Abstract A028: Progression of structural change in the breast cancer genome
Authors: Hartmaier Ryan J. , Lee Adrian V. 
Published: May 2015

Abstract
Purpose: Through the utilization of large-insert mate-pair sequencing, we strive to uncover structural variations (SVs) driving metastasis in breast cancer.
Background: Recent sequencing studies have shown that any given breast tumor harbors an immense number of point mutations. However, few are recurrent making identification of mutated ‘driver genes’ extremely difficult. Further, although the traditional paired-end sequencing methodology is extremely powerful at detecting point mutations, it is severely limited in detecting large structural changes (insertions, deletions) that occur near repetitive regions or ‘balanced’ events such as translocations or inversions. Current sequencing studies rely almost exclusively on tissues from primary disease rather than recurrent or metastatic disease. Together this suggests that current studies are likely missing (1) a large fraction of a major class of genetic events (i.e. SVs) and (2) the mutation events most likely to impact long term survivability (i.e. metastatic enriched).
Methods: Through the University of Pittsburgh Rapid Autopsy Program and the Health Sciences Tissue Bank, we obtained 26 fresh-frozen tissue specimens from 4 individuals originating from a combination of matched normal, primary tumor, recurrent tumor, lymph node metastases, and distant metastases. To overcome the limitations of traditional paired-end sequencing in detecting SVs, multiple large-insert (3-5, 5-8, & 8-12 kb) mate-pair sequencing was performed. Mate-pair sequencing is currently underway, however, one ‘patient set’ (adjacent normal, primary tumor, lymph node metastasis) has been fully completed. Alignment was conducted via NovoAlign while breakpoints were determined via Breakdancer and the recently published paired-end/split-read, integrative breakpoint caller, Lumpy-sv.
Additionally, to supplement the mate-pair sequencing findings and allow for detailed integrative analysis, genome-wide copy number profiling, RNA-seq, exome-seq, and targeted methyl-seq have either been completed or are in process.
Results: Analysis of the completed single ‘patient set’ has revealed SVs acquired by the primary tumor, shared between the primary and metastatic tumors, and specific to the metastasis. The most striking finding was a cluster of metastatic specific SVs involving chromosome 14, potentially the result of chromothripsis. These included intra-chromosomal SVs (including large inversions) and inter-chromosomal translocations (with chromosomes 8 & 19). Interestingly, the t(8;14) SV involves ASH2L, a member of the Set1 histone methyltransferase complex that is responsible for H3K4 mono- and di-methylation.
Conclusions: Extensive SV occurs during breast cancer tumorigenesis and metastasis, potentially the result of chromothripsis. As more samples and distant metastases are sequenced, orthologous datasets (RNA-seq) will be used to help prioritize specific metastatic specific SVs for functional studies.
Citation Format: Ryan J. Hartmaier, Adrian V. Lee. Progression of structural change in the breast cancer genome. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A028.