The UroVysion Bladder Cancer Recurrence Kit (UroVysion Kit) is designed to detect aneuploidy for chromosomes 3, 7, 17, and loss 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.

Device Description

The UroVysion Kit is based upon fluorescence in situ hybridization (FISH) DNA probe technology. The UroVysion probes are fluorescently labeled nucleic acid probes for use in in situ hybridization assays on urine specimens fixed on slides. The UroVysion Kit consists of a 4-color, four-probe mixture of DNA probe sequences homologous to specific regions on chromosomes 3, 7, 9, and 17. The UroVysion probe mixture consists of Chromosome Enumeration Probe (CEP®) 3 SpectrumRed™, CEP 7 SpectrumGreen™, CEP 17 SpectrumAqua™ and Locus Specific Identifier (LSI®) 9p21 SpectrumGold™.

AI/ML Overview

Here's an analysis of the acceptance criteria and the study that proves the UroVysion Bladder Cancer Recurrence Kit meets those criteria, based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

The document doesn't explicitly state quantitative acceptance criteria in a dedicated section. However, based on the "Substantial Equivalence vs. BTAstat Test" section, the key performance metric for substantial equivalence appears to be that the 95% Confidence Intervals (CIs) for UroVysion's agreement percentages (positive, negative, and overall) are greater than the BTAstat test's scores minus 15%.

For other aspects, the "acceptance criteria" are implied by the reported performance deemed satisfactory for marketing authorization.

Criteria Category	Acceptance Criteria (Implied/Derived)	Reported Device Performance
Hybridization Efficiency	≥ 87% (for clinical practice simulation)	87% (clinical study)
Analytical Specificity	No cross-hybridization to other chromosome loci	100% (No cross-hybridization observed in 42 metaphase spreads)
Interference	No interference from tested substances/microbes/therapeutic agents	No interference detected from any tested substance
Preservative Equivalence	Acceptable microbial inhibition; average percent variation of signals per nucleus < 50% between PreservCyt and Carbowax	PreservCyt acceptable for microbial inhibition; equivalent to Carbowax (avg. variation < 50%)
Reproducibility (Inter-site)	Mean number of signals for each probe varies within a narrow range; ability to correctly classify samples (e.g., >95% accurate)	Mean signal numbers vary within a narrow range; 95% of observations on specimen 2 correctly classified as 9p21 loss; 1 false positive out of 16 normal specimen evaluations
Specificity (Non-cancer pts)	Overall specificity considered high (e.g., >90%)	Overall Specificity: 93.0% (332/357), Unique Patients: 94.5% (260/275)
Positive Agreement vs. SOCO	95% CI > BTAstat score - 15%	95% CI for UroVysion positive agreement (58.1%-81.8%) > BTAstat positive agreement (50.0%) - 15% (35.0%)
Negative Agreement vs. SOCO	95% CI > BTAstat score - 15%	95% CI for UroVysion negative agreement (56.3%-74.4%) > BTAstat negative agreement (69.3%) - 15% (54.3%)
Overall Agreement vs. SOCO	95% CI > BTAstat score - 15%	95% CI for UroVysion overall agreement (60.2%-74.5%) > BTAstat overall agreement (62.5%) - 15% (47.5%)
Longitudinal Study (Recurrence)	Statistically significant difference in recurrence rate between FISH+/cysto:histo- and FISH-/cysto:histo- groups	Significant difference (p=0.014) observed: 41.7% recurrence in FISH+/cysto:histo- vs. 19.1% in FISH-/cysto:histo-
Automated System Equivalence	Equivalent results with manual vs. semi-automated methods	All tested compounds and preservatives performed within 2 standard deviations of controls, supporting equivalence

2. Sample Size Used for the Test Set and Data Provenance

Specificity Study (Non-cancer patients):
- Sample Size: 315 patient visits initially, resulting in 309 usable office visits and ultimately 275 unique patients yielding informative results. The data points used for specificity calculation totaled 357 (some patients had multiple conditions).
- Data Provenance: Multi-center, prospective study. Patients were "healthy volunteers and urology patients without prior history or clinical evidence of bladder cancer." (implicitly from the US, given FDA submission context).
Performance vs. Standard of Care (Bladder cancer recurrence monitoring):
- Sample Size: 309 patient visits initially, resulting in 251 usable office visits and 176 unique patients with informative FISH results.
- Data Provenance: Multi-center, prospective, longitudinal study. Patients had a "history of bladder cancer." (implicitly from the US, given FDA submission context).
Reproducibility: 4 specimens from bladder carcinoma cell lines.
Interference/Preservative: Three voided urine pools from normal healthy volunteers, spiked with various substances.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of Those Experts

The document does not explicitly state the number of experts used to establish the ground truth or their specific qualifications (e.g., "radiologist with 10 years of experience").

For the Performance vs. Standard of Care study, the comparative reference was "cystoscopy followed by histology." Histology is typically interpreted by pathologists. The document mentions "central pathologist" in the longitudinal study details (Page 18), but doesn't specify how many or their experience level.

4. Adjudication Method for the Test Set

The document does not explicitly detail an adjudication method (e.g., 2+1, 3+1) for the ground truth establishment.

For the "Performance vs. Standard of Care" study:
- "The comparative reference used for all percent agreement calculations was cystoscopy with histology confirmation for positive or suspicious cystoscopies."
- "If a patient had a positive cystoscopy but histology was absent (e.g., the lesion was fulgurated), then the specimen was considered positive for bladder cancer."
- "If a test had a suspicious cystoscopy but histology was absent, then the case was omitted from analysis."
  This indicates a defined hierarchy and decision rule for establishing the ground truth (cystoscopy + histology).

5. Multi Reader Multi Case (MRMC) Comparative Effectiveness Study

A MRMC study was not explicitly done comparing human readers with and without AI assistance, because the device is a Fluorescence in situ hybridization (FISH) DNA probe technology, not an AI-assisted diagnostic tool.

However, the study did compare the UroVysion Kit's performance to two other human-interpreted methods:

BTAstat test (lateral flow assay, antigen-specific antibodies, qualitative interpretation)
Cytology (visual interpretation of cells)

The study demonstrated the superiority of the UroVysion Kit over BTAstat and cytology, particularly in agreement of positive results across various tumor stages and grades. For example:

Overall Agreement of (+) Results:
- UroVysion: 71.0%
- BTAstat: 50.0%
- Cytology: 26.2%
For patients treated with BCG:
- UroVysion: 92.3%
- BTAstat: 69.2%
- Cytology: 30.8%

The basis for substantial equivalence was to demonstrate that the UroVysion Kit's 95% CIs for agreement were greater than the BTAstat test's score minus 15%, which it met across positive, negative, and overall agreement categories. This showcases a significant effectiveness improvement compared to a legally marketed predicate directly by the device itself, rather than human readers improving with AI assistance.

6. Standalone (Algorithm Only Without Human-in-the-Loop Performance) Study

The UroVysion Kit is a FISH assay that involves human visual interpretation by an analyst to recognize fluorescent signals. Therefore, its performance is inherently human-in-the-loop, and a standalone (algorithm only) performance study, in the context of AI, is not applicable. The detection relies on DNA probes and fluorescence, with an analyst visually counting signals, rather than an automated algorithm making the final call.

