B-acute Lymphoblastic Leukemia Research Articles

Aetio-pathological profile of anaemic children belonging to upper socioeconomic society in a tertiary care hospital of North India

Introduction: Anaemia is a major public health problem especially in the preschool age group. It is rampant even in children of upper and middle classes of the society.Objectives: To study the aetio-pathology of anaemia in children belonging to upper and upper middle socio-economic scale and to correlate it with their nutritional status. Method: It was a cross-sectional descriptive study on 165 preschool children aged 6-59 months who presented with pallor on clinical examination. They were advised to undergo a complete haemogram and further specific tests as required. Results: The most common cause of anaemia was nutritional, including iron deficiency (92%), combined iron and B12 deficiency (2%) and isolated B12 deficiency (0.6%). Thalassaemia was the next common cause (3%) followed by B-acute lymphoblastic leukaemia (1%) and microangiopathic haemolytic anaemia (0.6%). Maternal education and nutritional awareness had positive correlation with anaemia. Majority of children were normally nourished despite being anaemic, However, undernourished children were more severely anaemic. Conclusions: Nutritional anaemia is the commonest cause of anaemia even in children belonging to higher socio-economic classes.

Lineage Switching from Acute Myeloid Leukemia associated with Systemic Mastocytosis to B-Acute Lymphoblastic Leukemia: A Diagnostic Dilemma

Relapse acute leukemia is defined as the reappearance of more than 5% blast in the bone marrow. In most instances, blast expresses the specific lineage (myeloid or lymphoid), similar to a diagnosis. However, it rarely converts to a different lineage, either myeloid shifting to lymphoid or vice versa, during relapse. Thus, it is labelled as switching lineage acute leukemia, after excluding the criteria for mixed phenotypic acute leukemia, which can be a challenge to diagnose. We describe a 24-year-old gentleman with a known case of acute myeloid leukemia associated with systemic mastocytosis (SM); eight months later and after two complete cycles of chemotherapy, relapse occurred with a switched lineage to B-acute lymphoblastic leukemia. The laboratory investigation approaches and the challenges in the diagnosis are also discussed in this case report

Double Philadelphia chromosome: a rare and sole abnormality in pediatric B-acute lymphoblastic leukemia.

The present study describes a 7-year-old male child who had attended the Pediatric Oncology Clinic of the Regional Cancer Centre, Thiruvananthapuram, Kerala, India, and was pathologically confirmed to have B-Acute Lymphoblastic Leukemia (B-ALL). Conventional cytogenetics analysis at diagnosis showed the presence of a double Philadelphia chromosome and the karyotype of the case was 47, XY, t(9;22)(q34;q11.2), + der(22)t(9;22). FISH, done as a molecular confirmation of the translocation, t(9;22)(q34;q11.2), and this case showed an additional fusion signal that confirms the presence of double Ph. As far as we are aware, this represents the initial and only occurrence of an abnormality report regarding the double Philadelphia chromosome in pediatric B-ALL within India. The double Philadelphia chromosome in B-ALL has a very poor prognosis despite aggressive treatment with chemotherapy. This study reveals the importance of conventional and molecular cytogenetic analysis in risk stratification and prognosis prediction of pediatric B-ALL. The risk stratification based on the conventional and molecular cytogenetic analysis may be taken into consideration for deciding the treatment strategy for each patient.

Evaluating the Prognostic Utility of CAR-Hematotox Score in Predicting CAR T-Cell Toxicities in Children and Young Adults with R/R B- Acute Lymphoblastic Leukemia (B-ALL)

Evaluating the Prognostic Utility of CAR-Hematotox Score in Predicting CAR T-Cell Toxicities in Children and Young Adults with R/R B- Acute Lymphoblastic Leukemia (B-ALL)

Correlation of flow cytometric measurable residual disease (FC-MRD) with molecular MRD and clinical outcome at one year in adult B-acute lymphoblastic leukaemia (B-ALL) and acute myeloid leukaemia (AML)

Correlation of flow cytometric measurable residual disease (FC-MRD) with molecular MRD and clinical outcome at one year in adult B-acute lymphoblastic leukaemia (B-ALL) and acute myeloid leukaemia (AML)

Risk Factors for Hematotoxicity Following Tisagenlecleucel for Pediatric B-Acute Lymphoblastic Leukemia

Risk Factors for Hematotoxicity Following Tisagenlecleucel for Pediatric B-Acute Lymphoblastic Leukemia

Deciphering stage 0 hematogones by flow cytometry in follow-up bone marrow samples of pediatric B-Acute lymphoblastic leukemia cases: A potential mimicker of residual disease after anti CD19 therapy.

