Adult ALL Research Articles

Combination of Olverembatinib and VP Regimen As First-Line Therapy for Adult Patients with Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia

Backgrand ： Ph+ALL is the largest genetically defined subtype in adult ALL and is considered associated with high relapse risk and inferior clinical outcomes. The combination of tyrosine kinase inhibitors (TKI) with intensive chemotherapy has achieved a major step forward in the therapy of Ph+ALL in deep response and long-term survival. The upfront use of third generation TKI ponatinib has improved the frequency of complete molecular response (CMR) and the overall survival rate to a greater extent. More interesting, the data coming from MDACC showed that combination of ponatinib in the chemotherapy in Ph+ALL maybe challenge the need for stem cell transplantation (SCT) in most patients who achieved CMR (defined BCR-ABL1 transcript < 0·01%) within 3 months after induction therapy. But the cardiovascular adverse event(AE) of ponatinib was more and severely frequently occurred in the former reports. Olverembatinib,a new third generation of TKI, which can also suppress the mutation of T315I and showed promising results in the Ph+CML. Herein we designed the clinical trial to invest the efficiency and safety of Olverembatinib combine with VP regimen (OVP) on Ph+ALL patients as the first-line therapy. Motheds: In this multi-centers, phase 2, single-arm trial, adult patients with previously untreated Ph+ALL were sequentially enrolled. The patients had to be aged 18 years or older, have normal cardiac function (defined by ejection fraction above 50%), and have adequate organ function (serum creatinine≦3*ULN，BUN≦3*ULN，ALT≦3*ULN，AST≦3*ULN，serum bilirubin≦3*ULN). They received three cycles of OVP regimen(for 28 days each cycle :olverembatinib 40mg qod d1-28，Vindesine 4mg d1,8,15,22,Prednisone1mg/Kg/d d1-21,0.5mg/kg/dd22-28). Twelve intrathecal injections of cytarabine alternating with methotrexate were designed as central nervous system prophylaxis in the following therapy after the diagnosis. Findings: 32 patients were screened and 29 were eligible from June 21, 2022 until 21 July,2023. 3 Patients were withdrawn from the clinical trial(1 refused therapy after induction,1 early death due to Covid infection, 1 Protocol deviation ).Among all , Male 12 and Female 17. BCR/ABL1 transcript:M-p210 (11),m-p190（18）.The Median Age was 45(19-74) years old. The Median WBC was 24.8(1.06-308.00) *10E9/L. The treatment has resulted in a 100% overall response, including 96% (24/25) CR and 4% (1/25) CRp. As of data collection,23 finished 3cycles of OVP chemotherapy and 82.6% (19/23) got CMR.AS far the rest 6 patients, 1 got CMR after 1 cycle OVP,1 got CMR after 2cycles of OVP, the other 4 patients were undergoing induction therapy. The total CMR rate was 84% (21/25) at any time. The CMR rate was 36%（9/25） ,76%（19/25）and 82.6%(19/23) at 4weeks,8weeks and 12 weeks respectively. Among all the patients finished the 3 cycles, there were 4 patients not reached the CMR and all of them were M-bcr/abl-p210 protein status. The best response was MMR for 3 patients and CR for 1patient. The CMR rate was 70% (7/10) in M-bcr/abl-p210 transcript and 100% (13/13) in that of m-bcr/abl-p190. During a median follow-up of 241 days, one patient died within one month after transplantation, while all the remaining survived without relapse. During induction therapy, 4 patients had platelets consistently above normal values and 4 patients did not experience a further decrease in hemoglobin compared to baseline. The median time for the occurrence of the lowest values of platelets, hemoglobin, and neutrophils was 6th, 12th, and 11th respectively. The most common non-Hematology AE was liver function damage. AE events greater than or equal to grade 3 included 2 cases of elevated Alanine transaminase ,3 cases γ- GT elevation and 2 cases of triglyceride elevation. Other AEs were mild and less than level 3. Discussion: Ongoing efforts exist to optimize therapeutic options in the frontline for Ph+ALL. The future lies in using less cytotoxic and more targeted agents. The first results of our ongoing trial indicate that the OVP regimen is effective in achieving early high rate of CMR in patients with newly diagnosed Ph+ALL as first-line therapy. All these results were achieved with surprisingly few toxic effects.Until now,16 patients underwent ASCT. Will the excellent outcomes be preserved with longer？ Will there be a difference in long-term outcomes between patients who underwent ASCT and those who do not?The trial is ongoing. we need more time to observe.

Impact of Prior Response to Blinatumomab on Outcomes of Brexucabtagene Autoleucel (Brexu-cel) in Adult Patients with Relapsed or Refractory (r/r) B-Cell Acute Lymphoblastic Leukemia (B-ALL): Results from the Real-World Outcomes Collaborative of CAR-T in Adult ALL (ROCCA)

Introduction: Brexucabtagene autoleucel (brexu-cel) is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved for adults with relapsed or refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL). Blinatumomab is a CD19-directed bispecific T-cell engager that is also approved for r/r B-ALL, and often used as early salvage therapy. Data in the pediatric/AYA population suggest that patients who did not respond to blinatumomab may have inferior outcomes to CD19-directed CAR-T products compared to those who have achieved a response to blinatumomab or were blinatumomab-naïve (Myers et al. J Clin Oncol 2022). In this study, we evaluate the response to blinatumomab and subsequent response to brexu-cel in adults with r/r B-ALL. Methods: Retrospective data were collected from 25 centers across the U.S. as part of the real-world outcomes collaborative study of CAR-T in B-ALL (ROCCA). Consecutive patients treated with brexu-cel from 2021 to 2023 were categorized by blinatumomab exposure status. Those exposed were separated into “blinatumomab responders” (B-R), defined as patients achieving a CR/CRi in response to any number of cycles of blinatumomab, and “non-responders” (B-NR). Each cohort was compared for outcomes of interest. The primary outcome was CR/CRi rate at day 28 following brexu-cel administration. Secondary outcomes were duration of response (DOR), progression-free survival (PFS), and overall survival (OS). Survival outcomes were calculated from day of brexu-cel infusion and were not censored for allogeneic hematopoietic cell transplantation or maintenance therapy. All living patients were censored at data cutoff on June 30, 2023. Survival comparisons were made by log rank test. Results: Among 152 patients who received brexu-cel, the median follow-up time was 8.4 months. Eighty-eight (57%) of 152 r/r B-ALL brexu-cel recipients had received blinatumomab prior to apheresis. The median number of pre-apheresis blinatumomab cycles was 2 (range 1-12). The baseline characteristics of B-R, B-NR, and blinatumomab-naïve (B-NV) patients were similar, including pre-apheresis disease burden and receipt of maintenance therapy following brexu-cel infusion (Table 1). Seventy percent (N= 62) of the blinatumomab-exposed patients were B-R, while 30% (N=26) were B-NR. Rates of CR/CRi at day 28 following brexu-cel infusion were similar between B-R, B-NR, and B-NV patients (79% vs 84% vs 78%, respectively). Most of these remissions were negative for measurable residual disease (MRD), with similar MRD negativity rates in the three groups (table 2). 1-year DOR and PFS were significantly higher in the B-NV group compared to B-R or B-NR (77% vs 49% vs 50%, p<.0001; 60% vs 37% vs 30%, p<.0001). B-NV and B-R had better 1-year survival compared to B-NR (71% vs 65% vs 32%, p<.0001). There were more CD19-negative relapses following brexu-cel in B-NR (29%) and B-R (18%) compared to B-NV patients (8%), although this was not statistically significant. Conclusions: Brexu-cel induced deep responses in a majority of adults with r/r B-ALL, irrespective of prior exposure or response to blinatumomab. Similar to data published by the pediatric CAR-T cell groups, our data draw speculation that adult patients who did not respond to blinatumomab experience shorter overall survival following brexu-cel compared to those who responded to or did not receive blinatumomab. This should be confirmed in a larger, prospective clinical trial. Because this analysis could not account for patients who achieved durable remission after blinatumomab, these differences may not reflect superiority of sequencing brexu-cel before blinatumomab. The higher number of CD19-negative relapses in the blinatumomab-exposed cohort add to the interest in pursuing strategies to address antigen escape following CAR-T cell therapy directed against a previously targeted antigen.

