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. Author manuscript; available in PMC: 2022 Feb 9.
Published in final edited form as: Virchows Arch. 2019 Sep 3;476(3):423–429. doi: 10.1007/s00428-019-02654-1

Performance of novel non-invasive urine assay UroSEEK in cohorts of equivocal urine cytology

Maria Del Carmen Rodriguez Pena 1,2, Simeon U Springer 3,4, Diana Taheri 2,5, Lu Li 3,4, Aline C Tregnago 2, Marie-Lisa Eich 1,2, Isam-Eldin A Eltoum 1, Christopher J VandenBussche 2, Nickolas Papadopoulos 3,4, Kenneth W Kinzler 3,4, Bert Vogelstein 3,4, George J Netto 1,2
PMCID: PMC8827219  NIHMSID: NIHMS1751102  PMID: 31482302

Abstract

Urine cytology is an essential element of the diagnostic work up of hematuria. A significant proportion of cases continue to be placed in the “atypical” or “suspicious” categories of the Paris system for urine cytology, posing difficulty in patient management. We report on the performance of our recently described urine-based assay “UroSEEK” in cases with equivocal diagnosis in patients who are investigated for bladder cancer. Urine samples were collected from two cohorts. The first consisted of patients who presented with hematuria or lower urinary tract symptoms (early detection cohort) and the second of patients that are in follow-up for prior bladder cancer (surveillance cohort). Urine samples were analyzed for mutations in 11 genes and aneuploidy. In the early detection setting, we found high sensitivity and specificity (96% and 88%, respectively) and a strong negative predictive value of 99%. The assay performance was less robust in the surveillance cohort (sensitivity of 74%, specificity of 72%, and negative predictive value of 53%). UroSEEK demonstrated a notable lead time to cancer diagnosis. Seven cases in the early detection cohort and 71 surveillance cases were detected at least 6 months prior to clinical diagnosis. Our results suggest a potential role for UroSEEK assay in guiding management of patients with atypical urine cytology if confirmed in future prospective trials.

Keywords: DNA mutational analysis, Aneuploidy analysis, UroSEEK, Non-invasive urine assay, Atypical urine cytology, Urinary bladder neoplasms

Introduction

Bladder cancer (BC) is the most frequent urinary tract malignancy. Out of 158,220 new cases of urinary system cancer in the USA, 51% are of bladder origin, leading to 17,670 estimated bladder cancer deaths during 2019 [1]. Cystoscopy and urine cytology remain the gold standard for early detection and surveillance of BC. The latter has low sensitivity and specificity for low-grade lesions and low-intra and inter-observer reproducibility [2–4]. The Paris system for urine cytology is a standardized reporting system aiming to accurately diagnose high-grade urothelial carcinoma. It encompasses 7 categories: (I) non-diagnostic/unsatisfactory, (II) negative for high-grade urothelial carcinoma (NHGUC), (III) atypical urothelial cells (AUC), (IV) suspicious for high-grade urothelial carcinoma (SHGUC), (V) high-grade urothelial carcinoma (HGUC), (VI) low-grade urothelial neoplasm (LGUN), and (VII) other: primary and secondary malignancies and miscellaneous lesions [5]. Nevertheless, a significant proportion of cases remain indeterminate and are placed in the “atypical” or “suspicious” categories, posing difficulty for patient management. Piaton et al. performed a study examining the clinico-pathological findings of patients with atypical urothelial cells. They had a total of 534 urinary cytopathology specimens with 221 (41%) cases classified as “atypical urothelial cells, not otherwise specified” [6]. Rosenthal et al. reported 26% of cases classified as atypical urothelial cells of uncertain significance in their institution [7].

Molecular evidence has suggested distinct genetic alterations associated with non-muscleinvasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC). Signaling pathways consistently associated with NMIBC include the FGFR-3, H-RAS, and PIK3CA genes [8–11]. Alterations in tumor suppressor genes p53, p16, and Rb have been strongly associated with MIBC development [8, 12, 13]. More recently, our group and others have shown that activating mutations in the upstream promoter of the TERT gene are frequent genetic alterations in many tumors [14–16], including urothelial neoplasms and bladder cancer variants [17–21].

