Supplementary Table 3 HD and MPN samples.

Background Allogeneic stem cell transplant (allo-SCT) remains an important curative therapeutic modality in adults with high risk ALL. Reduced intensity conditioning (RIC) has extended the curative potential of allo-SCT to increasing numbers of older fit adults. There have been, however, no prospective randomised trials to guide the choice of the optimal RIC regimen in older adults with ALL. The UKALL14 trial previously reported outcomes for 249 adults >40y with ALL in CR1 who received fludarabine, melphalan and alemtuzumab (FMA) RIC allo-SCT. Overall survival (OS) at 4y was 55% with transplant-related mortality (TRM) 19.6%. The major cause of treatment failure was relapse (33.6% of patients by 4y) (Marks et al. 2022). Recent registry data has indicated that use of TBI is associated with improved disease outcomes (Giebel et al. 2017) in adult ALL patients receiving allo-SCT. We therefore performed a prospective, randomised comparison of the UK FMA RIC regimen with a cyclophosphamide plus 8Gy TBI RIC protocol (Cy/8TBI), with the goal of improving OS in high-risk adult ALL. Methods The FMA RIC regimen (fludarabine 30mg/m2 IV for 5d; melphalan 140mg/m2 IV single dose; alemtuzumab 30mg IV D-1 for sibling donor/20mg IV D-2 & -1 for unrelated donor) was compared to cyclophosphamide 50mg/kg for 2d plus 8Gy TBI (4# over 2d) and alemtuzumab (dosed as per FMA). Intrathecal prophylaxis was given for 2y post-SCT. Donor lymphocyte infusions were permitted for persistent minimal residual disease (MRD) or mixed donor T-cell chimerism from 3mo post-SCT. Primary endpoint was disease-free survival (DFS), with secondary endpoints including cumulative incidence of relapse (CIR), NRM, OS, GvHD rates and toxicity. Results Total 102 patients from 20 centres were randomised, with 89 proceeding to allo-SCT on study (45 FMA; 44 Cy/8TBI); 94 (92%) were in CR1 and 8 (7.8%) in CR2. Thirty patients (29.4%) had high risk cytogenetics (KMT2A-r; low hypodiploidy; complex karyotype), plus 31 (30.4%) Philadelphia positive cases. Median age of the entire cohort was 52y (IQR 46-59) with treatment arms being well balanced for pre-SCT characteristics. Total 31 (30%) and 71 (70%) patients received sibling and unrelated donor allo-SCT, respectively. Engraftment occurred in 85/89 (96%), with equivalent time to neutrophil and platelet engraftment between arms. With a median follow up of 41 months, 3y DFS by intention to treat (ITT) was 50% and 48% for FMA and Cy/8TBI, respectively (HR 1.12 [95% CI 0.64-1.95]; p=0.7). Neither cytogenetic risk nor pre-SCT MRD significantly affected DFS. At 3y, neither CIR (FMA 31% vs Cy/8TBI 36%; HR 1.23 [95% CI 0.63-2.42]; p=0.6), nor NRM (FMA 19% vs Cy/8TBI 16%; HR 0.81 [95% CI 0.32-2.07]; p=0.7) differed significantly between arms. OS at 3y (ITT) was also similar (FMA 61% vs Cy/8TBI 58%; HR 1.1; [95% CI 0.59-2.05]; p=0.8). Grade 2-4 aGvHD was seen in 2 (4.0%) and 5 (9.6%) patients with FMA and Cy/8TBI, respectively (p=0.44). No grade 3+ aGvHD was seen. Maximum grade 1 aGvHD occurred in 5 (10%) and 8 (15%) after FMA and Cy/8TBI, respectively. cGvHD developed in 4 (8%) FMA and 10 Cy/8TBI (19%) patients, with extensive in 6 (5.9%) patients, and no difference between arms (p=0.15). Total 97 and 102 SAEs were reported (in 30 and 26 patients) in the FMA and Cy/8TBI arms, respectively. Infections were not higher with Cy/8TBI (FMA 26 (27%); Cy/8TBI: 23 (23%)); nor were cardiac and pulmonary SAEs (FMA: 12 (12%); Cy/8TBI: 11 (11%)). Median total days of hospitalisation in year 1 did not differ between arms (FMA: 26 [IQR 22-33]; Cy/8TBI 27 [IQR 22-37]). During the study period, the most frequent causes of death in both arms were disease relapse (FMA 6 (32%); Cy/8TBI 9 (43%)) and infection (FMA 4 (21%); Cy/8TBI 8 (38%)). Conclusions This is the first prospective, randomised trial comparing RIC allo-SCT conditioning regimens in adult ALL. Cy/8TBI did not demonstrate superiority over a non-TBI FMA RIC protocol, achieving similar DFS and OS at 3y post-SCT. However, incorporation of 8Gy TBI into a RIC protocol designed for older adults was well tolerated, with no increases in acute or chronic GvHD, and no additional infectious or extramedullary toxicity. These data highlight the importance of performing randomised trials of innovative conditioning regimens, if outcomes for older patients receiving allo-SCT for ALL are to be improved. Importantly the ALL-RIC trial showcases feasibility and patient appetite for randomised transplant trials.

