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Diagnostic value of BRAFV600E-mutation analysis in fine-needle aspiration of thyroid nodules: a meta-analysis This article was published in the following Dove Press journal: OncoTargets and Therapy 27 April 2016 Number of times this article has been viewed
Xingyun Su 1 Xiaoxia Jiang 1 Xin Xu 1 Weibin Wang 1 Xiaodong Teng 2 Anwen Shao 3 Lisong Teng 1 Department of Surgical Oncology, Department of Pathology, First Affiliated Hospital, 3Department of Neurosurgery, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, People’s Republic of China 1 2
Abstract: Fine-needle aspiration (FNA) is a reliable method for preoperative diagnosis of thyroid nodules; however, about 10%–40% nodules are classified as indeterminate. The BRAFV600E mutation is the most promising marker for thyroid FNA. This meta-analysis was conducted to investigate the diagnostic value of BRAFV600E analysis in thyroid FNA, especially the indeterminate cases. Systematic searches were performed in PubMed, Web of Science, Scopus, Ovid, Elsevier, and the Cochrane Library databases for relevant studies prior to June 2015, and a total of 88 studies were ultimately included in this meta-analysis. Compared with FNA cytology, the synergism of BRAFV600E testing increased the diagnostic sensitivity from 81.4% to 87.4% and decreased the false-negative rate from 8% to 5.2%. In the indeterminate group, the mutation rate of BRAFV600E was 23% and varied in different subcategories (43.2% in suspicious for malignant cells [SMC], 13.77% in atypia of undetermined significance/follicular lesion of undetermined significance [AUS/FLUS], and 4.43% in follicular neoplasm/suspicious for follicular neoplasm [FN/SFN]). The sensitivity of BRAFV600E analysis was higher in SMC than that in AUS/FLUS and FN/SFN cases (59.4% vs 40.1% vs 19.5% respectively), while specificity was opposite (86.1% vs 99.5% vs 99.7% respectively). The areas under the summary receiver-operating characteristic curve also confirmed the diagnostic value of BRAFV600E testing in SMC and AUS/FLUS rather than FN/SFN cases. Therefore, BRAFV600E analysis can improve the diagnostic accuracy of thyroid FNA, especially indeterminate cases classified as SMC, and select malignancy to guide the extent of surgery. Keywords: thyroid cancer, fine-needle aspiration, BRAFV600E mutation, meta-analysis
Introduction
Correspondence: Lisong Teng Department of Surgical Oncology, First Affiliated Hospital, School of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou, Zhejiang 310003, People’s Republic of China Tel +86 571 8706 8873 Fax +86 571 8723 6628 Email [email protected]
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. 2495
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http://dx.doi.org/10.2147/OTT.S101800
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Su et al
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 BRAFV600E 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 BRAFV600E analysis could be routinely used in clinical practice is still controversial. Numerous researchers have proved that BRAFV600E-mutation testing is an effective diagnostic approach for thyroid FNA,11 while others believe that its utility is limited by low prevalence of BRAFV600E mutation in indeterminate nodules.12 Therefore, we conducted a structured meta-analysis to estimate the additional diagnostic yield of BRAFV600E-mutation analysis in thyroid FNA, and further evaluated the malignancy rate, BRAFV600E-mutation frequency, and diagnostic value of BRAFV600E 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 BRAFV600E-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 BRAFV600E-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
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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. The methodological quality of studies eligible for diagnostic analysis of FNA cytology and/or BRAFV600E 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 Table 1 Comparison between the British and Bethesda systems for classification of thyroid cytopathology Bethesda
British
Nondiagnostic or unsatisfactory Benign AUS/FLUS (atypia of undetermined significance/follicular lesion of undetermined significance) FN/SFN (follicular neoplasm/ suspicious for follicular neoplasm) SMC (suspicious for malignancy) Malignant
Thy1 (nondiagnostic) Thy2 (nonneoplastic) Thy3a (neoplasm possible, atypia/nondiagnostic) Thy3f (neoplasm possible, suggesting follicular neoplasm) Thy4 (suspicious of malignancy) Thy5 (malignant)
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BRAFV600E analysis in thyroid FNA
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 BRAFV600E 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 BRAFV600E-mutation rate, respectively. The characteristics of studies eligible for diagnostic analysis of FNA cytology and BRAFV600E 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 BRAFV600E 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.