7. Type of Ground Truth Used

Specificity Study: Ground truth was "healthy volunteers and urology patients without prior history or clinical evidence of bladder cancer."
Performance vs. Standard of Care Study: Ground truth for bladder cancer recurrence was established by cystoscopy followed by histology confirmation. If cystoscopy was positive but histology was absent (e.g., lesion fulgurated), it was considered positive. Suspicious cystoscopies without histology were omitted. This can be categorized as a form of clinical outcome/pathology-confirmed diagnosis.
Reproducibility and Analytical Specificity: Used cultured human bladder carcinoma cell lines (positive target) and normal lymphoblast cell lines (negative target), and metaphase spreads for structural analysis.

8. Sample Size for the Training Set

The document describes premarket clinical studies and does not refer to a "training set" in the context of machine learning. The studies are for device validation.

9. How the Ground Truth for the Training Set Was Established

As there is no mention of a "training set" for an AI algorithm, this question is not applicable to the provided document. The studies describe validation of a diagnostic kit based on established biological principles (FISH technology).

Summary

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JAN 2 2 2004

Image /page/0/Picture/1 description: The image shows a sequence of alphanumeric characters, specifically "KO33982-". The characters are written in a simple, sans-serif font, and they appear to be handwritten. The last character is a dash.

Vysis, Inc. 3100 Woodcreek Dr. Downers Grove, IL 60515 Tel: 630 271-7083 Fax: 630 271-7438 Contact: Kerry J. Flom, Ph.D.

510(k) Summary: Safety and Effectiveness Information for the UroVysion™ Bladder Cancer Recurrence Kit

December 19, 2003

Trade Name Vysis® UroVysion™ Bladder Cancer Recurrence Kit

Common or Usual Name Fluorescence in situ hybridization (FISH) reagents

Classification Name Class II IVD Device

Predicate Legally Marketed Device Bard® (Bion) BTAstat™ Test

Description of the Device

The UroVysion Kit is based upon fluorescence in situ hybridization (FISH) DNA probe technology. The UroVysion probes are fluorescently labeled nucleic acid probes for use in in situ hybridization assays on urine specimens fixed on slides. The UroVysion Kit consists of a 4-color, four-probe mixture of DNA probe sequences homologous to specific reaions on chromosomes 3. 7. 9. and 17. The UroVysion probe mixture consists of Chromosome Enumeration Probe (CEP®) 3 SpectrumRed™, CEP 7 SpectrumGreen™, CEP 17 SpectrumAqua™ and Locus Specific Identifier (LSI®) 9p21 SpectrumGold™.

Intended Use

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Different Technological Characteristics

Both the UroVysion Kit and the BTAstat test use the same specimen collection and preparation techniques in clinical practice. Thus, no new issues of safety with respect to patient care are introduced by the FISH technique; both the UroVysion Kit and the BTAstat test start with the same patient specimen (i.e., voided urine).

The major differences between the two tests are that they detect different substances and use different detection methods. Briefly, the UroVysion Kit uses DNA probes for specific regions on chromosomes 3, 7, 9 and 17 that bind to the target chromosomes by the DNA hybridization reaction. The actual binding mechanism of the UroVysion Kit is via specific complementary base pairing. In contrast, the BTAstat test is a lateral flow assay that detects the presence of bladder tumor associated antigen through antigen-specific antibodies. Also, the necessary visual interpretation of the results of the UroVysion Kit and of the BTAstat test is different. For the BTAstat test, urine is allowed to react with a colloidal gold-coniuqated antibody and the results are determined qualitatively by the presence or absence of a line on the test stick. For the UroVysion Kit, the analyst visually recognizes chromosomes 3, 7 and 17, and the 9p21 locus by the fluorescent signal carried by the DNA probe mixture.

Even though the technological characteristics are different between the BTAstat test (antigen test) and the UroVysion test (DNA probe test), both test are intended for use to monitor for the recurrence of bladder cancer from voided urine specimens. The overall performance of the UroVysion test was demonstrated to be substantially equivalent.

Safety and effectiveness issues evaluated for the UroVysion Kit included the following: prospective, comparative methods evaluation for monitoring bladder cancer recurrence; specificity evaluation in healthy and unhealthy patients (without previous diagnosis of bladder cancer); interference assessment; and reproducibility studies.

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Non-Clinical Parameters

Hybridization Efficiency

On the ProbeChek™ quality control slides run in conjunction with the clinical trials, 1.5% (4/261) of the targets failed due to lack of hybridization. These slides are prepared from cultured human bladder carcinoma (positive target) and normal lymphoblast (negative target) cell lines, and represent the best-case scenario for hybridization efficiency. Thus, under these conditions, the hybridization efficiency was found to be 98.5%, with <2% cells having no signal for any of the probes.

In a reproducibility study conducted on specimens prepared from human urine cell lines, 76 of 80 specimens yielded informative results on the first attempt. Of the 4 uninformative specimens. 3 were due to lack of hybridization. Therefore the hybridization efficiency was found to be 96.2%, based on the following definition:

% Hybridization Efficiency = 100-[hybridization failures/(informative results + hybridization failures)]*100

In a specificity study conducted on urine specimens from patients with no history of bladder cancer, 230 of 309 specimens vielded informative results on the first attempt and 18 of the uninformative results were due to lack of hybridization. resulting in a hybridization efficiency of 92.7% (see "Specificity: Technical Performance: Informative vs. Non-Informative Results" for more details). Similarly, in a clinical study conducted on urine specimens from patients with a history of bladder cancer, 175 of 251 specimens vielded informative results on the first attempt and 26 of the 76 uninformative results were due to lack of hybridization. The hybridization efficiency among these specimens was found to be 87%. Thus, under these conditions, which simulate the normal clinical practice, the hybridization efficiency was found to be ≥87% (see "Performance vs. Standard of Care: Technical Performance: Informative vs. Non-Informative Results" for more details).

Analytical Specificity

Locus specificity studies were performed with metaphase spreads according to standard Vysis QC protocols. A total of 42 metaphase spreads were examined sequentially by reverse DAPI banding to identify chromosomes 3, 7 and 17, and the 9p21 locus, followed by FISH. No cross-hybridization to other chromosome loci was observed in any of the 42 cells examined; hybridization was limited to the intended target regions of the four probes.

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Interference

Three voided urine pools (one male, one female, one male/female mix) from normal healthy volunteers were spiked with the substances listed in Table 1 and assayed with the UroVysion Kit to test for possible assay interference. Replicate samples for each urine pool were evaluated for each substance (i.e., 6 samples per substance tested); 25 consecutive cells were enumerated for each specimen. No interference was detected from any of the substances tested; results from all samples were negative (i.e., <4 abnormal cells as defined in this package insert). The highest concentrations tested for each substance are shown in Table 1.

