Functional Characterisation of Fusion Genes in Micropapillary Carcinomas of the Breast
Rachael Natrajan, Paul M Wilkerson, Caterina Marchio, Maryou B Lambros, Charlotte KY Ng, Chantal Topfer, Iwanka Kozarewa, Jarle Hakas, Konstantinos Mitsopoulos, David Hardisson, Christopher J Lord, Britta Weigelt, Alan Ashworth, Anna Sapino, Arul Chinnaiyan, Christopher A Maher, Jorge S Reis-Filho. Institute of Cancer Research, London, United Kingdom; University of Turin, Turin, Italy; Memorial Sloan-Kettering Cancer Center, New York, NY; Hospital Universitario La Paz, Madrid, Spain; University of Michigan, Ann Arbor, MI; Washington University, St Louis, MO
Background: Micropapillary carcinomas (MPCs) are a rare histological special type of breast cancer with an aggressive clinical behaviour. Some special types of breast cancer have been shown to be underpinned by recurrent fusion genes. The aims of this study were to determine i) whether MPCs harbour recurrent fusion genes and ii) if the fusion genes identified are of functional relevance.
Design: Five pure, microdissected MPCs were subjected to massively parallel RNA sequencing (Genome Analyser IIx sequencer). Fusion genes were nominated using Chimerascan version 4.0 and validated using RT-PCR and Sanger sequencing. The genomic breakpoints of the fusion genes were identified by long-range PCR (Roche 20Kb Expand Plus) using custom primers. Validated fusion genes were interrogated in an independent series of 11 MPCs, 16 grade and oestrogen receptor (ER)-matched invasive carcinomas of no special type (IC-NSTs), 53 grade III IC-NSTs and in publicly available datasets. The impact of forced expression of validated in-frame fusion genes was investigated using growth assays and CellTiter Glo (Promega), and RNA interference (RNAi) mediated silencing of recurrent partner genes of out-of-frame rearrangements was assessed in ER-positive breast cancer cell lines (i.e. MCF-7, BT474, T47D, ZR75.1 and SUM44).
Results: RNA-sequencing resulted in the identification of eight fusion genes that were successfully validated by orthogonal methods, of which two were in-frame (SLC2A1-FAF1 and BCAS4-AURKA). Forced expression of these in-frame fusion genes resulted in increased proliferation in a subset of ER-positive breast cancer cell lines. No recurrent fusion genes were identified in the cases analysed. RAE1 (2%), CDK12 (2%) and LASP1 (1%) were identified in external datasets as recurrent partners in out-of-frame fusion genes. RNAi silencing of CDK12 and LASP1 (out-of-frame fusion partners) resulted in a significant increase in cell viability.
Conclusions: Our findings suggest that MPCs are not underpinned by a highly recurrent fusion gene. Although present at low frequency or seemingly private genetic events, some of the fusion transcripts found in MPCs may play a role in tumour cell growth and viability.
Wednesday, March 6, 2013 9:30 AM
Poster Session V # 41, Wednesday Morning