Paul R. Hoban

Cyclin D1 Polymorphism and Expression in Patients with Squamous Cell Carcinoma of the Head and Neck

We have previously reported that the cyclin D1 (CCND1) GG870 genotype was associated with poorly differentiated tumors and reduced disease-free interval in patients with squamous cell carcinoma of the head and neck (SCCHN). We have now examined the association of this and a second CCND1 polymorphism with gene expression and outcome in SCCHN patients. Analysis of a CCND1 G/C1722 polymorphism revealed that CCND1 CC1722 genotype was associated with poorly differentiated tumors [P = 0.005; odds ratio (OR), 5.7; 95% CI, 1.7 to 19.2), and reduced disease-free interval (P = 0.003; Hazard Ratio (HR), 7.3; 95% CI, 1.1 to 27.2.) independently from the influence of CCND1 GG870 genotype. Patients whose tumors were negative for cyclin D1 were associated with reduced disease-free interval (P = 0.028; HR, 4.1; 95% CI, 1.4 to 14.2). Although G/C1722 genotypes were not associated with expression, we found a significant trend between reduced expression of cyclin D1 in patients with the CCND1 GG870 genotype (P = 0.04). Splicing of CCND1 mRNA in head and neck tissues was modulated by CCND1 A/G870 alleles, thus CCND1 transcript a was spliced equally from CCND1 A870 and G870 alleles, whereas CCND1 transcript b was spliced mainly from the CCND1 A870 allele. Our analysis has also identified differences in cyclin D1 genotype and protein expression and the pathogenesis of SCCHN in males and females. Thus, CCND1 CC1722 genotype was more common in female patients (P = 0.019; OR, 3.3; 95% CI, 1.3 to 10) and cyclin D1 expression was more frequent (chi-square1, 3.96; P = 0.046) and at higher levels (P = 0.004) in tumors from female patients. In summary, our data show that the two CCND1 polymorphic sites are independently associated with tumor biology and clinical outcome. CCND1 A/G870 alleles affect gene expression in head and neck tissues. We also provide preliminary evidence that the molecular genetics of SCCHN development may be influenced by patient gender.

Genetics and risk factors for basal cell carcinoma.

Nonmelanoma skin cancer (NMSC) is the commonest cancer in whites and its incidence is increasing worldwide. The prevalence of this cancer is predicted to equal that of all others combined and it was estimated that there were over 2 million cases diagnosed in the U.S.A. in 2004. Patients exhibit marked differences in clinical phenotype with variations in tumour numbers, rate of tumour accrual, site and histological subtype. Furthermore, patients are at increased risk of other cutaneous and noncutaneous cancers. The factors accounting for this variation are complex and still not completely understood. Clearly, ultraviolet light (UV) exposure is a major influence but its relationship to clinical phenotype is not yet clear. In addition, immunosuppression is a significant risk factor. Our group has identified high‐risk groups for the development of further basal cell carcinoma (BCC), namely patients with truncal BCC and those presenting with tumour clusters. This presentation will concentrate on these clinical subgroups as well as immunosuppressed patients. These groups represent significant management challenges and are areas where novel, nonsurgical treatment options may make a significant clinical impact in patient care. The risk factors predisposing to these clinical phenotypes will be discussed, including genetic factors and UV exposure. Potential clinical applications, including predictive indices, will be considered.

