Association of TERT Promoter Mutation 1,295,228 C>T With BRAF V600E Mutation, Older Patient Age, and Distant Metastasis in Anaplastic Thyroid Cancer

Abstract

Context:

The aggressive role of TERT promoter mutations has been well established in differentiated thyroid cancer but has not been established in anaplastic thyroid cancer (ATC).

Research Design:

We tested the mutation status by sequencing genomic tumor DNA and examined its relationship with clinicopathological characteristics of ATC.

Results:

Among 106 American and Chinese ATC samples, TERT 1,295,228 C>T (termed TERT C228T) mutation was found in 37 (34.9%) cases, TERT promoter mutation 1,295,250 C>T was found in four cases (3.8%), and the two mutations were mutually exclusive and collectively found in 41 cases (38.7%). TERT C228T occurred in 28 of 90 (31.1%) wild-type BRAF cases vs nine of 16 (56.3%) BRAF V600E cases, with an odds ratio of 2.85 (95% confidence interval, 0.96–8.42; P = .05). Patient age was 67.6 ± 13.6 vs 61.6 ± 11.4 years in the TERT C228T vs wild-type TERT patients (P = .02), demonstrating an association between TERT C228T and older patient age. This association was also seen within the American cohort. In this cohort, which had more available clinicopathological data, TERT C228T was associated with distant metastasis of the tumor; specifically, distant metastasis occurred in 15 of 18 (83.3%) TERT C228T patients vs eight of 26 (30.8%) wild-type TERT patients, with an odds ratio of 11.25 (95% confidence interval, 2.53–50.08; P = .001). No association was found with patient sex, tumor size, lymph node metastasis, and extrathyroidal invasion of ATC.

Conclusions:

This is the largest study on the aggressive role of TERT promoter mutations in ATC, demonstrating an association of TERT C228T with BRAF V600E, older patient age, and tumor distant metastasis in ATC.

Anaplastic thyroid cancer (ATC) is one of the most lethal human cancers. Although it is very rare, accounting for only 0.9% of the estimated 62 980 new cases of thyroid cancer in 2014 in the United States, it is a major cause of thyroid cancer-related mortality (1). ATC is characterized by a generally grave clinical course, with a 5- to 6-month median survival and a 1-year patient survival rate of 20% (2). Yet, individual patients with ATC often perform differently; some die very rapidly, others die relatively slowly, and still others may survive several years or even longer after the diagnosis. Thus, ATC tumors can behave differently, and their clinicopathological characteristics vary. For example, although distant metastasis of ATC, one of the most important aggressive tumor behaviors that affects profoundly the clinical outcomes of ATC (eg, patient mortality), occurs commonly, it is absent in many patients. The molecular mechanism underlying this spectrum of the variable behaviors of ATC is not clear.

Consequently, there is currently no molecular marker available that can help predict the behaviors of ATC (2).

ATC harbors various genetic alterations such as mutations in the BRAF, RAS, PIK3CA, PTEN, P53, and β-catenin genes (3). Genetic copy gains in major proto-oncogenes (4) and aberrant methylation of critical genes (3, 5) are also common in ATC. These genetic and epigenetic alterations result in signaling aberrations in major signaling pathways, particularly the MAPK and phosphatidylinositol-3-kinase pathways. In fact, these two pathways are dually aberrantly activated in virtually all cases of ATC (4), which represents a fundamental mechanism driving the tumorigenesis of ATC (3). It is possible that additional genetic abnormality, aside from these classical genetic alterations activating the MAPK and phosphatidylinositol-3-kinase pathways, may confer unique aggressive behaviors to ATC. The recently discovered TERT promoter mutation may be such a candidate genetic event. In our initial report on the discovery of TERT promoter mutations in thyroid cancer, we found an increasing prevalence of TERT promoter mutation 1,295,228 C>T (termed TERT C228T) from differentiated thyroid cancer (DTC) (including papillary thyroid cancer [PTC] and follicular thyroid cancer) to undifferentiated ATC, with a prevalence of 42.6% in the latter (6). This high prevalence of TERT C228T suggests a possible major role of this novel genetic event in the tumorigenesis of ATC. Another TERT promoter mutation, 1,295,250 C>T (termed TERT C250T), occurs infrequently in thyroid cancers. This study on TERT promoter mutation in thyroid cancer also demonstrated for the first time a significant association between TERT C228T and BRAF V600E mutation in PTC, which was confirmed in several subsequent studies (7–9). Studies by our group and others also demonstrated an association between TERT C228T and aggressive pathological characteristics of DTC (7–10). We have recently demonstrated a strong association between TERT C228T and recurrence of PTC, particularly when TERT C228T coexisted with BRAF V600E mutation (9).

