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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.
- Source :
-
Cancer cytopathology [Cancer Cytopathol] 2019 May; Vol. 127 (5), pp. 285-296. Date of Electronic Publication: 2019 Apr 25. - Publication Year :
- 2019
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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 × 10 <superscript>5</superscript> ). This was done to better reflect the clinical challenge of working with insufficient cytological material.<br />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&#95;2249del15 p.E746&#95;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&#95;1799GT>AA p.V600K mutations at various allele frequencies (AFs).<br />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&#95;1799GT>AA p.V600K showed a lower concordance rate for mutation detection and AF quantification.<br />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.<br /> (© 2019 American Cancer Society.)
- Subjects :
- ErbB Receptors genetics
Humans
Neoplasms genetics
Proto-Oncogene Mas
Proto-Oncogene Proteins B-raf genetics
Proto-Oncogene Proteins p21(ras) genetics
Reproducibility of Results
Biomarkers, Tumor genetics
Cytodiagnosis methods
DNA Mutational Analysis methods
High-Throughput Nucleotide Sequencing methods
Mutation
Neoplasms diagnosis
Subjects
Details
- Language :
- English
- ISSN :
- 1934-6638
- Volume :
- 127
- Issue :
- 5
- Database :
- MEDLINE
- Journal :
- Cancer cytopathology
- Publication Type :
- Academic Journal
- Accession number :
- 31021538
- Full Text :
- https://doi.org/10.1002/cncy.22134