The L84F polymorphic variant of human O6-methylguanine-DNA methyltransferase alters stability in U87MG glioma cells but not temozolomide sensitivity

First-line therapy for patients with glioblastoma multiforme includes treatment with radiation and temozolomide (TMZ), an oral DNA alkylating chemotherapy. Sensitivity of glioma cells to TMZ is dependent on the level of cellular O(6)-methylguanine-DNA methyltransferase (MGMT) repair activity. Several common coding-region polymorphisms in the MGMT gene (L84F and the linked pair I143V/K178R) modify functional characteristics of MGMT and cancer risk. To determine whether these polymorphic changes influence the ability of MGMT to protect glioma cells from TMZ, we stably overexpressed enhanced green fluorescent protein (eGFP)-tagged MGMT constructs in U87MG glioma cells. We confirmed that the wild-type (WT) eGFP-MGMT protein is properly localized within the nucleus and found that L84F, I143V/K178R, and L84F/I143V/K178R eGFP-MGMT variants exhibited nuclear localization patterns indistinguishable from WT. Using MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] proliferation and clonogenic survival assays, we confirmed that WT cells expressing eGFP-MGMT are resistant to TMZ treatment compared with control U87MG cells, and that each of the polymorphic eGFP-MGMT variants confers similar resistance to TMZ. However, upon exposure to O(6)-benzylguanine (O(6)-BG), a synthetic MGMT inhibitor, the L84F and L84F/I143V/K178R variants were degraded more rapidly than WT or I143V/K178R in a proteasome-dependent manner. Despite the increased O(6)-BG- stimulated protein turnover caused by the L84F alteration, cells expressing L84F eGFP-MGMT did not exhibit altered sensitivity to the combination of O(6)-BG and TMZ compared with WT cells. In conclusion, we demonstrated that the L84F polymorphic variant has altered protein turnover without modifying sensitivity of U87MG cells to TMZ or combined TMZ and O(6)-BG. These findings may provide a clue to determining the clinical significance of MGMT coding-region polymorphisms.     

Association of genetic variants of O6-methylguanine-DNA methyltransferase with risk of lung cancer in non-Hispanic Whites.

O6-methylguanine, a methylated damage lesion in DNA, correlates with spontaneous G:C --> A:T transition mutations and leads to activation of oncogene K-ras or dysfunction of the tumor suppressor gene p53. O6-methylguanine-DNA methyltransferase (MGMT) is critical for repairing damage to the O6-position of guanine. Therefore, we tested our hypothesis that genetic variants of MGMT are associated with increased lung cancer risk in a Caucasian population of 1,121 lung cancer patients and 1,163 matched cancer-free controls. We genotyped four potentially functional single nucleotide polymorphisms (SNPs) of MGMT: exon 3 codon 84C --> T (L84F), exon 5 codon 143A --> G (I143V), and two promoter SNPs 135G --> T and 485C --> A. The allele frequency distributions of the SNPs of codon 84C --> T and the promoter 135G --> T in the cases were borderline different from that in the controls. After defining the minor allele (T for codon 84C --> T and G for codon 143A --> G) as the variant allele, we categorized the MGMT genotypes as either 0 variants (84CC-143AA) or 1-4 variants. Compared with 0 variants, those with 1-4 variants showed a statistically significantly increased risk of lung cancer (P = 0.040). Further stratification analysis showed that this increased risk was more pronounced in women, current smokers, and non-small cell lung cancer. We did not find any association between the MGMT promoter SNPs and lung cancer risk. Our findings suggest that non-synonymous SNPs in MGMT are associated with modestly increased risk of lung cancer in Caucasians and need to be further investigated.     

Immunohistochemical analysis of DNA mismatch repair protein and O6-methylguanine-DNA methyltransferase in melanoma metastases in relation to clinical response to DTIC-based chemotherapy

