DOI:10.1111/cyt.12125
Pilot of BRAF mutation analysis in indeterminate, suspicious and malignant thyroid FNA cytology S. J. Johnson*, S. A. Hardy†, C. Roberts†, D. Bourn†, U. Mallick‡ and P. Perros§ *Department of Cellular Pathology, †Northern Genetics Service, ‡Northern Centre for Cancer Care, and §Department of Endocrinology, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK Accepted for publication 8 October 2013
S. J. Johnson, S. A. Hardy, C. Roberts, D. Bourn, U. Mallick and P. Perros Pilot of BRAF mutation analysis in indeterminate, suspicious and malignant thyroid FNA cytology Background: BRAF V600E mutation has been reported to show a high specificity for papillary thyroid carcinoma (PTC). Using this marker to upgrade ‘indeterminate’ or ‘suspicious’ thyroid fine needle aspiration (FNA) cytology to ‘malignant’ could potentially allow one-stage therapeutic total thyroidectomy. Methods: For a 14-month period, FNA cytology specimens in the Thy3–5 categories, which are the UK equivalents of indeterminate (Thy3a, atypical; Thy3f, follicular), suspicious for malignancy (Thy4) and malignant (Thy5) in the Bethesda System, underwent BRAF mutation testing by melt curve analysis. The results were correlated with histology. Results: We tested 123 cytology specimens of which 12 (9.8%) failed. The BRAF mutation rate in the remainder was 16.2% (18/111), with 93 showing the wild-type. Seventeen mutations were V600E and one was non-V600E. The rate of mutation increased significantly (P < 0.0001 if Thy3a and Thy3f were combined) with the cytology category: 1/42 Thy3a (2.4%), 1/36 Thy3f (2.8%), 4/15 Thy4 (26.7%), 12/18 Thy5 (66.7%). All BRAF mutations correlated with PTC on histology, except for one recurrent PTC without histology. One mutation-positive case with Thy3a cytology showed the target lesion to be a 10-mm follicular adenoma on histology with an immediately adjacent 4-mm micro-PTC, in a patient who did not require total thyroidectomy. Conclusion: BRAF mutational analysis by melt curve analysis is feasible in routine thyroid cytology, and in our series had a 100% specificity for PTC in subsequent histology. The application of BRAF analysis could be useful for indeterminate cytology, but we suggest that it would be most appropriate and cost-effective for Thy4/suspicious cases, for which it could enable one-stage therapeutic surgery in the context of multidisciplinary discussion. In contrast, the sensitivity is low and there is no role for avoiding diagnostic thyroid surgery if wild-type BRAF is found. Keywords: BRAF, mutation analysis, melt curve analysis, fine needle aspiration, FNA, cytology, papillary thyroid carcinoma
Introduction The utility of thyroid fine needle aspiration (FNA) cytopathology is often compromised by the ‘indeterminate’ and ‘suspicious’ categories, meaning that patients with these results usually require diagnostic
Correspondence: Dr S. J. Johnson, Department of Cellular Pathology, Royal Victoria Infirmary, Newcastle upon Tyne, NE1 4LP, UK Tel.: 0191 2825593; Fax: 0191 2825892; E-mail: [email protected]
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surgery for a histological diagnosis. For the majority of cases with non-neoplastic disease or a benign thyroid neoplasm, this is arguably unnecessary surgery, whereas, for the minority who have thyroid carcinoma proven on histology, a second operative procedure is needed to complete the surgical treatment. Thyroid cytology in the UK is reported in categories according to the Royal College of Pathologists guidance document.1 These categories map across to those of The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC),2 as shown in Table 1. The ‘indeterminate’ categories are Thy3a and Thy3f, and the © 2014 John Wiley & Sons Ltd Cytopathology 2014, 25, 146–154
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Table 1. UK system compared with the Bethesda System for reporting thyroid cytopathology UK category
The Bethesda System
Thy1 (non-diagnostic sample) Thy1c (non-diagnostic, cystic lesion) Thy2 (non-neoplastic) Thy2c (non-neoplastic, cystic lesion) Thy3a (neoplasm possible, atypia/non-diagnostic) Thy3f (neoplasm possible, suggesting follicular neoplasm) Thy4 (suspicious of malignancy)
Non-diagnostic or unsatisfactory (Class I) Non-diagnostic or unsatisfactory (Class I) Benign (Class II) Benign (Class II)
Thy5 (malignant)
AUS/FLUS (Class III) FN/SFN (Class IV) Suspicious for malignancy (Class V) Malignant (Class VI).
AUS/FLUS, atypia of undetermined significance/follicular lesion of undetermined significance; FN/SFN, follicular neoplasm/suspicious for follicular neoplasm.
‘suspicious’ samples are Thy4. Inter-observer agreement is worst for Thy3a and Thy4 samples in the UK system,3 and for atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) and follicular neoplasm/suspicious for follicular neoplasm (FN/SFN) in the Bethesda System.4 An ideal development would be a marker (or a panel of markers) that could be applied to indeterminate and suspicious thyroid cytology to predict accurately the benignity or malignancy of the nodule, enabling diagnostic surgery to be avoided or therapeutic surgery to be performed at one operation. Many such markers have been proposed, both immunocytochemical and molecular.5–11 Interest is currently high in the BRAF V600E mutation, which has almost 100% specificity for papillary thyroid carcinoma (PTC) (or PTC-derived poorly differentiated or anaplastic thyroid carcinoma), making it a potentially accurate marker for the reassignment of indeterminate or suspicious thyroid cytology to diagnostic cytology.5–8,11 The BRAF mutation analysis can be performed by various methods on DNA extracted from the cytology sample12,13 or, more recently, on immunohistochemistry on cell blocks.14 Previously, we have successfully performed BRAF mutation testing on archival cytological material.15 The aim of this follow-on 14-month study was to prospectively perform BRAF testing on cytology and © 2014 John Wiley & Sons Ltd Cytopathology 2014, 25, 146–154
compare the results with subsequent histological outcomes. The molecular results were not reported to clinicians or used for clinical decisions. Methods Case selection For a 14-month period (1 September 2011 to 29 October 2012), all thyroid cytology specimens reported as atypical (Thy3a, SNOMED M69700), suggesting follicular neoplasm (Thy3f, M69701) and suspicious of malignancy (Thy4, M69760) were retained. In addition, all cytology diagnostic for PTC or poorly differentiated thyroid carcinoma (PDC), whether on samples from the thyroid (Thy5, M80503, M80103), thyroid bed or cervical lymph nodes, was also retained. The malignant cases were included for comparison. These included cases reported within the Trust and some routinely referred in from local hospitals for multidisciplinary team (MDT) review. The original filed stained slides for each case were reviewed. When there was sufficient representative material on more than one slide, at least one such slide was retained for the files (and therefore the patient’s clinical record) and one or more slides were submitted for BRAF testing. The slides were not routinely marked to show the locations of cells. No additional material was taken from the patient as the original filed stained slides were used. BRAF analysis and reporting This was performed by Northern Genetics Service, located about one mile from the hospital’s Cellular Pathology Laboratory. BRAF mutation testing was by high-resolution melt curve analysis on the LightCycler 480 system (Roche Diagnostics, Burgess Hill, UK). Coverslips were removed by soaking in xylene and all the tissue from the microscope slides was removed with sterile pipette tips. Overnight proteinase K digestion was performed. DNA extraction was performed on the Qiagen (Manchester, UK) EZ1 BioRobot, which uses magnetic bead technology to extract the DNA. The same method was also used for formalin-fixed, paraffin-embedded tissue, which was supplied as 8–10-µm-thick curls. BRAF was assessed via high-resolution melt curve analysis on the LightCycler 480 system, as described by Nikiforov et al.16 Melt curve analysis assesses the dissocia-
