Journal article
Consistency and reproducibility of next-generation sequencing in cytopathology: A second worldwide ring trial study on improved cytological molecular reference specimens.
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Pisapia P
Department of Public Health, University of Naples Federico II, Naples, Italy.
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Malapelle U
Department of Public Health, University of Naples Federico II, Naples, Italy.
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Roma G
AccuRef Diagnostics, Applied Stem Cell, Inc, Milpitas, California.
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Saddar S
AccuRef Diagnostics, Applied Stem Cell, Inc, Milpitas, California.
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Zheng Q
AccuRef Diagnostics, Applied Stem Cell, Inc, Milpitas, California.
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Pepe F
Department of Public Health, University of Naples Federico II, Naples, Italy.
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Bruzzese D
Department of Public Health, University of Naples Federico II, Naples, Italy.
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Vigliar E
Department of Public Health, University of Naples Federico II, Naples, Italy.
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Bellevicine C
Department of Public Health, University of Naples Federico II, Naples, Italy.
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Luthra R
Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas.
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Nikiforov YE
Department of Pathology and Laboratory Medicine, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania.
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Mayo-de-Las-Casas C
Laboratory of Oncology, Pangaea Oncology, Barcelona, Spain.
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Molina-Vila MA
Laboratory of Oncology, Pangaea Oncology, Barcelona, Spain.
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Rosell R
Catalan Institute of Oncology, Badalona, Spain.
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Bihl M
Institute of Pathology, University Hospital Basel, Basel, Switzerland.
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Savic S
Institute of Pathology, University Hospital Basel, Basel, Switzerland.
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Bubendorf L
Institute of Pathology, University Hospital Basel, Basel, Switzerland.
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de Biase D
Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy.
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Tallini G
Anatomic Pathology, University of Bologna Medical Center, Bologna, Italy.
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Hwang DH
Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts.
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Sholl LM
Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts.
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Vander Borght S
Department of Pathology, University Hospitals Leuven, Leuven, Belgium.
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Weynand B
Department of Pathology, University Hospitals Leuven, Leuven, Belgium.
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Stieber D
National Health Laboratory, Dudelange, Luxembourg.
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Vielh P
National Health Laboratory, Dudelange, Luxembourg.
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Rappa A
Division of Pathology, European Institute of Oncology, Milan, Italy.
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Barberis M
Division of Pathology, European Institute of Oncology, Milan, Italy.
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Fassan M
Surgical Pathology Unit, Department of Medicine, University of Padua, Padua, Italy.
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Rugge M
Surgical Pathology Unit, Department of Medicine, University of Padua, Padua, Italy.
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De Andrea CE
Department of Pathology, University Clinic of Navarra, Pamplona, Spain.
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Lozano MD
Department of Pathology, University Clinic of Navarra, Pamplona, Spain.
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Lupi C
Department of Surgical, Medical, and Molecular Pathology and Critical Area, University of Pisa, Pisa, Italy.
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Fontanini G
Department of Surgical, Medical, and Molecular Pathology and Critical Area, University of Pisa, Pisa, Italy.
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Schmitt F
Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal.
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Dumur CI
Department of Pathology, Virginia Commonwealth University, Richmond, Virginia.
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Bisig B
Institute of Pathology, Lausanne University Hospital, Lausanne, Switzerland.
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Bongiovanni M
Institute of Pathology, Lausanne University Hospital, Lausanne, Switzerland.
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Merkelbach-Bruse S
Institute of Pathology and Center for Molecular Medicine, University of Cologne, Cologne, Germany.
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Büttner R
Institute of Pathology and Center for Molecular Medicine, University of Cologne, Cologne, Germany.
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Nikiforova MN
Department of Pathology and Laboratory Medicine, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania.
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Roy-Chowdhuri S
Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas.
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Troncone G
Department of Public Health, University of Naples Federico II, Naples, Italy.
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Published in:
- Cancer cytopathology. - 2019
English
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.
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Language
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Open access status
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closed
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Identifiers
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Persistent URL
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https://sonar.ch/global/documents/51004
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