B-cell Acute Lymphoblastic Leukemia Cases Research Articles

Unraveling Copy Number Alterations in Pediatric B-Cell Acute Lymphoblastic Leukemia: Correlation with Induction Phase Remission Using MLPA.

Acute Lymphoblastic Leukemia (ALL) is the most common malignancy occurring in children. Copy number alterations (CNA) like PAX5, CDKN2A/2B, PAR1 Region, ETV6, IKZF1, BTG1, and RB1 gene deletion are important genetic events that define and prognosticate B-cell ALL. Thus, this study aimed to evaluate associations of CNA with induction phase remission status in childhood B-cell ALL. This study was observational with a cross-sectional design at the Dharmais Cancer Hospital, Harapan Kita Mother and Children Hospital, and Tangerang Regional Public Hospital. We evaluated 74 pediatric B-cell ALL cases with 1-18-year-olds. Genomic DNA was analyzed by Multiplex Ligation Dependent Probe Amplification Assay (MLPA). This study used the P335 ALL-IKZF1 panel kit, which contains several ALL-related genes. The patient's clinical and laboratory characteristics were collected from medical records from January to December 2019. We observed gene copy number alteration in children with B-Cell ALL. PAX5 was the most commonly observed gene deletion, followed by CDKN21/2B, ETV6, IKZF1, BTG1, RB1, and PAR1 Region. Based on gene mutations, only the PAX5 had a significant association with the remission status of pediatric B-cell ALL (p-value <0.05; OR = 3.91). It showed that patients with PAX5 gene mutations have 3.9 times the risk of no remission and/or relapse compared to those without PAX5 gene mutations. Patients with mutations in the PAX5 gene have a higher chance of not achieving remission and/or experiencing relapse than those without such mutations. The MLPA method can be utilized for examining copy number alterations, which is valuable for achieving more precise stratification in diagnosis.. Further research is needed to expand upon this finding.

Abstract 5920: Inhibition of TBL1 and β-catenin interaction sensitizes acute lymphoblastic leukemia to chemotherapy

Abstract BACKGROUND: Relapse of T-cell acute lymphoblastic leukemia (T-ALL) and B-cell acute lymphoblastic leukemia (B-ALL) post-chemotherapy or post-immunotherapy remains a therapeutic problem warranting novel therapeutic approaches. Aberrant Wnt pathway activation is a common feature in ALL as Wnt activation through β-catenin is involved in self- renewal of drug resistant leukemia cells. A therapeutical approach to target Wnt signaling in T-ALL and B-ALL remains elusive. Here, we focus on the effect of transducin β-like protein 1 (TBL1) inhibitor tegavivint in combination with chemotherapy in T-ALL and B-ALL. We hypothesize that interrupting the binding of TBL1 to β-catenin abrogates leukemia cell survival and test if tegavivint sensitizes these cells to chemotherapeutic agents such as VDL (Vincristine, Dexamethasone, L-asparaginase) as part of preclinical evaluation of tegavivint in T- and B-cell ALL. METHODS: Four T-ALL cell lines one patient-derived T-ALL case, B-ALL cell lines and eight patient-derived B-ALL cases were used in vitro. We performed cytotoxicity studies using Annexin V/7AAD stainings and flow cytometry or Trypanblue counts to exclude dead cells, western blots, and co-immunoprecipitation assays to investigate effects of TBL-1 inhibition using tegavivint on β-catenin-mediated signaling. Tegavivint was injected intraperitoneally along with chemotherapeutic drugs. RESULTS: β-catenin and TBL1 were uniformly expressed in all T-ALL and B-ALL cells as determined by western blotting, albeit at different levels. Tegavivint decreases β-catenin protein expression as seen through western blot as well as decreases the binding of TBL1 to β-catenin as seen through co-immunoprecipitation (co-IP) in both the T-ALL and B-ALL samples. We also determined that tegavivint as a monotherapy induced apoptosis in both cell lines and patient samples at as little of a dose of 5nM after 24 hours of incubation using Annexin-V/DAPI staining by flow cytometry. We observed that tegavivint can sensitize all cases to chemotherapy (V: Vincristine, D: Dexamethasone, L:L-Asparaginase or Peg-Asparaginase) within 24 hours of incubation. Lastly, in vivo evaluation of tegavivint showed that tegavivint alone and in combination with VD chemotherapy significantly extends survival of NSG mice engrafted with patient-derived T-ALL and B-ALL compared to control treated mice. CONCLUSION: Our preliminary data shows that T-ALL and B-ALL are sensitive to TBL1-β-catenin inhibition using tegavivint as a monotherapy. Tegavivint can also sensitize T-ALL and B-ALL to chemotherapy (VDL). As our data supports our hypothesis that TBL-1-β-catenin inhibition is a new target in ALL therapy, further studies are in progress to preclinically evaluate this approach for clinical care. Citation Format: Samantha Hurwitz, Rachel Friedmann, Tatiana Fourfouris, Ki Jun Lee, Zesheng Wan, Chintan Parekh, Fariba Navid, Deepa Bhojwani, Kimberly Holloway, Yong-Mi Kim. Inhibition of TBL1 and β-catenin interaction sensitizes acute lymphoblastic leukemia to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5920.