CD19 is frequently targeted for immunotherapy in B cell malignancies, which may result in loss of CD19 expression in leukemic cells as an escape mechanism. Stage 0 hematogones (Hgs) are normal CD19-negative very early B cell precursors that can be potentially mistaken for CD19 negative residual leukemic cells by flow cytometry (FCM) in B cell acute lymphoblastic leukemia (BCP-ALL) cases treated with anti CD19 therapy. Our main objective was to characterize and study the incidence of stage 0 hematogones in follow-up bone marrow samples of pediatric BCP-ALL cases. We analyzed the flow cytometry standard files of 61 pediatric BCP-ALL cases treated with conventional chemotherapy and targeted anti-CD19 therapy, for identifying the residual disease and normal B cell precursors including stage 0 Hgs. A non-CD19 alternate gating strategy was used to isolate the B cells for detecting the residual disease and stage 0 Hgs. The stage 0 Hgs were seen in 95% of marrow samples containing CD19+ Hgs. When compared with controls and posttransplant marrow samples, the fraction of stage 0 Hgs was higher in patients receiving anti CD19 therapy (p = 0.0048), but it was not significant when compared with patients receiving chemotherapy (p = 0.1788). Isolated stage 0 Hgs are found in samples treated with anti-CD19 therapy simulating CD19 negative residual illness. Our findings aid in understanding the stage 0 Hgs and its association with CD19+ Hgs in anti CD19 therapy and conventional chemotherapy. This is crucial as it can be potentially mistaken for residual disease in patients treated with anti CD19 therapy.

Transforming CLL management with immunotherapy: Investigating the potential of CAR T-cells and bispecific antibodies

Immunotherapies, such as chimeric antigen receptor (CAR) T-cell therapy and bispecific antibodies or T-cell engagers, have revolutionized the treatment landscape for various B-cell malignancies, including B-acute lymphoblastic leukemia and many non-Hodgkin lymphomas. Despite their significant impact on these malignancies, their application in chronic lymphocytic leukemia (CLL) management is still largely under investigation. Although the initial success of CD19-directed CAR T-cell therapy was observed in 3 multiply relapsed CLL patients, with 2 of them surviving over 10 years without relapse, recent CAR T-cell therapy trials in CLL have shown reduced response rates compared to their efficacy in other B-cell malignancies. One of the challenges with using immunotherapy in CLL is the compromised T-cell fitness from persistent CLL-related antigenic stimulation, and an immunosuppressive tumor microenvironment (TME). These challenges underscore a critical gap in therapeutic options for CLL patients intolerant or resistant to current therapies, emphasizing the imperative role of effective immunotherapy. Encouragingly, innovative strategies are emerging to overcome these challenges. These include integrating synergistic agents like ibrutinib to enhance CAR T-cell function and persistence and engineering newer CAR T-cell constructs targeting diverse antigens or employing dual-targeting approaches. Bispecific antibodies are an exciting "off-the-shelf" prospect for these patients, with their investigation in CLL currently entering the realm of clinical trials. Additionally, the development of allogeneic CAR T-cells and natural killer (NK) cells from healthy donors presents a promising solution to address the diminished T-cell fitness observed in CLL patients. This comprehensive review delves into the latest insights regarding the role of immunotherapy in CLL, the complex landscape of resistance mechanisms, and a spectrum of innovative approaches to surmount therapeutic challenges.

Subclassification of B-acute lymphoblastic leukemia according to age, immunophenotype and microenvironment, predicts MRD risk in Mexican children from vulnerable regions.

The decisive key to disease-free survival in B-cell precursor acute lymphoblastic leukemia in children, is the combination of diagnostic timeliness and treatment efficacy, guided by accurate patient risk stratification. Implementation of standardized and high-precision diagnostic/prognostic systems is particularly important in the most marginalized geographic areas in Mexico, where high numbers of the pediatric population resides and the highest relapse and early death rates due to acute leukemias are recorded even in those cases diagnosed as standard risk. By using a multidimensional and integrated analysis of the immunophenotype of leukemic cells, the immunological context and the tumor microenvironment, this study aim to capture the snapshot of acute leukemia at disease debut of a cohort of Mexican children from vulnerable regions in Puebla, Oaxaca and Tlaxcala and its potential use in risk stratification. Our findings highlight the existence of a distinct profile of ProB-ALL in children older than 10 years, which is associated with a six-fold increase in the risk of developing measurable residual disease (MRD). Along with the absence of CD34+ seminal cells for normal hematopoiesis, this ProB-ALL subtype exhibited several characteristics related to poor prognosis, including the high expression level of myeloid lineage markers such as MPO and CD33, as well as upregulation of CD19, CD34, CD24, CD20 and nuTdT. In contrast, it showed a trend towards decreased expression of CD9, CD81, CD123, CD13, CD15 and CD21. Of note, the mesenchymal stromal cell compartment constituting their leukemic niche in the bone marrow, displayed characteristics of potential suppressive microenvironment, such as the expression of Gal9 and IDO1, and the absence of the chemokine CXCL11. Accordingly, adaptive immunity components were poorly represented. Taken together, our results suggest, for the first time, that a biologically distinct subtype of ProB-ALL emerges in vulnerable adolescents, with a high risk of developing MRD. Rigorous research on potential enhancing factors, environmental or lifestyle, is crucial for its detection and prevention. The use of the reported profile for early risk stratification is suggested.

CAR-T Cell Therapy in B-Cell Acute Lymphoblastic Leukemia.

