Stock, Wendy

Abstract Inotuzumab ozogamicin (InO) is an antibody-calicheamicin conjugate with high efficacy in lymphoid malignancies. It targets the B-cell surface protein CD22, which is expressed in most B-cell acute lymphoblastic leukemia (B-ALL) cases, albeit with variable intensity. However, factors governing CD22 expression and thus leukemia sensitivity to InO remain incompletely understood. Using multiomic characterization of 196 human B-ALL samples, coupled with ex vivo InO sensitivity profiling, we showed that early leukemia differentiation arrest at the pre–pro-B stage is associated with resistance to InO. Screening of 1639 transcription factor genes identified early B-cell factor 1 (EBF1) as a key regulator of CD22 expression (false discovery rate of 7.1 × 10−4). When comparing the assay for transposase-accessible chromatin with sequencing profiling results of the most InO-sensitive and -resistant cases (50% lethal concentration <10th vs >90th percentile, n = 18), the binding motif for EBF1 was strikingly enriched in regions with differential open chromatin status (P = 8 × 10−174). CRISPR interference targeting EBF1 binding sites at the CD22 locus led to an ∼50-fold reduction in cell surface CD22 expression and, consequently, an ∼22-fold increase in InO resistance in ALL cell lines. Interestingly, within BCR::ABL1 ALL, we observed intrasubtype heterogeneity linked to EBF1 transcriptional downregulation (P = 1.1 × 10−15) and/or somatic alteration (P = .004), which led to reduced CD22 expression (P = 8.3 × 10−11) and ex vivo and in vivo resistance to InO. Collectively, these findings point to the direct impact of EBF1 on CD22 expression during B-cell development, which, in turn, contributes to interpatient variability in InO response, even within the same subtype of B-ALL.

AbstractBackgroundAcute lymphoblastic leukemia (ALL) in adults is aggressive, with long‐term outcomes impacted by treatment resistance and toxicity. CD52 is expressed in most cases of B‐ and T‐lineage ALL. Alemtuzumab, a humanized immunoglobulin G1 monoclonal antibody that targets CD52, was identified as a potential agent to improve treatment efficacy without increasing toxicity.MethodsIn this phase 1/2 study (Cancer and Leukemia Group B [CALGB] 10102, NCT00061945), a course of single‐agent alemtuzumab was intercalated into CALGB 19802 backbone chemotherapy after the third course of intensive chemotherapy in those who were CD52+ at diagnosis. Phase 1 tested three dose levels of subcutaneous alemtuzumab (10, 20, and 30 mg 3 times weekly for 4 weeks/12 doses) and demonstrated that 30 mg was tolerable. Phase 2 established feasibility.ResultsThe study enrolled 295 evaluable patients (115 in phase 1, 180 in phase 2); 206 (69.8%) were CD52+. Among evaluable CD52+ patients, 43.7% (90/206) completed the first three treatment modules; 97.8% (88 of 90) were treated with alemtuzumab. Alemtuzumab was associated with cytomegalovirus viremia, which occurred in 23.3% (14 of 60) of patients during and 29.2% (19 of 65) after alemtuzumab treatment. With a median follow‐up of 101.2 months, median overall survival (OS) was 26.3 months (3‐year rate, 44%; 5‐year rate, 36%; 10‐year rate, 31%). Landmark analysis at the start of the fourth course of treatment demonstrated no difference in OS or disease‐free survival between patients who did and who did not receive alemtuzumab.ConclusionAlemtuzumab was feasible to administer in adults with ALL receiving intensive chemotherapy, but was without evidence of benefit.

