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Cancer Cytopathol. 2019 May;127(5):285-296. doi: 10.1002/cncy.22134. Epub 2019 Apr 25.

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

Author information

1
Department of Public Health, University of Naples Federico II, Naples, Italy.
2
AccuRef Diagnostics, Applied Stem Cell, Inc, Milpitas, California.
3
Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas.
4
Department of Pathology and Laboratory Medicine, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania.
5
Laboratory of Oncology, Pangaea Oncology, Barcelona, Spain.
6
Catalan Institute of Oncology, Badalona, Spain.
7
Rosell Cancer Institute, Quiròn-Dexeus University Institute, Barcelona, Spain.
8
Institute of Pathology, University Hospital Basel, Basel, Switzerland.
9
Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy.
10
Anatomic Pathology, University of Bologna Medical Center, Bologna, Italy.
11
Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts.
12
Department of Pathology, University Hospitals Leuven, Leuven, Belgium.
13
National Health Laboratory, Dudelange, Luxembourg.
14
Division of Pathology, European Institute of Oncology, Milan, Italy.
15
Surgical Pathology Unit, Department of Medicine, University of Padua, Padua, Italy.
16
Department of Pathology, University Clinic of Navarra, Pamplona, Spain.
17
Department of Surgical, Medical, and Molecular Pathology and Critical Area, University of Pisa, Pisa, Italy.
18
Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal.
19
Department of Pathology, Medical Faculty, Porto University, Porto, Portugal.
20
Department of Pathology, Virginia Commonwealth University, Richmond, Virginia.
21
Institute of Pathology, Lausanne University Hospital, Lausanne, Switzerland.
22
Institute of Pathology and Center for Molecular Medicine, University of Cologne, Cologne, Germany.

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.

KEYWORDS:

cytological molecular reference; cytology; lung cancer; molecular cytopathology; next-generation sequencing

PMID:
31021538
DOI:
10.1002/cncy.22134

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