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Research Article
 

Prevalence of BRAFV600E Mutation in Iranian Patients with Papillary Thyroid Carcinoma: A Single-Center Study



J. Mohammadi-Asl, B. Larijani, Z. Khorgami, S.M. Tavangar, V. Haghpanah and P. Mehdipour
 
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ABSTRACT

The aim of this study was to investigate the frequency of the BARFV600E mutation in Papillary Thyroid Carcinoma (PTC) in Iranian population through PCR-RFLP. Fifty formalin-fixed paraffin-embedded and 26 frozen thyroid tumors including 28 PTCs, two undifferentiated thyroid carcinoma and 46 benign thyroid tumors were evaluated. The BARFV600E mutation was detected in 20 of 28(71.4%) PTCs, but failed to distinguish the mutant allele in benign thyroid tumors. The age, sex, extrathyroid extension and lymph node metastases distribution did not, significantly, differ between the patients with and without the BARFV600E mutation. In conclusion, these findings might pave our way towards considering the BRAFV600E mutation in PTCs in the regions with high prevalence of this alteration as a molecular marker.

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  How to cite this article:

J. Mohammadi-Asl, B. Larijani, Z. Khorgami, S.M. Tavangar, V. Haghpanah and P. Mehdipour, 2009. Prevalence of BRAFV600E Mutation in Iranian Patients with Papillary Thyroid Carcinoma: A Single-Center Study. Journal of Applied Sciences, 9: 3593-3597.

DOI: 10.3923/jas.2009.3593.3597

URL: https://scialert.net/abstract/?doi=jas.2009.3593.3597
 

INTRODUCTION

Thyroid carcinoma is the most common endocrine malignancy. Recently, Kilfoy et al. (2009), reported that the world wide incidence rate of thyroid carcinoma varies between 0.7 to 3.4 and 1.8 to 11.8 per 100,000 populations for men and women, respectively in 19 different populations from 1998 to 2002. The thyroid cancer prevalence in Iran has been reported 1.0 and 3.5 per 100,000 population for male and female, respectively and the most frequent morphologic variant is Papillary Thyroid Carcinoma (PTC) with a frequency of 69.9% (Larijani et al., 2005). The BRAF mutations have been found in a variety of human cancers, most notably in melanoma. T1799A transversion (formerly known as T1796A) in exon 15, which is the most common BRAF mutation, accounts for more than 80% of BRAF mutations (Davies et al., 2002). The T1799A mutation results in constitution of Glutamate (E) for Valine (V) (V600E).

This missense mutation alters the conformation of the activation loop in the BRAF kinase domain and activates its kinase activity by simulating phosphorylation. This activation leads to tumorogenesis through the RAS-RAF- MEK-ERK-MAP kinase pathway (Davies et al., 2002). Although, the BRAFV600E mutation is reported to have association with more aggressive tumor phenotype and a higher risk of recurrence and persistent disease in patients with conventional PTC (Kebebew et al., 2007; Xing et al., 2005; Kim et al., 2006; Nikiforova et al., 2003; Xu et al., 2003; Lee et al., 2007), the reports are controversial (Kim et al., 2005; Liu et al., 2005; Ito et al., 2009). Preoperative detection of PTCs by a specific molecular cancer maker, such as BRAF mutation, would specially be helpful for predicting disease aggressiveness while determining the need for and extent of surgery, as well as, the need for adjuvant therapy and follow-up monitoring (Kebebew et al., 2007). As to our knowledge, there is no published data reporting BRAFV600E mutation in Iranian population. The aim of this study was to analyze the prevalence of the BRAFV600E mutation in Iranian PTC patients and its applicability in the diagnosis of PTC.

MATERIALS AND METHODS

Thyroid samples: This study was conducted in Department of Medical Genetics, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran from 2007 through 2008. A total of 50 Formalin Fixed Paraffin Embedded (FFPE ) samples were obtained from patients who underwent thyroidectomy surgery from 2006 to 2007 in referral Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran. The tissue samples of total of 50 patients, consisting of 25 malignant tumors and 25 non-malignant samples were reviewed by a pathologist. Also, a total of 26 fresh thyroid tissues were obtained from the patients who underwent surgery in 2008 in referral Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran. Tissue specimens were put in RNAlater (Ambion, Austin, USA) and frozen immediately at -80°C until use. Final Histologic classification was obtained finally from pathologic reports. The samples included 5 malignant and 21 non-malignant tumors. This study was approved by the Ethics and Research Committee of Tehran University of Medical Sciences and was conducted in accordance with the Declaration of Helsinki principles.

