Original article 185
TERT promoter mutations in sinonasal malignant melanoma: a study of 49 cases Mattias Jangarda,b,*, Abdlsattar Zebaryb,*, Boel Ragnarsson-Oldingb and Johan Hanssonb Sinonasal malignant melanoma (SNMM) comprises less than 1% of all melanomas and is located in the nasal cavity and the paranasal sinuses. The majority of SNMMs have unknown underlying oncogenic driver mutations. The recent identification of a high frequency of driver mutations in the promoter of the telomerase reverse transcriptase (TERT) gene in cutaneous melanoma led us to investigate whether these mutations also occur in SNMM. Our aim was to determine the TERT promoter mutation frequencies in primary SNMMs. Laser capture microdissection and manual dissection were used to isolate tumour cells from 49 formalin-fixed paraffin-embedded tissues. The tumours were screened for TERT promoter mutations by direct Sanger sequencing. Information on NRAS, BRAF and KIT mutation was available from an earlier study. Overall, 8% (4/49) of SNMMs harboured TERT promoter mutations. One of these mutated tumours had a coexistent NRAS mutation and one had a BRAF mutation. Our findings show that TERT
Introduction Sinonasal malignant melanoma (SNMM) is a rare disease that comprises less than 1% of all melanomas [1] and 1–9% of all malignant lesions of the nasal tract [2,3]. In the head and neck region, mucosal melanomas are most frequently found in the nasal cavity, followed by the paranasal sinuses and the oral cavity [4]. The incidence of SNMM has increased from 1960 through 2000 in Sweden for both sexes [5]. An increasing incidence of SNMM as well as a decreasing incidence of other sinonasal malignancies during the same time period in the USA have been reported by Turner and Reh [6]. The reason for this increase is still unknown [5]. SNMM patients have a poor 5-year survival rate of 20–28% [5,7]. We know that most of the primary SNMMs are localized at diagnosis, but radical surgical resection remains difficult [8]. Postoperative radiotherapy is used frequently, but it is still controversial whether this improves the local control rate [7,9]. Effective, alternative treatment options are not yet available for patients with these tumours, such as molecular targeted therapy, as the main driver mutations are still not known. Ultraviolet light is known to be the major carcinogen in the pathogenesis of cutaneous melanoma [10], but in contrast, the aetiology and predisposing factors of SNMM All supplementary data are available directly from the corresponding author. 0960-8931 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
promoter mutations are present in a moderate proportion of SNMM. No conclusion can be drawn on their potential influence on the clinical outcome or tumour progression. Melanoma Res 25:185–188 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. Melanoma Research 2015, 25:185–188 Keywords: mutation, sinonasal melanoma, TERT promoter a Department of Oto-Rhino-Laryngology, Head and Neck Surgery, Karolinska University Hospital and bDepartment of Oncology-Pathology, Karolinska Institute, Stockholm, Sweden
Correspondence to Mattias Jangard, MD, Department of Oncology-Pathology, Cancer Centrum Karolinska, Karolinska Institute, Stockholm 171 76, Sweden Tel: + 46 8 517 70000; fax: + 46 8 51774245; e-mail: [email protected] *Mattias Jangard and Abdlsattar Zebary contributed equally to the writing of this article. Received 15 September 2014 Accepted 5 February 2015
are still unknown. In a recent study from our group, we found that SNMM has a low frequency of BRAF, NRAS and KIT mutations [11], known driver mutations for cutaneous, vulvar and acral melanomas [12–18]. This indicates that SNMMs harbour other unknown underlying oncogenic driver mutations. The telomerase reverse transcriptase (TERT) gene is located on the short arm of the human chromosome 5. The core promoter spans 330 bp upstream of the translational start site [19]. Telomerase activity is important for the evasion of senescence and gain of limitless growth potential by cancer cells. The activation of telomerase, for example, through a TERT promoter mutation, is considered a driver mutation and an early event in carcinogenesis [20,21]. Other potentially carcinogenic biological effects of telomerase protein include enhanced cell proliferation, inhibition of apoptosis and regulation of DNA damage response and cellular proliferative life span, which could all be affected by TERT promoter mutations [22]. The role of telomerase in tumourigenesis is well established; however, the details of its dysregulation in cancer cells are not completely understood, particularly for melanoma. The TERT promoter mutations have been reported to create E-Twenty six (ETS)/ternary complex factors transcription-binding sites and this results in tumour-specific increased TERT expression. ETS transcription factors may also become DOI: 10.1097/CMR.0000000000000148
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited.