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Cohen et al18 Xing et al19 Domingues et al20 Pizzolanti et al21 Sapio et al22 Sapio et al23 Kim et al24 Bentz et al25 Jo et al26 Marchetti et al27 Nikiforov et al28 Zatelli et al29 Cantara et al30 Girlando et al31 Kim et al32 Kwak et al33 Moses et al34 Musholt et al35 Adeniran et al36 Kim et al37 Lee et al38 Moon et al39 Pelizzo et al40 Smith et al41 Yeo et al42 Cañadas-Garre et al43 Kang et al44 Kwak et al45 Lee et al46 Mancini et al47 Rossi et al48 Tomei et al49 Brahma et al50 Di Benedetto et al51 Koh et al52 Park et al53 Beaudenon-Huibregtse et al54 Crescenzi et al55 Eszlinger et al56
Study USA USA Portugal Italy Italy Italy South Korea USA South Korea Italy USA Italy Italy Italy South Korea South Korea USA Germany USA South Korea South Korea South Korea Italy USA South Korea Spain South Korea South Korea South Korea Italy Italy Italy Indonesia Italy South Korea South Korea USA Italy Germany
Country 1 1 1 1 2 2 1 1 1 1 2 1 1 1 1 1 1 6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 1 1 1 5 1 1
Centers, n Jan 2001–Jan 2003 – – Sep 2005–Jun 2006 – – Aug 2005–Jul 2006 1994–2004 June 2006–Dec 2006 1996–2008 – Oct 2008–Dec 2009 – – – Mar 2008–Jun 2008 Jun 2006–Jul 2008 Jan 2008–Jul 2009 Sep 2009–Nov 2010 Mar 2007–Feb 2009 July 2007–Dec 2009 Sep 2008–May 2009 Oct 2008–Sep 2009 – Jul 2009–Jan 2010 Jun 2006–Dec 2009 Apr 2008–Jul 2009 Jun 2009–Oct 2010 Aug 2008–Mar 2011 – – – Aug 2010–Jun 2011 – Jan 2009–Oct 2010 Jan 2011–May 2011 Jul 2010–Oct 2012 – 1995–2009
Enrollment period Retroa Prob Retro Pro Retro Retro Retro Retro Pro Retro Pro Pro Pro Pro Pro Retro Pro Pro Pro Pro Pro Retro Pro Retro Pro Pro Pro Retro Pro Pro Pro Retro Pro Pro Pro Retro Pro Pro Retro
Design – – – – – – – 40.9 – – – 50.7 51.2 – 50.6 45.6 51 – 52.6 – 50.3 49.4 47.8 – 51.27 49.8 – 48 49.5 55.1 52 – 46 – 48.6 – – – –
Mean age, years
Table 2 Characteristics of studies eligible for the diagnostic analysis of FNA cytology and BRAFV600E testing
– – – – – – – – 1 – – 1.1 – – 1.29 1.17 – – – – 1.46 0.95 – – 1.3 – – 0.92 – 2.38 – – .1 – 1.05 – – – –
Mean diameter, cm A B A A B B A A A A B A A A A A B A A A A B A A B A A A A A B A A A B B A A A
Final diagnosis
Direct sequencing + mutector assay Direct sequencing + colorimetric method PCR-RFLP Real-time AS-PCR Direct sequencing MASA Pyrosequencing LCPCR + FMCA Pyrosequencing Direct sequencing LCPCR + FMCA Direct sequencing DHPLC + direct sequencing Direct sequencing DPO-based multiplex PCR + direct sequencing DPO-based multiplex PCR Direct sequencing Direct sequencing SSCP analysis Pyrosequencing Pyrosequencing Direct sequencing Direct sequencing + MASA MCA Pyrosequencing PCR-RFLP AS-PCR + direct sequencing DPO-PCR + real-time PCR MEMO-PCR + direct sequencing High-resolution melting analysis Direct sequencing Pyrosequencing PCR-RFLP Direct sequencing DPO-PCR Real-time PCR + pyrosequencing Multiplex PCR Real-time sequencing High-resolution melting PCR + pyrosequencing
Genotyping method
Su et al Dovepress
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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.
– – – – –
Guo et al57 Johnson et al58 Liu et al59 Seo et al60 Seo et al61 Wan et al62 Zeck et al63 Eszlinger et al64 Krane et al65 Park et al66 Shi et al67
PRC UK PRC South Korea South Korea PRC USA Italy Germany South Korea USA
1 1 1 1 1 1 1 1 1 1 1
Nov 2010–Jul 2011 Sep 2011–Oct 2012 Sep 2012–Dec 2013 Dec 2010–Jan 2011 Dec 2010–Feb 2012 Mar 2013–Sep 2013 Apr 2011–Jan 2013 1995–2009 May 2011–Mar 2012 Jul 2011–Mar 2012 Jan 2011–Feb 2013
Pro Retro Pro Pro Pro Pro Pro Retro Pro Pro Retro
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BRAFV600E analysis in thyroid FNA
Synthesis of analysis results
Diagnostic value of FNA cytology, BRAFV600E-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 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 BRAFV600E-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 BRAFV600E testing was 99.5% (2,886 of 2,900). After BRAFV600E 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 BRAFV600E 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.
