Evaluation of the NOD/SCID xenograft model for glucocorticoid-regulated gene expression in childhood B-cell precursor acute lymphoblastic leukemia

Abstract Abstract 2493 Introduction. Glucocorticoids (GC) such as prednisolone (Pred) and dexamethasone (Dex) are critical drugs in multi-agent chemotherapy protocols used to treat acute lymphoblastic leukemia (ALL). The NOD/SCID ALL xenograft mouse model is a clinically relevant model in which the mice develop a systemic leukemia which retains the fundamental biological characteristics of the original disease. Here we report the results of a study evaluating the NOD/SCID xenograft model to investigate GC-induced gene expression. Methods. Cells from a GC-sensitive xenograft derived from a child with B-cell precursor ALL were inoculated into NOD/SCID mice. Engraftment, defined as the proportion of human vs mouse CD45+ cells in the peripheral blood, was monitored by serial weekly tail-vein sampling. When engraftment levels reached >50%, the mice were randomised and treated with either dexamethasone 15 mg/kg or vehicle control by intraperitoneal injection, and harvested at 0, 8, 24 or 48 h thereafter. The 48 hour groups received a second dose of vehicle or Dex at 24 hours. At the defined timepoints, the mice were euthanized and lymphoblasts harvested from the spleen. RNA was extracted, amplified and hybridised onto Illumina WG-6 V3 chips. The data was pre-processed using variance-stabilisation transformation, and quantile normalisation. Differential expression was determined using limma by comparing all treated groups to time 0, with the positive False Discovery Rate correction for multiple testing. Hierarchical clustering was used to compare groups to each other. The stability of results when reducing the number of replicates was assessed using the Recovery Score method. Functional analysis was performed using gene set enrichment analysis (GSEA) and comparison to publicly available microarray data using parametric GSEA. Results. The 8 hour Dex-treated timepoint had the most number of significantly differentially expressed genes (see Table), with fewer observed at the 24 and 48 hour Dex-treated timepoints. There was minimal significant differential gene expression across the time-matched controls. At the 8 hour timepoint, ZBTB16, a known GC-induced gene, was the most significantly upregulated gene. Other significantly differentially expressed genes included TSC22D3 and SOCS1, both downstream targets of the glucocorticoid receptor (upregulated), and BCL-2 and C-MYC (downregulated). GSEA at 8 hours revealed a significant upregulation of catabolic pathways and downregulation of pathways associated with cell proliferation, particularly C-MYC. GSEA at 24 hours revealed enrichment of pathways associated with NF-kB. Replicate analysis revealed that at the 8 hour Dex treated timepoint, a dataset with high signal and differential expression, using data from 3 replicates instead of 4 resulted in excellent recovery scores of >0.9. However at other timepoints with less signal very poor recovery scores were obtained using 3 replicates. We compared our data to publicly available datasets of GC-induced genes in ALL (Schmidt et al, Blood 2006; Rainer et al, Leukemia 2009) using parametric GSEA, which revealed that the 8 hour gene expression data obtained from the NOD/SCID xenograft model clustered with data from primary patient samples (Schmidt) rather than the cell line data (Rainer). The 24 and 48 hour datasets clustered separately from all other datasets by this method, reflecting fewer and predominantly downregulated gene expression at these timepoints. Conclusions: The NOD/SCID xenograft mouse model provides a reproducible experimental model system in which to investigate clinically-relevant mechanisms of GC-induced gene regulation in ALL; the 8 hour timepoint provides the highest number of significantly differentially expressed genes; time-matched controls are redundant and excellent recovery scores can be obtained with 3 replicates. Disclosures: No relevant conflicts of interest to declare.

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Mutation, methylation, and gene expression profiles in dup(1q)-positive pediatric B-cell precursor acute lymphoblastic leukemia

Leukemia ◽

10.1038/s41375-018-0092-2 ◽

2018 ◽

Vol 32 (10) ◽

pp. 2117-2125 ◽

Cited By ~ 3

Author(s):

Rebeqa Gunnarsson ◽

Sebastian Dilorenzo ◽

Kristina B Lundin-Ström ◽

Linda Olsson ◽

Andrea Biloglav ◽

...

