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/G(1229), C/G(3444), G/A(3944), CC(20965), CC(30056), F/f(30875), C/T(48200), T/t(65013)) with outcome or disability. ff(30875) frequency was lower in cases with EDSS > or = 6.0 than with scores < 6.0 (odds ratio = 0.38, 95% CI = 0.20-0.70). The association of ff(30875) 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).

RhoA signaling modulates cyclin D1 expression in human lung fibroblasts; implications for idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic Pulmonary Fibrosis (IPF) is a debilitating disease characterized by exaggerated extracellular matrix deposition and aggressive lung structural remodeling. Disease pathogenesis is driven by fibroblastic foci formation, consequent on growth factor overexpression and myofibroblast proliferation. We have previously shown that both CTGF overexpression and myofibroblast formation in IPF cell lines are dependent on RhoA signaling. As RhoA-mediated regulation is also involved in cell cycle progression, we hypothesise that this pathway is key to lung fibroblast turnover through modulation of cyclin D1 kinetic expression. METHODS: Cyclin D1 expression was compared in primary IPF patient-derived fibroblasts and equivalent normal control cells. Quantitative real time PCR was employed to examine relative expression levels of cyclin D1 mRNA; protein expression was confirmed by western blotting. Effects of Rho signaling were investigated using transient transfection of constitutively active and dominant negative RhoA constructs as well as pharmacological inhibitors. Cellular proliferation of lung fibroblasts was determined by BrdU incorporation ELISA. To further explore RhoA regulation of cyclin D1 in lung fibroblasts and associated cell cycle progression, an established Rho inhibitor, Simvastatin, was incorporated in our studies. RESULTS: Cyclin D1 expression was upregulated in IPF compared to normal lung fibroblasts under exponential growth conditions (p < 0.05). Serum deprivation inhibited cyclin D1 expression, which was restored following treatment with fibrogenic growth factors (TGF-beta1 and CTGF). RhoA inhibition, using a dominant negative mutant and a pharmacological inhibitor (C3 exotoxin), suppressed levels of cyclin D1 mRNA and protein in IPF fibroblasts, with significant abrogation of cell turnover (p < 0.05). Furthermore, Simvastatin dose-dependently inhibited fibroblast cyclin D1 gene and protein expression, inducing G1 cell cycle arrest. Similar trends were observed in control experiments using normal lung fibroblasts, though exhibited responses were lower in magnitude. CONCLUSION: These findings report for the first time that cyclin D1 expression is deregulated in IPF through a RhoA dependent mechanism that influences lung fibroblast proliferation. This potentially unravels new molecular targets for future anti-IPF strategies; accordingly, Simvastatin inhibition of Rho-mediated cyclin D1 expression in IPF fibroblasts merits further exploitation.

Overexpression of aurora B kinase (AURKB) in primary non-small cell lung carcinoma is frequent, generally driven from one allele, and correlates with the level of genetic instability.

Aurora kinases are key regulators of chromosome segregation during mitosis. We have previously shown by microarray analysis of primary lung carcinomas and matched normal tissue that AURKB (22 out of 37) and AURKA (15 out of 37) transcripts are frequently over-represented in these tumours. We now confirm these observations in a second series of 44 carcinomas and also show that aurora B kinase protein levels are raised in the tumours compared to normal tissue. Elevated levels of expression in tumours are not a consequence of high-level amplification of the AURKB gene. Using a coding sequence polymorphism we show that in most cases (seven out of nine) tumour expression is predominantly driven from one AURKB allele. Given the function of aurora B kinase, we examined whether there was an association between expression levels and genetic instability. We defined two groups of high and low AURKB expression. Using a panel of 10 microsatellite markers, we found that the group showing the higher level of expression had a higher frequency of allelic imbalance (P=0.0012). Analysis of a number of other genes that are strongly and specifically expressed in tumour over normal lung, including SERPINB5, TERT and PRAME, showed marked allelic expression imbalances in the tumour tissue in the context of balanced or only marginally imbalanced relative allelic copy numbers. Our data support a model of early carcinogenesis wherein defects in the process of inactivation of lung stem-cell associated genes during differentiation, contributes to the development of carcinogenesis.