Prognostic Value of MRD By Flow Cytometry in Adolescent and Adult Patients with Philadelphia Chromosome-Negative Acute Lymphoblastic Leukemia Treated with Pediatric-Inspired Regimen: A Post-Hoc Analysis from a Prospective Study of China

Background Minimal residual disease (MRD) is currently considered as one of the most powerful predictors in patients with Philadelphia chromosome-negative acute lymphoblastic leukemia (Ph - ALL).The time points and thresholds for MRD monitoring in pediatric Ph - ALL have been well established. However, surveillance strategy in adult ALL has not yet been standardized. In this study, we explored the prognostic value of MRD levels and monitoring time points by flow cytometry (FCM) in adolescent and adult patients with Ph - ALL treated with pediatric-inspired regimen. Methods This analysis includes adolescent and adult patients who were treated according to a pediatric-inspired regimen revised based on Children's Oncology Group protocol (CCG-1961). This prospective study was registered at Chinese Clinical Trial Registry Website with the registration number of ChiCTR-OOC-15006328. MRD was measured using bone marrow aspirates with eight-color multiparametric FCM on day 14 of induction, at end of induction (EOI, days 29-42) , post the first consolidation (post-C1), and throughout the treatment period. MRD was defined as the percentage of lymphoblasts present in the total nucleated cells counted. MRD levels of < 0.01% and ≥ 0.01% was considered as negative and positive, respectively.Patients who were MRD-positive were further stratified into three groups according to MRD level: MRD low positive (MRD-lp), ≥ 0.01% to < 0.1%; MRD high positive (MRD-hp), ≥ 0.1% to < 1%; and MRD very high positive (MRD-vhp), ≥ 1%. Results From 2014 to 2021, a total of 415 patients with a median age of 27 (range: 14-69) years were treated. Three hundred and twelve patients presented with B-precursor ALL and 103 with T-cell ALL. With a median follow-up time of 40.8 months, the median overall survival (OS) of the cohort has not yet been reached. There were 386, 389, and 346 BM samples available for MRD evaluation on day 14 of induction, at EOI, and post-C1, respectively. Figure 1A presents the distribution of MRD status over time. Figure 1B depicts the compositions of MRD levels at EOI stratified according to disease types. Notably, Ph-like ALL and early T-cell precursor (ETP) ALL were associated with extremely lower achievements of MRD negativity at EOI compared with other types. The 5-year OS rates of patients with MRD negativity, MRD-lp, MRD-hp, and MRD-vhp at EOI were 68.0% (95% CI: 60.2%-75.8%), 44.8% (95% CI: 28.9%-60.7%), 52.4% (95% CI: 34.2%-70.6%), and 36.7% (95% CI: 24.2%-49.2%), respectively ( P < 0.001) (Figure 2A). If patients were censored at the time of hematopoietic stem cell transplantation, the 5-year OS rates of these patients were 74.9% (95% CI: 65.1%-84.7%), 39.7% (95% CI: 18.7%-60.7%), 42.3% (95% CI: 11.7%-72.9%) and 0%, respectively ( P < 0.001) (Figure 2B). Patients with a negative MRD at EOI had a more favorable OS than those with a positive MRD at EOI. There was no statistically significant difference in terms of survival time between patients with MRD-lp and MRD-hp. Further, patients with MRD-vhp had the worst survival. The relapse probabilities had a similar pattern. Patients with MRD negativity at EOI had a lower 5-year CIR than those with MRD positivity at any levels. Compared to MRD-lp and MRD-hp patients, patients with MRD-vhp experienced a significantly higher rate of early relapse. However, with a longer follow-up, the 5-year cumulative incidence of relapse (CIR) did not differ significantly among patients with a positive MRD in any of the three groups, with a rate of 47.4%, 43.8%, and 49.1% in patients with MRD-lp, MRD-hp, and MRD-vhp, respectively (Figure 2C). Multivariate analysis showed that positive post-induction MRD was one of independent predictors of both OS and Disease-free survival. Conclusion Our study confirmed that MRD negativity at EOI is an independent prognostic factor of OS in adolescent and adult patients with Ph - ALL treated with pediatric-inspired regimen. Patients with MRD-lp and MRD-hp at EOI had a lower OS and a higher relapse incidence than those with MRD negativity. Therefore, an early and deep MRD clearance is important, and even extremely low MRD levels can predict poor prognosis.

Mitochondrial dsRNA from B-Precursor Acute Lymphoblastic Leukemia Cells Induce Formation of Cancer Associated Fibroblast in the Bone Marrow Leading to Formation of a Chemoprotective Bone Marrow Niche

Precursor B-cell Acute Lymphoblastic Leukemia (B-ALL) is typically a highly chemosensitive malignancy with the majority of children being cured by chemotherapy alone. Adult ALL is often chemosensitive at initial treatment, but long term disease free survival is less common. It is widely accepted that outcome in ALL closely relates to underlying genetic lesion (Moorman, Hematologica 2016) but no specific mechanisms by which ALL genetics influence outcome have been identified. We have previously demonstrated that an ‘activated mesenchymal stromal cell (MSC) niche’ develops in response to reactive oxygen species (ROS) -inducing chemotherapy in which mitochondria are actively transferred along tunnelling nanotubes to ‘rescue’ B-ALL cells from chemotherapy (Burt, et al Blood 2019). According to recent evidence that chemotherapy resistant ALL sub-clones may exist at diagnosis (Dobson et al Cancer Discovery 2020), we hypothesised that B-ALL cells may themselves directly activate bone marrow stromal cells to generate cancer associated fibroblasts (CAF) from MSC. To test this hypothesis, we first used our previously described in vitro niche model wherein either the HS27a MSC cell line or healthy donor (HD) MSCs are co-cultured with either B-ALL cell lines or primary patient B-ALL cells. Three of five B-ALL cell lines and 3 of 4 primary B-ALL samples tested, induced activation of the MSCs, as characterised by cytomorphological changes identified by confocal microscopy, up-regulation of CAF-relevant gene expression and increased IL6, IL8 and CCL2 protein secretion. The ability of the B-ALL cell lines to induce CAF correlated directly with the intrinsic ROS levels of the cell as determined by CellROX assay. In vivo, the CAF-inducing ALL cell lines also activated murine MSCs when established as xenografts in NOD-SCID gamma (NSG) mice. Immunohistochemistry of NSG murine femora revealed a very significant expansion in nestin+ cells after exposure to CAF-induing ALL cells compared to non-CAF-inducing controls. We established that the ALL cells activated MSC without contact, both from exposure of MSC to ALL cells in transwell and from exposure to B-ALL cell conditioned media (CM). RNA sequencing of HS27a stromal cells exposed to CAF-inducing B-ALL cell CM compared with non-CAF inducing controls unexpectedly showed that the most highly upregulated pathways were those involved in RNA sensing. Using a smaller gene panel selected from among the RNA-sensing pathway genes, we confirmed that this was also the case for primary, HD MSCs upon exposure to primary ALL patient samples. As a result, we sought evidence for the role of mitochondrial dsRNA, which has been shown to activate the anti-viral signalling pathway (Dhir et al, Nature 2018). Using the J2 anti-dsRNA monoclonal antibody in both confocal and flow cytometric assays, we observed very significantly higher levels of dsRNA in MSC which had been exposed to CAF inducing B-ALL cell CM than in non-CAF inducing B-ALL or MSC alone controls. We confirmed by RT-PCR for mitochondrial genes on dsRNA immunoprecipitated from CAF inducing B-ALL cell CM that the dsRNA was of mitochondrial origin. Using ELISA, we quantified dsRNA from total cell RNA extracted from paired primary B-ALL patient samples at diagnosis and in remission. We found significantly higher levels of dsRNA at diagnosis than in remission, with it being undetectable in most remission samples. Moreover, we confirmed by flow cytometry the presence of dsRNA in 6 B-ALL patient derived xenografts which was absent in the control NSG mice without leukemia. We used DMSO to degrade dsRNA from CAF inducing B-ALL cell CM, as is often done in Sanger sequencing and confirmed the degradation by ELISA. We then quantified gene expression of a selection of stromal cell activation marker genes, as well as genes in the RNA sensing pathway after exposure to either DMSO-treated B-ALL cell CM or non-treated control. There was a 2 to 4-fold reduction in expression of IL6, IL8 as well as of RIG-I, MX2, IFIH1, MAVS and OAS1 expression in DMSO-treated condition compared to control. We confirmed that IL6, IL8 and CCL2 protein secretion was also significantly abrogated in the test but not the control condition. We propose a model in which ROS-induced mitochondrial dysfunction in ALL cells leads to release of dsRNA which in turn activates bone marrow stromal cells leading to protective niche formation, despite lack of prior exposure to cytotoxic agents.

