Childhood B-cell Acute Lymphoblastic Leukemia Research Articles

Childhood B cell leukemia: Intercepting the paths to progression.

B-cell Acute Lymphoblastic Leukemia (B-ALL) is the most common pediatric cancer, arising most often in children aged 2-5 years. This distinctive age distribution hints at an association between B-ALL development and disrupted immune system function during a susceptible period during childhood, possibly triggered by early exposure to infection. While cure rates for childhood B-ALL surpass 90% in high-income nations, survivors suffer from diminished quality of life due to the side effects of treatment. Consequently, understanding the origins and evolution of B-ALL, and how to prevent this prevalent childhood cancer, is paramount to alleviate this substantial health burden. This article provides an overview of our current understanding of the etiology of childhood B-ALL and explores how this knowledge can inform preventive strategies.

Feasibility and Favorable Responses Following Investigational CAR T-Cell Therapy for Relapsed and Refractory Infant ALL

Infants with B-cell acute lymphoblastic leukemia (B-ALL) continue to have significantly worse outcomes compared to older children with B-ALL, and those with relapsed or refractory (R/R) infant ALL have especially dismal outcomes with conventional treatment. CD19-targeting chimeric antigen receptor (CAR) T-cell therapy has demonstrated remarkable success in the treatment of R/R childhood B-ALL, though the majority of reports have been in non-infant patients. Barriers to the successful implementation of CAR T-cell therapy in infant B-ALL include challenges related to apheresis, product manufacturing and disease-specific considerations such as lineage switch. We describe our experience utilizing two experimental CD19-CAR T-cell products, SCRI-CAR19 or SCRI-CAR19x22, for 19 patients with R/R infant B-ALL enrolled on three clinical trials. CAR T-cell products were successfully manufactured in 18/19 (94.7%) patients, with a median age of 22.5 months at enrollment (range, 14.5-40.1 months). Sixteen of 17 (94.1%) treated patients achieved a complete remission without detectable minimal residual disease. The 1-year leukemia free survival was 75% and 1-year overall survival was 76.5%, with a median follow up time of 35.8 months (range, 1.7-83.6 months). Cytokine release syndrome (CRS) occurred in 14/17 (82.4%) patients, with only 1 patient experiencing Grade 3 CRS. Neurotoxicity occurred in 2/17 (11.8%) patients with all events ≤ Grade 2. With the successful early clinical experience of CAR T-cell therapy in this population, more systematic evaluation specific to infant ALL is warranted.

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.

The Role of the JAK-STAT Pathway in Childhood B-Cell Acute Lymphoblastic Leukemia.

B-cell lymphoblastic leukemia is a hematologic neoplasm that poses a serious health concern in childhood. Genetic aberrations, such as mutations in the genes IL-7, IL7R, JAK1, JAK2, TLSP, CRLF2, and KTM2A or gene fusions involving BCR::ABL1, ETV6::RUNX1, and PAX5::JAK2, often correlate with the onset of this disease. These aberrations can lead to malfunction of the JAK-STAT signaling pathway, which is implicated in various important biological processes, including those related to immunology. Understanding the mechanisms underlying the malfunction of the JAK-STAT pathway holds potential for research on drugs targeting its components. Available drugs that interfere with the JAK-STAT pathway include fludarabine, ruxolitinib, and fedratinib.

Early-life infection depletes preleukemic cells in a mouse model of hyperdiploid B-cell acute lymphoblastic leukemia

AbstractEpidemiological studies report opposing influences of infection on childhood B-cell acute lymphoblastic leukemia (B-ALL). Although infections in the first year of life appear to exert the largest impact on leukemia risk, the effect of early pathogen exposure on the fetal preleukemia cells (PLC) that lead to B-ALL has yet to be reported. Using cytomegalovirus (CMV) infection as a model early-life infection, we show that virus exposure within 1 week of birth induces profound depletion of transplanted E2A-PBX1 and hyperdiploid B-ALL cells in wild-type recipients and in situ–generated PLC in Eμ-ret mice. The age-dependent depletion of PLC results from an elevated STAT4-mediated cytokine response in neonates, with high levels of interleukin (IL)-12p40–driven interferon (IFN)-γ production inducing PLC death. Similar PLC depletion can be achieved in adult mice by impairing viral clearance. These findings provide mechanistic support for potential inhibitory effects of early-life infection on B-ALL progression and could inform novel therapeutic or preventive strategies.