Expression and subcellular localization of cyclin D1 protein in epithelial ovarian tumour cells

SummaryThe expression of cyclin D1 protein in tumour sections from 81 patients with epithelial ovarian cancer was analysed using immunohistochemistry. The tumours that overexpressed cyclin D1 in more than 10% of neoplastic cells were considered positive. Thus overexpression of cyclin D1 was observed in 72/81 (89%) of the cases examined. Protein was detected in both the nucleus and the cytoplasm in 24/81 (30%) and localized exclusively in the cytoplasm in 48/81 (59%) of the tumours. Cyclin D1 was overexpressed in both borderline and invasive tumours. There was no association between protein overexpression and tumour stage and differentiation. Furthermore, no correlation between cyclin D1 expression and clinical outcome was observed. However, in tumours overexpressing cyclin D1 (n = 72), the proportion displaying exclusively cytoplasmic localization of protein was higher in those with serous compared with non-serous histology (P = 0.004, odds ratio 4.8, 95% confidence interval 1.4–19.1). Western analysis using a monoclonal antibody to cyclin D1 identified a 36 kDa protein in homogenates from seven tumours displaying cytoplasmic only and one tumour demonstrating both nuclear and cytoplasmic immunostaining. Using restriction fragment length polymorphism polymerase chain reaction and PCR-multiplex analysis, amplification of the cyclin D1 gene (CCNDI) was detected in 1/29 of the tumours demonstrating overexpression of cyclin D1 protein. We conclude that deregulation of CCND1 expression leading to both cytoplasmic and nuclear protein localization is a frequent event in ovarian cancer and occurs mainly in the absence of gene amplification.

Susceptibility and outcome in oral cancer : preliminary data showing an association with polymorphism in cytochrome P450 CYP2D6

Members of the cytochrome P450 and glutathione S-transferase supergene families are candidates for susceptibility and outcome in oral squamous cell cancer. We determined GSTM1, GSTM3, GSTT1, CYP1A1 and CYP2D6 genotypes in 100 Caucasian cases and 467 control individuals. The frequency of homozygosity for mutant CYP2D6 alleles was higher in the cases (P = 0.001, OR = 3.2, 95% CI = 1.6-6.5) than control individuals. In the cases, the frequency of homozygosity for mutant alleles was greater and that of homozygosity for wild-type CYP2D6 alleles was lower in those diagnosed at > or = 65 years (P = 0.009) than in those diagnosed at < or = 64 years. The older cases included relatively more women and patients who did not consume tobacco or alcohol. The association of CYP2D6 with outcome was assessed using the Coxs proportional hazards model. The time to first cervical node metastasis was shorter in heterozygotes and homozygotes for mutant CYP2D6 alleles compared with homozygotes for wild-type alleles after correction for age at diagnosis, gender, alcohol and tobacco consumption and tumour differentiation (P = 0.04, hazard ratio 3.6, 95% CI 1.1-12.5). The mechanism for the association of CYP2D6 alleles with susceptibility and outcome is unclear though the data are compatible with the view that homozygosity for mutant alleles confers impaired detoxication of an unknown carcinogen. No associations between GSTM1, GSTM3, GSTT1 or CYP1A1 genotypes and susceptibility or, time to node metastases were identified. We previously showed that CYP2D6 genotypes were not associated with susceptibility to squamous cell cancer in the pharynx or larynx. Therefore, the data presented suggest that susceptibility to squamous cell cancer in the various parts of the upper aerodigestive tract is associated with different genes and allelic variants.

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.

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.

Vitamin D receptor gene polymorphism is associated with reduced disability in multiple sclerosis.

Ultraviolet radiation (UVR) may contribute to multiple sclerosis (MS) outcome by a mechanism involving vitamin D and the vitamin D receptor (VDR). In 512 patients with MS duration of 10 or more years, we studied the association of VDR single nucleotide polymorphisms (A/G1229, C/G3444, G/A3944, CC20965, CC30056, F/f30875, C/T48200, T/t65013) with outcome or disability. ff30875 frequency was lower in cases with EDSS ≥ 6.0 than with scores < 6.0 (odds ratio = 0.38, 95% CI = 0.20–0.70). The association of ff30875 with outcome was not mediated by cumulative exposure to UVR as assessed by questionnaire; low exposure (odds ratio = 0.42, 95% CI = 0.14–1.34) and high exposure (odds ratio = 0.34, 95% CI = 0.16–0.73).

