Predicate Devices

Reference Devices

AI/ML (Artificial Intelligence / Machine Learning)

Unknown
The summary mentions a "proprietary EpiScore algorithm" used for the software cut-off, but does not provide enough detail to determine if this algorithm utilizes AI or ML techniques. It could be a rule-based or statistical algorithm without AI/ML components.

Therapeutic

No
The device is an in vitro diagnostic assay used for monitoring tumor recurrence and does not directly treat or diagnose a disease.

Diagnostic

Yes

The device qualitatively detects DNA methylation patterns associated with transitional cell carcinoma of the bladder to monitor for tumor recurrence. This function directly contributes to identifying or classifying a disease, which is the definition of a diagnostic device.

SaMD (Software as a Medical Device)

No

The device is an in vitro diagnostic assay that includes reagents for DNA extraction, digestion, and PCR amplification, in addition to analysis software. It is performed on a specific hardware system (ABI® 7500 Fast Dx Real-Time PCR system). Therefore, it is not a software-only medical device.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

Here's why:

Intended Use: The "Intended Use / Indications for Use" section explicitly states that the Bladder EpiCheck Kit is intended for the "qualitative detection of DNA methylation patterns... in human DNA" and is performed on "voided urine samples." This clearly indicates that the device is used to examine specimens derived from the human body to provide information for diagnostic purposes.
Device Description: The "Device Description" further reinforces this by describing the test as a "real-time PCR-based in vitro diagnostic assay" and detailing how it analyzes DNA extracted from urine samples.
Performance Studies: The document includes detailed descriptions of clinical and analytical performance studies, which are standard requirements for demonstrating the safety and effectiveness of IVD devices.
Predicate Device: The mention of a "Predicate Device" (K013785; Abbott Molecular UroVysion™ Bladder Cancer Kit) is a strong indicator that this device is being compared to an already cleared IVD device, a common practice in the regulatory pathway for new IVDs.

The definition of an In Vitro Diagnostic device generally includes reagents, instruments, and systems intended for use in the diagnosis of disease or other conditions, including a determination of the state of health, in order to cure, mitigate, treat, or prevent disease or its sequelae. The Bladder EpiCheck Kit fits this definition by analyzing human urine samples to monitor for bladder cancer recurrence.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

Bladder EpiCheck Kit is intended for the qualitative detection of DNA methylation patterns of 15 loci in Human DNA that are associated with transitional cell carcinoma of the bladder. The test is performed on voided urine samples and run on the ABI® 7500 Fast Dx Real-Time PCR system.

Bladder EpiCheck Kit is indicated for use as a non-invasive method to monitor for tumor recurrence in conjunction with cystoscopy in patients previously diagnosed with Non-Muscle Invasive Bladder Cancer.

Product codes

MMW

Device Description

The Bladder EpiCheck Test is a real-time PCR-based in vitro diagnostic assay intended for the qualitative detection of DNA methylation patterns associated with transitional cell carcinoma of the bladder to monitor for tumor recurrence (in conjunction with cystoscopy) in patients previously diagnosed with non-muscle invasive bladder cancer (NMIBC).

The assay consists of a panel of 15 novel DNA methylation (covalent addition of methyl (CH3) groups to the C5 position of the pyrimidine ring of cytosines, typically in a CpG dinucleotide) biomarkers that were found to distinguish between patients with bladder cancer and patients without bladder cancer. The Bladder EpiCheck Test differentiates between methylated and non-methylated DNA, creating a unique platform for methylation profiling of urine specimens towards the detection of bladder cancer recurrence in patients previously diagnosed with the disease. The test is comprised of reagents for end-to-end (sample-to-answer) processing of urine samples (reagents for DNA extraction, DNA digestion, PCR amplification, and analysis software), and is performed using the Applied Biosystems® 7500 Fast Dx Real-Time PCR system.

A voided urine specimen is centrifuged, and the cells (both normal and cancerous if present) are separated from the urine supernatant. DNA is then extracted from the cell pellet using the Bladder EpiCheck Extraction kit (P/N NX899090-01C). The extracted DNA is digested using a methylation-sensitive restriction enzyme mix. which cleaves DNA at specific recognition sequences if they are unmethylated. Methylated DNA is protected from enzymatic digestion and therefore remains intact.

Real-Time PCR Amplification:
Using reagents provided in the Bladder EpiCheck Test kit, the digested DNA samples and test controls are mixed together with PCR primers and probe into 8 unique PCR multiplexes reactions and assayed using the ABI 7500 Fast Dx Real-Time PCR system. The panel of 15 biomarkers is amplified according to their residual levels of methylation. Real-time PCR amplification is performed in a 96-well plate, requires 2 assay-control samples (Undigested Control and No Template Control) and can test up to 10 patient samples. Each sample is analyzed in 8 wells, corresponding to a single column of the plate. In each well. 3-4 biomarkers are amplified. The panel of 15 biomarkers are used to determine whether a sample is bladder cancer positive or bladder cancer negative. In addition. 3 internal controls (Internal Reference, Digestion Control, and Digestion Control Reference) are used to verify successful digestion and amplification processes.

Data Analysis:
The Bladder EpiCheck software (P/N NX899090-03C) accepts as an input (Source File) the *.SDS output file of the ABI7500 DX real-time PCR machine, and outputs for each patient's sample a qualitative diagnosis (positive/negative for recurrence of bladder cancer) based on the patient's EpiScore. The software implements the Bladder EpiCheck algorithm, which starts from the raw fluorescence data of the real-time PCR run and ends in the diagnosis (positive/negative) of each patient. The software enables the operator.

Result Reporting:
The Bladder EpiCheck Test Kit is used to calculate the associated methylation level for each of the 15 biomarkers based on the associated Cq (Cycle of quantitation, which is the PCR cycle at which the marker's amplification curve reached a predefined signal threshold). These methylation levels are translated to marker scores that are based on reference methylation levels obtained from a large set of bladder cancer negative and positive patients. These 15 marker scores are then combined into a single number: the EpiScore, a number between 0 and 100 representing the overall methylation level of the sample at the panel biomarkers. The resulting EpiScore is then compared to a prespecified threshold; if it is above or equal to the threshold (60), the test is called "Positive"; otherwise, it is called "Negative".

If the sample analysis is failed (e.g., insufficient DNA, incomplete digestion) or the analysis of the entire PCR plate failed (e.g., one of the plate controls failed), the output for the sample will be "Invalid". In some cases of failure, the sample/plate run may be repeated a second time, and in other cases the user will be instructed to collect a new sample from the patient.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

bladder

Indicated Patient Age Range

The study included subjects aged 22 and above.

Intended User / Care Setting

Not Found

Description of the training set, sample size, data source, and annotation protocol

The assay cut-off and proprietary EpiScore algorithm for the Bladder EpiCheck software were validated on a set of urine samples collected from control patients with a history of bladder cancer and bladder cancer positive patients confirmed by cystoscopy and pathology. The total sample size for the software algorithm development included 178 samples which were collected in two separate sets. The first set of samples served for the cut-off definition. The set included 109 samples, 40 of which were collected from control patients with a history of bladder cancer, and 69 collected from Urothelial Cell Carcinoma (UCC) positive patients confirmed by pathology. The second samples set served for the validation of the cut-off. The set included 67 samples, of which 51 were collected from control patients with a history of bladder cancer, and 16 collected from UCC positive patients confirmed by pathology.

Description of the test set, sample size, data source, and annotation protocol

A multi-center, prospective, IRB-approved longitudinal study was conducted at 11 academic and urology specialty medical centers in the U.S. and Canada to establish the performance of Bladder EpiCheck as compared to the predicate device (UroVysion Bladder Cancer Recurrence Kit). Performance of the Bladder EpiCheck kit was also assessed to a gold standard (GS) using both clinical cytology and combined cystoscopy/pathology data.

A total of 674 subject were enrolled in the study. Eligible subjects were any male or female subject aged 22 and above, diagnosed with incident or recurrent UCC and undergoing surveillance for bladder cancer recurrence, who had urothelial cell carcinoma tumor resected within 12 months from baseline visit and had a plan for cystoscopy surveillance. The study included up to 3 visits: baseline and up to 2 additional surveillance visits, where voided urine specimens were collected. In total, voided urine specimens were collected from 583 subjects. For subjects who experienced a recurrence (as determined by positive cytology /pathology), the first positive visit was used (i.e., the visit at which the diagnosis of recurrence was established). For the non-recurring subjects, the last negative visit was used for subjects with multiple surveillance visits.

DNA extracted from urine specimens was tested using the Bladder EpiCheck test and compared to the previously discussed gold standard (GS). Classification by the GS was as follows: a subject was considered "positive" if the interpretation for either cytology or the combined cystoscopy/pathology results were positive, and a subject was considered "negative" if both cytology and the combined cystoscopy/pathology results were negative. In total, valid Bladder EpiCheck and GS results were obtained from 449 subjects.

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

Clinical Cutoff (Training and Feasibility Data):
The assay cut-off and proprietary EpiScore algorithm for the Bladder EpiCheck software were validated on a set of urine samples collected from control patients with a history of bladder cancer and bladder cancer positive patients confirmed by cystoscopy and pathology. The total sample size for the software algorithm development included 178 samples. The first set of samples (109 samples) served for the cut-off definition (40 control patients, 69 UCC positive patients). The second samples set (67 samples) served for the validation of the cut-off (51 control, 16 UCC positive). The derived Bladder EpiCheck algorithm sensitivity and specificity compared to pathology outcome demonstrated a sensitivity of 94% for UCC recurrence and specificity of 84%.

Method Comparison:
A multi-center, prospective, IRB-approved longitudinal study was conducted at 11 academic and urology specialty medical centers in the U.S. and Canada.
Total subjects enrolled: 674. Valid Bladder EpiCheck and Gold Standard (GS) results obtained from 449 subjects.
The Bladder EpiCheck test performed with an accuracy of 78.8% ([74.8%; 82.4%]) (354/449), a sensitivity of 66.7% ([58.4%; 74.0%]) (92/138), and a specificity of 84.2% ([79.8%; 87.9%]) (262/311).
Comparison to Predicate Device (UroVysion Bladder Cancer Recurrence Kit): Valid Bladder EpiCheck, UroVysion, and GS results were obtained from 352 samples. The Bladder EpiCheck was slightly more sensitive (+4.82%) and slightly less specific (-2.97%) than the predicate device, showing non-inferiority.

Specificity in Urology Patients without Bladder Cancer:
A multi-center, prospective study with 147 subjects. The specificity of Bladder EpiCheck in this study population was 98.0%.

Clinical Specificity - Cross Reactivity with Other Cancers:
Assessed using 147 banked remnant deidentified urine samples from patients with different types of cancers. The specificity was found to be 95% (140/147: Prostate (3), RCC (2), Uterine (1), pancreas (1)) (95% confidence interval of 90.4%-98.1%).

