B-cell Acute Lymphoblastic Leukemia Patients Research Articles

Gimema ALL2418: Interim Analysis of a Phase Iia Study of Feasibility and Effectiveness of Inotuzumab Ozogamicin in Adult Patients with B-Cell Acute Lymphoblastic Leukemia with Positive Minimal Residual Disease before Any Hematopoietic Stem Cell Transplantation

Background: Persistence of minimal residual disease (MRD) is associated with a high rate of treatment failure, relapse, and death in B-cell acute lymphoblastic leukemia (B-ALL). Particularly, patients who remain MRD positive before an allogeneic hematopoietic stem cell transplant (HSCT) have an expected disease-related death rate due to the disease of about 60%. The T cell-engager CD19-directed therapy blinatumomab demonstrated considerable efficacy in this setting, however resistant or unsuitable patients have no alternative therapy. Inotuzumab Ozogamicin (IO) is a CD22-directed antibody-drug conjugate with remarkable efficacy in B-ALL with overt disease. Furthermore, IO does not need a high effective immune system to exert activity. Methods: In this multicenter phase 2 trial (NCT03610438), we are investigating the efficacy of IO in obtaining MRD negativity in two cohorts of 38 Ph+ and 38 Ph- B-ALL patients. IO was administered at the dose of 0.5 mg/sqm on days 1, 8, and 15 of a 28-days cycle. Patients who did not obtain MRD negativity after the 1st IO cycle had the option to receive the 2nd IO cycle maintaining the same dosage. Responding Ph- patients received short-term maintenance with low-dose chemotherapy (alternating vincristine, cyclophosphamide, prednisone, methotrexate, and 6-mercaptopurine), responding PH+ patients received tyrosine kinase inhibitor (TKI) for up to 12 weeks, allowing proper IO washout before HSCT. HSCT ineligible patients entered into low-dose chemotherapy or TKI maintenance for up to 2 years. For the primary endpoint, MRD was centrally assessed at baseline, after course 1, after course 2, and eventually before transplant with high sensitive molecular methods for BCR-ABL1 or V(d)J fusion transcripts. Here, we present the results of an unplanned interim analysis that was generated to evaluate the continuation of the study considering the slow accrual due to the Sars-COV2 pandemic. Results: To date, 51 patients underwent screening procedures, and 43 were considered eligible. We report data from the first 39 patients treated in the study. Nineteen out of 39 patients had Ph- B-ALL and 20/39 Ph+ B-ALL. Eighteen out of 39 (46%) patients were male. The median age was 55 years (22-84), 15/39 patients (38%) were older than 65 years. Demographics were comparable between Ph+ and Ph- patients. Within Ph+, ALL 39% of patients harbored p190 and 61% p210 fusion transcript. Within Ph- B-ALL, 1 patient harbored ALL1/AF4 and 1 patient harbored ALL1/ENL fusion transcript. Most of the patients received 1 (16/39, 42%) or 2 (19/39, 50%) previous lines of therapy; 5 patients (13%) were resistant to blinatumomab, and all of them had Ph- B-ALL. Twelve out of 19 (63%) Ph- B-ALL patients received only 1 IO cycle, 7/19 (37%) received 2 cycles; 5/20(25%) Ph+ B-ALL received only 1 IO cycle, and 15/20 (75%) received 2 cycles. To date, central MRD monitoring is available for 20 patients (12 Ph- and 8 Ph+ B-ALL, ongoing for the remaining cases). Overall, MRD negativity was obtained in 7/20 patients (35%); Particularly, in Ph- B-ALL cohort 5/12 (42%) patients achieved MRD negativity, 4/12 (38%) achieved disease reduction at low level MRD positivity (<10-4); in Ph+ B-ALL cohort, 2/8 (25%) patients achieved MRD negativity. One out of 5 (20%) blinatumomab pre-treated patients obtained MRD negative response. IO was an effective bridge to HSCT for 10/39 patients (26%; in patients <65 years old, 9/24 patients, 37%); Specifically, all 7 patients who obtained MRD negativity after IO, a patient with low-positive MRD and 2 patients with non yet available MRD data were bridged to HSCT. Accordingly, with the caveat of a short median follow-up (2.8 months, range 0.6-20.0 months), overall survival in the study population seems promising (figure 1, median OS not reached). Overall, adverse events collected during the study were limited in number and grading. Also, myelotoxicity was manageable. Out of 39 patients, we documented 1 veno-occlusive disease (2.5%) that occurred within 20 days from the last IO dose and before any HSCT. Conclusions: IO administration demonstrated activity with a good safety profile in the setting of MRD positive B-ALL, including blinatumomab resistant patients. Importantly, IO was capable to bridge 37% of eligible patients to HSCT, augmenting the chance to achieve a cure. The continuation of the study will deliver mature efficacy and survival data for a proper evaluation of this promising strategy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Multi-Omics Reveal Plasma Molecular Signatures of B-ALL Patients during CAR-T Cell Therapy

Background: The Chimeric antigen receptor (CAR) T cell therapy for hematological malignancies has revolutionized anticancer therapy. However, CAR-T cells can cause immunity-related adverse events, such as cytokine release syndrome and neurotoxicity; the efficacy of CAR-T cell therapy also varies substantially with different patients. Poor understanding of plasma proteomic and metabolomic features during chimeric antigen receptor (CAR) T cell therapy may affect the exploitation of therapeutic potential in the management of hematological malignancies. Methods: We longitudinally performed proteomic and metabolomic profiling of plasma from 20 patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) on CAR-T cell therapy. Blood samples were collected from B-ALL patients at different time points, including before fludarabine/cyclophosphamide lymphodepletion (Pre-FC), Day 0 (D0), Day 7 (D7), Day 14 (D14), Day 28 (D28), and Peak-IL-6 during CAR-T cell therapy. The plasma samples from 22 age- and gender-matched healthy controls (HC) were used as controls. Results: Compared with HC, 239, 217, 282, 260 and 261 proteins were markedly different in terms of expression level in these five groups (Pre-FC, D0, D7, D14, D28 versus HC), respectively (Figure 1). The pathways of differentially expressed proteins (DEPs) were highly enriched in processes implicated in actin binding, cadherin binding, regulation of ion transport, platelet aggregation and regulation of actin cytoskeleton, with actin binding obtaining the most significant change (Figure 1). 104, 139, 175, 223 and 87 metabolites were significantly different in these five groups, respectively (Figure 1). Interestingly, the alterations of plasma metabolites became more extensive on the 14th day after CAR-T cell infusion, indicating that the change of metabolites may lag behind that of proteins. Furthermore, CAR-T cell therapy reshaped plasma proteome and metabolome with discrete kinetics, as represented by upregulated acute-phase response and triglycerides metabolism and downregulated actin cytoskeleton organization, retrograde endocannabinoid signaling and glycosylphosphatidylinositol (GPI)-anchor biosynthesis during the therapy. We also identified sets of covarying molecules that were involved in acute inflammatory response, epithelial mesenchymal transition, glutathione metabolism and tryptophan metabolism in concert with the dynamics of circulating IL-6 in patients with cytokines storm. Subgroup analysis further revealed numerous prognostic biomarkers, such as proteins related to allograft rejection, which were down-regulated in patients who achieved stable complete remission. Conclusions: This study provided a dynamic landscape of plasma proteome and metabolome in B-ALL patients during CAR-T cell therapy and offered novel insights into CAR-T cell therapy-related toxicities and prognostic heterogeneity. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Better Prognosis of Dominant-Negative Type of IKZF1 Deletion in BCR-ABL-positive Adult B-Cell Acute Lymphoblastic Leukemia

