Conditioning Intensity and Peri-Transplant Flow Cytometric MRD Dynamics in Adult AML

In acute myeloid leukemia (AML), measurable residual disease (MRD) before or after allogeneic hematopoietic cell transplantation (HCT) is an established, independent indicator of poor outcome. To address how peri-HCT MRD dynamics could refine risk assessment across different conditioning intensities, we analyzed 810 adults transplanted in remission after myeloablative conditioning (MAC; n=515) or non-MAC (n=295) who underwent multiparameter flow cytometry-based MRD testing before and 20-40 days after allografting. Patients without pre- and post-HCT MRD (MRDneg/MRDneg) had the lowest risks of relapse and highest relapse-free survival (RFS) and overall survival (OS). Relative to those patients, outcomes for MRDpos/MRDpos and MRDneg/MRDpos patients were poor regardless of conditioning intensity. Outcomes for MRDpos/MRDneg patients were intermediate. Among 161 patients with MRD before HCT, MRD was cleared more commonly with a MAC (85/104 [81.7%]) than non-MAC (33/57 [57.9%]) regimen (P=0.002). Although non-MAC regimens were less likely to clear MRD, if they did the impact on outcome was greater. Thus, there was a significant interaction between conditioning intensity and "MRD conversion" for relapse (P=0.020), RFS (P=0.002), and OS (P=0.001). Similar findings were obtained in the subset of 590 patients receiving HLA-matched allografts. C-statistic values were higher (indicating higher predictive accuracy) for peri-HCT MRD dynamics compared to the isolated use of pre-HCT MRD status and post-HCT MRD status for prediction of relapse, RFS, and OS. Across conditioning intensities, peri-HCT MRD dynamics improve risk assessment over isolated pre- or post-HCT MRD assessments.

Conditioning Intensity and Probability of Live Birth after Blood or Marrow Transplantation (BMT): a BMTSS Report

We examine the impact of conditioning intensity (low intensity: non-myeloablative/reduced intensity vs. high intensity: myeloablative) and total body irradiation (TBI) on the probability of live birth after blood or marrow transplantation (BMT). Study participants were drawn from the BMT Survivor Study (BMTSS), and included 1,607 survivors transplanted 1974-2014 at age ≤45, with survival ≥2y post-BMT and age at study ≥18. Closest-age, same-sex biologic siblings (n=172) were 1:1 matched with 172 survivors. Survivors and siblings self-reported information on sociodemographic, chronic health conditions, and pregnancies. Within survivor analysis: The association between the primary exposure variable (No TBI/low-intensity conditioning; 200-800cGy TBI/low-intensity conditioning; No TBI/ high-intensity conditioning; >800cGy TBI/ high-intensity conditioning) and the odds of no post-BMT live birth was examined using multivariable logistic regression, adjusting for clinical and demographic variables. Median age at BMT was 31y (IQR=0-45), and median length of follow-up was 14.3y (IQR=2.4-41.4); 39.3% were autologous BMT recipients and 46.6% were female. Overall, 120 (8.7%) survivors reported post-BMT live births. Receipt of >800cGy TBI/ high-intensity conditioning (OR=3.7, 95%CI=1.9-7.0; ref: no TBI/low-intensity conditioning) was associated with higher odds of reporting no live birth post-BMT. In contrast, 200-800cGy TBI/low-intensity conditioning (OR=1.3, 95%CI=0.5-3.3), and no TBI/high-intensity conditioning (OR=0.9, 95%CI=0.5-1.7) were at similar risk of reporting post-BMT live birth as no TBI/low-intensity conditioning. Comparison with biologic siblings: Using conditional logistic regression, we found that BMT survivors were more likely to report no live birth (OR=2.0, 95%CI: 1.2-3.3) compared with siblings. These findings could inform conditioning intensity options for patients wishing to preserve fertility post-BMT.

Complex Clonal Evolution Can Occur Following Transplantation for Transfusion Dependent Thalassaemia in the Context of Mixed Myeloid Chimerism and Reduced Conditioning Regimens

