Saiful Miah

Distinct MicroRNA Alterations Characterize High- and Low-Grade Bladder Cancer

Urothelial carcinoma of the bladder (UCC) is a common disease that arises by at least two different molecular pathways. The biology of UCC is incompletely understood, making the management of this disease difficult. Recent evidence implicates a regulatory role for microRNA in cancer. We hypothesized that altered microRNA expression contributes to UCC carcinogenesis. To test this hypothesis, we examined the expression of 322 microRNAs and their processing machinery in 78 normal and malignant urothelial samples using real-time rtPCR. Genes targeted by differentially expressed microRNA were investigated using real-time quantification and microRNA knockdown. We also examined the role of aberrant DNA hypermethylation in microRNA downregulation. We found that altered microRNA expression is common in UCC and occurs early in tumorogenesis. In normal urothelium from patients with UCC, 11% of microRNAs had altered expression when compared with disease-free controls. This was associated with upregulation of Dicer, Drosha, and Exportin 5. In UCC, microRNA alterations occur in a tumor phenotype-specific manner and can predict disease progression. High-grade UCC were characterized by microRNA upregulation, including microRNA-21 that suppresses p53 function. In low-grade UCC, there was downregulation of many microRNA molecules. In particular, loss of microRNAs-99a/100 leads to upregulation of FGFR3 before its mutation. Promoter hypermethylation is partly responsible for microRNA downregulation. In conclusion, distinct microRNA alterations characterize UCC and target genes in a pathway-specific manner. These data reveal new insights into the disease biology and have implications regarding tumor diagnosis, prognosis and therapy.

An evaluation of urinary microRNA reveals a high sensitivity for bladder cancer.

Background:Urinary biomarkers are needed to improve the care and reduce the cost of managing bladder cancer. Current biomarkers struggle to identify both high and low-grade cancers due to differing molecular pathways. Changes in microRNA (miR) expression are seen in urothelial carcinogenesis in a phenotype-specific manner. We hypothesised that urinary miRs reflecting low- and high-grade pathways could detect bladder cancers and overcome differences in genetic events seen within the disease.Methods:We investigated urinary samples (n=121) from patients with bladder cancer (n=68) and age-matched controls (n=53). Fifteen miRs were quantified using real-time PCR.Results:We found that miR is stable within urinary cells despite adverse handling and detected differential expression of 10 miRs from patients with cancer and controls (miRs−15a/15b/24-1/27b/100/135b/203/212/328/1224, ANOVA P<0.05). Individually, miR-1224-3p had the best individual performance with specificity, positive and negative predictive values and concordance of 83%, 83%, 75% and 77%, respectively. The combination of miRs-135b/15b/1224-3p detected bladder cancer with a high sensitivity (94.1%), sufficient specificity (51%) and was correct in 86% of patients (concordance).Conclusion:The use of this panel in patients with haematuria would have found 94% of urothelial cell carcinoma, while reducing cystoscopy rates by 26%. However, two invasive cancers (3%) would have been missed.

Hypermethylation of CpG Islands and Shores around Specific MicroRNAs and Mirtrons Is Associated with the Phenotype and Presence of Bladder Cancer

Purpose: To analyze the role and translational potential for hypermethylation of CpG islands and shores in the regulation of small RNAs within urothelial cell carcinoma (UCC). To examine microRNAs (miR) and mirtrons, a new class of RNA located within gene introns and processed in a Drosha-independent manner. Experimental design: The methylation status of 865 small RNAs was evaluated in normal and malignant cell lines by using 5-azacytidine and microarrays. Bisulfite sequencing was used for CpG regions around selected RNAs. Prognostic and diagnostic associations for epigenetically regulated RNAs were examined by using material from 359 patients, including 216 tumors and 121 urinary samples (68 cases and 53 controls). Functional analyses examined the effect of silencing susceptible RNAs in normal urothelial cells. Results: Exonic/UTR-located miRs and mirtons are most susceptible to epigenetic regulation. We identified 4 mirtrons and 16 miRs with CpG hypermethylation across 35 regions in normal and malignant urothelium. For several miRs, hypermethylation was more frequent and dense in CpG shores than islands (e.g., miRs-9/149/210/212/328/503/1224/1227/1229), and was associated with tumor grade, stage, and prognosis (e.g., miR-1224 multivariate analysis OR = 2.5; 95% CI, 1.3–5.0; P = 0.006). The urinary expression of epigenetically silenced RNAs (miRs-152/328/1224) was associated with the presence of UCC (concordance index, 0.86; 95% CI, 0.80–0.93; ANOVA P < 0.016). Conclusions: Hypermethylation of mirtrons and miRs is common in UCC. Mirtrons appear particularly susceptible to epigenetic regulation. Aberrant hypermethylation of small RNAs is associated with the presence and behavior of UCC, suggesting potential roles as diagnostic and prognostic biomarkers. Clin Cancer Res; 17(6); 1287–96. ©2010 AACR.