Cell line samples, Laser Capture Microdissection (LCM) and DNA extraction: Previously extracted DNA of cell lines, ARO and 8305C (heterozygous for BRAFV600E mutation), 8505C (homozygous for BRAFV600E mutation) and finally TPC-1 (wild type BRAF gene), were used as positive (heterozygous and homozygous mutation) and negative controls, respectively. The 6 μM FFPE tissue sections were deparafinized and stained with Methyl Green (Sigma-Aldrich, St. Louis, MO) and then manually dissected or using laser capture microdissection system (Leica AS LMD, Wetzler, Germany). The DNA from dissected FFPE samples was extracted by using QIAamp DNA Micro Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. DNA from frozen tissues was extracted by conventional Phenol-Chloroform method. Quality and quantity of the extracted DNAs were determined by spectrophotometry (NanoDrop ND-1000, Wilmington, Delaware USA).

PCR-RFLP: We amplified BRAF exon 15 by PCR using oligonucleotide primer pairs: 5-TCA TGA AGA CCT CAC AGT AAA AAT-3 (forward) and 5-TGG ATC CAG ACA ACT GTT CAA-3 (reverse), as described previously by Takahashi et al. (2007). The PCR was carried out with the following conditions: initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 30 sec, annealing at 56°C for 30 sec and elongation at 72°C for 30 sec and a final extenstion at 72°C for 5 min. The PCR products were visualized on 8% polyacrylamid gels with Ethidium Bromide staining.

Then, the amplification products were digested by TspRI (New England Biolabs, Ipswich, MA) restriction endonuclease that cuts normal allele (ACA GTG AAA) but not the T1799A mutant sequence (ACA GAG AAA). The wild type BRAF fragment produced two 47 bp and 52 bp bands, while the mutant BRAF fragment was not digested and produced a 98 bp band. For confirmation of the results, we sequenced the PCR product in the 5 cell lines previously mentioned and some positive and negative mutant samples.

Statistical analysis: All the statistical analyses were performed by the SPSS software. Fisher exact test was used for association of BRAFV600E mutation with age and gender. The p-value of less than 0.05 considered to be significant. The True-Positive (TP), True-Negative (TN), False-Positive (FP) and False-Negative (FN) results were defined. Diagnosis sensitivity, specificity, Positive Predictive Value (PPV), Negative Predictive Value (NPV) and accuracy were calculated by using the following formulas:

Image for - Prevalence of BRAFV600E Mutation in Iranian Patients with Papillary Thyroid Carcinoma: A Single-Center Study

RESULTS

Overall, the prevalence of BRAFV600E mutation was 20 out of 30 (66.7%) in malignant thyroid carcinoma and 71.4% in PTCs. The prevalence of BRAFV600E mutation regarding to histologic classification of tumours was 72, 100 and 0% in Classic-Variant PTCs (CV-PTC), Tall-Cell variant PTCs (TC-PTC) and Follicular Variant PTC (FV-PTC), respectively (Table 1). It is worthy to note that in one TC-PTC patient the BRAFV600E mutation was present as homozygous. No BRAFV600E mutation was detected in the normal thyroid tissues surrounding the malignant tissue.

The age, sex, extrathyroid extension and lymph node metastases distribution of the patients with the malignant thyroid samples regarding to BRAFV600E mutation status are shown in Table 1.

Table 1: BRAFV600E mutation analysis of thyroid histology in 30 malignant and 46 benign thyroid tumors
Image for - Prevalence of BRAFV600E Mutation in Iranian Patients with Papillary Thyroid Carcinoma: A Single-Center Study
NS: Non significant, p>0.05

Table 2: Correlation between BRAFV600E mutation presence and malignancy detection in study results
Image for - Prevalence of BRAFV600E Mutation in Iranian Patients with Papillary Thyroid Carcinoma: A Single-Center Study
TP: True positive, TN: True negative, FP: False positive, FN: False negative, Sn: Sensitivity, Sp: Specificity, PPV: Positive predicted value, NPV: Negative predicted value

All the 46 benign specimens including 24 goiters, 15 follicular adenomas, 7 Hashimato’s thyroiditis, were BRAFV600E mutation negative. Diagnostic sensitivity, specificity, PPV, NPV and accuracy were 66.67, 100, 100, 82.18 and 86.84%, respectively (Table 2).

DISCUSSION

This study showed BRAFV600E mutation in 71.4% of PTC samples and it seems more common in TC-PTC (100%) comparing to CV-PTC (72%). Also there was no BRAFV600E mutation in benign thyroid lesions and in normal cells surrounding malignant tissues and this finding is consistent with other reports (Nikiforova et al., 2003; Xu et al., 2003; Xing et al., 2004).

