Trial Information

Evaluation of Non-Invasive Assay(s) for the Detection of Bladder Cancer

Bladder cancer is one of the most common malignancies worldwide. The rate of occurrence of
these tumors is highest in the developed countries, ranking as the sixth most frequent
neoplasm. Approximately 90% of malignant tumors arising in the bladder are of epithelial
origin, the majority being transitional cell carcinomas. Early stage bladder tumors have
been classified into two groups with distinct behaviors and different molecular profiles:
Low-grade tumors (always papillary and usually superficial), and high-grade tumors (either
papillary or non-papillary and often invasive). Clinically, superficial bladder tumors
(stages Ta, Tis and T1) account for 75-80% of bladder neoplasms, while the remaining 15-20%
are invasive (T2, T3, T4) or metastatic lesions at the time of presentation. Over 70% of
patients affected with superficial tumors will have one or more recurrences after initial
treatment, and about one-third of those patients will progress and eventually succumb to
their disease.

Previous publications from this Urology Department have introduced the idea of a
non-invasive, molecular-based assay for the detection and monitoring of bladder cancer
(Levesque et al. 1993; Fitzgerald et al. 1995). At the time of publication there was
limited knowledge of molecular changes underlying the different clinical pathways outlined
above and our assay was based on one gene (c-H-ras-1). In the last few years, it has become
clear that activation events associated with FGFR-3 can be found associated with 40%-60% of
low-grade, low-stage bladder tumors whilst p53 mutations are linked to a more aggressive
phenotype progressing via the CIS pathway. Mutations found in the c-H-ras-1 gene can
straddle both of these groups. We propose to assess the use of a multiple mutation-based
assay, using DNA from exfoliated cells in the urine of patients, to establish the
sensitivity and specificity in tumor detection compared to cystoscopy and cytology. In
addition, we propose to isolate free-DNA for use in molecular assays. The remaining urine
will be stored to evaluate biomarkers for the detection of tumor presence or progression
using protein-based analyses.

Bladder Cancer Patient Group:

All patients harboring tumors and scheduled to have a cystectomy or cystoscopy will be
eligible for this study. This will include patients at first presentation and those who are
in follow-up. Urine samples collected at the time of a procedure will be obtained from
catheterized urine in the operating room. Urine obtained in the clinic setting will be
obtained via voiding, as with standard urine sample collection. We propose to collect urine
from study participants who have a cystoscopy on each occasion that they visit the Urology
Department regarding the treatment and follow-up of their disease. The urine will be
collected at intervals over a two-year period.

Control Patient Group:

The cancer assays to be tested use DNA analysis and antibodies to specific proteins as well
as functional assays for proteins to attempt to identify bladder tumor presence. It is
important to know whether other changes, including kidney stones, cystitis etc. cause the
release of the same DNA or proteins into the urine as was found in cancer patients. The
frequency of false positive results will determine the utility of the assay in the cancer
detection arena. A patient group of age-matched controls will be recruited from the
aforementioned patient populations requiring a single urine donation at the time of their
scheduled clinic visit.

The primary objective of this study is to evaluate the utility of emerging technologies in
the detection of bladder tumor cells using non-invasive approaches utilizing voided urine
samples. This will include methodologies than can establish the sensitivity of detection of
specific mutations associated with bladder cancer progression and the utility of this
approach in complementing cystoscopy and cytology. The mutation status of amplified DNA
fragments will be established using multiple molecular techniques. We will also isolate
free-DNA from urine and perform extended-PCR to ascertain the proportional representation of
large DNA fragments i.e. >1,500bp as an indicator of apoptotic activity and evaluate
microRNA profiles from the urine samples.

Urine-borne exfoliated cells will be pelleted from urine collections using centrifugation
and DNA/RNA will be extracted. The urine supernatant will be run over a DNA affinity column
to capture free-DNA for analysis. Nucleic acids isolated from these procedures will be
analyzed using PCR and various molecular technologies to establish the mutation status at
different gene loci. The remaining urine will be frozen and used in protein detection
assays using a panel of biomarkers with putative prognostic significance.

Detection of tumor presence will be compared in urine specimens undergoing mutational
analysis and the current standard of treatment (cytology and cystoscopy together). As
cytology is not performed on all patients, fewer urine specimens are expected in the
cytology and cystoscopy group than in the mutational analysis group. Therefore a
comparative analysis between these two groups will be performed using an analysis of
variance (ANOVA) test where significance will be set at p<0.05.