Abstract Inotuzumab ozogamicin (InO) is an antibody-calicheamicin conjugate with striking efficacy in B-cell acute lymphoblastic leukemia (B-ALL). However, there is wide interpatient variability in treatment response, and the genetic basis of this variation remains largely unknown. Using a genome-wide CRISPR screen, we discovered that the loss of DNA nucleotidylexotransferase (DNTT) is a primary driver of InO resistance. Mechanistically, the downregulation of DNTT attenuated InO–induced DNA damage response, cell cycle arrest, and mitochondrial apoptotic priming, thereby ultimately leading to leukemia resistance to InO. Ex vivo leukemia InO sensitivity was highly associated with DNTT expression in ALL blasts with substantial intraleukemia heterogeneity as revealed by single-cell RNA sequencing. Among patients with B-ALL enrolled in the Children's Oncology Group trial AALL1621, we observed consistent DNTT downregulation in residual blasts following InO treatment. The selection of DNTT-low blasts by InO therapy was also recapitulated in vivo using patient-derived xenograft models. Collectively, our data indicate that DNTT is a key regulator of calicheamicin response in leukemia and thus a potential biomarker for individualizing InO therapy in B-ALL.

Background: The BeatAML Master Trial (BAMT) began in 2016 with a goal of determining the feasibility of a precision-medicine approach in treating acute myeloid leukemia (AML) in older adults ≥ 60 years of age where outcomes are poor. At the time, standard of care (SOC) for older patients with AML was intensive 7+3 chemotherapy (cytarabine + daunorubicin) if fit and hypomethylating agents (HMA: azacitidine or decitabine) if unfit. Besides confirming the feasibility, the early results (2019) from the Master Trial suggested that patients who received therapy on one of the sub-studies had a lower early death rate and superior overall survival compared to patients receiving off-protocol SOC, which was primarily single-agent HMA at the time. Since then, the 2019 approval of venetoclax (ven) + azacitidine for older unfit AML patients has been transformative and emerged as the SOC. Using data from the BAMT from 2016-2024, we suggest that knowing the comprehensive genomic profile of AML at the time of diagnosis is still beneficial in determining therapy that would lead to the optimal outcome and that development of targeted therapies and combination regimens with ven/HMA is still needed. Methods: This is a retrospective analysis of 1096 patients who consented and enrolled on the BAMT (NCT03013998) from Nov 2016 through Jun 2024. Patients ≥ 60 years with newly diagnosed AML were included. Patients underwent therapy on a BeatAML sub-study based on their genetics (N=433) or treatment off-protocol (N=663). We divided the patients according to what therapy they received: (1) Non-Intensive (Off-Protocol, N=121), (2) Ven/HMA (Off-Protocol, N=234), (3) Non-Intensive (Sub-Study, N=407), (4) Intensive (Sub-Study, N=26), and (5) Intensive (Off-Protocol, N=155). 94 patients received no treatment and 59 were unknown. Non-intensive therapies included primarily targeted agents or non-ven combinations. We analyzed baseline characteristics and demographics, overall survival (OS), and the impact of allogenic stem cell transplant (SCT). OS, using Kaplan-Meier method, was measured from the start of therapy to all-cause death, censoring patients at last follow-up. Results: Characteristics and demographics were similar between Groups 1, 2 and 3. Group 4 and 5 were treated with intensive chemotherapy and their characteristics are reflective of younger/fit patients appropriate for the intensive regimen. Groups 4 and 5 also had the longest median OS (mOS) of 40.2 months (95% CI: 13.9-not reached (NR)) and 42.3 months (95% CI: 25.1-NR) respectively. Group 3, which included patients treated with a non-intensive, targeted therapy on a sub-study, had an mOS of 14.0 months (95% CI: 12.3-16.7), which was similar to Group 2, treated with Ven/HMA, that had an mOS of 13.2 months (95% CI: 10.9-16.3). Lastly, patients in Group 1, who were treated primarily with HMA monotherapy, did poorly in comparison, with an mOS of 6.0 months (95% CI: 3.3-10.1). Additionally, we looked at the cumulative incidence rate (CIR) of SCT. The CIR in 12 months of Group 1 was 9.8% (95% CI: 6.9-13.4), Group 2: 5.4% (95% CI: 2.2-10.7), Group 3 and 4 combined: 35.0% (95% CI: 628.1-42.1), and Group 5: 13.1% (95% CI: 6.9-13.4). In a multi-variable analysis, after adjusting for typical favorable risk factors like NPM1 mutations and core binding factor, SCT was highly significant (p<0.0001) in improving OS with a hazard ratio of 0.23 (95% CI: 0.16-0.32), and Group 3 performed similarly to Group 2 (p=0.20). Conclusions: First, our results suggest that it is clinically relevant to correctly identify whether an intensive chemotherapy, targeted-therapy, or ven/HMA regimen is best suited for older AML patients, for which a precision-medicine trial is ideal for. Secondly, in this group of patients, our results show that patients treated with a non-intensive targeted therapy (Group 3) has a similar mOS to those treated with ven/HMA (Group 2). Especially after controlling for patient characteristics, patients in Group 2 performed similar to those in Group 3 (p=0.20). Patients in Group 3 were treated on Beat AML sub-studies which included targeted therapy alone or with non-venetoclax combinations, which is a strong rationale for continued development of targeted agents to optimize toxicity and quality of life with similar survival. Lastly, this data also supports the need to improve the ven/HMA SOC by developing combination regimens with targeted agents to further improve outcomes.

