IntroductionIdentification of molecular alterations in B cell precursor acute lymphoblastic leukemia (BCP-ALL) is critical for targeting leukemogenic drivers in BCP-ALL patients. Established driver lesions comprise structural or numerical chromosomal aberrations and define subgroups with specific gene expression signatures. Yet, up to one third of BCP-ALL patients lack recurrent genetic lesions to allow classification into distinct categories. We conceived a multi-omics study to characterize novel candidates in BCP-ALL patients.Patients and methodsWe analyzed diagnostic samples of 225 BCP-ALL patients (age: median 32 years, range 1-80 years), exploring sequence variants (whole exome sequencing, WES, n=50 / targeted gene panel sequencing, n=163 / variant calling from RNA-Seq, n=187), copy number alterations (WES, n=50 / multiplex ligation dependent probe amplification, n=123) and gene fusions (RNA-seq, n=187 / fusion break-point specific RT-PCR, n=225) together with their gene expression (RNA-Seq) and DNA methylation (Methylation Chip array, n=140) context. All patients were treated in population based German study trials (GMALL, ALL-BFM, COALL).ResultsWithin this BCP-ALL cohort (n=225), we could assign 180 patients to established molecular driver subgroups: Ph-like (n=42), DUX4/ERG (n=38), aneuploid (n=28), Ph-positive (n=23), ZNF384 (n=18), KMT2A (n=14), TCF3-PBX1 (n=6), ETV6-RUNX1 (n=4), MEF2D (n=4), PAX5-ETV6 (n=3).Among samples that lacked characteristics of any of the above subgroups (n=45), we identified a novel cluster of samples (n=17, median age: 21 years, range 9 - 64 years) by a distinct gene expression profile: all of these patients harbored PAX5 sequence variants. These patients had either homozygous mutations (n=14) or mutations occurring at two different positions in one patient(n=3). The predominant PAX5 amino acid change was p.P80R (n=15), occurring exclusively in this patient cluster. The PAX5 paired domain was affected at least once in all patients of this cluster and predictions (SWIFT, Polyphen and Condel) consistently indicated a deleterious effect on protein integrity for these mutations.RAS-pathway activating (n=15) and CDKN2A inactivating (n=15) alterations were identified in almost all patients of this patient cluster (n=17, further on named PAX5-plus), creating an exclusive pattern of co-occurrence, which was not observed in any of the other subgroups. RAS-pathway alterations were either established hotspot sequence variants (NRAS n=8, KRAS n=5) or NF1 inactivating alterations (n=2). CDKN2A alterations were copy number losses (n=14) and a homozygous sequence variant. Sequence variants affecting other PAX5 protein domains without RAS or CDKN2A alterations were enriched in the Ph-like cluster (n=6 of 42 samples).Unsupervised clustering of top differentially expressed genes and top differentially methylated CpG sites clearly separated the PAX5-plus subgroup (n=17) from established driver subgroups (n=180) and from the remaining unclassified samples (n=28). PAX5-plus patients showed differential expression of 1836 target genes (up: 548 / down: 1288, FDR<0.05). Gene set enrichment analysis revealed genes activated by PAX5 in pro-B cells (Revialla-I-Domingo R, 2012) to be down-regulated (NES -2.05, FDR=0.005) and genes repressed after PAX5 restoration in a PAX5 knock-down BCP-ALL model (Liu GJ, 2014) to be up-regulated in PAX5-plus samples (NES 4.16, FDR<0.001). Moreover, 70% of genes differentially expressed in PAX5-plus patients had been identified as PAX5 target genes by ChIP-Seq in murine pro B cells (Revilla-I-Domingo R, 2012), together establishing PAX5 mutations as bona-fide drivers of transcriptional deregulation in these patients.ConclusionWe have identified a novel subgroup, PAX5-plus BCP-ALL, among patients lacking alterations of previously described driver subgroups. PAX5-plus BCP-ALL is characterized by triple-hits of homozygous PAX5 sequence mutations, RAS pathway activating and CDKN2A inactivating alterations, which is in contrast to the established BCP-ALL subgroups being defined by chromosomal translocations or ploidy disorders. Defined DNA methylation and gene expression signatures specify this subgroup and relate deregulated transcription to PAX5 loss of function. DisclosuresNo relevant conflicts of interest to declare.
Read full abstract