The Vysis® UroVysion Bladder Cancer Recurrence Kit

FISH Assay (UroVysion) FDA Approved as Aid for Initial Bladder Cancer Diagnosis

On Jan. 25, 2005 the FDA approved a new indication for a fluorescence in situ hybridization (FISH) assay (UroVysion, made by Vysis, a division of Abbott Laboratories, Inc.), allowing its use as an aid for the initial diagnosis of bladder cancer in patients with hematuria and suspected of having bladder cancer.

The gene-based test detects aneuploidy for chromosomes 3, 7, and 17, and loss of the 9p21 locus via FISH in urine specimens.

The approval was based on the results of a multicenter study demonstrating that the FISH assay had significantly greater clinical sensitivity (68.6% vs 39.2%) but less specificity (77.7% vs 91.5%) than urine cytology in detecting bladder cancer compared with cytoscopy/histological (gold standard) testing methods.

The FDA notes that clinical interpretation of test results should be evaluated within the context of the patient’s medical history and other diagnostic test results. Positive FISH results in the absence of other signs and symptoms of bladder cancer recurrence may indicate the presence of other urinary tract–related cancers (ureteral, urethral, renal, and/or prostate in men), and further diagnostic testing should be performed.

A negative FISH result does not rule out all bladder cancer or its future development.

The FISH assay was first approved by the FDA in 2001 for use in conjunction with cytoscopy to monitor recurrent transitional cell carcinoma of the bladder.

Biomarker Test (Architect CTSTAT Myoglobin) Aids in Early Diagnosis of AMI

On Jan. 26, the FDA approved a myoglobin immunoassay (Architect CTSTAT Myoglobin, made by Abbott Laboratories, Inc.) to aid in the early diagnosis of acute myocardial infarction (AMI) and for the evaluation of thrombolytic therapy efficacy. The test is intended for use with the company’s Architect i2000 immunoassay and cl8300 immunochemistry systems.

In acute ischemic disease, a temporal pattern of increased release of myoglobin into the bloodstream occurs as a result of cardiac muscle damage. Serum myoglobin levels increase between two to four hours after an AMI, peaking at eight to 10 hours and then returning to baseline after 24 hours.

When assessed in conjunction with electrocardiogram results and other clinical data, measurement of myoglobin levels within two to 12 hours of an AMI can improve the efficacy of early diagnosis and aid in evaluating the success of thrombolytic therapy.

The approval of the myoglobin assay completes the acute cardiac triage menu for the Architect system, which includes tests for troponin–I and creatine kinase–MB that were approved by the FDA in July and August 2004, respectively.

Reviewed by Gary D. Vogin, MD

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Cellular Genomics

Cellular Genomics is a technology platform that uses known DNA sequence information to design products that can detect genetic changes associated with disease. This platform is especially useful for the detection of cancer since cancer does not occur without genetic change. Some examples of the genetic changes associated with cancer include chromosome aneuploidy, chromosome translocations/rearrangements, deletions, or amplifications. Aneuploidy – a condition where the number of chromosomes in a cell differs from the normal diploid number (two copies of each chromosome, 46 total) by loss or duplication of chromosomes.

Translocation – a condition where part of a chromosome is detached by breakage and then becomes attached to some other chromosome.

Deletion – a condition where a sequence of DNA along a chromosome is removed and the regions on either side become joined together.

Amplification — refers to the production of additional copies of a chromosomal sequence, which may then be present on the same or a different chromosome.

Direct examination of the genome (all of the DNA within a cell) is a sensitive and powerful means to detect cancer with minimal subjectivity. Vysis’ Cellular Genomics products, including the VysisÒ UroVysion Bladder Cancer Recurrence Kit, use this approach for disease management.

Fluorescence in situ hybridization (FISH) can be used as part of a Cellular Genomics-based approach to disease detection and management. With FISH, one uses known DNA sequence information to design short DNA molecules (probes) that will detect a specific gene or region of a chromosome. To make the probes visible under a microscope, fluorescent molecules of different colors are attached to each DNA probe.

A typical FISH procedure involves three key steps: specimen preparation, hybridization, and viewing.

1. Specimen preparation — since FISH is a microscopic procedure, the cells to be examined must be mounted on a microscope slide. There are numerous ways to accomplish this depending upon the tissue or sample type but all seek to affix the cells firmly to the slide. Once cells are fixed to the slide, they are pretreated to allow access of the fluorescent DNA probe to the chromosomes in each cell’s nucleus. Pretreatment typically involves a removal of cell protein by exposure to a protein-degrading enzyme (a protease) and several wash steps to remove other tissue debris.

