DiviTumTKa is an in vitro diagnostic device intended for the semi-quantitative measurement of thymidine kinase activity (TKa) in human serum. The assay is to be used as an aid in monitoring disease progression in previously diagnosed hormone receptor positive, metastatic postmenopausal female breast cancer patients. A TKa value of < 250 DuA is associated with the decreased likelihood of disease progression within 30 days or 60 days post testing. DiviTumTKa results should be used in conjunction with other clinical methods for monitoring breast cancer.

Device Description

Each DiviTum®TKa kit contains all necessary reagents, including calibrators and assay controls for analyses of 37 patient samples in duplicates. The kit components include Reagent A, Reagent B, Conjugate DiviTum®TKa, Control Low DiviTum®TKa, Control Medium DiviTum®TKa, Control High DiviTum®TKa, Calibrator 0 DiviTum®TKa through Calibrator 6 DiviTum®TKa, and Reaction Plate DiviTum®TKa. The Reaction Plate is a 96 well plate in sealed aluminum foil containing immobilized DNA strands, acting as a reaction chamber.

AI/ML Overview

Here's an analysis of the DiviTum®TKa device based on the provided FDA 510(k) summary, structured to address your specific questions:

Acceptance Criteria and Device Performance for DiviTum®TKa

1. Table of Acceptance Criteria and the Reported Device Performance

The acceptance criteria for DiviTum®TKa are primarily focused on its clinical effectiveness as an aid in monitoring disease progression, along with analytical performance. The key clinical acceptance relates to its ability to identify progression within 30 or 60 days.

Metric	Acceptance Criteria (Implied/Stated)	Reported Device Performance
Clinical Performance (Progression within 30 days)
Sensitivity (for progression)	N/A (performance studied)	52.4% (33/63) with 95% CI: 42.6 - 61.9
Specificity (for no progression)	N/A (performance studied)	80.7% (888/1101) with 95% CI: 78.1 - 83.1
Concordance	N/A (performance studied)	79.1% (921/1164) with 95% CI: 76.6 - 81.8
Probability of "No progression" for TKa < 250 DuA	N/A (performance studied)	96.7% (888/918)
Clinical Performance (Progression within 60 days)
Sensitivity (for progression)	N/A (performance studied)	48.7% (57/117) with 95% CI: 40.5 - 57.1
Specificity (for no progression)	N/A (performance studied)	81.9% (858/1047) with 95% CI: 79.3 - 84.6
Concordance	N/A (performance studied)	78.6% (915/1164) with 95% CI: 74.0 - 81.1
Probability of "No progression" for TKa < 250 DuA	N/A (performance studied)	93.5% (858/918)
Analytical Performance - Precision (Reproducibility & Repeatability)	Pre-defined acceptance criteria for acceptable variation (specific target %CV not explicitly stated in this summary for all studies, but results "fulfilled" them)	Multisite Reproducibility (%CV): <16.0% across samples (e.g., VS9 at 15.9%, VS12 at 16.0%) Single Site Within-Laboratory (%CV): <12.6% across samples (e.g., Sample 1 at 12.6%, Sample 5 at 11.0%)
Analytical Performance - Linearity	Linear correlation throughout the Analytical Measuring Interval (100 – 2000 DuA)	Slope: 0.96 (0.9123 to 1.008 CI), Intercept: 4.157 (-59.70 to 68.01 CI), R2: 0.996 (for range 61 - 2249 DuA)
Analytical Performance - High Dose Hook Effect	No Hook effect within expected range	No Hook effect up to approximately 100,000 DuA (50x ULOO)
Analytical Performance - Detection Limits	Acceptable LOB, LOD, LOQ	LOB: 33 DuA, LOD: 47 DuA, LOQ: 80 DuA
Analytical Performance - Analytical Measuring Interval (AMI)	Defined interval based on LoQ, linearity, and precision	100 DuA to 2000 DuA
Analytical Performance - Reagent Stability	Stable for 12 months at +2 to +8°C	Confirmed 12 months stability at +2 to +8°C
Analytical Performance - Assay Run Stability	%CV < 15% between assays run at different acceptable temperatures	%CV < 15% for temperatures +18℃, +23℃, and +28℃
Analytical Performance - Analytical Specificity (Interference)	No significant interference from 55 potential interfering substances when tested within normal reference values	None of 55 tested substances showed significant interference within normal reference values. Interference was observed for high lipemia, abnormally elevated Bilirubin (≥18 mg/dL), Cisplatin, or abnormally elevated ALP levels (>980 U/l).

