Establishment and chacterization of a novel cell line ICH-BCPALL-3 from B-cell precursor acute lymphoblastic leukemia with TCF3::HLF.

Establishment and chacterization of a novel cell line ICH-BCPALL-3 from B-cell precursor acute lymphoblastic leukemia with TCF3::HLF. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Establishment and chacterization of a novel cell line ICH-BCPALL-3 from B-cell precursor acute lymphoblastic leukemia with TCF3::HLF. Keisuke Kato, Ioannis Panagopoulos, Ai Yoshimi, Norihito Ikenobe, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5469901/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Nov, 2025 Read the published version in Human Cell → Version 1 posted 5 You are reading this latest preprint version Abstract In vitro models of acute leukemia are crucial for understandingits biology and developing effective treatments. The authors have established and characterized a novel cell line, ICH-BCPALL-3, which expresses the TCF3::HLF fusion from B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The karyotype of the cultured cells is 46,XY, der(1)(1qter->1q11::1p32->1q11::4q21->4qter), der(4)t(1;4)(q11;p32), add(8)(q24), del(17)(q24). Analysis of the diagnostic sample revealed deletions in RB1 , VPREB1 , and NR3C1 . The cell line showed additional deletions of VPREB1 , NR3C1 , and CDKN2A / 2B , as well as a gain of AKT1 . The loci for PAX5 and BTG1 were retained. Exome and Sanger sequencing identified nucleotide variants of ARID5B and NCOR1 in the diagnostic sample, as well as a KRAS variant (p.Lys117Asn) in the first recurrent sample and another KRAS variant (p.Asp119Gly) in the second recurrent sample and the cell line. Transcriptome analysis and RT-PCR confirmed that all examined samples contained a TCF3::HLF chimeric transcript. However, molecular cytogenetics did not verify the juxtaposition of TCF3 and HLF loci. Further long-range PCR analyses confirmed that genomic material containing HLF exon 4 was inserted into TCF3 intron 16. Using dimensional reduction techniques, we found that the current cell line shares an expression pattern with other TCF3::HLF -positive BCP-ALL cell lines. The cytotoxicity assay indicated that the cell line is sensitive to Aurora Kinase B inhibitor, but not to BCL2 inhibitor. This cell line is the first TCF3::HLF -positive BCP-ALL model without the t(17;19) translocation, facilitating research into leukemogenesis and the development of novel treatments for patients with poor prognosis associated with TCF3::HLF -positive BCP-ALL. B-cell precursor acute lymphoblastic leukemia TCF3:HLF oligoclonality cell line transcriptome cytotoxicity assay Figures Figure 1 Figure 2 Figure 3 Introduction Childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) includes various genetically defined subtypes, each with specific clinical characteristics and prognoses [ 1 ]. Modern treatment protocols require accurate classification of each case into its genomic subgroup, along with precise measurement of minimal residual disease (MRD) [ 1 ]. Further in-depth understanding of leukemogenesis tailored to each subtype enhances treatment strategies [ 2 ]. Thus, it is crucial to molecularly characterize each patient and to have a comprehensive understanding of novel insights into leukemogenesis [ 2 ]. The BCP-ALL subtype with the translocation t(17;19)(q21-q22;p13)/ TCF3::HLF accounts for less than 1% of childhood BCP-ALL cases [ 3 – 6 ]. It arises from an in-frame fusion between the TCF3 gene located on 19p13.3 and the HLF gene on 17q22, which is caused by the translocation. The resulting chimeric TCF3::HLF product functions as a transcription factor, utilizing the transactivation domains of TCF3 alongside a DNA-binding and dimerization basic leucine zipper (bZIP) domain from HLF [ 3 , 4 , 7 ]. Case series have shown that BCP-ALL with TCF3::HLF has distinct characteristics, including a precursor B-cell immunophenotype with co-expression of CD33, hypercalcemia, and symptomatic disseminated intravascular coagulation (DIC) [ 5 ]. Clinically, this subtype of BCP-ALL demonstrates a tendency for MRD persistence following induction therapy and is associated with an unfavorable prognosis, characterized by primary refractoriness or short intervals to relapse despite intensive chemotherapy [ 7 ]. As a result, BCP-ALL with TCF3::HLF is considered a clear indication for allogeneic hematopoietic cell transplantation (HCT) [ 5 , 8 ]. The development of preclinical models, such as patient-derived xenotransplantation models, has significantly advanced the creation of new and effective therapies, including immunotherapy and small molecule inhibitors [ 9 ]. However, BCP-ALL is heterogeneous, and not all subtypes can be successfully transplanted into xenograft models. Therefore, it remains vital to establish novel cell lines to study the tumorigenesis of specific categories of leukemia [ 10 ]. In this research, the authors encountered a BCP-ALL case with a pre-B ALL immunophenotype, which exhibited an obstinate clinical course. In this case, the authors confirmed the presence of the TCF3::HLF fusion gene and established a permanently growing cell line from the second recurrent sample. Method Clinical summary The index case is a 12-year-old male who was referred to the first author’s hospital due to hyperleukocytosis, with a peripheral white blood cell count of 99,500/mm³ at diagnosis. Hypercalcemia was not present. A bone marrow aspirate revealed a monotonous growth of peroxidase-negative lymphoblasts. The blasts were immunoreactive for CD10, CD19, CD33, TdT, CD79a, and surface immunoglobulin lambda chain, as well as the IgM chain, but were negative for CD20 and CD34. The karyotype was 46,XY. The initial analysis of the craniospinal fluid showed leukemic involvement. The patient was diagnosed with BCP-ALL with a pre-B immunophenotype. Initially, the patient received multi-agent chemotherapy, stratified for intermediate risk [8], and achieved complete remission after induction therapy. He completed the chemotherapy; however, one month after maintenance therapy ended, he developed an osteolytic lesion in the right femur, which was confirmed by biopsy to be an extramedullary bone recurrence. Concurrently, imaging and an open biopsy revealed a bilateral testicular recurrence. Following the report, he underwent multi-agent chemotherapy with minor modifications [11]. Unfortunately, the lesions did not resolve. Due to the leukemic cells displaying a mature B-cell-like immunophenotype, characterized by surface light chain expression, we opted for multi-agent chemotherapy for refractory mature B-cell lymphoma and focal irradiation [12]. This treatment resulted in complete remission. After consolidation treatment with inotuzumab ozogamicin, the patient underwent allogeneic bone marrow transplantation from an HLA 8/8 matched unrelated donor, using a preconditioning regimen that included total body irradiation, fludarabine, and melphalan. Given the high risk of relapse, we planned to administer blinatumomab to prevent recurrence [13]. However, a bone marrow aspiration confirmed a second recurrence 62 days after HCT, coinciding with the day we intended to start blinatumomab. Despite this, we proceeded with the treatment plan. Unfortunately, the patient succumbed to a rapidly deteriorating disease 64 days after HCT. Cell culture and flow cytometry We began by culturing mononuclear cells from the peripheral blood during the second relapse. The cells were placed in a plastic culture flask containing RPMI 1640 Medium, supplemented with GlutaMAX™ (Gibco™, Thermo Fisher Scientific, Waltham, MA, USA), 10% heat-inactivated fetal bovine serum (FBS, Gibco™), and penicillin/streptomycin. The culture was maintained in a humidified atmosphere at 37°C with 5% CO2. The authors analyzed the immunophenotype of the cultured cells passage 25 using flow cytometry, following methods previously reported [14]. Authenticity confirmation of the cell line We confirmed the authenticity of the cultured cell line by analyzing polymorphic markers, including AMELX / AMELY , CSF1R , D16S539, D13S317, D5S818, D7S820, TH , TPO , VWF , D2S1338, D3S1358, D8S1179, D18S51, D19S253, D20S438, and D21S11, using fluorescence-conjugated specific primer pairs ( Table S1 ). Since the second relapse sample contained both leukemic and HCT donor cells, we chose the testicular relapse sample as the reference for cell authenticity. The primer sequences are listed in Table S1. Next, we assessed the status of the immunoglobulin heavy ( IGH ) and light ( IGK , IGL ) chains, as well as the T cell receptor ( TRG , TRD ) genes in the initial diagnostic sample, the testicular recurrent sample, the second relapse sample, and the cultured cells at passage 30. This analysis was performed using polymerase chain reaction (PCR) with specific primer pools and a heteroduplex mobility assay, following the BIOMED-2 protocol [15]. The rearranged alleles identified on polyacrylamide gel electrophoresis were excised and directly sequenced after purification. Exome and transcriptome analyses Next, we analyzed diagnostic and recurrent testicular samples using exome sequencing according to the manufacturer's instructions. We prepared an exome library with the AmpliSeq Exome kit and sequenced the template constructed from this library using the Ion PI™ Hi Q™ Sequencing 200 Kit (Thermo Fisher Scientific). The variants detected were confirmed through Sanger sequencing and subcloning of the PCR product. To investigate the genotype of the current case, we performed transcriptome analysis on the diagnostic sample. An RNA sequencing library was created using the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech Laboratories, Inc., Mountain View, CA, USA) and the Ion Xpress™ Plus Fragment Library Kit (Thermo Fisher Scientific). The templates from this library were sequenced using the Ion PI™ Hi-Q™ Sequencing 200 Kit and the Ion Proton (Thermo Fisher Scientific). The single-end 200-bp reads in FASTQ format from the Ion Proton were analyzed using STAR-Fusion version 1.9.1-0, built on 64-bit Ubuntu 20.04 LTS [16]. We confirmed the candidate fusion gene through reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing of the amplified product. The primer sequences used were: forward primer 5'-CTACGACGGGGGTCTCCAC-3' and reverse primer 5'-CATGTTGTCCAGCCGCATCAG-3'. Genomic analysis on array comparative genomic hybridization The authors conducted a comprehensive analysis of genomic copy number alterations on the diagnostic sample and the established cell line at passage 13. This was performed using array comparative genomic hybridization (aCGH) with the CytoSure Consortium Cancer + Single Nucleotide Polymorphism arrays (Oxford Gene Technology IP Limited, Begbroke, Oxfordshire, UK), following the manufacturer's recommendations [17]. The resulting data were analyzed using Agilent Feature Extraction Software (Agilent Technologies; version 10.7.3.1) and CytoSure Interpret Software (version 4.9.40, Oxford Gene Technology) [17]. Cytogenetic and molecular cytogenetic analysis and additional transcriptome study and long range genomic PCR The authors conducted a molecular cytogenetic study to confirm the juxtaposition between the TCF3 and HLF loci. First, A metaphase spread from the cell line at passage 23 was analyzed, utilizing G-band karyotyping with Leishman's stain (Sigma-Aldrich, St. Louis, Missouri, USA) [18]. The karyotype was described according to the guidelines of the International System for Human Cytogenomic Nomenclature (2020) [19]. Additionally, the karyotype at passage ten was analyzed by spectral karyotyping (SKY) [20]. Then, the authors examined metaphase and interphase spreads using three-color fluorescence in situ hybridization with commercially available probes, specifically the CytoCell TCF3/PBX1 Plus Translocation Dual Fusion Probe and the CytoCell HLF Breakapart FISH Probe (Oxford Gene Technology), following the manufacturer's protocol [21]. Due to the inability of molecular cytogenetic analysis to confirm the juxtaposition of the TCF3 and HLF loci, RNA sequencing was performed once again. This sequencing was carried out according to the TruSeq paired-end RNA sequencing protocols using a Genome Analyzer IIx with a paired-end module. Total RNA was extracted from the ICH-BCPALL-3 cell line (passage 30) and sent to the Genomics Core Facility at the Norwegian Radium Hospital, Oslo University Hospital, for RNA sequencing [22]. The paired-end 150 bp reads in FASTQ format were analyzed using FusionCatcher version 1.33 [23]. To