By integrating short-read WGS and RNA-seq data with long-read RNA sequencing, we dissect the complex genomic architecture of PAX5 intragenic tandem multiplication (PAX5-ITM), revealing that these complex rearrangements result in in-frame transcripts that likely encode proteins with altered domains.

Children's Oncology Group AALL1331 utilized an intensive chemotherapy induction (Block 1) based on UK ALLR3 induction for children, adolescents, and young adults with acute lymphoblastic leukemia in first relapse, followed by risk-stratified therapy. High/intermediate-risk patients were subsequently randomized to receive two blocks of chemotherapy or two blocks of blinatumomab followed by a hematopoietic stem cell transplant. Low-risk patients were randomized to chemotherapy or chemotherapy cycles intercalated with three blinatumomab blocks. Patients who had early treatment failure were eligible to receive blinatumomab for up to two salvage cycles. We reviewed Block 1 responses, risk stratification, randomization rates, adverse events, event-free survival and overall survival for all enrolled patients. AALL1331 enrolled 661 patients: 24 died during Block 1 and 42 experienced early treatment failure. Overall, 531/661 (80.3%) attained complete remission with 586 risk-assigned and only 471 were randomized. Of 532 patients with bone marrow involvement, 290 (54.5%) were positive for minimal residual disease (≥0.01%) after Block 1. Grade 3, 4 or 5 adverse events occurred in Block 1 in 44.9%, 24.1%, and 3.6% of patients, respectively, with febrile neutropenia, infections, and sepsis being most frequent. Notably, 190 enrolled patients (28.7%) did not proceed with post-induction therapy, including 115 (17.4%) risk-stratified but not randomized. These patients had dismal survival. More effective and less toxic reinduction strategies are needed for B-cell acute lymphoblastic leukemia in first relapse. Trial registration number: NCT02101853.

PAX5 is known to be frequently mutated in childhood B-cell precursor acute lymphoblastic leukemia (B-ALL) via several molecular mechanisms, including chimeric fusion, focal deletion, and point mutations. Among these, patients with PAX5 internal tandem duplication (PAX5-ITD) are reported to have a poor outcome. Unlike other common ITDs (e.g., FLT3), PAX5-ITD often involves gain of more than one copy of involved exons, and the exact nature of these alterations remain elusive. Here we aim to investigate the following questions: 1) what are the typical copy number gains in DNA and RNA among PAX5-ITD cases? 2) are PAX5-ITD always in-frame? 3) are these aberrations a result of multi-step or single-step events? Short-read whole genome sequencing (WGS) and RNA sequencing (RNA-seq) and PacBio ISO-seq, a state-of-the-art long-read sequencing method, are used in this study. By integrative analysis of WGS and RNA-seq data from a relapse cohort (n=584) and a non-relapse cohort (n=1055), we identified PAX5 mutation rate of ∼30% in relapsed patients versus that of ∼20% in non-relapsed patients. Among the various PAX5 mutation types, the PAX5-ITD mutation shows the greatest degree of enrichment (P=3e-6) in relapsed patients (n=21), compared with those who remain in remission (n=5). Copy number gains in PAX5-ITD cases ranged from 3 to 25 and involved four exon combinations within the gene: E5, E3-E5, E2-E5 and E1-E5 and concordant copy number gains were detected between diagnosis tumor and relapse tumor. We observed a negative correlation between the number of copy gains and the length of duplicated genomic regions. Furthermore, 11 out of the 26 PAX5-ITD cases exhibited multiple structural variants (SVs) and 1 case harbored 2 somatic SNV in ITD region. To investigate the transcriptional consequences of these SV events, we performed ISO-seq on 7 PAX5-ITD cases with available specimen. By mapping SVs and SNVs to full-length RNA transcripts, we observed complex sequences of events driving the formation of ultimate PAX5-ITD in 3 cases. Besides, copy number analysis on full-length RNA transcripts revealed variable copy number gains within tumors due to alternative splicing. Additionally, we identified neo-splice sites in 2 cases, indicating that the splicing process was influenced by the altered DNA context created by SVs. Finally, our data indicated that all PAX5-ITDs can transcribe in-frame mRNAs, suggesting activating nature of these mutations. These findings provide valuable insights into the genomic complexity of PAX5-ITD and highlight the diverse transcriptional and splicing outcomes due to these alterations. Yanling Liu, Bensheng Ju, Li Dong, Melanie Loyd, Yuan Feng, Heather Mulder, Pandurang Kolekar, Li Fan, Hanxia Li, Scott Foy, Geoff Neale, Patrick A. Brown, William L. Carroll, Mignon L. Loh, John Easton, Xiaotu Ma. Uncovering genomic complexity of PAX5 internal tandem duplication using short-read and long-read sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2617.

