Reflex ImmunoCyt Testing for the Diagnosis of Bladder Cancer
in Patients with Atypical Urine Cytology
Anobel Y. Odishoa, Anna B. Berryb, Ardalan E. Ahmada, Matthew R. Cooperberga,
Peter R. Carrolla, Badrinath R. Konetyc,*
aDepartment of Urology and the Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA;
bDepartment of Pathology, University of California, San Francisco, San Francisco, CA, USA;cDepartment of Urology, University of Minnesota, Minneapolis,
of recurrence that necessitates intensive, invasive, and
costly long-term surveillance . Risk-stratified models
show 5-yr recurrence rates of 31–78% and 0.8–45%
progression rates, increasing from low- to high-risk disease
[1,2]. Current guidelines recommend that low-risk patients
undergo cystoscopy and cytology at 3 mo, follow-up in
patients undergo cystoscopy with urine cytology every
3 mo for the first 2 yr and annually thereafter .
E U R OP E A N U R O L O G Y 6 3 ( 2 0 1 3 ) 9 3 6 – 9 4 0
available at www.sciencedirect.com
journal homepage: www.europeanurology.com
Accepted April 3, 2012
Published online ahead of
print on April 14, 2012
Background: ImmunoCyt/uCyt (Scimedx, Denville, NJ, USA) is a well-established uri-
nary marker assay with high sensitivity for the diagnosis of urothelial carcinoma (UC)
and can function as a second-level test to arbitrate atypical reads of urine cytology.
Objective: To determine the utility of uCyt as a reflex test for atypical cytology in
patients undergoing a hematuria evaluation or surveillance with a history of UC.
Design, setting, and participants: The uCyt assay was performed as a second-level reflex
test on all voided urine cytology tests read as atypical between January 2007 and June
2010 in an academic medical center. Records were retrospectively reviewed. Three
hundred twenty-four patients underwent a total of 506 uCyt assays.
Intervention: Reflex uCyt assay on atypical urine cytology.
Outcome measurements and statistical analysis: The uCyt test characteristics include
Results and limitations: Reflex uCyt was performed on 506 atypical voided urine
samples that were followed by cystoscopy within 90 d. Reflex uCyt with a history of
UC showed a sensitivity of 73%, a specificity of 49%, and an NPV of 80%. In those with a
history of low-grade UC, reflex uCyt had a sensitivity of 75%, a specificity of 50%, and an
NPV of 82%, while in those with a history of high-grade UC, it had a sensitivity of 74%, a
specificity of 44%, and an NPV of 79%. Without prior history of UC, reflex uCyt had a
its retrospective design and interobserver variability inherent to cystoscopy, which was
used as the reference test.
Conclusions: When used as a reflex test on atypical urine cytology, negative uCyt may
predict a negative cystoscopy in select patients and modulate the urgency and further
work-up in those with no prior history or low-grade disease.
Published by Elsevier B.V. on behalf of European Association of Urology.
* Corresponding author. Department of Urology, University of Minnesota, 420 Delaware Street,
MMC 394, Minneapolis, MN 55455, USA. Tel. +1 612 625 1655; Fax: +1 612 624 4430.
E-mail address: email@example.com (B.R. Konety).
0302-2838/$ – seeback matter Published by ElsevierB.V. on behalf of European Association of Urology. http://dx.doi.org/10.1016/j.eururo.2012.04.019
Cystoscopy is an invasive procedure that can miss certain
lesions, such as the flat lesions of carcinoma in situ (CIS) .
In a pooled analysis of 14 studies, the sensitivity and
and 81%, respectively, compared against biopsy .
Urine cytology has been used >60 yr for the diagnosis of
UC. The test suffers from low sensitivity (38–51%), but its
makes it a useful adjunct for cystoscopy . Because of
factors such calculi, inflammation, instrumentation, or
infection, some samples cannot be categorized as either
benign or malignant and are considered ‘‘atypical.’’ Reliabili-
ty of results depends on sample quality and cytopathologist
experience. Urine cytology is diagnosed as atypical in as
many as 12% of voided samples and 28% of instrumented
underlies 23–68% of atypical cytology results [7–10]. Faced
with a poorly defined result with a high potential for
malignancy, clinicians have sparse data with which to make
appropriate and efficient treatment decisions.
