific markers.

5610437 HLA TISSUE TYPING AND HSCT FOR THE SICKLE CELL DISEASE COHORT AT THE ROYAL CHILDREN’S HOSPITAL (MELBOURNE, AUSTRALIA): AN AUDIT

Background: Sickle cell disease (SCD) is a serious and potentially life-threatening condition, of which the only established curative treatment is an allogeneic haematopoietic stem cell transplant (HSCT). HSCT is associated with significant risks, including up to a 10% risk of mortality in SCD1, and hence careful consideration of a patient’s suitability for HSCT is required by clinicians. For paediatric patients with SCD at the Royal Children’s Hospital (RCH), Melbourne, HSCT is considered on a case-by-case basis, with consideration being given to patient and family preferences in conjunction with the available international guidelines. There is currently no available guideline which provides a clear approach regarding the age at which HSCT should first be offered, the timing of which siblings should be investigated as potential donors, or whether HSCT should be offered in the absence of SCD complications. Aim: To determine how many patients from the SCD population treated at our centre have had human leucocyte antigen (HLA) tissue typing, how many proceeded to HSCT and the HSCT outcomes. Method: An audit was performed of the paediatric patients with SCD currently being treated at the RCH. Patient’s electronic medical records and laboratory results were accessed to determine if HLA tissue typing had been performed, if they had a HLA-matched sibling, and whether they had proceeded to HSCT. HSCT engraftment and mortality data was also reviewed. Results: There are currently 54 patients with SCD cared for at our centre. 12 of the 54 (22%) have had HLA typing performed for themselves and their siblings. Of these, eight (67%) do not have an HLA compatible sibling. Four patients (7%) have proceeded to BMT – three had matched sibling donors (MSD), and one patient underwent a haploidentical donor transplant in the absence of a MSD. Two of these patients have successfully engrafted, with >96% donor chimerism at 12 months. Unfortunately, one patient is deceased and the patient who received the haploidentical HSCT had failed engraftment. A fifth BMT is planned (MSD). Summary: Only a small proportion (22%) of SCD patients at our centre have had HLA typing performed for themselves and their siblings. 33% have a HLA-matched sibling, compared with <20% reported internationally2 (limited by small numbers). The small number of patients who have undergone HSCT in our centre demonstrated the variability in possible outcomes. HSCT offers the possibility of cure, but this is not certain, and comes with significant risks. Current ASH guidelines identify three indications for transplant: overt stroke, frequent pain and acute chest syndrome3. In Australia, children with SCD have access to a single disease modifying therapy, hydroxyurea. Voxelotor and Crizanlizumab are not currently approved for use, therefore the trajectory to transplant may be more pre-emptive. Before offering HLA tissue typing to every patient with a sibling, we must consider the potential barriers. Most notably, the significant risk of mortality and morbidity, the financial cost of transplant to the healthcare system compared to lifetime care of a patient with SCD, the availability of bone marrow transplant (BMT) unit beds and the potential implications for patients with malignant disease. Not insignificant, is the psychological cost to patients and their families who pin their hopes on a cure only to be disappointed if they are not an HLA match. References 1, 3. Aydin et al, American Society for Transplantation and Cellular Therapy 2021; 27: 1004.e1 - 1004.e8 2. Kanter et al, Blood Advances 2021; 5: 18