determine if genomic material from HLF exon 4 was inserted into the TCF3 intron 16, long-range PCR was carried out using primer pairs listed in Table S2 . Genomic PCR amplifications were conducted in a 25 μl reaction volume, which included 12.5 μl of Premix Ex Taq™ DNA Polymerase Hot Start Version (Takara Bio Europe/SAS, Saint-Germain-en-Laye, France), 100 ng of genomic DNA from the ICH-BCPALL-3 cell line, and 0.4 μM of each forward and reverse primer. The primer combinations utilized were TCF3-Intr16F1 + TCF3-Exon17R1 and TCF3-Intr16F2 + TCF3-Exon17R2. The PCR cycling conditions included an initial denaturation step at 94˚C for 30 seconds, followed by 35 cycles of 7 seconds at 98˚C and 2 minutes at 68˚C, and a final extension at 68˚C for 5 minutes. Three μl of the PCR products were stained with GelRed (Biotium, Fremont, CA, USA), analyzed by electrophoresis through a 1% agarose gel, and photographed. DNA gel electrophoresis was performed using lithium borate buffer [24]. The remaining PCR products were purified using the MinElute PCR Purification Kit (QIAGEN GmbH, Hilden, Germany) and directly sequenced using the dideoxy procedure with the BigDye terminator v1.1 cycle sequencing kit, following the manufacturer's recommendations (ThermoFisher Scientific). The primers used for cycle sequencing were the same as those employed in genomic PCR, along with additional primers HLF-1266R1, HLF-1300R1, HLF-1464F1, HLF-1423F1, HLF-4073F1, and HLF-4292F1 (see Table S2 ). Transcriptomic landscape of the established cell line The whole transcriptome of the established cell line was compared to those of other characterized cell lines, using the transcriptome data deposited in the public repository summarized in Table S3 [25]. The FASTQ-formatted data were pre-processed using fastp (version 0.24.0) [26], and then aligned to reference genome GRCh38.p13 using STAR (version 2.7.10b). The read count data were generated by RSEM (version 1.3.1) [27]. This read count data was then normalized with edgeR [28]. Initially, we investigated differentially expressed genes between five TCF3::HLF positive cell lines, including ICH-BCPALL-3, and TCF3::PBX1 positive cell lines. This analysis was performed using the edgeR package (version 4.0.16) on OlvTools (https://olvtools.com/, BxINFO, Tokyo, Japan). Genes with a log fold change greater than 1 and a false discovery rate (FDR) of less than 0.05 were considered significant. For functional enrichment analysis, we utilized the clusterProfiler package (version 4.10.1) to examine over-represented Gene Ontology (GO) terms and pathways on OlvTools [29]. It is when adjusted p-value is less than 0.05, GO terms within the Biological Process, Molecular Function, and Cellular Component categories were deemed significant. Similarly, of the 953 human-curated pathways analyzed, those with an adjusted p-value of less than 0.05 were considered significant as well. Next, we calculated similarity and difference in terms of the gene expression signature and classified the cell lines into the clusters using hierarchical clustering on iDEP (version 2.01) [30] and Uniform Manifold Approximation and Projection (UMAP) (version 0.2.10.0) [31]. Cytotoxicity assay The efficacy of sixty agents was evaluated on an established cell line, including cytotoxic agents typically used in leukemia chemotherapy and small molecules targeting proliferation signaling ( Table S4 ), based on studies by Goto A et al. and Szulkin A et al [32,33]. We seeded 10,000 cells from either passage 14 or passage 21 into 20 μL of culture medium containing test agents (StemSpan™ Serum-Free Expansion Medium II and StemSpan™ CC100, STEMCELL Technologies, Vancouver, Canada) in each well of a 384-well plate. The concentrations of each agent examined included the starting concentration, as well as 1:5, 1:25, and 1:125 dilutions. In the control wells, RPMI medium without the drug was added. After incubating for 4 days in a humidified environment at 37 °C and 5% CO2, the cell viability in each well was measured using the Cell Titer-Glo luminescent assay. The luminescence values were obtained using the Glo-Max plate reader (Promega, Madison, WI, USA) following the manufacturer's instructions. Drug sensitivity was expressed by the Drug Effect Score (DES), an index reflecting the concentration-weighted susceptibility of the test agents. For instance, DES100 indicates complete cell kill at all tested concentrations, whereas DES0 signifies no effect of the test drug at any concentration. Peripheral blood mononuclear cells (PBMC) from nine healthy volunteers were also included in the drug sensitivity test to provide reference values for DES. An agent was deemed efficacious if the DES for the cell line was greater than 10, and the ratio of the DES of the cell line to the DES of reference PBMC was greater than 1 [34]. Results Cell culture and flow cytometry Mononuclear cells were obtained and purified from peripheral blood during the second relapse using a density gradient. The cultured cells proliferated rapidly for over 70 passages, leading to the establishment of the cell line designated as ICH-BCPALL-3 ( Fig. 1a ). The doubling time for this established cell line is approximately 24 hours ( Fig. S1 ). The immunophenotype of the established cell line differed slightly from that of the initial diagnostic sample. The cultured cells were immunoreactive for CD10, CD11b (weak), CD13 (weak), CD19, CD22, CD24, CD33, CD66c, TdT, and cytoplasmic IgM, but were negative for CD20, CD34, surface IgM, and surface immunoglobulin lambda chain ( Table S5 and Fig. S2 ). Authenticity confirmation of the cell line The pattern of polymorphic markers was nearly identical to that of the first relapse sample, with the exception that the AMELY locus was deleted in the cell line ( Fig. S3 ). The diagnostic sample, the first recurrent sample, the second relapse sample, and the established cell line at passage 70 all demonstrated the same rearrangement patterns at the IGH , IGK , and IGL loci. Direct sequencing revealed IGHV3-43_IGHD3-10_IGHJ6, IGKV4-1_KDE, and IGLV1-47_IGLJ3 ( Fig. S4a ). The clone-specific insertions at the IGH V-D junction were GGTTTGAGG; for the IGH D-J junction, it was CCT; and for the IGL V-J junction, it was TG ( Fig. S4b ). These findings confirm the authenticity of the established cell line. Genomic analysis on array comparative genomic hybridization The analysis on aCGH confirmed the loss of the AMELY locus (chrY: 6,733,959-6,742,068 on GRCh37/hg19) in the cell line ( Figs. S5 and S6 ). The copy number alteration analysis using aGGH showed a homozygous loss at the RB1 locus, specifically at chr13:48879236-48942672 on GRCh37/hg19 for RB1 exons 2-10, chr13:48943195-48953428 for exons 12 and 13, and chr13:48954317-49159971 for exons 18-27. Additionally, there was a copy number neutral loss of heterozygosity at the BCL6 locus (chr3:183900308-188605455 on GRCh37/hg19) at the time of diagnosis ( Figs. S5 and S6 ). Interestingly, the RB1 locus was maintained in the established cell line (passage 13), as confirmed by copy number analysis based on exome data from both the diagnostic and recurrent testicular samples ( Figs. S5, S6, and S7a ). In terms of genes encoding transcription factors associated with lymphoid differentiation and immunoglobulin rearrangement, the loci for PAX5 and BTG1 were retained in the diagnostic sample. However, the locus containing VPREB1 and IGL was deleted (chr22:22389514-22517319 on aCGH ( Figs. S5 and S6 ). Furthermore, copy number analyses using aCGH and exome data indicated the deletion of the MLLT3 and CDKN2A/2B loci, which were observed only in the cell line (chr9:20624804-21994149, chr9:21994905-22005861, and chr9:22006328-22399305 on aCGH and chr9:20353464-20448297 and chr9:20456668-22009065 based on exome data) ( Figs. S5, S6, and S7b ). In addition, aCGH revealed a deletion of the NR3C1 gene located on chromosome 5 (142796870-143184287) in both the diagnostic sample and the cell line ( Figs. S5 and S6 ). Furthermore, the cell line exhibited a gain of the AKT1 gene, as well as a gain at 3q27, which includes the BCL6 and MECOM genes ( Fig. S6 ). Exome and transcriptome analyses Subsequently, exome analysis and Sanger sequencing identified somatic variants in ARID5B (NM_032199.3:c.39_44delinsGGAGAAG; NP_115575.1:p.Cys13TrpfsTer18) ( Fig. S8a ) and NCOR1 (NM_006311.4:c.5380C>T; NP_006302.2:p.Arg1794*) present at the time of diagnosis ( Fig. S8b ). To evaluate the clonal composition throughout the relapse and cell cultivation process, exome analysis was conducted on the testicular relapse sample, a second relapse sample, and the established cell line. This analysis revealed a KRAS amino acid substitution variant in the testicular relapse sample (NM_004985.5:c.356A>G; NP_004976.2:p.Asp119Gly). Impressively, we identified a different KRAS variant in the second recurrent bone marrow sample and the cultured cells (NM_004985.5:c.351A>T; NP_004976.2:p.Lys117Asn) ( Fig. S8c ). Subsequently, whole transcriptome analysis on the diagnostic sample and the cell line revealed junctional reads spanning the TCF3 and HLF genes using STAR-fusion and FusionCatcher. The RT-PCR and direct sequencing of the RT-PCR product revealed an in-frame fusion of TCF3 exon 16 and HLF exon 4 in the diagnostic sample, the second relapse sample, and the established cell line ( Fig. 1c ). The chimeric transcript contained an unidentified 11-base sequence, CGGACCGTCAG, between TCF3 exon 16 and HLF exon 4 ( Fig. 1c ). Cytogenetic and molecular cytogenetic analysis and additional transcriptome study and long range genomic PCR The G-band karyotype of the cultured cell at passage 30 was 46,XY,der(1)(1qter->1q11::1p32->1q11::4q21->4qter),der(4)t(1;4)(q11;p32),add(8)(q24),del(17)(q24) ( Fig. 1b ). The SKY analysis partially identified the unknown material, revealing the following finding: 46,XY,t(1;4)(q21;q11)(wcp1+,wcp4+),der(8)t(3;8)(p?;q?)(wcp3+,wcp8+). Notably, the abnormality in chromosome 17 was not confirmed during the SKY analysis ( Fig. S9 ). However, molecular cytogenetics performed on the metaphase spread, using a commercially available probe set, could not confirm the juxtaposition of the TCF3 and HLF loci in the cultured cells ( Fig. 1d ). To further characterize the present case, long-distance PCR was employed to amplify and sequence of TCF3 intron 16 and exon 17. This analysis ultimately demonstrated the insertion of a fragment composed of a portion of HLF intron 3 and a part of HLF exon 4 into TCF3 intron 16, with a segment of the inserted HLF exon 4 sequence being deleted ( Fig. 2, Fig. S10a, Fig. S10b, and Fig. 3 ). At the genomic junctions, there are sequences of unknown origin: a triplet (CGT) at the 5ˊ genomic junction between intron 16 of TCF3 and intron 3 and exon 4 of HLF , and a 14-base sequence (CACAAACCCCCAAA ) at the 3ˊ genomic junction between exon 4 of HLF and intron 16 of TCF3 ( Fig. 3) . The 11-base sequence CGGACCGTCAG between the TCF3 exon 16 and HLF exon 4 in the chimeric transcript consists of the last five bases (CGGAC) in the terminus of TCF3 intron 16, the unknown origin triplet (CGT), and a portion of HLF intron 3 (CAG) (indicated by box characters in Fig. 3 ). The genomic breakpoint in TCF3 is located near a CpG element and activation-induced cytidine deaminase (AID) consensus sequence lacking the classical RAG consensus sequence sites (CAC). The 5ˊ breakpoint of the inserted HLF material is situated directly on the CpG element, while the 3ˊ breakpoint of the HLF material is situated 11 base pairs away from the CpG element ( Fig. S10c ). Transcriptomic landscape of the established cell line The authors utilized whole transcriptome data to investigate differentially expressed genes between TCF3::HLF positive cell lines and TCF3::PBX1 positive cell lines. Among the genes that were upregulated in the TCF3::HLF positive cell lines are KLHL13 , CLSTN2 , HLF , HPGD , MMP25 , RUBCNL , B3GNT7 , C3AR1 , FCGR1A , KLRK , MCOLN2 , H2AC13 , and H2BC4 , as shown in the volcano plot and MA plot ( Fig. S11a and S11b, and Table S6 ). Conversely, the genes that were downregulated in TCF3::HLF positive cell lines include PHACTR3 , RORB , BEND4 , PBX1 , WNT16 , TCERG1L , and GREM1 , as illustrated in the volcano plot and MA plot ( Fig. S11a and S11b, and Table S6 ). The gene ontogeny analysis identified that several Gene Ontology (GO) terms are over-represented in TCF3::HLF -positive leukemia cell lines compared to TCF3::PBX1 -positive leukemia cell lines. These terms include: - **Molecular Function**: structural constituent