Given this background, in the present study we investigated specifically the role of TERT C228T in the aggressiveness of ATC, which has not been established previously.

Patients and Methods

Tumor samples and DNA isolation

The study included an American cohort of 49 ATC patients and a Chinese cohort of 57 ATC patients. The American cohort was overlapped with a previous study (6). The study was approved by institutional review boards or ethical committees of the authors' institutions, and informed patient consent was obtained where required. The pathological diagnosis of ATC tumors was made following World Health Organization criteria. DNA isolation was performed as previously described (11). Briefly, tissues dissected from paraffin-embedded primary ATC tumors were treated with xylene at room temperature for 8–10 hours to remove paraffin. Tissues were digested with 1% sodium dodecyl sulfate and 0.5 mg/mL proteinase K for 48 hours at 48°C. To facilitate the digestion, midinterval additions of a spiking aliquot of concentrated sodium dodecyl sulfate-proteinase K were added. DNA was isolated from the digested tissues following standard phenol-chloroform extraction and ethanol precipitation procedures. Isolated DNA was suspended and maintained in low Tris-EDTA buffer.

Identification of TERT promoter and BRAF V600E mutations

TERT promoter and BRAF V600E mutations were identified by Sanger's sequencing of genomic tumor DNA as described previously (6, 11). Briefly, identification of the BRAF V600E mutation was achieved by amplifying exon 15 of the BRAF gene using the primers and PCR conditions described previously (11). TERT C228T and TERT C250T were identified as we previously described (6). Using primers 5′-AGTGGATTCGCGGGCACAGA-3′ (sense) and 5′-CAGCGCTGCCTGAAACTC-3′ (antisense), a 235-bp PCR product of a region in the TERT promoter containing the hotspot of C228T mutation was produced. Each PCR tube contained 40–50 ng of genomic DNA. Because TERT promoter is exceptionally GC-rich, we enhanced the PCR efficiency using a GC-Rich PCR System (Roche Applied Science), following the manufacturer's instructions. Gel electrophoresis was used to confirm the quality of the PCR products. Sequencing PCR was subsequently performed using a Big Dye Terminator v3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems). DNA sequence was analyzed on an ABI Prism 3730 Automated Genetic Analyzer (Applied Biosystems). An independent PCR amplification/sequencing, both in forward and reverse directions, was performed to confirm a positive mutation result.

Statistical analysis

Wilcoxon-Mann-Whitney test was used for the comparison of two groups of continuous variables that were not normally distributed (expressed as medians and interquartile ranges), and independent t test was used for continuous variables that were normally distributed (expressed as means ± SD). Categorical data were summarized using frequencies and percentiles, and a comparison of two groups was performed using the Pearson χ² test or Fisher's exact test if the number was <5. Logistic regression was performed to assess odds ratios (ORs) and 95% confidence intervals (CIs). All reported P values were two sided, and a P ≤ .05 was considered to be significant. Analyses were performed using SPSS software version 11.5 (SPSS Inc).

