Exome Sequencing Identifies Pxdn and DSP As Recurrently Mutated Genes in Infant ALL Patients Carrying a MLL-AF4 Translocation

Background and purpose: Acute lymphoblastic leukemia (ALL) in infants (<1 year of age) carrying MLL translocations represent a highly aggressive subtype of leukemia with an exceedingly poor prognosis. Although MLL rearrangements have been considered to be causative for this type of leukemia, recent studies using mouse models suggest that the expression of chimeric MLL fusion genes alone may not be sufficient to induce leukemia. In an attempt to identify additional mutations that synergize with MLLfusion genes in driving leukemogenesis, we performed exome-sequencing in primary infant ALL patient samples (n=13) carrying the t(4;11)-translocation, giving rise to the MLL-AF4 fusion protein.

Materials and Methods: Bone marrow or peripheral blood samples from untreated infants diagnosed with t(4;11)+ ALL were collected at our laboratory as part of the international collaborative INTERFANT treatment protocols. Genomic DNA was extracted from ~5×10⁶leukemic cells. Exome capture has been performed using the Sure Select Human All Exon v2 kit (Agilent Technologies). The samples were prepped for sequencing according to the TruSeq v3 protocol (Illumina) prior to sequencing on the Hiseq2000 with a v3 flowcell for 100bp = 7bp index (Illumina). The reads have been aligned against the human reference genome build 19 (hg19) using a BWA and NARWHAL based pipeline. Sequential filtering selected for single nucleotide variations (SNVs) in canonical splice sites, insertions/deletions (indels) providing frameshifts, indels involving regions conserved between species (phyloP >3), nonsense mutations and nonsynonymous missense mutations involving regions conserved between species. All known annotated single nucleotide polymorphisms according to dbSNP build 138 and an in-house database of 1354 sequenced exomes were excluded. Recurrent mutations were validated by Sanger sequencing on an extended infant ALL patient cohort (n=122).

Results: The sequenced exomes had an average coverage depth of 102.5 reads per region or 109 per base. Exome sequencing revealed that, after filtering, t(4;11)+ infant ALL patients on average carried 71 variants (range 58-98, including rare germline variants and somatic mutations). To identify recurrent mutations we selected 23 genes in which two or more patients carried a mutation for further validation by Sanger sequencing. Although all 23 genes were successfully validated, no additional patients carrying these mutations were found. However, recurrent secondary mutations within the same functional region as the initial mutations were found in two genes: PXDN and DSP. The average incidences of the PXDN and DSP mutations amongst MLL-rearranged infant ALL patients were 32% and 20%, respectively. Interestingly, MLL-rearranged infant ALL patients carrying a PXDN mutation had a significantly (p=0.013) better prognosis compared with patients not carrying a PXDNmutation, with mean EFS rates of 6.4 vs. 3.2 years, respectively. Both gene products are involved in intracellular interactions, desmosome formation, extracellular matrix consolidation and phagocytosis and no prior associations with ALL have been made before.

Conclusion: We identified 23 genes with two or more mutations in our initial exome sequencing cohort. None of these mutations were found in additional patients in our validation cohort. Despite an average of 71 variations per patient, no recurrent mutations were identified and our study provides evidence supporting previous observations that infant ALL has one of the lowest mutation rates observed in human cancer. However, we identified additional mutations within the same functional region as the initial mutation in DSP and PXDN. Interestingly, PXDN mutations show an association with a favorable prognosis in MLL-R patients.