Diagnostic value of BRAFV600E-mutation analysis in indeterminate cases (Bethesda categories III–V) There were 43 studies included in the diagnostic analysis of BRAFV600E 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 BRAFV600E mutation. No threshold effect was detected, so the random-effect model was chosen to pool the diagnostic
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Table 3 Diagnostic analysis of FNA cytological examination and BRAFV600E-mutation analysis in all the FNA specimens Study
Year
Cohen et al18 Xing et al19 Domingues et al20 Pizzolanti et al21 Sapio et al22 Sapio et al23 Kim et al24 Bentz et al25 Jo et al26 Marchetti et al27 Nikiforov et al28 Zatelli et al29 Cantara et al30 Girlando et al31 Kim et al32 Kwak et al33 Moses et al34 Musholt et al35 Adeniran et al36 Kim et al37 Lee et al38 Moon et al39 Pelizzo et al40 Smith et al41 Yeo et al42 Cañadas-Garre et al43 Kang et al44 Kwak et al45 Lee et al46 Mancini et al47 Marchetti et al68 Rossi et al48 Tomei et al49 Brahma et al50 Di Benedetto et al51 Koh et al52 Park et al53 Beaudenon-Huibregtse et al54 Crescenzi et al55 Eszlinger et al56 Guo et al57 Johnson et al58
2004 2004 2005 2007 2007 2007 2008 2009 2009 2009 2009 2009 2010 2010 2010 2010 2010 2010 2011 2011 2011 2011 2011 2011 2011 2012 2012 2012 2012 2012 2012 2012 2012 2013 2013 2013 2013 2014 2014 2014 2014 2014
FNA
FNA + BRAF
BRAF
TP
FP
FN
TN
TP
FP
FN
TN
TP
FP
FN
TN
25 10 10 13 24 6 60 22 30 88 27 66 46 38 251 108 71 19 47 146 127 98 133 10 183 12 289 318 382 13 85 159 44 23 15 277 71 36 20 57 55 31
0 0 0 0 23 0 0 0 0 2 2 5 8 0 2 10 13 13 0 0 0 0 5 0 1 0 1 0 1 1 0 3 0 0 1 0 5 4 0 0 1 3
34 19 3 4 2 2 21 18 9 4 21 24 16 22 6 1 30 11 13 27 70 10 6 5 9 31 15 33 47 10 5 73 5 26 3 27 8 18 1 28 8 19
32 12 11 32 95 67 22 5 58 17 36 373 112 2 690 10 337 50 12 21 29 191 117 5 709 132 8 86 33 32 0 1,621 38 21 239 194 31 49 9 225 19 44
23 8 3 11 10 4 63 17 30 59 18 48 33 41 221 87 23 9 40 154 174 57 98 10 99 17 226 – 342 12 63 114 28 17 13 176 44 21 8 22 41 16
0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 0 0 0 0 1 1 0 0 0 0 0 2 – 0 0 0 0 0 0 0 3 1 0 0 0 0 0
36 22 10 6 16 4 18 20 10 32 30 42 45 19 47 22 78 21 20 19 24 51 59 5 93 31 78 – 87 11 22 172 21 32 5 141 37 35 13 43 22 28
32 14 11 32 118 123 22 5 58 19 38 378 157 2 688 20 95 63 12 20 28 191 113 5 710 160 7 – 34 33 0 93 38 21 240 198 35 53 9 188 20 42
30 12 10 15 25 6 73 24 38 88 33 73 50 51 253 109 75 23 55 167 183 105 138 11 185 23 291 192 398 16 32 193 44 25 17 287 76 37 20 57 57 29
0 0 0 0 23 0 0 0 0 2 2 5 8 0 6 10 13 13 0 0 0 0 5 0 1 0 3 85 1 1 0 4 0 0 1 3 5 4 0 0 1 3
29 17 3 2 1 2 8 16 2 4 15 17 12 9 4 0 27 7 5 6 15 3 3 4 7 25 13 1 31 7 15 42 5 24 1 30 3 19 1 28 6 17
32 12 11 32 95 67 22 5 58 17 36 373 112 2 686 10 336 50 12 21 29 191 124 5 709 162 6 169 33 32 0 1,672 38 21 239 198 31 49 9 225 19 44
(Continued)
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BRAFV600E analysis in thyroid FNA
Table 3 (Continued) Study
Year
FNA
FNA + BRAF
BRAF
TP
FP
FN
TN
TP
FP
FN
TN
TP
FP
FN
TN
Liu et al59
2014
109
8
11
171
88
0
32
179
113
8
7
171
Seo et al60 Seo et al61 Wan et al62 Zeck et al63 Eszlinger et al64 Krane et al65 Park et al66 Shi et al67
2014 2014 2014 2014 2015 2015 2015 2015
115 42 18 7 69 54 111 20
0 4 0 2 1 2 0 0
17 18 23 6 68 19 13 3
7 36 7 6 201 77 34 7
98 32 25 5 57 32 101 11
0 0 0 0 0 0 1 0
34 28 16 8 80 41 23 12
7 36 7 8 201 79 33 7
121 45 30 7 80 60 116 20
0 4 0 2 1 2 1 0
11 15 11 6 57 13 8 3
7 36 7 6 201 77 32 7
Abbreviations: FNA, fine-needle aspiration; TP, true positive; FP, false positive; FN, false negative; TN, true negative; –, data not available.
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). To evaluate the diagnostic value of BRAFV600E 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 BRAFV600Emutation 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 BRAFV600E 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).
