In an earlier proof-of-concept study we showed that elevated Mcm5 levels in urine cell sediments are highly predictive of bladder cancer 
. The prospective blinded observational trial reported here, involving a large patient cohort, confirms our initial observations that Mcm5 is a sensitive and specific biomarker for detection of UCC. Importantly, through multi-ROC analysis, we show here that the Mcm5 test, in combination with NMP22 at the established cut-point 10 U/mL, enhances diagnostic accuracy over NMP22 in isolation and identifies nearly all potentially life threatening disease.
Despite numerous studies over the last decade, the reported accuracy of the NMP22 test is highly variable. Many of the earlier studies recruited small to moderate numbers of subjects and reported high sensitivities and specificities, above 80% 
. However, a wide range in test performance has been observed in more recent studies with sensitivity ranging from 33% to 100% and specificity from 40% to 93% 
. A pooled analysis including more recent trials suggests a sensitivity of around 68% and a specificity of 79% 
. A recent large multi-institutional international trial revealed a marked variability in the performance of the NMP22 test across participating institutions with sensitivity and specificity ranging from 36% to 86% and 50% to 94% respectively 
. Variability has been attributed to many confounding factors including biological, analytical and epidemiological variables and methodological bias.
Our study represents the largest prospective observational trial ever undertaken using the NMP22 urinary biomarker. Notably, the performance at the 10 U/mL cut-point, with a sensitivity of 53% and specificity of 84%, is somewhat below that reported in the pooled analysis, but almost identical to the diagnostic performance reported in the Matritech supported large patient cohort trials using the NMP22 point-of-care proteomic assay 
. Interestingly we observed significantly greater diagnostic accuracy of NMP22 in females compared to males. Gender differences in NMP22 test performance have been previously noted 
however our data represent the largest study of this question and clearly establishes a clinically meaningful difference in test performance.
The analysis of false positive Mcm5 results in this study also revealed an unexpected difference between the male and female groups. The overall false positive rate in females was 38% compared to 26% in males. Rather than being related to benign pathology, the difference was most marked in the clear normal group. These findings require further investigation. Possible causes could be fungal contamination by vaginal flora (e.g. Candida
species) or mixing of menstrual endometrial contaminants in samples, both sources of extraneous MCM expressing cells. Patients with urinary calculi had the highest incidence of false positive Mcm5 results (44%). As previously reported, higher false positive rates are expected in patients with urinary calculi due to the associated mucosal injury, which exposes the underlying MCM expressing transit amplifying compartment of the transitional epithelium to the urinary tract 
. However, exclusion of patients with calculi from the ROC analysis did not make a significant improvement to the overall performance, presumably because they were a relatively small group (data not shown). Notably, other benign conditions such as urinary tract infection or benign prostatic hyperplasia were not associated with false positive Mcm5 results, in keeping with our proof-of-concept study 
. In contrast to the Mcm5 test, false positive NMP22 results were linked to urinary tract infection. The different aetiologies for false positives with Mcm5 and NMP22 may account for the improved performance observed when combining the two urinary biomarkers.
Decreasing urinary Mcm5 and NMP22 signals were observed with lower stage and grade of UCC, and this was associated with an increasing false negative rate for both tests. Expression of MCM proteins in bladder cancer is closely linked to grade 
and thus this trend is expected. The trend is also explained by the less spontaneous shedding of tumour cells seen in lower grade lesions due to stronger cell-cell and cell-matrix attachments. Commercial development of the Mcm5 test is currently underway and improvements in the assay design to enhance sensitivity are planned and thus reduced false negative rates in early stage, well-differentiated tumours are anticipated. The trend for higher grade and stage tumours to exhibit higher Mcm5 levels could provide a useful predictive clinical role e.g. to target imaging and rigid cystoscopic diagnostic procedures for high risk patients identified by urinary Mcm5. This potential role requires further study.
Current routine initial investigations for haematuria or other symptoms suggestive of bladder cancer include flexible cystoscopy and rigid white light cystoscopy. However an estimated 10–40% of tumours can be missed due to poor visualization as a result of inflammatory conditions or bleeding and flat urothelial lesions such as severe dysplasia and carcinoma in situ 
. Photodynamic diagnosis is a technique that can enhance tumour detection but its increased sensitivity is associated with higher false positive rates leading to additional unnecessary investigations, biopsies and thus increased cost 
. Urinary biomarkers also have potential to enhance tumour detection and identify tumours not visualized during initial endoscopy. A systematic review of the clinical effectiveness and cost-effectiveness of photodynamic diagnosis, cytology and urine biomarkers, including FISH, ImmunoCyt and NMP22, for detection and surveillance of bladder cancer has recently been undertaken 
. Urinary cytology had the lowest pooled sensitivity of the markers studied at 44% although specificity was highest at 96%. The range of reported sensitivity for cytology was 7–100%. Thus while our study reports low sensitivity for cytology this is not a unique finding. Pooled analyses performed by Mowatt et al showed similar diagnostic performance with NMP22 (sensitivity 84%, specificity 75%) and FISH (sensitivity 76%, specificity 85%) with ImmunoCyt slightly outperforming them (sensitivity 84% specificity 75%) 
. Notably, of eight diagnosis and follow-up strategies included in a probabilistic sensitivity analysis using combinations of photodynamic diagnosis, flexible cystoscopy, white light cystoscopy, cytology and urinary biomarkers, four were associated with around a 20% chance of being considered cost-effective. Three of these four strategies involved the use of either a biomarker or photodynamic diagnosis. Other urinary markers of bladder cancer such as Survivin 
, various urinary micro RNAs 
and epigenetic markers 
have also been shown to have great potential as diagnostic markers with sensitivity/specificity reported >90%. As yet these markers have not been evaluated in large-scale blinded observational studies thus the initial findings from these carefully controlled trials should be interpreted with caution.
In this study, the performance of Mcm5 is similar to that of NMP22 and both markers are significantly more accurate than urinary cytology. It also outperforms the performance of cytology from studies combined in a recent systematic review 
. The performance of Mcm5 falls below the reported accuracy of ImmunoCyt and some other novel approaches detailed above. It is worth noting that Mcm5 initially demonstrated an AUC of 0.93 in our earlier smaller study and it remains to be seen if the performance of Survivin and other novel markers is reproducible in large studies. Our current data suggest that an Mcm5 assay commercially developed for an end-user laboratory in combination with NMP22 could be used to modify diagnostic and surveillance care pathways to enhance the diagnostic accuracy in those at high risk (e.g. newly presenting visible haematuria patient) and reduce morbidity and cost of testing in low risk patients (e.g. newly presenting non-visible haematuria patient without a known risk factor or a patient with prior low grade non-muscle invasive tumour). Trials to evaluate modified against standard diagnostic pathways using a commercialised assay are currently in preparation.
In conclusion, we have demonstrated that immunofluorometric detection of Mcm5 in urine sediments is a sensitive and specific diagnostic test for bladder cancer. The test detects bladder cancers of all stages and grades. Through evaluation of different assay cut-points there could be a role for predicting high grade and stage disease. Importantly, urinary Mcm5 in combination with the urinary NMP22 measured with the FDA-approved Matritech NMP22® Test Kit, identifies nearly all life threatening disease.