2. Hybridization – once the cells have been pretreated, they are ready for hybridization. Hybridization is a process whereby a DNA probe is applied to the cells in a water-based solution so that it can seek out its target DNA sequence and bond (hybridize) to it. To start the hybridization process, and allow the DNA probes access to the chromosomal DNA, the cells have to be heated to sufficiently high temperature (72-75 °C) such that the normally double-stranded chromosomal DNA melts into its two separate strands of complementary DNA sequence. Strand separation by high temperature treatment is also known as “denaturing”. Once denatured, the chromosomal DNA is accessible to the fluorescent DNA probes (FISH probes) that float around in solution until they find their target DNA sequence. When two pieces of DNA come into contact, they will not naturally form a double-stranded molecule unless the strands have complementary DNA sequence. Since the probe DNA was designed to be complementary to a specific target DNA sequence, the probe will bond to it wherever it lies along a chromosome. To facilitate rapid and tight pairing of the probe and target DNAs, the temperature of the hybridization reaction is reduced to around 37-42 °C (this removes a lot of energy from the reaction environment that would otherwise keep the probe and target DNAs “bouncing around” in solution at high frequency, a situation that makes it very difficult for them to pair and form double-stranded molecules). Hybridization takes a minimum of two hours. After hybridization, the cell specimens are washed a few times to remove excess fluorescent probe DNA that was unable to hybridize to a target DNA sequence.
3. Viewing – Viewing is accomplished with an epi-fluorescent microscope, which is nothing more than a standard microscope that is equipped with a special type of light source and filters. The special light source provides lighting that is capable of exciting the fluorescent DNA probes so that their specific colors can be viewed through different light filters. Cells to be viewed are treated with a counterstain that binds to DNA non-specifically, painting each chromosome with a uniform “blue color” in all regions except where there is probe DNA bound to its target. This enables the viewer to see each chromosome, the margins of each cell’s nucleus, and the probe DNA’s fluorescent signal very clearly. Wherever a target DNA sequence lies along a chromosome is indicated by a specific color of fluorescent signal.

Goal of the Product
Provide a noninvasive, genomic DNA-probe based test to accurately detect the presence of bladder cancer.

Product Development
The Vysis® UroVysion Bladder Cancer Recurrence Kit (Vysis UroVysion Kit) was developed by an iterative process that methodically tested a set of DNA probes on bladder cells obtained from urine samples of patients with and without bladder cancer. The probes included nine that specifically identify a particular chromosome and one that identifies a specific region of chromosome 9 (the 9p21 locus). The set of probes was selected for testing based on reports in the scientific literature that associated changes in these DNAs (chromosome copy number changes or deletion of the locus) with bladder cancer. For example, the 9p21 region is important because it contains a proposed tumor suppressor gene, P16. Deletion of P16 is one of the most common alterations in urothelial carcinoma (bladder cancer) (Sokolova et al., 2000). At the end of the study, a set of four probes was selected that provided the greatest sensitivity for urothelial carcinoma detection (Sokolova et al., 2000).

Once the probe set was finalized, the product was placed into clinical trials to demonstrate its utility for a 510K submission to the US FDA. On August 1, 2001, the US FDA cleared for marketing the Vysis® UroVysion Bladder Cancer Recurrence Kit for monitoring the recurrence of bladder cancer.

Intended Use
The Vysis® UroVysion Kit is designed to detect aneuploidy for chromosomes 3, 7, 17, and loss (deletion) of the 9p21 locus via fluorescence in situ hybridization (FISH) in urine specimens from subjects with transitional cell carcinoma of the bladder. Results from the UroVysion Kit are intended for use as a noninvasive method for monitoring for tumor recurrence in conjunction with cystoscopy in patients previously diagnosed with bladder cancer.

