Pathology (December 2013) 45(7), pp. 637–644
ANATOMICAL PATHOLOGY
The prognostic significance of the BRAFV600E mutation in papillary thyroid carcinoma detected by mutation-specific immunohistochemistry PENELOPE A. MCKELVIE*{, FIONA CHAN*{, YONG YU*, PAUL WARING{, IRMA GRESSHOFF{, STEPHEN FARRELL{ AND RICHARD A. WILLIAMS* Departments of *Anatomical Pathology, {Surgery, St Vincent’s Hospital, Melbourne, and zDepartment of Pathology, University of Melbourne, Melbourne, Victoria, Australia; zthese authors contributed equally
Summary Aims: BRAFV600E mutation has been shown in a large metaanalysis to be an independent prognostic marker for papillary thyroid carcinoma (PTC) with poorer survival and higher recurrence rates. Methods: We studied prevalence of BRAFV600E mutation in 77 patients with PTC from an Australian cohort using competitive polymerase chain reaction (C-PCR) and immunohistochemistry (IHC) with BRAFV600E-specific antibody, VE1. Clinicopathological parameters, recurrence and mortality were analysed according to BRAFV600E mutation status. Results: Median follow-up was 84.5 months. BRAFV600E mutation was demonstrated in 65% of cases combining both C-PCR and IHC; in 71% (37/77) of tumours >1 cm and 52% (13/25) of microcarcinomas (45 years at diagnosis, size of primary tumour, extrathyroidal extension, lymph node or distant metastases or clinical stage at diagnosis. Conclusions: BRAFV600E mutation in PTC determined by IHC is associated with significantly increased risk of lymph node recurrence. Key words: BRAFV600E mutation, competitive PCR, lymph node recurrences, papillary thyroid carcinoma, prognostic factors, specific BRAFV600E mutation immunohistochemistry. Received 23 March, revised 20 May, accepted 22 May 2013
INTRODUCTION Thyroid carcinomas are the most common malignancies of the endocrine system and papillary thyroid carcinoma accounts for approximately 80–85% of these.1,2 The prognosis is generally very good with overall survival greater than 90% at 10 years.2 Nevertheless, up to 15% will present with recurrent disease within 10 years of diagnosis.3 The prevalence of thyroid cancer is increasing worldwide with the greatest rise between 1973 and 2002 reported in New South Wales, Australia, where there was an increase of 178% and 252% in men and women, respectively.1,4 Virtually all of the increase in thyroid cancer in the US has been due to a rise in incidence in papillary thyroid Print ISSN 0031-3025/Online ISSN 1465-3931 DOI: 10.1097/PAT.0000000000000008
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cancer (PTC).5 This rise has been attributed only in part to increased detection.1 Prognosis in PTC correlates with various tumour characteristics including size, extrathyroidal extension, lymph node and distant metastases as well as patient variables such as age and gender.1 The BRAF (the B-isoform of Raf kinase) oncogene has been shown to be mutated in various tumours including PTC.6,7 The most common mutation in BRAF is due to a T to A transversion at position 1796, resulting in a change from valine to glutamate at position 600 of the kinase activation segment (V600E mutation).6,7 This BRAFV600E mutation is found in about 49% (range 28–81%) of PTC, but is not found in benign lesions of thyroid nor medullary or follicular thyroid carcinomas.6,8 Many studies including two recent meta-analyses have shown the adverse prognostic effect of the BRAFV600E point mutation in papillary thyroid carcinoma.8–10 The largest meta-analyses of 5655 patients, including 27 different studies with negative and positive data, found that papillary thyroid carcinoma with the BRAFV600E mutation showed a 1.5–2.1fold increase in the risk of recurrent and persistent disease as well as extrathyroidal extension, lymph node metastases and higher TNM stages compared with patients with tumours showing wild-type BRAF gene.8 BRAFV600E point mutation can be detected by a variety of methods including mutation specific polymerase chain reaction, gene sequencing (direct DNA sequencing or pyrosequencing) and single-strand conformation polymorphism.11 Recently a mutation-specific antibody (clone VE1) has been developed, which allows immunohistochemical detection of the BRAFV600E protein in tissues of formalin fixed, paraffin embedded (FFPE) tissues.12–17 Its use has been reported in brain tumours,12,13 melanoma,12,18–21 papillary thyroid carcinoma,12,14,22 ovarian epithelial tumours,23,24 lung adenocarcinoma,24,25 hairy cell leukaemia,17 renal metanephric adenoma,25,26 in Langerhans cell histiocytosis15,16 and Erdheim–Chester disease,15,16 all diseases known to harbour BRAF mutations. One Australian group has already shown the adverse effect of BRAFV600E point mutation on disease-free survival in PTC with long follow-up data and analysis by both sequencing27 and more recently using specific BRAFV600E immunohistochemistry.22,27 We have undertaken a study of patients with papillary thyroid carcinoma, including detection of the BRAFV600E point mutation by mutation specific polymerase chain reaction, and compared this with detection by immunohistochemistry. This study seeks to confirm the prognostic value of the BRAFV600E mutation detected by IHC in a series of PTC with long clinical follow-up.
2013 Royal College of Pathologists of Australasia
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McKELVIE et al.
MATERIALS AND METHODS Patients Cases with a histological diagnosis of papillary thyroid carcinoma (PTC) between 1998 and 2011 were selected from the files of the Anatomical Pathology department of St Vincent’s Hospital, Melbourne. Data regarding follow-up, recurrence, lymph node and distant metastases, and treatment including iodine1,31 therapy was obtained from public hospital and private practice medical records. Disease-free survivors were identified as patients with no radiological (i.e., neck ultrasonography, chest computer assisted tomography or whole body radioactive iodine scan) or biochemical (i.e., serum thyroglobulin 85% of tumour cells. In cases where there was discordance between PCR and immunohistochemistry, SNaPshot PCR was performed on tumour dissected from sections on slides. SNaPshot PCR For details of SNaPshot PCR method see Supplementary Methods (http:// links.lww.com/PAT/A12). A H&E stained section was examined by a pathologist to determine and mark the tumour region. This was overlaid on an unstained slide, the tumour was scraped off the slide and DNA extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen) according to the manufacturer’s instructions. The region encompassing nucleotides 1798, 1799 and 1800 of the BRAF gene was amplified by PCR and the presence of a mutation was detected using a commercially available SNaPshot kit (Life Technologies, USA).33 Briefly, primers specifically designed to hybridise immediately adjacent to the mutation sites, were hybridised to the PCR product. Using all four fluorescently labelled dideoxynucleotides allowed the nucleotides at positions 1798, 1799 and 1800 to be determined after analysing the labelled primer-extension products on an ABI PRISM 3130xl genetic analyser (Applied Biosystems, USA). An estimate of mutation load (sensitivity) was determined by measuring the area under the mutant peak and comparing to the wild-type peak. Statistical analysis Statistical analysis was performed using Minitab 16. Data were reviewed and analysed by Sandy Clarke at the Statistical Consulting Centre, University of Melbourne. Means of continuous variables for the two groups (BRAF mutation positive and negative) were compared using independent samples t-tests. Frequencies of categorical variables for the two groups were compared using x2 tests, except for combinations with small frequencies when a Fisher’s exact test was used. A comparison of the progression-free survival for the BRAF positive and negative groups was made using a log-rank test. A Kaplan–Meier curve by group was also made. Statistical significance was set at p < 0.05.