AIMS:Fedratinib is a potent, oral, Janus kinase inhibitor for the treatment of myelofibrosis (MF). This report describes exposure-response (E-R) analyses of fedratinib based on pooled data from phase 2/3 studies in patients with intermediate-2 or high-risk MF, with or without prior ruxolitinib exposure. METHODS:Pharmacokinetic (PK) exposures were derived from the population PK analysis. Efficacy endpoints included spleen volume reduction ≥35% (SVR35) and total symptom score reduction ≥50% (TSS response). Safety endpoints included grade ≥3 anaemia or thrombocytopenia, any-grade nausea/vomiting and diarrhoea. The E-R models were developed using logistic regression analyses. RESULTS:Fedratinib exposure was positively associated with SVR35 (odds ratio [OR], 38.2; 95% confidence interval [CI], 12.4-118; P < 0.001) and TSS response (OR, 20.8; 95% CI, 6.27-69.2; P < 0.001), after adjusting for covariates. Baseline spleen volume was inversely associated with SVR35 (P = 0.029). Prior ruxolitinib exposure was not associated with SVR35 (P = 0.090) or TSS response (P = 0.326). Although numerically higher incidence of adverse events was observed in patients with higher fedratinib exposure, there was no statistically significant association between fedratinib exposure and any safety related endpoints. Prior ruxolitinib exposure was associated with experiencing grade ≥3 thrombocytopenia (P = 0.004). Lower baseline haemoglobin level (<10 g/dL) and platelet count (<100 × 109/L) were associated with experiencing grade ≥3 anaemia (P < 0.001) and thrombocytopenia (P < 0.001), respectively. Antiemetic prophylaxis was associated with lower rates of nausea/vomiting (P < 0.001). CONCLUSIONS:Fedratinib exposure was positively associated with spleen volume reduction and TSS responses, without having significant impact on safety. Fedratinib 400 mg once daily is an appropriate dose for patients with MF regardless of ruxolitinib exposure.

Despite extensive studies of the error profiles of SNVs, those of insertions/deletions (indels)/structural variants (SVs) remain elusive. Using ultra-deep sequencing, we show that the error rates of indel/SVs are >100-fold lower than those of SNVs, although repeat indels have high error rates of 1%. We validated this pattern in a cohort of 103 patients with relapsed B cell acute lymphoblastic leukemia (B-ALL). We analyzed repeat indels in 339 cancer driver genes and demonstrated that the number of repeat units is highly predictive of the error rate. We then analyzed minimal residual disease samples from 72 patients with relapsed B-ALL and demonstrated that our approach had positive detections in 61% of cases, outperforming clinical flow cytometry (51% detection). Overall, we established indel and SV error profiles in deep next-generation sequencing (NGS) data, enabling superior tumor detection at very low burdens, which has a significant impact on the clinical diagnosis and monitoring of human cancers and other diseases.

