Open Access

Analysis of the B-RafV600E mutation in cutaneous melanoma patients with occupational sun exposure

  • Authors:
    • Saverio Candido
    • Venerando Rapisarda
    • Andrea Marconi
    • Grazia Malaponte
    • Valentina Bevelacqua
    • Pietro Gangemi
    • Aurora Scalisi
    • James A. McCubrey
    • Roberta Maestro
    • Demetrios A. Spandidos
    • Concettina Fenga
    • Massimo Libra
  • View Affiliations

  • Published online on: January 14, 2014     https://doi.org/10.3892/or.2014.2977
  • Pages: 1079-1082
  • Copyright: © Candido et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY_NC 3.0].

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Sun-exposure is one of the risk factors associated with the development of a cutaneous neoplasm. In melanoma, the Ras-Raf-MEK-ERK (MAPK) signaling pathway is constitutively activated through multiple mechanisms, including B-Raf mutation. It has been hypothesized that B-Raf mutations in melanocytic lesions arise from DNA damage induced by ultraviolet (UV) radiation. However, it is still discussed if B-Raf mutations are associated with melanoma patients exposed to the sun. Therefore, in the present study, the known B-RafV600E mutation was analysed in melanoma samples from 30 indoor and 38 outdoor workers. B-RafV600E mutation was detected in 52 and 73% of outdoor workers and indoor workers, respectively. Of note, this mutation was identified in 12 of 14 (85%) melanoma of the trunk diagnosed in indoor workers and in 9 of 19 (47%) samples from outdoor workers (p=0.03). By analyzing melanomas of other body sites, no statistical difference in the frequency of B-RafV600E mutation was identified between the groups of workers. It appears that the mutation detected among indoor workers may be associated with a recreational or intermittent exposure to the sun, as usually the trunk is a sun-protected body site. Overall, these data indicate that the B-RafV600E mutation detected in melanoma is not associated with a chronic exposure to the sun. Mutations detected in other genes may also contribute to melanoma development in the subset of patients exposed to UV radiation.

Introduction

Cutaneous melanoma (CM) is the most aggressive form of skin cancer. Its incidence has increased dramatically worldwide over the last 50 years (1). In Europe the highest incidence rates have been reported in Scandinavia (ca. 15 cases per 100,000 inhabitants and year) and the lowest in Mediterranean countries (ca. 5–7 cases per 100,000 inhabitants and year). In a worldwide comparison, the highest incidence rates have been reported in Australia (40–60 cases per 100,000 inhabitants and year) (2,3). Epidemiologic studies suggest that melanoma is determined by a complex model of pathways that are activated by many factors including genetic factors, phenotypic characteristics, number of melanocytic nevi, anatomic sites (lower limbs in females, posterior trunk in males), family history of melanoma, and the interplay with environmental factors, in particular for cutaneous melanoma, intense and intermittent exposure to ultraviolet (UV) radiation, represent the main risk factors (4). It is well-established and recognized that a genetic predisposition exists for the development of melanoma and nevi, especially the clinically atypical variants (5). CM results from uncontrolled melanocytic proliferation, melanin-producing cells located in the basal layer of the epidermis where they have a protective role against UV radiation for the skin by distributing melanin pigment from melanosomes to keratinocytes (5). Several studies of outdoor workers have shown an excess risk of melanoma and other skin cancers (610). The UVB component of the solar spectrum is the main source of risk for the development of a cutaneous neoplasm. Moreover, the cumulative lifetime dose of UVB radiation seems to be the most important factor for determining carcinogenic potential (3). Intense sun exposure leads both to DNA damage and to immunosuppression, which together are held to mediate carcinogenesis, while photo-adaptation is thought to reduce DNA damage (11). Solar radiation is a highly prevalent occupational exposure in farm, fishery and construction workers, letter carriers, gardeners, lumbermen, skiing instructors and mountain guides (12). Sun-exposed workers in the EU range from 29% in Germany to 51% in Greece and 39% in Italy (3).

In melanoma, the Ras-Raf-MEK-ERK (MAPK) signaling pathway is constitutively activated through multiple mechanisms. Mutations of B-RAF have been proposed to contribute to melanoma development. V600E accounts for >60% of B-RAF mutations in melanoma and causes a substantial increase in B-Raf kinase activity (13). It has been suggested that B-RAF mutations in melanocytic lesions arise from DNA damage induced by UV radiation (14). However, it is still unclear if the B-RAFV600E mutations detected in melanoma patients results from their exposure to the sun. Therefore, the B-RAFV600E mutation was analysed in melanoma samples from patients with indoor and outdoor occupational activity.

