B-ALL Patients Research Articles

Stability Assessment of Fresh or Cryopreserved Whole Blood and Bone Marrow Samples from AML and B-ALL Patients to Monitor CD123 Receptor and Immune Cells Subsets

Introduction: Compounds targeting CD123 are currently under clinical development for the treatment of acute myeloid leukemia (AML) and high-risk myelodysplastic syndrome (MDS). As part of clinical trials, CD123 expression and immune cell subsets need be monitored by quantitative flow cytometry (qFC) in bone marrow (BM) aspirates and whole blood (WB) samples before and on-treatment. One critical parameter for analysis is the poor stability of the fresh biological BM or WB samples. No general rule can predict the stability of the expression of a given receptor on diseased or healthy cells thus the pre-analytical stability of each biomarker should be evaluated to design the sample logistics. The pre analytical stability of clinical samples is the main constraint for quantitative flow cytometry (qFC) in multicenter and worldwide clinical studies. The transportation of the samples is one of the limiting factor. The classical freezing method of peripherical whole blood (WB) or bone marrow (BM) isolated by Ficoll requires high skilled operators, is time consuming and may be difficult to set up at sampling site in hospitals. One option to make the clinical samples suitable for subsequent high dimensional analysis by qFC is to use a simple freezing method at -80°C. Our cryopreservation method could be a new option for easier preparation of clinical samples prior to centralized samples analysis. This study aims to evaluate the pre-analytical fresh or cryopreserved stability of each pharmacodynamic monitoring endpoint over time: CD123 expression and receptor density, T-cell subsets and activation profile. Methods: FreshBM and WB were collected in EDTA tubes from 5 AML/MDS patients at hospitals, in accordance with local regulations. The samples were stored at room temperature (RT) for 4, 24, 48 or 72 h before immunostaining with the standard operation procedures and analysis by qFC or mixed with the cryopreserved media (BioCytex) within 24 h post-collection before freezing at -80°C for months. After storage, the cryopreserved samples were thawed and immunostained before analysis by qFC. Using a qFC assay (Multiplex FC panel assay developed by BioCytex, Marseille, France), CD123 receptor density/cell was collected. T-cell and blast cell immunophenotypes were also assessed on fresh samples for time point 4, 24, 48, or 72 h post-collection but also for cryopreserved WB sample within the 24hs post collection. The assay profiled blasts, monocytes, plasmacytoid dendritic cells, basophils and T-cells. Results: CD123 density in blast and normal cells was stable at RT from 4 h up to 48 h after collection. The profiles of blast subsets were very similar in BM and WB samples from a particular patient, whether a single blast cell subset (CD45lowCD11b-CD34+CD38med) or several blast cell subsets (expressing CD34 and CD38 at different levels) were identified. Both T-cell distribution and activation markers were stable up to 48 h for BM and WB. A similar pattern of T-cell subsets was also observed in the BM and WB paired samples from the same donor. In addition, CD123 receptor density, immunophenotyping and percentage of blast subsets and T cells from fresh WB samples was comparable to paired cryopreserved samples with a new method and for at least 1 month. Conclusions: Based on these observations, we conclude that CD123 density, blast cells and T-cell subsets in WB and BM samples from AML patients were stable for 48 h in our experimental conditions. As a consequence, fresh WB and BM samples can be processed and analyzed by qFC within 48 h of sample collection. Furthermore, the new cryopreservation method showed sample stabilization for blasts and T cells immunophenotyping. This data opens new perspectives in clinical studies for AML This new strategy would also help to standardize preparation of clinical samples and to centralize sample analysis.

Blinatumomab and Inotuzumab Ozogamicin Sequential Use for the Treatment of Relapsed/Refractory Acute Lymphoblastic Leukemia: A Real-Life Campus All Study.

Blinatumomab (Blina) and inotuzumab ozogamicin (InO) has improved the outcome of relapsed/refractory B-lymphoblastic leukemia (R/R B-ALL). However, little is known about the outcome after recurrence and re-treatment with immunotherapy. We describe 71 R/R B-ALL patients treated for different relapses with Blina and InO. Blina was the first treatment in 57 patients and InO in 14. Twenty-seven patients had a previous allogeneic hematopoietic stem cell transplantation (allo-HSCT). In the Blina/InO group, after Blina, 36 patients (63%) achieved a complete remission (CR), with 42% of negative minimal residual disease (MRD-); after InO, a CR was achieved in 47 patients (82%, 34 MRD-). In the InO/Blina group, after InO, 13 cases (93%) reached a CR (6 MRD-); after Blina, a CR was re-achieved in 6 cases (43%, 3 MRD-). Twenty-six patients proceeded to allo-HSCT. In the Blina/InO group, the median overall survival (OS) was 19 months; the disease-free survival (DFS) after Blina was 7.4 months (11.6 vs. 2.7 months in MRD- vs. MRD+, p = 0.03) and after InO, 5.4 months. In the InO/Blina group, the median OS was 9.4 months; the median DFS after InO was 5.1 months and 1.5 months after Blina (8.7 vs. 2.5 months in MRD- vs. MRD+, p = 0.02). With a median follow-up of 16.5 months from the start of immunotherapy, 24 patients (34%) are alive and 16 (22%) are alive in CR. In our series of R/R B-ALL, Blina and InO treatment demonstrate efficacy for subsequent relapses in terms of MRD response, OS and DFS, and as a bridge to allo-HSCT.