Efficacy and Safety of AALL0232 Protocol in Adults with Acute Lymphoblastic Leukemia: Single Institution Experience

Background Over the past few decades, the outcomes for children with ALL have significantly improved with current regimens that yield a 5-year complete remission (CR) and event-free survival (EFS) rates of 98% and 70-80%, respectively. In contrast, the adult population has a CR of 85-90%, with a less satisfactory disease-free survival (DFS) rate of only 30-40% at 5-years. The feasibility of Children's Oncology Group (COG) AALL0232 protocol in older patients has not been established, especially adults older than 40 years and whether they will tolerate such unmodified pediatric based regimen. Therefore, we planned to assess our practice's outcomes in terms of the efficacy and safety of the COG AALL0232 protocol in adult ALL patients. Method This was a retrospective observational study that included patients ≥ 14 years with newly diagnosed ALL. Patients were excluded if they have secondary ALL. Our institute has adopted the AALL0232 protocol for both B-cell and T-cell ALL due to the similarities between the AALL0232 and CALGB 10403 protocols. MRD data was not included in our study since the cut-off for detection has been changed over the study period. Patient demographics and treatment-related outcomes will be reported using descriptive statistics. OS and RFS probabilities will be calculated using the Kaplan-Meier product-limit method from the date of pre-phase initiation. All P-values will be two-sided with P <0.05 indicating significance. Results Between 2014 and 2022, a total of 96 patients were included in our study. 74% had B-cell and 26% had T-cell ALL. The majority of the patients had standard risk ALL (23%). 65% of the patients were receiving AALL0232 protocol. Early death with AALL0232 protocol occurred in 11.1% of our population. The 2-year OS rate was 72.6% (95% CI 61.4-81.1) (Figure 1). The OS rate for B-cell ALL was 78.5% compared to 53.8% for T-cell ALL at 2-years (P =0.0912). The use of AALL0232 protocol was comparable to other protocols in terms of survival with a 2-year OS rate of 77% and 62.3%, respectively (P=0.0761). Adults with favorable cytogenetic risk had an OS rate of 100% compared to 67.6% for standard and 49.2% for poor risk (P =0.047). Adults younger than 40 years had a higher OS rate compared to those older than 40 (76.4% vs. 54.7%, P= 0.0187). RFS rate at 2-years was durable in 68.2% of the patients (95% CI 56.8-77.2) (Figure 2). RFS rates were 72.3% for B-cell and 54.8% for T-cell ALL. The AALL0232 Protocol had a significant impact on RFS compared to other protocols (73.6% vs. 56.5%, P= 0.0277) (Figure 3). Those who were younger than 40 had an RFS rate of 73.9%. The 2-year NRM occurred in 21.7% of the population. The AALL0232 protocol did not result in a higher NRM compared to other protocols (P= 0.492). Conclusion Although the use of AALL0232 protocol in the adult population has a remarkable impact on the rate of relapse, its effect on survival remains questionable especially for patients with T-cell ALL and poor cytogenetic risk. Another point of concern is the early death rate that was observed in our study. Further studies are needed to assess the overall impact of COG protocol in the adult population.

The Impact of Social Determinants of Health on Brexucabtagene Autoleucel Outcomes in Adults with Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia

Background Brexucabtagene autoleucel (brexu-cel) is an autologous anti-CD19 chimeric antigen receptor (CAR-T) cell therapy that recently received U.S. FDA approval as the first CAR-T cell therapy for adults with relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL). Social determinants of health (SDoH) are shown to significantly influence outcomes in B-ALL. However, how SDoH relate to and affect outcomes in patients with B-ALL receiving CAR-T therapy has not been well established. We studied the impact of SDoH on outcomes of adults with B-ALL receiving brexu-cel as part of the Real-world Outcomes Collaborative of CAR-T in Adult ALL (ROCCA). Methods This retrospective multicenter analysis spanning 25 U.S centers included adults (≥18 years) with r/r B-ALL treated with commercial brexu-cel between 2021-2023. Progression-free survival (PFS) and overall survival (OS) were calculated from the day of brexu-cel infusion and were not censored for hematopoietic cell transplant (HCT) or maintenance. Univariate and multivariate Cox proportional hazards models were used to evaluate the association of age, sex, pre-apheresis disease burden, and SDoH (referral source, insurance, distance from home to CAR-T site, marital status, and the social deprivation index) with progression-free survival (PFS) and overall survival (OS). The social deprivation index (SDI), a composite measure (including income, education, employment, housing, household characteristics, and transportation) used to quantify socio-economic variation in health outcomes, with higher SDI indicating greater social disadvantage, was estimated at the zip code level. (Butler et al., 2013) Results Of the 152 patients who received brexu-cel (Table 1), 57% were male. 51% identified as non-Hispanic White, with the remainder identifying as Hispanic (34%), Asian/Pacific Islander (7%), Black (6%), American Indian/Alaskan Native (1%), or mixed race (1%). A majority (46%) were referred for brexu-cel from private/community-based practices and 62% lived within 50 miles of the CAR-T center (36% lived >50 miles and 2% were unknown). Health insurance coverage included primarily public (49%) and private (42%); no patients were uninsured. High SDI (76-100% percentile) was present in 26%, while 14% had a low SDI (0-25 th percentile). In unadjusted analyses, there was no difference in PFS or OS between patients with high versus low SDI; closer distance to the CAR-T site (<50 miles versus >50 miles)) was associated with worse OS (HR 1.96; 95% CI 1.00, 3.84; p=0.05). Multivariate analysis (Table 2) revealed that male sex was associated with inferior PFS (HR 1.98; 95% CI 1.02, 3.85; p=0.04) and OS (HR 2.52; 95% CI 1.09, 5.84; p=0.03). There was no difference in PFS (HR 0.95, 95% CI 0.48-1.88, p=0.88) or OS (HR 0.80; 95% CI 0.33,1.92; p=0.62) when comparing Hispanic and non-Hispanic White patients. Additionally, there was no difference in PFS or OS based on any SDoH, including insurance type, marital status, referral source, caregiver type, and distance to transplant center. Conclusions In patients receiving brexu-cel, survival outcomes appear independent of SDoH. Contrary to previous reports suggesting inferior outcomes for Hispanic patients with B-ALL after receiving traditional systemic therapies, we observed comparable outcomes to non-Hispanic patients treated with brexu-cel. Distance from the CAR-T site and referrals from the community did not affect outcomes. Additionally, those with higher SDI had similar outcomes to those with a lower SDI, although this analysis was limited by sample size and the selection bias that exists in being referred for brexu-cel in the first place. The relatively lower logistical burden of CAR-T compared to allogeneic stem cell transplant or non-fixed duration systemic therapies may have mitigated the impact of SDoH on outcomes. These real-world data suggest that continuing to improve access to and treatment with brexu-cel may improve overall outcomes for disadvantaged populations with r/r B-ALL.

Role of Allogeneic Stem Cell Transplantation in Preventing Relapse in Adult BCR::ABL1-like Acute Lymphoblastic Leukemia