Keyword(s):

Gene Expression ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Expression Profiles ◽

Lymphoblastic Leukemia ◽

Gene Expression Profiles ◽

Cell Precursor

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Emerging molecular subtypes and therapeutic targets in B-cell precursor acute lymphoblastic leukemia

Frontiers of Medicine ◽

10.1007/s11684-020-0821-6 ◽

2021 ◽

Author(s):

Jianfeng Li ◽

Yuting Dai ◽

Liang Wu ◽

Ming Zhang ◽

Wen Ouyang ◽

...

Keyword(s):

Gene Expression ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Molecular Subtypes ◽

Lymphoblastic Leukemia ◽

Expression Patterns ◽

Therapeutic Targets ◽

Genetic Alterations ◽

Gene Expression Patterns ◽

Cell Precursor

AbstractB-cell precursor acute lymphoblastic leukemia (BCP-ALL) is characterized by genetic alterations with high heterogeneity. Precise subtypes with distinct genomic and/or gene expression patterns have been recently revealed using high-throughput sequencing technology. Most of these profiles are associated with recurrent non-overlapping rearrangements or hotspot point mutations that are analogous to the established subtypes, such as DUX4 rearrangements, MEF2D rearrangements, ZNF384/ZNF362 rearrangements, NUTM1 rearrangements, BCL2/MYC and/or BCL6 rearrangements, ETV6-RUNX1-like gene expression, PAX5alt (diverse PAX5 alterations, including rearrangements, intragenic amplifications, or mutations), and hotspot mutations PAX5 (p.Pro80Arg) with biallelic PAX5 alterations, IKZF1 (p.Asn159Tyr), and ZEB2 (p.His1038Arg). These molecular subtypes could be classified by gene expression patterns with RNA-seq technology. Refined molecular classification greatly improved the treatment strategy. Multiagent therapy regimens, including target inhibitors (e.g., imatinib), immunomodulators, monoclonal antibodies, and chimeric antigen receptor T-cell (CAR-T) therapy, are transforming the clinical practice from chemotherapy drugs to personalized medicine in the field of risk-directed disease management. We provide an update on our knowledge of emerging molecular subtypes and therapeutic targets in BCP-ALL.

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Evaluation of FOXP1 gene expression in pediatric B-cell precursor acute lymphoblastic leukemia patients at remission induction therapy

Iranian Journal of Pediatric Hematology & Oncology ◽

10.18502/ijpho.v10i4.4409 ◽

2020 ◽

Author(s):

Gholamhossein Tamaddon ◽

Mehran Bahraini ◽

Alieh Fazeli

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Cell Line ◽

Lymphoblastic Leukemia ◽

Remission Induction ◽

Control Group ◽

Cell Precursor ◽

New Diagnosis

Background: Transcription factors (TFs) play a key role in the development, therapy, and relapse of B-cell malignancies, such as B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Given the essential function of Forkhead box protein P1 (FOXP1) transcription factor in the early development of B-cells, this study was designed to evaluate FOXP1 gene expression levels in pediatric BCP-ALL patients and NALM6 cell-line. Materials and Methods: This case-control study was done on the NALM6 cell-line and bone marrow specimens of 23 pediatric BCP-ALL patients (median age: 7.5 years; range: 2.0 – 15.0 years) at different clinical stages including new diagnosis, 15th day after the treatment, and relapse. Also, 10 healthy children were included as the control group. FOXP1 gene expression was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). The correlation analysis was performed between the FOXP1 gene expression and patients’ demographic and laboratory characteristics. Results: The results showed that FOXP1 gene expression was significantly downregulated in the NALM-6 cell-line (median=0.05, P<0.001) and patients at new diagnosis (median=0.06, p<0.0001), and relapse (median=0.001, p<0.0001) phases, compared to the control group (median=0.08). FOXP1 gene expression on the 15th day of the treatment was significantly higher than its level at the new diagnosis stage (p<0.001). Moreover, FOXP1 gene was significantly downregulated in the relapse phase compared to the new diagnosis. Patients whose number of bone marrow blasts on the 15th day of the treatment was below 5% had higher FOXP1 gene expression at the diagnosis phase (Spearman’s correlation, P<0.05, r=-0.485) and higher ratio of diagnosis/day 15 (p<0.001, Mann-Whitney U test). Conclusions: FOXP1 levels could be a potential biomarker of therapy response in remission induction therapy for pediatric BCP-ALL patients.