RNA binding protein IGF2BP1 synergizes with ETV6-RUNX1 to drive oncogenic signaling in B-cell Acute Lymphoblastic Leukemia

BackgroundAcute lymphoblastic leukemia (ALL) is the most common pediatric hematological malignancy, with ETV6::RUNX1 being the most prevalent translocation whose exact pathogenesis remains unclear. IGF2BP1 (Insulin-like Growth Factor 2 Binding Protein 1) is an oncofetal RNA binding protein seen to be specifically overexpressed in ETV6::RUNX1 positive B-ALL. In this study, we have studied the mechanistic role of IGF2BP1 in leukemogenesis and its synergism with the ETV6::RUNX1 fusion protein.MethodsGene expression was analyzed from patient bone marrow RNA using Real Time RT-qPCR. Knockout cell lines were created using CRISPR-Cas9 based lentiviral vectors. RNA-Seq and RNA Immunoprecipitation sequencing (RIP-Seq) after IGF2BP1 pulldown were performed using the Illumina platform. Mouse experiments were done by retroviral overexpression of donor HSCs followed by lethal irradiation of recipients using a bone marrow transplant model.ResultsWe observed specific overexpression of IGF2BP1 in ETV6::RUNX1 positive patients in an Indian cohort of pediatric ALL (n=167) with a positive correlation with prednisolone resistance.IGF2BP1 expression was essential for tumor cell survival in multiple ETV6::RUNX1 positive B-ALL cell lines. Integrated analysis of transcriptome sequencing after IGF2BP1 knockout and RIP-Seq after IGF2BP1 pulldown in Reh cell line revealed that IGF2BP1 targets encompass multiple pro-oncogenic signalling pathways including TNFα/NFκB and PI3K-Akt pathways. These pathways were also dysregulated in primary ETV6::RUNX1 positive B-ALL patient samples from our center as well as in public B-ALL patient datasets. IGF2BP1 showed binding and stabilization of the ETV6::RUNX1 fusion transcript itself. This positive feedback loop led to constitutive dysregulation of several oncogenic pathways.Enforced co-expression of ETV6::RUNX1 and IGF2BP1 in mouse bone marrow resulted in marrow hypercellularity which was characterized by multi-lineage progenitor expansion and strong Ki67 positivity. This pre-leukemic phenotype confirmed their synergism in-vivo. Clonal expansion of cells overexpressing both ETV6::RUNX1 and IGF2BP1 was clearly observed. These mice also developed splenomegaly indicating extramedullary hematopoiesis.ConclusionOur data suggest a combined impact of the ETV6::RUNX1 fusion protein and RNA binding protein, IGF2BP1 in activating multiple oncogenic pathways in B-ALL which makes IGF2BP1 and these pathways as attractive therapeutic targets and biomarkers.

AKT inhibition generates potent polyfunctional clinical grade AUTO1 CAR T-cells, enhancing function and survival

BackgroundAUTO1 is a fast off-rate CD19-targeting chimeric antigen receptor (CAR), which has been successfully tested in adult lymphoblastic leukemia. Tscm/Tcm-enriched CAR-T populations confer the best expansion and persistence, but Tscm/Tcm...

ALL-544 Exploring the Genetic Landscape of B-ALL: A Comparative Analysis of Copy Number Alterations in Adult and Pediatric B-ALL Patients

ALL-544 Exploring the Genetic Landscape of B-ALL: A Comparative Analysis of Copy Number Alterations in Adult and Pediatric B-ALL Patients

Blinatumomab as salvage therapy in patients with relapsed/refractory B-ALL who have failed/progressed after anti-CD19-CAR T therapy