BPH and prostate cancer risk.

Introduction: With the exclusion of non-melanomatous skin malignancy, prostate cancer (PCa) is the second most prevalent cancer in men globally. It has been reported that the majority of men will develop benign prostatic hyperplasia (BPH) by the time they reach their 60s. Together, these prostatic diseases have a significant morbidity and mortality affecting over a billion men throughout the world. The risk of developing prostate cancer of men suffering BPH is one that has resulted in a healthy debate amongst the urological community. Here, we try to address this conundrum with clinical and basic science evidence. Materials and Methods: Data from an online search and contemporary data presented at international urological congresses was reviewed. Results: BPH and PCa can be linked together at a molecular and cellular level on genetic, hormonal, and inflammatory platforms suggesting that these prostatic diseases have common pathophysiological driving factors. Epidemiological studies are weighted towards the presence of BPH having a greater risk for a man to develop PCa in his lifetime; however, a conclusion of causality cannot be confidently stated. Conclusion: The future workload healthcare practitioners will face regarding BPH, and PCa will substantially increase. Further basic science and large epidemiological studies using a global cohort of men are required prior to the urological community confidently counseling their patients with BPH with regards to their PCa risk.

MicroRNA in Prostate Cancer: An Opportunity to Individualize Patient Care

MICRORNAS (miRs) are short, single stranded nucleic acids that regulate gene expression through a direct interaction with mRNAs. miRs are not translated into proteins and so are classified as noncoding RNAs. They represent one of the shortest but best understood noncoding RNA families. The first miR was identified in the nematode Caenorhabditis elegans in 1993. Termed lin-4, this miR was found to bind a protein coding gene necessary for adult cell fate. The importance of this discovery for human biology, and specifically for carcinogenesis, was not fully realized until the identification of miRs that play key roles in leukemia by Calin et al. Since 2004, there has been an exponential rise in interest regarding miRs in human cancer. Their short size makes them stable at room temperature and resistant to decay, while their role in key cellular functions makes them common targets of carcinogenic change. Consequently they are potentially ideal biomarkers and therapeutic targets. However, much work needs to be done before the realization of this potential, as demonstrated in this issue of the Journal by Li et al (page 1466). These authors report that miR-21 has prognostic and therapeutic properties in prostate cancer using a murine model and prostatectomy specimens. In terms of cancer, each miR may have either promalignant or antimalignant properties. These appear to be generic to all cells or specific to tissue types. This is analogous to other carcinogenic events, such as p53 mutation (common in most malignancies but rare in prostate cancer) or GSTP1 methylation (almost ubiquitous in prostate cancer but rarely found elsewhere). Examples of important miRs in prostate carcinogenesis include miRs-34a/ b/c, miR-141 and miRs-125b, each of which demonstrates important features of RNAs in the disease. Firstly, miRs play diverse procarcinogenic roles. These include pro-proliferative measures, those necessary for metastases or the suppression of apoptosis (eg reduced expression of miRs-34a/b/c allows the up-regulation of SIRT1, which in turn silences p53). These may be self-perpetuating (the miR-34/SIRT1 loop includes positive feedback as p53 drives miR-34

Does true Gleason pattern 3 merit its cancer descriptor

Nearly five decades following its conception, the Gleason grading system remains a cornerstone in the prognostication and management of patients with prostate cancer. In the past few years, a debate has been growing whether Gleason score 3 + 3 = 6 prostate cancer is a clinically significant disease. Clinical, molecular and genetic research is addressing the question whether well characterized Gleason score 3 + 3 = 6 disease has the ability to affect the morbidity and quality of life of an individual in whom it is diagnosed. The consequences of treatment of Gleason score 3 + 3 = 6 disease are considerable; few men get through their treatments without sustaining some harm. Further modification of the classification of prostate cancer and dropping the label cancer for Gleason score 3 + 3 = 6 disease might be warranted.