IntroductionIKZF1 codes Ikaros, which is a member of a family of zinc finger proteins that is required for normal lymphopoiesis. IKZF1 alterations in cases of B-cell acute lymphoblastic leukemia (B-ALL) were mainly analyzed in a pediatric cohort and showed an association with poor prognosis. In adults IKZF1 alterations are found in approximately 50% of B-ALL cases, and 65% of cases with BCR-ABL-positive B-ALL. IKZF1 deletion have several different types of deletions. Deletion of exons 4 to 7 (Δ4-7) leads to nonfunctional isoforms that do not bind DNA. However, they can still dimerize with residual normal isoforms, suppressing the function of the normal Ikaros protein expressed from the nondeleted allele, a so-called "dominant-negative” (DN) effect. Other deletions lead to loss of function (LOF) of Ikaros. The impact of DN and LOF type of IKZF1 deletion on prognosis in adult B-ALL patients has not been elucidated. We evaluated the impacts of IKZF1 deletion types on overall survival (OS) in our cohort. Patients and Methods Hokkaido Leukemia Net (HLN) is prospective cohort study collecting acute leukemia samples from hospitals of North Japan Hematology Study Group (NJHSG) covering Hokkaido, the northernmost island of Japan. In this study, we focused on newly diagnosed adult B-ALL patients. Bone marrow or peripheral blood samples from newly diagnosed B-ALL patients were screened by a fluorescence in situ hybridization (FISH) probe for detecting IKZF1 deletion (Hashiguchi J. J Mol Diagn. 2018). Then DNA specimens from IKZF1 deletion-positive patients were screened for the 4 most common intragenic IKZF1 deletions, Δ4-7, Δ2-7, Δ2-8 and Δ4-8, by polymerase chain reaction (PCR). Samples in which IKZF1 deletion was detected by FISH but in which the types of deletions were not identified were defined as "other". We categorized the patients into a DN group that had Δ4-7, LOF and other group, and wildtype (WT) group based on the types of IKZF1 deletion. This study was conducted in accordance with the Helsinki Declaration and was approved by the institutional review boards. Results Overall, 116 patients with newly diagnosed B-ALL were enrolled in HLN between August 2014 and December 2021 (age range, 17-90 years; median age ,59 years; 55 males and 61 females; 60 BCR-ABL-positive patients and 56 BCR-ABL-negative patients). IKZF1 deletion was detected in 53 cases including 6 cases with bi-allelic deletion. IKZF1 deletions were detected in 68.3% of BCR-ABL-positive B-ALL cases and 21.4% of BCR-ABL-negative B-ALL cases. DN/LOF and other ratio was larger in BCR-ABL-positive B-ALL cases than in BCR-ABL-negative B-ALL cases (20/21 vs 3/9). In patients with BCR-ABL-positive B-ALL, patients with the DN type of IKZF1 deletion had a significantly longer OS than that in the WT group. The 3-year OS rates in the DN, LOF and other, and WT groups were 100%, 60.4%, and 49.9%, respectively (Figure A). Distribution of age, sex, percentage of allogenic hematopoietic stem cell transplantation (allo-HSCT) were not significantly different between the three groups. Survival was analysed by univariate analysis including age, sex, WBC counts, complex karyotype, IKZF1 deletion type and allo-HSCT. Age≥65 was associated with poor OS (P=0.0078) and DN type of IKZF1 deletion was associated with favorable OS (P=0.026). Multivariate analysis showed that older age and DN type of IKZF1 deletion were independently associated with OS (age≥65: HR, 3.510; 95% CI, 1.327 to 9.289, P=0.011; DN type of IKZF1 deletion: HR, 0.200; 95% CI, 0.046 to 0.884, P=0.034; Cox regression). In patients with BCR-ABL-negative B-ALL, there was no statistically significant difference in OS between the three groups, whereas the LOF and other group showed a dismal prognosis. The 3-year OS rates in the DN, LOF and other, and WT groups were 100%, 0%, and 56.5%, respectively (Figure B). Conclusion The prognostic impact of IKZF1 deletion might differ in BCR-ABL-positive and BCR-ABL-negative B-ALL cases and might also depend on the type of deletion. DN type of IKZF1 deletion is independently associated with favorable prognosis in adult BCR-ABL-positive B-ALL patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Outcomes in Hispanic Patients with Philadelphia-Negative and Philadelphia-like Acute Lymphoblastic Leukemia with the Use of Blinatumomab

Introduction: In the United States, Hispanics have the highest incidence rate of B-cell acute lymphoblastic leukemia (ALL) amongst all minority groups. Hispanic adults with ALL have the lowest rates of progression-free survival and overall survival. Hispanic adults, for example, have an increased frequency of Philadelphia chromosome (Ph)-like ALL, a sub-type of ALL associated with significantly inferior survival outcomes In the last decade, use of Blinatumomab and other novel targeted immunotherapies were approved for treatment of relapsed or minimal residual disease positive disease (MRD +) B-cell ALL. Pivotal trial showing efficacy of blinatumomab in relapsed or MRD + setting had very limited number of Hispanic patients. Our aim is to evaluate survival outcomes and the efficacy of blinatumomab usage in Hispanic B-ALL including Philadelphia-negative (Ph negative) and Philadelphia-like (Ph-Like) patients treated at LAC-USC (Los Angeles County Hospital-University of Southern California). We report Hispanic B-cell ALL patients treated with blinatumomab outside of clinical trials Method: This is a retrospective review which included adults aged ≥18 years with newly diagnosed Philadelphia negative ALL (2016-2020) treated at the USC/Norris Comprehensive Cancer Centerand the Los Angeles County Hospital (LAC). Primary objectives was to investigate overall survival in Hispanic patients who were treated with blinatumomab. We stratified patient population into Philadelphia negative and Philadelphia-like B-cell ALL and assessed survival separately for blinatumomab administered for minimal residual positivity and for relapse disease. Most of Ph-Like patients were identified with CRLF2 rearrangement per institutional protocol. Results: 67 patients were B cell ALL, which included Ph-negative B cell ALL and Ph-like B cell ALL, 43 and 24 patients respectively. 50.7% were men and median age was 39 years. 38 patients were considered unfavorable risk due to cytogenetics risk factors and 44 patients received peg-asparaginase based induction. 30 (48%) of patients received blinatumomab, 15 in each ph-negative and ph-like group respectively. More patients received blinatumomab for relapsed disease as compared to MRD positive disease, 23 vs. 8 patients respectively. Mean overall survival for our patients was 68 months (SE=3.3). In ph-negative group, when blinatumomab was used for relapsed disease then 3 years survival probability was 0% compared to 91.5% for patients who did not receive blinatumomab (p-value=0.0001). On the other hand, for MRD positive disease, 3 years OS was 91.7% vs. 79.6% for patients who received blinatumomab compared to no blinatumomab (p-value=0.6). For ph-like group, when blinatumomab was used for relapsed disease then 3 years survival probability was 100% compared to 40% for patients who did not receive blinatumomab (p-value=0.2). For MRD positive disease, median OS was not reached for either group (p-value=0.6). Conclusion: Both relapse disease and MRD+ disease are considered poor prognostic indicator. Our study highlights that blinatumomab can decrease survival gap for MRD + disease and relapse disease for ph-like Hispanic patients and decrease survival gap in MRD + ph-negative Hispanic patients when compared to patients who did not need blinatumomab

IKZF1 Deletions Are Markers of Treatment Resistance in Adult Ph-Negative B-Cell Acute Lymphoblastic Leukemia Patients Treated within the Ongoing Risk-Adapted Pethema LAL19 Trial