Background: Mutations of isocitrate dehydrogenase (IDH) are recurrent in acute myeloid leukemia (AML) and the prevalence increases with age. The prognostic impact of IDH1 and IDH2 mutations remains controversial. IDH inhibitors are small molecules that bind within the IDH enzymatic active site, blocking aberrant 2-hydroxyglutarate production and inducing myeloid differentiation. While they can lead to differentiation syndrome, they generally have a more favorable side-effect profile, making them an attractive option for older patients. We aimed to describe the prevalence and prognostic impact of IDH mutations in newly diagnosed (ND) AML patients ≥ 60 years in a large cohort of patients treated on a Leukemia and Lymphoma Society-sponsored trial. Methods: We performed a retrospective observational analysis including patients ≥ 60 years with ND AML who were enrolled on the Beat AML clinical trial (NCT03013998) before May 10, 2023. Patients were treated with a variety of treatment regimens. Cytogenetic analysis and next-generation sequencing (FoundationOne®Heme) were obtained. Mutations were considered present at any detectable VAF. Cox proportional hazard models were used to describe the relative risk of each variable on death over time from the date of trial inclusion. Results: A total of 1023 patients with ND AML were identified. Patients had a median age of 72 (range 60-92) years and the majority were non-Hispanic White (83.2%), and male (57.6%). Ninety-nine (9.7%) patients were IDH1MUT, 193 (18.9%) patients were IDH2MUT including 10 (1.0%) patients with a co-occurring IDH1 and IDH2 gene mutations. Both IDH1MUT and IDH2MUT were associated with 2022 ELN favorable-risk (26.3% and 22.3% for IDH1 and IDH2 respectively, vs 15.5% overall (P<0.001)). IDH1MUT significantly co-occurred with DNMT3AMUT (42.4%), NPM1MUT (44.4%) and less frequently with TP53MUT (10.1%) (P<0.001 for all). IDH2MUT was associated with DNMT3AMUT (35.8%), NPM1MUT (31.1%), SRSF2MUT (38.3%), and less frequently with TET2MUT (6.7%) and TP53MUT (9.3%) (P<0.001 for all). Normal karyotype was higher in IDH1MUT (P<0.001) and IDH2MUT (P<0.001), whereas complex karyotype (P<0.001 for both) and core-binding factor AML (P=0.047, P=0.015) were lower in IDH1MUT and IDH2MUT. To evaluate the impact of IDH mutations in patients ≥ 60 years, we focused on patients evaluable for outcome treated with lower-intensity therapy (LIT) (N=674), and intensive chemotherapy (IC) (N=178), separately. Among the LIT treated patients, 96 patients received treatment with an IDH inhibitor, and 237 patients received a hypomethylating agent (HMA) plus venetoclax. For patients treated with LIT, the hazard ratios (HRs) of death over time were 0.58 [95% CI 0.47-0.72; P<0.002], 0.81 [95% CI 0.59-1.13; P=0.214] and 0.56 [95% CI 0.44-0.72; P<0.001] for IDHMUT, IDH1MUT, IDH2MUT respectively. Among patients receiving IC, the HRs were 1.05 [95% CI 0.68-1.69; P=0.777], 0.70 [95% CI 0.31-1.61; P=0.406] and 1.27 [95% CI 0.77-2.1; P=0.352] for IDHMUT, IDH1MUT, and IDH2MUT respectively. The HR of death for IDH1MUT patients treated with LIT with the addition of an IDH1 inhibitor (N=24) was significantly lower (HR 0.47 [95% CI 0.24-0.92; P=0.027] vs IDH1MUT patients treated without an IDH1 inhibitor, while an IDH2 inhibitor did not change survivial outcomes in patients with IDH2MUT (N=71). Discussion: To our knowledge this is the largest retrospective study characterizing the prevalence of IDH mutations in patients ≥ 60 years with AML. We demonstrated that IDH mutations were detected in ~27% of older patients with AML. Although there could have been selection bias as the Beat AML study had separate IDHMUT study arms, these findings are concordant with other studies showing an increase in frequency of IDH mutations with age. IDH2MUT was associated with a lower HR of death among patients treated with LIT, which was not seen in patients receiving IC. The high prevalence of IDH mutations suggests that IDH inhibitors used as single agents or in combination with lower-intensity therapies are an important class of drugs for this older patient population as they are generally well tolerated, that need further investigation.