Haemopoietic stem cell transplantation (HSCT) is a well-established treatment modality for the cure of transfusion dependent thalassaemia (TDT) and sickle cell disease (SCD). Clonal evolution has recently been identified as a concerning event in the setting of mixed chimerism and/or ineffective haemopoiesis following conventional bone marrow transplantation and gene therapy for haemoglobinopathies. This has so far been restricted to SCD only, with the presumption that despite both conditions sharing an ineffective erythropoietic marrow compartment, there may be inflammatory and hypoxic differences enabling clonal evolution, in addition to the different exposure to hydroxycarbamide as a therapeutic agent. However, there is a need to investigate whether this may also be an occurrence in TDT. From 2011 to 2021, over a ten-year period, sixty-five consecutive paediatric patients received a sibling HSCT (n=55) or a haploidentical HSCT (n=10) for TDT in our institution. Conditioning intensity was minimised at the start of this cohort in order to limit toxicity and late effects, abandoning the use of Bu/Cy, which resulted in approximately 50% of the patients having stable mixed chimerism long-term. Sibling HSCT was conditioned with fludarabine 160 mg/m 2, treosulfan 42 g/m 2, thiotepa 10 mg/kg and ATG (Thymoglobulin) or alemtuzumab, and received GvHD prophylaxis with ciclosporin and MMF. Haploidentical HSCT was conditioned with fludarabine 150 mg/m 2, cyclophosphamide 30 mg/kg, TBI 2 or 4 Gy, and ATG 4.5 mg/kg (thiotepa 10 mg/kg added if TBI 2 Gy only) with GvHD prophylaxis provided by two doses of post-transplantation cyclophosphamide 50 mg/kg, sirolimus and MMF. All patients had pre-transplantation endogenous haemopoiesis was suppressed pre-transplantation with hypertransfusions for a minimum of 8 weeks, and/or the use of hydroxycarbamide and azathioprine. GvHD prophylaxis was provided with ciclosporin and MMF. All patients were Pesaro class I or II at the time of transplantation. The median age was 5 years (2 - 19). The median survival was 26.8 months (2.6-101.8). The OS was 93.8% and DFS was 89.2%. Three patients in this cohort developed clonal evolution in the context of myeloid mixed chimerism identified due to the development of cytopenias and transfusion dependence. All patients had a complex karyotype and it involved deletion of chromosome 7: Patient 1 had a sibling BMT at 3 years and 2 months. Day +28 chimerism was >95% in whole blood and 95% in T cells. The myeloid fraction had a progressive reduction from day +60 onwards. At 15 months post-transplantation clonal evolution was identified [18% ring sideroblasts, 46;XY, del (7) (q22 q34) [5]/46;XY [5], chimerism 13% donor in whole marrow and 41% marrow T cells. Two months later he became red cell transfusion dependence. A second BMT with busulfan based conditioning resulted in long-term cure. Patient 2 had a sibling BMT at 3 years and 8 months of age. Day +28 chimerism was 99% donor in whole blood and 99% donor in T cells. Day +161 post-transplantation he started requiring erythropoietin support to maintain him transfusion independent and on day +217 we first identified in the bone marrow the appearance of myeloid mixed chimerism (75% donor in whole marrow and 91% marrow T cells) and complex clonal evolution involving -7 and FISH identified deletion of one copy of KMT2E (7q22) and MET (7q31) detected [16/200]. The patient is under monitoring at present with a progressive reduction of the size of the abnormal clones. Patient 3 had a haplo BMT at 5 years and 8 months. Day +28 chimerism was 97% donor in whole blood and 99% donor in T cells. She developed mixed myeloid chimerism following cessation of immunosuppression on day +206: 86% donor whole blood and 99% donor in T cells. At 22 months post-BMT she started to require transfusion and a complex clonal evolution involving deletion 7 in her bone marrow when the chimerism was 42% donor whole blood and 81% donor marrow T cells. FISH identified deletion of one copy of KMT2E(7q22) and MET(7q31) detected[22/100]. She is being prepared for a second bone marrow transplant. In conclusion, complex clonal evolution also occurs post HSCT in TDT, at least in the context of reduction of conditioning intensity and development of mixed myeloid chimerism. This finding warrants further investigation and may have significant implications for the design of both conventional HSCT and gene therapy strategies. Disclosures No relevant conflicts of interest to declare.

Validation of Pre-Transplantation Plasma ST2 Levels As a Prognostic Marker of 1-Year Non-Relapse Mortality after Allogeneic Hematopoietic Cell Transplantation