Abstract 2845: Novel genetic risk loci for B-cell acute lymphoblastic leukemia in African American children: Findings from the Admixture and Risk of Acute Leukemia (ADMIRAL) Study

Abstract Background: Racial and ethnic disparities in incidence and outcomes for childhood B-cell acute lymphoblastic leukemia (B-ALL) are well established. While genome-wide association studies (GWASs) among children of European (EUR) genetic ancestry have identified robust risk loci, comparable studies in children of African (AFR) genetic ancestry do not exist. Here we report on results from the first GWAS of B-ALL exclusive to AFR children. Methods: A total of 840 AFR cases (diagnosis age &amp;lt;21y) and 3360 sex-/ancestry-matched controls (from ADMIRAL and ZOE 2.0, a childhood caries study) were evaluated. We used 656 cases from the Children’s Oncology Group (COG) ALL frontline trials for discovery and 184 non-COG cases for replication. Samples were genotyped with the Illumina Global Diversity Array and imputed with the TOPMed reference panel. Ancestry was ascertained with RFMix-inferred global ancestry values and principal components analysis. Logistic regression models adjusted for cryptic population substructure evaluated variant risk associations. Discovery and replication results were meta-analyzed using an inverse variance-weighted fixed effects model. Results: We identified 7 genome-wide significant (GWS, P&amp;lt;5 × 10−8) variants, including 2 novel B-ALL risk loci with replication evidence (P&amp;lt;0.05) and striking per allele effect sizes: CNTN4 (meta-analysis odds ratio [OR]=2.09, P=1.4 × 10−11) and FAM174A (OR=2.94, P=1.2 × 10−15). Established B-ALL EUR risk loci were corroborated in these data (ARIDB5, IKZF1; P&amp;lt;5 × 10−8). Five additional novel loci were GWS after meta-analysis including at SLC37A4 (OR=2.02, P=1.1 × 10−8) and CBWD1 (OR=2.69, P=2.7 × 10−9), with suggestive associations in discovery data (P&amp;lt;5 × 10−6) and nominal replication (P&amp;lt;0.05). Notably, all novel risk alleles were low-frequency in reference AFR populations and AFR-specific (minor allele frequencies of 2-9% in 1000 Genomes AFR and &amp;lt;0.1% in non-AFR). Top variants had relevant in silico annotations; variants at CNTN4 and SLC37A4 had high predicted functional probability (&amp;gt;0.8, RegulomeDb). For example, annotations for a top CNTN4 signal suggest a potential leukemogenesis mechanism, overlapping a ChIP-seq peak for CEBPA in transdifferentiated B cells. In our replication data, a polygenic risk score (PRS) with 5 novel AFR-specific GWS variants from discovery had an appreciably stronger B-ALL risk association (top 25% vs rest: OR=2.03, P=1.2 × 10−4) compared to a published B-ALL PRS from a GWAS in EUR children (top 25% vs rest: OR=1.59, P=0.014). Conclusions: Despite the modest number of AFR B-ALL cases, we successfully detected and replicated genotype risk associations with considerable effect sizes. These results demonstrate the critical importance of increasing diversity in genomic studies of childhood ALL to promote discovery and reduce disparities. Citation Format: Cindy Im, Andrew Raduski, Lauren J. Mills, Rebecca A. Johnson, Andrew DeWan, Xiaomei Ma, Joseph L. Wiemels, Catherine Metayer, Jun J. Yang, Heather H. Nelson, Tianzhong Yang, Saonli Basu, Lucie M. Turcotte, Nathan Pankratz, Michael E. Scheurer, Logan G. Spector. Novel genetic risk loci for B-cell acute lymphoblastic leukemia in African American children: Findings from the Admixture and Risk of Acute Leukemia (ADMIRAL) Study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2845.