Introduction: Brexucabtagene autoleucel (brexu cel) is a CD19-targeted chimeric antigen receptor (CAR) T cell therapy approved for the treatment of adults with relapsed/refractory (R/R) acute lymphoblastic leukemia (ALL). Some patients (pts) proceed with a consolidative allogeneic hematopoietic cell transplant (HCT) after breux cel. We do not know which pts can safely avoid a consolidative HCT and its associated morbidity. Methods: We conducted a retrospective cohort study utilizing the Real-World Outcomes Collaborative of CAR-T in Adult ALL (ROCCA) dataset. ROCCA is a registry of brexu cel pts in 32 centers in North America. We defined complete remission (CR) as < 5% blasts in the bone marrow and no central nervous system (CNS) or extramedullary (EM) disease on or around D28 after brexu cel. We defined measurable residual disease negative (MRD-) CR as CR and no abnormal blasts in the bone marrow by multiparameter flow cytometry, quantitative polymerase chain reaction, ClonoSeq next generation sequencing (NGS), or other sequencing modalities with lower sensitivity. ClonoSeq NGS has a sensitivity of 10-6, while the other modalities have a sensitivity of at least 10-4. We defined progression free survival (PFS) as the time from CAR-T infusion to relapse or death, censored at the date of last follow-up. Statistical analyses were performed in R. Univariable Cox proportional hazards regression analyses were performed to determine the association of pre-specified variables with PFS. Variables known to be associated with CAR T outcomes in ALL were entered into a multivariable model. HCT was incorporated as a time-varying covariate. Results: During the study period, 281 pts were infused with brexu cel. We focused our analysis on 172 (61%) pts with confirmed MRD- CR on D28, as that is the group with clinical equipoise regarding maintenance strategy. 38 (22%) of those pts received a consolidative HCT, 30 (17%) received another form of maintenance therapy (primarily tyrosine kinase inhibitors for Philadelphia (Ph)-chromosome positive ALL), and 104 (60%) pts received no maintenance therapy. Pts who underwent consolidative HCT were younger (median age 35 years) than those who received no maintenance (42y) (p = 0.023) and were less likely to have a history of prior HCT (13% vs. 47%, p < 0.001). No differences were observed with respect to sex, TP53 mutation status, prior inotuzumab (ino) or blinatumomab (blina) exposure, disease burden prior to CAR, or development of any cytokine release syndrome (CRS) or any neurotoxicity with brexu cel. We performed univariable Cox proportional hazard regression analyses among pts in MRD- CR on D28, excluding those who received other maintenance therapy besides HCT; the median follow up was 8.1 months. In univariable analyses, D28 MRD negativity by ClonoSeq NGS (versus MRD negativity by other, less sensitive methods), development of CRS, and pre-CAR BM blasts < 5% were associated with improved PFS; prior ino exposure was negatively associated with PFS (p < 0.05). Age, sex, Ph status, TP53 status, and neurotoxicity development were not associated with PFS. In a multivariable model incorporating ALL type, prior HCT, prior ino/blina, and pre-CAR blast burden, as well as consolidative HCT as a time-varying covariate, only MRD negativity by ClonoSeq NGS with at least 10-6 sensitivity retained statistical significance (PFS hazard ratio 0.34, 95% confidence interval (CI) 0.15-0.77, p = 0.005). Pts who were MRD- by ClonoSeq NGS on D28 and received no maintenance had favorable 6m PFS of 76% (95% CI: 59-98%) and OS of 100% with a median follow up of 6.3 months in this subgroup. Among those in MRD- CR by NGS, HCT as a time-varying covariate was not associated with improved PFS (HR 0.6, p = 0.6). Conclusions: In a large real-world cohort of adults with R/R B-ALL infused with brexu cel, we identified practice variation regarding the use of consolidative HCT. Among brexu cel recipients who entered an MRD- CR, we identified ClonoSeq NGS MRD negativity as a novel predictive factor of favorable oncologic outcomes, even without a consolidative HCT. Similar results have been obtained in pediatric ALL patietns ftreated with 41BB-based tisagenleucleucel (Pulsipher et al., Blood Cancer Discovery 2022). Longer follow-up is needed to validate the safety of omitting consolidative HCT in such pts, but these results encourage the potential for definitive therapy with brexu cel when D28 ClonoSeq NGS MRD is negative.