Background Anti-CD19 chimeric antigen receptors (CARs) T-cell therapy has been shown to have excellent efficacy in patients with relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (ALL). But many patients are refractory to anti-CD19-CAR T-cell therapy or relapse again. Methods Five patients with R/R B-ALL did not respond to anti-CD19-CAR T-cell therapy or had a disease progression again after CAR-T cell therapy. They received a salvage therapy of Blinatumomab. The clinical response, CD19 expression on ALL cells, the proportion of CD3+ T cells, level of cytokine levels of interleukin-6 (IL-6), hematological toxicity, grade of cytokine release syndrome (CRS), and immune effector cell-associated neurotoxic syndrome (ICANS) were observed in salvage therapy of Blinatumomab. Results Four patients obtained CR/CRi, even in patients without high expression of CD19 in B-ALL cells, while the other patient received NR after Blinatumomab therapy. The CD19 expression on ALL cells, the proportion of CD3+ T cells, and CD3+CD8+ T cells were deficient in Pt 5, who obtained PR in Blinatumomab therapy. One patient (Pt 3) was diagnosed with grade 0 hematological toxicity. The other four patients were diagnosed with grades 2–3 of hematological toxicity. The CRS was grade 0/one patient, grade 1/three, and grade 2/one. The ICANS was grade 0/four patients, grade 1/one. Rhizopus microsporus pneumonia and cryptococcal encephalopathy in two patients were controlled during Blinatumomab therapy. Conclusions Blinatumomab could be an effective and safe salvage therapy in patients with R/R B-ALL who failed/progressed after anti-CD19-CAR T therapy, even in R/R B-ALL patients without high expression of CD19 in B-ALL cells, patients with CNS leukemia or co-infection. Key messages Some R/R B-ALL patients did not respond to anti-CD19 CAR T-cell therapy or had a disease progression again. Effective and safe salvage therapy for such patients remains to be explored. Blinatumomab could be an effective and safe salvage therapy in patients with R/R B-ALL who failed/progressed after anti-CD19-CAR T therapy, even in patients without high expression of CD19 in B-ALL cells. Blinatumomab could be an effective and safe salvage therapy in patients with R/R B-ALL who failed/progressed after anti-CD19-CAR T therapy, even in patients with CNS leukemia or co-infection.

Gain of chromosome 21 increases the propensity for P2RY8::CRLF2 acute lymphoblastic leukemia via increased HMGN1 expression.

Acute lymphoblastic leukemia (ALL) patients with a gain of chromosome 21, intrachromosomal amplification of chromosome 21 (iAMP21), or Down syndrome (DS), have increased expression of genes in the DS critical region (DSCR) of chromosome 21, including the high-mobility group nucleosome-binding protein 1, HMGN1. Children with DS are predisposed to develop hematologic malignancies, providing insight into the role of chromosome 21 in the development of leukemias. A 320-kb deletion in the pseudoautosomal region of the X/Y chromosome in leukemic cells, resulting in a gene fusion between the purinergic receptor and cytokine receptor-like factor-2 (P2Y Receptor Family Member 8 (P2RY8)::CRLF2), is a common feature in ~60% of DS-ALL and ~40% of iAMP21 patients, suggesting a link between chromosome 21 and P2RY8::CRLF2. In an Australian cohort of pediatric B-ALL patients with P2RY8::CRLF2 (n = 38), eight patients harbored gain of chromosome 21 (+21), and two patients had iAMP21, resulting in a significantly increased HMGN1 expression. An inducible CRISPR/Cas9 system was used to model P2RY8::CRLF2 and investigate its cooperation with HMGN1. This model was then used to validate HMGN1 as an influencing factor for P2RY8::CRLF2 development. Using Cas9 to cleave the DNA at the pseudoautosomal region without directed repair, cells expressing HMGN1 favored repair, resulting in P2RY8::CRLF2 generation, compared with cells without HMGN1. CRISPR/Cas9 P2RY8::CRLF2 cells expressing HMGN1 exhibit increased proliferation, thymic stromal lymphopoietin receptor (TSLPR) expression, and JAK/STAT signaling, consistent with cells from patients with P2RY8::CRLF2. Our patient expression data and unique CRISPR/Cas9 modeling, when taken together, suggest that HMGN1 increases the propensity for P2RY8::CRLF2 development. This has important implications for patients with DS, +21, or iAMP21.

Clinical Analysis of Pediatric T-Cell Acute Lymphoblastic Leukemia Using the MRD-Oriented Strategy System.

Pediatric T-cell acute lymphoblastic leukemia (T-ALL) has historically been associated with a poor prognosis. However, prognostic indicators and methods of treatment used for T-ALL remain controversial. A total of 136 children newly diagnosed with T-ALL between 2005 and 2018 were consecutively enrolled in this study. We assessed the effect of different prognostic factors, such as clinical characteristics, minimal residual disease (MRD), and the role of transplantation in postremission treatment, as the outcomes. Compared with B-ALL patients, patients with T-ALL are generally older, more likely to be male and have a higher white blood cell count. The complete remission (CR) rate was 95.6%, while the 5-year overall survival (OS), event-free survival (EFS), and cumulative incidence of relapse (CIR) were 74.3 ± 3.7%, 71.3 ± 3.9%, and 24.4 ± 3.8%, respectively. In the multivariate analysis, day 33 MRD ≥0.1% and hyperleukocytosis were associated with a significantly worse prognosis in the whole group. Transplantation resulted in a significant survival advantage, compared with chemotherapy, for high-risk (HR) patients (5-year CIR: 15.6 ± 10.2% vs. 55.6 ± 11.7%, P=.029). The prognosis of children with T-ALL was poor, and the MRD on day 33 was found to be an important predictive factor of clinical outcome at our center.