Results

TERT C228T and BRAF V600E mutations in ATC

A total of 106 American and Chinese ATC samples were analyzed for TERT promoter mutations and BRAF V600E mutation. Among these 106 samples, TERT C228T mutation was found in 37 (34.9%) cases, TERT C250T was found in four (3.8%) cases, and the two mutations were mutually exclusive and collectively found in 41 (38.7%) cases. BRAF V600E mutation was found in 16 (15.1%) cases. Due to the rarity of TERT C250T, we focused on the analysis of the far more common TERT C228T. Coexistence of TERT C228T and BRAF V600E mutations was found in nine (8.5%) cases. The two mutations tended to be associated with each other in the same tumor (Table 1). Specifically, TERT C228T occurred in 28 of 90 (31.1%) BRAF V600E-negative cases vs nine of 16 (56.3%) BRAF V600E-positive cases; inversely, BRAF V600E occurred in seven of 69 (10.1%) TERT C228T-negative cases vs nine of 37 (24.3%) TERT C228T-positive cases, with an OR of 2.85 (95% CI, 0.96–8.42; P = .05). This relationship between TERT C228T and BRAF V600E in ATC is consistent with the previously described association of the two mutations with each other in PTC (6–9). TERT C228T and BRAF V600E in ATC were each associated with older patient age (Table 2). Specifically, patient age was 62.4 ± 12.6 years in BRAF V600E-negative patients vs 70.6 ± 9.5 years in BRAF V600E-positive patients (P = .014). Patient age was 61.6 ± 11.4 vs 67.6 ± 13.6 years in TERT C228T-negative vs -positive cases (P = .016). No significant association was seen between TERT C228T and patient sex or tumor size (Table 2).

Table 1.

Association of TERT C228T With BRAF V600E Mutation in ATC

TERT C228T		BRAF V600E		OR (95% CI)	P Value
BRAF-Negative	BRAF-Positive	TERT-Negative	TERT-Positive
28/90 (31.1)	9/16 (56.3)	7/69 (10.1)	9/37 (24.3)	2.85 (0.96–8.42)	.05

Data are expressed as number of cases/total number (percentage).

Table 2.

Relationship Between BRAF V600E/TERT C228T and Clinicopathological Characteristics of ATC in All Patients

Clinicopathological Outcomes	Total	BRAF Status			TERT Status
		BRAF V600E	Wild-Type BRAF	P Value	TERT C228T	Wild-Type TERT	P Value
Total cases, n	106	16	90		37	69
Age at diagnosis (mean ± SD), y	63.7 ± 12.5	70.6 ± 9.5	62.4 ± 12.6	.014	67.6 ± 13.6	61.6 ± 11.4	.016
Sex (male), n (%)	41 (38.7)	6 (37.5)	35 (38.9)	.916	11 (29.7)	30 (43.5)	.166
Tumor size [median (interquartile range)], cm12	5.7 (3.6–7.0)14	6.0 (3.7–7.6)1	5.6 (3.6–7.0)13	.652	6.0 (3.6–7.5)2	5.5 (3.6–7.0)12	.486

Superscript numbers represent the number of missing cases in the category.

Association between TERT C228T and distant metastasis of ATC

We had 46 American cases of ATC patients that had sufficient clinicopathological data for further analyses. We took advantage of these data to investigate the relationship of TERT C228T with the characteristics of ATC. As shown in Table 3, as in the overall analysis of all 106 cases of ATC (Table 2), an association of TERT C228T in ATC with older patient age was also observed here. Specifically, patient age was 60.4 ± 11.2 vs 68.7 ± 12.5 years in TERT C228T-negative vs -positive patients (P = .023). Again, there was no association of TERT C228T with patient sex and tumor size as well as lymph node metastasis and extrathyroidal invasion. In contrast, there was a strong association between TERT C228T and distant metastasis of ATC. Specifically, distant metastasis occurred in eight of 26 (30.8%) patients with the wild-type TERT vs 15 of 18 (83.3%) patients with TERT C228T, corresponding to an OR of 11.25 (95% CI, 2.53–50.08; P = .001). Distant metastases involved the lungs in most of the patients and the bones, liver, and head in some patients.

Table 3.

Relationship Between TERT C228T and Clinicopathological Characteristics of ATC in the American Patients

Characteristics	Overall	TERT Status
		TERT C228T	Wild-Type TERT	P Value
Total cases, n	46	18	28
Age at diagnosis (mean ± SD), y	63.6 ± 12.3	68.7 ± 12.5	60.4 ± 11.2	.023
Sex (male)	18/46 (39.1)	8/18 (44.4)	10/28 (35.7)	.554
No. of tumors	36	17	19
Tumor size [median (interquartile range)], cm	5.7 (3.5–6.9)	5.0 (3.5–6.7)	6.0 (3.5–7.5)	.465
Lymph node metastasis	24/38 (63.2)	11/17 (64.7)	13/21 (61.9)	.859
Extrathyroidal invasion	40/42 (95.2)	18/18 (100.0)	22/24 (91.7)	.498
Distant metastasis	23/44 (52.3)	15/18 (83.3)	8/26 (30.8)	.001 (OR, 11.25; 95% CI, 2.53–50.08)

Data are expressed as number of cases/total number (percentage), unless described otherwise.