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
Table 4 Results of meta-analysis for diagnostic value of FNA cytology, BRAFV600E-mutation analysis, and the combined strategy in all FNA specimens Parameter FNA Pooled sensitivity Pooled specificity Pooled LR, + Pooled LR, Pooled DOR SROC AUC Q*
FNA + BRAF
BRAF
Result 95% CI
Heterogeneity, I2 Result 95% CI
Heterogeneity, I2 Result 95% CI
Heterogeneity, I2
0.814
0.803–0.824
93.5%
0.619
0.605–0.633
93%
0.874
0.865–0.884
92.5%
0.981
0.978–0.985
86.4%
0.997
0.995–0.998
14.1%
0.968
0.963–0.972
92.5%
23.868 14.139–40.293 87.7% 0.216 0.172–0.273 94.2% 127.73 75.082–217.28 76.1%
34.982 23.801–51.415 19.5% 0.433 0.384–0.489 91.8% 96.570 63.932–145.87 21.4%
22.353 13.027–38.355 93.1% 0.146 0.111–0.192 93% 187.92 110.24–320.35 76.4%
0.9551 0.8975
0.9207 0.8542
0.9744 0.9271
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.
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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.
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,
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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. BRAFV600E mutation is the most promising marker for thyroid nodules. A similar meta-analysis conducted by Jia et al of 16 studies suggested that BRAFV600E analysis had diagnostic value in indeterminate thyroid nodules,11 but another analysis of eight eligible studies found a low BRAFV600E-mutation rate within indeterminate
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OncoTargets and Therapy 2016:9
77.4% 17.9% 0 56.1% 0 0.7999 0.7358
0.328–0.477 0.982–0.999 3.336–14.691 0.576–0.835 6.100–34.320 0.401 0.995 7.001 0.694 14.469 76% 70.8% 64.1% 29.8% 0 0.7674 0.7079
0.556–0.631 0.784–0.918 1.625–7.259 0.462–0.637 3.944–14.598 0.594 0.861 3.434 0.542 7.588
Heterogeneity, I
0.8711 0.8015
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.
– –
0.128–0.278 0.983–1.000 3.611–25.379 0.522–1.030 4.966–44.156 0.195 0.997 9.573 0.733 14.808
Result 95% CI Heterogeneity, I Result 95% CI Result 95% CI Heterogeneity, I
86.4% 0 0 84.8% 0 0.416–0.468 0.994–0.999 8.175–18.406 0.551–0.683 15.388–37.242 0.442 0.997 12.267 0.613 23.939
Pooled sensitivity Pooled specificity Pooled LR, + Pooled LR, Pooled DOR SROC AUC Q*
cases, and thus the value of BRAFV600E-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 BRAFV600E analysis in thyroid FNA, especially those specific categories of indeterminate cases.
Result 95% CI
Abbreviations: TP, true positive; FP, false positive; FN, false negative; TN, true negative.
FN/SFN
23 9 15 45 6 15 19 31 71 34 2 5 10 94 13 12 12 392 10 104 10 32 7 33 30 157 12 13 9 15 28 4 42 49 14 4 17 5 119 35 27 4 4
2
TN
27 15 2 1 8 2 15 14 17 5 8 24 4 30 5 12 9 104 18 30 39 10 38 27 11 29 6 2 49 23 24 7 22 8 6 14 21 11 51 27 6 13 8
AUS/FLUS
FN
0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0
2
FP
5 2 2 1 13 7 18 7 1 2 10 50 16 13 1 10 52 17 2 30 14 5 57 79 6 14 5 4 32 21 1 16 5 6 6 22 10 12 37 6 1 17 1
SMC
TP
2
2004 2004 2007 2007 2008 2009 2009 2009 2009 2010 2010 2010 2010 2010 2010 2011 2011 2011 2011 2011 2011 2012 2012 2012 2012 2012 2013 2013 2013 2013 2014 2014 2014 2014 2014 2014 2014 2014 2015 2015 2015 2015 2015
Indeterminate
Cohen et al18 Xing et al19 Pizzolanti et al21 Sapio et al22 Kim et al24 Jo et al26 Marchetti et al27 Nikiforov et al28 Zatelli et al29 Cantara et al30 Girlando et al31 Kim et al32 Kwak et al33 Moses et al34 Musholt et al35 Adeniran et al36 Kim et al37 Nikiforov et al69 Patel et al70 Pelizzo et al40 Yeo et al42 Cañadas-Garre et al43 Kang et al44 Lee et al46 Mancini et al47 Rossi et al48 Brahma et al50 Di Benedetto et al51 Koh et al52 Park et al53 Beaudenon-Huibregtse et al54 Guo et al57 Johnson et al58 Liu et al59 Poller et al71 Seo et al60 Seo et al61 Wan et al62 Eszlinger et al64 Krane et al65 Le Mercier et al72 Park et al66 Shi et al67
BRAF
Parameter
Year
Table 6 Results of meta-analysis for diagnostic value of BRAFV600E mutation in indeterminate cases
Study
Heterogeneity, I2
Table 5 Diagnostic analysis of BRAFV600E-mutation analysis for indeterminate cases
73.1% 11.8% 0 85% 2.2%
BRAFV600E analysis in thyroid FNA
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Open Access Full Text Article
Diagnostic value of BRAFV600E-mutation analysis in fine-needle aspiration of thyroid nodules: a meta-analysis This article was published in the following Dove Press journal: OncoTargets and Therapy 27 April 2016 Number of times this article has been viewed
