Urine Cytology

Introduction
• Majority of UT malignancies are UC
– Urothelial carcinoma, 80-90%
– Mixed Carcinoma- UC (5%)
– Squamous cell carcinoma (5%)
– Adenocarcinoma (2%)
– Small Cell Carcinoma (1%)
• The main function of urine cytology is to
diagnose urothelial carcinoma (UC)
 Indications
1. Establish Dx in symptomatic patients-
hematuria
– Most common, low yield (5-10% malignancy)
2. Screen high risk patients (exposure to
industrial chemicals, metals, etc.)
3. Follow-up patients with Hx of UC
4. Complementary to cystoscopy and biopsy:
detect small and hidden lesions
(diverticuli, ureters, renal pelvis)
• Urine cytology is the most reliable method
Diagnostic Accuracy of Urine Cytology
• Number of Specimens
-Voided urine on 3 consecutive days
- 50% accuracy (1 specimen)
- 75-90% accuracy (3
specimens)
• Patient Population
- High risk and history of CA
• Tumor Grade > 90 %
• HGUC:
• LGUC: <50 %
Atypia in Urine Cytology
 Diagnostic Categories
• JH created a
template similar to
Gyn TBS:
1. Negative
2. AUC-US
3. AUC-H
4. LG neoplasm
5. HG neoplasm
6. Rosenthal,
Non-diagnostic
cancer cytopath
 Diagnostic Categories
1. Negative
2. AUC-US • Cleveland Clinic
(26%) 1. Negative
3. AUC-H (5%) 2. Atypical (14%)
4. LG neoplasm 3. Suspicious for HG UC
5. HG neoplasm (2%)
6. Non-diagnostic 5.

Positive
Rosenthal, cancer cytopath
 Diagnostic Categories
Preferred by Urologists

1. Negative for HGUC

2. Suspicious for HGUC
3. Positive for HGUC
 Should We Eliminate the
“Atypical” Category?

• Approx 10-20% of urines classified as

“atypical”
• Considerable inter-observer variability
among pathologists as to what constitutes
atypia
• Currently, most urologists interpret “atypia”
as negative or unhelpful
Arguments for Not Eliminating “Atypia”
• Significant proportion of malignant cases
would be missed if “atypia” was
eliminated
– Malignant rate on FU: 23-68%
• Ancillary studies such as FISH can be
helpful in those cases
 Variations in Atypical rate

• Inter-institutional:
– Reported wide variation: 2-31%
• Intra-departmental:
• ≈ 23,000 urine cases signed out by 12
cytopathologists at CC, over 3 yr
period
- All were cytopathology board certified
• Variable experience ranging from 2-26
 Reynolds, Elsheikh 2015
Atypical rate
30,0%
Group 1
25,0% Group 1
Group 1

20,0%
< 5 yrs 6-15 yrs > 15 yrs Group 1
Group 2

15,0% Group 2
Group 2

10,0% Group 2
Group 3

5,0% Group 3
Group 3
Group 3
0,0%
Group 1 Group 1 Group 1 Group 1 Group 2 Group 2 Group 2 Group 2 Group 3 Group 3 Group 3 Group 3

• Wide variation in Atypical rate: 8-28% (avg 14%)

• Higher rates are not related to level of experience
• More dependent on individual threshold for atypia
Urine Dx’s Categorized by Pathologist
BMH, Indiana 2009
 In Need of Standardization!
• Standard classification and terminology system
• Well defined and reproducible diagnostic criteria
• Uniform inter- and intra-
departmental communications
• Consistent prognostic and management
information leading to optimal patient
care
The Paris System for Reporting
Urinary Tract Cytology
 TPS Diagnostic Categories
• Negative for HGUC
• Atypical Urothelial Cells
• Suspicious for HGUC
• High Grade Urothelial Carcinoma
• Low Grade Urothelial Neoplasm
• Other malignancies, both primary and
secondary
 Urinary Tract Histology
• Superficial cell layer Bladder
one cell thick, superficial
squames-size or larger,
multinucleated
• Intermediate cell layer
approximately 5 cell layers,
parabasal-size cells
• Basal cell layer
one cell thick, cuboidal-
columnar
 Upper Urinary Tract Histology 2