Substances Tested for Assay Interference
Substance	Highest Concentration Tested
	Possible Urine Constituents
Albumin	1.0 g/dL
Ascorbic Acid	5 g/dL
Bilirubin (unconjugated)	2 mg/mL
Hemoglobin	100 mg/mL
IgG	10 mg/dL
Red Blood Cells (human)	1 x 106 cells/mL
White Blood Cells (human)	1 x 106 cells/mL
Sodium Chloride	730 mg/dL
Uric Acid	250 mg/dL
Caffeine	117 mg/dL
Ethanol	1% (v/v)
Nicotine	28 mg/dL
	Possible Microbial Contaminants
Candida albicans	2.5 x 1010 CFU/mL
Escherichia coli	2.5 x 1010 CFU/mL
Pseudomonas aerugenosa	2.5 x 1012 CFU/mL
	Therapeutic Agents
Acetaminophen	5.2 g/dL
Acetylsalicylic Acid	5.2 g/dL
Ampicillin	600 mg/dL
BCG	20 mg/dL
Doxorubicin-HCl	10 mg/dL
Mitomycin C	10 mg/dL
Nitrofurantoin	50 mg/dL
Phenazopyridine-HCl	200 mg/dL
Thiotepa	10 mg/dL
Trimethoprin	50 mg/dL
Preservatives
Vysis, Inc. standard: 2%	2% Carbowax/50% ethanol solution
Carbowax	(33 ml urine with 17 mL preservative)
UroCor, Inc. fixative	50/50 with urine
CytoRichRed (Autocyte)	50/50 with urine
Saccamono's solution	50/50 with urine
PreservCyt solution (Cytyc)	50/50 with urine
100% Ethanol	50/50 with urine

Table 1
Substances Tested for Assay Interference

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Specimen Preservative / Transport Media Studies

Cytyc PreservCyt® Microbial Challenge Study with the UroVysion Bladder Cancer Kit

Studies were conducted to assess the microbial inhibitory performance of PreservCyt in urine spiked to known microbial concentrations, and to evaluate the performance of the UroVysion Kit with urine samples containing PreservCyt or Carbowax preservatives. Cytyc PreservCyt is a commercially available specimen transport medium manufactured by Cytyc Corporation. Bladder carcinoma cells were spiked into pooled urine specimens obtained from donors with no history of bladder cancer. PreservCyt or Carbowax was added to aliquots of the spiked urine pools, and each aliguot was inoculated with one of the following microbial organisms: Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus), Enterococcus, or Candida albicans (C. albicans). The inoculated urine samples were stored at 2 to 8°C and 20 to 25°C for up to 72 hours.

PreservCyt was determined to be acceptable as a preservative if there was no increase greater than 13,000 CFU/mL from the initial inoculation up to 72 hours when stored at both 2 to 8°C and 20 to 25°C. The effect of PreservCyt on the performance of the UroVysion assay was determined by counting the number of signals per nucleus of the PreservCyt samples for each probe.

None of the microbial challenge organisms demonstrated an increase in microbial growth from 0 to 72 hours when stored with the PreservCyt or Carbowax preservative, and acceptable UroVysion assay performance results were obtained with urine samples containing both PreservCyt and Carbowax.

Validation of Cytyc Preservative for Use with the UroVysion Bladder Cancer Kit

Studies were conducted to determine the hybridization quality of the UroVysion Kit on urine specimens using Cytyc Preservative relative to Carbowax preservative, and to establish an alternative, commercially available preservative and transport medium for use with the UroVysion Kit.

Urine specimens from donors with no history of bladder cancer were divided into three categories: no preservative (urine only). Carbowax preservative, or PreservCyt preservative. Half of each urine sample was spiked with a bladder carcinoma cell line, and the other half remained unspiked. Samples were diluted with preservative and placed in simulated shipping conditions for up to seven days, and tested with the UroVysion Kit. The average number of signals per nucleus for each probe was calculated for each sample. The performance of PreservCyt was considered acceptable if the average percent variation of the signals per nucleus was less than 50 between PreservCyt and Carbowax.

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The results of this study demonstrated that PreservCyt and Carbowax were equivalent in the preservation of urine cells for analysis by the UroVysion assay. and that PreservCyt can be used as an alternative, commercially available preservative and transport medium for use with the UroVysion Kit.

Reproducibilitv

Reproducibility of Patient Samples

Conducting reproducibility studies on real patient urine specimens was not feasible, since one patient cell pellet does not vield enough cells to reasonably split the specimen between observers. Hence the reproducibility of results on the number of morphologically abnormal cells was not assessed.

Reproducibility of Bladder Carcinoma Cell Culture Specimens

To assess the reproducibility of the UroVysion assay, analyses of the signal distributions for CEP 3, CEP 7, CEP 17 and LSI 9p21 were assessed for inter-site (4) reproducibility on slides prepared from 4 different bladder carcinoma cell lines. Four specimens prepared from human bladder carcinoma cell lines with normal (one specimen) and abnormal (3 specimens) signal distribution were evaluated for CEP 3, CEP 7, CEP 17 and LSI 9p21 according to the instructions for analysis of quality control slides in this package insert (see "Interpretation of Results: Analysis of Quality Control Slides"). Each site assayed four replications of the same specimen on each of four assay days (a different specimen each day), using a single probe lot for all specimens. On each assay day, an additional "wild card" specimen was added to eliminate bias and was not included in the data analysis. Each specimen was evaluated by one observer at each site. Informative results were obtained in 95.0% (76/80) of the specimens on the first attempt. Hybridization of all replacement slides was successful.

The mean, standard deviation, and percent CV of the average number of signals for the four probes is shown in Table 2. As shown in this table, the mean number of signals for each probe varies within a narrow range. The absence of LSI 9p21 signals in specimen 2 causes a large %CV for this probe, but this specimen is still easily classified as having a loss of the 9p21 locus; in 95% of the observations on this specimen (19/20) the average number of LSI 9p21 signals was <0.2.

There were no false negative results in this study of human bladder carcinoma cell lines; all (48/48) evaluations of specimens 2, 3 and 4 (16 each) would have been classified as positive by the definition of ≥4 cells with gains of multiple chromosomes (3 or more signals for two or more of CEP 3, CEP 7 or CEP 17), or ≥12 cells with homozygous loss of 9p21 (0 LSI 9p21 signals). Of the 16 evaluations of the normal specimen, one would have been classified as positive using the above definition; this case showed 6 cells with gains of multiple chromosomes.

