Background
Bladder cancer (BCa) is a major burden for both patients and health care systems worldwide [
1]. At initial presentation, the majority of cases are non-muscle invasive tumors, which are not immediately life-threatening. However, more than 70 % of patients with operable BCa will have a recurrence during the first 2 years after diagnosis and the subsequent lesions can progress to being muscle invasive and metastatic [
2]. Thus, once treated, BCa patients are under continual surveillance with routine cystoscopy examinations for early detection of new BCa development. Due to the prolonged natural history of BCa, plus the prolonged and invasive nature of follow-up and treatment strategies, it is one of the most expensive malignancies to manage on a per-patient basis [
3]. Although Japan and other Asian countries have a somewhat lower incidence of BCa than Europe and the Americas, the clinico-pathological characteristics of the disease are similar. In Japan, around 12,000 patients are newly diagnosed and 5000 patients die from the disease annually. While the overall incidence trend has been relatively stable over the past decade [
4,
5], because the onset is most often seen in elderly patients, it is becoming a major social issue in an aging Japanese society.
In a series of previous studies, we have identified panels of protein biomarkers that are significantly associated with BCa [
6,
7]. Through several studies [
8‐
10] we have refined the target composition, and the immunoassay approach [
11], in order to develop a viable non-invasive assay for BCa detection. In this study, we tested the potential utility of the multiplex immunoassay for the detection of BCa in a Japanese cohort. Evaluation of 288 subjects obtained from two independent institutes confirmed a significant association of the tested biomarkers with the presence of BCa. The multiplex assay achieved a strong overall diagnostic performance achieving 85 % sensitivity and 81 % specificity (AUC 0.892). This retrospective phase II study confirms the potential of using urinary protein biomarker signatures for the non-invasive detection of BCa, and suggests that the described multiplex immunoassay could aid in Japanese urology patient management.
Discussion
Bladder cancer is among the most common worldwide malignancies, and due to the high recurrence rate, it is also one of the most burdensome for both patients and healthcare systems. Current guidelines for BCa diagnosis recommend cystoscopy coupled with voided urine cytology (VUC), but cystoscopy is an invasive procedure associated with discomfort and anxiety, infection and trauma [
21,
22]. Non-invasive VUC is used routinely as an adjunct to cystoscopy, but the subjective assay can be impaired by inter-observer variability and has poor sensitivity, especially for low-grade and low-stage tumors [
23,
24]. Urine-based, quantitative assays that can non-invasively detect BCa would improve the rapid diagnosis of BCa and help to avoid unnecessary invasive cystoscopy and biopsy procedures, however, while a number of such assays have been proposed, to date, such assays have lacked adequate accuracy to replace VUC, or to support or guide cystoscopy. The lack of accuracy of current urinary tests may be due to the fact that they typically rely on measuring a single biomarker. A shift from single biomarker assays to multiplex molecular signatures that reflect the molecular pathways of BCa provides an opportunity to develop assays with clinical utility across the breadth of disease states. The advent of high-throughput technologies has facilitated the discovery of more complex molecular signatures with diagnostic or prognostic potential, and a number of gene expression signature assays are now being incorporated into clinical practice [
25,
26].
