Mutagenesis vol. 19 no. 5 © UK Environmental Mutagen Society 2004; all rights reserved.
TP53 mutations in squamous-cell carcinomas of the conjunctiva: evidence for UV-induced mutagenesis
1Department of Ophthalmology, Makerere University, PO Box 7072, Kampala, Uganda, 2International Agency for Research on Cancer, World Health Organization, 150 Cours Albert Thomas, 69372 Lyon Cedex 08, France, 3Uganda Cancer Institute, PO Box 7051, Kampala, Uganda, 4Department of Epidemiology and Biostatistics, Karolinska Institutet, PO Box 281, S-17177 Stockholm, Sweden, 5Finnish Cancer Registry, Institute for Statistical and Epidemiological Cancer Research, Liisankatu 21 B, FIN-00170 Helsinki, Finland and Cancer Registry of Norway, Oslo, Norway
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Abstract |
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Squamous cell carcinoma of the conjunctiva is associated with sun exposure and often occurs in HIV-positive individuals. We have analysed TP53 mutations in 21 cases of squamous cell carcinoma and 22 controls with benign conjunctival lesions from a region (Uganda, Africa) with a high prevalence of heavy sun exposure and HIV infection. TP53 mutations were detected in 11 cases (52%) and 3 controls (14%). Seven of the mutations (6 in cases and 1 in controls) were CC
TT transitions, a molecular signature of mutagenesis by solar UV rays. A similar prevalence (56%) of TP53 mutations was found in 18 squamous cell carcinoma cases positive for epidermodysplasia verruciformis human papillomavirus types. The prevalence of CCTT transitions reported here is the highest observed in any cancer type and matches that of skin cancers in subjects with xeroderma pigmentosum, an inherited disease with hypersensitivity to UV damage. These results confirm at the molecular level the causal role of solar UV rays in the aetiology of squamous cell carcinoma of the conjunctiva and suggest that infection with epidermodysplasia verruciformis types of human papillomavirus may act as a cofactor to increase the sensitivity of conjunctiva cells to UV-induced mutagenesis. |
Introduction |
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Squamous cell carcinoma (SCC) of the conjunctiva is a tumour mainly reported in black Africans and associated with heavy sun exposure (Newton, 1996
). Solar UV is a well-characterized source of oncogenic DNA damage and in non-melanoma skin carcinomas mutations of the tumour suppressor gene TP53 are frequent, with a high prevalence of CT transitions including, in particular, tandem CCTT transitions. The latter are exceptionally rare in carcinomas of internal organs (IARC TP53 mutation database at: www.iarc.fr/p53) and are consistent with the mutagenic effects of UV in vivo due to inefficient repair of a common photoproduct, cyclobutane pyrimidine dimers (Daya-Grosjean et al., 1995). In addition, we have recently shown that a subgroup of cutaneous human papillomavirus types, termed epidermodysplasia verruciformis (EV) types (e.g. HPV5, 8, 19–25 and 36–38), but not the mucosal oncogenic HPV types (e.g. HPV16 and 18), are frequently detected in SCC of the conjunctiva in Uganda (odds ratio for the presence of EV HPV = 12.0, 95% confidence interval 1.7–85) (Ateenyi-Agaba et al., 2004).
Incidence rates of SCC of the conjunctiva have greatly increased in some sub-Saharan African countries in recent decades, probably related to the spread of HIV (Ateenyi-Agaba, 1995). An increased risk for SCC of the conjunctiva has also been reported in HIV-positive individuals in the USA (Goedert and Cote, 1995). In this respect, the involvement of an infection such as HPV in the aetiology of SCC of the conjunctiva would help explain the fact that the disease appears to be associated with immune impairment.
In this study we have assessed the presence of TP53 mutations in biopsy specimens from 21 cases of SCC of the conjunctiva and 22 controls affected by benign conjunctival lesions (i.e. 10 with pterygium, 8 with pingueculum, 3 with solar keratosis and 1 with pigmented naevus) from a region with a high prevalence of heavy sun exposure and HIV (Uganda, Africa) (Ateenyi-Agaba, 1995).
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Materials and methods |
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Specimens were obtained at the New Mulago Eye Department, Kampala, Uganda, from subjects enrolled in a pilot case–control study conducted between March 2000 and January 2001. All patients signed an informed consent form and the study was approved by the local ethical committee. SCC cases (mean age 33.5 years, range 23–49) were age frequency matched with controls with benign conjunctival lesions. HIV serology was not available for our study subjects, but was likely to be positive in a substantial proportion of them, notably among SCC cases.
Tissue specimens were histologically confirmed, deep frozen and shipped to IARC. DNA extraction and TP53 mutation analyses were performed using standard methods. Briefly, DNA was extracted and exons 5–9 of TP53, including splice junctions, were screened for the presence of a somatic mutation by denaturing high performance liquid chromatography (DHPLC) using primers and conditions described elsewhere (Dai et al., 2004). Exons with abnormal DHPLC profiles were further analysed by automated dideoxysequencing as described previously (Tanière et al., 2000). HPV detection was performed by means of PCR assays using different sets of primers, as described elsewhere (Ateenyi-Agaba et al., 2004).