Xingyun Su 1 Xiaoxia Jiang 1 Xin Xu 1 Weibin Wang 1 Xiaodong Teng 2 Anwen Shao 3 Lisong Teng 1 Department of Surgical Oncology, Department of Pathology, First Affiliated Hospital, 3Department of Neurosurgery, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, People’s Republic of China 1 2
Abstract: Fine-needle aspiration (FNA) is a reliable method for preoperative diagnosis of thyroid nodules; however, about 10%–40% nodules are classified as indeterminate. The BRAFV600E mutation is the most promising marker for thyroid FNA. This meta-analysis was conducted to investigate the diagnostic value of BRAFV600E analysis in thyroid FNA, especially the indeterminate cases. Systematic searches were performed in PubMed, Web of Science, Scopus, Ovid, Elsevier, and the Cochrane Library databases for relevant studies prior to June 2015, and a total of 88 studies were ultimately included in this meta-analysis. Compared with FNA cytology, the synergism of BRAFV600E testing increased the diagnostic sensitivity from 81.4% to 87.4% and decreased the false-negative rate from 8% to 5.2%. In the indeterminate group, the mutation rate of BRAFV600E was 23% and varied in different subcategories (43.2% in suspicious for malignant cells [SMC], 13.77% in atypia of undetermined significance/follicular lesion of undetermined significance [AUS/FLUS], and 4.43% in follicular neoplasm/suspicious for follicular neoplasm [FN/SFN]). The sensitivity of BRAFV600E analysis was higher in SMC than that in AUS/FLUS and FN/SFN cases (59.4% vs 40.1% vs 19.5% respectively), while specificity was opposite (86.1% vs 99.5% vs 99.7% respectively). The areas under the summary receiver-operating characteristic curve also confirmed the diagnostic value of BRAFV600E testing in SMC and AUS/FLUS rather than FN/SFN cases. Therefore, BRAFV600E analysis can improve the diagnostic accuracy of thyroid FNA, especially indeterminate cases classified as SMC, and select malignancy to guide the extent of surgery. Keywords: thyroid cancer, fine-needle aspiration, BRAFV600E mutation, meta-analysis
Introduction
Correspondence: Lisong Teng Department of Surgical Oncology, First Affiliated Hospital, School of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou, Zhejiang 310003, People’s Republic of China Tel +86 571 8706 8873 Fax +86 571 8723 6628 Email [email protected]
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. 2495
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http://dx.doi.org/10.2147/OTT.S101800
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Su et al
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 BRAFV600E 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 BRAFV600E analysis could be routinely used in clinical practice is still controversial. Numerous researchers have proved that BRAFV600E-mutation testing is an effective diagnostic approach for thyroid FNA,11 while others believe that its utility is limited by low prevalence of BRAFV600E mutation in indeterminate nodules.12 Therefore, we conducted a structured meta-analysis to estimate the additional diagnostic yield of BRAFV600E-mutation analysis in thyroid FNA, and further evaluated the malignancy rate, BRAFV600E-mutation frequency, and diagnostic value of BRAFV600E 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 BRAFV600E-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 BRAFV600E-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
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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. The methodological quality of studies eligible for diagnostic analysis of FNA cytology and/or BRAFV600E 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 Table 1 Comparison between the British and Bethesda systems for classification of thyroid cytopathology Bethesda
British
Nondiagnostic or unsatisfactory Benign AUS/FLUS (atypia of undetermined significance/follicular lesion of undetermined significance) FN/SFN (follicular neoplasm/ suspicious for follicular neoplasm) SMC (suspicious for malignancy) Malignant
Thy1 (nondiagnostic) Thy2 (nonneoplastic) Thy3a (neoplasm possible, atypia/nondiagnostic) Thy3f (neoplasm possible, suggesting follicular neoplasm) Thy4 (suspicious of malignancy) Thy5 (malignant)
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BRAFV600E analysis in thyroid FNA
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 BRAFV600E 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 BRAFV600E-mutation rate, respectively. The characteristics of studies eligible for diagnostic analysis of FNA cytology and BRAFV600E 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 BRAFV600E 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.