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 2(318), 3(429), 11(1552), 12(1665), 12(1686), 14(2199) and 23(3944) and intron 9(1336-135) and 15(2560+9)PTCH variants were sufficiently common for use in case-control studies, and if selected markers were associated with risk. Intron 15(2560+9) and exon 23(3944) variants were studied further. Their genotype frequencies were not significantly different in controls and cases, though frequency of the G(2560+9)-C(3944) 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.

Association between glutathione-S-transferase GSTP1 genotypes, GSTP1 over-expression, and outcome in epithelial ovarian cancer.

Ovarian cancer accounts for the majority of deaths from gynaecological malignancy, and polymorphisms in genes encoding the glutathione-S-transferase (GST) GSTP1 detoxifying enzymes may lead to variation in detoxification of carcinogens. We describe a study involving 81 women with invasive epithelial ovarian cancer. A number of important clinical variables and outcome data were obtained. GSTP1 genotyping was undertaken using PCR-based techniques, and GSTP1 expression was quantified using immunohistochemistry (IHC). A Cox's proportional hazard regression model was used to analyze the effects on outcome. We also independently examined 11 women with borderline or low malignant potential (LMP) tumors using IHC only. The mean age of the women was 61.5 years +/- 12 (1 SD) (range 36-88 years), the median overall survival was 26 months, and median progression free interval (PFI) 21 months. There was a significant association between GSTP1 (Val(104)/Val(104)) genotypes, and reduced survival (P = 0.05) and the GTP1 (Ile(104)/Val(104)) genotype appeared to have the best outcome (HR = 0.34, P = 0.045, 95% CI = 0.12-0.98). There was no significant association between the GSTP1 genotypes and any clinico-pathological parameters; there were also no associations between GSTP1 genotypes and response to postoperative chemotherapy. Specific nuclear GSTP1 over-expression was associated with less residual disease (P = 0.05); specific cytoplasmic GSTP1 over-expression with more favourable performance status (P = 0.014)). We found that 10/11 (91%) of the LMP (borderline) tumors over-expressed nuclear GSTP1 compared to only 52% of the invasive tumors (chi(2) ((1)) = 5.95, P = 0.015). There was no significant association between the level of GSTP1 expression and response to postoperative chemotherapy. The overall level of GSTP1 expression and the subcellular localization of GSTP1 expression were not associated with either survival or PFI. There was a significant association between the GSTP1 (Ile(104)/Ile(104)) genotypes and increased overall GSTP1 expression (P = 0.049), and the GSTP1 (Ile(104)/Val(104)) genotypes and reduced overall GSTP1 expression (P = 0.046). We speculate that GSTP1 Ile(104)/Val(104) genotypes are associated with improved outcome because the protein/enzyme, which is expressed, may provide a better balance between the effects of detoxification of carcinogens and the effects of metabolism of chemotherapy agents. In addition, over-expression of nuclear GSTP1 appears to be associated with more favorable ovarian tumor characteristics. In our preliminary study, we also reported a relationship between overall GSTP1 expression and certain GSTP1 genotypes. As far as we are aware, this is the first time that a relationship between the GSTP1 genotypes, GSTP1 expression and outcome has been described in ovarian cancer. Whether the genotype directly determines GSTP1 expression is at present unclear and the precise mechanism of this interaction is unknown.

Molecular analysis of region t(5;6)(q21;q21) in Wilms tumor.

