Detection of BRAF mutation in the cytocentrifugation supernatant fluid from fine-needle aspiration of thyroid lesions may enhance the diagnostic yield

Brown, Ashley E; Lim, Khin Sandar; Corpus, George; Hustek, Martha T; Tran, Tien Anh; Chang, Chung‐Che

doi:10.4103/1742-6413.200935

Cited by 13 publications

(15 citation statements)

References 23 publications

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“…Recent studies have reported on the feasibility of using residual liquid‐based cytology from thyroid FNA samples to perform molecular testing; this is akin to human papillomavirus testing from residual liquid‐based cytology in cervical cytology samples . A recent study has reported the detection of BRAF mutations by pyrosequencing in a subset of supernatants from FNA centrifuged needle rinses of thyroid nodules . In this study, we show that previously discarded supernatants from thyroid FNA needle rinses can provide substantial amounts of DNA for comprehensive mutational profiling by NGS for multiple genes, even when the corresponding FNA smear/cell block material is sparsely cellular and precludes further molecular testing.…”

Section: Discussionmentioning

confidence: 73%

“…The main concern regarding the use of supernatants from centrifuged FNA samples as a substrate for molecular analysis is the lack of a morphologic correlate that accounts for the tumor fraction in the sample. A recent study reported detecting BRAF mutations in FNA cytologic specimens but not in the corresponding centrifuged supernatant in a subset of its cases . However, the testing methodologies for the FNA cellular material and the supernatant material used in that study were different, and the difference in the analytic sensitivity of the 2 platforms used could explain the discordance seen in the paired samples.…”

Section: Discussionmentioning

confidence: 78%

“…Other studies have shown that substantial amounts of DNA can be extracted and amplified from the centrifuged supernatant fluid of pancreatic‐biliary FNA samples and can be used to detect KRAS mutations, even in samples containing few or no cells . Another study used pyrosequencing to detect BRAF mutations from supernatants derived from thyroid FNA samples in BRAF ‐positive tumors; however, the authors failed to identify mutations in a subset of supernatants corresponding to the BRAF ‐positive cellular FNA samples . These results raised some concern about the possibility of false‐negative results when one is performing molecular testing from a substrate that does not undergo direct microscopic evaluation.…”

Section: Introductionmentioning

confidence: 99%

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

Hannigan

Zalles

Mehrotra

et al. 2019

Cancer Cytopathology

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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.

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Section: Discussionmentioning

confidence: 73%

Section: Discussionmentioning

confidence: 78%

Section: Introductionmentioning

confidence: 99%

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

Hannigan

Zalles

Mehrotra

et al. 2019

Cancer Cytopathology

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“…To overcome the limitations of the FFPE CB and to better preserve the remaining tissue in the CB, various alternative tissue‐containing materials, such as smeared slides and liquid samples, have been studied . Several institutions have reported the successful use of FNA supernatant fluid (SN) for molecular testing . In the current study, we examined the use of traditionally discarded CytoLyt (Hologic Inc) SN for DNA‐based and RNA‐based NGS molecular testing and compared its performance with that of paired FFPE CB material.…”

Section: Introductionmentioning

confidence: 99%

Comparison of cytocentrifugation supernatant fluid and formalin‐fixed paraffin‐embedded tissue for targeted next‐generation sequencing