RESULTS Patients One hundred and thirty-one patients with a diagnosis of primary papillary thyroid carcinoma were identified retrospectively from a database of biopsy cases reported at St Vincent’s Hospital between the years 1998–2006 and included some later cases up to 2011. Of these, FFPE tissue was available in 77 cases. The cohort included 25 cases of papillary thyroid microcarcinoma. Sixty-seven patients underwent a total thyroidectomy or hemithyroidectomy followed by completion thyroidectomy, and ten underwent hemithyroidectomy alone. Therapeutic lymph node resection at diagnosis was performed in 29 patients (38%) (12 lateral, 7 central compartment, 10 central and lateral nodes). Fifty patients with total thyroidectomy had at least one ablative dose of radioactive iodine. Of these 50 patients, 22 (44%) had therapeutic lymph node dissection. Follow-up was obtained in 74 patients; 51 of 52 with tumours >1 cm and 23 of 25 with microcarcinoma, with a median follow-up of 84.5 months (range 20–192 months). The clinical and pathologicial features of the patients, categorised according to treatment received, are listed in Tables 1–3. The three groups include the entire group of 77 patients (74 with follow-up) (Table 1); 67 patients (66 with follow-up) treated with total thyroidectomy (Table 2) and 50 patients (all with follow-up) treated with total thyroidectomy and radioiodine ablation (Table 3).
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BRAFV600E MUTATION IN PTC DETECTED BY IMMUNOHISTOCHEMISTRY
Table 1
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Clinicopathological features of 77 patients with papillary thyroid carcinoma
Continuous variables, median (range) Age at diagnosis, years Follow-up, months Tumour diameter, mm Categorical variables, n (%) Gender Male Female Age, years 45 >45 Stage I II III IV Extrathyroidal extension Yes No Lymph node metastasis at diagnosis Yes No Distant metastasis at diagnosis Yes No Lymph node recurrence Yes No Death Yes No Recurrence, persistence, death Yes No Histological subtyping Classical Follicular Tall cell Microcarcinoma
Mutated BRAF (n ¼ 50)
Non-mutated BRAF (n ¼ 27)
PTC total (n ¼ 77)
43.5 (22–87) 84.5 (0–192) 15 (3.8–60)
46 (20–78) 86 (0–163) 12 (1.6–60)
46 (20–87) 86 (0–192) 15 (1.6–60)
19 (82.6%) 31 (57.4%)
4 (17.4%) 23 (42.6%)
23 54
0.034
26 (68.4%) 24 (61.5%)
12 (31.6%) 15 (38.5%)
38 39
0.5
32 2 6 10
19 0 1 7
(37.3%) (0%) (14.3%) (41.2%)
51 2 7 17
0.5
19 (67.9%) 31 (63.3%)
9 (32.1%) 18 (36.7%)
28 49
0.7
19 (65.5%) 31 (64.6%)
10 (34.5%) 17 (35.4%)
29 48
0.9
3 (42.9%) 47 (67.1%)
4 (57.1%) 23 (32.9%)
7 70
0.2
11 (91.7%) 39 (60.0%)
1 (8.3%) 26 (40.0%)
12 65
0.035
5 (83.3%) 45 (63.4%)
1 (16.7%) 26 (36.6%)
6 71
0.4
16 (69.6%) 34 (63.0%)
7 (30.4%) 20 (37.0%)
23 54
0.6
32 3 2 13
13 2 0 12
45 5 2 25
0.3
(62.7%) (100%) (85.7%) (58.8%)
(71.1%) (60.0%) (100%) (52.0%)
(28.9%) (40.0%) (0%) (48.0%)
p value
>0.9 0.7 0.7
All tests for continuous variables are t tests. All tests for categorical variables are chi-squared except for stage, death, distant metastasis and histological subtyping which use a Fisher’s exact test due to small numbers.
BRAFV600E mutation by C-PCR The BRAFV600E mutation was found by C-PCR in 53% (41 /77) cases studied, in 61.5% (32/52) of the carcinomas >1 cm diameter and 36% (9/25) of the microcarcinomas. BRAFV600E mutation detection by mutation-specific immunohistochemistry Sections of all 77 primary tumours were tested by immunohistochemistry. Of these, six cases had no tumour left in the block after initial C-PCR studies. Five of these tumours were BRAF wild-type by C-PCR and one had the BRAF V600E mutation. Sufficient tumour was present in sections of 71 primary tumours for evaluation by IHC. Seventy per cent of the cases (50/71) showed strong cytoplasmic staining with the VE1 antibody throughout the tumour (Fig. 1A,B), including nine cases that were BRAFV600E mutation negative by prior C-PCR. Four of these tumours were microcarcinomas. We did not find any cytoplasmic reactivity of normal thyroid epithelium with the VE1 antibody (Fig. 1C). Occasionally, we observed positive nuclear staining on normal thyroid epithelial
cells (Fig. 1C). In the majority of cases, we only examined one block of the carcinoma by immunohistochemistry and were not able to comment about clonality of the tumours. There was diffuse homogeneous staining throughout tumour in almost all cases. Nine cases had some focal variation in intensity of the immunoreactivity or even focal loss of staining, in areas of tumour around the defect due to the punch biopsy or in markedly cystic areas of tumour with large spaces in the tissue. Due to the localisation of these areas of variable intensity or focal staining loss to the areas with tissue defects or large cystic spaces, we interpreted this as artefactual rather than a true reflection of clonality. SNaPshot PCR results SNaPshot, performed from DNA obtained from paraffin sections, was deployed to resolve the discrepancies between C-PCR and IHC for the nine discordant cases. SNaPshot is an allele-specific PCR method with a sensitivity of 3% in our laboratory. In eight cases, SNaPshot PCR was positive in all cases that were positive by VE1 IHC and negative by C-PCR. These cases were deemed to be false negative C-PCR due to
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640 Table 2
McKELVIE et al.
Pathology (2013), 45(7), December
Clinicopathological features of 67 patients treated with total thyroidectomy
Continuous variables, median (range) Age at diagnosis, years Follow-up, months Tumour diameter, mm Categorical variables, n (%) Gender Male Female Age, years 45 >45 Stage I II III IV Extrathyroidal extension Yes No Lymph node metastasis at diagnosis Yes No Distant metastasis at diagnosis Yes No Lymph node recurrence Yes No Death Yes No Recurrence, persistence, death Yes No
Mutated BRAF (n ¼ 45)
Non-mutated BRAF (n ¼ 22)
PTC total (n ¼ 67)
44 (25–87) 88 (0–192) 15 (5–60)
46 (20–78) 92.5 (27–163) 13 (2–60)
46 (20–87) 88 (0–192) 15 (2–60)
19 (82.6%) 26 (59.1%)
4 (17.4%) 18 (40.9%)
23 44
0.052
23 (69.7%) 22 (64.7%)
10 (30.3%) 12 (35.3%)
33 34
0.7
29 2 6 8
14 0 1 7
(32.6%) (0%) (14.3%) (46.7%)
43 2 7 15
0.5
18 (69.2%) 27 (65.9%)
8 (30.8%) 14 (34.1%)
26 41
0.8
18 (66.7%) 27 (67.5%)
9 (33.3%) 13 (32.5%)
27 40
0.9
2 (33.3%) 43 (70.5%)
4 (66.7%) 18 (29.5%)
6 61
0.085
11 (91.7%) 34 (61.8%)
1 (8.3%) 21 (38.2%)
12 55
0.046
4 (80.0%) 41 (66.1%)
1 (20.0%) 21 (33.9%)
5 62
>0.9
15 (68.2%) 30 (66.7%)
7 (31.8%) 15 (33.3%)
22 45
0.9
(67.4%) (100%) (85.7%) (53.3%)
p value
0.9 0.5 >0.9
All tests for continuous variables are t tests. All tests for categorical variables are chi-squared except for stage, death and distant metastasis which use a Fisher’s exact test due to small numbers.