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

Accuracy: 78.8% [74.8%; 82.4%] (354/449)
Sensitivity: 66.7% [58.4%; 74.0%] (92/138)
Specificity: 84.2% [79.8%; 87.9%] (262/311)
PPV: 65.3% [57.1%; 72.6%] (92/141)
NPV: 85.1% [80.7%; 88.6%] (262/308)

Predicate Device(s)

K013785

Reference Device(s)

K082562

Predetermined Change Control Plan (PCCP) - All Relevant Information

Not Found

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.

Summary

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Image /page/0/Picture/0 description: The image contains the logo of the U.S. Food & Drug Administration (FDA). On the left, there is the Department of Health & Human Services logo. To the right of that is the FDA logo, which is a blue square with the letters "FDA" in white. To the right of the square is the text "U.S. FOOD & DRUG ADMINISTRATION" in blue.

May 4, 2023

Nucleix Ltd. % Janice Hogan Partner Hogan Lovells US LLP 1735 Market Street, Floor 23 Philadelphia, Pennsylvania 19103

Re: K203245

Trade/Device Name: Bladder EpiCheck Kit Regulation Number: 21 CFR 866.6010 Regulation Name: Tumor-Associated Antigen Immunological Test System Regulatory Class: Class II Product Code: MMW Dated: November 3, 2020 Received: November 3, 2020

Dear Janice Hogan:

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. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. 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. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. 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

1

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 Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about medical devices and radiation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely,

Zivana Tezak-fragale -S

Zivana Tezak Branch Chief Division of Molecular Genetics and Pathology OHT7: Office of In Vitro Diagnostics Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

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510(k) Number (if known) K203245

Device Name

Bladder EpiCheck Kit

Indications for Use (Describe)

Bladder EpiCheck Kit is intended for the qualitative detection of DNA methylation patterns of 15 loci in human DNA that are associated with transitional cell carcinoma of the bladder. The test is performed on voided urine samples and run on the ABI® 7500 Fast Dx Real-Time PCR system.

Bladder EpiCheck Kit is indicated for use as a non-invasive method to monitor for tumor recurrence in conjunction with cystoscopy in patients previously diagnosed with Non-Muscle Invasive Bladder Cancer.

Type of Use (Select one or both, as applicable)
X Prescription Use (Part 21 CFR 801 Subpart D)	Over-The-Counter Use (21 CFR 801 Subpart C)

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510(k) Summary for Bladder EpiCheck Test

1. Submitter

Nucleix, Ltd. Pekeris 3 Rehovot 7670203, Israel

Contact: Eli Frydman, Ph.D. President, EMEA Email: eli@nucleix.com

2. Submission Correspondent - US

Janice Hogan Hogan Lovells US LLP 1735 Market Street, Suite 2320 Philadelphia, PA 19103 Janice.hogan@hoganlovells.com

3. Device Identification

Name of Device:	Bladder EpiCheck® Test
Common or Usual Name:	Bladder EpiCheck
Classification Name	Tumor-associated antigen immunological test system
Classification Regulation:	866.6010
Product Code:	MMW
Device Class:	Class II
Classification Panel:	Immunology

4. Legally Marketed Predicate Device

Abbott Molecular UroVysion™ Bladder Cancer Kit (K013785)

5

ડ. Device Description

The Bladder EpiCheck Test is a real-time PCR-based in vitro diagnostic assay intended for the qualitative detection of DNA methylation patterns associated with transitional cell carcinoma of the bladder to monitor for tumor recurrence (in conjunction with cystoscopy) in patients previously diagnosed with non-muscle invasive bladder cancer (NMIBC).

The assay consists of a panel of 15 novel DNA methylation (covalent addition of methyl (CH3) groups to the C5 position of the pyrimidine ring of cytosines, typically in a CpG dinucleotide) biomarkers that were found to distinguish between patients with bladder cancer and patients without bladder cancer. The Bladder EpiCheck Test differentiates between methylated and non-methylated DNA, creating a unique platform for methylation profiling of urine specimens towards the detection of bladder cancer recurrence in patients previously diagnosed with the disease. The test is comprised of reagents for end-to-end (sample-to-answer) processing of urine samples (reagents for DNA extraction, DNA digestion, PCR amplification, and analysis software), and is performed using the Applied Biosystems® 7500 Fast Dx Real-Time PCR system.

A voided urine specimen is centrifuged, and the cells (both normal and cancerous if present) are separated from the urine supernatant. DNA is then extracted from the cell pellet using the Bladder EpiCheck Extraction kit (P/N NX899090-01C). The extracted DNA is digested using a methylation-sensitive restriction enzyme mix. which cleaves DNA at specific recognition sequences if they are unmethylated. Methylated DNA is protected from enzymatic digestion and therefore remains intact.

Real-Time PCR Amplification

Using reagents provided in the Bladder EpiCheck Test kit, the digested DNA samples and test controls are mixed together with PCR primers and probe into 8 unique PCR multiplexes reactions and assayed using the ABI 7500 Fast Dx Real-Time PCR system. The panel of 15 biomarkers is amplified according to their residual levels of methylation. Real-time PCR amplification is performed in a 96-well plate, requires 2 assay-control samples (Undigested Control and No Template Control) and can test up to 10 patient samples. Each sample is analyzed in 8 wells, corresponding to a single column of the plate. In each well. 3-4 biomarkers are amplified. The panel of 15 biomarkers are used to determine whether a sample is bladder cancer positive or bladder cancer negative. In addition. 3 internal controls (Internal Reference, Digestion Control, and Digestion Control Reference) are used to verify successful digestion and amplification processes.

Data Analysis

The Bladder EpiCheck software (P/N NX899090-03C) accepts as an input (Source File) the *.SDS output file of the ABI7500 DX real-time PCR machine, and outputs for each patient's sample a qualitative diagnosis (positive/negative for recurrence of bladder cancer) based on the patient's EpiScore. The software implements the Bladder EpiCheck algorithm, which starts from the raw fluorescence data of the real-time PCR run and ends in the diagnosis (positive/negative) of each patient. The software enables the operator.

6

who is running the test, to view and export reports of each patient, as well as to view the raw data of the real-time PCR run. The Bladder EpiCheck software is installed on a stand-alone PC: it does not require web connection. The software setup installs 3rd-partv specific templates, which contain all required data and steps for the PCR run on the ABI 7500 Dx Real-time PCR machine. The user only needs to input the sample's IDs in the third-party software and upload the output file once the PCR run is completed. The Bladder EpiCheck software then parses the source file, and translates its content to a unified internal format, Nucleix Real-time Information XML (NRIX). Once in NRIX format, the Bladder EpiCheck algorithm processes the signals, while removing background noise and anomalies, and interpolates the distinct cycle data. The algorithm identifies the sample's well location (controls and test) and validates their raw data and each calculation steps against predefined thresholds. Once the algorithm run is completed. the EpiScore and diagnosis are assigned to each sample unless an error occurred at any of the calculation steps. The software's user interface is designed to clearly correlate between the sample's location and their test results. A simple colorcoded scheme separates between negative, positive, or invalid results and from clinical samples and kit control samples (UC and NTC). Per sample view is also available to see the sample's fluorescence signals, a sigmoid-like amplification curve for each marker. The patient's qualitative results and the overall run summary report can be viewed in the software's user interface and exported as PDF file.

Result Reporting

The Bladder EpiCheck Test Kit is used to calculate the associated methylation level for each of the 15 biomarkers based on the associated Cq (Cycle of quantitation, which is the PCR cycle at which the marker's amplification curve reached a predefined signal threshold). These methylation levels are translated to marker scores that are based on reference methylation levels obtained from a large set of bladder cancer negative and positive patients. These 15 marker scores are then combined into a single number: the EpiScore, a number between 0 and 100 representing the overall methylation level of the sample at the panel biomarkers. The resulting EpiScore is then compared to a prespecified threshold; if it is above or equal to the threshold (60), the test is called "Positive"; otherwise, it is called "Negative".

If the sample analysis is failed (e.g., insufficient DNA, incomplete digestion) or the analysis of the entire PCR plate failed (e.g., one of the plate controls failed), the output for the sample will be "Invalid". In some cases of failure, the sample/plate run may be repeated a second time, and in other cases the user will be instructed to collect a new sample from the patient.

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6. Intended Use Statement

Bladder EpiCheck Kit is intended for the qualitative detection of DNA methylation patterns of 15 loci in Human DNA that are associated with transitional cell carcinoma of the bladder. The test is performed on voided urine samples and run on the ABI 7500 Fast Dx Real-Time PCR system.

Bladder EpiCheck Kit is indicated for use as a non-invasive method to monitor for tumor recurrence in conjunction with cystoscopy in patients previously diagnosed with nonmuscle invasive bladder cancer.

Special conditions for Use statement(s):

For prescription use only .
. For in vitro diagnostic use

Special instrument requirements:

Applied Biosystems™ 7500 Fast Dx Real-Time PCR Instrument, SDS Software ● version 1.4 (cleared by FDA, K082562)

7. Comparison of Technological Characteristics with The Predicate Device

Bladder EpiCheck is substantially equivalent to the Abbott Molecular UroVysion Bladder Cancer Kit (K013785). The Bladder EpiCheck test has the same general intended use as the previously cleared predicate device. Although the technological characteristics and principles of operation are different between Bladder EpiCheck and UroVysion, those changes do not raise any new questions of safety and efficacy. Both the Bladder EpiCheck test and UroVysion kit start with the same patient specimen type.

| Characteristic | Predicate
UroVysion Bladder Cancer
Recurrence Kit (K013785) | Subject Device
Bladder EpiCheck |
|------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Similarities | | |
| Indications for
Use | The UroVysion Bladder Cancer
Recurrence 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 Bladder
Cancer Recurrence Kit are intended
for use as a noninvasive method for
monitoring for tumor recurrence in
conjunction with cystoscopy in | Bladder EpiCheck Kit is intended
for the qualitative detection of DNA
methylation patterns of 15 loci in
Human DNA that are associated
with transitional cell carcinoma of
the bladder. The test is performed on
voided urine samples and run on the
ABI 7500 Fast Dx Real-Time PCR
system.

Bladder EpiCheck Kit is indicated
for use as a non-invasive method to
monitor for tumor recurrence in
conjunction with cystoscopy in |

Table 7.1 Similarities between the Predicate and Subject Device

8

| | patients previously diagnosed with
bladder cancer. | patients previously diagnosed with
Non-Muscle Invasive Bladder
Cancer. |
|----------------------------------|-----------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------|
| Measurement
Type | Qualitative | Same |
| Specimen Type | Voided Urine | Same |
| Sample
Collection
Method | Non-invasive collection of
biological samples
delivered into a non-sterile plastic
collection cap/tube | Same |
| Special
Conditions for
Use | Prescription Only | Same |

Table 7.2 Differences between the Predicate and Subject Device

| Characteristic | Predicate
UroVysion Bladder Cancer
Recurrence Kit (K013785) | Subject Device
Bladder EpiCheck |
|----------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| | Differences | |
| Principle of the
Procedure | Fluorescence In situ hybridization
(FISH) | Real-Time PCR Reaction (RT-
PCR) |
| Test Target | Chromosomal DNA | DNA Methylation Biomarkers |
| Extraction and
Assay
Preparation | Cells recovered from urine pellets
are fixed on slides. DNA is
denatured to its single stranded form
and subsequently allowed to
hybridize with the probes. | DNA is extracted from the cell
pellet recovered from urine.