Janaki

Harbhajanka

Michael

et al. 2019

Cancer Cytopathology

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BACKGROUND:The emergence of less invasive procedures coupled with the growth of molecular testing have created a need for clinical laboratories to optimize workflows to enable tissue preservation and ancillary testing. In the preparation of formalin-fixed paraffin-embedded cell blocks (FFPE CBs), there is a cytocentrifugation step for cell pellet extraction that results in postcentrifugation supernatant fluid (SN). This SN, which in most routine workflows is discarded, has been suggested to contain adequate cellular material for molecular testing. In the current study, the authors describe the use of DNA and RNA extracted from SN for the detection of clinically relevant biomarkers by next-generation sequencing (NGS). METHODS: After cell pellet removal, cytocentrifugation SN from 30 endobronchial fine-needle aspiration rinses that were positive for malignancy on FFPE CB were collected. DNA and RNA were extracted from the SN and tested using an in-house NGS Solid Tumor Focus Assay. The NGS results were compared with findings from corresponding FFPE samples. RESULTS: Testing was successful in all 30 samples. There was 100% concordance between variants observed in the SN and corresponding FFPE specimens, which included 50 single-nucleotide variants, 9 copy number amplifications, 3 structural variants, and 2 indels. Furthermore, there was excellent correlation (correlation coefficient, 0.93) between the variant allele frequency of mutations observed in SN compared with that noted in corresponding FFPE CBs. CONCLUSIONS:Cytocentrifugation SN is a valuable source for NGS, is comparable to FFPE that preserves tissue for other ancillary testing, and can reduce the failure rate of testing that may result from insufficient material being available in the CB.We thank team members from the cytology and molecular diagnostic laboratories at University Hospitals of Cleveland.

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“…However, due to the multitude of biomarkers requested, the limited volume tissue available on the cytology slides may not always be adequate. Recent studies have shown that substantial amounts of nucleic acid can be retrieved from the supernatant fluid of fine needle aspiration needle rinses after centrifugation and cell pelleting [23][24][25][26][27]. In routine clinical workflow, this supernatant fluid is typically discarded; however tumor DNA that may be left in this fluid can be effectively utilized for characterizing molecular changes in tumor.…”

Section: Introductionmentioning

confidence: 99%

Salvaging the supernatant: next generation cytopathology for solid tumor mutation profiling

et al. 2018

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With the expanding role of targeted therapy in patients with solid tumors, pathologists face the daunting task of having to maximize limited volume tissue obtained by fine needle aspiration for a variety of molecular tests. While most molecular studies on fine needle aspiration samples have been reported using cellular material, recent studies have shown that a substantial amount of DNA can be retrieved from the supernatant fluid of aspirate needle rinses after cell pelleting for cytospin or cell block preparations. In routine clinical workflow, the supernatant is discarded; however this fluid may provide a complementary source of DNA for tumor mutational profiling. In this study, we evaluated the post-centrifuged supernatant from 25 malignant and 10 benign fine needle aspiration needle rinses. The mean and median DNA yields from the supernatants were 445 ng and 176.4 ng (range, 15.1-2958 ng), respectively. Next generation sequencing using the Ion AmpliSeq Cancer Hotspot Panel v2 detected somatic mutations in all 25 malignant samples. No mutations were detected in any of the benign samples tested. When available, mutations detected in the supernatant fluid were compared to the next generation sequencing analysis performed on a prior or concurrent surgical specimen from the same patient and showed 100% concordance. In a subset of cases (n = 19) mutations in EGFR, KRAS, BRAF, PIK3CA, and NRAS were successfully confirmed by droplet digital PCR, providing an orthogonal platform for mutation analysis. In summary, in this study we show that post centrifuged supernatants from fine needle aspiration needle rinses can provide a robust substrate for expanded mutation profiling by next generation sequencing, as well as hotspot mutation testing by droplet digital PCR. The ability to detect somatic mutations from otherwise discarded supernatant fluids offers the ability to triage and effectively utilize limited volume fine needle aspiration samples when multiple molecular tests are requested, without the need to re-biopsy for additional tissue samples.

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Detection of BRAF mutation in the cytocentrifugation supernatant fluid from fine-needle aspiration of thyroid lesions may enhance the diagnostic yield

Cited by 13 publications

References 23 publications

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

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

Comparison of cytocentrifugation supernatant fluid and formalin‐fixed paraffin‐embedded tissue for targeted next‐generation sequencing

Salvaging the supernatant: next generation cytopathology for solid tumor mutation profiling

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