lesser sensitivity of the C-PCR method. One case was clearly positive by VE1 IHC, but was negative by both C-PCR and SNaPshot PCR. Overall, using the combination of BRAFV600E mutation results from C-PCR in 77 cases, VE1 IHC in 71 cases and SNaPshot PCR in nine cases, we found BRAFV600E mutations in 65% (50/77) of cases. Immunohistochemistry with the VE1 antibody was more sensitive than C-PCR, with nine additional cases being detected, eight confirmed by SNaPshot PCR. BRAFV600E mutation profile in primary and metastatic tumours Synchronous lymph node metastases were tested together with the primary thyroid tumour in eight cases. All of the tumours were concordant by C-PCR and IHC in both specimens. Four of these cases tested positive for the mutation and four were negative. Three patients had metachronous lymph node recurrences. Two patients were concordant for BRAF mutation by C-PCR and IHC in both the primary and recurrent tumours. In the third case, the metastasis tested positive by C-PCR and IHC. The primary tumour was a microcarcinoma which tested negative by C-PCR but there was no residual primary tumour for IHC studies. The block of the primary tumour in this case was 15 years old. It is possible that either the C-PCR on the primary tumour gave a false negative result due to the age of the block and relative insensitivity of the technique or that the
BRAF mutation was acquired by the metastasis, as has been reported by others.34,35 Association of BRAFV600E mutation with histological subtypes (Table 1) In our cohort, we had 52 (67.5%) tumours measuring >1 cm and 25 (32.5%) microcarcinomas, measuring 1 cm, 45 (86.5%) were classical papillary variant, five (9.6%) were follicular variant (FVPTC) and two (3.8%) tall cell variant. All of our FVPTC were infiltrating, three widely infiltrating through the gland, one encapsulated but invading through the capsule into normal thyroid parenchyma and one partly encapsulated with invasion of adjacent thyroid.30,31 Our series did not include any encapsulated FVPTC without invasion. BRAFV600E mutation was found more commonly (71%) in classical pattern PTC compared with follicular variant (60%), and both tall cell carcinomas had the BRAFV600E mutation. The relatively high prevalence of BRAFV600E mutation in our FVPTC most probably relates to the invasive nature of all of the five tumours in that group, since it has been reported by several studies that infiltrating FVPTC has a different biological behaviour from the encapsulated non-invasive FVPTC, and behaves more like classical PTC with frequent lymph node metastases.30,31,36,37 More recently, molecular profiling has shown that the encapsulated non-invasive type of FVPTC has genetic changes very close to follicular adenomas/carcinomas (such as high
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BRAFV600E MUTATION IN PTC DETECTED BY IMMUNOHISTOCHEMISTRY
Table 3
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Clinicopathological features of 50 patients treated with total thyroidectomy and radioiodine ablation
Continuous variables, median (range) Age at diagnosis, years Follow-up, months Tumour diameter, mm Categorical variables, n (%) Gender Male Female Age, years 45 >45 Stage I II III IV Extrathyroidal extension Yes No Lymph node metastasis at diagnosis Yes No Distant metastasis at diagnosis Yes No Lymph node recurrence Yes No Death Yes No Recurrence, persistence, death Yes No
Mutated BRAF (n ¼ 33)
Non-mutated BRAF (n ¼ 17)
PTC total (n ¼ 50)
41 (25–78) 102 (20–192) 15 (5–60)
40 (20–78) 86 (40–154) 15 (3–50)
43.5 (20–78) 100 (20–192) 15 (3–60)
15 (83.3%) 18 (56.3%)
3 (16.7%) 14 (43.8%)
18 32
0.052
19 (73.1%) 14 (58.3%)
7 (26.9%) 10 (41.7%)
26 24
0.3
21 2 4 6
10 0 1 6
(32.3%) (0%) (20.0%) (50.0%)
31 2 5 12
0.6
13 (65.0%) 20 (66.7%)
7 (35.0%) 10 (33.3%)
20 30
0.9
14 (63.6%) 19 (67.9%)
8 (36.4%) 9 (32.1%)
22 28
0.8
3 (50.0%) 30 (68.2%)
3 (50.0%) 14 (31.8%)
6 44
0.4
10 (100%) 23 (57.5%)
0 (0%) 17 (42.5%)
10 40
0.011
2 (100%) 31 (64.6%)
0 (0%) 17 (35.4%)
2 48
0.5
12 (70.6%) 21 (63.6%)
5 (29.4%) 12 (36.4%)
17 33
0.6
(67.7%) (100%) (80.0%) (50.0%)
p value
0.3 0.8 0.8
All tests for continuous variables are t tests. All tests for categorical variables are chi-squared except for stage, death and distant metastasis which use a Fisher’s exact test due to small numbers.
rate of RAS mutations and absence of BRAF mutations), compared with infiltrative FVPTC, which has a different profile, with higher BRAFV600E mutation, more similar to classical PTC.30,36 We did not find any statistically significant differences with regard to subtype, which most likely relates to our relatively small sample size in the subgroups and absence of encapsulated non-invasive FVPTC in our series.
and one BRAFV600E negative. All were 50 years or older (three were >65 years) and five had stage IV disease at diagnosis. Only five patients (7.5%; four BRAFV600E positive) died in the group with total thyroidectomy and two (both BRAFV600E positive) in the group with total thyroidectomy and radioactive iodine ablation.
DISCUSSION Association of BRAFV600E mutation with clinical features There was a significant association between BRAFV600E mutation and male sex ( p ¼ 0.034) for the whole group of 77 (Table 1), but no significant association was found between the BRAFV600E mutation and age >45 years at diagnosis, size of primary tumour, extrathyroidal extension, lymph node or distant metastases or clinical stage (AJCC) at diagnosis. However, there was a significantly increased risk of lymph node recurrence for the BRAFV600E positive group in all three groups, i.e., for the total cohort, those with total thyroidectomy and the group with total thyroidectomy and radioactive iodine ablation ( p values of 0.02, 0.046 and 0.011, respectively). For the latter group, there was a trend to decreased 10 year progression-free survival ( p ¼ 0.31) in those patients with BRAFV600E positive tumours (Fig. 2). Ten year progressionfree survival for the BRAF wild-type group was 83.3% and for the BRAFV600E positive group 56.2%. Disease specific mortality was low in the cohort of 77 with only six patients (7.8%), five with BRAFV600E positive tumours
Our series of 77 patients with PTC has demonstrated BRAFV600E mutation in 65% of PTC of all sizes with higher prevalence (71%) in those with a diameter greater than one centimetre compared with only 52% of the 25 microcarcinomas. The long follow-up in our study showed significantly higher lymph node recurrence for the BRAFV600E positive patients in the entire cohort of 77 patients as well as those treated with total thyroidectomy or total thyroidectomy and radioactive iodine ablation. Another Australian group has also recently demonstrated increased risk of clinically evident disease recurrence and/or death in their patients with BRAFV600E positive tumours.22,27 Our study also showed a trend to decreased 10 year progression-free survival for the BRAFV600E positive cohort compared to those with BRAFV600E negative tumours in those treated with total thyroidectomy and radioablation ( p ¼ 0.196). However, longer median followup (up to 15 years) for a small cohort, similar to that of Elisei et al., is required before statistically significant disease-specific mortality associated with BRAFV600E mutation
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642
McKELVIE et al.
