Bill Bowers

Glutathione S-transferase GSTM1 phenotypes and protection against cutaneous tumours

Multiple allelism at loci encoding detoxicating enzymes is associated with cancer risk. We have studied genetic variation at the glutathione S-transferase GSTM1 locus to see whether phenotypes confer altered susceptibility to basal cell carcinoma (BCC), squamous cell carcinoma (SCC), malignant melanoma (MM), or multiple skin tumours of different histological types. The frequency of GSTM1 null in cases and controls (52%) was similar, except for patients with two or more tumours of different types (71%, p = 0.033). GSTM1 A/B was reduced in frequency (p < 0.05) in patients with single or multiple BCC. Thus GSTM1 A/B may be protective, and effectiveness of detoxication may be a factor determining susceptibility to skin cancer.

Susceptibility to multiple cutaneous basal cell carcinomas: significant interactions between glutathione S-transferase GSTM1 genotypes, skin type and male gender

The factors that determine development of single and multiple primary cutaneous basal cell carcinomas (BCCs) are unclear. We describe a case-control study firstly, to examine the influence of allelism at the glutathione S-transferase GSTM1 and GSTT1 and cytochrome P450 CYP2D6 loci on susceptibility to these tumours and, secondly, to identify interactions between genotypes and relevant individual characteristics, such as skin type and gender. Frequency distributions for GSTM1 genotypes in cases and controls were not different, although the frequency of GSTM1 A/B was significantly lower (P = 0.048) in the multiple BCCs than in controls. We found no significant differences in the frequencies of GSTT1 and CYP2D6 genotypes in cases and controls. Interactions between genotypes were studied by comparing multinomial frequency distributions in mutually exclusive groups. These identified no differences between cases and controls for combinations of the putatively high risk GSTM1 null, GSTT1 null, CYP2D6 EM genotypes. Interactions between GSTM1 A/B and the CYP2D6 PM and GSTT1-positive genotypes were also not different. Frequency distributions of GSTM1 A/B with CYP2D6 EM in controls and multiple BCCs were significantly different (P = 0.033). The proportion of males in the multiple BCC group (61.3%) was greater than in controls (47.0%) and single BCC (52.2%), and the frequency of the combination GSTM1 null/male gender was significantly greater in patients with multiple tumours (P = 0.002). Frequency distributions of GSTM1 null/skin type 1 were also significantly different (P = 0.029) and the proportion of subjects who were GSTM1 null with skin type 1 was greater (P = 0.009) in the multiple BCC group. We examined the data for interactions between GSTM1 null/skin type 1/male gender by comparing frequency distributions of these factors in the single and multiple BCC groups. The distributions were almost significantly different (exact P = 0.051). No significant interactions between GSTT1 null or CYP2D6 EM and skin type 1 were identified. Comparisons of frequency distributions of smoking with the GSTM1 null, GSTT1 null and CYP2D6 EM genotypes identified no differences between patients with single and multiple tumours.

Cytochrome P450 CYP2D6 genotypes: association with hair colour, Breslow thickness and melanocyte stimulating hormone receptor alleles in patients with malignant melanoma.

