Clinicopathologic correlations of the BRAFV600E mutation, BRAF V600E immunohistochemistry, and BRAF RNA in situ hybridization in papillary thyroid carcinoma

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Original article

Clinicopathologic correlations of the BRAFV600E mutation, BRAF V600E immunohistochemistry, and BRAF RNA in situ hybridization in papillary thyroid carcinoma Yoon Yang Jung a , Jae Hyung Yoo b , Eon Sub Park b , Mi Kyung Kim b , Tae Jin Lee b , Bo Youn Cho d,e , Yun Jae Chung d,e , Kyung Ho Kang c,e , Hwa Young Ahn d,e , Hee Sung Kim b,e,∗ a

Department of Pathology and Translational Genomics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea Department of Pathology, Chung-Ang University Hospital, Chung-Ang University College of Medicine, Seoul, South Korea c Department of Surgery, Chung-Ang University Hospital, Chung-Ang University College of Medicine, Seoul, South Korea d Department of Internal Medicine, Division of Endocrinology, Chung-Ang University Hospital, Chung-Ang University College of Medicine, Seoul, South Korea e Thyroid Center, Chung-Ang University Hospital, Chung-Ang University College of Medicine, Seoul, South Korea b

a r t i c l e

i n f o

Article history: Received 6 May 2014 Received in revised form 13 October 2014 Accepted 15 October 2014 Keywords: Papillary carcinoma BRAF V600E mutation Immunohistochemistry RNA in situ hybridization

a b s t r a c t Background: The BRAFV600E mutation is the most common genetic alteration in papillary thyroid carcinoma (PTC). The aim of this study is to analyze the clinicopathologic correlations of the BRAFV600E mutation, BRAF V600E immunohistochemistry (IHC) and BRAF RNA in situ hybridization (ISH) in PTC. Methods: This study included 467 patients with PTC who underwent surgical resection. We studied the BRAFV600E mutation using real-time PCR and BRAF V600E and BRAF RNA ISH using tissue microarray (TMA). Results: The frequencies of a positive BRAFV600E mutation by real-time PCR, positive BRAF V600E IHC, and high BRAF RNA ISH were 84%, 86%, and 70%, respectively, in PTC. Conventional PTC had higher positive rates in all three tests than other histologic types. The BRAFV600E mutation, BRAF V600E IHC, low Ct, and high BRAF RNA ISH were significantly associated with lymph node metastasis. The BRAFV600E mutation was significantly associated with positive immunostaining for BRAF V600E mutant protein (P < 0.001) overall, with high BRAF RNA ISH only in the follicular variant (P = 0.035). No significant correlation was noted between BRAF V600E IHC and BRAF RNA ISH. The sensitivity of BRAF V600E IHC for the BRAFV600E mutation was 95%, and the specificity was 61% overall, 96% and 54% in the conventional type, and 85% and 70% in the follicular variant. Conclusions: Our results showed that positive BRAF V600E IHC significantly correlated with the BRAFV600E mutation. This suggests its clinical utility as a screening tool for the BRAFV600E mutation. In addition, a high BRAF RNA ISH score could be a candidate marker of aggressive behavior in BRAFV600E mutation-positive cases of PTC. © 2014 Elsevier GmbH. All rights reserved.

Introduction Papillary thyroid carcinoma (PTC) is the most common subtype of thyroid cancer, accounting for about 80% of all thyroid malignancies [2]. The BRAFV600E mutation is the most common type of BRAF mutation, and has been detected in 30–83% of PTCs [14]. The BRAF oncogene encodes the human gene for B-type Raf kinase. Over 30 mutations of BRAF associated with human cancers have been

∗ Corresponding author. Tel.: +82 2 6299 2758; fax: +82 2 6293 5630. E-mail address: [email protected] (H.S. Kim).

identified [7], the majority of which are located within the kinase domain. In an analysis of 22 BRAF mutants, 18 had elevated kinase activity and signaled to ERK in vivo. Three other mutants had reduced kinase activity toward MEK in vitro but, by activating CRAF in vivo, signaled to ERK in cells [26]. The T1799A point mutation in exon 15 of BRAF (thymidine-toadenine transversion) results in a valine-to-glutamate substitution at position 600 (V600E) and activates the RAS/RAF/MAPK signaling pathway by disrupting hydrophobic interactions between residues in both the activation loop and the ATP binding site [26]. This pathway is hyperactivated in about 30% of cancers including malignant melanoma, papillary thyroid carcinoma, pilocytic astrocytoma, adenocarcinoma of the lung, ovarian neoplasms, hairy cell

http://dx.doi.org/10.1016/j.prp.2014.10.005 0344-0338/© 2014 Elsevier GmbH. All rights reserved.

