NUTM1 Is a Rare but Recurrent Fusion Gene Partner in B Cell Precursor Acute Lymphoblastic Leukemia Associated with Increased Expression of Genes on Cytoband 10p12.31

Abstract INTRODUCTION In 20-25% of the pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL) patients, the driving cytogenetic aberration is unknown. It is important to identify more primary lesions in this remaining B-other group to provide better risk stratification and identify possible treatment options. In this study, we aimed to identify novel recurrent fusion genes in BCP-ALL through RNA sequencing. METHODS We used paired-end total RNA Illumina sequencing to detect fusion genes with STAR-fusion and FusionCatcher in a population-based ALL cohort (n=71). We used Affymetrix U133 Plus2 expression arrays in a larger population-based ALL cohort (n=661) and an infant ALL cohort (n=70) to compare gene expression levels. Fluorescent in situ hybridization (FISH) was performed using Cytocell NUTM1 break-apart probe set MPH4800. RESULTS We identified an in-frame SLC12A6-NUTM1 fusion transcript composed of exons 1-2 of SLC12A6 fused to exons 3 to 8 of NUTM1 by RNA sequencing. Both genes are located on 15q14 within 5.3 Kb distance on opposite strands, and the fusion could result from an inversion. The fusion transcript is predicted to encode almost the total NUTM1 protein including the acidic binding domain for the histone acetyltransferase EP300. The SLC12A6-NUTM1 fusion case showed high NUTM1 expression, while NUTM1 expression was absent in the remaining cases. Using gene expression profiling, we identified four additional pediatric and two non-KMT2A-rearranged infant BCP-ALL cases with high NUTM1 expression. In the population-based cohort reflecting all different cytogenetic subtypes, these cases were restricted to the B-other group without known sentinel cytogenetic abnormalities. FISH showed a NUTM1 break apart pattern in all four tested NUTM1-positive cases indicative of a balanced translocation. RNA sequencing confirmed an ACIN1-NUTM1 fusion in one of the infant cases. We conclude that NUTM1 is normally not expressed in leukemic lymphoblasts, and that its expression can be induced by a gene fusion. The karyotypes of the predicted NUTM1 fusion cases combined with RNA sequencing data suggest that different chromosomal rearrangements are involved, likely resulting in different NUTM1 fusion partners. In literature, BRD9-NUTM1, IKZF1-NUTM1, and CUX1-NUTM1 fusions were reported in pediatric B-other cases, and BRD9-NUTM1 and ACIN1-NUTM1 fusions were reported in non-KMT2A-rearranged infants. Our combined aberrant gene expression and FISH results indicate that NUTM1 fusions occur in 2.4% (5/210) of pediatric and in 28% (2/7) of infant BCP-ALL cases without a sentinel cytogenetic aberration. The recurrence of NUTM1 aberrations in BCP-ALL cases without a known driver and the resulting expression of NUTM1 suggests that this fusion could be a new oncogenic driver in leukemia. All seven patients with a NUTM1 fusion achieved continuous complete remission with a median follow-up time of 8.3 years (range 4.8-13.8 years), suggesting that NUTM1 fusions in BCP-ALL have a favorable prognosis. To get an insight in the underlying biology, we compared gene expression between NUTM1-positive and NUTM1-negative pediatric B-other cases. We identified 130 differentially expressed probe sets (FDR ≤0.01) with a peculiar enrichment of those located on chromosome band 10p12.31 (Bonferroni adjusted p=4.05E-04). The genes in cytoband 10p12.31, including BMI1, were variably upregulated in 6/7 NUTM1-positive cases and positively correlated to NUTM1 expression levels. The NUTM1 protein is capable of binding and hereby stimulating the histone acetyltransferase activity of the EP300 protein. The EP300 protein preferentially binds a risk allele of BMI1 associated with increased risk for BCP-ALL. The BMI1 protein has been shown to convert BCR-ABL1-positive progenitor cells into BCR-ABL1-positive BCP-ALL cells. Hence, we postulate that NUTM1 fusion proteins contribute to leukemogenesis by stimulating EP300, leading to upregulation of BMI1 and other 10p12.31 genes in BCP-ALL. CONCLUSION NUTM1 fusions are a rare but recurrent event in BCP-ALL that seems to have a good prognosis. The NUTM1 fusions result in expression of the normally silent NUTM1 gene and are associated with upregulation of a cluster of genes on 10p12.31 including the leukemogenic BMI1 gene. 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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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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Discovery of Novel Recurrent Mutations in Childhood Early T-Cell Precursor Acute Lymphoblastic Leukemia by Whole Genome Sequencing - a Report From the St Jude Children's Research Hospital - Washington University Pediatric Cancer Genome Project