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

Clinical Test Set:
- Number of patients: 454 patients (subjects).
- Total serum samples/test results: 1598 initially, with 52 excluded, resulting in 1546 test results for analysis (1164 during treatment, 382 from baseline).
- Data Provenance: The study used "left-over banked serum samples from metastatic breast cancer patients undergoing treatment." The document does not explicitly state the country of origin, but the "U.S. Collected" healthy post-menopausal female specimens suggest at least some data might be from the US. The retrospective or prospective nature of the collection of these banked samples is not specified, but the analysis of these archived samples is retrospective.
Analytical Test Sets:
- Precision studies involved various samples (pooled breast cancer serum, healthy blood donor serum) with replicate measurements. For example, the multisite study used 75 measurements per sample, and the single-site study used 79-80 measurements per sample.
- Linearity study used 10 intermediate concentrations with 4 replicates each.
- Detection limit studies used unspecified numbers of replicates for LOB, LOD, and LOQ calculations.
- Analytical specificity testing involved up to 55 substances.

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

The document does not specify the number or qualifications of experts used to establish the ground truth for disease progression in the clinical test set. It mentions that "clinical data on disease progression" was linked to the TKa measurements. This clinical data would typically be established by treating physicians using standard clinical methods (imaging, physician assessment, etc.), but the details of this process and the experts involved are not provided in this summary.

4. Adjudication Method for the Test Set

The document does not describe an explicit adjudication method (e.g., 2+1, 3+1) for establishing disease progression in the clinical test set. It relies on previously established "clinical data on disease progression."

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done, If So, What Was the Effect Size of How Much Human Readers Improve with AI vs without AI Assistance

This device (DiviTum®TKa) is an in vitro diagnostic (IVD) test for measuring a biomarker (thymidine kinase activity) in serum. It is not an imaging AI device that assists human readers in interpreting images. Therefore, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study focusing on human reader improvement with AI assistance is not applicable and was not performed or described in this summary.

6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) Was Done

Yes, the analytical and clinical performance studies presented describe the standalone performance of the DiviTum®TKa assay. The device measures TKa directly from serum samples and provides a quantitative result (DuA) which is then compared against a fixed cut-off of 250 DuA to aid in monitoring disease progression. There is no human-in-the-loop interpretation of the assay result itself described; the result is a direct measurement. However, the interpretation of what that result means for patient management requires a human clinician ("DiviTumTKa results should be used in conjunction with other clinical methods for monitoring breast cancer").

7. The Type of Ground Truth Used (Expert Consensus, Pathology, Outcomes Data, etc.)

The ground truth for the clinical study was outcomes data in the form of "disease progression." This was derived from "clinical data on disease progression" of breast cancer patients. While the specifics of how "disease progression" was defined (e.g., RECIST criteria, clinical signs, imaging changes) are not detailed in this summary, it represents an objective clinical outcome.

8. The Sample Size for the Training Set

The document refers to a "clinical validation study" but does not explicitly mention a separate "training set" for the clinical performance evaluation. The 454 patients (1546 samples) datasets were used for validation.

For the analytical performance, the device relies on a calibration curve for converting optical density readings to TKa units. Calibrators are included in each kit. The specific sample size for training the underlying algorithm if it involved machine learning beyond standard statistical models is not detailed, but given it's an enzyme immunoassay, it's unlikely to involve a large, separate machine learning "training set" in the common sense. The calibrators define the assay's conversion.

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

Given that DiviTum®TKa is an ELISA-based immunoassay, the "training set" more accurately refers to the development and calibration of the assay.

Analytical Calibration: Calibrators with "predetermined nominal values" are included in the kit. These values are established during the development and manufacturing process by Biovica using established "in-house measurement procedures and calibrators to support values assigned to the product calibrators." The definition of 1 DuA is linked to the TK activity of 1 pg/mL of recombinant TK1 (MyBiosource).
Clinical Reference Value ("Cut-off"): This was established by analyzing 123 serum samples from "apparently healthy post-menopausal females" in a reference study, with the ground truth being their healthy status. The 95th percentile of this healthy distribution (254 DuA) was used to set the initial clinical reference value. This was further "verified by published data" and evaluated using ROC analysis against the clinical outcomes (disease progression/no progression).