Background Most patients with myelofibrosis develop ruxolitinib intolerance or disease that is relapsed or refractory, and survival rates after ruxolitinib discontinuation are poor. We aimed to evaluate the safety and efficacy of fedratinib versus best available therapy (BAT) in patients with myelofibrosis previously treated with ruxolitinib. Methods FREEDOM2 was a multicentre, open-label, randomised, controlled, phase 3 trial in 86 clinics in 16 countries, in which patients aged at least 18 years with intermediate-2 or high-risk myelofibrosis that was relapsed or refractory or intolerant to ruxolitinib with Eastern Cooperative Oncology Group performance status 0-2 were stratified by spleen size by palpation, platelet count, and previous ruxolitinib treatment, and randomly assigned 2:1 by interactive response technology to receive fedratinib 400 mg per day (4 x 100 mg capsules orally once daily, open-label) or BAT. Patients received prophylactic antiemetics and thiamine supplementation, and symptomatic antidiarrhoeals as required. Primary endpoint was proportion of patients reaching spleen volume reduction (SVR) of at least 35% (SVR35) at end of cycle 6 in the intention-to-treat population. This manuscript reports the primary analysis of the trial; follow-up is ongoing. This trial is registered at clinicaltrials.gov, NCT03952039. Findings Between Sept 9, 2019 and June 24, 2022, of 316 patients screened, 201 were randomly assigned and treated (134 to fedratinib, 67 to BAT [including 52 receiving ruxolitinib]); 46 patients from the BAT group crossed over to fedratinib. Approximately half of enrolled patients were male (fedratinib 75 [56%] of 134; BAT 30 [45%] of 67) and most were White (fedratinib 106 [79%] of 134; BAT 58 [87%] of 67). At data cutoff (Dec 27, 2022), median survival follow-up was 645 weeks (IQR 379-1049). SVR35 at end of cycle 6 was seen in 48 (36%) of 134 patients receiving fedratinib versus four (6%) of 67 patients receiving BAT (30% difference; 95% CI 20-39; one-sided p-value <00001). During the first six cycles 53 (40%) of 134 patients in the fedratinib group and 8 (12%) of 67 patients in the BAT group had grade 3 or greater treatment-related adverse events, most frequently anaemia (fedratinib 12 [9%] of 134; BAT 6 [9%] of 67) and thrombocytopenia (fedratinib 16 [12%] of 134; BAT 2 [3%] of 67); one patient in the fedratinib group died from acute kidney injury suspected to be related to study drug (no treatment-related deaths in the BAT group). Gastrointestinal adverse events occurred more frequently in the fedratinib group compared with the BAT group, but were mostly grade 1-2 in severity and more frequent in early cycles, and were less frequent than in prior clinical trials. A total of 28 (21%) of 134 patients in the fedratinib group and 3 (4%) of 67 patients in the BAT group had thiamine levels below lower limit of normal per central laboratory assessment, with only one case of low thiamine in the fedratinib arm after the introduction of prophylactic thiamine supplementation. Interpretation Findings from FREEDOM2 support fedratinib as a second-line Janus kinase inhibitor option to reduce spleen size after ruxolitinib failure or intolerance in patients with myelofibrosis, and shows effective strategies for management of gastrointestinal adverse events and low thiamine concentrations through prophylaxis, monitoring, and treatment.

Abstract Infant acute lymphoblastic leukemia (ALL) is characterized by high frequency of rearrangements in KMT2A (KMT2Ar), associated with poor outcomes. Infants lacking KMT2Ar typically have superior outcomes, but remain understudied. Here, we use whole genome and transcriptome sequencing to define driver mutations and transcriptional phenotypes in non-KMT2Ar infant ALL. Based on two index cases, we initially suspected that some infants with high-risk clinical features harbored clinically undetected non-canonical alterations to the KMT2A gene; however, we find no such evidence in our data. Instead, we find that these infants acquire other clinically relevant features such as Ph-like expression signatures, fusions impacting ZNF384, TCF3, ETV6::RUNX1, PAX5 or NUTM1, and events in known tumor genes such as CDKN2A, NOTCH1, and others. By mapping transcriptional profiles between infant and childhood B-ALL, we find that - in the absence of KMT2Ar - infant ALL resembles well defined childhood B-ALL subtypes, sharing the same genetic drivers. NUTM1 fusions are particularly enriched in infants compared to older children, and are associated with decreased MHC-Class II expression and B-cell developmental signaling pathways. Ph-like transcriptional signatures were apparent in several infants and confirmed by machine learning driven classification. Overall, our data support a common developmental origin of ALL without KMT2Ar in infants and children. Citation Format: Matthew Zatzman, Jennifer Seelisch, Federico Comitani, Fabio Fuligni, Scott Davidson, Kyoko E. Yuki, Lisa-Monique Edwards, Ledia Brunga, Erin Guest, Stephen P. Hunger, Mignon L. Loh, Elizabeth A. Raetz, John Chen, Jack Bartram, Johann K. Hitzler, Patrick A. Brown, Sumit Gupta, Adam Shlien. Infant ALL without KMT2A rearrangements harbor clinically relevant alterations and share common origins with childhood ALL [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr B026.

