Application of a multiplex urinalysis test for the prediction of intravesical BCG treatment response: A pilot study

1.Introduction

Up to 60% of non-muscle invasive bladder cancer (NMIBC) cases that are treated by transurethral resection (TUR) will experience disease recurrence. Guidelines for NMIBC management include the recommendation for post-TUR intravesical instillation therapy [1, 2]. Intravesical Bacillus Calmette-Guerin (BCG), a live attenuated tuberculosis vaccine that acts as a non-specific immune system stimulant, has proven to be the most successful adjuvant treatment to date, assisting in the eradication of residual disease, reducing recurrence rates, and decreasing disease progression to muscle-invasive bladder cancer (MIBC) [1, 2]. However, despite considerable success, as many as 30% of patients will develop tumor recurrence and up to 15% can progress despite BCG therapy [3, 4]. Failure to intervene with definitive radical cystectomy prior to progression to MIBC is associated with a significant reduction in long-term survival probability [5, 6]. Thus, early identification of patients suited for bladder preservation with BCG treatment or for radical cystectomy is essential. At this time, the decision to preserve the bladder or to perform a cystectomy depends on models based on clinicopathological parameters [7, 8], but these tools have limited accuracy for predicting disease recurrence or progression [9, 10]. Furthermore, there is currently no established evaluation test available for the prediction of patient response to intravesical BCG.

Previously, we have identified a panel of protein biomarkers that are associated with bladder cancer [11, 12, 13, 14], and we have developed a multiplex immunoassay for the automated detection of the analyte panel in voided urine [15, 16, 17]. The test has been validated for non-invasive diagnosis, but in this study, we tested the potential clinical utility of the multiplex test for the prediction of BCG treatment response in a small prospective cohort.

2.Patients and methods

3.Results

The study population was comprised of 64 subjects with NMIBC who were scheduled to be treated with intravesical BCG. Among the 64 study subjects in this application, all 64 had high-grade disease with 23 with intermediate risk disease (i.e., ALL Ta high-grade < 3 cm) and the remaining 41 high risk disease (i.e., 29 with high-grade T1, 7 with high-grade Ta > 3 cm and 5 with CIS). The mean age of subjects was 65.8 ± 11.3 years, 78.1% of the subjects were men, 85.9% were Caucasian, and 54.7% of the subjects presented with Tis/Ta disease while 45.3% of the subjects presented with T1 disease (Table 1). Of the total 64 subjects, 11 (15.6%) were found to have post-treatment bladder cancer recurrence on follow-up. Median time to recurrence was 6 months (range 1–17 months). The recurrences were noted to be non-muscle invasive bladder cancer (NMIBC; stages Ta, Tis, T1) high-grade in 81.8% (n= 9), and muscle invasive bladder cancer (MIBC; stage ⩾ T2) high-grade in 18.2% (n= 2). Of these 11 subjects, two were noted to have a second recurrence.

For each of the 64 clinical samples, we reported the mean ± SD and range of each biomarker, along with the percentage of samples in which the biomarker was detectable (Table 2). Each individual biomarker was detectable in > 90% of the samples, except for A1AT (detected in 58% of samples). Increased urinary concentrations of MMP9 (P= 0.53), VEFGA (P= 0.20), CA9 (P= 0.19), SDC1 (P= 0.23), PAI1 (P= 0.19), ApoE (P= 0.21), A1AT (P= 0.04), ANG (P= 0.02) and MMP10 (P= 0.15) were observed in subjects with disease recurrence. The urinary concentration of IL8 was unchanged.

Figure 1.

Diagnostic performance of bladder cancer-associated molecular signature. The areas under the curves was 0.8971 (95% confidence interval, 0.8000–0.9942), with a sensitivity value of 81.8% and a specificity value of 84.9% for the prediction of disease recurrence.

Table 3 provides AUC data for each individual biomarker and for the combination of all 10 biomarkers in the OncuriaTM test. Using optimal cutoff values defined by the Youden index from this cohort, the 10-biomarker model resulted in an AUC of 0.8971 (95% confidence interval, 0.8000–0.9942), with a sensitivity value of 81.8% and a specificity value of 84.9% for the prediction of disease recurrence (Fig. 1). Patients with higher urinary levels of CA9 (HR: 3.44, 95% CI: 1.21–9.76; P= 0.02), ANG (HR: 42.89, 95% CI: 3.06–602.10; P= 0.005) and MMP10 (HR: 3.86, 95% CI: 1.06–14.05; P= 0.04) had a significantly higher risk of disease recurrence. Increased levels of PAI1 (HR: 3.54, 95% CI: 0.87–14.43; P= 0.08), APOE (HR: 4.64, 95% CI: 0.91–23.59; P= 0.06) and A1AT (HR: 3.72, 95% CI: 0.94–14.83; P= 0.06) approached significance (Table 4).