Through proteomic profiling of naturally micturated urine, we were able to identify a set of urinary proteins that were associated with the presence of BCa [
6,
7]. One advantage of profiling the urine component directly is the ability to compare samples collected from subjects with non-malignant conditions, a situation that is not feasible using surgically excised solid tissue samples. The candidate biomarkers were refined through independent validation studies [
27,
28], and a 10-biomarker panel was established for development. The diagnostic performance of the 10-protein assay was then tested in two independent cohorts, one [
9] comprised of 127 subjects (diagnostic sensitivity of 92 %, specificity of 97 %), and one [
10] comprised of 308 patients obtained from multiple institutions in the USA and Europe (sensitivity 74 %, specificity 90 %). Taken together, these studies illustrate the reproducibility of the diagnostic protein panel in terms of a robust biomarker panel that achieved similar performance data across multiple, independent cohorts. Based on these results, we investigated the feasibility of developing a multiplex assay that could accurately and simultaneously monitor the diagnostic biomarkers in an efficient format for potential clinical application. A custom-designed multiplex assay using MULTI-ARRAY® technology (meso scale diagnostics, LLC) was constructed, and the analytical performance was compared with data obtained from individual ELISA assays directed at each of the same 10 urinary proteins. The multiplex assay achieved excellent concordance, and improved detection range and technical sensitivity [
11]. The diagnostic performance of the assay was confirmed in an independent cohort of 200 subjects (sensitivity 85 %, specificity 81 %). The integration of multiplex molecular signatures into a single assay is beneficial for clinical translation through reduced sample volume, decreased processing time, low cost analysis and low reagent consumption, and the MULTI-ARRAY® assay can be readily implemented into a CLIA certified laboratory setting using existing instrumentation and skillsets.
Here, we extended the evaluation of the multiplex BCa diagnostic assay to a phase II, multicenter study with a cohort comprised of 288 Japanese subjects. For overall classification, the multiplex assay achieved a sensitivity of 84.8 % and specificity of 80.6 % (AUC of 0.892). Biomarker expression and assay performance increased with increasing tumor stage and grade. The results in the Japanese cohort were very much in line with our previous studies evaluating cohorts from the USA and Europe [
9‐
11], but as we refine and optimize the technical aspects of the assay and analyze additional samples, we expect to be able to further improve assay performance. While subtle differences in composition of optimal urinary diagnostic protein panels may exist in cohorts comprised of different ethnic populations, given the translation of an assay optimized in diverse cohorts to a Japanese population, these data are very encouraging, and further validate the concept that a multiplex panel of urinary protein biomarkers can assist the noninvasive diagnosis of BCa.
We recognize that the study has a number of limitations. The cohort was comprised of >50 % cases, but disease prevalence is typically considerably lower in routine urologic practice. While it was important to initially test enough cases to achieve statistical significance, it will be necessary to perform additional studies that reflect urology clinic presentation to assess predictive value of the assay. It will also be necessary to include more diverse controls that may be under-represented in our study cohort. To address these issues, we have launched a multi-institute, prospective clinical trial, which will assess the multiplex diagnostic assay in subjects with gross hematuria, microscopic hematuria and history of BCa on tumor surveillance. Such a study would minimize selection bias, better represent urological disease prevalence, and evaluate potential confounding comorbidities in the study population. Furthermore, in this study, we focused only on subjects with primary BCa, however, we have previously reported that the multiplex assay is also accurate for the detection of recurrent BCa (sensitivity 79 %, specificity 88 %), outperforming the Urovysion cytogenetic assay and VUC [
29]. The inclusion of Japanese patients on routine surveillance after primary BCa treatment will be an additional goal and will enable evaluation of potential prognostic utility of the assay. Finally, we have initiated the development of BCa diagnostic nomograms that incorporate biomarker data with relevant clinical information (e.g., age, sex, race, and tobacco history) in US cohort studies, and this can also be extended to future Japanese cohort studies.
Conclusion
Bladder cancer is a common neoplastic disease with a high rate of recurrence and progression, and the recurrence phenomenon makes it one of the most prevalent cancers worldwide. The development of robust non-invasive, urine-based assay for the detection of BCa is clinically urgent. In this study, we have been able to successfully translate a multiplex diagnostic assay derived from diverse cohort studies to the analysis of a Japanese population. The diagnostic assay achieved encouraging performance values and will be the focus of ongoing studies to investigate the potential added value of the multiplex assay if integrated into clinical decision making.
Authors’ contributions
OO, YM, KY, MM, SO, KF and KT provided samples reviewed ELISA results and drafted and reviewed manuscript. YS and HF organized the project and collated all data. YD organized and analyzed data. SG participated in study design and helped to draft the manuscript. CJR conceived the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.