Many voided urine biomarker assays, such as UroVysion
(Vysis, Downers Grove, IL, USA), NMP22, BTA TRAK (Poly-
medco, Cortlandt Manor, NY, USA), and ImmunoCyt/uCyt
UC diagnosis but have yet to gain widespread clinical
application in the context of atypical urine cytology. A triple
immunofluorescent monoclonal antibody assay, uCyt is a US
Food and Drug Administration–approved urine test for
antigens associated with UC. Fluorescein-labeled antibodies
labeled antibody 19A211 targets high-molecular-weight
carcinoembryonic antigen on exfoliated urothelial cells in
but this has improved in more recent studies to 77–91% and
68–83%, respectively [5,11].
We assessed whether the use of uCyt as a second-level
reflex test performed routinely in all patients with a reading
of atypical urine cytology can arbitrate results and improve
the sensitivity, specificity, negative predictive value (NPV),
and positive predictive value (PPV) of the uCyt assay
obtained in the context of atypical cytology. We used an
abnormal cystoscopy and positive biopsy within 90 d of
uCyt as reference standards. We also performed subgroup
analyses based on patient disease history.
2. Material and methods
Between January 2007 and June 2010, uCyt assays were performed
reflexively on all urine cytology samples at our institution that were read
as atypical . Urine cytology was performed as part of a hematuria
voided urine taken prior to instrumentation in patients without evidence
of urinary tract infection. Patients subsequently underwent work-up and
treatment based on current standards of care by their urologist.
After approval by the institutional review board, we retrospectively
demographics, disease history, uCyt results, and subsequent work-up
including white light cystoscopy, cytology, and biopsy. Cystoscopy and
biopsy results were considered pertinent to the uCyt assay if performed
within 90 d of the assay without a second urine test for bladder cancer
(cytology or uCyt) having been performed in the interim. Cystoscopy was
considered positive or abnormal if papillary lesions or any areas of
erythema distant from recent biopsy sites were identified.
trained cytopathologists. A test was considered positive if at least one cell
exhibiting characteristic red or green fluorescence was visible. Tumors
were graded using the 2004 World Health Organization/International
Society of Urological Pathology classification system . Statistical tests
were performed using Stata statistical software (StataCorp, College
Station, TX, USA).
Between January 2007 and June 2010, 636 urine cytology
specimens from 324 patients were read as atypical. Of these,
41 (6.4%) had inadequate cellularity or volume, and a reflex
uCyt assay was not performed. The analysis was limited to
506 uCyt assays that were followed by cystoscopy within
90 d. Figure 1 diagrams the clinical contexts in which these
assays were performed. Patient demographic and clinical
details are shown in Table 1. The mean age was 68.8 yr
(standard deviation [SD]: ?11.4yr)atthetimeoftesting,with
No history of
(n = 506)
within 90 d of uCyt
within 90 d of uCyt
History of UC
n = 394
n = 3
n = 99
n = 29
(n = 102)
(n = 10)
Fig. 1 – Distribution of clinical contexts in which uCyt was performed. UC = urothelial carcinoma.
EUROPEAN UR OLOGY 63 (2013) 936–940
no statistically significant difference in age between men and
women. Men made up 88% of the study population. Seventy-
eight percent of assays were performed in patients with a
history of UC (40% low grade, 56% high grade or CIS). Patients
without a history of UC who were undergoing a standard
hematuria evaluation made up 36% of the population and
underwent 142 (24%) of the reflex uCyt assays. Prior disease
five percent of patients had more than one uCyt assay
performed during the course of the study, with 51 undergoing
two assays and 63 having three or more done. Overall disease
prevalence was similar in those with and without a history of
UC. In 142 patients without a history of UC, 17 (12.0%) were
found to have biopsy-proven UC, while in 441 with a history of
UC, 55 (12.5%) were found have biopsy-proven recurrent UC at
the time of evaluation.
An abnormal cystoscopy within 90 d of ImmunoCyt was
used as the reference standard. Of the 506 reflex uCyt test
samples obtained, 372 (73.5%) were obtained on the same
day as the cystoscopy, and the remaining 132 (26.5%) were
obtained at a mean of 33.7 d (SD: ?25.0 d) from cystoscopy.
Test characteristics are detailed in Table 2. Overall, reflex uCyt
sensitivity was 75.2% and specificity was 49.3%, which led to a
PPV of 36.4 and an NPV of 83.7%. In those with no prior history
of UC (n = 102), sensitivity and specificity were improved to
85% and 59%, respectively, which led to a higher NPV of 94%.
