Pisapia, Pasquale ORCID: 0000-0002-6429-0620, Malapelle, Umberto ORCID: 0000-0003-3211-9957, Roma, Gianluca, Saddar, Sonika, Zheng, Qi, Pepe, Francesco ORCID: 0000-0002-2803-9586, Bruzzese, Dario, Vigliar, Elena, Bellevicine, Claudio ORCID: 0000-0002-7479-6457, 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 ORCID: 0000-0002-3711-8681, Dumur, Catherine I., Bisig, Bettina ORCID: 0000-0002-6840-375X, Bongiovanni, Massimo, Merkelbach-Bruse, Sabine, Buettner, Reinhard, Nikiforova, Marina N., Roy-Chowdhuri, Sinchita ORCID: 0000-0002-9447-7701 and Troncone, Giancarlo (2019). Consistency and reproducibility of next-generation sequencing in cytopathology: A second worldwide ring trial study on improved cytological molecular reference specimens. Cancer Cytopathol., 127 (5). S. 285 - 297. HOBOKEN: WILEY. ISSN 1934-6638

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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 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.

Item Type: Journal Article
Creators:
CreatorsEmailORCIDORCID Put Code
Pisapia, PasqualeUNSPECIFIEDorcid.org/0000-0002-6429-0620UNSPECIFIED
Malapelle, UmbertoUNSPECIFIEDorcid.org/0000-0003-3211-9957UNSPECIFIED
Roma, GianlucaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Saddar, SonikaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Zheng, QiUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Pepe, FrancescoUNSPECIFIEDorcid.org/0000-0002-2803-9586UNSPECIFIED
Bruzzese, DarioUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Vigliar, ElenaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Bellevicine, ClaudioUNSPECIFIEDorcid.org/0000-0002-7479-6457UNSPECIFIED
Luthra, RajyalakshmiUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Nikiforov, Yuri E.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Mayo-de-Las-Casas, ClaraUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Angel Molina-Vila, MiguelUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Rosell, RafaelUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Bihl, MichelUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Savic, SpasenijaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Bubendorf, LukasUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
de Biase, DarioUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Tallini, GiovanniUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Hwang, David H.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Sholl, Lynette M.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Vander Borght, SaraUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Weynand, BirgitUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Stieber, DanielUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Vielh, PhilippeUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Rappa, AlessandraUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Barberis, MassimoUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Fassan, MatteoUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Rugge, MassimoUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
De Andrea, Carlos E.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Lozano, Maria D.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Lupi, CristianaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Fontanini, GabriellaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Schmitt, FernandoUNSPECIFIEDorcid.org/0000-0002-3711-8681UNSPECIFIED
Dumur, Catherine I.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Bisig, BettinaUNSPECIFIEDorcid.org/0000-0002-6840-375XUNSPECIFIED
Bongiovanni, MassimoUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Merkelbach-Bruse, SabineUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Buettner, ReinhardUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Nikiforova, Marina N.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Roy-Chowdhuri, SinchitaUNSPECIFIEDorcid.org/0000-0002-9447-7701UNSPECIFIED
Troncone, GiancarloUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
URN: urn:nbn:de:hbz:38-149430
DOI: 10.1002/cncy.22134
Journal or Publication Title: Cancer Cytopathol.
Volume: 127
Number: 5
Page Range: S. 285 - 297
Date: 2019
Publisher: WILEY
Place of Publication: HOBOKEN
ISSN: 1934-6638
Language: English
Faculty: Unspecified
Divisions: Unspecified
Subjects: no entry
Uncontrolled Keywords:
KeywordsLanguage
LUNG-CANCER PATIENTS; GROWTH-FACTOR RECEPTOR; CELL-FREE DNA; TESTING GUIDELINE; LIQUID BIOPSY; ASSOCIATION; SMEARS; SUPERNATANT; VALIDATION; MUTATIONSMultiple languages
Oncology; PathologyMultiple languages
Refereed: Yes
URI: http://kups.ub.uni-koeln.de/id/eprint/14943

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