Pathology (2013), 45(7), December
A
B
C
Fig. 1 (A) Immunohistochemistry for BRAFV600E mutation using specific VE1 antibody in a case of classical pattern papillary thyroid carcinoma shows diffuse cytoplasmic reactivity. (B) High power shows papillary thyroid carcinoma using specific VE1 antibody. (C) Papillary thyroid carcinoma (BRAFV600E mutation þve) infiltrates normal gland using specific VE1 antibody.
can be demonstrated.38 A very recent retrospective multicentre international study demonstrated the association between BRAFV600E mutation with overall disease-specific mortality.39 However, this increase in mortality in patients with BRAFV600E mutation positive tumours was not independent of factors such as lymph node metastases, distant metastasis and extrathyroidal extension.39 Our study is the second to demonstrate the use of immunohistochemistry for BRAFV600E mutation to provide prognostic information in PTC.22 The study by Bullock et al. included 96 patients with molecular data, of which 71% of tumours showed BRAFV600E point mutation by IHC and 61% by sequencing.22 These authors also found that IHC was more sensitive than molecular techniques including massively parallel and Sanger sequencing, since 30% of their 10 discordant cases remained negative after repeat sequencing, by both methods. These authors suggest that specific BRAFV600E IHC may be the new gold standard for detection of BRAFV600E point mutation in PTC.22 Two previous studies used the VE1 BRAFV600E mutation-specific antibody in PTC, but neither had clinical follow-up.12,14 Koperek and colleagues found concordance in detection of the BRAFV600E mutation in 39/39 papillary thyroid cancers by capillary sequencing and specific immunohistochemistry using the same antibody.14 However, their study only involved sequencing of BRAF gene in those tumours >2 cm and those cases with equivocal or focal VE1 antibody staining (which were all negative by sequencing).14
Progression free survival
Survival percentage
100
80
60
40 BRAF V600E Negative Positive
20
0 0
20
40
60
80
100
120
140
160
180
Months
Method Chi-square DF P-value 1 0.196 Log-rank 1.67431 Fig. 2 Progression-free survival curve of 50 patients with papillary thyroid carcinoma treated with total thyroidectomy and radioactive iodine ablation.
Capper et al. studied 21 cases of PTC by immunohistochemistry and sequencing from FFPE blocks with all 21 showing positive immunoreactivity but only 18 of 21 (86%) showing BRAFV600E mutation by sequencing.12 Our data show that immunohistochemistry for BRAFV600E mutation is more sensitive than standard C-PCR using a commercial kit (Seegene) for detecting the BRAFV600E mutation. Of our nine false negative C-PCR cases, six involved paraffin blocks greater than 12 years of age. Quality of DNA is known to be reduced in FFPE blocks due to fragmentation of DNA, and therefore the immunohistochemical detection of BRAFV600E mutation is more likely to be more sensitive than DNA based techniques, particularly in screening older cases from paraffin blocks.40,41 Multiple methods have been used to determine the presence of BRAFV600E mutation, including direct sequencing of DNA after PCR amplification alone, followed by colorimetric method, DNA PCR single-strand conformation polymorphism (PCR-SSCP) direct sequencing, DNA PCR and fluorescence melting curve analysis and DNA-mutant allele-specific amplification (DNA-MASA) sequencing.9 For convenience, we used C-PCR by commercial kit which has a reported sensitivity of 56.3% in FFPE tissues of thyroid compared with 65% for standard sequencing.11 Since it is clinically important to identify the most accurate method for detection of the BRAFV600E mutation, particularly in FFPE tissues with reduced DNA quality, we propose that the immunohistochemical method using VE1 is highly sensitive, specific and rapid, compared to molecular techniques, for a standard anatomical pathology laboratory which does not have facilities for routine sequencing of all PTC. Indeed, the homogeneous staining with the VE1 in the majority of cases makes identification of tumour cells and their extent of invasion much easier to assess. Our immunohistochemical results were very similar to those of Bullock et al. with diffuse homogeneous cytoplasmic reactivity in the tumour epithelial cells.22 However, several authors have suggested that BRAFV600E point mutation may not be an early clonal event and that BRAFV600E may be clonally heterogeneous within a single tumour.42–44 These studies were based on pyrosequencing of macroscopically dissected tumours with correction for the lymphoid and stromal infiltrates, laser capture microdissection of four cases and subcloning of PTC cells in tissue culture.42,43 Gandolfi et al. showed no difference in allele frequency of the BRAFV600E point mutation between two groups of PTC with and without lymph node metastases.44 These authors, however, noted a puzzling discrepancy between IHC staining using the specific VE1 antibody and molecular data from pyrosequencing analysis
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BRAFV600E MUTATION IN PTC DETECTED BY IMMUNOHISTOCHEMISTRY
in their cases, as the numbers of positive cells seemed higher than expected from allele frequency. One possible explanation is that the BRAFV600E mutation is found only in the epithelial cells of PTC, not in the stromal cores or vessels as is highlighted by the IHC (Fig. 1A,B), but in the molecular studies to determine allele frequency in PTC, the samples must, even in macro- and micro-dissected tumour, contain variable amounts of negative stromal and vascular elements which may dilute the apparent allele frequency. Immunohistochemical detection of BRAFV600E mutation by the anatomical pathologist at the time of diagnosis of PTC offers immediate prognostic information that may alter management in individual patients.9 Since our data demonstrate significantly increased risk of lymph node recurrences in PTC with BRAFV600E mutation, the surgeon may then proceed to prophylactic lymph node resection of clinically uninvolved nodes. Only 38% of our patients underwent lymph node dissection, almost all of which was therapeutic for clinically evident metastasis. On the basis of newly developed recommendations,45 recent clinical studies of papillary thyroid carcinoma now include prophylactic lymph node dissection, at least of central lymph nodes.27,46 Prevalence of lymph node metastasis on microscopy in lymph nodes with negative preoperative ultrasound examination is high, ranging from 42% in small primary tumours measuring 3 cm.46,47 Our data showed a significant association of BRAFV600E mutation with male gender, but not with other important known clinicopathological prognostic factors such as age at diagnosis greater than 45 years, size of the primary tumour, extrathyroidal extension, lymph node or distant metastases at diagnosis or AJCC stage of disease at presentation. This is most likely due to the small numbers of patients in our study.27,48 Another Australian study found a BRAFV600E mutation prevalence of 45% in 76 patients with PTC and an association of BRAFV600E mutation with gender, stage, and subtype (classical versus follicular).49 However, the two cohorts are not directly comparable, since Smith et al.’s cohort contained no stage IV disease compared with 22% in ours.49 Furthermore, 47% of their group were follicular variant PTC compared with
ANATOMICAL PATHOLOGY
The prognostic significance of the BRAFV600E mutation in papillary thyroid carcinoma detected by mutation-specific immunohistochemistry PENELOPE A. MCKELVIE*{, FIONA CHAN*{, YONG YU*, PAUL WARING{, IRMA GRESSHOFF{, STEPHEN FARRELL{ AND RICHARD A. WILLIAMS* Departments of *Anatomical Pathology, {Surgery, St Vincent’s Hospital, Melbourne, and zDepartment of Pathology, University of Melbourne, Melbourne, Victoria, Australia; zthese authors contributed equally
