title: Acquisition of biologically relevant gene expression data by Affymetrix microarray analysis of archival formalin-fixed paraffin-embedded tumours.
abstract: Robust protocols for microarray gene expression profiling of archival formalin-fixed paraffin-embedded tissue (FFPET) are needed to facilitate research when availability of fresh-frozen tissue is limited. Recent reports attest to the feasibility of this approach, but the clinical value of these data is poorly understood. We employed state-of-the-art RNA extraction and Affymetrix microarray technology to examine 34 archival FFPET primary extremity soft tissue sarcomas. Nineteen arrays met stringent QC criteria and were used to model prognostic signatures for metastatic recurrence. Arrays from two paired frozen and FFPET samples were compared: although FFPET sensitivity was low ( approximately 50%), high specificity (95%) and positive predictive value (92%) suggest that transcript detection is reliable. Good agreement between arrays and real time (RT)-PCR was confirmed, especially for abundant transcripts, and RT-PCR validated the regulation pattern for 19 of 24 candidate genes (overall R(2)=0.4662). RT-PCR and immunohistochemistry on independent cases validated prognostic significance for several genes including RECQL4, FRRS1, CFH and MET - whose combined expression carried greater prognostic value than tumour grade - and cmet and TRKB proteins. These molecules warrant further evaluation in larger series. Reliable clinically relevant data can be obtained from archival FFPET, but protocol amendments are needed to improve the sensitivity and broad application of this approach.
authors.affiliation: Cancer Research UK, Department of Medical Oncology, Christie Hospital NHS Foundation Trust, Wilmslow Road, Withington, Manchester, M20 4BX, UK. kim.linton@christie.nhs.uk
authors.forename: K M
authors.initials: KM
authors.lastname: Linton
authors.forename: Y
authors.initials: Y
authors.lastname: Hey
authors.forename: E
authors.initials: E
authors.lastname: Saunders
authors.forename: M
authors.initials: M
authors.lastname: Jeziorska
authors.forename: J
authors.initials: J
authors.lastname: Denton
authors.forename: C L
authors.initials: CL
authors.lastname: Wilson
authors.forename: R
authors.initials: R
authors.lastname: Swindell
authors.forename: S
authors.initials: S
authors.lastname: Dibben
authors.forename: C J
authors.initials: CJ
authors.lastname: Miller
authors.forename: S D
authors.initials: SD
authors.lastname: Pepper
authors.forename: J A
authors.initials: JA
authors.lastname: Radford
authors.forename: A J
authors.initials: AJ
authors.lastname: Freemont
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics; D005557:Formaldehyde; D020869:Gene Expression Profiling; D006801:Humans; D009369:Neoplasms / Q000378:metabolism* / Q000473:pathology; D020411:Oligonucleotide Array Sequence Analysis; D016612:Paraffin Embedding; D011379:Prognosis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016707:Tissue Fixation
chemical_list: D014408:Biomarkers, Tumor; D005557:Formaldehyde