Please cite this article in press as: Y.Y. Jung, et al., Clinicopathologic correlations of the BRAFV600E mutation, BRAF V600E immunohistochemistry, and BRAF RNA in situ hybridization in papillary thyroid carcinoma, Pathol. – Res. Pract (2014), http://dx.doi.org/10.1016/j.prp.2014.10.005

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Fig. 1. BRAF V600E immunohistochemistry shows cytoplasmic localization of BRAF V600E protein in PTC. (A) Negative staining; (B) positive staining.

Fig. 2. BRAF mRNA expression level evaluated by RNA ISH in PTC. (A) Score 0; (B) score 1; (C) score 2; (D) score 4; (original magnification 400×).

Please cite this article in press as: Y.Y. Jung, et al., Clinicopathologic correlations of the BRAFV600E mutation, BRAF V600E immunohistochemistry, and BRAF RNA in situ hybridization in papillary thyroid carcinoma, Pathol. – Res. Pract (2014), http://dx.doi.org/10.1016/j.prp.2014.10.005

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leukemia, and colorectal carcinoma [4,8]. Previous studies have shown the association of BRAFV600E with aggressive clinicopathologic characteristics of PTC, such as extrathyroid extension and lymph node metastasis [16,21]. However, other studies found no correlation between this BRAF mutation and aggressive clinicopathologic features [17]. Initially, polymerase chain reaction (PCR) was the only widely used method for detection of the BRAF mutation; however, this method is expensive and time-consuming. Recently, several studies have shown the usefulness of immunohistochemical staining for detection of BRAFV600E mutation in various neoplasms [13,18,19,24]. This technique relies on the immunogen of the mouse anti-human BRAFV600E monoclonal antibody (Clone VE1), which is a synthetic peptide representing the BRAFV600E amino acid sequence from residue 596 to 606 (GLATEKSRWSG). Furthermore, the novel method of RNA in situ hybridization (ISH) directly visualizes the RNA transcripts. Although the efficacy of this technique is still being investigated, data from initial studies show that it can be a clinically useful option [10,15]. The aim of this study is to investigate the clinicopathologic correlations of the BRAFV600E mutation, BRAF V600E IHC, and BRAF RNA ISH, and the intercorrelations among the results of all three methods for detecting molecular alterations of BRAF in PTC. Materials and methods Patient selection The archives of the Chung-Ang University Hospital (Seoul, South Korea) were searched over a 24-month period (January 2011 to December 2012), and a total of 467 patients who had undergone surgical resection due to PTC and had tumor nodule measuring greater than 4 mm in the largest dimension were selected. The clinicopathologic and molecular data for each patient, including age at diagnosis, sex, tumor size, number of tumors, levels of thyroglobulin and antimicrosomal antibody, and BRAFV600E mutation status were obtained from our database. The presence of extrathyroidal extension, perineural invasion, and vascular invasion were also analyzed. In cases with multiple tumor nodules, the largest tumor nodule was defined as the dominant nodule. Non-neoplastic thyroid tissue was evaluated for the presence of lymphocytic thyroiditis. The age of the subjects ranged from 13 to 77 years at the time of diagnosis (median age: 46.0 years). The tumor size ranged from 0.4 to 6.5 cm (median size: 0.8 cm). All PTCs were classified and subtyped according to the World Health Organization criteria outlined in 2004 [23] and staged according to the AJCC staging manual [6]. This study was approved by the Institutional Review Board of Chung-Ang University Hospital (IRB No. C2013138 (1098)). Tissue microarray construction H&E-stained slides were observed and the appropriate tumor area was marked. The corresponding paraffin block was retrieved and the marked areas were matched. The cores of tumor areas were manually punched using a precision instrument (Labro TMA kit) and embedded into the recipient block with 60 microholes (diameter, 2 mm; depth, 5 mm) [20]. The microarray blocks were heated at 60 ◦ C for 30 min.

Fig. 3. Venn diagram showing overlap of positive BRAFV 600E mutation prediction by real-time PCR, BRAF V600E immunohistochemistry, and BRAF RNA in situ hybridization in PTC.