Ureter and Renal

Pelvis
•Lining cells are
larger and more
pleomorphic than
bladder (decreased
cell turnover &
exfoliation)
 Normal Urinary Tract
Cytology
• Superficial urothelial cells
– Marked variation in size and
shape (10-150 u), low N/C
– Often polygonal
– Abundant pale cytoplasm, well
defined borders
– Occasional vacuolization
– Round-oval nuclei, often
multinucleated
Normal Urinary Tract Cytology 2
• Deep urothelial cells
– Uniform in shape and
size (10-20 u)
– Scant to moderate dense
cytoplasm, distinct
borders, fine
vacuolization
– Central nuclei, finely
granular chromatin,
small nucleoli
 Normal Urinary Tract Cytology 3

• Voided urine is sparsely cellular, single cells-

degeneration
 Normal Urinary Tract Cytology 4

• Catheterization or washings specimens have many clusters

 TPS Diagnostic Categories
• Negative for HGUC
• Atypical Urothelial Cells
• Suspicious for HGUC
• High Grade Urothelial Carcinoma
• Low Grade Urothelial Neoplasm
• Other malignancies, both primary and
secondary
 High Grade Urothelial CA
 ften invasive,  mortality
 90% of pts dying of disease
present initially with
HGUC
• Cytology cannot reliably
separate CIS from invasive
CA
• High diagnostic accuracy
of cytology
–
 HGUC- TPS Criteria
Non-superficial and non-degenerated
(viable) urothelial cells
• High N/C ratio > 0.5-0.7 (required)
• Hyperchromasia, moderate-severe
(required)
– and one of the following:
• Irregular clumpy chromatin
• IrregularAtnuclear membranes
least 5-10 abnormal cells
– Based on pathologist’s level of comfort
– Voided vs. upper tract instrumented specimen
 HGUC
• Single cells and/or disorganized
clusters
• Irregular, hyperchromatic
- Pleomorphic bizarre cells, enlarged eccentric
nuclei, coarse dark chromatin
 TPS Diagnostic Categories
• Negative for HGUC
• Atypical Urothelial Cells
• Suspicious for HGUC
• High Grade Urothelial Carcinoma
• Low Grade Urothelial Neoplasm
• Other malignancies, both primary and
secondary
 Suspicious for HGUC- TPS Criteria
• Same criteria as HGUC:
- Viable deep urothelial cells
- High N/C ratio > 0.5-0.7
- Marked Hyperchromasia
- Irregular clumpy chromatin
- Irregular nuclear membranes

• Less than 5-10 abnormal cells

– Based on pathologist’s level of comfort
– Voided vs. upper tract instrumented specimen
 Coy Cells
• Suspicious for HGUC
• Opposite of “decoy cells”
– Often sparse in number
– Been compared to
“litigation cells” on Paps
– Small cells with
hyperchromatic irregular
nuclei
– India ink/coal black
nuclei
Differential Diagnosis of HGUC
• Human polyoma viral
infection
• Therapy effect
• Stones and reactive changes
• Other malignancies
 Human
Polyomavirus
• DNA virus (Papova)
• Immunocompromised and
healthy individuals
• Important cause of allograph
failure in renal transplant
recipients
• Decoy Cells- infected nuclei:
– Smudgy
– Washed out
 Polyoma Virus
• Diff DX is degenerated HG UC

Polyoma virus HG UC
Architecture Single cells Single cells &
clusters
Nuclear Smooth, round Marked irregularity
membrane
Chromatin Uniform, smudgy, Coarsely granular,
reticulated clumped
Polyoma HGUC
 Therapy Effect
• Cytoxan & Busulfan
– Systemic treatment of non
urothelial malignancies
– Hemorrhagic cystitis
– Severe cytologic atypia may
be indistinguishable from CA
– Atypia more bizarre than
usual HGUC
– Atypia often has degenerative
features
Photo from Modern Cytopathology.
Elsevier Science, 2004
 Therapy Effect 2 Photo from Murphy WM. Urinary
Cytopatholgy. ASCP Press,2000