DNA is digested using a
methylation-sensitive restriction
enzyme, which cleaves DNA at its
recognition sequence if it is
unmethylated. |
| Detection
Instrument | Epi-fluorescence microscope | FDA-cleared Applied Biosystems®
7500 Fast Dx Real-Time PCR
Instrument |
| Assay Controls | Control slides are run concurrently
with patient slides. | Controls are run concurrently with
patient samples.

Three controls consisting of
Internal Reference Control (IR), No
Template Control (NTC) and
Undigested Control (UC) |

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8. Performance Testing (Analytical)

Precision/Reproducibility 8.1

Laboratory-to-Laboratory

A laboratory-to-laboratory repeatability and reproducibility study was performed to assess variation of Bladder EpiCheck when performed in 3 different laboratories by 2 operators at each laboratory site over 3 non-consecutive run days using contrived human DNA samples. Six (6) contrived human DNA samples were created by blending gDNA from different sample sources (T24 cell line DNA (T24) and commercially available DNA (Cml.), representing different Bladder EpiCheck marker combinations) to represent four different EpiScore categories (Low Negative (LN), High Negative (HN), Low Positive (LP), and High Positive (HP)), including scores around the test cutoff (high negative and low positive). These 6 samples were tested in technical duplicates at 2 different inputs (Low = 4ng/PCR; High = 10ng/PCR). Overall performance characteristics (Agreement, PPA, and NPA) are shown in Tables 8.1.1, 8.1.2 and 8.1.3 below. There were no control (plate) failures (0/54); overall invalid rate was 0.2% (1/433; 95% CI 0.05%; 1.03%).

		Bladder EpiCheck Result
		Timepoint (Day)			Overall
		1	2		3
Negative
Samples	DNA
Input	%
(n/N)	95%
CI	%
(n/N)	95%
CI	%
(n/N)	95%
CI	%
(n/N)	95%
CI	%
(n/N)	95%
CI
T24 LN	High	100
%
(12/12)	[81.6
0%;
100%]	100
%
(12/12)	[81.60
%;
100%]	100%
(12/12)	[81.60
%;
100%]	100%
(36/36)	[93.01
%;
100%]	1.4%
(1/73)	[0.31
%;
5.91%]
	Low	100
%
(12/12)	[81.6
0%;
100%]	100
%
(12/12)	[81.60
%;
100%]	100%
(12/12)	[81.60
%;
100%]	100%
(36/36)	[93.01
%;
100%]
T24 HN	High	100
%
(12/12)	[81.6
0%;
100%]	100
%
(12/12)	[81.60
%;
100%]	100%
(12/12)	[81.60
%;
100%]	100%
(36/36)	[93.01
%;
100%]	0	--
	Low	100
%
(12/12)	[81.6
0%;
100%]	100
%
(12/12)	[81.60
%;
100%]	100%
(12/12)	[81.60
%;
100%]	100%
(36/36)	[93.01
%;
100%]
Cml.
HN	High	100
%
(12/12)	[81.6
0%;
100%]	91.7
%
(11/12)	[69.88
%;
98.12%]	100%
(12/12)	[81.60
%;
100%]	97.2%
(35/36)	[88.46
%;
99.38%]	0	--
	Low	100
%
(12/12)	[81.6
0%;
100%]	83.3
%
(10/12)	[60.08
%;
94.32%]	100%
(12/12)	[81.60
%;
100%]	94.4%
(34/36)	[84.53
%;
98.14%]
		12)	12)	%]		%]
All Negatives		100
%
(72/

| 196.3
8%;
100% | ૭5.8
%
(69)
| [90.02
%;
95.83
%] | 100%
(72/7
| 196.38
%:
100%] | 98.6%
(213/2
| 196.58
%:
99.44
%] | 0.5%
(1/21
| [0.10
%;
2.04
%] |
| Positive | | PPA | | PPA | | PPA | | PPA | | Invalid
Rate | |
| Sample
S | DNA
Input | %
(n/
N) | 95%
CI | %
(n/
N) | તે જેને જેવી જેવી સવલતો પ્રાપ્ય થયેલી છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામમાં પ્રાથમિક શાળા, પંચાયતઘર, આંગણવાડી તેમ જ દૂધની ડેરી જેવી સવલ
CI | 0/0
(n/N) | તે તે જેન્જી જેવી જેવી સવલતો પ્રાપ્ય થયેલી છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામમાં પ્રાથમિક શાળા, પંચાયતઘર, આંગણવાડી તેમ જ દૂધની ડેરી જેવ
CI | 0/0
(n/N) | તે તે જેની જેવી જેવી સવલતો પ્રાપ્
CI | %
(n/N
) | તે તે જેની જેવી જેવી સવલતો પ્રાપ્
CI |
| | High | 100
%
(12/
| [96.3
8%:
100% | 100
%
(12/
| [81.60
%:
100%] | 100%
(12/1
| [81.60
%:
100%] | 100%
(36/36
) | [93.01
%;
100%] | | |
| T24 LP | Low | 100
%
(12/
| 196.3
8%;
100% | 100
%
(12/
| [81.60
%:
100%] | 100%
(12/1
| [81.60
%:
100%] | 100%
(36/36
) | [93.01
%;
100%] | 0 | |
| | High | 100
%
(12/
| 196.3
8%:
100% | 100
%
(12/
| [81.60
%;
100%] | 100%
(12/1
| 81.60
%;
100%] | 100%
(36/36
) | [93.01
%;
100%] | 0 | -- |
| T24 HP | Low | 100
%
(12/
| 196.38
%:
100% | 100
%
(12/
| 81.60
%;
100%] | 100%
(12/12
) | [81.60
%:
100%] | 100%
(36/36) | [93.01
%:
100%] | | |
| Cml. LP | High | 100
%
(12/
| 196.38
%;
100% | 100
%
(12/
| 81.60
%:
100%] | 100%
(12/12
) | [81.60
%:
100%] | 100%
(36/36) | [93.01
%:
100%] | 0 | |
| | Low | 100
%
(12/
| 196.38
%:
100% | 100
0/0
(12/
| 81.60
%:
100%] | 100%
(12/12
) | [81.60
%:
100%] | 100%
(36/36) | [93.01
%:
100%] | | |
| All Positives | | 100
%
(72/
| 196.38
%;
100% | 100
0/0
(72/
| 196.38
%;
100%] | 100%
(72/72
) | 196.38
%:
100%] | 100%
(216/2
| 198.76
%;
100%] | 0 | |
| All Samples | | | Agreement | | Agreement | | Agreement | Agreement | | | Invalid
Rate |
| | | 0/0
(n/
N) | તે જેન્જીન
CI | 0%
(n/
N) | તે જેવી જેવી સવલતો પ્રાપ્ય થયેલી છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામનાં લ
CI | 0/0
(n/N) | વેરી જેવી જેવી સવલતો પ્
CI | 0/0
(n/N) | તે જેન્જી જેવી સવલતો પ્રાપ્
CI | 0/0
(n/N
œ | તે તે જેન્જી જેવી સવલતો પ્રાપ્
CI |
| Overall | | 100
%
(144/
| [98.16
%;
100%
] | 97.9
%
(141
/144 | [97.92
%;
99.16
%] | 100%
(144/1
| [98.16
%;
100%] | 99.3%
(429/4
| 198.28
0%
99.72
%] | 0.2%
(1/43
| [0.05
%;
1.03
%] |

Table 8.1.1 Interlaboratory Reproducibility per Sample Presented as Overall and by Dav

10

Table 8.1.2 Interlaboratory Reproducibility per Sample Presented as Overall and by
Operator

Bladder EpiCheck Result
Operator						Overall
	1	2	3	4	5	6
Nega
tive
Sam
ples/
Input	NPA
%
(n/N)
95%
CI	NPA
%
(n/N)
95%
CI	NPA
%
(n/N)
95%
CI	NPA
%
(n/N)
95%
CI	NPA
%
(n/N)
95%
CI	NPA
%
(n/N)
95%
CI	NPA
%
(n/N)
95%
CI