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High VLA-4 expression is associated with adverse outcome and distinct gene expression changes in childhood B-cell precursor acute lymphoblastic leukemia at first relapse

Haematologica ◽

10.3324/haematol.2011.047993 ◽

2011 ◽

Vol 96 (11) ◽

pp. 1627-1635 ◽

Cited By ~ 31

Author(s):

S. Shalapour ◽

J. Hof ◽

R. Kirschner-Schwabe ◽

L. Bastian ◽

C. Eckert ◽

...

Keyword(s):

Gene Expression ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Adverse Outcome ◽

Lymphoblastic Leukemia ◽

Cell Precursor ◽

Distinct Gene ◽

First Relapse

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Identification of Sphingosine Kinases As Therapeutic Targets in B-Lineage Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v120.21.1499.1499 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1499-1499

Author(s):

Craig T Wallington-Beddoe ◽

Kenneth F Bradstock ◽

Linda J Bendall

Keyword(s):

Gene Expression ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Xenograft Model ◽

Intracellular Location ◽

Gene Signatures ◽

Sphingosine Kinases ◽

B Lineage

Abstract Abstract 1499 Sphingosine 1-phosphate (S1P) is a bioactive lipid with roles in cell proliferation and survival. S1P is produced by the sphingosine kinases, SphK1 and SphK2. SphK1 is over expressed in a number of malignancies and evidence points overwhelmingly to a pro-survival role. The role of SphK2 is much less well defined and appears to be dependent on its intracellular location with some reports of opposite effects to those of SphK1. Little is known about the interaction of SphKs with intracellular signalling pathways. Here we assess the relevance of SphKs in B-lineage acute lymphoblastic leukemia (B-ALL). Gene expression signatures indicative of activation of SphK1 or SphK2 were not publicly available. Therefore we treated ALL cell lines with the SphK1 or SphK2 specific inhibitors SK1-I and ABC294640 respectively, and analysed gene expression by microarray. Although a signature for SphK1 was not obtained, two independent methods of analysis generated SphK2 gene signatures that segregated control and drug treated cell lines. The signatures consisted of 11 and 35 genes and included reduced expression of the NF-κB inhibitor TRIB3 (NF-κB activity is commonly up regulated in ALL), the C/EBP family transcription factor DDIT3, the protein phosphatase 1 regulatory subunit PPP1R15A and the sphingolipid/cholesterol transporter ABCA1, all validated by quantitative RT-PCR. These gene signatures were used to interrogate a large publicly available gene expression dataset (GSE28497) obtained from pediatric ALL patients at the time of diagnosis. SphK2 activity signatures were more highly expressed in ALL samples (p=0.001 and p=0.027 for the smaller and larger signatures respectively) than normal B-cell progenitors. Although SPHK1 or SPHK2 genes were not over expressed in this dataset, SphK1 protein levels were increased in ALL cell lines and in patient samples. The importance of SphK1 and SphK2 in the development of ALL was examined by transducing B-cell progenitors isolated from WT, SphK1−/− or SphK2−/− mice with the ALL associated p185 form of the oncogenic fusion gene BCR/ABL and injecting transduced cells into sub-lethally irradiated wild type C57BL/6 mice. Twenty-two of 29 mice receiving cells from WT animals developed ALL, with a mean survival of 52.6 days (95% CI 42.3–62.8 days). Mice receiving cells lacking Sphk1 or Sphk2 had a significantly reduced incidence of ALL development with 14 of 30 (mean survival 71.6 days, 95% CI 60.5–82.8 days) and 16 of 29 animals (mean survival of 68.7 days, 95% CI 57.9–79.5 days) respectively (p=0.001). Lymphoblasts with a B-cell progenitor phenotype (B220+, CD19+, IgM−, CD11b−) were present in blood films and livers from leukemic mice and cells recovered from these animals produced a rapidly fatal ALL in secondary recipients. The presence of BCR/ABL and the expected deletion of Sphk1 or Sphk2 were confirmed by PCR in all murine leukemias examined. We have previously reported that inhibitors of SphK1 and/or SphK2 inhibit proliferation and induce cell death in ALL cell lines, and that these agents can synergize with imatinib in Ph+ ALL cell lines. We have furthered these studies to demonstrate that patient derived ALL cells similarly respond to SphK1 and SphK2 inhibition in vitro. The SphK2 inhibitor ABC294640 (100mg/kg) reduced plasma S1P levels in mice consistent with our previous reports of reduced ALL burden following ABC294640 treatment, and using a NOD/SCID γc−/− xenograft model of human ALL we have now shown that ABC294640 extends survival of ALL-bearing mice (p=0.0012 for Ph− xenograft) and further extends the survival when combined with imatinib treatment in mice engrafted with a human Ph+ ALL patient sample (p=0.044). This is the first report to suggest that sphingosine kinase 2 activity is increased in ALL, providing support for targeting SphK2 as a therapeutic strategy. Loss of SphKs reduces the incidence of ALL in a murine model of BCR/ABL-driven disease and ABC294640 reduces disease and extends survival in a human xenograft model of ALL. This compound synergizes with a number of potential therapeutic agents and further extends survival in a xenograft model of Ph+disease when combined with imatinib. This has potential to translate into a useful anti-leukemic strategy. Disclosures: No relevant conflicts of interest to declare.

