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

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

  • Pisapia P Department of Public Health, University of Naples Federico II, Naples, Italy.
  • Malapelle U Department of Public Health, University of Naples Federico II, Naples, Italy.
  • Roma G AccuRef Diagnostics, Applied Stem Cell, Inc, Milpitas, California.
  • Saddar S AccuRef Diagnostics, Applied Stem Cell, Inc, Milpitas, California.
  • Zheng Q AccuRef Diagnostics, Applied Stem Cell, Inc, Milpitas, California.
  • Pepe F Department of Public Health, University of Naples Federico II, Naples, Italy.
  • Bruzzese D Department of Public Health, University of Naples Federico II, Naples, Italy.
  • Vigliar E Department of Public Health, University of Naples Federico II, Naples, Italy.
  • Bellevicine C Department of Public Health, University of Naples Federico II, Naples, Italy.
  • Luthra R Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas.
  • Nikiforov YE Department of Pathology and Laboratory Medicine, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania.
  • Mayo-de-Las-Casas C Laboratory of Oncology, Pangaea Oncology, Barcelona, Spain.
  • Molina-Vila MA Laboratory of Oncology, Pangaea Oncology, Barcelona, Spain.
  • Rosell R Catalan Institute of Oncology, Badalona, Spain.
  • Bihl M Institute of Pathology, University Hospital Basel, Basel, Switzerland.
  • Savic S Institute of Pathology, University Hospital Basel, Basel, Switzerland.
  • Bubendorf L Institute of Pathology, University Hospital Basel, Basel, Switzerland.
  • de Biase D Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy.
  • Tallini G Anatomic Pathology, University of Bologna Medical Center, Bologna, Italy.
  • Hwang DH Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts.
  • Sholl LM Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts.
  • Vander Borght S Department of Pathology, University Hospitals Leuven, Leuven, Belgium.
  • Weynand B Department of Pathology, University Hospitals Leuven, Leuven, Belgium.
  • Stieber D National Health Laboratory, Dudelange, Luxembourg.
  • Vielh P National Health Laboratory, Dudelange, Luxembourg.
  • Rappa A Division of Pathology, European Institute of Oncology, Milan, Italy.
  • Barberis M Division of Pathology, European Institute of Oncology, Milan, Italy.
  • Fassan M Surgical Pathology Unit, Department of Medicine, University of Padua, Padua, Italy.
  • Rugge M Surgical Pathology Unit, Department of Medicine, University of Padua, Padua, Italy.
  • De Andrea CE Department of Pathology, University Clinic of Navarra, Pamplona, Spain.
  • Lozano MD Department of Pathology, University Clinic of Navarra, Pamplona, Spain.
  • Lupi C Department of Surgical, Medical, and Molecular Pathology and Critical Area, University of Pisa, Pisa, Italy.
  • Fontanini G Department of Surgical, Medical, and Molecular Pathology and Critical Area, University of Pisa, Pisa, Italy.
  • Schmitt F Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal.
  • Dumur CI Department of Pathology, Virginia Commonwealth University, Richmond, Virginia.
  • Bisig B Institute of Pathology, Lausanne University Hospital, Lausanne, Switzerland.
  • Bongiovanni M Institute of Pathology, Lausanne University Hospital, Lausanne, Switzerland.
  • Merkelbach-Bruse S Institute of Pathology and Center for Molecular Medicine, University of Cologne, Cologne, Germany.
  • Büttner R Institute of Pathology and Center for Molecular Medicine, University of Cologne, Cologne, Germany.
  • Nikiforova MN Department of Pathology and Laboratory Medicine, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania.
  • Roy-Chowdhuri S Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas.
  • Troncone G Department of Public Health, University of Naples Federico II, Naples, Italy.
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  • 2019-04-26
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.
Language
  • English
Open access status
closed
Identifiers
Persistent URL
https://sonar.ch/global/documents/51004
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