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tion characteristics of double-stranded DNA during heating. Wild-type (WT) sample analysis produces a single melting peak at 63 °C. The presence of a mutation alters the melting temperature and, in the case of BRAF V600E, a second melting peak at 59 °C is observed. BRAF testing was batched and the results were made available after each batch had been run. This was to reduce costs for the study. The local clinicians involved in the investigation and treatment of thyroid nodules were all aware of the study, but the BRAF results were not reported to them nor were the results considered in treatment planning. Clinical management was according to usual local, regional and national protocols. Given that this work was part of the institutional service improvement programme, and the test is already commercially available, although in the authors’ view not fully validated, it was deemed that ethical approval was not required. Comparison with histology Histological outcomes were sought from the pathology database, including those for referred cytology specimens when available. A few of the early histology specimens were also tested for BRAF. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated using the non-failed BRAF test results and the histology outcome for the nodule that had been targeted by the FNA, excluding micro-PTCs found incidentally elsewhere in the thyroid specimen at histological examination. For the calculations, a true positive (TP) was taken as a case with a BRAF mutation on the cytology specimen and malignancy on histology; true negative (TN) as WT BRAF on cytology and benign (either non-neoplastic or a benign neoplasm) histology; false positive (FP) as BRAF mutation on cytology and benign histology; and false negative (FN) as WT BRAF on cytology and malignant histology. Sensitivity, specificity, PPV, NPV and accuracy were expressed as percentages. The results are all stated per specimen; some patients had more than one cytology specimen. Immunohistochemical staining for BRAF This has very recently been optimized in our laboratory for histology specimens and will be the subject of future publications, but was used for just one case in this study. It was performed on a Benchmark
Ultra (Tuscon, AZ, USA) staining platform using heat-induced epitope retrieval with high pH CC1 at 100 °C, OptiView DAB detection and OptiView amplification (Ventana, Tuscon, AZ, USA). The BRAF V600E antibody, clone VE1 (Spring Bioscience, Pleasanton, CA, USA), was used at a dilution of 1 : 3000. Positive staining was confirmed using malignant melanoma tissue with confirmed BRAF V600E mutation. Results Cytology and histology cases BRAF testing was carried out on 123 cytology specimens of which 45 were reported as atypical (Thy3a), 41 suggesting a follicular neoplasm (Thy3f), 16 suspicious of malignancy (Thy4) and 21 malignant (Thy5 or lymph node metastases or recurrence in the thyroid bed). The 123 specimens represented about 60% of the total number of Thy3–5 thyroid cytology samples in that time period. The cases that were not used for the study had insufficient cellular material to release a slide for testing. Subsequent histology was available for 97 of the cytology specimens whose outcomes included 26 non-neoplastic lesions, 21 follicular adenomas (benign neoplasm) and 50 carcinomas (36 PTC, seven follicular carcinomas, two medullary thyroid carcinomas, one anaplastic thyroid carcinoma and four poorly differentiated carcinomas of uncertain primary). BRAF analysis on cytology The test worked in 111 cytology specimens and failed in 12 (9.8%). Failed tests were those in which the melt curve analysis gave no results, or those below the threshold of detection, because of low DNA yield or failed DNA extraction. Of the 111 successful analyses, BRAF V600E mutation was found in 17 specimens, a non-V600E BRAF mutation in one specimen and WT in 93 specimens, giving a BRAF mutation rate of 16.2% (18/111). Correlation of the mutations with the cytology and subsequent histology is shown in Table 2. Excluding failed tests, the frequency of mutation increased with increasing Thy cytology category: 1/42 Thy3a (2.4%), 1/36 Thy3f (2.8%), 4/15 Thy4 (26.7%), 12/18 Thy5 (66.7%). Combining the Thy3 into one category, these proportions are significantly different from one another (P < 0.0001 on a chi-squared test). © 2014 John Wiley & Sons Ltd Cytopathology 2014, 25, 146–154
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Table 2. Correlation of cytology with BRAF result and histology or follow-up Histology outcome (n = 97) Cytology category Thy3a n = 45
Thy3f n = 41
Thy4 n = 16
Thy5 n = 21
Totals
No follow-up
Total malignant (n = 50)
BRAF result (n)
Follow-up cytology only
NN
FA
V600E mutation (1) WT (41) Failed (3) V600E mutation (1) WT (35) Failed (5) V600E mutation (3) Non-V600E mutation (1) WT (11) Failed (1) V600E mutation (12) WT (6) Failed (3) BRAF tests (123)
– 9 – – 1 – – – – – – – – 10
– 11 1 – 9 3 – – 2 – – – – 26
– 5 1 – 14 – – – 1 – – – – 21
Total PTC 1 6 – 1 3 – 3 1 4 1 11 4 1 36
PTC *
1 2 – 1† 1 – 2 1 3† – 8† 3 1 23
FVPTC
Others
– 4 – – 2 – 1 – 1 1 3 1 – 13
– 2 – – 4 2 – – 2 – – 2 2 14
– 8 1 – 4 – – – 2 – 1‡ – – 16
FA, follicular adenoma; FVPTC, follicular variant papillary thyroid carcinoma; NN, non-neoplastic; PTC, papillary thyroid carcinoma; WT, wild-type. *Follicular adenoma and adjacent micro-PTC (see text). † Number includes one tall cell variant PTC in each instance. ‡ Recurrent PTC.
BRAF results of cases with histological specimens All cytology specimens with BRAF mutations were followed by malignant histology, except for one, which was a recurrence in the thyroid bed in a patient with previous classical PTC; this was excluded from the calculations. As shown in Table 2, two (5.6%) of the 36 PTCs on histology had failed BRAF analysis on cytology. Of the remainder, 17 (50%) had BRAF mutation (16 V600E and one non-V600E mutation) and 17 (50%) had WT on cytology specimens. The 36 PTCs comprised 20 that were of classical or unspecified type, 13 follicular variant of PTC (FVPTC) and three tall cell variant (TCVPTC). Excluding failed tests, the BRAF mutation rate was found in 11/19 classical PTCs, 4/12 FVPTC and 2/3 TCVPTC. None of the follicular, medullary or anaplastic carcinoma cases had BRAF mutations in preceding cytology. Excluding failed tests and as with the results for cytology cases as a whole, the rate of mutation in cytology preceding histology of PTC progressively decreased from 73% of Thy5 (11/15), to 50% of Thy4 (4/8), to 18% of Thy3 (2/11). The proportion of histologically proven PTCs that were FVPTC © 2014 John Wiley & Sons Ltd Cytopathology 2014, 25, 146–154
appeared to decrease with increasing cytology category: 6/11 Thy3, 3/9 Thy4 and 4/16 Thy5. One particular case warrants further description and is indicated with an asterisk in Table 2. In this patient, two separate thyroid nodules in the same lobe were targeted on FNA. The 3–4-cm nodule yielded Thy3a cytology on initial and repeat cytology, with WT on genetic analysis; histology on the lobectomy showed this to be a dominant nodule in a multinodular goitre. A smaller 1-cm nodule was also aspirated on the second occasion, yielding Thy3a cytology, but BRAF V600E mutation was found on genetic testing; the histology was a 10-mm follicular adenoma with an immediately adjacent 4mm micro-PTC. This was a single focus of micro-PTC and, after MDT discussion, the patient has not required completion thyroidectomy. Immunostaining on this case is shown in Figure 1, using an antibody to BRAF V600E mutation that we have used successfully on histological sections, but not on cytology, highlighting negative staining (no BRAF mutation) in the follicular adenoma and positive staining (BRAF V600E mutation) in the immediately adjacent micro-PTC.