Introduction: Brexucabtagene Autoleucel (brexu-cel) is the first CD19-targeting chimeric antigen receptor (CD19.CAR) T-cell therapy approved for the treatment of relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL) in adult (≥18 yo) patients (pts) based on results from the ZUMA-3 trial. However, data on the safety and efficacy of CAR-T cells in older pts (≥60 yo) remains limited, with only 8 pts >65 yo enrolled in the ZUMA-3 trial. We evaluated the treatment characteristics and outcomes with use of brexu-cel amongst patients ≥60 yo in the Real-World Outcomes Collaborative for CAR-T in ALL (ROCCA) consortium database. Methodology: ROCCA is a consortium of over 30 U.S.-based institutions with a database of adult pts with B-ALL who received brexu-cel as standard of care therapy. We identified pts ≥60 yo and further stratified them into two cohorts: 60-69 yo and ≥70 yo. Outcomes of interest included OS, PFS, ORR, and toxicities (CRS and ICANS). Both OS and PFS were calculated from the day of brexu-cel infusion. Response assessment occurred on day +28, and MRD evaluation was per institutional practices. CRS and ICANS were graded according to the American Society for Transplantation and Cellular Therapy criteria. Results: Of 280 brexu-cel recipients in the ROCCA database, 73 (26%) were ≥60 yo, of which 58 (79%) were between 60-69 yo and 15 (21%) were ≥70 yo. Among pts ≥60 yo, the median age at infusion was 65 (range 60-81). Pts were heavily pretreated with a median of 3 prior lines of therapy (range 2-9). Prior therapies included: allogeneic HSCT (38%), inotuzumab ozogamicin (41%), and blinatumomab (62%). Forty pts (55%) had Ph-negative B-ALL, 24 (33%) had Ph+ B-ALL and 9 (12%) had Ph-like B-ALL. When grouped by HCT-CI equal to 0, 1-2 or ≥3, a higher proportion of pts ≥60 yo had HCT-CI ≥3 (19% vs. 7% in <60 yo; p=0.004). Pre-apheresis, 33 pts (45%) had active disease (>5% marrow blasts or extramedullary disease), 34 (47%) were in CR (of whom 14 were MRD-negative) and 6 had unknown disease status. Following brexu-cel, 43 (59%) achieved MRD-negative CR, 20 (27%) had MRD + or CR with unknown MRD status, 6 (8%) had refractory disease and 5 had no disease evaluation available. The ORR was similar between pts 60-69 and ≥70 yo (MRD-negative CR rate: 58% vs 61%, r/r to brexu-cel: 9% vs 6%) and was comparable to pts <60 yo (MRD-negative CR 66%; r/r disease of 9%). The rate of all grade ICANS was higher in pts ≥70 (77%) than in pts 60-69 (47%) or pts <60 (49%), with grade 1-2 ICANS occurring at a significantly higher rate in pts ≥70 (44% vs. 20% in 60-69 and 20% in <60, p=0.049). Grade 3-4 ICANS were similar between the groups: 33%, 27% and 29% in the ≥70, 60-69, and <60 groups, respectively. Conversely, rates of CRS were similar across age groups (80-84%). There was no statistically significant difference in the rate of grade 3-4 CRS which was 17% in pts ≥70, 4% in pts 60-69, and 11% in pts <60 yo. With a median follow-up of 332 days (range 8-936 days) from infusion, pts ≥60 yo had a median PFS of 395 days compared to 547 days in pts <60 yo. Median PFS was lower in the ³70 yo (231 days) compared to 677 in the 60-69 yo cohort. No statistically significant difference was found between the <60 and ≥60groups in 6- and 12-month PFS rates (6 months PFS: 66% vs 64%, p=0.77 and 12 months PFS: 57% vs 57%, p=1.00). When pts ≥60 yo were stratified by age, pts ≥70 yo had similar 6-month PFS compared to other age groups but significantly lower 12-month PFS (38%; vs. 64% in pts 