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Cohen et al18 Xing et al19 Domingues et al20 Pizzolanti et al21 Sapio et al22 Sapio et al23 Kim et al24 Bentz et al25 Jo et al26 Marchetti et al27 Nikiforov et al28 Zatelli et al29 Cantara et al30 Girlando et al31 Kim et al32 Kwak et al33 Moses et al34 Musholt et al35 Adeniran et al36 Kim et al37 Lee et al38 Moon et al39 Pelizzo et al40 Smith et al41 Yeo et al42 Cañadas-Garre et al43 Kang et al44 Kwak et al45 Lee et al46 Mancini et al47 Rossi et al48 Tomei et al49 Brahma et al50 Di Benedetto et al51 Koh et al52 Park et al53 Beaudenon-Huibregtse et al54 Crescenzi et al55 Eszlinger et al56
Study USA USA Portugal Italy Italy Italy South Korea USA South Korea Italy USA Italy Italy Italy South Korea South Korea USA Germany USA South Korea South Korea South Korea Italy USA South Korea Spain South Korea South Korea South Korea Italy Italy Italy Indonesia Italy South Korea South Korea USA Italy Germany
Country 1 1 1 1 2 2 1 1 1 1 2 1 1 1 1 1 1 6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 1 1 1 5 1 1
Centers, n Jan 2001–Jan 2003 – – Sep 2005–Jun 2006 – – Aug 2005–Jul 2006 1994–2004 June 2006–Dec 2006 1996–2008 – Oct 2008–Dec 2009 – – – Mar 2008–Jun 2008 Jun 2006–Jul 2008 Jan 2008–Jul 2009 Sep 2009–Nov 2010 Mar 2007–Feb 2009 July 2007–Dec 2009 Sep 2008–May 2009 Oct 2008–Sep 2009 – Jul 2009–Jan 2010 Jun 2006–Dec 2009 Apr 2008–Jul 2009 Jun 2009–Oct 2010 Aug 2008–Mar 2011 – – – Aug 2010–Jun 2011 – Jan 2009–Oct 2010 Jan 2011–May 2011 Jul 2010–Oct 2012 – 1995–2009
Enrollment period Retroa Prob Retro Pro Retro Retro Retro Retro Pro Retro Pro Pro Pro Pro Pro Retro Pro Pro Pro Pro Pro Retro Pro Retro Pro Pro Pro Retro Pro Pro Pro Retro Pro Pro Pro Retro Pro Pro Retro
Design – – – – – – – 40.9 – – – 50.7 51.2 – 50.6 45.6 51 – 52.6 – 50.3 49.4 47.8 – 51.27 49.8 – 48 49.5 55.1 52 – 46 – 48.6 – – – –
Mean age, years
Table 2 Characteristics of studies eligible for the diagnostic analysis of FNA cytology and BRAFV600E testing
– – – – – – – – 1 – – 1.1 – – 1.29 1.17 – – – – 1.46 0.95 – – 1.3 – – 0.92 – 2.38 – – .1 – 1.05 – – – –
Mean diameter, cm A B A A B B A A A A B A A A A A B A A A A B A A B A A A A A B A A A B B A A A
Final diagnosis
Direct sequencing + mutector assay Direct sequencing + colorimetric method PCR-RFLP Real-time AS-PCR Direct sequencing MASA Pyrosequencing LCPCR + FMCA Pyrosequencing Direct sequencing LCPCR + FMCA Direct sequencing DHPLC + direct sequencing Direct sequencing DPO-based multiplex PCR + direct sequencing DPO-based multiplex PCR Direct sequencing Direct sequencing SSCP analysis Pyrosequencing Pyrosequencing Direct sequencing Direct sequencing + MASA MCA Pyrosequencing PCR-RFLP AS-PCR + direct sequencing DPO-PCR + real-time PCR MEMO-PCR + direct sequencing High-resolution melting analysis Direct sequencing Pyrosequencing PCR-RFLP Direct sequencing DPO-PCR Real-time PCR + pyrosequencing Multiplex PCR Real-time sequencing High-resolution melting PCR + pyrosequencing
Genotyping method
Su et al Dovepress
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OncoTargets and Therapy 2016:9
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OncoTargets and Therapy 2016:9
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.
– – – – –
Guo et al57 Johnson et al58 Liu et al59 Seo et al60 Seo et al61 Wan et al62 Zeck et al63 Eszlinger et al64 Krane et al65 Park et al66 Shi et al67
PRC UK PRC South Korea South Korea PRC USA Italy Germany South Korea USA
1 1 1 1 1 1 1 1 1 1 1
Nov 2010–Jul 2011 Sep 2011–Oct 2012 Sep 2012–Dec 2013 Dec 2010–Jan 2011 Dec 2010–Feb 2012 Mar 2013–Sep 2013 Apr 2011–Jan 2013 1995–2009 May 2011–Mar 2012 Jul 2011–Mar 2012 Jan 2011–Feb 2013
Pro Retro Pro Pro Pro Pro Pro Retro Pro Pro Retro
– – – 48.4 50.3 49 – – – – –
– – – 1.11 1.9
A A B A B A A A A A A
Direct sequencing High-resolution MCA Pyrosequencing Real-time PCR Real-time PCR – miRInform test High-resolution melting analysis + pyrosequencing High-resolution melting PCR + pyrosequencing Real-time PCR/AS-PCR + MEMO sequencing Real-time PCR
BRAFV600E analysis in thyroid FNA
Synthesis of analysis results
Diagnostic value of FNA cytology, BRAFV600E-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 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 BRAFV600E-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 BRAFV600E testing was 99.5% (2,886 of 2,900). After BRAFV600E 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 BRAFV600E 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.
