Characterization of Translocations in Mesenchymal Hamartoma of the Liver by Targeted Next Generation Sequencing of Formalin-Fixed Paraffin-Embedded Tissue
James Mathews, Eric Duncavage, John Pfeifer. Washington University School of Medicine, St. Louis
Background: Mesenchymal hamartoma of the liver (MHL) is a rare primary tumor that occurs in the pediatric population, and is in some cases associated with subsequent development of undifferentiated embryonal sarcoma. Prior case studies have demonstrated a recurring t(11;19) translocation, calling into question the lesion's designation as a hamartoma. The translocation breakpoints on chromosomes 11 and 19 have been defined on the basis of cytogenetic studies, and have recently been shown to involve the MALAT1 gene on chromosome 11 and a gene poor region on termed MHLB1 on chromosome 19. Given the lack of detailed knowledge of the breakpoint regions, and the availability of only formalin fixed paraffin embedded (FFPE) tissue from most cases, we used targeted next generation sequencing (NGS) from archival FFPE tissue to provide more detail on the genetic events of MHL tumorigenesis.
Design: Sequencing libraries were prepared from 1 ug of genomic DNA extracted from 11 cases of MHL. Target enrichment was then performed using custom cDNA probes targeting MALAT1 plus 10Kb of flanking sequence and the 22Kb MHLB1 locus (71Kb total). The captured DNA was then sequenced on a single NGS lane using 2x101bp paired-end reads. Sequence data were aligned and translocation breakpoints identified using the Breakdancer software package.
Results: Sequencing data included 114,568,422 reads, 10% of which mapped to the target regions, resulting in 2,800x coverage. Reads that aligned to the capture regions were analyzed; those in which only one of the ends mapped to a target region were examined as potential translocation breakpoints. We identified putative translocations involving MALAT1 and regions of chromosomes 3, 4, 8, 16 and 19 in areas of Alu repeats and long interspersed elements. As a validation of the approach, Sanger sequencing was used to confirm the candidate translocation identified in a case known to harbor a t(11;19), and demonstrated a breakpoint involving MALAT1 and a region of chromosome 19 10 kb centromeric to the MHLB1 locus.
Conclusions: NGS can be used to characterize translocation breakpoints with single base pair resolution. In the case of MHL, the involvement of highly repetitive regions may indicate that homologous unequal recombination of similar but non-allelic sequences underlies the translocation event. The recurring involvement of MALAT1 (which encodes a non-translated RNA thought to be involved in the spliceosome) suggests a novel pathway of tumorigenesis due to aberrations in mRNA splicing.
Tuesday, March 20, 2012 1:00 PM
Poster Session IV # 240, Tuesday Afternoon