PAX5 Biallelic Genomic Alterations Define a Novel Subgroup of B-Cell
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Leukemia https://doi.org/10.1038/s41375-019-0430-z ARTICLE Acute lymphoblastic leukemia PAX5 biallelic genomic alterations define a novel subgroup of B-cell precursor acute lymphoblastic leukemia 1,2,3,4,5 1 3,4,6 1 1 Lorenz Bastian ● Michael P. Schroeder ● Cornelia Eckert ● Cornelia Schlee ● Jutta Ortiz Tanchez ● 1 2,7,8 1 1 Sebastian Kämpf ● Dimitrios L. Wagner ● Veronika Schulze ● Konstandina Isaakidis ● 1,3,4 5 1,3,4 9 1 Juan Lázaro-Navarro ● Sonja Hänzelmann ● Alva Rani James ● Arif Ekici ● Thomas Burmeister ● 1 10 11 3,4,12 13 Stefan Schwartz ● Martin Schrappe ● Martin Horstmann ● Sebastian Vosberg ● Stefan Krebs ● 13 14,15 3,4,12 3,4,16 5 Helmut Blum ● Jochen Hecht ● Philipp A. Greif ● Michael A. Rieger ● Monika Brüggemann ● 3,4,16 1,3,4,5 1,2,3,4,5 Nicola Gökbuget ● Martin Neumann ● Claudia D. Baldus Received: 6 September 2018 / Revised: 29 January 2019 / Accepted: 4 February 2019 © Springer Nature Limited 2019 Abstract Chromosomal rearrangements and specific aneuploidy patterns are initiating events and define subgroups in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Here we analyzed 250 BCP-ALL cases and identified a novel subgroup (‘PAX5- ’ n = fi 1234567890();,: 1234567890();,: plus , 19) by distinct DNA methylation and gene expression pro les. All patients in this subgroup harbored mutations in the B-lineage transcription factor PAX5, with p.P80R as hotspot. Mutations either affected two independent codons, consistent with compound heterozygosity, or suffered LOH predominantly through chromosome 9p aberrations. These biallelic events resulted in disruption of PAX5 transcriptional programs regulating B-cell differentiation and tumor suppressor functions. Homozygous CDKN2A/B deletions and RAS-activating hotspot mutations were highly enriched as cooperating events in the genomic profile of PAX5-plus ALL. Together, this defined a specific pattern of triple alterations, exclusive to the novel subgroup. PAX5-plus ALL was observed in pediatric and adult patients. Although restricted by the limited sample size, a tendency for more favorable clinical outcome was observed, with 10 of 12 adult PAX5-plus patients achieving long-term survival. PAX5-plus represents the first BCP-ALL subgroup defined by sequence alterations in contrast to gross chromosomal events and exemplifies how deregulated differentiation (PAX5), impaired cell cycle control (CDKN2A/B) and sustained proliferative signaling (RAS) cooperatively drive leukemogenesis. Introduction have been identified in pre-leukemic clones [4]char- acterizing chromosomal translocations or ploidy disorders Gross chromosomal events, such as specific aneuploidy as hallmark initiating events in leukemogenesis [5]. patterns or oncogenic gene fusions induced by chromo- A subset of these oncogenic gene fusions are already somal translocations, define molecular subgroups in B-cell targets for therapeutic interventions (e.g., ABL1-class precursor acute lymphoblastic leukemia (BCP-ALL) [1]. fusions) [1]. They inform biological programs as observed by Further steps of leukemic transformation include dele- distinct gene expression [2] and DNA methylation profiles tions of hematopoietic transcription factors (IKZF1, PAX5, [3], show an age-dependent prevalence pattern and and ETV6), which are co-occurring aberrations in 40% of impact treatment outcomes [1]. Some of these aberrations BCP-ALL cases, indicating that impairment of hemato- poietic differentiation promotes BCP-ALL development [6]. PAX5 has been characterized as haploinsufficient tumor Supplementary information The online version of this article (https:// suppressor in this context, both in BCP-ALL patient sam- doi.org/10.1038/s41375-019-0430-z) contains supplementary material, ples [6, 7] and mouse models [8–10]. Additional mutations which is available to authorized users. in genes, such as cell cycle regulators (CDKN2A/B) and * Claudia D. Baldus RAS/MAPK regulators (NRAS, KRAS, and NF1), are [email protected] acquired during leukemic development and contribute to an Extended author information available on the last page of the article individual disease phenotype [5]. L. Bastian et al. Up to 30% of BCP-ALL patients, especially at adult age, Variant calling and differential gene expression lack described initiating lesions, thus precluding the allo- analysis cation to molecular subgroups with specific functional and clinical characteristics and hampering the development of All sequences were aligned to the human genome build targeted therapeutic strategies. GRCh37.75 using the bcbio-nextgen pipeline v0.9.1a- To identify novel driver candidates and to characterize 7da8dce (https://github.com/bcbio/bcbio-nextgen) for WES their functional context, we analyzed 250 