186 Melanoma Research
2015, Vol 25 No 3
activated through dysregulation of mitogen-activated protein kinase (MAP-kinase) signalling, which is a known pathway in the pathogenesis of melanoma [23,24]. The increased expression of telomerase, when for example, TERT promoter mutations are present, may be useful as a target for therapy. There are several strategies of therapeutic telomerase inhibition in different cancers that are the subject of clinical trials, including small molecular inhibitors (enzyme inhibition), antisense oligonucleotides (targeting RNA template of telomerase), immunotherapy (using TERT peptides to elicit immune responses), gene therapy (TERT promoter-driven tumour cell lysis), and telomere-associated and telomeraseassociated proteins (disrupts the telomerase assembly resulting in nonfunctional telomerase), G-quadruplex stabilizers (blocking telomerases access to telomeres) and T-oligo approach (blocking of telomerase inducing DNA damage) [25]. Screening for promoter mutations represents a new way of finding significant alterations when mutations are missing in the coding sequences [23,24,26]. Huang et al. [24] recently reported a high prevalence of TERT promoter mutations in cutaneous malignant melanoma. Horn et al. [23] also reported a germline TERT promoter mutation in a kindred with familial cutaneous malignant melanoma. A recently published study by Egberts et al. [27] found a low frequency of TERT promoter mutations in mucosal melanoma, but the frequency in SNMM was not specifically reported. No former study has investigated the frequency of TERT promoter mutations in primary SNMM; therefore, the aim of the current study was to evaluate a large cohort for the presence of TERT promoter mutations and coexisting NRAS and BRAF mutations.
Materials and methods Tumour samples
We collected archival materials of formalin-fixed paraffinembedded tumour samples of 54 SNMMs from pathology departments throughout Sweden. All patients had been reported to the Swedish National Cancer Registry and diagnosed from 1986 to 2011. The clinical records and pathology reports for all patients were collected and reviewed. We obtained clinical information on diagnosis, classification, disease site, overall survival and other clinical features. All patients had true mucosal melanomas as no other cutaneous melanomas coexisted and the tumours were located in the mucosal areas of the nasal cavity. When data could not be appropriately determined, they were coded as missing. Five samples were excluded as the sections contained too few tumour cells for analysis. Altogether, 49 primary SNMMs were included in the study. These samples were analysed for KIT, NRAS and BRAF mutations in a recent study of our group [11]. The cutoff date for follow-up was 31 January 2013. The study
was approved by the Regional Research Ethics Committee, Karolinska Institutet, Stockholm, Sweden.
Laser capture microdissection and DNA extraction
We obtained 5 µm sections from the formalin-fixed paraffin-embedded blocks that were placed on plain slides. The sections were deparaffinized with two washes of xylene, rehydrated in decreasing concentrations of ethanol, rinsed with deionized water, briefly stained with haematoxylin, rinsed with deionized water and dehydrated in increasing concentrations of ethanol and two washes of xylene. Tumour cells were microdissected from sections by laser capture microdissection using the Arcturus PixCell LCM System (Arcturus Engineering, Mountain View, California, USA) according to the manufacturer’s recommendations. For 20 cases, the tumour cells were macrodissected and the DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Valencia, California, USA). Samples were incubated overnight with proteinase K-enriched digestion buffer (PicoPure DNA Extraction KIT, Arcturus Engineering) to extract the DNA from the dissected cells. Proteinase K was then inactivated by heating samples at 95°C for 10 min.
