The BRAF V600E Mutation Influences the Short- and Medium-Term Outcomes of Classic Papillary Thyroid Cancer,But Is Not an Independent Predictor of Unfavorable Outcome

Abstract

Introduction:

The prognostic usefulness of BRAF^V600E evaluation in papillary thyroid cancer (PTC) has been analyzed in many studies, with controversial conclusions.

Aim:

To analyze the clinical relevance of BRAF^V600E measurement in a homogenous series of PTC patients followed in a single institution.

Methods:

One hundred three classical variant PTC patients who underwent total thyroidectomy in the 3-year period between 2005 and 2008 were retrospectively selected, and BRAF^V600E assessment was performed using paraffin-embedded archival specimens in 2013. All patients were actively followed at our medical center, with an average follow-up of 55±13 months.

Results:

BRAF^V600E mutation-positive cancers (55.3%) were more frequently associated with lymph node metastasis (p=0.01) and advanced TNM stage (III–IV) (p=0.03). These findings were also confirmed in the subset of 42 microcarcinomas. BRAF^V600E -positive patients were also at a higher risk of persistent disease (OR 3.5 [95% confidence interval {CI} 1.2–10.3], p=0.03) in univariate but not multivariate analysis (OR 2.8 [CI 0.7–11.8], p=0.2). Lymph node involvement was an independent predictor of persistent disease (OR 30.9 [CI 6.0–159.0], p<0.0001). Kaplan-Meier curves confirmed a higher percentage of persistent/recurrent disease in BRAF^V600E -positive patients (p=0.02). However, the BRAF^V600E mutation did not change the recurrence rate of PTC in subgroup analyses on the basis of other established risk factors (p=0.2).

Conclusions:

BRAF^V600E -positive tumors were at higher risk of developing more aggressive behavior and were associated with less favorable outcomes in the short and medium term, but the BRAF^V600E mutation was not an independent predictor of unfavorable outcome. Therefore, its use as a prognostic marker in clinical practice is not advisable.

Introduction

Papillary thyroid cancer (PTC) is the most common endocrine neoplasm, and its incidence is increasing worldwide (1). Small PTCs (<2 cm) are primarily responsible for this increase, which has been attributed to the wider use of diagnostic techniques (2,3) that allow the diagnosis of indolent in situ cancers that previously went undiagnosed because they never reached clinical evidence. However, a true increase in the incidence of PTC is also a likely possibility because other thyroid cancer histotypes have not increased at the same rate (4,5). Moreover incidentally and nonincidentally discovered cancers have similar characteristics (6), and their mortality has remained unchanged in spite of great progress in diagnosis and treatment (7 –9). Although most small-PTC patients have excellent outcomes (>90% overall survival at 10 years), up to 15% of these patients have persistent or recurrent disease (10,11). Even papillary microcarcinomas (PTMCs), which are tumors smaller than 1 cm, may be associated with recognized risk factors for persistent/recurrent disease such as lymph node metastases, extrathyroidal extension, and multiple cancer foci (12 –14). Indeed, risk assessment and appropriate treatment of small PTCs are not clearly defined (15,16).

To improve risk stratification and clinical management, the presence of the BRAF^V600E mutation has been suggested as a prognostic marker of PTC outcomes. This genetic alteration is found in 29%–83% of PTCs (17) and consists of a T to A transversion that determines the substitution of valine for glutamate at position 600 of the B-RAF protein, which induces the constitutive activation of the enzyme (18). The prevalence of BRAF^V600E -positive PTCs may be influenced by environmental factors, as suggested by the great variability in different geographic areas (17,19,20) and by the significant increase in its prevalence in PTCs diagnosed over the last 15 years (21).

