CARCINOMA INSITU

History
1952 - Melicow and Hollowell - the first to describe CIS in grossly normal vesical mucosa and to document its multifocal nature. Koss[9] was the first to recognize the importance of urine cytology in diagnosing CIS in mucosa that could appear relatively unremarkable on cystoscopy. Subsequent mapping studies of cystectomy specimens and surveillance biopsies showed that apparently grossly unremarkable vesical mucosa often exhibited a spectrum of nuclear abnormalities culminating into CIS, invasive carcinoma, or both

TYPES
De novo CIS (Primary CIS) - <1% to 3% Concurrent CIS Secondary CIS - detected during follow-up in 90% of cases with urothelial neoplasms

Carcinoma in Situ
Velvety patch of erythematous mucosa confined to urothelium Quite often it is endoscopically invisible hence underdiagnosed CIS - ominous lesion that is difficult to identify and survey because of its occult and multicentric nature Histologically, it consists of poorly differentiated TCC confined to the urothelium Urine cytology - 80- 90 % positive

 Present in
25% of high-grade superficial 20-75% of muscle-invasive 40 - 83% progress to muscle-invasive cancer 20% specimens of cystectomy done for diffuse CIS have invasive disease

 Patients with marked urinary symptoms generally have a shorter interval preceding the development of muscle-invasive cancer.
About 20% of patients treated with cystectomy for diffuse carcinoma in situ are found to have microscopic muscle-invading cancer

 A variety of investigative approaches have confirmed carcinoma in situ's direct relationship to muscleinvading cancer.
Cytogenetic (loss of chromosome 17p) ( Olumni et al, 1990 ; Tsai et al, 1990 ; Knowles et al, 1994 ), molecular genetic immunohistologic

Esrig et al have shown that high proportions of both carcinoma in situ and deeply invasive bladder cancer have deletions and/or mutations of the TP53 gene and alterations of its protein product.
supports the contention that carcinoma in situ is a precursor lesion of invasive bladder cancer eliminates it as a precursor of low-grade papillary tumors in which TP53 abnormalities are rarely found (except in extremely young patients)

Risk Factors
Tobacco smoking - most important risk factor Analgesic abuse Occupational exposure to arylamine compound analogues. Long-term exposure to cyclophosphamide and pelvic irradiation may also act as predisposing factors

Genetic pathology
The most important are deletion/mutation of p53 gene - 'guardian of the genome,' located on 17p13.1 The other changes include loss of tumor suppressor genes, such as cyclin-dependent kinase inhibitor (CDKN2/p16), and deletion of 9q. Deletions in tumor suppressor genes such as p16INK4 and RB are responsible for unregulated clonal proliferation of these genetically abnormal cells and for absence of senescence in this population.

Clinical Features
sixth or seventh decade of life 3 times more common in men than women. Patients may be asymptomatic Irritative voiding symptoms, including frequency, urgency, and dysuria, are particularly associated with CIS in the absence of infection. Hematuria, if present, is typically microscopic

Cytology
Cytology is a useful diagnostic tool and has a high sensitivity (approaching 77%) for high-grade neoplasms of the urinary bladder, such as CIS, in which the cells are discohesive. The presence of severely atypical epithelial cells in urine in conjunction with normal cystoscopy is suggestive of CIS. Reactive atypia may prove to be a challenging differential diagnosis on cytology

CYSTOSCOPY
On standard white light cystoscopy - CIS may appear normal or erythematous and granular or erosion or just edema. Zaak et al - standard white light cystoscopy - miss nearly 53% of CIS Multiple cold cup biopsies from vesical mucosa that may seem relatively normal - in the presence of persistent unexplained bladder symptoms. 1414 5-ALAinduced fluorescence endoscopy based on the principal of preferential accumulation of photoactive porphyrins in neoplastic tissue or hexaminolevulinate fluorescence cystoscopy to detect CIS These methods may improve the sensitivity for cystoscopic sampling of CIS up to 95% to 68%.