60-69and 57% in pts <60, p=0.001). Median OS was not reached in any of the age groups, and there was no statistically significant difference in 6-month OS (72% in ≥ 70 vs. 82% in the 60-69 vs. 82% in <60, p=0.14) or 12-months OS (61% in ≥ 70 vs. 69% in 60-69 vs. 73% in <60, p=0.18). Conclusion: This is the largest study to date of older B-ALL pts receiving CAR-T cells. We observed that older pts, particularly those aged 60-69, have similar outcomes to younger pts. However, pts ≥70 yo had lower median and 12-month PFS when compared to younger patients. Additionally, pts ≥70 yo had higher rates of grades 1-2 ICANS. An important limitation of this analysis is the potential selection bias among clinicians when selecting older patients for this treatment. Longer follow-up, including inclusion of additional pts ≥70 yo is needed to confirm these results, and further studies are needed to identify the optimal approach for use of CAR T-cell therapy in patients in this age group.

Background: Patients with acute lymphoblastic leukemia (ALL) receiving L-asparaginase (ASP) based induction therapy have a high risk of venous thromboembolism (VTE). Studies investigating universal thromboprophylaxis strategies have yielded sub-optimal results. Risk prediction models are needed to identify high-risk patients who would be candidates for intensification of thromboprophylaxis, and lower risk patients who are unlikely to benefit. Aims: Derive and externally validate a risk prediction model for VTE in patients with ALL receiving ASP therapy. Methods: We conducted a multicenter, international, cohort study of patients with newly-diagnosed ALL receiving ASP-based induction therapy (≥18yrs). The derivation and external validation cohorts included 306 and 97 patients without VTE prior to ASP therapy, respectively. Patients on therapeutic anticoagulation at index were excluded. Patients were followed from date of first ASP dose for 100 days post ALL diagnosis or until VTE, therapeutic anticoagulation or death. Candidate predictors were documented at ALL diagnosis and included demographics, disease and treatment characteristics, VTE history, comorbidities, thromboprophylaxis, complete blood count, fibrinogen and d-dimer. D-dimer was only analyzed within the 7 derivation cohort centers who routinely tested this at ALL diagnosis with <20% missing values. Primary outcome was VTE at any site (centrally confirmed). Secondary outcomes included major bleeding by ISTH criteria. Cause-specific Cox proportional hazard regression was performed to identify VTE risk factors in the derivation cohort while treating death as a competing event. Variables with p-value <0.1 on univariable analysis were included in a multivariable model and subsequently used in the risk model if p-value <0.05. Lastly, the derived VTE risk prediction model was tested in the external validation cohort for association with time to VTE using cause-specific Cox proportional hazard model. All analyses were stratified by center and use of low molecular weight heparin prophylaxis (at any dose on the date of first ASP dose). Results: The cumulative incidence of VTE in the derivation cohort was 17% at 30 days (95% confidence interval [CI] 13% - 21%) and 22% at 90 days (95% CI 17% - 27%). The multivariable model included hyperlipidemia (hazard ratio [HR] 1.35; 95% CI 0.56 - 3.27), body mass index ≥ 25 kg/m² (HR 1.49; 95% CI 0.75 - 2.95), platelet count ≥ 25 x 10⁹/L (HR 1.42; 95% CI 0.67 - 3.00), increasing hemoglobin per 1 g/dL (HR 1.17; 95% CI 1.03 - 1.32) and d-dimer ≥ 1 µg FEU/mL (HR 2.68; 95% CI 1.08 - 6.66). Hemoglobin as a continuous variable and d-dimer ≥ 1 µg FEU/mL were included in the final model and used to create a risk score which was weighted by estimated log-HR sum of these risk factors. The lowest risk quartile (risk score below 2.1) successfully differentiated between a lower 30-day cumulative VTE incidence (4%, 95% CI 0.72% - 12%) compared with the other quartiles (20%, 95% CI 14% - 27%; HR 