B-cell Acute Lymphoblastic Leukemia Subtypes Research Articles

RanBALL: An Ensemble Random Projection Model for Identifying Subtypes of B-cell Acute Lymphoblastic Leukemia.

As the most common pediatric malignancy, B-cell acute lymphoblastic leukemia (B-ALL) has multiple distinct subtypes characterized by recurrent and sporadic somatic and germline genetic alterations. Identification of B-ALL subtypes can facilitate risk stratification and enable tailored therapeutic approaches. Existing methods for B-ALL subtyping primarily depend on immunophenotypic, cytogenetic and genomic analyses, which would be costly, complicated, and laborious in clinical practice applications. To overcome these challenges, we present RanBALL (an Ensemble Ran dom Projection-Based Model for Identifying B -Cell A cute L ymphoblastic L eukemia Subtypes), an accurate and cost-effective model for B-ALL subtype identification based on transcriptomic profiling only. RanBALL leverages random projection (RP) to construct an ensemble of dimension-reduced multi-class support vector machine (SVM) classifiers for B-ALL subtyping. Results based on 100 times 5-fold cross validation tests for >1700 B-ALL patients demonstrated that the proposed model achieved an accuracy of 93.35%, indicating promising prediction capabilities of RanBALL for B-ALL subtyping. The high accuracies of RanBALL suggested that our model could effectively capture underlying patterns of transcriptomic profiling for accurate B-ALL subtype identification. We believe RanBALL will facilitate the discovery of B-ALL subtype-specific marker genes and therapeutic targets, and eventually have consequential positive impacts on downstream risk stratification and tailored treatment design.

Ikaros sets the threshold for negative B-cell selection by regulation of the signaling strength of the AKT pathway

Inhibitory phosphatases, such as the inositol-5-phosphatase SHIP1 could potentially contribute to B-cell acute lymphoblastic leukemia (B-ALL) by raising the threshold for activation of the autoimmunity checkpoint, allowing malignant cells with strong oncogenic B-cell receptor signaling to escape negative selection. Here, we show that SHIP1 is differentially expressed across B-ALL subtypes and that high versus low SHIP1 expression is associated with specific B-ALL subgroups. In particular, we found high SHIP1 expression in both, Philadelphia chromosome (Ph)-positive and ETV6-RUNX1-rearranged B-ALL cells. As demonstrated by targeted knockdown of SHIP1 by RNA interference, proliferation of B-ALL cells in vitro and their tumorigenic spread in vivo depended in part on SHIP1 expression. We investigated the regulation of SHIP1, as an important antagonist of the AKT signaling pathway, by the B-cell-specific transcription factor Ikaros. Targeted restoration of Ikaros and pharmacological inhibition of the antagonistic casein kinase 2, led to a strong reduction in SHIP1 expression and at the same time to a significant inhibition of AKT activation and cell growth. Importantly, the tumor suppressive function of Ikaros was enhanced by a SHIP1-dependent additive effect. Furthermore, our study shows that all three AKT isoforms contribute to the pro-mitogenic and anti-apoptotic signaling in B-ALL cells. Conversely, hyperactivation of a single AKT isoform is sufficient to induce negative selection by increased oxidative stress. In summary, our study demonstrates the regulatory function of Ikaros on SHIP1 expression in B-ALL and highlights the relevance of sustained SHIP1 expression to prevent cells with hyperactivated PI3K/AKT/mTOR signaling from undergoing negative selection.

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 4907: RanBALL: Identifying B-cell acute lymphoblastic leukemia subtypes based on an ensemble random projection model