New tests for BC detection have been developed to capture genetic and protein alterations in urine and serum samples [17, 22–33]. Available FDA-approved tests for BC detection include the nuclear matrix protein 22 (NMP22) immunoassay test, and multi-target FISH (UroVysion) [34, 35].

UroSEEK is a novel non-invasive urine-based assay utilizing massively parallel sequencing applied to cellular DNA to detect frequently encountered bladder cancer mutations affecting TERT gene promoter and 10 additional genes (FGFR3, TP53, CDKN2A, ERBB2, HRAS, KRAS, PIK3CA, MET, VHL, MLL) combined with assessment of aneuploidy [8–11, 17, 30, 36–39].

We aim to assess the role of our recently described assay “UroSEEK” in patients who had equivocal urine cytology diagnosis. We analyzed two distinct early detection (ED) and surveillance cohorts of BC.

Material and methods

The study was approved by the Institutional Review Board. Urine samples were prospectively collected from the Johns Hopkins Hospital between 2013 and 2015 from the urology clinic. Patients with history of malignancy other than urothelial carcinoma were excluded. Samples were collected longitudinally and enriched for atypical and positive diagnoses. Study population included 2 cohorts: ED and surveillance. The ED cohort consisted of urine samples collected from patients without previous history of BC who presented with hematuria or lower urinary tract symptoms to be evaluated for malignancy. The surveillance cohort consisted of patients with known history of BC being followed with urine cytology and cystoscopy. Follow-up data was obtained from electronic medical records. All patients without detected tumor or recurrence had at least 11 months of follow-up. The mutational findings of the UroSEEK panel in a subset (105 early detection, 95 surveillance) of the current two larger cohorts were previously described in our initial report of the UroSEEK assay [36].

All urine cytology specimens were interpreted by a single group of board-certified cytopathologists. Only cases classified as category III (atypical urothelial cells) and IV (suspicious for high-grade urothelial carcinoma) according to The Paris System were included in the study. The two categories were combined and designated as “ATYP.”

Mutation analysis

The urine samples included two groups. The first group consisted of residual urinary cells after processing with standard BD Sure-Path liquid-based cytology protocols (Becton Dickinson and Company; Franklin Lakes, NJ, USA). To allow for standardofcare, residual Sure-Path fluids were kept refrigerated for 6–8 weeks before submission for DNA purification to allow for any potential need for repeat cytology processing of the same sample. The second group consisted of biobanked fresh urine in which 15–25 mL of voided urine samples were stored at 4 °C for up to 60 min prior to centrifugation (10 min at 500g). Afterwards, pellets were stored at minus 80 °C before DNA purification.

Purified DNA was assessed by “SafeSeqS,” a sequencing error-reduction technique capable of discriminating mutations from artefactual sequencing variants introduced during the analysis [39]. As detailed in Springer et al. [36], three separate assays were used to detect genetic alterations in urinary cell DNA. (I) UroSeqS: A multiplex PCR to detect alterations in regions of ten genes commonly mutated in BC: CDKN2A, ERBB2, FGFR3, HRAS, KRAS, MET, MLL, PIK3CA, TP53, and VHL. (II) TERTSeqS: A single amplification primer was used to amplify a 73 bp segment containing the region of the TERT promoter known to harbor mutations in BC and upper tract urothelial carcinoma. (III) Aneuploidy was assessed with FastSeqS, a technique which uses a single primer pair to amplify ~ 38,000 loci scattered throughout the genome [40]. Mutation and data analyses have been thoroughly described in our previous studies [18, 21, 36].