Mutation analysis
The TERT promoter region from base pair position + 65 to − 278 was screened for mutations using PCR and direct Sanger sequencing. Genomic DNA was subjected to initial and subsequently nested PCR to amplify TERT promoter regions. In the first PCR, the DNA was amplified in a 10 µl mixture reaction containing 2.5 mmol/l deoxynucleotide triphosphate, 5 U/µl platinum Taq DNA polymerase (Invitrogen, Carlsbad, California, USA), 50 pmol/µl of each primer, 10 × PCR buffer, 50 mmol/l MgCl2, and 10 µg/µl BSA and 6.67 µl betaine 3 mmol/l. Two microlitres of the first PCR reaction was used as a DNA template for the nested PCR. The DNA was extracted and purified from agarose gels using the QIAquick Gel Extraction Kit (Qiagen). Sequencing reactions were performed in a final volume of 20 µl using the BigDye Terminator V3.1 Cycle Sequencing kit (Applied Biosystems, Life Technologies Ltd, Paisley, UK). The sequencing products were purified by ethanol precipitation and automated DNA sequencing was performed using the ABI PRISM3130xl Genetic Analyzer (Applied Biosystems). All mutations were confirmed by a second independent PCR and sequencing reaction. The primers used for the amplification and sequencing of TERT promoter were as described by Rachakonda et al. [28] (Supplementary Table 1).
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited.
TERT-promotor in sinonasal melanoma Jangard et al. 187
Table 1
Patient and tumour characteristics Total (n = 49)
Characteristics Age at diagnosis (years) Median Mean Range Sex [n (%)] Male Female Anatomical site [n (%)] Nasal cavity Paranasal sinus Stage [n (%)] III IVa IVb Data missing Survivala [n (%)] Dead Alive Median (months) Range (months)
76 74 52–97 18 (36.7) 31 (63.3) 30 (61.2) 19 (38.8) 37 10 1 1
(76) (20) (2) (2)
6 (12.2) 43 (87.8) 33 2–164
a
Last updated on 1 January 2014.
Results Clinicopathological characteristics
Tumours were obtained from 31 women and 18 men (Table 1). The median age at diagnosis was 76 years. Overall, 30 of the tumours were located in the nasal cavity and 19 in the paranasal sinuses (nine in the maxillary sinuses, six in the ethmoid sinuses and four tumours invaded the surrounding structures: three involved the orbit; and one invaded the retromaxillary-infratemporal fossa). The majority of the patients were diagnosed in stage III according to UICC 7th edition. Only one patient had lymph node metastases in the neck region and none had distant metastases at diagnosis. Mutation analysis
Of the 49 primary SNMMs analysed, four (8%) harboured TERT promoter gene mutations and five (10%) were wild type. All the others, 40 (82%), had the − 245G > A alteration, a known single nucleotide polymorphism (SNP), rs2853669 [23,28]. Among tumours with mutations, two had the same mutations 1,295,228 and 1,295,229 GG > AA (228–229GG > AA) and one had the 1,295,250 G > A (250G > A) mutation and one had the 1295161 T > G (161T > G) mutation. All these mutations have been reported to create binding motifs for ETS/ternary complex factors [23,24,28]. Of the two Table 2
samples with mutations 228–229GG > AA, one had a coexisting BRAF mutation (V600K) and the other had a coexisting NRAS mutation (Q61H). Mutations in relation to clinicopathological features
Because of the small number of patients with mutated tumours, a statistical analysis could not be carried out between these and the remaining patients. There were three women and one man among the patients with TERT promoter gene mutations. Two tumours were localized in the nasal cavity and two in the maxillary sinuses. The youngest patient was diagnosed at the age of 52 years and the oldest was diagnosed at the age of 92 years. The survival varied from 19 to 121 months (Table 2).
Discussion In SNMM, there are only a few published reports on mutational data, with limited numbers of tumours [11]. In this study, we screened a large number of primary SNMM for TERT promoter gene mutations, which are common in cutaneous melanoma [23,24], but previously not analysed specifically in SNMM. To our knowledge, this is the largest study of its kind in SNMM. We identified mutations in 8% of the tumours, but because of the low number of cases, we could not carry out an analysis of clinicopathological features in relation to the mutations. Three of the tumour-associated mutations 228–229GG > AA and 250G > A are known to generate binding motifs for ETS transcription factors, resulting in increasing transcriptional activity from the TERT promoter by two- to four-fold [24]. The 161T > G mutation is also known to increase transcriptional activity in the same way, but only by 1.5-fold [23]. ETS transcription factors are also known to be downstream targets of RAS-RAF and MAP-kinase pathways and the TERT promoter mutations have been suggested to exert synergetic effects on NRAS and BRAF mutations in tumour cells. Interestingly, we found two coexisting mutations (NRAS and BRAF) in tumours with the TERT promoter mutation. The majority of the patients had the SNP rs2853669 (− 245A > G). In previous studies, this polymorphism has been reported to disrupt an ETSbinding site and is associated with low telomerase activity in patients with nonsmall cell lung cancer [29].
