© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
J Cutan Pathol 2014: 41: 504–508 doi: 10.1111/cup.12323 John Wiley & Sons. Printed in Singapore
Journal of Cutaneous Pathology
TERT promoter mutation is uncommon in acral lentiginous melanoma Background Melanoma is a heterogeneous group of diseases with distinct sets of genetic changes. Recurrent and mutually exclusive C>T or CC>TT transition mutations were identified in the promoter region of the reverse transcriptase catalytic subunit of the telomerase gene (TERT ) in melanoma recently, and it was suggested that they enhanced the expression of TERT gene and played important roles in the melanoma pathogenesis. These mono or di-nucleotide transitions were ultraviolet (UV)-signature mutations. Methods In this study, polymerase chain reaction and direct sequencing of TERT promoter using formalin-fixed and paraffin-embedded tissue was performed to investigate whether these UV-signature mutations were also present in acral lentiginous melanoma. Results TERT promoter mutation was identified in only 2 of the 32 cases (6%) of acral lentiginous melanomas while it was identified in 3 of the 9 cases (33%) of non-acral cutaneous melanomas. The difference was statistically significant (p = 0.028). Conclusions The rarity of TERT promoter mutation in the acral lentiginous melanoma was consistent with the supposed role of UV light in the melanoma pathogenesis and also corroborated the view that acral lentiginous melanomas have a different pathogenesis with the melanomas from sun-exposed sites. Keywords: acral lentiginous melanoma, TERT, UV light Liau J-Y, Tsai J-H, Jeng Y-M, Chu C-Y, Kuo K-T, Liang C-W. TERT promoter mutation is uncommon in acral lentiginous melanoma. J Cutan Pathol 2014; 41: 504–508. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Telomeres are repetitive DNA sequences at the ends of chromosomes that preserve genome integrity and stability.1 Telomeres shorten after repeated cell division. Telomere shortening results in genome instability because it induces chromosome end resection, fusion and breakage.1 Maintenance of telomeres is essential for malignant tumor formation. Two mechanisms of telomere maintenance have been identified in tumor cells: the activation of telomerase and alternative mechanisms of telomere
504
Jau-Yu Liau1,2 , Jia-Huei Tsai1,2 , Yung-Ming Jeng1,2 , Chia-Yu Chu3 , Kuan-Ting Kuo1 and Cher-Wei Liang1,2 1 Department
of Pathology, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan, 2 Graduate Institute of Pathology, College of Medicine, National Taiwan University, Taipei, Taiwan, and 3 Department of Dermatology, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan
Cher-Wei Liang, Department of Pathology, National Taiwan University Hospital, 7 Chung-Shan South Road, Taipei, Taiwan Tel: 886-2-23123456 ext. 65453 Fax: (886)-2-23934172 e-mail: [email protected] Accepted for publication February 28, 2014
lengthening (ALT).1 Telomerase is a ribonucleoprotein complex that adds repetitive DNA sequences to the telomeres. In normal adult tissue, telomerase activity is only present in limited cell types, including germ cells, bone marrow stem cells and basal cells of certain epithelia.2,3 The activation of telomerase in human tumors plays a key role in cellular ‘immortalization’.4 Telomerase activity was detected in 69% of cutaneous melanoma,5 but the mechanism of telomerase activity was unclear until recently.6,7
TERT promoter mutation and UV light Mutation in the promoter region of the reverse transcriptase catalytic subunit (TERT ) of telomerase was recently found in a large proportion of cutaneous melanoma.6,7 These mutations generate novel binding sites for E-twenty-six (ETS) transcription factors, and the mutations increase transcriptional activity from the TERT promoter two to 4-fold. The C>T or CC>TT transitions are consistent with ultraviolet (UV)-signature mutations, indicating that UV exposure is likely to be the major mechanism for the generation of these mutations. Melanomas from sites with different levels of sunexposure have been shown to harbor different sets of genetic alterations.8 Acral lentiginous melanomas usually do not have activating BRAF or NRAS mutations, but the C-KIT mutation rate is higher, in contrast to non-acral cutaneous melanomas. Acral lentiginous melanomas are also characterized by a high frequency of gene amplifications, such as C-KIT , CCND1, and CDK4. As acral sites are non or rarely sun-exposed, the existence and frequency of the TERT promoter mutations should be separately investigated. In this study, we performed TERT mutational analysis using formalin-fixed and paraffin-embedded (FFPE) tissue