Results

Early detection cohort

A total of 375 urine samples from 348 patients were assessed by cytology. Among these, 25 (7%) were positive and 236 (63%) were negative for malignancy. The remaining 114 (30%) samples from 107 patients were classified as ATYP on cytology and formed the ED cohort for this study.

Seventy-seven (72%) patients were male and 30 (28%) female. The mean age was 61 (range 28 to 93) years. The mean follow-up time was 17 months (range 0–40 months).

UroSEEK assay was positive in 35/114 (31%) samples. Mutations in the TERT promoter (TERTSeqS) were detected in 24 (21%), the multiplex assay (UroSeqS) detected mutations in 25 (22%), and aneuploidy (FastSeqS) was found in 14 (12%) samples. A total of 25/114 (22%) ATYP urine samples developed tumors while 89 (78%) did not. UroSEEK was positive in 24 (96%) of 25 samples with subsequent tumor detection. The distribution of UroSEEK assay components in these samples was as follows: TERT promoter mutations were detected in 15 (60%), UroSeqS detected mutations in 22 (88%), and aneuploidy was detected in 13 (52%). As shown in Fig. 1, samples with subsequent tumor diagnosis had a higher proportion of concurrent positivity in all three UroSEEK components, compared with those that did not develop tumor (38% vs 0%; p < 0.0001). In 3 out of the 25 cases that developed a tumor, the diagnosis of malignancy was established by urine cytology or cystoscopy, without obtaining a TUR biopsy, precluding further histologic classification. UroSEEK was positive in 3/3 urines subsequently diagnosed with low-grade urothelial tumors and 19/19 diagnosed with high-grade urothelial tumors. Table 1 provides a summary of alterations by each test component across diagnosis, grade, and stage categories.

Fig. 1.

Fig. 1

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Venn diagram depicting the distribution of UroSEEK’s components positivity in samples with subsequent tumor diagnosis (a) and those without (b)

Table 1.

UroSEEK’s component findings in the early detection cohort with equivocal urine cytology across diagnosis, grade and stage categories

n % TERTSeqS UroSeqS FastSeqS
Diagnosis
 CIS 2 9 1 50% 2 100% 0 0%
 LGTCC 3 14 3 100% 3 100% 2 67%
 HGTCC 7 32 4 57% 6 86% 6 86%
 INTCC 10 45 5 50% 9 90% 5 50%
Grade
 LG 3 14 3 100% 3 100% 2 67%
 HG 19 86 10 53% 17 89% 11 58%
Stage
 pTis 2 9 1 50% 2 100% 0 0%
 pTa 10 45 7 70% 9 90% 8 80%
 pT1 6 27 3 50% 5 83% 3 50%
 pT2 2 9 1 50% 2 100% 1 50%
 pT3 1 5 1 100% 1 100% 1 100%
 pT4 1 5 0 0% 1 100% 0 0%
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CIS, urothelial carcinoma in situ; LGTCC, non-invasive low-grade papillary urothelial carcinoma; HGTCC, non-invasive high-grade papillary urothelial carcinoma; INTCC, infiltrating high-grade urothelial carcinoma; LG, low grade; HG, high grade

*

Histologic tumor features are not available for 3/24 cases (detected by cytology or cystoscopy)

UroSEEK positivity preceded tumor diagnosis on average by 1 month (range 0–18 months). Seven cases had a lead time of at least 6 months. Performance characteristics of UroSEEK and its components are detailed in Table 2.

Table 2.

Performance characteristics of UroSEEK and its components in the early detection cohort with equivocal urine cytology diagnosis

UroSEEK TERTSeqS UroSeqS FastSeqS
Sensitivity 96% 60% 88% 52%
Specificity 88% 90% 97% 99%
Negative Predictive Value 99% 89% 97% 88%
Positive Predictive Value 69% 63% 88% 93%
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Surveillance cohort

A total of 717 urine samples from 496 patients were assessed by cytology. Among these, 84/717 (12%) were positive and 301/717 (42%) negative for malignancy. The remaining 332 (46%) samples from 249 patients were classified as ATYP on cytology and formed the surveillance cohort in this study.

