Molecular and Cellular Endocrinology 399 (2015) 288–295
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Molecular and Cellular Endocrinology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m c e
Telomerase in differentiated thyroid cancer: Promoter mutations, expression and localization Marina Muzza a,b,1, Carla Colombo a,b,1, Stefania Rossi c, Delfina Tosi d, Valentina Cirello a,e, Michela Perrino a,b, Simone De Leo a,b, Elisa Magnani f,g, Elisa Pignatti f,g, Beatrice Vigo a,b, Manuela Simoni f,g, Gaetano Bulfamante c,d, Leonardo Vicentini h, Laura Fugazzola a,e,* a
Endocrine Unit, Fondazione IRCCS Ca’ Granda, Milan, Italy Department of Clinical Sciences and Community Health, University of Milan, Italy c Division of Pathology, San Paolo Hospital, Milan, Italy d Department of Health Sciences, University of Milan, Italy e Department of Pathophysiology and Transplantation, University of Milan, Italy f Azienda USL of Modena, Italy g Department of Biomedicine, Metabolism and Neural Sciences, University of Modena and Reggio Emilia, Italy h Endocrine Surgery Unit, Fondazione IRCCS Ca’Granda, Milan, Italy b
A R T I C L E
I N F O
Article history: Received 3 July 2014 Received in revised form 16 October 2014 Accepted 21 October 2014 Available online 27 October 2014 Keywords: TERT Outcome BRAF RAS Genomics Epidemiology
A B S T R A C T
Telomerase-reverse-transcriptase (TERT) promoter mutations have been recently described in tumors. In the present large series, TERT mutations were found in 12% of papillary thyroid cancers (PTCs) and in 14% of follicular thyroid cancers (FTCs), and were found to significantly correlate with older age at diagnosis and poorer outcome. Interestingly, the prognostic value of TERT mutations resulted to be significantly stronger than that of BRAFV600E. Moreover, the outcome was not different among tumors with isolated TERT mutation and those with coexistent mutations (TERT/BRAF in PTCs or TERT/RAS in FTCs). TERT rs2853669 polymorphism was found in 44.4% of tumors. At WB, TERT was significantly more expressed in tumors than in normal samples, being the highest levels of expression recorded in TERT mutated cases. At IHC, in tumors and in metastatic lymph-nodes TERT staining was significantly higher in the cytoplasm than in the nucleus, whereas in normal tissue the degree of staining did not differ in the two cellular compartments. In conclusion, TERT mutations were shown to strongly correlate with a poorer outcome in differentiated thyroid tumors, and neither BRAF nor RAS mutation were found to confer an additional effect in the disease persistence. TERT protein was found to be more expressed in neoplastic than in normal tissues, and to display a different cellular localization, suggesting that it could contribute to thyroid cancer progression by mechanisms taking place in the cytoplasm. © 2014 Published by Elsevier Ireland Ltd.
1. Introduction Telomerase is a ribonucleoprotein polymerase that maintains telomere repeat TTAGGG at the ends of chromosomes, and consists of a protein component with reverse transcriptase activity, TERT, and a RNA component which serves as a template (Harrington et al., 1997). Telomerase expression is normally repressed in postnatal somatic cells, resulting in the progressive shortening of telomeres which leads to growth arrest (replicative senescence). Most cancer cells constitutively express telomerase, thus allowing telomeres
* Corresponding author. Endocrine Unit-Fondazione IRCCS Ca’ Granda, Via F. Sforza, Milan 35-20122, Italy. Tel.: +39 02 55033498; fax: +39 02 50320605. E-mail address: [email protected] (L. Fugazzola). 1 Equal contributors. http://dx.doi.org/10.1016/j.mce.2014.10.019 0303-7207/© 2014 Published by Elsevier Ireland Ltd.
maintenance and unlimited cellular proliferation (Skvortzov et al., 2009). It has been shown that the genetic mechanisms responsible for telomerase reactivation in tumors could include engagement of TERT alternative splicing, TERT gene amplification, and mutations in the TERT promoter (Skvortzov et al., 2009). In particular, the mutations −124 C > T (C228T) and −146 C > T (C250T) in the TERT promoter have been shown to occur in several tumors (Killela et al., 2013; Liu et al., 2013). These mutations generate de novo consensus binding motifs for E-twenty-six (ETS) transcription factors and in cancer cell lines have been shown to increase the transcriptional activity of the TERT promoter by two- to six-folds (Huang et al., 2013). These two TERT promoter mutations have been very recently reported in thyroid cancer cell lines and in thyroid tumors, either well differentiated (DTCs) or poorly differentiated and anaplastic (Landa et al., 2013; Liu et al., 2013, 2014; Melo et al., 2014; Vinagre et al.,
M. Muzza et al./Molecular and Cellular Endocrinology 399 (2015) 288–295
2013; Wang et al., 2014). In DTCs, an association between these TERT mutations (TERTMUT) and older age at diagnosis, male gender and tumor size (Liu et al., 2014; Melo et al., 2014; Vinagre et al., 2013), and a correlation with a reduced progression free survival and overall survival have been shown (Liu et al., 2013; Melo et al., 2014). Discordant data are available about the coexistence of TERT and BRAF or RAS mutations. In particular, TERTMUT were found to be significantly more frequent in BRAFV600E than in BRAFWT papillary thyroid cancers (PTCs) (Liu et al., 2013; Melo et al., 2014) and in RASMUT than in RASWT follicular thyroid cancers (FTCs) (Wang et al., 2014), but these data were confirmed only for advanced thyroid tumors in another series (Landa et al., 2013). The clinical impact of the association is still controversial and indeed, the coexistence of TERT and BRAF mutations in PTCs was found to correlate with more aggressive features in a Chinese series (Liu et al., 2014). On the other hand, in a series from Portugal, no differences in the outcome were noted between TERTMUT/BRAFMUT and TERTMUT/BRAFWT cases, indicating that BRAF per se does not have an addictive impact in the disease persistence (Melo et al., 2014). Very scarce, contradictory and not reliable data are available about TERT expression and cellular localization in the normal and neoplastic thyroid gland (Capezzone et al., 2009). Moreover, at the thyroid level no data exist about the possible modifying role of a common TERT polymorphism (rs2853669) located within a ETS binding site and shown in bladder cancer to be able to modify the effect of −124 C > T (C228T) and −146 C > T (C250T) mutations on survival and recurrence (Rachakonda et al., 2013). In the present study, we performed a comprehensive evaluation of TERT promoter, BRAFV600E and N- and H-RAS mutations in a large series of sporadic and familial tumors and in metastatic lymphnodes. The allelic frequency of rs2853669 SNP was also studied for the first time in thyroid cancer. Moreover, the expression and the localization of TERT was investigated by Western blot (WB) and immunohistochemistry (IHC). 2. Materials and methods 2.1. Patients tissue samples A large series of 254 primary thyroid tumors were included in this study. In particular, the following histologic types were analyzed: 182 PTCs (143 conventional, 34 follicular, 2 oncocytic and 3 diffuse sclerosing variants), 58 FTCs (42 conventional and 16 Hurtle variants), and 14 MTC. In addition, 3 diffuse toxic goiters, 3 multinodular goiters and 8 lymph-nodal metastases were studied. Moreover, 10 familial PTCs (fPTC) cases were included and analyzed for TERT mutations at the germline level. 2.2. Histological classification and outcome definition All specimens were reviewed by a senior pathologist (S.R.) to confirm the diagnosis. Tumors were classified and staged according to the 7th edition of the TNM staging (Compton et al., 2012). Criteria used to identify remission or persistent/recurrent disease were drawn on the bases of the European and American guidelines for the management of differentiated thyroid cancer (Cooper et al., 2009; Pacini et al., 2006) and have been previously reported in detail (Perrino et al., 2009).
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commercial kits (Puregene® Core Kit A, Qiagen, Germantown, MD, USA). All procedures performed for handling the tissues were approved by each Hospital Ethics Committee. An informed consent was obtained from all screened subjects.
2.4. Molecular analysis The TERT proximal promoter was amplified from tissue and blood DNA using the primers TERTPforward: 5′-AGTGGATTCGCG GGCACAGA-3′ and TERTPreverse: 5′-GCAGCGCTGCCTGAAACTC-3′. Exon 15 of the BRAF gene was analyzed using specific intronic primers, as previously described (Fugazzola et al., 2004). Exon 2 of N-RAS was PCR amplified by means of newly designed specific intronic primers, forward: 5′-ACCTTGGCAATAGCATTGCAT-3′ and reverse: 5′-TAGTGTGGTAACCTCATTTCC-3′, whereas exon 2 of H-RAS was PCR amplified using previously described primers (Moura et al., 2011). PCR products were purified and directly sequenced (ABI 3130, Perkin Elmer Applied Biosystem, Foster City, CA).
