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Updated: Nov 11, 2025
Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines
Published on: October 8, 2015
Molecular Testing for BRAFV600E and RAS Mutations from Cytoscrapes of Thyroid Fine Needle Aspirates: A Single-Center
Ojas Gupta1, Upasana Gautam1, Muralidaran Chandrasekhar1
1Department of Cytology and Gynecological Pathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India.
Context And Aim:
Molecular testing of thyroid FNA has been advocated in the indeterminate categories of The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) 2018. The utility of cytoscrapes of thyroid FNA samples for BRAF V600E and RAS mutations was evaluated in this pilot study.
Methods And Materials:
Thyroid FNA samples between 2015 and 2018 from TBSRTC categories 3-6 were included. DNA was extracted from one to two representative smears (cytoscrape). Real-time PCR for BRAF V600E and RAS (KRAS, NRAS, and HRAS) gene mutations was performed. Histopathology correlation was available in 44 cases.
Statistical Methods:
Chi-square test and calculation of sensitivity, specificity, and positive/negative predictive values were performed.
Results:
A total of 73 thyroid FNA cases and 11 nodal metastases of papillary thyroid carcinoma (PTC) were evaluated. The DNA yield ranged from 1.9 to 666 ng/μl (mean 128 ng/μl) in 80 cases and was insufficient in four cases. Overall, mutations were seen in 45 (56.25%) cases with BRAF V600E, NRAS, HRAS, and KRAS in 21 (46.7%), 19 (42.2%), 4, and 1 cases, respectively. BRAF V600E mutation was seen in PTC (11/18, 61%), nodal PTC metastases (5/10, 50%), and occasionally in TBSRTC category 3 (1/18, 5.5%). NRAS mutations were seen across all categories and were maximum in the AUS/FLUS group (6/18, 33%). BRAF V600E /RAS testing had an overall sensitivity, specificity, and positive and negative predictive values of 61.7%, 80%, 91.3%, and 38%, respectively, for the detection of malignancy. In indeterminate thyroid nodules, the sensitivity, specificity, PPV, and NPV were 56.2%, 80%, 81.8%, and 53.3%, respectively.
Conclusion:
BRAF V600E/RAS mutation testing from cytoscrapes are useful as a rule-in test for indeterminate thyroid nodules and provide molecular confirmation in nodal metastases of PTC.
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