Evaluation of the expression levels of BRAFV600E mRNA in primary tumors of thyroid cancer using an ultrasensitive mutation assay

Abstract

Background: The BRAF V600E gene encodes for the mutant BRAF V600E protein, which triggers downstream oncogenic signaling in thyroid cancer. Since most currently available methods have focused on detecting BRAF V600E mutations in tumor DNA, there is limited information about the level of BRAF V600E mRNA in primary tumors of thyroid cancer, and the diagnostic relevance of these RNA mutations is not known. Methods: Sixty-two patients with thyroid cancer and non-malignant thyroid disease were included in the study. Armed with an ultrasensitive technique for mRNA-based mutation analysis based on a two step RT-qPCR method, we analysed the expression levels of the mutated BRAF V600E mRNA in formalin-fixed paraffin-embedded samples of thyroid tissues. Sanger sequencing for detection of BRAF V600E DNA was performed in parallel for comparison and normalization of BRAF V600E mRNA expression levels. Results: The mRNA-based mutation detection assay enables detection of the BRAF V600E mRNA transcripts in a 10, 000-fold excess of wildtype BRAF counterparts. While BRAF V600E mutations could be detected by Sanger sequencing in 13 out of 32 malignant thyroid cancer FFPE tissue samples, the mRNA-based assay detected mutations in additionally 5 cases, improving the detection rate from 40.6 to 56.3%. Furthermore, we observed a surprisingly large, 3-log variability, in the expression level of the BRAF V600E mRNA in FFPE samples of thyroid cancer tissue. Conclusions: The expression levels of BRAF V600E mRNA was characterized in the primary tumors of thyroid cancer using an ultrasensitive mRNA-based mutation assay. Our data inspires further studies on the prognostic and diagnostic relevance of the BRAF V600E mRNA levels as a molecular biomarker for the diagnosis and monitoring of various genetic and malignant diseases.

Reliability of BRAF mutation detection using plasma sample

“…[ 9 ] Tumor tissue samples (resection or biopsy) are commonly used for tumor genotyping, which is abundant in tumor-derived DNA. [ 10 ] When tumor tissue samples are not available (e.g., in recurrent or metastatic cancer), liquid biopsy samples (e.g., plasma, urine, and etc) are mostly used as an alternative to determine the mutation status in tumor. [ 12 ] However, the reliability of tumor genotyping using liquid biopsy samples needs to be validated.…”

“…[ 9 ] When available, tumor tissue is a more reliable sample type for the testing of BRAF mutation status due to its high abundance of tumor DNA. [ 10 , 11 ] However, tissue sample is sometimes not available (e.g., in metastatic or recurrent cancer patients), and liquid biopsy sample (e.g., plasma, urine, etc) could serve as an alternative. [ 12 , 13 ] Liquid biopsy sample contains circulating tumor DNA (ctDNA) which derives from tumor cells and carries tumor-specific mutations, [ 13 ] making it possible to determine the gene mutation status in tumor using liquid biopsy samples.…”

Targeting Diagnosis of High‐Risk Papillary Thyroid Carcinoma Using Ultrasound Contrast Agent With the BRAFV600E Mutation

“…BRAF V600E mutation is not unique to PTC, which can also be manifested in other cancers. 45 If BRAF gene mutations are combined with other oncogene mutations, they may provide more accurate information on tumor invasion and prognostic risk stratification. Additionally, it is necessary to further explore the properties of BRAF V600E -targeted nanobubbles with animal experiments and clinical tests.…”

“…Of course, we also recognize the limitations of BRAF V600E mutation. BRAF V600E mutation is not unique to PTC, which can also be manifested in other cancers 45 . If BRAF gene mutations are combined with other oncogene mutations, they may provide more accurate information on tumor invasion and prognostic risk stratification.…”

Identification of novel natural drug candidates against BRAF mutated carcinoma; An integrative in-silico structure-based pharmacophore modeling and virtual screening process

“…One of the aggressive tumors, thyroid cancer, was also developed by the mutation in the BRAF gene. Most of the BRAF mutations occur in the position of the T1799A and others, including the mutation in the K601E in thyroid cancer ( Rowe et al, 2007 ) ( Tran et al, 2020 ). Through examining the total 75 samples, among whom 17 patients developed KRAS mutation and 26 were examined for BRAF mutation, it has been identified that BRAF mutation may lead to developing ovarian cancer in females ( Turashvili et al, 2018 ).…”