to investigate whether the TERT promoter mutation rate is different between acral lentiginous and non-acral melanomas. Materials and methods Cases of acral lentiginous and non-acral cutaneous melanomas with available archival material were retrieved from the Department of Pathology, National Taiwan University Hospital. The diagnosis was confirmed by histological review. Only primary tumors with a tumor thickness equal or greater than 2 mm or grossly metastatic tumors were analyzed to enrich tumor cells and reduce the influence of contaminated normal cells. After case selection, two to three 10-μm thick paraffin sections were cut from the block. The tumorous area from paraffin sections was dissected using sterilized razors under a microscope. Genomic DNA was extracted using a QIAamp DNA FFPE Tissue Kit (Qiagen, Santa Clarita, CA, USA) according to the manufacturer’s protocol. The samples were subjected to polymerase chain reaction (PCR) using a primer pair of the TERT promoter (forward: 5 -GGGGCCAGGGCTTCCCACGTG3 ; reverse: 5 -ACCCGTCCTGCCCCTTCACC3 ). The PCR was performed in an automatic DNA thermal cycler (PerkinElmer, Wellesley, MA, USA) with initial heating at 95 ◦ C for 2 min followed by 30 cycles at 95 ◦ C for 30 s, 68 ◦ C for 30 s, 72 ◦ C for 30 s, and, finally, 72 ◦ C for 10 min. After purification,
direct sequencing was performed in an automated ABI 3730 sequencer (Applied BioSystems, Foster City, CA, USA). This study was approved by the Institutional Research Ethnic Committee, and the specimens were anonymous and analyzed in a blind manner. For statistical analysis, comparison of TERT promoter mutation rate was performed using the Pearson Chi-square method. The result was considered significant if the p value was < .05. Results In total, 52 samples from 41 patients were analyzed. Thirty-two and nine patients had acral lentiginous and non-acral melanomas, respectively. Among the non-acral melanomas, five were superficial spreading and four were nodular melanomas. The acral lentiginous melanoma group encompassed 23 primary tumors and 17 metastases (11 lymph nodes and 6 systemic metastases). Eight patients had paired primary and metastatic tumors available for comparison. The non-acral melanoma group encompassed five primary tumors and seven metastases (five lymph nodes and two systemic metastases). Paired primary and metastatic tumors were available for three patients. TERT promoter mutation was detected in only 2 of the 32 acral lentiginous melanoma cases (6%, both C228T transitions), while it was found in 3 of the 9 non-acral cutaneous melanomas (33%, also C228T transitions). The mutations were confirmed by reverse sequencing. The difference between the mutation rates of acral and non-acral melanomas was statistically significant (p = .028). Histologically, both TERT promoter-mutated tumors exhibited prominent lentiginous junctional component and were not distinct from other wild type tumors. All tumor pairs showed concordant TERT promoter mutation status. In the acral lentiginous melanoma group, all eight tumor pairs were wild type. In the non-acral melanoma group, one tumor pair showed concordant C228T mutation, the other two were wild type. The demographic, histological, and molecular findings were summarized in Table 1. Representative hematoxylin and eosin stained sections and sequencing chromatograms were shown in Fig. 1. Discussion Melanomas are the most common lethal primary skin cancers. Although promising clinical responses have been shown for several small molecule inhibitors targeting the active oncoproteins, primary or acquired resistance is common and the prognosis of patients with metastatic disease is still poor. Histologically, cutaneous melanomas are classified
505
Liau et al. Table 1. Clinical, histological, and molecular findings
Sex, N (%) Male Female Mean age (range) Primary tumor, N (%) Upper extremity Lower extremity Trunk Tumor sample, N* Primary Metastasis Tumor stage at diagnosis (AJCC 7th) I II III IV Unavailable Mean Breslow thickness, mm (range) Ulceration Present Absent Unavailable Tumor subtype, N
TERT promoter mutation, N (%)
Acral lentiginous (N = 32)
Non-acral (N = 9)
17 (53%) 15 (47%) 66 (39–89)
4 (44%) 5 (56%) 45 (28–71)
5 (hand) (16%) 27 (foot) (84%) 0 (0%)
3 (33%) 2 (22%) 4 (44%)
23 17
5 7
2 14 15 1 0 6.2 (0.2–19)
0 3 2 1 3 7.3 (3.0–16)
17 12 3 Acral lentiginous, N = 32
3 3 3 Superficial spreading, N = 5 Nodular, N=4 3 (33%)†
2 (6.3%)
*Paired primary and metastatic tumors were available for 8 and 3 acral lentiginous and non-acral melanoma patients, respectively. † TERT promoter mutation was found in 2 and 1 superficial spreading and nodular melanomas, respectively.
