Gene expression profiling of formalin-fixed, paraffin-embedded familial breast tumours using the whole genome-DASL assay.

Author(s): Waddell N, Cocciardi S, Johnson J, Healey S, Marsh A, Riley J, da Silva L, Vargas AC, Reid L, Simpson PT, Lakhani SR, Chenevix-Trench G

Publication: J Pathol, 2010, Vol. 221, Page 452-61

PubMed ID: 20593485 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of storage and using FFPE instead of frozen breast tumor specimens on DASL assay success and results. Tumor cells were microdissected from 2-20 freshly cut sections and lysed in proteinase K before extraction of RNA with Tri Reagent.

   Summary of Findings:

   93% of the 92 FFPE specimens yielded sufficient RNA to use in the DASL assay, and 85% of the 101 specimens (including duplicates) passed DASL quality control. Interestingly, RNA from 7 of the 15 specimens that did not produce data of sufficient quality was arrayed in duplicate and worked in the replicate specimen. While some of the FFPE specimens stored 7 years or more failed DASL quality control, all of the specimens stored less than 7 years passed DASL quality control. Real-time qRT-PCR CT values, RNA fragment size (by bioanalyzer) and RIN did not predict DASL success, but average CT values using TaqMan assays were higher and RNA concentrations were lower for specimens that failed DASL quality control than those that passed. Technical replicates of FFPE specimens yielded DASL data that was very strongly correlated (r2=0.90-0.98), but these correlations were weaker than those observed between technical replicates of frozen specimens (r2=0.98-0.99). Importantly 3565 of 18,227 probes produced discordant data between technical replicates in at least 4 of 8 specimens for which replicates were available. Using a gene-list of 240 transcripts 12/15 FFPE specimens were assigned to the same cancer subtype as the frozen counterpart. Importantly, in 1 of 8 FFPE specimens that had technical replicates, the replicates were assigned to different subtypes.

   Biospecimens

   • Tissue - Breast

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	DASL
   RNA	Real-time qRT-PCR
   RNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Frozen
   Storage	Storage duration	<7 years > 7 years
   Real-time qRT-PCR Specific	Targeted nucleic acid	RPL13A
   Real-time qRT-PCR Specific	Detection method	SYBR TaqMan probes

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