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.

Lung cancer and molecular testing in small biopsies versus cytology: The Logics of Worlds

“…10 Despite its small size, a cytopathology specimen can routinely afford a correct, full-fidelity diagnosis and provide ample material for necessary ancillary testing in accordance with the standard of care. [11][12][13][14][15][16][17] As a diagnostic modality, a cytopathological specimen demonstrates equivalency to a surgical specimen specifically because of the veridical richness of cytomorphology and its relation to molecular pathology.…”

“…In fact, molecular cytopathology is often better than surgical pathology in many settings. 1,11,12,14,15,27 Cytological samples frequently demonstrate higher tumor yields than concurrent surgical biopsy specimens. Furthermore, diagnostic material can be apportioned among different media during ROSE, such as cell blocks, smears, and liquid-based preparations, and this allows for more diverse types of analysis during all phases of testing.…”

“…Furthermore, diagnostic material can be apportioned among different media during ROSE, such as cell blocks, smears, and liquid-based preparations, and this allows for more diverse types of analysis during all phases of testing. [14][15][16][17] In summary, recent history has demonstrated the superior ability of cytopathology specimens for obtaining an accurate diagnosis and performing the necessary theragnostic molecular testing.…”

“…New techniques developed in the last decade have allowed for high-quality molecular results to be obtained from a multiplicity of cytopathological specimens in addition to traditional surgical pathology specimens. [11][12][13][14][15]27 Interestingly, ROSE is increasingly important because of the triage possibilities afforded by postdiagnostic molecular testing. Cytomorphological analysis is now more evolved than ever and does not simply serve as an adequacy gesture for molecular analysis.…”

Predictive molecular pathology of lung cancer in Germany with focus on gene fusion testing: Methods and quality assurance

“…71 Currently, there are no ring trials especially designed for cytological specimens, but cell block preparations have been used successfully in some EQA schemes. German institutions have participated in the ring trials on cytological molecular reference specimens conducted by the Cancer Cytopathology Group, 87,88 and a ring trial on fusion testing in cytological specimens is currently in preparation.…”