as superficial spreading, nodular, acral lentiginous, and lentigo maligna melanomas based on the growth pattern of the junctional melanocytes, location and degree of solar damage. However, melanoma is a heterogeneous group of tumors with different genetic alterations. The traditional histology-based classification system has little or no prognostic or therapeutic relevancies and a molecular genetics-based classification scheme was proposed.8 In their seminal paper, Curtin et al.8 showed that melanomas from sites with different levels of sun exposure have distinct sets of genetic alterations. More than 80% of melanomas from non-acral skin without chronic sun-induced damage had mutations in either BRAF or NRAS genes. Acral melanomas, in contrast, had higher C-KIT mutation rate, a higher
506
degree of chromosomal aberrations and more gene amplifications. One of the most important findings in the melanoma research recently was the identification of recurrent mutations in the core promoter region of the TERT gene, which encodes the rate-limiting catalytic reverse transcriptase subunit of the telomerase ribonucleoprotein complex.6,7 The C>T transition at −124 bp (chr5, 1,295,228C>T, C228T) or −146 bp (1,295,250C>T, C250T) or less common CC>TT tandem mutations at −124 bp or −138 bp upstream the transcription start site generate identical 11-bp nucleotide stretch (5 CCCCTTCCGGG-3 ), which contains the general binding motif (GGAA/T, reverse complement) of ETS family transcription factors. This nucleotide stretch also contains the binding motif (CCGGAA, reverse complement) of the ETS subfamily TCF (ternary complex factor) transcription factors Elk1 and Elk4. These mono or di-nucleotide transitions are mutually exclusive and are consistent with the patterns of UV-induced mutations. In these two studies, more than 70% of cutaneous melanoma samples harbored one of these mutations, and TERT promoter mutation is thus, by far the most common genetic alterations in the melanoma. The ETS transcription factors are downstream targets of RAS-RAF-MAPK pathways and TERT promoter mutations are suggested to have synergistic effects with activating BRAF or NRAS mutations to promoter tumor cell proliferation.6 The high frequency of TERT promoter mutation and the higher than expected rate of concomitant mutations in the TERT promoter and BRAF suggested that TERT promoter mutations were driver mutations in the pathogenesis of melanoma.6 TERT promoter mutation was not identified in benign nevi. Consistent with the idea of UV-induced mutations, they were also recently found in a large proportion of cutaneous squamous cell carcinoma, basal cell carcinoma, atypical fibroxanthoma, and primary dermal pleomorphic sarcoma.9,10 However, the same mutations were also identified in cancers of internal organs, most notably primary glioblastoma, medulloblastoma, urothelial carcinoma, hepatocellular carcinoma, anaplastic carcinoma of the thyroid gland, squamous cell carcinoma of the tongue, and myxoid liposarcoma.7,11 – 18 In these tumors, TERT promoter mutations cannot be attributed to UV light and the causes of TERT promoter mutations need further studies. Recently, using FFPE tissue, TERT promoter mutations were detected in about one third of conjunctival melanoma but none of the uveal melanoma had TERT promoter mutation.19 The detected TERT promoter mutation rate in conjunctival melanomas was comparable to
TERT promoter mutation and UV light
Fig. 1. A) An acral lentiginous melanoma with prominent lentiginous junctional component. B) TERT promoter mutation (C228T) was detected in this tumor using bidirectional direct sequencing (upper panel: forward sequence, lower panel: reverse sequence with complement). C) A nodular type non-acral melanoma with (D) C228T mutation (upper panel: forward sequence, lower panel: reverse sequence with complement). Original magnification: (A) and (C): ×100.
the cutaneous melanoma in other studies.6,18 Conjunctival melanoma, like cutaneous melanoma, frequently harbor BRAF or NRAS mutations.20 They also behave more akin to cutaneous melanoma clinically.21 – 23 Uveal melanoma, in contrast, lack BRAF or NRAS mutations but frequently have GNAQ or GNA11 mutations.24 – 29 A subset of uveal melanoma with indolent behavior harbored SF3B1 (splicing factor 3B subunit 1) mutation, which was not found in cutaneous melanoma.30,31 These findings strongly suggested that the molecular pathogenesis of uveal melanoma is distinct from conjunctival or cutaneous melanomas. Similarly, TERT promoter mutations were detected in only 2 of the 32 cases of acral lentiginous melanomas in our series, despite that we had limited the analysis to relatively advanced tumors to enrich the tumor cells. However, TERT promoter mutation was detected in 33% (3/9) of the control group of non-acral melanomas, which is similar to the result of the previous study.6 In the study of Vinagre et al.,18 the overall TERT promoter mutation rate for cutaneous melanoma was 29% (16/56). However, the frequency of TERT promoter mutation appeared to be very different between histological types. TERT promoter mutation was