Supplementary Table 5 Antibodies

Supplementary Materials and Methods, Supplementary Figures S1-S12

Supplementary Table 6 qRT PCR Probes

Supplementary Table 1 VEGFAC Top Differentially Expressed Genes by Cluster

Background The UK Adult ALL CAR-T panel - established May 2023 - promotes equity of access and consistent eligibility assessment for nationally commissioned CAR-T therapies. This collaborative platform supports robust collection and assessment of real-world data (RWD). We previously reported RWD around use, toxicity and outcomes of adult ALL patients (pts) approved for brexu-cel therapy in the 1st 12mo of nationally funded access. Here we provide an updated analysis of UK CAR-T use for adult ALL and evaluate factors influencing outcome following brexu-cel infusion. Methods All pts approved for brexu-cel by panel between May 2023-April 2025 and with sufficient data were included. Retrospective data from 20 accredited immune effector cell centres were anonymised, collected on a standardised proforma and collated to support RWD analysis. Results Of 134 pts assessed, 92 were eligible for brexu-cel by national criteria (ITT cohort). Indications were post-allo-SCT relapse (60; 65.2%); 1st relapse within 12mo of CR1 (11; 12.0%); 2nd/subsequent relapse (7; 7.6%); relapse in non-SCT candidate (4; 4.4%); refractory disease (10; 10.8%). Median age (ITT) was 50.5y (IQR 40.5-59y), with 21 (22.8%) >60y. One-third (30; 33.3%) had tyrosine kinase activating fusions; 17 (18.9%) and 7 (7.8%) had v high and high-risk cytogenetics, respectively (per revised UKALL14 genetic risk). Median prior therapy lines were 2 (1-5), including prior allo-SCT, blinatumomab and inotuzumab in 61 (66.3%), 26 (28.3%) and 21 (22.8%), respectively. Of approved cases, 83 (88%) underwent apheresis (2 failed apheresis; 1 failed manufacture). Ultimately, 68 (73.9% of approved; 85% with product) underwent brexu-cel infusion. Median time from approval to infusion was 57.5d (IQR 43.5-74.5); 95.6% received bridging (84.6% 1 line only). At infusion 48 (73.8%) were in CR, with 53 (77.9%) having BM blasts <5% and 16 (24.6%) confirmed MRD negative. At D30 and D90 post-infusion, 58 (95.1%) and 49 (98%) were in CR, with 52 (85.2%) and 41 (82%) MRD negative, respectively. Of responders, 79% (95% CI 65.1-87.9) maintained response at 6mo. Five (7.4%) went on to allo-SCT. Grade 3+ CRS and ICANS occurred in 2.9% and 23.5% of cases, respectively. With median 12mo FU [95% CI 6.7-14.5] from infusion, 6mo relapse-free (RFS) and overall survival (OS) was 73.9% and 85.5%, respectively. Estimated median RFS was 17.1mo (95% CI 9.9-NR); median OS was not reached. Disease burden at infusion predicted outcome, with est median RFS for pts with ≤5% and >5% blasts of 20.1mo [95% CI 13.5-NR] and 4.2mo [95% CI 0.0-9.9] respectively (log-rank p<0.001). MRD-negativity at infusion was associated with improved RFS (median NR v 11.7mo [95% CI 7.3-20.0]; est 12mo RFS 90.0% [95% CI 47.3-98.5] v 50.0% [95% CI 33.6-64.3]; log-rank p=0.014). Prior EMD predicted worse RFS (median 4.7mo [95% CI 0-13.5] v 19.2mo [95% CI 11.3-NR]; log-rank p=0.004) and OS (median 9.0mo [95% 0.9-NR] v NR; log-rank p=0.021). Prior blinatumomab was associated with worse RFS (median 11.3mo [95% CI 3.3-NR] v 19.2mo [95% CI 9.9-NR]; log-rank p=0.046) and OS (median 14.2mo [95% CI 3.6-NR] v NR; log-rank p=0.042). OS was not impacted by prior inotuzumab exposure (any ino p=0.375; ino as bridging p=0.573). Relapse within 6mo of prior allo-SCT predicted poor outcomes post CAR-T (v relapse ≥6mo post-SCT: RFS p<0.001; OS p=0.015). In univariate Cox regression models, prior EMD (RFS HR 3.18 [1.38–7.33]; OS HR 3.22 [1.13–9.21]), >5% blasts (RFS HR 5.17 [2.35–11.39]; OS HR 4.04 [1.55–10.54]), and post-SCT relapse within 6mo (RFS HR 6.51 [2.16-19.63]; OS HR 4.70 [1.20-18.41]) predicted inferior outcomes. Adjusted for age, all retained significance (EMD: RFS HR 3.21 [1.39–7.43]; OS HR 3.04 [1.05–8.79]; >5% blasts: RFS HR 5.31 [2.39–11.82]; OS HR 4.30 [1.63–11.47]; post-SCT relapse <6mo: RFS HR 6.53 [2.12-20.08]; OS HR 4.50 [1.07-18.93]). Conclusion Here we present one of the largest RWD analyses of brexu-cel use in adult ALL. Brexu-cel was deliverable, tolerable, and efficacious, in a cohort notably differing from that trialled in ZUMA-3, including a higher proportion post-SCT, older median age, and more in CR at infusion. Outcomes were strongly influenced by disease burden at infusion and EMD status: the latter may be relevant in the frontline blinatumomab era (in which patterns of relapse may differ). Early post-SCT relapse (<6mo) was also associated with worse CAR-T outcomes, highlighting need for risk-adapted strategies in this subgroup.