Discussion

This is the largest study to investigate the TERT promoter mutation in ATC and its role in the aggressiveness of this cancer. The study demonstrated for the first time an association of TERT C228T with several high-risk factors of thyroid cancer, including BRAF V600E mutation, older patient age, and distant metastasis of the ATC tumor. All of the previous studies from our group and others have reported an association between TERT C228T and BRAF V600E in PTC (6–9), except for one study that reported a reciprocal relationship between TERT promoter and BRAF mutations in PTC (12). In the latter study, it was also shown that TERT promoter mutations were collectively associated with BRAF and RAS mutations when ATC and poorly differentiated thyroid cancers were lumped together for the analysis. The specific relationship between BRAF V600E and TERT promoter mutations in ATC alone was not known. In a previous study from our group, we were unable to demonstrate a significant association of TERT promoter mutation with BRAF V600E in a smaller cohort of ATC (6). Thus, the specific relationship between the TERT promoter mutation and BRAF V600E in ATC remains to be established. Our present study, focused on the largest series of ATC, for the first time demonstrates an association between TERT C228T and BRAF V600E mutation in this cancer. BRAF V600E mutation has been widely shown to play a role in the aggressiveness of thyroid cancer (3, 11). We have recently demonstrated that coexistence of TERT C228T and BRAF V600E was synergistically associated with the aggressiveness of PTC (9). The association of TERT promoter mutation with BRAF V600E mutation in ATC found in the present study thus suggests a potential role of this unique genetic combination in driving the pathogenesis and aggressiveness of some ATCs. Interestingly, coexistence of TERT promoter and BRAF V600E mutations is also common in melanoma (13, 14). Expression of TERT protein was higher in PTC that harbored both TERT promoter and BRAF V600E mutations (15). Thus, synergism between the two mutations may be a common mechanism in driving human cancer tumorigenesis and aggressiveness.

TERT promoter mutations were shown to be associated with older patient age in DTC (7–9, 15). It is interesting that TERT C228T was also found to be associated with older age of patients with ATC in the present study. Because old patient age is a well-established poor prognostic factor for thyroid cancers, including ATC, this association is in line with a role of the TERT promoter mutation in the aggressiveness of ATC. Unlike DTC in which TERT promoter mutations were associated with several aggressive tumor characteristics, such as large tumor size, extrathyroidal invasion, and lymph node metastasis (7–9), there was no such association between the TERT promoter mutation and these tumor characteristics of ATC in the present study. This can be explained by the fact that such aggressive tumor characteristics are extremely common in ATC, and their occurrence is virtually intrinsic in this aggressive tumor and may not be further affected by the TERT promoter mutation. The most striking finding in the present study was the strong association of TERT C228T with distant metastasis of ATC. Our group and others demonstrated a significant association of TERT C228T with distant metastasis of DTC (8, 9). The present study demonstrates for the first time a significant association of TERT C228T with distant metastasis of ATC. Distant metastasis is an aggressive behavior of thyroid cancer that is most commonly associated with poor clinical outcomes of thyroid cancer patients, such as mortality. This is also the case with ATC. Therefore, the association of TERT C228T with distant metastasis of ATC provides the strongest evidence thus far suggesting that TERT C228T plays an important role in the pathogenesis and aggressiveness of ATC. From a practical perspective, this finding suggests that in ATC patients it would be reasonable to search more vigilantly and monitor for possible distant metastasis if the tumor is positive for TERT C228T. This strong association of TERT C228T with distant metastasis of ATC may likely also translate into accelerated mortality of patients with this cancer. Indeed, a previous study demonstrated that ATC patients carrying TERT promoter mutations survived a shorter time than those without mutations, although this was not statistically significant (10). The role of TERT promoter mutations in ATC-related patient mortality is worth investigating further in future studies.