found in only 7% (1/14) of acral lentiginous melanomas, which was similar to our result, while they were common in superficial spreading and nodular melanomas (8/22 = 36% and 6/14 = 43%, respectively). Interestingly, the TERT promoter mutated acral lentiginous melanoma in that series also harbored BRAF V600E and NRAS mutations concomitantly, which was a very uncommon event, especially for acral lentiginous melanoma. No BRAF V600E or NRAS (exons 1 and 2) mutation was detected in the two TERT promoter mutated acral tumors in our series but BRAF V600E mutation was found in two of the three non-acral tumors (data not shown). In combination with our result, TERT promoter mutation was found in only 6–7% of acral lentiginous melanomas, a frequency that was one sixth of that of non-acral tumors. However, increased TERT copy number was found in about 30% of acral lentiginous melanomas in one study.32 Bastian et al.33 also showed that acral lentiginous melanoma including the in situ component was characterized by gene amplifications. One of the recurrently amplified loci was 5p15 (in 3 of 15 tumors, 20%), which contained the TERT gene. None of the superficial spreading melanoma analyzed harbored
507
Liau et al. amplification in this locus. These findings suggested that increased TERT gene dosage played a more important role than TERT promoter mutation for acral lentiginous melanoma. For cutaneous melanomas, the rarity of TERT promoter mutation in acral lentiginous melanoma is consistent with the supposed role of UV light in the pathogenesis, as acral sites are rarely sun-exposed. These findings corroborated the results of the previous array-based chromosomal genomic hybridization study that acral melanoma has a different pathogenesis compared with non-acral cutaneous melanoma, and are also in line with the finding that acral and mucosal melanomas have a higher degree of chromosomal aberrations and more frequent gene amplifications.8 In our study, the two acral lentiginous melanomas with TERT promoter mutation were primary
tumors, while all metastatic tumor samples analyzed were wild type. All eight tumor pairs showed concordant wild type result. The results suggested that TERT promomter mutation was present in the primary tumors and was not prerequisite for metastasis. Lack of C250T mutation, even in the nonacral melanoma group might be due to small number of positive cases, as the overall mutation rates were similar between our series and the study of Vinagre et al.18 Further studies to explore the potential roles of TERT promoter mutation and mutation spectrum in acral lentiginous melanoma are necessary. In conclusion, our results showed that TERT promoter mutation is uncommon in acral lentiginous melanoma. The mechanisms of telomere maintenance in acral lentiginous melanoma need further studies to achieve a better understanding.
References 1. G¨unes C, Rudolph KL. The role of telomeres in stem cells and cancer. Cell 2013; 152: 390. 2. H¨arle-Bachor C, Boukamp P. Telomerase activity in the regenerative basal layer of the epidermis inhuman skin and in immortal and carcinoma-derived skin keratinocytes. Proc Natl Acad Sci USA 1996; 93: 6476. 3. Norrback KF, Roos G. Telomeres and telomerase in normal and malignant haematopoietic cells. Eur J Cancer 1997; 33: 774. 4. Shay JW, Wright WE. Senescence and immortalization: role of telomeres and telomerase. Carcinogenesis 2005; 26: 867. 5. Parris CN, Jezzard S, Silver A, MacKie R, McGregor JM, Newbold RF. Telomerase activity in melanoma and non-melanoma skin cancer. Br J Cancer 1999; 79: 47. 6. Horn S, Figl A, Rachakonda PS, et al. TERT promoter mutations in familial and sporadic melanoma. Science 2013; 339: 959. 7. Huang FW, Hodis E, Xu MJ, Kryukov GV, Chin L, Garraway LA. Highly recurrent TERT promoter mutations in human melanoma. Science 2013; 339: 957. 8. Curtin JA, Fridlyand J, Kageshita T, et al. Distinct sets of genetic alterations in melanoma. N Engl J Med 2005; 353: 2135. 9. Scott GA, Laughlin TS, Rothberg PG. Mutations of the TERT promoter are common in basal cell carcinoma and squamous cell carcinoma. Mod Pathol 2014; 27: 516. 10. Griewank KG, Schilling B, Murali R, et al. TERT promoter mutations are frequent in atypical fibroxanthomas and pleomorphic dermal sarcomas. Mod Pathol 2014; 27: 502. 11. Killela PJ, Reitman ZJ, Jiao Y, et al. TERT promoter mutations occur frequently in gliomas and a subset of tumors derived from cells with low rates of self-renewal. Proc Natl Acad Sci USA 2013; 110: 6021. 12. Nonoguchi N, Ohta T, Oh JE, Kim YH, Kleihues P, Ohgaki H. TERT promoter mutations in primary and secondary glioblastomas. Acta Neuropathol 2013; 126: 931.