To ensure that the 90-d time frame did not bias results, we
further analyzed test characteristics for patients who had the
ImmunoCyt test performed on the same day as their
cystoscopy (73.5% of all tests). Results (Table 3) revealed test
characteristics that mirrored the overall study population.
Subgroup analysis revealed consistent test performance
between patients with a history of low-grade (n = 159) and
high-grade or CIS (n = 221) disease. Sensitivity was 75% for
low-grade and 74% for high-grade or CIS disease, with an
NPV of 82% and 79%, respectively. A low specificity (50% and
44%, respectively) led to lower test PPV.
To further characterize reflex uCyt, we evaluated test
results inpatients whohadUC proven by biopsywithin 90 d
of uCyt testing (Table 2). Of the 131 patients who
underwent a biopsy or transurethral resection (TUR) within
90 d of uCyt testing, 99 (76%) had a history of UC, and 29
(24%) underwent random bladder biopsies as part of a
comprehensive hematuria evaluation. The mean number
of days between ImmunoCyt and biopsy or TUR was 30.1
(SD: ?22.4). Overall sensitivity was 86.5% (85.5% for those
with prior history and 88.2% for those without). In this setting,
specificity ranged from 20% to 25%, resulting in low PPV
(57–63%) and NPV (53–60%).
ImmunoCyt test performance has been well studied since
its introduction by Fradet and Lockhard in 1997 . Recent
of 62–78%, with PPVs and NPV of 26–67% and 91–96%,
respectively [14–18]. The previously reported values are for
Table 3 – ImmunoCyt test characteristics with abnormal cystoscopy as the gold standard*
uCyt with cystoscopy within 90 d
No history of UC
With history of UC
High-grade UC or CIS
uCyt with cystoscopy on the same day
PPV = positive predictive value; NPV = negative predictive value; UC = urothelial carcinoma; CIS = carcinoma in situ.
*Ten patients with an unknown history of UC were excluded from this analysis.
Table 1 – Summary of patient characteristics
n = 324
Male, no. (%)
Female, no. (%)
Mean ageYr (SD)
p = 0.62
Disease history No. (%)
History of UC
No prior disease
SD = standard deviation; UC = urothelial carcinoma.
Table 2 – ImmunoCyt test characteristics with urothelial carcinoma proven by biopsy as the gold standard*
No.All positives, no. True positives, no. Sensitivity, % Specificity, %PPV, % NPV, %
uCyt with biopsy within 90 d
With history of UC
No history of UC
PPV = positive predictive value; NPV = negative predictive value; UC = urothelial carcinoma.
*Three patients with an unknown history of UC were excluded from this analysis.
EURO PEAN UROLOGY 63 ( 2013) 936–940
uCyt assays performed regardless of underlying cytologic
of atypical cytology. Tetu and colleagues, using a combina-
tion of cystoscopy and biopsy as the reference, performed
subgroup analyses focusing on just those patients with
atypical cytology. Of the 870 enrolled patients, 109 had a
cytology reported as ‘‘suspicious.’’ In this group, uCyt had a
sensitivity of 73%, a specificity of 34%, an NPV of 77%, and
a PPV of 30%. Although this is a lower specificity than the
overall group (62%), leading to a lower NPV, the authors
report that there was no statistically significant difference
in test performance between the overall group and the
‘‘suspicious’’ subgroup (p = 0.362) . In our study of
506 patients with atypical cytology, uCyt had comparable
of 84% and a PPV of 36%. Our larger study, focusing
exclusively on analyzing the utility of uCyt to arbitrate
atypical cytology, confirms the subgroup analyses from Tetu
et al. A sensitivity of 75% for atypical cytology is comparable
to overall published uCyt sensitivity.
overall uCyt. This may be the result of the general ambiguity
of the atypical category and the fact that these samples were
by definition not clearly differentiable. Work by Yoder and
colleagues implementing reflex fluorescence in situ hybrid-
ization using UroVysion as a reflex test against all negative
be aneffectivemechanism for improvingcytologic diagnosis
. UroVysion testing is generally more expensive and
as uCyt can be performed in a standard cytopathology
laboratory utilizing the same specimen, making it easier to
implement and more accessible.
