title: Comparison of triple-negative breast cancer molecular subtyping using RNA from matched fresh-frozen versus formalin-fixed paraffin-embedded tissue.
abstract: Triple negative breast cancer (TNBC) is a heterogeneous disease that lacks unifying molecular alterations that can guide therapy decisions. We previously identified distinct molecular subtypes of TNBC (TNBCtype) using gene expression data generated on a microarray platform using frozen tumor specimens. Tumors and cell lines representing the identified subtypes have distinct enrichment in biologically relevant transcripts with differing sensitivity to standard chemotherapies and targeted agents. Since our initial discoveries, RNA-sequencing (RNA-seq) has evolved as a sensitive and quantitative tool to measure transcript abundance.
  
  
  
  To demonstrate that TNBC subtypes were similar between platforms, we compared gene expression from matched specimens profiled by both microarray and RNA-seq from The Cancer Genome Atlas (TCGA). In the clinical care of patients with TNBC, tumor specimens collected for diagnostic purposes are processed by formalin fixation and paraffin-embedding (FFPE). Thus, for TNBCtype to eventually have broad and practical clinical utility we performed RNA-seq gene expression and molecular classification comparison between fresh-frozen (FF) and FFPE tumor specimens.
  
  
  
  Analysis of TCGA showed consistent subtype calls between 91% of evaluable samples demonstrating conservation of TNBC subtypes across microarray and RNA-seq platforms. We compared RNA-seq performed on 21-paired FF and FFPE TNBC specimens and evaluated genome alignment, transcript coverage, differential transcript enrichment and concordance of TNBC molecular subtype calls. We demonstrate that subtype accuracy between matched FF and FFPE samples increases with sequencing depth and correlation strength to an individual TNBC subtype.
  
  
  
  TNBC subtypes were reliably identified from FFPE samples, with highest accuracy if the samples were less than 4 years old and reproducible subtyping increased with sequencing depth. To reproducibly subtype tumors using gene expression, it is critical to select genes that do not vary due to platform type, tissue processing or RNA isolation method. The majority of differentially expressed transcripts between matched FF and FFPE samples could be attributed to transcripts selected for by RNA enrichment method. While differentially expressed transcripts did not impact TNBC subtyping, they will provide guidance on determining which transcripts to avoid when implementing a gene set size reduction strategy.
  
  
  
  NCT00930930 07/01/2009.
authors.affiliation: Medical Oncology Department, Dana-Farber Cancer Institute, Harvard Medical School and Broad Institute, Boston, 02215, MA, USA.
authors.forename: Bojana
authors.initials: B
authors.lastname: Jovanović
authors.affiliation: Center for Quantitative Sciences, Vanderbilt University School of Medicine, Nashville, 37232, TN, USA.
authors.forename: Quanhu
authors.initials: Q
authors.lastname: Sheng
authors.affiliation: Insight Genetics Incorporated, Nashville, 37217, TN, USA.
authors.forename: Robert S
authors.initials: RS
authors.lastname: Seitz
authors.affiliation: Insight Genetics Incorporated, Nashville, 37217, TN, USA.
authors.forename: Kasey D
authors.initials: KD
authors.lastname: Lawrence
authors.affiliation: Insight Genetics Incorporated, Nashville, 37217, TN, USA.
authors.forename: Stephan W
authors.initials: SW
authors.lastname: Morris
authors.affiliation: Insight Genetics Incorporated, Nashville, 37217, TN, USA.
authors.forename: Lance R
authors.initials: LR
authors.lastname: Thomas
authors.affiliation: Insight Genetics Incorporated, Nashville, 37217, TN, USA.
authors.forename: David R
authors.initials: DR
authors.lastname: Hout
authors.affiliation: Insight Genetics Incorporated, Nashville, 37217, TN, USA.
authors.forename: Brock L
authors.initials: BL
authors.lastname: Schweitzer
authors.affiliation: Center for Quantitative Sciences, Vanderbilt University School of Medicine, Nashville, 37232, TN, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Guo
authors.affiliation: Department of Biochemistry, Vanderbilt University, Nashville, 37232, TN, USA.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Pietenpol
authors.affiliation: Department of Biochemistry, Vanderbilt University, Nashville, 37232, TN, USA. brian.d.lehmann@vanderbilt.edu.|Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN, 37232, USA. brian.d.lehmann@vanderbilt.edu.
authors.forename: Brian D
authors.initials: BD
authors.lastname: Lehmann
mesh_terms: D005260:Female; D005557:Formaldehyde; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009363:Neoplasm Proteins / Q000235:genetics*; D016612:Paraffin Embedding; D012313:RNA / Q000235:genetics; D016707:Tissue Fixation / Q000379:methods; D064726:Triple Negative Breast Neoplasms / Q000145:classification / Q000235:genetics* / Q000473:pathology
chemical_list: D009363:Neoplasm Proteins; D005557:Formaldehyde; D012313:RNA
keywords: Formalin-fixed paraffin embedded; Fresh-frozen; RNA-seq; TNBCtype