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

Monitoring of donor chimerism (DC) may detect early relapse following allogeneic hematopoietic stem cell transplantation (allo-SCT) for acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Most centers use unfractionated peripheral blood or T-cells to monitor DC, although CD34+ DC may be more predictive. The limited adoption of CD34+ DC may be due to the lack of detailed, comparative studies. To address this knowledge gap, we compared peripheral blood CD34+ and CD3+ DC in 134 patients who underwent allo-SCT for AML or MDS. In July 2011, the Alfred Hospital Bone Marrow Transplantation Service adopted routine monitoring of DC in the lineage-specific CD34+ and CD3+ cell subsets from peripheral blood at 1, 2, 3, 4, 6, 9, and 12 months post-transplantation for AML or MDS. Immunologic interventions, including rapid withdrawal of immunosuppression, azacitidine, and donor lymphocyte infusion, were prespecified for CD34+ DC ≤80%. Overall, CD34+ DC ≤80% detected 32 of 40 relapses (positive predictive value [PPV], 68%; negative predictive value [NPV], 91%), compared with 13 of 40 relapses for CD3+ DC ≤80% (PPV, 52%; NPV, 75%). Receiver operating characteristic analysis showed the superiority of CD34+ DC, with the greatest value at day 120 post-transplantation. CD3+ DC provided additional value in only 3 cases, preceding CD34+ DC ≤80% by 1 month. We further show that the CD34+ DC sample can be used to detect NPM1mut, with the combination of CD34+ DC ≤80% and NPM1mut identifying the highest risk of relapse. Among the 24 patients in morphologic remission at the time of CD34+ DC ≤80%, 15 (62.5%) responded to immunologic interventions (rapid withdrawal of immunosuppression, azacitidine, or donor lymphocyte infusion) with recovery of CD34+ DC >80%, and 11 of these patients remained in complete remission for a median of 34 months (range, 28 to 97 months). In contrast, the other 9 patients did not respond to the clinical intervention and relapsed within a median of 59 days after detecting CD34+ DC ≤80%. The CD34+ DC was significantly higher in responders than in nonresponders (median, 72% versus 56%; P=.015, Mann-Whitney U test). Overall, monitoring of CD34+ DC was considered clinically useful (early diagnosis of relapse enabling preemptive therapy or predicting low risk of relapse) in 107 of 125 evaluable patients (86%). Our findings show that peripheral blood CD34+ DC is feasible and superior to CD3+ DC for predicting relapse. It also provides a source of DNA for measurable residual disease testing, which may further stratify the risk of relapse. If validated by an independent cohort, our results suggest that CD34+ should be used in preference to CD3+ DC for detecting early relapse and guiding immunologic interventions following allo-SCT for AML or MDS.

Effect of lethal total body irradiation on bone marrow chimerism, acute graft-versus-host disease, and tumor engraftment in mouse models: impact of different radiation techniques using low- and high-energy X-rays.

Effects of X‑ray energy levels used for myeloablative lethal total body irradiation (TBI) delivery prior to bone marrow transplantation (BMT) in preclinical mouse models were examined. In mouse models, single-fraction myeloablative TBI at alethal dose was delivered using two different X‑ray devices, either low (160 kV cabinet irradiator) or high energy (6MV linear accelerator), before semi-allogeneic hematopoietic stem-cell transplantation (HSCT) to ensure bone marrow (BM) chimerism, graft-versus-host disease (GVHD), and tumor engraftment. Recipient mice were clinically followed for 80days after bone marrow transplantation (BMT). Flow cytometry was performed to assess donor chimerism and tumor engraftment in recipient mice. Both X‑ray irradiation techniques delivered a10 Gy single fraction of TBI, presented alethal effect, and could allow near-complete early donor chimerism on day13. However, low-energy irradiation increased Tcells' alloreactivity compared to high-energy irradiation, leading to clinical consequences for GVHD and tumor engraftment outcomes. The alloreactive effect differences might be attributed to the distinction in inflammatory status of irradiated recipients at donor cell infusion (D0). Delaying donor cell administration (D1 after lethal TBI) attenuated Tcells' alloreactivity and clinical outcomes in GVHD mouse models. Different X‑ray irradiation modalities condition Tcell alloreactivity in experimental semi-allogeneic BMT. Low-energy X‑ray irradiator induces apost-TBI inflammatory burst and exacerbates alloreactive reactions. This technical and biological information should be considered in interpreting GVHD/ graft-versus-leukemia effect results in mice experimental models of BMT.

Bone marrow CD34+ molecular chimerism as an early predictor of relapse after allogeneic stem cell transplantation in patients with acute myeloid leukemia.

Minimal residual disease (MRD) monitoring is an important tool to optimally address post-transplant management of acute myeloid leukemia (AML) patients. We retrospectively analyzed the impact of bone marrow CD34+ molecular chimerism and WT1 on the outcome of a consecutive series of 168 AML patients submitted to allogeneic stem cell transplantation. The cumulative incidence of relapse (CIR) was significantly lower in patients with donor chimerism on CD34+ cells ≥ 97.5% and WT1 < 213 copies/ABL x 10^4 both at 1st month (p=0.008 and p<0.001) and at 3rd month (p<0.001 for both). By combining chimerism and WT1 at 3rd month, 13 patients with chimerism < 97.5% or WT1 > 213 showed intermediate prognosis. 12 of these patients fell in this category because of molecular chimerism < 97.5% at a time-point in which WT1 was < 213. Our results confirm that lineage-specific molecular chimerism and WT1 after allo-SCT (1st and 3rd month) are useful MRD markers. When considered together at 3rd month, CD34+ molecular chimerism could represent an earlier predictor of relapse compared to WT1. Further studies are necessary to confirm this preliminary observation.