Integrated multi-omics analyses reveal homology-directed repair pathway as a unique dependency in near-haploid leukemia

Whole chromosome losses resulting in near-haploid karyotypes are found in a rare subgroup of treatment-refractory acute lymphoblastic leukemia. To systematically dissect the unique physiology and uncover susceptibilities that can be exploited in near-haploid leukemia, we leveraged single-cell RNA-Seq and computational inference of cell cycle stages to pinpoint key differences between near-haploid and diploid leukemia cells. Combining cell cycle stage-specific differential expression with gene essentiality scores from a genome-wide CRISPR-Cas9-mediated knockout screen, we identified the homologous recombination pathway component RAD51B as an essential gene in near-haploid leukemia. DNA damage analyses revealed significantly increased sensitivity of RAD51-mediated repair to RAD51B loss in the G2/M stage of near-haploid cells, suggesting a unique role of RAD51B in the homologous recombination pathway. Elevated G2/M and G1/S checkpoint signaling was part of a RAD51B signature expression program in response to chemotherapy in a xenograft model of human near-haploid B-ALL, and RAD51B and its associated programs were overexpressed in a large panel of near-haploid B-ALL patients. These data highlight a unique genetic dependency on DNA repair machinery in near-haploid leukemia and demarcate RAD51B as a promising candidate for targeted therapy in this treatment-resistant disease.

Safety and efficacy of obecabtagene autoleucel (obe-cel, AUTO1), a fast-off rate CD19 CAR, in relapsed/refractory adult B-cell acute lymphoblastic leukemia (r/r B-ALL): Top line results of the pivotal FELIX study.

7000 Background: Obe-cel is an autologous CD19CAR with a fast off-rate CD19 binding domain designed to reduce CAR-T immunotoxicity and improve persistence. Its clinical activity has been tested in r/r paediatric and adult B-ALL trials (CARPALL, Ghorashian S et al., Nat Med 2019; ALLCAR19, Roddie C et al., JCO 2021) and more recently in adults with r/r B-cell malignancies (NCT02935257). Here, we present data from adult r/r B-ALL patients treated with obe-cel in the pivotal FELIX study (NCT04404660). Methods: FELIX is an open-label, multi-centre, global, single-arm Phase Ib/II study enrolling r/r B-ALL patients with morphological disease (≥5% BM blasts), measurable residual disease (MRD) (≥0.1% to < 5% BM blasts), or isolated extramedullary disease (Cohorts A, B and C). CAR-T products were generated using an automated closed process from fresh leukapheresate. Patients underwent bridging therapy as necessary and lymphodepletion with fludarabine (4x30mg/m2) and cyclophosphamide (2x500mg/m2). Patients received a target dose of 410E+6 CAR T cells as a split dose on Day 1 and Day 10. The dosing schedule is based on the % BM blasts performed locally prior to the pre-conditioning. Primary endpoint was overall remission rate (ORR) defined as proportion of patients achieving CR/CRi by central assessment. Results: As of 9th September 2022, a pre-specified interim analysis was conducted based on the first 50 patients infused in Cohort A who have been followed for 3 months or discontinued before month 3. The median age was 50 years (range 20-81), 22% had Ph+ B-ALL. The median number of prior lines of treatment was 2 (range 1-5), 42% underwent prior transplant. At screening, patients had a median of 55% BM blasts (range 6-96%) and 26% had EMD. The geometric mean of peak CAR expansion was 126147.6 copies/ug genomic DNA. Persistence was ongoing in majority of responders at last follow-up. Based on central assessment, the CR/CRi was 70% [95% CI: 55%, 82%] (p-value < 0.0001). As of 9th September 2022, a total of 92 patients received obe-cel and were evaluable for safety. 63% developed any grade CRS (3% Grade ≥3) at a median of 9 days post-infusion and a median duration of 5 days. Any grade ICANS was observed in 23% (8% Grade ≥3) at a median of 15 days post-infusion and of median duration 8 days. Other common Grade≥3 adverse events regardless of causality were febrile neutropenia (25%) and anaemia (20%). Conclusions: The pre-specified interim analysis of the FELIX study demonstrated that obe-cel for adult r/r B-ALL is safe with low rates of Grade >3 CRS and/or ICANS, even in patients with high burden disease. Obe-cel is effective with high CR/CRi rates and ongoing CAR T persistence in the majority of responders. The trial has completed dosing of all patients in Cohort A. Additional data and longer follow up will be reported at the conference. Clinical trial information: NCT04404660 .

A Comprehensive Analysis of the Expression Profiles of KCTD Proteins in Acute Lymphoblastic Leukemia: Evidence of Selective Expression of KCTD1 in T-ALL.