One hundred ninety-two (77%) patients were male and 57 (23%) female. The mean age was 64 (range 26 to 93) years. The mean follow-up time was 9 months (range 0–36 months).

UroSEEK assay was positive in 200/332 (60%) urine samples. Mutations in the TERT promoter (TERTSeqS) were detected in 174 (52%), the multiplex assay (UroSeqS) detected mutations in 137 (41%), and aneuploidy (FastSeqS) was detected in 157 (47%) out of 332 urinary samples. As shown in Fig. 2, samples from patients that recurred had a higher proportion of concurrent positivity in all three UroSEEK components compared with those that did not recur (55% vs 22%; p < 0.0001).

Fig. 2.

Fig. 2

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Venn diagram depicting the distribution of UroSEEK’s components positivity in samples from patients that recurred (a) and those that did not (b)

A total of 235/332 (71%) ATYP urine samples developed tumors while 97 (29%) did not. UroSEEK was positive in 173 (74%) of 235 samples with subsequent tumor detection. The distribution of UroSEEK assay components in these samples was as follows: Mutations in the TERT promoter were detected in 152 (65%), UroSeqS detected mutations in 126 (54%), and aneuploidy was detected in 138 (59%). Table 3 lists the mutations across diagnosis, grade, and stage categories. UroSEEK positivity preceded tumor recurrence diagnosis in average by 4 months, with a range between 0 and 26 months. Seventy-one cases had a lead time of at least 6 months and 6 of at least 20 months. Performance features of UroSEEK and each of the components are listed in Table 4.

Table 3.

UroSEEK findings in the surveillance cohort with equivocal urine cytology across diagnosis, grade and stage categories

n % UroSEEK TERTSeqS UroSeqS FastSeqS
Diagnosis
 URCa 3 1 0 0% 0 0% 0 0% 0 0%
 PUNLMP 3 1 1 33% 1 33% 1 33% 0 0%
 CIS 56 28 40 71% 37 66% 23 41% 33 59%
 LGTCC 58 29 47 81% 42 72% 34 59% 39 67%
 HGTCC 35 17 26 74% 22 63% 20 57% 20 57%
 INTCC 47 23 34 72% 32 68% 26 55% 28 60%
Grade
 LG 61 30 48 79% 43 70% 35 57% 39 64%
 HG 140 70 100 71% 91 65% 69 49% 81 58%
Stage
 pTis 56 28 40 71% 37 66% 23 41% 33 59%
 pTa 97 49 74 76% 65 67% 55 57% 59 61%
 pT1 24 12 15 63% 15 63% 13 54% 14 58%
 pT2 11 6 8 73% 7 64% 5 45% 5 45%
 pT3 8 4 7 88% 6 75% 4 50% 6 75%
 pT4 4 2 4 100% 4 100% 4 100% 3 75%
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URCa, urothelial carcinoma; PUNLMP, papillary urothelial neoplasm of low malignant potential; CIS, urothelial carcinoma in situ; LGTCC, non-invasive low-grade papillary urothelial carcinoma; HGTCC, non-invasive high-grade papillary urothelial carcinoma; INTCC, infiltrating high-grade urothelial carcinoma; LG, low grade; HG, high grade

*

Tumor details are not available for 33 cases (recurrence detected by cytology or cystoscopy)

**

Due to technical/artifact issues 2 cases were not graded and/or staged; 1 case was metastatic to lung

Table 4.

Performance characteristics of UroSEEK and its components in the surveillance cohort with equivocal urine cytology diagnosis

UroSEEK TERTSeqS UroSeqS FastSeqS
Sensitivity 74% 65% 54% 59%
Specificity 72% 77% 89% 80%
Negative predictive value 53% 47% 44% 45%
Positive predictive value 87% 87% 92% 88%
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Discussion

Current standard of care for bladder cancer surveillance consists of cystoscopy and urine cytology at 3-month intervals during the first 2 years [2]. While a definitive cytology diagnosis is actionable, the finding of atypia remains problematic in clinical management. Therefore, there is an overt need for a high-performing assay to resolve equivocal cases.

