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Carcinogenesis Advance Access originally published online on October 25, 2006
Carcinogenesis 2007 28(3):724-731; doi:10.1093/carcin/bgl191
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Analysis of skin cancer risk factors in immunosuppressed renal transplant patients shows high levels of UV-specific tandem CC to TT mutations of the p53 gene

Sophie Queille, Lionel Luron, Alain Spatz1, Marie Françoise Avril1,3, Vincent Ribrag1, Pierre Duvillard1, Christian Hiesse2, Alain Sarasin, Jean Pierre Armand1 and Leela Daya-Grosjean*

Laboratoire Génomes et Cancers, FRE2939 CNRS, Institut Gustave-Roussy, PRII, 39 Rue Camille Desmoulins 94805 Villejuif, France
1 Institut Gustave-Roussy, 39 Rue Camille Desmoulins 94805 Villejuif, France
2 Departement de nephrologie, Kremlin Bicêtre Hospital 94275 Le Kremlin Bicêtre, France
3 Present address: Departement de Dermatologie, Hopital Cochin-Saint Vincent de Paul 75006 Paris, France

*To whom correspondence and requests for reprints should be addressed at: Laboratoire Génomes et Cancers, FRE2939 CNRS, Institut Gustave-Roussy, PRII, 39 Rue Camille Desmoulins, 94805 Villejuif, France; Tel: 33 1 42 11 63 25; Fax: 33 1 42 11 50 08; E-mail: daya{at}igr.fr


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Immunosuppressed renal transplant recipients (RTRs) are predisposed to non-melanoma skin cancers (NMSCs), predominantly squamous cell carcinomas (SCCs). We have analyzed skin lesions from RTRs with aggressive tumors for p53 gene modifications, the presence of Human Papillomas Virus (HPV) DNA in relation to the p53 codon 72 genotype and polymorphisms of the XPD repair gene. We detected 24 p53 mutations in 15/25 (60%) NMSCs, 1 deletion and 23 base substitutions, the majority (78%) being UV-specific C to T transitions at bipyrimidine sites. Importantly, 35% (6/17) are tandem mutations, including 4 UV signature CC to TT transitions possibly linked to modulated DNA repair caused by the immunosuppressive drug cyclosporin A (CsA). We found 8 p53 mutations in 7/17 (41%) precancerous actinic keratosis (AK), suggesting that p53 mutations are early events in RTR skin carcinogenesis. Immunohistochemical analysis shows a good correlation between p53 accumulation and mutations. HPV DNA was detected in 78% of skin lesions (60% Basal Cell Carcinomas, 82%AK and 79% SCCs). Thus, immunosuppression has increased the risk of infections by HPVs, predominantly epidermodysplasia verruciformis, speculated to play a role in skin cancer development. No association is found between HPV status and p53 mutation. Moreover, p53 codon 72 or frequencies of three XPD genotypes of RTRs are comparable with control populations. The p53 mutation spectrum, presenting a high level of CC to TT mutations, shows that the UV component of sunlight is the major risk factor and modulated DNA repair by immunosuppressive drug treatment may be significant in the skin carcinogenesis of RTRs.

Abbreviations: AK, actinic keratosis; BCCs, basal cell carcinomas; CsA, cyclosporin A; HPV, human papillomas virus; NMSCs, non-melanoma skin cancers; RTRs, renal transplant recipients; SCCs, squamous cell carcinomas


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
The remarkable improvement in immunosuppressive therapy and surgical procedures for organ transplantation observed since the first successful kidney transplant achieved in 1954 now allows many organ types including kidney, heart, liver, lung, skin, cornea and bone marrow to be successfully transplanted. The survival of patients has increased substantially and it has also become clear with time that recipients present a higher risk of cardiovascular disease, infection and cancer (1). Renal transplant recipients (RTRs) submitted to lifelong immunosuppression, constitute the most important group of patients with long-term survival to have provided important information on the increased susceptibility to cancer. The cumulative risk of tumor development, which includes skin cancers, lymphomas and kaposi sarcoma (2,3) is 14% in the first 10 years after transplantation and increases to 40% at 20 years compared to 6% in the general population (4). Half of the tumors are non-melanoma skin cancers (NMSCs) predominantly squamous cell carcinomas (SCC), in contrast to the normal population where basal cell carcinomas (BCCs) prevail. The rapidly growing numerous NMSC, at multiple sun exposed body sites in RTRs, are more aggressive and present increased metastatic potential resulting in substantial morbidity with increased mortality.

