Genome-wide association study yields variants at 20p12.2 that associate with urinary bladder cancer.

Genome-wide association studies (GWAS) of urinary bladder cancer (UBC) have yielded common variants at 12 loci that associate with risk of the disease. We report here the results of a GWAS of UBC including 1670 UBC cases and 90 180 controls, followed by replication analysis in additional 5266 UBC cases and 10 456 controls. We tested a dataset containing 34.2 million variants, generated by imputation based on whole-genome sequencing of 2230 Icelanders. Several correlated variants at 20p12, represented by rs62185668, show genome-wide significant association with UBC after combining discovery and replication results (OR = 1.19, P = 1.5 × 10(-11) for rs62185668-A, minor allele frequency = 23.6%). The variants are located in a non-coding region approximately 300 kb upstream from the JAG1 gene, an important component of the Notch signaling pathways that may be oncogenic or tumor suppressive in several forms of cancer. Our results add to the growing number of UBC risk variants discovered through GWAS.

The role of microRNA-binding site polymorphisms in DNA repair genes as risk factors for bladder cancer and breast cancer and their impact on radiotherapy outcomes.

MicroRNAs (miRNAs) are involved in post-transcriptional regulation of gene expression through binding to messenger RNAs (mRNA) thereby promoting mRNA degradation or altered translation. A single-nucleotide polymorphism (SNP) located within a miRNA-binding site could thus alter mRNA translation and influence cancer risk and treatment response. The common SNPs located within the 3'-untranslated regions of 20 DNA repair genes were analysed for putative miRNA-binding sites using bioinformatics algorithms, calculating the difference in Gibbs free binding energy (ΔΔG) for each wild-type versus variant allele. Seven SNPs were selected to be genotyped in germ line DNAs both from a bladder cancer case-control series (752 cases and 704 controls) and 202 muscle-invasive bladder cancer radiotherapy cases. The PARP-1 SNP rs8679 was also genotyped in a breast cancer case-control series (257 cases and 512 controls). Without adjustment for multiple testing, multivariate analysis demonstrated an association with increased bladder cancer risk with PARP1 rs8679 (P(trend) = 0.05) while variant homozygotes of PARP1 rs8679 were also noted to have an increased breast cancer risk (P = 0.03). In the radiotherapy cases, carriers of the RAD51 rs7180135 minor allele had improved cancer-specific survival (hazard ratio 0.52, 95% confidence interval 0.31-0.87, P = 0.01). This is the first report of associations between DNA repair gene miRNA-binding site SNPs with bladder and breast cancer risk and radiotherapy outcomes. If validated, these findings may give further insight into the biology of bladder carcinogenesis, allow testing of the RAD51 SNP as a potential predictive biomarker and also reveal potential targets for new cancer treatments.

In vitro functional effects of XPC gene rare variants from bladder cancer patients.

The XPC gene is involved in repair of bulky DNA adducts formed by carcinogenic metabolites and oxidative DNA damage, both known bladder cancer risk factors. Single nucleotide polymorphisms (SNPs) in XPC have been associated with increased bladder cancer risk. Recently, rarer genetic variants have been identified but it is difficult to ascertain which are of functional importance. During a mutation screen of XPC in DNA from 33 bladder tumour samples and matched blood samples, we identified five novel variants in the patients' germ line DNA. In a case-control study of 771 bladder cancer cases and 800 controls, c.905T>C (Phe302Ser), c.1177C>T (Arg393Trp), c.*156G>A [3' untranslated region (UTR)] and c.2251-37C>A (in an intronic C>G SNP site) were found to be rare variants, with a combined odds ratio of 3.1 (95% confidence interval 1.0-9.8, P=0.048) for carriage of one variant. The fifth variant was a 2% minor allele frequency SNP not associated with bladder cancer. The two non-synonymous coding variants were predicted to have functional effects using analytical algorithms; a reduced recruitment of GFP-tagged XPC plasmids containing either c.905T>C or c.1177C>T to sites of 408 nm wavelength laser-induced oxidative DNA damage was found in vitro. c.*156G>A appeared to be associated with reduced messenger RNA stability in an in vitro plasmid-based assay. Although the laser microbeam assay is relevant to a range of DNA repair genes, our 3' UTR assay based on Green fluorescent protein(GFP) has widespread applicability and could be used to assess any gene. These assays may be useful in determining which rare variants are functional, prior to large genotyping efforts.