Treatment of refractory and relapsed (R/R) B acute lymphoblastic leukemia (B-ALL) is an unmet medical need in both children and adults. Studies carried out in the last two decades have shown that autologous T cells engineered to express a chimeric antigen receptor (CAR-T) represent an effective technique for treating these patients. Antigens expressed on B-cells, such as CD19, CD20, and CD22, represent targets suitable for treating patients with R/R B-ALL. CD19 CAR-T cells induce a high rate (80-90%) of complete remissions in both pediatric and adult R/R B-ALL patients. However, despite this impressive rate of responses, about half of responding patients relapse within 1-2 years after CAR-T cell therapy. Allo-HSCT after CAR-T cell therapy might consolidate the therapeutic efficacy of CAR-T and increase long-term outcomes; however, not all the studies that have adopted allo-HSCT as a consolidative treatment strategy have shown a benefit deriving from transplantation. For B-ALL patients who relapse early after allo-HSCT or those with insufficient T-cell numbers for an autologous approach, using T cells from the original stem cell donor offers the opportunity for the successful generation of CAR-T cells and for an effective therapeutic approach. Finally, recent studies have introduced allogeneic CAR-T cells generated from healthy donors or unmatched, which are opportunely manipulated with gene editing to reduce the risk of immunological incompatibility, with promising therapeutic effects.

Hypoxia-initiated Cysteine-rich protein 61 secretion promotes chemoresistance of acute B lymphoblastic leukemia cells.

The drug resistance is a major obstacle in acute B-lymphoblastic leukemia (B-ALL) treatment. Our previous study has indicated that increased levels of Cysteine-rich protein 61 (Cyr61) in the bone marrow can mitigate the chemosensitivity of B-ALL cells, though the specific source of Cyr61 in the bone marrow remains unknown. In this study, we aimed to investigate whether hypoxia can induce Cyr61 production in B-ALL cells, delineates the underlying mechanisms, and evaluates the effect of Cyr61 on the chemosensitivity of B-ALL cells under hypoxia conditions. The results indicate that hypoxia promotes Cyr61 production in B-ALL cells by activating the NF-κB pathway. Increased Cyr61 expression appears to reduce the chemosensitivity of B-ALL cell to vincristine (VCR) and daunorubicin (DNR) through autophagy under hypoxia. Notably, inhibition of Cyr61 restores the chemosensitivity of B-ALL cells to both chemotherapeutic agents. This study is the first time to report that hypoxia decreases the chemosensitivity of B-ALL cells by inducing Cyr61 production, suggesting that targeting Cyr61 or its associated pathways could potentially improve the clinical response of B-ALL patients.

DNA Copy Number Alterations and Copy Neutral Loss of Heterozygosity in Adult Ph-Negative Acute B-Lymphoblastic Leukemia: Focus on the Genes Involved.

The landscape of chromosomal aberrations in the tumor cells of the patients with B-ALL is diverse and can influence the outcome of the disease. Molecular karyotyping at the onset of the disease using chromosomal microarray (CMA) is advisable to identify additional molecular factors associated with the prognosis of the disease. Molecular karyotyping data for 36 patients with Ph-negative B-ALL who received therapy according to the ALL-2016 protocol are presented. We analyzed copy number alterations and their prognostic significance for CDKN2A/B, DMRTA, DOCK8, TP53, SMARCA2, PAX5, XPA, FOXE1, HEMGN, USP45, RUNX1, NF1, IGF2BP1, ERG, TMPRSS2, CRLF2, FGFR3, FLNB, IKZF1, RUNX2, ARID1B, CIP2A, PIK3CA, ATM, RB1, BIRC3, MYC, IKZF3, ETV6, ZNF384, PTPRJ, CCL20, PAX3, MTCH2, TCF3, IKZF2, BTG1, BTG2, RAG1, RAG2, ELK3, SH2B3, EP300, MAP2K2, EBI3, MEF2D, MEF2C, CEBPA, and TBLXR1 genes, choosing t(4;11) and t(7;14) as reference events. Of the 36 patients, only 5 (13.8%) had a normal molecular karyotype, and 31 (86.2%) were found to have various molecular karyotype abnormalities-104 deletions, 90 duplications or amplifications, 29 cases of cnLOH and 7 biallelic/homozygous deletions. We found that 11q22-23 duplication involving the BIRC3, ATM and MLL genes was the most adverse prognostic event in the study cohort.

Impact of inotuzumab ozogamicin on outcome in relapsed or refractory acute B-cell lymphoblastic leukemia patients prior to allogeneic hematopoietic stem cell transplantation and risk of sinusoidal obstruction syndrome/venous occlusive disease.