Abstract As the most common pediatric malignancy, B-cell acute lymphoblastic leukemia (B-ALL) has multiple distinct subtypes characterized by recurrent and sporadic somatic and germline genetic alterations like chromosomal alteration, transcription factor rearrangement or kinase inhibition. The treatment of B-ALL patients is personalized based on specific subtypes, as the treatment responses for different B-ALL subtypes may vary considerably. Identification of B-ALL subtypes can facilitate risk stratification and enable tailored therapeutic approaches. Existing methods for B-ALL subtyping primarily depend on immunophenotypic, cytogenetic and genomic analyses, which would be costly, complicated, and laborious in clinical practice applications. To overcome these challenges, we present RanBALL (an Ensemble Random Projection-Based Model for Identifying B-Cell Acute Lymphoblastic Leukemia Subtypes), an accurate and cost-effective model for B-ALL subtype identification based on transcriptomic profiling only. RanBALL leverages random projection (RP) to construct an ensemble of dimension-reduced multi-class classifiers for B-ALL subtyping. Specifically, the transcriptomic profiling features were projected onto low-dimensional spaces by random projection matrices whose elements conform to a distribution characterized by zero mean and unit variance. To ensure reliable and robust performance, we selected 20 subspace dimensions ranging from 600 to 2500, with intervals of 100. The transformed low dimensional data matrix was used for training an ensemble of multi-class support vector machine (SVM) classifiers, each corresponding to one of the RP matrices of various dimensions. The predicted probabilistic scores of each B-ALL subtype were integrated for determining the final decision. Results based on 10 times 10-fold cross validation tests for &amp;gt;1700 B-ALL patients demonstrated that the proposed model achieved an accuracy of 93.7%, indicating promising prediction capabilities of RanBALL for B-ALL subtyping. Furthermore, the 30% held-out tests suggested that the model was robust and consistent to maintain high confidence levels for accurate predictions. The high accuracies of RanBALL suggested that our model could effectively capture underlying patterns of transcriptomic profiling for accurate B-ALL subtype identification. To extend the impact of RanBALL, we have established a free and publicly available python package for RanBALL available at https://github.com/wan-mlab/RanBALL. We believe RanBALL will facilitate the discovery of B-ALL subtype-specific marker genes and therapeutic targets, and eventually have consequential positive impacts on downstream risk stratification and tailored treatment design. Citation Format: Lusheng Li, Hanyu Xiao, Joseph D. Khoury, Jieqiong Wang, Shibiao Wan. RanBALL: Identifying B-cell acute lymphoblastic leukemia subtypes based on an ensemble random projection model [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 4907.

Abstract 3952: Multiomic single-cell tumor evolution models of minimal residual disease in pediatric B-cell acute lymphoblastic leukemia

Abstract Detection of minimal residual disease (MRD) after initial diagnosis (IDX) and therapy is a strong prognostic marker for relapse in pediatric B-cell acute lymphoblastic leukemia (B-ALL). Previous studies have explored clonal genetic and gene expression changes between initial diagnosis and relapse. However, studying tumor cells present at MRD is challenging due to their low abundance. Examining the genomic profiles of leukemia cells that resist induction chemotherapy and understanding their evolutionary relationship to cells present at diagnosis may lead to a better understanding of the cells that eventually seed relapse. We analyzed somatic point mutations, indels, and copy number variants from 41 pediatric B-ALL patients with high-risk B-ALL. We specifically focused on patients from the following four common high-risk subtypes: KMT2A-rearranged, Ph+, Ph-like, and iAMP21, who had detectable disease at the end of induction. Leukemic burden at end-induction (day 29) ranged from 0.03% to 66.00%. Data were generated in partnership with the National Cancer Institute’s Human Tumor Atlas Network and the Children’s Oncology Group. Our data set of 41 patients includes 39 with paired initial diagnostic and end-induction samples that we used to create tumor evolution models based on single cells across disease stages. We produced single-cell multiome gene expression (scRNA) and chromatin accessibility (scATAC) profiles (10x Genomics) with a median of 7428 leukemic blast cells at IDX (range: 2490-14219) and 1907 at MRD (range: 240-10167) after filtering. We called somatic mutations from scRNA and scATAC using SComatic and Monopogen, and we detected copy number changes using Numbat and EpiAneufinder. We also designed a targeted DNA sequencing panel including 335 variants based on B-ALL literature and database searches to produce scDNA (Mission Bio) for 24 patients (16 IDX-MRD pairs) with a median of 2346 cells profiled at IDX (range: 340-8849) and 1828 at MRD (range: 596-5133). We built tumor phylogenies based on somatic SNVs and CNVs with COMPASS. Using multiple tools and complementary single-cell omics data types to detect somatic changes improved our confidence in the events used for tumor evolution tree construction. Our approach allowed us to define recurrent genomic events and patterns across pediatric B-ALL subtypes and to describe the clonal evolution of cells measured at initial diagnostic and end-induction disease stages. We were also able to link cell-specific mutation events to their transcriptional and epigenomic signatures, including mapping mutation events along the B-cell development trajectory and within differentially expressed gene pathways. Some cases showed dramatic shifts in subclonal structure between IDX and MRD that may be related to therapy-induced clonal selection, such as a loss of NRAS hotspot mutation. Citation Format: Steven M. Foltz, Avi Loren, Changya Chen, Rushabh Mehta, Elizabeth Li, Jason Xu, Fatemeh Alikarami, Kathrin M. Bernt, Kai Tan. Multiomic single-cell tumor evolution models of minimal residual disease in pediatric B-cell acute lymphoblastic leukemia [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 3952.