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13. Remke M, Ramaswamy V, Peacock J, et al. TERT promoter mutations are highly recurrent in SHH subgroup medulloblastoma. Acta Neuropathol 2013; 126: 917. 14. Landa I, Ganly I, Chan TA, et al. Frequent somatic TERT promoter mutations in thyroid cancer: higher prevalence in advanced forms of the disease. J Clin Endocrinol Metab 2013; 98: E1562. 15. Liu X, Bishop J, Shan Y, et al. Highly prevalent TERT promoter mutations in aggressive thyroid cancers. Endocr Relat Cancer 2013; 20: 603. 16. Rachakonda PS, Hosen I, de Verdier PJ, et al. TERT promoter mutations in bladder cancer affect patient survival and disease recurrence through modification by a common polymorphism. Proc Natl Acad Sci USA 2013; 110: 17426. 17. Kinde I, Munari E, Faraj SF, et al. TERT Promoter Mutations Occur Early in Urothelial Neoplasia and are Biomarkers of Early Disease and Disease Recurrence in Urine. Cancer Res 2013; 73: 7162. 18. Vinagre J, Almeida A, P´opulo H, et al. Frequency of TERT promoter mutations in human cancers. Nat Commun 2013; 4: 2185. 19. Griewank KG, Murali R, Schilling B, et al. TERT promoter mutations in ocular melanoma distinguish between conjunctival and uveal tumours. Br J Cancer 2013; 109: 497. 20. Griewank KG, Westekemper H, Murali R, et al. Conjunctival melanomas harbor BRAF and NRAS mutations and copy number changes similar to cutaneous and mucosal melanomas. Clin Cancer Res 2013; 19: 3143. 21. Zembowicz A, Mandal RV, Choopong P. Melanocytic lesions of the conjunctiva. Arch Pathol Lab Med 2010; 134: 1785. 22. Harooni H, Schoenfield LR, Singh AD. Current appraisal of conjunctival melanocytic tumors: classification and treatment. Future Oncol 2011; 7: 435.
23. Shields CL, Markowitz JS, Belinsky I, et al. Conjunctival melanoma: outcomes based on tumor origin in 382 consecutive cases. Ophthalmology 2011; 118: 389. 24. Van Raamsdonk CD, Bezrookove V, Green G, et al. Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi. Nature 2009; 457: 599. 25. Van Raamsdonk CD, Griewank KG, Crosby MB, et al. Mutations in GNA11 in uveal melanoma. N Engl J Med 2010; 363: 2191. 26. Cohen Y, Goldenberg-Cohen N, Parrella P, et al. Lack of BRAF mutation in primary uveal melanoma. Invest Ophthalmol Vis Sci 2003; 44: 2876. 27. Cruz F 3rd, Rubin BP, Wilson D, et al. Absence of BRAF and NRAS mutations in uveal melanoma. Cancer Res 2003; 63: 5761. 28. Weber A, Hengge UR, Urbanik D, et al. Absence of mutations of the BRAF gene and constitutive activation of extracellularregulated kinase in malignant melanomas of the uvea. Lab Invest 2003; 83: 1771. 29. Zuidervaart W, van Nieuwpoort F, Stark M, et al. Activation of the MAPK pathway is a common event in uveal melanomas although it rarely occurs through mutation of BRAF or RAS. Br J Cancer 2005; 92: 2032. 30. Harbour JW, Roberson ED, Anbunathan H, Onken MD, Worley LA, Bowcock AM. Recurrent mutations at codon 625 of the splicing factor SF3B1 in uveal melanoma. Nat Genet 2013; 45: 133. 31. Schilling B, Bielefeld N, Sucker A, et al. Lack of SF3B1 R625 mutations in cutaneous melanoma. Diagn Pathol 2013; 8: 87. 32. Puig-Butill´e JA, Badenas C, Ogbah Z, et al. Genetic alterations in RAS-regulated pathway in acral lentiginous melanoma. Exp Dermatol 2013; 22: 148. 33. Bastian BC, Kashani-Sabet M, Hamm H, et al. Gene amplifications characterize acral melanoma and permit the detection of occult tumor cells in the surrounding skin. Cancer Res 2000; 60: 1968.
J Cutan Pathol 2014: 41: 504–508 doi: 10.1111/cup.12323 John Wiley & Sons. Printed in Singapore
Journal of Cutaneous Pathology
TERT promoter mutation is uncommon in acral lentiginous melanoma Background Melanoma is a heterogeneous group of diseases with distinct sets of genetic changes. Recurrent and mutually exclusive C>T or CC>TT transition mutations were identified in the promoter region of the reverse transcriptase catalytic subunit of the telomerase gene (TERT ) in melanoma recently, and it was suggested that they enhanced the expression of TERT gene and played important roles in the melanoma pathogenesis. These mono or di-nucleotide transitions were ultraviolet (UV)-signature mutations. Methods In this study, polymerase chain reaction and direct sequencing of TERT promoter using formalin-fixed and paraffin-embedded tissue was performed to investigate whether these UV-signature mutations were also present in acral lentiginous melanoma. Results TERT promoter mutation was identified in only 2 of the 32 cases (6%) of acral lentiginous melanomas while it was identified in 3 of the 9 cases (33%) of non-acral cutaneous melanomas. The difference was statistically significant (p = 0.028). Conclusions The rarity of TERT promoter mutation in the acral lentiginous melanoma was consistent with the supposed role of UV light in the melanoma pathogenesis and also corroborated the view that acral lentiginous melanomas have a different pathogenesis with the melanomas from sun-exposed sites. Keywords: acral lentiginous melanoma, TERT, UV light Liau J-Y, Tsai J-H, Jeng Y-M, Chu C-Y, Kuo K-T, Liang C-W. TERT promoter mutation is uncommon in acral lentiginous melanoma. J Cutan Pathol 2014; 41: 504–508. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Telomeres are repetitive DNA sequences at the ends of chromosomes that preserve genome integrity and stability.1 Telomeres shorten after repeated cell division. Telomere shortening results in genome instability because it induces chromosome end resection, fusion and breakage.1 Maintenance of telomeres is essential for malignant tumor formation. Two mechanisms of telomere maintenance have been identified in tumor cells: the activation of telomerase and alternative mechanisms of telomere
