PAX5 Is a Frequently Altered Transcription Factor In Genetically Unclassified Childhood Common/B-Cell Precursor Acute Lymphoblastic Leukemia

Genetic lesions define treatment and prognosis in childhood common/B cell-precursor Acute Lymphoblastic Leukemia (c/pre-B ALL) (Pui CH et al., J Clin Oncol, 2011). However, up to 40% of childhood pre-B ALL cases, lack the presence of prognostically significant abnormalities; and relapses are common in this group (Mejerink J et al., Semin Hem, 2009). Molecular genetic characterization of genetically unclassified ALL is critical for a better understanding of the biology of this disease, to determine new prognostic markers and to optimize treatment for this group of patients. Several transcription factors, including EBF1, IKZF1 and PAX5, which regulate B-cell development, have recently been shown often to be altered in B-ALL (Mullighan CG et al., Nature, 2007; den Boer ML et al., Lancet, 2009). We decided to investigate the incidence of abnormalities involving B-cell transcription factors, in a cohort of patients with childhood c/pre-B ALL lacking significant prognostic genetic aberrations.

60 patients diagnosed with c/pre-B ALL in Great Ormond Street Hospital (London, UK) from 1994 to 2011 were included in the study. The median age was 3.43 (range: 1.14–12.24), with a median white blood cell count of 14.0 (range: 1.36–213.2). All patients showed an absence of high-hyperploidy/hypodiploidy, BCR-ABL1, MLL, ETV6-RUNX1 and E2A rearrangements, and also i(amp)21 by karyotype, fluorescence in-situ hybridization (FISH) and/or reverse transcriptase-polymerase chain reaction (RT-PCR) investigations. We performed FISH using commercially available probes and BAC clones, in parallel with RT-PCR, to detect deletions/rearrangements of the EBF1, IKZF1 and PAX5 gene regions. Direct sequence analysis was performed for all exons of the PAX5 gene. Real-time PCR was used to determine the level of expression of PAX5 and its transcriptional targets, mostly components of the pre-B cell receptor (pre-BCR) complex including CD19, CD79α, VpreB, BLNK and BLK genes. Western blotting was further performed for the identification of an abnormal PAX5 protein.

25/60 patients (42%) showed abnormalities involving PAX5, 2/60 patients (3%) showed deletions of IKZF1. No deletions/rearrangements of EBF1 were detected. Several forms of PAX5 deletions were observed: the entire loss of one PAX5 allele (32%), loss of 5'PAX5 (20%), loss of 3'PAX5 (12%) and loss of 3'PAX5 due to unbalanced rearrangements (8%). Balanced PAX5 rearrangements comprise 12% of the PAX5 alterations. Sequence analysis revealed 9 somatic mutations of PAX5 in 8/60 patients (13%): 6 frameshifts, 2 splice-site and 1 non-stop mutations. The mutations were located in functional exons of PAX5 and resulted in the initiation of premature stop-codons, exon skips, and the formation of abnormal transcripts. Interestingly, 2 patients with an identical splice site mutation, showed the same karyotypic abnormality of an isochromosome of the long arm of chromosome 9 leading to loss of the second PAX5 allele. Moreover, further investigation DNA and mRNA from remission samples demonstrated that in one case the mutation was acquired, and that in the second case the mutation was a germ-line variant. In total, 20% of patients with PAX5 abnormalities had bi-allelic alterations of the PAX 5 gene. PAX5, BLNK and BLK gene-expression levels were lower (p<0.05) in the group of patients with loss of one entire PAX5 allele. There were no significant changes in expression levels of the tested genes in patients with partial PAX5 deletions, rearrangements or mutations. Expression of an abnormal PAX5 protein was confirmed by western blot analysis.

The high incidence of PAX5 alterations in childhood c/pre-B ALL, without prognostically significant genetic abnormalities, suggests a pathogenetic role for PAX5 in these acute lymphoblastic leukemia cases. Down-regulation of expression of the BLNK and BLK genes could be one of the mechanisms by which abnormal PAX5 blocks B-cell differentiation and contributes to the initiation of leukemia.

No relevant conflicts of interest to declare.