Supplementary Table 2 Gene sets for GSEA

Poor outcome for older patients with ALL has multiple attributions, including a higher incidence of high-risk genetic features,1 and comorbidities as well as treatment intolerance.2, 3 The phase 2 clinical trial UKALL60+ (NCT01616238) was a collaboration between the UK National Cancer Research Institute Adult ALL Group and the Haemato-Oncology Foundation for Adults in the Netherlands (HOVON) to study treatment choices, quality of life (QoL) and outcomes in older patients with ALL. UKALL60+ offered four treatment "pathways": pathway A for BCR::ABL1+ ALL and pathways B, C, and D offering three choices of intensity for BCR::ABL1 negative ALL (Intensive, Intensive-plus and Non-Intensive, respectively), to be selected by investigator and patients. A registration-only choice (Pathway E) was also available. Details of treatment regimens are given in Figure S1. There were no exclusions for any comorbidities. The primary endpoint was complete remission (CR) after a 2-phase induction. Secondary endpoints included event-free survival (EFS) and overall survival (OS), the predictive value of MRD (Ig/TCR quantification, EuroMRD criteria),4 patient-reported outcomes, and the relationship between the baseline characteristics (Charlson index. ECOG, Karnofsky and Chemotherapy Risk Assessment Scale for High-Age Patients [CRASH] scores) and treatment option chosen. Between January 2013 and November 2018, 121 eligible patients, median age 69 (interquartile range [IQR]: 65–73, range: 55–83), of whom 107 had B-ALL and 14 T-ALL, were recruited at 34 sites (Table S1). Baseline characteristics are shown in Table 1 alongside the characteristics of the 65 patients aged over 60 years that were recruited to the contemporaneous UKALL14 trial, age 25–65 years. A consort diagram is shown in Figure S2. Fifty-one of 81 (63%) patients with BCR::ABL1 negative disease were allocated to pathway B, 11% (9/81) to pathway C, and the remaining 26% (21/81) to pathway D. At a median follow-up: 65.9 months (IQR: 38.1–80.9), CR rate after two phases of induction, was achieved by 92% (70% confidence interval [CI]: 82.1–97.2) on pathway A, 70.6% (70% CI: 62.6–77.6) on pathway B, 55.6% (70% CI: 33.6–75.9) on pathway C and 47.6% (70% CI: 34.5%–61%) of those on pathway D. No participant achieved CR on study later than end of induction. Molecular remission occurred in 5/25 (20%; A), 13/51 (25.5%; B), 2/9 (22.2%; C), and 1/21 (4.8%; D) with data available. Only 26/121 (21.5%) patients achieved molecular remission at any point. The relationship between MRD and outcome at the three study timepoints is given in Table S2. Ninety-six deaths were reported; 32 patients died without achieving CR (22/32, primary cause, ALL). Fifty-six patients died after relapse and eight died in CR (four from infection, three from second malignancies [small cell lung cancer, AML, and CMML] and one unknown). Survival data are shown in Table 1, with the corresponding Kaplan–Meier survival curves in Figure 1. At a median follow-up of 65.9 months (IQR: 38.1–80.9), the estimated 1-year EFS rates were: pathway A: 56.0% (95% CI: 34.8–72.7), pathway B: 54.9% (95% CI: 40.3–67.3), pathway C: 55.6% (95% CI: 20.4–80.5) and pathway D: 25.2% (95% CI: 9.2–45.1). The corresponding OS was: pathway A: 71.4% (95% CI: 49.2−85.2), pathway B: 64.7% (95% CI: 50.0–76.1), pathway C: 53.3% (95% CI: 17.7–79.6), and pathway D: 15.1% (95% CI: 3.8–33.6). The higher initial CR rate for patients with BCR::ABL positive ALL (pathway A) did not result in a markedly better 1-year EFS or OS than the BCR::ABL1 negative participants, regardless of pathway. The 3-year EFS (95% CI) were: pathway A: 27.0% (11.5–45.3), pathway B: 16.5% (7.7–28.3), pathway C: 41.7% (10.9–70.8), and pathway D: 15.1% (3.8–33.70) and OS (95% CI): pathway A: 33.0% (15.5–51.8), pathway B: 20.8% (10.8–33.0), pathway C: 55.3% (17.7–79.6) and pathway D: 15.1% (3.8–33.6). Three-year EFS for those achieving CR was 27.6% (18.2–37.8) and for those achieving molecular remission was 33.6% (16.4–51.7), as shown in Figure S3. Only 14 patients with T-ALL were recruited, with no discernable difference in outcome to B-ALL (Table S3). A description of the pathway E, registration-only cohort is given in the supplement. Adverse events (AEs), duration of hospitalization, treatment cessation by phase of therapy, are shown in Tables S4 and S5. Five of 121 (4.1%) patients (none of whom had achieved CR) suffered a fatal AE (three pathway C and one each pathways B and D), the causes being one cardiac arrest, three lung infections, and one febrile neutropenia. Grade 3/4 events were common (98/102; 96%), particularly infections (88/102; 86.3%). Patients in pathway C experienced significantly more grade 3/4 AEs during induction 1 (medians 16.0 [IQR: 8.0–20.5]) and induction 