Acute leukemia is the most common pediatric cancer. In most cases, this disease results from the malignant transformation of either the B-cell (B-ALL) or, less frequently, T-cell progenitors (T-ALL). Recently, a marked overexpression of KCTD15, a member of the emerging class of the potassium (K) channel tetramerization domain-containing proteins (KCTDs) has been detected in both patients and continuous cell lines as in vitro model systems. Because there is growing evidence of the key, yet diversified, roles played by KCTDs in cancers, we here report an exhaustive analysis of their expression profiles in both B-ALL and T-ALL patients. Although for most KCTDs, no significant alterations were found in these pathological states, for some members of the family, significant up- and down-regulations were detected in comparison with the values found in healthy subjects in the transcriptome analysis. Among these, particularly relevant is the upregulation of the closely related KCTD1 and KCTD15 in T-ALL patients. Interestingly, KCTD1 is barely expressed in both unaffected controls and B-ALL patients. Therefore, not only does this analysis represent the first study in which the dysregulation of all KCTDs is simultaneously evaluated in specific pathological contexts, but it also provides a promising T-ALL biomarker that could be suitable for clinical applications.

PAX5 fusion genes in acute lymphoblastic leukemia: A literature review.

Acute lymphoblastic leukemia (ALL) is a common cancer affecting children worldwide. The development of ALL is driven by several genes, some of which can be targeted for treatment by inhibiting gene fusions. PAX5 is frequently mutated in ALL and is involved in chromosomal rearrangements and translocations. Mutations in PAX5 interact with other genes, such as ETV6 and FOXP1, which influence B-cell development. PAX5/ETV6 has been observed in both B-ALL patients and a mouse model. The interaction between PAX5 and FOXP1 negatively suppresses the Pax5 gene in B-ALL patients. Additionally, ELN and PML genes have been found to fuse with PAX5, leading to adverse effects on B-cell differentiation. ELN-PAX5 interaction results in the decreased expression of LEF1, MB1, and BLNK, while PML-PAX5 is critical in the early stages of leukemia. PAX5 fusion genes prevent the transcription of the PAX5 gene, making it an essential target gene for the study of leukemia progression and the diagnosis of B-ALL.

Tracing back of relapse clones by Ig/TCR gene rearrangements reveals complex patterns of recurrence in pediatric acute lymphoblastic leukemia.

Relapse remained the major obstacle to improving the prognosis of children with acute lymphoblastic leukemia (ALL). This study aimed to investigate the changing patterns of Ig/TCR gene rearrangements between diagnosis and relapse and the clinical relevance and to explore the mechanism of leukemic relapse. Clonal Ig/TCR gene rearrangements were screened by multiplex PCR amplification in 85 paired diagnostic and relapse bone marrow (BM) samples from children with ALL. The new rearrangements presented at relapse were quantitatively assessed by the RQ-PCR approach targeting the patient-specific junctional region sequence in 19 diagnostic samples. The relapse clones were further back-traced to diagnostic and follow-up BM samples from 12 patients. Comparison of Ig/TCR gene rearrangements between diagnosis and relapse showed that 40 (57.1%) B-ALL and 5 (33.3%) T-ALL patients exhibited a change from diagnosis to relapse, and 25 (35.7%) B-ALL patients acquired new rearrangements at relapse. The new relapse rearrangements were present in 15 of the 19 (78.9%) diagnostic samples as shown by RQ-PCR, with a median level of 5.26 × 10-2 . The levels of minor rearrangements correlated with B immunophenotype, WBC counts, age at diagnosis, and recurrence time. Furthermore, back-tracing rearrangements in 12 patients identified three patterns of relapse clone dynamics, which suggested the recurrence mechanisms not only through clonal selection of pre-existing subclones but also through an ongoing clonal evolution during remission and relapse. Backtracking Ig/TCR gene rearrangements in relapse clones of pediatric ALL revealed complex patterns of clonal selection and evolution for leukemic relapse.

Minimal residual disease in acute leukemia based on the insight of molecular genetics monitoring

Patients with acute leukemia port 10 malignant cells at presentation. Following chemotherapy or stem cell transplant, patients in complete remission by conventional analyses may still harbor 106/108 malignant cells below the detection limit of standard clinical assessment. Minimal residual disease (MRD) monitoring is one of the most powerful predictors of disease-free and overall survival, particularly for children with acute lymphoblastic leukemia (cALL), the percent annual of cALL increase in the incidence of cALL in Saudi Arabia. Breakpoint fusion regions of chromosomal aberrations can be used as tumor-specific targets for MRD detection by polymerase chain reaction. Levels of MRD, measured at critical time points, significantly correlate with clinical outcomes. Previous works investigated the prognostic significance of leukemia-associated immunophenotypes (LAIPs) as an assessment of the index of MRD in 125 adult B-ALL patients by eight-colour flow cytometry. More advanced molecular and genetics studies are so necessary to identify the mechanisms and cellular structure of the minimal-level disease. Selecting molecular methods for minimal residual disease detection have a much higher sensitivity and precision (100-fold or more) than others. This review highlights the minimal residual disease molecular detection to demonstrate the characterization of the lymphoblastic leukemia gene. Precise MRD monitoring predicts disease relapse after chemotherapy or SCT, provides early intervention, and may result in the rescue of many patients and improvement in the probability of long-term disease-free survival.