Procedural Overview
Cells recovered from urine samples are fixed on slides. The DNA is denatured to its single stranded form and subsequently allowed to hybridize with the UroVysion DNA probes. Following hybridization, the unbound probe is removed by a series of washes, and the nuclei are counterstained with DAPI (4,6 diamidino-2-phenylindole), a DNA-specific stain that fluoresces blue. Specific hybridization of the UroVysion probes is viewed using a fluorescence microscope equipped with appropriate excitation and emission filters allowing visualization of the intense red, green, aqua, and gold fluorescent signals. Enumeration of the probes specific for chromosomes 3, 7, and 17, and the 9p21 region is conducted by microscopic examination of the nucleus, which is easily distinguished due to the DAPI staining

Clinical Trial Data
Cancer detection products are typically evaluated in terms of their sensitivity and specificity during clinical trials. Sensitivity is a measure of the product’s ability to detect various stages and grades of cancer. Specificity measures the product’s ability to detect true positives; in this case, this means true chromosome abnormalities.

Patients for the clinical trials were enrolled in 21 urology centers throughout the US and Canada. A total of 275 patients were examined, including 59 normal volunteers. Within unique patients, the test provided 94.5% specificity. Within the 59 normal volunteers, the test was 100% specific, indicating that the chance of a false positive with this test is very low. Sensitivity of the test was compared to a gold standard of cystoscopy and histology. Table 1 below summarizes the comparison data:

Table 1. Comparison of Vysis UroVysion vs. Cystoscopy/Histology for Detection of Bladder Cancer Recurrence by Stage and Grade*.

Agreement of (+) Results (%)
Ta, Grade 1 36/48 (75.0%)
Ta, Grade 2-3 11/20 (55.0%)
T1 10/12 (83.3%)
T2 3/3 (100%)
Tis/Cis 7/7 (100%)
All 36/49 (73.5%)
1 12/22 (54.5%)
2 7/9 (77.8%)
3 17/18 (94.4%)

*Biopsy was not performed in 11 cases. In addition, no stage was assigned in 3 cases and no grade in 2 cases.

The Vysis UroVysion Kit detects all stages and grades of bladder cancer but is highly sensitive for the more dangerous higher grade and stage types.

While cystoscopy/histology was the gold standard for comparison in these studies, the sensitivity and specificity of BTAstatä and standard cytology was also determined on the same samples. Sensitivity of BTAstatä was 30%, 83%, 83%, 67%, and 43%, respectively for TaG1, TaG2,3, T1, T2, and Tis (tumor in situ or carcinoma in situ). Sensitivity of cytology was 20%, 30%, 67%, 33%, and 33%, respectively, for TaG1, TaG2,3, T1, T2, and Tis. Specificity of BTAstatä was 18%, 44%, and 41%, respectively, for grade 1, 2, and 3 bladder cancer. Specificity of cytology was 18%, 44%, and 41%, respectively, for grade 1, 2, and 3 bladder cancer.

The Vysis UroVysion Kit performed extremely well in patients receiving BCG intravesical treatment. Test result agreement with gold standard cystoscopy/histology was 92%, indicating that the test results are unaffected by BCG-treatment, infection, or other genitourinary diseases that cause inflammation of the bladder lining.

Related Publications
Bubendorf L, Grilli M, Sauter G, Mihatsch MJ, Gasser TC, Dalquen P (2001) Multiprobe FISH for enhanced detection of bladder cancer in voided urine specimens and bladder washings. American Journal of Clinical Pathology 116: 79-86

Halling KC, King W, Sokolova IA, Meyer RG, Burkhardt HM, Halling AC, Cheville JC, Sebo TJ, Ramakumar S, Stewart CS, Pankratz S, O’Kane DJ, Seeling SA, Lieber MA, Jenkins RB (2000) A comparison of cytology and fluorescence in situ hybridization for the detection of urothelial carcinoma. Journal of Urology 164(5): 1768-1775

Sokolova IA, Halling KC, Jenkins RB, Burkhardt HM, Meyer RG, Seelig SA, King W (2000) The development of a multitarget, multicolor fluorescent in situ hybridization assay for the detection of urothelial carcinoma in urine. Journal of Molecular Diagnostics 2(3): 116-123

Medical Reimbursement
Several CPT codes (88271 x 4, 88274 or 88275, and 88365) apply to medical reimbursement for the Vysis UroVysion Kit. For more specifics on medical reimbursement, please contact the Vysis Medical Reimbursement Program at (800) 553-7042 x7061.

New Resources
The website should be up and running by 10/15/01. Much of the content development will be ongoing for the next few months.

This page contributed to WebCafe by
Scott A. Campbell, Ph.D., M.B.A.
Assoc. Product Manager
Vysis, Inc.
(800) 553-7042, ext 7071
(630) 271-7071
(630) 271-7028 FAX

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