DNA mismatch repair (MMR) deficiency and increased O6-methylguanine-DNA methyltransferase (MGMT) activity have been related to resistance to O6-guanine methylating agents in tumour cell lines. However, the clinical relevance of MMR and MGMT as drug resistance factors is still unclear. In a retrospective study, the expression levels of the MMR proteins, hMSH2, hMSH6 and hMLH1, were analysed by immunohistochemistry in melanoma metastases from 64 patients, who had received dacarbazine (DTIC) based chemotherapy. More than half of the melanoma patients had tumours with no nuclear staining for either hMSH2 or hMSH6 or both, while all tumours showed positive nuclear staining for hMLH1. The response rates were similar in patients with hMSH2 and/or hMSH6 positive tumours to these in patients with negative tumours. By combination of MMR with previously obtained MGMT data, only 2 of 12 responders had tumours with low MGMT and positive MMR expression. Still all except 3 of the non-responders were identified by having either high MGMT expression or absent staining for hMSH2 or hMSH6 or both in their tumours. However, there was no significant correlation of MMR expression alone or combined with MGMT levels with clinical response to DTIC-based chemotherapy in metastatic melanoma.     

Selected polymorphisms of DNA repair genes and risk of pancreatic cancer

BACKGROUND: Genetic variants of DNA repair genes may contribute to pancreatic carcinogenesis. O(6)-methylguanine-DNA methyltransferase (MGMT) is the major protein that removes alkylating DNA adducts, and apurinic/apyrimidinic endonuclease 1 (APE1) and X-ray repair cross-complementing group 1 (XRCC1) play important roles in the base excision repair pathway. METHODS: We investigated the association between polymorphisms of MGMT (Leu(84)Phe and Ile(143)Val), APE1 (Asp(148)Glu), and XRCC1 (Arg(194)Trp and Arg(399)Gln) and risk of pancreatic cancer in a case-control study. Exposure information from 384 patients with primary pancreatic ductal adenocarcinoma and 357 cancer-free healthy controls were collected and genomic DNAs were genotyped for five markers. Controls were frequency matched to patients by age at enrollment (+/-5 years), gender, and race. We estimated odds ratios (ORs) and 95% confidence intervals (CIs) by using unconditional logistic regression models. RESULTS: There was no significant main effect or interaction with smoking of these genetic variants on the risk of pancreatic cancer. However, the XRCC1(194) polymorphism had a significant interaction with the APE1(148) (p=0.005) or MGMT(84) polymorphism (p=0.02) in modifying the risk of pancreatic cancer. CONCLUSIONS: This study suggests that polymorphisms of genes involved in the repair of alkylating DNA adduct and DNA base damage may play a role in modulating the risk of pancreatic cancer. Larger studies are required to validate these preliminary findings. The mechanism of the combined genotype effects remains to be elucidated.     

O6-methylguanine-DNA methyltransferase depletion and DNA damage in patients with melanoma treated with temozolomide alone or with lomeguatrib

We evaluated the pharmacodynamic effects of the O⁶-methylguanine-DNA methyltransferase (MGMT) inactivator lomeguatrib (LM) on patients with melanoma in two clinical trials. Patients received temozolomide (TMZ) for 5 days either alone or with LM for 5, 10 or 14 days. Peripheral blood mononuclear cells (PBMCs) were isolated before treatment and during cycle 1. Where available, tumour biopsies were obtained after the last drug dose in cycle 1. Samples were assayed for MGMT activity, total MGMT protein, and O⁶-methylguanine (O⁶-meG) and N7-methylguanine levels in DNA. MGMT was completely inactivated in PBMC from patients receiving LM, but detectable in those on TMZ alone. Tumours biopsied on the last day of treatment showed complete inactivation of MGMT but there was recovery of activity in tumours sampled later. Significantly more O⁶-meG was present in the PBMC DNA of LM/TMZ patients than those on TMZ alone. LM/TMZ leads to greater MGMT inactivation, and higher levels of O⁶-meG than TMZ alone. Early recovery of MGMT activity in tumours suggested that more protracted dosing with LM is required. Extended dosing of LM completely inactivated PBMC MGMT, and resulted in persistent levels of O⁶-meG in PBMC DNA during treatment.     

