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.

Interleukin-17-positive mast cells influence outcomes from BCG for patients with CIS: Data from a comprehensive characterisation of the immune microenvironment of urothelial bladder cancer.

The tumour immune microenvironment is considered to influence cancer behaviour and outcome. Using a panel of markers for innate and adaptive immune cells we set out to characterise and understand the bladder tumour microenvironment of 114 patients from a prospective multicentre cohort of newly-diagnosed bladder cancer patients, followed-up for 4.33±1.71 years. We found IL-17-positive cells were significantly increased in primary and concomitant carcinoma in situ (CIS), p<0.0001, a highly malignant lesion which is the most significant single risk factor for disease progression. Further characterisation of the tumour immunophenotype identified IL-17+ cells as predominantly mast cells rather than T-cells, in contrast to most other tumour types. Expression of the IL-17-receptor in bladder tumours, and functional effects and gene expression changes induced by IL-17 in bladder tumour cells in vitro suggest a role in tumour behaviour. Finally, we assessed the effects of IL-17 in the context of patient outcome, following intravesical BCG immunotherapy which is the standard of care; higher numbers of IL-17+ cells were associated with improved event-free survival (p = 0.0449, HR 0.2918, 95% CI 0.08762-0.9721) in patients with primary and concomitant CIS (n = 41), we propose a model of IL-17+ Mast cells mechanism of action. Thus, in the context of bladder CIS, IL-17+ mast cells predict favourable outcome following BCG immunotherapy indicative of a novel mechanism of BCG immunotherapy in UBC and could form the basis of a stratified approach to treatment.

Impact of tissue processing, archiving and enrichment techniques on DNA methylation yield in rectal carcinoma.

BACKGROUND: Formalin fixation, duration of tissue storage and tissue enrichment techniques can affect DNA methylation yield but these effects have not been quantitatively measured. The aim is to investigate the relative impact of these conditions on DNA methylation in rectal cancer. METHODS: 10 rectal cancers with matched undissected fresh frozen tissues, laser capture microdissected (LCM) formalin-fixed paraffin-embedded (FFPE) tissues, manual macrodissected FFPE tissues, adjacent normal mucosa and stromal tissues were analysed for APC and LINE-1 methylation using bisulphite pyrosequencing. RESULTS: FFPE cancer tissues, which had been stored for at least 4 years showed similar APC and LINE-1 methylation changes to matched fresh frozen cancer tissues. Laser capture microdissection did not increase the degree of methylation detected compared to manual macrodissection. Analysis of stromal tissues showed that they had undergone significant methylation changes compared to adjacent macroscopically normal mucosa, but not to the same extent as cancer tissues. CONCLUSION: Reliable DNA methylation results can be obtained from FFPE rectal cancer tissues, which have been in long-term storage. Because only minor differences in methylation between macrodissected and LCM cancer tissues were found, our results do not support the routine use of LCM to enrich for cancer cells for DNA methylation studies.

Bfl-1 is a crucial pro-survival nuclear factor-κB target gene in Hodgkin/Reed-Sternberg cells.

Hodgkin/Reed-Sternberg (H/RS) cells are believed to represent clonal progeny of Germinal Centre B cells that have escaped negative selection by evading apoptosis. Aberrant constitutive activity of the transcription factor NF-κB plays a key role in the pathogenesis of Hodgkin's Lymphoma (HL), conferring a survival advantage on H/RS cells. Bfl-1 is a pro-survival NF-κB target gene from the Bcl-2 family of apoptosis-regulating proteins. Here, we report that bfl-1 (also known as A1 or GRS) is frequently expressed in primary H/RS cells from HL tumor biopsies and that elevated bfl-1 expression is a feature of H/RS derived cell lines. We show that bfl-1 is an NF-κB target gene in this cell context and that this regulation is effected through a p65-binding DNA element located in its promoter. We demonstrate that ectopic Bfl-1 can rescue cultured H/RS cells from apoptosis induced by pharmacological inhibitors of NF-κB, and that knockdown of bfl-1 potentiates the pro-apoptotic effect of these agents. These findings are the first indication that Bfl-1 plays a crucial role in setting the elevated threshold of resistance of this malignant cell type to apoptosis.