We evaluated 58 patients with relapsed or refractory (r/r) acute B-lymphoblastic leukemia (B-ALL; median age 42.5 years; range, 16-69 years), treated with inotuzumab ozogamicin (INO) between 2016-2022 and who received an allogeneic hematopoietic stem cell transplantation (allo-HCT) consecutively. Forty-seven (81%) of the 58 patients were heavily pretreated receiving intensive chemotherapy +/- tyrosine kinase inhibitor, blinatumomab in 24 (41%) and allo-HCT at first-line in 11 (19%) patients. Complete remission rate prior to allo-HCT was 84%. Median follow-up was 30.5 months and median overall survival (OS) measured from start of INO was 11.2 months. One- and 2-year OS rates were 50% (95% confidence interval [CI]: 38.4-56.1) and 36.7% (95% CI: 25.5-52.9), respectively. Sinusoidal obstruction syndrome/venous occlusive disease (SOS/ VOD) after allo-HCT occurred in 17 (29%) patients. Of those, nine (53%) patients died due to SOS/VOD and multi-organ failure. Two had received >2 INO cycles (3 cycles, 5 cycles, N=1, each), all others ≤2 INO cycles prior to allo-HCT. Logistic regression analysis revealed conditioning with double alkylators (P=0.038) and allo-HCT during first-line therapy (P=0.050) as significant risk factors for SOS/VOD and in trend allo-HCT ≤60 days from last INO application (P=0.07), whereas number of INO cycles before allo-HCT and time between last INO application and allo-HCT were not significant. Relapse/progressive disease occurred in 20 (34%) patients. Of those, five (25%) patients are still alive, whereas 15 succumbed of their disease. Treatment with INO seems to be an effective approach with successful bridge-to-transplant. However, risk of SOS/VOD is high, necessitating continuous monitoring and recognition of SOS/VOD risk factors.

Overexpression of Nrf2 in Mesenchymal Stem Cells Promotes Migration and Invasion of B- Acute Lymphoblastic Leukemia Cells

Background and objective:B -Acute lymphoblastic leukemia (B-ALL) is one of the most difficult cancers to treat in adults, especially recurrence and drug resistance are still the biggest challenges for B-ALL. In recent years, a new concept has been proposed, that is, the interaction between bone marrow microenvironment and leukemic cells can reduce the sensitivity of leukemic cells to chemotherapy. The components of bone marrow microenvironment (BMM) have been shown to mediate the occurrence and development of leukemia. There is growing evidence that leukemic cells teach the bone marrow microenvironment, change its function and establish leukemia protective interactions with stromal cells and immune cells. Bone marrow mesenchymal stem cells (Bone marrow mesenchymal stem cells, BM-MSCs) are stromal cells in leukemia microenvironment, which can promote the progression of leukemia under certain conditions. Nuclear factor erythroid related factor 2 (Nrf2) is an important transcription factor and a regulator of cellular antioxidant response, which regulates the expression of genes encoding antioxidant enzymes to prevent various oxidative changes. It has been confirmed that the high expression of Nrf2 in leukemic cells is related to drug resistance and poor prognosis of leukemia. However, the interaction mechanism of Nrf2 overexpression in MSCs on B-cell acute lymphoblastic leukemia (B-ALL) cells is not clear. We will for the first time propose the mechanism of Nrf2 overexpression in MSCs on the migration and invasion of B-ALL leukemic cells. Methods :Bone marrow nucleated cells were detected in 16 patients with complete remission (B-ALL/CR), 12 patients with relapsed/refractory (B-ALL/R/R) ALL and 10 normal controls. The expression of Nrf2 was detected byreal-time PCR and Western Blot. In vitro cell experiments, real-time PCR and Western Blot were used to detect the expression of Nrf2 in mesenchymal stem cells (MSCs) alone and in the co-culture of MSCs and cell lines (Nalm-6/RS4:11) express the situation Mouse model experiment in vivo, transwell chamber experiment in vitro, three-dimensional (3D) cell culture model, lentivirus transfection, WB and PCR were used to explore the possible mechanism of Nrf2 overexpression in mesenchymal stem cells on the migration and invasion of B-ALL cells. Results: In clinical samples, the expression of Nrf2 in B-ALL-R/R group was significantly higher than that in ALL-CR group and normal control group, suggesting that the expression of Nrf2 was closely related to the recurrence and drug resistance of ALL. In vitro cell experiment, the expression level of Nrf2 in MSCs co-cultured with cell line (NALM-6/RS4:11) was higher than that in MSC cultured alone(P < 0.05). By regulating the overexpression of Nrf2 in MSCs, it can significantly promote the growth, proliferation, migration and invasion of leukemia cells, and promote the expression of CXCR4 in tumor tissues. We further regulated the expression of Nrf2 in MSCs. The results showed that it significantly promoted the migration and invasion of NALM-6/RS4:11 cells. We concluded that the overexpression of Nrf2 in MSCs promotes the migration and invasion of leukemic cells by activating the activity of SDF-1-CXCR4 axis. In vivo experiments, we used tail vein injection of MSCs and B-ALL leukemia cells to explore the protective effect of Nrf2 overexpression in mesenchymal stem cells on leukemia cells and promote extramedullary infiltration of leukemia. The results showed that the infiltration degree of extramedullary organs of mice after combined infusion of two kinds of cells was significantly higher than that of leukemia cells alone, and the survival time of mice was significantly shortened after combined infusion. After statistical treatment, the difference was statistically significant (P < 0.05). Conclusion:The expression of Nrf2 in MSCs may be a key component in mediating the interaction between MSCs and ALL cells. This result may provide a new entry point for deepening all disease progression mechanisms and finding more promising targeted therapies.On the other hand, activation of PI3K/AKTERK1/2 signaling pathway in B-ALL cells further promoted the evolution of B-ALL. This may provide new ideas for findingpotential therapeutic targets for B-ALL extramedullary infi ltration, and help to further solve the current problem of B-ALL relapse and refractory.