Summary Aims: BRAFV600E mutation has been shown in a large metaanalysis to be an independent prognostic marker for papillary thyroid carcinoma (PTC) with poorer survival and higher recurrence rates. Methods: We studied prevalence of BRAFV600E mutation in 77 patients with PTC from an Australian cohort using competitive polymerase chain reaction (C-PCR) and immunohistochemistry (IHC) with BRAFV600E-specific antibody, VE1. Clinicopathological parameters, recurrence and mortality were analysed according to BRAFV600E mutation status. Results: Median follow-up was 84.5 months. BRAFV600E mutation was demonstrated in 65% of cases combining both C-PCR and IHC; in 71% (37/77) of tumours >1 cm and 52% (13/25) of microcarcinomas (45 years at diagnosis, size of primary tumour, extrathyroidal extension, lymph node or distant metastases or clinical stage at diagnosis. Conclusions: BRAFV600E mutation in PTC determined by IHC is associated with significantly increased risk of lymph node recurrence. Key words: BRAFV600E mutation, competitive PCR, lymph node recurrences, papillary thyroid carcinoma, prognostic factors, specific BRAFV600E mutation immunohistochemistry. Received 23 March, revised 20 May, accepted 22 May 2013
INTRODUCTION Thyroid carcinomas are the most common malignancies of the endocrine system and papillary thyroid carcinoma accounts for approximately 80–85% of these.1,2 The prognosis is generally very good with overall survival greater than 90% at 10 years.2 Nevertheless, up to 15% will present with recurrent disease within 10 years of diagnosis.3 The prevalence of thyroid cancer is increasing worldwide with the greatest rise between 1973 and 2002 reported in New South Wales, Australia, where there was an increase of 178% and 252% in men and women, respectively.1,4 Virtually all of the increase in thyroid cancer in the US has been due to a rise in incidence in papillary thyroid Print ISSN 0031-3025/Online ISSN 1465-3931 DOI: 10.1097/PAT.0000000000000008
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cancer (PTC).5 This rise has been attributed only in part to increased detection.1 Prognosis in PTC correlates with various tumour characteristics including size, extrathyroidal extension, lymph node and distant metastases as well as patient variables such as age and gender.1 The BRAF (the B-isoform of Raf kinase) oncogene has been shown to be mutated in various tumours including PTC.6,7 The most common mutation in BRAF is due to a T to A transversion at position 1796, resulting in a change from valine to glutamate at position 600 of the kinase activation segment (V600E mutation).6,7 This BRAFV600E mutation is found in about 49% (range 28–81%) of PTC, but is not found in benign lesions of thyroid nor medullary or follicular thyroid carcinomas.6,8 Many studies including two recent meta-analyses have shown the adverse prognostic effect of the BRAFV600E point mutation in papillary thyroid carcinoma.8–10 The largest meta-analyses of 5655 patients, including 27 different studies with negative and positive data, found that papillary thyroid carcinoma with the BRAFV600E mutation showed a 1.5–2.1fold increase in the risk of recurrent and persistent disease as well as extrathyroidal extension, lymph node metastases and higher TNM stages compared with patients with tumours showing wild-type BRAF gene.8 BRAFV600E point mutation can be detected by a variety of methods including mutation specific polymerase chain reaction, gene sequencing (direct DNA sequencing or pyrosequencing) and single-strand conformation polymorphism.11 Recently a mutation-specific antibody (clone VE1) has been developed, which allows immunohistochemical detection of the BRAFV600E protein in tissues of formalin fixed, paraffin embedded (FFPE) tissues.12–17 Its use has been reported in brain tumours,12,13 melanoma,12,18–21 papillary thyroid carcinoma,12,14,22 ovarian epithelial tumours,23,24 lung adenocarcinoma,24,25 hairy cell leukaemia,17 renal metanephric adenoma,25,26 in Langerhans cell histiocytosis15,16 and Erdheim–Chester disease,15,16 all diseases known to harbour BRAF mutations. One Australian group has already shown the adverse effect of BRAFV600E point mutation on disease-free survival in PTC with long follow-up data and analysis by both sequencing27 and more recently using specific BRAFV600E immunohistochemistry.22,27 We have undertaken a study of patients with papillary thyroid carcinoma, including detection of the BRAFV600E point mutation by mutation specific polymerase chain reaction, and compared this with detection by immunohistochemistry. This study seeks to confirm the prognostic value of the BRAFV600E mutation detected by IHC in a series of PTC with long clinical follow-up.
2013 Royal College of Pathologists of Australasia
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638
McKELVIE et al.
MATERIALS AND METHODS Patients Cases with a histological diagnosis of papillary thyroid carcinoma (PTC) between 1998 and 2011 were selected from the files of the Anatomical Pathology department of St Vincent’s Hospital, Melbourne. Data regarding follow-up, recurrence, lymph node and distant metastases, and treatment including iodine1,31 therapy was obtained from public hospital and private practice medical records. Disease-free survivors were identified as patients with no radiological (i.e., neck ultrasonography, chest computer assisted tomography or whole body radioactive iodine scan) or biochemical (i.e., serum thyroglobulin 85% of tumour cells. In cases where there was discordance between PCR and immunohistochemistry, SNaPshot PCR was performed on tumour dissected from sections on slides. SNaPshot PCR For details of SNaPshot PCR method see Supplementary Methods (http:// links.lww.com/PAT/A12). A H&E stained section was examined by a pathologist to determine and mark the tumour region. This was overlaid on an unstained slide, the tumour was scraped off the slide and DNA extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen) according to the manufacturer’s instructions. The region encompassing nucleotides 1798, 1799 and 1800 of the BRAF gene was amplified by PCR and the presence of a mutation was detected using a commercially available SNaPshot kit (Life Technologies, USA).33 Briefly, primers specifically designed to hybridise immediately adjacent to the mutation sites, were hybridised to the PCR product. Using all four fluorescently labelled dideoxynucleotides allowed the nucleotides at positions 1798, 1799 and 1800 to be determined after analysing the labelled primer-extension products on an ABI PRISM 3130xl genetic analyser (Applied Biosystems, USA). An estimate of mutation load (sensitivity) was determined by measuring the area under the mutant peak and comparing to the wild-type peak. Statistical analysis Statistical analysis was performed using Minitab 16. Data were reviewed and analysed by Sandy Clarke at the Statistical Consulting Centre, University of Melbourne. Means of continuous variables for the two groups (BRAF mutation positive and negative) were compared using independent samples t-tests. Frequencies of categorical variables for the two groups were compared using x2 tests, except for combinations with small frequencies when a Fisher’s exact test was used. A comparison of the progression-free survival for the BRAF positive and negative groups was made using a log-rank test. A Kaplan–Meier curve by group was also made. Statistical significance was set at p < 0.05.
