Annals of Surgical Oncology

, Volume 16, Issue 2, pp 240–245

Analysis of Differential BRAFV600E Mutational Status in High Aggressive Papillary Thyroid Microcarcinoma

Authors

  • Xiaolong Lee
    • Department of Thyroid & NeckTianjin Medical University Cancer Hospital
    • Department of Thyroid & NeckTianjin Medical University Cancer Hospital
  • Yifeng Ji
    • Department of Thyroid & NeckTianjin Medical University Cancer Hospital
  • Yang Yu
    • Department of Thyroid & NeckTianjin Medical University Cancer Hospital
  • Ying Feng
    • Department of Thyroid & NeckTianjin Medical University Cancer Hospital
  • Yigong Li
    • Department of Thyroid & NeckTianjin Medical University Cancer Hospital
  • Yan Zhang
    • Department of Thyroid & NeckTianjin Medical University Cancer Hospital
  • Wenyuan Cheng
    • Department of Thyroid & NeckTianjin Medical University Cancer Hospital
  • Wenchuan Zhao
    • Department of Thyroid & NeckTianjin Medical University Cancer Hospital
Head and Neck Oncology

DOI: 10.1245/s10434-008-0233-3

Cite this article as:
Lee, X., Gao, M., Ji, Y. et al. Ann Surg Oncol (2009) 16: 240. doi:10.1245/s10434-008-0233-3

Abstract

Papillary thyroid cancers often occur as microcarcinoma. Some papillary thyroid microcarcinoma (PTMC) have been considered to be high aggressive according to advanced disease stages, extrathyroidal extension, and severe cervical lymph node metastasis. Although several factors are thought to predict the occurrence of aggressiveness from PTMCs, the origin of aggressiveness has been rarely studied. To answer this question, the correlation between BRAFV600E mutation and high aggressive PTMCs was investigated. The clinicopathological characteristic of totally 64 cases of PTMCs was investigated and the BRAFV600E mutational status of them was identified. BRAFV600E mutation was exclusively detected in PTMCs (37.5%). The data provided no correlation between the occurrence of BRAFV600E mutations and clinicopathological parameters, such as sex, age, and tumor-like lesions combination. The prevalence of BRAFV600E mutation of PTMCs with high aggressiveness (advanced disease stages, extrathyroidal extension, and nodal metastasis) was significantly higher (< 0.05) than that of PTMCs without aggressive behavior. The BRAFV600E mutated PTMCs exhibited signs of higher aggressiveness than PTMCs without the mutation. BRAFV600E mutation may be a marker of high aggressiveness in PTMCs.

Papillary thyroid microcarcinomas (PTMCs) were defined by the World Health Organization in 1988 as papillary carcinoma measuring <10 mm in diameter. As living conditions and medical treatment have greatly improved, more PTMCs have been discovered in the early stage. Although most reports have shown that PTMCs followed an indolent course, our recent study have demonstrated that a significant number were associated with high aggressiveness, such as advanced disease stages, extrathyroidal extension, and cervical lymph node metastasis.

During the past few years, it has become evidence that aberrant signaling through the RAS–RAF–MEK cascade is crucial for the development of thyroid cancer. In the case of papillary thyroid carcinoma (PTC), the subtype that accounts for more than 80% of all thyroid cancers, mutations, and/or gene rearrangements in cell surface receptors or signaling molecules in this cascade. Of these abnormalities, activating mutations in BRAFV600E are particularly common in adults with PTC. In several studies, the presence of a BRAFV600E mutation has been associated with a more aggressive clinical course. Namba and colleagues1 and Nikiforova and colleagues2 reported that the BRAFV600E mutation was associated with tumor stages III and IV, and Xu and colleagues3, Namba and colleagues,1 and Kim and colleagues4 reported an association between BRAFV600E and metastatic tumors. In contrast, Puxeddu and colleagues5 did not find any association between the presence of BRAFV600E in PTC and tumor staging, local invasiveness, or lymph node metastasis.

In an attempt to clarify the origin of high aggressive PTMCs, we analyzed the association between the occurrence of BRAFV600E mutations and PTMCs to elucidate any possible relationship between the occurrence of BRAF mutations and some high aggressive clinicopathological features (advanced disease stages, extrathyroidal extension, and lymph node metastasis) of PTMCs.