Introduction IKZF1 (Ikaros) deletion has been related with poor outcome in pediatric B-cell precursor acute lymphoblastic leukemia (BCP ALL). However, its impact in adult patients (pts) treated with MRD-oriented trials has not been fully established. This study aimed to assess if IKZF1 deletions are markers of poor prognosis in adults with Ph-negative BCP ALL treated within the ongoing risk-adapted PETHEMA LAL19 trial (NCT04179929). The impact of IKZF1 loss in these pts and those from the EWALL Group (European Working group on ALL) is being evaluated in a HARMONY (Healthcare Alliance for Resourceful Medicine Offensive against Neoplasms in Hematology) project (Project Ref ALL2). Methods In the PETHEMA LAL19 trial, pts with poor end-induction MRD clearance (MRD>0.01%) or high-risk genetics (low-hypodiploid, KMT2Ar, TP53 biallelic alteration or IKZF1+CDKN2A/B deletion) are assigned to allogeneic stem cell transplantation, the remaining pts receive maintenance chemotherapy. All pts are analyzed by G-banding, FISH, NGS (DNA gene panel) and SNP array. Next generation flow cytometry is used for MRD assessment. Main outcome measures: i) Complete Remission (CR): absence of extramedullary disease, neutrophil level >1x109/L, platelet level >100x109/L, and bone marrow blast level < 5%; ii) Cumulative Incidence of Relapse (CIR) and non-relapse mortality (NRM): calculated using cumulative incidence rates to accommodate competing risks and compared by Gray's test; iii) Overall survival (OS): measured from the time of protocol entry to the time of death or last contact. Results 147pts were analyzed. Mean age 40y [range 18-60], 56% males, 10% CNS involvement, 78% common B, 15% pro-B and 7% pre-B ALL. Ph-like (12%) was the most common genetic subtype followed by KMT2Ar (11%), PAX5 P80R (8%), low-hypodiploid (7%), TCF3::PBX1 and high-hyperdiploid (6% each), iAMP21 and ETV6::RUNX1 (2% each). More uncommon biological entities such as ZNF384r, MEF2Dr, MYCr, IDH1 R132C and IDH2 R140Q were seen in single pts (<1% each).The remaining patients (41%) were B-other,no growth or normal karyotype. IKZF1 deletion (IK DEL) was observed in 66/147(45%) pts. Deletions involved the whole gene (37/66, 56%) or a subset of exons (partial gene deletions). The most recurrent partial gene deletions (n=29) were: loss of exons 4-7 (n=6, 21%), 4-8 (n=5, 17%), 2-8 and 1-7 (n=4, 14% each), 1-3 (n=3, 10%), 2&3 and 2-7 (n=2, 7% each). The IKZF1plus profile was observed in 37/147 (25%) pts. IK DEL were more frequent in low-hypodiploid (100%) and Ph-like ALL (88%), and less prevalent in KMT2Ar (19%), PAX5 P80R (18%) and TCF3::PBX1 ALL (11%), p<0.001. Median follow up was 11.4 months. IKDEL pts showed lower CR rate than pts without Ikaros loss (75% vs. 91%, p=0.027). The IKZF1plus signature showed similar CR rate to IK DEL without IKZF1plus profile (IKZF1not plus) (73% vs. 78%, p=ns). IKDEL pts showed poorer MRD clearance than WT individuals. Interestingly, this fact was mainly attributable to partial gene deletions (MRD≥0.01% partial 79% vs. whole 42% vs. WT 29%, p=0.003). IKZF1plus was also correlated with positive MRD, however IKZF1not plus pts showed higher frequency of MRD positivity than IKZF1plus pts (MRD≥0.01% plus 47% vs. not plus 71% vs. WT 29%, p=0.012). Pts with whole gene loss showed lower OS than pts with partial gene losses or IKZF1 WT (1y-OS 62% vs. 88% vs. 89%, p=0.012)(Figure 1A). Of note, relapse was not the main cause of death of IK DEL whole gene pts (n=26) as only 1 patient died due to disease progression. Instead, NRM was the main cause of death in whole gene-deleted pts (4 died in Ind-1, 3 in Ind-2, 1 due to TRM and 1 due to Covid19 infection). Accordingly, the 1y-CIR of IK DEL whole pts was lower than that of IKZF1 WT individuals, who showed similar CIR to IK DEL partial ones (1y-CIR whole 7% vs. WT 30% vs. partial 32%, p=0.059) (Figure 1B). Pts with IKZF1plus signature did not show differences in OS and CIR compared to IKZF1 WT or IKZF1not plus individuals (data not shown). Conclusions This preliminary data suggest that IK DEL may be marker of poor treatment response in adult Ph-negative BCP ALL. Further analyses including all patients of the HARMONY project may identify which isoforms or in which genetic ALL subtypes IKZF1 DEL are particularly deleterious. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Outcome and Molecular Measurable Residual Disease Monitoring in Adult B-Cell Acute Lymphoblastic Leukemia Patients with MEF2D Fusions

Oncogenic gene fusions caused by chromosomal rearrangements are the major hallmarks of B-cell acute lymphoblastic leukemia (B-ALL). With the application of next-generation sequencing technologies, several non-scarce recurrent fusion genes have been identified in B-ALL, and MEF2D fusions is the one. More clinical data is needed to clarify the incidence and prognosis. Furthermore, the prognostic significance of measurable residual disease (MRD) monitored by the quantitative detection of MEF2D fusions remains absent to date. A total of 357 adult Ph-negative B-ALL cases who were consecutively diagnosed and received treatment in our institute from 2009 to 2021, and have available bone marrow cDNA samples at diagnosis were retrospectively screened MEF2D fusions. All patients have been tested common fusion transcripts at the time of diagnosis. MEF2D fusions were tested by the multiplex TaqMan-based real time quantitative polymerase chain reaction technology. A total of 7 fusion partners were included. The split-out real time PCR was performed to identify the partner. Totally 16 patients were identified MEF2D fusions with the incidence of 4.5% in Ph-negative BCP-ALL. The distributions were as follows: 7 MEF2D-BCL9, 7 MEF2D-HNRNPUL1, 1 MEF2D-DAZAP1 and 1 MEF2D-FOXJ2. Their median transcript levels were 65.0% (range, 20.9%-335.5%) at diagnosis. The median age of these 16 patients were 30 (range, 17-60) years and 10 of them were male (63%). The median follow-up time of 16 patients with MEF2D fusions was 26 months (range, 8-156 months). 9 (61.1%) patients were alive at the last follow-up. 14 and 2 patients individually achieved complete remission (CR) after 1 and 2 courses of CODP ± L regimen for induction. Thereafter, 8 patients received hyper-CVAD-based chemotherapy alone and all of them relapsed after 1-6 cycles of consolidation, then 2 relapsed patients received allogeneic hematologic stem cell transplantation (allo-HSCT) as salvage treatment after achieving the 2nd CR (CR2); the remaining 8 patients received chemotherapy followed by allo-HSCT (matched sibling donor, n = 3; haploidentical related donor, n = 5) in CR1. The median follow-up time after HSCT was 19 (6-148) months, and 5 patients (4 CR1, 1 CR2) relapsed at a median of 9 (range 6-15) months post-HSCT. The 3-year relapse free survival [RFS] and overall survival (OS) rates of all 16 patients were 30.0% (95% confidence interval [CI]: 10.2%-53.0%) and 54.6% (95% CI: 23.9%-77.5%) (Fig 1). Bone marrow samples serially collected after treatment were retrospectively tested MEF2D fusion transcript for MRD monitoring. 58.3% (7/12) and 50.0% (6/12) of patients were PCR-negative at the time of achieving CR1 and after one cycle of consolidation, respectively. PCR-negative at the above both time-points was not related to relapse (P=1.0 and 0.45). For patients receiving allo-HSCT, the scheduled monitoring time-points were 0, 1, 2, 3, 4.5, 6, 9, 12, 18, 24 months post-HSCT then every 12 months thereafter. There was a tendency that PCR-negative at the time of receiving HSCT was related to a lower relapse rate (post-HSCT 2-year RFS rate, 75.0% [95% CI, 12.8%-96.1%] vs 20.0% [95% CI, 0.8%-58.2%], P=0.13). 5 patients who were in continuous CR were tested MEF2D fusion transcript at a median of 9 (range, 3-11) time-points after HSCT, 38 (97.4%) samples were PCR-negative. 5 patients who relapsed post-HSCT were tested MEF2D fusions at a median of 5 (range, 4-7) time-points before relapse, they all were in continuous PCR-negative from 1 to 2-6 months post-HSCT, then all became PCR-positive before the occurrence of relapse. Their intervals between the 1st PCR-positive and relapse were 1.5-6 months. As a result, occurrence of PCR-positive post-HSCT was significantly related to relapse (post-HSCT 2-year RFS rate, 100.0% (95% CI, 100%-100%) vs 33.0% (95% CI, 4.3%-67.2%), P=0.039, Fig 2). In conclusion, MEF2D fusions confer adverse prognostic significance to adult B-ALL, and relapse is a treatment challenge. PCR-negative of MEF2D fusion transcript at the time of achieving CR and after the 1st consolidation could not predict long-term CR, whereas PCR-positive after HSCT predict relapse. The benefit of urgent allo-HSCT after achieving CR and MEF2D fusion transcript levels directed intervention post-HSCT remains to be investigated. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

EP300-ZNF384 Rearrangement Drive B-Cell Acute Lymphoblastic Leukemia Development By Activating IL3-IL3RA Signaling

Abstract: EP300-ZNF384 chromosome rearrangement serves as an oncogenic driver in B-cell acute lymphoblastic leukemia development. However, the oncogenic mechanisms underlying EP300-ZNF384 remain largely elusive. Recent evidence shows that the gene expression signature in EP300-ZNF384-positive patients enriched in cytokine-cytokine receptor and JAK-STAT signaling. We reason the correlated cytokine-cytokine receptor and JAK-STAT signaling might play important roles in EP300-ZNF384 fusion primed B-cell acute lymphoblastic leukemia. To investigate this concept, we identified EP300-ZNF384 substantially induces the expression of IL3RA on cell membranes of B lineage cells. Further, we proved IL3 supplement promotes EP300-ZNF348 positive cells proliferation and colony formation by activating JAK-STAT signaling. IL3RA knockdown reduces the responsiveness of EP300-ZF384 positive cells to IL3 stimulation. Dual luciferase assay and ChIP-qPCR assay confirm that EP300-ZNF384 binds the promoter region of IL3RA and actives its transcription. Interestingly, IL3 expression was significantly upregulated in ZNF384 rearranged B-cell acute lymphoblastic leukemia patients comparing with B others through analyzing bioinformatics databases. These findings underscore the critical role of IL3-IL3RA signaling under EP300-ZNF384 driven B-cell acute lymphoblastic leukemia and imply EP300-ZNF384 positive patients might benefit from JAK inhibitors. Keywords: EP300-ZNF384, B-cell Acute Lymphoblastic leukemia, IL3RA, IL3 JAK, STAT

