Abstract P4-04-07: Heterogeneous gene fusions detected by RNASeq show enrichment of insulin signaling pathway genes in breast cancer
Authors: Varadan V , Agrawal V , Kamalakaran S , Banerjee N , Miskimen K , Vadodkar A , Abu-Khalaf M , Sikov W , Harris LN , Dimitrova N 
Published: August 2014

Abstract
Background: Recent discoveries of recurrent and targetable gene fusions in breast cancer suggest the need to characterize the functional significance of such genomic aberrations within larger cohorts. We quantify fusion transcript expression in patient samples using RNASeq and evaluate their functional significance using biological pathway enrichment analysis.
Methods: We sequenced transcriptomes of core biopsy RNA from 97 breast tumors obtained from brief-exposure preoperative clinical trials BrUOG 211A/211B. HER2- patients were treated with brief exposure to bevacizumab (B) or nab-paclitaxel (nP) followed by treatment with B/nP/carboplatin while HER2+ patients received brief exposure to trastuzumab (T) or nP followed by T/nP/carboplatin. Paired-end sequencing on 55 baseline biopsies and 42 post-exposure biopsies using amplified total RNA yielded 55 million reads on average per sample. We assigned RNASeq-based PAM50 subtypes for each of the samples using standard methodology. Fusion transcript abundance was evaluated using two independent pipelines, TopHat and deFuse, due to their complementary strategies in fusion detection. We eliminated fusions of genes with their respective pseudogenes as likely false positives arising due to alignment artifacts. TopHat fusion calls with total supporting reads ≥10 and deFuse calls with probability of fusion ≥0.7 were considered reliable.
Results: We identified high confidence gene fusions, detected by both TopHat and deFuse, in 30 of the 55 baseline biopsies (54.4%), with 3.3 fusions on average per sample and a maximum of 10. Fusions were predominantly associated with chromosomal aberrations (75%), with putative deletions responsible for 32% of fusions and translocations responsible for 43%. We find a high level of fusion transcript heterogeneity within breast cancers, detecting a total of 80 fusions across the 30 samples with only three fusions recurrent in two samples with high expression in each: MDN1-GAS5 in two basal breast cancers, KRAS-GRIP1 and ITPR2-CCDC91 in two LumB cancers. Several cancer-related genes were found to be fusion partners: AKT3-SMYD3, CREB1-PPP1R1C, FLOT2-TOP2A and FOXC1-ARID1B. Pathway analysis of the fusion genes at baseline revealed enrichment of proteasome (p = 0.000752), tight junction (p = 0.027), insulin signaling (p = 0.0284) and melanogenesis (p = 0.05) pathways after multiple testing correction (FDR≤0.25). We looked for modulation of gene fusions upon brief exposure to therapy in 18 patients and found a majority of the baseline fusion transcripts to be present post-brief exposure in 44% of the patients, irrespective of therapy regimen.
Conclusions: We find that gene fusions in breast cancer are highly heterogeneous but are enriched with cancer-related pathway genes. This is the first study to report a novel gene-lincRNA fusion transcript (MDN1-GAS5). We are currently validating the fusion calls using qRT-PCR. The heterogeneity of detected fusions suggests that multiple mechanisms could underlie the selective advantage of tumor cells expressing fusion transcripts. The brief-exposure preoperative paradigm provides a unique opportunity to evaluate modulation of fusion transcripts that can shed light on their functional importance.
Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-04-07.