Gut neuroendocrine tumor blood qPCR fingerprint assay: characteristics and reproducibility
-
Irvin M. Modlin
,
Ignat Drozdov
Abstract
Background: We have developed a PCR-based tool that measures a 51-gene panel for identification of gastroenteropancreatic (GEP) neuroendocrine neoplasms (NENs) in peripheral blood. This manuscript assesses the robustness (performance metrics) of this tool with a specific focus on the effects of individual parameters including collection, storage, acid suppressive medication [proton pump inhibitor (PPI)], age, sex, race and food on accuracy.
Methods: Performance metrics were evaluated using a gold standard (mRNA derived from three individual human neuroendocrine tumor cell lines) and clinical samples using qPCR.
Results: One hundred percent of the 51 transcripts were amplified in the gold standard (NEN cell line-derived mRNA) (CQ<35, average efficiency 1.94). The inter- and intra-assay variations were 1%–2%. In clinical samples, 50 of 51 targets (98%) were amplified. The inter- and intra-assay reproducibility ranged between 0.4% and 1.2%. The coefficient of variation (CV) was 5.3%. Expression of the reference gene, ALG9, was robust [low variation, low M-value, high (99.5%) PCR efficiency] and unaffected by sample processing. Test meals, long-term PPI use (>1 year), age, sex and ethnicity had no effect on the signature. Expression of two genes, ALP2 and CD59 correlated strongly with RNA integrity (R=0.72, p<0.001) and could be used to assess storage and processing.
Conclusions: The 51 marker gene signature was robust and reproducible, exhibiting acceptable inter- and intra-assay metrics (<5%). Feeding, PPI intake, age, sex and ethnicity do not affect the signature. Expression levels of APLP2 and CD59 are effective surrogate markers of proper sample collection and processing.
Daniele Alaimo and Steve Callahan for technical support.
Conflict of interest statement
Authors’ conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article. Research funding played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
Research funding: Funding for this project was provided by Clifton Life Sciences.
Employment or leadership: None declared.
Honorarium: None declared.
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Articles in the same Issue
- Masthead
- Masthead
- Editorial
- Point of care testing: evolving scenarios and innovative perspectives
- Review
- Point-of-care testing: where is the evidence? A systematic survey
- Mini Review
- Vulnerability of point-of-care test reagents and instruments to environmental stresses: implications for health professionals and developers
- Opinion Paper
- Twenty-five years of idiopathic calcium nephrolithiasis: has anything changed?
- Genetics and Molecular Diagnostics
- Optimizing the purification and analysis of miRNAs from urinary exosomes
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- Extensive study of human insulin immunoassays: promises and pitfalls for insulin analogue detection and quantification
- Absorptive chemistry based extraction for LC-MS/MS analysis of small molecule analytes from biological fluids – an application for 25-hydroxyvitamin D
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