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Diagnostic value of Xpert® Bladder Cancer Monitor in the follow-up of patients affected by non-muscle invasive bladder cancer: an update

Aims: Xpert® Bladder Cancer Monitor is a urinary marker based on the evaluation of five target mRNAs overexpressed in patients with bladder cancer (BC). The aim of our study is to update our results regarding the diagnostic accuracy of the Xpert® Bladder Cancer Monitor test in the follow-up of patients with non-muscle invasive bladder cancer (NMIBC). Methods: We conducted a prospective study on 1015 samples of 416 patients (mean age 72.2 ± 10.3 years) under follow-up for NMIBC. Patients underwent voided urinary cytology, the Xpert® Bladder Cancer Monitor test and cystoscopy and, if positive, a transurethral resection of the bladder. Xpert® Bladder Cancer Monitor was reported as negative or positive: cut-off total Linear Discriminant Analysis (LDA) = 0.5. Results: We identified 168 recurrent tumours: 126 (75%) were low-grade (LG) and 42 (25%) high-grade (HG). Overall sensitivity was 17.9% for cytology, 52.4% for Xpert® Bladder Cancer Monitor and 54.2% for the two tests combined. The sensitivity of cytology increased from 6.3% in LG to 52.4% in HG tumours whereas Xpert® Bladder Cancer Monitor showed a sensitivity ranging from 42.9% in LG to 80.9% in HG tumours. Combined cytology and Xpert® Bladder Cancer Monitor yielded an overall sensitivity of 45.2% for LG and 80.9% for HG tumours. Overall specificity was 98.5% for cytology and 78.4% for Xpert® Bladder Cancer Monitor and 78.2% for the two tests combined. The area under the curve (AUC) for Xpert® Bladder Cancer Monitor was 0.71; stratifying the patients according to the European Association of Urology risk groups, the AUC was 0.69, 0.67 and 0.85 for low, intermediate and high risk, respectively (p = 0.0003). Conclusion: Our data confirm a significantly higher sensitivity of Xpert® Bladder Cancer Monitor than for cytology in a larger patient cohort. The test performed very well in terms of specificity but could not reach the high value of cytology. Along with voided urinary cytology the test could allow to reduce cystoscopies in follow-up patients, reducing discomfort to the patients and costs.


Introduction
In 2018, worldwide there were 549,393 new cases of bladder cancer (BC), with 197,105 new cases in Europe. 1 BC requires a life-long follow-up with cytology and cystoscopy, resulting in a significant psychological burden for the patients 2 as well as an economic burden for society. 3 At the moment, no urinary marker for the followup of non-muscle invasive BC (NMIBC) patients which aims at replacing cystoscopy can be found in the European Association of Urology (EAU) Guidelines, on the one hand due to the risk of missing tumours in high-risk patients, on the other hand due to the low sensitivity in low grade recurrences. 4 Xpert ® Bladder Cancer Monitor is a new mRNAbased BC marker test based on the evaluation of five target mRNAs [ABL Proto-oncogene 1 (ABL1), Uroplakin 1B (UPK1B), Corticotropin Releasing Hormone (CRH), Annexin 10 (ANXA10), Insulin-like Growth Factor 2 (IGF2)], which are overexpressed in patients with BC. 5 In a previous cohort of 230 patients under follow-up for NMIBC at our institution, the test showed an overall sensitivity of 46.2%, a specificity of 77% and a fair diagnostic efficacy, with an area under the curve (AUC) of 0.65. 6 In other studies, Xpert ® Bladder Cancer Monitor showed excellent performances with higher sensitivity and specificity rates and with higher AUC values. 7,8 The aim of our study was to further evaluate the diagnostic value of Xpert ® Bladder Cancer Monitor in a larger cohort of patients followed for NMIBC, including those already analysed in the previous study. 6

Material and methods
After approval of the local institutional ethics committee (Ethic Committee of General Hospital of Bolzano, study registration number: 47-2017) and after written informed consent was obtained from the patients, 416 patients under follow-up for NMIBC in our outpatient department were enrolled in the present study.
Patients were routinely evaluated by voided urinary cytology, by the Xpert ® Bladder Cancer Monitor test and by cystoscopy. Any cystoscopically suspicious lesion was biopsied or removed trans-urethrally and specimens were evaluated according to the 2017 TNM classification of urinary BC and graded according to both the 1973 and the 2004 World Health Organization grade classification, as recommended by the European Association of Urology (EAU) Guidelines for NMIBC. 4,9,10 Of the voided urine of every patient admitted to the outpatient clinic for a routine follow-up control, 4.5 ml were added to the Xpert Urine Transport Reagent Kit, a RNA stabilizing reagent, and inverted three times in order to mix it properly. The residual urine was added to 15 ml Cytolyt fixation liquid (Hologic, Inc., Marlborough, MA, USA) in a Falcon tube and sent to the laboratory along with the stabilized urine for the Xpert ® Bladder Cancer Monitor test.