The causative factors generally implicated in the formation of skin cancers, namely solar UV radiation, skin type, life style and diverse genetic factors are obviously relevant in RTRs and enhanced tumor development may be associated with the molecular effects of immunosuppressive drugs such as cyclosporin A (CsA) (5,6) and azathioprine (7,8). CsA can inhibit removal of UVB-induced lesions (6) and UVB-induced apoptosis in normal human epidermal keratinocytes (9) and can also stimulate TGFbeta synthesis in cancerous cells promoting cancer invasiveness and metastasis (5,10). Recently, azathioprine has been shown to sensitize DNA to UVA radiation resulting in increased mutagenesis (11). Molecular epidemiological studies of the p53 tumor suppressor gene in sporadic NMSC have established the major role of the UV component of solar radiation in skin carcinogenesis (12–14). So far, only two studies have shown that p53 mutations in skin cancers of RTRs are correlated to unrepaired UV-induced DNA damage (15,16), p53 protein accumulation analyzed by immunohistochemistry (IHC) being more often used as an indicator of p53 mutations (17–20).

Impaired immune surveillance often results in susceptibility to many different types of infections including those by human papilloma virus (HPV), which are associated with a variety of benign and malignant skin lesions. HPV DNA is detectable in normal skin but is found more frequently in skin lesions (60–95%) of both immunocompetent patients (ICPs) and immunosuppressed RTRs (15,21–23). Nevertheless, the pathogenic role of HPV in NMSC development remains speculative. Interestingly, patients with the rare inherited disorder, Epidermodysplasia Verruciformi (EV), who present some immunodeficiency, develop numerous polymorphous HPV-associated cutaneous warts which develop into SCCs on sun-exposed body sites.

Specific gene polymorphisms have been associated with cancer predisposition and a p53 codon 72 polymorphism related to overrepresentation of the homozygous Arg-72 p53 has been linked to HPV-associated cervical tumors and SCCs from RTRs (16,24). In other studies, no positive association has been found for skin cancer predisposition in RTRs (25,26). Repair gene polymorphisms which could modulate DNA repair capacity may also have some impact on skin cancer predisposition, in particular the XPD gene implicated in nucleotide excision repair (NER) and transcription presents polymorphisms which may be linked with skin cancer risk (27–29).

The data on the different risk factors associated with the susceptibility to skin cancers in renal transplant patients is dispersed among many studies and is often contradictory and difficult to compare. No single study has analyzed for the many risk factors in the same set of samples and here we have had the opportunity to analyze precursor and malignant skin lesions, mainly SCCs, from renal transplant patients presenting numerous aggressive skin cancers referred to the Institut Gustave Roussy for specialized therapy. Our analysis of the different risk factors for skin cancer has shown that EV-HPV DNA is preponderant in the tumors and the XPD and p53 codon 72 genotypes are not significant for the skin cancer predisposition observed in transplant patients. The UV-specific p53 mutation spectrum characterized in these tumors confirms that solar UV damage is the major risk factor favoring skin cancer predisposition in RTRs together with side effects of the immunosuppressor drugs which may be implicated in the high frequency of CC to TT tandem mutations found here.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Tissue samples of renal transplant recipients
Twenty European Caucasian RTRs, presenting numerous aggressive skin cancers, were investigated in this study (Institut Gustave-Roussy, Villejuif, France). The Hospital Medical Ethics Committee (CCPPRB) approved the study protocol. Informed consent was obtained from all the patients. The study was conducted in accordance with the Declaration of Helsinki Principles. All patients were under immunosuppressive treatment with different combinations of drugs including CsA, Azathioprine (Imurel) and predinosone (Cortancyl). Age at transplantation was between 18 and 66 years and the first skin tumors appeared between 1 and 21 years after transplantation. SCC, BCC and actinic keratosis (AK), from renal transplant patients were obtained at the time of surgical resection. Healthy skin biopsies from four patients were also obtained. Half of each sample was fixed for histopathological and immunohistochemical analysis and the rest was snap frozen in liquid nitrogen and stored at –80°C.