Blood ◽

10.1182/blood.v118.21.68.68 ◽

2011 ◽

Vol 118 (21) ◽

pp. 68-68

Author(s):

Jinghui Zhang ◽

Li Ding ◽

Linda Holmfeldt ◽

Gang Wu ◽

Susan L. Heatley ◽

...

Keyword(s):

Gene Expression ◽

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Lymphoblastic Leukemia ◽

Whole Genome ◽

Structural Variants ◽

Cell Precursor ◽

Recurrent Mutations

Abstract Abstract 68 Early T-cell precursor acute lymphoblastic leukemia (ETP ALL) is characterized by an immature T-lineage immunophenotype (cCD3+, CD1a-, CD8- and CD5dim) aberrant expression of myeloid and stem cell markers, a distinct gene expression profile and very poor outcome. The underlying genetic basis of this form of leukemia is unknown. Here we report results of whole genome sequencing (WGS) of tumor and normal DNA from 12 children with ETP ALL. Genomes were sequenced to 30-fold haploid coverage using the Illumina GAIIx platform, and all putative somatic sequence and structural variants were validated. The frequency of mutations in 43 genes was assessed in a recurrence cohort of 52 ETP and 42 non-ETP T-ALL samples from patients enrolled in St Jude, Children's Oncology Group and AEIOP trials. Transcriptomic resequencing was performed for two WGS cases, and whole exome sequencing for three ETP ALL cases in the recurrence cohort. We identified 44 interchromosomal translocations (mean 4 per patient, range 0–12), 32 intrachromosomal translocations (mean 3, 0–7), 53 deletions (mean 4, 0–10) and 16 insertions (mean 1, 0–5). Three cases exhibited a pattern of complex rearrangements suggestive of a single cellular catastrophe (“chromothripsis”), two of which had mutations targeting mismatch and DNA repair (MLH3 and DCLRE1C). While no single chromosomal alteration was present in all cases, 10 of 12 ETP ALLs harbored chromosomal rearrangements, several of which involved complex multichromosomal translocations and resulted in the expression of chimeric in-frame novel fusion genes disrupting hematopoietic regulators, including ETV6-INO80D, NAP1L1-MLLT10, RUNX1-EVX1 and NUP214-SQSTM1, each occurring in a single case. An additional ETP case with the ETV6-INO80D fusion was identified in the recurrence cohort. Additionally, 51% of structural variants had breakpoints in genes, including those with roles in hematopoiesis and leukemogenesis, and genes also targeted by mutation in other cases (MLH3, SUZ12, RUNX1). We identified a high frequency of activating mutations in genes regulating cytokine receptor and Ras signalling in ETP ALL (67.2% of ETP compared to 19% of non-ETP T-ALL) including NRAS (17%), FLT3 (14%), JAK3 (9%), SH2B3 (or LNK; 9%), IL7R (8%), JAK1 (8%), KRAS (3%), and BRAF (2%). Seven cases (5 ETP, 2 non-ETP) harbored in frame insertion mutations in the transmembrane domain of IL7R, which were transforming when expressed in the murine cell lines, and resulted in enhanced colony formation when expressed in primary murine hematopoietic cells. The IL7R mutations resulted in constitutive Jak-Stat activation in these cell lines and primary leukemic cells expressing these mutations. Fifty-eight percent of ETP cases (compared to 17% of non-ETP cases) harbored mutations known or predicted to disrupt hematopoietic and lymphoid development, including ETV6 (33%), RUNX1 (16%), IKZF1 (14%), GATA3 (10%), EP300 (5%) and GATA2 (2%). GATA3 regulates early T cell development, and mutations in this gene were observed exclusively in ETP ALL. The mutations were commonly biallelic, and were clustered at R276, a residue critical for binding of GATA3 to DNA. Strikingly, mutations disrupting chromatin modifying genes were also highly enriched in ETP ALL. Genes encoding the the polycomb repressor complex 2 (EZH2, SUZ12 and EED), that mediates histone 3 lysine 27 (H3K27) trimethylation were deleted or mutated in 42% of ETP ALL compared to 12% of non-ETP T-ALL. In addition, alterations of the H3K36 trimethylase SETD2 were observed in 5 ETP cases, but not in non-ETP ALL. We also identified recurrent mutations in genes that have not previously been implicated in hematopoietic malignancies including RELN, DNM2, ECT2L, HNRNPA1 and HNRNPR. Using gene set enrichment analysis we demonstrate that the gene expression profile of ETP ALL shares features not only with normal human hematopoietic stem cells, but also with leukemic initiating cells (LIC) purified from patients with acute myeloid leukemia (AML). These results indicate that mutations that drive proliferation, impair differentiation and disrupt histone modification cooperate to induce an aggressive leukemia with an aberrant immature phenotype. The similarity of the gene expression pattern with that observed in the LIC of AML raises the possibility that myeloid-directed therapies might improve the outcome of ETP ALL. Disclosures: Evans: St. Jude Children's research Hospital: Employment, Patents & Royalties; NIH & NCI: Research Funding; Aldagen: Membership on an entity's Board of Directors or advisory committees.