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BRAF testing on histology specimens Seven histology specimens (from four patients) were also tested for BRAF mutation. The results are shown in Table 5, with correlating cytology specimen results. Testing failed in two. Discussion
Figure 1. Immunohistochemical staining for BRAF V600E mutation in a histological section of a follicular adenoma (right, negative) and immediately adjacent papillary thyroid microcarcinoma (left, positive).
Cytology specimen types Table 3 shows the cytology specimen types correlated with the BRAF analysis results. The 12 failed specimens comprised six directly spread slides previously stained with Giemsa-based stains (either Diff-Quikâ or May–Gr€ unwald– Giemsa), five slides that had previously been stained for immunocytochemistry (ICC) and one Papanicolaou-stained SurePathâ liquid-based cytology (LBC) slide preparation. Multiple cytology specimens from the same nodule Some of the cytology specimens represented repeat or multiple samples from the same patient. These results are shown in Table 4. There were 11 such patients, nine of whom had only one lesion sampled. Two patients had multiple lesions sampled: one was the case described above; the other was a patient with two samples of different sites showing the same tumour on histology (poorly differentiated carcinoma, uncertain primary, probably not thyroid). Accuracy calculations Excluding the failed tests, BRAF mutation in preoperative cytology predicted subsequent malignancy on histology with 100% specificity and PPV, 38.6% sensitivity, 60.9% NPV and 69.4% accuracy.
Prior to this study, we had shown that BRAF genetic testing was feasible in highly selected archival histology and routine cytology cases.15 The current study was designed to assess prospectively over at least a year as many routine cytology specimens as had sufficient material. The results show that the testing is feasible on the majority of specimens, and that all cases with BRAF V600E mutation had PTC on subsequent histology. The frequency of BRAF mutation in PTCs overall was 50% (17/34). The BRAF mutation rate was highest in TCVPTC (2/3) and lowest in FVPTC (4/ 12), which is in line with previous reports.17,18 FVPTCs are known often to harbour RAS mutations rather than BRAF, more akin to follicular neoplasms.19 No BRAF mutations were seen in non-PTC thyroid malignancies. BRAF mutation was detected in one Thy3a cytology sample, which correlated with a 4-mm PTC on histology, immediately adjacent to the targeted 10mm lesion (a follicular adenoma); this was only a single micro-PTC not warranting completion thyroidectomy. The original report on this Thy3a cytology was of colloid and follicular epithelial cells, but with some microfollicular patterning, and a follicular neoplasm could not be excluded. Although incidental micro-PTCs are usually excluded from calculations of accuracy in thyroid cytology, this micro-PTC was located with the target lesion and so has been included. Of clinical importance is that completion thyroidectomy was not required in this patient, and so over-reliance and action on the BRAF result in this case could have led to over-treatment. The frequency of mutation decreased according to the cytology category for all specimens and those preceding histological confirmation of PTC. The majority of PTCs (9/11) following Thy3a or Thy3f cytology and half of those after Thy4 cytology were not associated with BRAF mutation, compared with 4/15 (27%) of Thy5. A WT BRAF genetic result cannot be taken as reassuring, because it does not exclude the presence of PTC, and ultrasound follow-
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Table 3. Correlation of cytology specimen type with outcome of BRAF testing Outcome of BRAF testing
Specimen type Cytology slides
Histology cases
DQ DQ/MGG MGG DPAS Papanicolaou ICC Unknown LBC Total Curls of FFPE tissue
Total number
Failed (%)
WT
V600E mutation
Non-V600E mutation
87 3 11 2 4 10 5 1 123 7
5 – 1 – – 5 – 1 12 2
66 3 10 2 3 5 4 – 93 1
15 – – – 1 – 1 – 17 4
1 – – – – – – – 1 –
DPAS, diastase-treated periodic acid Schiff-stained smear; DQ, Diff-Quik-stained smear; DQ/MGG, Giemsa-stained smear, DQ or MGG not specified; FFPE, formalin-fixed, paraffin-embedded tissue; ICC, immunostained slide; LBC, SurePath liquidbased cytology, Papanicolaou-stained slide; MGG, May–Gr€ unwald–Giemsa-stained smear; WT, wild-type.
up, lobectomy or thyroidectomy should not be avoided in suspicious, indeterminate or positive cases just because BRAF testing is negative. FVPTC can be a problematic diagnosis on histological assessment, with known inter-observer variation, even between expert endocrine pathologists,20 and represented a higher proportion of PTCs after indeterminate cytology in our study than suspicious or malignant; none of the six with indeterminate cytology had BRAF mutations. Views on the management of FVPTC are also changing, especially for the encapsulated variant, which may not always require total thyroidectomy, especially when noninvasive.21,22 It is known that FVPTC is more challenging on pre-operative cytology than classical PTC and may be represented in various of the cytology categories,23,24 and there is less inter-observer agreement with UK Thy3a and Thy4 cytology, and the Bethesda System AUS/FLUS and FN/SLN, than other categories.3,4 An important question is therefore which cytology categories could warrant the additional cost of BRAF mutation analysis to alter operative management without risking potential over-treatment of encapsulated FVPTCs or incidental micro-PTCs. From our results in this study, the case is stronger for the Thy4 cytology category than for the Thy3a and Thy3f categories. The Portsmouth group have recently advocated co-testing (Roche CobasTM platform; Roche Diagnostics, Burgess Hill, UK) all Thy4/suspicious thyroid cytology for BRAF mutation, © 2014 John Wiley & Sons Ltd Cytopathology 2014, 25, 146–154
with three BRAF-positive Thy4 cases proceeding to one-stage total thyroidectomy after discussion and slide review by the MDT.12 The Thy3a category cases, in particular, may benefit best from repeat cytology to obtain a more cellular sample, especially in cases in which the initial cytology was not obtained at ultrasound, which will provide additional useful information on the lesion. Others have reported that BRAF testing can increase the accuracy of pre-operative thyroid cytology,8,25,26 especially in BRAF mutation prevalent areas or as part of a panel.27–31 The high specificity for PTC could guide the extent of surgery and lymph node dissection5,6,29 Others have found occasional false-positive cases, especially with highly sensitive analytical methods, such as dual-priming oligonucleotide-based multiplex polymerase chain reaction (PCR).25 A lower mutation rate has been noted in FVPTC, reducing the value of the test in this setting, with the comment that the highest mutation rate is seen in cases that are diagnostic on FNA, as we have shown here and before,15 and that BRAF-negative PTCs are more likely to yield indeterminate cytology.30 Value has been demonstrated, particularly in cases that are suspicious on ultrasound.31 Hyalinizing trabecular tumours can mimic PTC on cytology, leading to over-diagnosis, but have usefully been shown to be BRAF mutation negative.32 Otherwise, a WT BRAF result is not reassuring and has a low NPV. Others have shown that ICC for other markers [e.g. cytokeratin 19 (CK19) and HBME-1] may have
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Table 4. Multiple cytology samples from the same patients
Patient and samples 1. 2. 3. 4.