2.5. Immunohistochemical studies Paraffin blocks, including 19 PTCs, 5 FTCs and corresponding contralateral normal tissues, and 8 lymph-nodal metastases, were selected according to the following major criteria: (a) good morphology and (b) preservation of follicular structures (extensive fibrotic or hemorrhagic areas were excluded). From each paraffin block, 3 μm sections were obtained and tested for human TERT reactivity by means of a specific rabbit polyclonal antibody (Rockland Immunochemicals Inc. Gilbertsville, PA) at 1:2000 dilution, using an immunoperoxidase technique. The reaction was detected by Novolink Max polymer detection system (Novocastra Laboratories L.T.D., Leica Microsystem, Nussloch, Germany). In order to test the specificity and sensitivity of the antibody, control neoplastic tissues (gliomas) were also stained. The degree of positive staining for TERT was defined based on both the intensity of the staining and the percentage of stained cells. In particular, the intensity was defined as negative (0), low (1), medium (2) and high (3), whereas the percentage of stained cells were defined as none (0), 1–20% stained (1), 20–50% stained (2) and >50% stained (3).
2.6. Protein extraction and Western blotting Briefly, thyroid tissue was homogenized in lysis buffer (Cell Signaling Technology, Inc., Danvers, MA), supplemented with protease inhibitors (Roche, Basel, CH) and immunoblotting was performed with a rabbit anti-human TERT antibody (Rockland Immunochemicals Inc. Gilbertsville, PA) at 1:1000 dilution, and with an antiactin antibody (Novus Biologicals, Littleton, CO) or anti-GAPDH antibody (Ambion, Life Technologies, Foster City, CA). Chemiluminescence was detected using the Chemi-Doc-IT Imaging System (UVP, Upland, CA, USA). Band intensities were analyzed with the image analysis program NIH ImageJ and the TERT signal was normalized to actin signal.
2.7. Statistical analysis 2.3. Nucleic acids extraction Tissues were collected during surgery, immediately frozen and stored at −80 °C. To ensure a pure tumor tissue isolation in tumors T was more prevalent than TERT−146C>T in both histotypes (86.4% in PTCs and 62.5% in FTCs). No mutations were found in 14 MTCs and 6 thyroid nodular benign diseases (Table 1). The clinical features were available for all the cases included and the mean follow-up was of 78.9 months (median 74.5 months). Both patients with PTC and FTC were divided according to the TERT status (mutated/non-mutated) and several clinical parameters were compared. In PTC cases, TERT mutations were significantly associated at univariate analysis with an older age at diagnosis (P = 0.004), with a non-conventional PTC variant – i.e. follicular/oncocytic/diffuse sclerosing – (P = 0.008), and with persistent/relapsing disease (P = 0.002) (Table 2a). These 3 variables were confirmed at logistic regression
Table 2 (a) Clinical features of the 182 TERT mutated and wild-type (WT) papillary thyroid cancers (PTC); (b) Multivariate analysis using TERT promoter mutations as dependent variable. (a) PTC Age at diagnosis (years) Female gender Mean tumor diameter (mm) Tumor (T)
Multifocality Extrathyroidal invasion Lymph-nodes (N1) Stage
Histological variants
Outcome
Mean Range
T1 T2 T3 T4a
N1 I II III IV Conventional Follicular/ oncocytic/ diffuse sclerosing Persistence or recurrence
TERT mutated (n = 22) (12%)
TERT WT (n = 160) (88%)
P
57.6 29–82 14/22 (64%) 22
44.2 14–80 126/160 (79%) 19.3
0.004
7 (32%) 2 (9%) 12 (55%) 1 (4%) 11/22 (50%) 11 (50%)
57 (36%) 17 (11%) 82 (50%) 4 (3%) 77/160 (48%) 85 (53%)
11 (50%) 10 (45%) 2 (9%) 6 (27%) 4 (18%) 12/22 (55%) 10/22 (45%)
83 (52%) 106 (66%) 5 (3%) 31 (20%) 18 (11%) 131/160 (82%) 29/160 (18%)
10/22 (45%)
29/160 (18%)
0.19 0.34 0.8
0.95 0.9 0.9 0.09
0.008
0.002
(b) Variable
Odds ratio
95% CI
P
Older age Non-conventional variants* Persistence/recurrence
4.6848 2.9663 3.4007
1.4768–14.8615 1.0816–8.1348 1.2909–8.9588
0.008 0.03 0.01
Statistically significant values are reported in bold. * follicular/oncocytic/diffuse sclerosing variants.
analysis to be independently correlated with TERT mutations (P = 0.008, P = 0.03 and P = 0.01, respectively) (Table 2b). In FTC cases, TERT mutations were significantly associated with an older age at diagnosis (P = 0.04), with a worst stage (P = 0.02) and with a poorer outcome (P = 0.03) at univariate analysis, whereas at logistic regression TERT mutations were found to significantly correlate only with persistent disease (P = 0.03). Finally, there were no significant differences in the distribution of TERT mutated and nonmutated cases according to the histological variant (classic or Hurtle cells) (Table 3). After pooling all the differentiated cases (PTCs + FTCs), TERT mutations were confirmed to be significantly associated with an older age at diagnosis (P = 0.002) and a worst outcome (P = 0.006). At the logistic regression analysis, either age or outcome were found to be independently associated with TERT mutations (P = 0.003 and P = 0.002, respectively) (Supplemental Table S1). 3.2. TERT polymorphisms in primary tumors The overall allelic frequency of rs2853669 polymorphism in thyroid cancers was 44.4%, without significant differences between
Table 3 (a) Clinical features of the 58 TERT mutated and wild-type (WT) follicular thyroid cancers (FTC); (b) Multivariate analysis using TERT promoter mutations as dependent variable. (a) FTC Age at diagnosis (years) Female gender Mean tumor diameter (mm) Tumor (T)
Multifocality Extrathyroid invasion Lymph-nodes (N1) Stage
Histological variants Outcome
Mean Range
TERT mutated (n = 8) (13.8%)
TERT WT (n = 50) (86.2%)
P
66 48–81 6/8 (75%) 54
56 18–85 25/50 (50%) 40.9
0.04
T1 T2 T3 T4
I II III IV Conventional Hurtle Persistence or recurrence
0 3 4 1 1/8 (12.5%) 5/8 (62.5%)
21 7 19 3 9/50 (18%) 24/50 (48%)
7/8 (87.5%) 0 2 (25%) 3 (37.5%) 3 (37.5%) 6/8 (75%) 2/8 (25%) 5/8 (62.5%)
46/50 (92%) 20 (20%) 7 (14%) 18 (36%) 5 (10%) 36/50 (72%) 14/50 (28%) 11/50 (22%)
1 0.1 0.08
0.9 0.7 0.7 0.02
1 0.03
(b) Variable
Odds ratio
95% CI
P
Age Stage Persistence/recurrence
– – 5.27
– – 1.0962–25.4103
NS NS 0.03
Statistically significant values are reported in bold.
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TERTMUT (41.6%) or TERTWT (46.8%) cases. The presence of rs2853669 was not correlated with either any clinical parameter or the outcome (data not shown).
3.3. Correlation of TERT promoter, BRAF and RAS mutations in primary tumors The analysis of BRAFV600E mutations in PTCs revealed an overall prevalence of 35.2% (64/182 cases). TERT mutations were found in 10/64 (15.6%) BRAFMUT cases and in 12/118 (10.1%) BRAFWT tumors (P = 0.34) (Table 4A). PTCs were divided according to the presence of a mutation in TERT, in BRAF or in both genes, and the prevalence of persistence/remission was compared (Table 4B). Interestingly, persistent disease was significantly more frequent in BRAFWT/ TERTMUT than in BRAFMUT/TERTWT cases (50 vs 17%, P = 0.02). Of note, no significant differences in the outcome was found between BRAFWT/ TERTMUT and BRAFMUT/TERTMUT cases. In FTCs, RAS mutations were found in 14/58 cases (24.1%). In particular, 11 tumors had a N-RAS mutation (10 Q61R and 1 Q61K) and 3 cases had a H-RAS mutation (2 Q61R and 1 Q61K). TERT mutations had a similar frequency among RASMUT and RASWT tumors (14.3 vs 13.6%; P = 1) (Table 4A1). FTCs were divided according to the presence of a mutation in TERT, in RAS or in both genes, and the prevalence of persistence/remission was compared (Table 4B). The prevalence of persistent disease resulted to be significantly higher in RASMUT/TERTMUT than in RASMUT/TERTWT cases (100 vs 8%, P = 0.03).