Identification of an alternative short ARID5B isoform associated with B-ALL survival

Utilizing RNA sequence (RNA-Seq) splice junction data from a cohort of 1841 B-cell acute lymphoblastic leukemia (B-ALL) patients we define transcriptionally distinct isoforms of ARID5B, a risk-associated gene identified in genome wide association studies (GWAS), which associate with disease survival. Short (S) and long (L) ARID5B transcripts, which differ in an encoded BAH-like chromatin interaction domain, show remarkable correlation to the isoform splicing pattern. Testing of the ARID5B proximal promoter of the S & L isoforms indicated that both are functionally independent in luciferase reporter assays. Increased short isoform expression is associated with decreased event-free and overall survival. The abundance of short and long transcripts strongly correlates to B-ALL prognostic stratification, where B-ALL subtypes with poor outcomes express a higher proportion of the S-isoform. These data demonstrate that the analysis of independent promoters and alternative splicing events are essential for improved risk stratification and a more complete understanding of disease pathology.

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.

Clonal Heterogeneity and Evolution during Treatment in High Hyperdiploid B-Cell Acute Lymphoblastic Leukemia

Introduction: Acute lymphoblastic leukemia (ALL), the most common pediatric malignancy, is caused by the accumulation of multiple genomic aberrations in developing lymphocytes. In children, high hyperdiploidy (HeH) is the most prevalent subtype of B-cell ALL (B-ALL), which is associated with a superior outcome. However, because of the high incidence of HeH B-ALL in children, this subtype is responsible for about 25% of all relapses. The prediction of relapse remains difficult, especially in these good risk subtypes. Recent studies indicate that ALL is a heterogeneous disease, with multiple leukemia clones at diagnosis, each having a distinct set of mutations. We hypothesize that these different subclones have a different sensitivity to treatment and that samples with more heterogeneity are more prone to relapse. We used single-cell sequencing to determine the clonal heterogeneity of HeH B-ALL at diagnosis and followed the clonal evolution during chemotherapy treatment. Methods: Bone marrow samples of 11 patients with HeH B-ALL were collected at diagnosis, during treatment (for 9 cases) and at relapse (for 1 case) (Table 1). Samples during treatment were enriched for leukemia cells using magnetic antibodies targeting cell membrane markers (CD10, CD19, CD34) with the MARS® gentle sorter. We performed targeted single-cell DNA sequencing with the Mission Bio Tapestri® Platform, using our custom amplicon panel of 414 amplicons across 39 commonly mutated genes. This method detected single nucleotide variants (SNVs) and small insertions/deletions (indels) based on targeted PCR amplification in about 4000 cells per sample. Results: We sequenced diagnostic samples from 11 HeH B-ALL patients. For 9 patients we also tested samples during treatment, either at day 15 of treatment (1 case) or at the end of induction (EOI) (8 cases), and for 3 cases an additional sample at the end of consolidation (EOC). Furthermore, for one patient, we analyzed samples of 3 subsequent relapses. In total, 117,148 single cells were processed, with an average of 4500 cells per sample. We detected multiple subclones in almost all diagnostic samples (9/11), ranging from 2 to 10 subclones per case. Most frequently mutated genes were KRAS, NRAS, PTPN11 and FLT3, as well as NSD2. Four cases had 2 or more different KRAS and/or NRAS subclones. Three cases had a subclone with a KRAS/ NRAS mutation co-occurring with a NSD2 mutation. For the patients with multiple subclones at diagnosis, we subsequently performed single-cell analysis on one or more follow-up samples, either during treatment or at relapse. In the samples taken at EOI, we detected residual leukemia cells in all 8 cases: multiple subclones were detected in 5 cases and only one subclone persisted in 3 cases. Residual leukemia cells were also detected in all samples collected at EOC, though in a lesser amount as compared to the previous timepoint sequenced. Interestingly, in the matched diagnosis-relapse case, we found that a minor subclone with a NSD2, PTPN11and KRAS mutation became the dominant clone at first relapse (Figure 1). Conclusions: Single-cell DNA sequencing reveals a strong genetic heterogeneity in HeH B-ALL based on a variety of mutations in KRAS, NRAS, PTPN11, FLT3 and NSD2. RAS mutations and FLT3 mutations are mutually exclusive in subclones, whereas NSD2 mutations and RAS mutations are frequently co-occurring in the same subclone. This cooperation is highlighted in our relapse patient, having a minor subclone with a KRAS and NSD2 mutation at diagnosis that became the largest subclone at relapse. Even though few leukemia cells persisted during treatment, we were able to genetically identify the residual subclones with a potentially higher therapy resistance.