The phase 3b FREEDOM trial (ClinicalTrials.gov: NCT03755518) evaluates efficacy/safety of fedratinib in intermediate- or high-risk myelofibrosis patients with platelet count ≥50 × 109/L, previously treated with ruxolitinib. The trial design included protocol specified strategies to mitigate the risk for gastrointestinal (GI) adverse events (AEs), thiamine supplementation, and encephalopathy surveillance. Due to COVID-19, accrual was cut short with 38 patients enrolled. In the efficacy evaluable population (n = 35), nine (25.7%; 95% confidence interval 12.5-43.3) patients achieved primary endpoint of ≥35% spleen volume reduction (SVR) at end of cycle (EOC) 6; and 22 (62.9%) patients showed best overall response of ≥35% SVR up to end of treatment. Sixteen (44.4%) patients showed ≥50% reduction in total symptom score at EOC6 (n = 36). Compared to previously reported JAKARTA-2 trial, rates of GI AEs were lower, and no patient developed encephalopathy. Overall, FREEDOM study showed clinically relevant spleen and symptom responses with fedratinib, and effective mitigation of GI AEs.

Improvements in survival have been made over the past two decades for childhood acute myeloid leukemia (AML), but the approximately 40% of patients who relapse continue to have poor outcomes. A combination of checkpoint-inhibitor nivolumab and azacitidine has demonstrated improvements in median survival in adults with AML. This phase I/II study with nivolumab and azacitidine in children with relapsed/refractory AML (NCT03825367) was conducted through the Therapeutic Advances in Childhood Leukemia & Lymphoma consortium. Thirteen patients, median age 13.7 years, were enrolled. Patients had refractory disease with multiple reinduction attempts. Twelve evaluable patients were treated at the recommended phase II dose (established at dose level 1, 3 mg/kg/dose). Four patients (33%) maintained stable disease. This combination was well tolerated, with no dose-limiting toxicities observed. Grade 3–4 adverse events (AEs) were primarily hematological. Febrile neutropenia was the most common AE ≥ grade 3. A trend to improved quality of life was noted. Increases in CD8+ T cells and reductions in CD4+/CD8+ T cells and demethylation were observed. The combination was well tolerated and had an acceptable safety profile in pediatric patients with relapsed/refractory AML. Future studies might explore this combination for the maintenance of remission in children with AML at high risk of relapse.

Infants less than 1 year old diagnosed with KMT2A-rearranged (KMT2A-r) acute lymphoblastic leukemia (ALL) are at high risk of failure to achieve remission, relapse, and death due to leukemia, despite intensive therapies. Infant KMT2A-r ALL blasts are characterized by DNA hypermethylation. Epigenetic priming with DNA methyltransferase inhibitors increases the cytotoxicity of chemotherapy in preclinical studies. The Children's Oncology Group trial AALL15P1 tested the safety and tolerability of 5 days of azacitidine treatment immediately prior to the start of chemotherapy on day 6, in four post-induction chemotherapy courses for infants with newly diagnosed KMT2A-r ALL. The treatment was well-tolerated, with only two of 31 evaluable patients (6.5%) experiencing dose-limiting toxicity. Whole genome bisulfite sequencing of peripheral blood mononuclear cells demonstrated decreased DNA methylation in 87% of samples tested following 5 days of azacitidine treatment. Event-free survival was similar to that in prior studies of newly diagnosed infant ALL. Azacitidine is safe and results in decreased DNA methylation of peripheral blood mononuclear cells in infants with KMT2A-r ALL, but the incorporation of azacitidine to enhance cytotoxicity did not impact survival. Clinicaltrials.gov identifier: NCT02828358.