Table 4 (A) The prevalence of BRAFV600E mutations in papillary thyroid cancers (PTCs) was 35.2% (64/182 cases); (A1) The prevalence of N- and H-RAS mutations in follicular thyroid cancers (FTCs) was 24.1% (14/58 cases); (B) In PTCs, a poorer outcome was significantly more frequent in BRAFWT/TERTMUT than in BRAFMUT/TERTWT cases; in FTCs a poorer outcome was significantly more frequent in RASMUT/TERTMUT than in RASMUT/ TERTWT tumors. A PTCs
TERTMUT (n = 22)
TERTWT (n = 160)
P
BRAFMUT (n = 64) BRAFWT (n = 118)
10 12
54 106
0.34
A1 FTCs
TERTMUT (n = 8)
TERTWT (n = 50)
P
RASMUT (n = 14) RASWT (n = 44)
2 6
12 38
1
B PTCs
Remission
Persistence
P
BRAFMUT/TERTMUT BRAFWT/TERTWT BRAFWT/TERTMUT BRAFMUT/TERTWT BRAFMUT/TERTMUT BRAFMUT/TERTWT BRAFWT/TERTMUT BRAFMUT/TERTMUT
6 84 6 45 6 45 6 6
4 (40%) 22 (21%) 6 (50%) 9 (17%) 4 (40%) 9 (17%) 6 (50%) 4 (40%)
0.22 0.02 0.19 0.69
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3.4. TERT promoter and BRAF mutations in lymph-nodal metastases We had the opportunity to include in the study 12 metastatic lymph-nodes. The molecular pattern was identical among the primary tumor and the lymph-node metastases in 9 cases (5 BRAFV600E/TERTWT, 1 BRAFWT/TERT−124C>T, and 3 BRAFWT/TERTWT), while it was discrepant in 3 patients. In particular, we observed the acquisition in the lymph node metastasis of a TERT or a BRAFV600E mutation (BRAFWT/TERTWT primary tumor vs BRAFWT/TERT−124C>T lymph node metastasis in 2 cases, and BRAFWT/TERT−124C>T primary tumor vs BRAFV600E/TERT−124C>T lymph node metastasis in one patient). 3.5. TERT promoter and BRAF germline mutations in familial cases Neither BRAF nor TERT mutations were found at the germline level in the 10 familial PTC cases examined (Table 1). 3.6. Western blot analysis At WB analysis, tumors were found to harbor an enhanced TERT expression with respect to normal tissues (Fig. 1). The mean value of densitometric analysis (TERT/housekeeping expression) was of 0.89 for tumors and 0.25 for normal tissues (P = 0.0001). Moreover, TERTMUT tumors had a higher expression of the protein, though not at a significant level, with respect to TERTWT cases (1.14 vs 0.75, P = 0.2). Besides the expected band of 127 kDa, additional bands were visualized with this antibody, as already reported in experiments on HeLa cells (Wu et al., 2006). They could be non-specific or match with TERT isoforms. In particular, a 95 kDa band was identified which could correspond with the beta deletion variant (Listerman et al., 2013). 3.7. Immunohistochemistry analyses At IHC, either normal and tumor tissues displayed a TERT staining though a different pattern was observed between tumor tissue and the contralateral normal thyroid tissue (Table 5 and Fig. 2, panels A and B). In particular, tumor tissues displayed a prevalent cytoplasmic staining, whereas normal tissues had both a cytoplasmic and a nuclear TERT positivity. In tumors, the mean intensity of staining was 1.7 in the cytoplasm and 0.9 in the nucleus (P = 0.02), and the mean percentage of immunoreactive cells was 1.3 in the cytoplasm and 0.6 in the nucleus (P = 0.0008). On the other hand, in normal tissues no statistically significant differences were noted neither in the intensity of staining nor in the percentage of immunoreactive cells between cytoplasm and nucleus (1.7 vs 2 and 1.6 vs 1.8, respectively, P = NS). In some tumors a typical perinuclear positivity was observed, located at the apical pole of the nucleus, towards the cytoplasm (Fig. 2, panel C). No differences in TERT staining were seen between TERTMUT and TERTWT tumors. The differential expression of TERT between normal and metastatic cells was further confirmed in lymph-nodes. In neoplastic lymph-nodes the staining was exclusively cytoplasmic (1.25 for either mean intensity of staining and mean percentage of immunoreactive cells). In both B and T lymphocytes the staining was present in both compartments, but more prevalent in the nucleus (1 vs 2.25 for both criteria, P = 0.01) (Table 5 and Fig. 2, panel D).
FTCs
Remission
Persistence
P
RASMUT/TERTMUT RASWT /TERTWT RASWT/TERTMUT RASMUT/TERTWT RASMUT/TERTMUT RASMUT/TERTWT RASMUT/TERTMUT RASWT/TERTMUT
0 27 3 11 0 11 0 3
2 (100%) 11 (41%) 3 (50%) 1 (8%) 2 (100%) 1 (8%) 2 (100%) 3 (50%)
0.1
4. Discussion
0.08
In the present series of 240 DTCs, TERT promoter mutations were found in 12% of PTCs and in 14% of FTCs. Similar or higher frequencies were found in previous series (Landa et al., 2013; Liu et al., 2013, 2014; Melo et al., 2014; Vinagre et al., 2013) (Table 1). No TERT mutations were documented either in MTCs or, at the germline level, in familial PTC cases, as previously reported (Liu et al., 2013; Melo
Statistically significant values are reported in bold.
0.03 0.46
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Fig. 1. Western blot analysis with TERT antibody (Rockland Immunochemicals Inc. Gilbertsville, PA) of a representative cohort of TERTMUT (#1p, 2p, 3p and 7p) and TERTWT (#4p, 5p, 6 p, 8p and 9p) PTCs, and of TERTMUT FTCs (#1f and 2f). Normal contralateral thyroid tissues (#1n, 2n, 6n, 7n and 9n), corresponding to the above mentioned PTCs, are also shown. The band of 127 kDa corresponds to TERT. An anti-actin blot is also shown as a protein loading control. In the lower part of the figure, the densitometric analysis of TERT/housekeeping gene in a total of 7 TERTMUT PTCs, 11 TERTWT PTCs, 3 TERTMUT FTCs, and 11 normal tissues is reported. PTC and p: papillary thyroid cancer; FTC and f: follicular thyroid cancer; n: normal tissue; bis: replicate experiment on the same tumor tissue.