BRAF test result
Cytology result Thy3–5
V600E Mutation
Wild Type
Failed Test
2 1
1
Same lesion, separate times Same lesion, separate times Same lesion, separate times Same lesion, separate times fluid with 1st sample 5. Same lesion, separate times two samples at 2nd FNA
3a, 3f Both 3f 4, 5 3f, 4 (fluid), 5
6. 7. 8. 9.
Same lesion, separate times Same lesion, solid and cystic Same lesion, solid and cystic Two slides from same lesion SurePath and Diff Quik 10. 3–4-cm nodule in lower pole two samples 1-cm nodule upper pole
Both 4 Both 3a 3a (fluid), 5 Both 5
11. Same tumour in lymph node and mediastinal mass Thyroid, same patient
Both malignant
1
3f
1
2 2
1 (fluid)
3a, 3a, 3f
2
1 1 (fluid) 1 (fluid) 1
1
Both 3a
1
1 1 1 (SP)
2
3a
1 1
Histology Outcome FVPTC Nodular HT PTC PTC (TCV) Hyperplastic nodule in multinodular goitre FVPTC None FVPTC PD/anaplastic carcinoma* Dominant nodule in multinodular goitre 10-mm FA; adjacent 4-mm microPTC PD carcinoma unknown origin* Non-neoplastic
FVPTC, follicular variant of papillary thyroid carcinoma; HT, Hashimoto’s thyroiditis; PTC, papillary thyroid carcinoma; TCV, tall cell variant; SP, SurePath; PD, poorly differentiated; FA, follicular adenoma. *Patient No. 9, probable thyroid origin; Patient 11, probable non-thyroid origin.
Table 5. Histology cases tested for BRAF mutation Histology specimens PTC PTC PTC FA
Thyroid Lymph node metastasis Thyroid Lymph node metastasis Thyroid Lymph node metastasis Thyroid
BRAF mutation result on FFPE curls
BRAF mutation result on pre-operative cytology specimen
V600E V600E Fail Fail V600E V600E WT
No cytology from thyroid V600E mutation in lymph node V600E mutation No cytology from lymph node V600E mutation No cytology from lymph node WT
mutation mutation
mutation mutation
FA, follicular adenoma; FFPE, formalin-fixed, paraffin-embedded; PTC, papillary thyroid carcinoma; WT, wild-type.
a better sensitivity than BRAF mutation for malignancy.9,11 For clinical application, the BRAF result would need to be turned around within a week or two, which would increase our cost to about £50 per sample, compared with the batched method for the study, which cost £25 per sample. Molecular testing of indeterminate cytology specimens could, how-
ever, decrease the cost of thyroid nodule evaluation overall by avoiding two-stage surgery in cases of thyroid cancer.33 Certain cytology specimen types had a higher failure rate for BRAF mutation testing, especially the SurePath LBC case, as noted previously.15 There was a 50% failure rate for slides on which ICC had previously been performed, suggesting that, until © 2014 John Wiley & Sons Ltd Cytopathology 2014, 25, 146–154
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methodology improvements can be made, these slides are best avoided for BRAF testing by this method. Interestingly, there was not always correlation in BRAF results when more than one cytology sample was available from a single lesion. This mostly reflected one of the tests failing, but, in six cases, there was more than one non-failed specimen. We have reported this mismatch previously in samples from the same lesion.15 The reason for this is not entirely clear, but it may reflect the proportion of tumour in the sample. The importance of pre-analytical steps has recently been emphasized7 and manual dissection may be helpful.13,26 BRAF test results on the few histology samples correlated very well with the results on preceding cytology. This study has limitations, especially the small numbers of cases and long turnaround time for BRAF results, but reflects real-life prospective collection of cases from one department. In summary, we have found that BRAF mutational analysis by melt curve analysis is feasible in routine thyroid cytology specimens and shows 100% specificity for PTC in subsequent histology, potentially allowing for one-stage therapeutic surgery after less than diagnostic cytology suspicious for malignancy. Mutation was seen more frequently in diagnostic than indeterminate cytology but sensitivity is low and there is no role for avoiding diagnostic thyroid surgery if WT BRAF is found. Careful cost-effectiveness analysis is needed with consideration as to which cytology specimens should undergo testing.
Acknowledgments We thank Anna Long for performing immunohistochemistry for BRAF and Steve Brabazon for assistance with photography. We are also grateful to colleagues in local pathology hospitals for help with tracing histology results.
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15. Hardy S, Mallick UK, Perros P et al. Development of molecular testing for thyroid cytology specimens. Cytopathology 2011;22:i–xvi. 16. Nikiforov YE, Steward DL, Robinson-Smith TM et al. Molecular testing for mutations in improving the fineneedle aspiration diagnosis of thyroid nodules. J Clin Endocrinol Metab 2009;94:2092–8. 17. Proietti A, Giannini R, Ugolini C et al. BRAF status of follicular variant of papillary thyroid carcinoma and its relationship to its clinical and cytological features. Thyroid 2010;20:1263–70. 18. Min HS, Lee C, Jung KC. Correlation of immunohistochemical markers and BRAF mutation status with histological variants of papillary thyroid carcinoma in the Korean population. J Korean Med Sci 2013;28:534–41. 19. Lee SR, Jung CK, Kim TE et al. Molecular genotyping of follicular variant of papillary thyroid carcinoma correlates with diagnostic category of fine-needle aspiration cytology: values of ras mutation testing. Thyroid 2013;23:1416–22. 20. Elsheikh TM, Asa SL, Chan JK et al. Interobserver and intraobserver variation among experts in the diagnosis of thyroid follicular lesions with borderline nuclear features of papillary thyroid carcinoma. Am J Clin Pathol 2008;130:736–44. 21. Liu J, Singh B, Tallini G et al. Follicular variant of papillary thyroid carcinoma: a clinicopathologic study of a problematic entity. Cancer 2006;107:1255–64. 22. Rivera M, Tuttle RM, Patel S et al. Encapsulated papillary thyroid carcinoma: a clinico-pathologic study of 106 cases with emphasis on its morphologic subtypes (histologic growth pattern). Thyroid 2009;19:119–27. 23. Chang HY, Lin JD, Chou SC, Chao TC, Hsueh C. Clinical presentations and outcomes of surgical treatment of follicular variant of the papillary thyroid carcinomas. Jpn J Clin Oncol 2006;36:688–93. 24. Kurian EM, Dawlett M, Wang J, Gong Y, Guo M. The triage efficacy of fine needle aspiration biopsy for follicular variant of papillary thyroid carcinoma using the Bethesda reporting guidelines. Diagn Cytopathol 2012;40 (suppl1):E69–E73.