Background: The safety and efficacy of the bispecific T-cell engaging antibody blinatumomab (blina) was compared to intensive chemotherapy for the treatment of relapsed pediatric B-cell acute lymphoblastic leukemia (B-ALL) at first relapse treated on the phase 3 international Children's Oncology Group trial AALL1331. Patients treated with blina had improved survival and lower toxicity (Brown JAMA 2021, Hogan JCO 2023). Blina is now FDA-approved for pediatric B-ALL. However, blina was not universally effective, and disease-free survival on the high-risk blina arm was only 54% at two years. A deeper understanding of the mechanism of action of blina may allow us to further improve outcomes. We hypothesize that blina response and resistance are driven by endogenous T-cell function in addition to cellular and secreted factors that influence T-cell activity. Methods: Bone marrow (BM) and peripheral blood (PB) samples were collected at pre-defined timepoints and separated into cell and plasma components for cryopreservation. We classified patients as responders (R) and non-responders (NR) based on clinical outcome (second relapse). The Olink platform was used to quantify 3072 secreted proteins from the plasma samples of each patient. Protein quantities were compared between response groups by differential expression analysis. Bulk mononuclear cell mRNA expression was measured in a subset of patients by Nanostring, and multiparameter flow cytometry was employed to identify cellular subpopulations. Single cell RNA sequencing (scRNA-seq) was performed on a cohort of cases to further characterize cell subpopulations and their gene expression signatures. Results: Comprehensive secreted protein analysis on 19 patients revealed elevated levels of T-cell leukemia/lymphoma protein 1A (TCL1A) in R (n=11) vs NR (n=8) in pre-infusion BM plasma (5.5 fold increase (5.5x); p=5.6E-5). TCL1A was also increased in PB plasma of R compared to NR during blina infusion, most pronounced at hour 48 (4.5x;p=1.5E-3). Pro-inflammatory cytokines were elevated in PB of R vs NR, including CXCL9 (1.7x;p=9.8E-3), IL-2 (2.6x;p=1.8E-2), and IL-2RA (1.9x,p=8.9E-3) while the immune checkpoint associated protein CTLA4 was found to be elevated in NR vs R (2.2x,p=2.1E-2) at hour 48 of blina infusion. Analysis of mRNA confirmed upregulation of inflammatory cytokines in the plasma of R compared to NR at hour 48 including CXCL9 (19x;p=6.6E-2). scRNA-seq of pre-blina bone marrow samples from 4 R and 3 NR identified a relative abundance of hematopoietic stem and progenitor cells (HSPC) in the bone marrow of responders (12% vs 3%). In comparing proportions of T cell and myeloid-derived suppressor cells (MDSC), we identified a preponderance of MDSC in NR (T:MDSC ratio 1.1 vs 1.6). Multiparameter flow cytometry from 25 R and 9 NR identified an increased abundance of naïve CD8+ T cells in R (21% vs 14% of CD3+ cells), whereas increased expression of inhibitory receptors CTLA-4, LAG-3, and TIM-3 on CD8+ T-cells was observed in NR (~1% vs ~5%). Conclusions: In comparing patients with relapsed B-ALL treated with blinatumomab and chemotherapy on the Phase III clinical trial AALL1331, we made the novel observation that the protein TCL1A is significantly higher in responders compared to non-responders, both before and during blina infusion. This is distinct from proinflammatory cytokine elevation which is only seen after blina initiation. We are investigating TCL1A as a candidate pre-treatment predictor of blina response in a validation cohort. Interestingly, TCL1A is known to be expressed in early stages of lymphocyte development. Taken together with identification of increased naïve cells and fewer MDSCs, this may suggest that responders have a more activating immune phenotype. As blina is being integrated into frontline treatment, it is paramount that we understand the factors that influence response and resistance. Ultimately, these data will help guide risk allocation and treatment interventions to improve responses to blina with the overall goal of reducing toxicities and improving survival for patients with B-ALL.