BRAF is a component of the MAPK signaling pathway, which plays a central role in the regulation of cell proliferation; when up-regulated, it promotes tumorigenesis in many human cancers, including thyroid cancer (22). Most clinical studies have found an association between the presence of the BRAF^V600E mutation and both clinicopathological features of cancer aggressiveness (extrathyroidal extension, lymph node metastasis, larger size, and advanced TNM stage) and poor clinical outcomes (recurrence/persistent disease and mortality) (17,23 –28). Other studies, however, did not find this association (29 –32). Additionally, in PTMCs and low-risk PTCs, the most frequent type of thyroid cancers, the prognostic role of the BRAF^V600E mutation is controversial (33 –38).

To further evaluate the clinical relevance of BRAF^V600E measurement in PTC, study, we report data obtained from a continuous and homogenous series of 103 classical variant PTCs at a single institution; the patient treatment procedures were identical, and both the clinicopathological characteristics and the outcomes were recorded.

Materials and Methods

Patients

We retrospectively selected a consecutive series of patients with thyroid cancer who underwent total thyroidectomy at the Surgical Oncology Unit of our Medical Center from 2005 through 2008. Central lymph node dissection was performed when suspicious nodes were detected at preoperative ultrasound or during surgery (therapeutic dissection), or when preoperative diagnosis of thyroid cancer was cytologically assessed (prophylactic dissection). Within this series, 103 patients were selected according to the following criteria: 1) histological diagnosis of classical variant PTC according to the World Health Organization criteria (defined by the presence of true papillae with a central fibrovascular core and a lining of cuboidal cells with ground glass nuclei, nuclear pseudoinclusions, and nuclear grooves) (39), and 2) available data on postsurgical follow-up and current disease status. Postoperative treatment included thyrotropin (TSH) suppression at appropriate levels according to current guidelines (15,16).

Intrathyroidal unifocal microcarcinomas without lymph node metastases were not ablated with ¹³¹I (n=21). Therefore, postsurgical radioiodine ablation (1110–3700 MBq of ¹³¹I) and subsequent whole-body scans were performed in 82 of 103 patients (79.6%).

All patients were actively followed up at our thyroid clinic (at least two visits per year) for an average of 55.0±13.3 months (median 52.8, range 36.0–88.7). Patients who underwent ¹³¹I ablation were considered free of disease when their neck ultrasound and whole-body scan were negative and when their serum thyroglobulin levels were less than 2 ng/mL after human recombinant (rh)TSH administration or less than 5.0 ng/mL after levothyroxine (LT₄) withdrawal, with undetectable anti-thyroglobulin antibodies. In the 21 patients who did not undergo ¹³¹I ablation, the criteria for remission were negative neck ultrasound and low serum thyroglobulin levels with no increase over time. Patients that did not meet the described criteria at last control were considered affected by persistent disease.

BRAF^V600E assessment was performed using archival formalin fixed paraffin-embedded (FFPE) PTC specimens in 2013. Therefore, the presence of the BRAF^V600E mutation was unknown to clinicians taking care of the patients and did not influence diagnostic and therapeutic procedures.

DNA extraction and BRAF detection

DNA was isolated from FFPE blocks sectioned at 10 μm thickness by microtome and collected in 1.5 mL tubes. These microsectioned specimens were treated by xylene to remove the paraffin. Genomic DNA was extracted using a 5 Prime DNA FFPE Tissue kit (5 PRIME, Inc. Gaithersburg, MD) according to the manufacturer's instructions.

Polymerase chain reaction and DNA sequencing

Isolated DNA was amplified by polymerase chain reaction (PCR) using a specific pair of primers for exon 15 of the BRAF gene: 5′-TCA TAA TGC TTG CTC TGA TAG G-3′ (forward) and 5′-GGC CAA AAA TTT AAT CAG TGG A-3′ (reverse). Primer pairs were synthesized by MWG-Biotech (Ebersberg, Germany). The PCR thermal program included initial denaturation at 95°C for 5 minutes followed by 40 cycles of denaturation at 94°C for 30 seconds, annealing at 57°C for 45 seconds and elongation at 72°C for 45 seconds. After the last cycle, a final extension at 72°C for 10 minutes was performed. Specific amplification of the 222-bp fragment was verified by visualizing 5 μL of PCR product on a 2% agarose TBE gel using a Gel Doc image analysis system (Bio-Rad, Hercules, CA). PCR-amplified products were sequenced by Eurofins MWG Operon (Ebersberg, Germany).

