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Abstract
Purpose:
The prevalence rate of the BRAF mutation in papillary thyroid cancer (PTC) is as high as about 52 to 83% in Korea. Preoperative detection of BRAF mutation on fine needle aspiration cytology (FNAC) slides may help the surgeon make better therapeutic decisions. The present study aims to assess the feasibility of the mutant allele specific amplification (MASA) and restriction fragment length polymorphism (RFLP) method with using conventional FNAC slides and we also wanted to evaluate the clinical role of preoperatively detecting BRAF mutation.
Methods:
We extracted the genomic DNA from 59 FNAC slides and performed direct sequencing (DS) for detecting BRAF mutation. We could use only 17 slides for the MASA method and 6 slides for the RFLP method due to the shortage of extracted DNA. Additionally, we retrospectively analyzed the cases for which a histological diagnosis could be made.
Results:
Genomic DNA was extracted from 23 out of the 59 FNAC slides. The BRAF mutation status could be assessed via DS in 33 out of the 59 FNAC slides. The concordance between the MASA method and DS and the RFLP method and DS was 36.3% and 66.7% respectively. The positive and negative predictive value of the 13 indeterminate nodules was 87.5% and 20%, respectively. We could not find any association between the BRAF mutations and the alleged risk factors of PTC.
Conclusion:
We believe that the purity and the amount of the DNA template must be increased to detect BRAF mutation with using a FNAC slide. Preoperative detection of the BRAF mutation on a FNAC slide may refine the cytological diagnosis, but the application of assessing BRAF mutation as a prognostic marker is debatable.
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Fig. 2
BRAF exon15 PCR amplification. Which samples have enough and pure DNA to be sequenced might not be determined on electrophoresis gel.
Fig. 3
MASA products showing low sensitivity for BRAF mutation. Faint bands on No. 1, 3, 16 and 17 indicate the presence of BRAF mutation (Underlined numbers of samples have been proved for positive BRAF mutation by direct sequencing).
Table 1.
Results of direct sequencing with fine needle aspiration cytology slides
| No.∗ | Direct sequencing | No. | Direct sequencing | No. | Direct sequencing |
|---|---|---|---|---|---|
| 1 | Mutant type | 21 | Mutant type | 41 | X† |
| 2 | Mutant type | 22 | Wild | 42 | Wild |
| 3 | Mutant type | 23 | X | 43 | X |
| 4 | Mutant type | 24 | Mutant type | 44 | X |
| 5 | X† | 25 | X | 45 | Mutant type |
| 6 | Mutant type | 26 | Wild | 46 | X |
| 7 | Mutant type | 27 | Mutant type | 47 | X |
| 8 | X | 28 | Mutant type | 48 | X |
| 9 | Mutant type | 29 | Mutant type | 49 | Mutant type |
| 10 | Mutant type | 30 | X | 50 | X |
| 11 | Mutant type | 31 | Mutant type | 51 | X |
| 12 | X | 32 | Wild | 52 | X |
| 13 | Mutant type | 33 | Wild | 53 | X |
| 14 | Wild | 34 | Wild | 54 | Mutant type |
| 15 | Wild | 35 | Wild | 55 | X |
| 16 | Mutant type | 36 | X | 56 | X |
| 17 | Wild | 37 | Mutant type | 57 | Mutant type |
| 18 | X | 38 | Mutant type | 58 | X |
| 19 | X | 39 | X | 59 | X |
| 20 | X | 40 | X |
Fig. 4
RFLP products showing insecure results for BRAF mutation (Underlined numbers of samples have been proved for positive BRAF mutation by direct sequencing).
Table 2.
Association between cytological diagnosis and BRAF mutation status
| Cytological diagnosis | BRAF status∗ | |||
|---|---|---|---|---|
| Mutant type | Wild type | No information | Total | |
| Benign | 1† | 2 | 4 | 7 |
| Suspicious for FN‡ | 1§ | 2 | 5 | 8 |
| Suspicious for PTC∥ | 8 | 3 | 4 | 15 |
| Malignant (PTC) | 13 | 3 | 9 | 25 |
| Inadequate | 0 | 0 | 4 | 4 |
| Total | 23 | 10 | 26 | 59 |
Table 3.
Results of the cytological diagnosis and the number of patients who received surgery in Seoul National University Hospital
| Cytological diagnosis | Number of patients received surgery (n=46) | Number of lost patients (n=11) |
|---|---|---|
| Benign | 3 | 3 |
| Suspicious for FN∗ | 8 | 0 |
| Suspicious for PTC† | 12 | 3 |
| Malignant (PTC) | 20 | 4 |
| Inadequate | 3 | 1 |
Table 4.
BRAF mutation status of fine needle aspiration cytology slides with malignant and indeterminate result on cytology and postoperative histological diagnosis
| Cytological diagnosis | BRAF status | Histological diagnosis (Number) |
|---|---|---|
| Malignant | Mutant | PTC∗ (11) |
| Wild | PTC (2) | |
| Suspicious PTC | Mutant | PTC (7) |
| Wild | PTC (2), Minimal invasive FTC† (1) | |
| Suspicious FN‡ | Mutant | Nodular hyperplasia (1) |
| Wild | Minimal invasive FTC (1), Nodular hyperplasia (1) |
Table 5.
Association between BRAF mutation status and clinico pathological characteristics in 22 papillary thyroi cancer patients
| BRAF mutation (＋) | BRAF mutation (−) | P value | |
|---|---|---|---|
| Number | 18 | 4 | |
| Female：Male | 14：4 | 14：1 | 0.905∗ |
| Age (range) | 51.3 (34∼74) | 43.5 (39∼49) | 0.233† |
| Tumor size (cm) | 1.33±0.76 | 1.05±0.7 | 0.494† |
| LN metastasis | 6 (33.3%) | 2 (50%) | 0.602∗ |
| Perithyroidal extension | 14 (77.8%) | 3 (75%) | 1.000∗ |
| Multiplicity | 7 (38.9%) | 3 (75%) | 0.293∗ |
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