Concomitant inhibition of the thioredoxin system and nonhomologous DNA repair potently sensitizes Philadelphia-positive lymphoid leukemia to tyrosine kinase inhibitors.

Breakpoint cluster region-Abelson (BCR::ABL1) gene fusion is an essential oncogene in both chronic myeloid leukemia (CML) and Philadelphia-positive (Ph+) B-cell acute lymphoblastic leukemia (B-ALL). While tyrosine kinase inhibitors (TKIs) are effective in up to 95% of CML patients, 50% of Ph+ B-ALL cases do not respond to treatment or relapse. This calls for new therapeutic approaches for Ph+ B-ALL. Previous studies have shown that inhibitors of the thioredoxin (TXN) system exert antileukemic activity against B-ALL cells, particularly in combination with other drugs. Here, we present that peroxiredoxin-1 (PRDX1), one of the enzymes of the TXN system, is upregulated in Ph+ lymphoid as compared to Ph+ myeloid cells. PRDX1 knockout negatively affects the viability of Ph+ B-ALL cells and sensitizes them to TKIs. Analysis of global gene expression changes in imatinib-treated, PRDX1-deficient cells revealed that the nonhomologous end-joining (NHEJ) DNA repair is a novel vulnerability of Ph+ B-ALL cells. Accordingly, PRDX1-deficient Ph+ B-ALL cells were susceptible to NHEJ inhibitors. Finally, we demonstrated the potent efficacy of a novel combination of TKIs, TXN inhibitors, and NHEJ inhibitors against Ph+ B-ALL cell lines and primary cells, which can be further investigated as a potential therapeutic approach for the treatment of Ph+ B-ALL.

BRD4 PROTAC degrader MZ1 exhibits anti-B-cell acute lymphoblastic leukemia effects via targeting CCND3

ABSTRACTIntroductionB-cell acute lymphoblastic leukemia (B-ALL) is the most prevalent malignant tumor affecting children. While the majority of B-ALL patients (90%) experience successful recovery, early relapse cases of B-ALL continue to exhibit high mortality rates. MZ1, a novel inhibitor of Bromodomains and extra-terminal (BET) proteins, has demonstrated potent antitumor activity against hematological malignancies. The objective of this study was to examine the role and therapeutic potential of MZ1 in the treatment of B-ALL.MethodsIn order to ascertain the fundamental mechanism of MZ1, a sequence of in vitro assays was conducted on B-ALL cell lines, encompassing Cell Counting Kit 8 (CCK8) assay, Propidium iodide (PI) staining, and Annexin V/PI staining. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) were employed to examine protein and mRNA expression levels. Transcriptomic RNA sequencing (RNA-seq) was utilized to screen the target genes of MZ1, and lentiviral transfection was employed to establish stably-expressing/knockdown cell lines.ResultsMZ1 has been observed to induce the degradation of Bromodomain Containing 4 (BRD4), Bromodomain Containing 3 (BRD3), and Bromodomain Containing 2 (BRD2) in B-ALL cell strains, leading to inhibited cell growth and induction of cell apoptosis and cycle arrest in vitro. These findings suggest that MZ1 exhibits cytotoxic effects on two distinct molecular subtypes of B-ALL, namely 697 (TCF3/PBX1) and RS4;11 (MLL-AF4) B-ALL cell lines. Additionally, RNA-sequencing analysis revealed that MZ1 significantly downregulated the expression of Cyclin D3 (CCND3) gene in B-ALL cell lines, which in turn promoted cell apoptosis, blocked cell cycle, and caused cell proliferation inhibition.ConclusionOur results suggest that MZ1 has potential anti-B-ALL effects and might be a novel therapeutic target.

Transcription factor networks link B-lymphocyte development and malignant transformation in leukemia.

