Consistency and reproducibility of next-generation sequencing in cytopathology: A second worldwide ring trial study on improved cytological molecular reference specimens

• Type: Article in International Scientific Journal
• Date: 2019

Authors

Pisapia, P (Author) Other; Malapelle, U (Author) Other; Roma, G (Author) Other; Saddar, S (Author) Other; Zheng, Q (Author) Other; Pepe, F (Author) Other; Bruzzese, D (Author) Other; Vigliar, E (Author) Other; Bellevicine, C (Author) Other; Luthra, R (Author) Other; Nikiforov, YE (Author) Other; Mayo de Las Casas, C (Author) Other; Angel Molina Vila, MA (Author) Other; Rosell, R (Author) Other; Bihl, M (Author) Other; Savic, S (Author) Other; Bubendorf, L (Author) Other; de Biase, D (Author) Other; Tallini, G (Author) Other; Hwang, DH (Author) Other; Sholl, LM (Author) Other; Vander Borght, S (Author) Other; Weynand, B (Author) Other; Stieber, D (Author) Other; Vielh, P (Author) Other; Rappa, A (Author) Other; Barberis, M (Author) Other; Fassan, M (Author) Other; Rugge, M (Author) Other; De Andrea, CE (Author) Other; Lozano, MD (Author) Other; Lupi, C (Author) Other; Fontanini, G (Author) Other; Fernando Schmitt (Author) FMUP; Dumur, CI (Author) Other; Bisig, B (Author) Other; Bongiovanni, M (Author) Other; Merkelbach Bruse, S (Author) Other; Buettner, R (Author) Other; Nikiforova, MN (Author) Other; Roy Chowdhuri, S (Author) Other; Troncone, G (Author) Other

Journal

Title: Cancer Cytopathology

Vol. 127

Pages: 285-296

ISSN: 1934-662X

Publisher: Wiley-Blackwell

Indexing

ISI Web of Knowledge - 33 Citations

Scopus - 38 Citations

Other information

Authenticus ID: P-00Q-KKJ

DOI: 10.1002/cncy.22134

Abstract (EN): Background Artificial genomic reference standards in a cytocentrifuge/cytospin format with well-annotated genomic data are useful for validating next-generation sequencing (NGS) on routine cytopreparations. Here, reference standards were optimized to be stained by different laboratories before DNA extraction and to contain a lower number of cells (2 x 10(5)). This was done to better reflect the clinical challenge of working with insufficient cytological material. Methods A total of 17 worldwide laboratories analyzed customized reference standard slides (slides A-D). Each laboratory applied its standard workflow. The sample slides were engineered to harbor epidermal growth factor receptor (EGFR) c.2235_2249del15 p.E746_A750delELREA, EGFR c.2369C>T p.T790M, Kirsten rat sarcoma viral oncogene homolog (KRAS) c.38G>A p.G13D, and B-Raf proto-oncogene, serine/threonine kinase (BRAF) c.1798_1799GT>AA p.V600K mutations at various allele frequencies (AFs). Results EGFR and KRAS mutation detection showed excellent interlaboratory reproducibility, especially on slides A and B (10% and 5% AFs). On slide C (1% AF), either the EGFR mutation or the KRAS mutation was undetected by 10 of the 17 laboratories (58.82%). A reassessment of the raw data in a second-look analysis highlighted the mutations (n = 10) that had been missed in the first-look analysis. BRAF c.1798_1799GT>AA p.V600K showed a lower concordance rate for mutation detection and AF quantification. Conclusions The data show that the detection of low-abundance mutations is still clinically challenging and may require a visual inspection of sequencing reads to detect. Genomic reference standards in a cytocentrifuge/cytospin format are a valid tool for regular quality assessment of laboratories performing molecular studies on cytology with low-AF mutations.

Language: English

Type (Professor's evaluation): Scientific

No. of pages: 12