of chromatin, GTPase regulator activity, nucleoside-triphosphatase regulator activity, protein heterodimerization activity, transmembrane signaling receptor activity, structural molecule activity, calcium channel activity, calcium-release channel activity, intracellular ligand-gated monoatomic ion channel activity, and ligand-gated calcium channel activity ( Fig S12a and Table S7a ). - **Cellular Component**: nucleosome and MHC class II protein complex ( Fig S12b and Table S7b ). - **Biological Process**: regulation of cell adhesion, transmembrane receptor protein tyrosine kinase signaling pathway, chemotaxis, cell chemotaxis, defense response to bacterium, nucleosome assembly, nucleosome organization, mucosal immune response, and organ- or tissue-specific immune response ( Fig S12c and Table S7c ). Additionally, pathway analysis indicated that two pathways, the Senescence Associated Secretory Phenotype (SASP) and histone modifications, are over-represented in TCF3::HLF -positive leukemia cell lines compared to TCF3::PBX1 -positive leukemia cell lines ( Fig S13 and Table S8 ). The authors then sought to classify the cell lines using mathematical methods based on the transcriptome dataset. Nonlinear dimensionality reduction algorithms, specifically UMAP, demonstrated that TCF3::HLF -positive leukemia cell lines are distinct from other cell lines ( Fig S14 ). Hierarchical cluster analysis further revealed a unique cluster of five TCF3::HLF -positive leukemia cell lines, as illustrated by the dendrogram ( Fig S15 ). Cytotoxicity assay The cytotoxicity assay revealed that ICH-BCLALL-3 is significantly vulnerable to the cytotoxic agents, including SN-38 (derivative of irinotecan), cytarabine, etoposide, docetaxel, topotecan, and gemcitabine ( Table S9 ). Furthermore, the cell line is sensitive to small molecule inhibitors for cell signal transduction pathways, including inhibitors for mitogen-activated protein kinase kinase 1/2 (MEK1/2), trametinib and selumetinib, inhibitors for mechanistic/mammalian target of rapamycin complex 1 (mTORC1), everolimus and rapamycin, dual inhibitors for phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR), PI-103 and PF-04691502, an inihibitor for checkpoint kinase 1, AZD7762, and an inhibitor for Aurora Kinase B (AURKB) inhibitors, barasertib ( Table S9 ). Discussion Accurate molecular diagnosis is essential for the clinical management of BCP-ALL [1]. Whole transcriptome sequencing provides precise molecular diagnoses, allowing for proper patient stratification, although it does not apply to every patient. Consequently, BCP-ALL cases with rare molecular classifiers that cannot be confirmed using existing diagnostic tests face the risk of being assigned inappropriate treatment plans. In this particular case, the absence of hypercalcemia and severe disseminated intravascular coagulation (DIC) led to the decision not to conduct RT-PCR screening for TCF3::HLF , resulting in a missed opportunity for accurate diagnosis and stratification. While positive expression of CD33 may suggest the presence of TCF3::HLF [5], the mature B-cell-like immunophenotype observed complicated the diagnostic process. Clinicians should be cautious and consider TCF3::HLF -positive BCP-ALL when encountering a BCP-ALL case that expresses CD33, because BCP-ALL with TCF3::HLF is extremely unfavorable without HCT [5,6,8]. Molecular and cell physiological analyses revealed a complex execution of abnormal genetic alterations [6]. Comprehensive molecular analysis of BCP-ALL featuring the TCF3::HLF fusion demonstrated numerous molecular changes, including alterations in the BCL2 , LMO2 , PAX5 , and RAS pathway genes, alongside TCF3::HLF . These changes lead to distinctive clinical presentations, such as resistance to chemotherapy [6]. Moreover, the transition from pro-B cells to pre-B cells is significantly disrupted due to deletions of several genes that encode transcription factors critical for lymphoid differentiation and immunoglobulin rearrangement [6]. These include PAX5 , BTG1 , VPREB1 , and NCOR1 [6]. In line with previous studies, this case also exhibited alterations in VPREB1 and NCOR1 in the diagnostic sample ( Fig.S8 ). Notably, alterations in ARID5B and RB1 were also observed in the diagnostic sample ( Fig. S5 , Fig. S7, and Fig. S8 ). These genes are involved in regulating epigenetic processes and the cell cycle, suggesting they contribute to the unique clinical features of this case [35,36]. More interestingly, these alterations were not present in the recurrent samples ( Fig. S6 ), but deletion of CDKN2A/2B emerged in the cell line ( Fig.S6 and Fig.S7 ). Furthermore, the first recurrent sample exhibited the KRAS mutation c.356A>G, resulting in the p.Asp119Gly alteration, while the second recurrent sample showed the KRAS mutation c.351A>T, leading to the p.Lys117Asn change ( Fig.S8 ). These events (alterations of ARID5B , NCOR1 , RB1 , CDKN2A/2B , and KRAS ) are regarded as secondary event, highlighting the oligoclonal nature of TCF3::HLF positive leukemia as indicated in the previous study [6] and the strong driver capabilities of the TCF3::HLF oncoprotein. The heterogeneous clonal composition and progression of the disease explain why alterations in the ARID5B , NCOR1 , and RB1 genes were not present in the recurrent sample, while changes in CDKN2A/2B and KRAS emerged. Furthermore, these findings indicate that alterations in the epigenetic machinery, cell cycle regulators, and signaling pathways related to cell proliferation collectively contribute to the progression of TCF3::HLF -positive BCP-ALL under the TCF3::HLF driver capacity. Additionally, the current case exhibited copy number neutral loss of heterozygosity at the 3q27 locus, which contains the BCL6 gene, at the time of diagnosis ( Fig. S5 ). There was also a gain of 3q27 harboring the MECOM and BCL6 genes in the cell line ( Fig. S6 ). The exact significance of the 3q27 abnormality remains unclear; however, these genetic alterations may contribute to the unusual immunophenotype seen in this case, because alteration of BCL6 is frequently encountered in mature B-cell neoplasm. Furthermore, the present case has a hemizygous deletion of NR3C1 , which may lead to refractoriness to chemotherapy [37]. Finally, the cell line demonstrated a gain of AKT1 ( Fig. S6 ), which could facilitate the progression of leukemic cells [38]. Overall, this cell line provides a unique opportunity to explore the relationship between these genetic abnormalities and clinical characteristics within the context of TCF3::HLF tumorigenesis, potentially advancing the development of molecular targeted therapies. The present study has molecularly characterized the breakpoints on TCF3 in a cell line exhibiting the TCF3::HLF fusion gene. This raises interest in the mechanism responsible for the insertion that creates the TCF3::HLF fusion. To date, four TCF3::HLF -positive cell lines have been reported: YCUB-2, HAL-01, UOC-B1, and Endo-kun, all of which contain the t(17;19) karyotype [39-42]. In contrast, the current cell line exhibited neither der(19) nor t(17;19) on karyotyping, nor any juxtaposition of the TCF3 locus with the HLF locus in molecular cytogenetic analyses. Further molecular studies revealed that this cell line contained an insertion of HLF exon 4 material into TCF3 intron 16. A similar observation was noted in the study conducted by Salim M et al [43]. The breakpoint of TCF3 typically occurs near CpG sites, a characteristic feature of translocations that happen in lymphoid progenitors at the pro-/pre-B stage, as demonstrated in the studies by Tsai et al [44]. Therefore, it is intriguing to confirm whether the current case demonstrates that the TCF3 breakpoints are located near CpG sites. A study by Liu et al. found that methylated cytosines within the WRCG motif (where W = A or T, and R = A or G) in high cytosine density string are crucial in producing double-strand breaks, which are essential for translocation [45]. The formation of single strands during transcription or due to thermal dynamic fluctuations increases the deamination of methylated cytosine in the WRCG motif by AID [45]. Additionally, the long-lasting T:G mismatch leads to double-strand breaks, facilitating translocation [45]. In the cell line being studied, the breakpoint of TCF3 intron 16 is located very close to the WRCG motif (specifically AGCG , positioned near the end of upstream TCF intron 16, highlighted in bold italic letters in Fig. 3 ). According to the theory proposed by Liu D, the cytosine in the TCF3 intron 16 sequences on the chromosome 19 + strand is thought to have undergone methylation, resulting in its conversion to thymine (T) by AID in this case (the red bold C , to which " Me " was added in Fig. 3 ). In this case, it was observed that GAC sequences are present at the end of the upstream TCF3 intron 16 sequence and at the beginning of the downstream TCF3 intron 16 sequence (highlighted with a red background in Fig. 3 ). This suggests that the putative double-strand break in TCF3 intron 16 has created a three-base overhang of GAC on the - strand and a GTC overhang on the + strand of TCF3 intron 16 (highlighted with a blue background in Fig. 3 ). These overhangs have fused with the inserted HLF material and were repaired into a double strand through the classical non-homologous end-joining system [46]. The genomic 5' breakpoint of the inserted HLF material is located on a CpG site; however, the 3' breakpoint is not ( Fig. S10c ). There are no RAG consensus sequence sites (CAC) or repetitive sequences near the genomic 3' breakpoint of the inserted HLF material according to the database (https://genome.ucsc.edu/). Therefore, the root cause of the double-strand break formation associated with the HLF insertion remains partially unclear. The TCF3::HLF fusion gene found in this case contains a stop codon in the inserted HLF exon 4 sequence, suggesting that it is functional. The authors have discovered that the current cell line, ICH-BCPALL-3, shares an expression signature with four other TCF3::HLF -positive BCP-ALL cell lines, as shown by hierarchical clustering and UMAP analysis ( Figs. S13 and S14 ). This finding supports the idea that ICH-BCPALL-3 is a reliable in vitro model for TCF3::HLF -positive BCP-ALL, which can be used to develop novel agents aimed at improving the prognosis of this condition. Furthermore, the expression data analysis identified several over-represented cellular processes and signaling pathways, including the transmembrane receptor protein tyrosine kinase signaling pathway and histone modifications ( Table S7c and Table S8 ). The study also revealed potential active agents for TCF3::HLF -positive BCP-ALL, such as inhibitors targeting the PI3K/Akt/mTOR pathway, including everolimus, rapamycin, PI-103, and PF-04691502 ( Table S9 ) [38,47,48]. The observed susceptibility to PI3K/Akt/mTOR pathway inhibitors strongly correlates with the pronounced over-representation of the transmembrane receptor protein tyrosine kinase signaling pathway identified in the gene ontology analysis of ICH-BCLALL-3. This compelling connection underscores the pivotal role of this signaling pathway in the context of targeted therapeutic strategies. ( Fig. S12c and Table S7c ). Indeed, inhibitors of the PI3K/Akt/mTOR pathway have been tested in in vitro assays and early-phase trials for BCP-ALL [38, 47, and 48], highlighting their promising potential for clinical application in TCF3::HLF -positive BCP-ALL. フォームの始まりフォームの終わりThe sensitivity of the ICH-BCPALL-3 cell line to MEK1/2 inhibitors, such as trametinib and selumetinib, likely stems from somatic variants of the KRAS (Table S9) [49]. However, the potential oligoclonal composition of KRAS in this model raises concerns, as it could contribute to the early onset of resistance to these crucial MEK1/2 inhibitors. Regarding the Bcl-2 inhibitor venetoclax, two previous studies have independently confirmed that clinical cases with the TCF3::HLF fusion exhibit vulnerability to this drug [6,50]. However, the ICH-BCPALL-3 cell line does not show the expected efficacy of venetoclax when evaluated using the current cytotoxicity assay ( Table S9 ). Evidence suggests that increased mitochondrial activity may contribute to venetoclax resistance [51]. Nevertheless, the current study has not confirmed a significant over-representation of mitochondrial activity in the gene ontology analysis and pathway analysis. This lack of confirmation may be due to the inclusion of other TCF-HLF -positive BCP-ALL cell lines. As