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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
Author(s) -
Pisapia Pasquale,
Malapelle Umberto,
Roma Gianluca,
Saddar Sonika,
Zheng Qi,
Pepe Francesco,
Bruzzese Dario,
Vigliar Elena,
Bellevicine Claudio,
Luthra Rajyalakshmi,
Nikiforov Yuri E.,
MayodeLasCasas Clara,
MolinaVila Miguel Angel,
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,
MerkelbachBruse Sabine,
Büttner Reinhard,
Nikiforova Mari.,
RoyChowdhuri Sinchita,
Troncone Giancarlo
Publication year - 2019
Publication title -
cancer cytopathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.29
H-Index - 57
eISSN - 1934-6638
pISSN - 1934-662X
DOI - 10.1002/cncy.22134
Subject(s) - kras , medicine , mutation , cytopathology , molecular diagnostics , dna sequencing , concordance , cancer , pathology , genetics , biology , gene , cytology , colorectal cancer
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 × 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.