Introduction: Accurate assessment of the risk of non-relapse mortality (NRM) is important for making the shared decision about treatment with allogeneic hematopoietic cell transplantation (allo-HCT). We have shown that the pre-transplantation plasma level of suppression of tumorigenicity 2 (ST2)-a protein that is released to the bloodstream upon inflammation, cellular stress and endothelial damage-was associated with NRM after myeloablative allo-HCT [Gjærde et al., ASH Annual Meeting 2020, abstract #1524]. In an expanded cohort of both myeloablative- and non-myeloablative conditioned patients, we aimed to validate the value of pre-transplant ST2 in predicting 1-year NRM after allo-HCT. Methods: Pre-transplantation plasma ST2 levels were measured by enzyme-linked immunosorbent assays in 374 adult patients who underwent allo-HCT at Rigshospitalet between July 2015 and December 2019 (Table 1), using stored plasma samples collected at a median (Q1, Q3) of 23 (21, 24) days before allo-HCT. All patients were followed-up for at least 1 year after transplant. NRM was defined as all deaths in relapse-free patients. Given our sample size and outcome proportion, we could include four parameters in a logistic regression model of 1-year NRM to avoid severe overfitting [Riley et al., BMJ, 2020]. Based on our current clinical risk assessment practice, we included age (linear), comorbidity index (HCT-CI [Sorror et al., Blood, 2005], linear) and conditioning intensity (myeloablative vs. non-myeloablative) in a base model, to which we added the pre-transplantation ST2 level (linear) and assessed its incremental prognostic value [Steyerberg et al., Epidemiology, 2019]. The internal validity of the full model was estimated by bootstrapping [Steyerberg et al., J Clin Epidemiol, 2001]. Results: The median (Q1, Q3) pre-transplantation plasma ST2 level was 20.4 (15.2, 27.2) ng/mL. NRM at 1-year was 9% (N = 33). The main causes of NRM were organ failure (39%), infection (23%) and acute graft-versus-host disease (21%). Relapse risk at 1-year was 18%. The patients who constituted the 33 cases of 1-year NRM had a 2.7 ng/mL higher median pre-transplantation ST2 level than the remaining 341 patients (95% bootstrap confidence interval [CI] of the difference: -1.9, 6.2 ng/mL, Figure Panel A). In the full logistic regression model-including age, HCT-CI, conditioning intensity and ST2-ST2 was associated with 1-year NRM with an odds ratio of 1.32 (CI: 1.05, 1.65) per 10 ng/mL increase. Adding ST2 to the base model increased the model likelihood ratio χ 2 from 12.1 to 17.3 (p = 0.02), i.e. ST2 added a fraction of 30% (12.1/17.3) of new predictive information to age, HCT-CI and conditioning intensity. However, the ability of the full model to discriminate cases of NRM at 1-year remained poor with minimal improvement after adding ST2 (AUC up to 0.675 from 0.674 in the base model). The bootstrap-corrected AUC (the expected AUC of the full model used in a new population) was 0.63. Moreover, bootstrap-corrected estimates of predicted vs. observed risk revealed slight model miscalibration: lower predicted risks were generally underestimated, while higher predicted risks were overestimated (Figure Panel B). Conclusion: Pre-transplantation plasma levels of ST2 was a prognostic biomarker of 1-year NRM after allo-HCT, adding new predictive information to age, HCT-CI and conditioning intensity. However, internal validation of the full ST2-based prediction model revealed poor overall performance, precluding further validation and use of the model in clinical practice. When identifying prognostic biomarkers, investigation of overall predictive performance (in addition to already known prognostic factors) is needed before clinical usefulness can be evaluated. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Conditioning Intensity and Probability of Live Birth after Blood or Marrow Transplantation (BMT) - a Report from the BMT Survivor Study (BMTSS)