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Biology of t(6;11) Fusion Proteins and Their Role in MLL-Rearranged Acute Leukemia Lineage Determination

Blood ◽

10.1182/blood-2019-123070 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5033-5033

Author(s):

Arpita Kundu ◽

Eric Kowarz ◽

Jennifer Reis ◽

Rolf Marschalek

Keyword(s):

Gene Expression ◽

Acute Lymphoblastic Leukemia ◽

High Risk ◽

Acute Leukemia ◽

Fusion Proteins ◽

Lymphoblastic Leukemia ◽

Chromosomal Translocation ◽

Fusion Genes ◽

Disease Phenotype ◽

Acute Leukemias

Chromosomal translocations are genetic rearrangements where a chromosomal segment is transferred to a non-homologous chromosome which give rise to novel chimeras. Chromosomal rearrangements play a significant role in the development of acute leukemias (acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML)). Chromosomal translocation events occurring at 11q23 involving the KMT2A or Mixed-Lineage Leukemia (MLL) gene (n=102) can be diagnosed in about 5-10% of all acute leukemia patients (Marschalek Ann Lab Med 2016), especially prevalent in infant acute leukemias (up to 70% of cases). Different chromosomal translocation partner genes (such as AF4, AF6, AF9orENL and ELL) account for the majority of leukemia cases and have their genomic breakpoints within a major breakpoint cluster region (BCR intron 9-11; Meyer et. al. Leukemia 2018). Some rearrangements are specifically associated with particular disease phenotype e.g. the majority of ALL patients (~ 90%) are mainly caused by the following gene fusions, MLL-AF4, MLL-AF9, MLL-ENL. We are interested in a rare but yet drastic chromosomal translocation t(6;11)(q27;q23) which fuses KMT2A/MLL to Afadin (AFDN/AF6) gene. This chromosomal rearrangement has a very poor prognosis (survival-rate is ~10%) and is predominantly diagnosed in patients with high-risk AML. In this project, we investigate the molecular consequences of two different MLL-AF6 fusions and their corresponding reciprocal AF6-MLL fusions. MLL-AF6 fusions are mainly occurring within MLL intron 9 to 11 and are associated with an AML disease phenotype, while the same fusion occurring within the minor breakpoints region in MLL intron 21 until exon (ex) 24 are mainly diagnosed with T-ALL (T-cell acute lymphoblastic leukemia) disease phenotype. The molecular mechanism that determines the resulting disease phenotype is yet unknown. Therefore, we cloned all of these t(6;11) fusion proteins in order to investigate the functional consequences of the two different breakpoints (MLLex1-9::AF6ex2-30, AF6ex1::MLLex10-37; MLLex1-21::AF6ex2-30, AF6ex1::MLLex22-37). All 4 fusion genes were introduced into our inducible Sleeping Beauty system (Ivics et. al. Mobile DNA 2010; Kowarz et. al. Biotechnol J. 2015) and stably transfected reporter cell lines. Basically, these 4 fusion proteins differ only in the presence or absence of their Plant homeodomain 1-3/Bromodomain (PHD1-3/BD) domain (see Figure 1). The PHD domain regulates the epigenetic and transcriptional regulatory functions of wildtype MLL. Subsequently, we analyzed gene expression differences