Cytogenetic Profiling In Paediatric Acute Leukaemia; A Report On 746 Newly Diagnosed Paediatric Cases Analyzing The Spectrum Of Recurring Chromosomal Rearrangements In B Cell Lymphoblastic And Acute Myeloid Leukaemia.

Cytogenetics is evolving and different molecular mechanisms we know now have proved to be of diagnostic and prognostic significance in both acute lymphoid (ALL) and myeloid leukaemia (AML). This study aims to find out and compare the occurrence of different cytogenetics in paediatric acute leukaemia. This is a cross-sectional study of diagnosed B-ALL and AML patients presenting at The Indus Hospital. We studied FISH and karyotype in BALL and FISH in AML patients. FISH analysis shows a total of 69 (12.8%) of B ALL patients had cytogenetic abnormalities. BCR-ABL1 was positive in 5.1%, ETV6/RUNX1T1 in 8.6% and KMT2A in 2.3% individuals. Karyotype reveals hyper diploidy in 24.3%, Monosomy in 1.94%, and t (1:19) and t (17:19) were observed in 5.8% and 0.24% cases respectively. FISH analysis in AML cases reveal positivity of t (8:21) in 26.4%, INV (16) in 6.1% while PML-RARA t(15:17) was done on morphological suspicion in 17 cases; all of which showed positivity; making 7.9% of the total AMLs. The study demonstrated a wide spectrum of heterogeneity in paediatric acute leukaemia. Hyperdiploidy was the most common cytogenetic abnormality. We report a lower incidence of t (12:21), compared to the world. We showed a higher prevalence of RUNX1/RUNX1T1 in young children. The prevalence of core binding factor AML was 32.5%.

Multiparameter flow cytometry and ClonoSEQ correlation to evaluate precursor B-lymphoblastic leukemia measurable residual disease.

Measurable residual disease (MRD) detection for precursor B-lymphoblastic leukemia (B-ALL) has become the standard of care. However, the testing methodology has not been standardized. We aim to correlate COG multiparameter flow cytometry (MFC) and ClonoSEQ techniques to assess the test characteristics, to study abnormal immunophenotype for B-ALL MRD, and to observe B-ALL clonal evolution and the impact of blinatumomab therapy on MFC testing. MFC and molecular reports were retrieved from electronic medical records and data was reviewed. Included in this study were 74 bone marrow samples collected from 31 B-ALL patients at our institution between January 2021 and March 2022. COG MFC and ClonoSEQ results were concordant in 59/74 samples (80%) with positive concordant results in 12 samples (16%) and negative concordant results in 47 samples (64%). Discordant results were seen in 15/74 samples (20%), with 14 samples (19%) showing ClonoSEQ + /MFC- results and only 1 sample (1%) showing MFC + /ClonoSEQ- result. ClonoSEQ + /MFC- cases had MRD values ranging from 1 to 1400 cells/million nucleated cells with 86% of cases showing MRD values of < 100 cells/million nucleated cells. Newly identified dominant sequences were detected using ClonoSEQ in 2/31 patients (6%) during follow-up. All 14 bone marrow samples from 8 patients, who had gone through blinatumomab immunotherapy, were MRD negative by MFC, but 3 cases were MRD positive by ClonoSEQ. Our results show strong correlation between COG MFC and ClonoSEQ (r = 0.96), and both methods are complementary. Clonal evolution may occur, and blinatumomab immunotherapy may impact MFC B-ALL MRD evaluation.

Impact of TP53 gene variants on prognosis and survival of childhood acute lymphoblastic leukemia

The tumor suppressor protein 53 (TP53) gene is one of the most studied genes in cancer. Although TP53 variants are rare events in acute leukemia, recent observations showed that relapse samples might harbor TP53 variants. Here, we aimed to determine TP53 variants (hotspot region, exon 4–11) in childhood acute lymphoblastic leukemia (B and T-ALL) patients (n = 94) including diagnostic-relapse pairs (n = 15) by amplicon sequencing and evaluate the clinical impact of these variants. In total, nine different (E298*, R283C, R273H, L252F, C229F, I195T, E286G, c.955_956insC, and c.920-1G > C) variants were identified in 17 (18%) childhood ALL patients. c.(920-1G> C) splice site variant and c.(955_956insC) insertion were reported for the first time. In diagnose-relapse pair samples, we identified acquired and/or loss of TP53 variants in the samples at the time of relapse. TP53 variants were found to be more common in T-ALL (37%) than in B-ALL patients (9%). Pathogenic TP53 variants were associated with a shorter overall survival time (p = 0.001).TP53 variants were found to be associated with inferior outcomes in our cohort and can be an independent risk factor for poor prognosis in childhood acute leukemia patients. Identification of low-frequent variants with next-generation sequencing approaches enables better knowledge of the clonal dynamics of ALL.