Background Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL) is characterized by the activation of several kinase pathways that promote treatment resistance leading to poor survival of patients at all ages. Despite the use of tyrosine kinase inhibitors in the Ph-like ABL-class subtype (15% Ph-like ALL), there is not a consensus approach for treatment of most patients (JAK-class especially), and the role of allogeneic stem cell transplantation (alloSCT) has scarcely been explored so far (i.e., mandatory vs. MRD-oriented). Objective To assess the outcome of Ph-like patients enrolled in the ongoing ALL19 trial from the Programa Español de Tratamientos en Hematología (PETHEMA) for adults (18-60y) with Ph-negative ALL, and to evaluate the effectiveness of alloSCT in first complete remission (CR). Methods In the ALL19 trial, patients are allocated to alloSCT according both to the end of induction (EOI) measurable residual disease (MRD) level and to high-risk genetic markers such as low-hypodiploidy, KMT2A rearrangement, TP53 biallelic alteration and concomitant deletion of IKZF1 and CDKN2A/B. Therefore, BCR::ABL1-like subtype was not a criteria for early transplantation unless meeting either poor MRD clearance at EOI or concomitant deletion of IKZF1+CDKN2A/B. Patients with MRD≥0.01% on day+35 (EOI) and/or high-risk genetics were assigned to early alloSCT (preceded by 1 cycle of consolidation chemotherapy) while those with good MRD clearance and standard risk genetics received early and delayed consolidation (3 cycles each) and maintenance therapy. Bone marrow or peripheral blood samples were analyzed by G-banding+FISH, SNP array (750K Affymetrix, Thermo Fisher) and next generation sequencing (NGS, with a custom DNA gene panel, Illumina) at diagnosis in 4 reference laboratories. MRD was centrally assessed by next generation flow cytometry with a sensitivity up to 2x10 -6. Results Twenty-four Ph-like out of 248 adult BCP ALL (10%) were detected. Baseline characteristics were similar between Ph-like and the remaining BCP-ALL. Most Ph-like ALL patients showed the JAK-class profile (20/24 [83%]: 17/24 CRLF2 rearrangement[r], 1/24 JAK2r, 1 BLNK::DNTT and 1 JAK2 R683 mutation), 3/24 belonged to the ABL-class subtype (2 ABL2r, 1 NUP214::ABL1) and 1 patient was identified by RNAseq (gene expression profiling similar to BCR::ABL1 ALL) without identifying any rearrangement. Regarding secondary genetic alterations, 17/24 (71%) patients showed the IKZF1 plus profile, 10/23 (43%) harbored JAK2 R683 mutation, 4/23 N/KRAS mutations, 2/23 CRLF2 mutation and 1/23 showed mutations in several genes such as IKZF1, NR3C1 or FLT3, among others. There were no significant differences in the probability of achieving CR between Ph-like and the remaining BCP ALL (15/20 [75%] vs. 137/162 [85%], p=0.334). However, EOI MRD level of Ph-like individuals was significantly poorer (MRD<0.01% Ph-like 4/15 [27%] vs. other BCP-ALL 85/136 [63%], p=0.007). By intention to treat, most Ph-like patients (17/19, 89%) were allocated to alloSCT (vs. 91/153 [59%] in the other BCP-ALL, p=0.011) due to either EOI MRD≥0.01% (9/18), IKZF1+CDKN2A/B concomitant deletions (3/18) or both (6/18). The 2-y overall survival (OS) in both groups was not significantly different (57% [95% CI, 27%-78%] vs. 67% [55%-77%, p=0.304]) (Figure 1). Interestingly, most Ph-like patients died due to treatment-related toxicity (2 transplant-related mortality, 1 CART-related, 1 during induction, 1 in CR and 1 by COVID-19 infection). The 2-y cumulative incidence of relapse (CIR) was also similar (Ph-like 27% [8%-50%] vs. other BCP-ALL 35% [25%-45%], p=0.946) (Figure 1). Conclusions Despite the short follow up of this series, our results show that Ph-like ALL patients have poorer EOI MRD clearance and higher rate of alloSCT realization than the remaining BCP ALL patients, without significant differences in outcome. This suggests that early alloSCT in first CR might overcome the poor prognosis of Ph-like ALL patients. Funding Funded in part by: PI10/01417 y FIS PI19/01183 Ministerio de Salud Carlos III, JCyL_SA118P20, FMM21/002_AP176752021, “La Caixa” Foundation, Pfizer Spain, Fundación PETHEMA. This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement No IMI001-07. The JU receives support from the European Union's Horizon 2020 research and innovation programme and EFPIA.

Mutually-Exclusive Pathways between IKZF1plus and RAS Mutations and TP53 double-Hit Alterations Lead Relapse in B-Other, Ph-like and Hyperdiploidy Genetic Groups in Adult B-Cell Acute Lymphoblastic Leukemia

Introduction: Adult B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a hematologic malignancy characterized by a high genetic heterogeneity and high relapse rate. Despite treatment advances have led to high cure rates in pediatric BCP-ALL (90%), improving survival in adults remains a challenge. Identification of genetic alterations driving relapse and both new prognostic and therapeutic biomarkers are important to improve survival in adults with BCP-ALL. Aim: To identify genetic alterations that lead to relapse in adult BCP-ALL patients (18-72 years) based on the tumor genetic landscape. Methods: Forty-five adult BCP-ALL patients (median age: 43 years; range 18-72 years) were studied at diagnosis and relapse by massive sequencing using a custom panel that allows detection of single nucleotide variants (SNVs) and insertions/deletions (INDELs) in 150 genes involved in BCP-ALL, in addition to copy number variations (CNVs), gene fusions and aneuploidy. Thirty-eight percent of patients belonged to the BCP-other ALL group, 16% were Philadelphia-positive (Ph+), 16% were classified as Philadelphia-like (Ph-like), 11% belonged to the high hyperdiploidy group, 8% had KMT2A rearrangements ( KMT2Ar), 4% TCF3::PBX1 fusion, 3% showed hypodiploidy, 2% low hyperdiploidy and 2% de novo MYC and BCL2 double-hit. Patients were treated according to the Spanish Adult-PETHEMA high-risk ALL protocols. Results: The most frequently deleted gene at diagnosis was IKZF1 (25/45, 56%). Among these cases, 72% (20/25) had IKZF1plus genetic profile, characterized by co-occurrence of IKZF1 deletions with at least one deletion in CDKN2A/B, PAX5 or PAR1, without ERG deletion. The IKZF1plus genetic profile was associated with specific genetic subgroups: Ph+ (5/7, 71%), Ph-like (5/7, 71%) and B-other (10/17, 59%). Moreover, it was significantly related with the presence of minimal/measurable residual disease (MRD) after induction (16/18, 89%) p < 0.001 and persistence at relapse in most patients (18/20, 90%; p < 0.001). Interestingly, among those 5 patients initially diagnosed with IKZF1 deletion but who did not meet the criteria for the IKZF1plus profile, 2 of them acquired the IKZF1plus profile at relapse. This suggests a possible cooperation of these genetic deletions contributing to therapy resistance and disease progression in these genetic subgroups. Eighty-six percent (6/7) of Ph+ patients who received imatinib treatment presented ABL1 mutations that were detected exclusively at relapse. Of these cases, 67% (4/6) were also associated with IKZF1plus at relapse (Figure 1A). Two mutually exclusive genetic patterns were identified at relapse in adult B-other and Ph-like patients. Thus, patients with IKZF1plus profile did not show RAS pathway mutations (59% and 57%, respectively), while patients with RAS pathway mutations lack in IKZF1plus profile (18% and 29%, respectively) (Figure 1B). Moreover age-related differences in the adult B-other ALL patient group were observed. The presence of mutations in RAS pathway was associated with younger age (<40 years) (3/10, 30% vs. 0/7, 0%), whereas older patients (≥40 years) predominated in the IKZF1plus profile (6/7, 86% vs. 3/10, 30%). TP53 alterations were detected at diagnosis and/or relapse in 11 patients (24%), most of which (9/11, 82%) had a heterozygous mutation plus a deletion or a homozygous mutation (double-hit). The high hyperdiploidy subgroup was enriched in TP53 mutations (4/5, 80%), most of which were already present at diagnosis (3/4, 75%). These findings indicate that genetic biomarkers usually linked to a favorable prognosis, such as high hyperdiploidy, can be associated with unfavorable genetic markers, leading to poorer outcomes in this group of patients (Figure 1C). Both the IKZF1plus profile and TP53 alterations were associated with poorer overall survival (OS) rates (P=0.034 and P=0.040, respectively) in adult BCP-ALL (Figure 2). Conclusion: Our results highlight the clinical importance of the TP53 alterations and the IKZF1plus profile as prognostic factors in adult BCP-ALL, suggesting they might help guiding more personalized therapeutic strategies to improve outcomes in this group of BCP-ALL patients. Grants: SACYL(GRS2386/A/21,GRS2385/A/21,GRS2527/A/22,GRS2506/A/22), JCyL SA118P20, FMM21/002 AP176752021, Pfizer 69383919.

