Development of a Molecular Assay for Detection and Quantification of the BRAF Variation in Residual Tissue From Thyroid Nodule Fine-Needle Aspiration Biopsy Specimens

Key Points Question Is the residual tissue from thyroid fine-needle aspiration (FNA) biopsies clinically valuable in molecular assay to aid diagnosis of thyroid nodules with indeterminate FNA findings? Findings This diagnostic study developed a molecular assay using digital polymerase chain reaction (dPCR) for sensitive detection and absolute quantification of BRAF V600E variation in the residual tissue from routine thyroid FNA biopsies. The dPCR testing showed better sensitivity than immunohistochemical staining and good concordance between residual tissue of FNA biopsies and matched surgical specimens. Meaning These findings suggest that the molecular assay by dPCR can be widely implemented to detect BRAF V600E in residual FNA biopsy specimens to improve the management of thyroid nodules harboring BRAF variation in a cost-effective manner.


Thyroid Cancer Cells Harboring Wild-type and Variant BRAF
Thyroid cancer cell lines FTC-133 and BCPAP 1 , respectively harboring wild-type BRAF and variant BRAF V600E (kindly provided by Dr. Shilpa Thakur, NIH, MD, US), were used for extraction of genomic DNA as the negative and positive controls for verifying the detection of BRAF variations. FTC-133 cells were cultured in DMEM growth medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) and BCPAP cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific) with 5% FBS, each containing 100 U/mL penicillin and 100 mg/mL streptomycin in a humidified 5% CO2 incubator. The cell authentication of two cell lines originated from human thyroid follicular (FTC-133) and papillary cancers (BCPAP) were confirmed by short tandem repeat profiling analysis by TCAG Genetic Analysis Facility (The Centre for Applied Genomics, Toronto, CAN).

Genomic DNA Extraction from Thyroid Cancer Cells, FFPE and FNA Biopsies
Monolayer FTC-133 or BCPAP cell culture were detached at 90% confluence, then fixed in CytoLyte solution overnight at room temperature to mimic the procedure of preparation of FNA biopsies. FTC-133 or BCPAP cells and the remaining material of FNA biopsies preserved in CytoLyte solution were pelleted first and re-suspended in 200 µl of PBS for DNA isolation using the DNeasy Blood & Tissue Kits (Qiagen, Hilden, Germany), according to manufacturer's instructions. FFPE tissue blocks were sectioned at 10-μm thickness, and DNA extraction was performed with QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany). All patient samples were de-identified. The DNA isolation and subsequent testing were all blinded to the patients' demographics, diagnosis, and disease status. The extracted DNA elute was stored at -20°C. DNA concentration was measured using NanoDrop 8000 (Thermo Fisher Scientific).

Immunohistochemistry for the BRAF V600E Variation
Immunohistochemistry (IHC) staining was performed using FFPE tissue sections (4 μm)  Sinai Hospital and all H&E or immunostaining images were acquired via ZEISS ZEN (blue edition). Slides were evaluated by an experienced pathologist who was blinded to the BRAF mutational status. BRAF V600E immunostaining was scored by combining percentage scale and overall intensity level of cells staining positive 4 . The percentage scale was determined by the percentage of cells staining positive in cytoplasm according to: 0 (≤ 10%), 1 (11-30%), 2 (31-50%), 3 (51-70%), or 4 (> 70%), and the intensity level of stained cells was graded as 1= weak or focal cytoplasmic staining, 2 = moderate or 3 = strong. The total score value ranges from 0 to 7. Equivocal or no staining in tumor cells and non-specific staining of colloids were considered negative.

Assessment of Sensitivity, Specificity, and Positive and Negative Predictive Values
The accuracy and reproducibility of dPCR detection of BRAF V600E was further assessed using serial dilutions of DNA samples from FTC-133 cells mixed with 1%, 5% and 10% of DNA from BCPAP cells, respectively, and serial dilutions of DNA samples from clinical specimens. BRAF VAF was calculated by BRAF variant alleles (copies) vs. the total copies of BRAF variant alleles and BRAF wild-type alleles in each reaction from all triplicate wells. The analytical specificity was validated with a temperature gradient to ensure 6-FAM LNA probe to specifically detect

Availability of Methods and Materials
Further information and requests for dPCR assays should be directed to and will be fulfilled by