Terminal deoxynucleotidyl transferase expression in different subtypes of childhood B-cell acute lymphoblastic leukemia

The lack of expression of terminal deoxynucleotidyl transferase (TdT) is frequently associated with KMT2A-rearranged subtype of pediatric acute lymphoblastic leukemia (ALL). However, this association has not been investigated extensively in the Asian population. A retrospective analysis of TdT expression in pediatric B-cell ALL (B-ALL) was performed in patients treated using the Taiwan Pediatric Oncology Group (TPOG) ALL 2002 and 2013 protocols. Among the 331 patients with B-ALL, 12 patients showed TdT negativity at initial diagnosis. Among these, eight patients showed KMT2A rearrangement (66.7%). Other patients showing negative TdT expression had ETV6::RUNX1, MEF2D-rearranged, and other B-ALL subtypes. However, in the context of KMT2A-rearranged B-ALL (n = 20), only eight patients showed TdT negativity. The 5-year event-free survival and overall survival of patients with and without TdT expression were 83.8% versus 46.8% (P <0.001) and 86.3% versus 55.4% (P = 0.004), respectively. Moreover, several aberrant markers, such as CD2, CD56, CD7, and CD117, were rarely expressed in the B-ALL samples, and if expressed, they were enriched in specific genetic subtypes. The results of this study indicate that immunophenotypic features are correlated with specific genetic subtypes of childhood B-ALL.

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.

CircRNAome of Childhood Acute Lymphoblastic Leukemia: Deciphering Subtype-Specific Expression Profiles and Involvement in TCF3::PBX1 ALL.

Childhood B-cell acute lymphoblastic leukemia (B-ALL) is a heterogeneous disease comprising multiple molecular subgroups with subtype-specific expression profiles. Recently, a new type of ncRNA, termed circular RNA (circRNA), has emerged as a promising biomarker in cancer, but little is known about their role in childhood B-ALL. Here, through RNA-seq analysis in 105 childhood B-ALL patients comprising six genetic subtypes and seven B-cell controls from two independent cohorts we demonstrated that circRNAs properly stratified B-ALL subtypes. By differential expression analysis of each subtype vs. controls, 156 overexpressed and 134 underexpressed circRNAs were identified consistently in at least one subtype, most of them with subtype-specific expression. TCF3::PBX1 subtype was the one with the highest number of unique and overexpressed circRNAs, and the circRNA signature could effectively discriminate new patients with TCF3::PBX1 subtype from others. Our results indicated that NUDT21, an RNA-binding protein (RBP) involved in circRNA biogenesis, may contribute to this circRNA enrichment in TCF3::PBX1 ALL. Further functional characterization using the CRISPR-Cas13d system demonstrated that circBARD1, overexpressed in TCF3::PBX1 patients and regulated by NUDT21, might be involved in leukemogenesis through the activation of p38 via hsa-miR-153-5p. Our results suggest that circRNAs could play a role in the pathogenesis of childhood B-ALL.

Association between genetic variants of membrane transporters and the risk of high-grade hematologic adverse events in a cohort of Mexican children with B-cell acute lymphoblastic leukemia.

Advances in the understanding of the pathobiology of childhood B-cell acute lymphoblastic leukemia (B-ALL) have led towards risk-oriented treatment regimens and markedly improved survival rates. However, treatment-related toxicities remain a major cause of mortality in developing countries. One of the most common adverse effects of chemotherapy in B-ALL is the hematologic toxicity, which may be related to genetic variants in membrane transporters that are critical for drug absorption, distribution, and elimination. In this study we detected genetic variants present in a selected group genes of the ABC and SLC families that are associated with the risk of high-grade hematologic adverse events due to chemotherapy treatment in a group of Mexican children with B-ALL. Next generation sequencing (NGS) was used to screen six genes of the ABC and seven genes of the SLC transporter families, in a cohort of 96 children with B-ALL. The grade of hematologic toxicity was classified according to the National Cancer Institute's Common Terminology Criteria for Adverse Events (CTCAE) version 5.0, Subsequently, two groups of patients were formed: the null/low-grade (grades 1 and 2) and the high-grade (grades 3 to 5) adverse events groups. To determine whether there is an association between the genetic variants and high-grade hematologic adverse events, logistic regression analyses were performed using co-dominant, dominant, recessive, overdominant and log-additive inheritance models. Odds ratio (OR) and 95% confidence intervals (95% CI) were calculated. We found two types of associations among the genetic variants identified as possible predictor factors of hematologic toxicity. One group of variants associated with high-grade toxicity risk: ABCC1 rs129081; ABCC4 rs227409; ABCC5 rs939338, rs1132776, rs3749442, rs4148575, rs4148579 and rs4148580; and another group of protective variants that includes ABCC1 rs212087 and rs212090; SLC22A6 rs4149170, rs4149171 and rs955434. There are genetic variants in the SLC and ABC transporter families present in Mexican children with B-ALL that can be considered as potential risk markers for hematologic toxicity secondary to chemotherapeutic treatment, as well as other protective variants that may be useful in addition to conventional risk stratification for therapeutic decision making in these highly vulnerable patients.