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Transcriptional landscape of B cell precursor acute lymphoblastic leukemia based on an international study of 1,223 cases

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1814397115 ◽

2018 ◽

Vol 115 (50) ◽

pp. E11711-E11720 ◽

Cited By ~ 46

Author(s):

Jian-Feng Li ◽

Yu-Ting Dai ◽

Henrik Lilljebjörn ◽

Shu-Hong Shen ◽

Bo-Wen Cui ◽

...

Keyword(s):

Gene Expression ◽

Acute Lymphoblastic Leukemia ◽

High Risk ◽

B Cell ◽

Rna Sequencing ◽

Large Scale ◽

Lymphoblastic Leukemia ◽

International Study ◽

Disease Classification ◽

Cell Precursor

Most B cell precursor acute lymphoblastic leukemia (BCP ALL) can be classified into known major genetic subtypes, while a substantial proportion of BCP ALL remains poorly characterized in relation to its underlying genomic abnormalities. We therefore initiated a large-scale international study to reanalyze and delineate the transcriptome landscape of 1,223 BCP ALL cases using RNA sequencing. Fourteen BCP ALL gene expression subgroups (G1 to G14) were identified. Apart from extending eight previously described subgroups (G1 to G8 associated with MEF2D fusions, TCF3–PBX1 fusions, ETV6–RUNX1–positive/ETV6–RUNX1–like, DUX4 fusions, ZNF384 fusions, BCR–ABL1/Ph–like, high hyperdiploidy, and KMT2A fusions), we defined six additional gene expression subgroups: G9 was associated with both PAX5 and CRLF2 fusions; G10 and G11 with mutations in PAX5 (p.P80R) and IKZF1 (p.N159Y), respectively; G12 with IGH–CEBPE fusion and mutations in ZEB2 (p.H1038R); and G13 and G14 with TCF3/4–HLF and NUTM1 fusions, respectively. In pediatric BCP ALL, subgroups G2 to G5 and G7 (51 to 65/67 chromosomes) were associated with low-risk, G7 (with ≤50 chromosomes) and G9 were intermediate-risk, whereas G1, G6, and G8 were defined as high-risk subgroups. In adult BCP ALL, G1, G2, G6, and G8 were associated with high risk, while G4, G5, and G7 had relatively favorable outcomes. This large-scale transcriptome sequence analysis of BCP ALL revealed distinct molecular subgroups that reflect discrete pathways of BCP ALL, informing disease classification and prognostic stratification. The combined results strongly advocate that RNA sequencing be introduced into the clinical diagnostic workup of BCP ALL.