Statistical analysis

Quantitative data are shown as the mean±SD, and numbers and percentages are provided for qualitative data. Percentages were compared using χ² tests, and t-test was used for continuous variables. Univariate and multivariate analyses were performed using logistic regression. The results are reported with odds ratio (ORs) and 95% confidential intervals (CIs). Survival curves were analyzed using the Kaplan-Meier method, statistical significance was determined using the log-rank test and p values <0.05 were considered statistically significant. Statistical analyses were performed using SPSS software, version 13.0 for Windows (SPSS Inc., Chicago, IL).

Results

Clinical and histopathological features at presentation

Table 1 summarizes the clinical and histopathological features of the 103 patients (F=86, M=17, F/M ratio=5.1) included in the study. The mean age at diagnosis was 44.2±14.9 years (median 45, range 15.2–78.2). The mean tumor size was 1.5±0.9 cm (median 1.4, range 0.3–4.0 cm). PTMCs were 42 of 103 cases (40.8%). Multifocality, extrathyroidal extension (including both minimal and large invasion), and lymph node metastases were present in 39.8%, 30.0%, and 35.0% of the patients, respectively. None of the patients exhibited distant metastasis at diagnosis. According to TNM classification (VII edition), 73 cases (70.9%) were classified as stage I, three (2.9%) were classified as stage II, 21 (20.4%) were classified as stage III, and six (5.8%) were classified as stage IV (Table 1).

Table 1.

Association Between BRAF ^V600E Mutation, Clinicopathological Characteristics at Diagnosis, and Outcomes in 103 Papillary Thyroid Cancer Patients

	All	BRAF⁺	BRAF⁻	p ^a
N	103	57 (55.3)^b	46 (44.7)
Patients at presentation
Male sex	17 (16.5)	10 (17.5)	7 (15.2)	0.8
Mean age±SD, years	44.2±14.9	44.8±11.5	43.4±16.8	0.6
>45 years	51 (49.5)	30 (52.6)	21 (45.7)	0.6
Mean tumor size±SD, cm	1.5±0.9	1.64±0.9	1.42±0.8	0.2
>2 cm	37 (35.9)	21 (36.8)	16 (34.8)	0.8
PTMC	42 (40.8)	20 (35.1)	22 (47.8)	0.2
Multifocality	41 (39.8)	22 (38.6)	19 (41.3)	0.8
Extrathyroidal invasion	31 (30.0)	19 (33.3)	12 (26.1)	0.5
LNM	36 (35.0)	26 (45.6)	10 (21.7)	0.01
TNM stage III–IV	27 (26.2)	20 (35.1)	7 (15.2)	0.03
Outcome
Follow-up±SD, months	55.0±13.3	56.9±14.1	52.7±11.6	0.1
¹³¹I therapy	82 (79.6)	50 (87.7)	32 (69.6)	0.03
Recurrent/persistent disease during follow-up	27 (26.2)	20 (35.1)	7 (15.2)	0.03
Persistent disease at last control	22 (21.4)	17 (29.8)	5 (10.9)	0.03

p- Values refer to the comparison between BRAF^V600E -positive and BRAF^V600E -negative patients.

Values in parentheses are percentages.

LNM, lymph node metastasis; PTMC, papillary microcarcinoma; SD, standard deviation.