et al., 2014; Vinagre et al., 2013), indicating that these genetic alterations are not involved in the pathogenesis of these diseases. In the present PTCs cohort, TERT promoter mutations were found to strongly and independently correlate with an older age at diagnosis and with a poorer outcome, in accordance with previous studies (Liu et al., 2013, 2014; Melo et al., 2014; Vinagre et al., 2013). The association of TERT mutations with older age at diagnosis is consistent with the knowledge that telomeres become progressively shorter during lifetime, and thyroid follicular cells in older subjects are thus predicted to harbor short telomeres. When these cells begin to proliferate in response to an oncogenic event (e.g. BRAFV600E), the presence of very short telomeres leads to telomere dysfunction, triggering genomic instability and telomerase activation by TERT promoter mutations (Londoño-Vallejo, 2008). Accordingly, TERT mutations have
been found to correlate with shorter telomeres and age >45 years in a cohort of PTC patients (Liu et al., 2013). Moreover, in the present series, the non-conventional PTC variants (follicular/oncocytic/ diffuse sclerosing) were found to independently correlate with TERT mutations, consistent with previous data (Liu et al., 2013). The overall prevalence of BRAFV600E was 35.2% in the present series of PTCs. TERT mutations were found in 15.6 and 10.1% in BRAFMUT and BRAFWT cases, respectively. Interestingly, persistence was significantly more frequent in BRAF W T /TERT MUT than in BRAF MUT / TERT W T cases (P = 0.02), likely indicating that TERT promoter mutations have a more relevant impact on disease outcome than BRAFV600E. Since this finding is in contrast with a Chinese series reporting a limited effect for TERT promoter mutations alone (Liu et al., 2014), further studies on larger series are likely needed to define
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Table 5 Immunohistochemical data in papillary and follicular* thyroid cancers in TERTMUT (mutated) and TERTWT (wild-type), and in metastatic lymph-nodes. T , tumor; N, normal thyroid or lymph-nodal tissue; L, lymph-node. Intensity of the staining/number of immunoreactive cells are both reported for a better identification of the degree of staining. In particular, the intensity of staining was defined as negative (0), low (1), medium (2) and high (3), while the percentage of immunoreactive cells were defined as none (0), 1–20% stained (1), 20–50% stained (2) and >50% stained (3). TERT MUT
T1*
N1
T2
N2
T3
N3
T4
N4
T5*
N5
T6
N6
T7
N7
T8
N8
T9*
N9
Cytoplasm Nucleus
1/1 1/1
2/2 2/2
3/2 0/0
2/2 1/1
1/1 1/1
1/1 2/2
2/1 0/0
2/2 3/2
1/1 1/1
2/2 2/2
1/1 0/0
2/2 1/1
1/1 3/1
-
3/2 3/1
-
1/1 2/1
1/1 2/1
TERT WT
T10
N10
T11
N11
T12*
N12
T13
N13
T14*
N14
T15
N15
T16
N16
T17
N17
T18
N18
T19
N19
Cytoplasm Nucleus
1/1 0/0
3/1 2/2
1/1 0/0
2/2 2/2
1/1 1/1
2/2 2/2
2/1 0/0
2/2 2/2
1/1 1/1
1/1 2/2
1/1 1/1
3/1 2/2
3/1 1/1
2/2 3/2
1/3 1/3
1/1 2/2
2/1 2/1
1/1 2/1
3/2 0/0
2/2 3/3
Metastatic lymph-nodes
L1
N1
L2
N2
L3
N3
L4
N4
Cytoplasm Nucleus
2/2 0/0
1/1 2/2
1/1 0/0
1/1 3/3
1/1 0/0
1/1 2/2
1/1 0/0
1/1 2/2
* correspond to follicular thyroid cancers as reported in the legend of the Table.
the role of TERT mutations when separated from BRAFV600E. Nevertheless, and consistent with a previous report (Melo et al., 2014), in our series no statistically significant difference in the outcome was found between BRAFWT/TERTMUT and BRAFMUT/TERTMUT, indicating that BRAF mutation does not confer an additional effect in the disease persistence. In the present FTCs cohort, TERT promoter mutations were found to strongly and independently correlate with a poorer outcome and RAS mutations were found in 24.1% of cases, without differences between TERTMUT and TERTWT. Even though the statistical significance was not reached, tumors with TERT mutations alone tended to have a persistent disease more frequently than those with RAS mutations alone (50 vs 8%, P = 0.08), indicating, as above reported for PTCs, the relevant role of TERT mutations in the degree of aggressiveness of the disease. Consistent with these findings, there were no statistically significant differences in the percentage of recurrent/persistent cases between RASWT/TERTMUT and RASMUT/
TERTMUT FTCs, indicating that RAS mutations do not contribute in the worsening of the outcome. On the contrary, RASMUT/TERTMUT were significantly more recurrent/persistent than RASMUT/TERTWT cases (P = 0.03), suggesting that TERT mutation can worsen the outcome of RAS mutated cases. This is the first study including metastatic lymph-nodes from PTCs, which were found to harbor TERT mutations in 25% of cases. In 3/12 patients we found a discrepant pattern between the primary tumor and the lymph node metastases, with the acquisition of a TERT or a BRAF mutation in the metastasis. This finding is consistent with data obtained in thyroid cancer (Vasko et al., 2005) and in melanoma and could be due to the selection of mutant alleles during tumor progression or to the heterogeneous pattern of tumoral cells in the primary tumor with only some subclones able to metastatize (Lin et al., 2011). In this context, thyroid cancer has been shown to consist of a mixture of BRAFV600E and BRAFWT cells, suggesting that BRAF mutation in PTC is a later subclonal event (Guerra et al., 2012).
Fig. 2. Expression of TERT at immunohistochemistry in exemplificative cases. Panel A: papillary thyroid cancer. Note the TERT staining in the cytoplasm (40×); panel B: normal thyroid tissue. Note the TERT positivity in the nucleus and in the cytoplasm with a «dot» pattern of distribution (200×); panel C: papillary thyroid cancer. Note that TERT positivity is located at the apical part of the nucleus (200×); panel D: papillary thyroid cancer metastatic lymph node. Note on the left the nuclear staining of lymphocytes and on the right the cytoplasmic staining of metastatic cells (200×).
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The relevance of the present study relies on the combination of genetic data with expression and localization studies. In particular, WB data have been obtained for the first time in human thyroid tumor tissues, whereas IHC analyses for TERT have been previously performed in several normal and neoplastic human tissues, with discordant results concerning the immunolocalization of hTERT, reported to be exclusively nuclear (Ito et al., 2005; Lantuejoul et al., 2004; Preto et al., 2004) or both cytoplasmic and nuclear (Gasinska et al., 2013; Kyo et al., 2003; Mavrommatis et al., 2005). The discrepancies are likely to be mainly due to the different antibodies (Ab) used, since around 15 different monoclonal and polyclonal Ab are available (Fabricius et al., 2009), which are directed against different portions of the protein. Accurate studies concluded that among the commercially available anti-hTERT Ab, only the RCK-hTERT antibody, used in the present study, is suitable for hTERT protein detection (Wu et al., 2006). Consistent with the postulated role of telomerase in malignancies, at WB analysis, a higher expression of TERT was found in tumors with respect to normal tissues. TERT promoter mutations are predicted to be one of the mechanisms responsible for the up-regulation of TERT protein expression (Huang et al., 2013; Vinagre et al., 2013), and accordingly TERTMUT tumors displayed a mildly increased expression of the protein with respect to TERTWT cases. At IHC, TERT staining has been demonstrated to be both nuclear and cytoplasmic in normal tissues and in normal lymph-nodes, whereas tumors and metastatic lymph-nodes displayed a significantly higher staining in the cytoplasm, both concerning the intensity of staining and the percentage of stained cells. This latter finding was surprising since the critical locus of action of the enzyme is the nucleus. Nevertheless, due to the absence of previous IHC data for TERT in thyroid cancer performed with this Ab, we can just speculate on the meaning of the cytoplasmic TERT localization in this tumor. Interestingly, telomerase has been shown to shuttle dynamically between different subcellular localizations as the consequence of cellular stresses, such as irradiation or anticancer treatments, and in particular oxidative stress, which is believed to be an important event in the molecular pathogenesis of thyroid cancer (Ahmed et al., 2008; Xing, 2012). Thus, we hypothesize that, whereas in normal follicular cells and in lymphocytes TERT has both a cytoplasmic and nuclear localization, in neoplastic follicular cells TERT mainly localizes, due to oxidative stress, in the cytoplasm. Interestingly, in some cases a polar localization of TERT was identified, consistent with the migration of the protein. Further studies are needed to confirm present data, and to identify if TERT has a specific localization within the cytoplasm. In conclusion, TERT mutations were shown to correlate with a poorer outcome in a large series of differentiated thyroid tumors. The prognostic value of TERT was found to be stronger than that of BRAFV600E in PTCs. In this context, it should be underlined that while the correlation of BRAFV600E with the persistence/remission of disease is still controversial (Fugazzola et al., 2006; Xing et al., 2013), the association of TERT promoter mutations with a worst outcome has been confirmed in the studies available to date. Moreover, while the predictive role of BRAFV600E is limited to PTCs, TERT mutations are indicator of prognosis also in FTCs and in de-differentiated tumors. According to its postulated role in carcinogenesis, TERT protein was found to be significantly more expressed in tumors than in normal tissues. Finally, the prevalent cytoplasmic localization found in thyroid tumor cells is consistent with the recent evidence that, besides telomeres maintenance, TERT plays several functions involved in different biological processes, such as gene expression regulation, DNA damage response and stress protection (Chiodi and Mondello, 2012). These data suggest that telomerase could contribute to cell survival and cancer progression by other mechanisms besides telomeres maintenance, possibly taking place in extranuclear compartments.
Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.mce.2014.10.019.
