Next‐generation sequencing in residual liquid‐based cytology specimens for cancer genome analysis

Abstract

Background: Cancer genome profiling of cytology specimens using next-generation sequencing (NGS) requires adequate and good-quality DNA. Genomic examination of cytology samples was conventionally performed on cell block (CB) or smear specimens than on residual liquid-based cytology (LBC) specimens, which are high-quality DNA sources even after long-term storage. Methods: We estimated tumor fractions of 37 residual LBC specimens, including 30 fine needle aspiration (FNA) samples from the thyroid (12 papillary thyroid carcinomas and two malignant lymphomas), lymph node (13 metastatic carcinomas and one malignant lymphoma), and breast cancer (one phyllodes tumor and one invasive ductal carcinoma), two pancreatic carcinoma samples, and five liquid (ascites, pleural effusion, and cerebrospinal fluid) samples. The DNA was extracted from all samples and subjected to NGS using a customized cancer gene panel comprising 28 cancerrelated genes. Results: NGS analysis revealed somatic mutations corresponding to pathological diagnosis with adequate variant allele frequency (VAF) in 24 LBC specimens, which had significantly higher tumor fraction (72.5% ± 4.9%). Ten cases, including the five fluid samples, had very small tumor fractions (7.5% ± 2.3%) to obtain sufficient VAF. Other two samples had high tumor fractions but showed very low VAF, indicating the presence of fusion genes. The remaining one sample yielded no DNA recovery. Conclusion: The residual LBC specimens collected by FNA from the thyroid gland and lymph node were verified to carry high tumor fraction and could serve as an alternate source for molecular testing to screen and diagnose cancers without the use of CB or smears.

How limited molecular testing can also offer diagnostic and prognostic evaluation of thyroid nodules processed with liquid‐based cytology: Role of TERT promoter and BRAF V600E mutation analysis

“…To demonstrate the feasibility of a 4‐genotype classification for PTC, we analyzed BRAF V600E and TERT promoter mutations on our cytology samples, specifically using LBC specimens from indeterminate, SFM, and PM cases. Whereas the accuracy and feasibility of LBC versus conventional cytology for molecular testing has been extensively confirmed by several groups, including ours, 38‐42,49‐50,58‐61 to the best of our knowledge, this is the second study, after a publication by Decaussin‐Petrucci and colleagues, documenting the evaluation of TERT promoter mutations on LBC stored material 62 . Although Decaussin‐Petrucci et al found that TERT promoter mutations were rare, but very specific for malignancy (5.5%) in indeterminate cytology, the current series confirmed a scant number of TERT mutated cases (4 of 356 cases; 1.1%) only among the malignant category and, despite the limited number of TERT ‐positive cases, all of them exhibited pleomorphic nuclei and tall cell features.…”

Performance of a 50‐gene next generation sequencing panel with post‐centrifuge supernatant cytology fluid in non‐small‐cell lung cancer

“…These results are in agreement with similar published studies, which have consistently shown concordance between results of molecular testing on FFPE specimens and on liquid cytology specimens, including supernatant with cytopreservative 3,8 and without cytopreservative 7,8 and resuspended cell pellets derived from centrifuged cytology fluid. [9][10][11] Only base pair substitutions and one small deletion were detected in this study. The NGS assay utilized is not designed to detect large insertions or deletions, copy number variations, or gene rearrangements, potentially limiting its utility in nonsmall-cell carcinoma of the lung, where the latter two in particular are recurring genetic alterations.…”

“…A 28-gene NGS panel was successfully performed on the remaining samples, and mutations were detected in 24 of these samples. 11 Though long term storage of cytology specimens is not likely a viable practice for most labs, it is conceivable that short term storage of these specimens is more feasible, and they could function as a backup specimen source for molecular analysis if needed, without the upfront cost of DNA isolation.…”

Thyroid Lobectomy for Low to Intermediate Risk Differentiated Thyroid Cancer

“…Preoperative molecular analysis today can aid in decision-making for the diagnosis of follicular and indeterminate lesions (Bethesda cytology classes IV and III). With progress in the future, the determination of molecular markers for excellent prognosis may aid in selecting low-risk cancers for active surveillance, with surgery for tumors with markers for poorer prognosis [105,106]. Higher-risk lesions, such as those harboring BRAF and TERT mutations, may benefit from more extensive surgery [107,108].…”