Summary

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July 29, 2022

Biovica International AB Joakim Arwidson Regulatory and QA Director Dag Hammarskjölds väg 54B Uppsala, 75237 Sweden

Re: K202852

Trade/Device Name: DiviTumTKa Regulation Number: 21 CFR 866.6010 Regulation Name: Tumor-Associated Antigen Immunological Test System Regulatory Class: Class II Product Code: QTE Dated: April 28, 2022 Received: May 2, 2022

Dear Joakim Arwidson:

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 statutes and regulations administered by other Federal agencies. You must comply with all the Act's

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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 (OS) 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 mediation-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,

Ying Mao, Ph.D. Branch Chief Division of Immunology and Hematology Devices OHT7: Office of In Vitro Diagnostics Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K202852

Device Name DiviTum®TKa

Indications for Use (Describe)

Type of Use (Select one or both, as applicable):

☑ Prescription Use (Part 21 CFR 801 Subpart D)
☐ Over-The-Counter Use (21 CFR 801 Subpart C)

CONTINUE ON A SEPARATE PAGE IF NEEDED.

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Traditional 510(k) Submission. DiviTum®TKa A.6 510(k) Summary DiviTum®TKa 510(K) OWNER: Biovica International AB ADDRESS: Dag Hammarskjölds Väg 54B, 752 37 Uppsala, Sweden PHONE NUMBER: + 46 (0) 18 44 44 830 EMAIL: info@biovica.com NAME OF CONTACT PERSON: Joakim Arwidson DATE THE SUMMARY WAS PREPARED: April 7th, 2021

A. 510(K) Number

K202852

PURPOSE OF SUBMISSION B.

New Device

C. MEASURAND

Thymidine Kinase Activity (TKa)

D. TYPE OF TEST

Semiquantitative, enzyme immunoassay

E. APPLICANT

Biovica International AB

F. PROPRIETARY AND ESTABLISHED NAMES

DiviTum®TKa DiviTum®

G. REGULATORY INFORMATION

Regulation Section 21 CFR §866.6010, Tumor - Associated antigen immunological test system

Classification Class II

Product Code QTE

Panel Immunology (82) (Assay) Clinical Chemistry (75) (Calibrators and Controls)

H. INTENDED USE

1. Intended Use(s)

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Traditional 510(k) Submission, DiviTum®TKa A.6 510(k) Summary DiviTum®TKa

60 days post testing. DiviTumTKa results should be used in conjunction with other clinical methods for monitoring breast cancer.

Warnings:

DiviTum®TKa should not be used for cancer screening or diagnosis. .
. DiviTum®TKa is not for serial testing because the test result at given timepoint does not compare to the test result at previous timepoint, but to a fixed cut-off value.
Indication(s) for Use 2. Same as above.
1. Special Conditions for Use Statement(s) For prescription use only.
1. Special Instrument Requirements ELISA Plate Reader at 405 and 630 nm.

I. DEVICE DESCRIPTION

Each DiviTum®TKa kit contains all necessary reagents, including calibrators and assay controls for analyses of 37 patient samples in duplicates.

Component	Quantity	Description
Reagent A	1 vial, lyophilized	Forms Reaction buffer A after reconstitution,containing necessary enzymes for the enzymereaction.
Reagent B	1 vial, lyophilized	Forms Reaction buffer B after reconstitution,containing the substrate BrdU.
ConjugateDiviTum®TKa	1 vial, lyophilized	Forms antibody-enzyme conjugate solution.after reconstitution with Conjugate Buffer
Control LowDiviTum®TKa	1 vial, lyophilized	With pre-determined value, for assay runquality control. To be reconstituted withReaction Buffer before use
Control MediumDiviTum®TKa	1 vial, lyophilized
Control HighDiviTum®TKa	1 vial, lyophilized
Calibrator 0DiviTum®TKa	1 vial, lyophilized	With pre-determined value, for generatingcalibration curve. To be reconstituted withReaction Buffer before use
Calibrator 1DiviTum®TKa	1 vial, lyophilized