Chromosomal instability in aneuploid acute lymphoblastic leukemia associates with disease progression

Chromosomal instability (CIN) lies at the core of cancer development leading to aneuploidy, chromosomal copy-number heterogeneity (chr-CNH) and ultimately, unfavorable clinical outcomes. Despite its ubiquity in cancer, the presence of CIN in childhood B-cell acute lymphoblastic leukemia (cB-ALL), the most frequent pediatric cancer showing high frequencies of aneuploidy, remains unknown. Here, we elucidate the presence of CIN in aneuploid cB-ALL subtypes using single-cell whole-genome sequencing of primary cB-ALL samples and by generating and functionally characterizing patient-derived xenograft models (cB-ALL-PDX). We report higher rates of CIN across aneuploid than in euploid cB-ALL that strongly correlate with intraclonal chr-CNH and overall survival in mice. This association was further supported by in silico mathematical modeling. Moreover, mass-spectrometry analyses of cB-ALL-PDX revealed a “CIN signature” enriched in mitotic-spindle regulatory pathways, which was confirmed by RNA-sequencing of a large cohort of cB-ALL samples. The link between the presence of CIN in aneuploid cB-ALL and disease progression opens new possibilities for patient stratification and offers a promising new avenue as a therapeutic target in cB-ALL treatment.

Genomic Determinants of Therapy Response in ETV6::RUNX1 Leukemia

ETV6::RUNX1 leukemia is the second most common childhood B-cell acute lymphoblastic leukemia subtype. Although it has a low overall relapse risk, a significant proportion of relapses occur within this subtype due to its relatively high incidence. No consistent genomic biomarkers have been identified that predict therapy response beyond early therapy response as measured by minimal residual disease at the end of induction. In this study, our aim was to use multiomic data from 175 ETV6::RUNX1 leukemias treated according to the NOPHO ALL2000 and ALL2008 protocols to identify genomic features that predict therapy response at presentation. Cases were classified into three responder groups based on MRD at end-of-induction (EOI) therapy: fast responders with negative MRD, intermediate responders with positive but &amp;lt;0.1% MRD, and slow responders with &amp;gt;0.1% MRD. Response data was also collected at mid-induction (day 15) and end-of-consolidation (EOC), when available. Diagnostic and remission DNA were analyzed using array-based copy number data (n=142) or whole genome sequencing (WGS) (n=33), and diagnostic bulk RNA sequencing (n=51) was complemented with single cell multiomics (RNA, surface protein and VDJ sequencing) data from both healthy (n=8) and leukemic bone marrow cells upon chemotherapy (n=8). Overall, the frequency of somatic genetic changes was not significantly associated with treatment response suggesting that genetic diversification through elevated random mutagenesis events may not underlie the drug resistant phenotype. However, analysis of mutational signatures revealed that fast-responding leukemic blasts frequently exhibited the APOBEC mutational signature and had higher cell cycle activity (Figure 1A). Accordingly, we show that expression of A3B is highly specific to cycling cells also in healthy bone marrow hematopoietic progenitor cells, pro- and pre-B cells. We also examined RAG-mediated genetic instability that manifests in focal structural variations and has highest activity in ETV6::RUNX1leukemia (Figure 1B). We confirmed several recurrent RAG off-target sites, including significant enrichment of the known risk locus TBX1LR1 in slow responding cases. Strikingly, slow-responding leukemias had also frequent rearrangements in the immunoglobulin kappa genes and the kappa-deleting elements, despite their pro-B-like transcriptome state upon diagnosis. Based on the single cell multiomics profiles, we found that upon treatment the blast phenotype shifts towards an immature-B-like transcriptome, with concomitant expression of Ig light chains with productive sequence, thereby permitting activation of BCR signaling that provides a survival advantage. At CNV level, we found that the chromosomal alterations related to the translocation event associated with treatment response. Specifically, slow responders had more often the amplification of der(21)t(12;21) chromosome, impacting expression of USP5, TIGAR and SLC2A3, associated in earlier studies to chemoresistance and tumorigenesis. In comparison, fast responders were often missing a region of chromosome 12p with lower expression of several genes including oncogenic KRAS. In summary, combining multiomic and response data, we characterized genetic alterations and mutational processes that affect the treatment response in ETV6::RUNX1leukemia. Our findings highlight the genetic and transcriptomic heterogeneity of this common pediatric leukemia subtype and its implications for treatment sensitivity, paving the way for better classification of the disease at presentation.