Pro-Active 4SCAR2.0 Therapy with NGS-MRD Disease Monitoring in High-Risk B Cell Leukemia Management

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HCT) is a standard treatment for high-risk relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). However, about 30-40% of patients still relapse after HCT. Chimeric antigen receptor-modified T cell (CAR-T) therapy holds great promise as a salvage approach. Although CD19-targeted CAR-Ts have demonstrated a rapid and significant anti-tumor effect in patients with B-cell malignancies, many still suffer disease relapse due to the decrease or loss of CD19 antigen. Therefore, novel curative approaches are needed. PURPOSE: The aim of this study is to determine the best timing for applying CAR-T therapy in high-risk B-ALL patients based on high-sensitivity analysis of tumor cells by next-generation sequencing (NGS) detecting minimal residual disease (MRD). An innovative CAR-T therapy concept, CAR2.0, has been proposed, which uses multi-antigen-targeted CAR-Ts to prevent antigen escape and prolong remission. Based on NGS-MRD detection, patients with potential tumor relapse can be identified early and managed accordingly. On the other hand, if extended negative NGS-MRD is found, the patient is deemed at low risk and no further treatment is needed. The study proposes to treat high-risk B-ALL patients in the first remission period with CAR2.0 followed by continued disease monitoring by NGS-MRD to avoid HCT. METHODS: B-ALL patients who have high tumor burden and high-risk genetic indication is enrolled with the understanding that CAR-T therapy will be applied in the first remission and disease will be continuously monitored by NGS-MRD. The tumor cells from initial diagnosis will be analyzed for target antigens by immunostaining and to detect clonally rearranged immunoglobulin gene sequences by NGS-MRD with sensitivity ≤10-6. Patients will receive 4SCAR19 (CD19scFv-CD28/CD27/CD3ζ-iCasp9) therapy in the first remission and will be followed up for CAR-Ts, B cells and IgG/IgA/IgM in the blood, and bi-monthly NGS-MRD for residual tumor cells in the bone marrow (BM). A booster CAR-T treatment targeting additional tumor antigen such as CD22 will be applied 2 months later. A consolidation CAR-T treatment based on known target antigens will be applied 6 months after the first CAR-T infusion. The cyclophosphamide/fludarabine chemo-conditioning will be included prior to the first CAR-T treatment only. The CAR-T infusion dose is 1-5x106 CAR-Ts/kg. The quality of apheresis cells, efficiency of gene transfer, T cell proliferation, CAR-T infusion dose and blood CAR copies are quantitatively documented. The end point for this study is the establishment of NGS-MRD negativity for 1 year, with continued follow-up for 5 years. RESULTS: A 20 years old female was diagnosed with BCR/ABL1 P190 positive B-ALL, with 82.52% tumor blasts in the BM. NGS identified two high-frequency IgH clones at 66.29% and 30.41%. The patient received induction chemotherapy (VTCP, CAM, VP+MTX) combined with TKI therapy (Nilotinib) and achieved complete remission (CR) after the first round of treatment based on both flow cytometry and NGS MRD analyses. During the fourth round of chemotherapy (Methotrexate-Cytarabine-Dexamethasone), the patient suffered severe oral and intestinal mucositis, along with kidney and liver toxicity and required week-long dialysis. A decision was made to stop chemotherapy and start the first 4SCAR19 therapy of 3.53x106 CAR-Ts/kg, while maintaining the TKI therapy. The CAR-T cells in blood, the B cell count and Ig levels were monitored, and a booster CD22/CD19 dual CAR-Ts (4SCAR2219) was given at a dose of 3.35 x106 CAR-Ts/kg on day 96 (see figure). Consistent with 4SCART safety profile, the patient experienced no greater than grade 1 CRS. NGS-MRD detection of tumor cell clones remained negative on day 96 after the first CAR-T infusion and at 6 months into the remission. In summary, the 4SCAR2.0 therapy regimen appeared safe and effective in the innovative NGS-MRD/4SCAR2.0 B-ALL study regimen with in vivo persistency for more than three months. The simultaneous expansion of CD19 mono-specific and CD22/CD19 bi-specific CAR-Ts supports a stable maintenance treatment with good life quality as compared with conventional chemotherapy. CONCLUSIONS: The combination of 4SCAR2.0 with NGS-MRD detection of tumor clones in the management of high-risk B-ALL appears to be safe and may be a logical option for patients not elected for HCT therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Inferior Outcome in Non-Adult Young Adolescent B-Cell Acute Lymphoblastic Leukemia Exhibiting Obesity

Background: Previously, obesity has been associated with increased risk of relapse in aggressive lymphoproliferative disorders [Teegaparavu. ASH.2021]. The mechanism is largely unknown. However, attenuated chemosensitivity driven by unbalanced adiponectin/leptin regulation and obesity specific tumor microenvironment defects represent interesting "reversible” factors. Additionally, it is possible that B-cells aberrantly proliferate under obesity induced cytokine/chemokine influence. In pediatric patients (pt), obesity correlates with suboptimal Measurable Residual Disease (MRD) after induction. High insulin levels and PI3K/ATK/mTOR activation render anti-apoptotosis and chemorefractoriness [Bresin. Cell Death & Disease. 2016]. In this study, we investigate the effect of obesity on overall survival (OS) and event free survival (EFS) in a cohort of adult B-cell Acute Lymphoblastic Leukemia (B-cell ALL) older than 40 years with and without pre-treatment obesity. Methods: After IRB approval, B-cell ALL patients (pt) treated under standard induction protocols were selected at the Instituto de Cancerologia in Mexico and Baylor College of Medicine Institutions from 2010 to 2019. Obesity was diagnosed according to standard definitions based on body mass index (BMI) to include class I, II and III subgroups (BMI>30). Detectable modification in OS and EFS were estimated by using the Kaplan Meier Method. Descriptive statistics summarized results. To assess the independent effect of intervening variables on outcomes of interest, cox regression model was performed. SAS software was used for analysis. Results: 46/71(64.7%) and 31/71 (43.6%) pt exhibited or not obesity. Median age for obese and not obese pt was 52 years (y) vs 56 y, respectively. WBC, LDH and blast at disease onset were 43.2 K/UL v 20.1 K/UL, p=0.02; 1009 U/L v 600 U/L, p= 0.2; 62.8% v 60.6% for obese vs not obese pt, respectively. Incidence of high risk genomic did not statistically differ when accounting for t (9; 22), hypodyploid karyotype, P53 and 11q23 abnormalities. OS was 779 days (d) v 6429 d, in pt with and without obesity, p=0.03 [HR=0.43, CI 0.2-0.93] [Fig 1]. When considered relapse risk, EFS was 252 d v 936 d, in pt with and without obesity, respectively, p= 0.04 [HR=0.50, CI 0.22-0.99] [Fig 2]. Univariate analysis revealed that high BMI [p=0.001] and higher WBC [p=0.04] were predictor for OS and EFS. Cox regression model demonstrated that high WBC independently predicted inferior OS and EFS, p=0.002, each. Conclusions: Obesity is associated with inferior OS and EFS in adult non- AYA pt diagnosed with B-cell ALL. High BMI >30 is associated with enhance risk of relapse, which may explain, at least in part inferior OS in our pt cohort. Higher WBC at disease onset suggests that obesity is linked with blast proliferation efficiency. Deeper understanding of obesity induced metabolic abnormalities leading to B-cell ALL chemorefractoriness is needed. Weight reduction via caloric restriction and exercise may represent interesting interventions to improve clinical outcome of leukemic pt with high BMI. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Final Results of Phase I/II Study of Donor-Derived CAR T Cells Engineered with Sleeping Beauty in Pediatric and Adult Patients with B-Cell Acute Lymphoblastic Leukemia Relapsed Post-HSCT