by the MACE-Seq (Massive Analyses of cDNA Ends). MACE data revealed fundamental differences in gene expression profiles when analyzing the two different sets of t(6;11) fusion genes. The resulting profiles have similarities to either AML or T-ALL and might give a rational explanation for the different lineages in these t(6;11) patients. Altogether, these results notably indicate that our study will provide a novel insight into this type of high-risk leukemia and subsequently will be useful for developing of novel and appropriate therapeutic strategies against acute leukemia. Disclosures No relevant conflicts of interest to declare.

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Single-cell transcriptomics predicts relapse in MLL-rearranged acute lymphoblastic leukemia in infants

10.1101/2020.04.14.20056580 ◽

2020 ◽

Author(s):

Tito Candelli ◽

Pauline Schneider ◽

Patricia Garrido Castro ◽

Luke A. Jones ◽

Rob Pieters ◽

...

Keyword(s):

Gene Expression ◽

Acute Lymphoblastic Leukemia ◽

Risk Stratification ◽

Single Cell ◽

Rna Sequencing ◽

Lymphoblastic Leukemia ◽

Leukemic Cells ◽

List Type ◽

Relapse Risk ◽

Single Cell Rna Sequencing

AbstractInfants with MLL-rearranged acute lymphoblastic leukemia (ALL) undergo intense therapy to counter a highly aggressive leukemia with survival rates of only 30-40%. The majority of patients initially show therapy response, but in two-thirds of cases the leukemia returns, typically during treatment. Accurate relapse prediction would enable treatment strategies that take relapse risk into account, with potential benefits for all patients. Through analysis of diagnostic bone marrow biopsies, we show that single-cell RNA sequencing can predict future relapse occurrence. By analysing gene modules derived from an independent study of the gene expression response to the key drug prednisone, individual leukemic cells are predicted to be either resistant or sensitive to treatment. Quantification of the proportion of cells classified by single-cell transcriptomics as resistant or sensitive, accurately predicts the occurrence of future relapse in individual patients. Strikingly, the single-cell based classification is even consistent with the order of relapse timing. These results lay the foundation for risk-based treatment of MLL-rearranged infant ALL, through single-cell classification. This work also sheds light on the subpopulation of cells from which leukemic relapse arises. Leukemic cells associated with high relapse risk are characterized by a smaller size and a quiescent gene expression program. These cells have significantly fewer transcripts, thereby also demonstrating why single-cell analyses may outperform bulk mRNA studies for risk stratification. This study indicates that single-cell RNA sequencing will be a valuable tool for risk stratification of MLL-rearranged infant ALL, and shows how clinically relevant information can be derived from single-cell genomics.Key PointsSingle-cell RNA sequencing accurately predicts relapse in MLL-rearranged infant ALLIdentification of cells from which MLL-rearranged infant ALL relapses occur