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Between-Site Reproducibility
		Number of Signals
Specimen	Statisticsb	CEP 3	CEP 7	CEP 17	LSI 9p21
	Mean	2.21	2.12	2.14	2.19
	S.D.	0.15	0.12	0.12	0.21
1	C.V. (%)	6.79%	5.52%	5.66%	9.66%
	Range	2.08-2.68	1.92-2.40	1.96-2.52	2.00-2.92
	n	16	16	16	16
	Mean	3.95	4.31	3.42	0.03
	S.D.	0.10	0.25	0.16	0.07
2	C.V. (%)	2.49%	5.76	4.76%	220.44%
	Range	3.84-4.16	3.76-4.84	3.16-3.72	0.00-0.24
	n	16	16	16	16
	Mean	4.28	3.55	3.42	3.86
	S.D.	0.32	0.34	0.25	0.47
3	C.V. (%)	7.58%	9.47%	7.21%	12.14%
	Range	3.88-5.04	3.12-4.24	3.04-3.96	3.16-4.72
	n	16	16	16	16
	Mean	3.18	3.88	3.84	3.85
	S.D.	0.15	0.10	0.10	0.15
4	C.V. (%)	4.63%	2.45%	2.70%	3.90%
	Range	2.96-3.52	3.64-4.04	3.64-4.12	3.56-4.24
	n	16	16	16	16

Table 2

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Specificity

Study Summary

A multi-center, prospective study was conducted to establish the specificity of the UroVysion test in urine from healthy volunteers and urology patients without prior history or clinical evidence of bladder cancer.

Technical Performance: Informative vs. Non-Informative Results

A total of 315 patient visits were conducted in conjunction with this trial, resulting in 309 usable office visits. The 6 unusable visits included one that failed to meet the study eligibility criteria, 4 with insufficient urine volume, and in 1 cases urine was not sent to the testing laboratory. FISH assay and analysis on the 309 usable office visits resulted in informative results in 230 specimens on the first attempt. Of the 79 specimens that failed to yield informative results on the first attempt, only 18 were due to hybridization failures. The hybridization efficiency for the first assay attempt was 93%. The remaining non-informative assays were the result of poor specimen quality (e.g., insufficient number of cells) or technical error (e.g., oil under coverslip). Repeat assays were conducted on 67 specimens; 12 of these 79 specimens had insufficient volume remaining to repeat the assay. Of the 67 repeat assays, 45 yielded informative results, leaving 34 specimens classified as "non-informative" (including 12 cases with insufficient volume for repeat assay). In summary, 89% of the cases yielded an informative result on the first or second attempt. Since several patients' health conditions fell into multiple categories, the 275 patient specimens vielding informative results represented 357 data points. The patient population is summarized by category in Table 3.

Table 3
Patient Population
Condition	# of Patients
Healthy Donors	59
Non-Smokers	50
Smokers	9
Non-GU Benign Diseases	48
Non-GU Cancer	3
GU Diseases	184
BPH	58
Microhematuria	15
Interstitial Cystitis	11
Inflammation/Infection: Other	17
STD	2
Other	81
GU Cancer (non-bladder)	61
Prostate	58
Renal	3
GU Trauma	2
Total:	357

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Specificity

The overall specificity of the UroVysion test in this patient population was 93.0% (332/357). The overall specificity was calculated based on all patients and all conditions; patients with medical conditions falling in multiple categories and/or multiple conditions within the same category were counted for each individual condition. A summary of the overall specificity and the specificity by category is shown in Table 4. To eliminate the potential bias of including multiple data points for any particular patient, the specificity was also calculated on "unique cases", where each patient was counted only once, regardless of the number of medical conditions present. The specificity among the unique cases was 94.5% (260/275, Table 4).

Table 4
Summary: UroVysion Kit Specificity
Overall Specificity	93.0% (332/357)
Unique Patients	94.5% (260/275)
Healthy vs. Non-Healthy
Healthy	100% (59/59)
Non-Healthy	93.1 (201/216)
Smokers vs. Non-Smokers1
Smokers	95.2% (40/42)
Non-Smokers	94.7% (234/247)
Individual Categories2
Healthy Donors	100% (59/59)
Healthy non-smokers	100% (50/50)
Healthy smokers	100% (9/9)
Non-GU Benign Diseases	91.7% (44/48)
Non-GU Cancer3	66.7% (2/3)
GU Diseases	91.9% (169/184)
BPH	91.4% (53/58)
Microhematuria	86.7% (13/15)
Interstitial Cystitis	90.7% (10/11)
Inflammation/Infection: Other	100% (17/17)
STD	100% (2/2)
Other	91.4% (74/81)
GU Cancer (non-bladder)	91.8% (56/61)
Prostate	91.4% (53/58)
Renal	100% (3/3)
GU Trauma	100% (2/2)

260/275, Table 4).

Smoking status unknown in 1 patient.

2 Some non-healthy patients had health conditions falling into multiple

disease categories, resulting in totals >275 for individual disease categories.

3 Non-GU cancers included breast (1), colon (1), and leukemia (1)

Based on the patient population in this study, the UroVysion test demonstrated an overall specificity of 93.0% (332/357), with a 100% specificity (59/59) among healthy patients. The specificity among unique cases was 94.5% (260/275). The false positive results found in 15 patients represented the following categories (note that some patients had health conditions falling into multiple disease categories); non-Special 510(k): Device Modification Attachment 5 Vysis® UroVysion™ Bladder Cancer Recurrence Kit Page 66 Volume I

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genitourinary (GU) benign diseases (3), non-GU cancer (2), GU diseases (15), and GU cancer (5). These results indicate that the test is highly specific in this patient group, which reinforces the fact that FISH does not generate artificial aneuploidy determinations; the FISH probes react only with the intended chromosomes.

Performance vs. Standard of Care

Study Summary

A multi-center, prospective, longitudinal study was conducted to further define the performance characteristics of the UroVysion Kit relative to cystoscopy followed by histology, the standard of care for monitoring for disease recurrence in patients previously diagnosed with bladder cancer. The comparative reference used for all percent agreement calculations was cystoscopy with histology confirmation for positive or suspicious cystoscopies. If a patient had a positive cystoscopy but histology was absent (e.g., the lesion was fulgurated), then the specimen was considered positive for bladder cancer. If a test had a suspicious cystoscopy but histology was absent, then the case was omitted from analysis. A total of 309 patient visits were conducted at 21 investigation sites, resulting in 251 usable office visits. The 58 unusable visits included 17 that did not meet the eligibility criteria, 16 with insufficient urine volume, 10 with suspicious cystoscopies but no histology, and in 15 cases urine was not sent to the testing laboratories. Urine processing and analysis were conducted at one centralized testing laboratory. FISH assay and analysis on the 251 usable office visits resulted in 234 informative results, representing 176 unique patients. For patients who experienced a recurrence during the trial (as determined by cystoscopy and/or histology), the first positive visit was used (i.e., the visit at which the diagnosis of recurrence was established). For the non-recurring patients, the last negative visit was used for those patients with more than one visit. The demographics for the 176 unique patients are summarized in Table 5.

Table 5
Patient Demographics
Sex
Male	132
Female	44
Race
Caucasian	153
African American	3
Hispanic	3
Other	13
Unknown	4
Age
Range	36 - 98 years
Average	71 years

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Technical Performance: Informative vs. Non-Informative Results

FISH assays on 70% (175/251) of the eligible study specimens were informative on the first attempt. Of the 76 specimens that failed to vield informative results on the first attempt, only 26 were due to hybridization failures. The hybridization efficiency for the first assay attempt was 87%. The remaining non-informative assays were the result of poor specimen quality (e.g., insufficient number of cells) or technical error (e.g., broken slide).