DNA extraction
Frozen samples were reduced to a fine powder and DNA was extracted as described previously (30). Blood samples were also obtained from 37 RTRs including 30 patients with skin cancers and 7 without skin malignancies to date. Lymphocytes were prepared using Histopaque 10771 (Sigma, Saint Quentin Fallavier, France). DNA was extracted from lymphocytes using the QIAmp DNA Mini Kit (Qiagen, Courtaboeuf, France).

Human papillomavirus DNA detection and genotyping
All samples were analyzed for the presence of HPV DNA using a degenerate PCR-based method using the following conditions. The EV-associated HPV belonging to group B1 were characterized with the outer primers (CP62/CP69) and internal nested primers, (CP65/CP68) described by Berkhout et al. (22). Primers described by Harwood et al. (31) were used for the specific amplification of the HPV-EV clusters a1 (EN1F/EN1R), a2 (EN2F/EN2R) and b1 + b2 (EN3F/EN3R). For amplification of the cutaneous HPV types, the outer primers HVP2/B5 described by Shamanin et al. (23,32) were used in combination with the nested primer pairs CN1F/CN1R, CN2F/CN2R, CN3F/CN3R described by Harwood et al. (31) specific for the subgroups E, A2 and A4, respectively. The cutaneous HPV type group B2 was detected by the single round primer set C4F/C4R described by Harwood et al. (31). The mucosal HPV DNA was detected using the MY09/11 outer primer sets described by Manos et al. (33) and GP5/GP6 as nested primers (34). All PCR amplifications were performed exactly as described for the different primer pairs (27) using Taq polymerase (Qiagen, Courtaboeuf, France). PCR products were purified after separation in 1.5% agarose gels using the QIAquick Gel extraction kit (QiAquick Gel Extraction kit; Qiagen, Courtaboeuf, France). The purified products were directly sequenced using the ABI prism Dye terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystem) with both forward and reverse primers. The products were analyzed on an ABI Prism 310 (Perkin–Elmer) automated sequencer. After sequence alignment, the consensus sequence was compared with those of known HPV types available through the GenBank database (National Center for Biotechnology Information, NIH, Bethesda, MD). In accordance with established guidelines, a nucleotide sequence was regarded as an HPV type if it shared over 90% homology with a known type and a related type if the sequence homology was <90%.

PCR-single strand conformation polymorphism analysis
The coding region of the p53 gene was amplified using seven sets of primers covering exons 4, 5, 6, 7, 8, 9 and 8–9 where the majority of mutations have been located. Genomic DNA (200–500 ng) was incubated in a total volume of 25 µl containing 100 µM each dNTP, 0.2 µM primers, 1.5 U of Taq polymerase (Qiagen, Courtaboeuf, France), 5 µCi of [{alpha}-33P]dATP (Amersham Pharmacia Biotech, Saclay, France) buffered at pH 8.7 in Tris–HCl, KCl, (NH4)2SO4 with 1.5 mM MgCl2. Cycle parameters were 2 min at 94°C followed by 30 cycles of 94°C for 1 min, 40–55°C for 1 min and 72°C for 1 min (Gene Amp PCR system 9600; Perkin-Elmer, Courtaboeuf, France). An extension period of 10 min at 72°C was added at the end of the program. The reactions were stopped by a 3-fold dilution in stop solution [95% (v/v) formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol]. The PCR products were denatured for 5 min at 100°C, quick-chilled on ice for 3 min and were loaded immediately on 0.5x MDE Gels (Tebu, Le Perray en Yvelines, France) with or without 10% glycerol and run at 4–10 W for 13–24 h at room temperature or at 8°C in a cold room (for gels without glycerol). After autoradiography, shifted bands were cut out of the gel and DNA was eluted in distilled water and reamplified. PCR products were purified by treatment with shrimp alkaline phosphatase and exonuclease I and then sequenced using the Thermosequenase kit (Amersham Pharmacia, Saclay, France).