a result, the mechanisms behind venetoclax resistance in the ICH-BCPALL-3 cell line remain unclear and require further investigation. Finally, the authors identified that ICH-BCPALL-3 is sensitive to the AURKB inhibitor barasertib ( Table S9 ). AURKB is a protein that plays a crucial role in regulating cell division, and its inhibition can lead to polyploidy and cellular senescence. Overexpression of this protein in cancer cells is associated with cancer progression and resistance to radiotherapy and chemotherapy. Notably, barasertib also demonstrates activity in the TCF3::HLF -positive BCP-ALL cell line, YCUB-2, offering hope for its potential incorporation into clinical usage [32]. In vitro models play a crucial role in uncovering detailed molecular signatures and developing innovative therapies [9,10]. The previous and current drug sensitivity assays indicated that BCP-ALL with the TCF3::HLF fusion may be susceptible to AURKB inhibitors. However, this finding needs to be validated across a broader range of preclinical models before it can be applied to human clinical trials. The distinct molecular characteristics of TCF3::HLF -positive BCP-ALL highlight the significance of the current model, allowing researchers to evaluate the efficacy of molecular targeting agents in ICH-BCPALL-3, which will soon be available. Declarations Data availability statement : This published article and its supplementary information files include all data generated or analyzed during this study. The fastq-formatted exome data and transcriptome data will be deposited in the Sequence Read Archive of DNA Data Bank of Japan. The cell line, ICH-BCPALL-3 will be available from RIKEN BioResource Research Center (Tsukuba, Japan). The cell number is RCB6089. Funding This investigation was supported by a subsidy from the Science and Technology Promotion Locating Electronic Power Plant from the Ministry of Education, Culture, Sports, and Technology (provided to K.K.) and a grant, JSPS KAKENHI (Grant Number JP24K11571, provided to K.K.). Conflict of interest disclosure The authors declare no conflicts of interest in the current study. Ethics approval The experimental design and procedure were approved by the Institutional Review Board of Ibaraki Children's Hospital (IRB approval number 1IRB-33) in accordance with Helsinki Declaration . Informed consent We have obtained the informed consent from the guardian of the patient. References Jeha S, Choi J, Roberts KG, Pei D, Coustan-Smith E, Inaba H, et al. Clinical significance of novel subtypes of acute lymphoblastic leukemia in the context of minimal residual disease-directed therapy. Blood Cancer Discov. 2021;2:326-337. doi: 10.1158/2643-3230.BCD-20-0229. 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Venetoclax resistance in acute lymphoblastic leukemia is characterized by increased mitochondrial activity and can be overcome by co-targeting oxidative phosphorylation. Cell Death Dis. 2024;15:475. doi: 10.1038/s41419-024-06864-7. Supplementary Files FigureS10submitted.pptx FigureS11submitted.pptx FigureS12submitted.pptx FigureS13submitted.pptx FigureS14submitted.pptx FigureS15submitted.pptx FigureS1submitted.pptx FigureS2submitted.pdf FigureS3submitted.pptx FigureS4submitted.pptx FigureS5submitted.pdf FigureS6submitted.pdf FigureS7submitted.pptx FigureS8submitted.pptx FigureS9submitted.pptx TableS1submitted.docx TableS2submitted.docx TableS3submitted.xlsx TableS4submitted.xlsx TableS5submitted.xlsx TableS6submitted.xlsx TableS7submitted.xlsx TableS8submitted.xlsx TableS9submitted.xlsx Cite Share Download PDF Status: Published Journal Publication published 19 Nov, 2025 Read the published version in Human Cell → Version 1 posted Editorial decision: Accept as is 22 Apr, 2025 Reviewers agreed at journal 18 Apr, 2025 Reviewers invited by journal 18 Apr, 2025 Editor assigned by journal 18 Apr, 2025 First submitted to journal 11 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Igakuiki","correspondingAuthor":false,"prefix":"","firstName":"Minori","middleName":"","lastName":"Tamai","suffix":""},{"id":444970111,"identity":"39e344df-0ba4-4947-a571-7244fccc2b34","order_by":14,"name":"Takeshi Inukai","email":"","orcid":"","institution":"Yamanashi Daigaku - Igakubu Campus: Yamanashi Daigaku Igakubu Daigakuin Sogo Kenkyubu Igakuiki","correspondingAuthor":false,"prefix":"","firstName":"Takeshi","middleName":"","lastName":"Inukai","suffix":""},{"id":444970112,"identity":"6703d223-c258-44b3-bc3d-92cb9a027a07","order_by":15,"name":"Koshi Akahane","email":"","orcid":"","institution":"Yamanashi Daigaku - Igakubu Campus: Yamanashi Daigaku Igakubu Daigakuin Sogo Kenkyubu Igakuiki","correspondingAuthor":false,"prefix":"","firstName":"Koshi","middleName":"","lastName":"Akahane","suffix":""}],"badges":[],"createdAt":"2024-11-17 12:52:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5469901/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5469901/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s13577-025-01318-4","type":"published","date":"2025-11-19T15:59:03+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81104887,"identity":"e9e88a07-b7af-42d7-b8b0-a1538daded21","added_by":"auto","created_at":"2025-04-22 09:24:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":590630,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Upper left: Wright-Giemsa staining of the cultured cells reveals large nuclei with two to three nucleoli and scant cytoplasm containing vacuoles. (B) Upper right: Karyotype of the cultured cell metaphase spread at passage 23 shows no abnormalities observed on chromosomes 17 and 19. The loci for HLF and TCF3 are marked. (C) Lower right: A partial chromatogram displays the sequence around the breakpoints of the \u003cem\u003eTCF3:HLF\u003c/em\u003e chimeric transcript, which contains 11 unidentified bases between the TCF3 exon 16 and HLF exon 4 sequences. (D) Lower left: A metaphase spread from the cultured cells, hybridized with blue-fluorochrome labeled HLF probe, green-fluorochrome labeled TCF3 probe, and red-fluorochrome labeled PBX1 probe, indicates that the HLF locus is not juxtaposed to the TCF3 locus.\u003c/p\u003e","description":"","filename":"Figure1submitted.png","url":"https://assets-eu.researchsquare.com/files/rs-5469901/v1/0d79fcd846dc0d8bd9d8f162.png"},{"id":81104886,"identity":"61af437a-2f0b-4875-b07a-9a1b4df7e243","added_by":"auto","created_at":"2025-04-22 09:24:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":122633,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Upper: The partial chromatogram of the long-range genomic PCR product from cultured cells displays the 5´-end genomic breakpoint of the inserted HLF material. Notably, an unidentified three-base insertion, \"CGT,\" was found between the sequence of TCF3 intron 16 and the HLF material. (B) Lower: The sequence around the 3´-end genomic breakpoint of the inserted HLF material reveals an unidentified sequence, “CACAAACCCCCAAA,” located between HLF exon 4 and TCF3 intron 16 sequences.\u003c/p\u003e","description":"","filename":"Figure2submitted.png","url":"https://assets-eu.researchsquare.com/files/rs-5469901/v1/71d2c4a0839e34fda150de59.png"},{"id":81104905,"identity":"37487c22-862b-47b2-ae6d-7f05a5f68878","added_by":"auto","created_at":"2025-04-22 09:24:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":55322,"visible":true,"origin":"","legend":"\u003cp\u003eThe diagram illustratesthe genomic sequences surrounding the TCF3 breakpoint of the fusion gene. The boxed sequences include\"cggac\" located in intron 16 of TCF3, \"CGT\" from an insertion sequence of unknown origin, and \"CAG\" in intron 3 of HLF. Together, these sequences form the insertion sequence\"CGGACCGTCAG,\" which is found between exon 16 of TCF3 and exon 4 of HLF in the chimeric transcript. Additionally, the sequence \"GAC,\" highlighted with a magenta background, and \"GTC,\" highlighted with a light blue background, represent a putative three-base overhang at the TCF3 breakpoint.\u003c/p\u003e","description":"","filename":"Figure3submitted.png","url":"https://assets-eu.researchsquare.com/files/rs-5469901/v1/107946a331cdba71d3f0f730.png"},{"id":96650397,"identity":"41ef22e1-db69-47d6-a8c5-f1e866eb68b0","added_by":"auto","created_at":"2025-11-24 16:11:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1932593,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5469901/v1/db270f16-61a1-46dd-a8ad-06bea3f1daed.pdf"},{"id":81104890,"identity":"aa97f5ba-a713-4c3e-86ad-369ba94b9fee","added_by":"auto","created_at":"2025-04-22 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lymphoblastic leukemia (BCP-ALL) includes various genetically defined subtypes, each with specific clinical characteristics and prognoses [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Modern treatment protocols require accurate classification of each case into its genomic subgroup, along with precise measurement of minimal residual disease (MRD) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Further in-depth understanding of leukemogenesis tailored to each subtype enhances treatment strategies [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Thus, it is crucial to molecularly characterize each patient and to have a comprehensive understanding of novel insights into leukemogenesis [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe BCP-ALL subtype with the translocation t(17;19)(q21-q22;p13)/\u003cem\u003eTCF3::HLF\u003c/em\u003e accounts for less than 1% of childhood BCP-ALL cases [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. It arises from an in-frame fusion between the \u003cem\u003eTCF3\u003c/em\u003e gene located on 19p13.3 and the \u003cem\u003eHLF\u003c/em\u003e gene on 17q22, which is caused by the translocation. The resulting chimeric \u003cem\u003eTCF3::HLF\u003c/em\u003e product functions as a transcription factor, utilizing the transactivation domains of TCF3 alongside a DNA-binding and dimerization basic leucine zipper (bZIP) domain from HLF [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Case series have shown that BCP-ALL with \u003cem\u003eTCF3::HLF\u003c/em\u003e has distinct characteristics, including a precursor B-cell immunophenotype with co-expression of CD33, hypercalcemia, and symptomatic disseminated intravascular coagulation (DIC) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Clinically, this subtype of BCP-ALL demonstrates a tendency for MRD persistence following induction therapy and is associated with an unfavorable prognosis, characterized by primary refractoriness or short intervals to relapse despite intensive chemotherapy [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. As a result, BCP-ALL with \u003cem\u003eTCF3::HLF\u003c/em\u003e is considered a clear indication for allogeneic hematopoietic cell transplantation (HCT) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe development of preclinical models, such as patient-derived xenotransplantation models, has significantly advanced the creation of new and effective therapies, including immunotherapy and small molecule inhibitors [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, BCP-ALL is heterogeneous, and not all subtypes can be successfully transplanted into xenograft models. Therefore, it remains vital to establish novel cell lines to study the tumorigenesis of specific categories of leukemia [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In this research, the authors encountered a BCP-ALL case with a pre-B ALL immunophenotype, which exhibited an obstinate clinical course. In this case, the authors confirmed the presence of the \u003cem\u003eTCF3::HLF\u003c/em\u003e fusion gene and established a permanently growing cell line from the second recurrent sample.\u003c/p\u003e"},{"header":"Method","content":"\u003cp\u003e\u003cstrong\u003eClinical summary\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe\u0026nbsp;index case is a 12-year-old male who was referred to the first author\u0026rsquo;s hospital due to hyperleukocytosis,\u0026nbsp;with a\u0026nbsp;peripheral white blood cell count of 99,500/mm\u0026sup3; at diagnosis. Hypercalcemia was not\u0026nbsp;present.\u0026nbsp;A\u0026nbsp;bone marrow aspirate revealed a monotonous growth of peroxidase-negative lymphoblasts. The blasts were immunoreactive for CD10, CD19, CD33, TdT, CD79a, and surface immunoglobulin lambda chain, as well as the IgM chain,\u0026nbsp;but were\u0026nbsp;negative for CD20 and CD34. The karyotype was 46,XY. The initial analysis of the craniospinal fluid\u0026nbsp;showed\u0026nbsp;leukemic involvement.