Materials and Methods

Materials from 64 patients with 64 tumors of the thyroid were collected from Tianjin Medical University Cancer Hospital. The histological classification was made by three pathologists without information on BRAF data. Whenever there was a discrepancy, the case was discussed together under a multiple head microscope and a consensus diagnosis was reached.

Date on sex, age, nodular goiter, Hashimoto thyroiditis, variant, disease stage, extrathyroidal extension, and lymph node metastasis were available in all the 64 patients. In all the cases, 40 samples were paraffin-embedded and 24 samples were fresh tissue stored at −80 centigrade. The DNA material was retrieved from paraffin blocks and fresh tissue after careful microdissection performed by an experienced pathologist.

DNA Extraction

Genomic DNA was extracted from paraffin-embedded tissue and fresh tissue. For paraffin-embedded tissue, the tumor areas were marked using 4–mm-thick hematoxylin and eosin-stained sections as a guide. Then the marked areas were matched with dewaxed but unstained 20–mm-thick sections. The tumor areas were dissected from the unstained slides and transferred into Eppendorf tubes. After the dissection, the blocks were cut into 4-mm sections for hematoxylin and eosin staining to confirm the tumor continuity. All samples, including fresh tissue, were then digested with proteinase K for more than 24 hours at 56 centigrade and DNA was isolated from the digested tissue using a Tissue SV mini kit (Bao Biosystems, Dalian, China).

PCR-RFLP and Sequencing

The BRAF mutation reported recently in thyroid carcinomas is confined to exons 11 and 15. We therefore amplified BRAF exon 15 by polymerase chain reaction (PCR) by using the following primers: forward, 5′-GCTTGCTCTGATAGGAAAATG AG-3′; reverse, 5′-GATACTCAGCAGCATCTCAGG-3′. PCRs conditions were: initial denaturation at 95 centigrade for 5 minutes, followed by 40 cycles of denaturation at 94 centigrade for 20 seconds, annealing at 56 centigrade for 20 seconds, and elongation at 72 centigrade for 20 seconds, and a final extension at 72 centigrade for 10 minutes. The denatured PCR products were electrophoresed and digestion of the 237-base pair (bp) PCR fragment with restriction endonuclease TspRI yielded one major band of 117 bp. The V600E mutation abolished the restriction sites, resulting in a prominent band of 237 bp from the mutant allele and residual bands from the normal allele (Fig. 1).
https://static-content.springer.com/image/art%3A10.1245%2Fs10434-008-0233-3/MediaObjects/10434_2008_233_Fig1_HTML.gif
Fig. 1

M Marker (100–2000 bp); * BRAFV600E mutation case. Polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis and sequence of codon 600 of the BRAF gene. Results of PCR-RFLP of the wild-type and mutant BRAF gene. The V600E mutation abolished the restriction sites, resulting in a prominent band of 237 bp from the mutant allele and residual bands from the normal allele

To confirm the reliability of the PCR-restriction fragment length polymorphism (RFLP) results, we sequenced all the 24 mutated samples in a PTC-225 Peltier Thermal Cycler using ABI Prism BigDye Terminator Cycle Sequencing Kits (Bao Biosystems, Dalian, China) and AmpliTaq DNA polymerase (FS enzyme) (Bao Biosystems). Single-pass sequencing was performed on each template using the forward primer. The fluorescence-labeled fragments obtained were purified from unincorporated terminators by ethanol precipitation. Samples were then resuspended in distilled water and subjected to electrophoresis in an ABI 3730xl sequencer (Bao Biosystems). All 24 sequences so determined confirmed the BRAFV600E status indicated by the PCR-RFLP analysis (Fig. 2).
https://static-content.springer.com/image/art%3A10.1245%2Fs10434-008-0233-3/MediaObjects/10434_2008_233_Fig2_HTML.gif
Fig. 2

Sequence of the V600E BRAF gene. Because the 1799A mutant allele of the BRAF gene and the normal 1799T allele were both present, the sequencing result show a mixture of T and A at the 1799 site

Statistical Analysis

Statistical analyses were performed with SPSS software (version 13.0; SPSS Inc., Chicago, IL). Chi-square or Fisher exact tests were used when comparing frequencies between groups. All numeric data were expressed as means ± SD, and differences between group means were compared by the independent sample Student’s t test or the Mann–Whitney U test. Probability values <0.05 were considered statistically significant.