Analysis of the p53 codon 72 polymorphism
PCR-single strand conformation polymorphism (SSCP) analysis of exon 4 was performed on lymphocyte DNA using the protocol described above for the p53 mutation analysis of skin lesions. Shifted bands have been sequenced to verify the p53 codon 72 genotype.

Analysis of the XPD polymorphisms
Exons 23, 6–7 and 10 of the XPD gene were amplified from lymphocyte DNA using primers described previously (24). PCR products were purified and sequenced using ABI Prism Dye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Courtaboeuf, France) with both forward and reverse primers.

p53 immunohistochemical analysis in skin lesions
The majority of the lesions analyzed for p53 mutations were also screened for p53 protein accumulation by IHC. Thus, thin sections (5 mm) of formalin-fixed and paraffin-embedded archival material were examined using the Mab DO-7 (Dako, Copenhagen, Denmark) p53 antibody, directed against a short N-terminal segment. Mab DO-7 used at a dilution of 1:100 and incubated for 1 h at room temperature after microwave pretreatment in citrate buffer. Antigen localization was achieved by using the alkaline phosphatase method (Dako, Copenhagen, Denmark). Negative controls were incubated with PBS, and no positive staining was observed. The slides were analyzed with no prior knowledge of the p53 gene status. Immunohistochemical staining was recorded using a semi-quantitative grading, considering the proportion of tumor cells showing unequivocal positive reaction in the tumor cell nuclei or in the cytoplasm. Intensity was graded as follows: 0, no staining; 1, staining in <10% of the tumor cells; 2, staining in 10–50% of the tumor cells; 3, staining in >50% of the tumor cells.

Statistical analysis
The statistical analysis was performed using {chi}2 testing or Fisher-exact testing when frequencies were smaller than five. A P-value <0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
HPV status of skin lesions
HPV DNA is detected in 78% (32/41) of skin lesions from RTRs and little difference is seen between levels found in pre-cancerous AK, BCCs and SCCs (Table I). EV types are significantly predominant in all types of skin lesions particularly in SCCs. Cutaneous HPV are observed as mixed infections with the EV types only in SCCs and their precursor lesions, the AK. The mucosal HPV DNA is only detected with EV-HPV in one AK. We find no correlation between HPV and p53 status as HPV DNA is detected in skin lesions carrying mutated or wild-type p53 (Table I). HPV DNA was not detected in healthy skin biopsies of four patients and normal skin samples from other patients were unavailable.


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  		Table I Distribution of HPV groups and correlation between HPV and p53 status in different skin lesions from RTRs

 
Analysis of P53 immunohistochemical expression
P53 protein was analyzed by IHC in 23 NMSC and 17 AK from RTRs. Figure 1 shows typical examples of p53 nuclear expression in different skin lesions from RTRs. Results, expressed by grading the number of p53 positive cells, are presented in Table II. 13/23 NMSC and only 1/17 AK precursor lesions present >50% of p53 stained cells, the majority of the AK lesions presenting <10% of p53 positive cells.


Figure 1
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  		Fig. 1 Immunohistochemical staining of p53 expression: (A) no staining in normal skin; (B) p53 nuclear staining in the lower third of the epidermis in Actinic Keratosis; (C) p53 nuclear staining throughout the Squamous Cell Carcinoma section, note hair follicule in the center is not reactive.