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.

1. Microdeletion 13q12.2 in B cell acute lymphoblastic leukemia: Little but important!

Chromosomal microarray analysis (CMA) is instrumental for identification of recurrent focal deletions in patients with B cell acute lymphoblastic leukemia (B-ALL). These microdeletions include genes involved in B cell differentiation, cell cycle control and transcriptional regulation often with prognostic or therapeutic significance. Recently, a 13q12.2 microdeletion with breakpoints upstream of FLT3 and within intronic regions of the PAN3 gene was reported. Functionally, this microdeletion resulted in enhancer hijacking and constitutive activation of FLT3 in pediatric B-ALL. Seven institutions performed internal database searches to identify similar deletions. In total, 10 patients were identified, all with B-ALL, confirming the specificity for this diagnosis. This cohort included seven pediatric cases (3 male, 4 female; ages 2-14 yo, mean 5) and 3 adult cases (1 male, 2 female; ages 22, 39, 42). Subtypes included two high hyperdiploid, one hypodiploid, one iAMP21, one P2RY8::CLRF, one unbalanced rearrangement involving TFE3, one PAX5::PML, three B-ALL NOS.? Deletions ranged in size from?6.7 kb ? 136 kb (mean 76 kb); each with proximal breakpoints between FLT3 and PAN3 and 9/10 with distal breakpoints within intron 5 of the PAN3 gene. Our study is the first to report the microdeletion in adults, and unlike the discovery cohorts, our patients showed no sex bias. Our data does not confirm a strong association of the deletion with high hyperdiploidy, since 8/10 had other B-ALL subtypes. Constitutive activation of FLT3 may have significant clinical implications for prognosis and treatment; thus, detection of 13q12.2 microdeletion at diagnosis or disease relapse is paramount.

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.

B‐cell acute lymphoblastic leukemia with iAMP21 in a patient with Down syndrome due to a constitutional isodicentric chromosome 21

Pediatric B-cell acute lymphoblastic leukemia (B-ALL) is associated with various specific cytogenetic and molecular markers that have significant influence on treatment and prognosis. A subset of children has a much higher risk of developing B-ALL due to constitutional genetic alterations such as trisomy 21 (Down's syndrome). In these patients, B-ALL is often associated with specific genomic profiles leading to leukemic transformation. In rare cases, constitutional structural chromosomal abnormalities involving chromosome 21, such as the der(15;21) Robertsonian translocation and a ring 21 chromosome, have been associated with intrachromosomal amplification of chromosome 21 (iAMP21) B-ALL. Here, we report the development of B-ALL in a child with Down's syndrome who carries a constitutional isodicentric chromosome 21 [idic(21)], described previously by Putra et al., 2017. This idic(21) appeared to be unstable during mitosis, leading to somatic rearrangements consistent with iAMP21 amplification, resulting in the development of leukemia. In this case, a single constitutional structural chromosome 21 rearrangement resulted in a B-ALL with Down syndrome-associated genomic lesions as well as genomic lesions not common to the Down syndrome subtype of B-ALL. Our findings highlight the need for counseling of individuals with constitutional structural chromosome 21 rearrangements regarding their risks of developing a B-ALL.