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New oncogenic subtypes in pediatric B-cell precursor acute lymphoblastic leukemia

Blood ◽

10.1182/blood-2017-05-742643 ◽

2017 ◽

Vol 130 (12) ◽

pp. 1395-1401 ◽

Cited By ~ 24

Author(s):

Henrik Lilljebjörn ◽

Thoas Fioretos

Keyword(s):

Gene Expression ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Molecular Subtypes ◽

Lymphoblastic Leukemia ◽

Cell Precursor ◽

Mutational Spectrum ◽

Molecular Studies

Abstract Until recently, 20% to 30% of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) could not be classified into any of the established molecular subtypes. Recent molecular studies of such cases have, however, further clarified their mutational spectrum and identified new oncogenic subtypes consisting of cases with DUX4 rearrangements, ETV6-RUNX1–like gene expression, MEF2D rearrangements, and ZNF384 rearrangements. In this review, we describe these new subtypes, which account for up to 50% of previously unclassified pediatric BCP-ALL cases.

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Genomic Determinants of Response to Blinatumomab in Relapsed/Refractory (R/R) B-Cell Precursor Acute Lymphoblastic Leukemia in Adults

Blood ◽

10.1182/blood-2018-99-113604 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1552-1552 ◽

Cited By ~ 2

Author(s):