Dual-priming oligonucleotide-based PCR analysis was performed using Anyplex BRAFV600E Real-time Detection (v2.0) system (Seegene, Seoul, South Korea). Each PCR reaction mixture contained 2 ␮l 5× BRAF primer, 3 ␮l 8-methoxypsoralen solution, 5 ␮l extracted DNA, and 10 ␮l 2× Anyplex PCR master mix for a total volume of 20 ␮l. PCR was performed using a GeneAmp 7500 Real-time PCR System (Applied Biosystem, Foster City, CA, USA). Reactions underwent an initial 15 min incubation at 94 ◦ C, followed by 35 cycles of denaturation at 94 ◦ C for 30 s, annealing at 62 ◦ C for 30 s, and extension at 72 ◦ C for 60 s, and a final extension at 72 ◦ C for 10 min. If the Ct value of the internal control or V600E was ≥30 or undetermined, it was interpreted as negative. If the Ct was ≤13, it was regarded as positive for the BRAFV600E mutation. The Ct value was calculated as the difference in the cycle threshold between the target (BRAFV600E ) and the internal control. Immunohistochemistry Four ␮m-thick sections of the formalin-fixed, paraffinembedded tissue microarray blocks were prepared for immunohistochemistry. These sections were incubated with mouse anti-human BRAFV600E monoclonal antibody (Clone VE1) (1:50, Spring Bioscience, CA, USA) using the Ventana Benchmark XT automated staining system (Ventana Medical Systems, Tucson, AZ) as recently described [13]. As controls, PTC tissue which had a V600E mutation previously detected by capillary sequencing, and PTC tissues which showed strong, moderate, or no V600E protein expression, as well as tissues lacking the V600E mutation, were stained with every batch. Cytoplasmic staining was scored as positive or negative according to the intensity. Faint or weak cytoplasmic staining was considered negative. Moderate or strong cytoplasmic staining was considered positive (Fig. 1). The slides were read by two pathologists (Y.Y.J., H.S.K). In case of a discrepancy, the case was discussed using a multihead microscope until a consensus was reached.

Real-time PCR RNA in situ hybridization (ISH) H&E-stained slides were reviewed and the appropriate areas were marked. QIAmp DNA mini kits (QIAGEN, Chatsworth, CA, USA) were used for genomic DNA extraction.

Tissue microarray (TMA) blocks were used for RNA ISH. BRAF RNA transcripts were detected using the RNA ISH 2.0 FFPE assay

Please cite this article in press as: Y.Y. Jung, et al., Clinicopathologic correlations of the BRAFV600E mutation, BRAF V600E immunohistochemistry, and BRAF RNA in situ hybridization in papillary thyroid carcinoma, Pathol. – Res. Pract (2014), http://dx.doi.org/10.1016/j.prp.2014.10.005

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Table 1 Clinicopathologic correlations of the BRAFV600E mutation and BRAF V600E IHC in PTC. BRAFV600E mutation Total