Polymorphisms in MGMT and DNA repair genes and the risk of esophageal adenocarcinoma

Rates of adenocarcinoma of the esophagus (EAC) and esophago-gastric junction (EGJAC) have increased rapidly in recent decades. The primary risk factors, gastro-esophageal acid reflux and smoking, are potentially genotoxic through the generation of N-nitroso compounds. The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) is the major cellular defense against alkylating DNA damage. We compared patients with EAC (n = 263) or EGJAC (n = 303) with matched population controls (n = 1,337) for the frequency of 5 MGMT single nucleotide polymorphisms (SNPs) (rs12269324, rs12268840, L84F, I143V, K178R), as well as SNPs in DNA repair genes ERCC1 (N118N), XRCC1 (Q399R) and XPD (K751Q). Relative risks were estimated using multivariable logistic regression. Potential biological interaction was assessed through the synergy index S. Each MGMT SNP conferred increased risks of EAC but not EGJAC; strongest associations were found for the 2 variant MGMT alleles rs12268840 and I143V (p = 0.005 and p < 0.001, respectively). Homozygous carriers of MGMT rs12268840 with frequent acid reflux had significantly higher risks of EAC (OR 15.5, 95% CI 5.8-42) than expected under an additive model, consistent with biological interaction (S = 3.3, 95% CI 1.1-10). Modest, nonsignificant interactions with smoking were also observed. Homozygous variant ERCC1 genotype was associated with reduced risks of EAC (OR 0.6, 95% CI 0.4-1.1), while the homozygous variant XRCC1 genotype conferred higher risks of EGJAC (OR 1.6, 95% CI 1.1-2.4). No associations with EAC or EGJAC were observed with XPD (rs13181). In summary, MGMT SNPs are associated with increased risks of EAC. Exposure to acid reflux, and possibly smoking, confer markedly higher risks among homozygous variant genotype carriers.     

Correlation of BRAF and NRAS mutation status with outcome,site of distant metastasis and response to chemotherapy in metastatic melanoma

Background:

The prognostic significance of BRAF and NRAS mutations in metastatic melanoma patients remains uncertain, with several studies reporting conflicting results, often biased by the inclusion of patients treated with BRAF and MEK (MAPK) inhibitors. We therefore interrogated a historical cohort of patients free of the confounding influence of MAPK inhibitor therapy.

Methods:

Patients with available archival tissue first diagnosed with metastatic melanoma between 2002 and 2006 were analysed. Mutational analysis was performed using the OncoCarta Panel. Patient characteristics, treatment outcome and survival were correlated with BRAF/NRAS mutation status.

Results:

In 193 patients, 92 (48%) melanomas were BRAF-mutant, 39 (20%) were NRAS-mutant and 62 (32%) were wild-type for BRAF/NRAS mutations (wt). There was no difference in response to chemotherapy based on mutation status (35–37%). The distant disease-free interval (DDFI) was significantly shorter in patients with wt melanoma (27.9 months vs 35.1 for BRAF and 49.1 for NRAS) although this was not significant in multivariate analysis. Survival from stage IV melanoma diagnosis was not significantly different based on mutation status. The DDFI was significantly shorter in patients with BRAF^V600K/R versus BRAF^V600E melanoma in univariate and multivariate analyses.

Conclusions:

BRAF and NRAS mutation status does not influence survival in metastatic melanoma.     

Role of MC1R variants in uveal melanoma

Variants of the melanocortin-1 receptor (MC1R) gene have been linked to sun-sensitive skin types and hair colour, and may independently play a role in susceptibility to cutaneous melanoma. To assess the role of MC1R variants in uveal melanoma, we have analysed a cohort of 350 patients for the changes within the major region of the gene displaying sequence variation. Eight variants were detected - V60L, D84E, V92M, R151C, I155T, R160W, R163Q and D294H - 63% of these patients being hetero- or homozygous for at least one variant. Standard melanoma risk factor data were available on 119 of the patients. MC1R variants were significantly associated with hair colour (P=0.03) but not skin or eye colour. The frequency of the variants detected in the 350 patients was comparable with those in the general population, and comparison of the cumulative tumour distribution by age at diagnosis in carriers and noncarriers provided no evidence that MC1R variants confer an increased risk of uveal melanoma. We interpret the data as indicating that MC1R variants do not appear to be major determinants of susceptibility to uveal melanoma.     