Patients and methods

Patients

The subjects enrolled in this study included 68 consecutive patients diagnosed with cutaneous melanoma between October 1999 and June 2010. Tumor biopsy-specimens were isolated from 52 males and 16 females having at least 10 years of work history before the diagnosis of melanoma. All melanoma samples were collected by the Department of Bio-medical Sciences, University of Catania, Catania, Italy. The local scientific ethics committee approved all the procedures. The patients provided a written informed consent for the study. Medical files of each patient were analyzed for their occupational activity. Accordingly, patients were divided in two groups on the basis of their indoor or outdoor activity. Thirty-eight patient were outdoor workers, while 30 were indoor workers. In the group of outdoor workers there were: 11 farmers, 14 construction workers, 10 road paving workers, 2 beach attendant and 1 fisher man. In the group of indoor work there were: 11 office workers, 12 teachers, 1 Ph.D. student, 4 factory workers and 2 physicians. The duration of sun exposure for all outdoor workers was estimated as >6 h per day. Clinical patient characteristics are reported in Table I. DNA was isolated from each melanoma sample with the QIAgen Tissue kit (Qiagen, Valencia, CA, USA).

Table I

Clinical characteristics of melanoma patients.

Table I

Clinical characteristics of melanoma patients.

Clinical featuresOutdoor (38)
n (%)
Indoor (30)
n (%)
P-value
Gender
 Male34 (89)18 (60)0.004
 Female4 (11)12 (40)
Age
 ≤5521 (55)16 (53)NS
 >5517 (45)14 (47)
Tumor type
 Primary melanoma25 (66)18 (60)NS
 Metastatic melanoma13 (34)12 (40)
Clark’s level
 III13 (34)13 (43)NS
 IV11 (29)17 (57)
 V1 (3)-
Breslow thickness
 ≤2.00 mm13 (34)10 (33)NS
 2.01–5.00 mm14 (37)10 (33)
 ≥5.00 mm11 (29)10 (33)
Tumor site
 Trunk19 (50)14 (47)NS
 Head and Necka12 (32)9 (30)
 Limbs7 (18)7 (23)

a Including nose and scalp. NS, not significant.

B-RAFV600E mutation analysis

All DNA samples were screened in duplicate for B-RAFV600E mutation within exon 15 as previously described (15).

‘Cosmic Catalogue of Mutations in Cancer’ analysis

The website of the ‘Cosmic Catalogue of Mutations in Cancer’ (http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/) was explored imputing the following key words: ‘B-RAFV600E’, ‘melanoma’, ‘skin’, ‘intermittent sun exposure’ and ‘chronic sun exposure’.

Statistical analysis

Potential relationships between B-RAFV600E mutation and other patient characteristics were examined by the Chi-square test or Fisher’s exact test. A p-value <0.05 by a two tailed test was defined to be statistically significant.

Results

Clinical characteristics of melanoma patients are summarized in Table I. No differences in the main pathologic features between outdoor workers and indoor workers were observed except for the gender. The number of male outdoor workers was significantly higher compared to that of indoor workers (89 vs. 60%; p=0.004) (Table I). B-RAFV600E mutation was detected in 42 of 68 (61%) melanoma samples. Specifically, 21 out 42 (50%) mutations were observed in melanoma of the trunk, 29% in melanoma of the head and neck site and 21% of the limbs. B-RAFV600E mutation was detected in 20 of 38 (52%) outdoor workers and in 22 of 30 (73%) indoor workers. However, this difference was not statistically significant (Table II). In Table II, the distribution of B-RAFV600E mutation according to the tumor sites in both groups of outdoor and indoor workers is also shown. Notably, B-RAFV600E mutation was detected in 12 of 14 (85%) melanoma of the trunk diagnosed in indoor workers; while 47% of melanoma samples, occurred in outdoor workers, displayed this mutation in the same site (p=0.03). Similarly, higher number of mutations was observed in melanoma of the head and neck diagnosed among indoor workers when compared with those occurred among outdoor workers (78 vs. 42%). However, this difference was not statistically significant. B-RAFV600E mutation was detected in 43% of melanoma of the limbs among indoor workers; while 86% of mutations were detected in melanoma of the same sites among outdoor workers. No statistical difference was observed (Table II).