Rapid advances in genomics have opened unprecedented possibilities to explore the mutational landscapes in malignant diseases, such as B-cell acute lymphoblastic leukemia (B-ALL). This disease is manifested as a severe defect in the production of normal blood cells due to the uncontrolled expansion of transformed B-lymphocyte progenitors in the bone marrow. Even though classical genetics identified translocations of transcription factor-coding genes in B-ALL, the extent of the targeting of regulatory networks in malignant transformation was not evident until the emergence of large-scale genomic analyses. There is now evidence that many B-ALL cases present with mutations in genes that encode transcription factors with critical roles in normal B-lymphocyte development. These include PAX5, IKZF1, EBF1, and TCF3, all of which are targeted by translocations or, more commonly, partial inactivation in cases of B-ALL. Even though there is support for the notion that germline polymorphisms in the PAX5 and IKZF1 genes predispose for B-ALL, the majority of leukemias present with somatic mutations in transcription factor-encoding genes. These genetic aberrations are often found in combination with mutations in genes that encode components of the pre-B-cell receptor or the IL-7/TSLP signaling pathways, all of which are important for early B-cell development. This review provides an overview of our current understanding of the molecular interplay that occurs between transcription factors and signaling events during normal and malignant B-lymphocyte development.

Biochemical and Hematologic Profiles in B-Cell Acute Lymphoblastic Leukemia Children.

B-cell acute lymphoblastic leukemia is the most common type of leukemia found in children. Timely diagnosis, white blood cell count, age of onset, and sex are considered the most important prognostic factors in childhood leukemia. Hematological and biochemical profiles are crucially important to infer the health of leukemia patient pre-chemotherapy and post-chemotherapy treatment. In the current study 200 cases were taken and evaluated for hematological (complete blood count and white blood differential count) and biochemical parameters (renal function tests, liver function tests, serum electrolytes and serum proteins) by comparison with normal reference values. Most of the cases were male under 5 years of age. Hematology parameters including red blood cells, hemoglobin and platelet levels were relatively low whereas white blood cells level was high in cases as compared with normal reference value. Sex-wise and age-wise comparison of biochemical profile showed significant difference among B-cell acute lymphoblastic leukemia cases whereas hematological profile did not show any visible difference.

Phenotypic and genotypic infidelity in B-lineage neoplasms, including transdifferentiation following targeted therapy: Report from the 2021 SH/EAHP Workshop.

Session 2 of the 2021 Society for Hematopathology and European Association for Haematopathology Workshop collected examples of lineage infidelity and transdifferentiation in B-lineage neoplasms, including after targeted therapy. Twenty cases were submitted. Whole-exome sequencing and genome-wide RNA expression analysis were available on a limited subsample. A diagnosis of B-cell acute lymphoblastic leukemia (B-ALL) was rendered on at least 1 biopsy from 13 patients. There was 1 case of acute myeloid leukemia (AML); the remaining 6 cases were mature B-cell neoplasms. Targeted therapy was administered in 7 cases of B-ALL and 4 cases of mature B-cell neoplasms. Six cases of B-ALL underwent lineage switch to AML or mixed-phenotype acute leukemia at relapse, 5 of which had rearranged KMT2A. Changes in maturational state without lineage switch were observed in 2 cases. Examples of de novo aberrant T-cell antigen expression (n = 2) were seen among the mature B-cell lymphoma cohort, and their presence correlated with alterations in tumor cell gene expression patterns. This cohort of cases enabled us to illustrate, discuss, and review current concepts of lineage switch and aberrant antigen expression in a variety of B-cell neoplasms and draw attention to the role targeted therapies may have in predisposing neoplasms to transdifferentiation as well as other, less expected changes in maturational status.

Intrachromosomal amplification of chromosome 21 in Egyptian pediatric B-lineage acute lymphoblastic leukemia: a hospital-based observational study

Abstract Objective This study was conducted to evaluate the clinicopathologic features as well as the association between the intrachromosomal amplification of chromosome 21 (iAMP21) and the clinical outcome in Egyptian patients with B-cell acute lymphoblastic leukemia (B-ALL) attending/admitted in the Department of Pediatric Oncology of our university hospital. Patients and methods This was a prospective study conducted on 25 newly diagnosed and five relapsed pediatric patients with B-ALL (23 males and seven females), with a mean age of 7.76 ± 5.11 years. Bone marrow aspiration was done to diagnose and evaluate remission of participants, and fluorescence in situ hybridization (FISH) analysis using the ETV6-RUNX1 probes was used to detect RUNX1 amplification. Results iAMP21 was identified in 28% of newly diagnosed patients with B-ALL and in 80% of relapsed cases with B-ALL. There was a significant association between positive iAMP21 cases and failure to attain complete remission at day 28 (P=0.004). A significant relationship among the 14 patients monitored for disease outcome was found between the presence of iAMP21 and occurrence of relapse or death in newly diagnosed, or failure to achieve complete remission or death in relapsed cases (P=0.036). Conclusion There was a higher incidence of iAMP21 among our Egyptian pediatric B-ALL cases and unfavorable effect of iAMP21 on patients’ prognosis even with coexistence of ETV6-RUNX1.