25. Kim SW, Lee JI, Kim JW et al. BRAFV600E mutation analysis in fine-needle aspiration cytology specimens for evaluation of thyroid nodule: a large series in a BRAFV600E-prevalent population. J Clin Endocrinol Metab 2010;95:3693–700. 26. Marchietti I, Iervasi G, Mazzanti CM et al. Detection of the BRAFV600E mutation in fine needle aspiration cytology of thyroid papillary microcarcinoma cells selected by manual microdissection: an easy tool to improve the pre-operative diagnosis. Thyroid 2012;22:292–8. 27. Domingues R, Mendoncßa E, Sobrinho L, Bugalho MJ. Searching for RET/PTC rearrangements and BRAF V599E mutation in thyroid aspirates might contribute to establish a preoperative diagnosis of papillary thyroid carcinoma. Cytopathology 2005;16:27–31. 28. Nikiforov YE, Ohori NP, Hodak SP et al. Impact of mutational testing on the diagnosis and management of patients with cytologically indeterminate thyroid nodules: a prospective analysis of 1056 FNA samples. J Clin Endocrinol Metab 2011;96:292–8. 29. Yip L, Nikiforova MN, Carty SE et al. Optimizing surgical treatment of papillary thyroid carcinoma associated with BRAF mutation. Surgery 2009;146:1215–23. 30. Moon WJ, Choi N, Choi JW, Kim SK, Hwang TS. BRAF mutation analysis and sonography as adjuncts to fineneedle aspiration cytology of papillary thyroid carcinoma: their relationships and roles. Am J Roentgenol 2012;198:668–74. 31. Moon HJ, Kim EK, Chung WY et al. Diagnostic value of BRAF(V600E) mutation analysis of thyroid nodules according to ultrasonographic features and the time of aspiration. Ann Surg Oncol 2011;18:792–9. 32. Kim T, Oh YL, Kim KM, Shin JH. Diagnostic dilemmas of hyalinizing trabecular tumours on fine needle aspiration cytology: a study of seven cases with BRAF mutation analysis. Cytopathology 2011;22:407–13. 33. Yip L, Farris C, Kabaker AS et al. Cost impact of molecular testing for indeterminate thyroid nodule fine-needle aspiration biopsies. J Clin Endocrinol Metab 2012; 97:1905–12.
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Pilot of BRAF mutation analysis in indeterminate, suspicious and malignant thyroid FNA cytology S. J. Johnson*, S. A. Hardy†, C. Roberts†, D. Bourn†, U. Mallick‡ and P. Perros§ *Department of Cellular Pathology, †Northern Genetics Service, ‡Northern Centre for Cancer Care, and §Department of Endocrinology, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK Accepted for publication 8 October 2013
S. J. Johnson, S. A. Hardy, C. Roberts, D. Bourn, U. Mallick and P. Perros Pilot of BRAF mutation analysis in indeterminate, suspicious and malignant thyroid FNA cytology Background: BRAF V600E mutation has been reported to show a high specificity for papillary thyroid carcinoma (PTC). Using this marker to upgrade ‘indeterminate’ or ‘suspicious’ thyroid fine needle aspiration (FNA) cytology to ‘malignant’ could potentially allow one-stage therapeutic total thyroidectomy. Methods: For a 14-month period, FNA cytology specimens in the Thy3–5 categories, which are the UK equivalents of indeterminate (Thy3a, atypical; Thy3f, follicular), suspicious for malignancy (Thy4) and malignant (Thy5) in the Bethesda System, underwent BRAF mutation testing by melt curve analysis. The results were correlated with histology. Results: We tested 123 cytology specimens of which 12 (9.8%) failed. The BRAF mutation rate in the remainder was 16.2% (18/111), with 93 showing the wild-type. Seventeen mutations were V600E and one was non-V600E. The rate of mutation increased significantly (P < 0.0001 if Thy3a and Thy3f were combined) with the cytology category: 1/42 Thy3a (2.4%), 1/36 Thy3f (2.8%), 4/15 Thy4 (26.7%), 12/18 Thy5 (66.7%). All BRAF mutations correlated with PTC on histology, except for one recurrent PTC without histology. One mutation-positive case with Thy3a cytology showed the target lesion to be a 10-mm follicular adenoma on histology with an immediately adjacent 4-mm micro-PTC, in a patient who did not require total thyroidectomy. Conclusion: BRAF mutational analysis by melt curve analysis is feasible in routine thyroid cytology, and in our series had a 100% specificity for PTC in subsequent histology. The application of BRAF analysis could be useful for indeterminate cytology, but we suggest that it would be most appropriate and cost-effective for Thy4/suspicious cases, for which it could enable one-stage therapeutic surgery in the context of multidisciplinary discussion. In contrast, the sensitivity is low and there is no role for avoiding diagnostic thyroid surgery if wild-type BRAF is found. Keywords: BRAF, mutation analysis, melt curve analysis, fine needle aspiration, FNA, cytology, papillary thyroid carcinoma
Introduction The utility of thyroid fine needle aspiration (FNA) cytopathology is often compromised by the ‘indeterminate’ and ‘suspicious’ categories, meaning that patients with these results usually require diagnostic
Correspondence: Dr S. J. Johnson, Department of Cellular Pathology, Royal Victoria Infirmary, Newcastle upon Tyne, NE1 4LP, UK Tel.: 0191 2825593; Fax: 0191 2825892; E-mail: [email protected]
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surgery for a histological diagnosis. For the majority of cases with non-neoplastic disease or a benign thyroid neoplasm, this is arguably unnecessary surgery, whereas, for the minority who have thyroid carcinoma proven on histology, a second operative procedure is needed to complete the surgical treatment. Thyroid cytology in the UK is reported in categories according to the Royal College of Pathologists guidance document.1 These categories map across to those of The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC),2 as shown in Table 1. The ‘indeterminate’ categories are Thy3a and Thy3f, and the © 2014 John Wiley & Sons Ltd Cytopathology 2014, 25, 146–154
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Table 1. UK system compared with the Bethesda System for reporting thyroid cytopathology UK category
The Bethesda System
Thy1 (non-diagnostic sample) Thy1c (non-diagnostic, cystic lesion) Thy2 (non-neoplastic) Thy2c (non-neoplastic, cystic lesion) Thy3a (neoplasm possible, atypia/non-diagnostic) Thy3f (neoplasm possible, suggesting follicular neoplasm) Thy4 (suspicious of malignancy)
Non-diagnostic or unsatisfactory (Class I) Non-diagnostic or unsatisfactory (Class I) Benign (Class II) Benign (Class II)
Thy5 (malignant)
AUS/FLUS (Class III) FN/SFN (Class IV) Suspicious for malignancy (Class V) Malignant (Class VI).
AUS/FLUS, atypia of undetermined significance/follicular lesion of undetermined significance; FN/SFN, follicular neoplasm/suspicious for follicular neoplasm.