Introduction Non-viral engineering of allogeneic T cells is a promising alternative option to produce a fit CAR T cell product. We recently proposed the use of Sleeping Beauty (SB) transposon to engineer donor-derived cytokine-induced killer (CIK) cells (CARCIK-CD19) for the treatment of B-cell acute lymphoblastic leukemia (B-ALL) patients relapsed after allogeneic hematopoietic stem cell transplantation (HSCT) (Magnani C et al.: JCI 2020). Here we report the final results of a phase I/II study evaluating the safety and efficacy of CARCIK-CD19 cells (FT01CARCIK, Clinical trial.gov, NCT03389035). Methods We conducted a multi-center, phase I/II dose-escalation (n=12) and dose expansion (n=9) trial in patients relapsed after HSCT. After approval by the Agenzia Italiana del Farmaco (AIFA), 6 additional patients were treated on a compassionate base use and were considered in the final analysis. CARCIK-CD19 cells were manufactured as described (Magnani C et al.: JCI 2020). CARCIK-CD19 were obtained by HLA-identical sibling (n=7, 26%), matched unrelated (n= 10, 37%), and haploidentical donors (n=10, 37%). After lymphodepletion with Fludarabine (30 mg/m2/day x 4 days) and Cyclophosphamide (500 mg/m2/day x 2 days), patients received a single CARCIK-CD19 infusion. Bridging therapy was allowed before CAR-T cell infusion. The Bayesian Optimal Interval (BOIN) design was used for the dose escalation part of the trial. The primary objectives were to define the Maximum Tolerated Dose (MTD) and the recommended Phase II dose as well as the safety. Secondary objectives included the rate of complete hematologic response (CR), duration of response (DOR), event-free (EFS), and overall survival (OS). DOR and EFS were censored when patient underwent a new anticancer therapy. Results A total of 27 patients (4 children and 23 adults) were infused between February 2018 and August 2021. A successful production of CARCIK-CD19 cells was achieved for all patients. The first 9 patients were treated with increasing numbers of CARCIK cells, in groups of three patients per dose level (1x106 /kg, 3x106/kg and 7.5x106/kg), whereas the remaining 18 patients (67%) received the highest planned dose level of 15x106 cells/Kg, as no dose limiting toxicity (DLT) was observed. The median number of prior therapies was 4 (range, 2-8) with a median time interval from HSCT to relapse of 8 months. We enrolled 25 patients (93%) with >5% bone marrow (BM) blasts with a median of 50% (range, 5-100%) at enrollment and 4% (range, 0-96%) after lymphodepletion. Two patients in the compassionate use group, presented extramedullary diseases without BM involvement. CRS occurred in 9 patients (4 patients with grade I and 5 with grade II) and immune effector cell-associated neurotoxicity-ICAN (grade 3) in 2 patients at the two highest doses. Although 10 out of 27 had experienced GvHD after the previous HSCT, GvHD never occurred after treatment with CARCIK-CD19. Nine patients had infections, of which 6 of grade 3 or higher. CR was achieved by 18 out of 27 patients (66.7%, 95%CI=46.0-83.5%) and by 16 out of 21 patients treated with the two highest doses (76.2%, 95%CI=52.8-91.8%). Fourteen (77.8%) of the overall responders and 13 of the responders at the highest doses (81.3%) were MRD-negative. With a median follow up of 2.8 years (range, 0.05-4.4 years, last update as of July 2022), a significantly better EFS was observed in patients treated with the highest doses (p=0.0265). For the 21 patients treated at the two highest dose levels, the median EFS and OS were 4 and 12 months, respectively (Figure 1). At 6 months, the EFS and OS were 41.5% (SE=11.6) and 71.4% (SE=9.9), respectively. The median DOR of the 16 patients in CR was 9.5 months, with a 6-months DOR of 54.4% (SE=13.8). By univariate analysis, EFS was significantly associated with the % of blasts after lymphodepletion (≥ 5% vs <5%) (p=0.0245). Conclusions In this phase I/II dose-finding study, SB-engineered CAR T cells demonstrated an excellent safety profile with anti-leukemia activity in heavily pretreated patients with B-ALL relapsed after HSCT. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

SC-gtCAR19, a Non-Viral Genome Targeting Anti-CD19 CAR-T Candidate in Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia: Updated Results from Long-Term Follow-up

Introduction It has been made great commercial success in hematological malignancies by chimeric antigen receptor (CAR) T cell therapy. However, high rate of adverse events (AEs) still remains an issue. Non-viral Genome Targeting CAR (gtCAR) delivery technology, which uses CRISPR-Cas9 to insert CAR DNA to TRAC locus specifically in human T cells, produces moderate CAR-T cells compared with conventional ones. Earlier results of SC-gtCAR19, an anti-CD19 gtCAR-T candidate from an investigator initiated Phase I clinical trial (ChiCTR2000031942) demonstrated a favorable benefit-risk profile in relapsed/refractory B-Cell Acute Lymphoblastic Leukemia (r/r B-ALL) patients. Reported here are updated long-term follow-up results. Objective The primary objective is to assess safety. Secondary objective is to evaluate efficacy. Level of CAR-T cell in blood was explored. Method This is a single center, open-label and single-arm trial. Patients or allogeneic donors underwent leukapheresis and their CD3+ T cells were selected and modified by non-viral vector to produce SC-gtCAR19. A standard lymphodepletion chemotherapy was performed. The median dose was 3.89×106CAR+ cells/kg (0.58-8.31×106/kg). AEs were graded according to CTCAE v5.0. Cytokine Release Syndrome (CRS) and neurological events (NEs) were graded by ASTCT consensus grading. Relapse defined as bone marrow (BM) blast or B-ALL related mutation gene positive from nonexistence regardless of specific value, and extramedullary disease (EMD) occurrence. Result As of May 1st 2022, twenty-two r/r B-ALL patients received SC-gtCAR19 (Table 1). The median age was 29 (13-61), and the median number of previous regimen was 5 (2-16). The median baseline BM blast after preconditioning was 44.1% (0.3-99%), particularly 73% of patents with high disease burden (>25% leukemic blast). Five patients (23%) once had stem cell transplantation (SCT), and 14 (64%) have B-ALL related chromosomal and molecular abnormalities. One patient has EMD. No patient has been treated by any other CD19 related therapy before enrollment. The most common AEs of any grade were anaemia (100%), pyrexia (91%), neutrophil count decreased (91%), and white blood cell count decreased (86%) (Table 2). CRS occurred in 20 patients (91%), and 4 of them (18%) with grade 3 CRS. The median time to CRS onset was 7 days (IQR 7-14), and the median duration of any grade CRS was 3 days (IQR 2-5). Three patents developed NEs, one was grade 2 and 2 were grade 3 (temporary seizure, related to infection and patient's cerebral infarction before enrollment). No fatal or life-threatening events happened. No Tocilizumab administered, four patients used vasopressors and 2 steroids for treatment of CRS. All patients were eligible for efficacy analysis. At Day 28, the overall response rate was 100%, with 9 (41%) patients achieving complete remission (CR), ten (45%) achieving complete remission with incomplete haematological recovery (CRi), and 3 (14%) with blast-free hypoplastic. Among them, twenty patients (91%) achieved minimum residual disease (MRD) negative CR, and those 2 patients with MRD positive CR were assessed undetectable BM MRD in Day 90. Patient with EMD, the nodal lesions in her breast disappeared at Day 60. At a median follow-up of 19.9 months (4.5-26.6), nineteen patients relapsed, within which 7 (36.8%) antigen escape relapse. The median duration of remission for 19 patients achieving CR/CRi was 8 months (2.5-NE). All patients received a consolidation treatment by Lenalidomide 3 months after infusion on condition of CR status until disease progression/death, and all of them did not receive SCT afterwards before relapse. As of the data cutoff, two patients (9%) had ongoing CR with median follow-up of 22.4 months. The median relapse-free survival for 19 patients was 8.9 months (2.9-NE) (Figure 1). The median OS was 24.2 months (3.7-NE) (Figure 2). CAR T cell level measured by cell copies percentage per peripheral blood mononuclear cells peaked at median 12 days (IQR 10-13) (Figure 3). The median peak percentage of copies was 53% (IQR 26.5-72.1%). Conclusion This study is the first clinical trial applied a gtCAR-T candidate for r/r B-ALL treatment. SC-gtCAR19 has a better risk-tolerance. Despite TCR gene knocked-out, gtCAR-T cell proliferation and its function both short and long term may not be influenced. We illustrate that this novel approach is feasible in terms of manufacture and clinical application. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Humanized Nanobody Anti-CD72 CAR-T Cells Efficiently Eliminate B-Cell Malignancies Via Improved Affinity for CD72 but Induce Persistent Antigen Downregulation In Vivo

