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 × 105 ). 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.

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

Gabriella Fontanini;
2019

Abstract

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 × 105 ). 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.
Pisapia, Pasquale; Malapelle, Umberto; Roma, Gianluca; Saddar, Sonika; Zheng, Qi; Pepe, Francesco; Bruzzese, Dario; Vigliar, Elena; Bellevicine, Claudio; Luthra, Rajyalakshmi; Nikiforov, Yuri E.; Mayo‐de‐las‐casas, Clara; Angel Molina‐Vila, Miguel; Rosell, Rafael; Bihl, Michel; Savic, Spasenija; Bubendorf, Lukas; de Biase, Dario; Tallini, Giovanni; Hwang, David H.; Sholl, Lynette M.; Vander Borght, Sara; Weynand, Birgit; Stieber, Daniel; Vielh, Philippe; Rappa, Alessandra; Barberis, Massimo; Fassan, Matteo; Rugge, Massimo; De Andrea, Carlos E.; Lozano, Maria D.; Lupi, Cristiana; Fontanini, Gabriella; Schmitt, Fernando; Dumur, Catherine I.; Bisig, Bettina; Bongiovanni, Massimo; Merkelbach‐bruse, Sabine; Büttner, Reinhard; Nikiforova, Marina N.; Roy‐chowdhuri, Sinchita; Troncone for the Molecular Cytopathology Meeting Group, Giancarlo
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11568/1021274
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