Late relapse after hematopoietic stem cell transplantation for acute leukemia: a retrospective study by SFGM-TC

Late relapse (LR) after allogeneic hematopoietic stem cell transplantation (AHSCT) for acute leukemia is a rare event (nearly 4.5%) and raises the questions of prognosis and outcome after salvage therapy. We performed a retrospective multicentric study between January 1, 2010 and December 31, 2016, considering data from the French national retrospective register ProMISe (SFGM-TC (French Society for Bone Marrow Transplantation and Cellular Therapy)). We included patients presenting LR, defined as a relapse occurring at least two years after AHSCT. We used the Cox model to identify prognosis factors associated with LR. During the study period, 7,582 AHSCT were performed in 29 centers and 33.8% of patients relapsed. Among them, 319 (12.4%) were considered as LR, representing an incidence of 4.2% from the entire cohort. The full dataset was available for 290 patients, including 250 (86.2%) with acute myeloid leukemia, and 40 (13.8%) with acute lymphoid leukemia. Median delay from AHSCT to LR was 38.2 months (29.2-49.7) and 27.2% of patients had extramedullary involvement at LR (17.2% exclusively and 10% associated with medullary involvement). One-third of patients had persistent full donor chimerism at LR. Median overall survival (OS) after LR was 19.9 months (5.6-46.4). The most common salvage therapy was induction regimen (55.5%), with complete remission being obtained for 50.7%. Ninety-four patients (38.5%) underwent a second AHSCT, with a median OS of 20.4 months (7.1-49.1). Non-relapse mortality after second AHSCT was 18.2%. We identified in the Cox model some of the associated factors with delay of LR: the disease status not in first complete remission at first HSCT (odds ratio (OR) 1.31, 1.04-1.64, p=0.02) and the use of post-transplant cyclophosphamide (OR 2.23, 1.21-4.14, p=0.01). Chronic GVHD appeared to be a protective factor (OR 0.64, 0.42-0.96, p=0.04). Prognosis of LR is better than early relapses, with a median OS after LR of 19.9 months. Salvage therapy associated with a second AHSCT improves outcome and is feasible, without creating excess toxicity.

Post-transplant cyclophosphamide and early mixed donor Chimerism in myeloid malignancies; a single-center experience

BackgroundAllogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the only curative option for high-risk myeloid malignancies. Post-transplant cyclophosphamide (PT-Cy) has proven to be effective for graft versus host disease (GVHD) prophylaxis. Given that graft-versus-tumor (GVT) effect plays a major role in reducing the risk of disease relapse, the application of PT-Cy must balance the risk of relapse. Mixed chimerism (MC) refers to a state of concurrent presence of recipient and donor cells post allo-HSCT which may precede relapse disease. ObjectiveWe investigated the impact of PT-Cy on early MC (EMC) and disease relapse in patients with a myeloid malignancy post allo-HSCT. Study designThis retrospective single-center study included patients that underwent allo-HSCT between 2015 and 2021. Patient and disease characteristics were collected from the electronic health records. EMC was defined as <95% donor cells at day 90–120 post allo-HSCT. ResultsA total of 144 patient that received an allo-HSCT were included in the study. One hundred and eight (75%) patients received PT-Cy as part of the GVHD prophylaxis regimen. The majority underwent allo-HSCT for acute myeloid leukemia (62%) or myelodysplastic syndrome (31%). Sixty-five percent received allo-HSCT from a matched unrelated donor transplant and 65% received a myeloablative conditioning regimen. A lower rate of chronic GVHD (p = 0.03) and a higher rate of EMC (p = 0.04) were observed in patients that received PT-Cy. PT-Cy was not associated with overall survival (OS) and relapse-free survival (RFS). Multivariable analysis identified measurable residual disease status (p = 0.003), hematopoietic cell transplantation-specific comorbidity index (p = 0.012) and chronic GVHD (p = 0.006) as independent prognostic variables for OS. AML-adverse risk (p = 0.004) and EMC (p = 0.018) were independently prognostic for RFS. While EMC overall was not significantly associated with higher risk of relapse, EMC was associated with shorter RFS within adverse-risk AML patients. ConclusionOur study shows that PT-Cy was associated with an increased risk of EMC. The predictive value of EMC for relapse remains unclear and may depend on the underlying disease, which should be validated in a larger cohort.