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

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.

• Authors: Pasquale Pisapia, Umberto Malapelle, Gianluca Roma, Sonika Saddar, Qi Zheng, Francesco Pepe, Dario Bruzzese, Elena Vigliar, Claudio Bellevicine, Rajyalakshmi Luthra, Yuri E Nikiforov, Clara Mayo-de-Las-Casas, Miguel Angel Molina-Vila, Rafael Rosell, Michel Bihl, Spasenija Savic, Lukas Bubendorf, Dario de Biase, Giovanni Tallini, David H Hwang, Lynette M Sholl, Sara Vander Borght, Birgit Weynand, Daniel Stieber, Philippe Vielh, Alessandra Rappa, Massimo Barberis, Matteo Fassan, Massimo Rugge, Carlos E De Andrea, Maria D Lozano, Cristiana Lupi, Gabriella Fontanini, Fernando Schmitt, Catherine I Dumur, Bettina Bisig, Massimo Bongiovanni, Sabine Merkelbach-Bruse, Reinhard Büttner, Marina N Nikiforova, Sinchita Roy-Chowdhuri, Giancarlo Troncone, Molecular Cytopathology Meeting Group
• Journal: Cancer cytopathology (Cancer Cytopathol) Vol. 127 Issue 5 Pg. 285-296 (05 2019) ISSN: 1934-6638 [Electronic] United States
• PMID: 31021538 (Publication Type: Journal Article)
• Copyright: © 2019 American Cancer Society.

Chemical References

• Biomarkers, Tumor
• KRAS protein, human
• MAS1 protein, human
• Proto-Oncogene Mas
• EGFR protein, human
• ErbB Receptors
• BRAF protein, human
• Proto-Oncogene Proteins B-raf
• Proto-Oncogene Proteins p21(ras)

Topics

• Biomarkers, Tumor (genetics)
• Cytodiagnosis (methods)
• DNA Mutational Analysis (methods)
• ErbB Receptors (genetics)
• High-Throughput Nucleotide Sequencing (methods)
• Humans
• Mutation
• Neoplasms (diagnosis, genetics)
• Proto-Oncogene Mas
• Proto-Oncogene Proteins B-raf (genetics)
• Proto-Oncogene Proteins p21(ras) (genetics)
• Reproducibility of Results