Amino acid substitution polymorphisms of the DNA repair gene MGMT and the susceptibility to cervical carcinoma

In the current study, we examined the association between polymorphisms in the O(6)-methylguanine-DNA methyltransferase gene (MGMT) and the risk for cervical carcinoma. We prospectively selected 1012 patients, including 539 with carcinoma and 473 with cervical intra-epithelial neoplasia and 800 healthy women from five hospitals in Zhejiang Province, China. Three single-nucleotide polymorphisms (Leu84Phe, Ile143Val and Lys178Arg) were genotyped, and their association with other epidemiological risk factors was examined. Compared with the MGMT Lys178Lys (AA) or Ile143Ile (AA) genotypes, women homozygous for the Arg178Arg (GG) or Val 143Val (GG) genotypes had a significantly increased risk for cervical carcinoma both in the overall carcinoma group and in the high-risk human papillomavirus-positive group. Compared with using Leu84Leu (CC), Phe84Phe (TT) and Leu84Phe (CT) which did not increase the risk for cervical carcinoma. In addition, using 84Leu (C)-143Ile (A)-178Lys (A) as reference, women carrying 84Phe (T)-143Val (G)-178Arg (G) had a 1.87-fold higher risk for cervical carcinoma (95% confidence interval 1.07-3.27). Similar results were observed for squamous cell carcinomas. The effect of the combination of Arg178Arg (GG) and Lys178Arg (AG) genotypes and the 84Phe (T)-143Val (G)-178Arg (G) haplotype was more pronounced in women infected with high-risk human papillomavirus, an early onset of sexual activity, multiple sexual partners, an early age of the first full-term pregnancy and high parity. These findings suggest that polymorphism in MGMT increases the susceptibility of women to cervical carcinoma, especially in those with high-risk sexual and reproductive histories.     

Lack of Influence of MGMT Codon Leu84Phe and Codon Ileu143Val Polymorphisms on Esophageal Cancer Risk in the Kashmir Valley

The enzyme encoded by the MGMT gene is involved in the repair of alkylated lesions formed in DNA bycarcinogenic nitrosamines. Since dietary items consumed by the Kashmiri population contain high concentrationsof these agents, it is biologically plausible that MGMT polymorphic variants may be associated with their riskof esophageal cancer. The present study was performed to assess whether non-synonymous SNPS at codonLeu84Phe and codon Ileu143Val of the MGMT gene, close to the active site of the protein, might be linked topredisposition of Kashmiris to esophageal cancer. Genotyping was carried out by polymerase chain reactionrestrictionfragment length polymorphism on 92 cases and 77 healthy controls. Codon 84 and codon 143 SNPs ofthe MGMT gene were not associated with any increase in risk. While the frequency of the Phe allele at codon 84in cases was (0.16), slightly higher than controls (0.12), the difference was not statistically significant. Similarly,the frequency of Valine allele in cases at codon 143 (0.08) and controls (0.09) was nearly equal. Moreover, nosignificant association of MGMT genotypes with the clinicopatholgic variables of esophageal cancer patients wasobserved. In conclusion, MGMT variants at codon 84 and codon143 may not be involved in the susceptibility ofthe Kashmiri population to esophageal cancer.     

Common ERBB2 polymorphisms and risk of breast cancer in a white British population: a case-control study

IntroductionAbout two-thirds of the excess familial risk associated with breast cancer is still unaccounted for and may be explained by multiple weakly predisposing alleles. A gene thought to be involved in low-level predisposition to the disease is ERBB2 (HER2). This gene is involved in cell division, differentiation, and apoptosis and is frequently amplified in breast tumours. Its amplification correlates with poor prognosis. Moreover, the coding polymorphism I655V has previously been associated with an increased risk of breast cancer.MethodsWe aimed to determine if common polymorphisms (frequency ≥ 5%) in ERBB2 were associated with breast cancer risk in a white British population. Five single-nucleotide polymorphisms (SNPs) were selected for study: SNP 1 near the promoter, SNP 2 in intron 1, SNP 3 in intron 4, SNP 4 in exon 17 (I655V), and SNP 5 in exon 27 (A1170P). We tested their association with breast cancer in a large case–control study (n = 2192 cases and 2257 controls).ResultsThere were no differences in genotype frequencies between cases and controls for any of the SNPs examined. To investigate the possibility that a common polymorphism not included in our study might be involved in breast cancer predisposition, we also constructed multilocus haplotypes. Our set of SNPs generated all existing (n = 6) common haplotypes and no differences were seen in haplotype frequencies between cases and controls (P = 0.44).ConclusionsIn our population, common ERBB2 polymorphisms are not involved in predisposition to breast cancer.     