Postsurgical follow-ups and outcome

Thirteen (12.6%) patients had persistent disease after surgery, and recurrence was observed in 14 (13.6%) patients after a median disease-free interval of 19.8 months. Among these 27 patients, 10 had a serum thyroglobulin >2.0 ng/mL after rhTSH administration or >5.0 ng/mL after LT₄ withdrawal without morphological evidence of disease; 16 patients had lymph node metastases identified by neck ultrasound and confirmed by performing a thyroglobulin assay of the washout liquid from the fine-needle biopsy; one patient had distant metastases identified by whole-body scan and computed tomography. Five out of these 27 patients with persistent/recurrent disease decided to follow a conservative approach with no further treatment, whereas of the remaining 22 patients, 16 underwent surgical excision followed by radioiodine treatment, and six patients were treated only with one or more radioiodine administration. Among these 27 patients, at the time of the last visit, five were disease free, while 22 of 103 patients (21.4%) were not disease free.

The parameters associated with persistent disease at last control are listed in Table 2. The univariate analysis indicated that male sex, tumor size, lymph node metastasis, advanced stage at diagnosis, and presence of the BRAF^V600E mutation were all associated with persistent disease. The multivariate analysis indicated that, after adjusting for age at diagnosis, sex, tumor size, lymph node metastasis, and the presence of the BRAF^V600E mutation, only the presence of lymph node metastases (OR 30.9 [CI 6.0–159.0], p<0.0001) was an independent factor associated with persistent disease at the last follow-up.

Table 2.

Univariate and Multivariate Analysis for Persistent Disease at Last Follow-Up Visit

	Univariate analysis		Multivariate analysis ^a
Parameters	OR [CI]	p	OR [CI]	p
Male sex	4.6 [1.5–13.9]	0.007	3.6 [0.8–16.5]	0.1
Age at diagnosis	1.0 [0.9–1.1]	0.87	1.0 [0.9–1.0]	0.5
>45 years	0.4 [0.1–1.1]	0.07
Tumor size	2.4 [1.3–4.2]	0.003	1.7 [0.8–3.5]	0.2
Multifocality	1.7 [0.7–4.4]	0.3
Extrathyroidal invasion	2.4 [0.9–6.3]	0.08
LNM	40.6 [8.6–192.0]	<0.0001	30.9 [6.0–159.0]	<0.0001
TNM stage III–IV	3.1 [1.2–8.5]	0.02
BRAF ^V600E mutation	3.5 [1.2–10.3]	0.03	2.8 [0.7–11.8]	0.2

Adjusted for sex, age at diagnosis, tumor size, LNM, and BRAF^V600E mutation.

CI, 95% confidence interval.

Comparison of BRAF-negative and BRAF-positive patients

In our series of 103 classical PTCs, the prevalence of the BRAF^V600E mutation was 55.3% (57 of 103; Table 1). At presentation, BRAF^V600E -positive cancers, in comparison to BRAF^V600E -negative cancers, were more frequently (64.9% vs. 50.0%) associated with two or more risk factors (male sex, age >45 years, size >2 cm, multifocality, extrathyroidal extension, lymph node metastasis) (13,40). This difference, however, was not statistically significant (p=0.16). In the univariate analysis, the presence of lymph node metastases at diagnosis (p=0.01), advanced cancer stage (p=0.03), and recurrent/persistent disease during the follow-up (p=0.03) were more frequent in BRAF^V600E -positive patients (Table 1). In the multivariate analysis, however, only lymph node metastasis was significantly different between BRAF^V600E -positive and BRAF^V600E -negative cancers: OR 2.9 [CI 1.1–7.3]; p=0.03 (Table 3).

Table 3.

Association Between BRAF ^V600E Mutation and Clinicopathological Characteristics at Diagnosis in 103 Papillary Thyroid Cancer Patients

	Univariate analysis		Multivariate analysis ^a
Parameters	OR [CI]	p	OR [CI]	p
Male sex	1.2 [0.4–3.4]	0.75
Age at diagnosis	1.0 [0.9–1.1]	0.61
>45 years	0.9 [0.4–2.0]	0.86
Tumor size	1.4 [0.8–2.2]	0.2
Multifocality	0.9 [0.4–2.0]	0.78
Extrathyroidal invasion	1.4 [0.6–3.3]	0.43
LNM	3.0 [1.3–7.2]	0.01	2.9 [1.1–7.3]	0.03
TNM stage III–IV	3.0 [1.1–8.0]	0.03

Adjusted for sex, age at diagnosis, tumor size, multifocality, extrathyroidal extension, and LNM.