Manufacturing results of tisagenlecleucel for acute lymphoblastic leukemia: a survey by the CAR-T cell therapy taskforce of the Japan Society of Transfusion Medicine and Cell Therapy

The frequency of the manufacturing failure of chimeric antigen receptor (CAR)-T cell therapy in clinical practice is unknown. To clarify the current state of how likely CAR-T cell production is to succeed or fail for B-cell acute lymphoblastic leukemia (B-ALL), we analyzed cases in which the production of tisagenlecleucel was performed for patients with B-ALL at 15 facilities in Japan from October 2019 to March 2022. Total 81 patients (47 males and 34 females) were analyzed. The median age at apheresis was 13 years (1-25) with a median number of prior treatments of 4 (1-9). The numbers of patients with histories of allogeneic transplantation, inotuzumab ozogamicin, or blinatumomab treatments were 51 (63.0%), 26 (32.1%), and 37 (45.7%), respectively. The median blast percentage and CD3+ cell counts in peripheral blood were 0% (0-91.5), and 611/µl (35-4,210) at apheresis, and the median number of CD3+ cells shipped was 2.2×109 (0.5-8.3). While cases with a history of heavy prior treatment before apheresis were included, no manufacturing failures were observed. Continuing to monitor the status of manufacturing failures is necessary as the number of B-ALL cases treated with CAR-T cell therapy increases.

Thrombocytopenia and Low CD4/CD8 Ratio at Apheresis Along with Prior Bendamustine Use Increase Risk of CAR-T Cell Manufacturing Failure

[Introduction] Chimeric antigen receptor T (CAR-T) cell therapy targeting CD19 has shown good outcomes in patients with relapsed or refractory (r/r) B-cell malignancies, and the number of patients awaiting this therapy is increasing dramatically. Successful CAR-T cell therapy can be achieved only after a number of steps are optimized. Of these, it is essential to ensure that CAR-T cells are manufactured without failure. Although manufacturing failure rates range from 1 to 13%, its causes and risk factors remain unclear. Therefore, we sought to determine risk factors for CAR-T cell manufacturing failure in a real-world setting. [Methods] This retrospective study, conducted by the Japan Society of Transfusion Medicine and Cell Therapy, enrolled patients who underwent CAR-T cell production for tisagenlecleucel (tisa-cel) using T cells harvested at 23 Japanese institutions from October 2019 to March 2022. Clinical factors, including patient characteristics, laboratory tests at apheresis, and apheresis procedures, were compared between successful and unsuccessful case groups. Categorical and continuous variables were compared between groups using Fisher's exact test and two-tailed unpaired Student's t-test, respectively. In comparison between groups, CD4/CD8 ratio was log-transformed to normalize skewed distribution. We evaluated the effect of each factor on the manufacturing outcome using a logistic regression model. p values <0.05 were considered statistically significant. [Results] We analyzed 519 cases, including 438 diffuse large B-cell lymphoma (DLBCL) and 81 B-cell acute lymphoblastic leukemia (B-ALL) cases. Whereas no manufacturing failure was observed among B-ALL cases, there were 30 manufacturing failures (6.8%) among DLBCL cases. Then, we compared clinical factors between DLBCL cases that failed (n = 30) and that succeeded (n = 378), excluding those employed in clinical trials for out-of-spec products. The number of chemotherapy cycles performed prior to apheresis (median 13 vs. 10; p = 0.01) and the proportion of patients with previously treated with 3 or more cycles of bendamustine (43.3 vs. 10.9%; p < 0.01) were significantly higher in the failed group than the successful group. Regarding cell counts in peripheral blood at apheresis, platelet count (1.20 vs. 1.70 × 105/μL; p = 0.01) and CD4/CD8 ratio (0.30 vs. 0.56; p < 0.01) were significantly lower in the failed group than the successful group (Figure 1). For T-cell collection, Spectra Optia® was used in 403 cases (98.8%), and there were no significant differences in the failure rate in terms of the device type or collection program. There were no significant differences in the total number of CD3+ cells (3.1 vs. 2.9 ×109; p = 0.51) or proportion of CD3+ cells to total nucleated cells (55.6 vs. 46.4%; p = 0.14) shipped for tisa-cel production between the failed and successful groups. Multivariate logistic analysis revealed that history of 3 or more cycles of bendamustine therapy prior to apheresis (odds ratio [OR], 3.19; 95% confidence interval [CI], 1.04-9.82; p = 0.04), low platelet counts (OR, 0.52 per 105/μL; 95% CI, 0.28-0.96; p = 0.04), and low CD4/CD8ratio (less than one third) (OR, 3.06; 95% CI 1.23-7.59; p = 0.02) in peripheral blood at apheresis were risk factors for CAR-T manufacturing failure. [Conclusions] This study showed that manufacturing failure remains an obstacle to CAR-T cell therapy. Repeated use of bendamustine prior to apheresis, low platelet counts, and low CD4/CD8 ratio in peripheral blood at apheresis were associated with increased risk of manufacturing failure. Avoiding these risk factors before apheresis and risk-adopted therapeutic strategy after apheresis may help to optimize CAR-T cell therapy for patients with r/r B-cell malignancies. 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