504
Jau-Yu Liau1,2 , Jia-Huei Tsai1,2 , Yung-Ming Jeng1,2 , Chia-Yu Chu3 , Kuan-Ting Kuo1 and Cher-Wei Liang1,2 1 Department
of Pathology, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan, 2 Graduate Institute of Pathology, College of Medicine, National Taiwan University, Taipei, Taiwan, and 3 Department of Dermatology, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan
Cher-Wei Liang, Department of Pathology, National Taiwan University Hospital, 7 Chung-Shan South Road, Taipei, Taiwan Tel: 886-2-23123456 ext. 65453 Fax: (886)-2-23934172 e-mail: [email protected] Accepted for publication February 28, 2014
lengthening (ALT).1 Telomerase is a ribonucleoprotein complex that adds repetitive DNA sequences to the telomeres. In normal adult tissue, telomerase activity is only present in limited cell types, including germ cells, bone marrow stem cells and basal cells of certain epithelia.2,3 The activation of telomerase in human tumors plays a key role in cellular ‘immortalization’.4 Telomerase activity was detected in 69% of cutaneous melanoma,5 but the mechanism of telomerase activity was unclear until recently.6,7
TERT promoter mutation and UV light Mutation in the promoter region of the reverse transcriptase catalytic subunit (TERT ) of telomerase was recently found in a large proportion of cutaneous melanoma.6,7 These mutations generate novel binding sites for E-twenty-six (ETS) transcription factors, and the mutations increase transcriptional activity from the TERT promoter two to 4-fold. The C>T or CC>TT transitions are consistent with ultraviolet (UV)-signature mutations, indicating that UV exposure is likely to be the major mechanism for the generation of these mutations. Melanomas from sites with different levels of sunexposure have been shown to harbor different sets of genetic alterations.8 Acral lentiginous melanomas usually do not have activating BRAF or NRAS mutations, but the C-KIT mutation rate is higher, in contrast to non-acral cutaneous melanomas. Acral lentiginous melanomas are also characterized by a high frequency of gene amplifications, such as C-KIT , CCND1, and CDK4. As acral sites are non or rarely sun-exposed, the existence and frequency of the TERT promoter mutations should be separately investigated. In this study, we performed TERT mutational analysis using formalin-fixed and paraffin-embedded (FFPE) tissue to investigate whether the TERT promoter mutation rate is different between acral lentiginous and non-acral melanomas. Materials and methods Cases of acral lentiginous and non-acral cutaneous melanomas with available archival material were retrieved from the Department of Pathology, National Taiwan University Hospital. The diagnosis was confirmed by histological review. Only primary tumors with a tumor thickness equal or greater than 2 mm or grossly metastatic tumors were analyzed to enrich tumor cells and reduce the influence of contaminated normal cells. After case selection, two to three 10-μm thick paraffin sections were cut from the block. The tumorous area from paraffin sections was dissected using sterilized razors under a microscope. Genomic DNA was extracted using a QIAamp DNA FFPE Tissue Kit (Qiagen, Santa Clarita, CA, USA) according to the manufacturer’s protocol. The samples were subjected to polymerase chain reaction (PCR) using a primer pair of the TERT promoter (forward: 5 -GGGGCCAGGGCTTCCCACGTG3 ; reverse: 5 -ACCCGTCCTGCCCCTTCACC3 ). The PCR was performed in an automatic DNA thermal cycler (PerkinElmer, Wellesley, MA, USA) with initial heating at 95 ◦ C for 2 min followed by 30 cycles at 95 ◦ C for 30 s, 68 ◦ C for 30 s, 72 ◦ C for 30 s, and, finally, 72 ◦ C for 10 min. After purification,
direct sequencing was performed in an automated ABI 3730 sequencer (Applied BioSystems, Foster City, CA, USA). This study was approved by the Institutional Research Ethnic Committee, and the specimens were anonymous and analyzed in a blind manner. For statistical analysis, comparison of TERT promoter mutation rate was performed using the Pearson Chi-square method. The result was considered significant if the p value was < .05. Results In total, 52 samples from 41 patients were analyzed. Thirty-two and nine patients had acral lentiginous and non-acral melanomas, respectively. Among the non-acral melanomas, five were superficial spreading and four were nodular melanomas. The acral lentiginous melanoma group encompassed 23 primary tumors and 17 metastases (11 lymph nodes and 6 systemic metastases). Eight patients had paired primary and metastatic tumors available for comparison. The non-acral melanoma group encompassed five primary tumors and seven metastases (five lymph nodes and two systemic metastases). Paired primary and metastatic tumors were available for three patients. TERT promoter mutation was detected in only 2 of the 32 acral lentiginous melanoma cases (6%, both C228T transitions), while it was found in 3 of the 9 non-acral cutaneous melanomas (33%, also C228T transitions). The mutations were confirmed by reverse sequencing. The difference between the mutation rates of acral and non-acral melanomas was statistically significant (p = .028). Histologically, both TERT promoter-mutated tumors exhibited prominent lentiginous junctional component and were not distinct from other wild type tumors. All tumor pairs showed concordant TERT promoter mutation status. In the acral lentiginous melanoma group, all eight tumor pairs were wild type. In the non-acral melanoma group, one tumor pair showed concordant C228T mutation, the other two were wild type. The demographic, histological, and molecular findings were summarized in Table 1. Representative hematoxylin and eosin stained sections and sequencing chromatograms were shown in Fig. 1. Discussion Melanomas are the most common lethal primary skin cancers. Although promising clinical responses have been shown for several small molecule inhibitors targeting the active oncoproteins, primary or acquired resistance is common and the prognosis of patients with metastatic disease is still poor. Histologically, cutaneous melanomas are classified