2 (15.0 [9.0–17.5]) compared to patients in pathways A (9.0 [6.0–11.0], p = 0.045 [induction 1] and 6.0 [3.0–8.0], p = 0.030 [induction 2]) and D (6.5 [5.5–10.0], p = 0.026 [induction 1] and 5.5 [3.5–8.5], p = 0.026 [induction 2]). More events were also seen for pathway B (10.0 [8.0–13.0], p = 0.023) than pathway D. Only 21/106 (19.8%) patients completed all protocol treatment. Discontinuation was highest during inductions 1 and 2; 27/106 (25.5%) and 11/106 (10.4%) mainly due to refractory/relapsed disease (19/38; 50%). Relapsed/refractory ALL was also the main reason for discontinuation of therapy at other timepoints (33/47; 70.2%) across all arms. Only five of 106 (4.7%) overtly stopped therapy due to toxicity. A comparison of patient characteristics across the pathways is shown in Table 1. Participants on pathway D were significantly older than those on pathway B (median 73 years [IQR: 70–78] vs. 67 [IQR: 62–70], p = 0.0001), and had greater comorbidity; 9/21 (45%) with a Charleston Index of 7 or more in pathway D compared to only 8/51 (16.7%) in pathway B. The greater frailty of the pathway D group was also evident when comparing baseline QoL measures and comorbidities, with significantly lower physical functioning compared to pathway B; medians 60.0 (IQR: 53.3–80) versus 86.7 (IQR: 66.7–100), p = 0.014 (Tables S6 and S7). No patient with a Charleston index score of 7 or above was allocated to pathway C (p = 0.013). Major, age-associated comorbidities were common across the entire study cohort and included cardiac disease 27/121 (22.3%), diabetes 17/121 (14.0%), hypertension 39/121 (32.2%), and other cancer 22/121 (18.1%), eight of which were previous breast cancer and seven previous hematological malignancy. Significant differences were seen in duration of inpatient stay, analysed by percentage of total treatment period spent in hospital (p = 0.026). Patients receiving pathways B and C spent more treatment-time in hospital compared to pathways A and D, with the effect most pronounced during induction (p = 0.0001) where pathway B and C participants were inpatients for 62.1% (46.3–96.7) and 75.8% (68.8–83.0), respectively compared to pathway A and D participants at 22.8% (IQR: 9.8–55.6) and 31.1% (IQR: 14.5–51.5), respectively. QoL was compared by pathway—summarized in supplementary results. We saw no indication that the least intensive pathway D provided a better QoL, with scores for some scales numerically lower than those of pathways A–C (Figure S4A–D). Any decreases in QoL from baseline were generally seen at the end of induction phases, with improvements in the FACT scores seen by the end of consolidation 1 and maintenance 1 (Table S8). Physical function scores, as assessed by QLQ-C30, remained reduced throughout while sensory and motor neuropathy scores increased at later points during therapy. We compared the EFS and OS of UKALL60+ cohort with that of the 65 patients aged 60–65 years, treated on the full intensity adult ALL trial UKALL14 in an overlapping recruitment timeframe. Unsurprisingly, patients in UKALL14 had lower ECOG scores (40.0% vs. 60.9% ECOG 0, p = 0.0068). Fewer had baseline comorbidities (67.7% vs. 85.0%, p = 0.0059)—particularly notable for cardiac morbidities (6.2% vs. 22.5% p = 0.0046). Although CR rates were higher; UKALL14 55/63 (87.3%) vs. UKALL60+ 82/118 (69.2%), as shown in Table 1, EFS and OS rates at 3 years were 20.5% (11.5–31.3) and 25.2% (15.2–36.5) for UKALL14 and 18.8% (12.2–26.5) and 23.2% (15.9–31.4) for UKALL60+ (Figure 1C,D). However, the type of events differed, with only 18/55 (32.7%) being relapse, 9/55 deaths without remission, and 26/55 (47.2%) deaths in remission in UKALL14 compared to 56/95 (58.9%), 32/95 (33.7%), and 7/95 (7.4%) respectively, for UKALL60+. Among the UKALL14 60–65 year-old cohort, 28/65 had received allo-SCT, resulting in death in remission in 3/28 (46%).5 Among patients treated on pathway B, there was no difference in outcome between patients with high/very high-risk versus standard-risk genetics (EFS HR: 1.35 [0.68–2.67], p = 0.39) whereas there was a difference in outcome by genetic risk for patients aged 60–65 years old treated on UKALL14 (HR: 2.59 [1.12–5.98], p = 0.026). Our survival data are broadly commensurate with data from a GMALL cohort of similar median age6 where OS at 3 years was 32%. However, the UKALL60+ population were in worse health overall; 27.5% had a Charlson Index >7 compared to the GMALL cohort, with only 11% scoring >3, commensurate with the GMALL exclusion criteria for comorbidities. By contrast to the GMALL early death rate of 14%, we observed a low treatment-related mortality. UKALL60+ treatments were successfully planned to minimize harm, but this did not improve OS. It was unexpected that the 3-year EFS and OS of 20.5% and 25.2% for those aged 60–65 