Subtype Dependent Drug Resistance Alterations in Relapsed Childhood B-ALL: A Children's Oncology Group Study

Introduction The survival rate for children with B-cell acute lymphoblastic leukemia (B-ALL) now approaches 90%. However, given the poor prognosis of those who relapse, B-ALL remains a leading cause of cancer related mortality in children. To discover underlying mechanisms of treatment failure, previous genomic profiling has identified somatic alterations in 12 genes that drive treatment resistance. However, these studies were underpowered to detect association between these alterations and known genetic subtypes (e.g., hyperdiploid, ETV6::RUNX1, TCF3::PBX1, etc.) that would indicate cooperativity in driving clonal evolution. Results We performed whole genome sequencing (WGS) of diagnosis/remission/relapse specimens of 181 B-ALL cases (age 0-18 years) enrolled on a recently completed clinical trial for relapsed B-ALL (AALL1331). Somatic aberrations including SNV, Indel, structural variations (SV), copy number variations (CNV) were identified from WGS and fusion genes were detected from RNAseq data. By integrating these cases with 146 previously published cases (totaling N=327 with WGS/WES; N=255 with WGS), we aimed to identify novel resistance drivers and to discover associations between genetic subtypes and resistance drivers. Here, we report two novel somatic alterations to be enriched in relapsed B-ALL (Table 1), an intergenic deletion in chr1 (named ERASER) and FLT3 promoter alteration (which results in overexpression of FLT3). Importantly, we also discovered statistically significant (with FDR corrected P value<0.2) associations between resistance drivers and subtypes that confirmed previously known associations such as NR3C1/NR3C2 in ETV6::RUNX1 and FLT3 promoter alteration in hyperdiploid ALL. We also discovered novel associations including NSD2 in TCF3::PBX1, CREBBP in hyperdiploid, ERASER in ETV6::RUNX1, TP53 in KMT2Ar, mismatch repair deficiency (MMR) in TCF3::PBX1, NT5C2 in PAX5 alt and DUX4, and FLT3 promoter alteration in ZNF384 subtypes. In addition to these novel findings, our data also revealed high frequency nested P2RY8::CRLF2 fusions in other subtypes, which was validated using single cell sequencing. Conclusion Together, our study features NGS of the largest cohort of relapsed childhood ALL samples, identifies novel resistance markers for ETV6::RUNX subtype, and uncovers novel associations between resistance drivers and subtypes. These finding may shape future clinical management of childhood ALL. 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

PAX5 alterations in B-cell acute lymphoblastic leukemia.

PAX5, a master regulator of B cell development and maintenance, is one of the most common targets of genetic alterations in B-cell acute lymphoblastic leukemia (B-ALL). PAX5 alterations consist of copy number variations (whole gene, partial, or intragenic), translocations, and point mutations, with distinct distribution across B-ALL subtypes. The multifaceted functional impacts such as haploinsufficiency and gain-of-function of PAX5 depending on specific variants have been described, thereby the connection between the blockage of B cell development and the malignant transformation of normal B cells has been established. In this review, we provide the recent advances in understanding the function of PAX5 in orchestrating the development of both normal and malignant B cells over the past decade, with a focus on the PAX5 alterations shown as the initiating or driver events in B-ALL. Recent large-scale genomic analyses of B-ALL have identified multiple novel subtypes driven by PAX5 genetic lesions, such as the one defined by a distinct gene expression profile and PAX5 P80R mutation, which is an exemplar leukemia entity driven by a missense mutation. Although altered PAX5 is shared as a driver in B-ALL, disparate disease phenotypes and clinical outcomes among the patients indicate further heterogeneity of the underlying mechanisms and disturbed gene regulation networks along the disease development. In-depth mechanistic studies in human B-ALL and animal models have demonstrated high penetrance of PAX5 variants alone or concomitant with other genetic lesions in driving B-cell malignancy, indicating the altered PAX5 and deregulated genes may serve as potential therapeutic targets in certain B-ALL cases.