Yaqi Zhao ◽

Ibrahim Aldoss ◽

Chunxu Qu ◽

Guido Marcucci ◽

Anthony S. Stein ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

Acute Lymphoblastic Leukemia ◽

T Cell ◽

B Cell ◽

High Frequency ◽

Research Funding ◽

Lymphoblastic Leukemia ◽

Cell Precursor ◽

B Lymphoid

Abstract Introduction: Blinatumomab, a CD19/CD3 bi-specific T-cell engager monoclonal antibody that re-directs CD3-positive T cells towards CD19-positive B cells, has shown promise in the treatment of R/R B-cell precursor acute lymphoblastic leukemia (B-ALL), with superior survival rates compared to salvage chemotherapy. However, many patients do not respond or subsequently relapse, and the mechanisms underlying resistance are unclear. The goals of this study were to characterize the genomic features associated with response to blinatumomab. Methods: We studied 29 patients (pts; median age 28, range 18-70) with R/R B-ALL who were treated with up to 5 cycles of blinatumomab, and predominantly of Hispanic ancestry (66%). Overall, 17 pts (59%) achieved remission with blinatumomab whilst 12 showed no response. Among the 17 responders, 7 (41%) subsequently relapsed or progressed during treatment. We analyzed leukemic blasts obtained before and after blinatumomab treatment whenever available: pre-blinatumomab R/R (n=28), post-blinatumomab refractory (n=5), post-blinatumomab relapsed (n=3) (Figure 1). Leukemia and matched remission samples were studied using transcriptome sequencing (n=34), whole genome sequencing (n=28), whole exome sequencing (n=19) and Infinium Omni2.5Exome-8 (SNP array, n=19). Results: Seventeen of 29 pts (59%) were Ph-like ALL. Twelve of 17 Ph-like ALL pts had high CRLF2 expression, among these we identified P2RY8-CRLF2 (n=4) and IGH-CRLF2 (n=8). Within the remaining 5 Ph-like ALL cases, two pts harbored NUP214-ABL1, two IGH-EPOR and one TERF-JAK2. Fifteen of the 17 (88%) Ph-like ALL cases were of Hispanic ancestry. The prevalence of other known subtypes was relatively low: BCR-ABL1 7%, hypodiploid 7%, KMT2A 3%, TCF3-PBX1 3% and B-other 21%. We observed a high response rate of 83.3% (10/12 cases) in Ph-like_CRLF2 pts, whilst the frequency of response was 60% (3/5, including two IGH-EPOR) for Ph-like_non-CRLF2 pts, and 33% (4/12) for the other subtypes (Ph-like ALL vs. others, P=0.029) (Figure 1). Unsupervised hierarchical clustering of pre-blinatumomab samples identified 3 clusters based on response to blinatumomab: cluster 1 contained non-responders, clusters 2 and 3 were largely made up of responders. By gene expression profiling using CIBERSORT we found reduced infiltration of cytotoxic CD8+ T-cells in cluster 1 compared to clusters 2 and 3 (6.1% vs. 14.9%, P=0.014), which was inversely correlated with the presence of CD4+ T cells (17.9% vs. 11.5%). GSEA showed enrichment for the IFNγ response, JAK-STAT signaling, chemokine and cytokine signaling in responders. In non-responders, differential gene expression analysis identified up-regulation of the H3K4 demethylase KDM5B, an oncogene associated with progression and chemoresistance of glioma and neuroblastoma. We observed a high frequency of alterations affecting B-lymphoid development (IKZF1, PAX5 and EBF1) in the pre-blinatumomab samples (20 of 22, 91%), which were maintained during progression or relapse. The frequency of B-lymphoid alterations did not differ significantly between responders and non-responders (13 of 14, 93% vs. 7 of 8, 88%). Alterations affecting the cell cycle (CDKN2A/B, TP53, RB1) were observed at a high frequency in pre-blinatumomab samples (15 of 22, 68%), with CDKN2A/B deletions enriched in responders compared to non-responders (11 of 14, 79% vs. 2 of 8, 25%; P=0.026). We also observed a high prevalence of alterations affecting epigenetic modifiers (ARID1B, CREBBP, KDM6A, KMT2D, TRRAP, SMARCA4) in pre-blinatumomab samples (17 of 22, 77%), with no difference between responders and non-responders (10 of 14, 71% vs. 7 of 8, 88%; P=0.61). Of the post-blinatumomab R/R samples available for study (n=8), CD19 expression was negative (n=1), dim (n=2) or positive (n=5). In contrast to previous reports of CD19 escape in CAR T-cell treated patients, there was no evidence of aberrantly spliced CD19 mRNA species, CD19 mutation or deletion in the three negative/dim cases. Conclusion: We show that a heightened immune response through the infiltration of cytotoxic T-cells and activation of IFNγ and JAK-STAT signaling in leukemic cells is an important determinant of response to blinatumomab. Importantly, blinatumomab is a valid therapeutic approach for patients harboring high-risk CRLF2 and EPOR-rearrangements. CD19 escape is not associated with genetic alterations at the CD19 locus. Figure. Figure. Disclosures Stein: Amgen Inc.: Speakers Bureau; Celgene: Speakers Bureau. Mullighan:Loxo Oncology: Research Funding; Cancer Prevention and Research Institute of Texas: Consultancy; Amgen: Honoraria, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau; Abbvie: Research Funding. Forman:Mustang Therapeutics: Other: Licensing Agreement, Patents & Royalties, Research Funding.

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Clinical Relevance of Tyrosine Kinase Fusion Genes in Pediatric BCR-ABL1-like Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v126.23.3735.3735 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3735-3735 ◽

Cited By ~ 1

Author(s):

Judith M. Boer ◽

Aurélie Boeree ◽

João R.M. Marchante ◽

Berna Beverloo ◽

Gabriele Escherich ◽

...

Keyword(s):