Summary of TERT promoter mutations and coexisting BRAF and NRAS mutations in primary SNMM (n = 49) Sex
Age
Site
Survival (months)
Nucleotide change
1 2 3
Male Female Female
92 76 82
Nasal cavity Maxillary sinus Nasal cavity
69 121 113
161T > G 250G > A 228–229GG > AA
4
Female
52
Maxillary sinus
19
228–229GG > > AA
Case
SNMM, sinonasal mucosal melanoma; TERT, telomerase reverse transcriptase.
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited.
BRAF and NRAS mutations Amino acid change
NRAS Q61H BRAF V600K
188 Melanoma Research
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Rachaconda et al. [28] have also found a reduction in promoter activity in urothelial carcinoma with the same SNP, and suggest that the rs2853669 polymorphism possibly exerts a protective effect on survival and recurrence.
8 9
10
Conclusion
Our results indicate that TERT promoter mutations can occur in SNMM and should be included in the molecular characterization of such tumours as these alterations may become therapeutic targets in the near future. In the present analysis, we could not draw any conclusions on the potential influence on the clinical outcome nor tumour progression in the patients with SNMM harbouring TERT promoter mutations.
Acknowledgements The authors thank Dr Lena Kanter and Dr Lars Olding for re-examining tumour histopathology. This study was financed by grants from the Swedish Cancer Society, the Radiumhemmet Research Funds, the Karolinska Institutet Research Funds and the ACTA Otolaryngologica Foundation. The Ministry of Higher Education and Scientific Research in Iraqi-Kurdistan Regional Government is acknowledged for financial support (AZ).
11
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13 14
15
16
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19
20
Conflicts of interest
There are no conflicts of interest.
References 1
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Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited.
TERT promoter mutations in sinonasal malignant melanoma: a study of 49 cases Mattias Jangarda,b,*, Abdlsattar Zebaryb,*, Boel Ragnarsson-Oldingb and Johan Hanssonb Sinonasal malignant melanoma (SNMM) comprises less than 1% of all melanomas and is located in the nasal cavity and the paranasal sinuses. The majority of SNMMs have unknown underlying oncogenic driver mutations. The recent identification of a high frequency of driver mutations in the promoter of the telomerase reverse transcriptase (TERT) gene in cutaneous melanoma led us to investigate whether these mutations also occur in SNMM. Our aim was to determine the TERT promoter mutation frequencies in primary SNMMs. Laser capture microdissection and manual dissection were used to isolate tumour cells from 49 formalin-fixed paraffin-embedded tissues. The tumours were screened for TERT promoter mutations by direct Sanger sequencing. Information on NRAS, BRAF and KIT mutation was available from an earlier study. Overall, 8% (4/49) of SNMMs harboured TERT promoter mutations. One of these mutated tumours had a coexistent NRAS mutation and one had a BRAF mutation. Our findings show that TERT
Introduction Sinonasal malignant melanoma (SNMM) is a rare disease that comprises less than 1% of all melanomas [1] and 1–9% of all malignant lesions of the nasal tract [2,3]. In the head and neck region, mucosal melanomas are most frequently found in the nasal cavity, followed by the paranasal sinuses and the oral cavity [4]. The incidence of SNMM has increased from 1960 through 2000 in Sweden for both sexes [5]. An increasing incidence of SNMM as well as a decreasing incidence of other sinonasal malignancies during the same time period in the USA have been reported by Turner and Reh [6]. The reason for this increase is still unknown [5]. SNMM patients have a poor 5-year survival rate of 20–28% [5,7]. We know that most of the primary SNMMs are localized at diagnosis, but radical surgical resection remains difficult [8]. Postoperative radiotherapy is used frequently, but it is still controversial whether this improves the local control rate [7,9]. Effective, alternative treatment options are not yet available for patients with these tumours, such as molecular targeted therapy, as the main driver mutations are still not known. Ultraviolet light is known to be the major carcinogen in the pathogenesis of cutaneous melanoma [10], but in contrast, the aetiology and predisposing factors of SNMM All supplementary data are available directly from the corresponding author. 0960-8931 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