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.

Clinical and Prognostic Impact of Copy Number Alterations and Associated Risk Profiles in a Cohort of Pediatric B-cell Precursor Acute Lymphoblastic Leukemia Cases Treated Under ICiCLe Protocol.

Copy number alteration (CNA) status and CNA risk profiles of IKZF1plus, UK-ALL CNA risk groups and MRplus scores, were evaluated for clinical and prognostic impact in a cohort of 493 B-cell acute lymphoblastic leukemia cases diagnosed and treated under the Indian Collaborative Childhood Leukemia group (ICiCLe) protocol trial. Overall CNA frequency was 59% with 60% of cases showing 2-loci deletion. CDKN2A/B deletion was most common CNA (36.3%), while IKZF1 deletion and IKZF1plus profile were noted in 19.5% and 13.4% of cases, respectively. IKZF1 deletions and other CNA risk profiles were significantly associated with poor (PR)/high risk (HR) clinical and genetic profile parameters (P < 0.001). In addition, the 3-year OS, event-free survival (EFS) was significantly poor with high relapse rate (RR) of 38.6%, 46.5%, and 35.2% for IKZF1 deletions, IKZF1plus profiles, and UK-ALL CNA-intermediate risk (IR)+PR risk groups, respectively (P < 0.001). Integrated evaluation of UK-ALL CNA risk profile with ICiCLe trial risk stratification groups revealed a worse overall survival, EFS, and RR of 63.3%, 43.2%, and 35.2% for CNA-IR+PR profile compared to CNA-good risk profile (81.3%, 65.0%, and 21.0%; P < 0.001). Hence, routine CNA testing in our setting is must to identify standard risk and IR cases likely to benefit from HR treatment.

HYPERDIPLOID KARYOTYPE ASSOCIATED WITH PHILADELPHIA CHROMOSOME IN A PEDIATRIC PATIENT WITH ACUTE LYMPHOBLASTIC LEUKEMIA

An exacerbated proliferation of B-cell progenitors in bone marrow can evolve to a Precursor B-cell Acute Lymphoblastic Leukemia (B-ALL). Molecular and chromosomal abnormalities affect differentiation and proliferation of lymphoid precursors. Chromosomal alterations (hypodiploidy and hyperdiploidy), rearrangements involving KMT2A gene and the translocations BCR-ABL (Ph+), TCF3-PBX1 and ETV6-RUNX1 can be used as biomarkers to predict prognostic. The association of hyperdiploidy with chromosomal translocations is observed in 1%‒4% of hyperdiploidy patients in B-ALL. The aim of this study was to describe a rare case of B-ALL in an adolescent with BCR-ABL and hyperdiploidy. Immunophenotyping was used to characterize the leukemia type. Molecular tests were performed to investigate the presence of recurrent chromosomal translocations in B-ALL. G-banding and Fluorescence in situ hybridization (FISH) were carried out to characterize the karyotype. A 16-year-old girl was admitted in Hospital Sarina Rolim, São Paulo in February 2019 after experiencing 3 months of recurrent limb pain along with loss of weight, skin pallor and muscle weakness. The peripheral blood count showed 8.800 white blood cells/mm 3 , platelets count 90.000, hemoglobin 10 g/dL and hematocrit 30%. The immunophenotyping showed: CD19++/+++, CD20−/+/++, CD10+/++/+++, CD66C+/++/+++, CD123−/+/++, CD9+/++/+++, CD127−/+, CD44+++. The RT-PCR was negative for TCF3-PBX1, ETV6-RUNX rearrangements. It was detected the presence of Ph+ (P190). The results were consistent with B-ALL. The karyotype was: 58≍61,XX,+2,+4,+6,+8,t(9;22)(q34;q22),+10,+11,+12,+13,+19,+20,+21,+22,+22,+der(22)[cp16]/46,XX[1]. The patient was treated according to ALL BFM 2009 protocol associated with imatinib 400 mg/day. During the follow-up of minimal residual disease (D33), the patient presented hematological and infectious toxicity stage IV, requiring hospitalization due to febrile neutropenia and sepsis. Also, exhibited aseptic necrosis of the femoral head and underwent orthopedic surgery. The treatment has been completed in February 2021. In July 2022, the patient had no evidence of disease recidive but she acquired modest motor sequelae due to the orthopedic surgery. Hyperdiploidy occurs in 30% of pediatric B-ALL. The most frequent chromosome gains are X, 4, 6, 10, 14, 17, 18 and 21. However, BCR-ABL translocation comprises 2%‒5% of childhood ALL. There are only a few case reports of patients presenting BCR-ABL associated to hyperdiploidy. The outcome of patients can change according to these variations in genetic factors. Although BCR-ABL is associated with poor prognosis, the outcome has improved with implementation of tyrosine kinase inhibitors in Ph+ patients'therapy. We described an unusual case of pediatric B-ALL with hyperdiploid and Ph+ karyotype. This study and the literature review demonstrate the importance of cytogenetic and molecular tests during the diagnosis aiding to choose the treatment and improving the survival for ALL pediatric patients.