SFPQ-ABL1 and BCR-ABL1 use different signaling networks to drive B-cell acute lymphoblastic leukemia

AbstractPhiladelphia-like (Ph-like) acute lymphoblastic leukemia (ALL) is a high-risk subtype of B-cell ALL characterized by a gene expression profile resembling Philadelphia chromosome–positive ALL (Ph+ ALL) in the absence of BCR-ABL1. Tyrosine kinase–activating fusions, some involving ABL1, are recurrent drivers of Ph-like ALL and are targetable with tyrosine kinase inhibitors (TKIs). We identified a rare instance of SFPQ-ABL1 in a child with Ph-like ALL. SFPQ-ABL1 expressed in cytokine-dependent cell lines was sufficient to transform cells and these cells were sensitive to ABL1-targeting TKIs. In contrast to BCR-ABL1, SFPQ-ABL1 localized to the nuclear compartment and was a weaker driver of cellular proliferation. Phosphoproteomics analysis showed upregulation of cell cycle, DNA replication, and spliceosome pathways, and downregulation of signal transduction pathways, including ErbB, NF-κB, vascular endothelial growth factor (VEGF), and MAPK signaling in SFPQ-ABL1–expressing cells compared with BCR-ABL1–expressing cells. SFPQ-ABL1 expression did not activate phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling and was associated with phosphorylation of G2/M cell cycle proteins. SFPQ-ABL1 was sensitive to navitoclax and S-63845 and promotes cell survival by maintaining expression of Mcl-1 and Bcl-xL. SFPQ-ABL1 has functionally distinct mechanisms by which it drives ALL, including subcellular localization, proliferative capacity, and activation of cellular pathways. These findings highlight the role that fusion partners have in mediating the function of ABL1 fusions.

Near-Haploidy and Low-Hypodiploidy in B-Cell Acute Lymphoblastic Leukemia: When Less Is Too Much.

Simple SummaryB-cell acute lymphoblastic leukemia (B-ALL) is characterized by an uncontrolled proliferation of blood cells in the bone marrow. A small fraction of B-ALL patients shows abnormally low chromosome numbers, defined as hypodiploidy, in leukemic cells. Hypodiploidy with less than 40 chromosomes is a rare genetic abnormality in B-ALL and is associated to an extremely poor outcome, with low survival rates both in pediatric and adult cases. In this review, we describe the main clinical and genetic features of hypodiploid B-ALL subtypes with less than 40 chromosomes, the current treatment protocols and their clinical outcomes. Additionally, we discuss the potential cellular mechanisms involved on the origin of hypodiploidy, as well as its leukemogenic impact. Studies aiming to decipher the biological mechanisms involved in hypodiploid subtypes of B-ALL with less than 40 chromosomes are crucial to improve the poor survival rates in these patients. Hypodiploidy with less than 40 chromosomes is a rare genetic abnormality in B-cell acute lymphoblastic leukemia (B-ALL). This condition can be classified based on modal chromosome number as low-hypodiploidy (30–39 chromosomes) and near-haploidy (24–29 chromosomes), with unique cytogenetic and mutational landscapes. Hypodiploid B-ALL with <40 chromosomes has an extremely poor outcome, with 5-year overall survival rates below 50% and 20% in childhood and adult B-ALL, respectively. Accordingly, this genetic feature represents an adverse prognostic factor in B-ALL and is associated with early relapse and therapy refractoriness. Notably, half of all patients with hypodiploid B-ALL with <40 chromosomes cases ultimately exhibit chromosome doubling of the hypodiploid clone, resulting in clones with 50–78 chromosomes. Doubled clones are often the major clones at diagnosis, leading to “masked hypodiploidy”, which is clinically challenging as patients can be erroneously classified as hyperdiploid B-ALL. Here, we summarize the main cytogenetic and molecular features of hypodiploid B-ALL subtypes, and provide a brief overview of the diagnostic methods, standard-of-care treatments and overall clinical outcome. Finally, we discuss molecular mechanisms that may underlie the origin and leukemogenic impact of hypodiploidy and may open new therapeutic avenues to improve survival rates in these patients.