Table II

Distribution of B-RAFV600E mutation according to tumor sites in the groups of outdoor and indoor workers.

Table II

Distribution of B-RAFV600E mutation according to tumor sites in the groups of outdoor and indoor workers.

Tumor site B-RAFV600E mutationOutdoor
n (%)
Indoor
n (%)
P-valuea
TrunkYes9 (47)12 (85)0.03
No10 (53)2 (15)
Total1914
Head and neckYes5 (42)7 (78)0.18
No7 (58)2 (22)
Total129
LimbsYes6 (86)3 (43)0.26
No1 (14)4 (57)
Total77
All sitesYes20 (52)22 (73)0.08
No18 (48)8 (27)
Total3830

a Fisher’s exact test, Two-tailed.

To investigate whether B-RAFV600E mutations may occur differentially in melanoma patients chronically exposed to sun compared with those intermittently exposed to sun, further studies were performed by analyzing the ‘Cosmic Catalogue of Mutations in Cancer’. Higher B-RAFV600E mutation rate was detected in melanoma from patients with an intermittent exposure to sun when compared with those chronically exposed to sun (47 vs. 16%; p<0.0001) (Table III).

Table III

Distribution of B-RAFV600E mutation according to chronic exposure to sun and intermittent exposure to sun.a

Table III

Distribution of B-RAFV600E mutation according to chronic exposure to sun and intermittent exposure to sun.a

B-RAFV600E mutationChronic exposure to sun
n (%)
Intermittent exposure to sun
n (%)
P-valueb
Yes9 (16)30 (47)<0.0001
No46 (84)34 (53)
Total5564

a Cosmic Catalogue of Mutations in Cancer analysis;

b Fisher’s exact test, Two-tailed.

Discussion

Risk factors associated with the increased incidence of melanoma are still debated. Genetic behavior and sun exposure represent the main players for cutaneous melanoma development (5,16). However, intermittent exposure to UV exposure has been demonstrated to have an increased risk of skin cancer (17).

In recent years, a growing body of evidence supports a role of MAPK pathway in melanoma cell proliferation and survival (13,18). In this disease, the most frequent genetic alteration, which accounts for >60% of melanomas is the alteration of B-RAF, with a glutamic acid for valine substitution at codon 600 in exon 15 (Val600Glu; B-RAFV600E); this mutation introduces a conformational change in protein structure due to glutamic acid that acts as a phosphomimetic between the Thr598 and Ser601 phosphorylation sites, leading to constitutive activation of the protein with a large increase in the basal kinase activity; the resulting hyperactivity of the MAP kinase pathway promotes tumor development (13,18).

To understand whether the B-rafV600E mutation is associated to chronic or intermittent sun exposure, in the present study we analyzed this mutation in melanoma from patients with and without occupational sun exposure assuming that indoor workers have an intermittent exposure to the sun. B-RAFV600E was mutated in 20 of 38 (52%) outdoor workers and in 22 of 30 (73%) indoor workers. Although, no statically significant difference was recorded, these data are in agreement with previous findings by Curtin et al (20) showing that B-RAFV600E mutation is not associated with a chronic sun exposure. Accordingly, most recent data indicated that occupational sun exposure did not increase risk of melanoma (9,19).

Our study also analysed the B-RAFV600E mutation according to the tumor site between indoor and outdoor workers. This mutation was detected more frequently in melanoma of the trunk from indoor workers compared with outdoor workers. We can argue that the B-RAFV600E mutation detected among indoor workers may be associated with a recreational or intermittent exposure to the sun, as usually the trunk is a less frequently exposed body site. In fact, it was suggested that chronic exposure to the sun may induce photoadaption with increased melanisation and epidermal thickening (11). The higher frequency of B-RAFV600E mutation melanoma of the trunk in indoor workers is in line with previous data since this mutation was detected more frequently in melanoma of the trunk (20,21). Therefore, we can speculate that the melanocytes of melanoma patients, intermittently exposed to the sun, have an increased susceptibility to proliferate and acquire B-RAF mutations. Further analysis were performed by exploring the Cosmic Catalogue of Mutations in Cancer confirming that higher B-RAFV600E mutation rate is observed in melanoma from patients with an intermittent exposure to sun when compared with those chronically exposed to sun (47 vs. 16%; p<0.0001). In the study conducted by Whiteman et al (4) it has been shown that patients with chronic sun exposure may preferentially develop melanoma of the head and neck. Similarly, in our series melanoma of the head and neck was diagnosed in 12 outdoor workers and in 9 indoor workers, however, this difference did not reach significance due to the small number of the samples.