Mesorectal radiotherapy for early stage rectal cancer: A novel target volume.

With the introduction of population-based bowel cancer screening, rectal cancer is diagnosed at earlier stages, yet standard treatment still requires the same extensive surgery that is used for more advanced stages. Organ preserving treatment is rapidly developing and is subject of investigation in numerous clinical trials. The STAR-TREC trial is an international, multi-centre randomised trial investigating organ preservation using (chemo)radiotherapy. Patients with small mrT1-3bN0V0M0 tumours are randomized between three arms: standard TME, organ preservation with SCRT or with CRT. In this trial, the clinical target volume has been tailored to the early staged disease of the included patients. This mesorectal irradiation volume includes the mesorectum and pre-sacral lymph nodes at the level of the tumour, two centimetres below and cranially up to the S2-3 interspace level. In contrast to conventional irradiation volumes, the lateral lymph nodes and the nodes along the superior rectal artery are excluded. As a result, the dose to the bowel, bladder, anal sphincter and the neurovascular plexus in the lower pelvis is substantially decreased, especially when combined with modern irradiation techniques, such as dynamic arc therapy. These lower doses are expected to lead to decreasing acute and late toxicity and beneficial functional outcomes. The implementation of this novel target volume will be accompanied by an extensive quality assurance program in the STAR-TREC trial. We describe the rationale behind the novel, mesorectal only radiotherapy treatment used in the STAR-TREC trial specifically tailored for early stage disease, with the goal of organ preservation.

MRE11 as a Predictive Biomarker of Outcome After Radiation Therapy in Bladder Cancer.

PURPOSE: Organ-confined muscle-invasive bladder cancer is treated with cystectomy or bladder preservation techniques, including radiation therapy. There are currently no biomarkers to inform management decisions and aid patient choice. Previously we showed high levels of MRE11 protein, assessed by immunohistochemistry (IHC), predicted outcome after radiation therapy, but not cystectomy. Therefore, we sought to develop the MRE11 IHC assay for clinical use and define its relationship to clinical outcome in samples from 2 major clinical trials. METHODS AND MATERIALS: Samples from the BCON and BC2001 randomized controlled trials and a cystectomy cohort were stained using automated IHC methods and scored for MRE11 in 3 centers in the United Kingdom. RESULTS: Despite step-wise creation of scoring cards and standard operating procedures for staining and interpretation, there was poor intercenter scoring agreement (kappa, 0.32; 95% confidence interval, 0.17-0.47). No significant associations between MRE11 scores and cause-specific survival were identified in BCON (n = 132) and BC2001 (n = 221) samples. Reoptimized staining improved agreement between scores from BCON tissue microarrays (n = 116), but MRE11 expression was not prognostic for cause-specific survival. CONCLUSIONS: Manual IHC scoring of MRE11 was not validated as a reproducible biomarker of radiation-based bladder preservation success. There is a need for automated quantitative methods or a reassessment of how DNA-damage response relates to clinical outcomes.

Phase 2 Neoadjuvant Treatment Intensification Trials in Rectal Cancer: A Systematic Review.