years treated on UKALL14 data did not differ from that of the UKALL60+ recruits, with completely overlapping survival curves (Figure 1). The outcomes are similar to those reported in an EBMT study of 418 patients aged over 55 years receiving alloSCT; 5-year LFS of 34% but a 51% nonrelapse mortality.7 Taken together, these data suggest even the most intensive treatments including alloSCT do not generate excellent outcomes for most older patients with ALL. We were surprised to see no evidence of better QoL for the recipients of the least intensive pathway D, despite a significant reduction in length of hospital stay. In summary, the UKALL60+ pathways proved safe for initial cytoreduction, but the survival outcomes in this representative population of older patients with ALL was unsatisfactory. Nonchemotherapy approaches should be employed at the earliest opportunity for this patient group. QoL should always be measured, as the investigators assumptions do not necessarily reflect patients' experience. Bela Patel: Trial design, trial management group member, manuscript writing. Amy A. Kirkwood: Trial design, lead statistician, data analysis, manuscript writing. Clare J. Rowntree: Trial management group member, site principal investigator. Krisztina Z. Alapi: Central lab manager, specimen processing, MRD analysis. Emilio Barretta: Central analysis of genetics. Laura Clifton-Hadley: Trial management group member, lead trial co-ordination. Tom Creasey: Central analysis of genetics. SooWah Lee: Specimen processing, MRD analysis. David I. Marks: Trial management group member, site principal investigator. Anthony V. Moorman: Trial management group member, central coordination of genetics. Nicholas Morley: Trial management group member, site principal investigator. Pip Patrick: Trial management group member, senior trial coordination. Zaynab Rana: Trial management group member, trial coordination. Anita Rijneveld: Site principal investigator, lead for HOVON. John A. Snowden: Trial management group member, quality of life lead. Adele K. Fielding: Trial concept and design, trial management group chair, trial chief investigator, manuscript writing, trial funding. The authors declare no conflict of interest. Cancer Research UK CRUK/A13920 to AKF, CRUK/A21019 to AKF/AVM, and an unrestricted educational support grant from Jazz Pharma. The data that support the findings of this study are available from the corresponding author upon reasonable request. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Experts from the European Leukemia Net (ELN) working group for adult acute lymphoblastic leukemia have identified an unmet need for guidance regarding management of adult ALL from diagnosis to aftercare. The group has previously summarized their recommendations regarding diagnostic approaches, prognostic factors and assessment of ALL (cross-reference). The current recommendation summarizes clinical management. It covers treatment approaches including the use of new immunotherapies, application of MRD for treatment decisions, management of specific subgroups and challenging treatment situations as well as late effects and supportive care. The recommendation provides guidance for physicians caring for adult ALL patients which has to be complemented by regional expertise preferably provided by national academic study groups.

Cancer associated fibroblasts (CAF) arising from bone marrow-derived mesenchymal stromal cells (MSC) are prominent in B-precursor acute lymphoblastic leukaemia (B-ALL). We have previously shown that CAF formation is triggered by exposure to reactive oxygen species-inducing chemotherapy and that CAF support chemoresistance by donating mitochondria to the cancer cells, through tunnelling nanotubes. In the present study, we show that exposure of MSC to ALL cell lines, patient-derived xenografts and primary cells or their conditioned media can also trigger CAF formation. Using bulk RNA sequencing in cell lines, we show that the MSC to CAF transition is accompanied by a robust interferon pathway response and we have validated this finding in primary cells. Using confocal microscopy and flow cytometry, we identify the take-up of leukaemia cell-derived mitochondrial dsRNA by MSC as a proximate trigger for the MSC to CAF transition. We show that inhibition of dsRNA formation in ALL cells by treatment with low-dose ethidium or the mitochondrial transcription inhibitor IMT1 or degradation of dsRNA in conditioned media by 100°C exposure ablates the ability of the ALL conditioned media to stimulate MSC to CAF transition. Our data reveal a novel and previously undescribed mechanism by which cancer cells induce a CAF phenotype in stromal cells, showing how B-ALL cells can directly induce the previously described niche-mediated protection within the bone marrow.

