Diagnostic value of BRAF^V600E-mutation analysis in fine-needle aspiration of thyroid nodules: a meta-analysis

Introduction

Thyroid cancer is the most common endocrine malignancy, with favorable outcome after early detection and treatment.^1,2 Fine-needle aspiration (FNA) guided by ultrasound is a routine and reliable approach for preoperative evaluation of thyroid nodules. Approximately 10%–40% of FNA specimens yield indeterminate results, and the majority of them turn out to be benign after diagnostic surgery, and thus a sizable portion of indeterminate specimens lead to unnecessary thyroidectomy.^3–7 The Bethesda System for Reporting Thyroid Cytopathology divides indeterminate nodules into three subgroups: atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS), follicular neoplasm/suspicious for follicular neoplasm (FN/SFN), and suspicious for malignant cells (SMC).⁸ The indeterminate thyroid nodule is the most intractable problem in clinical management, which highlights the urgency to develop effective ancillary testing to identify cancerous nodules for timely and appropriate management.

Great progress has been achieved in the understanding of molecular mechanisms of thyroid cancer, and various mutations have been identified in the early stage of thyroid cancer, such as BRAF, RAS, PI3K, and PTEN.⁹ These genetic alterations are excellent candidates for disease hallmarks, since 60%–70% of thyroid cancers harbor at least one genetic mutation.⁹ The BRAF^V600E mutation appears to be the most promising biomarker specific for papillary thyroid cancer (PTC),⁹ which aberrantly activates the tumor-initiating MAPK pathway and drives the carcinogenesis and progression of thyroid cancer.^9,10

Whether BRAF^V600E analysis could be routinely used in clinical practice is still controversial. Numerous researchers have proved that BRAF^V600E-mutation testing is an effective diagnostic approach for thyroid FNA,¹¹ while others believe that its utility is limited by low prevalence of BRAF^V600E mutation in indeterminate nodules.¹² Therefore, we conducted a structured meta-analysis to estimate the additional diagnostic yield of BRAF^V600E-mutation analysis in thyroid FNA, and further evaluated the malignancy rate, BRAF^V600E-mutation frequency, and diagnostic value of BRAF^V600E testing in different categories of indeterminate nodule.

Materials and methods

Search strategy and selection criteria

Systematic searches were performed in the PubMed, Web of Science, Scopus, Ovid, Elsevier, and Cochrane Library databases for relevant articles prior to June 2015. The search terms were: ([thyroid cancer] or [thyroid neoplasm] or [thyroid tumor]), (BRAF), and ([FNA] or [fine needle aspiration]). The references of available articles were also reviewed. Study selection consisted of initial screening of titles or abstracts and second screening of full texts. Studies were included if they met the following criteria: 1) research article rather than review, system review, case report, editorial, or comments; 2) the material for BRAF^V600E-mutation analysis was obtained by FNA; 3) the final diagnosis was based on a definite gold standard, such as surgical histology, unequivocal histocytopathology, or reliable clinical follow-up; 4) the data were available to construct 2×2 tables or analyze malignancy rate or BRAF^V600E-mutation prevalence.

Data extraction and quality assessment

The following items were extracted: study by author name(s), country, number of centers, enrollment period, study design, mean age of patients, mean diameter of nodules, reference standard of final diagnosis, and genotyping method. Most research classified cytological results according to the Bethesda system⁸ or the British Thyroid Association,^13,14 as shown in Table 1. In this meta-analysis, FNA cases classified as AUS/FLUS (Thy3a) and FN/SFN (Thy3f) were regarded as cytologically negative and lesions diagnosed as SMC (Thy4) were cytologically positive. Final diagnosis was based on histopathologic examination after surgery or a combination of cytological examination and clinical follow-up. Then, patient numbers for true-positive, false-positive, false-negative, and true-negative results were extracted to construct the 2×2 tables.

Table 1 Comparison between the British and Bethesda systems for classification of thyroid cytopathology

The methodological quality of studies eligible for diagnostic analysis of FNA cytology and/or BRAF^V600E testing was assessed according to the Quality Assessment of Diagnostic Studies 2, which comprises four domains: patient selection, index test, reference standard, and flow and timing.¹⁵ A series of questions was used to judge the risk of bias and applicability concerns as low, high, or unclear risk.

