Homocygous PAX5Sequence Mutations Define a Novel Subtype of B Cell Precursor Acute Lymphoblastic Leukemia

Introduction

Identification 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 methods

We 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).

Results

Within 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.

Conclusion

We 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.