Background The UK National Adult ALL CAR-T panel, established in 05/2023, ensures robust assessment of eligibility and equitable access to Brexu-cel as the first EMA licensed, nationally approved CAR-T therapy for R/R adult B-ALL (≥26 yrs) in the NHS. The panel is uniquely positioned to facilitate real world data (RWD) collection from point of eligibility determination. Here we evaluate use, toxicity and outcomes for patients (pts) approved within the first 12 months. Methods All pts approved for Brexu-cel between 05/2023 and 05/2024 with sufficient data were included in the analysis. Retrospective data recorded by JACIE-FACT IEC centres were anonymised, collected on a standardised datasheet and collated to support RWD analysis. Results Of 75 pts screened against nationally approved criteria, 54 (72%) were deemed eligible, with sufficient data available for analysis in 51 (ITT cohort). Indication was post-allo-HSCT relapse in 62.7% (32/51) of approved cases. Six were not apheresed (1 progressive disease, 2 deaths from disease, 3 clinical decision), for an apheresis rate of 88% in the ITT cohort. Successful apheresis and manufacture was achieved in 44 (97.7%) and infusion proceeded in 36 (70.6% approved; 81.8% with available product). Reasons for non-infusion included death fromdisease (n=4),progression (n=2) and consent withdrawal (n=2). Thirty-three (64.7%) were male. Median age of approved and infused pts was 52.0 (IQR 43.0-59.0) and 51.5 (IQR 43.5-59.5) years, respectively; 12/51 (23.5%) ITT and 9/36 (25.0%) infused were ≥60 years of age. By UKALL14 revised genetic risk status, 20/51 (39.2%) were standard risk, 5/51 (9.8%) high, 12/51 (23.5%) very high, and 13/51 (25.5%) had tyrosine kinase activating fusions (1/51 unknown). Median prior lines of therapy were 2 (1-4) with 33/51 (64.7%) having prior allo-HSCT, 9/51 (17.6%) prior blinatumomab and 15/51 (29.4%) prior inotuzumab. Seventeen (33.3%) had comorbidity index (HCT-CI) 3+ at point of eligibility. Bridging therapy was delivered in 34/36 (94.4%) of infused pts. At infusion, 22/36 (61.1%) were in CR with 24/36 (66.7%) having ≤5% BM blasts and 8/36 (22.2%) MRD negative. Extramedullary disease featured in 5/36 (13.9%), with no cases of active CNS disease. Median approval-to-infusion time was 60 days (IQR 45.0-77.5). Within 30 days of infusion there was one death due to G5 ICANS. A further 3 pts had insufficient follow up (FU) for D30 response assessment. Of the remainder, 32/32 (100%) were in CR/CRi post-infusion, with 78.1% (25/32) MRD negative (3.1% (1/32) MRD positive; 18.8% (6/32) MRD unknown). Two further deaths occurred before D90 (n=1 soft tissue infection; n=1 unspecified). Six had insufficient FU for D90 response assessment. Of those remaining alive with sufficient FU at D90, 23/24 (95.8%) remained in CR/CRi, with ongoing MRD negativity confirmed in 18/23 (78.3%; 5/23 (21.7%) MRD unknown). With median FU from approval of 7.3 months (95% CI 6.2-10.1) and 8.2 months (95% CI 6.2-10.1) for infused and ITT cohorts respectively, 86.1% (31/36) of infused and 76.5% (39/51) ITT pts remained alive. Estimated OS from approval date was 88.4% (95% CI 71.9-95.5) and 81.0% (95% CI 56.6-92.5) at 6 and 12 months respectively for infused, and 77.2% (95% CI 62.5-86.7) and 72.0% (95% CI 54.1-83.9) for ITT cohorts. At 6 months, RFS for those infused was 82.1% (95% CI 60.8-92.5) (median RFS 11.5 months (95% CI 7.3-NR)), with only one progressing to allo-HSCT. Blasts >5% (vs ≤5%) at infusion was associated with worse RFS (x2 11.56; p=0.003). Of 36 infused pts, 94.4% (34/36) experienced CRS, but only 5.6% (2/36) G3+ events. Median CRS duration was 5 days (IQR 4-6). Neurotoxicity occurred in 61.1% (22/36) with G3+ ICANS in 30.6% (11/36) and 7/36 (19.4%) requiring ITU stay for neurotoxicity. Median ICANS duration was 3 days (IQR 2-4). Conclusions The UK adult ALL CAR-T access scheme uniquely supports consistent patient assessment, collaborative discussion and cross-centre analysis of Brexu-cel use and outcomes from the point of eligibility. In a cohort with median age exceeding that of ZUMA 3, Brexu-cel delivers high rates of MRD negative CR at D30 and D90, with acceptable procedural mortality (8.3% at D90) and ITT survival outcomes comparable to those for infused pts in ZUMA 3. Despite lack of CNS disease and low disease burden in most at infusion, rates of G3+ neurotoxicity mandate consideration during patient selection, consent and therapy delivery.

