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Updated: Jul 26, 2026
Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines
Published on: October 8, 2015
Papillary thyroid carcinoma: morphological features and association with normalized BRAFV600E allelic frequency;
Maria Soledad Martínez-Martin1,2,3, María Eugenia Galán-García2, Eduardo José Araujo-Ruano1
1Pathology Unit, Faculty of Medicine, University of Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain.
Background:
Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy. The BRAF V600E (c·1799T > A; p.Val600Glu) mutation results in oncogenic activation of the MAP-kinase pathway and plays a key role in the development and aggressiveness of PTC. Significant morphological differences have been observed between BRAF V600E-mutated and non-mutated PTCs. The BRAF V600E mutation has been associated with adverse clinicopathological features, including lymph node metastasis, recurrence, tumor size, extrathyroidal invasion, and unfavorable outcomes, particularly when the mutant allele fraction is high. However, the relationship between the proportion of mutated neoplastic cells and the morphological characteristics of PTC remains unclear. This study aims to investigate the potential connection between the proportion of mutated neoplastic tumor cells and the morphological characteristics of PTC, and to evaluate the impact of normalizing the allele frequency to the percentage of neoplastic tumor cells in the tissue used for DNA extraction.
Methods:
Eighty-five cases of PTC, spanning a 12-year period, were analysed with a focus on specific morphological features, including infiltrative tumor margin, plumps pink cells, desmoplasia and tumor size. Additional features included surgical margin status, lymph node and distant metastasis, multifocality, psammoma bodies, fibrosis, encapsulation and nuclear atypia. DNA mutation analysis was performed using pyrosequencing, and the percentage of neoplastic cells (MNC) was estimated from the slide used for DNA extraction. Normalization of the allele frequency was performed using a corresponding formula. Tumors were classified into three groups: subclonal ( < 30% MNC), heterogeneous (30-80%) and clonal ( > 80%). Statistical analysis, including interquartile range (IQR) calculations, was conducted.
Results:
The frequency of mutated cases (BRAF V600E ≥10%) was 57 out of 85 cases (67%). The variables infiltrative tumor margins (p < 0.001), plump pink cells (p = 0.007), and desmoplasia (p = 0.002) showed statistically significant associations with clonality, with the first remaining independently significant in the multivariate logistic regression analysis (p < 0.001). Surgical margin status, distant metastasis, and tumor size also showed significance. Similar results were obtained using IQR analysis for the first three variables and for surgical margin status. The remaining variables did not reach statistical significance.
Conclusions:
The presence of infiltrative tumor margin, plump pink cells, and myxoid desmoplasia, may serve as active markers associated with the normalized BRAF V600E mutation. These findings suggest that the mutation acts as a driver in both early and late stages of PTC development. Furthermore, a direct relationship between tumor size and clonality underscores the role of BRAF V600E mutation in tumor progression and morphological evolution.