11

| T24
LN
High | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | [93.0%;
100%
(36/36)] | | | | | | | | | | | | | |
|----------------------------------|------------------|--------------------------------|------------------|--------------------------------|-----------------------|--------------------------------|------------------|--------------------------------|-----------------------------|--------------------------------|-----------------------------|-----------------------------|---------------------------------|------|----|---------|----|------|----|---------|----|------|-------|---------|----|---------|
| T24
LN
Low | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | [93.0%;
100%
(36/36)] | | | | | | | | | | | | | |
| T24
HN
High | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | [93.0%;
100%
(36/36)] | | | | | | | | | | | | | |
| T24
HN
Low | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | [93.0%;
100%
(36/36)] | | | | | | | | | | | | | |
| Cml.
HN
Low | 100%
(6/6) | [68.9%;
100%] | 83.3%
(5/6) | [49.8%;
96.19%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | [88.5%;
99.38%
(35/36)] | | | | | | | | | | | | | |
| Cml.
HN
Low | 100%
(6/6) | [68.9%;
100%] | 66.7%
(4/6) | [34.7%;
88.27%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | [84.5%;
98.14%
(34/36)] | | | | | | | | | | | | | |
| All
Neg | 100%
(36/36) | [93.0%;
100%] | 91.7%
(33/36) | [80.9%;
96.62%] | 100%
(36/36) | [93.0%;
100%] | 100%
(36/36) | [93.0%;
100%] | 100%
(36/36) | [93.0%;
100%] | 100%
(36/36) | [93.0%;
100%] | [96.6%;
99.44%
(213/216)] | | | | | | | | | | | | | |
| Posit
ive
Sample/
Input | | PPA
%
(n/
CI
N) | | PPA
%
(n/
CI
N) | | PPA
%
(n/
CI
N) | | PPA
%
(n/
CI
N) | PPA
%
(n/
CI
N) | | PPA
%
(n/
CI
N) | PPA
%
(n/
CI
N) | | | | | | | | | | | | | | |
| T24
LP
High | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | [93.0%;
100%
(36/36)] | | | | | | | | | | | | | |
| T24
LP
Low | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | [93.0%;
100%
(36/36)] | | | | | | | | | | | | | |
| T24
HP
High | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | [93.0%;
100%
(36/36)] | | | | | | | | | | | | | |
| T24
HP
Low | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | 100%
(6/6) | [68.9%;
100%] | [93.0%;
100%
(36/36)] | | | | | | | | | | | | | |
| Cml.
LP | Input | 100% | N) | [68.9%; | CI | 100% | N) | [68.9%; | CI | 100% | N) | [68.9%; | CI | 100% | N) | [68.9%; | CI | 100% | N) | [68.9%; | CI | 100% | (n/N) | [68.9%; | CI | [93.0%; |
| T24
LP
High | 100
(6/6
) | [68.9
%
100
%] | 100
(6/6
) | [68.9
%
100
%] | 100
(6/6
) | [68.9
%
100
%] | 100
(6/6
) | [68.9
%
100
%] | 100
(6/6
) | [68.9
%
100
%] | 100%
(36/36) | [93.0
%
100
%] | | | | | | | | | | | | | | |
| T24
LP
Low | 100
(6/6
) | [68.9
%
100
%] | 100
(6/6
) | [68.9
%
100
%] | 100
(6/6
) | [68.9
%
100
%] | 100
(6/6
) | [68.9
%
100
%] | 100
(6/6
) | [68.9
%
100
%] | 100%
(36/36) | [93.0
%
100
%] | | | | | | | | | | | | | | |
| T24
HP
High | 100
(6/6
) | [68.9
%
100
%] | 100
(6/6
) | [68.9
%
100
%] | 100
(6/6
) | [68.9
%
100
%] | 100
(6/6
) | [68.9
%
100
%] | 100
(6/6
) | [68.9
%
100
%] | 100%
(36/36) | [93.0
%
100
%] | | | | | | | | | | | | | | |
| T24
HP
Low | 100
(6/6
) | [68.9
%
100
%] | 100
(6/6
) | [68.9
%
100
%] | 100
(6/6
) | [68.9
%
100
%] | 100
(6/6
) | [68.9
%
100
%] | 100
(6/6
) | [68.9
%
100
%] | 100%
(36/36) | [93.0
%
100
%] | | | | | | | | | | | | | | |
| Cml.
LP
High | 100
(6/6
) | [68.9
%
2%;
100
%] | 100
(6/6
) | [68.9
%
2%;
100
%] | 100
(6/6
) | [68.9
%
2%;
100
%] | 100
(6/6
) | [68.9
%
2%;
100
%] | 100
(6/6
) | [68.9
%
2%;
100
%] | 100%
(36/36) | [93.0
%
100
%] | | | | | | | | | | | | | | |
| Cml.
HP
Low | 100
(6/6
) | [68.9
%
2%;
100
%] | 100
(6/6
) | [68.9
%
2%;
100
%] | 100
(6/6
) | [68.9
%
2%;
100
%] | 100
(6/6
) | [68.9
%
2%;
100
%] | 100
(6/6
) | [68.9
%
2%;
100
%] | 100%
(36/36) | [93.0
%
100
%] | | | | | | | | | | | | | | |
| All
Pos | 100
(36/36) | [93.0
%
100
%] | 100
(36/36) | [93.0
%
100
%] | 100
(36/36) | [93.0
%
100
%] | 100
(36/36) | [93.0
%
100
%] | 100
(36/36) | [93.0
%
100
%] | 100%
(216/216) | [98.8
%
100
%] | | | | | | | | | | | | | | |
| All
Samples | | Agreement
%
(n/ | 95%
CI | | Agreement
%
(n/ | 95%
CI | | Agreement
%
(n/ | 95%
CI | | Agreement
%
(n/N) | 95%
CI | | | | | | | | | | | | | | |

12

N)	N)	N)	N)	N)	N)
Overall	100%
(72/72)	[96.4%;
100%]	95.8%
(69/72)	[90.0%;
95.83%]	100%
(72/72)	[96.4%;
100%]	100%
(72/72)	[96.4%;
72/72]	100%
(72/72)	[96.4%;
100%]	100%
(72/72)	[96.4%;
100%]	99.3%
(429/432)	[98.3%;
99.72%]

Table 8.1.3 Interlaboratory Reproducibility per Sample Presented as Overall and by Laboratory

		Bladder EpiCheck Result
		Laboratory
		1	2	3
Negati
ve
Sampl
es	DN
A
Inp
ut	NPA		NPA		NPA	NPA
		%
(n/N)	95% CI	%
(n/N)	95% CI	%
(n/N)	95% CI	%
(n/N)	95% CI
T24
LN	High	100%
(12/12)	[81.60
%;
100%]	100%
(12/12)	[81.60
%;
100%]	100%
(12/12)	[81.60
%;
100%]	100%
(36/36)	[93.01
%;
100%]
	Low	100%
(12/12)	[81.60
%;
100%]	100%
(12/12)	[81.60
%;
100%]	100%
(12/12)	[81.60
%;
100%]	100%
(36/36)	[93.01
%;
100%]
T24
HN	High	100%
(12/12)	[81.60
%;
100%]	100%
(12/12)	[81.60
%;
100%]	100%
(12/12)	[81.60
%;
100%]	100%
(36/36)	[93.01
%;
100%]
	Low	100%
(12/12)	[81.60
%;
100%]	100%
(12/12)	[81.60
%;
100%]	100%
(12/12)	[81.60
%;
100%]	100%
(36/36)	[93.
01%
100
%]
Cml.
HN	High	91.7%
(11/12)	[69.88
%;
98.12%
]	100%
(12/12)	[81.60
%;
100
%]	100%
(12/12)	[81.60
%;
100
%]	97.2%
(35/36)	[88.
46%
;
99.3
8%]
	Low	83.3%
(10/12)	[60.08
%;
94.32%
]	100%
(12/12)	[81.60
%;
100
%]	100%
(12/12)	[81.60
%;
100
%]	94.4%
(34/36)	[84.
53%
;
98.1
4%]
All Negatives		95.8%
(69/72)	[90.02
%;
95.83%
]	100%
(72/72)	[96.
38%
;
100
%]	100%
(72/72)	[96.
38%
;
100
%]	98.6%
(213/216)	[96.
58%
;
99.4
4%]
Positiv
e
Sampl
es	DN
A
Inp
ut	PPA		PPA		PPA	PPA
		%
(n/N)	95% CI	%
(n/N)	95% CI	%
(n/N)	95% CI	%
(n/N)	95% CI
T24 LP	High	100%
(12/12)	[81.60
%;
100%]	100%
(12/12)	[81.
60%
100%]	100%
(12/12)	[81.
60%
100%]	100%
(36/36)	[93.
01%
;

13


	Low	100%
(12/12)	[81.60%;
100%]	100%
(12/12)	[81.60%;
100%]	100%
(12/12)	[81.60%;
100%]	100%
(36/36)	[93.01%;
100%]
T24
HP	High	100%
(12/12)	[81.60%;
100%]	100%
(12/12)	[81.60%;
100%]	100%
(12/12)	[81.60%;
100%]	100%
(36/36)	[93.01%;
100%]
	Low	100%
(12/12)	[81.60%;
100%]	100%
(12/12)	[81.60%;
100%]	100%
(12/12)	[81.60%;
100%]	100%
(36/36)	[93.01%;
100%]
Cml.
LP	High	100%
(12/12)	[81.60%;
100%]	100%
(12/12)	[81.60%;
100%]	100%
(12/12)	[81.60%;
100%]	100%
(36/36)	[93.01%;
100%]
	Low	100%
(12/12)	[81.60%;
100%]	100%
(12/12)	[81.60%;
100%]	100%
(12/12)	[81.60%;
100%]	100%
(36/36)	[93.01%;
100%]
All Positives		100%
(72/72)	[96.38%;
100%]	100%
(72/72)	[96.38%;
100%]	100%
(72/72)	[96.38%;
100%]	100%
(216/216)	[98.76%;
100%]

All Samples		Agreement %
(n/N)	95% CI	Agreement %
(n/N)	95% CI	Agreement %
(n/N)	95% CI	Agreement %
(n/N)	95% CI
Overall		97.9%
(141/144)	[97.92%;
99.16%]	100%
(144/144)	[98.16%;
100%]	100%
(144/144)	[98.16%;
100%]	99.3%
(429/432)	[98.28%;
99.72%]

An additional laboratory-to-laboratory reproducibility study, using clinical samples, was performed at 3 laboratories to determine precision between laboratories. The four (4) clinical samples used represent Low Negative (LN; ES ~~20), High Negative (HN; ES~~45), Low Positive (HP; ES~~75), and High Positive (HP; ES~~90) at 2 DNA input levels. One operator ran the test at each laboratory location on 3 different, non-consecutive workdays with an interval of at least 3 days between runs. A single reagent lot was used for this study. Overall performance characteristics (agreement, PPA, and NPA) are described in Tables 8.1.4 and 8.1.5 below. There were no control (plate) failures (0/18).

Table 8.1.4 Interlaboratory Reproducibility per Sample Presented as Overall and by Day

	Bladder EpiCheck Result
	Laboratory	Overall

14

			1	2
			NPA	NPA	NPA	NPA
Negative
Sample	DNA
Input	%
(n/N	90%
CI	0/0
(n/N)	90%
CI	0/0
(n/N)	90%
CI	0/0
(n/N)	90%
CI
LN	High	100%
(6/6)	[78.51
%;
100%]	100%
(6/6)	[78.51
%;
100%]	100%
(6/6)	[78.51
%;
100%]	100%
(18/18)	[91.64
%;
100%]
	Low	100%
(6/6)	[78.51
%;
100%]	100%
(6/6)	178.51
%;
100%]	100%
(6/6)	178.51
%:
100%	100%
(18/18)	[91.64
%;
100%]
HN	High	100%
(6/6)	[78.51
%;
100%]	83.3%
(5/6)	[57.47
%;
94.87
%]	83.3%
(5/6)	[57.47
%:
94.87
%]	88.9%
(16/18)	175.99
%:
95.29
%]
	Low	66.7
%
(4/6)	[40.94
%;
85.23
%]	100%
(6/6)	178.51
%;
100%]	83.3%
(5/6)	[57.47
%;
94.87
%]	83.3%
(15/18)	[69.42
%:
91.68
%]
All Negatives		91.7
%
(22/2

                                                                                                                                                                     | 175.1

%:
98.2% | 95.8%
(23/24) | [80.4
%:
99.7%

| 91.7%
(22/24) | [75.1
%;
98.2% | 93.1%
(67/72) | 88.2
%;
96.0% |
| | | PPA | | PPA | | PPA | | PPA | |
| Positive
Sample | DNA
Input | %
n/N | 90%
CI | 0/0
(n/N) | 90%
CI | 0/0
(n/N) | 90%
CI | 0/0
(n/N) | 90%
CI |
| LP | High | 100%
(6/6) | [78.51
%;
100%] | 100%
(6/6) | [78.51
%;
100%] | 100%
(6/6) | [78.51
%;
100% | 100%
(18/18) | [91.64
%;
100%1 |
| | Low | 100%
(6/6) | [78.51
%;
100%] | 100%
(6/6) | [78.51
%;
100%] | 100%
(6/6) | 178.51
%;
100%] | 100%
(18/18) | [91.64
%:
100%] |
| Hb | High | 100%
(6/6) | [78.51
%;
100%] | 100%
(6/6) | [78.51
%;
100%] | 100%
(6/6) | [78.51
%:
100%] | 100%
(18/18) | [91.64
%:
100%] |
| | Low | 100%
(6/6) | [78.51
%;
100%] | 100%
(6/6) | [78.51
%;
100%] | 100%
(6/6) | [78.51
%:
100% | 100%
(18/18) | [91.64
%:
100%1 |
| All Positives | | 100%
(24/2