The clinical need for undergraduate urology

Urologists have highlighted the lack of urological exposure amongst undergraduate students, voicing concerns that it may adversely affect patient care in the future, particularly with a global aging population. 4,5 This viewpoint has been mirrored by newly qualifi ed doctors, as only 9.7 per cent of UK foundation trainees (junior doctors) deemed their undergraduate urological exposure adequate. 4 This article refl ects upon the importance of this issue and also discusses our experience of an outcomebased teaching plan to address this need.

Identification and Diagnostic Performance of a Small RNA within the PCA3 and BMCC1 Gene Locus That Potentially Targets mRNA

Background: PCA3 is a long noncoding RNA (lncRNA) with unknown function, upregulated in prostate cancer. LncRNAs may be processed into smaller active species. We hypothesized this for PCA3. Methods: We computed feasible RNA hairpins within the BMCC1 gene (encompassing PCA3) and searched a prostate transcriptome for these. We measured expression using qRT-PCR in three cohorts of prostate cancer tissues (n = 60), exfoliated urinary cells (n = 484 with cancer and n = 166 controls), and in cell lines (n = 22). We used in silico predictions and RNA knockup to identify potential mRNA targets of short transcribed RNAs. Results: We predicted 13 hairpins, of which PCA3-shRNA2 was most abundant within the prostate transcriptome. PCA3-shRNA2 is located within intron 1 of PCA3 and appears regulated by androgens. Expression of PCA3-shRNA2 was upregulated in malignant prostatic tissues, exfoliated urinary cells from men with prostate cancer (13–273 fold change; t test P < 0.003), and closely correlated to PCA3 expression (r = 0.84–0.93; P < 0.001). Urinary PCA3-shRNA2 (C-index, 0.75–0.81) and PCA3 (C-index, 0.78) could predict the presence of cancer in most men. PCA3-shRNA2 knockup altered the expression of predicted target mRNAs, including COPS2, SOX11, WDR48, TEAD1, and Noggin. PCA3-shRNA2 expression was negatively correlated with COPS2 in patient samples (r = −0.32; P < 0.001). Conclusion: We identified a short RNA within PCA3, whose expression is correlated to PCA3, which may target mRNAs implicated in prostate biology. Impact: This short RNA is stable ex vivo, suggesting a role as a robust biomarker. We identify cytoplasmic enrichment of this RNA and potential targeting of mRNAs implicated in prostate carcinogenesis. Cancer Epidemiol Biomarkers Prev; 24(1); 268–75. ©2014 AACR.

MicroRNA and urothelial cell carcinoma

Predicting the behaviour of upper tract urothelial cancer (UTUC) is difficult but likely to be helped by measuring multiple variables. These variables may be clinical, radiological or pathological or, more excitingly, molecular biomarkers. Although to date no molecular markers have been used within the clinical setting for the prediction of UTUC [1], many people (across all urological cancers) are searching for robust and accurate biomarkers.

RE: “What factors influence British medical students’ career intentions?” Location and social relationships

We read with great interest the article by Ibrahim et al. (2014) regarding factors influencing British medical students’ in their future career intentions. The article highlighted that undergraduate experience and prestige, both significantly influenced British medical students’ future career aspirations. The Foundation Programme is the first 2-years of UK postgraduate medical training consisting of a training programme with six 4-monthly specialities. Recruitment for the 21 foundation schools is an annual national process in which applicants rank their choice of deanery and are accordingly given their rotations depending on the overall score they obtain on their online application. Recently our group assessed the factors influencing final-year medical students in their choice of their Foundation Programme. All 361 participants from 9 medical schools – Edinburgh, Swansea, Imperial, Peninsula, Leicester, Keele, Sheffield, Liverpool and Glasgow – had applied to the Foundation Programme. Twelve choices (year 1 specialities, year 2 specialities, undergraduate experience, prestige of foundation school, competitiveness, availability of academic post, location, family, partner, friends, finance and recommendations) were ranked in a 5-point Likert scale. Additionally, all respondents stated the single most important factor influencing their choice of foundation school. 67.6% of respondents either strongly agreed or agreed that undergraduate experience was an important factor for foundation school choice. When asked to give the single most important factor when embarking on their choice of foundation training school, 44% chose location with 26% choosing partner, family or friends. Only 3% and 1.6% of finalyear medical students stated that undergraduate experience and prestige was the most important factor in the choice of foundation school, respectively. Our study confirms that under-graduate experience is an important factor for the choice of future career intentions of British medical students. However, it appears that geographical and social factors are also important issues that shape early post-graduate training for British medical students. Saiful Miah, Suresh Venugopal & Karl Pang, Department of Urology, Royal Hallamshire Hospital, Sheffield, UK. E-mail: s.miah@sheffield.ac.uk