RESULTS Patients One hundred and thirty-one patients with a diagnosis of primary papillary thyroid carcinoma were identified retrospectively from a database of biopsy cases reported at St Vincent’s Hospital between the years 1998–2006 and included some later cases up to 2011. Of these, FFPE tissue was available in 77 cases. The cohort included 25 cases of papillary thyroid microcarcinoma. Sixty-seven patients underwent a total thyroidectomy or hemithyroidectomy followed by completion thyroidectomy, and ten underwent hemithyroidectomy alone. Therapeutic lymph node resection at diagnosis was performed in 29 patients (38%) (12 lateral, 7 central compartment, 10 central and lateral nodes). Fifty patients with total thyroidectomy had at least one ablative dose of radioactive iodine. Of these 50 patients, 22 (44%) had therapeutic lymph node dissection. Follow-up was obtained in 74 patients; 51 of 52 with tumours >1 cm and 23 of 25 with microcarcinoma, with a median follow-up of 84.5 months (range 20–192 months). The clinical and pathologicial features of the patients, categorised according to treatment received, are listed in Tables 1–3. The three groups include the entire group of 77 patients (74 with follow-up) (Table 1); 67 patients (66 with follow-up) treated with total thyroidectomy (Table 2) and 50 patients (all with follow-up) treated with total thyroidectomy and radioiodine ablation (Table 3).
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BRAFV600E MUTATION IN PTC DETECTED BY IMMUNOHISTOCHEMISTRY
Table 1
639
Clinicopathological features of 77 patients with papillary thyroid carcinoma
Continuous variables, median (range) Age at diagnosis, years Follow-up, months Tumour diameter, mm Categorical variables, n (%) Gender Male Female Age, years 45 >45 Stage I II III IV Extrathyroidal extension Yes No Lymph node metastasis at diagnosis Yes No Distant metastasis at diagnosis Yes No Lymph node recurrence Yes No Death Yes No Recurrence, persistence, death Yes No Histological subtyping Classical Follicular Tall cell Microcarcinoma
Mutated BRAF (n ¼ 50)
Non-mutated BRAF (n ¼ 27)
PTC total (n ¼ 77)
43.5 (22–87) 84.5 (0–192) 15 (3.8–60)
46 (20–78) 86 (0–163) 12 (1.6–60)
46 (20–87) 86 (0–192) 15 (1.6–60)
19 (82.6%) 31 (57.4%)
4 (17.4%) 23 (42.6%)
23 54
0.034
26 (68.4%) 24 (61.5%)
12 (31.6%) 15 (38.5%)
38 39
0.5
32 2 6 10
19 0 1 7
(37.3%) (0%) (14.3%) (41.2%)
51 2 7 17
0.5
19 (67.9%) 31 (63.3%)
9 (32.1%) 18 (36.7%)
28 49
0.7
19 (65.5%) 31 (64.6%)
10 (34.5%) 17 (35.4%)
29 48
0.9
3 (42.9%) 47 (67.1%)
4 (57.1%) 23 (32.9%)
7 70
0.2
11 (91.7%) 39 (60.0%)
1 (8.3%) 26 (40.0%)
12 65
0.035
5 (83.3%) 45 (63.4%)
1 (16.7%) 26 (36.6%)
6 71
0.4
16 (69.6%) 34 (63.0%)
7 (30.4%) 20 (37.0%)
23 54
0.6
32 3 2 13
13 2 0 12
45 5 2 25
0.3
(62.7%) (100%) (85.7%) (58.8%)
(71.1%) (60.0%) (100%) (52.0%)
(28.9%) (40.0%) (0%) (48.0%)
p value
>0.9 0.7 0.7
All tests for continuous variables are t tests. All tests for categorical variables are chi-squared except for stage, death, distant metastasis and histological subtyping which use a Fisher’s exact test due to small numbers.
BRAFV600E mutation by C-PCR The BRAFV600E mutation was found by C-PCR in 53% (41 /77) cases studied, in 61.5% (32/52) of the carcinomas >1 cm diameter and 36% (9/25) of the microcarcinomas. BRAFV600E mutation detection by mutation-specific immunohistochemistry Sections of all 77 primary tumours were tested by immunohistochemistry. Of these, six cases had no tumour left in the block after initial C-PCR studies. Five of these tumours were BRAF wild-type by C-PCR and one had the BRAF V600E mutation. Sufficient tumour was present in sections of 71 primary tumours for evaluation by IHC. Seventy per cent of the cases (50/71) showed strong cytoplasmic staining with the VE1 antibody throughout the tumour (Fig. 1A,B), including nine cases that were BRAFV600E mutation negative by prior C-PCR. Four of these tumours were microcarcinomas. We did not find any cytoplasmic reactivity of normal thyroid epithelium with the VE1 antibody (Fig. 1C). Occasionally, we observed positive nuclear staining on normal thyroid epithelial
cells (Fig. 1C). In the majority of cases, we only examined one block of the carcinoma by immunohistochemistry and were not able to comment about clonality of the tumours. There was diffuse homogeneous staining throughout tumour in almost all cases. Nine cases had some focal variation in intensity of the immunoreactivity or even focal loss of staining, in areas of tumour around the defect due to the punch biopsy or in markedly cystic areas of tumour with large spaces in the tissue. Due to the localisation of these areas of variable intensity or focal staining loss to the areas with tissue defects or large cystic spaces, we interpreted this as artefactual rather than a true reflection of clonality. SNaPshot PCR results SNaPshot, performed from DNA obtained from paraffin sections, was deployed to resolve the discrepancies between C-PCR and IHC for the nine discordant cases. SNaPshot is an allele-specific PCR method with a sensitivity of 3% in our laboratory. In eight cases, SNaPshot PCR was positive in all cases that were positive by VE1 IHC and negative by C-PCR. These cases were deemed to be false negative C-PCR due to
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640 Table 2
McKELVIE et al.
Pathology (2013), 45(7), December
Clinicopathological features of 67 patients treated with total thyroidectomy
Continuous variables, median (range) Age at diagnosis, years Follow-up, months Tumour diameter, mm Categorical variables, n (%) Gender Male Female Age, years 45 >45 Stage I II III IV Extrathyroidal extension Yes No Lymph node metastasis at diagnosis Yes No Distant metastasis at diagnosis Yes No Lymph node recurrence Yes No Death Yes No Recurrence, persistence, death Yes No
Mutated BRAF (n ¼ 45)
Non-mutated BRAF (n ¼ 22)
PTC total (n ¼ 67)
44 (25–87) 88 (0–192) 15 (5–60)
46 (20–78) 92.5 (27–163) 13 (2–60)
46 (20–87) 88 (0–192) 15 (2–60)
19 (82.6%) 26 (59.1%)
4 (17.4%) 18 (40.9%)
23 44
0.052
23 (69.7%) 22 (64.7%)
10 (30.3%) 12 (35.3%)
33 34
0.7
29 2 6 8
14 0 1 7
(32.6%) (0%) (14.3%) (46.7%)
43 2 7 15
0.5
18 (69.2%) 27 (65.9%)
8 (30.8%) 14 (34.1%)
26 41
0.8
18 (66.7%) 27 (67.5%)
9 (33.3%) 13 (32.5%)
27 40
0.9
2 (33.3%) 43 (70.5%)
4 (66.7%) 18 (29.5%)
6 61
0.085
11 (91.7%) 34 (61.8%)
1 (8.3%) 21 (38.2%)
12 55
0.046
4 (80.0%) 41 (66.1%)
1 (20.0%) 21 (33.9%)
5 62
>0.9
15 (68.2%) 30 (66.7%)
7 (31.8%) 15 (33.3%)
22 45
0.9
(67.4%) (100%) (85.7%) (53.3%)
p value
0.9 0.5 >0.9
All tests for continuous variables are t tests. All tests for categorical variables are chi-squared except for stage, death and distant metastasis which use a Fisher’s exact test due to small numbers.