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The NOD/SCID Xenograft Model Provides Clinically-Relevant Insights Into Glucocorticoid-Induced Gene Expression In Childhood B-Cell Precursor Acute Lymphoblastic Leukemia (ALL)

Blood ◽

10.1182/blood.v116.21.2493.2493 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2493-2493

Author(s):

Vivek A Bhadri ◽

Mark J Cowley ◽

Warren Kaplan ◽

Richard B Lock

Keyword(s):

Gene Expression ◽

Acute Lymphoblastic Leukemia ◽

Mouse Model ◽

B Cell ◽

Differential Expression ◽

Differentially Expressed Genes ◽

Lymphoblastic Leukemia ◽

Xenograft Model ◽

Differentially Expressed ◽

Cell Precursor

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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The Heterogeneous Fusion Gene Landscape in Pediatric Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v128.22.4081.4081 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4081-4081

Author(s):

Yanara Marincevic-Zuniga ◽

Johan Dahlberg ◽

Sara Nilsson ◽

Amanda Raine ◽

Jonas Abrahamsson ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Acute Lymphoblastic Leukemia ◽

Gene Fusion ◽

Conflicts Of Interest ◽

Fusion Gene ◽

Lymphoblastic Leukemia ◽

Fusion Genes ◽

Fusion Transcripts ◽

Pediatric All

Abstract Background: Next generation sequencing allows for the detection of expressed fusion transcripts across the transcriptome and has spurred the discovery of many novel chimeric transcripts in various cancers. Structural chromosomal rearrangements that lead to fusion transcripts are a hallmark of acute lymphoblastic leukemia (ALL) and serve as markers for diagnosis and stratification of pediatric ALL patients into prognostically relevant subgroups. Improved delineation of structural alterations in ALL could provide additional information for prognosis in ALL and for improved stratification of patients into treatment groups. Methods: To identify novel fusion transcripts in primary pediatric ALL cells we performed whole transcriptome sequencing of 134 BCP and T-ALL patient samples collected at diagnosis. Our study include samples from patients with the well-known ALL subtypes t(12;21)ETV6-RUNX1, high hyperdiploid (51-67 chromosomes), t(9;22)BCR-ABL1, 11q23/MLL and dic(9;20), in addition to patients with undefined karyotype or non-recurrent cytogenetic aberrations ("undefined" and "other") (n=58). FusionCatcher was used for the detection of somatic fusion genes, followed by a stringent filtering pipeline including gene fusion validation by Sanger sequencing in order to reduce the number of false positives. Principal component analysis (PCA) of patients with fusion genes was performed using genome wide gene expression levels and DNA methylation levels (Infinium HumanMethylation450 bead array). Results: We identified and validated 60 unique fusion events in almost half of the analyzed patients (n=69). Of the identified fusion genes, 60% have not previously been reported in ALL or other forms of cancer. The majority of the fusion genes were found in a single patient, but 23% were recurrent, including known ALL fusion genes (n=10) and novel fusion genes (n=7). We found that BCP-ALL samples displayed a higher number of validated fusion genes (54%) compared to the T-ALL samples (28%) moreover in BCP-ALL patients with "other" and "undefined" karyotypes, we detected fusion genes in 71% and 61% of the samples, respectively. High hyperdiploid patients had the lowest rate of validated fusion genes (24%) compared to the other well-known subtypes, where we detected subtype-associated fusion genes in 97% of cases. We also identified promiscuous fusion gene partners, such as ETV6, RUNX1, PAX5 and ZNF384 that fused with up to five different genes. Interestingly, PCA revealed molecularly distinct gene expression and DNA methylation signatures associated with these fusion partners. Conclusion: RNA-sequencing of pediatric ALL cells revealed a detailed view of the heterogeneous fusion gene landscape, identifying both known and novel fusion genes. By grouping samples based on recurrent gene fusion partners we are able to find shared gene expression and DNA methylation patterns compared to other subtypes of ALL, suggesting a shared molecular etiology within these distinct subgroups, offering novel insights into the delineation of fusion genes in ALL. 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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Frequency of p190 and p210 BCR-ABL Fusions Genes in Acute Lymphoblastic Leukemia in a Long Group of Adults and Childhood