Thiotepa & Mitomycin

C Intravesical Rx of sup
UC Repair-like changes
BCG Vaccine
Treatment of CIS
Granulomas, mild atypia
Radiation Change
Extreme cytomegaly,
multinucleation, but low N/C ratio
 Lithiasis
• Papillary clusters common
• Smooth bordered clusters
• Centrally placed nuclei,
smooth nuclear membranes,
finely granular chromatin
• Hyperchromatic smudgy
nuclei (degenerative
changes)
 Lithiasis 2
• Inflammation & debris
in background may be
misinterpreted as tumor
diathesis
• May be impossible
to distinguish
from LGUC
• Occasionally marked cytologic atypia, including
nuclear pleomorphism, coarsely granular
chromatin, mitotic figures  false-positive
 Lithiasis 3
• Important source of false positive Dx
for LGUC and HGUC
• Clinical history not reliable: filling defect
in upper UT stone vs. neoplasm
• Persistent atypical features
(weeks) aggressively worked up
for neoplasia
 TPS Diagnostic Categories
• Negative for HGUC
• Atypical Urothelial Cells
• Suspicious for HGUC
• High Grade Urothelial Carcinoma
• Low Grade Urothelial Neoplasm
• Other malignancies, both primary and
secondary
 Mild Nuclear
Atypia

• Single cells with enlarged and irregular

nuclei (no significant hyperchromasia)
• Most common and most frustrating
 Atypical Urothelial Cells (AUC)-TPS

Definition:
1.Atypia that falls short of “Suspicious” or
“HGUC”
2.Degenerative changes where nature and
degree of atypia cannot be explained
 AUC- TPS
Criteria
1. Non-degenerated, Non-superficial urothelial cells
• High N/C ratio > 0.5 (required)
and one of the following:
• Hyperchromasia, mild-moderate
- Compared to benign urothelial or
squamous cell nuclei
• Nuclear Irregularity,
significant
• Irregular clumpy chromatin,
AUC

• Nuclear irregularity, high NC ratio, but no significant

hyperchromasia, compared to benign urothelial cells
 AUC

High NC ratio, mild hyperchromasia

High NC ratio, nuclear irregularity
 AUC- TPS
Criteria
2. Degenerated non-
superficial urothelial cells
• High N/C ratio and
hyperchromasia
• Extensive degeneration of
nuclei and/or incomplete
cytoplasm
 AUC- Exclusions

• Mere presence of
degeneration does
not equate to AUC
• Excludes atypia
secondary to known
conditions:
• Polyoma virus, stones,
reactive/repair, therapy,
instrumentation, etc.
 Negative AUC

Lost nuclear membrane

I
n
t
a
c
t

n
 TPS Diagnostic Categories
• Negative for HGUC

• Low Grade Urothelial Neoplasm

• Other malignancies, both primary and
secondary
 Low Grade Urothelial
CA

• Predominately papillary
• Capacity to invade
(<20%)
• Rarely metastasizes
• Progression < 15%
 Low Grade Urothelial CA 2
• Cytologic diagnosis of LGUC is problematic
– Minimal shedding of neoplastic cells
– Subtle cytologic alterations, difficult to
distinguish from reactive changes, i.e. stones,
instrumentation
– No discriminating cytologic features
between PUNLMP and LGUC
– Wide range of sensitivities 0-73% (Avg 25-
40%)
 Mcroskey 2015
• Compared biopsy proven LGUC cytologies
(98 cases) to negative cytologies (53 cases)
• Instrumented urine specimens
• Evaluated 17 published cytologic features
• All cases were examined blinded to histology
• No single cytologic feature was found to be
helpful in DDX, except for papillary
clusters with fibrovascular cores (2/98
cases)
 LGUC Benign

Few cells with enlarged slightly irregular

 Clusters in voided urine
• Papillary clusters (without
fibrovascular core are not
associated with increased risk
of neoplasia
• Should place less reliance
on presence or shape of
clusters
• More emphasis on
nuclear features
(Deshpande & Mckee, Cancer Cytopathol,
2005)
Low Grade Urothelial Neoplasm- TPS Criteria
• 3D papillary clusters (extreme nuclear
overlapping) with fibrovascular cores- very rare
Cell Block

• Diagnosis should also be qualified as Neg for HGUC

 LGUN
TPS
Criteria

• Tight papillary clusters with atypia and extreme

nuclear overlapping or ocean of cells-
instrumented May consider LGUN in presence
of mass or correlated LGUN biopsy- Neg for
 Differential Diagnosis of
LG Urothelial CA

• Reactive/reparative changes
• Upper urinary tract
sampling
• Instrumentation effect
• Lithiasis
• Reactive changes
and repair
 Upper Urinary Tract specimens
• Direct sampling of upper UT is effective in
detecting HGUC, but poor for low grade
lesions
• Sensitivity: LGUC 37% vs. HGUC 80% Barkan
2015
• Normal upper UT epithelium shows more atypia
than lower UT and occasionally more than
LGUC
N/C ratio, nuclear irregularities, papillary
clusters
 Renal pelvis/ureter brushing