Repeat assays were conducted on 70 specimens; six of the 76 specimens had insufficient volume remaining to repeat the assay. Of the 70 repeat assays, 59 vielded informative results, leaving 17 specimens classified as "non-informative" (including the 6 cases with insufficient volume for repeat assay). In summary. over 93% of the cases vielded an informative result on the first or second attempt.

Performance vs. Standard of Care

Of the eligible patients with informative FISH results, 62 were positive by cystoscopy/histology. A breakdown of the number of tumors by stage and grade is shown in Table 6.

Table 6Number of Tumors, by Stage and Grade
Tumor GradeTumor
Stage	ND	1	2	3	Unknown	Total
ND	11	0	0	0	0	11
Ta	O	20	്	6	0	32
T1	0	0	2	3		ട്
T2	0	0	0	2		ﻟﻠﺘﺨﻠﻴﻔﺔ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘ
Tis	O	O	O	7	0	1
Unknown	0	2	r	O	0	3
Total	11	22	ರಿ	18	2	62

ND = not assigned or no biopsy

Table 7 shows the performance of the UroVysion Kit, relative to cystoscopy / histology, by tumor stage and grade for all cases with biopsy information available. The UroVysion Kit showed greatest agreement of positive results (100%) among the most severe tumors (T2 and Tis), when compared to cystoscopy/histology.

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Table 7 Comparison of UroVvsion vs. Cystoscopy/Histology for Detection of Bladder Cancer Recurrence by Tumor Stage and Grade* Agreement of (+) Results (%)

Stage:
All	36/48 (75.0%)
Ta, Grade 1	11/20 (55.0%)
Ta, Grade 2,3	10/12 (83.3%)
T1	5/6 (83.3%)
T2	3/3 (100%)
Tis	7/7 (100%)
Grade:
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.

Table 8 shows a comparison of the performance of the UroVysion Kit relative to cystoscopy followed by histology. Overall, FISH analysis with the UroVysion Kit demonstrated a percent agreement of positive results of 71.0% and a percent agreement of negative results of 65.8% when compared to the results of cystoscopy, followed by histology in the case of positive or suspicious cystoscopy (Note: A positive cystoscopy without a biopsy was considered positive in this analysis).

Table 8
Comparison of UroVysion vs. Cystoscopy/Histology
for Detection of Bladder Cancer Recurrence
Custo/Histo

			ે જિણા તરીકે છ
		+		Total
દાદમ	+	44	39	83
		18	75	03
	Total	62	114	176
			SER FRE ( ) " = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = =	AL AALY

Agreement of (+) results = 71.0% (95% Cl = 58.1% - 81.8%) Agreement of (-) results = 65.8% (95% Cl = 56.3% - 74.4%) Overall Agreement = 67.6% (95% CI = 60.2% - 74.5%) (+) Predictive Value = 53.0% (95% CI = 41.7%-64.1%) (-) Predictive Value = 80.6% (95% CI = 71.1% - 88.1%) Prevalence = 35.2% (95% CI = 28.2% - 42.8%) p = < 0.0001 (Fisher's Exact Test)

{12}------------------------------------------------

The positive and negative predictive values of the UroVysion Test could be determined for prevalence rates of 10%, 20% and 30%; these are presented in Table 9. This extrapolation assumed a percent agreement of positive results of 71.0% and a percent agreement of negative results of 65.8% (Table 8).

Table 9
Hypothetical Positive Predictive and Negative Predictive Values of the
UroVysion Test

Bladder CancerRecurrence Prevalence	PPV	NPV
10%	18.7%	95.3%
20%	34.2%	90.1%
30%	47.1%	84.1%

Table 10 shows a comparison of the performance of the UroVysion Kit relative to cystoscopy/ histology in patients who had received their last treatment with intravesical BCG within 3 months of FISH testing. The mean time duration of BCG treatment was 1.3 months (range 0.4-3.4 months). The mean time between the last BCG treatment and FISH testing among these patients was 1.3 months; the range was 0 (treatment ongoing at the time of FISH testing) to 3 months. Three of the 12 true positive cases were Tis, three were stage Ta grade 1, three were stage Ta grade 3, two were stage T1 grade 3, and one was stage T2 grade 3 (muscle invasive); the one false negative case was stage Ta grade 1.

Table 10 Comparison of FISH vs. Cystoscopy/Histology for Detection of Bladder Cancer Recurrence in Patients on BCG Therapy within 3 Months Cysto/Histo

FISH	Cysto/Histo
	+	-	Total
+	12	10	22
-	1	16	17
Total	13	26	39
Agreement of (+) results = 92.3% (95% CI = 64.0% - 99.8%)Agreement of (-) results = 61.5 % (95% CI = 40.6% - 79.8%)Overall Agreement = 71.8% (95% CI = 55.1% - 85.0%)(+) Predictive Value = 54.5% (95% CI = 32.2% - 75.6%)(-) Predictive Value = 94.1% (95% CI = 71.3% - 99.9%)Prevalence = 33.3% (95% CI = 19.1% - 50.2%)p = 0.0014 (Fisher's Exact Test)

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Substantial Equivalence vs. BTAstat Test

Substantial Equiralone vol a nhove, the performance of the UroVysion test was m the climour study documed a a Astat test to establish substantial equivalence of also comparou to the specimens from each of the 176 unique patients (first positive, last negative office visit) were also analyzed by the BTAstat test. positive, last nogatro on the study specimens and results are included for information purposes.

Tables 11 and 12 show the percent agreement of results of the UroVysion test, the BTAstal test and cytology by tumor stage and tumor grade. The UroVysion test showed greater percent agreement of positive results for all turnor stages, including 100% agreement for T2 and Tis tumors.

Table 11
Percent Agreement of (+) Results Analysis by Tumor Stage
Ta, 1 - Total: 20 Cases
55.0%	FISH	11 Positive	9 Negative
20.0%	Cytology	4 Positive	16 Negative
30.0%	BTAstat	6 Positive	14 Negative
Ta 2,3 - Total: 12 Cases
83.3%	FISH	10 Positive	2 Negative
33.3%	Cytology	4 Positive	8 Negative
83.3%	BTAstat	10 Positive	2 Negative
T1 - Total: 6 Cases
83.3%	FISH	5 Positive	1 Negative
66.7%	Cytology	4 Positive	2 Negative
83.3%	BTAstat	5 Positive	1 Negative
T2 - Total: 3 Cases
100%	FISH	3 Positive	0 Negative
33.3%	Cytology	1 Positive	2 Negative
66.7%	BTAstat	2 Positive	1 Negative
Tis- Total: 7 Cases
100%	FISH	7 Positive	0 Negative
33.3%	Cytology	2 Positive	4 Negative
42.9%	BTAstat	3 Positive	4 Negative

NOTE: Three (3) cases were considered Unknown by Central Pathology for Tumor Stage

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Table 12
Percent Agreement of (+) Results Analysis by Tumor Grade
Grade 1 - Total: 22 Cases
54.5%	FISH	12 Positive	10 Negative
18.2%	Cytology	4 Positive	18 Negative
27.3%	BTAstat	6 Positive	16 Negative
Grade 2 - Total: 9 Cases
77.8%	FISH	7 Positive	2 Negative
44.4%	Cytology	4 Positive	5 Negative
77.8%	BTAstat	7 Positive	2 Negative
Grade 3 - Total: 18 Cases
94.4%	FISH	17 Positive	1 Negative
41.2%	Cytology	7 Positive	10 Negative 1 inconclusive
72.2%	BTAstat	13 Positive	5 Negative

NOTE: Two (2) cases were considered Unknown by Central Pathology for Tumor Grade.