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Results |
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Eleven (52.4%) of 21 cases had TP53 mutations in exons 5–9, including one (sample 1) with two independent mutations (Table I). TP53 mutations were also detected in three (13.6%) controls (two with pterygium and one with pingueculum). Seven mutations in SCC cases [samples 1 (with two mutations), 2, 5, 7, 10 and 11] and one (sample 14, pterygium) in controls were CC
TT transitions, representing 50% of the mutations in this series (Table I). These mutations are known to have various consequences on the TP53 open reading frame, including single amino acid substitutions (samples 1, 2, 5, 7, 11 and 14), introduction of a STOP codon (sample 1) or destruction of a splice junction (sample 10). Several CCTT mutations spanned adjacent codons and had complex consequences, as for example in sample 1, which contains two distinct CCTT mutations. The first spans codons 195–196 and induces a silent substitution at codon 195 and a nonsense mutation at codon 196. The second one affects the second and third bases of codon 241, resulting in a SerPhe substitution.
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The majority of SCC cases (18/21, 86%) tested positive for EV HPV types, mainly HPV38. TP53 mutations were detected in 10 (56%, samples 1–5 and 7–11) of the 18 EV HPV-positive, and one (sample 6) of the three EV HPV-negative cases. Nine (41%) of the 22 controls were EV HPV-positive; in only one of them (sample 14, pterigium) was TP53 found to be mutated (Table II).
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Discussion |
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The prevalence of tandem CC
TT transitions reported in this series is the highest ever reported in any cancer type. In non-melanoma skin cancers the overall prevalence of these mutations does not exceed 6% (Rees, 1994), but high prevalence (50%) of these mutations has been observed in skin cancer patients with xeroderma pigmentosum (XP), a rare DNA repair deficiency syndrome characterized by hypersensitivity to UV (Dumaz et al., 1993; Sato et al., 1993; Giglia-Mari and Sarasin, 2003). Eye cancer is second only to skin cancer in patients with XP in Africa and elsewhere (Jacyk, 1999).
DNA of EV HPV types, in particular HPV38, was detected in the vast majority of SCC (18/21), including 10 with TP53 mutations. The simultaneous presence of EV HPV and TP53 mutations has also been reported in non-melanomatous carcinomas of the skin (Padlewska et al., 2001; Caldeira et al., 2004). Conversely, TP53 mutations are not found in cancer of the cervix uteri (Olivier et al., 2002) and in the fraction of head and neck cancers (Dai et al., 2004) where mucosal types of HPV are aetiologically involved. The early gene products, E6 and E7, of HPV types 16 and 18 play a key role in the malignant transformation of the mucosae of the genital and upper aerodigestive tracts. In particular, HPV16 E6 has been shown to inactivate the product of TP53, p53, promoting its degradation (Dai et al., 2004). Hence, TP53 mutations are not detected in cancer biopsies positive for mucosal high risk HPVs and expressing the E6 gene (Dai et al., 2004). Not so, apparently in SCC and skin carcinomas, where EV HPV types may be aetiologically involved but via a mechanism(s) different from p53 inactivation. Indeed, the E6 proteins of the EV HPV types are unable to promote p53 degradation (Caldeira et al., 2004).
In conclusion, the high frequency and the types of TP53 mutations that we report in our study provide strong molecular support to a role of solar radiation in the aetiology of SCC of the conjunctiva. The presence of many CCTT transitions in SCC resembles the findings in skin carcinomas of XP patients, but it is unclear whether host factors (e.g. genetic factors and HIV-related immune impairment) or characteristics of the conjunctival epithelium (i.e. the thinnest epithelium in the body) account for the similarity between SCC and XP patients. EV HPV types may also be part of the aetiopathogenesis of SCC through mechanisms that differ from those described for mucosal types of HPV, such as HPV16 and 18. In particular, these results raise the hypothesis that EV types of HPV may contribute to carcinogenesis by down-regulating the cellular responses to DNA-damaging agents, making infected cells more sensitive to mutagenesis by UV.
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Acknowledgments |
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We thank Binta Kahwa (Jinja Hospital, Uganda) and Henry Wabinga (Makerere University, Uganda) for participation in the early phases of this study. This study was supported by grants from the Swedish International Development Cooperation Agency (SIDA) for collaboration between Makerere University (Kampala, Uganda) and the Karolinska Institutet (Stockholm, Sweden).
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Notes |
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6 To whom correspondence should be addressed at: Finnish Cancer Registry, Institute for Statistical and Epidemiological Cancer Research, Liisankatu 21 B, FIN-00170 Helsinki, Finland. Tel: +358 9 135 33274; Fax: +358 9 135 5378; Email: elisabete.weiderpass{at}cancer.fi
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References |
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Received on June 17, 2004; accepted on July 22, 2004.
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