promoter mutations are present in a moderate proportion of SNMM. No conclusion can be drawn on their potential influence on the clinical outcome or tumour progression. Melanoma Res 25:185–188 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. Melanoma Research 2015, 25:185–188 Keywords: mutation, sinonasal melanoma, TERT promoter a Department of Oto-Rhino-Laryngology, Head and Neck Surgery, Karolinska University Hospital and bDepartment of Oncology-Pathology, Karolinska Institute, Stockholm, Sweden
Correspondence to Mattias Jangard, MD, Department of Oncology-Pathology, Cancer Centrum Karolinska, Karolinska Institute, Stockholm 171 76, Sweden Tel: + 46 8 517 70000; fax: + 46 8 51774245; e-mail: [email protected] *Mattias Jangard and Abdlsattar Zebary contributed equally to the writing of this article. Received 15 September 2014 Accepted 5 February 2015
are still unknown. In a recent study from our group, we found that SNMM has a low frequency of BRAF, NRAS and KIT mutations [11], known driver mutations for cutaneous, vulvar and acral melanomas [12–18]. This indicates that SNMMs harbour other unknown underlying oncogenic driver mutations. The telomerase reverse transcriptase (TERT) gene is located on the short arm of the human chromosome 5. The core promoter spans 330 bp upstream of the translational start site [19]. Telomerase activity is important for the evasion of senescence and gain of limitless growth potential by cancer cells. The activation of telomerase, for example, through a TERT promoter mutation, is considered a driver mutation and an early event in carcinogenesis [20,21]. Other potentially carcinogenic biological effects of telomerase protein include enhanced cell proliferation, inhibition of apoptosis and regulation of DNA damage response and cellular proliferative life span, which could all be affected by TERT promoter mutations [22]. The role of telomerase in tumourigenesis is well established; however, the details of its dysregulation in cancer cells are not completely understood, particularly for melanoma. The TERT promoter mutations have been reported to create E-Twenty six (ETS)/ternary complex factors transcription-binding sites and this results in tumour-specific increased TERT expression. ETS transcription factors may also become DOI: 10.1097/CMR.0000000000000148
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited.
186 Melanoma Research
2015, Vol 25 No 3
activated through dysregulation of mitogen-activated protein kinase (MAP-kinase) signalling, which is a known pathway in the pathogenesis of melanoma [23,24]. The increased expression of telomerase, when for example, TERT promoter mutations are present, may be useful as a target for therapy. There are several strategies of therapeutic telomerase inhibition in different cancers that are the subject of clinical trials, including small molecular inhibitors (enzyme inhibition), antisense oligonucleotides (targeting RNA template of telomerase), immunotherapy (using TERT peptides to elicit immune responses), gene therapy (TERT promoter-driven tumour cell lysis), and telomere-associated and telomeraseassociated proteins (disrupts the telomerase assembly resulting in nonfunctional telomerase), G-quadruplex stabilizers (blocking telomerases access to telomeres) and T-oligo approach (blocking of telomerase inducing DNA damage) [25]. Screening for promoter mutations represents a new way of finding significant alterations when mutations are missing in the coding sequences [23,24,26]. Huang et al. [24] recently reported a high prevalence of TERT promoter mutations in cutaneous malignant melanoma. Horn et al. [23] also reported a germline TERT promoter mutation in a kindred with familial cutaneous malignant melanoma. A recently published study by Egberts et al. [27] found a low frequency of TERT promoter mutations in mucosal melanoma, but the frequency in SNMM was not specifically reported. No former study has investigated the frequency of TERT promoter mutations in primary SNMM; therefore, the aim of the current study was to evaluate a large cohort for the presence of TERT promoter mutations and coexisting NRAS and BRAF mutations.
