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

Autores: Pisapia, P.; Malapelle, U.; Roma, G.; Saddar, S.; Zheng, Q. ; Pepe, F.; Bruzzese, D. ; Vigliar, E.; Bellevicine, C.; Luthra, R. ; Nikiforov, Y. E. ; Mayo-de-Las-Casas, C. ; Molina-Vila, M. A.; Rosell, R. ; Bihl, M. ; Savic, S.; Bubendorf, L.; de Biase, D.; Tallini, G.; Hwang, D. H. ; Sholl, L. M.; Vander Borght, S.; Weynand, B.; Stieber, D. ; Vielh, P. ; Rappa, A. ; Barberis, M.; Fassan, M.; Rugge, M.; De Andrea, Carlos Eduardo; Lozano Escario, María Dolores; Lupi, C.; Fontanini, G.; Schmitt, F. ; Dumur, C. I.; Bisig, B.; Bongiovanni, M. ; Merkelbach-Bruse, S. ; Buttner, R. ; Nikiforova, M. N.; Roy-Chowdhuri, S. ; Troncone, G. (Autor de correspondencia)
Título de la revista: CANCER CYTOPATHOLOGY
ISSN: 1934-662X
Volumen: 127
Número: 5
Páginas: 285 - 296
Fecha de publicación: 2019
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.