‘suspicious’ samples are Thy4. Inter-observer agreement is worst for Thy3a and Thy4 samples in the UK system,3 and for atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) and follicular neoplasm/suspicious for follicular neoplasm (FN/SFN) in the Bethesda System.4 An ideal development would be a marker (or a panel of markers) that could be applied to indeterminate and suspicious thyroid cytology to predict accurately the benignity or malignancy of the nodule, enabling diagnostic surgery to be avoided or therapeutic surgery to be performed at one operation. Many such markers have been proposed, both immunocytochemical and molecular.5–11 Interest is currently high in the BRAF V600E mutation, which has almost 100% specificity for papillary thyroid carcinoma (PTC) (or PTC-derived poorly differentiated or anaplastic thyroid carcinoma), making it a potentially accurate marker for the reassignment of indeterminate or suspicious thyroid cytology to diagnostic cytology.5–8,11 The BRAF mutation analysis can be performed by various methods on DNA extracted from the cytology sample12,13 or, more recently, on immunohistochemistry on cell blocks.14 Previously, we have successfully performed BRAF mutation testing on archival cytological material.15 The aim of this follow-on 14-month study was to prospectively perform BRAF testing on cytology and © 2014 John Wiley & Sons Ltd Cytopathology 2014, 25, 146–154
compare the results with subsequent histological outcomes. The molecular results were not reported to clinicians or used for clinical decisions. Methods Case selection For a 14-month period (1 September 2011 to 29 October 2012), all thyroid cytology specimens reported as atypical (Thy3a, SNOMED M69700), suggesting follicular neoplasm (Thy3f, M69701) and suspicious of malignancy (Thy4, M69760) were retained. In addition, all cytology diagnostic for PTC or poorly differentiated thyroid carcinoma (PDC), whether on samples from the thyroid (Thy5, M80503, M80103), thyroid bed or cervical lymph nodes, was also retained. The malignant cases were included for comparison. These included cases reported within the Trust and some routinely referred in from local hospitals for multidisciplinary team (MDT) review. The original filed stained slides for each case were reviewed. When there was sufficient representative material on more than one slide, at least one such slide was retained for the files (and therefore the patient’s clinical record) and one or more slides were submitted for BRAF testing. The slides were not routinely marked to show the locations of cells. No additional material was taken from the patient as the original filed stained slides were used. BRAF analysis and reporting This was performed by Northern Genetics Service, located about one mile from the hospital’s Cellular Pathology Laboratory. BRAF mutation testing was by high-resolution melt curve analysis on the LightCycler 480 system (Roche Diagnostics, Burgess Hill, UK). Coverslips were removed by soaking in xylene and all the tissue from the microscope slides was removed with sterile pipette tips. Overnight proteinase K digestion was performed. DNA extraction was performed on the Qiagen (Manchester, UK) EZ1 BioRobot, which uses magnetic bead technology to extract the DNA. The same method was also used for formalin-fixed, paraffin-embedded tissue, which was supplied as 8–10-µm-thick curls. BRAF was assessed via high-resolution melt curve analysis on the LightCycler 480 system, as described by Nikiforov et al.16 Melt curve analysis assesses the dissocia-
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tion characteristics of double-stranded DNA during heating. Wild-type (WT) sample analysis produces a single melting peak at 63 °C. The presence of a mutation alters the melting temperature and, in the case of BRAF V600E, a second melting peak at 59 °C is observed. BRAF testing was batched and the results were made available after each batch had been run. This was to reduce costs for the study. The local clinicians involved in the investigation and treatment of thyroid nodules were all aware of the study, but the BRAF results were not reported to them nor were the results considered in treatment planning. Clinical management was according to usual local, regional and national protocols. Given that this work was part of the institutional service improvement programme, and the test is already commercially available, although in the authors’ view not fully validated, it was deemed that ethical approval was not required. Comparison with histology Histological outcomes were sought from the pathology database, including those for referred cytology specimens when available. A few of the early histology specimens were also tested for BRAF. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated using the non-failed BRAF test results and the histology outcome for the nodule that had been targeted by the FNA, excluding micro-PTCs found incidentally elsewhere in the thyroid specimen at histological examination. For the calculations, a true positive (TP) was taken as a case with a BRAF mutation on the cytology specimen and malignancy on histology; true negative (TN) as WT BRAF on cytology and benign (either non-neoplastic or a benign neoplasm) histology; false positive (FP) as BRAF mutation on cytology and benign histology; and false negative (FN) as WT BRAF on cytology and malignant histology. Sensitivity, specificity, PPV, NPV and accuracy were expressed as percentages. The results are all stated per specimen; some patients had more than one cytology specimen. Immunohistochemical staining for BRAF This has very recently been optimized in our laboratory for histology specimens and will be the subject of future publications, but was used for just one case in this study. It was performed on a Benchmark
Ultra (Tuscon, AZ, USA) staining platform using heat-induced epitope retrieval with high pH CC1 at 100 °C, OptiView DAB detection and OptiView amplification (Ventana, Tuscon, AZ, USA). The BRAF V600E antibody, clone VE1 (Spring Bioscience, Pleasanton, CA, USA), was used at a dilution of 1 : 3000. Positive staining was confirmed using malignant melanoma tissue with confirmed BRAF V600E mutation. Results Cytology and histology cases BRAF testing was carried out on 123 cytology specimens of which 45 were reported as atypical (Thy3a), 41 suggesting a follicular neoplasm (Thy3f), 16 suspicious of malignancy (Thy4) and 21 malignant (Thy5 or lymph node metastases or recurrence in the thyroid bed). The 123 specimens represented about 60% of the total number of Thy3–5 thyroid cytology samples in that time period. The cases that were not used for the study had insufficient cellular material to release a slide for testing. Subsequent histology was available for 97 of the cytology specimens whose outcomes included 26 non-neoplastic lesions, 21 follicular adenomas (benign neoplasm) and 50 carcinomas (36 PTC, seven follicular carcinomas, two medullary thyroid carcinomas, one anaplastic thyroid carcinoma and four poorly differentiated carcinomas of uncertain primary). BRAF analysis on cytology The test worked in 111 cytology specimens and failed in 12 (9.8%). Failed tests were those in which the melt curve analysis gave no results, or those below the threshold of detection, because of low DNA yield or failed DNA extraction. Of the 111 successful analyses, BRAF V600E mutation was found in 17 specimens, a non-V600E BRAF mutation in one specimen and WT in 93 specimens, giving a BRAF mutation rate of 16.2% (18/111). Correlation of the mutations with the cytology and subsequent histology is shown in Table 2. Excluding failed tests, the frequency of mutation increased with increasing Thy cytology category: 1/42 Thy3a (2.4%), 1/36 Thy3f (2.8%), 4/15 Thy4 (26.7%), 12/18 Thy5 (66.7%). Combining the Thy3 into one category, these proportions are significantly different from one another (P < 0.0001 on a chi-squared test). © 2014 John Wiley & Sons Ltd Cytopathology 2014, 25, 146–154
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Table 2. Correlation of cytology with BRAF result and histology or follow-up Histology outcome (n = 97) Cytology category Thy3a n = 45
Thy3f n = 41
Thy4 n = 16
Thy5 n = 21
Totals
No follow-up
Total malignant (n = 50)
BRAF result (n)
Follow-up cytology only
NN
FA
V600E mutation (1) WT (41) Failed (3) V600E mutation (1) WT (35) Failed (5) V600E mutation (3) Non-V600E mutation (1) WT (11) Failed (1) V600E mutation (12) WT (6) Failed (3) BRAF tests (123)
– 9 – – 1 – – – – – – – – 10
– 11 1 – 9 3 – – 2 – – – – 26
– 5 1 – 14 – – – 1 – – – – 21
Total PTC 1 6 – 1 3 – 3 1 4 1 11 4 1 36
PTC *
1 2 – 1† 1 – 2 1 3† – 8† 3 1 23
FVPTC
Others
– 4 – – 2 – 1 – 1 1 3 1 – 13
– 2 – – 4 2 – – 2 – – 2 2 14
– 8 1 – 4 – – – 2 – 1‡ – – 16
FA, follicular adenoma; FVPTC, follicular variant papillary thyroid carcinoma; NN, non-neoplastic; PTC, papillary thyroid carcinoma; WT, wild-type. *Follicular adenoma and adjacent micro-PTC (see text). † Number includes one tall cell variant PTC in each instance. ‡ Recurrent PTC.