The present study on TERT promoter mutations in ATC, together with the previous studies on DTC, strongly suggests that, regardless of the type of thyroid cancer—whether well differentiated or undifferentiated—TERT promoter mutation is a universal predictor for poorer prognosis of thyroid cancer. This role of TERT promoter mutation can be explained by the increased TERT promoter activities conferred by TERT promoter mutations through creation of a binding consensus for E26 transformation specific transcription factors (13, 16), leading to amplified transcription of the TERT gene and expression of TERT protein. TERT normally maintains the length of chromosomes by adding repeats of telomeres (TAAGGG) to them, thus increasing the immortality of cells, and also promotes cell proliferation and decreases apoptosis (17–19). The MAPK pathway is important in up-regulating the E26 transformation specific transcription factors (20–22). There are wide genetic alterations that can activate the MAPK pathway in ATC (4). The resulting overactivated MAPK pathway presumably can synergize with TERT promoter mutation to promote TERT expression. This explains the very poor clinical outcomes of PTC with coexistence of TERT C228T and BRAF V600E mutations (9). This mechanism is likely also important in ATC.

In large studies, TERT C228T was shown to be far more common than TERT C250T in DTC (6–8). In small cohorts of ATC, TERT C228T was also shown to be much more common than TERT C250T (6, 12). The present study on an expanded large cohort of ATC confirms and establishes that TERT C228T is the dominant TERT promoter mutation also in this most aggressive thyroid cancer, just as the BRAF V600E is the dominant BRAF mutation in thyroid cancer. From an evolutionary perspective, this dominance of TERT C228T over TERT C250T in occurrence seems to make good sense because the former was shown to more strongly confer the TERT promoter increased activities and more forcefully drive cell migration than TERT C250T did (23). Given this role of the TERT C228T mutation in ATC, it may be an important novel therapeutic target in this cancer; targeting TERT in TERT C228T-positive cases in the treatment of ATC may prove to be an effective therapeutic strategy for this cancer. Microenvironmental changes associated with the BRAF V600E, such as aberrations in the thrombospondin-1 system, were indicated to be an important molecular mechanism in the pathogenesis of ATC (24). In animal models, targeting BRAF V600E in such ATC cells as an adjuvant therapy to thyroidectomy demonstrated a survival benefit to the animals (25). Thus, it may prove to be even more effective to therapeutically target both BRAF V600E and TERT in ATC harboring the two mutations.

In contrast, the absence of TERT promoter and BRAF mutations may signify a less aggressive disease course of ATC. The present study included the case of an ATC patient who has been living well without ATC recurrence more than 4 years after surgical removal of a 9-cm ATC tumor and radiation therapy. We previously reported the pathological characteristics and clinical course of this unusual patient in a case report (26). The tumor in this patient was a histologically typical ATC, but it was well-circumscribed without extrathyroidal invasion, and none of the 73 lymph nodes that were removed were positive for metastasis. Interestingly, this case harbored neither TERT promoter mutation nor BRAF V600E mutation as examined in the present study. There have been many case reports on similar long-term survivors of ATC (27–34). It would be interesting to know whether such cases of ATC, like our case, also do not harbor TERT promoter mutations.

Sanger sequencing has a relatively low resolution in detecting mutations. Thus, the presence of abundant ramified tumor-associated macrophages in ATC, as demonstrated previously (35), could potentially cause an underestimate of mutation rates detected using the Sanger sequencing approach in the present study.

In summary, this is the largest study on the TERT promoter mutation in ATC and its role in the aggressiveness of this cancer, which for the first time demonstrates an association of TERT C228T with several high-risk characteristics of ATC, including BRAF V600E mutation, old patient age, and distant metastasis of the tumor. The study has thus far provided the strongest evidence suggesting an important role of TERT promoter C228T in the tumorigenesis and aggressiveness of ATC. The study also supports TERT C228T being a potentially novel prognostic marker and therapeutic target for ATC. Further studies are warranted to investigate these interesting aspects of ATC.