                                                                                                                                                                          | 186.4

%;
100% | 100%
(24/24) | 186.4
%;
100% | 100%
(24/24) | [86.4
%:
100% | 100%
(72/72) | [97.8
%;
100%7 |
| | | | Agreement | Agreement | | Agreement | | Agreement | |
| All Samples | | %
(n/N
) | 90%
CI | %
(n/N) | 90%
CI | %
(n/N) | 90%
CI | %
(n/N) | 90%
CI |
| Overall | | તે જેવી જેવી જેવી સવલતો પ્રાપ્ય થયેલી છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામ
%
(46/4
8) | [90.4
%;
98.3% | 97.9%
(47/48) | [93.3
%;
99.4%
1 | 95.8%
(46/48) | [90.4
%;
98.3% | 96.5%
(139/144) | [94.0
%;
98.0% |

Table 8.1.5 Interlaboratory Reproducibility per Sample Presented as Overall and by Day

Bladder EpiCheck Result
	Timepoint (Day)
1	2	3	Overall

15

Negative	DNA Input	NPA		NPA		NPA		NPA		Invalid Rate
Sample		% (n/N)	90% CI	% (n/N)	90% CI	% (n/N)	90% CI	% (n/N)	90% CI	% (n/N)	90% CI
LN	High	100% (6/6)	[78.51%; 100%]	100% (6/6)	[78.51%; 100%]	100% (6/6)	[78.51%; 100%]	100% (18/18)	[91.64%; 100%]	0	--
	Low	100% (6/6)	[78.51%; 100%]	100% (6/6)	[78.51%; 100%]	100% (6/6)	[78.51%; 100%]	100% (18/18)	[91.64%; 100%]
HN	High	100% (6/6)	[78.51%; 100%]	66.7% (4/6)	[40.94%; 85.23%]	100% (6/6)	[78.51%; 100%]	88.9% (16/18)	[75.99%; 95.29%]	5.3% (2/38)	[2.2%; 12.0%]
	Low	100% (4/4)	[70.89%; 100%]	75% (6/8)	[52.37%; 89.11%]	83.3% (5/6)	[57.47%; 94.87%]	83.3% (15/18)	[69.42%; 91.68%]
All Negatives		100% (22/22)	[85.3%; 100%]	84.6% (22/26)	[67.6%; 94.1%]	95.8% (23/24)	[80.4%; 99.7%]	93.1% (67/72)	[88.2%; 96.0%]	2.7% (2/74)	[1.1%; 6.3%]
Positive	DNA Input	PPA		PPA		PPA		PPA		Invalid Rate
Sample		% (n/N)	90% CI	% (n/N)	90% CI	% (n/N)	90% CI	% (n/N)	90% CI	% (n/N)	90% CI
LP	High	100% (6/6)	[78.51%; 100%]	100% (6/6)	[78.51%; 100%]	100% (6/6)	[78.51%; 100%]	100% (18/18)	[91.64%; 100%]	0	--
	Low	100% (6/6)	[78.51%; 100%]	100% (6/6)	[78.51%; 100%]	100% (6/6)	[78.51%; 100%]	100% (18/18)	[91.64%; 100%]
HP	High	100% (6/6)	[78.51%; 100%]	100% (6/6)	[78.51%; 100%]	100% (6/6)	[78.51%; 100%]	100% (18/18)	[91.64%; 100%]	2.7% (1/37)	[0.8%; 8.6%]
	Low	100% (6/6)	[78.51%; 100%]	100% (5/5)	[75.27%; 100%]	100% (7/7)	[81.00%; 100%]	100% (18/18)	[91.64%; 100%]
All Positives		100% (24/24)	[86.4%; 100%]	100% (23/23)	[85.9%; 100%]	100% (25/25)	[86.9%; 100%]	100% (72/72)	[97.8%; 100%]	1.4% (1/73)	[0.4%; 4.5%]
All Samples			Agreement		Agreement		Agreement		Invalid Rate
		% (n/N)	90% CI	% (n/N)	90% CI	% (n/N)	90% CI	% (n/N)	90% CI	% (n/N)	90% CI
Overall		100%	[96.6%;	91.8%	[85.4%;	98.0% (48/49)	[93.4%;	96.5% (139/144)	[94.0%; 98.0%]	2.0% (3/14)	[1.0%;

16

				00
40/	100	. J/G	ﺮ
1
ﻟﻠﻘﻀﺎﺀ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮ	1.7		4.1
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
11
46	0/01	Al	0/01	%1	%1

Operator-to-Operator/Day-to-Day

An operator-to-operator and day-to-day repeatability and reproducibility study was performed to assess the within-run, between-run, and between-operator variation of Bladder EpiCheck when performed by different operators over 5 non-consecutive days. Healthy donor urine pools were spiked with bladder cancer-derived cell line material at different input levels to formulate 4 test samples spanning the measurable range of EpiScore values. Overall performance characteristics (agreement, PPA, and NPA) are summarized in Tables 8.1.6, 8.1.7, and 8.1.8 below. There were no invalid results and 1 control (plate) failure out of 21 plates (1/21 = 4.8% [95% CI 1.1%; 18.8%].

| Sample
Category | Bladder EpiCheck Result
Timepoint (Day) | | | | | Overall |
|--------------------|-------------------------------------------------|-------------------------------------------------|-------------------------------------------------|-------------------------------------------------|-------------------------------------------------|-----------------------------------------------------|
| | 1 | 2 | 3 | 4 | 5 | |
| Negative
Sample | NPA | NPA | NPA | NPA | NPA | NPA |
| | % (n/ N) CI | % (n/ N) CI | % (n/ N) CI | % (n/ N) CI | % (n/ N) CI | % (n/N ) 95% CI |
| LN | 100
%
[78.7%;
(10/
10)
100%] | 100
%
[78.7%;
(10/
10)
100%] | 100
%
[78.7%;
(10/
10)
100%] | 100
%
[78.7%;
(10/
10)
100%] | 100
%
[78.7%;
(10/
10)
100%] | 100
%
(50/5
0)
[94.9%;
100%] |
| HN | 100
%
[78.7%;
(10/
10)
100%] | 100
%
[78.7%;
(10/
10)
100%] | 90
%
[59.5%;
(9/10)
99.3%] | 100
%
[78.7%;
(10/
10)
100%] | 100
%
[78.7%;
(10/
10)
100%] | 98%
(49/5
0)
[91.5%;
99.6%] |
| All
Negatives | 100
%
[83.9%;
(20/
20)
100%] | 100
%
[83.9%;
(20/
20)
100%] | 95
%
[76.4%;
(19/
20)
99.1%] | 100
%
[83.9%;
(20/
20)
100%] | 100
%
[83.9%;
(20/
20)
100%] | 99%
(99/1
00)
[94.6%;
99.8%] |
| Positive
Sample | PPA | PPA | PPA | PPA | PPA | PPA |
| | % (n/ N) CI | % (n/ N) CI | % (n/ N) CI | % (n/ N) CI | % (n/ N) CI | % (n/N ) 95% CI |
| LP | 100
%
[78.7%;
(10/
10)
100%] | 100
%
[78.7%;
(10/
10)
100%] | 100
%
[78.7%;
(10/
10)
100%] | 100
%
[78.7%;
(10/
10)
100%] | 90
%
[59.5%;
(9/10)
99.3%] | 98%
(49/5
0)
[91.5%;
99.6%] |
| HP | 100
%
[78.7%;
(10/
10)
100%] | 100
%
[78.7%;
(10/
10)
100%] | 100
%
[78.7%;
(10/
10)
100%] | 100
%
[78.7%;
(10/
10)
100%] | 100
%
[78.7%;
(10/
10)
100%] | 100
%
(50/5
0)
[94.9%;
100%] |
| All
Positives | 100
%
[83.9%;
(20/
20)
100%] | 100
%
[83.9%;
(20/
20)
100%] | 100
%
[83.9%;
(20/
20)
100%] | 100
%
[83.9%;
(20/
20)
100%] | 95
%
[76.4%;
(19/
20)
99.1%] | 99%
(99/1
00)
[94.6%;
99.8%] |

Table 8.1.6 Within Site Precision per Sample Presented as Overall and by Day (5 Days)

17

	20)	%]

		Agreeme
nt		Agreeme
nt		Agreeme
nt		Agreeme
nt		Agreeme
nt		Agreement
All
Samples
	%
(n/
N)	95 % CI	%
(n/
N)	95 % CI	%
(n/
N)	95 % CI	%
(n/
N)	95 % CI	%
(n/
N)	95 % CI	%
(n/N)	95% CI
Overall	100 %
(40/

| [91.2%;
100 %] | 100 %
(40/
| [91.2%;
100 %] | 97.5%
(39/
| [87.1%;
99.6 %] | 100 %
(40/
| [91.2%;
100 %] | 97.5%
(39/
| [87.1%;
99.6 %] | 99%
(198/
| [96.4%;
99.7%] |

Table 8.1.7 Within Site Precision per Sample Presented as - Overall and by Operator 1 per Day (5 Days)

| Sample
Category | Timepoint
t 1 | Timepoint
t 2 | Timepoint
t 3 | Timepoint
t 4 | Timepoint
t 5 | Overall | | | | |
|--------------------|----------------------------------|-----------------------------|----------------------------------|-----------------------------|----------------------------------|----------------------|----------------------------------|-----------------------|-------------------------|-------------------|
| Negative
Sample | NPA
%
(n/
N)
CI | NPA
%
(n/
N)
CI | NPA
%
(n/
N)
CI | NPA
%
(n/
N)
CI | NPA
%
(n/
N)
CI | NPA
%
(n/N) | 95% CI | | | |
| LN | 100
%
(5/5
)
100%] | [56.
6%;
100%] | 100
%
(5/5
)
100%] | [56.
6%;
100%] | 100
%
(5/5
)
100%] | [56.
6%;
100%] | 100
%
(5/5
)
100%] | [56.
6%;
100%] | 100%
(25/2
5) | [86.7%;
100%] |
| HN | 100
%
(5/5
)
100%] | [56.
6%;
100%] | 100
%
(5/5
)
100%] | [56.
6%;
100%] | 100
%
(5/5
)
100%] | [56.
6%;
100%] | 100
%
(5/5
)
100%] | [56.
6%;
100%] | 100%
(25/2
5) | [86.7%;
100%] |
| All
Negatives | 100
%
(10/
10)
100%] | [78.
7%;
100%] | 100
%
(10/
10)
100%] | [78.
7%;
100%] | 100
%
(10/
10)
100%] | [78.
7%;
100%] | 100
%
(10/
10)
100%] | [78.
7%;
100%] | 100%
(50/5
0) | [94.9%;
100%] |
| Positive
Sample | PPA
%
(n/
N)
CI | PPA
%
(n/
N)
CI | PPA
%
(n/
N)
CI | PPA
%
(n/
N)
CI | PPA
%
(n/
N)
CI | PPA
%
(n/N) | 95% CI | | | |
| LP | 100
%
(5/5
)
100%] | [56.
6%;
100%] | 100
%
(5/5
)
100%] | [56.
6%;
100%] | 100
%
(5/5
)
100%] | [56.
6%;
100%] | 80
%
(4/5
)
100%] | [37.
6%;
95.4%] | 96%
(24/
25) | [80.5%;
99.7%] |
| HP | 100
%
(5/5
)
100%] | [56.
6%;
100%] | 100
%
(5/5
)
100%] | [56.
6%;
100%] | 100
%
(5/5
)
100%] | [56.
6%;
100%] | 100
%
(5/5
)
100%] | [56.
6%;
100%] | 100
%
(25/
25) | [86.7%;
100%] |
| All
Positives | 100
%
(10/
10)
100%] | [78.
7%;
100%] | 100
%
(10/
10)
100%] | [78.
7%;
100%] | 100
%
(10/
10)
100%] | [78.
7%;
100%] | 90
%
(9/1
0)
100%] | [59.
5%;
99.3%] | 98%
(49/
50) | [91.5%;
99.6%] |