American Journal of HematologyEarly View IMAGES IN HEMATOLOGY Pre-B acute lymphoblastic leukemia presenting with NPM1 and FLT3 mutations Alesia A. Khan, Corresponding Author Alesia A. Khan [email protected] Hematological Malignancy Diagnostic Service, Leeds Teaching Hospitals, Leeds, UK Correspondence Brunangelo Falini, Institute of Hematology and Center for Haemato-Oncological research (CREO), University of Perugia and Santa Maria della Misericordia Hospital, Perugia, Italy. Email: [email protected] Alesia A. Khan, Hematological Malignancy Diagnostic Service, Leeds Teaching Hospitals, Leeds, UK. Email: [email protected]Search for more papers by this authorDaniel James, Daniel James Centre for Blood Research, Hull University Teaching Hospitals NHS Trust, Hull, UKSearch for more papers by this authorVibeke Andresen, Vibeke Andresen Centre for Cancer Biomarkers (CCBIO), Department of Clinical Science, University of Bergen, Bergen, NorwaySearch for more papers by this authorJulie Atkey, Julie Atkey Hematological Malignancy Diagnostic Service, Leeds Teaching Hospitals, Leeds, UKSearch for more papers by this authorRachel Bradbury, Rachel Bradbury Hematological Malignancy Diagnostic Service, Leeds Teaching Hospitals, Leeds, UKSearch for more papers by this authorCatherine Cargo, Catherine Cargo Hematological Malignancy Diagnostic Service, Leeds Teaching Hospitals, Leeds, UKSearch for more papers by this authorRichard Dillon, Richard Dillon Department of Medical and Molecular Genetics, Guy's and King's Hospitals, London, UKSearch for more papers by this authorBjørn Tore Gjertsen, Bjørn Tore Gjertsen Centre for Cancer Biomarkers (CCBIO), Department of Clinical Science, University of Bergen, Bergen, Norway Department of Medicine, Hematology Section, Haukeland University Hospital, Bergen, NorwaySearch for more papers by this authorAntony R. Goldstone, Antony R. Goldstone Centre for Blood Research, Hull University Teaching Hospitals NHS Trust, Hull, UKSearch for more papers by this authorRichard Leach, Richard Leach Hematological Malignancy Diagnostic Service, Leeds Teaching Hospitals, Leeds, UKSearch for more papers by this authorDaniel Lock, Daniel Lock Hematological Malignancy Diagnostic Service, Leeds Teaching Hospitals, Leeds, UKSearch for more papers by this authorMayanka Narayanan, Mayanka Narayanan Centre for Blood Research, Hull University Teaching Hospitals NHS Trust, Hull, UKSearch for more papers by this authorNigel Russell, Nigel Russell Department of Medical and Molecular Genetics, Guy's and King's Hospitals, London, UKSearch for more papers by this authorEleni-Anna Verigou, Eleni-Anna Verigou Centre for Blood Research, Hull University Teaching Hospitals NHS Trust, Hull, UKSearch for more papers by this authorSimone Green, Simone Green Centre for Blood Research, Hull University Teaching Hospitals NHS Trust, Hull, UKSearch for more papers by this authorAdele K. Fielding, Adele K. Fielding Centre for Blood Research, Hull University Teaching Hospitals NHS Trust, Hull, UK Centre for Blood research, University of York, York, UKSearch for more papers by this authorBrunangelo Falini, Corresponding Author Brunangelo Falini [email protected] orcid.org/0000-0002-7198-5965 Institute of Hematology and Center for Haemato-Oncological research (CREO), University of Perugia and Santa Maria della Misericordia Hospital, Perugia, Italy Correspondence Brunangelo Falini, Institute of Hematology and Center for Haemato-Oncological research (CREO), University of Perugia and Santa Maria della Misericordia Hospital, Perugia, Italy. Email: [email protected] Alesia A. Khan, Hematological Malignancy Diagnostic Service, Leeds Teaching Hospitals, Leeds, UK. Email: [email protected]Search for more papers by this author Alesia A. Khan, Corresponding Author Alesia A. Khan [email protected] Hematological Malignancy Diagnostic Service, Leeds Teaching Hospitals, Leeds, UK Correspondence Brunangelo Falini, Institute of Hematology and Center for Haemato-Oncological research (CREO), University of Perugia and Santa Maria della Misericordia Hospital, Perugia, Italy. Email: [email protected] Alesia A. Khan, Hematological Malignancy Diagnostic Service, Leeds Teaching Hospitals, Leeds, UK. Email: [email protected]Search for more papers by this authorDaniel James, Daniel James Centre for Blood Research, Hull University Teaching Hospitals NHS Trust, Hull, UKSearch for more papers by this authorVibeke Andresen, Vibeke Andresen Centre for Cancer Biomarkers (CCBIO), Department of Clinical Science, University of Bergen, Bergen, NorwaySearch for more papers by this authorJulie Atkey, Julie Atkey Hematological Malignancy Diagnostic Service, Leeds Teaching Hospitals, Leeds, UKSearch for more papers by this authorRachel Bradbury, Rachel Bradbury Hematological Malignancy Diagnostic Service, Leeds Teaching Hospitals, Leeds, UKSearch for more papers by this authorCatherine Cargo, Catherine Cargo Hematological Malignancy Diagnostic Service, Leeds Teaching Hospitals, Leeds, UKSearch for more papers by this authorRichard Dillon, Richard Dillon Department of Medical and Molecular Genetics, Guy's and King's Hospitals, London, UKSearch for more papers by this authorBjørn Tore Gjertsen, Bjørn Tore Gjertsen Centre for Cancer Biomarkers (CCBIO), Department of Clinical Science, University of Bergen, Bergen, Norway Department of Medicine, Hematology Section, Haukeland University Hospital, Bergen, NorwaySearch for more papers by this authorAntony R. Goldstone, Antony R. Goldstone Centre for Blood Research, Hull University Teaching Hospitals NHS Trust, Hull, UKSearch for more papers by this authorRichard Leach, Richard Leach Hematological Malignancy Diagnostic Service, Leeds Teaching Hospitals, Leeds, UKSearch for more papers by this authorDaniel Lock, Daniel Lock Hematological Malignancy Diagnostic Service, Leeds Teaching Hospitals, Leeds, UKSearch for more papers by this authorMayanka Narayanan, Mayanka Narayanan Centre for Blood Research, Hull University Teaching