HPE

Unequivocal severe cytologic atypia involve either the full or partial thickness of the urothelium umbrella cells may be present occasionally. The urothelium - hyperplastic or denuded secondary to the discohesive nature of the neoplastic cells or artifactually because of hot wire loop biopsy techniques. Loss of polarity leading to nuclear overcrowding may be evident at low magnification. The cytoplasm may be eosinophilic or amphophilic, and the nuclear cytoplasmic (N/C) ratio may not always be altered. The nuclei - enlarged, pleomorphic, hyperchromatic, with irregular notched nuclear contours and have coarse chromatin and prominent nucleoli. Atypical mitoses may be present even in the more superficial layers

Histologic Patterns of Urothelial Carcinoma in Situ

Pattern Description

Large-cell pleomorphic CIS

Large-cell nonpleomorphic CIS Small-cell CIS Clinging CIS Cancerization of the urothelium

Easily recognized because of loss of polarity, nucleomegaly, and nuclear pleomorphism; nuclear/cellular ratio is unaltered because of abundant cytoplasm
Often mimics reactive atypia; although nuclei are enlarged and hyperchromatic, the cells are monomorphic with abundant cytoplasm

Enlarged nuclei with chromatin abnormalities; cells appear small because of scant cytoplasm
Denuded urothelium with patchy to single layer of cytologically malignant cells Presence of atypical urothelium within otherwise normal-appearing urothelium: pagetoid spread or undermining/overriding

A) Large-cell pleomorphic pattern. Loss of polarity is present and the nuclei of the urothelial cells are 5 times larger than the lymphocytes in the stroma. Marked variation is seen in the size and shape of the nuclei of the neoplastic cells B) Large-cell nonpleomorphic pattern. The neoplastic cells have abundant amphophilic cytoplasm and a relatively monomorphic appearance. A mitotic figure is seen in the superficial layer Clinging pattern: a denuded layer of atypical cells is present

C)

D) Pagetoid spread: clusters of neoplastic cells are seen within the urothelium and normal urothelium is seen towards the surface

Immunohistochemistry
A panel comprising
cytokeratin 20 (CK 20) - superficial umbrella cell layer CD44 - the basal and parabasal cell layer p53 Negative All 3 immunostains in the panel must be used in conjunction with morphologic evaluation. Ki67 staining index - higher in CIS than in dysplasia or reactive atypia.

Overview
New biomarkers for bladder cancer Cell-based detection
Cytology FISH (UroVysion) Telomerase

Protein based detection

BTA stat/TRAK NMP22 ImmunoCyt Survivin

Diagnosis, surveillance, treatment response

Why are new biomarkers attractive? Cystoscopy gold standard for diagnosis and surveillance Invasive, expensive, time consuming Up to 10% of significant lesions still missed by cystoscopy

Surveillance Methods
Cystocopy
0-2 years Every 3 months 2-4 years Every 6 moths 5 years + Anually

Cytology
Obtained at time of cystocpy Butonly 40% complete recommended surveillance protocol1

Biomarkers for Urothelial Cancer

Early detection important 25% of bladder tumors are high grade High risk of recurrence High risk of progression for high grade tumors Compared against cystoscopy (considered gold standard)

Bladder Cancer Survival by Stage

Stage 5-year Survival T0 92% T1 83% T2 89% T3a 78% T3b 62% T4 50% Metastatic <2%
Early detection is important!

Urinary Cytology

Cytology
Cells readily available in urine Relies on abnormal cellular morphology Reasonable specificity (87-95%) Poor sensitivity (1248%) Greater sensitivity for high grade disease Interpreter dependent

Bladder Tumor Antigen (BTA)Stat/TRAK

Detect complement factor H and complement factor H-related protein produced by bladder cancer BTA stat is qualitative and point-of-care BTA TRAK quantitative by ELISA

BTA Stat
220 patients with recurrent bladder cancer3 67% sensitive, 95% specificity 107 with h/o bladder cancer
70% specific

445 patients with history of bladder cancer4 undergoing surveillance

56% sensitive 86% specific

BTA Stat
False positives can be high
BPH Hematuria UTI Urolithiasis Recent BCG

Cytology ultimately more specific in head to head comparisons

Nuclear Matrix Protein (NMP) 22

NMPs are involved with DNA structure, replication and gene transcription NMP22 Regulator of mitosis Expression increased in tumor cells Released into urine after cell death ELISA and point-of-care tests available

Surveillance: NMP22 vs. Cytology

231 patients with history of bladder cancer5
49% sensitive 92% specific

70% sensitive for CIS 90% sensitive for high grade tumors False positives seen with inflammatory conditions