Materials and methods Tumour samples
We collected archival materials of formalin-fixed paraffinembedded tumour samples of 54 SNMMs from pathology departments throughout Sweden. All patients had been reported to the Swedish National Cancer Registry and diagnosed from 1986 to 2011. The clinical records and pathology reports for all patients were collected and reviewed. We obtained clinical information on diagnosis, classification, disease site, overall survival and other clinical features. All patients had true mucosal melanomas as no other cutaneous melanomas coexisted and the tumours were located in the mucosal areas of the nasal cavity. When data could not be appropriately determined, they were coded as missing. Five samples were excluded as the sections contained too few tumour cells for analysis. Altogether, 49 primary SNMMs were included in the study. These samples were analysed for KIT, NRAS and BRAF mutations in a recent study of our group [11]. The cutoff date for follow-up was 31 January 2013. The study
was approved by the Regional Research Ethics Committee, Karolinska Institutet, Stockholm, Sweden.
Laser capture microdissection and DNA extraction
We obtained 5 µm sections from the formalin-fixed paraffin-embedded blocks that were placed on plain slides. The sections were deparaffinized with two washes of xylene, rehydrated in decreasing concentrations of ethanol, rinsed with deionized water, briefly stained with haematoxylin, rinsed with deionized water and dehydrated in increasing concentrations of ethanol and two washes of xylene. Tumour cells were microdissected from sections by laser capture microdissection using the Arcturus PixCell LCM System (Arcturus Engineering, Mountain View, California, USA) according to the manufacturer’s recommendations. For 20 cases, the tumour cells were macrodissected and the DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Valencia, California, USA). Samples were incubated overnight with proteinase K-enriched digestion buffer (PicoPure DNA Extraction KIT, Arcturus Engineering) to extract the DNA from the dissected cells. Proteinase K was then inactivated by heating samples at 95°C for 10 min.
Mutation analysis
The TERT promoter region from base pair position + 65 to − 278 was screened for mutations using PCR and direct Sanger sequencing. Genomic DNA was subjected to initial and subsequently nested PCR to amplify TERT promoter regions. In the first PCR, the DNA was amplified in a 10 µl mixture reaction containing 2.5 mmol/l deoxynucleotide triphosphate, 5 U/µl platinum Taq DNA polymerase (Invitrogen, Carlsbad, California, USA), 50 pmol/µl of each primer, 10 × PCR buffer, 50 mmol/l MgCl2, and 10 µg/µl BSA and 6.67 µl betaine 3 mmol/l. Two microlitres of the first PCR reaction was used as a DNA template for the nested PCR. The DNA was extracted and purified from agarose gels using the QIAquick Gel Extraction Kit (Qiagen). Sequencing reactions were performed in a final volume of 20 µl using the BigDye Terminator V3.1 Cycle Sequencing kit (Applied Biosystems, Life Technologies Ltd, Paisley, UK). The sequencing products were purified by ethanol precipitation and automated DNA sequencing was performed using the ABI PRISM3130xl Genetic Analyzer (Applied Biosystems). All mutations were confirmed by a second independent PCR and sequencing reaction. The primers used for the amplification and sequencing of TERT promoter were as described by Rachakonda et al. [28] (Supplementary Table 1).
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited.
TERT-promotor in sinonasal melanoma Jangard et al. 187
Table 1
Patient and tumour characteristics Total (n = 49)
Characteristics Age at diagnosis (years) Median Mean Range Sex [n (%)] Male Female Anatomical site [n (%)] Nasal cavity Paranasal sinus Stage [n (%)] III IVa IVb Data missing Survivala [n (%)] Dead Alive Median (months) Range (months)
76 74 52–97 18 (36.7) 31 (63.3) 30 (61.2) 19 (38.8) 37 10 1 1
(76) (20) (2) (2)
6 (12.2) 43 (87.8) 33 2–164
a
Last updated on 1 January 2014.