Hospitals NHS Trust, Hull, UKSearch for more papers by this authorNigel Russell, Nigel Russell Department of Medical and Molecular Genetics, Guy's and King's Hospitals, London, UKSearch for more papers by this authorEleni-Anna Verigou, Eleni-Anna Verigou Centre for Blood Research, Hull University Teaching Hospitals NHS Trust, Hull, UKSearch for more papers by this authorSimone Green, Simone Green Centre for Blood Research, Hull University Teaching Hospitals NHS Trust, Hull, UKSearch for more papers by this authorAdele K. Fielding, Adele K. Fielding Centre for Blood Research, Hull University Teaching Hospitals NHS Trust, Hull, UK Centre for Blood research, University of York, York, UKSearch for more papers by this authorBrunangelo Falini, Corresponding Author Brunangelo Falini [email protected] orcid.org/0000-0002-7198-5965 Institute of Hematology and Center for Haemato-Oncological research (CREO), University of Perugia and Santa Maria della Misericordia Hospital, Perugia, Italy Correspondence Brunangelo Falini, Institute of Hematology and Center for Haemato-Oncological research (CREO), University of Perugia and Santa Maria della Misericordia Hospital, Perugia, Italy. Email: [email protected] Alesia A. Khan, Hematological Malignancy Diagnostic Service, Leeds Teaching Hospitals, Leeds, UK. Email: [email protected]Search for more papers by this author First published: 03 April 2024 https://doi.org/10.1002/ajh.27301Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat CONFLICT OF INTEREST STATEMENT B.F. holds a patent on NPM1 mutants (number 102004901256449). The remaining authors declare no competing financial interests. Open Research DATA AVAILABILITY STATEMENT All data and information concerning this study will be made available from the corresponding authors upon reasonable request. REFERENCES 1Arber DA, Orazi A, Hasserjian RP, et al. International consensus classification of myeloid neoplasms and acute Leukemias: integrating morphologic, clinical, and genomic data. Blood. 2022; 140(11): 1200-1228. 10.1182/blood.2022015850 CASPubMedWeb of Science®Google Scholar 2Khoury JDSE, Abla O, Akkari Y, Alaggio R, Apperley JF, et al. The 5th edition of the world health organization classification of haematolymphoid tumours: myeloid and histiocytic /dendritic neoplasms. Leukemia. 2022; 36: 1703-1719. 10.1038/s41375-022-01613-1 PubMedWeb of Science®Google Scholar 3Falini B, Brunetti L, Sportoletti P, Martelli MP. NPM1-mutated acute myeloid leukemia: from bench to bedside. Blood. 2020; 136(15): 1707-1721. 10.1182/blood.2019004226 PubMedWeb of Science®Google Scholar 4Falini B, Mecucci C, Tiacci E, et al. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N Engl J Med. 2005; 352(3): 254-266. 10.1056/NEJMoa041974 CASPubMedWeb of Science®Google Scholar 5Falini B, Martelli MP, Brunetti L, Gjertsen BT, Andresen V. The NPM1 mutant defines AML irrespective of blast count. Am J Hematol. 2023; 98(7): E187-E189. 10.1002/ajh.26946 CASPubMedWeb of Science®Google Scholar 6Falini B, Dillon R. Criteria for diagnosis and molecular monitoring of NPM1-mutated AML. Blood Cancer Discov. 2024; 5: 8-20. 10.1158/2643-3230.BCD-23-0144 PubMedGoogle Scholar 7Martelli MP, Manes N, Pettirossi V, et al. Absence of nucleophosmin leukaemic mutants in B and T cells from AML with NPM1 mutations: implications for the cell of origin of NPMc+ AML. Leukemia. 2008; 22(1): 195-198. 10.1038/sj.leu.2404857 CASPubMedWeb of Science®Google Scholar 8O'Donnell MR, Tallman MS, Abboud CN, et al. Acute myeloid leukemia, version 3.2017, NCCN clinical practice guidelines in oncology. J Natl Compr Canc Netw. 2017; 15(7): 926-957. 10.6004/jnccn.2017.0116 PubMedWeb of Science®Google Scholar 9Grafone T, Palmisano M, Nicci C, Storti S. An overview on the role of FLT3-tyrosine kinase receptor in acute myeloid leukemia: biology and treatment. Oncol Rev. 2012; 6(1):e8. 10.4081/oncol.2012.e8 PubMedGoogle Scholar 10Engen C, Hellesoy M, Grob T, et al. FLT3-ITD mutations in acute myeloid leukaemia - molecular characteristics, distribution and numerical variation. Mol Oncol. 2021; 15(9): 2300-2317. 10.1002/1878-0261.12961 CASPubMedWeb of Science®Google Scholar 11Alcalay M, Tiacci E, Bergomas R, et al. Acute myeloid leukemia bearing cytoplasmic nucleophosmin (NPMc+ AML) shows a distinct gene expression profile characterized by up-regulation of genes involved in stem-cell maintenance. Blood. 2005; 106(3): 899-902. 10.1182/blood-2005-02-0560 CASPubMedWeb of Science®Google Scholar 12Martelli MP, Pettirossi V, Thiede C, et al. CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice. Blood. 2010; 116(19): 3907-3922. 10.1182/blood-2009-08-238899 CASPubMedWeb of Science®Google Scholar 13Haferlach C, Mecucci C, Schnittger S, et al. AML with mutated NPM1 carrying a normal or aberrant karyotype show overlapping biologic, pathologic, immunophenotypic, and prognostic features. Blood. 2009; 114(14): 3024-3032. 10.1182/blood-2009-01-197871 CASPubMedWeb of Science®Google Scholar 14Okabe A, Guirales F, Zhao D, Tirado CA. FLT3 gene involvement in B-cell acute lymphoblastic leukemia (B-ALL). J Assoc Genet Technol. 2021; 47(1): 6-14. PubMedGoogle Scholar Early ViewOnline Version of Record before inclusion in an issue ReferencesRelatedInformation