Real-World Data in a Prospective Clinical Observation Study for Newly Diagnosed Adult Acute Lymphoblastic Leukemia, the JALSG ALL-CS-12 : Advanced Age Is a Poor Prognostic Factor

Background In the past 20 years, many clinical trials with pediatric type chemotherapy have improved outcome in adult ALL patients. However, they were selected patients with good performance status, less comorbidity, and tolerance to intensive chemotherapy. In the real-world, many ALL patients did not receive intensive chemotherapy for various reasons especially in elderly patients. Because the aging of the population is increasing remarkably in Japan, it is important to clarify the current overall treatment results and consider new treatment strategies for all ALL patients. Real-world data in ALL including patients not participating in the clinical trials have rarely been reported. Therefore, Japan Adult Leukemia Study Group (JALSG) conducted JALSG ALL-CS-12 study, a prospective clinical observation study for newly diagnosed adult ALL with large number of patients. Methods All newly diagnosed ALL patients according to the 2008 edition of WHO classification aged 15 years and older were prospectively registered by JALSG after informed consent. The study was approved by institutional ethical board and conducted in accordance with the Declaration of Helsinki. Results Nine-hundred and fifty-four ALL patients were accrued between April 2012 and August 2017. Nineteen patients were excluded for misdiagnosis including CML blast crisis, history of chemotherapy, lack of data, and duplicate registration. Among the remaining 935 patients, 209 were over 65 years old patients generally not focused on clinical trials. Four-hundred and sixty-nine were Philadelphia chromosome negative B-ALL (Ph-), 130 were T-ALL, 293 were Ph +ALL, and 44 were Burkitt leukemia. Median age was 45 years (range 15-89) in B-ALL (Ph-), 33 (15-80) in T-ALL, 60 (15-89) in Ph +ALL, and 60 (15-91) in Burkitt leukemia with significantly younger in T-ALL patients as has been reported previously. Chemotherapy was not performed in 23 patients in B-ALL (Ph-), 3 in T-ALL, 4 in Ph +ALL, and 3 in Burkitt leukemia. Fifteen of them were over 70 years old. CR rate was comparable in four disease cohorts, 81.7% in B-ALL (Ph-), 78.7% in T-ALL, 88.7% in Ph +ALL and 74.4% in Burkitt leukemia, respectively. However, in the patients with 75 years or older, CR rate was low, 42.4% in B-ALL (Ph-), 64.5% in T-ALL, 64.5% in Ph +ALL and 42.9% in Burkitt leukemia, respectively. Cumulative incidence of relapse at 5 years was 38.8% in B-ALL (Ph-), 20.8% in T-ALL, 43.8% in Ph +ALL, and 50.9% in Burkitt leukemia, respectively (p<0.05). 5-year event free survival (EFS) and overall survival (OS) rates were 46.1% and 52.6% in B-ALL (Ph-), 55.4% and 61.6% in T-ALL, 44.5% and 52.5% in Ph +ALL, and 26.2% and 36.0% in Burkitt leukemia, respectively (p<0.05). When the age of the patients was classified in increments of 10 years, EFS was worse as the age increased in B-ALL (Ph-), T-ALL, and Ph +ALL (table). Conclusions JALSG ALL-CS-12 study is the large prospective cohort study and provides important information that is less to the inherent selection bias. The incidence and median age of the four types of leukemia cohorts in Japan were comparable to those in European reports. Although CR rate was acceptable despite the inclusion of patients outside of clinical trials, EFS was strongly dependent on age. Post-remission treatment and management of complications are assignments to be solved especially for elderly ALL patients. Acknowledgement We thank Ms. Ryoko Fujiyoshi in Nagasaki data center for collecting CRFs.

Brexucabtagene Autoleucel in Adults with Relapsed/Refractory B-Cell ALL: Outcomes and Novel Insights from the Real-World Outcomes Collaborative of CAR T in Adult ALL (ROCCA)

Introduction In October 2021, brexucabtagene autoleucel (brexu-cel) received U.S. FDA approval as the first CAR T-cell therapy for adults with relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL) based on the 55-patient ZUMA-3 Phase II study. We subsequently established ROCCA and now report on the largest cohort of patients treated with commercial brexu-cel for r/r B-ALL to date. Methods Adults (18+) with r/r B-ALL infused with commercial brexu-cel across 25 U.S. institutions were included. ASTCT consensus criteria were used to score CRS and ICANS. Methodologies for assessing MRD (minimal threshold of 10 -4) included flow cytometry, NGS, or qPCR depending on institution practice. Duration of remission (DOR) was calculated from time of complete response (CR); progression-free survival (PFS) and overall survival (OS) were calculated from day of brexu-cel infusion and were not censored for hematopoietic cell transplant (HCT) or maintenance. All living patients were censored at the time of last follow-up prior to data lock, which occurred on June 30, 2023. Results Among 152 infused, the median age was 46 (range, 18-81), 57% were male, and 34% were Hispanic. Most (67%) had Ph- ALL, were heavily pre-treated (median 4 prior lines), and entered apheresis with high disease burden (57%). At time of apheresis, 23% of patients only had MRD+ disease and 15% were in complete molecular remission. While 82% developed CRS, the majority was grade 1-2, with 9% of the overall cohort experiencing grade 3-4 CRS. In contrast, 55% developed ICANS, with 32% of the entire cohort experiencing grade 3-4 ICANS. Eight patients (5%) died of toxicity/infection prior to D+28 response assessment. Among 133 patients with response assessment, 120 (90%) achieved morphologic CR, of whom 82% were MRD-, 15% were MRD+, and 3% MRD unknown. The median follow-up for survivors was 8.4 months; 45 patients have relapsed and 42 patients died. Median DOR was not reached. Median PFS and OS were 8.6 months and 15.6 months, respectively. Estimated PFS and OS of the entire cohort at 6-months were 61% (95% CI, 52-68) and 81% (95% CI, 73-87) and at 12-months were 47% (95% CI, 37-56) and 63% (95% CI, 53-72), respectively. We found no association between pre-CAR disease burden and post-CAR PFS/OS. However, patients with MRD- response to CAR had superior PFS relative to patients with MRD+ CR (median 14 months vs. 5 months, P=0.002). Forty-four patients received post-CAR consolidation/maintenance therapy while in CR: 25 allogeneic HCT, 15 TKI, 2 POMP, and 3 other/unknown. To examine the effect of consolidation/maintenance following brexu-cel, we performed a landmark analysis of PFS limited to patients alive and in CR at 2-months post-CAR infusion (N=113) and suggests superior PFS in patients receiving either HCT or other forms maintenance, relative to those receiving no further therapy following brexu-cel ( P=0.055). We then investigated post-CAR MRD-response in combination with receipt of post-CAR consolidation/maintenance and found that even among patients achieving MRD-negative response, post-CAR consolidation/maintenance led to superior PFS (Figure 1). Conclusions Among 152 adults treated with commercial brexu-cel for r/r B-ALL across 25 U.S. institutions, we found very high response rates (CR/CRi: 90%; 82% MRD-) consistent with the Zuma-3 data. While rates of severe CRS are low, grade 3-4 ICANS was observed in 32% of patients and warrants further investigation We demonstrate the prognostic impact of achieving an MRD-negative CR and an emerging role for consolidation/maintenance therapies to enhance the durability of response following brexu-cel in adults r/r B-ALL.

A Dual Role of SOX11 Expression in the Formation of T-Cell Acute Lymphoblastic Leukemia in Mouse Models