Because the presence of the BRAF^V600E mutation has been suggested as a marker for stratifying PTMC into the categories of low risk versus increased risk of recurrence (41), we independently analyzed the 42 PTMCs according to the presence of this mutation. Also, in this cohort, BRAF^V600E was significantly associated with the same parameters as those in the overall series of PTCs (data not shown).

After a similar follow-up period (56.9±14.1 vs. 52.7±11.7 months for PTC patients with or without the BRAF^V600E mutation, respectively; p=0.1), the BRAF^V600E -positive cancers had a significantly higher risk of recurrence (OR 5.8 [CI 1.2–27.9], p=0.03) in spite of more frequent ¹³¹I treatment. The Kaplan-Meier curves estimated a mean time to recurrence of 65.7 months in BRAF^V600E -positive patients versus 77.1 months in BRAF^V600E -negative patients (log rank p test=0.02; Fig. 1A), with a higher risk of persistent disease at the last visit (OR 3.5 [CI 1.2–10.3], p=0.03; Table 2). However, at the last follow-up, only the presence of lymph node metastasis was independently predictive of persistent disease, and the Kaplan-Meier curves created for the BRAF^V600E mutation were not significantly different between the two groups (log rank p test=0.14).

FIG. 1.

Kaplan-Meier survival curves for the postsurgical persistent or recurrent disease. (A) Curves plotted for the presence (BRAF^V600E ) or the absence (BRAF WT) of the mutation. (B) Curves for all BRAF^V600E -negative patients (BRAF WT, solid line) and BRAF^V600E -positive patients with fewer than two (dashed line) or two or more (dotted line) risk factors.

To better visualize the specific role of the BRAF^V600E mutation in determining cancer recurrence, we subdivided the 57 BRAF^V600E -positive cancers according to the absence or the presence of two or more established risk factors. BRAF^V600E -positive cancers with fewer than two risk factors (n=20) behaved like BRAF^V600E -negative cancers according to the Kaplan-Meier analysis and had a significantly (p=0.05) lower recurrence rate than did the BRAF^V600E -positive cancers associated with two or more risk factors (n=37, Fig. 1B). Additionally, among PTCs with two or more risk factors, the presence or absence of the BRAF^V600E mutation did not cause a difference in the cancer recurrence rate according to the Kaplan-Meier analysis (p=0.2).

Discussion

The BRAF^V600E mutation is the most common genetic alteration in PTC. This mutation can initiate thyroid cancer development and progression in laboratory animals (42), but in some instances, it is also suggested to be a secondary event in the pathogenesis of thyroid cancer (43,44). In most clinical studies, the presence of the BRAF^V600E mutation is related to characteristics of cancer aggressiveness (17,23 –28), an observation confirmed in our study carried out exclusively in patients with the classical variant of PTC. The BRAF^V600E mutation was associated with many recognized risk factors for unfavorable outcomes that is, male sex, tumor size, lymph node metastases, and advanced TNM stages (III–IV).

A strength of the present study is that it is a single-center series, with identical follow-up criteria and homogeneity of histopathological features (all classical variant PTCs with no follicular, sclerosing, or tall cell variants that are often associated with a different biology and progression of the cancer). The retrospective design, the limited number of patients, and the short follow-up are the major limitations.

The strong synergism between the presence of BRAF^V600E and aggressive clinicopathological features confirms the role of this mutation in PTC biology. The mechanisms of increased aggressiveness in BRAF^V600E -positive thyroid cancers are unknown. In addition to hyperactivation of the MAPK pathway, another possible molecular mechanism is the induction of matrix metalloproteinase (MMP) in BRAF^V600E -mutated tumors (24). MMPs are proteinases that degrade extracellular matrix. Their expression is associated with lymph node metastasis, extrathyroidal and vascular invasion (45), and therefore with the promotion of tumor spread and invasion.