\u0026nbsp;The patient\u0026nbsp;was diagnosed with BCP-ALL\u0026nbsp;with\u0026nbsp;a pre-B immunophenotype.\u0026nbsp;Initially,\u0026nbsp;the patient received multi-agent chemotherapy, stratified\u0026nbsp;for\u0026nbsp;intermediate risk [8], and achieved complete remission after induction therapy.\u0026nbsp;He\u0026nbsp;completed the chemotherapy; however, one month after maintenance therapy\u0026nbsp;ended, he developed an osteolytic lesion in the right femur, which\u0026nbsp;was confirmed by biopsy to be an\u0026nbsp;extramedullary bone recurrence. Concurrently, imaging and\u0026nbsp;an\u0026nbsp;open biopsy revealed a bilateral testicular recurrence.\u0026nbsp;Following the report, he underwent multi-agent chemotherapy with minor modifications [11].\u0026nbsp;Unfortunately, the lesions\u0026nbsp;did not resolve.\u0026nbsp;Due to the leukemic cells displaying a\u0026nbsp;mature B-cell-like immunophenotype,\u0026nbsp;characterized by\u0026nbsp;surface light chain expression, we\u0026nbsp;opted for\u0026nbsp;multi-agent chemotherapy for refractory mature B-cell lymphoma and focal irradiation [12].\u0026nbsp;This treatment resulted in\u0026nbsp;complete remission. After consolidation treatment with inotuzumab ozogamicin,\u0026nbsp;the patient\u0026nbsp;underwent allogeneic bone marrow transplantation from an HLA 8/8 matched unrelated donor, using a preconditioning regimen\u0026nbsp;that included\u0026nbsp;total body irradiation, fludarabine, and melphalan.\u0026nbsp;Given the high risk of relapse, we planned to administer blinatumomab to prevent recurrence [13]. However, a bone marrow aspiration confirmed a second recurrence 62 days after HCT,\u0026nbsp;coinciding with the day\u0026nbsp;we intended to start blinatumomab.\u0026nbsp;Despite this, we proceeded with the treatment plan. Unfortunately,\u0026nbsp;the patient\u0026nbsp;succumbed to\u0026nbsp;a rapidly deteriorating disease 64 days after HCT.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture and flow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe began\u0026nbsp;by culturing mononuclear cells from the peripheral blood during the second relapse. The cells were placed in a plastic culture flask containing RPMI 1640 Medium, supplemented with GlutaMAX\u0026trade; (Gibco\u0026trade;, Thermo Fisher Scientific, Waltham, MA, USA), 10% heat-inactivated fetal bovine serum (FBS, Gibco\u0026trade;), and penicillin/streptomycin. The culture was maintained in a humidified atmosphere at\u0026nbsp;37\u0026deg;C with 5% CO2. The authors analyzed the immunophenotype of the cultured cells passage 25\u0026nbsp;using\u0026nbsp;flow cytometry, following\u0026nbsp;methods previously reported [14].\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthenticity confirmation of the cell line\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe confirmed the authenticity of the cultured cell line by analyzing polymorphic markers, including \u003cem\u003eAMELX\u003c/em\u003e/\u003cem\u003eAMELY\u003c/em\u003e, \u003cem\u003eCSF1R\u003c/em\u003e, D16S539, D13S317, D5S818, D7S820, \u003cem\u003eTH\u003c/em\u003e, \u003cem\u003eTPO\u003c/em\u003e, \u003cem\u003eVWF\u003c/em\u003e, D2S1338, D3S1358, D8S1179, D18S51, D19S253, D20S438, and D21S11, using\u0026nbsp;fluorescence-conjugated specific primer pairs (\u003cstrong\u003eTable S1\u003c/strong\u003e). Since the second relapse sample contained both leukemic and HCT donor cells, we chose the testicular relapse sample as the reference for cell authenticity. The primer sequences are listed in Table S1. Next, we assessed the status of the immunoglobulin heavy (\u003cem\u003eIGH\u003c/em\u003e) and light (\u003cem\u003eIGK\u003c/em\u003e, \u003cem\u003eIGL\u003c/em\u003e) chains, as well as the T cell receptor (\u003cem\u003eTRG\u003c/em\u003e, \u003cem\u003eTRD\u003c/em\u003e) genes in the initial diagnostic sample, the testicular recurrent sample, the second relapse sample, and the cultured cells at passage 30. This analysis was performed using polymerase chain reaction (PCR) with specific primer pools and a heteroduplex mobility assay, following the BIOMED-2 protocol [15]. The rearranged alleles identified on polyacrylamide gel electrophoresis were excised and directly sequenced after purification.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExome and transcriptome analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we analyzed diagnostic and recurrent testicular samples using exome sequencing according to the manufacturer\u0026apos;s instructions. We prepared an exome library with the AmpliSeq Exome kit and sequenced the template constructed from this library using the Ion PI\u0026trade; Hi Q\u0026trade; Sequencing 200 Kit (Thermo Fisher Scientific). The variants detected were confirmed through Sanger sequencing and subcloning of the PCR product. To investigate the genotype of the current case, we performed transcriptome analysis on the diagnostic sample. An RNA sequencing library was created using the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech Laboratories, Inc., Mountain View, CA, USA) and the Ion Xpress\u0026trade; Plus Fragment Library Kit (Thermo Fisher Scientific). The templates from this library were sequenced\u0026nbsp;using the Ion PI\u0026trade; Hi-Q\u0026trade; Sequencing 200 Kit and the Ion Proton (Thermo Fisher Scientific). The single-end 200-bp reads in FASTQ format from the Ion Proton were analyzed using STAR-Fusion version 1.9.1-0, built on 64-bit Ubuntu 20.04 LTS [16]. We confirmed the candidate fusion gene through reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing of the amplified product. The primer sequences used were: forward primer 5\u0026apos;-CTACGACGGGGGTCTCCAC-3\u0026apos; and reverse primer 5\u0026apos;-CATGTTGTCCAGCCGCATCAG-3\u0026apos;.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenomic analysis on array comparative genomic hybridization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors conducted a comprehensive analysis of genomic copy number alterations\u0026nbsp;on the diagnostic sample and the established cell line at passage 13. This was performed using array comparative genomic hybridization (aCGH) with the CytoSure Consortium Cancer + Single Nucleotide Polymorphism arrays (Oxford Gene Technology IP Limited, Begbroke, Oxfordshire, UK), following the manufacturer\u0026apos;s recommendations [17]. The resulting data were analyzed using Agilent Feature Extraction Software (Agilent Technologies; version 10.7.3.1) and CytoSure Interpret Software (version 4.9.40, Oxford Gene Technology) [17].\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytogenetic and molecular cytogenetic analysis and additional transcriptome study and long range genomic PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors conducted a molecular cytogenetic study to confirm the juxtaposition between the \u003cem\u003eTCF3\u003c/em\u003e and \u003cem\u003eHLF\u003c/em\u003e loci. First, A metaphase spread from the cell line at passage 23 was analyzed, utilizing G-band karyotyping with Leishman\u0026apos;s stain (Sigma-Aldrich, St. Louis, Missouri, USA) [18]. The karyotype was described according to the guidelines of the International System for Human Cytogenomic Nomenclature (2020) [19]. Additionally, the karyotype at passage ten was analyzed by spectral karyotyping (SKY) [20].\u003c/p\u003e\n\u003cp\u003eThen, the authors examined metaphase and interphase spreads using three-color fluorescence in situ hybridization with commercially available probes, specifically the CytoCell TCF3/PBX1 Plus Translocation Dual Fusion Probe and the CytoCell HLF Breakapart FISH Probe (Oxford Gene Technology), following the manufacturer\u0026apos;s protocol [21]. Due to the inability of molecular cytogenetic analysis to confirm the juxtaposition of the TCF3 and HLF loci, RNA sequencing was performed once again. This sequencing was carried out according to the TruSeq paired-end RNA sequencing protocols using a Genome Analyzer IIx with a paired-end module. Total RNA was extracted from the ICH-BCPALL-3 cell line (passage 30) and sent to the Genomics Core Facility at the Norwegian Radium Hospital, Oslo University Hospital, for RNA sequencing [22]. The paired-end 150 bp reads in FASTQ format were analyzed using FusionCatcher version 1.33 [23]. To determine if genomic material from \u003cem\u003eHLF\u003c/em\u003e exon 4 was inserted into the \u003cem\u003eTCF3\u003c/em\u003e intron 16, long-range PCR was carried out using primer pairs listed in \u003cstrong\u003eTable S2\u003c/strong\u003e. Genomic PCR amplifications were conducted in a 25 \u0026mu;l reaction volume, which included 12.5 \u0026mu;l of Premix Ex Taq\u0026trade; DNA Polymerase Hot Start Version (Takara Bio Europe/SAS, Saint-Germain-en-Laye, France), 100 ng of genomic DNA from the ICH-BCPALL-3 cell line, and 0.4 \u0026mu;M of each forward and reverse primer. The primer combinations utilized were TCF3-Intr16F1 + TCF3-Exon17R1 and TCF3-Intr16F2 + TCF3-Exon17R2. The PCR cycling conditions included an initial denaturation step at 94˚C for 30 seconds, followed by 35 cycles of 7 seconds at 98˚C and 2 minutes at 68˚C, and a final extension at 68˚C for 5 minutes. Three \u0026mu;l of the PCR products were stained with GelRed (Biotium, Fremont, CA, USA), analyzed by electrophoresis through a 1% agarose gel, and photographed. DNA gel electrophoresis was performed using lithium borate buffer [24]. The remaining PCR products were purified using the MinElute PCR Purification Kit (QIAGEN GmbH, Hilden, Germany) and directly sequenced using the dideoxy procedure with the BigDye terminator v1.1 cycle sequencing kit, following the manufacturer\u0026apos;s recommendations (ThermoFisher Scientific). The primers used for cycle sequencing were the same as those employed in genomic PCR, along with additional primers HLF-1266R1, HLF-1300R1, HLF-1464F1, HLF-1423F1, HLF-4073F1, and HLF-4292F1 (see \u003cstrong\u003eTable S2\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscriptomic landscape of the established cell line\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe whole transcriptome of the established cell line was compared to those of other characterized cell lines, using the transcriptome data deposited in the public repository summarized in \u003cstrong\u003eTable S3\u003c/strong\u003e [25]. The FASTQ-formatted data were pre-processed using fastp (version 0.24.0) [26], and then aligned to reference genome GRCh38.p13 using STAR (version 2.7.10b). The read count data were generated by RSEM (version 1.3.1) [27]. This read count data was then normalized with edgeR [28]. Initially, we investigated differentially expressed genes between five \u003cem\u003eTCF3::HLF\u003c/em\u003e positive cell lines, including ICH-BCPALL-3, and \u003cem\u003eTCF3::PBX1\u003c/em\u003e positive cell lines. This analysis was performed using the edgeR package (version 4.0.16) on OlvTools (https://olvtools.com/, BxINFO, Tokyo, Japan). Genes with a log fold change greater than 1 and a false discovery rate (FDR) of less than 0.05 were considered significant. For functional enrichment analysis, we utilized the clusterProfiler package (version 4.10.1) to examine over-represented Gene Ontology (GO) terms and pathways on OlvTools [29]. It is when adjusted p-value is less than 0.05, GO terms within the Biological Process, Molecular Function, and Cellular Component categories were deemed significant. Similarly, of the 953 human-curated pathways analyzed, those with an adjusted p-value of less than 0.05 were considered significant as well. Next, we calculated similarity and difference in terms of the gene expression signature and classified the cell lines into the clusters using hierarchical clustering on iDEP (version 2.01) [30] and Uniform Manifold Approximation and Projection (UMAP) (version 0.2.10.0) [31].