We previously identified associations between polymorphism in the cytochrome P450 CYP2D6 gene and outcome in several cancers. We have now examined the hypothesis that homozygosity for the mutant alleles, CYP2D6*4 and CYP2D6*3, is associated with susceptibility and outcome in malignant melanoma. Outcome was assessed by Breslow thickness. We first confirmed previous reports that these mutant alleles are associated with increased susceptibility to malignant melanoma. For example, the frequency of homozygosity for CYP2D6*4 was significantly greater (P = 0.006, chi-squared 1 d.f. = 7.4, odds ratio 2.2, 95% confidence interval 1.2, 3.9) in cases (9.1%) than in control individuals (4.3%). The frequency of homozygosity for the mutant alleles was next examined in the malignant melanoma cases grouped on the basis of characteristics associated with malignant melanoma risk. Homozygosity was significantly more common (P = 0.038) in cases with red/blonde hair than in those with brown/black hair. We found no associations between the CYP2D6 genotype and sex, skin type or eye colour. The possible association of CYP2D6 with outcome was assessed by comparing genotype frequencies in patients with tumours of Breslow thickness < 1.5 mm with those whose tumours were > or = 1.5 mm. In patients with red/blonde, but not brown or black hair, homozygosity for CYP2D6*4 was significantly associated with thicker lesions in a multivariate model (P = 0.036). We further examined the association of CYP2D6*4 homozygosity with red/blonde hair by classifying patients on the basis of homo- or heterozygosity for wild-type or val92met, asp294his or asp84glu melanocyte stimulating hormone receptor (MC1R) alleles. None of the nine patients with brown/black hair with the asp294his allele were homozygotes for CYP2D6*4. By contrast, in the patients with red/blonde hair, three of five cases with asp294his were homozygotes for the mutant CYP2D6 allele. The difference in the frequency of CYP2D6*4 homozygotes in the red/blonde cases with wild-type MC1R alleles compared with those with asp294his was significant (exact P = 0.029). No associations between val92his or asp84glu and CYP2D6 alleles were identified.

Basal cell carcinomas: association of allelic variants with a high-risk subgroup of patients with the multiple presentation phenotype.

Previous studies have shown that patients who present at first or a later presentation with a cluster of new basal cell carcinoma (BCC) comprise a subgroup, termed multiple presentation phenotype (MPP), that is at increased risk of developing further lesions. In this study, we examined the hypothesis that patients who develop multiple clusters are a high-risk subgroup. We found, in a total group of 926 BCC patients, 32 patients with 2-5 BCC clusters (multiple cluster MPP) and 113 cases with only one cluster (single cluster MPP). Multiple cluster MPP cases had mean of 11.3 BCC compared with 3.7 in single cluster MPP cases during similar follow-up. Ultraviolet (UV) exposure in these groups was similar. We determined whether the multiple cluster MPP was associated with characteristics associated with sensitivity to UV or glutathione S-transferase (GST) GSTT1, GSTM1, cytochrome P450 (CYP) CYP2D6, tumour necrosis factor (TNF)-alpha and vitamin D receptor (VDR) genotypes previously associated with BCC presentational phenotypes. While the frequencies of blue eyes and male gender were greater in multiple cluster than single cluster cases, these differences were not significant. In multiple cluster cases, mean age at first presentation with single tumours occurred earlier and the frequencies of CYP2D6 extensive metabolizer (EM) (94.4%) and GSTT1 null (41.2%) were significantly greater (P = 0.028 and P = 0.004) than in single cluster cases (67.1% and 14.3%, respectively). The odds ratios for the individual associations of CYP2D6 EM and GSTT1 null with the multiple cluster MPP were relatively larger; 15.5 and 7.39, respectively. TNF-alpha and VDR genotypes were not associated with multiple cluster MPP. We propose that the MPP is not the consequence of excessive UV exposure but rather reflects the presence of a distinct BCC subgroup which is defined by combinations of risk genes.

Glutathione S-transferase GSTP1 and cyclin D1 genotypes: association with numbers of basal cell carcinomas in a patient subgroup at high-risk of multiple tumours.