The PTCs are usually curable with standard surgical and adjuvant radioiodine treatment but unfortunately lymph node metastases are found in 30 to 65% of cases at the time of initial diagnosis and 15% of tumors with lymph node metastases also display very aggressive behavior, characterized by local invasion, distant metastasis, treatment resistance and increased mortality (Mazzaferri and Kloos, 2001). The use of molecular markers for diagnosis and prognosis of thyroid cancers has been explored extensively. The V600E mutation in BRAF kinase appears to be an attractive molecular marker for thyroid cancer diagnosis and prognosis as it has been found to be the most common genetic events and specific for PTCs (Rowe et al., 2006). The aim of present study was to determine the prevalence of the BRAFV600E mutation in an Iranian population and its applicability for PTC detection.

This study showed that the BRAFV600Emutation was much more common in PTC patients of this study compared to western countries. As meta-analysis by Lee et al. (2007) most studies reported that BRAFV600E mutation is common in PTCs but the reported frequencies vary from 30 to 83% (mean 49%). Some investigators have suggested that diagnostic technical methods and geographic factors may account for the differences in reported prevalences for BRAFV600E mutation in PTCs (Kebebew et al., 2007).

Guan et al. (2009) recently reported that there is an association between BRAFV600E mutation and Iodine intake. According to their report BRAFV600E mutation was found in 69% of PTCs in high iodine content in natural drinking water but it was found only in 53% of PTCs in normal Iodine content in natural drinking water in China (Guan et al., 2009). Present results for BRAFV600E mutation prevalence in PTCs is more similar to those published for Asian (Korean and Chinese) populations (Guan et al., 2009; Kim et al., 2004). The higher prevalence of BRAFV600E mutation in high Iodine intake in China is in agreement with our results. In agreement to this study reports from Korea, a country with very high iodine intake, indicated that BRAFV600E mutation is highly prevalent (52-83%) in PTC (Kim et al., 2004, 2005; Chung et al., 2006). In contrast, the prevalence of BRAFV600E mutation in western countries, Such as Spain and Italy showed low prevalence (37-42%) (Lee et al., 2007). According to the results we can propose that the high prevalence of BRAFV600E mutation may be due to high Iodine intake in Iran.

The higher frequency of BRAFV600E mutation in the present study might be due to the probable increase in iodine intake after starting a mass programme of iodine supplementation implemented since 1983 in Iran (Larijani et al., 2005). However, this proposal requires definition by further studies. Although, we found that BRAFV600E mutation occurred slightly more in females, this was not statistically significant (p>0.05). The BRAFV600E mutation was observed in 73.3% of PTC patients older than 40 years old and in 69.2% of PTC patients younger than 40 years old. BRAFV600E mutation prevalence was slightly more common in older patients than in younger patients, but this was not also a significant difference (p>0.05). Present results is in agreement with the results of a meta-analysis study on 1168 PTC patients that showed there were not significant association between BRAFV600E mutation and age or gender (Lee et al., 2007).

The results of present research showed that there is not significant association between BRAFV600E and extrathyroidal extension or lymph node metastases that is in agreement with some previous reports (Lee et al., 2007; Ito et al., 2009).

In this study, we confirmed that BRAFV600E mutation was specific for PTC (100%), because no benign thyroid samples were found to harbor BRAFV600E mutation and all the samples that were positive for BRAFV600E mutation were malignant. This result is consistent with previous reports (Rowe et al., 2006; Xing, 2005). In the present study, the diagnostic sensitivity and specificity were 66.67 and 100%, respectively. Chung et al. (2006) reported the sensitivity of 83.0% and a specificity of 96.0% for direct DNA sequencing method and the sensitivity of 79.6% and a specificity of 80.0% for PCR-RFLP method for detection of PTC in Fine Needle Aspiration Biopsy (FNAB) samples.

High specificity in our study indicates the advantage of BRAFV600E mutation for PTCs detection. However, the method based on PCR-RFLP is enough sensitive to detect BRAFV600E mutation even in the samples of FFPE tissue and when the DNA yield was low (Kumagai et al., 2007). The lower sensitivity might be explained by the fact that diagnostic sensitivity of BRAFV600E mutation strictly depends upon its prevalence in PTC patients.

In conclusion, these findings might pave our way towards considering the BRAFV600E mutation in PTCs in the regions with high prevalence of this alteration as a molecular marker.

ACKNOWLEDGMENT

We want to thank K. Hamatani (Hiroshima, Japan) for the donating of cell lines DNA and core facilities to perform microdisection of FFPE samples.

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