18

| All
Samples | Agreeme
nt
%
(n/
N) | Agreeme
nt
95% CI | Agreeme
nt
%
(n/
N) | Agreeme
nt
95% CI | Agreeme
nt
%
(n/
N) | Agreeme
nt
95% CI | Agreeme
nt
%
(n/
N) | Agreeme
nt
95% CI | Agreeme
nt
%
(n/
N) | Agreeme
nt
95% CI | Agreement
%
(n/
N) | Agreement
95% CI |
|----------------|---------------------------------|-------------------------|---------------------------------|-------------------------|---------------------------------|-------------------------|---------------------------------|-------------------------|---------------------------------|-------------------------|-----------------------------|---------------------|
| Overall | 100
%
(20/
20) | [83.9%;
100%] | 100
%
(20/
20) | [83.9%;
100%] | 100
%
(20/
20) | [83.9%;
100%] | 100
%
(20/
20) | [83.9%;
100%] | 95
%
(19/
20) | [76.4%;
99.1%] | 99%
(99/
100) | [94.6%;
99.8%] |

Table 8.1.8 Within Site Precision per Sample Presented as - Overall and by Operator 2 per Day (5 Days)

| Sample

Category	Operator 2					Overall
	Timepoint
t 1	Timepoint
t 2	Timepoint
t 3	Timepoint
t 4	Timepoint
t 5
Negative
Sample	NPA					NPA
	95%
(n/
N)	95%
(n/
N)	95%
(n/
N)	95%
(n/
N)	95%
(n/
N)	95% CI
(n/N)
LN	100%
[56.6%;
(5/5)
100%]	100%
[56.6%;
(5/5)
100%]	100%
[56.6%;
(5/5)
100%]	100%
[56.6%;
(5/5)
100%]	100%
[56.6%;
(5/5)
100%]	100%
[86.7%;
(25/25)
100%]
HN	100%
[56.6%;
(5/5)
100%]	100%
[56.6%;
(5/5)
100%]	80%
[37.6%;
(4/5)
95.4%]	100%
[56.6%;
(5/5)
100%]	100%
[56.6%;
(5/5)
100%]	96%
[80.5%;
(24/25)
99.7%]
All
Negatives	100%
[78.7%;
(10/10)
100%]	100%
[78.7%;
(10/10)
100%]	90%
[59.5%;
(9/10)
99.3%]	100%
[78.7%;
(10/10)
100%]	100%
[78.7%;
(10/10)
100%]	98%
[91.5%;
(49/50)
99.6%]
Positive
Sample	PPA					PPA
	95%
(n/
N)	95%
(n/
N)	95%
(n/
N)	95%
(n/
N)	95%
(n/
N)	95% CI
(n/N)
LP	100%
[56.6%;
(5/5)
100%]	100%
[56.6%;
(5/5)
100%]	100%
[56.6%;
(5/5)
100%]	100%
[56.6%;
(5/5)
100%]	100%
[56.6%;
(5/5)
100%]	100%
[86.7%;
(25/25)
100%]
HP	100%
[56.6%;
(5/5)
100%]	100%
[56.6%;
(5/5)
100%]	100%
[56.6%;
(5/5)
100%]	100%
[56.6%;
(5/5)
100%]	100%
[56.6%;
(5/5)
100%]	100%
[86.7%;
(25/25)
100%]
All
Positives	100%
[78.7%;
(10/10)
100%]	100%
[78.7%;
(10/10)
100%]	100%
[78.7%;
(10/10)
100%]	100%
[78.7%;
(10/10)
100%]	100%
[78.7%;
(10/10)
100%]	100%
[94.9%;
(50/50)
100%]
All
Samples	Agreement					Agreement
	95% CI	95% CI	95% CI	95% CI	95% CI	95% CI

19

| | (n/
N) | %
CI | (n/
N) | %
CI | (n/
N) | %
CI | (n/
N) | %
CI | (n/
N) | %
CI | (n/
N) | |
|---------|-----------------|------------------|-----------------|------------------|----------------|-------------------|-----------------|------------------|-----------------|------------------|-----------------|-------------------|
| | 100%
(20/20) | [83.9%;
100%] | 100%
(20/20) | [83.9%;
100%] | 95%
(19/20) | [76.4%;
99.1%] | 100%
(20/20) | [83.9%;
100%] | 100%
(20/20) | [83.9%;
100%] | 99%
(99/100) | [94.6%;
99.8%] |
| Overall | | | | | | | | | | | | |

Lot-to-Lot/Instrument-to-Instrument

A lot-to-lot and instrument-to-instrument repeatability and reproducibility study was performed to assess the variability of Bladder EpiCheck when performed using 3 unique kit lots and 3 unique PCR instruments. Healthy donor urine pools were spiked with bladder cancer-derived cell line material at different DNA concentration levels to create 4 test samples spanning the range of EpiScore values. A minimum of 70 replicates were tested for each sample type. Overall agreement is summarized in Table 8.1.9 below.

Table 8.1.9 Within Site Precision - Overall and by Lot. Instrument, and EpiScore Value

Condition	% (n/N)	Wilson Score 95% CI
Overall	100.0% (294/294)	99.09%; 100.0%
Lot
1	100.0% (175/175)	98.48%; 100.0%
2	100.0% (59/59)	95.62%; 100.0%
3	100.0% (60/60)	95.69%; 100.0%
Instrument
1	100.0% (214/214)	98.75%; 100.0%
2	100.0% (40/40)	93.66%; 100.0%
3	100.0% (40/40)	93.66%; 100.0%
EpiScore Value
Low Negative	100.0% (74/74)	96.47%; 100.0%
High Negative	100.0% (75/75)	96.52%; 100.0%
Low Positive	100.0% (70/70)	96.28%; 100.0%
High Positive	100.0% (75/75)	96.52%; 100.0%

Analytical Sensitivity 8.2

Limit of Blank (LoB)

A Limit of Blank study was conducted to determine the residual levels of nonspecific background signal in the absence of template DNA. Sixty (60) replicates of non-template control (without DNA) were tested across 2 reagent lots - a total of 120 test results. Mean Cq results were 44.75 and 44.86 for Lots 1 and 2, respectively. Using a non-parametric approach, the resulting LoB was determined to be a Cq value equal to 45.

Functional Limit of Detection (fLoD)

The functional Limit of Detection (fLoD) refers to the lowest total DNA that is

20

required for the assay and was determined by testing DNA extracted from positive clinical specimens mixed with DNA extracted from pooled healthy individuals (negative clinical specimens) to create a clinical specimen with an EpiScore near the clinical cutoff (Low Positive). This sample was tested across a range of 5 concentrations (two-fold dilutions) around and below the assay cut-off (1.2 ng/well, 0.65 ng/well, 0.30 ng/well, 0.15 ng/well, and 0.075 ng/well). A blank sample (0 ng/well) was also tested. Each concentration was tested in replicates of 30 using one lot of reagents and one instrument - a total of 330 test measurements. The fLoD was determined from a PROBIT regression model as the concentration at which, with 95% probability and 90% CI, the measurement result yielded a "positive" classification. Hit Rate and PROBIT results are summarized in Tables 8.2.1 and 8.2.2, respectively. Using this approach, the estimated fLoD was determined to be 0.186 ng/well (2.23 ng/sample).

DNA Conc	Detection	Hit Rate	90% Confidence Interval
(ng/PCR well)	(n/N)	%	Lower	Upper
0.84	30/30	100	94.8%	100%
0.42	30/30	100	94.8%	100%
0.27	30/30	100	94.8%	100%
0.15	26/30	86.7	76.8%	92.7%
0.075	17/30	56.7	45.0%	67.6%

	Table 8.2.1 Hit Rate
--	----------------------	--	--	--

Table 8.2.2 Probability Estimated from the PROBIT Model
---------------------------------------------------------

| Probability | Sample Type | Concentration
(ng/well) | 90% Confidence Intervals | |
|-------------|-------------|----------------------------|--------------------------|-------|
| | | | Lower | Upper |
| 0.95 | Clinical | 0.186 | 0.88 | 0.98 |
| 0.95 | Contrived | 0.202 | 0.89 | 0.98 |
| 0.75 | Clinical | 0.104 | 0.65 | 0.83 |
| 0.75 | Contrived | 0.081 | 0.63 | 0.85 |
| 0.5 | Clinical | 0.070 | 0.35 | 0.65 |
| 0.5 | Contrived | 0.043 | 0.28 | 0.72 |
| 0.25 | Clinical | 0.047 | 0.10 | 0.47 |
| 0.25 | Contrived | 0.023 | 0.06 | 0.59 |

Tumor Limit of Detection (tLoD)

The Tumor Limit of Detection (tLoD) refers to the minimum amount of tumor DNA in the lowest total volume of DNA that can be detected and was determined by testing DNA extracted from bladder cancer positive patient specimens and mixing with DNA extracted from healthy urine donor samples in a ratio gradient of 7 positive DNA spike-in levels (30%, 20%, 15%, 12%, 8%, and 4%). These samples were tested using 2 reagent lots in 30 replicates total. Samples with 100%, 50% and 0% positive DNA spike-ins were also tested, but only in 10 replicates total. The total DNA concentration was kept constant at 1-2x fLoD. The tLoD was calculated from a PROBIT regression model as the concentration at which with 95% probability the measurement result yields a "positive" classification. Hit rate and PROBIT estimation results are summarized in Tables

21

8.2.3 and 8.2.4, respectively. Based on this model, the tLoD was determined to be 7.5%, which equates to a minimum amount of tumor DNA in a clinical sample of ~0.17 ng.