Results

Clinicopathological Characteristics of Patients with PTMCs

The 64 PTMCs patients whose samples yielded analyzable DNA all underwent total thyroidectomy with routine central neck node dissection (Table 1). All of these patients had tumors ≤10 mm in diameter. The frequencies of extrathyroidal extension and lymph node metastasis in these patients were 25% and 28.1%, respectively. None of these patients was positive for distant metastases. Two patients underwent modified lateral neck dissection due to suspected lateral neck metastasis, and the presence of lateral neck metastasis in each was confirmed by postoperative pathological examination. Forty-eight patients had stage T1 tumors, none had stage T2, 15 had stage T3, and 1 had stage T4a. In addition, the frequencies of nodular goiter and Hashimoto thyroiditis in these patients were 21.8% and 12.5%, respectively. The follow-up period after operation ranged from 9 to 30 months. No patient had clinical recurrence during the observation period.
Table 1

Analysis of BRAFV600E mutation in PTMCs

 

BRAF mutation

p

Positive (n = 24)

Negative (n = 40)

Age (yr)

49.1 ± 10.4

45.1 ± 11.2

0.533

Male:female ratio

2:22

6:34

0.358

Nodular goiter

6 (25%)

8 (20%)

0.432

Hashimoto thyroiditis

0

8 (20%)

>0.99

Extrathyroidal extension

12 (50%)

4 (10%)

0.001*

Nodes metastasis

12 (50%)

6 (15%)

0.003*

Tall cell variant

2 (8.3%)

2 (5%)

0.483

T1:T3/4 ratio

12:12

36:4

0.001*

* Statistically significant

Correlation of Clinicopathological Characteristics and BRAFV600E Mutation in PTMCs

BRAFV600E mutation frequency in PTMCs was 37.5% (24/64). Date from Table 1 indicated that there was no significant correlation between BRAFV600E mutation and some clinicopathological characteristics in PTMCs, such as sex, age, nodular goiter, and Hashimoto thyroiditis (p > 0.05), although a background of nodular goiter tended to be present at a higher frequency in patients with BRAFV600E PTMCs. Among all the cases, 50% (12/24) of tumors in T3 or T4 stage were in the mutated group and only 10% (4/40) of tumors in T3 or T4 stage were in the wild-type group (p < 0.05), which showed that BRAFV600E mutation may have correlation with advanced disease stages of PTMCs. Also the statistically significance existed when PTMCs with extrathyroidal extension and nodes metastasis.

BRAFV600E Mutational Status in Metastatic Lymph Nodes

To assess the correlation between the BRAF mutational status of primary tumors and that of metastatic tumors in lymph nodes, we also performed BRAFV600E mutational analyses on the lymph node samples of 18 PTMCs, 12 of which were BRAFV600E-positive and 6 BRAFV600E-negative. The 6 BRAFV600E-negative PTMCs did not have the BRAFV600E mutation in the metastatic lymph nodes. Ten of 12 BRAFV600E PTMCs had the BRAFV600E mutation in their metastatic lymph nodes and 2 had wild-type BRAF (Table 2).
Table 2

Comparison of BRAFV600E mutational status of PTMCs of the thyroid and associated metastatic carcinomas of the lymph nodes

Case no.