We have previously described the physical localization of a constitutional t(5;6)(q21;q21) in a patient (tumor cell sample designated as MA214) with bilateral Wilms tumor (WT). We have now physically refined the breakpoints and identified putative gene targets within this region. The translocation breakpoints are contained within a 2.5-Mbp region on 5q21 containing four candidate genes and a 1.3-Mbp region on 6q21 that contains three candidate genes. To explore the role of this region in WT genesis, we have performed loss of heterozygosity (LOH) analysis with markers flanking the translocation breakpoints in tumor from MA214 and a panel of sporadic WT. Alleles were retained for all informative markers used in the MA214 tumor. In sporadic tumors LOH was found in 6 of 63 (9.5%) and 5 of 62 (8%) informative cases for flanking markers D6S301 and D6S1592 on 6q21. LOH was found in 3 of 58 (5.2%) and 2 of 54 (3.6%) for flanking markers D5S495 and D5S409 on 5q21. These preliminary data suggest LOH at the t(5;6)(q21;q21) region is unlikely to be a mechanism for tumor development in MA214, but may be important for a subgroup of sporadic WT.

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.

Subcellular localisation of cyclin D1 protein in colorectal tumours is associated with p21(WAF1/CIP1) expression and correlates with patient survival.

We investigated the expression of the cell cycle regulatory proteins cyclin D1 and p21(WAF1/CIP1) (p21) in human colorectal carcinomas using immunohistochemistry. Cyclin D1 was not detected in normal colonic epithelium; however, expression was observed in 74/126 (58.7%) of the tumour samples studied. Protein was detected in the nucleus in 22/126 (17.4%) and exclusively in the cytoplasm in 52/126 (41.3%) tumours. Nuclear expression of cyclin D1 was associated with poorly differentiated tumours (p = 0.035) and was more common in right- than in left-sided tumours (p = 0.005). Tumours displaying either, expression of cytoplasmic, (p = 0.05, HR 0.56, 95% CI 0.31-1.0) or nuclear (p = 0.021, HR 0.24, 95% CI 0.07-0.81) cyclin D1 were associated with improved patient survival compared with tumours negative for cyclin D1. p21 protein was strongly expressed mainly in the upper crypts of normal colonic epithelial cells, but in 63/126 (50%) of the tumour samples studied p21 expression was absent. Patients with tumours in which >50% of cells expressed p21 had improved survival compared to patients whose tumours were negative or had < or =50% of cells expressing p21 (p = 0.06, HR 0.33, 95% CI 0.1-1.0). We also observed a significant association between cyclin D1 subcellular localisation and p21 expression: 21/22 (95.5%) tumours expressing cyclin D1 in the nucleus also expressed p21, whereas only 17/52 (32.7%) of the tumours displaying exclusive cytoplasmic cyclin D1 staining were positive for p21 (p < 0.001). These data highlight the significance of exclusive cytoplasmic expression of cyclin D1 in colorectal cancer and lend support to recent in vitro studies suggesting that p21 protein may modulate the subcellular localisation of the cyclin D1 protein. Thus, deregulated expression of the cyclin D1 and p21 proteins are important in colorectal tumourigenesis and have implications for patient prognosis.

Glutathione S-transferase GSTM1 and GSTT1 genotypes in ovarian cancer: association with p53 expression and survival.

The objective of this study was to determine whether the association between GSTM1 null/GSTTI null and survival in ovarian cancer is mediated by the influence of these genes on p53 expression. In 81 women with pure invasive ovarian cancer, GSTM1 null and GSTT1 null genotypes were identified using polymerase chain reaction and p53 expression was assessed using immunohistochemistry. The association of these factors with survival was examined using Cox's proportional hazards regression models. Performance status (P < 0.001), operative stage (P = 0.004), residual disease (P = 0.001), histologic subtype (P = 0.05), tumor grade (P = 0.007), and the combined GSTMI null/GSTTl null genotype (P = 0.023) were all individually associated with survival. p53 expression was not associated with survival (P = 0.45). In a multivariate analysis, the effects of GSTM1 null/GSTT1 null on survival were lost when residual disease and tumor grade were included. The effects of p53 expression on survival were unchanged when residual disease, tumor grade, operative stage, and performance score were included. GSTM1 null/GSTT1null did not influence the effects of p53 expression on survival and vice versa. The GSTM1 null/GSTT1 null genotype was associated with response to primary chemotherapy (P = 0.007) but p53 expression was not. We conclude that the association of GSTM1 null/GSTTl null with survival appears to be mediated through different mechanisms to p53 expression in ovarian cancer and in addition, may be a better predictor of outcome.