Risk Factors and Outcomes for Hematotoxicity Following Tisagenlecleucel for Pediatric B-Acute Lymphoblastic Leukemia

Background: Hematotoxicity following chimeric antigen receptor (CAR) T-cell therapy is increasingly recognized as a distinct adverse event. The CAR-HEMATOTOX score has been developed as a prognostic tool for subsequent development of prolonged cytopenias in adults with lymphoma. However, risk factors in children and young adults (CAYA) with B-acute lymphoblastic leukemia (B-ALL) receiving CAR T cell therapy are less well-defined. We sought to describe incidence, outcomes, and risk factors for prolonged severe neutropenia in CAYA treated with tisagenlecleucel. Methods: This was a multi-institutional retrospective study involving CAYA ≤26 years of age with B-ALL treated with tisagenlecleucel who underwent leukapheresis and manufacturing between August 2017 and March 2020, with data collected through the Pediatric Real World CAR Consortium (PRWCC). Of 185 patients infused, 41 patients were excluded from analysis due to missing data (n=10), refractory disease/death from leukemia (n=27) or non-relapse mortality (n=4) prior to day 35 post-CAR T. Severe prolonged neutropenia was defined as absolute neutrophil count (ANC) <500 cells/mm 3 for ≥30 days. For those with severe neutropenia pre-CAR T, day 0 of severe neutropenia was considered the day of CAR T-cell infusion. Statistical analyses included comparisons using Mann-Whitney and Fisher's exact tests, receiver operating characteristic (ROC) curves to determine cut points with optimal sensitivity and specificity, univariate logistic regression, Kaplan-Meier curves for recovery from neutropenia, overall survival (OS) and relapse free survival (RFS). Results: Of 144 patients, 23 (15.9%) met the definition of severe prolonged neutropenia and 121 (84%) did not. For lymphodepleting chemotherapy, 139 (96.5%) received standard fludarabine (30mg/m 2/dose x 4) and cyclophosphamide (500mg/m 2 x 2). Pre- and post-infusion characteristics are shown in Table 1. Pre-infusion, higher pre-infusion C-reactive protein (CRP), ferritin, bone marrow disease burden, and lower ANC and platelet counts were associated with severe prolonged neutropenia. Post-infusion, any grade cytokine release syndrome (CRS), severe CRS, CRS with hemophagocytic lymphohistiocytosis (HLH)-like manifestations, any grade neurotoxicity, peak CRP and ferritin, use of tocilizumab and steroids, and presence of infections within 30 days were associated with severe prolonged neutropenia. With univariate logistic regression, pre-infusion inflammatory markers, cytopenias, and bone marrow disease burden associated with severe prolonged neutropenia and of these, disease burden >15% had the highest odds ratio (Figure 1). Post-infusion infections and inflammatory toxicities, particularly CRS with HLH-like toxicities, as well as peak CRP, and peak ferritin, were associated with severe prolonged neutropenia. Peak ferritin >3800 ng/ml had the highest odds ratio. In patients with severe prolonged neutropenia, OS was inferior (p<0.0001) and RFS trended towards being inferior (p=0.052), compared to those without. Groups with or without severe prolonged neutropenia were also subdivided into >15% or ≤15% pre-infusion bone marrow disease burden subgroups. OS was similar between patients without severe prolonged neutropenia that had >15% or ≤15% pre-infusion disease burden, but patients with severe prolonged neutropenia and >15% disease burden had inferior OS, suggesting prolonged severe neutropenia may have independent prognostic value. Conversely, RFS was inferior in patients in the subgroups with >15% disease burden compared to those with ≤15%, regardless of occurrence of severe prolonged neutropenia. Discussion/Conclusions: We describe incidence, risk factors and outcomes for the development of severe prolonged neutropenia in CAYA with B-ALL following tisagenlecleucel. While there are similarities in risk factors with the CAR-HEMATOTOX score that has been validated in adults, in CAYA with B-ALL high disease burden pre-infusion and severe inflammation post-infusion were identified as the strongest risk factors for prolonged neutropenia. Identification of risk factors unique to the CAYA B-ALL population allows for tailored risk mitigation in higher risk patients and may help improve outcomes in CAYA at increased risk of prolonged severe neutropenia following tisagenlecleucel.

Clinical Implications of Germline Predisposition Gene Variants in Patients with Refractory or Relapsed B Acute Lymphoblastic Leukemia