4.40, 95% CI 1.54 - 12.5) which were defined as high risk (score ≥2.1). The risk score's negative predictive value (NPV) for VTE at 30 days was 96% and positive predictive value (PPV) was 20%. The validation cohort (n=97 from 3 external cohorts with routine d-dimer measurement) included 16 (16.5%) patients in the low-risk group and 81 (83.5%) in the high-risk group, with a 30-day VTE incidence of 6.3% (95% CI 0% - 17%) and 21% (95% CI 12% - 30%), respectively (HR 6.22; 95% CI 0.85 - 45.8; p = 0.073)). The NPV (93.7%) and PPV (21%) were comparable to the derivation cohort. The 30-day incidence of major bleeding was 0% (95% CI NR - NR) in low-risk patients (HR 1.36; 95% CI 0.27 - 6.77) and 2.0% (95% CI 0.55% - 5.4%) in the high-risk group. Conclusion: Patients with ALL receiving ASP induction therapy have a 17% VTE risk at 30 days. We derived and externally validated a VTE risk prediction model based upon d-dimer and hemoglobin. This model successfully identifies at least 16.5% of patients with ALL who have a low VTE risk after ASP treatment. Using this model, novel prevention strategies which may be associated with cost and risk could now be tailored to patients with higher risk. Prospective studies are needed to validate the model's performance and role in VTE risk management.

Introduction: TP53 mutations are found in 18% of adult patients (pts) with acute lymphoblastic leukemias (ALL), and portends poor prognosis (Saygin, Blood Cancer Discov 2024). TP53 mutations are often associated with low hypodiploidy and Li Fraumeni syndrome (germline) in children. However, we and others have shown that TP53-mutant (TP53-m) ALL arises from pre-existing TP53-m clonal hematopoiesis in adults as opposed to germline origin. The impact of co-occurring mutations, TP53 allelic frequency (AF) and different types of treatment on outcomes of TP53-m ALL are not fully characterized. We set out to investigate the clinical and molecular predictors of outcomes in adults with TP53-mutant ALL. Methods: We retrospectively studied 107 patients with TP53-m ALL treated at seven different academic centers in North America. Genetic profiling of pre-treatment bone marrow samples was performed with a comprehensive panel covering 147 myeloid and lymphoid genes. We also performed RNA-sequencing to identify fusions that define ALL subtypes. Single nucleotide polymorphism (SNP) array was used to define low hypodiploidy. Results: The median age at diagnosis was 59 years (range, 10-80), 42% were female, 70% White. 89% of pts had B-ALL while 11% had T-lineage ALL. 24% of pts were classified to have therapy-related ALL based on their history of exposure to genotoxic therapy for another cancer. 45% of pts had hypodiploid karyotype, 79% of whom were low-hypodiploid. 7% had hyperdiploid karyotype and 5% had BCR::ABL1. 11 pts had two different TP53 variants. 90% of TP53 variants involved the DNA binding domain. Most frequently observed co-occurring mutations involved CDKN2A (20%), NOTCH1 (12%) and RB1 (10%) genes. 71% of pts were treated with standard cytotoxic chemotherapy, while the remaining pts received newer antibody-based therapies (i.e., inotuzumab, blinatumomab) or venetoclax in first-line setting. 11% of pts received allogeneic hematopoietic cell transplant (HCT) in first complete remission (CR1) while 3% received HCT after relapse (CR2 or CR3). When patients with vs without low hypodiploidy were compared, we did not observe a significant difference in overall survival (OS) as outcomes were poor in both groups (median, 17 vs 19 months, p= 0.89). However, pts who had TP53 AF >0.5 had significantly worse OS when compared to pts with TP53 VAF <0.5 (13 vs 25 months, p= 0.03). Conclusions: TP53-m ALL is a very high-risk disease subtype and its dismal outcome is independent from presence of co-existing low hypodiploidy. Loss of heterozygosity, characterized by TP53 AF >0.5 is associated with even worse survival. New therapies are urgently needed to improve outcomes in this subset.