Table 13 shows a comparison of the performance of the BTAstaf test relative to cystoscopy/histology among the unique patients (first positive, last negative office visit). Overall, analysis with the BTAstat test demonstrated a percent agreement of positive results of 50.0% and a percent agreement of negative results of 69.3% when compared to the results of cystoscopy followed by histology in the case of positive or suspicious cystoscopy. (Note: A positive cystoscopy without a biopsy was considered positive in this analysis). In a comparison of the UroVysion Kit with cystoscopy/ histology on the same dataset (Table 8), the UroVysion Kit showed a percent agreement of positive results of 71.0% and a percent agreement of negative results of 65.8% (Table 8).

Table 13 Comparison of BTAstat vs. Cystoscopy/Histology for Detection of Bladder Cancer Recurrence Cysto/Histo

BTAstat	Cysto/histo
	+	-	Total
+	31	35	66
-	31	79	110
Total	62	114	176

Agreement of (+) results = 50.0% (95% Cl = 37.0% - 63.0%) Agreement of (-) results = 69.3% (95% Cl = 60.0% - 77.6%) Overall Agreement = 62.5% (95% Cl = 54.9% - 69.7%) (+) Predictive Value = 47.0% (95% CI = 34.6% - 59.7%) (-) Predictive Value = 71.8% (95% CI = 62.4% - 80.0%) Prevalence = 35.2% (95% Cl = 28.2% - 42.8%)

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Figure 1 compares the percent agreement of results for FISH, BTAstat and rigare Toompares thent visits), relative to cystoscopy/histology. The UroVysion of they ( tail lower 95% CI for percent agreement of positive, negative and overall results was 58.1%. 56.3% and 60.2%, respectively. On the overall results was out with the BTAstat test, the scores minus 15% conceptialing addition and 47.5%, respectively. Thus, the criteria for substantial were 55.0%, 34.5% and 47.0%, responsely, with BTAstat test were met; the 95% Cls oquivalonsion are greater than the BTAstat scores minus 15%. This is represented graphically in Figure 1; the error bars represent the upper and lower reprosemed graphical test results and the test score minus 15% for the 55% Of the the U16 vygain, as shown in the figure, in each case the 95% Cl for UroVysion is greater than the BTAstat score minus 15%.

Image /page/15/Figure/2 description: This image is labeled as "Figure 1" and presents a comparison of three detection methods. The comparison is relative to Cystoscopy/Histology. The text is in bold font and centered on the page.

Image /page/15/Figure/3 description: This bar graph compares the agreement between UroVysion, BTAstat, and Cytology. The y-axis represents the percentage, ranging from 0 to 100. The x-axis shows three categories: Agreement (+), Agreement (-), and Overall Agreement. For Agreement (+), UroVysion is around 70%, BTAstat is around 50%, and Cytology is around 25%. For Agreement (-), UroVysion is around 65%, BTAstat is around 70%, and Cytology is around 90%. For Overall Agreement, UroVysion is around 68%, BTAstat is around 63%, and Cytology is around 67%.

A summary of the percent agreement of the three detection methods in the group of patients treated with BCG within the last 3 months is shown in Figure 2 (unique natient visits). In this group, the UroVysion test's two tail lower 95% Cl for percent agreement of positive, negative and overall results was 64.0%. 40.6% and 55.1%, respectively. On the corresponding dataset assayed with the BTAstat test, the scores minus 15% were 54.2%, 27.3% and 36.3%, respectively. Thus, the criteria for substantial equivalence of the UroVysion assay to the BTAstat test were met; the 95% Cls for UroVysion are greater than the BTAstat scores minus 15%. This is represented graphically in Figure 2; the error bars represent the upper and lower 95% CIs for the UroVysion test results and the test score minus 15% for the BTAstat test results. Again, as shown in the figure, test ooor minus 10% Cl for UroVysion is greater than the BTAstat score minus 15%.

Special 510(k): Device Modification Vysis® UroVysion™ Bladder Cancer Recurrence Kit Volume I

Attachment 5 Page 73

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Image /page/16/Figure/1 description: This image is labeled Figure 2 and contains a title describing a comparison of three detection methods relative to cystoscopy/histology. The study focuses on patients treated with BCG within 3 months of their study visit. The text is centered and uses a clear, readable font.

Image /page/16/Figure/2 description: This image is a bar graph comparing the agreement between UroVysion, BTAstat, and Cytology. The y-axis represents the percentage, ranging from 0 to 120. The x-axis shows three categories: Agreement (+), Agreement (-), and Overall Agreement. For Agreement (+), UroVysion is around 90%, BTAstat is around 70%, and Cytology is around 30%. For Agreement (-), UroVysion is around 60%, BTAstat is around 40%, and Cytology is around 85%. For Overall Agreement, UroVysion is around 70%, BTAstat is around 50%, and Cytology is around 70%.

The UroVysion test and the BTAstat test were each compared to cytology on patients positive for recurrence, as determined by cystoscopy/histology, the results are shown in Tables 14 and 15. Cytology did not pick up any cases that were negative by FISH (Table 14). Cytology was positive in 2 cases found negative by BTAstat (Table 15).

Table 14 Comparison of FISH vs. Cytology Results in Patients Positive for Recurrence Cytology

	Cytology
FISH	+	-	Total
+	16	27	43
-	0	18	18
Total	16	45	61

Note: One (1) Tis case was scored inconclusive for cytology and not included in this table.

Table 15 Comparison of BTAstat vs. Cytology Results in Patients Positive for Recurrence

		Cytology
		t		Total
ದ್	——	15	16	31
3			29	30
	Total	16	45	61
			1 1 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1	mont in

Note: One (1) Tis case was scored inconclusive for cytology and not included in this table.

Special 510(k): Device Modification Vysis® UroVysion™ Bladder Cancer Recurrence Kit Volume I

Attachment 5 Page 74

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The results for the percent agreement of results for the UroVysion test (FISH), the The results for the poroent agreemmarized in Table 16 (per patient office visit).

Table 16

Summary: Methods Comparison
		FISH	BTAstat	Cytology
Overall	Agreement of (+) Results	71.0%	50.0%	26.2%
	Agreement of (-) Results	65.8%	69.3%	89.1%
	Overall Agreement	67.6%	62.5%	66.7%
BCG	Agreement of (+) Results	92.3%	69.2%	30.8%
	Treatment Agreement of (-) Results	61.5%	42.3%	84.6%
	Overall Agreement	71.8%	51.3%	66.7%

Table 17 shows a head-to-head comparison of the results from the UroVysion Table 11 onews a noad to n those cases (unique office visits) with informative results for both tests. The concordance between the two tests was 61.9%.