Gene Expression ◽

Acute Lymphoblastic Leukemia ◽

Tyrosine Kinase ◽

B Cell ◽

Signaling Pathway ◽

Ex Vivo ◽

Lymphoblastic Leukemia ◽

Fusion Genes ◽

Rt Pcr ◽

Cell Precursor

Abstract Background Patients with pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) with the BCR-ABL1 fusion gene form a small high-risk patient group with a poor prognosis. Approximately 15% of BCP-ALL are characterized by a gene expression signature similar to that of BCR-ABL1-positive disease and an unfavorable prognosis. This BCR-ABL1-like group shows a high frequency of B-cell development gene aberrations, especially IKZF1 deletions and tyrosine kinase-activating lesions (Den Boer et al. Lancet Oncol 2009; Mullighan et al. N Engl J Med 2009; Roberts et al. Cancer Cell 2012, N Engl J Med 2014; Van der Veer et al. Blood 2013). Aims To evaluate the clinical value of tyrosine kinase fusions in newly diagnosed children with B-cell precursor ALL, we studied their frequency, prognosis and drugability in a Dutch/German cohort. Methods This study comprised 204 children with BCP-ALL in three Dutch trials (DCOG ALL-8, 9, 10) and two German trials (COALL 06-97, 07-03) including 92 previously described BCR-ABL1-like cases identified by hierarchical clustering and 112 non-BCR-ABL1-like B-other cases. Molecular characterization included RT-PCR and FISH to detect fusions involving ABL1, PDGFRB, JAK2 and CSF1R, gene expression analysis, and copy number analysis. Results We identified 12 tyrosine kinase-activating fusion genes among 73 tested BCR-ABL1-like cases (16%) and none among 87 tested B-other cases. Eight fusions activated the ABL signaling pathway: 4 EBF1-PDGFRB, ZMIZ1-ABL1, RCSD1-ABL2, SSBP1-CSF1R, and one case with split ABL1 and an unknown fusion partner. Four fusions activated the JAK signaling pathway: 2 PAX5-JAK2, BCR-JAK2, and TERF2-JAK2. The gene fusions were confirmed by RT-PCR or targeted locus amplification. Gene expression of the involved tyrosine kinase was high in each of the fusion cases. IKZF1 deletions occurred more frequently in tyrosine kinase fusion cases compared with non-BCR-ABL1-like B-other cases (55% vs. 32%; p=0.2), and were enriched for rare, i.e. other than exon 4-7 or full deletion, variants (45% vs. 18%; p=0.05). In the remaining BCR-ABL1-like cases, the frequency of rare IKZF1 variants was similar to that in B-other (17%). Single deletion of exon 16 of EBF1 occurred in the EBF1-PDGFRB fusions and was rare among the remaining BCR-ABL1-like (0/77) and B-other cases (2/105). High CRLF2 expression co-occurred only in the BCR-JAK2 fusion case. The cumulative incidence of relapse (CIR) in the BCR-ABL1-like group with tyrosine kinase fusions (8-yr CIR 40% ± 18%) was comparable with that in the remaining BCR-ABL1-like group (8-yr CIR 36% ± 6%), and worse than in the B-other group (8-yr CIR 19% ± 4%; overall Gray p=0.04). Of the 12 tyrosine kinase fusion cases, four were late responders who only achieved remission after day 33 of induction therapy, and one was a non-responder resulting in early death. This non/late response rate was significantly higher in the tyrosine kinase fusion cases compared with non-BCR-ABL1 -like B-other (42% vs. 9%, p=0.008) and also higher compared with the remaining, fusion-negative BCR-ABL1-like cases (42% vs. 17%, p=0.06). Leukemic cells from three EBF1-PDGFRB patients were sensitive to 15 and 30 µM imatinib in ex vivo cultures, compared with lack of cytotoxic response in four EBF1-PDGFRB-negative samples, two of which even showed growth on imatinib. Combination of imatinib with 100 µg/ml prednisolone resulted in further growth inhibition in 2/3 EBF1-PDGFRB patients' ex vivo cultures. Conclusions Tyrosine kinase fusion genes were found in 16% of DCOG/COALL BCR-ABL1-like cases, representing ~3% of total BCP-ALL. BCR-ABL1-like cases with tyrosine kinase fusions were characterized by poor initial response to treatment, had an unfavorable clinical outcome compared with non-BCR-ABL1-like B-other ALL cases and a similar unfavorable outcome compared with tyrosine kinase fusion-negative BCR-ABL1-like cases. Imatinib worked additive to prednisolone in EBF1-PDGFRB patients' cells, indicating that this inhibitor may be clinically used in combination with at least prednisone. These results are in line with promising results of refractory EBF1-PDGFRB-positive and other ABL class fusion patients successfully treated with imatinib added to consolidation chemotherapy (Lengline et al. Haematologica 2013; Weston et al. J Clin Oncol 2013; Roberts et al. N Engl J Med 2014). Disclosures No relevant conflicts of interest to declare.

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