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

Authors: Kidd, M.; Drozdov, I. A.; Matar, S.; Gurunlian, N.; Ferranti, N. J.; Malczewska, A.; Bennett, P.; Bodei, L.; Modlin, I. M.
Article Title: Utility of a ready-to-use PCR system for neuroendocrine tumor diagnosis
Abstract: Background 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. Aims 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). Methods 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. Results 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. Conclusion 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. © 2019 Kidd et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Keywords: clinical article; controlled study; human tissue; diagnostic accuracy; gene expression; cohort analysis; clinical protocol; prediction; neuroendocrine tumor; algorithm; messenger rna; blood sampling; diagnostic value; pilot study; laboratory; tumor diagnosis; complementary dna; chromogranin a; molecular diagnosis; human; article; chemiluminescence immunoassay
Journal Title: PLoS ONE
Volume: 14
Issue: 6
ISSN: 1932-6203
Publisher: Public Library of Science  
Date Published: 2019-06-27
Start Page: e0218592
Language: English
DOI: 10.1371/journal.pone.0218592
PUBMED: 31247038
PROVIDER: scopus
PMCID: PMC6597157
Notes: Article -- Source: Scopus
Citation Impact
MSK Authors
  1. Lisa   Bodei
    100 Bodei