Abstract 5497: Venetoclax and dinaciclib elicit synergisticpreclinicalefficacy against high-risk B-cell leukemia

Abstract Hypodiploid acute lymphoblastic leukemia (ALL) is an aggressive blood cancer with a poor prognosis despite intensive chemotherapy or stem cell transplant. Children and adolescents with positive end-of-induction minimal residual disease (MRD) have an overall survival lower than 30%. However, data regarding therapeutic alternatives for this disease is nearly non-existent, emphasizing the critical need for new or adjunctive therapies that can improve outcomes. We previously reported on the therapeutic efficacy of venetoclax (ABT-199) in hypodiploid B-ALL but with limitations as monotherapy (Diaz-Flores, Cancer Research, 2019). Accordingly, hypodiploid B-ALL patient derived xenografts treated with Venetoclax developed therapeutic resistance over time. To better understand the mechanism of resistance that arises following inhibition of BCL-2, we first generated a hypodiploid cell line (NALM-16) deficient in BCL-2. Given that BCL-2 plays an essential role in B-cell function standard CRISPR/Cas9 knockout strategies greatly impaired survival. We overcame this limitation by using a magnetic levitation system (the LeviCell by LevitasBio). Analysis of the surviving clones identified an increase in MCL1 (Fig. 1A). We then harnessed unbiased drug screen (&amp;gt;1800 bioactive compounds) followed by selection of synergistic drug combinations identified Dinaciclib as an efficient drug combination with Venetoclax in eliminating leukemic cells in vitro. Interestingly, mechanistic studies identified that Dinaciclib, through transcription regulation (RNApol II) significantly modulates MCL-1. Data using knock-outs and knock-ins of MCL-1 indicated how MCL-1 only becomes essential for survival when Venetoclax is present. Preclinical studies using patient derived xenografts indicated that, while none of the drug monotherapies achieved complete blast elimination, venetoclax + dinaciblib led to complete resolution of the leukemic burden within all solid organs analyzed (Fig. 1B). This combination therapy was relatively well tolerated. This study also indicates that high BCL-2 and MCL-1 levels with concomitant BIM, BAD and/or PUMA levels, represent biomarkers for response to this combination. In summary, our study identified a highly synergistic drug combination, venetoclax and dinaciclib, for the treatment of hypodiploid B ALL, an aggressive leukemia with few effective current therapies. Finally, the promising results presented in this study may prompt further studies to support the inclusion of hypodiploid and other B-ALL patients to clinical trials combining phase I/II drugs against BCL-2 (mainly venetoclax) and CDK9 (dinaciclib, alvocidib, flavopiridol, SNS-032) or MCL-1 (MIK-665). Citation Format: Ernesto Diaz-Flores, Holly Pariury, Mignon Loh. Venetoclax and dinaciclib elicit synergisticpreclinicalefficacy against high-risk B-cell leukemia. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5497.

Abstract 5655: preclinical development of a novel anti-tslpr bispecific antibody 1b7/cd3 targeting crlf2-rearranged ph-like b-all

