Gerard Nuovo, MD, a pathologist who was formerly a professor (now retired) at The Ohio State University College of Medicine, Columbus, cautions that gene-expression assays using FFPE tissues may generate unreliable results due to variation within the samples themselves. “The interference with the RNA amplification will differ from case to case based on variables such as tissue thickness, tumor necrosis, formalin fixation time, etc., which invariably causes problems when interpreting the data,” he noted.

Furthermore, tumor heterogeneity could pose a problem for the assay, Dr Nuovo said. “[P]arts of a cancer that metastasize may have a different RNA (and/or DNA) profile from the rest of the tumor,” he wrote in an email to Cancer Therapy Advisor. “When one grinds the tissue up, it is impossible to see such observations. The assumption is that one can detect the products of the ‘aggressive cancer cells’ and not be concerned with the products of the ‘less aggressive cancer cells.’ But this may not always be accurate.”

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The Metastatic Assay skirts this issue because it classifies a patient sample as either positive or negative for metastatic-like biology based on a predefined threshold. To find out how tumor heterogeneity would affect the assay results, researchers tested multiple biopsies from the same patient. “Although marginal differences were observed in assay score within a patient,” Dr Knight said, “the overall classification of the patient into positive [or] negative remained consistent.”

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Using an objective measure like gene expression could help counterbalance the potential subjectivity involved in histopathological assessment, such as the Gleason score, which can vary depending on the pathologist.3

“How we would probably see this in the clinic is in combination with something like CAPRA [cancer of the prostate risk assessment],” commented Dr Knight, because combining CAPRA results with the Metastatic Assay improves the predictive ability of either test alone.4 “It’s an independent predictor, but it’s also additive,” she noted.

Disclosure: This study was funded by Almac Diagnostics.


  1. Medlow PW, Steele CJ, McCavigan AM, et al. Analytical validation of a prognostic prostate cancer gene expression assay using formalin fixed paraffin embedded tissue. BMC Med Genomics. 2018;11(1):125.
  2. Walker SM, Knight LA, McCavigan AM, et al. Molecular subgroup of primary prostate cancer presenting with metastatic biology. EurUrol. 2017;72(4):509-518. 
  3. Salmo EN. An audit of inter-observer variability in Gleason grading of prostate cancer biopsies: the experience of central pathology review in the North West of England. Integr Cancer Sci Therap. 2015;2(2)104-106. doi: 10.15761/ICST.1000123
  4. Jain S, Lyons CA, Walker SM, et al. Validation of a metastatic assay using biopsies to improve risk stratification in patients with prostate cancer treated with radical radiation therapy. Ann Oncol. 2017;29(1):215-222.