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytotoxicity assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe efficacy of sixty agents was evaluated on an established cell line, including cytotoxic agents typically used in leukemia chemotherapy and small molecules targeting proliferation signaling (\u003cstrong\u003eTable S4\u003c/strong\u003e), based on studies by Goto A et al. and Szulkin A et al [32,33]. We seeded 10,000 cells from either passage 14 or passage 21 into 20 \u0026mu;L of culture medium containing test agents (StemSpan\u0026trade; Serum-Free Expansion Medium II and StemSpan\u0026trade; CC100, STEMCELL Technologies, Vancouver, Canada) in each well of a 384-well plate. The concentrations of each agent examined included the starting concentration, as well as 1:5, 1:25, and 1:125 dilutions. In the control wells, RPMI medium without the drug was added. After incubating for 4 days in a humidified environment at 37 \u0026deg;C and 5% CO2, the cell viability in each well was measured using the Cell Titer-Glo luminescent assay. The luminescence values were obtained using the Glo-Max plate reader (Promega, Madison, WI, USA) following the manufacturer\u0026apos;s instructions. Drug sensitivity was expressed by the Drug Effect Score (DES), an index reflecting the concentration-weighted susceptibility of the test agents. For instance, DES100 indicates complete cell kill at all tested concentrations, whereas DES0 signifies no effect of the test drug at any concentration. Peripheral blood mononuclear cells (PBMC) from nine healthy volunteers were also included in the drug sensitivity test to provide reference values for DES. An agent was deemed efficacious if the DES for the cell line was greater than 10, and the ratio of the DES of the cell line to the DES of reference PBMC was greater than 1 [34].\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCell culture and flow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMononuclear cells were obtained and purified from peripheral blood during the second relapse using a density gradient. The cultured cells proliferated rapidly for over 70 passages, leading to the establishment of the cell line designated as ICH-BCPALL-3 (\u003cstrong\u003eFig. 1a\u003c/strong\u003e). The doubling time for\u0026nbsp;this established cell line is approximately 24 hours (\u003cstrong\u003eFig. S1\u003c/strong\u003e). The immunophenotype of the established cell line differed\u0026nbsp;slightly from that\u0026nbsp;of the initial diagnostic sample. The cultured cells were immunoreactive for CD10, CD11b (weak), CD13 (weak), CD19, CD22, CD24, CD33, CD66c, TdT, and cytoplasmic IgM, but were\u0026nbsp;negative for CD20, CD34, surface IgM, and surface immunoglobulin lambda chain (\u003cstrong\u003eTable S5\u003c/strong\u003e and \u003cstrong\u003eFig. S2\u003c/strong\u003e). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthenticity confirmation of the cell line\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe pattern of polymorphic markers was nearly identical to that of the first relapse sample, with the exception that the \u003cem\u003eAMELY\u003c/em\u003e locus was deleted in the cell line (\u003cstrong\u003eFig. S3\u003c/strong\u003e). The diagnostic sample, the first recurrent sample, the second relapse sample, and the established cell line at passage 70 all demonstrated the same rearrangement patterns at the \u003cem\u003eIGH\u003c/em\u003e, \u003cem\u003eIGK\u003c/em\u003e, and \u003cem\u003eIGL\u003c/em\u003e loci. Direct sequencing revealed IGHV3-43_IGHD3-10_IGHJ6, IGKV4-1_KDE, and IGLV1-47_IGLJ3 (\u003cstrong\u003eFig. S4a\u003c/strong\u003e). The clone-specific insertions at the \u003cem\u003eIGH\u003c/em\u003e V-D junction were GGTTTGAGG; for the \u003cem\u003eIGH\u003c/em\u003e D-J junction, it was CCT; and for the \u003cem\u003eIGL\u003c/em\u003e V-J junction, it was TG (\u003cstrong\u003eFig. S4b\u003c/strong\u003e). These findings confirm the authenticity of the established cell line.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenomic analysis on array comparative genomic hybridization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe analysis on aCGH confirmed the loss of the \u003cem\u003eAMELY\u003c/em\u003e locus (chrY: 6,733,959-6,742,068 on GRCh37/hg19) in the cell line (\u003cstrong\u003eFigs. S5\u003c/strong\u003e and \u003cstrong\u003eS6\u003c/strong\u003e). The copy number alteration analysis using aGGH showed a homozygous loss at the \u003cem\u003eRB1\u003c/em\u003e locus, specifically at chr13:48879236-48942672 on GRCh37/hg19 for \u003cem\u003eRB1\u003c/em\u003e exons 2-10, chr13:48943195-48953428 for exons 12 and 13, and chr13:48954317-49159971 for exons 18-27. Additionally, there was a copy number neutral loss of heterozygosity at the \u003cem\u003eBCL6\u003c/em\u003e locus (chr3:183900308-188605455 on GRCh37/hg19) at the time of diagnosis (\u003cstrong\u003eFigs. S5 and S6\u003c/strong\u003e). Interestingly, the \u003cem\u003eRB1\u003c/em\u003e locus was maintained in the established cell line (passage 13), as confirmed by copy number analysis based on exome data from both the diagnostic and recurrent testicular samples (\u003cstrong\u003eFigs. S5, S6, and S7a\u003c/strong\u003e). In terms of genes encoding transcription factors associated with lymphoid differentiation and immunoglobulin rearrangement, the loci for \u003cem\u003ePAX5\u003c/em\u003e and \u003cem\u003eBTG1\u003c/em\u003e were retained in the diagnostic sample. However, the locus containing \u003cem\u003eVPREB1\u003c/em\u003e and \u003cem\u003eIGL\u003c/em\u003e was deleted (chr22:22389514-22517319 on aCGH (\u003cstrong\u003eFigs. S5 and S6\u003c/strong\u003e). Furthermore, copy number analyses using aCGH and exome data indicated the deletion of the \u003cem\u003eMLLT3\u003c/em\u003e and \u003cem\u003eCDKN2A/2B\u003c/em\u003e loci, which were observed only in the cell line (chr9:20624804-21994149, chr9:21994905-22005861, and chr9:22006328-22399305 on aCGH and chr9:20353464-20448297 and chr9:20456668-22009065 based on exome data) (\u003cstrong\u003eFigs. S5, S6, and S7b\u003c/strong\u003e). In addition, aCGH revealed a deletion of the \u003cem\u003eNR3C1\u003c/em\u003e gene located on chromosome 5 (142796870-143184287) in both the diagnostic sample and the cell line (\u003cstrong\u003eFigs. S5 and S6\u003c/strong\u003e). Furthermore, the cell line exhibited a gain of the \u003cem\u003eAKT1\u003c/em\u003e gene, as well as a gain at 3q27, which includes the \u003cem\u003eBCL6\u003c/em\u003e and \u003cem\u003eMECOM\u003c/em\u003e genes (\u003cstrong\u003eFig. S6\u003c/strong\u003e). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExome and transcriptome analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSubsequently, exome analysis and Sanger sequencing identified somatic variants in \u003cem\u003eARID5B\u003c/em\u003e (NM_032199.3:c.39_44delinsGGAGAAG; NP_115575.1:p.Cys13TrpfsTer18) (\u003cstrong\u003eFig. S8a\u003c/strong\u003e) and \u003cem\u003eNCOR1\u003c/em\u003e (NM_006311.4:c.5380C\u0026gt;T; NP_006302.2:p.Arg1794*) present at the time of diagnosis (\u003cstrong\u003eFig. S8b\u003c/strong\u003e). To evaluate the clonal composition throughout the relapse and cell cultivation process, exome analysis was conducted on the testicular relapse sample, a second relapse sample, and the established cell line. This analysis revealed a \u003cem\u003eKRAS\u003c/em\u003e amino acid substitution variant in the testicular relapse sample (NM_004985.5:c.356A\u0026gt;G; NP_004976.2:p.Asp119Gly). Impressively, we identified a different \u003cem\u003eKRAS\u003c/em\u003e variant in the second recurrent bone marrow sample and the cultured cells (NM_004985.5:c.351A\u0026gt;T; NP_004976.2:p.Lys117Asn) (\u003cstrong\u003eFig. S8c\u003c/strong\u003e). Subsequently, whole transcriptome analysis on the diagnostic sample and the cell line revealed junctional reads spanning the \u003cem\u003eTCF3\u003c/em\u003e and \u003cem\u003eHLF\u003c/em\u003e genes using STAR-fusion and FusionCatcher. The RT-PCR and direct sequencing of the RT-PCR product revealed an in-frame fusion of \u003cem\u003eTCF3\u003c/em\u003e exon 16 and HLF exon 4 in the diagnostic sample, the second relapse sample, and the established cell line (\u003cstrong\u003eFig. 1c\u003c/strong\u003e). The chimeric transcript contained an unidentified 11-base sequence, CGGACCGTCAG, between \u003cem\u003eTCF3\u003c/em\u003e exon 16 and \u003cem\u003eHLF\u003c/em\u003e exon 4 (\u003cstrong\u003eFig. 1c\u003c/strong\u003e). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytogenetic and molecular cytogenetic analysis and additional transcriptome study and long range genomic PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe G-band karyotype of the cultured cell at passage 30 was 46,XY,der(1)(1qter-\u0026gt;1q11::1p32-\u0026gt;1q11::4q21-\u0026gt;4qter),der(4)t(1;4)(q11;p32),add(8)(q24),del(17)(q24) (\u003cstrong\u003eFig. 1b\u003c/strong\u003e).\u0026nbsp;The SKY analysis partially identified the unknown material, revealing the following finding: 46,XY,t(1;4)(q21;q11)(wcp1+,wcp4+),der(8)t(3;8)(p?;q?)(wcp3+,wcp8+). Notably,\u0026nbsp;the abnormality in chromosome 17 was not confirmed during the SKY analysis (\u003cstrong\u003eFig. S9\u003c/strong\u003e). However, molecular cytogenetics performed on the metaphase spread, using a commercially available probe set, could not confirm the juxtaposition of the \u003cem\u003eTCF3\u003c/em\u003e and \u003cem\u003eHLF\u003c/em\u003e loci in the cultured cells (\u003cstrong\u003eFig. 1d\u003c/strong\u003e). To further characterize the present case, long-distance PCR was employed to amplify and sequence of \u003cem\u003eTCF3\u003c/em\u003e intron 16 and exon 17. This analysis ultimately demonstrated the insertion of a fragment composed of a portion of \u003cem\u003eHLF\u003c/em\u003e intron 3 and a part of \u003cem\u003eHLF\u003c/em\u003e exon 4 into \u003cem\u003eTCF3\u003c/em\u003e intron 16, with a segment of the inserted \u003cem\u003eHLF\u003c/em\u003e exon 4 sequence being deleted (\u003cstrong\u003eFig. 2, Fig. S10a, Fig. S10b, and Fig. 3\u003c/strong\u003e). At the genomic junctions, there are sequences of unknown origin: a triplet (CGT) at the 5ˊ genomic junction between intron 16 of \u003cem\u003eTCF3\u003c/em\u003e and intron 3 and exon 4 of \u003cem\u003eHLF\u003c/em\u003e, and a 14-base sequence (CACAAACCCCCAAA ) at the 3ˊ genomic junction between exon 4 of \u003cem\u003eHLF\u003c/em\u003e and intron 16 of \u003cem\u003eTCF3\u003c/em\u003e (\u003cstrong\u003eFig. 3)\u003c/strong\u003e. The 11-base sequence CGGACCGTCAG between the \u003cem\u003eTCF3\u003c/em\u003e exon 16 and \u003cem\u003eHLF\u003c/em\u003e exon 4 in the chimeric transcript consists of the last five bases (CGGAC) in the terminus of \u003cem\u003eTCF3\u003c/em\u003e intron 16, the unknown origin triplet (CGT), and a portion of \u003cem\u003eHLF\u003c/em\u003e intron 3 (CAG) (indicated by box characters in \u003cstrong\u003eFig. 3\u003c/strong\u003e). The genomic breakpoint in \u003cem\u003eTCF3\u003c/em\u003e is located near a CpG element and activation-induced cytidine deaminase (AID) consensus sequence lacking the classical RAG consensus sequence sites (CAC). The 5ˊ breakpoint of the inserted \u003cem\u003eHLF\u003c/em\u003e material is situated directly on the CpG element, while the 3ˊ breakpoint of the \u003cem\u003eHLF\u003c/em\u003e material is situated 11 base pairs away from the CpG element (\u003cstrong\u003eFig. S10c\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscriptomic landscape of the established cell line\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors utilized whole transcriptome data to investigate differentially expressed genes between \u003cem\u003eTCF3::HLF\u003c/em\u003e positive cell lines and \u003cem\u003eTCF3::PBX1\u003c/em\u003e positive cell lines. Among the genes that were upregulated in the \u003cem\u003eTCF3::HLF\u003c/em\u003e positive cell lines are \u003cem\u003eKLHL13\u003c/em\u003e, \u003cem\u003eCLSTN2\u003c/em\u003e, \u003cem\u003eHLF\u003c/em\u003e, \u003cem\u003eHPGD\u003c/em\u003e, \u003cem\u003eMMP25\u003c/em\u003e, \u003cem\u003eRUBCNL\u003c/em\u003e, \u003cem\u003eB3GNT7\u003c/em\u003e, \u003cem\u003eC3AR1\u003c/em\u003e, \u003cem\u003eFCGR1A\u003c/em\u003e, \u003cem\u003eKLRK\u003c/em\u003e, \u003cem\u003eMCOLN2\u003c/em\u003e, \u003cem\u003eH2AC13\u003c/em\u003e, and \u003cem\u003eH2BC4\u003c/em\u003e, as shown in the volcano plot and MA plot (\u003cstrong\u003eFig. S11a and S11b, and Table S6\u003c/strong\u003e). Conversely, the genes that were downregulated in \u003cem\u003eTCF3::HLF\u003c/em\u003e positive cell lines include \u003cem\u003ePHACTR3\u003c/em\u003e, \u003cem\u003eRORB\u003c/em\u003e, \u003cem\u003eBEND4\u003c/em\u003e, \u003cem\u003ePBX1\u003c/em\u003e, \u003cem\u003eWNT16\u003c/em\u003e, \u003cem\u003eTCERG1L\u003c/em\u003e, and \u003cem\u003eGREM1\u003c/em\u003e, as illustrated in the volcano plot and MA plot (\u003cstrong\u003eFig. S11a and S11b, and Table S6\u003c/strong\u003e). The gene ontogeny analysis identified that several Gene Ontology (GO) terms are over-represented in \u003cem\u003eTCF3::HLF\u003c/em\u003e-positive leukemia cell lines compared to \u003cem\u003eTCF3::PBX1\u003c/em\u003e-positive leukemia cell lines. These terms include: - **Molecular Function**: structural constituent of chromatin, GTPase regulator activity, nucleoside-triphosphatase regulator activity, protein heterodimerization activity, transmembrane signaling receptor activity, structural molecule activity, calcium channel activity, calcium-release channel activity, intracellular ligand-gated monoatomic ion channel activity, and ligand-gated calcium channel activity (\u003cstrong\u003eFig S12a and Table S7a\u003c/strong\u003e). - **Cellular Component**: nucleosome and MHC class II protein complex (\u003cstrong\u003eFig S12b and Table S7b\u003c/strong\u003e). - **Biological Process**: regulation of cell adhesion, transmembrane receptor protein tyrosine kinase signaling pathway, chemotaxis, cell chemotaxis, defense response to bacterium, nucleosome assembly, nucleosome organization, mucosal immune response, and organ- or tissue-specific immune response (\u003cstrong\u003eFig S12c and Table S7c\u003c/strong\u003e). Additionally, pathway analysis indicated that two pathways, the Senescence Associated Secretory Phenotype (SASP) and histone modifications, are over-represented in \u003cem\u003eTCF3::HLF\u003c/em\u003e-positive leukemia cell lines compared to \u003cem\u003eTCF3::PBX1\u003c/em\u003e-positive leukemia cell lines (\u003cstrong\u003eFig S13 and Table S8\u003c/strong\u003e). The authors then sought to classify the cell lines using mathematical methods based on the transcriptome dataset. Nonlinear dimensionality reduction algorithms, specifically UMAP, demonstrated that \u003cem\u003eTCF3::HLF\u003c/em\u003e-positive leukemia cell lines are distinct from other cell lines (\u003cstrong\u003eFig S14\u003c/strong\u003e). Hierarchical cluster analysis further revealed a unique cluster of five \u003cem\u003eTCF3::HLF\u003c/em\u003e-positive leukemia cell lines, as illustrated by the dendrogram (\u003cstrong\u003eFig S15\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytotoxicity assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The cytotoxicity assay revealed that ICH-BCLALL-3 is significantly vulnerable to the cytotoxic agents, including SN-38 (derivative of irinotecan), cytarabine, etoposide, docetaxel, topotecan, and gemcitabine (\u003cstrong\u003eTable S9\u003c/strong\u003e). Furthermore, the cell line is sensitive to small molecule inhibitors for cell signal transduction pathways, including inhibitors for mitogen-activated protein kinase kinase 1/2 (MEK1/2), trametinib and selumetinib, inhibitors for\u0026nbsp;mechanistic/mammalian target of rapamycin complex 1 (mTORC1), everolimus and rapamycin,\u0026nbsp;dual inhibitors for phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR), PI-103 and PF-04691502, an inihibitor for\u0026nbsp;checkpoint kinase 1, AZD7762, and an inhibitor for Aurora Kinase B (AURKB) inhibitors, barasertib (\u003cstrong\u003eTable S9\u003c/strong\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAccurate\u0026nbsp;molecular diagnosis is essential for the clinical management of BCP-ALL [1]. Whole transcriptome sequencing provides precise molecular diagnoses, allowing for proper patient stratification, although it does not apply to every patient. Consequently, BCP-ALL cases with rare molecular classifiers that cannot be confirmed using existing diagnostic tests face the risk of being assigned inappropriate treatment plans. In this particular case, the absence of hypercalcemia and severe disseminated intravascular coagulation (DIC) led to the decision not to conduct RT-PCR screening for \u003cem\u003eTCF3::HLF\u003c/em\u003e, resulting in a missed opportunity for accurate diagnosis and stratification. While positive expression of CD33 may suggest the presence of \u003cem\u003eTCF3::HLF\u003c/em\u003e [5], the mature B-cell-like immunophenotype observed complicated the diagnostic process. Clinicians should be cautious and consider \u003cem\u003eTCF3::HLF\u003c/em\u003e-positive BCP-ALL when encountering a BCP-ALL case that expresses CD33, because BCP-ALL with \u003cem\u003eTCF3::HLF\u003c/em\u003e is extremely unfavorable without HCT [5,6,8].\u003c/p\u003e\n\u003cp\u003eMolecular and cell physiological analyses revealed a complex execution of abnormal genetic alterations [6]. Comprehensive molecular analysis of BCP-ALL featuring the \u003cem\u003eTCF3::HLF\u003c/em\u003e fusion demonstrated numerous molecular changes, including alterations in the \u003cem\u003eBCL2\u003c/em\u003e, \u003cem\u003eLMO2\u003c/em\u003e, \u003cem\u003ePAX5\u003c/em\u003e, and RAS pathway genes, alongside \u003cem\u003eTCF3::HLF\u003c/em\u003e. These changes lead to distinctive clinical presentations, such as resistance to chemotherapy [6]. Moreover, the transition from pro-B cells to pre-B cells is significantly disrupted due to deletions of several genes that encode transcription factors critical for lymphoid differentiation and immunoglobulin rearrangement [6]. These include \u003cem\u003ePAX5\u003c/em\u003e, \u003cem\u003eBTG1\u003c/em\u003e, \u003cem\u003eVPREB1\u003c/em\u003e, and \u003cem\u003eNCOR1\u003c/em\u003e [6]. In line with previous studies, this case also exhibited alterations in \u003cem\u003eVPREB1\u003c/em\u003e and \u003cem\u003eNCOR1\u003c/em\u003e in the diagnostic sample (\u003cstrong\u003eFig.S8\u003c/strong\u003e). Notably, alterations in \u003cem\u003eARID5B\u003c/em\u003e and \u003cem\u003eRB1\u003c/em\u003e were also observed in the diagnostic sample (\u003cstrong\u003eFig. S5 , Fig. S7, and Fig. S8\u003c/strong\u003e). These genes are involved in regulating epigenetic processes and the cell cycle, suggesting they contribute to the unique clinical features of this case [35,36]. More interestingly, these alterations were not present in the recurrent samples (\u003cstrong\u003eFig. S6\u003c/strong\u003e), but deletion of \u003cem\u003eCDKN2A/2B\u003c/em\u003e emerged in the cell line (\u003cstrong\u003eFig.S6 and Fig.S7\u003c/strong\u003e). Furthermore, the first recurrent sample exhibited the \u003cem\u003eKRAS\u003c/em\u003e mutation c.356A\u0026gt;G, resulting in the p.Asp119Gly alteration, while the second recurrent sample showed the \u003cem\u003eKRAS\u003c/em\u003e mutation c.351A\u0026gt;T, leading to the p.Lys117Asn change (\u003cstrong\u003eFig.S8\u003c/strong\u003e). These events (alterations of \u003cem\u003eARID5B\u003c/em\u003e, \u003cem\u003eNCOR1\u003c/em\u003e, \u003cem\u003eRB1\u003c/em\u003e, \u003cem\u003eCDKN2A/2B\u003c/em\u003e, and \u003cem\u003eKRAS\u003c/em\u003e) are regarded as secondary event, highlighting the oligoclonal nature of \u003cem\u003eTCF3::HLF\u003c/em\u003e positive leukemia as indicated in the previous study [6] and the strong driver capabilities of the \u003cem\u003eTCF3::HLF\u003c/em\u003e oncoprotein. The heterogeneous clonal composition and progression of the disease explain why alterations in the \u003cem\u003eARID5B\u003c/em\u003e, \u003cem\u003eNCOR1\u003c/em\u003e, and \u003cem\u003eRB1\u003c/em\u003e genes were not present in the recurrent sample, while changes in \u003cem\u003eCDKN2A/2B\u003c/em\u003e and \u003cem\u003eKRAS\u003c/em\u003e emerged. Furthermore, these findings indicate that alterations in the epigenetic machinery, cell cycle regulators, and signaling pathways related to cell proliferation collectively contribute to the progression of \u003cem\u003eTCF3::HLF\u003c/em\u003e-positive BCP-ALL under the \u003cem\u003eTCF3::HLF\u003c/em\u003e driver capacity.\u0026nbsp;Additionally, the current case exhibited copy number neutral loss of heterozygosity at the 3q27 locus, which contains the \u003cem\u003eBCL6\u003c/em\u003e gene, at the time of diagnosis (\u003cstrong\u003eFig. S5\u003c/strong\u003e). There was also a gain of 3q27 harboring the \u003cem\u003eMECOM\u003c/em\u003e and \u003cem\u003eBCL6\u003c/em\u003e genes in the cell line (\u003cstrong\u003eFig. S6\u003c/strong\u003e). The exact significance of the 3q27 abnormality remains unclear; however, these genetic alterations may contribute to the unusual immunophenotype seen in this case, because alteration of BCL6 is frequently encountered in mature B-cell neoplasm. Furthermore, the present case has a hemizygous deletion of \u003cem\u003eNR3C1\u003c/em\u003e, which may lead to refractoriness to chemotherapy [37]. Finally, the cell line demonstrated a gain of \u003cem\u003eAKT1\u0026nbsp;\u003c/em\u003e(\u003cstrong\u003eFig. S6\u003c/strong\u003e), which could facilitate the progression of leukemic cells [38]. Overall, this cell line provides a unique opportunity to explore the relationship between these genetic abnormalities and clinical characteristics within the context of \u003cem\u003eTCF3::HLF\u003c/em\u003e tumorigenesis, potentially advancing the development of molecular targeted therapies.\u003c/p\u003e\n\u003cp\u003eThe present study has molecularly characterized the breakpoints on \u003cem\u003eTCF3\u003c/em\u003e in a cell line exhibiting the \u003cem\u003eTCF3::HLF\u003c/em\u003e fusion gene. This raises interest in the mechanism responsible for the insertion that creates the \u003cem\u003eTCF3::HLF\u003c/em\u003e fusion. To date, four \u003cem\u003eTCF3::HLF\u003c/em\u003e-positive cell lines have been reported: YCUB-2, HAL-01, UOC-B1, and Endo-kun, all of which contain the t(17;19) karyotype [39-42]. In contrast, the current cell line exhibited neither der(19) nor t(17;19) on karyotyping, nor any juxtaposition of the \u003cem\u003eTCF3\u003c/em\u003e locus with the \u003cem\u003eHLF\u003c/em\u003e locus in molecular cytogenetic analyses. Further molecular studies revealed that this cell line contained an insertion of \u003cem\u003eHLF\u003c/em\u003e exon 4 material into \u003cem\u003eTCF3\u003c/em\u003e intron 16. A similar observation was noted in the study conducted by Salim M et al [43]. The breakpoint of \u003cem\u003eTCF3\u003c/em\u003e typically occurs near CpG sites, a characteristic feature of translocations that happen in lymphoid progenitors at the pro-/pre-B stage, as demonstrated in the studies by Tsai et al [44].\u0026nbsp;Therefore,\u0026nbsp;it is intriguing to confirm whether the current case demonstrates that the \u003cem\u003eTCF3\u003c/em\u003e breakpoints are located near CpG sites. A study by Liu et al. found that methylated cytosines within the WRCG motif (where W = A or T, and R = A or G) in high cytosine density string are\u0026nbsp;crucial in producing double-strand breaks, which are essential for translocation\u0026nbsp;[45]. The formation of\u0026nbsp;single strands during transcription or due to thermal dynamic fluctuations increases\u0026nbsp;the deamination of methylated cytosine in the WRCG motif by AID [45]. Additionally,\u0026nbsp;the long-lasting T:G mismatch leads to double-strand breaks, facilitating translocation [45]. In the cell line being studied, the breakpoint of \u003cem\u003eTCF3\u003c/em\u003e intron 16 is located very close to the WRCG motif (specifically \u003cstrong\u003eAGCG\u003c/strong\u003e, positioned near the end of upstream TCF intron 16, highlighted in bold italic letters in \u003cstrong\u003eFig. 3\u003c/strong\u003e). According to the theory proposed by Liu D, the cytosine in the \u003cem\u003eTCF3\u003c/em\u003e intron 16 sequences on the chromosome 19 + strand is thought to have undergone methylation, resulting in its conversion to thymine (T) by AID in this case (the red bold \u003cstrong\u003eC\u003c/strong\u003e, to which \u0026quot;\u003cstrong\u003eMe\u003c/strong\u003e\u0026quot; was added in \u003cstrong\u003eFig. 3\u003c/strong\u003e). In this case, it was observed that GAC sequences are present at the end of the upstream \u003cem\u003eTCF3\u003c/em\u003e intron 16 sequence and at the beginning of the downstream \u003cem\u003eTCF3\u003c/em\u003e intron 16 sequence (highlighted with a\u0026nbsp;red background in \u003cstrong\u003eFig. 3\u003c/strong\u003e). This suggests that the\u0026nbsp;putative double-strand break in \u003cem\u003eTCF3\u003c/em\u003e intron 16 has created a three-base overhang of GAC on the - strand and a GTC overhang on the + strand of \u003cem\u003eTCF3\u003c/em\u003e intron 16 (highlighted with a blue background in \u003cstrong\u003eFig. 3\u003c/strong\u003e). These overhangs have\u0026nbsp;fused with the inserted \u003cem\u003eHLF\u003c/em\u003e material and were repaired into\u0026nbsp;a double strand through the classical non-homologous end-joining system [46]. The genomic 5\u0026apos; breakpoint of the inserted \u003cem\u003eHLF\u003c/em\u003e material is located on a CpG site; however, the 3\u0026apos; breakpoint is\u0026nbsp;not (\u003cstrong\u003eFig. S10c\u003c/strong\u003e). There are no RAG consensus sequence sites (CAC) or repetitive sequences near the genomic 3\u0026apos; breakpoint of the inserted \u003cem\u003eHLF\u003c/em\u003e material according to\u0026nbsp;the database (https://genome.ucsc.edu/). Therefore, the root cause of the double-strand break formation associated with the \u003cem\u003eHLF\u003c/em\u003e insertion remains partially\u0026nbsp;unclear. The \u003cem\u003eTCF3::HLF\u003c/em\u003e fusion gene found in this case contains\u0026nbsp;a stop codon in the inserted \u003cem\u003eHLF\u003c/em\u003e exon 4 sequence, suggesting that it is functional.