Limited prognostic factors have been associated with overall survival (OS) post-relapse in childhood Acute Lymphoblastic Leukemia (ALL). Patients enrolled on 12 Children’s Oncology Group frontline ALL trials (1996–2014) were analyzed to assess for additional prognostic factors associated with OS post-relapse. Among 16,115 patients, 2053 (12.7%) relapsed. Relapse rates were similar for B-ALL (12.5%) and T-ALL (11.2%) while higher for infants (34.2%). Approximately 50% of B-ALL relapses occurred late (≥36 months) and 72.5% involved the marrow. Conversely, 64.8% of T-ALL relapses occurred early (<18 months) and 47.1% involved the central nervous system. The 5-year OS post-relapse for the entire cohort was 48.9 ± 1.2%; B-ALL:52.5 ± 1.3%, T-ALL:35.5 ± 3.3%, and infant ALL:21.5 ± 3.9%. OS varied by early, intermediate and late time-to-relapse; 25.8 ± 2.4%, 49.5 ± 2.2%, and 66.4 ± 1.8% respectively for B-ALL and 29.8 ± 3.9%, 33.3 ± 7.6%, 58 ± 9.8% for T-ALL. Patients with ETV6::RUNX1 or Trisomy 4 + 10 had median time-to-relapse of 43 months and higher OS post-relapse 74.4 ± 3.1% and 70.2 ± 3.6%, respectively. Patients with hypodiploidy, KMT2A-rearrangement, and TCF3::PBX1 had short median time-to-relapse (12.5-18 months) and poor OS post-relapse (14.2 ± 6.1%, 31.9 ± 7.7%, 36.8 ± 6.6%). Site-of-relapse varied by cytogenetic subtype. This large dataset provided the opportunity to identify risk factors for OS post-relapse to inform trial design and highlight populations with dismal outcomes post-relapse.

Introduction: Myelofibrosis (MF) is a clonal myeloproliferative neoplasm (MPN) characterized by a hyperactive JAK-STAT pathway, splenomegaly, and constitutional symptoms. JAK inhibitors (JAKi) have been the mainstay in MF treatment over the past decade. Pro-inflammatory cytokines, the levels of which have prognostic value (Tefferi et al. J Clin Oncol 2011), have been shown to be upregulated in MF. However, there is paucity of data on chronic immune dysregulation in pathogenesis of MF. Here, we report the multiplatform biomarker analysis including immune changes from phase 3 (FREEDOM2) trial in patients with MF previously exposed to ruxolitinib (Rux) and treated with fedratinib vs. best available therapy (BAT) (primary efficacy: 35.8% vs 6% responders at the end of cycle 6 (EOC6) in fedratinib vs BAT, respectively). Of note, 78% of the patients on BAT continued RUX therapy until EOC6, allowing direct comparison between RUX and fedratinib. Methods: In the FREEDOM2 trial, patients (N=201) were randomized into two arms, fedratinib (400 mg/day) or BAT in a 2:1 ratio, respectively. The primary endpoint was the proportion of patients with ≥35% reduction of spleen volume (SVR35) on MRI at the end of cycle 6 (EOC6). In the FREEDOM2 biomarker cohort, mutational analysis was performed in enriched CD66b+ cells using the MLL myeloid panel targeting 74 genes. Rules-Based Medicine's (RBM) HumanMAP v2.0 panel targeting 85 cytokines was used to measure changes in serum cytokine levels from C1D1 to EOC6. Custom Q2 immune panels to detect myeloid and lymphoid subsets were used for immunophenotyping of peripheral blood samples. Results: Baseline mutations in FREEDOM2 were commonly found MPN mutations that include JAK2 V617F (71%), ASXL1 (43%), CALR (22%), TET2 (22%) and MPL (11%). However, baseline mutation status was not a predictor of response. Patients receiving fedratinib demonstrated significant down-regulation of several pro-inflammatory cytokines including CRP, ENRAGE, IL-18, IL-16, TIMP-1, and increase of anti-inflammatory cytokines including adiponectin, MMP2, and CEA (Wilcoxon signed rank test, BH adjusted p <0.001) at EOC6 vs C1D1. In addition, the