Overall, these data support the notion that B-RAFV600E mutation detected in melanoma from outdoor workers is not associated with a chronic exposure to the sun. In contrast, indoor workers, that may be exposed to intermittent sunbathing, are more susceptible to developing a melanoma harboring B-RAFV600E mutation. These findings may have important therapeutic implications as melanoma patients with B-RAF mutations may benefits from B-RAF inhibition (18). Indication for a direct UV mutagenic effect in melanoma development remains still controversial as the nucleotide exchange detected in the B-RAF gene (T/A) is not classically linked to UV mutagenesis signature attributable to cytidine to thymidine (C>T) transitions. As proposed before, it is possible that B-RAF mutations could arise from error prone replication of UV-damaged DNA (14). However, a potential mechanism of melanoma development after UV-damage is described in Fig. 1. Mutations detected in other genes may also contribute to melanoma development in a subset of patients exposed to UV radiation (22).

Acknowledgements

This study was supported in part by a grant from the Italian League Against Cancer (Catania, Italy).

References

1 

Siegel R, Naishadham D and Jemal A: Cancer statistics, 2013. CA Cancer J Clin. 63:11–30. 2013. View Article : Google Scholar

2 

Garbe C and Blum A: Epidemiology of cutaneous melanoma in Germany and worldwide. Skin Pharmacol Appl Skin Physiol. 14:280–290. 2001. View Article : Google Scholar : PubMed/NCBI

3 

Kütting B and Drexler H: UV-induced skin cancer at workplace and evidence-based prevention. Int Arch Occup Environ Health. 83:843–854. 2010.PubMed/NCBI

4 

Whiteman DC, Stickley M, Watt P, Hughes MC, Davis MB and Green AC: Anatomic site, sun exposure, and risk of cutaneous melanoma. J Clin Oncol. 24:3172–3177. 2006. View Article : Google Scholar : PubMed/NCBI

5 

Ko JM, Velez NF and Tsao H: Pathways to melanoma. Semin Cutan Med Surg. 29:210–217. 2010. View Article : Google Scholar

6 

Perez-Gomez B, Pollán M, Gustavsson P, Plato N, Aragonés N and López-Abente G: Cutaneous melanoma: hints from occupational risks by anatomic site in Swedish men. Occup Environ Med. 61:117–126. 2004. View Article : Google Scholar : PubMed/NCBI

7 

Bouchardy C, Schüler G, Minder C, et al: Cancer risk by occupation and socioeconomic group among men - a study by the Association of Swiss Cancer Registries. Scand J Work Environ Health. 28(Suppl 1): 1–88. 2002.PubMed/NCBI

8 

Håkansson N, Floderus B, Gustavsson P, Feychting M and Hallin N: Occupational sunlight exposure and cancer incidence among Swedish construction workers. Epidemiology. 12:552–557. 2001.PubMed/NCBI

9 

Newton-Bishop JA, Chang YM, Elliott F, et al: Relationship between sun exposure and melanoma risk for tumours in different body sites in a large case-control study in a temperate climate. Eur J Cancer. 47:732–741. 2011. View Article : Google Scholar : PubMed/NCBI

10 

Chang YM, Barrett JH, Bishop DT, et al: Sun exposure and melanoma risk at different latitudes: a pooled analysis of 5700 cases and 7216 controls. Int J Epidemiol. 38:814–830. 2009. View Article : Google Scholar : PubMed/NCBI

11 

Bataille V, Bykov VJ, Sasieni P, Harulow S, Cuzick J and Hemminki K: Photoadaptation to ultraviolet (UV) radiation in vivo: photoproducts in epidermal cells following UVB therapy for psoriasis. Br J Dermatol. 143:477–483. 2000. View Article : Google Scholar : PubMed/NCBI

12 

Radespiel-Tröger M, Meyer M, Pfahlberg A, Lausen B, Uter W and Gefeller O: Outdoor work and skin cancer incidence: a registry-based study in Bavaria. Int Arch Occup Environ Health. 82:357–363. 2009.PubMed/NCBI