 


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  		Table II P53 immunohistochemical analysis in non-melanoma skin cancer and actinic keratosis from RTRs

 
P53 mutations in skin lesions from RTRs
The p53 gene alterations in exons 4–9 were analyzed in 42 skin lesions (17 AK and 25 NMSC) from 20 RTRs. p53 mutations were found in 41% (7/17) of the AK, in 60% (15/25) of the NMSC including 50% (2/4) BCCs and 61% (13/21) of SCCs. Among the total of 24 p53 modifications (tandem mutations are taken as one event), we have identified in 15 NMSC, 1 deletion and 23 base substitutions (Table III). 78% (18/23) of these base substitutions are UV-specific, being C to T transitions located at dipyrimidine sites. Interestingly, 35% (6/17) of the base substitutions are tandem mutations (tandem mutations are taken as one event) including 4 CC to TT transitions considered to be the veritable UV signature (Table IV). We have also detected 13% of G to T base substitutions and two transversions, one C to G and one T to A (Table IV). Results of p53 IHC show that p53 expression is consistent with the presence of the p53 mutations detected by PCR–SSCP (Table III). The majority of the base substitutions lead to important accumulation of p53 protein. In SCC from two different patients (TL and GR) two base substitutions on codon 196 result in a premature stop which in one patient (TL) shows a weak accumulation of p53. However, in patient GR, despite the stop codon, a strong p53 accumulation is detected. The 1bp deletion found in the SCC from patient VC, results in a frameshift mutation and no p53 accumulation is detected for this truncated protein. p53 mutations are detected in 41% (7/17) of the precancerous AK from RTRs (Table III), the majority also being the C to T transitions located at bipyrimidine sites. p53 protein expression is lost by the deletion of 10 bp in patient HA, due to the frameshift mutation giving rise to a premature stop codon. The relationship between p53 mutations and p53 accumulation in the benign AK is less consistent than in NMSC as only four of the seven AK with p53 base substitutions leading to amino acid changes, were found to have p53 protein accumulation.


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  		Table III p53 mutations and p53 immunohistochemical analysis in different skin lesions from RTRs

 
Gene polymorphisms
p53 codon 72 polymorphisms. Lymphocyte DNA from 30 RTRs patients presenting aggressive skin cancers were analyzed for the p53 codon 72 genotypes by PCR–SSCP and sequencing of all different exon 4 polymorphic bands. Our data [Table V, (A)] show that there are no differences in genotype or allele frequency between RTRs with skin tumors and control individuals of the same Northern European Caucasian population.

XPD gene polymorphisms. Lymphocyte DNA from 30 patients all presenting skin cancers were analyzed for three XPD polymorphisms (exons 6, 10 and 23). In Table V, (B), it can be seen that our results show no difference in distribution of the XPD genotypes and allele frequencies between control populations and RTRs. Our sample numbers were too small for a statistical analysis.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In this study we have carried out a detailed molecular analysis of different risk factors implicated in the formation of skin cancers form a cohort of 20 French RTRs who were referred for treatment to our cancer center for treatment of numerous aggressive skin cancers. These patients underwent kidney transplantation at ages between 18 and 66 years and their skin tumors appeared with a latency of 1–21 years, the median latency being between 5 and 15 years. Interestingly, two patients aged over 60 years at the date of transplant developed a skin tumor within a year, indicating enhanced susceptibility to skin tumor development with age. SCCs and their precursor lesions, the AK, can occur up to 200 times more frequently in transplant patients with a 4:1 ratio of SCCs to BCCs which is reversed compared to that found in the general population. We find a 5-fold incidence of SCCs compared to BCCs amongst our samples. Nevertheless, the incidence of BCCs in transplant patients can still be up to 10 times higher than that seen in control populations (1). It is interesting to note that UV hypersensitive Xeroderma pigmentosum XP patients who present some defective immunosurveillance (35–37) and are predisposed to skin cancer (38) also present more SCCs than BCCs, which may be also related to their defect in DNA repair.