Interleukin-9 Promotes Chemoresistance in B-ALL Which Correlates with Decreased Bim and c-Raf Protein Expression

Background: According to the National Cancer Institute, B-cell acute lymphoblastic leukemia (B-ALL) is the most common cancer of children and adolescents (ALL, NCI, PDQ, accessed 8/2/2021). Recently, obesity has been identified as a risk factor which is associated with poor survival outcomes (Butturini et al., 2007; Eissa et al., 2017; Ethier et al., 2012) which is concerning due to the obesity rates in children and adolescents having tripled since the 1970's (Ogden et al., 2006; Ogden et al., 2020). Indeed, survival rates in obese pediatric patients with B-ALL can decline by as much as 30% relative to outcomes observed in lean patients, with obese patients more commonly presenting with treatment-related adverse events (Butturini et al., 2007; Eissa et al., 2017; Ethier et al., 2012). A hallmark of obesity is the accumulation of adipocytes, an endocrine cell type which can promote chemoresistance (Ehsanipour et al., 2013; Sheng et al., 2016; Mittelman., 2021). The mechanistic understanding of how adipocytes promote chemoresistance in B-ALL is still under investigation and further insight into this relationship could lead to the rational design of effective therapeutic strategies for obese patients with limited treatment options.Methods: A cytokine/chemokine array was performed on adipocyte and stromal cell secretomes to identify potential adipocyte-secreted inflammatory mediators, which may promote chemoresistance in human B-ALL cells. Once candidate cytokines were identified, we performed in vitro assays to measure how the addition or neutralization of proteins of interest impacted the proliferation, activation of signaling pathways, steady-state mitochondrial protein levels, and survival of human B-ALL cells in the absence or presence of chemotherapy treatment. Additionally, we mined publicly available databases to determine how protein-coding genes of interest were associated with patient survival. Furthermore, we have used the diet-induced murine model of obesity to determine how targeting candidate cytokines impact B-ALL pathogenesis.Results: We have made the novel finding that interleukin-9 (IL-9) levels are higher in adipose-rich microenvironments and activates pro-survival pathways that promote chemoresistance in human B-ALL cells. We have found that obese mice lacking IL-9 are more resistant to B-ALL development due to significant increases in survival outcomes compared to lean mice transplanted with B-ALL cells. Furthermore, we have discovered that human B-ALL cells upregulate the interleukin-9 receptors (IL-9R) when exposed to the adipocyte secretome. This potential feedback loop may increase the responsiveness of leukemia cells to local IL-9 levels. These observations were supported by our data mining results, which revealed that IL-9R gene expression levels were higher in more aggressive subtypes of B-ALL, including Ph-like B-ALL. When human B-ALL cells were treated with recombinant IL-9 (rIL-9), chemoresistance to methotrexate and doxorubicin was observed. Mechanistically, rIL-9 treatment of human B-ALL cells also downregulated the protein expression of the pro-apoptotic mitochondrial-associated protein Bim and pro-proliferative protein Raf. In all, our experiments have identified IL-9 as an adipocyte-enriched cytokine, which promotes pan-chemoresistance in human B-ALL cells. Furthermore, we have shown that this effect maybe mediated in part by suppressing the protein of expression of pro-apoptotic and proliferative proteins.Conclusions: To our knowledge, our results represent the first reports of IL-9 mediated chemoresistance in human B-ALL and the first to demonstrate that IL-9 regulates the protein homeostasis of anti- and pro-apoptotic mitochondrial proteins. In ongoing studies, we are conducting in vitro and murine studies with parental and IL-9R-deficient B-ALL cells to determine how B-ALL pathogenesis and chemosensitivity are impacted. Subsequent studies will be conducted in lean and obese mice transplanted with B-ALL cells who receive chemotherapy treatment alone or in combination with IL-9 neutralizing antibody administration. DisclosuresLee: PureTech Health: Research Funding. Henry: PureTech Health: Research Funding.