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Contents lists available at ScienceDirect
Molecular and Cellular Endocrinology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m c e
Telomerase in differentiated thyroid cancer: Promoter mutations, expression and localization Marina Muzza a,b,1, Carla Colombo a,b,1, Stefania Rossi c, Delfina Tosi d, Valentina Cirello a,e, Michela Perrino a,b, Simone De Leo a,b, Elisa Magnani f,g, Elisa Pignatti f,g, Beatrice Vigo a,b, Manuela Simoni f,g, Gaetano Bulfamante c,d, Leonardo Vicentini h, Laura Fugazzola a,e,* a
Endocrine Unit, Fondazione IRCCS Ca’ Granda, Milan, Italy Department of Clinical Sciences and Community Health, University of Milan, Italy c Division of Pathology, San Paolo Hospital, Milan, Italy d Department of Health Sciences, University of Milan, Italy e Department of Pathophysiology and Transplantation, University of Milan, Italy f Azienda USL of Modena, Italy g Department of Biomedicine, Metabolism and Neural Sciences, University of Modena and Reggio Emilia, Italy h Endocrine Surgery Unit, Fondazione IRCCS Ca’Granda, Milan, Italy b
A R T I C L E
I N F O
Article history: Received 3 July 2014 Received in revised form 16 October 2014 Accepted 21 October 2014 Available online 27 October 2014 Keywords: TERT Outcome BRAF RAS Genomics Epidemiology
A B S T R A C T
Telomerase-reverse-transcriptase (TERT) promoter mutations have been recently described in tumors. In the present large series, TERT mutations were found in 12% of papillary thyroid cancers (PTCs) and in 14% of follicular thyroid cancers (FTCs), and were found to significantly correlate with older age at diagnosis and poorer outcome. Interestingly, the prognostic value of TERT mutations resulted to be significantly stronger than that of BRAFV600E. Moreover, the outcome was not different among tumors with isolated TERT mutation and those with coexistent mutations (TERT/BRAF in PTCs or TERT/RAS in FTCs). TERT rs2853669 polymorphism was found in 44.4% of tumors. At WB, TERT was significantly more expressed in tumors than in normal samples, being the highest levels of expression recorded in TERT mutated cases. At IHC, in tumors and in metastatic lymph-nodes TERT staining was significantly higher in the cytoplasm than in the nucleus, whereas in normal tissue the degree of staining did not differ in the two cellular compartments. In conclusion, TERT mutations were shown to strongly correlate with a poorer outcome in differentiated thyroid tumors, and neither BRAF nor RAS mutation were found to confer an additional effect in the disease persistence. TERT protein was found to be more expressed in neoplastic than in normal tissues, and to display a different cellular localization, suggesting that it could contribute to thyroid cancer progression by mechanisms taking place in the cytoplasm. © 2014 Published by Elsevier Ireland Ltd.
1. Introduction Telomerase is a ribonucleoprotein polymerase that maintains telomere repeat TTAGGG at the ends of chromosomes, and consists of a protein component with reverse transcriptase activity, TERT, and a RNA component which serves as a template (Harrington et al., 1997). Telomerase expression is normally repressed in postnatal somatic cells, resulting in the progressive shortening of telomeres which leads to growth arrest (replicative senescence). Most cancer cells constitutively express telomerase, thus allowing telomeres
* Corresponding author. Endocrine Unit-Fondazione IRCCS Ca’ Granda, Via F. Sforza, Milan 35-20122, Italy. Tel.: +39 02 55033498; fax: +39 02 50320605. E-mail address: [email protected] (L. Fugazzola). 1 Equal contributors. http://dx.doi.org/10.1016/j.mce.2014.10.019 0303-7207/© 2014 Published by Elsevier Ireland Ltd.
maintenance and unlimited cellular proliferation (Skvortzov et al., 2009). It has been shown that the genetic mechanisms responsible for telomerase reactivation in tumors could include engagement of TERT alternative splicing, TERT gene amplification, and mutations in the TERT promoter (Skvortzov et al., 2009). In particular, the mutations −124 C > T (C228T) and −146 C > T (C250T) in the TERT promoter have been shown to occur in several tumors (Killela et al., 2013; Liu et al., 2013). These mutations generate de novo consensus binding motifs for E-twenty-six (ETS) transcription factors and in cancer cell lines have been shown to increase the transcriptional activity of the TERT promoter by two- to six-folds (Huang et al., 2013). These two TERT promoter mutations have been very recently reported in thyroid cancer cell lines and in thyroid tumors, either well differentiated (DTCs) or poorly differentiated and anaplastic (Landa et al., 2013; Liu et al., 2013, 2014; Melo et al., 2014; Vinagre et al.,
M. Muzza et al./Molecular and Cellular Endocrinology 399 (2015) 288–295
2013; Wang et al., 2014). In DTCs, an association between these TERT mutations (TERTMUT) and older age at diagnosis, male gender and tumor size (Liu et al., 2014; Melo et al., 2014; Vinagre et al., 2013), and a correlation with a reduced progression free survival and overall survival have been shown (Liu et al., 2013; Melo et al., 2014). Discordant data are available about the coexistence of TERT and BRAF or RAS mutations. In particular, TERTMUT were found to be significantly more frequent in BRAFV600E than in BRAFWT papillary thyroid cancers (PTCs) (Liu et al., 2013; Melo et al., 2014) and in RASMUT than in RASWT follicular thyroid cancers (FTCs) (Wang et al., 2014), but these data were confirmed only for advanced thyroid tumors in another series (Landa et al., 2013). The clinical impact of the association is still controversial and indeed, the coexistence of TERT and BRAF mutations in PTCs was found to correlate with more aggressive features in a Chinese series (Liu et al., 2014). On the other hand, in a series from Portugal, no differences in the outcome were noted between TERTMUT/BRAFMUT and TERTMUT/BRAFWT cases, indicating that BRAF per se does not have an addictive impact in the disease persistence (Melo et al., 2014). Very scarce, contradictory and not reliable data are available about TERT expression and cellular localization in the normal and neoplastic thyroid gland (Capezzone et al., 2009). Moreover, at the thyroid level no data exist about the possible modifying role of a common TERT polymorphism (rs2853669) located within a ETS binding site and shown in bladder cancer to be able to modify the effect of −124 C > T (C228T) and −146 C > T (C250T) mutations on survival and recurrence (Rachakonda et al., 2013). In the present study, we performed a comprehensive evaluation of TERT promoter, BRAFV600E and N- and H-RAS mutations in a large series of sporadic and familial tumors and in metastatic lymphnodes. The allelic frequency of rs2853669 SNP was also studied for the first time in thyroid cancer. Moreover, the expression and the localization of TERT was investigated by Western blot (WB) and immunohistochemistry (IHC). 2. Materials and methods 2.1. Patients tissue samples A large series of 254 primary thyroid tumors were included in this study. In particular, the following histologic types were analyzed: 182 PTCs (143 conventional, 34 follicular, 2 oncocytic and 3 diffuse sclerosing variants), 58 FTCs (42 conventional and 16 Hurtle variants), and 14 MTC. In addition, 3 diffuse toxic goiters, 3 multinodular goiters and 8 lymph-nodal metastases were studied. Moreover, 10 familial PTCs (fPTC) cases were included and analyzed for TERT mutations at the germline level. 2.2. Histological classification and outcome definition All specimens were reviewed by a senior pathologist (S.R.) to confirm the diagnosis. Tumors were classified and staged according to the 7th edition of the TNM staging (Compton et al., 2012). Criteria used to identify remission or persistent/recurrent disease were drawn on the bases of the European and American guidelines for the management of differentiated thyroid cancer (Cooper et al., 2009; Pacini et al., 2006) and have been previously reported in detail (Perrino et al., 2009).
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commercial kits (Puregene® Core Kit A, Qiagen, Germantown, MD, USA). All procedures performed for handling the tissues were approved by each Hospital Ethics Committee. An informed consent was obtained from all screened subjects.
2.4. Molecular analysis The TERT proximal promoter was amplified from tissue and blood DNA using the primers TERTPforward: 5′-AGTGGATTCGCG GGCACAGA-3′ and TERTPreverse: 5′-GCAGCGCTGCCTGAAACTC-3′. Exon 15 of the BRAF gene was analyzed using specific intronic primers, as previously described (Fugazzola et al., 2004). Exon 2 of N-RAS was PCR amplified by means of newly designed specific intronic primers, forward: 5′-ACCTTGGCAATAGCATTGCAT-3′ and reverse: 5′-TAGTGTGGTAACCTCATTTCC-3′, whereas exon 2 of H-RAS was PCR amplified using previously described primers (Moura et al., 2011). PCR products were purified and directly sequenced (ABI 3130, Perkin Elmer Applied Biosystem, Foster City, CA).
