Whole transcriptome RNA-Seq analysis of breast cancer recurrence risk using formalin-fixed paraffin-embedded tumor tissue.
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.
Genomic Health Inc, Redwood City, California, United States of America.
Dominick
D
Sinicropi
Kunbin
K
Qu
Francois
F
Collin
Michael
M
Crager
Mei-Lan
ML
Liu
Robert J
RJ
Pelham
Mylan
M
Pho
Andrew
A
Dei Rossi
Jennie
J
Jeong
Aaron
A
Scott
Ranjana
R
Ambannavar
Christina
C
Zheng
Raul
R
Mena
Jose
J
Esteban
James
J
Stephans
John
J
Morlan
Joffre
J
Baker
D001483:Base Sequence; D014408:Biomarkers, Tumor / Q000235:genetics; D001943:Breast Neoplasms / Q000235:genetics*; D021901:DNA, Intergenic / Q000235:genetics; D005260:Female; D005557:Formaldehyde; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007438:Introns / Q000235:genetics; D009364:Neoplasm Recurrence, Local / Q000235:genetics*; D016612:Paraffin Embedding; D016016:Proportional Hazards Models; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm; D012307:Risk Factors; D017423:Sequence Analysis, RNA; D016707:Tissue Fixation
D014408:Biomarkers, Tumor; D021901:DNA, Intergenic; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D005557:Formaldehyde