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.

Genetic polymorphism and clinical outcome: identification of individuals at risk of a poor clinical outcome.

Susceptibility and outcome in complex disorders such as asthma and cancer appear to be determined, at least in part, by genetic polymorphism. However, while our ability to identify new allelic variants and study them in case and control populations has greatly improved, considerable difficulties remain in elucidating how many genes determine particular clinical phenotypes. This is because most studies have concentrated on study of single genes in relatively small study groups. The important issues of gene-gene interactions (epistasis) and high-risk subgroups have not yet been adequately addressed. We now describe a general approach, using patients with head and neck cancers as an example. Our purpose is to demonstrate candidate gene selection, statistical approaches, and identification of patient subgroups.

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

The 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 (CCND1 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.

Cyclin D1, glutathione S-transferase, and cytochrome P450 genotypes and outcome in patients with upper aerodigestive tract cancers: assessment of the importance of individual genes using multivariate analysis.

GST, CYP, and CCND1 genotypes have been associated with outcome in several cancers. Accordingly, we have examined, in patients with one squamous cell carcinoma (SCC) of the head and neck, associations between GSTM1, GSTT1, GSTM3, GSTP1, CYP2D6, CYP1A1, CYP2E1, and CCND1 genotypes and the outcome parameters, tumor extension, histological grade, and presence of nodes. We used logistic regression to study, first, each gene individually and, second, in a step-wise model that included all of the genes. Different genes were associated with each outcome parameter. Thus, GSTT1 null was associated with T3/T4 lesions in the oral cavity/pharyngeal (P = 0.029), but not laryngeal, SCC cases. GSTT1 null was also associated with histological differentiation (G3) in the oral cavity/pharyngeal, but not laryngeal, SCC cases, although this association only approached significance (P = 0.069). CCND1 GG was associated with G3 tumors in the oral cavity/pharyngeal (P = 0.011), but not laryngeal, SCC cases. The combination of GSTT1 null/CCND1 GG was also associated with G3 tumors. CYP2D6 PM and HET were associated with lymph node involvement in the laryngeal, but not oral/pharynx, SCC cases. Genes that were individually associated with outcome were also associated with the parameter in the step-wise routine. The GSTT1 null frequency was greater in 39 patients with second primary tumors than in those with one lesion (P = 0.014). The data demonstrate site-dependent associations between GSTT1 null, CCND1 GG, and CYP2D6 PM and tumor extension, differentiation, and nodes.

Analysis of cyclin D1 (CCND1) allelic imbalance and overexpression in sporadic human pituitary tumors.

Cyclin D1 plays an important role in the regulation of cell progression through G1 of the cell cycle and has been demonstrated to have oncogenic properties. Using RFLP-PCR, an A/G polymorphism within the cyclin D1 (CCND1) gene was analyzed in 151 sporadic human pituitary tumors, of which 60 were informative at this locus. Further analysis showed that in 15 of 60 (25%) tumors, there was evidence of allelic imbalance, which is indicative of gene amplification. Allelic imbalance was observed more frequently in invasive tumors (11 of 29 tumors; 38%) than in their noninvasive counterparts (4 of 31 tumors; 13%; P = 0.02). Forty-six of the tumors informative for the polymorphism were available for immunohistochemical analysis. Cyclin D1 expression (nuclear and/or cytoplasmic) was detected in 25 of 46 (54%) tumors. Of these cases, expression of nuclear cyclin D1 was detected in 9 of 46 (20%) tumors, whereas 16 of 46 (35%) tumors showed cyclin D1 staining exclusively confined to the cytoplasm. Neither nuclear staining nor cytoplasmic staining was observed in any of the normal pituitaries or in the negative control. Expression of cyclin D1 was observed in significantly more nonfunctional tumors (18 of 27 tumors; 67%) than in somatotrophinomas (7 of 19 tumors; 37%; P = 0.046). Nuclear cyclin D1 expression was observed more frequently in nonfunctional tumors (8 of 27 tumors; 30%) than in somatotrophinomas (1 of 19 tumors; 5%; P = 0.04). There was no correlation between cyclin D1 expression and tumor grade or between allelic imbalance of CCND1 and cyclin D1 expression. We conclude that amplification of CCND1 occurs in pituitary tumors and that the overexpression of cyclin D1 may be an early event in tumorigenesis. Cyclin D1 overexpression occurring in the absence of CCND1 allelic imbalance suggests that additional mechanisms responsible for deregulated cyclin D1 expression are involved in human pituitary tumorigenesis.

