TY - JOUR
T1 - RNA-seq from archival FFPE breast cancer samples
T2 - Molecular pathway fidelity and novel discovery
AU - Pennock, Nathan D.
AU - Jindal, Sonali
AU - Horton, Wesley
AU - Sun, Duanchen
AU - Narasimhan, Jayasri
AU - Carbone, Lucia
AU - Fei, Suzanne S.
AU - Searles, Robert
AU - Harrington, Christina A.
AU - Burchard, Julja
AU - Weinmann, Sheila
AU - Schedin, Pepper
AU - Xia, Zheng
N1 - Publisher Copyright:
© 2019 The Author(s).
PY - 2019/12/19
Y1 - 2019/12/19
N2 - Background: Formalin-fixed, paraffin-embedded (FFPE) tissues for RNA-seq have advantages over fresh frozen tissue including abundance and availability, connection to rich clinical data, and association with patient outcomes. However, FFPE-derived RNA is highly degraded and chemically modified, which impacts its utility as a faithful source for biological inquiry. Methods: True archival FFPE breast cancer cases (n = 58), stored at room temperature for 2-23 years, were utilized to identify key steps in tissue selection, RNA isolation, and library choice. Gene expression fidelity was evaluated by comparing FFPE data to public data obtained from fresh tissues, and by employing single-gene, gene set and transcription network-based regulon analyses. Results: We report a single 10 μm section of breast tissue yields sufficient RNA for RNA-seq, and a relationship between RNA quality and block age that was not linear. We find single-gene analysis is limiting with FFPE tissues, while targeted gene set approaches effectively distinguish ER+ from ER- breast cancers. Novel utilization of regulon analysis identified the transcription factor KDM4B to associate with ER+ disease, with KDM4B regulon activity and gene expression having prognostic significance in an independent cohort of ER+ cases. Conclusion: Our results, which outline a robust FFPE-RNA-seq pipeline for broad use, support utilizing FFPE tissues to address key questions in the breast cancer field, including the delineation between indolent and life-threatening disease, biological stratification and molecular mechanisms of treatment resistance.
AB - Background: Formalin-fixed, paraffin-embedded (FFPE) tissues for RNA-seq have advantages over fresh frozen tissue including abundance and availability, connection to rich clinical data, and association with patient outcomes. However, FFPE-derived RNA is highly degraded and chemically modified, which impacts its utility as a faithful source for biological inquiry. Methods: True archival FFPE breast cancer cases (n = 58), stored at room temperature for 2-23 years, were utilized to identify key steps in tissue selection, RNA isolation, and library choice. Gene expression fidelity was evaluated by comparing FFPE data to public data obtained from fresh tissues, and by employing single-gene, gene set and transcription network-based regulon analyses. Results: We report a single 10 μm section of breast tissue yields sufficient RNA for RNA-seq, and a relationship between RNA quality and block age that was not linear. We find single-gene analysis is limiting with FFPE tissues, while targeted gene set approaches effectively distinguish ER+ from ER- breast cancers. Novel utilization of regulon analysis identified the transcription factor KDM4B to associate with ER+ disease, with KDM4B regulon activity and gene expression having prognostic significance in an independent cohort of ER+ cases. Conclusion: Our results, which outline a robust FFPE-RNA-seq pipeline for broad use, support utilizing FFPE tissues to address key questions in the breast cancer field, including the delineation between indolent and life-threatening disease, biological stratification and molecular mechanisms of treatment resistance.
KW - Archival tissue
KW - Breast Cancer
KW - Estrogen receptor
KW - FFPE
KW - Formalin fixed paraffin embedded
KW - KDM4B
KW - RNA sequencing
KW - RNA-Seq, genomics
KW - RNA-seq
KW - Regulon
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U2 - 10.1186/s12920-019-0643-z
DO - 10.1186/s12920-019-0643-z
M3 - Article
C2 - 31856832
AN - SCOPUS:85076946320
SN - 1755-8794
VL - 12
JO - BMC Medical Genomics
JF - BMC Medical Genomics
IS - 1
M1 - 195
ER -