2.5. Immunohistochemical studies Paraffin blocks, including 19 PTCs, 5 FTCs and corresponding contralateral normal tissues, and 8 lymph-nodal metastases, were selected according to the following major criteria: (a) good morphology and (b) preservation of follicular structures (extensive fibrotic or hemorrhagic areas were excluded). From each paraffin block, 3 μm sections were obtained and tested for human TERT reactivity by means of a specific rabbit polyclonal antibody (Rockland Immunochemicals Inc. Gilbertsville, PA) at 1:2000 dilution, using an immunoperoxidase technique. The reaction was detected by Novolink Max polymer detection system (Novocastra Laboratories L.T.D., Leica Microsystem, Nussloch, Germany). In order to test the specificity and sensitivity of the antibody, control neoplastic tissues (gliomas) were also stained. The degree of positive staining for TERT was defined based on both the intensity of the staining and the percentage of stained cells. In particular, the intensity was defined as negative (0), low (1), medium (2) and high (3), whereas the percentage of stained cells were defined as none (0), 1–20% stained (1), 20–50% stained (2) and >50% stained (3).
2.6. Protein extraction and Western blotting Briefly, thyroid tissue was homogenized in lysis buffer (Cell Signaling Technology, Inc., Danvers, MA), supplemented with protease inhibitors (Roche, Basel, CH) and immunoblotting was performed with a rabbit anti-human TERT antibody (Rockland Immunochemicals Inc. Gilbertsville, PA) at 1:1000 dilution, and with an antiactin antibody (Novus Biologicals, Littleton, CO) or anti-GAPDH antibody (Ambion, Life Technologies, Foster City, CA). Chemiluminescence was detected using the Chemi-Doc-IT Imaging System (UVP, Upland, CA, USA). Band intensities were analyzed with the image analysis program NIH ImageJ and the TERT signal was normalized to actin signal.
2.7. Statistical analysis 2.3. Nucleic acids extraction Tissues were collected during surgery, immediately frozen and stored at −80 °C. To ensure a pure tumor tissue isolation in tumors T was more prevalent than TERT−146C>T in both histotypes (86.4% in PTCs and 62.5% in FTCs). No mutations were found in 14 MTCs and 6 thyroid nodular benign diseases (Table 1). The clinical features were available for all the cases included and the mean follow-up was of 78.9 months (median 74.5 months). Both patients with PTC and FTC were divided according to the TERT status (mutated/non-mutated) and several clinical parameters were compared. In PTC cases, TERT mutations were significantly associated at univariate analysis with an older age at diagnosis (P = 0.004), with a non-conventional PTC variant – i.e. follicular/oncocytic/diffuse sclerosing – (P = 0.008), and with persistent/relapsing disease (P = 0.002) (Table 2a). These 3 variables were confirmed at logistic regression
Table 2 (a) Clinical features of the 182 TERT mutated and wild-type (WT) papillary thyroid cancers (PTC); (b) Multivariate analysis using TERT promoter mutations as dependent variable. (a) PTC Age at diagnosis (years) Female gender Mean tumor diameter (mm) Tumor (T)
Multifocality Extrathyroidal invasion Lymph-nodes (N1) Stage
Histological variants
Outcome
Mean Range
T1 T2 T3 T4a
N1 I II III IV Conventional Follicular/ oncocytic/ diffuse sclerosing Persistence or recurrence
TERT mutated (n = 22) (12%)
TERT WT (n = 160) (88%)
P
57.6 29–82 14/22 (64%) 22
44.2 14–80 126/160 (79%) 19.3
0.004
7 (32%) 2 (9%) 12 (55%) 1 (4%) 11/22 (50%) 11 (50%)
57 (36%) 17 (11%) 82 (50%) 4 (3%) 77/160 (48%) 85 (53%)
11 (50%) 10 (45%) 2 (9%) 6 (27%) 4 (18%) 12/22 (55%) 10/22 (45%)
83 (52%) 106 (66%) 5 (3%) 31 (20%) 18 (11%) 131/160 (82%) 29/160 (18%)
10/22 (45%)
29/160 (18%)
0.19 0.34 0.8
0.95 0.9 0.9 0.09
0.008
0.002
(b) Variable
Odds ratio
95% CI
P
Older age Non-conventional variants* Persistence/recurrence
4.6848 2.9663 3.4007
1.4768–14.8615 1.0816–8.1348 1.2909–8.9588
0.008 0.03 0.01
Statistically significant values are reported in bold. * follicular/oncocytic/diffuse sclerosing variants.
analysis to be independently correlated with TERT mutations (P = 0.008, P = 0.03 and P = 0.01, respectively) (Table 2b). In FTC cases, TERT mutations were significantly associated with an older age at diagnosis (P = 0.04), with a worst stage (P = 0.02) and with a poorer outcome (P = 0.03) at univariate analysis, whereas at logistic regression TERT mutations were found to significantly correlate only with persistent disease (P = 0.03). Finally, there were no significant differences in the distribution of TERT mutated and nonmutated cases according to the histological variant (classic or Hurtle cells) (Table 3). After pooling all the differentiated cases (PTCs + FTCs), TERT mutations were confirmed to be significantly associated with an older age at diagnosis (P = 0.002) and a worst outcome (P = 0.006). At the logistic regression analysis, either age or outcome were found to be independently associated with TERT mutations (P = 0.003 and P = 0.002, respectively) (Supplemental Table S1). 3.2. TERT polymorphisms in primary tumors The overall allelic frequency of rs2853669 polymorphism in thyroid cancers was 44.4%, without significant differences between
Table 3 (a) Clinical features of the 58 TERT mutated and wild-type (WT) follicular thyroid cancers (FTC); (b) Multivariate analysis using TERT promoter mutations as dependent variable. (a) FTC Age at diagnosis (years) Female gender Mean tumor diameter (mm) Tumor (T)
Multifocality Extrathyroid invasion Lymph-nodes (N1) Stage
Histological variants Outcome
Mean Range
TERT mutated (n = 8) (13.8%)
TERT WT (n = 50) (86.2%)
P
66 48–81 6/8 (75%) 54
56 18–85 25/50 (50%) 40.9
0.04
T1 T2 T3 T4
I II III IV Conventional Hurtle Persistence or recurrence
0 3 4 1 1/8 (12.5%) 5/8 (62.5%)
21 7 19 3 9/50 (18%) 24/50 (48%)
7/8 (87.5%) 0 2 (25%) 3 (37.5%) 3 (37.5%) 6/8 (75%) 2/8 (25%) 5/8 (62.5%)
46/50 (92%) 20 (20%) 7 (14%) 18 (36%) 5 (10%) 36/50 (72%) 14/50 (28%) 11/50 (22%)
1 0.1 0.08
0.9 0.7 0.7 0.02
1 0.03
(b) Variable
Odds ratio
95% CI
P
Age Stage Persistence/recurrence
– – 5.27
– – 1.0962–25.4103
NS NS 0.03
Statistically significant values are reported in bold.
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TERTMUT (41.6%) or TERTWT (46.8%) cases. The presence of rs2853669 was not correlated with either any clinical parameter or the outcome (data not shown).
3.3. Correlation of TERT promoter, BRAF and RAS mutations in primary tumors The analysis of BRAFV600E mutations in PTCs revealed an overall prevalence of 35.2% (64/182 cases). TERT mutations were found in 10/64 (15.6%) BRAFMUT cases and in 12/118 (10.1%) BRAFWT tumors (P = 0.34) (Table 4A). PTCs were divided according to the presence of a mutation in TERT, in BRAF or in both genes, and the prevalence of persistence/remission was compared (Table 4B). Interestingly, persistent disease was significantly more frequent in BRAFWT/ TERTMUT than in BRAFMUT/TERTWT cases (50 vs 17%, P = 0.02). Of note, no significant differences in the outcome was found between BRAFWT/ TERTMUT and BRAFMUT/TERTMUT cases. In FTCs, RAS mutations were found in 14/58 cases (24.1%). In particular, 11 tumors had a N-RAS mutation (10 Q61R and 1 Q61K) and 3 cases had a H-RAS mutation (2 Q61R and 1 Q61K). TERT mutations had a similar frequency among RASMUT and RASWT tumors (14.3 vs 13.6%; P = 1) (Table 4A1). FTCs were divided according to the presence of a mutation in TERT, in RAS or in both genes, and the prevalence of persistence/remission was compared (Table 4B). The prevalence of persistent disease resulted to be significantly higher in RASMUT/TERTMUT than in RASMUT/TERTWT cases (100 vs 8%, P = 0.03).