Diagnostic value of BRAFV600E-mutation analysis in indeterminate cases (Bethesda categories III–V) There were 43 studies included in the diagnostic analysis of BRAFV600E 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 BRAFV600E mutation. No threshold effect was detected, so the random-effect model was chosen to pool the diagnostic
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Table 3 Diagnostic analysis of FNA cytological examination and BRAFV600E-mutation analysis in all the FNA specimens Study
Year
Cohen et al18 Xing et al19 Domingues et al20 Pizzolanti et al21 Sapio et al22 Sapio et al23 Kim et al24 Bentz et al25 Jo et al26 Marchetti et al27 Nikiforov et al28 Zatelli et al29 Cantara et al30 Girlando et al31 Kim et al32 Kwak et al33 Moses et al34 Musholt et al35 Adeniran et al36 Kim et al37 Lee et al38 Moon et al39 Pelizzo et al40 Smith et al41 Yeo et al42 Cañadas-Garre et al43 Kang et al44 Kwak et al45 Lee et al46 Mancini et al47 Marchetti et al68 Rossi et al48 Tomei et al49 Brahma et al50 Di Benedetto et al51 Koh et al52 Park et al53 Beaudenon-Huibregtse et al54 Crescenzi et al55 Eszlinger et al56 Guo et al57 Johnson et al58
2004 2004 2005 2007 2007 2007 2008 2009 2009 2009 2009 2009 2010 2010 2010 2010 2010 2010 2011 2011 2011 2011 2011 2011 2011 2012 2012 2012 2012 2012 2012 2012 2012 2013 2013 2013 2013 2014 2014 2014 2014 2014
FNA
FNA + BRAF
BRAF
TP
FP
FN
TN
TP
FP
FN
TN
TP
FP
FN
TN
25 10 10 13 24 6 60 22 30 88 27 66 46 38 251 108 71 19 47 146 127 98 133 10 183 12 289 318 382 13 85 159 44 23 15 277 71 36 20 57 55 31
0 0 0 0 23 0 0 0 0 2 2 5 8 0 2 10 13 13 0 0 0 0 5 0 1 0 1 0 1 1 0 3 0 0 1 0 5 4 0 0 1 3
34 19 3 4 2 2 21 18 9 4 21 24 16 22 6 1 30 11 13 27 70 10 6 5 9 31 15 33 47 10 5 73 5 26 3 27 8 18 1 28 8 19
32 12 11 32 95 67 22 5 58 17 36 373 112 2 690 10 337 50 12 21 29 191 117 5 709 132 8 86 33 32 0 1,621 38 21 239 194 31 49 9 225 19 44
23 8 3 11 10 4 63 17 30 59 18 48 33 41 221 87 23 9 40 154 174 57 98 10 99 17 226 – 342 12 63 114 28 17 13 176 44 21 8 22 41 16
0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 0 0 0 0 1 1 0 0 0 0 0 2 – 0 0 0 0 0 0 0 3 1 0 0 0 0 0
36 22 10 6 16 4 18 20 10 32 30 42 45 19 47 22 78 21 20 19 24 51 59 5 93 31 78 – 87 11 22 172 21 32 5 141 37 35 13 43 22 28
32 14 11 32 118 123 22 5 58 19 38 378 157 2 688 20 95 63 12 20 28 191 113 5 710 160 7 – 34 33 0 93 38 21 240 198 35 53 9 188 20 42
30 12 10 15 25 6 73 24 38 88 33 73 50 51 253 109 75 23 55 167 183 105 138 11 185 23 291 192 398 16 32 193 44 25 17 287 76 37 20 57 57 29
0 0 0 0 23 0 0 0 0 2 2 5 8 0 6 10 13 13 0 0 0 0 5 0 1 0 3 85 1 1 0 4 0 0 1 3 5 4 0 0 1 3
29 17 3 2 1 2 8 16 2 4 15 17 12 9 4 0 27 7 5 6 15 3 3 4 7 25 13 1 31 7 15 42 5 24 1 30 3 19 1 28 6 17
32 12 11 32 95 67 22 5 58 17 36 373 112 2 686 10 336 50 12 21 29 191 124 5 709 162 6 169 33 32 0 1,672 38 21 239 198 31 49 9 225 19 44
(Continued)
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BRAFV600E analysis in thyroid FNA
Table 3 (Continued) Study
Year
FNA
FNA + BRAF
BRAF
TP
FP
FN
TN
TP
FP
FN
TN
TP
FP
FN
TN
Liu et al59
2014
109
8
11
171
88
0
32
179
113
8
7
171
Seo et al60 Seo et al61 Wan et al62 Zeck et al63 Eszlinger et al64 Krane et al65 Park et al66 Shi et al67
2014 2014 2014 2014 2015 2015 2015 2015
115 42 18 7 69 54 111 20
0 4 0 2 1 2 0 0
17 18 23 6 68 19 13 3
7 36 7 6 201 77 34 7
98 32 25 5 57 32 101 11
0 0 0 0 0 0 1 0
34 28 16 8 80 41 23 12
7 36 7 8 201 79 33 7
121 45 30 7 80 60 116 20
0 4 0 2 1 2 1 0
11 15 11 6 57 13 8 3
7 36 7 6 201 77 32 7
Abbreviations: FNA, fine-needle aspiration; TP, true positive; FP, false positive; FN, false negative; TN, true negative; –, data not available.
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). To evaluate the diagnostic value of BRAFV600E 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 BRAFV600Emutation 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 BRAFV600E 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).