Total

Wild type

Mutant

BRAF V600E IHC P

Negative

Positive

P

N

%

N

%

N

%

N

%

N

%

467

100

74

16

393

84

65

14

402

86

Age

≤45 years >45 years

233 234

50 50

35 39

15 17

198 195

85 83

0.626

33 32

14 14

200 202

86 86

0.879

Size

≤2 cm >2 cm

433 34

93 7

66 8

15 24

367 26

85 76

0.203

57 8

13 24

376 26

87 76

0.118*

Subtype

Conventional Follicular variant Others

402 50 15

86 11 3

46 23 5

11 46 33

356 27 10

89 54 67

<0.001*

41 20 4

10 40 27

361 30 11

90 60 73

<0.001*

Extrathyroidal extension

Absent Present

323 144

69 31

53 21

16 15

270 123

84 85

0.618

45 20

14 14

278 124

86 86

0.990

Multiplicity

Absent Present

272 195

58 42

47 27

17 14

225 168

83 86

0.316

40 25

15 13

232 170

85 87

0.562

LNM

Absent Present

196 271

42 58

42 32

21 12

154 239

79 88

0.005

36 29

18 11

160 242

82 89

0.018

CLT

Absent Present

317 150

68 32

45 29

14 19

272 121

86 81

0.156

42 23

13 15

275 127

87 85

0.543

Lymphatic invasion

Absent Present

462 5

99 1

72 2

16 40

390 3

84 60

0.180*

63 2

14 40

399 3

86 60

0.144*

Perineural invasion

Absent Present

433 34

93 7

72 2

17 6

361 32

83 94

0.098

62 3

14 9

371 31

86 91

0.605*

Blood vessel invasion

Absent Present

464 3

99 1

73 1

16 33

391 2

84 67

0.405*

65 0

14 0

399 3

86 100

1.000*

pN

pN0 pN1a pN1b

213 198 56

46 42 12

44 20 10

21 10 18

169 178 46

79 90 82

0.012

37 19 9

17 10 16

176 179 47

83 90 84

0.066

AJCC stage

I II III IV

309 17 118 23

66 4 25 5

50 4 15 5

16 24 13 22

259 13 103 18

84 76 87 78

0.443*

42 5 14 4

14 29 12 17

267 12 104 19

86 71 88 83

0.238*

Ki67 labeling index

≤5 >5

412 52

89 11

68 5

17 10

344 47

83 90

0.199

61 3

15 6

351 49

85 94

0.075

Tg Ab level

Normal High

350 80

81 19

50 12

14 15

300 68

86 85

0.870

44 13

13 16

306 67

87 84

0.381

Antimicrosomal Ab level

Normal High

350 79

82 18

46 17

13 22

304 62

87 78

0.057

45 12

13 15

305 67

87 85

0.581

* Fisher’s exact test. Ab, antibody; CLT, chronic lymphocytic thyroiditis; IHC, immunohistochemistry; LNM, lymph node metastasis; Tg, thyroglobulin

(Advanced Cell Diagnostics, Hayward, CA, USA) according to the manufacturer’s instructions. Briefly, 4 ␮m FFPE tissue sections were pretreated by heating and protease application prior to hybridization with a BRAF target probe. The detailed procedure was previously described [25]. Brown punctuated dots were used for scoring. Probes to the endogenous ubiquitin C (UBC) were used as positive control. The cases were categorized into five grades, 0–4, according to the number of brown punctuated dots in the nucleus and/or cytoplasm (Fig. 2). The grades were assigned as follows: no staining (score 0), staining that was difficult to see under the 40× objective lens in more than 10% of tumor cells (score 1), staining that was difficult to see under the 20× objective lens, but was easily seen under the 40× objective lens in more than 10% of tumor cells (score 2), staining that was difficult to see under the 10× objective lens, but was easily seen under the 20× objective in more than 10% of tumor cells (score 3), and staining that was easy to see under the 10× objective lens in more than 10% of tumor cells (score 4). The slides were read by two pathologists (Y.Y.J., H.S.K). In case of a discrepancy, the case

was discussed through a multihead microscope until a consensus was reached. Statistical analysis Statistical analysis was performed with the SPSS software (SPSS standard version 18.0; SPSS Inc., Chicago, IL, USA). A 2 test or Fisher’s exact test was applied as appropriate to evaluate the statistical significance. P < 0.05 was considered statistically significant. Results Clinicopathologic correlations of BRAFV600E mutation by real-time PCR, BRAF V600E IHC, and BRAF RNA ISH in PTC The BRAFV600E mutation was detected in 265 (89%) of 302 cases of conventional subtype PTC by real-time PCR. Twenty-seven (54%) of 50 cases and 10 (67%) of 15 cases carried the BRAFV600E mutation in follicular variant PTC and other subtypes of PTC, respectively.

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Table 2 Clinicopathologic correlation of the Ct value for the BRAFV600E mutation in PTC. Ct≤7.45

Total

Total

Ct >7.45

P

N

%

N

%

N

%

371

100

189

51

182

49

Age

≤45 years >45 years

188 183

51 49

103 86

55 47

85 97

45 53

0.133

Size

≤2 cm >2 cm

347 24

93 7

171 18

49 75

176 6

51 25

0.015

Subtype

Conventional Follicular variant Others

340 24 7

92 6 2

181 7 1

53 29 14

159 17 6

47 71 86

0.003*

Extrathyroidal extension

Absent Present

257 114

69 31

121 68

47 60

136 46

53 40

0.025

Multiplicity

Absent Present

210 161

57 43

115 74

55 46

95 87

45 54

0.093

LNM

Absent Present

142 229

38 62

63 126

44 55

79 103

56 45

0.046

CLT

Absent Present

257 114

69 31

147 42

57 37

110 72

43 63

<0.001

Lymphatic invasion

Absent Present

368 3

99 1

187 2

51 67

181 1

49 33

1.000*

Perineural invasion

Absent Present

345 26

93 7

176 13

51 50

169 13

49 50

0.921

Blood vessel invasion

Absent Present

369 2

99 1

188 1

51 50

181 1

49 50

1.000*

pN

pN0 pN1a pN1b

157 173 41

42 47 11

70 93 26

45 54 63

87 80 15

55 46 37

0.060

AJCC stage

I II III IV

244 12 98 17

66 3 26 5

118 9 51 11

48 75 52 65

126 3 47 6

52 25 48 35

0.190

Ki67 labeling index

≤5 >5

325 44

88 12

167 22

51 50

158 22

49 50

0.863

Tg Ab level

Normal High

285 63

82 18

156 19

55 30

129 44

45 70

<0.001

Antimicrosomal Ab level

Normal High

289 58

83 17

156 19

54 33

133 39

46 67

0.003

* Fisher’s exact test. Ab, antibody; CLT, chronic lymphocytic thyroiditis; LNM, lymph node metastasis; Tg, thyroglobulin