Genetic ancestry, differential gene expression, and survival in pediatric B-cell acute lymphoblastic leukemia.

Black children have lower incidence yet worse survival than White and Latinx children with B-cell acute lymphoblastic leukemia (B-ALL). It is unclear how reported race/ethnicity (RRE) is associated with death in B-ALL after accounting for differentially expressed genes associated with genetic ancestry. Using Phase 1 and 2 NCI TARGET B-ALL cases (N= 273; RRE-Black=21, RRE-White=162, RRE-Latinx=69, RRE-Other=9, RRE-Unknown=12), we estimated proportions of African (AFR), European (EUR), and Amerindian (AMR) genetic ancestry. We estimated hazard ratios (HR) and 95% confidence intervals (95% CI) between ancestry and death while adjusting for RRE and clinical measures. We identified genes associated with genetic ancestry and adjusted for them in RRE and death associations. Genetic ancestry varied within RRE (RRE-Black, AFR proportion: Mean: 78.5%, Range: 38.2%-93.6%; RRE-White, EUR proportion: Mean: 94%, Range: 1.6%-99.9%; RRE-Latinx, AMR proportion: Mean: 52.0%, Range: 1.2%-98.7%). We identified 10, 1, and 6 differentially expressed genes (padjusted <0.05) associated with AFR, AMR, and EUR ancestry proportion, respectively. We found AMR and AFR ancestry were statistically significantly associated with death (AMR each 10% HR: 1.05, 95% CI: 1.03-1.17, AFR each 10% increase HR: 1.03, 95% CI:1.01-1.19). RRE differences in the risk of death were larger in magnitude upon adjustment for genes associated with genetic ancestry for RRE-Black, but not RRE-Latinx children (RRE-Black HR: 3.35, 95% CI: 1.31, 8.53; RRE-Latinx HR: 1.47, 0.88-2.45). Our work highlights B-ALL survival differences by RRE after adjusting for ancestry differentially expressed genes suggesting other factors impacting survival are important.

Clinicopathologic and genetic evaluation of B-lymphoblastic leukemia with intrachromosomal amplification of chromosome 21 (iAMP21) in adult patients

Intrachromosomal amplification of chromosome 21 (iAMP21) defines a rare provisional entity of B-cell acute lymphoblastic leukemia (B-ALL) in the current WHO classification and has been described as specific for pediatric patients with a median age at diagnosis of 9–10 years. We report two adult cases of B-ALL with iAMP21, one 31-year-old woman and one 40-year-old man, identified by karyotyping and next generation sequencing (NGS), with fluorescence in situ hybridization (FISH) pattern meeting diagnostic criteria for iAMP21. Both patients were treated on high-risk chemotherapeutic regimen followed by stem cell transplant. In contrast to reported high relapse rate within the first three years in pediatric population, our adult patients are alive in remission, with the interval from diagnosis to last follow up of 2.95 and 3.96 years. Our cases illustrate the importance of screening for iAMP21 in adult population when ETV6-RUNX1 FISH testing is not routinely performed for adult patients.