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that accounts for 10%-15% of pediatric and 25% of adult ALL cases. Intensified chemotherapy and bone marrow transplants have improved the survival of T-ALL patients. However, around 20% of patients still suffer from primary resistant or relapsed T-ALL. Therefore, improving our knowledge of T-ALL biology and developing precision oncology therapeutics is essential for further progress in T-ALL treatment. SRY-related HMG-box 11 (SOX11) is a transcription factor that regulates the development and survival of multiple embryonic tissues and is crucial for the nervous system. SOX11 is expressed in a multitude of tumor types, where it has been associated with either increased or decreased cell proliferation, metastatic potential, and with either good or bad prognosis, depending on cancer type. Despite being absent in the T-cell lineage, SOX11 is expressed in a subset of T-ALLs, where it plays an unknown role. Based on having a malignantly restricted expression, we hypothesize that SOX11 might be oncogenic in T-ALL. Aim: Identify the role of aberrant SOX11 expression in (pre)leukemic T-cells. Methods: Using a conditional Rosa26-SOX11 overexpression mouse model, we generated mice with T-cell specific (using Lck-cre) SOX11 expression (SOX11 Lck). To assess the self-renewal potential of SOX11 Lck thymocytes, we performed thymic transplantations on sublethally irradiated mice expressing a different allele of CD45. To study SOX11 in a leukemic context, we crossed the SOX11 Lck mice with two established T-ALL mouse models that are either driven by the overexpression of Lmo2 (SOX11 Lck-Lmo2 CD2) or by the loss of the tumor suppressor Pten (SOX11/Pten Lck). An aging cohort of these T-ALL models was followed, and tumor samples were collected. Thymi of 8-weeks old mice were used to study the impact of SOX11 in a pre-leukemic context, and T-cell tumors arising in these models were also used for RNA-sequencing. Using a retroviral strategy, we overexpressed SOX11 in human cord blood stem cells, which we differentiated towards the T-cell lineage ex vivo by co-culturing them on OP9 stromal cells expressing Delta-like Notch ligand 4. Furthermore, we transduced Jurkat cells with a SOX11 expression vector, or with an empty vector, and injected the cells into immunocompromised mice to assess the effect of SOX11 on tumor growth in vivo. Results: In SOX11 Lck mice, we found a significant increase in the immature double-negative 3 (DN3) thymic fraction, with a concomitant decrease in the amount of more mature double-positive CD4 +CD8 + (DP) thymocytes (Fig. A). A possible explanation for this phenotype would be the establishment of a stem-like self-renewal program similar to the one driven by Lmo2. However, by performing thymic transplantations into sublethally irradiated mice, we concluded that SOX11 does not confer self-renewal properties to thymocytes. Additionally, we aged a cohort of SOX11 Lck mice and did not observe formation of T-cell malignancies up to 500 days of age. Given that the expression of SOX11 does not suffice to produce T-cell tumors, we generated the SOX11 Lck-Lmo2 CD2 and SOX11/Pten Lck T-ALL mouse models. In SOX11 Lck-Lmo2 CD2 mice, we found a synergistic effect of the co-expression of both genes on the expansion of DN3 thymocytes (Fig. A), which led to accelerated leukemia formation (Fig. B). By sequencing the RNA of pre-leukemic and leukemic DN3 cells, we observed that SOX11 is mostly regulating cell adhesion, morphology, and cell-cell interactions to promote this oncogenic phenotype in Lmo2-driven T-ALL. On the other hand, SOX11/Pten Lck mice suffered from delayed leukemogenesis (Fig. B), and this tumor suppressor role might be associated with the decrease in DP cells observed in the pre-leukemic stage. Lastly, we set out to validate these findings in a human context. The ex vivo differentiation of human hematopoietic stem cells toward the T-cell lineage showed that SOX11 strongly accelerates early commitment to double-negative T-cell precursors. Also, we observed that Jurkat T-ALL cells engraft faster in immunocompromised mice when expressing SOX11. Conclusions: By generating multiple models of SOX11 expression in the T-cell lineage, we showed that SOX11 impacts T-cell differentiation, both in mice and humans, and that it can promote or hinder T-ALL formation depending on the oncogenic driver.

Bridging Therapy in Adults with Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia Receiving Standard of Care Brexucabtagene Autoleucel: Results from the Real-World Outcomes Collaborative of CAR T in Adult ALL (ROCCA)

Introduction: Brexucabtagene autoleucel (brexu-cel) is an autologous anti-CD19 chimeric antigen receptor (CAR) T cell therapy approved for adults with relapsed/refractory B-cell acute lymphoblastic leukemia (R/R B-ALL). Bridging therapy, defined as anti-leukemia therapy given between apheresis and lymphodepleting chemotherapy, was permitted on the pivotal ZUMA-3 trial. The optimal bridging therapy is unknown in this setting. Here we report patient and disease characteristics in those receiving bridging therapies prior to brexu-cel and the impact of bridging on clinical outcomes in a large multicenter cohort. Methods and Results: ROCCA includes 25 US institutions contributing retrospective data from 152 B-ALL patients treated with standard of care brexu-cel between 2021-2023. Ninety-nine patients (65%) received bridging therapy, while 53 patients (35%) received no bridging therapy. Bridging therapy consisted of cytotoxic chemotherapy ( n=65, 66%), immunotherapy ( n=26, 26%; 23 inotuzumab, 3 blinatumomab), tyrosine kinase inhibitors (TKI) ( n=20, 20%; 16 ponatinib), steroids only ( n=10, 10%), and intrathecal chemotherapy ( n=22, 22%). The median age of the cohort was 46 (IQR 32-61). Patients were heavily pre-treated with a median of 4 prior lines of therapy. The median days from apheresis to infusion was 33 (IQR 26-42). 119 patients had known pre-apheresis blast counts; the bridging group had higher pre-apheresis blast burden, with 21 of 76 patients (28%) having ≥ 50% blasts compared to only 5 of 43 patients (12%) in the no bridging group ( p=0.02). In addition, more patients who received bridging had pre-apheresis extramedullary disease (32% vs. 15%, p=0.02). All other baseline characteristics were similar between the two groups (Table 1). Fifty-four (54%) patients had response assessment after bridging therapy and prior to brexu-cel infusion. In patients who received inotuzumab for pre-apheresis active disease, defined as ≥ 5% blasts, 6 of 13 (46%) who had post-bridging assessment achieved a complete response (CR; 5 measurable residual disease (MRD)+, 1 MRD-), with a mean proportional reduction in blast burden of 46% from baseline. In contrast, after chemotherapy bridging, only 2 of 16 patients (13%) with pre-apheresis active disease had a CR. Patients receiving chemotherapy bridging had a mean proportional increase in blast burden of 4.4%. In recipients of TKI bridging, 2 of 4 patients (50%) with pre-apheresis active disease had a CR. Compared to no bridging, patients in the bridging group had similar rates of grade 3 or 4 CRS (9% bridging vs. 8% no bridging, p=0.73) and grade 3 or 4 ICANS (34% vs. 26%, p=0.32). In univariate analysis, there was no statistically significant difference in day 28 response with 63 patients (72%) achieving MRD- complete response in the bridging group as compared to 36 patients (78%) in the no bridging group ( p=0.60). Non-relapse mortality was similar between the two groups (17% bridging vs. 9% no bridging, p=0.20). Overall survival (OS) and progression-free survival (PFS) were superior in the no bridging group: 1-year OS 74.7% vs 56.8% (log-rank p=0.04), and 1-year PFS 57.3% vs 41.7% (log-rank p=0.04). In a multivariate analysis stratified by bridging and pre-apheresis disease burden, patients with pre-apheresis blasts ≥ 5% and bridging had worse survival than ≥ 5% blasts and no bridging: 1-year OS 45.3% vs. 87.5% and 1-year PFS 27.7% vs. 64.3% (Figure 1). However, patients with < 5% blasts had similar survival regardless of bridging. Conclusions: In our real-world analysis, adults with R/R B-ALL receiving bridging therapy had a higher disease burden at baseline. Despite greater baseline disease, there were no significant differences in toxicity and day 28 response in those receiving bridging therapy vs. not. While survival was inferior in patients with pre-apheresis active disease who received bridging, this may be confounded by the degree of tumor burden. In patients with < 5% blasts pre-apheresis, bridging did not impact survival. Furthermore, inotuzumab bridging demonstrated the most significant disease debulking. However, the comparisons in this study are limited by the variability in practice for post-bridging disease reassessment and the small proportion of patients with active disease who did not receive bridging. A more uniform approach to peri-CAR disease assessment would enable more robust study of optimal bridging strategies.

Use of Ponatinib Alone or Combined with Other Therapies in Relapsed/Refractory Ph-like Acute Lymphoblastic Leukemia. a Campus ALL Real-Life Study