Table 17
Concordance of FISH vs. BTAstat
BTAstat

	DTAstat
FISH	+	-	Total
+	41	42	83
-	25	68	93
Total	66	110	176

An analysis of the discordant results is presented in Table 18. Of the cases positive / In and negative by BTAstat, 21 (50.0%) were positive by either cytology or by For and histology, or both, including 4 Tis tumors and 1 T2 tumor (Table 18). Of oyotooopymiology, mochogy, and positive by BTAstat, only 3 (12.0%) were positive by one or both of the comparative methods.

Table 18FISH vs. BTAstat: Discordant Analysis
	FISH "+" / BTAstat "-"	FISH "-" / BTAstat "+"
N	42	25
Cytology "+"	7 (16.7%)	1 (4.0%)
Cysto/Histo "+"	15 (35.7%)	2 (8.0%)
Ta	6	1
T1	--	--
T2	1	--
Tis	4	--
Unk	1	1
Pos/No Biopsy	3	--
Cytology "+" orCysto/Histo "+"	21 (50.0%)	3 (12.0%)

Note: One (1) case showed both positive cytology and positive cystoscopy/histology in the FISH "+"/BTAstat "-" group.

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Longitudinal Study

As a continuation of the multi-center prospective study described above, office visit information (without FISH or BTAstat testing) was subsequently collected for patients who had not experienced a relapse (i.e., cystoscopy/ histology negative) for a period of approximately 1 year from their last visit during the main phase of the trial. Of the 114 eligible patients, office visit information was collected from 105. A total of 335 patient visits were reported, resulting in 299 usable office visits, representing 104 unique patients (Note: for 1 patient the only office visit reported was an ineligible visit). The 36 unusable visits included 21 that did not meet eligibility criteria and 15 with suspicious cystoscopies but no histology. For patients who experienced a recurrence (as determined by cystoscopy/histology), the first positive visit was used. For non-recurring patients, the last negative visit was used for those patients with more than one visit.

The results showed recurrence in a greater percentage of patients in the FISH positive, cystoscopy/histology negative group than in the FISH negative, cystoscopy/histology negative group; the difference was statistically significant (p=0.014, x2, 1 df). The results are summarized in Table 19.

	Table 19Longitudinal Study Summary
	FISH -/cysto:histo -	FISH+/cysto:histo -
% Recurrence	19.1% (13/68)	41.7% (15/36)
	p = 0.014*
Follow-up time(months):
No recurrence	14.3±3.9	13.5±3.4
Recurrence	11.0±5.8	6.9±4.4
Recurrence Details^:
Stage
Ta G1	5	3
Ta G2,3	0	1
T1	2	0
Tis	0	1
Grade
1	5	5
2	1	1
3	1	1

, 1 df

^Biopsy was not performed in 8 cases (4 FISH+/cysto:histo-, 4 FISH-/cysto:histo-). Slides were not provided by collection site for assessment by the central pathologist in 6 cases (4 FISH+/cysto:histo-, 2 FISH-/cysto:histo-). No stage was assigned in 2 FISH+/cysto:histo- cases.

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Probability estimates for non-recurrence at various intervals were r robablify octimate product-limit method for right-censored data (i.e., Kaplan-Meier). Analysis of homogeneity between the two patient groups Kaplan-Meller). Analysio of nomogenewy was determined using the log-rank (anticipatory positives, and trac-hegan in Figure 3, both methods show and Wilcoxon chi-square etails.maintained throughout the follow-up period that a Statistical difference traso:histo - and the UroVysion - /cysto:histo groups.

Figure 3

Recurrence-Free Survival for Patients in the UroVysion - /cysto:histo - vs. UroVysion + /cysto:histo - Groups

Image /page/19/Figure/4 description: This image is a survival plot comparing two groups: "UroVysion +/cysto:histo" and "UroVysion -/cysto:histo". The y-axis represents the survival probability, ranging from 0.0 to 1.0, while the x-axis represents time in months, ranging from 0 to 25. The plot shows that the "UroVysion -/cysto:histo" group has a higher survival probability than the "UroVysion +/cysto:histo" group. A table below the plot shows the results of statistical tests between the two groups, including Log-Rank (Chi-Square = 8.7454, DF = 1, Prob>ChiSq = 0.0031) and Wilcoxon (Chi-Square = 10.6166, DF = 1, Prob>ChiSq = 0.0011).

{20}------------------------------------------------

HYBrite/VP 2000 Validation

The VP2000 is considered to be a class I, exempt device according to 21 CFR & THE VF 2000 is considered to be a labor, and 21 CFR § 864.3875 Automated tissue ob4.3800 Automation of the VP2000 is consistent with both of the above processor. The function of the VT 2008 is consister modifications the device is paragraphs from the OF A. "Indocution OEM device bought and sold by Zeiss during the past decade as a class I device for cytology laboratories.

The paragraphs from the CFR are reproduced below:

21 CFR § 864.3800 Automated slide stainer. (a) Identification. An automated slide stainer is a device used to stain histology, An adomatod ollustically slides for diagnosis. (b) Classification. Class I. The device is exempt from the premarket notification procedures in Subpart E of Part 807 of this chapter.

21 CFR § 864.3875 Automated tissue processor. (a) Identification. An automated tissue processor is an automated system used to process tissue specimens for examination through fixation, dehydration, and infiltration. (b) Classification. Class I. The device is exempt from the premarket notification procedures in Subpart E of Part 807 of this chapter.

A validation study was conducted to determine if the recommended specimen / validation otably for the UroVysion Kit performed the same pretreatment protocol and technician or by semi-automated using the VP2000 Sample Processor and HYBrite instruments.

Study specimens consisted of three human urine pools prepared from voided urine specimens obtained from normal donors. Study specimens used in the anne Specificans obtains obtains (see Appendix B for protocol and , tody intentional were also used as part of this study. Each of the 29 substances stady report, were aliquots of each of the three pools at two different which word oplical in three separate VP-2000 and HYBrite instruments and compared to results obtained in the manual study.

Quality evaluations from samples of the 23 different compounds and 6 &uality of charactions it equivalent results using the UroVysion Kit and FISH Pretreatment Kit for all concentrations tested and across all three instrument set-ups.

Normal urine pools (unspiked) and manual assay results from the Interference Study Protocol, 99-402R were used as controls. All compounds and Otudy 1 10.000, 00 Trod in Table 20 performed within 2 standard deviations or preservatives from hools, supporting the conclusion that the manual and semiautomated methods are equivalent.

