Preanalytical variables and performance of diagnostic RNA-based gene expression analysis in breast cancer.
Author(s): Poremba C, Uhlendorff J, Pfitzner BM, Hennig G, Bohmann K, Bojar H, Krenn V, Brase JC, Haufe F, Averdick M, Dietel M, Kronenwett R, Denkert C
Publication: Virchows Arch, 2014, Vol. 465, Page 409-17
PubMed ID: 25218890 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effects of cold ischemia time and temperature, duration of formalin fixation, tumor cell content, and duration and temperature of section storage on the diagnostic EndoPredict real time qRT-PCR assay scores using breast cancer tissue specimens.
Conclusion of Paper
A cold ischemia time of 24 h at 20°C or fixation for 10 days resulted in decreased EndoPredict (EP) scores compared to specimens fixed for 5 or less days with cold ischemia times of 12 h or less. Generally, tumor cell content did not affect EP score, but 5 of the 38 tumors examined had a disagreement in the risk classification based on the EP score of the microdissected (100% tumor cell content) versus whole section (15-95% tumor cell content), but these 5 specimens had EP score very close to the cutoff level. Storing prepared tissue sections on slides for 12 months at either 4°C or 20°C had no impact on EP scores or resulting risk classifications, however, a decrease in RNA yield over time was observed.
Studies
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Study Purpose
The purpose of this study was to determine the effects of cold ischemia time and temperature (4°C, 20°C, or 37°C), duration of formalin fixation, tumor cell content, and duration and temperature of section storage (4°C or 20°C) on the diagnostic EndoPredict real time qRT-PCR assay results using breast cancer tissue specimens. One breast tumor was used for the cold ischemia time experiment, a second tumor was used for the time in fixative experiment, 38 tumor specimens were used in the tumor cell content experiment, and 10 tumor specimens were used in the section storage experiment. RNA isolation was performed using the magnetic bead-based VERSANT Tissue Preparation Reagents and System.
Summary of Findings:
The specimen held at 20°C for 24 h prior to formalin fixation showed a decreased EP score compared to specimens subjected to 10 min cold ischemia, but shorter cold ischemia times (1 and 12 h) and cold ischemia at 4°C for 24 h (37°C was not tested) did not alter the specimen EP scores. The EP score was unaffected by fixation durations ranging from 1 h to 5 d. However, when the specimens were fixed for 10 d, the EP score significantly decreased by 1.2 units compared to the reference score (20 h fixation). Generally, there was a very strong correlation between the EP scores from the manually microdissected sections (100% tumor cells) and the whole sections (15-95% tumor cell content). 5 of the 38 tumors examined had a disagreement in the risk classification based on the EP score of the microdissected versus whole section, but these 5 specimens had EP score very close to the cutoff level. Importantly, combining the EP score with tumor size and nodal status to get the EPclin score resulted in discrepant risk classification for just 2 of 38 specimens. Storing prepared tissue sections on slides for 12 months at either 4°C or 20°C had no impact on EP scores or resulting risk classifications, however, a decrease in RNA yield over time was observed.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Morphology H-and-E microscopy RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Cold ischemia time 1 h
12 h
24 h
10 min
Storage Storage temperature 4°C
20°C
37°C
Storage Storage duration 0 d
12 months
Biospecimen Aliquots and Components Cell capture method Manually microdissected
Not microdissected
Biospecimen Aliquots and Components Biospecimen heterogeneity A range of tumor cell contents examined
Biospecimen Preservation Time in fixative 1 h
6 h
20 h
2 d
5 d
10 d