Susceptibility to basal cell carcinoma: associations with PTCH polymorphisms.

Loss of function of the human patched gene (PTCH) is common and critical in basal cell carcinoma (BCC) development. Indirect evidence suggests polymorphism in PTCH mediates BCC risk. We studied 659 BCC cases and 300 controls to determine if exon 2318, 3429, 111552, 121665, 121686, 142199 and 233944 and intron 91336‐135 and 152560+9PTCH variants were sufficiently common for use in case‐control studies, and if selected markers were associated with risk. Intron 152560+9 and exon 233944 variants were studied further. Their genotype frequencies were not significantly different in controls and cases, though frequency of the G2560+9‐C3944 haplotype was lower in all cases (odds ratio = 0.44, p = 0.009) and those stratified by BCC site and rate of development of further tumours. This association was not mediated by the extent of UVR exposure. We confirmed the robustness of these findings by showing these associations demonstrated similar odds ratios in two groups of randomly selected cases and controls, and using the false positive report probability (FPRP) approach described by Wacholder et al. (2004) . The FPRP value (0.168) was in the noteworthy category. These data, showing for the first time that PTCH polymorphism mediates susceptibility, are compatible with reports showing that PTCH haploinsufficiency influences development of BCC precursor lesions.

Environment, phenotype and genetics: risk factors associated with BCC of the skin

Basal cell carcinoma is the most common cancer in Caucasians and its incidence is rising. Whilst not life threatening, the tumor can cause substantial morbidity and because of long follow-up, it places a major burden on healthcare agencies worldwide. Patients with the disease demonstrate wide phenotypic diversity with respect to tumor numbers, rate of tumor appearance and site. The factors involved in patient susceptibility to basal cell carcinoma are not well understood although exposure to ultraviolet radiation in sunlight appears critical. In this review we discuss the role of environmental and genetic factors on predisposition to basal cell carcinoma and illustrate how stratification of the basal cell carcinoma cohort into high-risk subgroups is helpful in identifying factors significant in disease susceptibility.

Induced expression of human CCND1 alternative transcripts in mouse Cyl-1 knockout fibroblasts highlights functional differences.

Splicing of human cyclin D1 (CCND1) mRNA producing transcripts a and b is modulated by a common polymorphism (A → G) located in a conserved splice donor region at nucleotide 870. CCND1 A/G870 genotype is associated with tumour progression and clinical outcome in a variety of cancers. Although in vitro expression of cyclin D1 transcript a (CCND1tra) has been widely investigated, few studies have examined the expression of CCND1 transcript b (CCND1trb). We have studied the effects of inducible expression of human CCND1trb in comparison with human CCND1tra in a mouse fibroblast knock‐out for cyclin D1 (MEFCyl‐1−/−). Inducible expression was in stable clones isolated from MEFCyl‐1−/− transfectants. Induction of CCND1tra produced a 36‐kDa protein, which led to a significant increase in the proportion of cells in S‐phase, as detected by BrdU incorporation after 32 hr, compared to non‐induced cells (p = 0.012). Clones induced to express CCND1tra exhibited a significantly increased ability to grow in serum depleted (2% FCS) medium compared to non‐induced clones (p = 0.0004). Induced expression of CCND1trb in MEFCyl‐1−/− transfectants produced a 31‐kDa protein and resulted in no significant difference in DNA synthesis, neither did the cells acquire the ability to grow in serum‐depleted conditions compared to non‐induced cells. Induction of CCND1trb significantly enhanced the ability of MEFCyl‐1−/− transfectants to form colonies in soft agar, (average 30‐fold increase) compared to non‐induced clones or those induced to express CCND1tra. Our data supports the emerging view that CCND1 alternate transcripts encode proteins with differing independent biological functions. We suggest that CCND1tra encodes a protein involved in regulating mitogen responsive, anchorage‐dependent G1 progression, whereas CCND1trb modulates the ability of the cell to grow in an anchorage‐independent manner.