BRAFV600E mutational status

Tumor foci of thyroid

Metastatic lymph nodes

1

Positive

Positive

3

Negative

Negative

4

Positive

Positive

5

Positive

Positive

6

Negative

Negative

10

Positive

Positive

12

Positive

Negative

20

Negative

Negative

21

Positive

Positive

25

Negative

Negative

30

Positive

Positive

32

Positive

Positive

35

Negative

Negative

39

Positive

Positive

46

Positive

Negative

55

Negative

Negative

60

Positive

Positive

61

Positive

Positive

Discussion

Thyroid cancers ≤10 mm in diameter, usually papillary (PTMCs), represent up to 43% of all thyroid cancers.6 This cancer generally has a benign prognosis, and cancer-related death has rarely been reported.7,8 However, persistence/recurrence of disease has been reported in some studies with a low prevalence ranging from 1.4–6%811 and in others with a higher prevalence ranging from 13–26%.7,12 The value of the BRAFV600E mutation as a prognostic factor has been evaluated in many studies.1317 This oncogenic event may be an initiating or very early step in tumor development, perhaps by facilitating the acquisition of secondary genetic events through induction of genomic instability.18 However, attempts to show a relationship between the BRAFV600E mutation and the clinicopathological outcomes of PTMCs have yielded mixed results.35,1921 In the present study, we evaluated the origins of high aggressive PTMCs by analyzing BRAFV600E mutation status in the individual tumors in a series of PTMCs. Our results showed that BRAFV600E mutation was indentified in 50% of high aggressive PTMCs. In our study population, we found that age distribution, gender ratio, variants, nodular goiter, and Hashimoto thyroiditis combination did not differ significantly between patients with and without the BRAFV600E mutation. However, extrathyroidal extension, tumor stages, and lymph node metastasis differed significantly. These findings seem to indicate that the presence of the BRAFV600E mutation is related to the degree of PTMCs malignancy in the Chinese population.

High aggressive PTMCs are becoming more common, but their origin is not clear. High aggressive PTMCs may be an important predictor of behavior, because if a given high aggressive PTMCs has resulted from intrathyroidal metastasis, there is likely to be an increased risk of further metastases.22,23 In some study, approximately 60% of high aggressive PTC cases had BRAFV600E mutational status, which supports the notion that a large percentage of high aggressive PTC are the result of the BRAFV600E mutation.10,23,24 When we analyzed BRAFV600E mutational status according to high aggressive PTMCs, we found that BRAFV600E-mutated cases were more frequent in patients with penetration of capsule than in those without. Our results showed that 50% of tumors in T3 or T4 stage were in the mutated group and only 10% (4/40) of tumors in T3 or T4 stage were in the wild-type group, which indicated that BRAFV600E mutational status can be used to predict the presence of extrathyroidal metastasis.

Lymph node metastasis, as a sign of high aggressiveness, has been reported to be a risk factor for persistence/recurrence of disease and also for cancer-related mortality in many studies.8,2530 Furthermore, the lymphatic drainage system of the thyroid gland supports this possibility because an abundant network of intralobular lymphatic vessels anastomoses and penetrates into the capsule throughout the gland, and this would favor spread to other parts of the gland.22,23 Our results showed that BRAFV600E mutation was more frequent and statistically significant when PTMCs combined with T3/4 stage, extrathyroid extension, and nodes metastasis, which indicated that BRAFV600E mutation may be a predictor of high aggressiveness. Besides, the BRAFV600E mutation of PTMCs was reported to show a clear high aggressive subtype-related pattern, which supported the relationship between BRAFV600E mutational status and poor behavior.31,32 Our results confirmed that BRAFV600E mutation was more prevalent in tall cell PTMCs than in the other variant of PTMCs, although cases of tall cell variant were too few to show statistically significant differences.

The BRAF mutated PTMCs of our series did exhibit signs of clinical aggressiveness (advanced disease stages, extrathyroidal extension, and lymph node metastasis), although we cannot insist that all the tumors of BRAF mutational status are of high aggressiveness because the use of mutational status as a marker of clonality has limitations.33 However, we did observe a significant association between BRAFV600E mutation status and prognostic indicators, and some trends were observed. For example, 66.7% (12/18) of PTMC patients with lateral neck lymph node metastases were positive for the BRAFV600E mutation. In addition, 75% (12/16) of patients who experienced extrathyroid extension harbored the BRAFV600E mutation. Our data contrast with those of other groups,14 who reported a negative association between BRAF mutations and tumor staging. In fact, because PTMCs are associated with an extremely good prognosis after surgical removal, large numbers of patients with very long-term follow-up are needed to identify the clinical relevance of the BRAFV600E mutation in PTMCs. Despite the limitations, our data suggest that larger studies are warranted to examine the relationships between the BRAFV600E mutation and extrathyroid extension, stages, and/or lateral neck node metastases in patients with PTMCs.

In conclusion, we have shown that the BRAFV600E mutation was found in 50% of high aggressive PTMC patients from a Chinese study population, which suggests that high aggressive tumors arise in a significant subset of BRAFV600E mutational PTMCs. Our results further indicate that this mutation is associated with prognostic factors in Chinese patients with PTMCs.

Acknowledgements

The authors thank Drs. Baocun Sun for providing material, and Lili Yang and Feng Fei for grant support to Xiaolong Lee, Ming Gao, Yifeng Ji, Yang Yu, Ying Feng, Yigong Li, Yan Zhang, Wenyuan Cheng, and Wenchuan Zhao.

Copyright information

© Society of Surgical Oncology 2008