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
Table 4 Results of meta-analysis for diagnostic value of FNA cytology, BRAFV600E-mutation analysis, and the combined strategy in all FNA specimens Parameter FNA Pooled sensitivity Pooled specificity Pooled LR, + Pooled LR, Pooled DOR SROC AUC Q*
FNA + BRAF
BRAF
Result 95% CI
Heterogeneity, I2 Result 95% CI
Heterogeneity, I2 Result 95% CI
Heterogeneity, I2
0.814
0.803–0.824
93.5%
0.619
0.605–0.633
93%
0.874
0.865–0.884
92.5%
0.981
0.978–0.985
86.4%
0.997
0.995–0.998
14.1%
0.968
0.963–0.972
92.5%
23.868 14.139–40.293 87.7% 0.216 0.172–0.273 94.2% 127.73 75.082–217.28 76.1%
34.982 23.801–51.415 19.5% 0.433 0.384–0.489 91.8% 96.570 63.932–145.87 21.4%
22.353 13.027–38.355 93.1% 0.146 0.111–0.192 93% 187.92 110.24–320.35 76.4%
0.9551 0.8975
0.9207 0.8542
0.9744 0.9271
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.
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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.
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,
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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. BRAFV600E mutation is the most promising marker for thyroid nodules. A similar meta-analysis conducted by Jia et al of 16 studies suggested that BRAFV600E analysis had diagnostic value in indeterminate thyroid nodules,11 but another analysis of eight eligible studies found a low BRAFV600E-mutation rate within indeterminate
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OncoTargets and Therapy 2016:9
77.4% 17.9% 0 56.1% 0 0.7999 0.7358
0.328–0.477 0.982–0.999 3.336–14.691 0.576–0.835 6.100–34.320 0.401 0.995 7.001 0.694 14.469 76% 70.8% 64.1% 29.8% 0 0.7674 0.7079
0.556–0.631 0.784–0.918 1.625–7.259 0.462–0.637 3.944–14.598 0.594 0.861 3.434 0.542 7.588
Heterogeneity, I
0.8711 0.8015
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.
– –
0.128–0.278 0.983–1.000 3.611–25.379 0.522–1.030 4.966–44.156 0.195 0.997 9.573 0.733 14.808
Result 95% CI Heterogeneity, I Result 95% CI Result 95% CI Heterogeneity, I
86.4% 0 0 84.8% 0 0.416–0.468 0.994–0.999 8.175–18.406 0.551–0.683 15.388–37.242 0.442 0.997 12.267 0.613 23.939
Pooled sensitivity Pooled specificity Pooled LR, + Pooled LR, Pooled DOR SROC AUC Q*
cases, and thus the value of BRAFV600E-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 BRAFV600E analysis in thyroid FNA, especially those specific categories of indeterminate cases.
Result 95% CI
Abbreviations: TP, true positive; FP, false positive; FN, false negative; TN, true negative.
FN/SFN
23 9 15 45 6 15 19 31 71 34 2 5 10 94 13 12 12 392 10 104 10 32 7 33 30 157 12 13 9 15 28 4 42 49 14 4 17 5 119 35 27 4 4
2
TN
27 15 2 1 8 2 15 14 17 5 8 24 4 30 5 12 9 104 18 30 39 10 38 27 11 29 6 2 49 23 24 7 22 8 6 14 21 11 51 27 6 13 8
AUS/FLUS
FN
0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0
2
FP
5 2 2 1 13 7 18 7 1 2 10 50 16 13 1 10 52 17 2 30 14 5 57 79 6 14 5 4 32 21 1 16 5 6 6 22 10 12 37 6 1 17 1
SMC
TP
2
2004 2004 2007 2007 2008 2009 2009 2009 2009 2010 2010 2010 2010 2010 2010 2011 2011 2011 2011 2011 2011 2012 2012 2012 2012 2012 2013 2013 2013 2013 2014 2014 2014 2014 2014 2014 2014 2014 2015 2015 2015 2015 2015
Indeterminate
Cohen et al18 Xing et al19 Pizzolanti et al21 Sapio et al22 Kim et al24 Jo et al26 Marchetti et al27 Nikiforov et al28 Zatelli et al29 Cantara et al30 Girlando et al31 Kim et al32 Kwak et al33 Moses et al34 Musholt et al35 Adeniran et al36 Kim et al37 Nikiforov et al69 Patel et al70 Pelizzo et al40 Yeo et al42 Cañadas-Garre et al43 Kang et al44 Lee et al46 Mancini et al47 Rossi et al48 Brahma et al50 Di Benedetto et al51 Koh et al52 Park et al53 Beaudenon-Huibregtse et al54 Guo et al57 Johnson et al58 Liu et al59 Poller et al71 Seo et al60 Seo et al61 Wan et al62 Eszlinger et al64 Krane et al65 Le Mercier et al72 Park et al66 Shi et al67
BRAF
Parameter
Year
Table 6 Results of meta-analysis for diagnostic value of BRAFV600E mutation in indeterminate cases
Study
Heterogeneity, I2
Table 5 Diagnostic analysis of BRAFV600E-mutation analysis for indeterminate cases
73.1% 11.8% 0 85% 2.2%
BRAFV600E analysis in thyroid FNA
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