change in levels of many cytokines correlated with the clinical endpoint (SVR35) demonstrating that the therapeutically relevant changes were mediated by fedratinib (Pearson rho P<0.05) (Figure 1). Importantly, fedratinib was superior to the BAT in its ability to alter levels of key cytokines (p <0.05) at EOC6. PD-1 expression has been shown to be upregulated in CD4+ and CD8+ T cells in MF patients and is associated with poor overall survival. However, RUX did not have a significant effect on PD1+ T cells (Veletic et al. Haematologica 2021). Interestingly, flow cytometric analysis revealed that fedratinib decreased exhausted CD8+ T cells (PD1+) up to 40% (∆ median change -11.4%,p<0.001)) (figure 2). Importantly, the reduction in exhausted T cells correlated with the reduction in spleen volume with fedratinib treatment ( R= -0.51, P=0.00011; Figure 2). Consistent with this, we also observed median increases in NK (+3%, p<0.001) and NK-T (+13.8%, p<0.001) populations when compared to the BAT arm. Conclusions: To our knowledge, this is the first comprehensive study detailing cytokine and immune changes in patients with MF in a clinical trial. In addition, our data for the first time demonstrates the impact of fedratinib on the immune system and its ability to reduce exhausted T cells, highlighting the immune modulatory mechanism of fedratinib. Notably, the cytokine and immune changes correlated with the primary endpoint and were not observed in the BAT arm demonstrating superior efficacy of fedratinib in previously RUX exposed patients. Furthermore, since majority of the patients in the BAT arm continued to receive RUX, these findings highlight the mechanistic differences between fedratinib and RUX on clinical response in MF (SVR35). Overall, our data highlight multiple novel avenues to achieve therapeutic efficacy in MF and positions fedratinib as an effective therapy with a distinctive mechanism of action. Additional analyses on the effect of fedratinib treatment on myeloid immune subsets and transcriptomic changes are underway to gain comprehensive mechanistic understanding.

BACKGROUND:Previous studies have identified racial and ethnic disparities in childhood acute lymphocytic leukaemia survival. We aimed to establish whether disparities persist in contemporaneous cohorts and, if present, are attributable to differences in leukaemia biology or insurance status. METHODS:Patients with newly diagnosed acute lymphocytic leukaemia in inpatient and outpatient centres in the USA, Canada, Australia, and New Zealand, aged 0-30 years, who had race or ethnicity data available, enrolled on eight completed Children's Oncology Group trials (NCT00103285, NCT00075725, NCT00408005, NCT01190930, NCT02883049, NCT02112916, NCT02828358, and NCT00557193) were included in this secondary analysis. Race and ethnicity were categorised as non-Hispanic White, Hispanic, non-Hispanic Black, non-Hispanic Asian, and non-Hispanic other. Event-free survival and overall survival were compared across race and ethnicity groups. The relative contribution of clinical and biological disease prognosticators and insurance status was examined through multivariable regression models, both among the entire cohort and among those with B-cell lineage versus T-cell lineage disease. FINDINGS:Between Jan 1, 2004, and Dec 31, 2019, 24 979 eligible children, adolescents, and young adults with acute lymphocytic leukaemia were enrolled, of which 21 152 had race or ethnicity data available. 11 849 (56·0%) were male and 9303 (44·0%) were female. Non-Hispanic White patients comprised the largest racial or ethnic group (13 872 [65·6%]), followed by Hispanic patients (4354 [20·6%]), non-Hispanic Black patients (1517 [7·2%]), non-Hispanic Asian (n=1071 [5·1%]), and non-Hispanic other (n=338 [1·6%]). 