Utility of a ready-to-use PCR system for neuroendocrine tumor diagnosis

• Published in: PloS one Vol. 14; no. 6; p. e0218592
• Main Authors: Kidd, Mark, Drozdov, Ignat A., Matar, Somer, Gurunlian, Nicole, Ferranti, Nicholas J., Malczewska, Anna, Bennett, Philip, Bodei, Lisa, Modlin, Irvin M.
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 27.06.2019; Public Library of Science (PLoS)
• Subjects: Algorithms; Assaying; Automation; Biochemistry; Biological markers; Biology and Life Sciences; Biomarkers; Biomarkers, Tumor - blood; Biomarkers, Tumor - genetics; Blood; Cancer; Case-Control Studies; Chromogranin A - blood; Diagnosis; Diagnostic systems; Gene Expression; Genes; Genetic research; Genomics; Humans; Laboratories; Lung cancer; Medical diagnosis; Medical research; Medical schools; Medicine and Health Sciences; Messenger RNA; Neuroendocrine tumors; Neuroendocrine Tumors - blood; Neuroendocrine Tumors - diagnosis; Neuroendocrine Tumors - genetics; Novels; Physical Sciences; Pilot Projects; Polymerase Chain Reaction - instrumentation; Polymerase Chain Reaction - methods; Polymerase Chain Reaction - statistics & numerical data; Primers; Prospective Studies; Reproducibility; Reproducibility of Results; Research and Analysis Methods; RNA; RNA - genetics; Statistical methods; Systematic review; Tumors; New York; United Kingdom > UK; United States > US; Connecticut
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0218592

Multigene-based PCR tests are time-consuming and limiting aspects of the protocol include increased risk of operator-based variation. In addition, such protocols are complex to transfer and reproduce between laboratories. Evaluate the clinical utility of a pre-spotted PCR plate (PSP) for a novel multigene (n = 51) blood-based gene expression diagnostic assay for neuroendocrine tumors (NETs). A pilot study (n = 44; 8 controls and 36 NETs) was undertaken to compare CQ, normalized gene expression and algorithm-based output (NETest score). Gene expression was then evaluated between matched blood:tumor tissue samples (n = 7). Thereafter, two prospective sets (diagnostic: n = 167; clinical validation: n = 48, respectively) were evaluated for diagnostic and clinical utility value. Two independent molecular diagnostics facilities were used to assess assay reproducibility and inter-laboratory metrics. Samples were collected (per CLIA protocol) processed to mRNA and cDNA and then either run per standard assay (liquid primers) or on PSPs. Separately, matching plasma samples were analyzed for chromogranin A (CgA). Statistics included non-parametric testing, Pearson-concordance, Predictive Modeling and AUROC analyses. In the pilot study (n = 44), CQ values were highly concordant (r: 0.82, p<0.0001) and normalized gene expression data significantly related (p<0.0001) (Pearson-pairwise correlation). NETest values were not different (49.7±33 standard vs. 48.5±31.5 PSP) and the overall concordance in output 96%. Predictive modelling confirmed this concordance (F1 score = 0.95). Gene expression levels were highly correlated between blood and tumor tissue (R: 0.71-0.83). In the diagnostic cohort (n = 30 controls, n = 87 non-NET controls, n = 50 NET), NETest was significantly lower (p<0.0001) in controls (11±6.5) and non-NET controls (13±18) than NETs (61±31). The AUROCs were 0.93-0.97 and the diagnostic accuracy was 90-97.5%. As a diagnostic, the PSP-NETest was significantly better than CgA (accuracy: 56%, p<0.0001). For clinical samples, the PSP generated robust and accurate (>96%) scores and was significantly better (p<0.0001) than CgA. The assay protocol was consistent (r: 0.97) and reproducible (co-efficient of variation: 1.3-4.2%) across the two facilities. The PSP protocol for the NETest has been established and prospectively tested in clinical samples. It is highly reproducible, has similar metrics (CV, categorization by control or NET) to the standard PCR assay and generates clinically concordant (>96%) NETest results. Moreover, it functions significantly more accurately than CgA.