Tumor DNA Fraction	Hit Rate	Hit Rate	90% Confidence Interval
(%)	(n/N)	(%)	Lower	Upper
100	10/10	100	85.9%	100%
50	10/10	100	85.9%	100%
30	30/30	100	94.8%	100%
20	30/30	100	94.8%	100%
15	30/30	100	94.8%	100%
12	30/30	100	94.8%	100%
8	29/30	96.7	89.5%	99.0%
4	11/30	36.7	26.4%	48.4%
0	0/10	0	0%	14.1%

Table 8.2.3 Hit Rate

Table 8.2.4 Probability Estimated from the PROBIT Model

| Probability | Sample Type | Tumor DNA Fraction
(%) | 90% Confidence Limits | |
|-------------|-------------|---------------------------|-----------------------|-------|
| | | | Lower | Upper |
| 0.95 | Clinical | 7.5 | 85.0% | 99.0% |
| | Contrived | 12.7 | 89.0% | 98.0% |

Methylation Limit of Detection (mLoD)

The Methylation Limit of Detection (mLoD) refers to the minimal detectable level of methylation across the assay's target loci and was determined using a mixture of methylated and unmethylated artificial plasma DNA to create a gradient of different levels of methylation. This study took place across 2 phases, performed using contrived DNA samples.

In Study 1 (Phase I), a preliminary mLoD was established using a single PCR mix (Mix A, which consisted of biomarkers BE-1 and BE-2), as it represented the lower end of the sensitivity range of the assay. A gradient of 10 admixtures (10%, 5%, 2.5%, 1%, 0.5%, 0.25%, 0.1%, 0.05%, 0.025%, and 0.01%), along with a 0% methylation sample, were tested using one reagent lot and between 16 and 60 replicates, depending on the methylation level - a total of 378 total measurements. Undigested 100% methylated UC was also run as a positive control. Hit rate and PROBIT results for both biomarkers are summarized in Tables 8.2.5 and 8.2.6, respectively. Based on interpolation from the PROBIT estimation, the higher of the two mLoD values was used as the preliminary mLoD: 0.348%.

In Study 2 (Phase II), the preliminary mLoD value determined in Phase I was confirmed for the other 13 biomarkers in the assay using a subset of the admixture gradients used in Phase I (3 methylation levels around the preliminary LoD: 0.05%, 0.1%, and 0.25%) and tested using one reagent lot and 60 replicates per methylation level. Undigested 100% methylated UC was also run as a positive

22

control. Hit rate results for the other 13 biomarkers are summarized in Table 8.2.7 below. Based on the results, biomarkers BE-4 through BE-15 had hit rates of 100.0% at values below the preliminary mLoD. The one exception, BE-3, underwent a follow-up root analysis, and preparation of mix B (containing biomarkers BE-3 and BE-4) was repeated across the entire range of tested ratios from Phase II. Results from the repeat preparation showed acceptable hit rates below the preliminary mLoD.

Marker	Methylation (%)	Hit Rate % (n/N)	Upper One-Sided 95% CL
BE-1	0.01	10.00% (3/30)	25.62%
	0.025	10.00% (3/30)	25.62%
	0.05	6.67% (4/60)	15.93%
	0.1	40.00% (24/60)	52.63%
	0.25	65.00% (39/60)	75.83%
	0.5	100.0% (30/30)	100.0%
	1	100.0% (30/30)	100.0%
	2.5	100.0% (16/16)	100.0%
	5	100.0% (16/16)	100.0%
	10	100.0% (16/16)	100.0%
BE-2	0.01	10.00% (3/30)	25.62%
	0.025	76.67% (23/30)	88.21%
	0.05	78.33% (47/60)	86.88%
	0.1	98.33% (59/60)	99.71%
	0.25	98.33% (59/60)	99.71%
	0.5	100.0% (30/30)	100.0%
	1	100.0% (30/30)	100.0%
	2.5	100.0% (16/16)	100.0%
	5	100.0% (16/16)	100.0%
	10	100.0% (16/16)	100.0%

Table 8.2.5 Hit Rate with Upper One-Sided 95% Confidence Interval – per Marker and Methylation % (Phase I)

Table 8.2.6 Probability Estimated from PROBIT Model

| Marker | Probability | Methylation
% | 95% Fiducial
Limits |
|--------|-------------|------------------|------------------------|
| BE-1 | 0.90 | 0.34872 | 0.30102; 0.42287 |
| BE-2 | 0.90 | 0.06681 | 0.05337: 0.09068 |

Table 8.2.7 Hit Rate with Upper One-Sided 95% Confidence Limit - Per Marker and per Methylation % (Phase II)

Marker	Methylation %	Hit Rate % (n/N)	Upper One-Sided 95% CL
BE-3	0.05	39.17% (47/120)	46.66%
BE-3	0.1	39.17% (47/120)	46.66%
BE-3	0.25	56.90% (66/116)	64.22%
BE-4	0.05	53.33% (32/60)	63.55%
BE-4	0.1	78.33% (47/60)	85.76%

23

	0.25	98.21% (55/56)	99.60%
BE-5	0.05	35.00% (21/60)	45.58%
	0.1	80.00% (48/60)	87.11%
	0.25	96.43% (54/56)	98.81%
BE-6	0.05	47.50% (57/120)	54.97%
	0.1	75.83% (91/120)	81.65%
	0.25	92.24%
(107/116)	95.43%
BE-7	0.05	50.00% (30/60)	60.39%
	0.1	70.00% (42/60)	78.69%
	0.25	98.21% (55/56)	99.60%
BE-8	0.05	46.67% (28/60)	57.17%
	0.1	70.00% (42/60)	78.69%
	0.25	94.44% (51/54)	97.76%
BE-9	0.05	10.00% (6/60)	18.19%
	0.1	36.67% (22/60)	47.27%
	0.25	91.07% (51/56)	95.59%
BE-
10	0.05	26.67% (16/60)	36.91%
	0.1	41.67% (25/60)	52.27%
	0.25	87.50% (49/56)	93.08%
BE-
11	0.05	71.67% (41/60)	80.14%
	0.1	91.67% (55/60)	95.86%
	0.25	100.0% (55/55)	100.0%
BE-
12	0.05	48.33% (29/60)	58.79%
	0.1	88.33% (53/60)	93.55%
	0.25	100.0% (56/56)	100.0%
BE-
13	0.05	65.00% (39/60)	74.28%
	0.1	93.33% (56/60)	96.97%
	0.25	100.0% (56/56)	100.0%
BE-
14	0.05	50.85% (30/59)	61.28%
	0.1	73.33% (44/60)	81.57%
	0.25	100.0% (56/56)	100.0%
BE-
15	0.05	48.28% (28/58)	58.90%
	0.1	81.67% (49/60)	88.45%
	0.25	96.43% (54/56)	98.81%

8.3 Analytical Specificity

Digestion Restriction Efficiency

Digestion restriction efficiency was established by evaluating the digestion of all 15 Bladder EpiCheck markers using 100% unmethylated synthetic (plasmid) DNA tested across a series of dilution levels for a total of 30 replicates based on the relationship of the ΔCq between the non-digestible IR marker (100% copies in the reaction) and digested targets (BE markers 1-15). Digestion efficiency for all 15 markers was shown to exceed 99.9%, with an average efficiency of 99.997%.

An additional study was performed to assess the impact of hemi-methylated

24

molecules have on the enzymatic digestion step, as well as the contamination risk, in the Bladder EpiCheck assay protocol performed using synthetic DNA molecules of a representative locus (BE-14) containing all different permutations of the targeted cut site. Results showed that hemi-methylated DNA does represent a digestion efficiency profile similar to unmethylated DNA; however, at best, this reflects a very small sub-population of target molecules, so the effect should be negligible.

Digestion Restriction Efficiency - Exclusivity

Digestion restriction efficiency (Exclusivity) was established by evaluating the digestion of nonmethylated markers to avoid false positive results using a commercially available hypo-methylated human genomic DNA sample (99.99% for all 15 biomarkers, thus further ensuring that no non-specific signal is generated in the qPCR step.

Primer Probe Specificity

In-Silico PCR analysis for each locus (Informative markers BE-1 to BE-15 and the Internal Reference (IR)) obtained a single hit using the forward and reverse primer sequences. Additionally, confirmatory PCR was performed using each primer pair, followed by gel electrophoresis. This showed only one product amplified per primer pair, this confirming the in-silico analysis.

8.4 Robustness (Guardbanding)

Robustness of the Bladder EpiCheck was evaluated using 4 contrived specimens (T24 cell line DNA spiked into DNA extracted from healthy volunteer urine pools) representing the full range of EpiScore values (LN, HN, LP, and HP). Each sample was run in quintuplicates across a series of 13 conditions with variations to PCR denaturation and annealing temperatures, digestion temperature, and/or digestion time. Overall agreement was shown to be 98.5% [96.77%; 99.31%].

A supplementary study was conducted to further demonstrate the robustness of the Bladder EpiCheck assay using 4 clinical specimens (DNA extracted from clinical specimens adjusted using DNA extracted from healthy urine donor pools) that represent the full range of EpiScore values (LN, HN, LP, and HP). Each sample was tested in quintuplicates across 5 different assay conditions

25

(including the control condition). Overall agreement was shown to be 99.3% [96.9%; 99.8%], thus confirming the robustness of the assay.

8.5 Interference

Performance of Bladder EpiCheck was evaluated with 27 potentially interfering substances (including common urine constituents, microbial contaminants, therapeutic agents, and laboratory preservatives), which were tested across 3 sample pools (2 negative pools from healthy individuals, and one positive pool of healthy urine spiked with bladder cancer cell-line material) at 2 concentrations, with each sample run in 3 replicates. Preliminary results suggested that uric acid and ampicillin were both potential interferents, so a dose response analysis was conducted, which showed that interference either did not repeat itself or was present at elevated levels that are not clinically meaningful. Therefore, no evidence of interference caused by the substances tested at clinically relevant physiological ranges exists.

8.6 Real-Time and In-Use Stability

In-use and real-time stability testing of 3 lots of the Bladder EpiCheck kit stored at the recommended storage condition of -20°C/2-8°C/25°C (depending on the reagents storage specifications) was conducted at six separate time points. Each lot was tested using 4 contrived urine pools representing the full range of EpiScore values (LN, HN, LP, and HP) tested in 7 replicates at each timepoint. For in-use stability assessment, during each time point, all kits from the same lot (n=3) underwent the same freeze/thaw and handling procedures. For real-time stability assessment, at each time point, "fresh" (unused) Extraction and test kits were used. Each "run" included the Undigested (UC) and Non-Template (NTC) Controls. For T=0, a total of 84 samples were tested (4 sample pools tested in each of 3 kit lots in replicates of 7. For T=1 through T=5. a total of 168 samples were tested (4 samples pools tested in each of 3 kit lots in replicates of 7 spanning both studies). Results showed that there was no significant change in performance when testing kits and controls up to 486 days.

8.7 Freeze-Thaw Stability

Freeze/Thaw stability testing of three lots of the Bladder EpiCheck Test kit stored at the recommended storage condition of -20°C was conducted at three separate test points (T = 1. 8 and 12 cycles). Each lot was tested using 4 contrived urine pools (T24 cell line DNA spiked into DNA extracted from healthy volunteer urine pools) representing the full range of EpiScore values (LN. HN, LP, and HP) with 5 replicates per sample. Results showed no significant performance changes and low variability in EpiScore value between the 3 timepoints. The stability claim of 10 cycles was then confirmed in a supplementary study using 4 clinical samples representing the full range of EpiScore values (LN, HN, LP, and HP) with 5 replicates per sample over 2 timepoints (1 cycle and 12 cycles).