Two novel high-risk adult B-cell acute lymphoblastic leukemia subtypes with high expression of CDX2 and IDH1/2 mutations

The genetic basis of leukemogenesis in adults with B-cell acute lymphoblastic leukemia (B-ALL) is largely unclear, and its clinical outcome remains unsatisfactory. This study aimed to advance the understanding of biological characteristics, improve disease stratification, and identify molecular targets of adult B-ALL. Adolescents and young adults (AYA) (15 to 39 years old, n = 193) and adults (40 to 64 years old, n = 161) with Philadelphia chromosome-negative (Ph−) B-ALL were included in this study. Integrated transcriptomic and genetic analyses were used to classify the cohort into defined subtypes. Of the 323 cases included in the RNA sequencing analysis, 278 (86.1%) were classified into 18 subtypes. The ZNF384 subtype (22.6%) was the most prevalent, with 2 novel subtypes (CDX2-high and IDH1/2-mut) identified among cases not assigned to the established subtypes. The CDX2-high subtype (3.4%) was characterized by high expression of CDX2 and recurrent gain of chromosome 1q. The IDH1/2-mut subtype (1.9%) was defined by IDH1 R132C or IDH2 R140Q mutations with specific transcriptional and high-methylation profiles. Both subtypes showed poor prognosis and were considered inferior prognostic factors independent of clinical parameters. Comparison with a previously reported pediatric B-ALL cohort (n = 1003) showed that the frequencies of these subtypes were significantly higher in AYA/adults than in children. We delineated the genetic and transcriptomic landscape of adult B-ALL and identified 2 novel subtypes that predict poor disease outcomes. Our findings highlight the age-dependent distribution of subtypes, which partially accounts for the prognostic differences between adult and pediatric B-ALL.

Recognition of Philadelphia chromosome‐like acute lymphoblastic leukemia as part of routine diagnostic work‐up

Philadelphia chromosome (Ph)-like acute lymphoblastic leukemia (ALL) is a biologically and clinically challenging subtype of B-cell ALL which has been incorporated into the 2016 revision of the World Health classification of acute leukemia. It is independently associated with poor outcome. As it can only be reliably detected by expression profiling, it is difficult to diagnose with routine methods. Its recognition has become of greater importance due to prognostication and even more due to the new diagnostic options given by targeted therapies. There is still no standardized diagnostic test enabling its prompt recognition. Here, we introduce our approach how to detect it by combination of widely available techniques. 179 ALL patients diagnosed in our center during the last 8years were included. Data on immunophenotype and cytogenetics were used to select patients with potentially Ph-like ALL (65/179). CRLF2 gene rearrangement (CRLF2-r) was tested by FISH in 59/65 patients, and next-generation sequencing was done by Archer FusionPlex ALL kit in 34 patients. TSLPR expression was determined in 20 patients. Philadelphia chromosome-like aberrations were confirmed in 9 patients. In 10% of tested samples, CRLF2-r was confirmed. Due to a lack of material, NGS was done only in a half of potentially Ph-like cases. In 10%, other Ph-like fusions were found by NGS. The obtained frequencies, and genetic and patients' characteristics are in concordance with the literature data, ensuring a reliable detection of this challenging ALL subtype. The proposed algorithm allows detection of Ph-like ALL at reasonable cost and acceptable workload.

IGF2BP3 Associates with Proliferative Phenotype and Prognostic Features in B-Cell Acute Lymphoblastic Leukemia.