Introduction. Ponatinib is a third-generation tyrosine kinase inhibitor (TKI) employed in relapsed/refractory (R/R) and, more recently, also in newly diagnosed Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL). Ph-like ALL is a genetically heterogenous subgroup that shares a common transcriptional profile, similar to that of true Ph+ ALL, though lacking the BCR::ABL1 rearrangement. Ph-like ALL is characterized by a poor prognosis, a high relapse rate and a worse overall survival if treated with standard chemotherapy. Several molecular pathways are involved, including JAK/STAT, CRLF2 and ABL-class mutations, providing a heterogeneous genetic landscape with very limited data on the subsequent clinical outcome. Few isolated cases of successful treatment of R/R Ph-like ALL with ponatinib have been reported, including also patients with lesions different from ABL-class mutations. Currently, ponatinib prescription in Italy is not allowed for Ph-like ALL. Patients and Methods. Between January 2019 and July 2023, 17 patients were candidate to receive ponatinib on a nominate compassionate use basis; data were collected in the context of the Campus ALL network in Italy. The criteria for inclusion were a diagnosis of Ph-like ALL, with either hematological or molecular evidence of disease, and a treatment period of at least 28 consecutive days of ponatinib. The Ph-like signature was based on the BCR/ABL1 like-predictor (Chiaretti et al, BJH 2018) and, whenever possible, targeted RNA sequencing. Final data were collected for 15 patients (2 patients were excluded due to a treatment period <28 days) treated with ponatinib either as a single agent or in association with immuno and/or chemotherapy. Results. All patients had common ALL. Eight of the 15 cases were classified as high risk or very high risk ALL at diagnosis; 10 were males, the median age was 28 years (14-66) and the median white blood count at diagnosis was 25.6 x10 9/l (2.7-317 x10 9/l). Targeted RNA sequencing was carried out in 10/15 cases: 7 presented gene fusions ( ABL-class mutations in 2, JAK2 mutations in 2, and CRLF2::P2RY8, IKZF1::DDC and RB1::RCBTB2 in 1 case each); 2 additional cases had a CRLF2 rearrangement, evaluated by FISH. Copy number aberrations analysis by multiplex ligation-dependent probe amplification (MLPA) technology was performed in 12/15 cases: 5 cases had a IKZF1 plus signature, 6 had a IZKF1 loss, while 1 case was IKZF1 wild type. Ponatinib was started in first (n=2) or second (n=5) hematologic relapse in 7 patients (46%), with 3 also having an extramedullary disease; 7 additional patients (46%) were treated in first (n=5) or second (n=2) minimal residual disease (MRD) persistence/recurrence; finally, 1 patient was refractory to 3 subsequent lines of treatment. In 8/15 patients ponatinib was used as single agent or in association with steroids or intrathecal chemotherapy only, in 4 it was administered in combination with chemotherapy and in 3 with blinatumomab; 7 patients started ponatinib after having failed an allogeneic transplant (6 hematologic relapses and 1 molecular relapse). A complete hematological and molecular remission (MRD- CR) was achieved in 6 patients (40%); notably, 4 of these 6 cases carried a gene fusion (1 JAK2 rearrangement, 1 ABL-class fusion, 1 IKZF1::DDC and 1 RB1::RCBTB2); 2 further patients in hematologic relapse at the start of treatment achieved a MRD+ CR (1 patient had a ABL-class gene fusion). Thus, the overall response rate was 53%. Four patients were refractory, while 2 maintained a stable disease. After ponatinib-based treatment, 6 patients were allografted and 1 received a CAR-T cell infusion. The toxicity profile was mild: 3 patients developed a transaminitis, 1 also had a fungal pneumonia, and 1 an increase in pancreatic enzymes. At a median follow-up of 3 months (1-5), 10 patients are alive, 8 being in continuous complete remission. Conclusions. Treatment with ponatinib showed promising results in terms of CR achievement in a heavily pre-treated population of Ph-like ALL patients, with an acceptable toxicity profile. In a pre-transplant setting, ponatinib was effective as a bridge to cellular therapies in 7 out of 8 patients as intention-to-treat (ITT), thus suggesting that this strategy may represent an effective bridge to further therapies. To our knowledge, this is the largest cohort of adult Ph-like ALL cases treated with ponatinib so far reported.

Chromothripsis and Genomic Complexity As Pejorative Prognostic Markers in Pediatric and Adult T-ALL

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplasm that accounts for 25% and 15% of adult and pediatric ALL, respectively. The prognosis is particularly poor in patients with relapsed disease, justifying the research for novel predictive markers of relapse.To date, a significant series of adult and pediatric T-ALL with analysis using high-resolution SNP-array is still missing. Methods: Here, we report a comprehensive high-resolution SNP-array profiling in a large well-characterized cohort of 317 T-ALL patients all uniformly treated according to the French FRALLE and GRAALL protocols. SNP-array results have been correlated with clinico-biological data including age range, immunophenotype, oncogenetic and survival data. Results: The SNP-array analysis detected at least one genomic imbalance (CNVs and UPDs combined) in 304/317 (96%) T-ALL cases with a mean of 6.3 (range 0-47) genomic imbalances per sample. Among genomic imbalances detected by SNP-array analysis, we identified 35 recurrent genomic imbalances (i.e. observed in at least 2% of T-ALL patients). The most common recurrent CNV was the del(9p) including CDKN2A/B at 9p21 (~70%). Unexpectedly, the second more frequent CNV was del(13q) minimally including RB1 and/or DLEU1 at 13q14 (~14%). The third more frequent CNV was the del(6q) minimally encompassing CASP8AP2 at 6q15 (~11%). The following recurrent CNVs were: del(12p) minimally including ETV6 and/or CDKN1B at 12p13 (~9%), del(18p) minimally including PTPN2 at 18p11 (~9%), del(1p) minimally including RPL22 at 1p36 (~9%), del(17q) minimally including NF1/SUZ12 at 17q11 (~8%). Two regions were identified as being recurrently affected by UPDs: 9p21 ( CDKN2A/B) (~28%), and 12q14 ( SH2B3) (~2%). Chromothripsis was detected in 6 (~2%) cases. The present study revealed relative minor differences in the genomic landscape of T-ALL according to age range consisting primarily of higher incidence of del(1p36)/ RPL22, del(13q14)/ RB1/DLEU1 del(12p13)/ ETV6/CDKN1B and del(18p11)/ PTPN2 and lower incidence of del(9p21) and UPD(9p21)/ CDKN2A/B in T-ALL older than 30 years. Comparison of SNP-array results according to immunophenotype and oncogenetic mainly highlighted the specific cytogenetic pattern of the SIL-TAL1 subgroup which exhibited a low genomic complexity with a lower number of genomic imbalances per sample and a lower diversity in genomic imbalances. Using the “survcutpoint” approach, we determined that a cutoff of 15 alterations was the most significant threshold to stratify the patients into a high- and a low-risk group of relapse according to the number of alterations detected with our SNP-array approach. Compared to patients harboring less than 15 alterations (n=296), patients with ≥ 15 genomic imbalances (n=21) had a significantly shorter event-free survival (EFS) and increased cumulative incidence of relapse (CIR)(4y-EFS: 38% vs. 64%; hazard ratio (HR): 2.1, 95%CI (1.2 - 3.7); P=.008, 4y-CIR: 53% vs. 26%; subdistribution hazard ratio (SHR): 2.9, 95%CI (1.5 - 5.4); P=.001) (Figure 1A). Additionally, survival analysis revealed the poor outcome conferred by the presence of chromothripsis (n=6) as well as gain(6q27)/ MLLT4 (n=10) and del(16p13)/ CREBBP (n=15) despite the low number of affected cases (4y-EFS: 17% vs. 63%; HR: 3.1, 95%CI (1.3 - 7.8); P=.01 and 4y-CIR: 83% vs. 26%; SHR: 5.0, 95%CI (2.2 - 11.3); P <.001 for chromothripsis; 4y-EFS: 30% vs. 63%; HR: 2.5, 95%CI (1.2 - 5.4); P=.002 and 4y-CIR: 57% vs. 27%; SHR: 2.8, 95%CI (0.96 - 8.1); P=.06 for gain (6q27)/ MLLT4; 4y-EFS: 40% vs. 63%; HR: 2.5, 95%CI (1.3 - 4.8); P=.005 and CIR in the FRALLE cohort; CIR: 4y-CIR: 50% vs. 21%; SHR: 5.4, 95%CI (1.5 - 20.0); P=.001 for del(16p13)/ CREBBP) (Figure 1B). Conclusion: Our study demonstrated that the whole genome analysis of imbalances provides new insights to refine the risk stratification in T-ALL. Notably, genomic complexity (≥ 15 genomic imbalances) and chromothripis demonstrated an association with an inferior outcome in terms of EFS and CIR in pediatric and adult T-ALL.

Impact of Specialized Treatment Setting on Survival in Adolescent and Young Adult ALL.

Unlike children with ALL who receive cancer care primarily at specialized cancer centers (SCCs; National Cancer Institute and/or Children's Oncology Group centers), adolescents and young adults (AYAs; 15-39 years) receive care in a variety of settings. Using population-based data, we describe where AYAs with ALL receive treatment and determine associations with overall survival (OS). Data from the 2004 to 2018 California (CA, n = 2,283), New York (NY, n = 795), and Texas (TX, n = 955) state cancer registries were used to identify treatment setting of AYAs with newly diagnosed ALL. Multivariable Cox proportional hazards regression models evaluated associations with OS. Seventy percent were older than 18 years, and 65% were male. A majority in CA (63%) and TX (64%) were Hispanic while most in NY were non-Hispanic White (50%). Treatment at an SCC occurred in 48.2% (CA), 44.4% (NY), and 19.5% (TX). Across states, AYAs who were older or uninsured were less likely to receive treatment at an SCC. Treatment at an SCC was associated with superior OS in CA (hazard ratio [HR], 0.73; 95% CI, 0.63 to 0.85) and TX (HR, 0.61; 95% CI, 0.45 to 0.83); a nonsignificant association was seen in NY (HR, 0.83; 95% CI, 0.64 to 1.08). Only 20%-50% of AYA patients with ALL received frontline treatment at SCCs. Treatment of ALL at an SCC was associated with superior survival, highlighting the importance of policy efforts to improve access and reduce inequities in AYA ALL care.