Abstract Acute lymphocytic leukemia (ALL) is the most common form of leukemia in childhood and adolescence. Outcomes of relapsed/refractory adult B-ALL remain dismal with long-term survival of less than 10%. Although targeted therapies such as blinatumomab, inotuzumab ozogamicin, and CD19 CAR T cell therapy are a significant treatment advance for patients with B-ALL, there is still an unmet medical need for novel therapies for B-ALL. CRLF2-rearranged B-ALL, a subtype of Philadelphia chromosome-like B-ALL, is a high-risk disease subset with poor long-term outcomes. In an effort to identify new therapeutic targets for B-ALL, we validated by flow cytometry that patients with CRLF2-rearranged Ph-like ALL overexpress thymic stromal lymphopoietin receptor (TSLPR) at levels comparable with CD19. We subsequently generated antibodies against TSLPR from immunized H2L2 Harbor mice through single B cell cloning technology. The lead antibody was further engineered to a CD3-redirecting bispecific antibody (BsAb) using Xencor’s CD3e bispecific antibody technology to create 1B7/CD3 BsAb with an Fc-silent region. 1B7/CD3 BsAb exhibits high affinity binding to both human and cynomolgus monkey CD3e and TSLPR by BLI. Additionally, UHPLC-SEC analysis of 1B7/CD3 shows that this BsAb is stable without any aggregation or degradation present after five weeks incubation at 37 °C. In cell-based analyses,1B7/CD3 BsAb demonstrate potent antigen-specific T cell activation and tumor lysis activity against TSLPR+-REH and MHH-CALL4 cell lines as well as a primary B-ALL patient sample, but none against human peripheral blood mononuclear cells (PBMC) collected from healthy donors. Animal tumor models suggest that 1B7/CD3 triggers a dose-dependent tumor regression or growth inhibition in PBMC humanized TSLPR-REH CDX and BOS-1 PDX models across donors. Correspondingly, T cell activation and expansion were examined by CD69 and CD3+ T cells with the best effect observed at the 1mg/kg dose. 1B7/CD3 exhibits durable PK with T1/2 in NSG mice of up to ten days. Finally, 1B7/CD3 BsAb demonstrated a tolerable safety margin in a two-dose exploratory toxicity study in cynomolgus monkeys with MTD equal to or less than 1mg/kg. Consistent with clinical observation, we observed a transient increase of six major cytokines (IFNγ, IL-6, IL-8, IL-10, MCP-1 and TNFα) 4hr post the initial dosing, but no notable induction of cytokine levels after the second dosing (except for IL-8). Thus, our preclinical data provide the framework for the clinical evaluation of 1B7/CD3 BsAb in patients with CRLF2-rearranged B-ALL. Citation Format: Ze Tian, chunhua shi, Amin AI-Shami, GuoJun Yong, Jason K Allen, Jill Wardell Olson, Melinda G Smith, Qing Chang, Qing Shi, Junping You, Michelle A Gonzalez, Timothy E Lofton, Jasbir Kaur, Qi Zhang, DongXing Zha, Nitin Jain, Marina Y Konopleva, Timothy Heffernan, Jeffrey J Molldrem. preclinical development of a novel anti-tslpr bispecific antibody 1b7/cd3 targeting crlf2-rearranged ph-like b-all. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5655.

Abstract 6719: Osteopontin-c is worse prognostic marker and mediate key steps associated with central nervous system infiltration in B-cell acute lymphoblastic leukemia cells

Abstract B-cell acute lymphoblastic leukemia (B-ALL) is the most common pediatric malignance, but 35% of patients still relapse after chemotherapy treatment, mainly in the central nervous system (CNS). In order to search for new biomarkers that could predict CNS infiltration and relapse, many studies are trying to find gene products aberrantly expressed in these patients, including osteopontin (OPN). The OPN primary transcript undergoes alternative splicing, generating at least five OPN splicing isoforms (OPN-SI), named OPNa, OPNb, OPNc, OPN4 and OPN5, that have tumor specific roles and have been reported as biomarkers in solid tumors. However, knowledge regarding OPN-SI in B-ALL is scarce. This study aimed to investigate the expression patterns and roles of OPN-SI in CNS infiltration in B-ALL. OPN-SI transcript levels were analyzed by quantitative real time PCR (qRT-PCR) assays using RNA extracted from 54 B-ALL pediatric patient blood samples or from RS4,11 cell line. OPNc transcript knock-down in RS4;11 cells (a B-ALL cell line containing the MLL gene fusion) was performed using anti-OPNc or control scrambled antisense DNA oligomers (anti-OPNc-ASOs and Scbl-ASO, respectively). These oligomers were transfected in RS4;11 cells and the in vitro effects of OPNc knock-down were evaluated by several functional assays, such as cell viability by MTT analysis, cell adhesion, and transmigration and invasion assays using a boyden chamber device. Among tested OPN-SI, OPNc is the most highly expressed OPN-SI in B-ALL patient samples containing the MLL fusion gene, when compared to those samples harboring the ETV6-RUNX1 fusion gene (p&amp;lt;0,005). High OPNc expression levels were significantly associated with poor prognostic features, such as high-risk classification (p&amp;lt;0,005), CNS infiltration and high white blood cell counting (p&amp;lt;0,05). The RS4;11 cells in which OPNc was knocked-down decreased their OPNc expression levels in 85% in relation to control Scbl-ASO transfected cells (p&amp;lt;0,005). These cells decreased their viability in 41% (p&amp;lt;0,05), besides displaying a 55% decrease in RS4;11 cell adhesion to matrigel. In addition, these cells decreased their capacity to transmigrate (54%) and invade the matrigel (51%) in comparison to control cells. In summary, these results suggest that OPNc transcript levels are associated with B-ALL worse prognostic features and that it may mediate some key steps involved on CNS infiltration mechanistic. Hence, our data provide important basis for the potential use of OPNc as a biomarker for B-ALL CNS relapse and for the involvement of this isoform on some steps that can mediate CNS invasion, opening new opportunities to improve B-ALL pediatric patients’ survival rates and quality of life. Citation Format: Ana Clara Ana Clara Santos da Fonseca Bastos, Luciana Bueno Ferreira, Mariana Emerenciano, Etel Rodrigues Pereira Gimba. Osteopontin-c is worse prognostic marker and mediate key steps associated with central nervous system infiltration in B-cell acute lymphoblastic leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6719.