Background: Approximately 50% of pediatric B-cell acute lymphoblastic leukemia (B-ALL) and non-Hodgkin lymphoma patients treated with CD19 chimeric antigen receptor (CAR)-T cells relapse. CD22 CAR-T cells are curative only rarely. New immunotherapeutic targets are urgently needed. Our laboratory recently identified CD72 as a target on B-cell malignancies and developed a fully synthetic nanobody against CD72 (NbD4), demonstrating that NbD4 CAR-T cells (nanoCARs) are potent in vitro and in vivo (Nix et al., Cancer Discovery (2021)). We previously described the development of a humanized version of this nanobody binder (H24) that had improved efficacy in preliminary studies (Nix et al., ASH 2021). As we move CD72 nanoCARs toward clinical translation, in this follow-up study we aimed to compare H24 vs. NbD4 by 1) tumor clearance kinetics; 2) T-cell immunophenotypes; 3) in vivo mechanism of relapse; and 4) binding affinity to CD72. Methods: CD4/CD8 CAR-T cells were generated by lentiviral transduction. Single time point (24 hr) in vitro cytotoxicity was assessed across a range of effector:tumor (E:T) ratios. CAR-T elimination kinetics was determined by IncuCyte live cell imaging. CD45RA and CD62L flow cytometry was used to assess T-cell immunophenotypes. In vivo studies were performed by implanting 5e5 luciferase-labeled Jeko cells into NSG mice on Day -4, and administration of 3e6 CAR-T cells on Day 0. At sacrifice, flow cytometry was performed to evaluate CD72 expression on relapsed tumors. Relapsed Jeko was re-implanted into naïve NSG mice for 31 days. Binding affinity of recombinant NbD4 or H24 to biotinylated CD72 extracellular domain was determined by bio-layer interferometry. Results: In single time point assays, H24 nanoCARs eliminated SEM B-ALL cells with similar efficacy to CD19 CAR-Ts (not significant) and were more potent than NbD4 nanoCARs at a 1:10 E:T ratio (72% vs 44% cytotoxicity; p = 3e-4). Against a Jeko mantle cell lymphoma model, at a 1:1 E:T ratio, H24 nanoCARs were more potent than both CD19 CAR-Ts (96% vs 87%; p < 1e-4) and NbD4 nanoCARs (96% vs 70%; p < 1e-4). In live cell imaging assays, H24 nanoCARs eliminated SEM faster than CD19 CAR-Ts (Fig. 1a). After SEM co-culture, H24 nanoCARs secreted more IL-2 and IFNγ compared to NbD4 (IL-2 p < 1e-4; IFNγ p = 4e-4) and CD19 CAR-Ts (IL-2 p = 4e-4; IFNγ p = 3e-4). Versus SEM, both pre- and post-tumor proportions of naïve T cells (CD45RA+/CD62L+) were similar for H24, NbD4, and CD19 CAR-Ts. Bulk RNA-seq after exposure to SEM revealed that H24 nanoCARs upregulate genes relevant to T cell activation and effector T cell function, compared to CD19 CAR-Ts. In in vivo Jeko studies, we treated mice with NbD4 or H24 nanoCARs and extracted tumors after relapse. We found that both nanoCARs led to CD72 loss on tumors (Fig. 1b). Surprisingly, however, upon re-implantation into a new cohort of NSG mice, H24 relapse Jeko continued to maintain low CD72 expression, while re-implanted NbD4 relapse Jeko had restoration of CD72 in the absence of anti-CD72 immune pressure (H24 vs NbD4 CD72 expression p = 4e-2). Given more potent anti-tumor efficacy and more persistent CD72 suppression in murine models, we hypothesized that H24 has a higher binding affinity for CD72. Indeed, H24 demonstrated a dissociation constant (KD) versus CD72 of 33.0 ± 0.04 nM, while NbD4 was 50.4 ± 0.05 nM. These results support the notion that relatively low-affinity binders can still generate potent CAR-Ts, when compared to published values of KD <1 nM for the anti-CD19 scFv FMC63. Conclusions: We have developed a new, humanized anti-CD72 cellular therapy known as H24 nanoCARs. Humanizing the framework regions, while keeping the complementary determining regions (CDRs) identical to NbD4, was intended to reduce immunogenicity. We were surprised that eight amino acid substitutions in the framework regions, with no changes to CDRs, improved antitumor efficacy and binding affinity for CD72. These improved characteristics motivate our planned clinical translation of H24-based nanoCARs. We also found that CD72 downregulation occurs after CD72 nanoCAR treatment in mice, and that increased potency of H24 correlates with longer-term CD72 suppression on tumors. These findings will inform expected outcomes in future clinical trials and motivate further studies to prevent CD72 downregulation after nanoCAR treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Efficacy of Chidamide Maintenance Therapy after CAR-T Therapy for Refractory or Relapsed B-Cell Acute Lymphoblastic Leukemia

Introduction Anti-CD19 chimeric antigen receptor (CAR) T-cells therapy has achieved unprecedented efficacy in refractory or relapsed (R/R) B-cell acute lymphoblastic leukemia (B-ALL) patients, with up to a 90% complete remission (CR) rate. However, a large body of clinical data has shown that the relapse rate post CD19 CAR-T therapy remains high. Our published study of 254 patients with R/R B-ALL treated with CD19 CAR-T showed the median time to relapse following CAR-T cells infusion was 100 days. Patients who received consolidative allogeneic hematopoietic stem cell transplantation (allo-HSCT) post CAR-T therapy demonstrated much higher overall survival (OS) and leukemia-free survival (LFS) compared to those who received CAR-T therapy alone (1-year OS of 77.8% vs. 35.4%, p<0.0001; 1-year LFS of 70.9% vs. 33.3%, p<0.0001, respectively). However, there are still many patients who cannot undergo allo-HSCT, including those who have relapsed following allo-HSCT and elderly patients. Prolonging disease-free survival (DFS) after CAR-T therapy in these patients remains a challenge for physicians. Studies have shown that the histone deacetylase (HDAC) inhibitor chidamide can up-regulate T-cell activity and down-regulate derived suppressor cell (MDSC) expression. Chidamide also promotes the expression of CD22 on the surface of B-cell tumor cells in vitro and in vivo, and enhances CAR-T function. Here we explore the efficacy of chidamide as maintenance therapy post CAR-T in B-ALL. Patients and methods We analyzed 19 patients with R/R B-ALL who received CD19 CAR-T at our center (NCT04546893) between October 2019 and July 2022. All patients achieved CR on day 30 following CD19 CAR-T cells infusion and were unable to bridge to allo-HSCT within 3 months. We administered chidamide as maintenance therapy to all 19 patients between 30 to 90 days after CAR-T infusion. Chidamide was continuously administered until the patient relapsed, transferred to transplant, or had intolerable side effects. Results On Day 30 post CD19 CAR-T, 19/19 (100%) patients achieved CR or CR with incomplete count recovery (CRi)(Figure 1). Sixteen of 18 (88.89%) who had bone marrow involvement achieved minimal residual disease (MRD)-negative CR. The median time from CAR-T cell infusion to start chidamide therapy was 30 days (range: 30-90 days) and the median duration of chidamide administration was 90 days (range: 12-420 days). No patient experienced cytokine release syndrome (CRS) or encephalopathy while taking chidamide. All side effects that occurred were mild. Two patients experienced nausea and vomiting and one patient had mild bone marrow suppression. No other side effects occurred. The median follow-up time was 12 months (range: 1-33 months), the 12-month OS rate was 83.1% and the 12-month DFS rate was 62.4% (Figure 2). Five patients received allo-HSCT following chidamide maintenance and 13 patients who did not undergo transplantation had 12-month OS of 83.3%, 12-month DFS of 62.9%. Six patients were monitored for the proportion of CAR-T cells in T lymphocytes following chidamide therapy. In all six patients, the proportion of CAR-T cells increased but to different degrees. Conclusions For those patients with R/R B-ALL for whom allo-HSCT is not available following CD19 CAR-T therapy, HDAC inhibitor chidamide as maintenance therapy is a viable option to reduce the risk of relapse rate following CD19 CAR-T therapy. This study demonstrated that chidamide could prolong OS and DFS in some patients who achieved CR after CD19 CAR-T therapy. Chidamide was safe, inexpensive and convenient to take orally. The inclusion of more patients and extended observation time is needed for further evaluation of chidamide maintenance therapy following CAR-T cell therapy in patients with R/R B-ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Activina: A Key Factor Mediating Chemoresistance in B-Cell Type Acute Lymphoblastic Leukemia