In our series, the strong association between BRAF status and lymph node metastasis could be confirmed. Since BRAF mutation evaluation can be available preoperatively in fine-needle aspiration biopsies, it might be useful to guide the extent of surgery and, specifically, to decide whether or not to perform prophylactic central lymph node dissection in patients with PTC and clinically and ultrasound-negative neck nodes. However, the evidence supporting this view remains controversial (46,47).

In most cases, PTCs exhibit a very indolent behavior, and persistent or recurrent disease, rather than cancer-related mortality, is the predominant cause of concern in clinical practice. Our data confirm that short- to medium-term outcomes are less favorable in mutation-positive patients in comparison to mutation- negative patients. Even in the medium to long term (>5 years), and in spite of more aggressive treatment (decided only on the basis of traditional risk factors and independently of the presence of the BRAF^V600E mutation, unknown to the treating physicians), BRAF^V600E -positive tumors showed a less favorable trend in the Kaplan-Meyer curves for persistent or recurrent disease.

In our cohort, the BRAF^V600E mutation lost significance as an indicator of persistent disease in the multivariate analysis. Therefore, the presence of the BRAF^V600E mutation is not an independent factor of thyroid cancer recurrence. Moreover, age was not an independent risk factor for persistent disease, possibly because of the small number of cases or the short follow-up period. Only lymph node metastasis, a prognostic factor for thyroid cancer recurrence but not cancer-related death (48,49), was a significant factor.

A recent multicenter retrospective study found an association between the presence of the BRAF^V600E mutation and cancer-related mortality (28). Even in that study, however, when lymph node metastasis, extrathyroidal extension, and distant metastases were included in the analysis, the statistical significance of the association between BRAF^V600E and cancer-related death was lost (28).

A debated issue is the usefulness of BRAF^V600E mutation detection as a prognostic factor in PTCs. In fact, an evaluation of the traditional clinicopathological risk factors allows risk stratification of PTCs that is not significantly improved by BRAF^V600E mutation detection (50).

The independent role of the BRAF^V600E mutation in determining PTC aggressiveness is also questioned by the observation that although approximately 50% of PTCs are BRAF^V600E positive, only a subset of them exhibit aggressive behavior. In our series, the outcome of BRAF^V600E -positive cancers in terms of recurrence rate was significantly affected by the associated clinicopathological risk factors (Fig. 1B). In contrast, the outcome of tumors with two or more risk factors was not influenced by the presence of the BRAF^V600E mutation. These observations imply that the role of the BRAF^V600E mutation is not predominant and that additional genomic factors, either in the cancer or in the host or in both, cooperate in determining cancer aggressiveness.

The limited relationship between the presence of the BRAF^V600E mutation and cancer aggressiveness could also be the consequence of the quantitative presence of the BRAF^V600E mutation in the tumor. Within the same cancer, malignant cells are heterogeneous; recent data suggest that only when more than 25% of the alleles contain the BRAF^V600E mutation will the tumor become aggressive (51). Moreover, recent evidence suggests that BRAF^V600E activation of the mitogenic MAPK/ERK pathway is also influenced by the activity of the SPRY2 regulatory feedback pathway (52). Therefore, qualitative BRAF^V600E mutation testing does not provide enough information and cannot predict the clinical behavior of PTC. This is a reasonable conclusion considering the heterogeneity of cancer cells and the complexity of their genetic abnormalities.

In conclusion, the current evidence does not support the cost-effectiveness of the qualitative detection of the BRAF^V600E mutation in clinical practice. In any case, multicentric and prospective studies are needed to further clarify the prognostic role of this molecular tool. At present, the determination of the BRAF^V600E mutation status should be considered one among the many markers that indicate a higher risk of more aggressive PTC biology, although this is not true for all BRAF^V600E -positive PTCs.

Better understanding of PTC biology may increase the significance of BRAF^V600E evaluation in the future.

Footnotes

Author Disclosure Statement

The authors declare that no competing financial interests exist.

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