lesser sensitivity of the C-PCR method. One case was clearly positive by VE1 IHC, but was negative by both C-PCR and SNaPshot PCR. Overall, using the combination of BRAFV600E mutation results from C-PCR in 77 cases, VE1 IHC in 71 cases and SNaPshot PCR in nine cases, we found BRAFV600E mutations in 65% (50/77) of cases. Immunohistochemistry with the VE1 antibody was more sensitive than C-PCR, with nine additional cases being detected, eight confirmed by SNaPshot PCR. BRAFV600E mutation profile in primary and metastatic tumours Synchronous lymph node metastases were tested together with the primary thyroid tumour in eight cases. All of the tumours were concordant by C-PCR and IHC in both specimens. Four of these cases tested positive for the mutation and four were negative. Three patients had metachronous lymph node recurrences. Two patients were concordant for BRAF mutation by C-PCR and IHC in both the primary and recurrent tumours. In the third case, the metastasis tested positive by C-PCR and IHC. The primary tumour was a microcarcinoma which tested negative by C-PCR but there was no residual primary tumour for IHC studies. The block of the primary tumour in this case was 15 years old. It is possible that either the C-PCR on the primary tumour gave a false negative result due to the age of the block and relative insensitivity of the technique or that the
BRAF mutation was acquired by the metastasis, as has been reported by others.34,35 Association of BRAFV600E mutation with histological subtypes (Table 1) In our cohort, we had 52 (67.5%) tumours measuring >1 cm and 25 (32.5%) microcarcinomas, measuring 1 cm, 45 (86.5%) were classical papillary variant, five (9.6%) were follicular variant (FVPTC) and two (3.8%) tall cell variant. All of our FVPTC were infiltrating, three widely infiltrating through the gland, one encapsulated but invading through the capsule into normal thyroid parenchyma and one partly encapsulated with invasion of adjacent thyroid.30,31 Our series did not include any encapsulated FVPTC without invasion. BRAFV600E mutation was found more commonly (71%) in classical pattern PTC compared with follicular variant (60%), and both tall cell carcinomas had the BRAFV600E mutation. The relatively high prevalence of BRAFV600E mutation in our FVPTC most probably relates to the invasive nature of all of the five tumours in that group, since it has been reported by several studies that infiltrating FVPTC has a different biological behaviour from the encapsulated non-invasive FVPTC, and behaves more like classical PTC with frequent lymph node metastases.30,31,36,37 More recently, molecular profiling has shown that the encapsulated non-invasive type of FVPTC has genetic changes very close to follicular adenomas/carcinomas (such as high
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BRAFV600E MUTATION IN PTC DETECTED BY IMMUNOHISTOCHEMISTRY
Table 3
641
Clinicopathological features of 50 patients treated with total thyroidectomy and radioiodine ablation
Continuous variables, median (range) Age at diagnosis, years Follow-up, months Tumour diameter, mm Categorical variables, n (%) Gender Male Female Age, years 45 >45 Stage I II III IV Extrathyroidal extension Yes No Lymph node metastasis at diagnosis Yes No Distant metastasis at diagnosis Yes No Lymph node recurrence Yes No Death Yes No Recurrence, persistence, death Yes No
Mutated BRAF (n ¼ 33)
Non-mutated BRAF (n ¼ 17)
PTC total (n ¼ 50)
41 (25–78) 102 (20–192) 15 (5–60)
40 (20–78) 86 (40–154) 15 (3–50)
43.5 (20–78) 100 (20–192) 15 (3–60)
15 (83.3%) 18 (56.3%)
3 (16.7%) 14 (43.8%)
18 32
0.052
19 (73.1%) 14 (58.3%)
7 (26.9%) 10 (41.7%)
26 24
0.3
21 2 4 6
10 0 1 6
(32.3%) (0%) (20.0%) (50.0%)
31 2 5 12
0.6
13 (65.0%) 20 (66.7%)
7 (35.0%) 10 (33.3%)
20 30
0.9
14 (63.6%) 19 (67.9%)
8 (36.4%) 9 (32.1%)
22 28
0.8
3 (50.0%) 30 (68.2%)
3 (50.0%) 14 (31.8%)
6 44
0.4
10 (100%) 23 (57.5%)
0 (0%) 17 (42.5%)
10 40
0.011
2 (100%) 31 (64.6%)
0 (0%) 17 (35.4%)
2 48
0.5
12 (70.6%) 21 (63.6%)
5 (29.4%) 12 (36.4%)
17 33
0.6
(67.7%) (100%) (80.0%) (50.0%)
p value
0.3 0.8 0.8
All tests for continuous variables are t tests. All tests for categorical variables are chi-squared except for stage, death and distant metastasis which use a Fisher’s exact test due to small numbers.
rate of RAS mutations and absence of BRAF mutations), compared with infiltrative FVPTC, which has a different profile, with higher BRAFV600E mutation, more similar to classical PTC.30,36 We did not find any statistically significant differences with regard to subtype, which most likely relates to our relatively small sample size in the subgroups and absence of encapsulated non-invasive FVPTC in our series.
and one BRAFV600E negative. All were 50 years or older (three were >65 years) and five had stage IV disease at diagnosis. Only five patients (7.5%; four BRAFV600E positive) died in the group with total thyroidectomy and two (both BRAFV600E positive) in the group with total thyroidectomy and radioactive iodine ablation.
DISCUSSION Association of BRAFV600E mutation with clinical features There was a significant association between BRAFV600E mutation and male sex ( p ¼ 0.034) for the whole group of 77 (Table 1), but no significant association was found between the BRAFV600E mutation and age >45 years at diagnosis, size of primary tumour, extrathyroidal extension, lymph node or distant metastases or clinical stage (AJCC) at diagnosis. However, there was a significantly increased risk of lymph node recurrence for the BRAFV600E positive group in all three groups, i.e., for the total cohort, those with total thyroidectomy and the group with total thyroidectomy and radioactive iodine ablation ( p values of 0.02, 0.046 and 0.011, respectively). For the latter group, there was a trend to decreased 10 year progression-free survival ( p ¼ 0.31) in those patients with BRAFV600E positive tumours (Fig. 2). Ten year progressionfree survival for the BRAF wild-type group was 83.3% and for the BRAFV600E positive group 56.2%. Disease specific mortality was low in the cohort of 77 with only six patients (7.8%), five with BRAFV600E positive tumours
Our series of 77 patients with PTC has demonstrated BRAFV600E mutation in 65% of PTC of all sizes with higher prevalence (71%) in those with a diameter greater than one centimetre compared with only 52% of the 25 microcarcinomas. The long follow-up in our study showed significantly higher lymph node recurrence for the BRAFV600E positive patients in the entire cohort of 77 patients as well as those treated with total thyroidectomy or total thyroidectomy and radioactive iodine ablation. Another Australian group has also recently demonstrated increased risk of clinically evident disease recurrence and/or death in their patients with BRAFV600E positive tumours.22,27 Our study also showed a trend to decreased 10 year progression-free survival for the BRAFV600E positive cohort compared to those with BRAFV600E negative tumours in those treated with total thyroidectomy and radioablation ( p ¼ 0.196). However, longer median followup (up to 15 years) for a small cohort, similar to that of Elisei et al., is required before statistically significant disease-specific mortality associated with BRAFV600E mutation
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McKELVIE et al.
