Abstract 213: Integrated proteogenomic analysis of laser capture microdissected breast tumors
Authors: Sridhara Viswanadham , Liu Tao , Gritsenko Marina A. , Sturtz Lori A. , Kovatich Albert J. , Petyuk Vladislav A. , Deyarmin Brenda , McDermott Jason E. , Shukla Anil K. , Moore Ronald J. , Monroe Matthew E. , Webb-Robertson Bobbie-Jo M. , Hooke Jeffrey A. , Fantacone-Campbell Leigh , Kumar Praveen Kumar Raj , 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. , Shriver Craig D. , Hu Hai , Rodland Karin D. 
Published: July 2017

Abstract
Introduction: Molecular characteristics of breast tumors play an important role in determining patients’ survival outcome. Here, we report preliminary findings of proteogenomic profiling of 50 breast tumors using RNA-Seq and mass-spectrometry (MS) based proteomic technologies. An additional 60 tumors are being analyzed, including WGS for all samples. We are also collecting patient survival outcome data.
Methods: Cases used in this study were drawn from the Clinical Breast Care Project, where patients were consented using an IRB-approved protocol. A total of 50 breast tumors were selected and processed by laser capture microdissection (LCM). This cohort includes 36 Caucasian Americans (CA) and 8 African Americans (AA), and the age of the patients is 57 ± 13 years. Protein and RNA were extracted using the Illustra triplePrep kit, which isolates DNA from the same cells as well. Quantitative global proteomics and phosphoproteomics analyses were performed using isobaric TMT 6-plex labeling with the “universal reference” strategy and IMAC enrichment of phosphopeptides. Mass spectrometry data were acquired using a Q-Exactive instrument and analyzed using Proteome Discoverer with Byonic node. Phosphopeptide abundance was normalized to abundance measurements of the parent protein for all of the phosphorylation analyses. Phosphoproteomic data was also searched for the presence of O-GlcNAc modifications. RNA-Seq analyses were done on Illumina HiSeq and the data were analyzed using GSNAP.
Results: There were 19 Luminal A, 7 Luminal B, 8 HER2-enriched, and 16 basal-like subtypes based on the PAM50 algorithm. In the global proteomics data, we were able to quantitate &gt;8600 proteins. Unsupervised clustering on the highly varying proteins across the samples resulted in two primary clusters, with one being luminal-enriched. The other cluster contains a basal-like tumor sub-cluster and a sub-cluster of mixed subtypes. Differential protein expression analyses between the two primary clusters confirmed known markers (e.g., overexpression of KRT8/KRT18 in luminal-enriched cluster). The luminal-enriched cluster is primarily CA with post-menopausal status.
A similar search of the phosphoproteomic data yielded quantitation of &gt;12500 phosphopeptides. Unsupervised clustering of the phosphoproteins resulted in four primary groups, with one being basal-enriched and another being luminal-enriched. We also observed &gt;50 overexpressed phosphopeptides. While some of these phosphosites have been previously reported (e.g., on RANBP2), other phosphosites appeared to be novel (e.g., on IRF2BP2).
Conclusion: Analysis of LCM breast tumors using proteogenomic technologies resulted in basal- and luminal-enriched clusters, thus enabling us to study protein and phosphopeptide markers across multiple platforms.
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: Viswanadham Sridhara, Tao Liu, Marina A. Gritsenko, Lori A. Sturtz, Albert J. Kovatich, Vladislav A. Petyuk, Brenda Deyarmin, Jason E. McDermott, Anil K. Shukla, Ronald J. Moore, Matthew E. Monroe, Bobbie-Jo M. Webb-Robertson, Jeffrey A. Hooke, Leigh Fantacone-Campbell, Praveen Kumar Raj Kumar, 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, Craig D. Shriver, Hai Hu, Karin D. Rodland. Integrated proteogenomic analysis of laser capture microdissected breast tumors [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 213. doi:10.1158/1538-7445.AM2017-213