Genes Regulating B-Cell Development and Differentiation Are Mutated in 40% of Pediatric Acute Lymphoblastic Leukemia.
Authors: Mullighan Charles G. , Goorha Salil , Radtke Ina , Miller Christopher B. , Coustan-Smith Elaine , Dalton James D. , Girtman Kevin , Mathew Susan , Ma Jing , Pounds Stanley B. , Su Xiaoping , Pui Ching-Hon , Relling Mary V. , Evans William E. , Shurtleff Sheila A. , Downing James R. 
Published: October 2019

Chromosomal aberrations are a hallmark of acute lymphoblastic leukemia (ALL) but alone fail to induce leukemia. To identify cooperating oncogenic lesions, we performed genome-wide analysis of leukemic blasts from 242 pediatric ALL patients using high-resolution 100K and 250K Affymetrix single nucleotide polymorphism arrays, and genomic DNA sequencing. Remarkably, our analyses identified deletion, amplification, point mutation and structural rearrangement in genes encoding regulators of B lymphocyte development in over 40% of B-progenitor ALL. PAX5, which encodes a transcription factor critical for B cell commitment and differentiation, was the most frequent target of somatic mutation, being altered in 31.7% of cases. The most frequent PAX5 mutations were copy number alterations with mono-allelic loss in 53 cases, biallelic loss in 3 cases, and an internal amplification in 1 case. PAX5 deletions and the amplification were confirmed by FISH and/or RT-PCR, and were present in over 90% of blasts. Twenty-five of the mono-allelic deletions were confined to PAX5, with the majority deleting only a subset of PAX5 exons. Thus, of the 57 cases with PAX5 copy number changes, the majority had haploinsufficiency of PAX5 (n=30), or generated hypomorphic alleles that produce proteins that lack the DNA-binding domain (n=20) or the transcriptional activation domain (n=7). Four cases contained cryptic PAX5 translocations: PAX5-ETV6 (n=2), PAX5-FOXP1 and PAX5-ZNF521 (1 case each). In addition to the structural alterations, sequencing identified 14 B-ALLs with PAX5 point mutations. Mutations were identified in the DNA-binding paired domain, homeodomain and transactivation domains. The mutations were somatically acquired, and present in a dominant clone. Gel-shift and transcriptional reporter assays demonstrated reduced DNA binding and/or transcriptional activity for each of the identified PAX5 fusion proteins and point mutants. In addition to the PAX5 mutations, deletions were also detected in the B cell regulatory genes Ikaros (20 cases), Aiolos (3), EBF (8), E2A (1), LEF1 (3), and RAG1/2 (11). Importantly, genomic sequencing of Ikaros and EBF revealed no point mutations. Although the high frequency of mutations in genes regulating B-cell development was unexpected, even more surprising was the marked difference in the frequency and type of mutations among the various genetic subtypes of ALL. Hypodiploid ALLs had alterations in B-cell development genes in 100% of cases, with broad deletions of one PAX5 allele, mutation of the other PAX5 allele in 50% of the cases, and mono-allelic deletion of other regulators of B-cell development, frequently with multiple genes affected within a single case. In contrast ETV6-RUNX1 cases exhibited a much more restricted pattern of mutations, with 27% showing focal PAX5 deletion without alterations of the retained allele. At the other end of the spectrum were hyperdiploid ALLs, with only rare cases harboring a deletion of one of these genes. These data demonstrate that disruption of pathways controlling B cell development and differentiation contributes to the pathogenesis of ALL. Moreover, the approach used provides a rational roadmap for the application of genome-wide approaches to the identification of new molecular lesions in cancer.