Pathology (2013), 45(7), December
A
B
C
Fig. 1 (A) Immunohistochemistry for BRAFV600E mutation using specific VE1 antibody in a case of classical pattern papillary thyroid carcinoma shows diffuse cytoplasmic reactivity. (B) High power shows papillary thyroid carcinoma using specific VE1 antibody. (C) Papillary thyroid carcinoma (BRAFV600E mutation þve) infiltrates normal gland using specific VE1 antibody.
can be demonstrated.38 A very recent retrospective multicentre international study demonstrated the association between BRAFV600E mutation with overall disease-specific mortality.39 However, this increase in mortality in patients with BRAFV600E mutation positive tumours was not independent of factors such as lymph node metastases, distant metastasis and extrathyroidal extension.39 Our study is the second to demonstrate the use of immunohistochemistry for BRAFV600E mutation to provide prognostic information in PTC.22 The study by Bullock et al. included 96 patients with molecular data, of which 71% of tumours showed BRAFV600E point mutation by IHC and 61% by sequencing.22 These authors also found that IHC was more sensitive than molecular techniques including massively parallel and Sanger sequencing, since 30% of their 10 discordant cases remained negative after repeat sequencing, by both methods. These authors suggest that specific BRAFV600E IHC may be the new gold standard for detection of BRAFV600E point mutation in PTC.22 Two previous studies used the VE1 BRAFV600E mutation-specific antibody in PTC, but neither had clinical follow-up.12,14 Koperek and colleagues found concordance in detection of the BRAFV600E mutation in 39/39 papillary thyroid cancers by capillary sequencing and specific immunohistochemistry using the same antibody.14 However, their study only involved sequencing of BRAF gene in those tumours >2 cm and those cases with equivocal or focal VE1 antibody staining (which were all negative by sequencing).14
Progression free survival
Survival percentage
100
80
60
40 BRAF V600E Negative Positive
20
0 0
20
40
60
80
100
120
140
160
180
Months
Method Chi-square DF P-value 1 0.196 Log-rank 1.67431 Fig. 2 Progression-free survival curve of 50 patients with papillary thyroid carcinoma treated with total thyroidectomy and radioactive iodine ablation.
Capper et al. studied 21 cases of PTC by immunohistochemistry and sequencing from FFPE blocks with all 21 showing positive immunoreactivity but only 18 of 21 (86%) showing BRAFV600E mutation by sequencing.12 Our data show that immunohistochemistry for BRAFV600E mutation is more sensitive than standard C-PCR using a commercial kit (Seegene) for detecting the BRAFV600E mutation. Of our nine false negative C-PCR cases, six involved paraffin blocks greater than 12 years of age. Quality of DNA is known to be reduced in FFPE blocks due to fragmentation of DNA, and therefore the immunohistochemical detection of BRAFV600E mutation is more likely to be more sensitive than DNA based techniques, particularly in screening older cases from paraffin blocks.40,41 Multiple methods have been used to determine the presence of BRAFV600E mutation, including direct sequencing of DNA after PCR amplification alone, followed by colorimetric method, DNA PCR single-strand conformation polymorphism (PCR-SSCP) direct sequencing, DNA PCR and fluorescence melting curve analysis and DNA-mutant allele-specific amplification (DNA-MASA) sequencing.9 For convenience, we used C-PCR by commercial kit which has a reported sensitivity of 56.3% in FFPE tissues of thyroid compared with 65% for standard sequencing.11 Since it is clinically important to identify the most accurate method for detection of the BRAFV600E mutation, particularly in FFPE tissues with reduced DNA quality, we propose that the immunohistochemical method using VE1 is highly sensitive, specific and rapid, compared to molecular techniques, for a standard anatomical pathology laboratory which does not have facilities for routine sequencing of all PTC. Indeed, the homogeneous staining with the VE1 in the majority of cases makes identification of tumour cells and their extent of invasion much easier to assess. Our immunohistochemical results were very similar to those of Bullock et al. with diffuse homogeneous cytoplasmic reactivity in the tumour epithelial cells.22 However, several authors have suggested that BRAFV600E point mutation may not be an early clonal event and that BRAFV600E may be clonally heterogeneous within a single tumour.42–44 These studies were based on pyrosequencing of macroscopically dissected tumours with correction for the lymphoid and stromal infiltrates, laser capture microdissection of four cases and subcloning of PTC cells in tissue culture.42,43 Gandolfi et al. showed no difference in allele frequency of the BRAFV600E point mutation between two groups of PTC with and without lymph node metastases.44 These authors, however, noted a puzzling discrepancy between IHC staining using the specific VE1 antibody and molecular data from pyrosequencing analysis
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BRAFV600E MUTATION IN PTC DETECTED BY IMMUNOHISTOCHEMISTRY
in their cases, as the numbers of positive cells seemed higher than expected from allele frequency. One possible explanation is that the BRAFV600E mutation is found only in the epithelial cells of PTC, not in the stromal cores or vessels as is highlighted by the IHC (Fig. 1A,B), but in the molecular studies to determine allele frequency in PTC, the samples must, even in macro- and micro-dissected tumour, contain variable amounts of negative stromal and vascular elements which may dilute the apparent allele frequency. Immunohistochemical detection of BRAFV600E mutation by the anatomical pathologist at the time of diagnosis of PTC offers immediate prognostic information that may alter management in individual patients.9 Since our data demonstrate significantly increased risk of lymph node recurrences in PTC with BRAFV600E mutation, the surgeon may then proceed to prophylactic lymph node resection of clinically uninvolved nodes. Only 38% of our patients underwent lymph node dissection, almost all of which was therapeutic for clinically evident metastasis. On the basis of newly developed recommendations,45 recent clinical studies of papillary thyroid carcinoma now include prophylactic lymph node dissection, at least of central lymph nodes.27,46 Prevalence of lymph node metastasis on microscopy in lymph nodes with negative preoperative ultrasound examination is high, ranging from 42% in small primary tumours measuring 3 cm.46,47 Our data showed a significant association of BRAFV600E mutation with male gender, but not with other important known clinicopathological prognostic factors such as age at diagnosis greater than 45 years, size of the primary tumour, extrathyroidal extension, lymph node or distant metastases at diagnosis or AJCC stage of disease at presentation. This is most likely due to the small numbers of patients in our study.27,48 Another Australian study found a BRAFV600E mutation prevalence of 45% in 76 patients with PTC and an association of BRAFV600E mutation with gender, stage, and subtype (classical versus follicular).49 However, the two cohorts are not directly comparable, since Smith et al.’s cohort contained no stage IV disease compared with 22% in ours.49 Furthermore, 47% of their group were follicular variant PTC compared with