Blood ◽

10.1182/blood.v128.22.5273.5273 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5273-5273 ◽

Cited By ~ 3

Author(s):

Rosa Maria Arana-Trejo ◽

Gregorio Ignacio ◽

Raquel Amador-Sánchez ◽

Jorge Cruz-Rico ◽

Maria-Paula Hernández ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

De Novo ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Fusion Transcript ◽

Age Group ◽

Fusion Genes ◽

Rt Pcr ◽

Pcr Multiplex ◽

The Impact

Abstract The Ph chromosome is a translocation (9;22)(q34;q11), that results in the constitutive activation of the BCR/ABL tyrosine kinase. The incidence of BCR/ABL in Acute Lymphoblastic Leukemia (ALL) increases with age, from less than 5% in younger children to 20-30% in older adults, with a peak incidence in patients aged 35-50 years. BCR/ABL1 has diverse breakpoints, in adult patients with Ph+ ALL the p190BCR/ABL transcript e1a2/e3a2 may be documented in 50-70%; p210BCR/ABL b2a2/b3a2 in 15-30% of patients and <1% having both breakpoint. Childhood patients with Ph+ ALL fusion genes present p190BCR/ABL transcipt e1a2/e3a2 in 90% and the remaining present other fusion transcrit or co-expression of both p190 and p210 BCR-ABL. OBJETIVE. The aim of this study was identify the occurrence of fusion genes to p190 and p210 BCR-ABL rearrangements in adult and childhood patients with ALL. METHODS. We include between 2008-2015 870 patients with ALL de novo from seven different hospitals from México, the 45% (394) were childood and the rest 55% (476) were adults. All patients were studied to RT-PCR multiplex and nested in RNA for fusion transcripts 190 and p210 BCR-ABL, at diagnosis, according to the international BIOMED-1 protocol. RESULTS. From 870 patients with ALL, the most frequent subtype FAB were L2 (87%) and second L1 (13%). The immunophenotype by B-ALL was to 80%, bilineal in 5% and the rest have not data. The overall incidence to BCR-ABL in both children and adults with ALL were to 17% [147/870]. The analysis by age group were; in 476 adults with ALL, their average age was 37 years old (range 17-84 years) and their incidence of BCR-ABL positive was 20% (95/476 cases). The distributions by type of fusion transcript were 83% p190 and 17% p210; we did not observe co-expression of transcripts to BCR-ABL. In children patients the average age was 9 years old (range 0.1-16 years), the incidence of BCR-ABL was 13.2% (52/394 cases). The distributions by type of fusion transcript to BCR-ABL were p190 78.8%; p210 13.4% and their co-expression by both isoforms 8%. CONCLUSION. The 20% frequency for BCR-ABL1 in adults with ALL is concordant with others reports published, with values from 17% to 37% with predominancy of p190 (83%). In our pediatric patients group with ALL, document a frequency of 13.2% by BCR-ABL1 positive; it is higher than other populations reporting 5-10%. The distributions of fusion transcript p190 and p210 coincides with previous prevalence estimates in other countries where p190 transcript was the most frequent. But the coexpression of both isoforms [p190/p210] were 8% it has not been reported in this age group with ALL. In conclusion, we recommend to identify the BCR-ABL transcript type in every patients with ALL at diagnosis, using a RT-PCR verified method for P190/p210 and followed the patient by mesure the impact clinical and will be adjust the treatment like o plus the cytogenetic studies. Disclosures No relevant conflicts of interest to declare.

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