Table 4 (A) The prevalence of BRAFV600E mutations in papillary thyroid cancers (PTCs) was 35.2% (64/182 cases); (A1) The prevalence of N- and H-RAS mutations in follicular thyroid cancers (FTCs) was 24.1% (14/58 cases); (B) In PTCs, a poorer outcome was significantly more frequent in BRAFWT/TERTMUT than in BRAFMUT/TERTWT cases; in FTCs a poorer outcome was significantly more frequent in RASMUT/TERTMUT than in RASMUT/ TERTWT tumors. A PTCs
TERTMUT (n = 22)
TERTWT (n = 160)
P
BRAFMUT (n = 64) BRAFWT (n = 118)
10 12
54 106
0.34
A1 FTCs
TERTMUT (n = 8)
TERTWT (n = 50)
P
RASMUT (n = 14) RASWT (n = 44)
2 6
12 38
1
B PTCs
Remission
Persistence
P
BRAFMUT/TERTMUT BRAFWT/TERTWT BRAFWT/TERTMUT BRAFMUT/TERTWT BRAFMUT/TERTMUT BRAFMUT/TERTWT BRAFWT/TERTMUT BRAFMUT/TERTMUT
6 84 6 45 6 45 6 6
4 (40%) 22 (21%) 6 (50%) 9 (17%) 4 (40%) 9 (17%) 6 (50%) 4 (40%)
0.22 0.02 0.19 0.69
291
3.4. TERT promoter and BRAF mutations in lymph-nodal metastases We had the opportunity to include in the study 12 metastatic lymph-nodes. The molecular pattern was identical among the primary tumor and the lymph-node metastases in 9 cases (5 BRAFV600E/TERTWT, 1 BRAFWT/TERT−124C>T, and 3 BRAFWT/TERTWT), while it was discrepant in 3 patients. In particular, we observed the acquisition in the lymph node metastasis of a TERT or a BRAFV600E mutation (BRAFWT/TERTWT primary tumor vs BRAFWT/TERT−124C>T lymph node metastasis in 2 cases, and BRAFWT/TERT−124C>T primary tumor vs BRAFV600E/TERT−124C>T lymph node metastasis in one patient). 3.5. TERT promoter and BRAF germline mutations in familial cases Neither BRAF nor TERT mutations were found at the germline level in the 10 familial PTC cases examined (Table 1). 3.6. Western blot analysis At WB analysis, tumors were found to harbor an enhanced TERT expression with respect to normal tissues (Fig. 1). The mean value of densitometric analysis (TERT/housekeeping expression) was of 0.89 for tumors and 0.25 for normal tissues (P = 0.0001). Moreover, TERTMUT tumors had a higher expression of the protein, though not at a significant level, with respect to TERTWT cases (1.14 vs 0.75, P = 0.2). Besides the expected band of 127 kDa, additional bands were visualized with this antibody, as already reported in experiments on HeLa cells (Wu et al., 2006). They could be non-specific or match with TERT isoforms. In particular, a 95 kDa band was identified which could correspond with the beta deletion variant (Listerman et al., 2013). 3.7. Immunohistochemistry analyses At IHC, either normal and tumor tissues displayed a TERT staining though a different pattern was observed between tumor tissue and the contralateral normal thyroid tissue (Table 5 and Fig. 2, panels A and B). In particular, tumor tissues displayed a prevalent cytoplasmic staining, whereas normal tissues had both a cytoplasmic and a nuclear TERT positivity. In tumors, the mean intensity of staining was 1.7 in the cytoplasm and 0.9 in the nucleus (P = 0.02), and the mean percentage of immunoreactive cells was 1.3 in the cytoplasm and 0.6 in the nucleus (P = 0.0008). On the other hand, in normal tissues no statistically significant differences were noted neither in the intensity of staining nor in the percentage of immunoreactive cells between cytoplasm and nucleus (1.7 vs 2 and 1.6 vs 1.8, respectively, P = NS). In some tumors a typical perinuclear positivity was observed, located at the apical pole of the nucleus, towards the cytoplasm (Fig. 2, panel C). No differences in TERT staining were seen between TERTMUT and TERTWT tumors. The differential expression of TERT between normal and metastatic cells was further confirmed in lymph-nodes. In neoplastic lymph-nodes the staining was exclusively cytoplasmic (1.25 for either mean intensity of staining and mean percentage of immunoreactive cells). In both B and T lymphocytes the staining was present in both compartments, but more prevalent in the nucleus (1 vs 2.25 for both criteria, P = 0.01) (Table 5 and Fig. 2, panel D).
FTCs
Remission
Persistence
P
RASMUT/TERTMUT RASWT /TERTWT RASWT/TERTMUT RASMUT/TERTWT RASMUT/TERTMUT RASMUT/TERTWT RASMUT/TERTMUT RASWT/TERTMUT
0 27 3 11 0 11 0 3
2 (100%) 11 (41%) 3 (50%) 1 (8%) 2 (100%) 1 (8%) 2 (100%) 3 (50%)
0.1
4. Discussion
0.08
In the present series of 240 DTCs, TERT promoter mutations were found in 12% of PTCs and in 14% of FTCs. Similar or higher frequencies were found in previous series (Landa et al., 2013; Liu et al., 2013, 2014; Melo et al., 2014; Vinagre et al., 2013) (Table 1). No TERT mutations were documented either in MTCs or, at the germline level, in familial PTC cases, as previously reported (Liu et al., 2013; Melo
Statistically significant values are reported in bold.
0.03 0.46
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Fig. 1. Western blot analysis with TERT antibody (Rockland Immunochemicals Inc. Gilbertsville, PA) of a representative cohort of TERTMUT (#1p, 2p, 3p and 7p) and TERTWT (#4p, 5p, 6 p, 8p and 9p) PTCs, and of TERTMUT FTCs (#1f and 2f). Normal contralateral thyroid tissues (#1n, 2n, 6n, 7n and 9n), corresponding to the above mentioned PTCs, are also shown. The band of 127 kDa corresponds to TERT. An anti-actin blot is also shown as a protein loading control. In the lower part of the figure, the densitometric analysis of TERT/housekeeping gene in a total of 7 TERTMUT PTCs, 11 TERTWT PTCs, 3 TERTMUT FTCs, and 11 normal tissues is reported. PTC and p: papillary thyroid cancer; FTC and f: follicular thyroid cancer; n: normal tissue; bis: replicate experiment on the same tumor tissue.