• Negative cystoscopy, biopsy & followup

 Instrumentation Effect
• Catheterized urine and bladder wash specimens
• Large pseudopapillary groups and 3D clusters
• Nuclear overlap and crowding
• Low N/C ratio
• Finely granular chromatin with even distribution
• Well defined cytoplasmic borders
• Nuclear palisading at periphery of clusters with
abundant cytoplasm (cytoplasmic collar)
ThinPrep

Instrumentation effect
 How Long is Cytology Abnormal
after Cystoscopy?

• Evaluated 48 patients
• Examined urine before, immediately after,
1, 2, 7, 14 and 28 days
• Instrumentation effect was transient,
mostly disappearing within 1 day after
cystoscopy
 TPS Diagnostic Categories
• Negative for HGUC
• Atypical Urothelial Cells
• Suspicious for HGUC
• High Grade Urothelial Carcinoma
• Low Grade Urothelial Neoplasm
• Other malignancies, both primary and
secondary
 Negative for HGUC-TPS Criteria
• If there is a known cause for “atypia”- it’s
Negative
– Reactive urothelial cells
– Instrumentation effect
– Upper urinary tract specimens
– Changes associated with lithiasis
– Polyoma viral cytopathic effect
– Post-therapy effects
– Clusters without fibrovascular cores or atypia
 Therapy

Reactive Repair

Polyoma
Stones

Instrumentation

Upper tract NEGATIVE

Clusters
 Negative for HGUC-TPS Criteria
• This category also
includes
“LGUN”
Sample Dx:
- Negative for HGUC
-Changes consistent/suggestive
of LGUN
 Nuclear / cytologic atypia
NFM AUC-Suspicious HGUC
8%-30%

low
moderate/high certain
Slide courtesy of Probability of high grade UC
D. Rosenthal, MD
Ancillary Tests for Detecting & Monitoring UC
Test Sensitivity Specificity Lab Comment
% (range) % (range)
Urine cytology 54 (35-68) 95 (83-100)
DNA ploidy 62 (45-86) 89 (76-100) IA, FCM
BTA 60 (32-100) 77 (40-96) POC, Ref ⇈ False +

NMP22 67 (47-81) 72 (60-86) Ref

ImmunoCyt 50-100 69-79 ⇈ False +
Telomerase 74 (62-93) 79 (60-99) Ref ⇈ False +
Microsatellite 83-95 83-100 Ref
Analysis
FISH 69-87 85-97 Ref
 FISH (UroVysion)
• A 4-probe set that targets the
common chromosomal
abnormalities in UC
• FISH+ results:
– Polysomy 3, 7, 17
• Gain of 2 or more
chromosomes
• Seen mostly in HGUC, but not
LGUC
– Deletion 9p21
•
 FISH 2
• FDA approved for surveillance of patients
with hematuria and history of UC
• Recommended in hematuria pts with other
risk factors such as smoking hx and age > 45
• High sensitivity 69-93%, esp. for
HGUC, lower for LG UC
• ? FISH positive AUC treated as Susp/ HGUC
 FISH 3
Impressive Sensitivity results:
• Surveillance UC patients:
– FISH +/ cystoscopy-/ cytology-
65% recurrent CA (within 29
months)
– FISH- 13% recurrent CA
• Hematuria surveillance:
– FISH+/cytology- (30%) 60% UC
• Post BCG therapy
– FISH+ approx 10 times more likely to develop
invasive cancer
 False-Positive FISH
• Be careful about significance of FISH+ in
upper tract cytlogy
– Limited value for upper tract tumor surveillance
– High false + (Johannes, J Urol. 2010)
• Polyoma virus can cause false + FISH
(approx 15%)
– Usually in pts with high viral titers (renal
transplant)
 FISH vs. Cytology
• FISH more sensitive but less specific
than urine cytology
• PPV of urine cytology in HGUC > 90%
– PPV of FISH: as low as 50%
– Cytology= 7-10 times cheaper (Murphy 2009)
• Combined FISH & Cytology 
98% sensitivity and > 95%
specificity
• FISH-neg patients (low risk) may be
allowed extended time intervals between
cystoscopies