NMP22: Prospective, Multicenter Trial

668 patients with h/o bladder cancer NMP22 compared with cytology and cystoscopy Cystoscopy found 91% recurrent tumors 8/9 tumors missed by cystoscopy, positive for NMP22, 7 were high grade 3/9 detected by cytology Overall sensitivity=50% Overall specificity=87%

Sensitivity of NMP22 and Cytology by Tumor Stage

Stage
Tis Ta T1 T2 T3 T4 Overall

NMP22
Sensitivity (95%) 50 (16-84) 36 (23-51) 65 (38-86) 88 (47-100) 100 (3-100) 100 (16-100) 50 (40-60)

Cytology
Sensitivity (95%) 38 (9-76) 6 (1-17) 13 (2-38) 0 (0-41) 0 (0-98) 0 (0-84) 12.2 (7-20)

Sensitivity of NMP22 and Cytology by Tumor Grade

Grade
Well differentiated Moderately differentiated Poorly differentiated

NMP22
Sensitivity (95%) 32 (18-49) 44 (20-70) 75 (57-89)

Cytology
Sensitivity (95%) 5 (1-18) 0 (0-23) 19 (7-38)

Fluorescence In Situ Hybridization (FISH) UroVysion

 LOH chromosome

Molecular Abnormalities in Urothelial Carcinoma

Common in low grade, noninvasive lesions Loss of p16 tumor suppressor gene 9p21

DNA aneuploidy
High grade tumors Several and variable chromosomal gains/losses Chromosomes 7,9,17 particularly involved

p53, p27, pRB mutations

Fluorescence In Situ Hybridization (FISH)

Urothelial carcinoma has a high frequency of genetic abnormalities Fluorescently labeled DNA probes that hybridize to chromosomal abnormalities Identifies specific genetic changes at a particular DNA region in a given cell

UroVysion FISH
Two types of probes used, 4 probe cocktail Centromeric numeration probes (CEPs) detect abnormal chromosome number
Chromosomes 3, 7, 17

Locus specific probes (LSPs) detect specific mutations in known tumor suppressor gene
p16 tumor suppressor gene

FISH (UroVysion)

Morphologically abnormal cells

FISH shows marked aneuploidy chromosomes 3, 7

FISH and Surveillance

176 patients with h/o TCC8 62 recurrences on surveillance FISH sensitivity 71% BTA stat and cytology 50% and 26%, respectively FISH negative in 160/175 controls=94.5% specificity

FISH Sensitivity by Grade9

Grade 1 Cytology FISH p-value 27% 36% 0.564 Grade 2 54% 76% 0.059 Grade 3 71% 97% 0.003

FISH Specificity
Cytology FISH p-value 98% 96% 0.564

Positive FISH, Negative Cystoscopy10cysto as gold Is specificity underestimated using

standard? What is the risk of recurrence with positive FISH? Recurrence free survival at 29 months

FISH (+)

38% 95%

N=55

FISH (-)

N=155

FISH and Treatment Response11 37 patients

superficial bladder Ca treated with BCG

12 FISH (+)

25 FISH (-)

12/12 recurred
7/12 muscle invasive
p=<0.001

13/25 recurred
2/25 muscle invasive

FISH
Pattern of positivity may be predictive
Positivity by loss of 9p21 may predict low grade tumors with low risk of progression Aneuploidy suggests higher grade with high risk of recurrence and progression

ImmunoCyte
IHC for bladder cancer cellular markers 3 fluorescently labeled antibodies 2 mucin-like proteins HW form of CEA 341 patients with h/o superficial TCC13 81% sensitivity, 75% specificity Cytology 23% sensitive, 93% specific More sensitive than cytology for low grade, superficial disease Significant interobserver variability

Survivin
Inhibitor of apoptosis Commonly overexpressed in tumors Sensitivity ranges from 64-100% Survivin and Surveillance14
Positive in all 46 patients with new or recurrent tumors Negative in 32/35 patients with no visible disease (91% specificity)

Telomerase
Expressed by bladder tumors Maintains telomere length to allow infinite cell divisions Cellular telomerase measure in 2 ways
TRAP assay measures telomerase activity RT-PCR for telomerase expression

70-100% sensitivity, 60-70% specificity15