Objectives:Gene variants are important factors in prognosis of the patients with hematological malignancies. In current study, our team investigate the relationship between blood and immune system germline predisposition variants and refractory/relapsed acute B lymphoblastic leukemia in patients. Methods:The study included 75 patients with refractory/recurrent acute B-lymphoblastic leukemia between January 2020 and December 2022. The first group of 41 patients, median age 13.5 years (range: 2-66 years), had not had a bone marrow transplant. The second group of 34 patients with a median age of 28.5 (range: 4-51 years old, once had a bone marrow transplant. More than 700 genetic susceptibility genes related to blood and immune system diseases were detected in all patient samples using high-throughput DNA sequencing technology to detect the type and number of Class I and Class II gene variants. The average sequencing depth was 150×. Class I refers to the gene variants that are clearly significant in guidelines or authoritative research literature. Class II are genetic variants that software analysis suggests may be harmful. Results:In the first group, 41 patients had an average number of Class I genetic susceptibility gene variants of 3.8 and Class II genetic susceptibility gene variants of 14.5 per patient. A total of 94 Class I genetic susceptibility gene variants were found, Thirty-two of them were recurrent more than 3 times that including HIF1A、SERPINE1、CFTR、CHEK2、ELANE、IFIH1、IRF7、JAK3、NCF2、PRF1、TGFBR2、TNFRSF13B、ATM、CYP3A4、ITGA2、NUDT15、THBD、BTLA、DNASE1、MPEG1、ADAMTS13、EP300、FANCA、KIT、BRCA2、F7、MLH1、STK11、TP53、YARS2、CYP2C19、TNFAIP3. The functions of these genes involve in combined immunodeficiency, autoinflammatory disease, complement deficiency, immune deficiency, T-cell dysfunction and antibody deficiency. After remission by CART cell therapy, twenty-three of the 41 patients underwent haploidentical hematopoietic stem cell transplantation, among which 5 patients relapsed after transplantation, three of the five donors of relapsed patients were also found HIF1A and SERPINE1 gene variants. In the second group of 34 patients, the average number of Class I genetic susceptibility gene variants in each patient was 3, and the average number of class 2 genetic susceptibility gene variants was 14. A total of 53 class 1 genetic susceptibility gene variants were found, Eleven of them were recurrent more than 3 times that Including BTLA、MPEG1 、KIT、SERPINE1、STK11、TP53、UGT1A1、EP300、FANCA、YARS2、F7,and four donors also carried the above gene variants, which were EP300. MPEG1, SERPINE1, YARS2 . After 34 patients achieved remission by CART cell therapy, 9 patients underwent the second hematopoietic stem cell transplantation (related half-matched), and 3 of them relapsed after the second transplantation (average 2.3 months). Two of the 3 donors of relapsed patients after second hematopoietic stem cell transplantation were also found BTLA and MPEG1 gene variants. Conclusions:Our results have shown that genetic susceptibility gene variants related to blood and immune system diseases are associated with refractory or relapsed acute B lymphoblastic leukemia. The number of genetic susceptibility gene variants may be related to the relapse of leukemia. 32 Class I genetic susceptibility gene variants may be related to the relapse of leukemia during chemotherapy, and 11 class I genetic susceptibility gene variants may be associated with relapse after bone marrow transplantation. Donor carrying genetic susceptibility gene variants (HIF1A and SERPINE1, EP300, MPEG1, YARS2, BTLA) may be associated with relapse after transplantation. Genetic susceptibility gene variants may also be helpful in the selection of bone marrow transplant donors. Keywords Acute lymphoblastic leukemia, germline predisposition gene, hematopoietic stem cell transplant, , relapse, hematological malignancy

Graft-Versus-Host Disease after Anti-CD19 Chimeric Antigen Receptor T-Cell Infusion Post Allogeneic Hematopoietic Cell Transplantation: A Transplant Complications and Paediatric Disease Working Parties EBMT Joint Study

Background Anti-CD19 chimeric antigen receptor T-cells (CART) have been incorporated into the therapeutic landscape of B-acute lymphoblastic leukemia (B-ALL) and B-non-Hodgkin's lymphoma (B-NHL). The manufacturing process of commercially available autologous CART in patients relapsing after allogeneic hematopoietic cell transplant (allo-HCT) might include T-cells of donor origin. In this setting, there is limited data on graft-versus-host disease (GvHD) as an off-target effect in patients treated with CART after allo-HCT. We hereby report on a large, retrospective, EBMT registry-based study on GvHD in patients treated with CART therapy after allo-HCT. Methods Inclusion criteria were B-ALL and B-NHL adult and pediatric allo-HCT patients, treated with a first anti-CD19 CART (axicabtagene ciloleucel [axi-cel] and tisagenlecleucel [tisa-cel]) from 2018 to August 2022. The primary study endpoints were the cumulative incidences (CI) of new acute GvHD (aGvHD) and chronic GvHD (cGvHD). Secondary endpoints were the 1-year GvHD relapse-free survival (GRFS), non-relapse mortality (NRM), and overall survival (OS). Overall data was analyzed in a descriptive manner. Results A total of 257 allo-HCT patients treated with anti-CD19 CART were included. One hundred seventy-two patients (66.9%) were ≥18 years old. Tisa-cel was the therapy of choice in 184 patients (71.6%), whereas axi-cel was used in 73 patients (28.4%). More than half of the cohort (57.6%) underwent allo-HCT from unrelated donors. Notably, 109 patients (46.6%) and 38 patients (15%) had previously develop aGvHD and cGvHD between allo-HCT and CART infusion. Table 1 describes data on baseline patient, allo-HCT and CART characteristics of the whole cohort and of patients developing aGvHD and cGvHD. In total, 3 patients developed new aGvHD and 6 patients developed new cGvHD after CART therapy. The 100-day CI of new aGvHD was 1.6% (95% CI, 0.4-4.2) and the 12-month CI of new cGvHD was 2.8% (95% CI, 1.1-5.7). No GvHD was observed in the pediatric cohort. The median time from allo-HCT to CART infusion was 15.8 months (range, 3.8-220.3) and the median times from CART to the development of aGvHD and cGvHD were respectively 44 days (range, 8-81) and 144 days (range, 5-182). The 1-year GRFS and NRM were 52.1% (95% CI 45.6-59.4) and 4.7% (95% CI 2.5-8.1) respectively (Figure 1). With a median follow up of 18.8 months (95% CI, 16.2-23.7), the 1-year OS was 76.8% (95% CI 71.5-82.4). The most frequent cause of death was disease-related in 56 patients (74.6%). Conclusion Together, in this large cohort including adult and pediatric allo-HCT patients treated with tisa-cel and axi-cel after allo-HCT, these data show that the incidence of both aGvHD and cGvHD is very low. Of note, GvHD was not observed in the pediatric cohort. Furthermore, no excessive NRM was observed compared to previously published methods of treating relapse post allo-HCT, being disease progression a major hurdle to optimal outcomes in this cohort of patients.