Statistical analysis

The threshold effect was calculated by the Spearman correlation coefficient, and P<0.05 indicated the existence of a threshold effect. Nonthreshold heterogeneity was assessed by the Cochran Q test and inconsistency index (I²). I²>50% suggested significant heterogeneity, and a random-effect model (DerSimonian–Laird method) was chosen.^16,17 Metaregression analysis was used to identify the possible sources of nonthreshold heterogeneity. The following covariates were considered in the metaregression analysis: country, number of centers (single or multiple), sample size (<100, 100–500, 500–1,000, or >1,000), study design (prospective or retrospective), reference standard (histology or cytology plus clinical follow-up), and genotyping method. If P<0.05, the covariate was to be regarded as the source of nonthreshold heterogeneity.

The pooled sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic odds ratio (DOR) with 95% confidence interval (CI) were computed to estimate diagnostic accuracy. DOR combined the data of sensitivity and specificity into a single indicator ranging from 0 to infinity, reflecting the discriminatory performance of testing. The summary receiver-operating characteristic (SROC) curve was a mathematical model for the plot of sensitivity (1 – specificity). The Q index indicated the point at which sensitivity was equal to specificity. The areas under the SROC curve (AUCs) calculated the inherent capacity of the diagnostic test. If the AUC closed to 1, the diagnostic method was thought to be perfect.

The threshold effect, pooled diagnostic features, and metaregression were calculated by Meta-Disc (version 1.4; Ramony Cajal Hospital, Madrid, Spain). Pooled rates of malignancy and BRAF^V600E mutation were calculated by R statistical software (version 3.2.1; R Foundation for Statistical Computing, Vienna, Austria). Quality assessment was conducted using Review Manager (version 5.2; Cochrane Collaboration). P<0.05 was considered statistically significant.

Results

Search results and quality assessment

The search process is shown in Figure 1. A total of 1,261 articles were initially identified, and 1,130 of these were excluded after reviewing titles and abstracts. The remaining 131 articles were investigated in detail. In accordance with the selection criteria mentioned in the Materials and methods section, 43 articles were excluded after reading the full texts. Finally, 88 studies published from 2004 to 2015 were included in this meta-analysis. Among these, 51 studies were included in the analysis of diagnostic accuracy, and at the same time 37 studies and 62 studies were available for analysis of malignancy rate and BRAF^V600E-mutation rate, respectively.

Figure 1 Flowchart of study-selection process. Abbreviation: FNA, fine-needle aspiration.

The characteristics of studies eligible for diagnostic analysis of FNA cytology and BRAF^V600E testing are summarized in Table 2. As shown in Figure 2, about a third of studies had a high risk of bias in patient selection, because 14 of them did not enroll the samples consecutively or at random and eleven excluded a number of patients inappropriately. Twelve studies did not receive the same reference standard, since some patients were diagnosed by histopathology and others by FNA cytology plus clinical follow-up. Also, 17 studies did not include all patients, due to the unsatisfactory FNA or failure of BRAF^V600E testing. As a result, nearly half of the studies harbored a high risk of bias in flow and timing. Fortunately, the risk of bias in the index test and reference standard was relatively low.

Table 2 Characteristics of studies eligible for the diagnostic analysis of FNA cytology and BRAFV600E testing Notes: aRetrospective; bprospective; A, histopathologic examination after surgery; B, combination of cytological examination and clinical follow-up. Abbreviations: PCR-RFLP, polymerase chain reaction–restriction fragment-length polymorphism; AS, allele-specific; MASA, mutant allele-specific amplification; LCPCR, LightCycler PCR; FMCA, fluorescent melting-curve analysis; SSCP, single-strand conformational polymorphism; DHPLC, denaturing high-performance liquid chromatography; DPO, dual-priming oligonucleotide; MEMO, 3′-modified oligonucleotide; PRC, People’s Republic of China; FNA, fine-needle aspiration; –, data not available.