O6-Methylguanine DNA methyltransferase protein expression in tumor cells predicts outcome of temozolomide therapy in glioblastoma patients

O⁶-Methylguanine DNA methyltransferase (MGMT) is implicated as a major predictive factor for treatment response to alkylating agents including temozolomide (TMZ) of glioblastoma multiforme (GBM) patients. However, whether the MGMT status in GBM patients should be detected at the level of promoter methylation or protein expression is still a matter of debate. Here, we compared promoter methylation (by methylation-specific polymerase chain reaction) and protein expression (by Western blot) in tumor cell explants with respect to prediction of TMZ response and survival of GBM patients (n = 71). Methylated MGMT gene promoter sequences were detected in 47 of 71 (66%) cases, whereas 37 of 71 (52%) samples were scored positive for MGMT protein expression. Although overall promoter methylation correlated significantly with protein expression (χ² test, P < .001), a small subgroup of samples did not follow this association. In the multivariate Cox regression model, a significant interaction between MGMT protein expression, but not promoter methylation, and TMZ therapy was observed (test for interaction, P = .015). In patients treated with TMZ (n = 42), MGMT protein expression predicted a significantly shorter overall survival (OS; hazard ratio [HR] for death 5.53, 95% confidence interval [CI] 1.76–17.37; P = .003), whereas in patients without TMZ therapy (n = 29), no differences in OS were observed (HR for death 1.00, 95% CI 0.45–2.20; P = .99). These data suggest that lack of MGMT protein expression is superior to promoter methylation as a predictive marker for TMZ response in GBM patients.     

Lack of association between Caucasian lung cancer risk and O6-methylguanine-DNA methyltransferase-codon 178 genetic polymorphism

The formation of DNA adducts is thought to be a critical step for the induction of chemically induced cancer. O(6)-Methylguanine-DNA methyltransferase (MGMT) is a ubiquitously expressed enzyme that repairs DNA adducts formed by alkylating carcinogens. Thus, genetic polymorphisms of the MGMT that could result in differences in MGMT activity are potential risk factors for cancer. In the present study, we established a convenient and reliable genotyping method for the MGMT codon 178 polymorphism, a Lys (AAG) to Arg (AGG) substitution, using restriction fragment length polymorphism (RFLP), and studied differences in the distribution of this polymorphism in 92 Caucasian lung cancer patients and 85 controls. Frequencies of the "A" and "G" alleles (MGMT codon 178, AAG and AGG, respectively) were 0.91 and 0.09, respectively. The genetic polymorphism of the MGMT codon 178 was linked with that of the MGMT codon 143 (P < 0.05). The distribution of the MGMT codon 178 genetic polymorphism was not significantly different between lung cancer patients and controls. Thus, our study suggests that the MGMT codon 178 (and possibly 143) polymorphisms do not appear to markedly affect lung cancer risk for this population. In addition, we found an apparent 10bp-deletion in the intron before exon 5 by DNA sequencing. Because this "deletion" was observed in all sequenced samples (N = 20), the previously reported human (Caucasian) MGMT gene sequence should be revised to exclude this 10bp segment.     

Analysis of O(6)-methylguanine-DNA methyltransferase in melanoma tumours in patients treated with dacarbazine-based chemotherapy

In a retrospective study we analysed the levels of the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) in melanoma metastases in patients receiving dacarbazine (DTIC) either as a single drug or as part of combination chemotherapy regimens, and related the expression levels to the clinical response to treatment. Biopsies of subcutaneous and lymph node metastases obtained before chemotherapy in 65 patients with disseminated malignant melanoma were examined for MGMT protein levels by immunohistochemistry using a monoclonal anti-human MGMT antibody. All patients received chemotherapy with DTIC, given either as a single drug or in combination with vindesine and in some cases cisplatin. DTIC as single agent was given to 44 patients, while 21 received combination chemotherapy. Objective responses to chemotherapy were seen in 12 patients, while 53 patients failed to respond to treatment. The expression of MGMT was determined according to the proportion of antibody-stained tumour cells, using a cut-off level of 50%. In 12 of the patients more than one metastasis was analysed, and in seven of these cases the MGMT expression differed between tumours in the same individual. Among the responders a larger proportion (six out of 12, 50%) had tumours containing less than 50% MGMT-positive tumour cells than among the non-responders (12 out of 53, 23%). These data are consistent with the hypothesis that MGMT contributes to resistance to DTIC-based treatment, although the difference between responders and non-responders with respect to the proportion of MGMT-positive tumour cells was not statistically significant (P = 0.077).     