Inotuzumab Use: A Single-Center Experience in B-Cell Acute Lymphoblastic Leukemia

Introduction: Inotuzumab Ozogamicin (InO) is a humanized anti-CD22 monoclonal antibody conjugated to calicheamicin, a cytotoxic antibiotic agent used in B-Acute Lymphoblastic Leukemia (B-ALL). The conjugate is internalized and produces DNA cleavage and subsequent apoptosis. InO has shown better results than conventional chemotherapy. Patients and methods: We present the results of a single-center experience with a small cohort of 13 patients diagnosed with ALL who received InO outside of clinical trials. Results: Of the 13 patients, 69% (9) were male. The median age was 38 years old (range 16-64). 10 patients younger than 55 years 9 patients had relapsed diseases, 3 refractory diseases and one patient was newly diagnosed. Regardless of relapsed disease, 7 patients used InO as a first line of salvage and 2 as a second line. In refractory disease, 1 patient used InO as the second salvage and 2 as third or more. White blood cells range from 1,500 to 285,000 mm3. Peripheral blood blast from 50-80%. Before InO administration, all the patients had bilirubin lower than 2 mg/DL (range 0.15-1.26 mg/dL), an ALT (alanine aminotransferase) medium value of 34 U/dl, and an AST (aspartate aminotransferase) medium value of 33 U/dL. The most common type of leukemia was CALLA in 8 patients: B-ALL Phi positive in 2, Lymphoid blast crisis in1, pre-B ALL 1, and t (8;22) 1 patient. 10 patients had CD-22 positivity in flow cytometry, and 3 had negative results. There was no relation between the intensity of CD 22 and the rate of response. 11 patients received InO as monotherapy, and 2 were associated with Mini-HyperCVAD-InO. 7 patients received 1 cycle and 6 patients received 2 cycles of InO; the average dose received was 2,7 mg/m2. 7 (53%) patients in complete response (CR) plus MDR (Minimal Residual disease) negative after treatment, 4 progressive diseases and 2 missing. Three patients received HCT (hematopoietic cell transplantation) post-InO, and two are waiting in complete remission. One patient relapsed while she was waiting for HCT and died. 4 patients received InO as a salvage after relapse post-HCT. All these patients had progressive diseases and died. In the patients that used InO as a bridge to HCT, the average time to transplant was 94 days. Regardless of Veno-Occlusive Disease (VOD), 1 patient developed it after InO treatment; no patients had VOD associated with HCT. Overall survival: 38 months; progression-free survival: 4.1 months. Mortality rate was 53% (7 patients). Causes were: 4 with progressive disease, 1 hemorrhagic stroke, 1 septic shock, and 1 suicide. Conclusion: We show our small local experience using this new drug in B-ALL patients. Patients who use InO as a bridge to HCT could have a good outcome. We had not found good results when InO was used in relapse post-HCT. There was no relation between the intensity of CD22 at flow cytometry and the rate of responses. The incidence of VOD was low. Rates of response are comparable to other real-world reports. This small experience could be the beginning of a multicenter Real-World study in Latin America.

Patients with Down Syndrome and High-Risk B-Acute Lymphoblastic Leukemia Demonstrate Improved Outcomes on a Modified Chemotherapy Regimen: A Report from Children's Oncology Group Study AALL1131