8.8 Shipping Stability

12 Extraction Kit boxes and 12 Test kit boxes will be used to evaluate the packing

26

tolerance due to transportation (sudden changes in temperature for 24 hours, vibration, pressure, and drop tolerance). From the 12 boxes, two boxes will be used to evaluate the functionality of the kits (i.e., extracting DNA and prepare the DNA for the PCR analysis) and to test the performance of the kit (quantitative and qualitative results). In addition, a single control box (i.e., test kit that won't go through any transportation simulation) will be used as control. The performance of the kit will be tested on urine samples collected from 4 healthy and 4 Bladder Cancer (n=8) subjects. Each sample will be tested in five replicates by the kits underwent the transportation analysis (n=40) and in duplicate by the control kit (n=16). Overall, Positive, and Negative agreement were 100%.

8.9 Sample Stability

30 clinical specimens, collected from 15 bladder cancer patients and 15 normal donors across the full range of EpiScore values, were divided into 3 parts and used to evaluate the stability of fresh urine, urine pellet, and extracted DNA. Each sample was tested immediately and after storage. Each clinical sample was divided into different aliquots and tested at 3 timepoints depending on the specimen type (Fresh urine: 0, 5, and 6 days; Pelleted urine: 0, 14, and 21 days; DNA: 0, 30, and 60 days) after storage at the recommended conditions. Agreement results are shown in Tables 8.9.1, 8.9.2, and 8.9.3 for urine, pelleted urine, and DNA, respectively. Results confirm a stability claim of 5 days for fresh urine at 2-8°C, urine pellets for 19 days at -20°C, and DNA for 30 days at -20°C.

Timepoint	EpiCheck Result
	% (n/N)	Wilson Score 95% CI
Overall	99.01% (100/101)	95.68%; 99.78%
0 Days (T₀)	100% (34/34)	92.63%; 100%
5 Days (T₁)	100% (33/33)	92.42%; 100%
6 Days (T₂)	97.06% (33/34)	87.84%; 99.34%

Table 8.9.1 Bladder EpiCheck Results, Overall and by Timepoint - Fresh Urine

Table 8.9.2 Bladder EpiCheck Results, Overall and by Timepoint – Urine Pellet
	Overall	Baseline	3 Months	6 Months
Negative	95%	95%	95%	95%
Positive	5%	5%	5%	5%

Timepoint	EpiCheck Result
	% (n/N)	Wilson Score 95% CI
Overall	100.0% (90/90)	97.08%; 100.0%
0 Days (T₀)	100.0% (30/30)	91.73%; 100.0%
14 Days (T₁)	100.0% (30/30)	91.73%; 100.0%
21 Days (T₂)	100.0% (30/30)	91.73%; 100.0%

Table 8.9.3 Bladder EpiCheck Results, Overall and by Timepoint - Extracted DNA

Timepoint	EpiCheck Result
	% (n/N)	Wilson Score 95% CI
Overall	98.25% (112/114)	94.84%; 99.42%
0 Days (T₀)	100.0% (38/38)	93.35%; 100.0%
30 Days (T₁)	100.0% (38/38)	93.35%; 100.0%

27

60 Days (T2)	94.87% (36/38)	85.28%, 98.24%
--------------	----------------	----------------

8.10 DNA Extraction Efficiency

Forty-seven clinical urine specimens, collected from bladder cancer patients and healthy donors, were used for this study. Each specimen was pelleted, split into 10mL urine equivalents, and DNA was extracted from each split specimen using 4 Bladder EpiCheck Extraction lots. Performance of the Bladder EpiCheck kits was evaluated by invalid rate and Bladder EpiCheck test performance and tested in the Bladder EpiCheck test using one ABI 7500 Fast Dx Real-Time PCR system and analyzed using Bladder EpiCheck software (Ver. 1.9.19). A total of 1 sample failed after extraction, leading to an overall sample failure rate of 2.1%. Study results showed an overall, positive, and negative agreement of 100%, justifying that the Bladder EpiCheck DNA Extraction Kit met the extraction capabilities for use with the Bladder EpiCheck test.

9. Performance Testing (Clinical)

9.1 Clinical Cutoff (Training and Feasibility Data)

The assay cut-off and proprietary EpiScore algorithm for the Bladder EpiCheck software were validated on a set of urine samples collected from control patients with a history of bladder cancer and bladder cancer positive patients confirmed by cystoscopy and pathology. The total sample size for the software algorithm development included 178 samples which were collected in two separate sets. The first set of samples served for the cut-off definition. The set included 109 samples, 40 of which were collected from control patients with a history of bladder cancer, and 69 collected from Urothelial Cell Carcinoma (UCC) positive patients confirmed by pathology. The second samples set served for the validation of the cut-off. The set included 67 samples, of which 51 were collected from control patients with a history of bladder cancer, and 16 collected from UCC positive patients confirmed by pathology. The derived Bladder EpiCheck algorithm sensitivity and specificity compared to pathology outcome demonstrated a sensitivity of 94% for UCC recurrence and specificity of 84%.

9.2 Method Comparison

A multi-center, prospective, IRB-approved longitudinal study was conducted at 11 academic and urology specialty medical centers in the U.S. and Canada to establish the performance of Bladder EpiCheck as compared to the predicate device (UroVysion Bladder Cancer Recurrence Kit). Performance of the Bladder EpiCheck kit was also assessed to a gold standard (GS) using both clinical cytology and combined cystoscopy/pathology data.

A total of 674 subject were enrolled in the study. Eligible subjects were any male or female subject aged 22 and above, diagnosed with incident or recurrent UCC and undergoing surveillance for bladder cancer recurrence, who had urothelial cell carcinoma tumor resected within 12 months from baseline visit and had a plan for cystoscopy surveillance. The study included up to 3 visits: baseline and

28

up to 2 additional surveillance visits, where voided urine specimens were collected. In total, voided urine specimens were collected from 583 subjects. For subjects who experienced a recurrence (as determined by positive cytology /pathology), the first positive visit was used (i.e., the visit at which the diagnosis of recurrence was established). For the non-recurring subjects, the last negative visit was used for subjects with multiple surveillance visits.

DNA extracted from urine specimens was tested using the Bladder EpiCheck test and compared to the previously discussed gold standard (GS). Classification by the GS was as follows: a subject was considered "positive" if the interpretation for either cytology or the combined cystoscopy/pathology results were positive, and a subject was considered "negative" if both cytology and the combined cystoscopy/pathology results were negative. In total, valid Bladder EpiCheck and GS results were obtained from 449 subjects. The Bladder EpiCheck test performed with an accuracy of 78.8% ([74.8%; 82.4%]), a sensitivity of 66.7% ([58.4%; 74.0%]), and a specificity of 84.2% ([79.8%; 87.9%]). Further details of the performance characteristics of the Bladder EpiCheck test compared to the established GS is found in Table 9.2.1. Additionally, performance of the Bladder EpiCheck test was evaluated in several subgroup analyses, including cancer grade (High, Low, No grade) and stage (CIS, T1, T2, Ta). Performance results from these analyses were comparable to the overall performance characteristics of the Bladder EpiCheck test.

	Gold Standard Result
	Negative
N	Positive
N	Total GS
N
EpiCheck Result
Negative	262	46	308
Positive	49	92	141
Total EpiCheck	311	138	449
Accuracy	78.8% [74.8%; 82.4%] (354/449)
Sensitivity	66.7% [58.4%; 74.0%] (92/138)
Specificity	84.2% [79.8%; 87.9%] (262/311)
PPV	65.3% [57.1%; 72.6%] (92/141)
NPV	85.1% [80.7%; 88.6%] (262/308)

Table 9.2.1 Bladder EpiCheck Performance vs. Gold Standard

Performance of the Bladder EpiCheck test was also compared to the predicate device with respect to the GS sample definition. In total, valid Bladder EpiCheck, UroVysion, and GS results were obtained from 352 samples. Based on the results (Table 9.2.2), the Bladder EpiCheck was slightly more sensitive (+4.82%) and slightly less specific (-2.97%) than the predicate device, thus showing that the Bladder EpiCheck test was non-inferior to the predicate. An additional analysis was performed by cancer grade, which showed better sensitivity (+5.00%) and similar specificity (0.00%) between the two assays.

29

| Gold
Standard

Result	Bladder EpiCheck Result
	UroVysion Result		UroVysion Result		Total
	Negative	Positive	Negative	Positive	N
Negative	195	18	26	30	269
Positive	21	8	12	42	83
Total	216	26	38	72	352

Metric				% (n/N)	95% CI
Sensitivity	EpiCheck			65.1% (54/83)	54.8%; 75.3%
	Urovysion			60.2% (50/83)	49.7%; 70.8
		Difference		4.82%	-5.7%; 15.3%
Specificity		EpiCheck		79.2% (213/269)	74.3%; 84.0%
		Urovysion		82.2% (221/269)	77.6%; 86.7%
	Difference		-2.97%		-7.8%; 1.9%

Table 9.2.2. Bladder EpiCheck and UroVysion vs. Gold Standard - Matched Cases

9.3 Specificity in Urology Patients without Bladder Cancer

A multi-center, prospective study was conducted to establish the specificity of Bladder EpiCheck test in urology patients without prior history or clinical evidence of bladder cancer. The study population consisted of 147 subjects, aged 22 and above, visiting the urology clinic for any non-bladder cancer disorders. Subjects were excluded from the study if they had known current or prior diagnosis of both invasive and non-muscle invasive bladder cancer. The specificity of Bladder EpiCheck in this study population was 98.0%. Covariates analysis by sex, age group, ethnicity, race, smoking status, reason for visit at clinic, non-cancer genitourinary disease type, presence of hematuria, BPH, UTI, and STD were performed. No statistical significance was found, indicating that there was no impact of any of the tested covariates on the applicability of the primary analysis results on the population tested.

9.4 Clinical Specificity - Cross Reactivity with Other Cancers

The effect of potential cross-reactivity was assessed using banked remnant deidentified urine samples collected from patients diagnosed with different types of cancers, specifically cancers that are more likely to shed cancer cells into the bladder such as renal cell carcinoma (RCC) and prostate cancer as well as other solid and hematology cancer. 147 urine samples were assessed. The most frequent cancer category was genitourinary, with 54 subjects (37%) including prostate and renal cell carcinoma, followed by gastrointestinal with 26 subjects (18%); 15 subjects (10%) had gynecological cancers and 10 subjects had hematological cancers (7%). 42 subjects (29%) had other solid cancer types. The study population had a mean age of 65 years old (ranging from 27 to 89) with a similar rate of sexes. Nearly all subjects were non-smokers or former smokers, with 12% current smokers. In this study, the specificity was found to be 95% (140/147: Prostate (3), RCC (2), Uterine (1), pancreas (1)) (95% confidence interval of 90.4%-98.1%), confirming the high specificity of Bladder EpiCheck in patients

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with cancer other than bladder cancer.

10. Conclusion

The analytical and clinical performance supports that the Nucleix EpiCheck Test is substantially equivalent with the predicate device.