Disruption of the E2 gene is a common and early event in the natural history of cervical human papillomavirus infection: a longitudinal cohort study.

Integration of high-risk human papillomavirus (HPV) types into the host-cell genome disrupts the HPV regulatory E2 protein, resulting in a loss of negative feedback control of viral oncogene expression; this disruption has been considered a critical event in the pathogenesis of cervical neoplasia, and a potential biomarker of progressive disease. However, using serial samples taken from a cohort of young women who were recruited soon after they first had sexual intercourse, we show that disruption of the E2 gene is a common and early event in the natural history of incident cervical HPV infections. The E2 gene was significantly more likely to be disrupted in women who tested positive for HPV18 in their baseline sample than in those who tested positive for HPV16 [26% versus 58%; relative risk, 2.26; 95% confidence interval (CI), 1.38-3.71; chi(2), 9.23; 1 degree of freedom (df); P = 0.002]. Among women with an intact E2 gene in their baseline sample, the median time to first detection of E2 disruption was also shorter for those who tested positive for HPV18 than HPV16 (5.7 versus 10.9 months; hazards ratio, 1.93; 95% CI, 0.84-4.44; chi(2), 2.49; 1 df; P = 0.11). This tendency for HPV18 to integrate early, coupled with the substantial reduction in viral load in HPV18-positive samples in which E2 is disrupted, may explain why HPV18-associated disease is often reported to be characterized by minor cytologic changes, which underestimate the severity of the underlying histologic abnormality.

Measurement of the humoral immune response following an incident human papillomavirus type 16 or 18 infection in young women by a pseudovirion-based neutralizing antibody assay.

We have evaluated a neutralizing antibody assay which uses human papillomavirus (HPV) type 16 (HPV-16) and HPV-18 pseudovirions carrying a secretory alkaline phosphatase reporter gene and which can potentially measure functionally relevant HPV type-specific neutralizing antibodies. The reproducibility of the assay was excellent; for HPV-16, the intra- and interassay kappa values were 0.95 and 0.90, respectively; and for HPV-18, the corresponding values were 0.90 and 0.90. This assay was used to describe the kinetics of the neutralizing antibody response in a cohort of 42 young women who were recruited soon after first intercourse and who first tested positive for HPV-16 DNA or HPV-18 DNA, or both, during follow-up. Most women seroconverted following the first detection of type-specific HPV DNA and remained seropositive until the end of follow-up. Our findings are broadly consistent with those of two other cohort studies which have measured the serological response following an incident infection by using the technically simpler virus-like-particle-based enzyme-linked immunosorbent assay.

A retinoid-related molecule that does not bind to classical retinoid receptors potently induces apoptosis in human prostate cancer cells through rapid caspase activation.

Synthetic retinoid-related molecules, such as N-(4-hydroxyphenyl)retinamide (fenretinide) and 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) induce apoptosis in a variety of malignant cells. The mechanism(s) of action of these compounds does not appear to involve retinoic acid receptors (RARs) and retinoid X receptors (RXRs), although some investigators disagree with this view. To clarify whether some retinoid-related molecules can induce apoptosis without involving RARs and/or RXRs, we used 4-[3-(1-heptyl-4,4-dimethyl-2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)-3-oxo-E-propenyl] benzoic acid (AGN193198) that neither binds effectively to RARs and RXRs nor transactivates in RAR- and RXR-mediated reporter assays. AGN193198 potently induced apoptosis in prostate, breast, and gastrointestinal carcinoma cells and in leukemia cells. AGN193198 also abolished growth (by 50% at 130-332 nM) and induced apoptosis in primary cultures established from prostatic carcinoma (13 patients) and gastrointestinal carcinoma (1 patient). Apoptosis was induced rapidly, as indicated by mitochondrial depolarization and DNA fragmentation. Molecular events provoked by AGN193198 included activation of caspase-3, -8, -9, and -10 (by 4-6 h) and the production of BID/p15 (by 6 h). These findings show that caspase-mediated induction of apoptosis by AGN193198 is RAR/RXR-independent and suggest that this compound may be useful in the treatment of prostate cancer.