Abstract
RNA biomarkers discovered by RT-PCR-based gene expression profiling of archival formalin-fixed paraffin-embedded (FFPE) tissue form the basis for widely used clinical diagnostic tests; however, RT-PCR is practically constrained in the number of transcripts that can be interrogated. We have developed and optimized RNA-Seq library chemistry as well as bioinformatics and biostatistical methods for whole transcriptome profiling from FFPE tissue. The chemistry accommodates low RNA inputs and sample multiplexing. These methods both enable rediscovery of RNA biomarkers for disease recurrence risk that were previously identified by RT-PCR analysis of a cohort of 136 patients, and also identify a high percentage of recurrence risk markers that were previously discovered using DNA microarrays in a separate cohort of patients, evidence that this RNA-Seq technology has sufficient precision and sensitivity for biomarker discovery. More than two thousand RNAs are strongly associated with breast cancer recurrence risk in the 136 patient cohort (FDR <10%). Many of these are intronic RNAs for which corresponding exons are not also associated with disease recurrence. A number of the RNAs associated with recurrence risk belong to novel RNA networks. It will be important to test the validity of these novel associations in whole transcriptome RNA-Seq screens of other breast cancer cohorts.
Highlights
Recent major advances in DNA sequencing, ‘‘ generation sequencing (NGS)’’, provide massively parallel throughput, and data volumes that eclipse the nucleic acid information content possible with other technologies, making feasible unprecedented extensive genome analyses of groups of individuals, including analyses of sequence differences, polymorphisms, mutations, copy number variations, epigenetic variations and transcript abundance (RNA-Seq) [1,2,3]
There was a mixture of chemotherapy and hormonal therapy usage
Estrogen receptor (ER) status was not included in patient records
Summary
Recent major advances in DNA sequencing, ‘‘ generation sequencing (NGS)’’, provide massively parallel throughput, and data volumes that eclipse the nucleic acid information content possible with other technologies, making feasible unprecedented extensive genome analyses of groups of individuals, including analyses of sequence differences, polymorphisms, mutations, copy number variations, epigenetic variations and transcript abundance (RNA-Seq) [1,2,3]. Many thousands of FFPE tissue blocks associated with mature clinical records exist in hospital pathology archives These can be used for tumor gene expression profiling and enable rapid clinical biomarker discovery in studies that are statistically well-powered [12,13,14,15]. We have carried out RNA-Seq analysis on FFPE tumor RNA from a cohort of 136 breast cancer patients with tumor tissue at the time of resection and clinical outcomes for disease recurrence This tumor RNA was originally screened by RT-PCR in the biomarker discovery phase of the development of the 21 gene breast cancer recurrence risk assay [4,16]. This study suggests associations between hundreds of both coding and non-coding RNAs and breast cancer recurrence risk
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