Lessons from mouse models in the impact of risk factors on the genesis of childhood B-cell leukemia.

B-cell acute lymphoblastic leukemia (B-ALL) stands as the primary contributor to childhood cancer-related mortality on a global scale. The development of the most conventional forms of this disease has been proposed to be conducted by two different steps influenced by different types of risk factors. The first step is led by a genetic insult that is presumably acquired before birth that transforms a healthy cell into a preleukemic one, which is maintained untransformed until the second step takes place. This necessary next step to leukemia development will be triggered by different risk factors to which children are exposed after birth. Murine models that recap the stepwise progression of B-ALL have been instrumental in identifying environmental and genetic factors that contribute to disease risk. Recent evidence from these models has demonstrated that specific environmental risk factors, such as common infections or gut microbiome dysbiosis, induce immune stress, driving the transformation of preleukemic cells, and harboring genetic alterations, into fully transformed leukemic cells. Such models serve as valuable tools for investigating the mechanisms underlying preleukemic events and can aid in the development of preventive approaches for leukemia in child. Here, we discuss the existing knowledge, learned from mouse models, of the impact of genetic and environmental risk factors on childhood B-ALL evolution and how B-ALL prevention could be reached by interfering with preleukemic cells.

Cytokine network imbalance in children with B-cell acute lymphoblastic leukemia at diagnosis

Immune imbalance has been proved to be involved in the pathogenesis of hematologic neoplasm. However, little research has been reported altered cytokine network in childhood B-cell acute lymphoblastic leukemia (B-ALL) at diagnosis. Our study aimed to evaluate the cytokine network in peripheral blood of newly diagnosed pediatric patients with B-ALL. Serum levels of interleukin (IL)-2, IL-4, IL-6, IL-10, tumor necrosis factor (TNF), interferon (IFN)-γ, and IL-17A in 45 children with B-ALL and 37 healthy control children were measured by cytometric bead array, while the level of transforming growth factor-β1 (TGF-β1) in the serum was measured by enzyme-linked immunosorbent assay. Patients showed a significant increase in IL-6 (p < 0.001), IL-10 (p < 0.001), IFN-γ (p = 0.023) and a significant reduction in TGF-β1 (p = 0.001). The levels of IL-2, IL-4, TNF and IL-17A were similar in the two groups. Higher concentrations of pro-inflammatory cytokines were associated with febrile in patients without apparent infection by using unsupervised machine learning algorithms. In conclusion, our results indicated a critical role for aberrant cytokine expression profiles in the progression of childhood B-ALL. Distinct cytokine subgroups with different clinical features and immune response have been identified in patients with B-ALL at the time of diagnosis.

Flow cytometric assessment of leukemia-associated monocytes in childhood B-cell acute lymphoblastic leukemia outcome

Flow cytometric assessment of leukemia-associated monocytes in childhood B-cell acute lymphoblastic leukemia outcome

DNMT3A low-expression is correlated to poor prognosis in childhood B-ALL and confers resistance to daunorubicin on leukemic cells