505
Liau et al. Table 1. Clinical, histological, and molecular findings
Sex, N (%) Male Female Mean age (range) Primary tumor, N (%) Upper extremity Lower extremity Trunk Tumor sample, N* Primary Metastasis Tumor stage at diagnosis (AJCC 7th) I II III IV Unavailable Mean Breslow thickness, mm (range) Ulceration Present Absent Unavailable Tumor subtype, N
TERT promoter mutation, N (%)
Acral lentiginous (N = 32)
Non-acral (N = 9)
17 (53%) 15 (47%) 66 (39–89)
4 (44%) 5 (56%) 45 (28–71)
5 (hand) (16%) 27 (foot) (84%) 0 (0%)
3 (33%) 2 (22%) 4 (44%)
23 17
5 7
2 14 15 1 0 6.2 (0.2–19)
0 3 2 1 3 7.3 (3.0–16)
17 12 3 Acral lentiginous, N = 32
3 3 3 Superficial spreading, N = 5 Nodular, N=4 3 (33%)†
2 (6.3%)
*Paired primary and metastatic tumors were available for 8 and 3 acral lentiginous and non-acral melanoma patients, respectively. † TERT promoter mutation was found in 2 and 1 superficial spreading and nodular melanomas, respectively.
as superficial spreading, nodular, acral lentiginous, and lentigo maligna melanomas based on the growth pattern of the junctional melanocytes, location and degree of solar damage. However, melanoma is a heterogeneous group of tumors with different genetic alterations. The traditional histology-based classification system has little or no prognostic or therapeutic relevancies and a molecular genetics-based classification scheme was proposed.8 In their seminal paper, Curtin et al.8 showed that melanomas from sites with different levels of sun exposure have distinct sets of genetic alterations. More than 80% of melanomas from non-acral skin without chronic sun-induced damage had mutations in either BRAF or NRAS genes. Acral melanomas, in contrast, had higher C-KIT mutation rate, a higher
506
degree of chromosomal aberrations and more gene amplifications. One of the most important findings in the melanoma research recently was the identification of recurrent mutations in the core promoter region of the TERT gene, which encodes the rate-limiting catalytic reverse transcriptase subunit of the telomerase ribonucleoprotein complex.6,7 The C>T transition at −124 bp (chr5, 1,295,228C>T, C228T) or −146 bp (1,295,250C>T, C250T) or less common CC>TT tandem mutations at −124 bp or −138 bp upstream the transcription start site generate identical 11-bp nucleotide stretch (5 CCCCTTCCGGG-3 ), which contains the general binding motif (GGAA/T, reverse complement) of ETS family transcription factors. This nucleotide stretch also contains the binding motif (CCGGAA, reverse complement) of the ETS subfamily TCF (ternary complex factor) transcription factors Elk1 and Elk4. These mono or di-nucleotide transitions are mutually exclusive and are consistent with the patterns of UV-induced mutations. In these two studies, more than 70% of cutaneous melanoma samples harbored one of these mutations, and TERT promoter mutation is thus, by far the most common genetic alterations in the melanoma. The ETS transcription factors are downstream targets of RAS-RAF-MAPK pathways and TERT promoter mutations are suggested to have synergistic effects with activating BRAF or NRAS mutations to promoter tumor cell proliferation.6 The high frequency of TERT promoter mutation and the higher than expected rate of concomitant mutations in the TERT promoter and BRAF suggested that TERT promoter mutations were driver mutations in the pathogenesis of melanoma.6 TERT promoter mutation was not identified in benign nevi. Consistent with the idea of UV-induced mutations, they were also recently found in a large proportion of cutaneous squamous cell carcinoma, basal cell carcinoma, atypical fibroxanthoma, and primary dermal pleomorphic sarcoma.9,10 However, the same mutations were also identified in cancers of internal organs, most notably primary glioblastoma, medulloblastoma, urothelial carcinoma, hepatocellular carcinoma, anaplastic carcinoma of the thyroid gland, squamous cell carcinoma of the tongue, and myxoid liposarcoma.7,11 – 18 In these tumors, TERT promoter mutations cannot be attributed to UV light and the causes of TERT promoter mutations need further studies. Recently, using FFPE tissue, TERT promoter mutations were detected in about one third of conjunctival melanoma but none of the uveal melanoma had TERT promoter mutation.19 The detected TERT promoter mutation rate in conjunctival melanomas was comparable to
TERT promoter mutation and UV light
Fig. 1. A) An acral lentiginous melanoma with prominent lentiginous junctional component. B) TERT promoter mutation (C228T) was detected in this tumor using bidirectional direct sequencing (upper panel: forward sequence, lower panel: reverse sequence with complement). C) A nodular type non-acral melanoma with (D) C228T mutation (upper panel: forward sequence, lower panel: reverse sequence with complement). Original magnification: (A) and (C): ×100.