We previously described associations between basal cell carcinoma (BCC) numbers and allelic variants at loci that mediate host response to ultraviolet radiation (UV). These associations were largely exerted in cases with the multiple presentation phenotype (MPP). This phenotype describes patients who present at their first or a later presentation with a cluster of BCC (2-10 new BCC). Remaining BCC cases have the single presentation phenotype (SPP) and may develop more than one BCC but only have single new lesions at any presentation. We proposed that the MPP cases comprise a high-risk group as they suffer significantly more lesions than SPP cases. We are attempting to determine, in the total BCC case group and subgroups, how many genes influence BCC numbers and their relative importance. In this study, we assessed the influence of two further candidates, glutathione S-transferase GSTP1 and cyclin D1 (CCND1), on tumour numbers in a total group of 457 patients comprising MPP and SPP cases. The relative importance of these genes in comparison with occupational UV exposure and host response (skin type) was also considered. We found that the frequencies of GSTP1 genotypes based on the Ile105 and Val105-expressing alleles and CCND1 AA, AG, GG genotypes were similar in MPP and SPP cases and that there were no significant associations between GSTP1 or CCND1 genotypes and BCC numbers in the total or SPP groups. However, in the MPP cases, GSTP1 Val105/Val105 was associated with more tumours (P = 0.05, reference GSTP1 Ile105/Ile105). Inclusion of skin type and indoor/outdoor occupation in the negative binomial regression models did not alter the associations of these genotypes with tumour numbers. DNA from 258 cases was analysed to identify GSTP1*A (Ile105-Ala114), GSTP1*B (Val105-Ala114), GSTP1*C (Val105-Val114) and GSTP1*D (Ile105-Val114). In SPP cases, there was no association between BCC numbers and GSTP1 BB, though the association with GSTP1 BC approached significance (P = 0.09). In MPP cases, GSTP1 BC was associated with BCC numbers (P = 0.03). We also found that the interaction term, GSTP1 Val105/Val105 with CCND1 AA, was associated with BCC numbers in the total (P = 0.001) and MPP (P = 0.006) but not SPP (P = 0.68) groups. In a stepwise model including GSTP1 Val105/Val105, CCND1 AA and their interaction terms as well as GSTM1, GSTT1 and CYP2D6 genotypes, skin type 1 and gender, the combination of genotypes was the best predictor of BCC numbers. These data suggest that study of further genes involved in cell-cycle control and protection from oxidative stress will be useful, particularly in high-risk subgroups.

The melanocyte stimulating hormone receptor polymorphism: Association of the V92M and A294H alleles with basal cell carcinoma

Allelic variants in the melanocyte stimulating hormone receptor (MC1R) gene are susceptibility/outcome candidates for cutaneous basal cell carcinoma (BCC). We identified the val92met (V92M) and asp294his (A294H) alleles in 311 cases and 190 controls. The cases included four homo- and 53 heterozygotes for V92M and 12 heterozygotes for A294H and two compound heterozygotes (V92M/A294H). Allele frequencies were similar in controls. In the cases, we found no association between the alleles and skin type though A294H was more common in those with red hair (4/19) than with other hair colours (6/163) (P = 0.012). V92M was not associated with BCC numbers. Cases with A294H had fewer BCC in comparison with those without the allele though the difference was not significant. After inclusion of red hair in the model, A294H was significantly associated with fewer tumours. While MCIR alleles are attractive candidates for BCC, the variants studied did not influence susceptibility. The association with outcome was relatively weak. The large number of MC1R alleles and their low frequency, make assessment of the importance of this gene in the pathogenesis of skin cancers difficult.

Polymorphism in Detoxifying Enzymes and Susceptibility to Skin Cancer

ates, isomerization of urocanic acid and activation of a surprising range of cytokine genes within keratinocytes. The DNA photoproducts (1 8) and UVB-generated oxygen intermediates (19) have been implicated as damaging to cutaneous immunity. The possibility exists that polyrnorphisms at loci that encode excision and repair genes or at loci regulating free radical quenching may exist and could have a profound effect on cutaneous immunity. Similarly, polymorphisms at cytokine gene loci may contribute to UVB-S. Finally, UVB radiation produces multiple, mechanistically distinct immune aberrations. Locally, at the irradiated site, UVB radiation (1) impairs induction of CH, (2) promotes hapten-specific tolerance and (3) creates a sustained immunosuppressive microenvironment (20). Systemically, UVB radiation (1) impairs both delayed and CH to a variety of antigens (21) and (2) induces active, nonspecific unresponsiveness. Our studies have revealed genetics factors operating in only one of these aberrations, but we suspect that, in time, other polymorphic genetic factors will be identified that contribute to the damaging immune effects of UVB radiation and thereby alter an individual’s risk of developing sunlight-induced skin cancer.