Background: The observed inter-individual variability in the probability of live birth after BMT is due to exposure to gonadotoxic agents (including total body irradiation [TBI]), age at exposure to gonadotoxic agents, sex of the BMT recipient, and post-BMT morbidity. The last decade has seen an increase in use of non-myeloablative (NMA) conditioning to reduce the risk of early post-treatment mortality. However, the impact of TBI in the context of conditioning intensity (NMA vs. myeloablative conditioning [MAC]) on the probability of post-BMT live birth remains unknown. We addressed this gap by utilizing the BMTSS - a multi-institutional collaborative designed to understand the burden of morbidity after BMT. Methods: This study included 1,607 BMT survivors who underwent BMT between 1974 and 2014 at age ≤45, had survived ≥2y, and were ≥18y of age at study. This study also included pair-matched nearest-age, same-sex biologic siblings (≥18y of age at study) to control for genetic or environmental factors that could affect fertility. Survivors and their siblings completed the BMTSS survey. Sociodemographic characteristics (race/ethnicity, annual household income, availability of health insurance, level of education), chronic health conditions, medical assistance for fertility assistance and details about all pregnancies and their outcomes were retrieved from the BMTSS survey. Clinical characteristics were obtained from the institutional BMT databases and/or participants' medical record. Within survivor analysis: Potential risk factors for not reporting a live birth after BMT were analyzed using multivariable logistic regression. Exposure to TBI and conditioning intensity were consolidated to create four distinct exposure groups: no_TBI/NMA (least intense), TBI/NMA, no_TBI/MAC, TBI/MAC (most intense). Matched-pair comparison with biologic siblings: We matched 172 survivors with their closest-age, same-sex, biological siblings and used conditional logistic regression to determine the failure to report live birth in BMT survivors when compared with their siblings. Results: In this cohort of 1,607 survivors, 599 (37.3%) were autologous BMT recipients, and 765 (47.6%) were female. Median age at BMT was 30y, and at study participation was 45y (IQR: 18-73); median length of follow-up from BMT to study was 14.4y (IQR: 2.4-41.4). The primary indications for BMT included HL/NHL (30.6%), AML/MDS (24.3%), CML (13.4%), ALL (12.2%), and other diagnoses (13.5%). Of the 1,607 survivors, 120 (7.5%) reported one or more live births after BMT. Within survivor comparison: Multivariable analysis (Figure) revealed that receipt of TBI/MAC (OR=2.9, 95%CI: 1.5-5.8; ref: no_TBI/ NMA) was associated with an increased risk of not reporting a post-BMT live birth. Of note, TBI/NMA (OR=1.5, 95%CI, 0.5-5.0), and no_TBI/ MAC (OR=0.9, 95%CI, 0.4-2.0) were at a similar risk of reporting a post-BMT live birth as no_TBI/NMA. Other factors associated with increased risk of not reporting a post-BMT live birth included older age at BMT (>35y: OR=3.5, 95%CI, 1.4-7.9; reference: <12y), lower income ($50k-100k: OR=2.0, 95%CI, 1.1-3.6; <$50k: OR=3.2, 95%CI, 1.6-6.4; reference: >$100k), no medical interventions to facilitate pregnancy (OR=2.7, 95%CI, 1.6-4.6), and history of pre-BMT live birth (OR=2.7, 95%CI, 1.6-4.6). Matched-pair comparison with biologic siblings: Multivariable conditional logistic regression revealed that BMT survivors were less likely to report live birth (OR=0.5, 95%CI: 0.3-0.8) compared to their siblings. However, once pregnant, they were also less likely to report a miscarriage (OR=0.4. 95%CI: 0.2-0.8). Conclusion: While full dose TBI in the setting of myeloablative conditioning is associated with a lower probability of live birth after BMT, lower doses of TBI in the setting of myeloablative or reduced intensity conditioning yield a similar probability of live birth as no TBI. These findings provide evidence for alternative conditioning regimens for patients who wish to have children after BMT. Figure 1 Figure 1. Disclosures Weisdorf: Fate Therapeutics: Research Funding; Incyte: Research Funding. Forman: Lixte Biotechnology: Consultancy, Current holder of individual stocks in a privately-held company; Mustang Bio: Consultancy, Current holder of individual stocks in a privately-held company; Allogene: Consultancy. Arora: Syndax: Research Funding; Pharmacyclics: Research Funding; Kadmom: Research Funding.

Temporal Factors Modulate Haloperidol-Induced Conditioned Catalepsy

Repeated pairings of a neutral context and the effects of haloperidol give rise to conditioned catalepsy when the context is subsequently presented in a drug-free test. In order to confirm whether this response is based on Pavlovian processes, we conducted two experiments involving two manipulations that affect conditioning intensity in classical conditioning procedures: time of joint exposure to the conditioned and the unconditioned stimulus, and the length of the inter-stimulus interval (ISI). The results revealed that both an increase in the length of context-drug pairings during conditioning and a reduced ISI between drug administration and context exposure increased conditioned catalepsy. These results are discussed in terms of the temporal peculiarities of those procedures that involve drugs as the unconditioned stimulus along with the role of Pavlovian conditioning in context-dependent catalepsy.

Prognostic value of measurable residual disease monitoring by next-generation sequencing before and after allogeneic hematopoietic cell transplantation in acute myeloid leukemia

Given limited studies on next-generation sequencing-based measurable residual disease (NGS-MRD) in acute myeloid leukemia (AML) patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT), we longitudinally collected samples before and after allo-HSCT from two independent prospective cohorts (n = 132) and investigated the prognostic impact of amplicon-based NGS assessment. Persistent mutations were detected pre-HSCT (43%) and 1 month after HSCT (post-HSCT-1m, 20%). All persistent mutations at both pre-HSCT and post-HSCT-1m were significantly associated with post-transplant relapse and worse overall survival. Changes in MRD status from pre-HSCT to post-HSCT-1m indicated a higher risk for relapse and death. Isolated detectable mutations in genes associated with clonal hematopoiesis were also significant predictors of post-transplant relapse. The optimal time point of NGS-MRD assessment depended on the conditioning intensity (pre-HSCT for myeloablative conditioning and post-HSCT-1m for reduced-intensity conditioning). Serial NGS-MRD monitoring revealed that most residual clones at both pre-HSCT and post-HSCT-1m in patients who never relapsed disappeared after allo-HSCT. Reappearance of mutant clones before overt relapse was detected by the NGS-MRD assay. Taken together, NGS-MRD detection has a prognostic value at both pre-HSCT and post-HSCT-1m, regardless of the mutation type, depending on the conditioning intensity. Serial NGS-MRD monitoring was feasible to compensate for the limited performance of the NGS-MRD assay.