the cutaneous melanoma in other studies.6,18 Conjunctival melanoma, like cutaneous melanoma, frequently harbor BRAF or NRAS mutations.20 They also behave more akin to cutaneous melanoma clinically.21 – 23 Uveal melanoma, in contrast, lack BRAF or NRAS mutations but frequently have GNAQ or GNA11 mutations.24 – 29 A subset of uveal melanoma with indolent behavior harbored SF3B1 (splicing factor 3B subunit 1) mutation, which was not found in cutaneous melanoma.30,31 These findings strongly suggested that the molecular pathogenesis of uveal melanoma is distinct from conjunctival or cutaneous melanomas. Similarly, TERT promoter mutations were detected in only 2 of the 32 cases of acral lentiginous melanomas in our series, despite that we had limited the analysis to relatively advanced tumors to enrich the tumor cells. However, TERT promoter mutation was detected in 33% (3/9) of the control group of non-acral melanomas, which is similar to the result of the previous study.6 In the study of Vinagre et al.,18 the overall TERT promoter mutation rate for cutaneous melanoma was 29% (16/56). However, the frequency of TERT promoter mutation appeared to be very different between histological types. TERT promoter mutation was
found in only 7% (1/14) of acral lentiginous melanomas, which was similar to our result, while they were common in superficial spreading and nodular melanomas (8/22 = 36% and 6/14 = 43%, respectively). Interestingly, the TERT promoter mutated acral lentiginous melanoma in that series also harbored BRAF V600E and NRAS mutations concomitantly, which was a very uncommon event, especially for acral lentiginous melanoma. No BRAF V600E or NRAS (exons 1 and 2) mutation was detected in the two TERT promoter mutated acral tumors in our series but BRAF V600E mutation was found in two of the three non-acral tumors (data not shown). In combination with our result, TERT promoter mutation was found in only 6–7% of acral lentiginous melanomas, a frequency that was one sixth of that of non-acral tumors. However, increased TERT copy number was found in about 30% of acral lentiginous melanomas in one study.32 Bastian et al.33 also showed that acral lentiginous melanoma including the in situ component was characterized by gene amplifications. One of the recurrently amplified loci was 5p15 (in 3 of 15 tumors, 20%), which contained the TERT gene. None of the superficial spreading melanoma analyzed harbored
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Liau et al. amplification in this locus. These findings suggested that increased TERT gene dosage played a more important role than TERT promoter mutation for acral lentiginous melanoma. For cutaneous melanomas, the rarity of TERT promoter mutation in acral lentiginous melanoma is consistent with the supposed role of UV light in the pathogenesis, as acral sites are rarely sun-exposed. These findings corroborated the results of the previous array-based chromosomal genomic hybridization study that acral melanoma has a different pathogenesis compared with non-acral cutaneous melanoma, and are also in line with the finding that acral and mucosal melanomas have a higher degree of chromosomal aberrations and more frequent gene amplifications.8 In our study, the two acral lentiginous melanomas with TERT promoter mutation were primary
tumors, while all metastatic tumor samples analyzed were wild type. All eight tumor pairs showed concordant wild type result. The results suggested that TERT promomter mutation was present in the primary tumors and was not prerequisite for metastasis. Lack of C250T mutation, even in the nonacral melanoma group might be due to small number of positive cases, as the overall mutation rates were similar between our series and the study of Vinagre et al.18 Further studies to explore the potential roles of TERT promoter mutation and mutation spectrum in acral lentiginous melanoma are necessary. In conclusion, our results showed that TERT promoter mutation is uncommon in acral lentiginous melanoma. The mechanisms of telomere maintenance in acral lentiginous melanoma need further studies to achieve a better understanding.
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