13 

Russo AE, Torrisi E, Bevelacqua Y, et al: Melanoma: molecular pathogenesis and emerging target therapies (Review). Int J Oncol. 34:1481–1489. 2009.PubMed/NCBI

14 

Thomas NE, Berwick Mb and Cordeiro-Stone M: Could BRAF mutations in melanocytic lesions arise from DNA damage induced by ultraviolet radiation? J Invest Dermatol. 126:1693–1696. 2006. View Article : Google Scholar : PubMed/NCBI

15 

Libra M, Malaponte G, Navolanic PM, Gangemi P, Bevelacqua V, Proietti L, et al: Analysis of BRAF mutation in primary and metastatic melanoma. Cell Cycle. 4:1382–1384. 2005. View Article : Google Scholar : PubMed/NCBI

16 

Gilchrest BA, Eller MS, Geller AC and Yaar M: The pathogenesis of melanoma induced by ultraviolet radiation. N Engl J Med. 340:1341–1348. 1999. View Article : Google Scholar : PubMed/NCBI

17 

Gandini S, Sera F, Cattaruzza MS, Pasquini P, Picconi O, Boyle P and Melchi CF: Meta-analysis of risk factors for cutaneous melanoma: II. Sun exposure. Eur J Cancer. 41:45–60. 2005. View Article : Google Scholar : PubMed/NCBI

18 

Maira F, Catania A, Candido S, Russo AE, McCubrey JA, Libra M, Malaponte G and Fenga C: Molecular targeted therapy in melanoma: a way to reverse resistance to conventional drugs. Curr Drug Deliv. 9:17–29. 2012. View Article : Google Scholar : PubMed/NCBI

19 

Vuong K, McGeechan K and Armstrong BK; AMFS Investigators; GEM Investigators. Cust AE: Occupational sun exposure and risk of melanoma according to anatomical site. Int J Cancer. Nov 13–2013.(Epub ahead of print). View Article : Google Scholar

20 

Curtin JA, Fridlyand J, Kageshita T, et al: Distinct sets of genetic alterations in melanoma. N Engl J Med. 353:2135–2147. 2005. View Article : Google Scholar : PubMed/NCBI

21 

Deichmann M, Krahl D, Thome M, Wüst K, Hassanzadeh J and Helmke B: The oncogenic B-raf V599E mutation occurs more frequently in melanomas at sun-protected body sites. Int J Oncol. 29:139–145. 2006.

22 

Hodis E, Watson IR, Kryukov GV, et al: A landscape of driver mutations in melanoma. Cell. 150:251–263. 2012. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

2014-March
Volume 31 Issue 3

Print ISSN: 1021-335X
Online ISSN:1791-2431

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Candido S, Rapisarda V, Marconi A, Malaponte G, Bevelacqua V, Gangemi P, Scalisi A, McCubrey JA, Maestro R, Spandidos DA, Spandidos DA, et al: Analysis of the B-RafV600E mutation in cutaneous melanoma patients with occupational sun exposure. Oncol Rep 31: 1079-1082, 2014.
APA
Candido, S., Rapisarda, V., Marconi, A., Malaponte, G., Bevelacqua, V., Gangemi, P. ... Libra, M. (2014). Analysis of the B-RafV600E mutation in cutaneous melanoma patients with occupational sun exposure. Oncology Reports, 31, 1079-1082. https://doi.org/10.3892/or.2014.2977
MLA
Candido, S., Rapisarda, V., Marconi, A., Malaponte, G., Bevelacqua, V., Gangemi, P., Scalisi, A., McCubrey, J. A., Maestro, R., Spandidos, D. A., Fenga, C., Libra, M."Analysis of the B-RafV600E mutation in cutaneous melanoma patients with occupational sun exposure". Oncology Reports 31.3 (2014): 1079-1082.
Chicago
Candido, S., Rapisarda, V., Marconi, A., Malaponte, G., Bevelacqua, V., Gangemi, P., Scalisi, A., McCubrey, J. A., Maestro, R., Spandidos, D. A., Fenga, C., Libra, M."Analysis of the B-RafV600E mutation in cutaneous melanoma patients with occupational sun exposure". Oncology Reports 31, no. 3 (2014): 1079-1082. https://doi.org/10.3892/or.2014.2977