Despite previous attempts to develop non-invasive bladder cancer detection assays, their integration into routine patient management remains a challenge. This is in part due to their less than optimal performance characteristics and high technical expertise requirements. We recently reported on the utility of the urine-based UroSEEK assay in BC early detection and surveillance settings with high sensitivity and specificity when combined with cytology. UroSEEK has also demonstrated a superior capability compared with cytology for detection of low-grade tumors, capturing molecular alterations in 67% of urines that were either negative or atypical on cytology [36].

In this report, we detail UroSEEK’s performance in a large cohort of cases with atypical cytology. In the early detection setting, we found high sensitivity and specificity (96% and 88%; respectively) and a strong negative predictive value of 99%. The assay performance was less robust in the surveillance cohort (sensitivity of 74%, specificity of 72%, and negative predictive value of 53%). Of note, a higher proportion of samples with tumor detection (or recurrence) had concomitant positivity in all 3 UroSEEK components compared with those without.

Our findings suggest that in atypical cytology, UroSEEK may yield favorable performance characteristics compared with UroVysion, for which a sensitivity of 44.64% and specificity of 81.82% has been reported [41]. Like UroVysion, UroSEEK’s component FastSeqS detects numerical chromosomal abnormalities. Interestingly, FastSeqS alone had a superior sensitivity (52% and 59% for ED and surveillance, respectively) and a comparable specificity (99% and 80%, respectively) to UroVysion.

UroSEEK demonstrated a notable lead time to clinical cancer diagnosis. Seven cases in the ED cohort and 71 surveillance cases were detected at least 6 months prior to clinical diagnosis.

The error-reducing technical approach of the massively parallel sequencing method (SafeSeqS), the prospective nature of the large single institutional cohort, and the consistency in guideline-based review by a single group of subspecialized expert cytopathologists are some of the strengths of our study. The fact that we could not address the role of UroSEEK in the setting of prospective management in patients with sequential equivocal cytology diagnoses could be seen as a weakness. This should be addressed in a future study, as well as separately assessing the performance of UroSEEK in each of the two equivocal Paris system categories III and IV.

Our promising results suggest a potential for the clinical application of UroSEEK assay in cases of atypical cytology. If further validated in larger multi-institutional cohorts, the impact of incorporating UroSEEK in the decision-making algorithm regarding the need and frequency of cystoscopy should be assessed.

Funding information

Support provided by Henry and Marsha Laufer, Virginia and D.K. Ludwig Fund for Cancer Research, the Commonwealth Foundation, John Templeton Foundation, Conrad R. Hilton Foundation and grants from the NIH (T32 GM007309/GM/NIGMS NIH HHS/United States; P30 CA077598/CA/NCI NIH HHS/United States; P30 CA006973/CA/NCI NIH HHS/United States; R01 ES019564/ES/NIEHS NIH HHS/United States). All sequencing was performed at the Sol Goldman Sequencing Facility at Johns Hopkins.

Footnotes

Conflict of interest Nickolas Papadopoulos, Ken W Kinzler, and Bert Vogelstein: Founders of Personal Genome Diagnostics and PapGene and advice Sysmex-Inostics. Kinzler and Vogelstein also advise Eisai. Vogelstein is also an advisor to Camden Partners. These companies and others have licensed technologies from Johns Hopkins that are related to the work described in this paper. These licenses are associated with equity or royalty payments to Papadopoulos, Kinzler, Netto, and Vogelstein. Additional patent applications on the work described in this paper may be filed by Johns Hopkins University. The terms of these arrangements are managed by the university in accordance with its conflict of interest policies. The other authors declare that no competing interests exist.

Informed consent The Institutional Review Board approved this study.

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