Simple SummaryAlthough the prognosis of acute lymphoblastic leukemia (ALL) has improved significantly during the past decades, ALL remains a major cause of pediatric cancer mortality, and more accurate risk-stratification is required. We investigated IGF2BP3, which has previously been associated with aggressive cancers, and found high and subtype-specific expression of IGF2BP3 in B-cell ALL, that was associated with good outcome in high-risk patients. Results suggest that IGF2BP3 could be useful to improve stratification and prognosis of B-ALL.The oncofetal protein insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) belongs to a family of RNA-binding proteins involved in localization, stability, and translational regulation of target RNAs. IGF2BP3 is used as a diagnostic and prognostic marker in several malignancies. Although the prognosis of pediatric B-cell acute lymphoblastic leukemia (B-ALL) has improved, a subgroup of patients exhibits high-risk features and suffer from disease recurrence. We sought to identify additional biomarkers to improve diagnostics, and we assessed expression of IGF2BP3 in a population-based pediatric cohort of B-ALL using a tissue microarray platform. The majority of pediatric B-ALL cases were positive for IGF2BP3 immunohistochemistry and were associated with an increased proliferative phenotype and activated STAT5 signaling pathway. Two large gene expression data sets were probed for the expression of IGF2BP3—the highest levels were seen among the B-cell lymphomas of a germinal center origin and well-established (KMT2A-rearranged and ETV6-RUNX1) and novel subtypes of B-ALL (e.g., NUTM1 and ETV6-RUNX1-like). A high mRNA for IGF2BP3 was associated with a proliferative “metagene” signature and a high expression of CDK6 in B-ALL. A low expression portended inferior survival in a high-risk cohort of pediatric B-ALL. Overall, our results show that IGF2BP3 shows subtype-specificity in expression and provides prognostic utility in high-risk B-ALL.

Non-Coding RNA Signatures of B-Cell Acute Lymphoblastic Leukemia.

Non-coding RNAs (ncRNAs) comprise a diverse class of non-protein coding transcripts that regulate critical cellular processes associated with cancer. Advances in RNA-sequencing (RNA-Seq) have led to the characterization of non-coding RNA expression across different types of human cancers. Through comprehensive RNA-Seq profiling, a growing number of studies demonstrate that ncRNAs, including long non-coding RNA (lncRNAs) and microRNAs (miRNA), play central roles in progenitor B-cell acute lymphoblastic leukemia (B-ALL) pathogenesis. Furthermore, due to their central roles in cellular homeostasis and their potential as biomarkers, the study of ncRNAs continues to provide new insight into the molecular mechanisms of B-ALL. This article reviews the ncRNA signatures reported for all B-ALL subtypes, focusing on technological developments in transcriptome profiling and recently discovered examples of ncRNAs with biologic and therapeutic relevance in B-ALL.

B-ALL Complexity: Is Targeted Therapy Still A Valuable Approach for Pediatric Patients?

Simple SummaryB-ALL is the more frequent childhood malignancy. Even though significant improvements in patients’ survival, some pediatric B-ALL have still poor prognosis and novel strategies are needed. Recently, new genetic abnormalities and altered signaling pathways have been described, defining novel B-ALL subtypes.Innovative targeted therapeutic drugs may potentially show a great impact on the treatment of B-ALL subtypes, offering an important chance to block multiple signaling pathways and potentially improving the clinical management of B-ALL younger patients, especially for the new identified subtypes that lack efficient chemotherapeutic protocols. In this review, we shed light on the up-to-date knowledge of the novel childhood B-ALL subtypes and the altered signaling pathways that could become new druggable targets.B-cell acute lymphoblastic leukemia (B-ALL) is a hematologic malignancy that arises from the clonal expansion of transformed B-cell precursors and predominately affects childhood. Even though significant progresses have been made in the treatment of B-ALL, pediatric patients’ outcome has to be furtherly increased and alternative targeted treatment strategies are required for younger patients. Over the last decade, novel approaches have been used to understand the genomic landscape and the complexity of the molecular biology of pediatric B-ALL, mainly next generation sequencing, offering important insights into new B-ALL subtypes, altered pathways, and therapeutic targets that may lead to improved risk stratification and treatments. Here, we will highlight the up-to-date knowledge of the novel B-ALL subtypes in childhood, with particular emphasis on altered signaling pathways. In addition, we will discuss the targeted therapies that showed promising results for the treatment of the different B-ALL subtypes.