4.Discussion

A comprehensive review of the literature regarding potential predictive biomarkers of BCG response reveals limited results. In 2003, investigators reported that the failure to detect urinary IL-2 during a BCG induction course correlated with the time to disease recurrence and progression [24]. Similarly, Kamat and others developed the CyPRIT assay and noted that an increase in nine cytokines, including IL-2, induced after a BCG treatment correlated with disease recurrence, and that combinatorial changes in the cytokine panel could best predict disease recurrence [19]. Monitoring cytokine profile changes is promising as a test of immune response once BCG treatment is initiated and may guide BCG treatment frequency or continuance in the individual.

Previous studies have assessed the ability of the diagnostic FISH test (UroVysionTM, Abbott Molecular Inc.) to predict whether patients with NMIBC would incur disease recurrence or progression after BCG treatment. UroVysion testing was applied at the beginning and the end of the BCG induction cycle (typically involving 6 weekly instillations), and urine cytology and cystoscopy were performed six weeks after cycle completion. In a multivariate analysis, the presence of high-grade disease and a positive UroVysion test after BCG initiation were significant predictors of disease recurrence [25, 26]. Again, in these studies, the urine-based results were only informative after the initiation of BCG treatment, and so are not predictive of BCG outcome prior to the first cycle of instillation. Therefore, the information would not be available to guide the clinical management of the individual patient regarding the decision to embark on an intravesical BCG treatment regimen. In a small prospective study, Lotan and others noted a positive FISH test prior to BCG to be associated with recurrence and progression, hazard ratio HR 2.59 (95% CI 1.42–4.73) [27].

Here, we evaluated the performance of the diagnostic OncuriaTM test to predict the response to BCG therapy, prior to treatment initiation. In this pilot study, we modified the established diagnostic algorithm, which weights biomarker values to produce a risk score, to the fit the predictive scenario. The 10 biomarkers that comprise the OncuriaTM test were reliably detected in almost all of the 64 urine samples, and the levels of 9 of the biomarkers were elevated in pre-treatment urine samples from subjects who had a subsequent bladder cancer recurrence after the completion of BCG treatment. As shown in diagnostic applications, it is the combination of the biomarker values through a weighted analytical algorithm that provides a model with sufficient predictive power for potential clinical application. In this case, the test shows promise for the evaluation of patients with respect to the decision to undergo BCG treatment.

The biomarkers that compose the bladder cancer diagnostic signature have a varied range of reported functions including angiogenesis, breakdown of extracellular matrix, serine protein inhibitor, catalyze the reversible hydration of carbon dioxide, lipoprotein metabolism and cell binding/signaling (Table 5) with the two primary functional groups being extracellular matrix remodeling (MMP9 and MMP10) and angiogenesis (IL8, VEGFA and ANG). Angiogenesis, the development of new blood vessels from existing blood vessels, is essential for normal growth and development of tissues and organs. A balance of pro-angiogenic factors and anti-angiogenic factors tightly controls this process [28, 29, 30]. However in solid tumors, the balance may favor pro-angiogenic factors, ensuring nutrients are provided to the rapidly dividing cancer cells, thus allowing the support of the abnormal growth seen in tumors [31]. Recent studies also suggest ANG and PAI1 can breakdown the extracellular matrix [32, 33], allowing cancer cells to invade and metastasize [34]. The extent of tumor vascularization differs between malignancies, and has been shown to correlate directly with metastatic potential [35].

We recognize that the study has several limitations. First, as a pilot study, with only 64 total subjects the study is small and underpowered. Second, defining truly independent disease recurrence and distinguishing new lesions from regrowth from a previous tumor resection site can be challenging. In future studies, we would propose noting the location of the primary tumor, as well as the location and timing of the recurrence, realizing that a second tumor within the previous tumor bed at 3 months follow-up may not necessarily be due to a lack of BCG response, while recurrences outside of the tumor bed may be more indicative of BCG failure. The evaluation of the test in a larger, prospective cohort with more information on lesion characteristics before and after BCG treatment may determine whether the urinary biomarker profile indicates residual disease or a field change in the urothelial or immune landscape which may be predictive of the response to intravesical BCG.

The development of an accurate and robust test that can predict BCG treatment response would benefit both patients and healthcare systems. The identification of predictive molecular signatures has the potential to better tailor treatment regimens for individual patients, avoiding potentially significant delays in clinical management. In this pilot study, the multiplex OncuriaTM test achieved encouraging predictive performance. The test uses established technology facilitating uptake in clinical laboratories once validated for defined applications. Additional studies are underway to evaluate the potential added value of the test in clinical decision making.

Ethics approval and consent to participate

MD Anderson Cancer Center local ethics review board approved.

Consent for publication

Not applicable.

Availability of data and materials

Reasonable requests for data will be made available for review.

Competing interests

Dr. Charles Rosser is an officer of Nonagen Bioscience. No financial or commercial conflicts of interest were declared by other co-authors.

Funding

This work was supported by research grants R01 CA1988887 (CJR) and R01 CA206584 (SG).

Author contributions

Conception, interpretation or analysis of data: KM.

Interpretation or analysis of data, revision for important intellectual content: AK.

Interpretation or analysis of data: IP, RC, YS.

Revision for important intellectual content: AG and SG.

Conception, supervision, preparation of the manuscript: CJR and HF.