Background: Inotuzumab Ozogamicin (InO) is an antibody-cytotoxin conjugate with high activity in B-cell acute lymphoblastic leukemia (B-ALL). InO targets the B-cell surface protein CD22, which is expressed in most B-ALL cases, albeit with great variability in expression levels. Emerging evidence suggest that this variation is associated with the stage of leukemia differentiation arrest [PMID: 21348573]. However, the exact relationship between CD22 expression and leukemia sensitivity to InO, particularly in the context of leukemia developmental state, remains unclear. Furthermore, our understanding of the molecular mechanisms regulating CD22 transcription in B-ALL is still incomplete. Methods: To comprehensively investigate the genomic basis of variability in leukemia CD22 expression and thus B-ALL sensitivity to InO, we performed a multi-omic characterization of 196 primary human B-ALL samples, using whole transcriptome sequencing (RNAseq), assay for transposase-accessible chromatin with sequencing (ATAC-seq), coupled with ex vivo profiling of InO sensitivity. Additionally, genomic editing studies were performed in Nalm6 and REH B-ALL cell lines, and patient derived xenograft (PDX) models were used for in vitro and in vivo validations. Results: Applying digital cytometry to leukemia bulk RNAseq data to infer the B-ALL developmental state, we found that blast cells with early Pre-pro-B-like signature had low levels of CD22 expression and were resistant to InO. As B-cell differentiation is controlled by stage-specific transcription factors (TFs), we performed a targeted CRISPR library screen of 1,639 TF genes and identified EBF1 as a key activator of CD22 expression (FDR=7.1x10-4). In primary B-ALL samples, EBF1 expression was significantly correlated with CD22 expression (R=0.69, P<0.0001) and leukemia response to InO (measured as LC50, R=-0.42, P<0.0001). In addition, TF footprint analysis of leukemia ATAC-seq data identified EBF1 as the most differentially activated TF comparing InO-sensitive and -resistant B-ALL cases (P=8x10-174). Blocking the EBF1 binding sites within the CD22 promoter by CRISPR interference resulted in a 50-fold and 49-fold decrease in cell surface CD22 expression, and a 22-fold and 25.7-fold increase in InO LC50 for Nalm6 and REH, respectively, confirming the direct impact of EBF1 on CD22 transcription and InO sensitivity. Because BCR::ABL1-positive ALL showed the greatest variability in InO sensitivity among all B-ALL subtypes, we further investigate the role of the EBF1/CD22 axis on InO sensitivity within this subtype. Unsupervised clustering of gene expression profiles revealed two distinct groups, namely BCR::ABL1 ALL with multilineage (BCR::ABL1-M) or lymphoid only (BCR::ABL1-L) involvement [PMID: 38153913]. Compared to BCR::ABL1-L, BCR::ABL1-M displayed markedly reduced CD22 expression (P=8.3×10-11) and resistance to InO (P=1.9×10-4), likely due to the lower expression and frequent somatic alteration of EBF1. Finally, we established PDX models for representative cases of BCR::ABL1-M and BCR::ABL1-L. Strikingly, InO treatment did not affect leukemia progression in the BCR::ABL1-M PDX model, whereas the BCR::ABL1-L PDX model achieved complete remission and has remained leukemia free to date. Conclusion: Collectively, these data demonstrate the direct impact of EBF1 on CD22 expression during B-cell development, and explain the heterogeneity of InO sensitivity, even within the same molecular B-ALL subtype.