Prognostic significance of cyclin D1 gene (CCND1) polymorphism in epithelial ovarian cancer.

We have investigated the influence of CCND1 genotype on clinical outcome in 138 women with epithelial ovarian cancer. CCND1 genotypes were identified from peripheral blood DNA by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis. Patient CCND1 genotypes were compared with clinical details including FIGO tumor stage, residual tumor volume, tumor histology and differentiation, response to chemotherapy, progression free interval, and survival. We observed no association between patient CCND1 genotypes and tumor characteristics or response to chemotherapy. There was no significant difference in overall survival and progression free interval (PFI) among women with different CCND1 genotypes. However, analysis of data from patients who responded to postoperative chemotherapy revealed that women with CCND1 AA genotype were associated with early disease progression (P = 0.020, HR 4.58, 95% CI 1.27-16.48) and reduced survival (P = 0.026, HR 4.48, 95% CI 1.19-16.79) compared with those with CCND1 AG and GG genotypes. These data show that CCND1 genotype does not influence overall prognosis in a cohort of epithelial ovarian cancer patients, however, it is associated with disease progression in a subgroup of patients following initial response to chemotherapy.

Polymorphism within the cyclin D1 gene is associated with prognosis in patients with squamous cell carcinoma of the head and neck.

We have examined the correlation of a frequent A/G polymorphism within exon 4 of the cyclin D1 gene (CCND1) with genetic susceptibility and clinical outcome in 384 patients with squamous cell carcinoma (SCC) of the head and neck. CCND1 genotype frequencies were similar in the cases and 191 controls. Furthermore, the CCND1 genotype was not associated with susceptibility to SCC of the larynx, pharynx, or oral cavity. The influence of the CCND1 genotype on clinical outcome was also assessed. We found no correlation between genotype and tumor size (T1-T4), the involvement of nodes at presentation, or patient age and gender. However, the distribution of CCND1 genotypes in cases with poorly differentiated tumors was significantly different to that in patients with well-/moderately differentiated tumors (P = 0.016; chi2(2) = 8.71). Homozygosity for CCND1*G (GG genotype) was associated with poorly differentiated tumors (G3). We used Cox's proportional hazards model to investigate the influence of the CCND1 genotype on disease-free interval. CCND1 GG was associated with reduced disease-free interval [P = 0.001; hazard ratio (HR) = 2.95; 95% confidence interval (CI) = 1.54-5.63]. This remained significant after correction for tumor differentiation (P = 0.013; HR = 2.34; 95% CI = 1.2-4.6) and tumor stage (P = 0.005; HR = 2.64; 95% CI = 1.34-5.19). Analysis of the data from patients with tumors at different sites showed that the CCND1 GG genotype was associated with reduced disease-free interval in laryngeal (P = 0.004; HR = 3.63; 95% CI = 1.44-8.83) and pharyngeal (P = 0.006; HR = 3.48; 95% CI = 1.43-8.46) tumors. This is the first report of an association between CCND1 polymorphism and prognosis in SCC of the head and neck. These data show that the CCND1 GG genotype is an independent prognostic indicator of disease-free interval and supports initial observations in non-small cell lung cancer, that polymorphism within CCND1 influences tumor behavior.