Our data, in line with numerous investigations (15,21–23) studying the role of HPV in the development of the NMSC in RTRs and ICPs shows a high prevalence of EV-HPV DNA in the different skin lesions, particularly in SCCs. HPV DNA was not detected in control skin biopsies. There is no significant correlation between HPV types found in AK or NMSC from the same patients (DCA and GR). The presence of cutaneous HPV DNA is rare and confined to SCCs (2/19) and the AK (2/17) and always occurs as mixed infections with EV-HPV DNA (Table I). Mucosal HPV DNA was only detected in one AK also with EV-HPV DNA. The preponderance of EV-HPV DNA is in line with epidemiological evidence suggesting a significant role in SCC development. Their oncogenic function has not been clearly elucidated and unlike the high-risk ano-genital HPVs, EV-HPV DNA remains extrachromosomal and is rarely integrated in the genome of skin cancer cells. Up till recently, only SCCs from EV patients were found to present active EV-HPV gene transcription but a recent study has demonstrated active EV-HPV expression in malignant keratinocytes of SCCs from both immunosuppressed and immunocompetent individuals (39). Moreover, the carcinogenic potential of the early EV-HPV genes has been shown in transgenic mouse models (40). HPV can also modulate different cellular UV responses such as inhibition of UV-induced apoptosis, which may induce and facilitate the persistence of UV-induced DNA damage and lead to mutation and cancer development. Thus, E6 proteins from cutaneous HPV types can selectively block the transcription of UV-induced p53-dependent pro-apoptotic genes (41) or inhibit apoptosis by a p53 independent pathway (42). UV is also found to stimulate the promoter activity of EV-HPV types 5 and 8 (43,44). Taken together, these findings suggest that the EV-HPV we have shown here to be strongly associated with SCCs from our RTRs, might act as important cofactors with UV irradiation in skin cancer development.

This analysis of the p53 codon 72 genotypes in 30 RTRs, all North European Caucasians, shows a genotype distribution typical of the general population in agreement with the study also analyzing North European RTRs who had or not developed skin cancers (26). We find no relation between the latency of tumor appearance and the p53 codon 72 genotype of the RTRs. In fact, Arg homozygous RTRs have not developed skin cancer more rapidly or in larger numbers than the Arg/Pro and Pro/Pro RTRs (clinical data not shown).

The data on p53 mutations and protein expression indicates the major role of solar UV-induced DNA lesions in the skin carcinogenesis of RTRs. We find that the premutagenic UV lesions are located equally on the transcribed and non-transcribed strand of the p53 gene indicating that there is no strand-specific repair deficiency in the RTRs (Table III). The majority of the p53 alterations are C to T transitions mainly located at bipyrimidine sites, which are specific targets for UV-induced lesions (Table III). There is no intra-individual difference in mutations found in benign and malignant lesions as can be seen for patients DCA and GR (Table III) for which both their AK and NMSC present UV-specific mutations. The G to T transversions we see here may be attributed to the oxidation of guanine residues by cellular reactive oxygen species (ROS) generated by the UV component of sunlight, notably UVA (45,46). However, as they are located at Py-Py sites, it is more likely that UV-induced CPDs are the premutagenic lesions and that they are not induced by endogenously generated ROS. Moreover, the G to T frequency we find is similar to that detected in NMSC from the normal population indicating that UVA generated ROS are not synergistically mutagenic with 6-thioguanine DNA lesions, resulting from use of the immunosuppressive drug azathioprine (11). UV-induced oxidative damage is implicated in skin carcinogenesis (47,48) but our data reveal the that UV photoproducts are responsible for the majority of mutations that we have detected here which are located on the same hot spot codons (codons 248, 196, 282) as those found in NMSC from the general population (13). Our p53 mutation frequency is comparable to those from the two studies by McGregor et al. (15,16) of the molecular analysis of p53 mutations in 23 and 45 NMSC from RTRs (60% versus 48%, P = 0.3978 and 60% versus 35,5%, P = 0,08511; {chi}2-test).