Introduction: Children with Down syndrome (DS) have a 20-fold increased risk of developing B-acute lymphoblastic leukemia (B-ALL), and demonstrate an increased risk of both relapse and treatment-related mortality (TRM). The Children's Oncology Group (COG) High-Risk (HR) B-ALL trial AALL0232 was permanently closed to DS HR patients due to excessive TRM, primarily due to overwhelming sepsis during times of severe neutropenia. On the successor HR B-ALL trial AALL1131, a primary objective for DS patients was therefore to reduce TRM by enhancing supportive care and modifying some elements of therapy. Methods: DS HR patients were treated on a single non-randomized stratum, with several modifications compared to non-DS HR patients. The majority of DS HR patients received a three-drug Induction, with only slow early responders (M2/M3 marrow at Induction day 15) receiving a single dose of anthracycline (daunorubicin 50 mg/m 2). Interim Maintenance 1 (IM-1) consisted of four IV courses of intermediate dose (ID; 2,000 mg/m 2) rather than high-dose methotrexate (HD MTX; 5,000 mg/m 2), with leucovorin rescue beginning earlier, at hour 30 and advancing if tolerated to hour 36, rather than at hour 42. Maintenance modifications included administration of vincristine/steroid pulses every 12 instead of every 4 weeks, and equal duration of therapy for boys and girls. Results: Patient characteristics (Table 1) were similar among the 280 DS HR patients and 5,612 non-DS HR patients except for more frequent bone marrow minimal residual disease (BM MRD) >0.01% in DS vs non-DS patients both at end of Induction (EOI; 54.1% vs 34.9%, p <0.0001) and at end of consolidation (EOC; 23.9% vs 12.0%, p=0.001). DS HR patients were also less likely to have favorable or unfavorable cytogenetics (p <0.0001). In terms of tolerability of the modified IM-1 phase, the duration was significantly longer in DS vs non-DS patients (mean 80.0 ± 16.6 days vs 74.0 ± 14 days, p <0.0001), and delays over 14 days were significantly more frequent (9.7% vs 4.7%, p=0.001). Nevertheless severe toxicities during IM-1 were low, with only 2 DS HR cases of grade 4 mucositis, 2 grade 4 infections, and 1 grade 5 event (sudden death, not otherwise specified). AALL1131 successfully achieved an induction death rate <10% in DS HR patients, although the rate remained significantly higher than in non-DS patients (3.6% vs 1.7%, p=0.035). Event-free survival (EFS) and overall survival (OS) in DS HR patients were improved compared to outcomes on older trials but remained significantly lower than non-DS patients (Figure 1), with 5-year EFS 69.1 ± 3.1% vs 79.5 ± 0.6% (p <0.0001), and 5-year OS 83.3 ± 2.5% vs 88.4 ± 0.5% (p=0.0005). Among DS HR patients, outcomes were particularly poor among those with EOC MRD >0.01% compared to those with EOC MRD <0.01% (5-year EFS 25.8 + 9.9% vs 62.2 + 6.6%, p <0.0021; 5-year OS 54.0 + 12.2% vs 88.1 + 4.5%, p <0.0003). Conclusion: AALL1131 achieved the goal of decreasing induction mortality for DS HR patients, and the modified IM-1 phase utilizing ID MTX with early leucovorin rescue was well-tolerated, with minimal grade 4-5 toxicities, despite a longer mean duration and more frequent treatment delays compared to non-DS patients receiving HD MTX. DS HR patients continued to demonstrate disparities in EOI and EOC MRD levels, as well as in 5-year EFS and OS. Innovative strategies utilizing targeted therapies and immunotherapy are needed to further improve outcomes for patients with DS HR ALL.

Machine Learning Models for Hematogone Enumeration and Differentiation between Hematogones and Leukemic-Blasts in B-Acute Lymphoblastic Leukemias

Background: Hematogone (htg) counts are usually low in bone marrows but often increase in reactive or neoplastic conditions. When increased, htgs resemble leukemic-blasts in B-lymphoblastic leukemias (B-ALL) phenotypically. The differentiation between htgs and leukemic-blasts is critical as hematogone counts in marrows with reactive conditions may increase around or beyond B-ALL leukemic-blast diagnostic level and, conversely, leukemic-blast counts may be low in some B-ALL marrow samples. Additionally, current hematogone enumeration by the gating approach is operator dependent. Objective: To enumerate htgs and differentiate htgs from leukemic-blasts using machine-learning (ML) models. Research Methods: Cases with reported htgs (CReH), B-ALL, and controls (548, 124, and 771 marrow samples, respectively) were selected using key word search from historical flow cytometry (FC) results. Datasets for analysis were from FC-panel: FS_SS_CD5_CD10_CD20_ CD19_CD45. Three preliminary models, the htg enumeration model (HEM), immature cell enumeration models for B-lineage (IEM_B), and htg similarity model (HSM), were used (data not published). HSM classified a given htg-population or leukemic-blast-population into three categories: Htg-p (htg-population), Borderline, and non-Htg-p. Results: HEM distinguished CReHs with median 530 from the controls with median 5 (figure 1). Only six (<1%) of the controls had >500 htgs. IEM_B was used for separating hgt-populations in CReHs or leukemic-blast-populations in B-ALL cases from other cell populations. HSM classified 100 (95.2%) out of 105 hgt-populations from CReHs (≥1000 htgs) as Htg-p, and, due to fluorescence intensity downward shift, 4 as non-Hgt-p and 1 as Borderline. Conversely, HSM classified 113(92%) out of 123 leukemic-blast-populations from B-ALL as non-Hgt-p, 6 (5%) as Hgt-p and 4 (3%) as Borderline. Multiple scatter plots (figure 1a) illustrate two-dimensional distributions of a leukemic cluster classified as non-Hgt-p in a B-ALL case compared with those of a standard hematogone cluster. Conclusion: In an experimental setting, HEM is an efficient/high-throughput alternative for hgt-enumeration with high-consistency and is operator-independent. In the majority of B_ALL cases, HSM was able to differentiate leukemic-blast-populations from htg-populations. Future models will use additional markers, including leukemia-specific ones, to increase models' specificity.