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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×).
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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
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
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
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
ARTICLE IN PRESS Pathology – Research and Practice xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Pathology – Research and Practice journal homepage: www.elsevier.com/locate/prp
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
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
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
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