Cytology
The tubes were centrifuged for 10 min at 2000 rev/ min. The resulting cell pellets were resuspended in ThinPrep vials that contained PreservCyt solution and processed by the TP 5000 System (Hologic, Marlborough, MA, USA). Cytological evaluation was performed using the Papanicolaou staining procedure and the Paris System for Reporting Urinary Cytology. 11 The latter classifies the cytological specimens accordingly into negative for high grade urothelial carcinoma (NHGUC), atypical urothelial cells, suspicious for high grade urothelial carcinoma (SHGUC), high grade urothelial carcinoma (HGUC), low grade intraepithelial neoplasia (LGUN) and not diagnostic. For the statistical analysis NHGUC and atypical urothelial cells were grouped as negative, SHGUC, HGUC and LGUN as positive.

Xpert ® Bladder Cancer Monitor
According to the manufacturer, the Xpert ® Bladder Cancer Monitor, performed on the Cepheid GeneXpert Instrument System, is a qualitative in vitro diagnostic test created to monitor the recurrence of BC in patients previously diagnosed with BC.
The test measures the level of five target mRNAs, which are overexpressed in BC: ABL1, UPK1B, CRH, ANXA10, IGF2.
ABL1 is a protein-tyrosine kinase, which is found overexpressed in the progression of several solid tumours. An increased ABL1 expression was found in urine specimens of patients with BC, probably due to increased urothelial cellularity in these subjects. UPK1B is a structural protein of urothelial cells and can be high in BC patients. CRH is secreted from the hypothalamus and regulates biological and psychological response to stress, playing a role in regulation of several human cancers' development; ANXA10 is a journals.sagepub.com/home/tau 3 member of calcium-dependent phospholipidbinding protein family and plays a role in the regulation of cellular growth, IGF2 mRNA is often upregulated in BC and the level of IGF2 protein is high in urine samples of BC patients; 12 CRH, ANXA10 and IGF2 were already identified as BC markers in a prospective study of gene expression pattern in urine samples of BC patients. 13 Xpert ® Bladder Cancer Monitor detects these five mRNA targets, by means of real-time, reverse transcription-polymerase chain reaction (RT-PCR). The results are interpreted by the GeneXpert Instrument System from measured fluorescent signals and embedded calculation algorithms; Test Result, LDA totals and Analyte Results are shown on the Test Report. A cut-off is set at a LDA of ⩾0.5.

Statistical analysis
The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the Xpert ® score have been evaluated and area under the receiver operating characteristic curve and its confidence interval were calculated. In addition to the Xpert ® score the predictiveness of demographic characteristics (such as age and sex) were evaluated with respect to cystoscopy using classification and regression trees. 14

Results
One thousand and fifteen samples were obtained from 416 patients (89 women and 327 men). Mean age of the patients included in this prospective study was 72.2 ± 10.3 years. An average of two urine samples (from one to eight) for each patient were analysed over a period of 3-48 months after diagnosis. Thirty-one samples (3%) had to be excluded due to a not diagnostic Xpert ® Bladder Cancer Monitor test and/or cytology.     Figure 2).

Discussion
The Xpert ® Bladder Cancer Monitor is a test presented in October 2016. It evaluates the level of five target mRNAs in voided urine, by using a RT-PCR and thereby identifies patients with NMIBC recurrence. In the present study the cases were stratified according to the EAU risk tables in low, intermediate and high risk (respectively, 25.4%, 37.4% and 37.2% of the cases).
The AUCs of Xpert ® Bladder Cancer Monitor in these three groups are respectively 0.69, 0.67 and 0.84 (Figure 2), showing the significantly higher performance of the test in high-risk patients (p = 0.0003), suggesting a possible tailored use of this urinary marker in a specific subgroup of patients. Currently, no other analysis of the performance of this urinary marker has been conducted in a specific group of patients.  18 the cost of an office cystoscopy ranges from €163 to €520, so far reducing the number of cystoscopies could be an economical advantage.
Although urinary cytology remains the marker with the highest specificity, it seems obvious that the use of the Xpert ® Bladder Cancer Monitor test improves sensitivity in contrast to cytology alone; even though the combined use of cytology and Xpert ® Bladder Cancer Monitor does not seem much better than each of the two alone, by analysing the subgroups they show to be complementary and allow to increase the diagnostic accuracy. Therefore, cytology and Xpert ® Bladder Cancer Monitor should be used together to increase the interval between cystoscopies in the follow-up, if both are negative.
However, further prospective studies with a larger number of patients and longer follow-up are needed to validate the prognostic value of Xpert ® Bladder Cancer Monitor and its potential role in risk stratification of BC patients in terms of predictor of a recurrence or of response to BCG therapy.

Conclusions
Our data confirm a significantly higher sensitivity of Xpert ® Bladder Cancer Monitor than of cytology in a larger patient cohort. The test performed very well in terms of specificity but could not reach the high value of cytology, while PPV and NPV performed approximately the same for both tests. Along with voided urinary cytology the test could allow to reduce cystoscopies in follow-up patients, thus reducing discomfort to the patients and costs.

Significant conclusions
Our data confirm a significantly higher sensitivity of Xpert ® Bladder Cancer Monitor than of cytology in a larger patient cohort. The test performed very well in terms of specificity but could not reach the high value of cytology.