PURPOSE: Multiple phase 2 trials of neoadjuvant treatment intensification in locally advanced rectal cancer have reported promising efficacy signals, but these have not translated into improved cancer outcomes in phase 3 trials. Improvements in phase 2 trial design are needed to reduce these false-positive signals. This systematic review evaluated the design of phase 2 trials of neoadjuvant long-course radiation or chemoradiation therapy treatment intensification in locally advanced rectal cancer. METHODS AND MATERIALS: The PubMed, EMBASE, MEDLINE, and Cochrane Library databases were searched for published phase 2 trials of neoadjuvant treatment intensification from 2004 to 2016. Trial clinical design and outcomes were assessed, with statistical design and compliance rated using a previously published system. Multivariable meta-regression analysis of pathologic complete response (pCR) was conducted. RESULTS: We identified 92 eligible trials. Patients with American Joint Committee on Cancer stage II and III equivalent disease were eligible in 87 trials (94.6%). In 43 trials (46.7%), local staging on magnetic resonance imaging was mandated. Only 12 trials (13.0%) were randomized, with 8 having a standard-treatment control arm. Just 51 trials (55.4%) described their statistical design, with 21 trials (22.8%) failing to report their sample size derivation. Most trials (n=84, 91.3%) defined a primary endpoint, but 15 different primary endpoints were used. All trials reported pCR rates. Only 38 trials (41.3%) adequately reported trial statistical design and compliance. Meta-analysis revealed a pooled pCR rate of 17.5% (95% confidence interval, 15.7%-19.4%) across treatment arms of neoadjuvant long-course radiation or chemoradiation therapy treatment intensification and substantial heterogeneity among the reported effect sizes (I2 = 55.3%, P<.001). Multivariable meta-regression analysis suggested increased pCR rates with higher radiation therapy doses (adjusted P=.025). CONCLUSIONS: Improvement in the design of future phase 2 rectal cancer trials is urgently required. A significant increase in randomized trials is essential to overcome selection bias and determine novel schedules suitable for phase 3 testing. This systematic review provides key recommendations to guide future treatment intensification trial design in rectal cancer.

Post-transcriptional regulation of MRE11 expression in muscle-invasive bladder tumours.

Predictive assays are needed to help optimise treatment in muscle-invasive bladder cancer, where patients can be treated by either cystectomy or radical radiotherapy. Our finding that low tumour MRE11 expression is predictive of poor response to radiotherapy but not cystectomy was recently independently validated. Here we investigated further the mechanism underlying low MRE11 expression seen in poorly-responding patients. MRE11 RNA and protein levels were measured in 88 bladder tumour patient samples, by real-time PCR and immunohistochemistry respectively, and a panel of eight bladder cancer cell lines was screened for MRE11, RAD50 and NBS1 mRNA and protein expression. There was no correlation between bladder tumour MRE11 protein and RNA scores (Spearman's rho 0.064, p=0.65), suggesting MRE11 is controlled post-transcriptionally, a pattern confirmed in eight bladder cancer cell lines. In contrast, NBS1 and RAD50 mRNA and protein levels were correlated (p=0.01 and p=0.03, respectively), suggesting primary regulation at the level of transcription. MRE11 protein levels were correlated with NBS1 and RAD50 mRNA and protein levels, implicating MRN complex formation as an important determinant of MRE11 expression, driven by RAD50 and NBS1 expression. Our findings of the post-transcriptional nature of the control of MRE11 imply that any predictive assays used in patients need to be performed at the protein level rather than the mRNA level.

Imatinib radiosensitizes bladder cancer by targeting homologous recombination.

Radiotherapy is a major treatment modality used to treat muscle-invasive bladder cancer, with patient outcomes similar to surgery. However, radioresistance is a significant factor in treatment failure. Cell-free extracts of muscle-invasive bladder tumors are defective in nonhomologous end-joining (NHEJ), and this phenotype may be used clinically by combining radiotherapy with a radiosensitizing drug that targets homologous recombination, thereby sparing normal tissues with intact NHEJ. The response of the homologous recombination protein RAD51 to radiation is inhibited by the small-molecule tyrosine kinase inhibitor imatinib. Stable RT112 bladder cancer Ku knockdown (Ku80KD) cells were generated using short hairpin RNA technology to mimic the invasive tumor phenotype and also RAD51 knockdown (RAD51KD) cells to show imatinib's pathway selectivity. Ku80KD, RAD51KD, nonsilencing vector control, and parental RT112 cells were treated with radiation in combination with either imatinib or lapatinib, which inhibits NHEJ and cell survival assessed by clonogenic assay. Drug doses were chosen at approximately IC40 and IC10 (nontoxic) levels. Imatinib radiosensitized Ku80KD cells to a greater extent than RAD51KD or RT112 cells. In contrast, lapatinib radiosensitized RAD51KD and RT112 cells but not Ku80KD cells. Taken together, our findings suggest a new application for imatinib in concurrent use with radiotherapy to treat muscle-invasive bladder cancer. Cancer Res; 73(5); 1611-20. ©2012 AACR.