BRAF IHC is positive in 86% of cases overall, 90% in the conventional type, and 60% in the follicular variant. BRAF RNA ISH is high in 70% overall, 71% in the conventional type, and 58% in the follicular variant. No significant association was noted between the presence of the BRAFV600E mutation and the age the patient. The tumor size ranged from 0.4 to 6.5 cm (mean: 1.0 cm; median: 0.8 cm) in patients carrying the mutation, and 0.4 to 2.8 cm (mean: 1.1 cm; median: 1.0 cm) in wild-type cases (P = 0.046). The BRAFV600E mutation was associated with the conventional subtype (P < 0.001), lymph node metastasis (P = 0.005), and pN stage (P = 0.012). BRAF V600E IHC was associated with both the conventional subtype (P < 0.001) and lymph node metastasis (P = 0.018). The clinicopathologic correlations of the BRAFV600E mutation and BRAF V600E IHC for all subjects are summarized in Table 1. Next, we evaluated the correlation between clinicopathologic factors and Ct level (Table 2). Lower Ct values were associated with larger (>2 cm) tumor size (P = 0.015), the conventional subtype (P = 0.003), extrathyroidal extension (P = 0.025), lymph node metastasis (P = 0.046), the absence of chronic lymphocytic

thyroiditis (P < 0.001), a normal Tg Ab level (P < 0.001), and a normal antimicrosomal Ab level (P = 0.003). Clinicopathologic correlations of the BRAF RNA ISH score were analyzed for subjects that were positive for the BRAFV600E mutation based on real-time PCR or BRAF IHC (Table 3). In patients with the BRAFV600E mutation on real-time PCR, higher RNA ISH scores were associated with younger age (P = 0.006), conventional type (P = 0.007), lymph node metastasis (P = 0.004), and higher pN stage (P = 0.010). A higher score in the BRAF RNA ISH was associated with younger age (P = 0.002), lymph node metastasis (P = 0.009) and higher pN stage (P = 0.013) in BRAF IHF positive cases. Intercorrelations among the BRAFV600E real-time PCR, BRAF V600E IHC, and BRAF RNA ISH in PTC BRAF V600E IHC staining showed significant association with the BRAFV600E real-time PCR (P < 0.001). Subgroup analysis according to the histologic type showed consistently significant association. BRAF RNA ISH, however, was not significantly associated with the BRAFV600E real-time PCR either overall or for the histologic subtypes, except for the follicular variant subtype

Please cite this article in press as: Y.Y. Jung, et al., Clinicopathologic correlations of the BRAFV600E mutation, BRAF V600E immunohistochemistry, and BRAF RNA in situ hybridization in papillary thyroid carcinoma, Pathol. – Res. Pract (2014), http://dx.doi.org/10.1016/j.prp.2014.10.005

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Table 3 Clinicopathologic correlations of BRAF RNA ISH with real-time PCR BRAFV600E mutant cases or BRAF IHC positive cases in PTC. BRAFV600E mutant (real-time PCR)