BackgroundLittle is known about DNMT3A expression and its prognostic significance in childhood B cell acute lymphoblastic leukemia (B-ALL).MethodsWe determined DNMT3A mRNA expression in 102 children with B-ALL. Correlations with relapse-free survival (RFS) and common clinical characteristics were analyzed. DNMT3A was stably knocked out by CRISPR/Cas9 gene editing technology in Reh and 697 B-ALL cell lines. Cell proliferation activity after treated with daunorubicin (DNR) was determined by CCK8 assay in DNMT3A KO Reh and 697 cell lines.ResultsDNMT3A expression in B-ALL patients who were in continuous complete remission (CCR) was higher than in those who got relapse (P = 0.0111). Receiver operating characteristic curve showed prognostic significance of DNMT3A expression (P = 0.003). Low expression of DNMT3A (≤ 0.197) was significantly correlated with poor RFS (P < 0.001) in children with B-ALL. Knock-out of DNMT3A in Reh and 697 cell lines significantly increased IC50 of DNR (P = 0.0201 and 0.0022 respectively), indicating elevated resistance to DNR.ConclusionLow expression of DNMT3A associates with poor prognosis in children with B-ALL. Knock-out of DNMT3A confers resistance to DNR on leukemic cells.

Significance of CEBPE Gene Promoter Polymorphism (Rs2239630 G > A ) Assessment in Childhood B-cell Acute Lymphoblastic Leukemia.

A significant association has been reported between CEBPE gene promoter polymorphisms (rs2239630 G > A ) and the incidence of B-cell acute lymphoblastic leukemia (B-ALL). However, no previous study on this issue has been included among the Egyptian cohort of pediatric patients with B-ALL. Therefore, this study was designed to address the associations between CEBPE polymorphisms and susceptibility to B-ALL, as well as its impact on the outcome of B-ALL Egyptian patients with B-ALL. In the current study, we evaluated the rs2239630 polymorphism in 225 pediatric patients and 228 controls to assess the association of different rs2239630 genotypes with childhood susceptibility to B-ALL and the impact on the outcome of the patients. The frequency of the A allele was significantly higher in the cases of B-ALL compared with the control group ( P = 0.004). By analyzing different genotypes for the predictive value of disease development, the GA and AA genotypes have been identified to be the highest among multivariate factors with an odds ratio of 3.330 (95% CI: 1.105-10.035). Likewise, the A allele was significantly associated with the shortest overall survival. CEBPE gene promoter polymorphism (rs2239630 G > A ) AA is frequently associated with B-ALL; and has the worst overall survival among the 3 genotypes, followed by the GA and GG genotypes ( P < 0.001).

Whole genome sequencing provides comprehensive genetic testing in childhood B-cell acute lymphoblastic leukaemia

Childhood B-cell acute lymphoblastic leukaemia (B-ALL) is characterised by recurrent genetic abnormalities that drive risk-directed treatment strategies. Using current techniques, accurate detection of such aberrations can be challenging, due to the rapidly expanding list of key genetic abnormalities. Whole genome sequencing (WGS) has the potential to improve genetic testing, but requires comprehensive validation. We performed WGS on 210 childhood B-ALL samples annotated with clinical and genetic data. We devised a molecular classification system to subtype these patients based on identification of key genetic changes in tumour-normal and tumour-only analyses. This approach detected 294 subtype-defining genetic abnormalities in 96% (202/210) patients. Novel genetic variants, including fusions involving genes in the MAP kinase pathway, were identified. WGS results were concordant with standard-of-care methods and whole transcriptome sequencing (WTS). We expanded the catalogue of genetic profiles that reliably classify PAX5alt and ETV6::RUNX1-like subtypes. Our novel bioinformatic pipeline improved detection of DUX4 rearrangements (DUX4-r): a good-risk B-ALL subtype with high survival rates. Overall, we have validated that WGS provides a standalone, reliable genetic test to detect all subtype-defining genetic abnormalities in B-ALL, accurately classifying patients for the risk-directed treatment stratification, while simultaneously performing as a research tool to identify novel disease biomarkers.