Attachment 5 Page 78

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Substance	Concentrations	Results- Manual vsSemi-Automation
	Possible Urine Constituents
Albumin	0.5 g/dL and 1.0 g/dL	Equivalent.
Ascorbic Acid	2.5 g/dL and 5 g/dL	Equivalent.
Bilirubin (unconjugated)	1 mg/mL and 2 mg/mL	Equivalent.
Hemoglobin	50 mg/mL and 100 mg/mL	Equivalent.
IgG	5 mg/dL and 10 mg/dL	Equivalent.
Red Blood Cells (human)	$5 x 10^5$ cells/mL and $1 x 10^6$ cells/mL	Equivalent.
White Blood Cells (human)	$5 x 10^5$ cells/mL and $1 x 10^6$ cells/mL	Equivalent.
Sodium Chloride	365 mg/dL and 730 mg/dL	Equivalent.
Uric Acid	125 mg/dL and 250 mg/dL	Equivalent.
Caffeine	58.5 mg/dL and 117 mg/dL	Equivalent.
Ethanol	0.5% (v/v) and 1% (v/v)	Equivalent.
Nicotine	14 mg/dL and 28 mg/dL	Equivalent.
	Possible Microbial Contaminants
Candida albicans	$1.25 x 10^{10}$ CFU/mL and $2.5 x 10^{10}$ CFU/mL	Equivalent.
Escherichia coli	$1.25 x 10^{10}$ CFU/mL and $2.5 x 10^{10}$ CFU/mL	Equivalent.
Pseudomonas aerugenosa	$1.25 x 10^{10}$ CFU/mL and $2.5 x 10^{12}$ CFU/mL	Equivalent.
	Therapeutic Agents
Acetaminophen	2.6 g/dL and 5.2 g/dL	Equivalent.
Acetylsalicylic Acid	2.6 g/dL and 5.2 g/dL	Equivalent.
Ampicillin	300 mg/dL and 600 mg/dL	Equivalent.
BCG	10 mg/dL and 20 mg/dL	Equivalent.
Doxorubicin-HCI	5 mg/dL and 10 mg/dL	Equivalent.
Mitomycin C	5 mg/dL and 10 mg/dL	Equivalent.
Nitrofurantoin	25 mg/dL and 50 mg/dL	Equivalent.
Phenazopyridine-HCI	100 mg/dL and 200 mg/dL	Equivalent.
Thiotepa	5 mg/dL and 10 mg/dL	Equivalent.
Trimethoprin	25 mg/dL and 50 mg/dL	Equivalent.
	Preservatives
Vysis, Inc. standard:2% Carbowax	2% Carbowax/50% ethanol solution (33 mlurine with 17 mL preservative)	Equivalent.
UroCor, Inc. fixative	50/50 with urine	Equivalent.
CytRichRed (Autocyte)	50/50 with urine	Equivalent.
Saccamono's solution	50/50 with urine	Equivalent.
PreservCyt solution (Cytyc)	50/50 with urine	Equivalent.
100% Ethanol	50/50 with urine	Equivalent.

Table 20 Manual versus Semi-Automation Study Results

Conclusions

The nonclinical and clinical studies described in this document demonstrate that the nonomical and UroVysion Kit is safe and effective. The performance of the IroVysion Kit is also supported by the Vysis Quality Control procedures. When Gro Frontision Kit is used as instructed in the package insert, the above 1 statements describe its performance.

{22}------------------------------------------------

Image /page/22/Picture/1 description: The image shows the logo for the Department of Health & Human Services (HHS). The logo consists of a circular seal with the words "DEPARTMENT OF HEALTH & HUMAN SERVICES USA" arranged around the perimeter. Inside the circle is an abstract symbol resembling an eagle or bird in flight, composed of three curved lines representing the wings and a stylized tail.

Public Health Service

Food and Drug Administration 2098 Gaither Road Rockville MD 20850

JAN 2 2 2004

Kerry J. Flom, Ph.D. Senior Director Clinical Research and Regulatory Submissions Vysis, Inc. 3100 Woodcreek Drive Downers Grove, IL 60515

Re: K033982

Trade/Device Name: Vysis® UroVysion™ Bladder Cancer Kit Regulation Number: 21 CFR 866.6010 Regulation Name: Tumor-associated antigen immunological test system Regulatory Class: Class II Product Code: MMW Dated: December 19, 2003 Received: December 23, 2003

Dear Dr. Flom:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to such additional controls. Existing major regulations affecting your device can be found in Title 21, Code of Federal Regulations (CFR), Parts 800 to 895. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); and good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820).

{23}------------------------------------------------

Page 2

This letter will allow you to begin marketing your device as described in your Section 510(k) premarket notification. The FDA finding of substantial equivalence of your device to a legally marketed predicate device results in a classification for your device and thus, permits your device to proceed to the market.

If you desire specific information about the application of labeling requirements to your device, or questions on the promotion and advertising of your device, please contact the Office of In Vitro Diagnostic Device Evaluation and Safety at (301) 594-3084. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21CFR Part 807.97). You may obtain other general information on your responsibilities under the Act from the Division of Small Manufacturers, International and Consumer Assistance at its toll-free number (800) 638-2041 or (301) 443-6597 or at its Internet address http://www.fda.gov/cdrh/dsma/dsmamain.html.

Sincerely yours,

Fiven Autman, M.D.

Steven I. Gutman, M.D., M.B.A. Director Office of In Vitro Diagnostic Device Evaluation and Safety Center for Devices and Radiological Health

Enclosure

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ATTACHMENT 3

Indications for Use Statement

K033982 510(k) Number (if known):

Vysis® UroVysion™ Bladder Cancer Kit DEVICE NAME:

INDICATIONS FOR USE:

The UroVysion Bladder Cancer Recurrence Kit (UroVysion Kit) is designed to detect aneuploidy for chromosomes 3, 7, 17, and loss 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 Uro Vysion Kit are intended for use as a noninvasive method for monitoring for turnor recurrence in conjunction with cystoscopy in patients previously diagnosed with bladder cancer.

Note: No change from the current, cleared indications for use (K013785)

Donald J. B

(Division Sign-OH) (Office of In Vitro Diagnostic Devices Evaluation and Safety 510(k) Number 4033882

(PLEASE DO NOT WRITE BELOW THIS LINE - CONTINUE ON ANOTHER PAGE IF NEEDED)

Concurrence of CDRH, Office of Device Evaluation (ODE)

Prescription Use > (Per 21 CFR 801.109) OR

Over-The-Counter-Use (Optimal Format 1-2-96)

Special 510(k): Device Modification Vysis® UroVysion™ Bladder Cancer Recurrence Kit Volume I

Attachment 3 Page 55

Regulation Number and Section

§ 866.6010 Tumor-associated antigen immunological test system.

(a)
Identification. A tumor-associated antigen immunological test system is a device that consists of reagents used to qualitatively or quantitatively measure, by immunochemical techniques, tumor-associated antigens in serum, plasma, urine, or other body fluids. This device is intended as an aid in monitoring patients for disease progress or response to therapy or for the detection of recurrent or residual disease.(b)
Classification. Class II (special controls). Tumor markers must comply with the following special controls: (1) A guidance document entitled “Guidance Document for the Submission of Tumor Associated Antigen Premarket Notifications (510(k)s) to FDA,” and (2) voluntary assay performance standards issued by the National Committee on Clinical Laboratory Standards.