FCM marker importance for MRD assessment in T-cell acute lymphoblastic leukemia: An AIEOP-BFM-ALL-FLOW study group report.

T-lineage acute lymphoblastic leukemia (T-ALL) accounts for about 15% of pediatric and about 25% of adult ALL cases. Minimal/measurable residual disease (MRD) assessed by flow cytometry (FCM) is an important prognostic indicator for risk stratification. In order to assess the MRD a limited number of antibodies directed against the most discriminative antigens must be selected. We propose a pipeline for evaluating the influence of different markers for cell population classification in FCM data. We use linear support vector machine, fitted to each sample individually to avoid issues with patient and laboratory variations. The best separating hyperplane direction as well as the influence of omitting specific markers is considered. Ninety-one bone marrow samples of 43 pediatric T-ALL patients from five reference laboratories were analyzed by FCM regarding marker importance for blast cell identification using combinations of eight different markers. For all laboratories, CD48 and CD99 were among the top three markers with strongest contribution to the optimal hyperplane, measured by median separating hyperplane coefficient size for all samples per center and time point (diagnosis, Day 15, Day 33). Based on the available limited set tested (CD3, CD4, CD5, CD7, CD8, CD45, CD48, CD99), our findings prove that CD48 and CD99 are useful markers for MRD monitoring in T-ALL. The proposed pipeline can be applied for evaluation of other marker combinations in the future.

Super Enhancer Regulatory Gene FYB1 Promotes the Progression of T Cell Acute Lymphoblastic Leukemia by Activating IGLL1.

Arising from T progenitor cells, T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignant tumor, accounting for 15% of childhood ALL and 25% of adult ALL. Composing of putative enhancers in close genomic proximity, super enhancer (SE) is critical for cell identity and the pathogenesis of multiple cancers. Belonging to the cytosolute linker protein group, FYB1 is essential for TCR signaling and extensively studied in terms of tumor pathogenesis and metastasis. Dissecting the role of FYN binding protein 1 (FYB1) in T-ALL holds the potential to improve the treatment outcome and prognosis of T-ALL. In this study, SEs were explored using public H3K27ac ChIP-seq data derived from T-ALL cell lines, AML cell lines and hematopoietic stem and progenitor cells (HSPCs). Downstream target of FYB1 gene was identified by RNA-seq. Effects of shRNA-mediated downregulation of FYB1 and immunoglobulin lambda-like polypeptide 1 (IGLL1) on self-renewal of T-ALL cells were evaluated in vitro and/or in vivo. As an SE-driven gene, overexpression of FYB1 was observed in T-ALL, according to the Cancer Cell Line Encyclopedia database. In vitro, knocking down FYB1 led to comprised growth and enhanced apoptosis of T-ALL cells. In vivo, downregulation of FYB1 significantly decreased the disease burden by suppressing tumor growth and improved survival rate. Knocking down FYB1 resulted in significantly decreased expression of IGLL1 that was also an SE-driven gene in T-ALL. As a downstream target of FYB1, IGLL1 exerted similar role as FYB1 in inhibiting growth of T-ALL cells. Our results suggested that FYB1 gene played important role in regulating self-renewal of T-ALL cells by activating IGLL1, representing a promising therapeutic target for T-ALL patients.

Molecular characterization of TCF3::PBX1 chromosomal breakpoints in acute lymphoblastic leukemia and their use for measurable residual disease assessment

The translocation t(1;19)(q23;p13) with the resulting chimeric TCF3::PBX1 gene is the third most prevalent recurrent chromosomal translocation in acute lymphoblastic leukemia and accounts for 3–5% of cases. The molecular background of this translocation has been incompletely studied, especially in adult cases. We characterized the chromosomal breakpoints of 49 patients with TCF3::PBX1 and the corresponding reciprocal PBX1::TCF3 breakpoints in 15 cases at the molecular level, thus providing an extensive molecular overview of this translocation in a well-defined study patient population. Breakpoints were found to be remarkably clustered not only in TCF3 but also in PBX1. No association with DNA repeats or putative cryptic recombination signal sequence sites was observed. A simplified detection method for breakpoint identification was developed and the feasibility of patient-specific chromosomal break sites as molecular markers for detecting measurable residual disease (MRD) was explored. A highly sensitive generic real-time PCR for MRD assessment using these breakpoint sequences was established that could serve as a useful alternative to the classical method utilizing rearranged immune gene loci. This study provides the first extensive molecular data set on the chromosomal breakpoints of the t(1;19)/TCF3::PBX1 aberration in adult ALL. Based on the obtained data a generic MRD method was developed that has several theoretical advantages, including an on average higher sensitivity and a greater stability of the molecular marker in the course of disease.

Early T-Cell Precursor Leukemia: A High-Risk Subtype of Acute Lymphoblastic Leukemia, Single Center Experience in Jordan

Background and ObjectivesEarly T-cell precursor (ETP) acute lymphoblastic leukemia/lymphoma (ALL/LBL) is a newly recognized entity of T-lymphoblastic leukemia/lymphoma. The optimal therapeutic approaches to adult patients are poorly studied. Patients and MethodsWe compared the outcomes of adult's patents with ETP-ALL/LBL who received frontline chemotherapy regimens with other T-ALL/LBL immunophenotypic subtypes. Patients with ETP-ALL/LBL were identified based on CD1a (-), CD8 (-), CD5 (-) (dim), and positivity for 1 or more stem cell or myeloid antigens. ResultsSixty-nine patients were included between the years 2010 and 2021 (19 ETP-T-ALL/LBL; 50 non ETP- T-cell ALL/LBL). The median age was 26 year (IQR: 21, 33). Fifty-six patients presented as ALL, while 16 with lymphoblastic lymphoma. Forty-seven patients achieved complete remission, and 43 were alive at last encounter. The complete remission rate in patients with ETP-ALL/LBL was lower than that of non-ETP-ALL/LBL patients (32% vs. 68%; P = .2), and the MRD at end of induction was significantly higher (26% vs. 6.2%, P < .001), and more likely to receive allo-SCT consolidation in CR1 (95% vs. 40%, P < .001). After a median follow-up of survivors of 48 months (range: 32-74 months), the median overall survival for patients with ETP-ALL/LBL was not reached versus 11.5 months for the non-ETP-ALL/LBL patients (P = .014)). Twenty-six patients receive allo-SCT in CR1. There was no significant difference in overall survival (79% vs. 70%; P = .49) between both transplant-cohorts in both groups. ConclusionETP-ALL/LBL represents a high-risk disease subtype of adult ALL. Novel treatment strategies are needed to improve treatment outcomes in this patient's population

Genetic alterations and MRD refine risk assessment for KMT2A-rearranged B-cell precursor ALL in adults: a GRAALL study

AbstractKMT2A-rearranged (KMT2A-r) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is widely recognized as a high-risk leukemia in both children and adults. However, there is a paucity of data on adults treated in recent protocols, and the optimal treatment strategy for these patients is still a matter of debate. In this study, we set out to refine the prognosis of adult KMT2A-r BCP-ALL treated with modern chemotherapy regimen and investigate the prognostic impact of comutations and minimal residual disease (MRD). Of 1091 adult patients with Philadelphia-negative BCP-ALL enrolled in 3 consecutive trials from the Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL), 141 (12.9%) had KMT2A-r, with 5-year cumulative incidence of relapse (CIR) and overall survival (OS) rates of 40.7% and 53.3%, respectively. Molecular profiling highlighted a low mutational burden in this subtype, reminiscent of infant BCP-ALL. However, the presence of TP53 and/or IKZF1 alterations defined a subset of patients with significantly poorer CIR (69.3% vs 36.2%; P = .001) and OS (28.1% vs 60.7%; P = .006) rates. Next, we analyzed the prognostic implication of MRD measured after induction and first consolidation, using both immunoglobulin (IG) or T-cell receptor (TR) gene rearrangements and KMT2A genomic fusion as markers. In approximately one-third of patients, IG/TR rearrangements were absent or displayed clonal evolution during the disease course, compromising MRD monitoring. In contrast, KMT2A-based MRD was highly reliable and strongly associated with outcome, with early good responders having an excellent outcome (3-year CIR, 7.1%; OS, 92.9%). Altogether, our study reveals striking heterogeneity in outcomes within adults with KMT2A-r BCP-ALL and provides new biomarkers to guide risk-based therapeutic stratification.