B-Cell Acute Lymphoblastic Leukemia (B-ALL) is the most common cancer in children. Multidrug high-dose chemotherapy has increased the 5-year event free survival rate to more than 80%. However, the prognosis of refractory/relapsed (r/r) ALL remains dismal. Increasing evidence indicates that treatment failure could be linked to the protective role of leukemic bone marrow (BM) microenvironment. The identification of the molecules involved in the crosstalk between leukemic cells and the BM niche would be crucial to establish niche-targeted therapies to be combined with conventional drugs. Recently, we identified ActivinA (ActA) as a new potentially targetable leukemia-promoting factor, which is significantly overexpressed in the BM plasma of B-ALL patients. We demonstrated that ActA is able to potentiate the migratory and invasive ability of B-ALL cells in vitro and enhance both BM engraftment and metastatic potential of leukemic cells in a xenograft mouse model. Literature data indicate that ActA exerts a pro-tumoral role in several solid tumors, by promoting cancer cell migration, proliferation and chemoresistance. The aim of this work was to ascertain whether ActivinA could mediate resistance to standard chemotherapy agents routinely used in pediatric B-ALL, such as Vincristine (VCR), Dexamethasone (Dex) and Asparaginase (ASNase). To investigate the role of ActA on B-ALL cell chemoresistance, we stimulated or not 697 cells with ActA for 24h. Cells were then treated with increasing concentrations of Dex, ASNase and VCR for 72h to obtain a dose-response curve. Cell viability was evaluated by flow cytometry and used to calculate the inhibitory dose 50 (IC50). Interestingly, the IC50 of ActA stimulated 697 cells for Dex and ASNase was in both cases about ten-fold higher than NS controls. In the case of VCR-treated cells, we did not detect any difference in IC50. These data suggest that ActA is able to convey resistance to Dex and ASNase-induced cell death, while sparing VCR-based leukemic cell killing, possibly due to different mechanisms of action. Validation experiments, with selected doses of Dex and ASNase (7 µg/mL and 1 U/mL, respectively), confirmed the ability of ActA to induce chemoresistance. In detail, the median viability of ActA stimulated 697 cells after 3 days of treatment with Dex was 64.15% (range=51.95-73.75%). This percentage was significantly higher compared to NS cells (median viability=41.95%, range=31.95-60.93%, Wilcoxon test: p<0.0001). Alongside, the viability of ActA stimulated cells (median=53.40%, range=38.70-75.00%) was also significantly increased (median=31.40%, range=22.45-54.25%, p=0.001) after ASNase treatment. To understand the molecular mechanisms underlying Acta-mediated chemoprotection, we firstly evaluated in our experimental setting the percentage of 697 cells expressing the active form of Caspase-3, by flow cytometry. Our results highlight that the activation of Caspase-3 was significantly reduced in ActA pre-stimulated cells treated with Dex (mean fold change (FC) ActA/NS=0.48±0.22, p=0.0003, One-sample t test) and ASNase (mean FC=0.49±0.062, p<0.0001), compared to NS cells. Then, we focused on the impact of ActA on anti- and pro-apoptotic factors playing a key role in drug-mediated apoptosis. By western blot, we demonstrated that Bcl-2 was significantly increased upon Dex treatment in ActA pre-stimulated cells (median FC ActA+Dex/NS untreated cells=0.63, range=0.49-1.12, Wilcoxon test: p<0.05) compared to NS cells (median FC NS+Dex/NS untreated cells=0.43, range=0.26-0.74). In contrast, we did not observe any difference with ASNase. Furthermore, we demonstrated that ActA was able to decrease the drug-induced cleavage activation of pro-apoptotic factors BAX and BAK. Finally, we investigated by confocal microscopy the levels of intracellular Reactive Oxygen Species (ROS), well-known key elements of the drug-induced oxidative stress, resulting in the activation of apoptosis. Interestingly, upon Dex treatment we observed a reduction of ROS levels of 59.7%±4.6% (mean±sd) in ActA-stimulated cells compared to NS. In the case of ASNase treatment the mean reduction was 46.9%±8.8%. Overall, our data suggest that ActA plays a pivotal role in B-ALL cell chemoprotection and that its targeting may be a promising strategy to be used in combination with standard chemotherapy in r/r ALL patients.

Minimal Residual Disease Negativity after Blinatumomab Is Predictive of Survival in B-Cell Acute Lymphoblastic Leukemia: Data from a Real-Life Study

Introduction The novel pediatric-like and TKI-based induction regimens induce up to 90 % of complete hematologic response in, respectively, Ph'-negative and Ph'-positive B-Cell Acute Lymphoblastic Leukemia (B-ALL). Minimal residual disease (MRD) strongly correlates with relapse and clearance of MRD is the goal of treatment. Blinatumomab is a bispecific T-cell engager that redirects T-Cells to kill CD19+ cells. Data from clinical trials show that the MRD-positive patients responding to Blinatumomab achieve a better survival than the non-responsive. This retrospective study from real-life demonstrates a superior outcome for MRD-responsive patients to blinatumomab over non-responsive patients. Patients and methods Data from adults treated with blinatumomab in six centers were analyzed to identify factors correlated to survival. Each cycle of blinatumomab was administered in 4 weeks at 15 mg/m2/day by continuous infusion followed by a 2-week interval. Intrathecal prophylaxis with ARA-C, Methotrexate and Dexamethasone was performed between cycles. Twenty-nine (87 %) patients received levetiracetam for seizure-prophylaxis. MRD was measured after each cycle by RT-qPCR with a sensitivity of 10-4 for clonal IgH and for BCR-ABL. As MRD response was considered clonal IgH or BCR-ABL < 10-4. Relapse-free survival (RFS) and overall survival (OS) were estimated by the Kaplan-Meier analysis and compared by the log-rank across the sub-groups using 95 % confidence intervals. The significant variables were compared in multivariate analysis by the log-logistic regression model. Toxicity was graded according to CTCAEs v.4 scale. Results From January 2016 to February 2021 34 B-ALL patients received Blinatumomab. The median age was 37 (18-72). Male to female ratio was 14/20. B-ALL was Ph'+ in 9 and Ph'- in 25 patients, respectively. Indications to blinatumomab were, respectively, relapsed/refractory B-ALL in 19 and MRD-positive in 15 patients. SNC disease was negative before blinatumomab in all patients. Blinatumomab was administered, respectively, as 2nd line in 26 and as > 2nd line of therapy in 8 patients. Overall, 24 patients received hematopoietic transplantation (HCT): 10 before and 14 after blinatumomab, respectively. The cycles of blinatumomab were 1, 2, 3 and 4, respectively, in 10, 13, 5, and 5 patients. The response to blinatumomab was, respectively, complete remission without MRD in 17 (50 %), complete remission with MRD in 8 (23 %), refractory in 4 (12 %) and extramedullary progression in 2 (6 %) patients. Three patients (9 %) had to suspend treatment in 1st cycle because of grade III-IV neuro-toxicity. The response was obtained after 1 and 2 cycles in 18 (72 %) and 7 patients (28 %), respectively. Among the MRD-responsive, MRD-negativity was obtained after 1 and 2 cycles in 16 (93 %) and 1 (7 %) patient, respectively. At a median follow-up of 16 months (1-47) 15 patients (44 %) had relapsed, 15 (44 %) were alive and in complete remission and 4 (12 %) died of transplant-related mortality. The median RFS was 12.5 months (1-39). The median OS was 15.5 months (1-46). The median RFS was 21 vs 4.4 months among MRD-responsive and non-responsive patients, respectively (p = .0004). The median OS was 27 vs 8.7 months among MRD-responsive and non-responsive patients, respectively (p = .008). Apart from MRD-response, the other variables significant for RFS were: blinatumomab as ≤ 2nd line (p = .0008), blinatumomab administered pre-HCT (p = .002) and MRD as an indication to treatment (p = .001). Apart from MRD-response, the other variable significant for OS was MRD as an indication (p = .01). By multivariate analysis only the MRD-response resulted significant for RFS (p = .0009) and OS (p = .02). MRD-response resulted significantly for RFS and OS also by censoring at HCT (p < .0001 and .005, respectively) [Figure 1]. Clinically significant toxicity was: grade II cytokine-release syndrome in 2 patients, grade III and IV neuro-toxicity in 2 and 1 patient, respectively. Overall, the percentage of patients experiencing grade III-IV neuro-toxicity, which occurred in 1st cycle, was 9 %. Conclusion This retrospective study in adult B-ALL Ph'- and Ph'+ patients, confirms the ability of blinatumomab to reach the MRD-negativity in a substantial proportion of patients. MRD-responsive gain a significant survival advantage over non-responsive patients. This advantage was observed both for transplanted and non-transplanted patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Integrating RNA-seq and scRNA-seq to explore the biological significance of NAD + metabolism-related genes in the initial diagnosis and relapse of childhood B-cell acute lymphoblastic leukemia.