Enteral feeding outcomes after chemoradiotherapy for oropharynx cancer: a role for a prophylactic gastrostomy?

To determine the outcomes of patients managed with different routes of enteral feeding during chemoradiotherapy for oropharynx cancer. The hospital and dietetic records of consecutive patients with oropharynx squamous cell carcinoma treated between January 2007 and June 2009 with concurrent chemoradiotherapy were reviewed retrospectively. One hundred and four patients were analysed. Seventy-one received a prophylactic gastrostomy, 21 were managed with a strategy of NG tube as required and 12 received a therapeutic gastrostomy. Patients with a prophylactic gastrostomy commenced enteral feeding a median of 24 days after commencing radiotherapy, compared with a median of 41 days (p<0.001) for the NG as required group. Comparing prophylactic gastrostomy, NG as required and therapeutic gastrostomy, median number of unplanned inpatient days were 6, 14 and 7, respectively (p<0.01 for prophylactic gastrostomy vs. NG as required). Mean percentage weight loss at the end of treatment (6.1% vs. 7.1% vs. 5.2%, respectively) and at 6 months post-radiotherapy (11.7%, 14.3% and 8.9%) were similar in all groups (p=0.23). There was no significant difference in type of diet post-radiotherapy between prophylactic gastrostomy and NG as required groups (p=0.22). Median duration of enteral feeding was 181, 64 and 644 days, respectively (p<0.01 for prophylactic gastrostomy vs. NG as required). Use of a prophylactic gastrostomy (p<0.01) and higher T stage (p<0.01) were associated with increased duration of enteral feeding on a multivariate analysis. These data reinforce concerns regarding the detrimental impact of prophylactic gastrostomy placement upon long-term enteral feed dependence.

MRE11 expression is predictive of cause-specific survival following radical radiotherapy for muscle-invasive bladder cancer.

Radical radiotherapy and surgery achieve similar cure rates in muscle-invasive bladder cancer, but the choice of which treatment would be most beneficial cannot currently be predicted for individual patients. The primary aim of this study was to assess whether expression of any of a panel of DNA damage signaling proteins in tumor samples taken before irradiation could be used as a predictive marker of radiotherapy response, or rather was prognostic. Protein expression of MRE11, RAD50, NBS1, ATM, and H2AX was studied by immunohistochemistry in pretreatment tumor specimens from two cohorts of bladder cancer patients (validation cohort prospectively acquired) treated with radical radiotherapy and one cohort of cystectomy patients. In the radiotherapy test cohort (n = 86), low tumor MRE11 expression was associated with worse cancer-specific survival compared with high expression [43.1% versus 68.7% 3-year cause-specific survival (CSS), P = 0.012] by Kaplan-Meier analysis. This was confirmed in the radiotherapy validation cohort (n = 93; 43.0% versus 71.2%, P = 0.020). However, in the cystectomy cohort (n = 88), MRE11 expression was not associated with cancer-specific survival, commensurate with MRE11 being a predictive marker. High MRE11 expression in the combined radiotherapy cohort had a significantly better cancer-specific survival compared with the high-expression cystectomy cohort (69.9% versus 53.8% 3-year CSS, P = 0.021). In this validated immunohistochemistry study, MRE11 protein expression was shown and confirmed as a predictive factor associated with survival following bladder cancer radiotherapy, justifying its inclusion in subsequent trial designs. MRE11 expression may ultimately allow patient selection for radiotherapy or cystectomy, thus improving overall cure rates.