MGMT gene variants,temozolomide myelotoxicity and glioma risk. A concise literature survey including an illustrative case

Temozolomide may cause thrombocytopenia or neutropenia in 3–4% of glioblastoma patients, respectively. However, pancytopenia is rarely reported. MGMT (O6-methylguanine-DNA-methyltransferase) enzyme repairs temozolomide-induced DNA mutations and associates both with antitumour efficacy and myelosuppression. Many studies on the effects of MGMT gene-methylation on temozolomide’s effects exist, but much fewer publications concerning MGMT variants were documented. A full sequencing of the MGMT gene was performed in a female glioblastoma patient, who developed pancytopenia following temozolomide treatment. Results indicated the presence of all the rs2308321 (I143 V), rs2308327 (K178R) and rs12917 (L84F) MGMT-variants, which were previously associated with temozolomide myelotoxicity. rs12917 (L84F) variant was reported as associating with lesser risk of gallbladder tumours, yet with higher risk of non-Hodgkin lymphomas related with exposure to chlorinated solvents or hair dyes. DNA repair proteins may exert diverging effects on DNA injuries caused by different chemicals and therefore exerting complex effects on myelotoxicity, antitumour activity and carcinogenesis.     

Predictive value of the MGMT promoter methylation status in metastatic melanoma patients receiving first-line temozolomide plus bevacizumab in the trial SAKK 50/07

The O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation status is a predictive parameter for the response of malignant gliomas to alkylating agents such as temozolomide. First clinical trials with temozolomide plus bevacizumab therapy in metastatic melanoma patients are ongoing, although the predictive value of the MGMT promoter methylation status in this setting remains unclear. We assessed MGMT promoter methylation in formalin-fixed, primary tumor tissue of metastatic melanoma patients treated with first-line temozolomide and bevacizumab from the trial SAKK 50/07 by methylation-specific polymerase chain reaction. In addition, the MGMT expression levels were also analyzed by MGMT immunohistochemistry. Eleven of 42 primary melanomas (26%) revealed a methylated MGMT promoter. Promoter methylation was significantly associated with response rates CR + PR versus SD + PD according to RECIST (response evaluation criteria in solid tumors) (p<0.05) with a trend to prolonged median progression-free survival (8.1 versus 3.4 months, p>0.05). Immunohistochemically different protein expression patterns with heterogeneous and homogeneous nuclear MGMT expression were identified. Negative MGMT expression levels were associated with overall disease stabilization CR+PR+SD versus PD (p=0.05). There was only a poor correlation between MGMT methylation and lack of MGMT expression. A significant proportion of melanomas have a methylated MGMT promoter. The MGMT promoter methylation status may be a promising predictive marker for temozolomide therapy in metastatic melanoma patients. Larger sample sizes may help to validate significant differences in survival type endpoints.     

Single-nucleotide polymorphisms in the RB1 gene and association with breast cancer in the British population

A substantial proportion of the familial risk of breast cancer may be attributable to genetic variants each contributing a small effect. pRb controls the cell cycle and polymorphisms within it are candidates for such low penetrance susceptibility alleles, since the gene has been implicated in several human tumours, particularly breast cancer. The purpose of this study was to determine whether common variants in the RB1 gene are associated with breast cancer risk. We assessed 15 tagging single-nucleotide polymorphisms (SNPs) using a case-control study design (n< or = 4474 cases and n < or = 4560 controls). A difference in genotype frequencies was found between cases and controls for rs2854344 in intron 17 (P-trend = 0.007) and rs198580 in intron 19 (P-trend = 0.018). Carrying the minor allele of these SNPs appears to confer a protective effect on breast cancer risk (odd ratio (OR) = 0.86 (0.76-0.96) for rs2854344 and OR = 0.80 (0.66-0.96) for rs198580). However, after adjusting for multiple testing these associations were borderline with an adjusted P-trend = 0.068 for the most significant SNP (rs2854344). The RB1 gene is not known to contain any coding SNPs with allele frequencies > or = 5% but several intronic variants are in perfect linkage disequilibrium with the associated SNPs. Replication studies are needed to confirm the associations with breast cancer.