Figure 2 Methodological quality of studies included, assessed by the Quality Assessment of Diagnostic Studies 2 criteria.

Synthesis of analysis results

Diagnostic value of FNA cytology, BRAF^V600E-mutation analysis, and combined strategy in all the thyroid FNA specimens

Spearman correlation coefficients for FNA cytology, BRAF^V600E testing and combined strategy were 0.032 (P=0.826), 0.254 (P=0.078), and 0.064 (P=0.661), respectively; therefore, no threshold effect existed in the analysis. However, there was substantial nonthreshold heterogeneity (I²>50%, P<0.05), so the random-effect model was chosen to pool the diagnostic features. A total of 51 studies were included in this part of the analysis,^18–68 but one was excluded because it had no false-positive or true-negative case to calculate the diagnostic index (Table 3).⁶⁸

Table 3 Diagnostic analysis of FNA cytological examination and BRAFV600E-mutation analysis in all the FNA specimens Abbreviations: FNA, fine-needle aspiration; TP, true positive; FP, false positive; FN, false negative; TN, true negative; –, data not available.

Based on the feasible FNA cytology results from 50 studies, pooled sensitivity, specificity, PLR, NLR, and DOR were 0.814 (95% CI 0.803–0.824), 0.981 (95% CI 0.978–0.985), 23.868 (95% CI 14.139–40.293), 0.216 (95% CI 0.172–0.273), and 127.73 (95% CI 75.082–217.28) (Table 4). The AUC of the SROC curve was 0.9551 (standard error [SE] 0.0127), with a Q-value of 0.8975 (SE 0.0178) (Figure 3A). Data for the BRAF^V600E-mutation test were unavailable in one study,⁴⁵ and 49 studies with 9,361 patients were finally analyzed. Pooled sensitivity, specificity, PLR, NLR, and DOR were 0.619 (95% CI 0.605–0.633), 0.997 (95% CI 0.995–0.998), 34.982 (95% CI 23.801–51.415), 0.433 (95% CI 0.384–0.489), and 96.570 (95% CI 63.932–145.87) (Table 4). The AUC of the SROC was 0.9207 (SE 0.0233), with a Q-value of 0.8542 (SE 0.0268) (Figure 3B). Also, the positive predictive value of BRAF^V600E testing was 99.5% (2,886 of 2,900). After BRAF^V600E analysis was combined with FNA cytology, sensitivity increased to 0.874 (95% CI 0.865–0.884), the DOR and AUC improved to 187.92 (95% CI 110.24–320.35) and 0.9744 (SE 0.0062), respectively, with a Q-value of 0.9271 (SE 0.0107) (Table 4, Figure 3C). The synergism between FNA cytology and BRAF^V600E testing also decreased the false-negative rate from 8% in FNA cytology to 5.2%, but increased the false-positive rate from 3% to 5% at the same time.

Table 4 Results of meta-analysis for diagnostic value of FNA cytology, BRAFV600E-mutation analysis, and the combined strategy in all FNA specimens Note: *The Q index indicates the point at which sensitivity is equal to specificity. Abbreviations: FNA, fine-needle aspiration; CI, confidence interval; LR, likelihood ratio; DOR, diagnostic odds ratio; SROC, summary receiver-operating characteristic; AUC, area under the curve.

Figure 3 Summary receiver-operating characteristic (SROC) curve and area under the curve (AUC). Notes: FNA cytology (A), BRAFV600E-mutation analysis (B), and combination of BRAFV600E mutation and FNA cytology (C). *The Q index indicates the point at which sensitivity is equal to specificity. Abbreviations: FNA, fine-needle aspiration; SE, standard error.