Association of glutathione S-transferase GSTM1 and GSTT1 null genotypes with clinical outcome in epithelial ovarian cancer.

Epithelial ovarian cancer is generally associated with a poor outcome, although the mechanisms that determine survival and progression-free interval (PFI) are unclear. Data from ovarian tumors showing associations between (a) null genotypes at the glutathione S-transferase GSTM1 and GSTT1 loci and expression of p53 protein and (b) outcome and expression of p53 suggest that polymorphism at these loci is a factor determining outcome. Accordingly, we have studied the association between the GSTM1 null and GSTT1 null genotypes and survival and PFI in 148 women with epithelial ovarian cancer. Although we did not find an association between individual genotypes and outcome, women with both GSTM1 null and GSTT1 null genotypes demonstrated poorer survival (P = 0.001) and reduced PFI (P = 0.003). Thus, no cases with both these genotypes survived past 42 months postdiagnosis. In contrast, 43% of the women without this combination survived beyond this time. Because response to chemotherapy is a major factor determining outcome in ovarian cancer, we also examined the data for associations between the glutathione S-transferase genotypes and response to such treatment. Thus, in 78 patients treated with chemotherapy, the combination of GSTM1 null and GSTT1 null was associated with unresponsiveness to primary chemotherapy (P = 0.004); none of the eight patients with both these genotypes responded, compared with 38 of 70 (54%) of patients with other genotype combinations. The effect of the combination of genotypes on survival and PFI was lost in a multivariate model that included response to chemotherapy as a confounding factor. This suggests that the combination of GSTM1 null/GSTT1 null is associated with outcome because of its influence on response to chemotherapy. These preliminary findings may provide a basis for the selection of patients for treatment with chemotherapeutic agents.

Physical localisation of the breakpoints of a constitutional translocation t(5;6)(q21;q21) in a child with bilateral Wilms' tumour.

A 6 month old boy presented with bilateral Wilms' tumour. Cytogenetic analysis of the lymphocytes from the patient showed a de novo balanced translocation t(5;6)(q21;q21), which was also present in the tumour material as the sole cytogenetic abnormality. To facilitate the identification of the translocation breakpoints, we have established a lymphoblastoid cell line (MA214L) from the patient which maintains the translocation in culture. We have used Genethon microsatellite markers as sequence tagged sites (STSs) to isolate yeast artificial chromosome (YAC) clones to 5q and 6q from human genomic libraries. Using fluorescence in situ hybridisation (FISH) on metaphase preparations of MA214L, we have physically defined the translocation breakpoints between YAC clones on each chromosome arm. The genetic distance separating the flanking YACs on 6q21 is 3 cM, while that on 5q21 is 4 cM. To date this is the first report of these chromosomal regions being implicated in Wilms' tumourigenesis.

High levels of loss at the 17p telomere suggest the close proximity of a tumour suppressor.

High levels of loss of distal markers on 17p13.3 in breast cancer suggested the presence within the region of at least one tumour-suppressor gene. Here we describe the derivation of two biallelic polymorphisms from the 17p telomeric yeast artificial chromosome (YAC) TYAC98. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and multiplex PCR analysis demonstrated that the high level of allelic imbalance observed in breast tumours represented loss of constitutional heterozygosity (LOH) and that this LOH extended to the telomere. Lung carcinoma (but not Wilms' tumour)-derived DNA again revealed a high level of loss of subtelomeric 17p sequences. Telomeric microsatellite polymorphisms from other chromosome arms did not show such elevated loss in either tumour type. This suggested that the 17p loss observed did not reflect a general telomeric instability and provided further evidence for the presence of a breast cancer tumour-suppressor gene in the distal region of 17p13.3.