Breast Micropapillary Carcinomas: RNA-Seq and Mutation Profiling
Caterina Marchio, Daniel Nava Rodrigues, Paul Wilkerson, Maryou B Lambros, Britta Weigelt, Anna Sapino, Alan Mackay, Christopher Maher, Rachael Natrajan, Jorge S Reis-Filho. University of Turin, Turin, Italy; The Institute of Cancer Research, London, United Kingdom; Cancer Research UK London Research Institute, London, United Kingdom; Washington University School of Medicine, St Louis
Background: Micropapillary carcinomas (MPCs) are a rare special type of breast cancer, characterised by specific morphological features and an aggressive clinical behaviour. Genomics studies have demonstrated that MPCs harbour a constellation of gene copy number changes that are distinct from that of grade- and ER-matched invasive ductal carcinomas of the breast. The aims of this study were to investigate whether MPCs would harbour recurrent fusion genes and to characterise the repertoire of mutations affecting known oncogenes in MPCs.
Design: Twenty-two (15 pure and 7 mixed) MPCs of the breast were microdissected. RNA and DNA were extracted. Six pure MPCs were subjected to massively parallel RNA sequencing. cDNA libraries were prepared according to standard mRNA prep Illumina protocols and run on the Genome Analyser IIx sequencers. Data were aligned to the genome and transcriptome using Bowtie. Mate-pairs supporting novel chimaeric transcripts were identified using Chimerascan version 4.0. Fusion genes identified were validated using RT-PCR and Sanger sequencing. Somatic mutation profiling was performed in all 22 MPCs using the Sequenom OncoCarta Panel v1.0 covering hotspot mutations in 19 oncogenes. The results were validated using Sanger sequencing.
Results: Twelve high-confidence fusion genes were found in four MPCs. Three of the chimaeric transcripts (i.e. SLC2A1-FAF1, ELMO2-RAE1, BCAS4-AURKA) were present in a single tumour and mapped to regions of amplification. All chimaeric transcripts were confirmed using RT-PCR and Sanger sequencing. An independent series of 12 MPCs and other types of breast cancer (n=160) were screened for fusions by RT-PCR. No recurrent fusions were identified. Forced expression of two of the in-frame fusion genes (SLC2A1-FAF1 and ELMO2-RAE1) and their partner genes in MCF7 cells resulted in increased proliferation, whilst forced expression of BCAS4-AURKA had no effect on cell growth and proliferation. Sequenom MassARRAY analysis led to the identification of a single mutation (i.e. PIK3CA H1047R) in one case, which was validated by Sanger sequencing.
Conclusions: A proportion of breast MPCs harbour intra-chromosomal fusion transcripts that appear to be private events, but may play a role in tumour proliferation. Neither recurrent fusion genes nor mutations in the genes assessed by Oncocarta v1.0 are likely to account for the characteristic morphological features and aggressive clinical behaviour of MPCs.
Tuesday, March 20, 2012 8:45 AM
Platform Session: Section B, Tuesday Morning