BCP-ALL first and STAR-aligner for RNA-Seq data [11]. Somatic muta- diagnosis samples across age groups for gene fusions and tions were detected in WES and corresponding target gene gene expression profiles, DNA methylation profiles, copy panel sequencing using bcbio-nextgen employing Mutect number alterations, single nucleotide variants (SNVs), and [12], Freebayes [13], VarDict [14], and VarScan [15]. small insertions/deletions (Indels). Integration of these Mutations were required to be reported by two of the four multi-omics data enabled us to identify a novel BCP-ALL callers with a variant allele frequency (VAF) of >10%, a subgroup defined by PAX5 sequence variants with different read depth of at least 10 with at least three reads supporting modes of inactivation of the second allele, CDKN2A/B the mutation and a VAF below 5% in the remission control. deletions, and RAS-activating mutations. For the 206-gene target panel, mutations were detected by Varscan [15] or Platypus [16]. Ensemble Variant Effect predictor [17] was used to annotate variants. Mutations Patients and methods were required to have a read depth of at least 30 and a VAF > 10%. Population variants were excluded based on dbSNP Patient materials and standard diagnostics 135, COSMIC v75 and gnomAD annotations. Gene panel sequencing achieved an average target coverage of >300×. Bone marrow or peripheral blood samples at first diagnosis Only mutations confirmed by gene panel sequencing were of BCP-ALL were obtained from adult and pediatric patients reported and included in the manuscript. Gene fusions were enrolled into trials of population-based German study detected from RNAseq data using deFuse v0.7.0 [18] and cohorts (GMALL, AIOP-BFM, and COALL). Patients and/ FusionCatcher v0.99.7c [19]. Expression quantification was or their families gave informed consent for biological obtained with Stringtie [20]. Differential expression analy- research on their banked samples. Studies were approved by sis was performed by ANOVA based on TPM. GSEA the institutional ethic review boards of the study groups (v3.0) software [21] was used in preranked mode for gene and the institutional ethic review board of the Charité - set enrichment analysis. For input, ranked gene lists were Universitätsmedizin, Berlin, Germany. Bone marrow sam- obtained by median log-fold change between each mole- ples obtained during remission (minimal residual disease cular subgroup and all other samples of the cohort. level < 10−2) were used as germline control. Further sample processing and clinical standard procedures are detailed in Copy number analyses Supplementary patient materials and methods. DNA copy number variations (CNV) were assessed by Sequencing analyses DNA hybridization to SNP-Arrays (CytoScan HD Array; Life Technologies, Carlsbad, CA) and analysis using the For whole-exome sequencing (WES) three samples per lane Chromosome Analysis Suite (ChAS; Life Technologies, were processed on an Illumina HiSeq4000 platform using Carlsbad, CA). Virtual karyotypes were derived from WES Low input Exome-Seq Human v5 + UTRs (Agilent, Santa data using CopywriteR [22] to calculate log-ratio counts in Clara, California) with an average coverage of 141.6 Mio bins of 20–50 kb between diagnosis and remission control. mapped reads/sample (MMRS). Panel sequencing was CNVkit [23] was used to call copy number variations from performed using one of two customized biotinylated RNA CopywriteR output. Copy number status in nine regions oligo pools (both SureSelect, Agilent, Santa Clara, Cali- most frequently affected by CNVs in BCP-ALL were fornia). For samples with WES data, target regions were assessed by Multiplex Ligation dependent Probe Amplifi- selected to validate mutations form WES (362 kbp target cation (MLPA) using the SALSA MLPA P335 ALL-IKZF1 size). For the remaining samples, target genes were selected probemix and Coffalyser Software (both: MRC Holland, to cover genes with central functions in normal or malignant Amsterdam, The Netherlands). hematopoiesis and frequent mutations in leukemia (Sup- plementary Table S1; 206 target genes). Panel sequencing DNA methylation profiling was performed on HiSeq2000 or HiSeq1500 platforms. For RNA sequencing (RNA-Seq), six samples per lane were DNA methylation status was obtained by DNA methylation sequenced with an average of 64 MMRS. chip array (Infinium Human Methylation 450 K or EPIC PAX5 biallelic genomic alterations define a novel subgroup of B-cell precursor acute lymphoblastic. BeadChip, Illumina; San Diego, CA) and analyzed within fusion (n = 19), Near haploid - High Hyperdiploid (NH- Genomics Suite (Partek, St. Louis, USA) using SWAN HeH, n = 16), Low Hypodiploid - Near Triploid (LH–NT, normalization to obtain corrected beta values. Differentially n = 14), KMT2A fusions (n = 14), TCF3-PBX1 (n = 6), methylated