A valuable adjunct to FNA diagnosis of papillary thyroid carcinoma: In‐house PCR assay for BRAF T1799A (V600E)

Abstract

The diagnostic approach to thyroid nodules generally starts with FNA cytology. However, approximately one-fifth of cytologic evaluations yield indeterminate cytological findings but only 20% of cases with indeterminate thyroid nodule cytology have a cancer diagnosis, emphasizing the need for an effective ancillary test based on FNA material to help prevent unnecessary surgery. Detection of BRAFV600E mutation, the genetic signature of papillary thyroid carcinoma (PTC) in FNA material provides an invaluable diagnostic adjunct to overcome the limitations of FNA cytology. There are many ways to detect V600E, such as direct DNA sequencing, allele-specific PCR and hybridization-based colorimetric methods. In this study, a newer simple PCR method is presented that removes requirements for sequencing special equipment and commercial kits. Two forward primers including the mutant sequence specific (F2), and one common reverse (R) primer were optimized to generate a 241 bp fragment (F1R), an internal PCR control, and a 141 bp fragment (F2R) denoting the presence of V600E. Sensitivity studies revealed that the assay is capable of detecting V600E even in 1 ng of DNA. Direct sequencing data of 241 bp F1R fragment proved the specificity of the assay. For validation studies of the sequence specific multiplex PCR assay, archival FNA slides were used in a group of thyroid lesions including PTC, follicular carcinoma, follicular adenoma, Hashimoto thyroiditis, and benign thyroid nodules. The newer PCR-based method presented in this study is a practical, inexpensive one-step assay to detect the BRAF T1796A mutation on FNA samples.

Impact of BRAFV600E Mutation on Event-Free Survival in Patients with Papillary Thyroid Carcinoma: A Retrospective Study in a Romanian Population

“…BRAF V600E mutation represents a very specific marker for PTC, also referred to as the “genetic signature of PTC” [ 24 , 25 , 26 ]. As this mutation appears to play an important role in PTC tumorigenesis, it has been postulated that it might also have a prognostic value.…”

Assessment of cellularity, genomic DNA yields, and technical platforms for BRAF mutational testing in thyroid fine‐needle aspirate samples

“…Recently, several studies have been published on the feasibility of successfully testing for BRAF mutations using cytologic materials. 7,9,[14][15][16][17][18][19][20] It has been shown that detection of BRAF mutations can be helpful in facilitating accurate diagnosis in thyroid FNA cytology. 5,13 A successful cytologic diagnosis relies on adequate sample with good cellularity.…”

The Utility of Fine-Needle Aspiration in the Diagnosis and Management of Follicular Thyroid Neoplasms:One Institution’s 10-Year Experience

“…Most of the studies were done on histological section while only a few have involved cytologic material. These markers include Ret-PTC translocation, BRAF mutation, K-ras and others [19][20][21][22][23][24]. In summary, unless larger studies are done specifically utilizing molecular markers in FNA material, clinical and cytological features remain the mainstay for diagnosis of follicular thyroid lesions.…”