Diagnostic value of BRAF^V600E-mutation analysis in indeterminate cases (Bethesda categories III–V)

There were 43 studies included in the diagnostic analysis of BRAF^V600E testing in the indeterminate thyroid nodules (Table 5).^{18,19,21,22,24,26–37,40,42–44,46–48,50–54,57–62,64–67,69–72} Our data showed that 23% of indeterminate nodules harbored the BRAF^V600E mutation. No threshold effect was detected, so the random-effect model was chosen to pool the diagnostic features: sensitivity 0.442 (95% CI 0.416–0.468), specificity 0.997 (95% CI 0.994–0.999), PLR 12.267 (95% CI 8.175–18.406), NLR 0.613 (95% CI 0.551–0.683), and DOR 23.939 (95% CI 15.388–37.242) (Table 6; Figure 4A and B). The AUC of the SROC was 0.8711 (SE 0.0414), with a Q-value of 0.8015 (SE 0.0410) (Figure 4C).

Table 5 Diagnostic analysis of BRAFV600E-mutation analysis for indeterminate cases Abbreviations: TP, true positive; FP, false positive; FN, false negative; TN, true negative.

Table 6 Results of meta-analysis for diagnostic value of BRAFV600E mutation in indeterminate cases Note: *The Q index indicates the point at which sensitivity is equal to specificity. “–’’ indicates the AUC of the SROC was not significant in FN/SFN cases, since the lower limit of the AUC was less than 0.5. Abbreviations: FNA, fine-needle aspiration; CI, confidence interval; LR, likelihood ratio; DOR, diagnostic odds ratio; SROC, summary receiver-operating characteristic; AUC, area under the curve.

Figure 4 Forest plots. Notes: Sensitivity (A), specificity (B), and summary receiver-operating characteristic (SROC) curve and area under the curve (AUC) (C) of BRAFV600E-mutation analysis in cases classified as indeterminate by FNA cytology. *The Q index indicates the point at which sensitivity is equal to specificity. Abbreviations: FNA, fine-needle aspiration; CI, confidence interval; SE, standard error.

To evaluate the diagnostic value of BRAF^V600E testing in different categories of indeterminate nodules, we separated the indeterminate cases into three different and more specific categories according to the Bethesda system. Studies with sample sizes fewer than ten were excluded to avoid potential bias. The malignancy rates of FN/SFN and AUS/FLUS were 30.55% and 34.99%, while 90.35% of SMC cases turned out to be malignant (Table 7). Besides that, the BRAF^V600E-mutation rate varied among these groups: it existed in 43.2% of SMC cases, but only 13.77% in AUS/FLUS and 4.43% in FN/SFN patients (Table 7). Furthermore, the sensitivity of BRAF^V600E testing was higher in SMC (0.594, 95% CI 0.556–0.631) than AUS/FLUS (0.401, 95% CI 0.328–0.477) and FN/SFN (0.195, 95% CI 0.128–0.278), while specificity was higher in the AUS/FLUS (0.995, 95% CI 0.982–0.999) and FN/SFN (0.997, 95% CI 0.983–1.000) groups than the SMC group (0.861, 95% CI 0.784–0.918) (Table 6). The AUC of the SROC was 0.7674 (SE 0.0564) with a Q-value of 0.7079 (SE 0.0474) in the SMC group, and 0.7999 (SE 0.0897) with a Q-value of 0.7358 (SE 0.0783) in the AUS/FLUS group, but was not significant in FN/SFN cases, since the lower limit of the AUC was less than 0.5 (Figure 5).

Table 7 Malignancy rate and BRAFV600E-mutation prevalence in three categories of indeterminate cases Abbreviations: CI, confidence interval; SMC, suspicious for malignant cells; FN/SFN, follicular neoplasm/suspicious for FN; AUS/FLUS, atypia of undetermined significance/follicular lesion of undetermined significance.

Figure 5 Summary receiver-operating characteristic (SROC) curve and area under the curve (AUC) of SMC cases (A), AUS/FLUS cases (B) and FN/SFN cases (C). Note: *The Q index indicates the point at which sensitivity is equal to specificity. Abbreviations: SMC, suspicious for malignant cells; AUS/FLUS, atypia of undetermined significance/follicular lesion of undetermined significance; FN/SFN, follicular neoplasm/suspicious for FN; SE, standard error.

