Abstract 3372: Differential exon usage in the HER2 subtype of breast cancer identified with RNA-seq and proteomic data
Authors: Kumar Praveen Kumar Raj , Liu Tao , Sturtz Lori A. , Kovatich Albert , Gritsenko Marina A. , Petyuk Vladislav A. , Deyarmin Brenda , Sridhara Viswanadham , Craig James , McDermott Jason E. , Shukla Anil K. , Moore Ronald J. , Monroe Matthew E. , Webb-Robertson Bobbie-Jo M. , Hooke Jeffrey A. , Fantacone-Campbell Leigh , Kvecher Leonid , Liu Jianfang , Kane Jennifer , Melley Jennifer , Somiari Stella , Iida Joji , Benz Stephen C. , Golovato Justin , Rabizadeh Shahrooz , Soon-Shiong Patrick , Smith Richard D. , Mural Richard J. , Rodland Karin D. , Shriver Craig D. , Hu Hai 
Published: July 2017

Abstract
Background: Heterogeneity between different breast cancer tumors plays an important role in patients’ survival outcomes, and such tumor heterogeneity unveiled by transcriptome and proteome are often in disagreement. We hypothesize that alternative splicing of mRNA could explain heterogeneity within intrinsic breast cancer subtypes.
Methods: Cases used in this study were drawn from the Clinical Breast Care Project where breast cancer patients were consented using an IRB-approved protocol. Fifty breast tumors were selected and processed by laser microdissection. RNA and protein were extracted from tissues using the Illustra triplePrep kit (GE Healthcare). Paired-end mRNA sequencing was performed using the Illumina HiSeq platform. Paired-end reads were preprocessed using PRINSEQ and splice-aligned to the genome using GSNAP software. Gene counts were measured using HTSeq while Exon counts used the DEXSeq. Differential expression was called at 10% false discovery rate. Proteome Discoverer with Byonic node was used for analyzing the quantitative global proteomics dataset, and we were able to quantitate 8600 proteins. All other analyses were performed using Perl and R.
Results: The number of sequencing reads for the 50 cases ranged from 60 million to 410 million reads. After preprocessing, an average of 93% of reads was mapped, and 36,700 genes were identified among 50 tumor samples. PAM50 algorithm was used for intrinsic subtype calls, yielding 16 Basal, 8 Her2+, 19 Luminal A (LA) and 7 Luminal B tumors. Unsupervised clustering of the 8 Her2+ cases using the PAM50 genes and highly varying proteins resulted in different clustering patterns, with the latter clustering 6 of the 8 Her2+ cases with two distinct 3-case sub-clusters. Between these two sub-clusters, there were 10 differentially expressed (DEX) genes and 25 DEX proteins, but none of the 25 proteins were mapped to the 10 DEX genes. This motivated us to investigate the DEX exons, and we found 7,076 DEX exons between the Her2+ sub-clusters, and 9 of the 25 DEX proteins were matched to genes bearing DEX exons. For comparison, we performed the same analyses between similarly clustered Basal and LA sub-clusters. Even though the number of DEX genes between Basal (8) and LA (18) sub-clusters were comparable to that between the Her2+ sub-clusters, DEX exons were much lower for both subtypes (616 & 157), and none of the DEX proteins (3 & 7) mapped to DEX exons or genes for either subtype.
Conclusions: Our findings imply that there is more proteomic-level heterogeneity in the Her2+ subtype than in Basal and LA subtypes, which could be due to alternative usage of exons (alternative splicing). If such heterogeneity is associated with patients’ survival outcomes then our results will further stress the importance of alternative splicing in breast cancer.
The views expressed in this article are those of the author and do not reflect the official policy of the Department of Defense, or U.S. Government.
Citation Format: Praveen Kumar Raj Kumar, Tao Liu, Lori A. Sturtz, Albert Kovatich, Marina A. Gritsenko, Vladislav A. Petyuk, Brenda Deyarmin, Viswanadham Sridhara, James Craig, Jason E. McDermott, Anil K. Shukla, Ronald J. Moore, Matthew E. Monroe, Bobbie-Jo M. Webb-Robertson, Jeffrey A. Hooke, Leigh Fantacone-Campbell, Leonid Kvecher, Jianfang Liu, Jennifer Kane, Jennifer Melley, Stella Somiari, Joji Iida, Stephen C. Benz, Justin Golovato, Shahrooz Rabizadeh, Patrick Soon-Shiong, Richard D. Smith, Richard J. Mural, Karin D. Rodland, Craig D. Shriver, Hai Hu. Differential exon usage in the HER2 subtype of breast cancer identified with RNA-seq and proteomic data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3372. doi:10.1158/1538-7445.AM2017-3372