BRAF results of cases with histological specimens All cytology specimens with BRAF mutations were followed by malignant histology, except for one, which was a recurrence in the thyroid bed in a patient with previous classical PTC; this was excluded from the calculations. As shown in Table 2, two (5.6%) of the 36 PTCs on histology had failed BRAF analysis on cytology. Of the remainder, 17 (50%) had BRAF mutation (16 V600E and one non-V600E mutation) and 17 (50%) had WT on cytology specimens. The 36 PTCs comprised 20 that were of classical or unspecified type, 13 follicular variant of PTC (FVPTC) and three tall cell variant (TCVPTC). Excluding failed tests, the BRAF mutation rate was found in 11/19 classical PTCs, 4/12 FVPTC and 2/3 TCVPTC. None of the follicular, medullary or anaplastic carcinoma cases had BRAF mutations in preceding cytology. Excluding failed tests and as with the results for cytology cases as a whole, the rate of mutation in cytology preceding histology of PTC progressively decreased from 73% of Thy5 (11/15), to 50% of Thy4 (4/8), to 18% of Thy3 (2/11). The proportion of histologically proven PTCs that were FVPTC © 2014 John Wiley & Sons Ltd Cytopathology 2014, 25, 146–154
appeared to decrease with increasing cytology category: 6/11 Thy3, 3/9 Thy4 and 4/16 Thy5. One particular case warrants further description and is indicated with an asterisk in Table 2. In this patient, two separate thyroid nodules in the same lobe were targeted on FNA. The 3–4-cm nodule yielded Thy3a cytology on initial and repeat cytology, with WT on genetic analysis; histology on the lobectomy showed this to be a dominant nodule in a multinodular goitre. A smaller 1-cm nodule was also aspirated on the second occasion, yielding Thy3a cytology, but BRAF V600E mutation was found on genetic testing; the histology was a 10-mm follicular adenoma with an immediately adjacent 4mm micro-PTC. This was a single focus of micro-PTC and, after MDT discussion, the patient has not required completion thyroidectomy. Immunostaining on this case is shown in Figure 1, using an antibody to BRAF V600E mutation that we have used successfully on histological sections, but not on cytology, highlighting negative staining (no BRAF mutation) in the follicular adenoma and positive staining (BRAF V600E mutation) in the immediately adjacent micro-PTC.
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BRAF testing on histology specimens Seven histology specimens (from four patients) were also tested for BRAF mutation. The results are shown in Table 5, with correlating cytology specimen results. Testing failed in two. Discussion
Figure 1. Immunohistochemical staining for BRAF V600E mutation in a histological section of a follicular adenoma (right, negative) and immediately adjacent papillary thyroid microcarcinoma (left, positive).
Cytology specimen types Table 3 shows the cytology specimen types correlated with the BRAF analysis results. The 12 failed specimens comprised six directly spread slides previously stained with Giemsa-based stains (either Diff-Quikâ or May–Gr€ unwald– Giemsa), five slides that had previously been stained for immunocytochemistry (ICC) and one Papanicolaou-stained SurePathâ liquid-based cytology (LBC) slide preparation. Multiple cytology specimens from the same nodule Some of the cytology specimens represented repeat or multiple samples from the same patient. These results are shown in Table 4. There were 11 such patients, nine of whom had only one lesion sampled. Two patients had multiple lesions sampled: one was the case described above; the other was a patient with two samples of different sites showing the same tumour on histology (poorly differentiated carcinoma, uncertain primary, probably not thyroid). Accuracy calculations Excluding the failed tests, BRAF mutation in preoperative cytology predicted subsequent malignancy on histology with 100% specificity and PPV, 38.6% sensitivity, 60.9% NPV and 69.4% accuracy.
Prior to this study, we had shown that BRAF genetic testing was feasible in highly selected archival histology and routine cytology cases.15 The current study was designed to assess prospectively over at least a year as many routine cytology specimens as had sufficient material. The results show that the testing is feasible on the majority of specimens, and that all cases with BRAF V600E mutation had PTC on subsequent histology. The frequency of BRAF mutation in PTCs overall was 50% (17/34). The BRAF mutation rate was highest in TCVPTC (2/3) and lowest in FVPTC (4/ 12), which is in line with previous reports.17,18 FVPTCs are known often to harbour RAS mutations rather than BRAF, more akin to follicular neoplasms.19 No BRAF mutations were seen in non-PTC thyroid malignancies. BRAF mutation was detected in one Thy3a cytology sample, which correlated with a 4-mm PTC on histology, immediately adjacent to the targeted 10mm lesion (a follicular adenoma); this was only a single micro-PTC not warranting completion thyroidectomy. The original report on this Thy3a cytology was of colloid and follicular epithelial cells, but with some microfollicular patterning, and a follicular neoplasm could not be excluded. Although incidental micro-PTCs are usually excluded from calculations of accuracy in thyroid cytology, this micro-PTC was located with the target lesion and so has been included. Of clinical importance is that completion thyroidectomy was not required in this patient, and so over-reliance and action on the BRAF result in this case could have led to over-treatment. The frequency of mutation decreased according to the cytology category for all specimens and those preceding histological confirmation of PTC. The majority of PTCs (9/11) following Thy3a or Thy3f cytology and half of those after Thy4 cytology were not associated with BRAF mutation, compared with 4/15 (27%) of Thy5. A WT BRAF genetic result cannot be taken as reassuring, because it does not exclude the presence of PTC, and ultrasound follow-
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Table 3. Correlation of cytology specimen type with outcome of BRAF testing Outcome of BRAF testing
Specimen type Cytology slides
Histology cases
DQ DQ/MGG MGG DPAS Papanicolaou ICC Unknown LBC Total Curls of FFPE tissue
Total number
Failed (%)
WT
V600E mutation
Non-V600E mutation
87 3 11 2 4 10 5 1 123 7
5 – 1 – – 5 – 1 12 2
66 3 10 2 3 5 4 – 93 1
15 – – – 1 – 1 – 17 4
1 – – – – – – – 1 –
DPAS, diastase-treated periodic acid Schiff-stained smear; DQ, Diff-Quik-stained smear; DQ/MGG, Giemsa-stained smear, DQ or MGG not specified; FFPE, formalin-fixed, paraffin-embedded tissue; ICC, immunostained slide; LBC, SurePath liquidbased cytology, Papanicolaou-stained slide; MGG, May–Gr€ unwald–Giemsa-stained smear; WT, wild-type.