Heterogeneity test

Heterogeneity was present in our meta-analysis, and Spearman correlation coefficients suggested no significant threshold effect. To explore sources of heterogeneity, we assessed multiple variables by metaregression, including country, number of centers, sample size, study design, reference standard, and genotyping method. The results indicated that country and sample size were possible sources of heterogeneity (data not shown). Other covariates that may have caused heterogeneity, such as enrollment period, age, sex, nodule diameter, size of needle, use of blinding method, and differences in operating protocol, were not analyzed here, due to the loss of partial data.

Discussion

Thyroid cancer is on a rapid increase these days, partially due to advancing diagnostic methods. The majority of cases have an excellent prognosis, with 30-year survival rate exceeding 90% after thyroidectomy and/or radioiodine ablation.² Preoperative diagnosis is of indisputable value in distinguishing thyroid cancer from benign nodules. FNA biopsy is a conventional technique to identify malignant thyroid nodules preoperatively and effectively, which has also been demonstrated in our meta-analysis. However, the extensive use of this approach is influenced by its inherent limitations, such as size or location of nodule, quantity and quality of obtained material, technical skill of the cytopathologist, and the overlap of cytomorphological features between malignant and benign nodules. Therefore, a fraction of cases are classified as nondiagnostic or indeterminate, and about 15%–30% of them get malignant pathology after diagnostic surgery.^8,73 Since the occurrence of malignancy is too high for just watchful waiting, numerous patients with indeterminate diagnosis accept unnecessary surgical intervention. BRAF^V600E mutation is the most promising marker for thyroid nodules. A similar meta-analysis conducted by Jia et al of 16 studies suggested that BRAF^V600E analysis had diagnostic value in indeterminate thyroid nodules,¹¹ but another analysis of eight eligible studies found a low BRAF^V600E-mutation rate within indeterminate cases, and thus the value of BRAF^V600E-mutation testing remains controversial.¹² However, the number of studies these two analyses included was limited, and did not systematically stratify the indeterminate categories. Therefore, we designed a more comprehensive meta-analysis to evaluate the diagnostic yield of BRAF^V600E analysis in thyroid FNA, especially those specific categories of indeterminate cases.

Consistent with previous research, our meta-analysis showed that BRAF^V600E analysis had high specificity and positive predictive value. As a rule-in test, a positive result of BRAF^V600E analysis indicates a high probability of malignancy so that therapeutic surgery is recommended, but the negative result cannot exclude malignancy, and further evaluations, such as follow-up ultrasound or repeat FNA, are needed. When we combined BRAF^V600E-mutation testing with FNA cytological examination, sensitivity increased by 6% and the false-negative rate decreased from 8% to 5.2%, while the false-positive rate increased from 3% to 5% at the same time. However, BRAF^V600E testing had relatively low sensitivity of 44.2% in the indeterminate group. Also, the yield and usefulness of BRAF^V600E analysis can be greatly varied with the prevalence of BRAF^V600E mutation in different subcategories of indeterminate nodules. BRAF^V600E mutation was present in 43.2% of SMC cases regarded as cytologically positive in our meta-analysis, but only 13.77% in AUS/FLUS and 4.43% in FN/SFN cases. Therefore, it was reasonable that BRAF^V600E analysis did best in SMC lesions (sensitivity 59.4%, specificity 86.1%) and also had certain diagnostic value in AUS/FLUS nodules (sensitivity 40.1%, specificity 99.5%), but no significant benefit in the FN/SFN group, which needs other diagnostic approaches with high sensitivity.

