Analytical validation of a prognostic prostate cancer gene expression assay using formalin fixed paraffin embedded tissue

Paul Wallace Medlow, Christopher James Steele, Andrena Marie McCavigan, Wesley Reardon, Christopher Michael Brown, Shauna May Lambe, Felipe Augusto Andre Ishiy, Steven Michael Walker, Gemma Elizabeth Logan, Olaide Yaqeen Raji, Viktor Berge, Betina Katz, Elaine Williamson Kay, Katherine Sheehan, Ronald William Watson, Denis Paul Harkin, Richard Darragh Kennedy, Laura Anne Knight*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

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Abstract

Background: There is a clear need for assays that can predict the risk of metastatic prostate cancer following curative procedures. Importantly these assays must be analytically robust in order to provide quality data for important clinical decisions. DNA microarray based gene expression assays measure several analytes simultaneously and can present specific challenges to analytical validation. This study describes the analytical validation of one such assay designed to predict metastatic recurrence in prostate cancer using primary formalin fixed paraffin embedded tumour material. Methods: Accuracy was evaluated with a method comparison study between the assay development platform (Prostate Disease Specific Array) and an alternative platform (Xcel™ microarray) using 50 formalin-fixed, paraffin-embedded prostate cancer patient samples. An additional 70 samples were used to establish the assay reportable range. Determination of assay precision and sensitivity was performed on multiple technical replicates of three prostate cancer samples across multiple variables (operators, days, runs, reagent lots, and equipment) and RNA/cDNA inputs respectively using the appropriate linear mixed model. Results: The overall agreement between the development and alternative platform was 94.7% (95% confidence interval, 86.9-98.5%). The reportable range was determined to be 0.150 to 1.107 for core needle biopsy samples and - 0.214 to 0.844 for radical prostatectomy samples. From the precision study, the standard deviations for assay repeatability and reproducibility were 0.032 and 0.040 respectively. The sensitivity study demonstrated that a total RNA input and cDNA input of 50 ng and 3.5 μg respectively was conservative. Conclusion: The Metastatic Assay was found to be highly reproducible and precise. In conclusion the studies demonstrated an acceptable analytical performance for the assay and support its potential use in the clinic.

Original languageEnglish
Article number125
Number of pages11
JournalBMC Medical Genomics
Volume11
Issue number1
DOIs
Publication statusPublished - 27 Dec 2018

Bibliographical note

Funding Information:
We acknowledge the support of each of the collaborative biobanks, including the Welsh Cancer Biobank/Cardiff University Health, Irish Prostate Cancer Research Consortium Biobank, the Northern Ireland Biobank and The Prostate Biobank associated with Oslo University Hospital, for sample acquisition and collation of clinical data. The Northern Ireland Biobank which is funded by HSC Research and Development Division of the Public Health Agency in Northern Ireland and Cancer Research UK through the Belfast CR-UK Centre and the Northern Ireland Experimental Cancer Medicine Centre; additional support was received from the Friends of the Cancer Centre. In addition, we would also like to acknowledge Joanna Fay (RCSI, Beaumont) for administrative and technical support of pathology review.

Publisher Copyright:
© 2018 The Author(s).

Keywords

  • Analytical validation
  • Metastatic
  • Prognostic
  • Prostate cancer
  • Recurrence

ASJC Scopus subject areas

  • Genetics
  • Genetics(clinical)

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