5-year event-free survival was 87·4% (95% CI 86·7-88·0%) among non-Hispanic White patients compared with 82·8% (81·4-84·1%; hazard ratio [HR] 1·37, 95% CI 1·26-1·49; p<0·0001) among Hispanic patients and 81·8% (79·3-84·0; HR 1·45, 1·28-1·65; p<0·0001) among non-Hispanic Black patients. Non-hispanic Asian patients had a 5-year event-free survival of 88·1% (95% CI 85·5-90·3%) and non-Hispanic other patients had a survival of 82·8% (76·4-87·6%). Inferior event-free survival among Hispanic patients was substantially attenuated by disease prognosticators and insurance status (HR decreased from 1·37 [1·26-1·49; p<0·0001] to 1·11 [1·00-1·22; p=0·045]). The increased risk among non-Hispanic Black patients was minimally attenuated (HR 1·45 [1·28-1·65; p<0·0001] to 1·32 [1·14-1·52; p<0·0001]). 5-year overall survival was 93·6% (91·5-95·1%) in non-Hispanic Asian patients, 93·3% (92·8-93·7%) in non-Hispanic White patients, 89·9% (88·7-90·9%) in Hispanic, 89·7% (87·6-91·4%) in non-Hispanic Black patients, 88·9% (83·2-92·7%) in non-Hispanic other patients. Disparities in overall survival were wider than event-free survival (eg, among non-Hispanic other patients, the HR for event-free survival was 1·43 [1·10-1·85] compared with 1·74 [1·27-2·40] for overall survival). Disparities were restricted to patients with B-cell acute lymphocytic leukaemia, no differences in event-free survival or overall survival were seen in the T-cell acute lymphocytic leukaemia group. INTERPRETATION:Substantial disparities in outcome for B-cell acute lymphocytic leukaemia persist by race and ethnicity, but are not observed in T-cell acute lymphocytic leukaemia. Future studies of relapsed patients, access to and quality of care, and other potential aspects of structural racism are warranted to inform interventions aimed at dismantling racial and ethnic disparities. FUNDING:National Cancer Institute and St Baldrick's Foundation.

Remarkable complete response rates have been shown with tisagenlecleucel, a chimeric antigen receptor (CAR) T-cell therapy targeting CD19, in patients up to age 26 years with refractory/ relapsed B-cell acute lymphoblastic leukemia; it is US Food and Drug Administration approved for this indication. Currently, patients receive a single dose of tisagenlecleucel across a wide dose range of 0.2 to 5.0 x 106 and 0.1 to 2.5 x 108 CAR T cells per kg for patients <= 50 and >50 kg, respectively. The effect of cell dose on survival and remission is not yet well established. Our primary goal was to determine if CAR T-cell dose affects overall survival (OS), event-free survival (EFS), or relapse-free-survival (RFS) in tisagenlecleucel recipients. Retrospective data were collected from Pediatric Real World CAR Consortium member institutions and included 185 patients infused with commercial tisagenlecleucel. The median dose of viable transduced CAR T cells was 1.7 x 106 CAR T cells per kg. To assess the impact of cell dose, we divided responders into dose quartiles: 0.134 to 1.300 x 106 (n = 48 [27%]), 1.301 to 1.700 x 106 (n = 46 [26%]), 1.701 to 2.400 x 106 (n = 43 [24%]), and 2.401 to 5.100 x 106 (n = 43 [24%]). OS, EFS, and RFS were improved in patients who received higher doses of tisagenlecleucel (P = .031, .0079, and .0045, respectively). Higher doses of tisagenlecleucel were not associated with increased toxicity. Because the current tisagenlecleucel package insert dose range remains broad, this work has implications in regard to targeting higher cell doses, within the approved dose range, to optimize patients' potential for long-standing remission.