BRAF RNA ISH score

0–2

Total

3–4

BRAF IHC positive P

N

%

N

118

30

275

70

47 71

24 36

151 124

76 64

113 5

31 19

254 21

%

0–2

3–4

P

N

%

N

119

30

283

70

%

0.006

45 74

23 37

155 128

78 63

0.002

69 81

0.214

114 5

30 19

262 21

70 81

0.231

101 14 4

28 47 36

260 16 7

72 53 64

0.084*

Age

≤45 years >45 years

Size

≤2 cm >2 cm

Subtype

Conventional Follicular variant Others

99 15 4

28 56 40

257 12 6

72 44 60

0.007*

Extrathyroidal extension

Absent Present

89 29

33 24

181 94

67 76

0.060

89 30

32 24

189 94

68 76

0.113

Multiplicity

Absent Present

70 48

31 29

155 120

69 71

0.587

72 47

31 28

160 123

69 72

0.462

LNM

Absent Present

59 59

38 25

95 180

62 75

0.004

59 60

37 25

101 182

63 75

0.009

CLT

Absent Present

84 34

31 28

188 87

69 72

0.578

86 33

31 26

189 94

69 74

0.280

Lymphatic invasion

Absent Present

118 0

30 0

272 3

70 100

0.557*

119 0

30

280 3

70 100

0.558*

Perineural invasion

Absent Present

108 10

30 31

253 22

70 69

0.875

109 10

29 32

262 21

71 68

0.736

Blood vessel invasion

Absent Present

118 0

30 0

273 2

70 100

1.000*

118 1

30 33

281 2

70 67

1.000*

pN

pN0 pN1a pN1b

64 45 9

38 25 20

105 133 37

62 75 80

0.010

65 45 9

37 25 19

111 134 38

63 75 81

0.013

AJCC stage

I II III IV

80 6 28 4

31 46 27 22

179 7 75 14

69 54 73 78

0.463*

82 4 28 5

31 33 27 26

185 8 76 14

69 67 73 74

0.885*

Ki67 labeling index

≤5 >5

105 11

31 23

239 36

69 77

0.316

105 12

30 24

246 37

70 76

0.434

Tg Ab level

Normal High

88 23

29 34

212 45

71 66

0.466

91 21

30 31

215 46

70 69

0.795

Antimicrosomal Ab level

Normal High

90 20

30 32

214 42

70 68

0.678

90 21

30 31

215 46

70 69

0.766

* Fisher’s exact test. Ab, antibody; CLT, chronic lymphocytic thyroiditis; IHC, immunohistochemistry; ISH, in situ hybridization; LNM, lymph node metastasis; Tg, thyroglobulin.

(P = 0.035) (Table 4). No significant association was noted between BRAF V600E IHC and BRAF RNA ISH, either overall or for each histologic subtype (data not shown).

subtype. Conversely, the follicular variant showed higher specificity (70%) and NPV (80%) than conventional subtype.

Overlap of the BRAFV600E mutation by real-time PCR, BRAF V600E IHC, and BRAF RNA ISH in PTC

Discussion

A Venn diagram was generated, illustrating the overlap of cases of positive BRAFV600E mutation by real-time PCR, positive BRAF V600E immunostaining, and higher BRAF RNA ISH scores (score 3–4) (Fig. 3). A total of 264 (57%) of 467 subjects had positive results in all three; 109 (23%) were positive for the BRAFV600E mutation by real-time PCR and IHC, but negative for BRAF RNA ISH. We evaluated the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of BRAF V600E IHC for the detection of the BRAFV600E mutation (Table 5). Overall, sensitivity was 95%, PPV was 93%, and accuracy was 90%. For the conventional type, sensitivity (96%), PPV (94%), and accuracy (91%) were all higher compared to the follicular variant

Our study showed that the BRAFV600E mutation was significantly associated with the conventional subtype of PTC and lymph node metastasis. Our findings were consistent with the previous notion that the BRAFV600E mutation is known to be an independent prognostic factor for recurrent and persistent disease, such as lymph node metastasis and advanced tumor stage [11]. Our results support that it can potentially be used as a predictor for poor clinical outcome and as a useful molecular marker for risk stratification in PTC patients [28]. Unlike in previous studies that have revealed an association between BRAFV600E mutation and greater tumor size [12], our study did not find an association between the BRAFV600E mutation and larger tumor size; however, lower Ct value correlated with larger tumor size.

Please cite this article in press as: Y.Y. Jung, et al., Clinicopathologic correlations of the BRAFV600E mutation, BRAF V600E immunohistochemistry, and BRAF RNA in situ hybridization in papillary thyroid carcinoma, Pathol. – Res. Pract (2014), http://dx.doi.org/10.1016/j.prp.2014.10.005

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0.099 100 55 4 6 0 46 0 5 0.035 71 41 15 12 29 59 6 17 0.198 85 90 99 257 15 10 17 29 0.856 IHC, immunohistochemistry; ISH, in situ hybridization.

84 84 118 275 23 51 0–2 3–4 BRAF RNA ISH score

16 16

0 91 0 10 100 9 16 340 61 6% 31 93 20 373 69 7% 45 29 Negative Positive BRAF V600E IHC

%

%

P

<0.001

25 21

% N

39 94

<0.001

16 7

80 23

4 23

20 77

<0.001

4 1

% N % N

Wild type Mutant

N % N

Wild type

N

Mutant Wild type Mutant

N

Wild type

N

Conventional type Overall

Histologic subtype

Table 4 Correlation between the BRAFV600E mutation detected by BRAF V600E IHC and BRAF RNA ISH in PTC.

