Abstract 4143: Targeted RNA sequencing for expression analysis of breast cancer patient samples using a biomarker gene panel.
Authors: Oades Kahuku , Vo Lien , Lee Jerry , Landers Mark , Wang Yipeng , Lee Byung-In , Monforte Joseph 
Published: August 2014

Background: Gene expression profiling has been shown to be effective in the analysis of postoperative tumor samples in various cancers. However, for studies requiring the analysis of small specimens, such as core biopsies, the limited amount of available material makes multi-gene analyses exceedingly difficult. Moreover, microarray-based analyses provide somewhat limited dynamic range and multi-gene qRT-PCR analyses often require in excess of 100 ng of input RNA per sample. To this end, we describe the development of targeted RNA-sequencing methodology which combines the power of a universal RNA amplification with NGS for ultra-deep expression analysis of multiple target genes, enabling &lt;100 ng of sample input for multi-gene analysis in a single tube format. Methods: The gene expression patterns of triple-negative breast cancer FFPE samples were analyzed using a 96-gene breast cancer biomarker panel across three different platforms. Affymetrix Human Gene ST 1.0 microarrays and a pre-developed qRT-PCR panel were employed for analysis. For Targeted RNA-seq analysis, the 96-gene panel was amplified using a universal, single-tube “XP-PCR” amplification strategy followed by sequence analysis using the Ion-Torrent Personal Genome Machine. NGS-based expression analysis was performed by counting the frequency of expression tag sequences and the performance of the three expression analysis platforms were compared. Results: The performance of the three gene expression platforms was analyzed according to two different parameters. Gene pairs from four well-characterized expression modules were compared, demonstrating a positive correlation and association with expected clinical biomarker characteristics in all three platforms. Targeted RNA-seq provided the most sensitivity in terms of detection rates with &lt;100 ng FFPE RNA input and provides unlimited dynamic range with increased sequencing depth. In particular, low expressing genes, undetectable by qRT-PCR analysis from 1,000 ng input FFPE RNA, were detected and eligible for expression analysis with a significant number of sequencing reads. In addition to expression analysis, alternative transcription/splicing analysis is possible from sequence analysis of the target transcripts using targeted RNA-seq. Conclusion: By combining universally-primed pre-amplification and NGS in multi-gene expression analysis, targeted RNA-seq provides a robust, sensitive gene expression analysis methodology for a variety of clinical sample specimens. Compression issues typically associated with high pre-amplification cycles were not observed here, resulting in higher gene detection rates, at lower sample input amounts leading to high-sensitivity expression analysis.

Citation Format: Kahuku Oades, Lien Vo, Jerry Lee, Mark Landers, Yipeng Wang, Byung-In Lee, Joseph Monforte. Targeted RNA sequencing for expression analysis of breast cancer patient samples using a biomarker gene panel. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4143. doi:10.1158/1538-7445.AM2013-4143