up, lobectomy or thyroidectomy should not be avoided in suspicious, indeterminate or positive cases just because BRAF testing is negative. FVPTC can be a problematic diagnosis on histological assessment, with known inter-observer variation, even between expert endocrine pathologists,20 and represented a higher proportion of PTCs after indeterminate cytology in our study than suspicious or malignant; none of the six with indeterminate cytology had BRAF mutations. Views on the management of FVPTC are also changing, especially for the encapsulated variant, which may not always require total thyroidectomy, especially when noninvasive.21,22 It is known that FVPTC is more challenging on pre-operative cytology than classical PTC and may be represented in various of the cytology categories,23,24 and there is less inter-observer agreement with UK Thy3a and Thy4 cytology, and the Bethesda System AUS/FLUS and FN/SLN, than other categories.3,4 An important question is therefore which cytology categories could warrant the additional cost of BRAF mutation analysis to alter operative management without risking potential over-treatment of encapsulated FVPTCs or incidental micro-PTCs. From our results in this study, the case is stronger for the Thy4 cytology category than for the Thy3a and Thy3f categories. The Portsmouth group have recently advocated co-testing (Roche CobasTM platform; Roche Diagnostics, Burgess Hill, UK) all Thy4/suspicious thyroid cytology for BRAF mutation, © 2014 John Wiley & Sons Ltd Cytopathology 2014, 25, 146–154
with three BRAF-positive Thy4 cases proceeding to one-stage total thyroidectomy after discussion and slide review by the MDT.12 The Thy3a category cases, in particular, may benefit best from repeat cytology to obtain a more cellular sample, especially in cases in which the initial cytology was not obtained at ultrasound, which will provide additional useful information on the lesion. Others have reported that BRAF testing can increase the accuracy of pre-operative thyroid cytology,8,25,26 especially in BRAF mutation prevalent areas or as part of a panel.27–31 The high specificity for PTC could guide the extent of surgery and lymph node dissection5,6,29 Others have found occasional false-positive cases, especially with highly sensitive analytical methods, such as dual-priming oligonucleotide-based multiplex polymerase chain reaction (PCR).25 A lower mutation rate has been noted in FVPTC, reducing the value of the test in this setting, with the comment that the highest mutation rate is seen in cases that are diagnostic on FNA, as we have shown here and before,15 and that BRAF-negative PTCs are more likely to yield indeterminate cytology.30 Value has been demonstrated, particularly in cases that are suspicious on ultrasound.31 Hyalinizing trabecular tumours can mimic PTC on cytology, leading to over-diagnosis, but have usefully been shown to be BRAF mutation negative.32 Otherwise, a WT BRAF result is not reassuring and has a low NPV. Others have shown that ICC for other markers [e.g. cytokeratin 19 (CK19) and HBME-1] may have
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Table 4. Multiple cytology samples from the same patients
Patient and samples 1. 2. 3. 4.
BRAF test result
Cytology result Thy3–5
V600E Mutation
Wild Type
Failed Test
2 1
1
Same lesion, separate times Same lesion, separate times Same lesion, separate times Same lesion, separate times fluid with 1st sample 5. Same lesion, separate times two samples at 2nd FNA
3a, 3f Both 3f 4, 5 3f, 4 (fluid), 5
6. 7. 8. 9.
Same lesion, separate times Same lesion, solid and cystic Same lesion, solid and cystic Two slides from same lesion SurePath and Diff Quik 10. 3–4-cm nodule in lower pole two samples 1-cm nodule upper pole
Both 4 Both 3a 3a (fluid), 5 Both 5
11. Same tumour in lymph node and mediastinal mass Thyroid, same patient
Both malignant
1
3f
1
2 2
1 (fluid)
3a, 3a, 3f
2
1 1 (fluid) 1 (fluid) 1
1
Both 3a
1
1 1 1 (SP)
2
3a
1 1
Histology Outcome FVPTC Nodular HT PTC PTC (TCV) Hyperplastic nodule in multinodular goitre FVPTC None FVPTC PD/anaplastic carcinoma* Dominant nodule in multinodular goitre 10-mm FA; adjacent 4-mm microPTC PD carcinoma unknown origin* Non-neoplastic
FVPTC, follicular variant of papillary thyroid carcinoma; HT, Hashimoto’s thyroiditis; PTC, papillary thyroid carcinoma; TCV, tall cell variant; SP, SurePath; PD, poorly differentiated; FA, follicular adenoma. *Patient No. 9, probable thyroid origin; Patient 11, probable non-thyroid origin.
Table 5. Histology cases tested for BRAF mutation Histology specimens PTC PTC PTC FA
Thyroid Lymph node metastasis Thyroid Lymph node metastasis Thyroid Lymph node metastasis Thyroid
BRAF mutation result on FFPE curls
BRAF mutation result on pre-operative cytology specimen
V600E V600E Fail Fail V600E V600E WT
No cytology from thyroid V600E mutation in lymph node V600E mutation No cytology from lymph node V600E mutation No cytology from lymph node WT
mutation mutation
mutation mutation
FA, follicular adenoma; FFPE, formalin-fixed, paraffin-embedded; PTC, papillary thyroid carcinoma; WT, wild-type.
a better sensitivity than BRAF mutation for malignancy.9,11 For clinical application, the BRAF result would need to be turned around within a week or two, which would increase our cost to about £50 per sample, compared with the batched method for the study, which cost £25 per sample. Molecular testing of indeterminate cytology specimens could, how-
ever, decrease the cost of thyroid nodule evaluation overall by avoiding two-stage surgery in cases of thyroid cancer.33 Certain cytology specimen types had a higher failure rate for BRAF mutation testing, especially the SurePath LBC case, as noted previously.15 There was a 50% failure rate for slides on which ICC had previously been performed, suggesting that, until © 2014 John Wiley & Sons Ltd Cytopathology 2014, 25, 146–154
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methodology improvements can be made, these slides are best avoided for BRAF testing by this method. Interestingly, there was not always correlation in BRAF results when more than one cytology sample was available from a single lesion. This mostly reflected one of the tests failing, but, in six cases, there was more than one non-failed specimen. We have reported this mismatch previously in samples from the same lesion.15 The reason for this is not entirely clear, but it may reflect the proportion of tumour in the sample. The importance of pre-analytical steps has recently been emphasized7 and manual dissection may be helpful.13,26 BRAF test results on the few histology samples correlated very well with the results on preceding cytology. This study has limitations, especially the small numbers of cases and long turnaround time for BRAF results, but reflects real-life prospective collection of cases from one department. In summary, we have found that BRAF mutational analysis by melt curve analysis is feasible in routine thyroid cytology specimens and shows 100% specificity for PTC in subsequent histology, potentially allowing for one-stage therapeutic surgery after less than diagnostic cytology suspicious for malignancy. Mutation was seen more frequently in diagnostic than indeterminate cytology but sensitivity is low and there is no role for avoiding diagnostic thyroid surgery if WT BRAF is found. Careful cost-effectiveness analysis is needed with consideration as to which cytology specimens should undergo testing.
Acknowledgments We thank Anna Long for performing immunohistochemistry for BRAF and Steve Brabazon for assistance with photography. We are also grateful to colleagues in local pathology hospitals for help with tracing histology results.
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