%

P

Follicular variant

%

P

Others

Mutant

P

0.001

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Our study revealed a higher frequency of the BRAFV600E mutation (84% overall, 89% in the conventional type, and 54% in the follicular variant) compared to previous studies. Similar to our results, other studies conducted with Korean populations also show a relatively high frequency of the BRAFV600E mutation (58–83%), compared to studies in other ethnic groups [32,33]. It was suggested that the BRAF mutation is especially involved in carcinogenesis in PTCs in Koreans [33]. Previously, the utility of IHC for the detection of the BRAFV600E mutation in various neoplasms has been reported for cutaneous melanoma, PTC, colorectal carcinoma, and lung adenocarcinoma [1,13,18,24]. Especially good reproducibility was reported for melanoma [19]. We evaluated the diagnostic utility of BRAF V600E IHC as a screening tool for detecting the BRAFV600E mutation. BRAF V600E IHC significantly correlates with the BRAFV600E mutation. It showed high sensitivity (95%) and accuracy (90%) for overall PTC cases, with higher sensitivity (96%) and accuracy (91%) for the conventional subtype, compared to follicular variant PTC. Using IHC decreases the turn-around-time for results and improves the cost effectiveness of BRAFV600E mutation analysis, when compared to PCR-based methods [1]. The diagnostic utility of BRAF V600E IHC was confirmed using surgically resected PTC tissue. In thyroid fine needle aspiration (FNA) specimen, molecular analysis for the BRAFV600E mutation has significant diagnostic value in cases of atypia of unknown significance (AUS) [22], and preoperative molecular testing can help distinguish between benign and malignant lesions [30]. In surgical specimen of PTC, the BRAFV600E mutation is reported as an independent predictor of central compartment lymph node metastasis [9]. The detection can be a cost-effective measure, as it bypasses the need for two-stage thyroidectomy [29]. The PCR-based assay for the BRAFV600E mutation is performed with cytological specimens when clinically indicated, but there is a long wait time for results. Instead, we propose IHC staining for BRAF V600E in the cell block of FNA specimen. Using immunostaining for BRAF V600E, combined with classical PTC markers, including HBME-1, CK19, and galectin-3, will aid rapid and accurate diagnosis in diagnostically difficult cases. Zimmermann et al. [31] validated BRAF V600E IHC in FNA biopsies of PTCs with high reliability. Although realtime PCR is the most accurate and reliable method, the shorter turn-around-time and low cost of BRAF V600E IHC makes it a worthwhile screening tool, especially on pre-operative cytology or core-biopsy. Recently, a novel RNA ISH technique for formalin-fixed paraffinembedded tissue was introduced and is currently under validation. Our study evaluated the clinical utility of BRAF RNA ISH for detection of the BRAFV600E mutation in PTCs. Using this method, gene expression information is obtained for tissues, and singlemolecules can be visualized in individual cells [27]. In contrast, real-time PCR lacks the gene expression measurement in tissue context and is prone to interference from unintended cell types. The diagnostic utility of RNA ISH has been validated for detection of high-risk HPV virus E6/E7 mRNA in oropharyngeal squamous cell carcinoma [3,25], Her2 in gastric carcinoma [10], and EpCAM, CD44, and KRT17 in esophageal squamous cell carcinoma [5]. In our study, BRAF RNA ISH scores correlated with positive BRAF V600E IHC in the mutant group. However, it lacked suitability for a screening test. Although we did not obtain any statistically significant relevance between the PCR-based assay and RNA ISH, it is worth noting the significant association between the Ct value of the BRAFV600E mutation and the RNA ISH score. The Ct value reflects the number of cycles required for the fluorescent signal to cross the threshold, and it is inversely proportional to the amount of target nucleic acid in the sample. Thus, a lower Ct level means a higher amount of the target nucleic acid in the sample, indicating a strong positive reaction. Our study revealed that the lower Ct value significantly

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Table 5 Diagnostic utility of BRAF V600E IHC for detection of the BRAFV600E mutation in PTC.

Total Conventional subtype Follicular variant

Prevalence (%)

Sensitivity (%)

Specificity (%)

PPV (%)

NPV (%)

Accuracy (%)

84 89 54

95 96 85

61 54 70

93 94 77

69 61 80

90 91 78

IHC, immunohistochemistry; PPV, positive predictive value; NPV, negative predictive value.