\u003c/p\u003e\n\u003cp\u003eThe authors have discovered that the current cell line, ICH-BCPALL-3, shares an expression signature with four other \u003cem\u003eTCF3::HLF\u003c/em\u003e-positive BCP-ALL cell lines, as shown by hierarchical clustering and UMAP analysis (\u003cstrong\u003eFigs. S13 and S14\u003c/strong\u003e). This finding supports the idea that ICH-BCPALL-3 is a reliable in vitro model for \u003cem\u003eTCF3::HLF\u003c/em\u003e-positive BCP-ALL, which can be used to develop novel agents aimed at improving the prognosis of this condition. Furthermore, the expression data analysis identified several over-represented cellular processes and signaling pathways, including the transmembrane receptor protein tyrosine kinase signaling pathway and histone modifications (\u003cstrong\u003eTable S7c and Table S8\u003c/strong\u003e). The study also revealed potential active agents for \u003cem\u003eTCF3::HLF\u003c/em\u003e-positive BCP-ALL, such as inhibitors targeting the PI3K/Akt/mTOR pathway, including everolimus, rapamycin, PI-103, and PF-04691502 (\u003cstrong\u003eTable S9\u003c/strong\u003e) [38,47,48]. The observed susceptibility to PI3K/Akt/mTOR pathway inhibitors strongly correlates with the pronounced over-representation of the transmembrane receptor protein tyrosine kinase signaling pathway identified in the gene ontology analysis of ICH-BCLALL-3. This compelling connection underscores the pivotal role of this signaling pathway in the context of targeted therapeutic strategies. (\u003cstrong\u003eFig. S12c and Table S7c\u003c/strong\u003e). Indeed, inhibitors of the PI3K/Akt/mTOR pathway have been tested in in vitro assays and early-phase trials for BCP-ALL [38, 47, and 48], highlighting their promising potential for clinical application in \u003cem\u003eTCF3::HLF\u003c/em\u003e-positive BCP-ALL.\u0026nbsp;フォームの始まりフォームの終わりThe sensitivity of the ICH-BCPALL-3 cell line to MEK1/2 inhibitors, such as trametinib and selumetinib, likely stems from somatic variants of the \u003cem\u003eKRAS\u003c/em\u003e (Table S9) [49]. However, the potential oligoclonal composition of \u003cem\u003eKRAS\u003c/em\u003e in this model raises concerns, as it could contribute to the early onset of resistance to these crucial MEK1/2 inhibitors. Regarding the Bcl-2 inhibitor venetoclax, two previous studies have independently confirmed that clinical cases with the \u003cem\u003eTCF3::HLF\u003c/em\u003e fusion exhibit vulnerability to this drug [6,50]. However, the ICH-BCPALL-3 cell line does not show the expected efficacy of venetoclax when evaluated using the current cytotoxicity assay (\u003cstrong\u003eTable S9\u003c/strong\u003e). Evidence suggests that increased mitochondrial activity may contribute to venetoclax resistance [51]. Nevertheless, the current study has not confirmed a significant over-representation of mitochondrial activity in the gene ontology analysis and pathway analysis. This lack of confirmation may be due to the inclusion of other \u003cem\u003eTCF-HLF\u003c/em\u003e-positive BCP-ALL cell lines. As a result, the mechanisms behind venetoclax resistance in the ICH-BCPALL-3 cell line remain unclear and require further investigation. Finally, the authors identified that ICH-BCPALL-3 is sensitive to the AURKB inhibitor barasertib (\u003cstrong\u003eTable S9\u003c/strong\u003e). AURKB is a protein that plays a crucial role in regulating cell division, and its inhibition can lead to polyploidy and cellular senescence. Overexpression of this protein in cancer cells is associated with cancer progression and resistance to radiotherapy and chemotherapy. Notably, barasertib also demonstrates activity in the \u003cem\u003eTCF3::HLF\u003c/em\u003e-positive BCP-ALL cell line, YCUB-2, offering hope for its potential incorporation into clinical usage [32].\u003c/p\u003e\n\u003cp\u003eIn vitro models play a crucial role\u0026nbsp;in uncovering detailed molecular signatures and developing innovative\u0026nbsp;therapies [9,10]. The previous and current drug sensitivity assays indicated that BCP-ALL with the \u003cem\u003eTCF3::HLF\u003c/em\u003e fusion may be susceptible to AURKB\u0026nbsp;inhibitors. However, this finding needs to be validated across a broader range of\u0026nbsp;preclinical models before it can be applied to human clinical trials. The\u0026nbsp;distinct\u0026nbsp;molecular characteristics of \u003cem\u003eTCF3::HLF\u003c/em\u003e-positive BCP-ALL highlight the significance of the current model, allowing researchers to evaluate the efficacy of molecular targeting agents in ICH-BCPALL-3, which will soon be available.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e: This published article and its supplementary information files include all data generated or analyzed during this study. The fastq-formatted exome data and transcriptome data will be deposited in the Sequence Read Archive of DNA Data Bank of Japan. The cell line, ICH-BCPALL-3 will be available from RIKEN BioResource Research Center (Tsukuba, Japan). The cell number is RCB6089.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis investigation was supported by a subsidy from the Science and Technology Promotion Locating Electronic Power Plant from the Ministry of Education, Culture, Sports, and Technology (provided to K.K.) and a grant, JSPS KAKENHI (Grant Number JP24K11571, provided to K.K.).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest disclosure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest in the current study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental design and procedure were approved by the Institutional Review Board of Ibaraki Children\u0026apos;s Hospital (IRB approval number 1IRB-33) in accordance with \u003cem\u003eHelsinki Declaration\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe have obtained the informed consent from the guardian of the patient.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJeha S, Choi J, Roberts KG, Pei D, Coustan-Smith E, Inaba H, et al. Clinical significance of novel subtypes of acute lymphoblastic leukemia in the context of minimal residual disease-directed therapy. Blood Cancer Discov. 2021;2:326-337. doi: 10.1158/2643-3230.BCD-20-0229.\u003c/li\u003e\n\u003cli\u003eLee SHR, Yang W, Gocho Y, John A, Rowland L, Smart B, et al. Pharmacotypes across the genomic landscape of pediatric acute lymphoblastic leukemia and impact on treatment response. Nat Med. 2023;29:170-179. doi:10.1038/s41591-022-02112-7.\u003c/li\u003e\n\u003cli\u003eInaba T, Roberts WM, Shapiro LH, Jolly KW, Raimondi SC, Smith SD, et al. Fusion of the leucine zipper gene HLF to the E2A gene in human acute B-lineage leukemia. Science. 1992;257:531-4. doi: 10.1126/science.1386162.\u003c/li\u003e\n\u003cli\u003eHunger SP, Ohyashiki K, Toyama K, Cleary ML. Hlf, a novel hepatic bZIP protein, shows altered DNA-binding properties following fusion to E2A in t(17;19) acute lymphoblastic leukemia. 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Cell Death Dis. 2024;15:475. doi: 10.1038/s41419-024-06864-7.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"human-cell","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"huce","sideBox":"Learn more about [Human Cell](http://link.springer.com/journal/13577)","snPcode":"13577","submissionUrl":"https://www.editorialmanager.com/huce/default2.aspx","title":"Human Cell","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"B-cell precursor acute lymphoblastic leukemia, TCF3:HLF, oligoclonality, cell line, transcriptome, cytotoxicity assay","lastPublishedDoi":"10.21203/rs.3.rs-5469901/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5469901/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn vitro models of acute leukemia are crucial for understandingits biology and developing effective treatments. The authors have established and characterized a novel cell line, ICH-BCPALL-3, which expresses the \u003cem\u003eTCF3::HLF\u003c/em\u003efusion from B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The karyotype of the cultured cells is 46,XY, der(1)(1qter-\u0026gt;1q11::1p32-\u0026gt;1q11::4q21-\u0026gt;4qter), der(4)t(1;4)(q11;p32), add(8)(q24), del(17)(q24). Analysis of the diagnostic sample revealed deletions in \u003cem\u003eRB1\u003c/em\u003e, \u003cem\u003eVPREB1\u003c/em\u003e, and \u003cem\u003eNR3C1\u003c/em\u003e. The cell line showed additional deletions of \u003cem\u003eVPREB1\u003c/em\u003e, \u003cem\u003eNR3C1\u003c/em\u003e, and \u003cem\u003eCDKN2A\u003c/em\u003e/\u003cem\u003e2B\u003c/em\u003e, as well as a gain of \u003cem\u003eAKT1\u003c/em\u003e. The loci for \u003cem\u003ePAX5\u003c/em\u003eand \u003cem\u003eBTG1\u003c/em\u003e were retained. Exome and Sanger sequencing identified nucleotide variants of \u003cem\u003eARID5B\u003c/em\u003e and \u003cem\u003eNCOR1\u003c/em\u003e in the diagnostic sample, as well as a \u003cem\u003eKRAS\u003c/em\u003e variant (p.Lys117Asn) in the first recurrent sample and another \u003cem\u003eKRAS\u003c/em\u003e variant (p.Asp119Gly) in the second recurrent sample and the cell line. Transcriptome analysis and RT-PCR confirmed that all examined samples contained a \u003cem\u003eTCF3::HLF\u003c/em\u003echimeric transcript. However, molecular cytogenetics did not verify the juxtaposition of \u003cem\u003eTCF3\u003c/em\u003e and \u003cem\u003eHLF\u003c/em\u003e loci. Further long-range PCR analyses confirmed that genomic material containing \u003cem\u003eHLF\u003c/em\u003e exon 4 was inserted into \u003cem\u003eTCF3\u003c/em\u003e intron 16. Using dimensional reduction techniques, we found that the current cell line shares an expression pattern with other \u003cem\u003eTCF3::HLF\u003c/em\u003e-positive BCP-ALL cell lines. The cytotoxicity assay indicated that the cell line is sensitive to Aurora Kinase B inhibitor, but not to BCL2 inhibitor. This cell line is the first \u003cem\u003eTCF3::HLF\u003c/em\u003e-positive BCP-ALL model without the t(17;19) translocation, facilitating research into leukemogenesis and the development of novel treatments for patients with poor prognosis associated with \u003cem\u003eTCF3::HLF\u003c/em\u003e-positive BCP-ALL.\u003c/p\u003e","manuscriptTitle":"Establishment and chacterization of a novel cell line ICH-BCPALL-3 from B-cell precursor acute lymphoblastic leukemia with TCF3::HLF.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-22 09:23:47","doi":"10.21203/rs.3.rs-5469901/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept as is","date":"2025-04-22T22:51:51+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-04-18T22:01:46+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-18T20:56:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-18T12:13:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Human Cell","date":"2025-04-12T02:39:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"human-cell","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"huce","sideBox":"Learn more about [Human Cell](http://link.springer.com/journal/13577)","snPcode":"13577","submissionUrl":"https://www.editorialmanager.com/huce/default2.aspx","title":"Human Cell","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"90444e58-6a81-4773-8cf9-b3f3b1b365ac","owner":[],"postedDate":"April 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-24T16:07:35+00:00","versionOfRecord":{"articleIdentity":"rs-5469901","link":"https://doi.org/10.1007/s13577-025-01318-4","journal":{"identity":"human-cell","isVorOnly":false,"title":"Human Cell"},"publishedOn":"2025-11-19 15:59:03","publishedOnDateReadable":"November 19th, 2025"},"versionCreatedAt":"2025-04-22 09:23:47","video":"","vorDoi":"10.1007/s13577-025-01318-4","vorDoiUrl":"https://doi.org/10.1007/s13577-025-01318-4","workflowStages":[]},"version":"v1","identity":"rs-5469901","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5469901","identity":"rs-5469901","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}