Urine cytology is a highly specific test for diagnosis of
high-grade UC, routinely reported at >95%. However, it is
notoriously unreliable for low-grade disease, which cytolog-
ically resembles nonneoplastic urothelium; uCyt was
designed to improve urine cytology test performance in
those with low-grade disease. In this study, however,
subgroup analyses revealed consistent test performance
across disease grade. In patients with a history of low-grade
disease but atypical cytology, a negative uCyt has a
sufficiently high predictive value to safely delay cystoscopy.
recur at 37%, with 0% progression in their cohort, whereas
high-risk tumors recurred more often (54%), with a higher
rate of progression to invasive disease (15%) . Although
some have proposed alternative screening regimens that
cy, they have yet to be integrated into clinical practice
[17,21,22]. Sylvester and colleagues showed that multifac-
torial analysis of patient characteristics more accurately
predicts recurrence and progression risk , and incorpo-
ration of uCyt and other urinary marker data may improve
Mian and colleagues described 100% sensitivity in
detecting recurrent CIS using uCyt in a small group of
patients, showing its potential for both low- and high-risk
disease . Others have taken patient preferences into
account, using patient survey data to determine that any
urinary marker assay would need a minimum sensitivity of
75–90% for approximately 90% of patients to prefer it to
In those with no prior history of disease, sensitivity was
highest at 85%, with an NPV of 94%. uCyt can provide
important information in evaluating hematuria, particu-
larly in the patient with negative imaging and cystoscopy
but atypical cytology. Schmitz-Dra ¨ger and colleagues
studied 189 patients being evaluated for microscopic
hematuria with ImmunoCyt and noted an NPV of 99.4%
It is important to note in that even with an atypical
cytology, NPV remained high.
There were some limitations in the current study.
Approximately 6% of samples were of inadequate quality
for uCyt analysis, the result of inadequate collection and
processing early in theinstitutional experience, and therate
rapidly decreased over time. Although all patients with
atypical cytology did undergo reflex ImmunoCyt testing,
further evaluation andmanagement was atthe discretionof
the treating urologist. In addition, cystoscopy was used as
the reference standard, which is less reliable than a
histologically confirmed diagnosis. This method suffers
from the inherent variability and subjectivity of cystoscopy
but reflects its performance and utilization in practice.
A 90-d window within which a cystoscopy was considered
relevant was used. There may be a chance of interval
development of new lesions, but >74% of uCyt assays were
performed on the same day as the cystoscopy. In addition,
imaging of the upper tract was not independently evaluated
in this study. Although we did analyze data using biopsy as
the reference, these data are biased by the fact that only
those with positive or suspicious lesions on cystoscopy
underwent biopsy, introducing assignment bias. Using
biopsy results as a standard, the sensitivity of uCyt was
relatively high at 86.5%.
a negative ImmunoCyt result can be used to predict a
negative cystoscopy in select patients. The high NPV can be
used to modify the urgency and nature of further urologic
work-up, both in those without a history of UC and in those
with a history of low-grade UC in which a routine follow-up
schedule with fewer cystoscopies can be maintained despite
a reading of atypical cytology. A prospective study assessing
cytology in a standardized pathway will help identify an
optimal method of avoiding unnecessary work-up in these
patients while ensuring identification of those with disease.
Author contributions: Badrinath R. Konety had full access to all the data
in the study and takes responsibility for the integrity of the data and the
accuracy of the data analysis.
Study concept and design: Odisho, Konety, Carroll, Cooperberg.
Acquisition of data: Odisho, Ahmad, Berry.
EUROPEAN UR OLOGY 63 (2013) 936–940
Analysis and interpretation of data: Odisho, Berry, Cooperberg, Carroll, Download full-text
Drafting of the manuscript: Odisho, Konety.
Critical revision of the manuscript for important intellectual content:
Odisho, Berry, Cooperberg, Carroll, Konety.
Statistical analysis: Odisho.
Obtaining funding: None.
Administrative, technical, or material support: Konety, Carroll.
Supervision: Cooperberg, Carroll, Konety.
Other (specify): None.
Financial disclosures: Badrinath R. Konety certifies that all conflicts of
interest, including specific financial interests and relationships and
affiliations relevant to the subject matter or materials discussed in the
manuscript (eg, employment/affiliation, grants or funding, consultan-
cies, honoraria, stock ownership or options, expert testimony, royalties,
or patents filed, received, or pending), are the following: None.
Funding/Support and role of the sponsor: None.
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