correlated with the higher RNA ISH score, implying that high RNA ISH scores correlated with the relatively higher amounts of target gene. The higher RNA ISH score (score 3–4) was associated with younger age (P < 0.001), the conventional subtype (P = 0.008), and lymph node metastasis (P = 0.004). Thus, we predict that the correlated low Ct level and high RNA ISH score can be used to predict aggressive behavior, even after PTC diagnosis and confirmation of BRAF V600E mutation status. This finding proves that the RNA ISH score and the Ct value can be used as a predictor of poor prognosis and may be useful in treatment optimization for PTC patients. In conclusion, our study showed the clinical significance of the BRAFV600E mutation and the clinical usefulness of BRAF V600E IHC for detecting it. Moreover, we have found that lower Ct levels and high RNA ISH scores, which correlated with each other, were significantly associated with clinicopathologic features of poor prognosis. BRAF V600E IHC can be used as a screening test for the detection of the BRAFV600E mutation, and a high RNA ISH score may be used to predict a higher level of gene expression and aggressive behavior in PTCs. Acknowledgements This Research was supported by Chung-Ang University Research Grants in 2013. References [1] K. Affolter, W. Samowitz, S. Tripp, M.P. Bronner, BRAF V600E mutation detection by immunohistochemistry in colorectal carcinoma, Genes Chromosomes Cancer 52 (2013) 748–752. [2] S.K. Baek, K.Y. Jung, S.M. Kang, S.Y. Kwon, J.S. Woo, S.H. Cho, E.J. Chung, Clinical risk factors associated with cervical lymph node recurrence in papillary thyroid carcinoma, Thyroid 20 (2010) 147–152. [3] J.A. Bishop, X.J. Ma, H. Wang, Y. Luo, P.B. Illei, S. Begum, J.M. Taube, W.M. Koch, W.H. Westra, Detection of transcriptionally active high-risk HPV in patients with head and neck squamous cell carcinoma as visualized by a novel E6/E7 mRNA in situ hybridization method, Am. J. Surg. Pathol. 36 (2012) 1874–1882. [4] H. Davies, G.R. Bignell, C. Cox, P. Stephens, S. Edkins, S. Clegg, J. Teague, H. Woffendin, M.J. Garnett, W. Bottomley, N. Davis, E. Dicks, R. Ewing, Y. Floyd, K. Gray, S. Hall, R. Hawes, J. Hughes, V. Kosmidou, A. Menzies, C. Mould, A. Parker, C. Stevens, S. Watt, S. Hooper, R. Wilson, H. Jayatilake, B.A. Gusterson, C. Cooper, J. Shipley, D. Hargrave, K. Pritchard-Jones, N. Maitland, G. Chenevix-Trench, G.J. Riggins, D.D. Bigner, G. Palmieri, A. Cossu, A. Flanagan, A. Nicholson, J.W. Ho, S.Y. Leung, S.T. Yuen, B.L. Weber, H.F. Seigler, T.L. Darrow, H. Paterson, R. Marais, C.J. Marshall, R. Wooster, M.R. Stratton, P.A. Futreal, Mutations of the BRAF gene in human cancer, Nature 417 (2002) 949–954. [5] Q. Du, W. Yan, V.H. Burton, S.M. Hewitt, L. Wang, N. Hu, P.R. Taylor, M.D. Armani, S. Mukherjee, M.R. Emmert-Buck, M.A. Tangrea, Validation of esophageal squamous cell carcinoma candidate genes from high-throughput transcriptomic studies, Am. J. Cancer Res. 3 (2013) 402–410. [6] S.B. F.A. Edge, D.R. Byrd, F.L. Greene, C.C. Compton, A. Trotti III (Eds.), AJCC Cancer Staging Manual, seventh ed., Springer-Verlag, New York, NY, 2009. [7] M.J. Garnett, R. Marais, Guilty as charged: B-RAF is a human oncogene, Cancer Cell 6 (2004) 313–319. [8] R. Hoshino, Y. Chatani, T. Yamori, T. Tsuruo, H. Oka, O. Yoshida, Y. Shimada, S. Ari-i, H. Wada, J. Fujimoto, M. Kohno, Constitutive activation of the 41-/43kDa mitogen-activated protein kinase signaling pathway in human tumors, Oncogene 18 (1999) 813–822. [9] G.M. Howell, M.N. Nikiforova, S.E. Carty, M.J. Armstrong, S.P. Hodak, M.T. Stang, K.L. McCoy, Y.E. Nikiforov, L. Yip, BRAF V600E mutation independently predicts central compartment lymph node metastasis in patients with papillary thyroid cancer, Ann Surg Oncol 20 (2013) 47–52. [10] H.J. Joo, S.S. Han, J.T. Kwon, E.S. Park, Y.Y. Jung, H.K. Kim, Epidural intracranial metastasis from benign leiomyoma: a case report with literature review, Clin. Neurol. Neurosurg. 115 (2013) 1180–1183.

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Please cite this article in press as: Y.Y. Jung, et al., Clinicopathologic correlations of the BRAFV600E mutation, BRAF V600E immunohistochemistry, and BRAF RNA in situ hybridization in papillary thyroid carcinoma, Pathol. – Res. Pract (2014), http://dx.doi.org/10.1016/j.prp.2014.10.005