Results Clinicopathological characteristics
Tumours were obtained from 31 women and 18 men (Table 1). The median age at diagnosis was 76 years. Overall, 30 of the tumours were located in the nasal cavity and 19 in the paranasal sinuses (nine in the maxillary sinuses, six in the ethmoid sinuses and four tumours invaded the surrounding structures: three involved the orbit; and one invaded the retromaxillary-infratemporal fossa). The majority of the patients were diagnosed in stage III according to UICC 7th edition. Only one patient had lymph node metastases in the neck region and none had distant metastases at diagnosis. Mutation analysis
Of the 49 primary SNMMs analysed, four (8%) harboured TERT promoter gene mutations and five (10%) were wild type. All the others, 40 (82%), had the − 245G > A alteration, a known single nucleotide polymorphism (SNP), rs2853669 [23,28]. Among tumours with mutations, two had the same mutations 1,295,228 and 1,295,229 GG > AA (228–229GG > AA) and one had the 1,295,250 G > A (250G > A) mutation and one had the 1295161 T > G (161T > G) mutation. All these mutations have been reported to create binding motifs for ETS/ternary complex factors [23,24,28]. Of the two Table 2
samples with mutations 228–229GG > AA, one had a coexisting BRAF mutation (V600K) and the other had a coexisting NRAS mutation (Q61H). Mutations in relation to clinicopathological features
Because of the small number of patients with mutated tumours, a statistical analysis could not be carried out between these and the remaining patients. There were three women and one man among the patients with TERT promoter gene mutations. Two tumours were localized in the nasal cavity and two in the maxillary sinuses. The youngest patient was diagnosed at the age of 52 years and the oldest was diagnosed at the age of 92 years. The survival varied from 19 to 121 months (Table 2).
Discussion In SNMM, there are only a few published reports on mutational data, with limited numbers of tumours [11]. In this study, we screened a large number of primary SNMM for TERT promoter gene mutations, which are common in cutaneous melanoma [23,24], but previously not analysed specifically in SNMM. To our knowledge, this is the largest study of its kind in SNMM. We identified mutations in 8% of the tumours, but because of the low number of cases, we could not carry out an analysis of clinicopathological features in relation to the mutations. Three of the tumour-associated mutations 228–229GG > AA and 250G > A are known to generate binding motifs for ETS transcription factors, resulting in increasing transcriptional activity from the TERT promoter by two- to four-fold [24]. The 161T > G mutation is also known to increase transcriptional activity in the same way, but only by 1.5-fold [23]. ETS transcription factors are also known to be downstream targets of RAS-RAF and MAP-kinase pathways and the TERT promoter mutations have been suggested to exert synergetic effects on NRAS and BRAF mutations in tumour cells. Interestingly, we found two coexisting mutations (NRAS and BRAF) in tumours with the TERT promoter mutation. The majority of the patients had the SNP rs2853669 (− 245A > G). In previous studies, this polymorphism has been reported to disrupt an ETSbinding site and is associated with low telomerase activity in patients with nonsmall cell lung cancer [29].
Summary of TERT promoter mutations and coexisting BRAF and NRAS mutations in primary SNMM (n = 49) Sex
Age
Site
Survival (months)
Nucleotide change
1 2 3
Male Female Female
92 76 82
Nasal cavity Maxillary sinus Nasal cavity
69 121 113
161T > G 250G > A 228–229GG > AA
4
Female
52
Maxillary sinus
19
228–229GG > > AA
Case
SNMM, sinonasal mucosal melanoma; TERT, telomerase reverse transcriptase.
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BRAF and NRAS mutations Amino acid change
NRAS Q61H BRAF V600K
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Rachaconda et al. [28] have also found a reduction in promoter activity in urothelial carcinoma with the same SNP, and suggest that the rs2853669 polymorphism possibly exerts a protective effect on survival and recurrence.
8 9
10
Conclusion
Our results indicate that TERT promoter mutations can occur in SNMM and should be included in the molecular characterization of such tumours as these alterations may become therapeutic targets in the near future. In the present analysis, we could not draw any conclusions on the potential influence on the clinical outcome nor tumour progression in the patients with SNMM harbouring TERT promoter mutations.
Acknowledgements The authors thank Dr Lena Kanter and Dr Lars Olding for re-examining tumour histopathology. This study was financed by grants from the Swedish Cancer Society, the Radiumhemmet Research Funds, the Karolinska Institutet Research Funds and the ACTA Otolaryngologica Foundation. The Ministry of Higher Education and Scientific Research in Iraqi-Kurdistan Regional Government is acknowledged for financial support (AZ).
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Conflicts of interest
There are no conflicts of interest.
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