Nicotinamide Adenine Dinucleotide (NAD) depletion is reported to be a potential treatment for B-cell Acute Lymphoblastic Leukemia (B-ALL), but the mechanism of NAD metabolism-related genes (NMRGs) in B-ALL relapse remains unclear. Transcriptome data (GSE3912), and single-cell sequencing data (GSE130116) of B-ALL patients were downloaded from Gene Expression Omnibus (GEO) database. NMRGs were sourced from Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome databases. Further, the differentially expressed NMRGs (DE-NMRGs) were selected from the analysis between initial diagnosis and relapse B-ALL samples, which further performed functional enrichment analyses. The biomarkers were obtained through random forest (RF) algorithm and repeated cross validation. Additionally, cell type identification by estimating relative subsets of RNA transcripts (CIBERSORT) algorithm was used to evaluate the immune cell differences between the initial diagnosis and relapse samples, and the correlations between biomarkers and gene markers of differential immune cells were analyzed. Furthermore, single cell RNA sequencing was conducted in the GSE130116 dataset to find key cell clusters. In addition, according to biomarkers expressions, cell clusters were categorized into high and low biomarker expression groups, and Gene Set Enrichment Analysis (GSEA) analysis was performed on them. Finally, the cell clusters with the highest expression of biomarkers were selected to explore the roles of biomarkers in different cell clusters and identify transcription factors (TFs) influencing biological markers. 23 DE-NMRGs were screened out, which were mainly enriched in nucleoside phosphate metabolic process, nucleotide metabolic process, and Nicotinate and nicotinamide metabolism. Moreover, 3 biomarkers (NADSYN1, SIRT3, and PARP6) were identified from the machine learning. CIBERSORT results demonstrated that four types of immune cells (B Cells naive, Monocyte, Neutrophils, and T cells CD4 memory Activated) were significantly different between the initial diagnosis and the relapse B-ALL samples, and there were strong correlations between biomarkers and differential immune cells such as positive correlation between NADSYN1 and B Cells naive. The single cell analyses showed that the biomarkers were highly expressed in common myeloid progenitors (CMP), granulocyte-macrophage progenitor (GMP), and megakaryocyte-erythroid progenitor (MEP) cell clusters. Gene set enrichment analysis (GSEA) results indicated that 55 GO terms and 3 KEGG pathways were enriched by the genes in high and low biomarker expression groups. It was found that TF CREB3L2(+) was significantly reduced in the high expression group, which may be the TF affecting biomarkers in the high expression group. This study identified NADSYN1, SIRT3, and PARP6 as the biomarkers of B-ALL, explored biological significance of NMRGs in the initial diagnosis and relapse of B-ALL, and revealed mechanism of biomarkers at the level of the single cell.

Blinatumomab for treating pediatric B-lineage acute lymphoblastic leukemia: A retrospective real-world study.

Blinatumomab was shown to be safe and effective for consolidation therapy in B-cell acute lymphoblastic leukemia (B-ALL). This study aimed to investigate the effectiveness and safety of blinatumomab in pediatric B-ALL patients in a real-world setting. This was a retrospective, observational study that included patients who initiated blinatumomab treatment between October 1, 2020 and June 20, 2022. Patients with B-ALL diagnosis, age below 18 years, and at least one blinatumomab treatment cycle were included. Treatment-related toxicities were assessed. Totally 23 pediatric patients were included in this study, with a median age of 6 years (range, 2 to 11 years). Blinatumomab therapy was applied for MRD-positive (disease ≥0.01%, n = 3) or chemotherapy-ineligible (n = 20) B-ALL cases. The median follow-up time was 9 months, and all evaluable patients achieved complete molecular remission with undetectable MRD. Four relapsed B-ALL cases proceeded to hematopoietic stem cell transplantation (HSCT) without further bridging therapy, while the others underwent maintenance chemotherapy after blinatumomab treatment. Grade ≥3 febrile neutropenia, white blood cell decrease and seizure were observed in 57%, 48% and 4.3% of patients, respectively. One case discontinued therapy due to neurologic toxicities. Elevated cytokine levels were observed in 4 patients. In all 23 patients, increased T-cell and low B-cell counts (<10/μl) were detected during blinatumomab therapy. These encouraging results suggest blinatumomab in pediatric B-ALL patients with MRD+ or chemotherapy-related toxicities is effective and safe in the short run, although long-term follow-up is still needed.

ALL-374 Mature B Cells, B-Cell Progenitors, and Plasma Cells in B-Cell Acute Lymphoblastic Leukemia Patients Enrolled in the RALL-2016 Study

The numbers of normal, mature B cells (MBC), B-cell progenitors (BCP), and plasma cells (PC) in B-cell acute lymphoblastic leukemia (B-ALL) patients provide the possibility of an antigen-associated immune response. The aim of the study was to assess the amount of MBC, BCP, and PC in B-ALL patients enrolled in the RALL-2016 study at different stages of the protocol. This study included data from 2016 to 2020 from patients enrolled in the Russian multicenter study RALL-2016 (NCT03462095). The amounts of MBC, BCP, and PC and minimal residual disease (MRD) status were assessed in the bone marrow (BM) by flow cytometry. We assessed the MRD status and the numbers of MBC, BCP, and PC in 88 patients with B-ALL at all points of the protocol and maintenance therapy. Analysis of MRD status was performed by 6- or 10-color flow cytometry. To analyze the dynamics of changes in the number of B cells depending on the MRD status, on the 70th day of therapy, regression dependences were done. Differences between groups of patients and donors (n=5) were estimated with ANOVA. During therapy, the numbers of MBC (P<0.0001) and BCP (P<0.0001) significantly changed and did not depend on the MRD status (P>0.05) on the 70th day of therapy (P<0.0001). The counts of MBC and BCP were less in B-ALL patients than in donors at all stages of therapy. There was no change in the number of normal PC. The obtained results indicate that the cytostatic treatment used in the RALL-2016 protocol had a minimal effect on differentiated B cells. In this way, a stable number of the most differentiated B cells can contribute to the maintenance of humoral immunity. BCP were more sensitive to cytostatic effects and were not recovered for more than a year after the end of treatment. The absence of changes in PC count may indicate the resistance of this cell population to ongoing chemotherapy.

Expression and Clinical Significance of Spi-B in B-cell Acute Lymphoblastic Leukemia.

Spi-B, a member of the E26 transformation-specific (ETS) family of transcription factors, plays an important role in B cell differentiation. Spi-B also functions in development of diffuse large B-cell lymphoma; thus, we hypothesized that it may participate in leukemogenesis of B-cell acute lymphoblastic leukemia (B-ALL). To test this hypothesis, we first generated an anti-Spi-B monoclonal antibody that recognized Spi-B on formalin-fixed, paraffin-embedded tissue sections. This antibody, designated S28-5, selectively stained B cell nuclei at the pre-plasma cell stage (including centrocytes and centroblasts in germinal centers) and nuclei of plasmacytoid dendritic cells, but not fully differentiated plasma cells, T cells, macrophages, or follicular dendritic cells. Employing S28-5, we then performed immunohistochemical staining of bone marrow aspiration biopsy specimens obtained from B-ALL patients (n=62). Cases that showed stronger nuclear S28-5 signals than T-cell ALL were scored positive. In 26 (42%) of 62 specimens, leukemic cells showed nuclear Spi-B expression, and positivity was associated with patient age at diagnosis, and serum uric acid and creatinine levels. Moreover, Spi-B-positive patients demonstrated significantly shorter overall survival than did Spi-B-negative patients. These results suggest that Spi-B expression may serve as a prognostic indicator of B-ALL.

A convergent malignant phenotype in B-cell acute lymphoblastic leukemia involving the splicing factor SRRM1.

A significant proportion of infant B-cell acute lymphoblastic leukemia (B-ALL) patients remains with a dismal prognosis due to yet undetermined mechanisms. We performed a comprehensive multicohort analysis of gene expression, gene fusions, and RNA splicing alterations to uncover molecular signatures potentially linked to the observed poor outcome. We identified 87 fusions with significant allele frequency across patients and shared functional impacts, suggesting common mechanisms across fusions. We further identified a gene expression signature that predicts high risk independently of the gene fusion background and includes the upregulation of the splicing factor SRRM1. Experiments in B-ALL cell lines provided further evidence for the role of SRRM1 on cell survival, proliferation, and invasion. Supplementary analysis revealed that SRRM1 potentially modulates splicing events associated with poor outcomes through protein-protein interactions with other splicing factors. Our findings reveal a potential convergent mechanism of aberrant RNA processing that sustains a malignant phenotype independently of the underlying gene fusion and that could potentially complement current clinical strategies in infant B-ALL.