BRAF^V600E mutation is specific to PTC or anaplastic thyroid cancer arising from PTC, and more common in conventional and tall-cell PTC than follicular-variant PTC (FVPTC), which results in the discrepancy of BRAF^V600E test in different indeterminate subgroups. The FN/SFN category is mainly constituted of FVPTC, follicular thyroid cancer (FTC), adenomatoid hyperplasia, and follicular adenoma,⁷⁴ which harbors low prevalence of BRAF^V600E mutation and is hard for BRAF^V600E testing to determine malignancy, so FVPTC and FTC may be the main source of false-negative results. The molecular profiles of FVPTC and FTC are similar, with frequent RAS and rare BRAF mutation.^75,76 RAS mutation, mutually exclusive with BRAF mutation, is the most frequent genetic mutation in indeterminate nodules, and provides important diagnostic information for BRAF^V600E-negative nodules.^69,77 An et al reported that single RAS-mutation analysis had a sensitivity of 93.3% and specificity of 75.0% in indeterminate nodules, and the combination of RAS and BRAF mutation provided additional diagnosis value for 60%–70% indeterminate thyroid nodules.⁷⁸ Other genetic alterations, such as RET/PTC and PAX8/PPARG, also contribute to the definite diagnosis of indeterminate nodules.^69,79,80 Therefore, an expanded panel can be more effective, which is also recommended by the revised American Thyroid Association management guidelines.⁷³ As some mutations also present in benign nodules, the accompanying increase in false-positive rate should not be neglected. For instance, RAS mutation and PAX8/PPARG translocation are also found in follicular adenoma.^79,81 Additionally, some thyroid cancer does not have definitive molecular mutation, and other efficient rule-out testing with high negative predictive value should be explored.

The clinical management decision is directly based on the malignant risk, ranging from repeat FNA to diagnostic lobectomy to total thyroidectomy. Uncertain diagnosis may lead to delayed treatment or unnecessary intervention. Based on the Bethesda classification, malignancy rates for FN/SFN and SMC nodules are 15%–30% and 60%–75%, respectively, and are much more variable in AUS/FLUS cases (7%–48%).⁸ In our analysis, the malignancy rate of the SMC group was higher than that recorded in the Bethesda classification, and this discrepancy might have resulted from continuous improvement in FNA technique, since the data for the Bethesda system were collected several years ago. BRAF^V600E mutation is a strong indicator for malignancy, and total thyroidectomy should be proposed as the first-line treatment for BRAF^V600E-positive nodules to decrease the recurrence and avoid complications caused by standard two-stage surgery. Nevertheless, BRAF^V600E testing is relatively insufficient for AUS/FLUS and even has no effect in FN/SFN patients, due to the low prevalence of BRAF^V600E mutation, but their malignant occurrence (30.55% and 34.99%) was too high to perform clinical observation. Other approaches, such as core-needle biopsy and immunohistochemistry, are also required to confidently guide the management. Several multicenter studies have reported that BRAF^V600E mutation is associated with aggressive clinicopathological characteristics and predicts recurrence and mortality for PTC patients.^82–89 Therefore, more aggressive surgery, such as prophylactic central lymph-node dissection and closer follow-up, should be considered in the management of BRAF^V600E-positive thyroid cancer.

Despite its achievements, our meta-analysis had several limitations. Firstly, there was significant nonthreshold heterogeneity, partly caused by country and sample size of different studies, but other possible covariates were unable to be analyzed due to the paucity of data. The heterogeneity from country may be due to the different BRAF^V600E prevalence in worldwide populations, eg, it is up to 80% in South Korea, which is much higher than other regions.²⁴ Secondly, about a third of the studies had a high risk of bias in patient selection, and nearly half had a high risk of bias in flow and timing, which may affect the reliability of our results.

Conclusion

This meta-analysis demonstrated that BRAF^V600E analysis using residual material obtained from routine FNA could improve diagnostic accuracy and reduce false-negative rates. Besides, BRAF^V600E analysis had certain diagnostic value in SMC and AUS/FLUS cases, especially the SMC group, selecting cases with high malignancy possibility and guiding intraoperative or postoperative management, though its value in FN/SFN cases was doubtful, and expanded panels containing other diagnostic markers are recommended. Therefore, more studies of high quality are needed to balance the advantages and disadvantages of BRAF^V600E testing for patients and to select the most suitable population for this diagnostic method.

Acknowledgments

This study was supported by grants from the National Natural Science Foundation of China (81202141 and 81272676), Key Project of Scientific and Technological Innovation of Hangzhou (20131813A08), National Science and Technology Major Project of the Ministry of Science and Technology of China (2013ZX09506015), and Medical Science and Technology Project of Zhejiang Province (2011ZDA009).

Disclosure

The authors report no conflicts of interest in this work.

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