Molecular Diagnosis Using Residual Liquid-Based Cytology Materials for Patients with Nondiagnostic or Indeterminate Thyroid Nodules

BACKGROUND: Molecular testing is recommended as an adjunct to improve the preoperative diagnosis of fine-needle aspiration (FNA) of thyroid nodules. Centrifuged supernatants from FNA samples, which are typically discarded, have recently emerged as a novel liquid-based biopsy for molecular testing. This study evaluates the use of thyroid FNA supernatants for detecting clinically relevant mutations. METHODS: Supernatants from thyroid FNA samples (n = 156) were evaluated. A 50-gene next-generation sequencing (NGS) assay was used, and mutation analysis results from a subset of samples were further compared with those of paired FNA smears and/or cell blocks. RESULTS: All 156 samples yielded adequate DNA (median, 135 ng; range, 11-3180 ng), and 129 of these samples (83%) were successfully sequenced by NGS. The most frequently detected somatic mutations included BRAF and RAS mutations, which were followed by RET, TP53, PTEN, CDKN2A, and PIK3CA mutations. Eleven of 31 cases with an indeterminate cytologic diagnosis and 9 of 12 cases that were suspicious for malignancy had somatic mutations, including the BRAF V600E mutation, which is highly definitive for papillary thyroid carcinoma (PTC). Seven of the 9 indeterminate and suspicious cases with the BRAF V600E mutation had surgical follow-up, and they were all confirmed to be PTC. A comparison of the mutation profiles derived from supernatants with those of paired smears and/or cell blocks in a small subset of cases (n = 8) showed 100% concordance. CONCLUSIONS: This study provides evidence that FNA supernatants can be used as a surrogate for thyroid molecular testing to improve diagnostic accuracy in indeterminate nodules, provide prognostic/predictive information, and improve overall patient management.

Section: Discussionmentioning

confidence: 99%

Centrifuged supernatants from FNA provide a liquid biopsy option for clinical next‐generation sequencing of thyroid nodules

Hannigan

Zalles

Mehrotra

et al. 2019

“…Recent studies have explored the usefulness of the molecular testing of residual LBC from nongynecological FNA samples, with promising results. Several studies have been published to date describing the usefulness of extracting nucleic acids from residual LBC solution from thyroid FNA specimens for mutation analysis using high‐resolution melting polymerase chain reaction (PCR), pyrosequencing, and next‐generation sequencing (NGS), and evaluating gene rearrangements using quantitative PCR . Wei et al described using residual PreservCyt (Cytyc Corporation, Marlborough, Massachusetts) specimens from FNA samples and body fluids (either fresh unconcentrated specimens or residual PreservCyt specimens) to extract DNA for multigene NGS analysis .…”

mentioning

confidence: 99%

Molecular testing of residual cytology samples: Rethink, reclaim, repurpose

Roy‐Chowdhuri

2018

“…Extraction kits vary in the input amount, extraction volume, and chemistry used, and differing results in terms of the quantity and type of DNA extracted among studies that use different kits are to be expected. 29 Studies in which nucleic acid was analyzed after extraction from thyroid FNA needle rinses are listed in Table 5, 10,18,20,23,25,30 along with their respective nucleic acid extraction kits and associated methodologies, all of which used manual processing. Among the studies, only one 18 used the same extraction kit as that in the current study, and the authors reported neither the quantity nor the quality of nucleic acids extracted.…”

Section: Nucleic Acid Extractionmentioning

confidence: 99%

“…In a study that used an ethanol-based fixative with a small (<0.2%) concentration of formalin (SurePath; Becton Dickinson and Company), Kwon et al reported an elevated rate of p.Val600Glu mutations in the BRAF gene in TBSRTC category III specimens compared with the current study. 30 This suggests discrepant morphologic diagnoses between the 2 studies, which may be mitigated by careful monitoring of diagnostic AUS rates and AUS:malignant ratios. 31 It is also important to establish the lower limit of tumor cellularity at which genetic alterations may be detected with confidence.…”

Section: Other Preanalytic Variablesmentioning

confidence: 99%

Preanalytic variables in quality and quantity of nucleic acids extracted from FNA specimens of thyroid gland nodules collected in CytoLyt: Cellularity and storage time

Heymann

Yoxtheimer

Park

et al. 2020

Background Morphologic and genetic analysis of thyroid nodules may be performed from a single vial. Preanalytic variables that affect nucleic acid extracted from a single vial are evaluated. Methods Thyroid fine‐needle aspiration (FNA) specimens collected in CytoLyt were evaluated. A ThinPrep slide was prepared. Extracted nucleic acids were analyzed using Oncomine Comprehensive Panel, version 2, after Ion AmpliSeq library preparation. A pathologist and a cytotechnologist enumerated specimen cellularity. Results Fifty‐six samples were collected from 55 nodules in 53 patients. Bethesda category correlated with cellularity (P = .01), and storage time (median, 43 days; range, 7‐77 days) was longer for specimens in categories II and III than for those in categories IV and VI (P = .01). The mean specimen DNA concentration was 4.5 ng/µL (range, 0‐23.8 ng/µL), and 25 (45%) had concentrations >3.3 ng/µL. The mean specimen RNA concentration was 4.8 ng/µL (range, 0‐42.4 ng/µL), and 31 (55%) had concentrations >1.4 ng/µL. Nucleic acid quantity increased with epithelial cellularity. Storage time weakly correlated with the quantity of extracted DNA, independent of cellularity, but not extracted RNA. Greater proportions of cell‐free DNA and lesser proportions of long, intact RNA fragments were extracted from a subset of samples with longer storage time. Among 15 single nucleotide variants, the median mutant allelic fraction was 15.1%. One false‐negative result was identified. Five specimens subsequently determined to harbor a genetic alteration failed quality metrics. Conclusions Cellularity and storage time affect the quantity and quality of nucleic acid extracted from thyroid FNA specimens collected in CytoLyt. Further investigation will serve to quantify the magnitude of such effects and to elucidate other contributing factors.