et al., 2014; Vinagre et al., 2013), indicating that these genetic alterations are not involved in the pathogenesis of these diseases. In the present PTCs cohort, TERT promoter mutations were found to strongly and independently correlate with an older age at diagnosis and with a poorer outcome, in accordance with previous studies (Liu et al., 2013, 2014; Melo et al., 2014; Vinagre et al., 2013). The association of TERT mutations with older age at diagnosis is consistent with the knowledge that telomeres become progressively shorter during lifetime, and thyroid follicular cells in older subjects are thus predicted to harbor short telomeres. When these cells begin to proliferate in response to an oncogenic event (e.g. BRAFV600E), the presence of very short telomeres leads to telomere dysfunction, triggering genomic instability and telomerase activation by TERT promoter mutations (Londoño-Vallejo, 2008). Accordingly, TERT mutations have
been found to correlate with shorter telomeres and age >45 years in a cohort of PTC patients (Liu et al., 2013). Moreover, in the present series, the non-conventional PTC variants (follicular/oncocytic/ diffuse sclerosing) were found to independently correlate with TERT mutations, consistent with previous data (Liu et al., 2013). The overall prevalence of BRAFV600E was 35.2% in the present series of PTCs. TERT mutations were found in 15.6 and 10.1% in BRAFMUT and BRAFWT cases, respectively. Interestingly, persistence was significantly more frequent in BRAF W T /TERT MUT than in BRAF MUT / TERT W T cases (P = 0.02), likely indicating that TERT promoter mutations have a more relevant impact on disease outcome than BRAFV600E. Since this finding is in contrast with a Chinese series reporting a limited effect for TERT promoter mutations alone (Liu et al., 2014), further studies on larger series are likely needed to define
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293
Table 5 Immunohistochemical data in papillary and follicular* thyroid cancers in TERTMUT (mutated) and TERTWT (wild-type), and in metastatic lymph-nodes. T , tumor; N, normal thyroid or lymph-nodal tissue; L, lymph-node. Intensity of the staining/number of immunoreactive cells are both reported for a better identification of the degree of staining. In particular, the intensity of staining was defined as negative (0), low (1), medium (2) and high (3), while the percentage of immunoreactive cells were defined as none (0), 1–20% stained (1), 20–50% stained (2) and >50% stained (3). TERT MUT
T1*
N1
T2
N2
T3
N3
T4
N4
T5*
N5
T6
N6
T7
N7
T8
N8
T9*
N9
Cytoplasm Nucleus
1/1 1/1
2/2 2/2
3/2 0/0
2/2 1/1
1/1 1/1
1/1 2/2
2/1 0/0
2/2 3/2
1/1 1/1
2/2 2/2
1/1 0/0
2/2 1/1
1/1 3/1
-
3/2 3/1
-
1/1 2/1
1/1 2/1
TERT WT
T10
N10
T11
N11
T12*
N12
T13
N13
T14*
N14
T15
N15
T16
N16
T17
N17
T18
N18
T19
N19
Cytoplasm Nucleus
1/1 0/0
3/1 2/2
1/1 0/0
2/2 2/2
1/1 1/1
2/2 2/2
2/1 0/0
2/2 2/2
1/1 1/1
1/1 2/2
1/1 1/1
3/1 2/2
3/1 1/1
2/2 3/2
1/3 1/3
1/1 2/2
2/1 2/1
1/1 2/1
3/2 0/0
2/2 3/3
Metastatic lymph-nodes
L1
N1
L2
N2
L3
N3
L4
N4
Cytoplasm Nucleus
2/2 0/0
1/1 2/2
1/1 0/0
1/1 3/3
1/1 0/0
1/1 2/2
1/1 0/0
1/1 2/2
* correspond to follicular thyroid cancers as reported in the legend of the Table.
the role of TERT mutations when separated from BRAFV600E. Nevertheless, and consistent with a previous report (Melo et al., 2014), in our series no statistically significant difference in the outcome was found between BRAFWT/TERTMUT and BRAFMUT/TERTMUT, indicating that BRAF mutation does not confer an additional effect in the disease persistence. In the present FTCs cohort, TERT promoter mutations were found to strongly and independently correlate with a poorer outcome and RAS mutations were found in 24.1% of cases, without differences between TERTMUT and TERTWT. Even though the statistical significance was not reached, tumors with TERT mutations alone tended to have a persistent disease more frequently than those with RAS mutations alone (50 vs 8%, P = 0.08), indicating, as above reported for PTCs, the relevant role of TERT mutations in the degree of aggressiveness of the disease. Consistent with these findings, there were no statistically significant differences in the percentage of recurrent/persistent cases between RASWT/TERTMUT and RASMUT/
TERTMUT FTCs, indicating that RAS mutations do not contribute in the worsening of the outcome. On the contrary, RASMUT/TERTMUT were significantly more recurrent/persistent than RASMUT/TERTWT cases (P = 0.03), suggesting that TERT mutation can worsen the outcome of RAS mutated cases. This is the first study including metastatic lymph-nodes from PTCs, which were found to harbor TERT mutations in 25% of cases. In 3/12 patients we found a discrepant pattern between the primary tumor and the lymph node metastases, with the acquisition of a TERT or a BRAF mutation in the metastasis. This finding is consistent with data obtained in thyroid cancer (Vasko et al., 2005) and in melanoma and could be due to the selection of mutant alleles during tumor progression or to the heterogeneous pattern of tumoral cells in the primary tumor with only some subclones able to metastatize (Lin et al., 2011). In this context, thyroid cancer has been shown to consist of a mixture of BRAFV600E and BRAFWT cells, suggesting that BRAF mutation in PTC is a later subclonal event (Guerra et al., 2012).
Fig. 2. Expression of TERT at immunohistochemistry in exemplificative cases. Panel A: papillary thyroid cancer. Note the TERT staining in the cytoplasm (40×); panel B: normal thyroid tissue. Note the TERT positivity in the nucleus and in the cytoplasm with a «dot» pattern of distribution (200×); panel C: papillary thyroid cancer. Note that TERT positivity is located at the apical part of the nucleus (200×); panel D: papillary thyroid cancer metastatic lymph node. Note on the left the nuclear staining of lymphocytes and on the right the cytoplasmic staining of metastatic cells (200×).
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The relevance of the present study relies on the combination of genetic data with expression and localization studies. In particular, WB data have been obtained for the first time in human thyroid tumor tissues, whereas IHC analyses for TERT have been previously performed in several normal and neoplastic human tissues, with discordant results concerning the immunolocalization of hTERT, reported to be exclusively nuclear (Ito et al., 2005; Lantuejoul et al., 2004; Preto et al., 2004) or both cytoplasmic and nuclear (Gasinska et al., 2013; Kyo et al., 2003; Mavrommatis et al., 2005). The discrepancies are likely to be mainly due to the different antibodies (Ab) used, since around 15 different monoclonal and polyclonal Ab are available (Fabricius et al., 2009), which are directed against different portions of the protein. Accurate studies concluded that among the commercially available anti-hTERT Ab, only the RCK-hTERT antibody, used in the present study, is suitable for hTERT protein detection (Wu et al., 2006). Consistent with the postulated role of telomerase in malignancies, at WB analysis, a higher expression of TERT was found in tumors with respect to normal tissues. TERT promoter mutations are predicted to be one of the mechanisms responsible for the up-regulation of TERT protein expression (Huang et al., 2013; Vinagre et al., 2013), and accordingly TERTMUT tumors displayed a mildly increased expression of the protein with respect to TERTWT cases. At IHC, TERT staining has been demonstrated to be both nuclear and cytoplasmic in normal tissues and in normal lymph-nodes, whereas tumors and metastatic lymph-nodes displayed a significantly higher staining in the cytoplasm, both concerning the intensity of staining and the percentage of stained cells. This latter finding was surprising since the critical locus of action of the enzyme is the nucleus. Nevertheless, due to the absence of previous IHC data for TERT in thyroid cancer performed with this Ab, we can just speculate on the meaning of the cytoplasmic TERT localization in this tumor. Interestingly, telomerase has been shown to shuttle dynamically between different subcellular localizations as the consequence of cellular stresses, such as irradiation or anticancer treatments, and in particular oxidative stress, which is believed to be an important event in the molecular pathogenesis of thyroid cancer (Ahmed et al., 2008; Xing, 2012). Thus, we hypothesize that, whereas in normal follicular cells and in lymphocytes TERT has both a cytoplasmic and nuclear localization, in neoplastic follicular cells TERT mainly localizes, due to oxidative stress, in the cytoplasm. Interestingly, in some cases a polar localization of TERT was identified, consistent with the migration of the protein. Further studies are needed to confirm present data, and to identify if TERT has a specific localization within the cytoplasm. In conclusion, TERT mutations were shown to correlate with a poorer outcome in a large series of differentiated thyroid tumors. The prognostic value of TERT was found to be stronger than that of BRAFV600E in PTCs. In this context, it should be underlined that while the correlation of BRAFV600E with the persistence/remission of disease is still controversial (Fugazzola et al., 2006; Xing et al., 2013), the association of TERT promoter mutations with a worst outcome has been confirmed in the studies available to date. Moreover, while the predictive role of BRAFV600E is limited to PTCs, TERT mutations are indicator of prognosis also in FTCs and in de-differentiated tumors. According to its postulated role in carcinogenesis, TERT protein was found to be significantly more expressed in tumors than in normal tissues. Finally, the prevalent cytoplasmic localization found in thyroid tumor cells is consistent with the recent evidence that, besides telomeres maintenance, TERT plays several functions involved in different biological processes, such as gene expression regulation, DNA damage response and stress protection (Chiodi and Mondello, 2012). These data suggest that telomerase could contribute to cell survival and cancer progression by other mechanisms besides telomeres maintenance, possibly taking place in extranuclear compartments.
Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.mce.2014.10.019.
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