Our p53 mutation analysis also has revealed a significantly higher frequency of tandem mutations than that found in NMSC from the general immunocompetent population (35% versus 12%; P < 0.01; {chi}2-test) (Table III and IV). These are notably CC to TT mutations considered to be the veritable UV signature and found very rarely (<1%) in other types of human cancers. Surprisingly, McGregor's studies (15,16) analyzing a similar number of skin tumors did not reveal any tandem mutations in the p53 gene. The deamination-bypass mechanism is accepted as the origin of UV-induced mutations. Thus, within a UV-induced pyrimidine dimer, deamination of the cytosine or 5-methyl cytosine gives rise to C to T mutations by insertion of an A opposite the U or T by the DNA damage bypass polymerase eta (49). Tandem CC to TT mutations arise when increased deamination occurs which can be linked to latency in replication related to decreased repair. Indeed, it has been shown that when a UVB-irradiated CpG methylated supF shuttle vector is allowed time for deamination before replication, it presents an increase in CC to TT tandem transitions probably due to double deamination events (50). In vivo and in vitro studies have shown that CsA can interfere with repair of UVB-induced DNA damage and apoptosis (6,9,51,52) and we have also observed that CsA treatment accentuates the sensitivity to UV irradiation in diploid human skin fibroblasts (data not shown). The tandem mutations in NMSC of RTRs may thus be related to their CsA treatment, which might slow replication and repair. This would result in a higher frequency of cells harboring unrepaired lesions in which deamination has time to take place and lead to the formation of tandem CC to TT mutations. However, the overall cellular repair capacity of the patients seems to be normal as the frequency of mutated p53 in the RTRs skin tumors we have observed is comparable to that found in skin cancers from the general population. Interestingly, a very high frequency of tandem mutations are observed amongst modifications of key genes (<90% in the p53 gene) in NMSC from DNA repair deficient XP patients (38,53–55). Moreover, we have not detected a specific signature mutation spectrum, which could be attributed to the genotoxicity of immunosuppressive drugs such as CsA or azathioprine used for treatment of our patients. Mutagenesis studies with shuttle vectors in the presence of the immunosuppressive drugs could elucidate whether lowered immunosurveillance slows down elimination of damaged or mutated cells facilitating the induction of tandem mutation.


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  		Table IV Distribution of single and tandem p53 mutations in non melanoma skin cancers from RTRs

 
Our study has indicated that UV irradiation plays a crucial role in the skin carcinogenesis of RTRs and we find no relationship between the p53 codon 72 genotype status and p53 mutations in RTRs. Moreover, HPV (Table I) are found in tumors independently of their status regarding wild-type or mutated p53. Our attention has also been drawn to the importance of other gene polymorphisms, which may be involved in cancer predisposition, in particular, that of the NER gene, XPD. We have analyzed for three specific polymorphisms that have been particularly studied in relation to DNA repair and cancer development; a C to A silent polymorphism in exon 6; a G to A polymorphism in exon 10; and an A to C polymorphism in exon 23. Our results indicate that there is no difference in the XPD genotypes and allele frequencies between RTRs and control population (Table V). Among the different studies on XPD polymorphisms in skin tumors a recent study on a large cohort of Danish patients aged 50–64 years indicates that only the exon 6 homozygous Arg 156 polymorphism has a weak association with risk of BCC but with a strong age dependency (56).


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  		Table V Distribution of p53 codon 72 and XPD polymorphisms associated with RTRs skin cancer risk

 
In conclusion, our study of skin tumors from RTRs shows the predominant role of the UV component of sunlight in the enhanced skin carcinogenesis of the RTRs. UV irradiation of the skin reduces local and systematic responses, which together with the drug-induced immunosuppression of patients facilitates infection by HPV, in particular the EV-HPV types specifically associated with SCCs. HPV, which can modify essential cell functions controlling genome stability may also contribute to the rapid accumulation of deleterious gene mutations implicated in skin carcinogenesis. Whether, the HPV play an essential role in initiation or progression or merely have a ‘hit and run’ effect has yet to be defined. Our data on the molecular alterations of the p53 gene and in particular the high level of the UV hallmark CC to TT tandem mutations support an accumulative effect of the many parameters involved in enhancing skin cancer formation observed in renal transplant patients, particularly those of UV light and immunosuppressive drugs.


   Acknowledgments
 
We would like to thank Drs M. Merad and O. Rixe for their help in the initiation of this project. We also thank Professor Charpentier, Director of the Nephrology Department, Kremlin Bicêtre Hospital, for his support in this work. Sophie Queille was supported by a grant from the Association de Recherche sur le Cancer (Villejuif, France). The work was funded by a PHRC (Programme Hospitalier de Recherche Clinique) grant No. IDF 96003.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 31, 2006; revised September 21, 2006; accepted October 3, 2006.


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