Prognostic Assessment of BRAF Mutation in Preoperative Thyroid Fine-Needle Aspiration Specimens

Rita Abi-Raad, MD; Manju L. Prasad, MD; Jingwei Zheng, MD; Pei Hui, MD, PhD; Berrin Ustun, MD; Kevin Schofield, CT(ASCP); Guoping Cai, MD; Adebowale J. Adeniran, MD


Am J Clin Pathol. 2021;156(1):100-108. 

In This Article

Materials and Methods

Study Population

This study was approved by the institutional review board. The pathology files were retrospectively searched for all thyroid FNA cases reviewed in the Department of Pathology at our institution between October 2009 and August 2014. All FNAs were performed under ultrasound guidance by either endocrinologists or radiologists. The aspirated material was divided into air-dried direct smears stained with Diff-Quik (Dade Behring) and alcohol-fixed smears for Papanicolaou staining. The needle was then rinsed in Cyto-Rich Red solution (Thermo-Fisher Scientific) for Thin-Prep slide (Hologic) and subsequent BRAF analysis. Cases with available BRAF mutation status and surgical follow-up were included in the study. Only PTC was included in the analysis; metastatic tumors to the thyroid, non-PTC primary thyroid malignancy, and PTC recurrence were excluded. Due to the recent reclassification of tumors as noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), cases diagnosed as such on surgical resection were excluded from the analysis. To avoid confounding effects of multiple PTCs harboring different BRAF mutation statuses, the analysis was restricted to patients with unilateral and unifocal disease, while bilateral and multifocal PTCs were excluded from the analysis Figure 1. All patients subsequently underwent one- or two-stage total thyroidectomy, near-total thyroidectomy, or lobectomy with or without lymph node dissection.

Figure 1.

Flowchart of the patients represented in the study. FNA, fine-needle aspiration; NIFTP, noninvasive follicular thyroid neoplasm with papillary-like nuclear features; PTC, papillary thyroid carcinoma.

Of 15,367 thyroid FNA specimens reviewed during the study period, a total of 956 cases were referred for BRAF mutation analysis, and 878 FNAs (91.8%) were found to have adequate DNA for the assay. Of those, 855 cases were FNAs from primary thyroid tumors, whereas 23 were recurrences and therefore excluded. A total of 452 patients with primary tumors underwent subsequent surgery: 349 patients were found to have a primary thyroid malignancy, including 340 PTCs. Of the PTC cases, 61 and 128 were found to have bilateral and multifocal disease, respectively, and were excluded from the analysis. In total, 151 cases with equivocal and positive cytologic diagnosis were included in the study cohort (see Figure 1 for details).

BRAF Mutation Analysis

BRAF testing was performed as described previously.[11] Briefly, DNA was extracted from FNA specimens and amplified by polymerase chain reaction using the primers flanking the region of V600E mutation of BRAF, and the resulting product was analyzed by single-strand conformational polymorphism on electrophoresis. The presence or absence of V600E mutation was determined by comparison with a known V600E-mutated control specimen. At our institution, testing for BRAF mutation status is reflexed on cases with cytology diagnosis of PTC, suspicious for PTC and follicular lesion of undetermined significance (FLUS), or category VI, V, and III of The Bethesda System for Reporting Thyroid Cytopathology. Most of those cases were reviewed at the daily intradepartmental cytopathology consensus conference by at least three board-certified cytopathologists.

Data Collection and Analysis

Patients' electronic medical records (EMRs) and pathology reports were reviewed for sex, age at diagnosis, type of surgery, size of tumor, presence of lymphovascular invasion (LVI), gross and/or microscopic extrathyroidal extension (ETE), presence of lymph node (LN) metastasis, and adjuvant treatments, including postoperative 131I therapy. The date of initial surgery, last date of follow-up, and status at last follow-up were recorded. The status at last follow-up was recorded as no evidence of disease, alive with disease, or follow-up not available. Disease persistence/recurrence was confirmed through EMR and/or biopsy-proven recurrence. Follow-up time was defined as the time from the initial thyroid surgery to the first evidence of disease recurrence in patients who had recurrence and to the most recent clinical evaluation for those who remained free of disease. BRAF mutation status was correlated with cytologic, histologic, and clinical findings.

Data were stratified by BRAF mutation status and analyzed against the clinicopathologic characteristics such as age, sex, tumor characteristics, LN status and American Joint Committee on Cancer (AJCC) stage. Statistical analysis with χ2 test or Fisher exact test was performed wherever appropriate. Independent samples t test was used to compare tumor size. A P value less than .05 was considered significant.