The Landscape of Secondary Genetic Rearrangements in Pediatric Patients with B-Cell Acute Lymphoblastic Leukemia with t(12;21)

The most frequent chromosomal rearrangement in childhood B-cell acute lymphoblastic leukemia (B-ALL) is translocation t(12;21)(p13;q22). It results in the fusion of the ETV6::RUNX1 gene, which is active in the regulation of multiple crucial cellular pathways. Recent studies hypothesize that many translocations are influenced by RAG-initiated deletions, as well as defects in the RAS and NRAS pathways. According to a "two-hit" model for the molecular pathogenesis of pediatric ETV6::RUNX1-positive B-ALL, the t(12;21) translocation requires leukemia-causing secondary mutations. Patients with ETV6::RUNX1 express up to 60 different aberrations, which highlights the heterogeneity of this B-ALL subtype and is reflected in differences in patient response to treatment and chances of relapse. Most studies of secondary genetic changes have concentrated on deletions of the normal, non-rearranged ETV6 allele. Other predominant structural changes included deletions of chromosomes 6q and 9p, loss of entire chromosomes X, 8, and 13, duplications of chromosome 4q, or trisomy of chromosomes 21 and 16, but the impact of these changes on overall survival remains unclarified. An equally genetically diverse group is the recently identified new B-ALL subtype ETV6::RUNX1-like ALL. In our review, we provide a comprehensive description of recurrent secondary mutations in pediatric B-ALL with t(12;21) to emphasize the value of investigating detailed molecular mechanisms in ETV6::RUNX1-positive B-ALL, both for our understanding of the etiology of the disease and for future clinical advances in patient treatment and management.

The prognostic significance of hematogones in childhood B-cell acute lymphoblastic leukemia.

Recent studies have demonstrated hematogones (HGs) expansion to be associated with favorable outcomes in hematological diseases, especially in patients with acute myeloid leukemia and patients undergoing hematopoietic stem cell transplantation. Acute lymphoblastic leukemia (ALL) is the most common form of cancer in children. As of now, minimal residual disease (MRD) remains the most compelling independent prognostic factor in childhood ALL. There is need for more prognostic tools for evaluating relapse risk. The goal of this study was to assess the prognostic value of HGs on relapse-free survival (RFS) and overall survival (OS) in childhood ALL. In this prospective cohort study, a total of 122 subjects with definitive diagnosis of precursor B lymphoblastic leukemia were evaluated. Flow cytometric HG detection was performed in bone marrow aspirates after induction and consolidation therapy. The median follow-up period of patients was 35.5±9.4 (SD) months. Patients who had at least 1.0% HGs had a significantly better RFS (p=.023). Moreover, univariate and multivariate analyses confirmed that positive HGs were independently associated with longer RFS (unadjusted model: hazard ratio=0.33, 95% CI=0.12-0.91, p=.031; adjusted model: hazard ratio=0.30, 95% CI=0.11-0.82, p=.020). Along with the role of MRD, our study shows the significance of HGs as an independent prognostic factor. The results indicate the independent prognostic value of HGs on RFS after adjustment for other prognostic factors, and can be beneficial for risk stratification and treatment modifications amongst pediatric B-cell ALL patients.

B-Cell Acute Lymphoblastic Leukemia with iAMP21 in a Patient with Constitutional Ring Chromosome 21

Pediatric B-cell acute lymphoblastic leukemia (B-ALL) is associated with various specific cytogenetic and molecular markers that significantly influence treatment and prognosis. Intrachromosomal amplification of chromosome 21 (iAMP21) defines a rare distinct cytogenetic subgroup of childhood B-ALL, which is characterized by amplification of region 21q22.12 comprising the RUNX1 gene. Constitutional structural chromosomal abnormalities involving chromosome 21 confer an increased risk for B-ALL with iAMP21. Here, we report the development of B-ALL with iAMP21 in a 9-year-old child with a constitutional ring chromosome 21, r(21)c, uncovered after B-ALL diagnosis. Cytogenetic and microarray analysis of the post-therapy sample revealed an abnormal chromosome 21 lacking a satellite and having a deletion of the terminal 22q22.3 region, consistent with a constitutional ring chromosome 21, r(21)(p11.2q22). On a retrospective analysis, this ring chromosome was observed in the normal cells in the pre-treatment diagnostic specimen. Constitutional ring chromosome 21 may remain undetected in patients with mild or no neurodevelopmental phenotype, posing an unknown lifelong risk of developing B-ALL with iAMP21. Individuals with constitutional structural chromosome 21 rearrangements such as ring 21 require a close surveillance and long-term follow-up studies to establish their risk of B-ALL relapse and possibility of developing other malignancies. Germline analysis is recommended to all pediatric patients with iAMP21-related B-ALL to rule out structural chromosome 21 rearrangements and to elucidate molecular mechanisms of iAMP21 formation.