Table 1 - Components of DiviTum®TKa kit

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Traditional 510(k) Submission, DiviTum®TKaA.6 510(k) Summary DiviTum®TKa
Calibrator 2DiviTum®TKa	1 vial, lyophilized
Calibrator 3DiviTum®TKa	1 vial, lyophilized
Calibrator 4DiviTum®TKa	1 vial, lyophilized
Calibrator 5DiviTum®TKa	1 vial, lyophilized
Calibrator 6DiviTum®TKa	1 vial, lyophilized
Reaction PlateDiviTum®TKa	One 96 well plate in sealedaluminum foil	Contains immobilized DNA strands, acts asreaction chamber

J. SUBSTANTIAL EQUIVALENCE INFORMATION:

1. Predicate Device Name(s): CYFRA 21-1 (soluble cytokeratin 19 fragments)
1. Predicate 510(k) Number(s): K100831
Comparison with Predicate: 3.

Table 2- Summary Predicate Comparison - Similarities

Device Name	DiviTum® TKa	CYFRA 21-1 EIA Kit
Device Type	In vitro diagnostic	Same
Classification	Class II	Same
Regulation	21 CFR § 866.6010, Tumor -Associated antigen immunologicaltest system	Same
Clinical function	Aid in monitoring diseaseprogression in cancer patients	Same
Type of specimen	Serum samples	Same
Intended clinicalenvironment	Clinical and hospital laboratory	Same
Type of test	Enzyme-linked immunosorbent assay(ELISA)	Same

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Traditional 510(k) Submission. DiviTum®TKa A.6 510(k) Summary DiviTum®TKa

Interpretation ofresults	Calibration curve	Same
Supply ofcalibrators andcontrols	Supplied with kit	Same
Special conditionfor use statement	For prescription use only	Same
Specialinstrumentrequirements	ELISA Plate Reader	Same

Table 3 - Summary Predicate Comparison - Differences

Device Name	DiviTum®TKa	CYFRA 21-1 EIA Kit
Intended use	DiviTumTKa is an in vitro diagnostic device intended for the semi-quantitative measurement of thymidine kinase activity (TKa) in human serum. The assay is to be used as an aid in monitoring disease progression in previously diagnosed hormone receptor positive, metastatic postmenopausal female breast cancer patients. A TKa value of < 250 DuA is associated with the decreased likelihood of disease progression within 30 days or 60 days post testing. DiviTumTKa results should be used in conjunction with other clinical methods for monitoring breast cancer.	The CYFRA 21-1 EIA kit is intended for the quantitative determination of soluble cytokeratin 19 fragments in human serum. The assay is to be used as an aid in monitoring disease progression during the course of disease and treatment in lung cancer patients. Serial testing for patient CYFRA 21-1 assay values should be used in conjunction with other clinical methods used for monitoring lung cancer.
Reference value	250 DuA	Relative percentage change
Target analyte	TK activity (TKa)	CYFRA 21-1 (soluble Cytokeratin 19 fragments)

STANDARD/GUIDANCE DOCUMENT REFERENCED: K.

CLSI EP05-A3 Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline -Third Edition

CLSI EP06-2nd edition: Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline

CLSI EP07-3rd edition: Interference Testing in Clinical Chemistry

CLSI EP17-A2: Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline –Second Edition

CLSI EP25-A: Evaluation of Stability of In Vitro Diagnostic Reagents. Approved Guideline.

CLSI EP28-A3c Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory; Approved Guidelines – Third Edition

CLSI EP34-1st edition: Establishing and verifying an extended measuring interval through specimen dilution and spiking

CLSI EP37-1st edition: Supplemental Tables for Interference Testing in Clinical Chemistry

ISO 15223-1 Fourth edition 2021-07. Medical devices - Symbols to be used with information to be supplied by the manufacturer - Part 1: General requirements.

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Traditional 510(k) Submission. DiviTum®TKa

A.6 510(k) Summary DiviTum®TKa

ISO 17511: 2020: In vitro diagnostic medical devices - Requirements for establishing metrological traceability of values assigned to calibrators, trueness control materials and human samples

L. TEST PRINCIPLE:

The DiviTum®TKa is a multi-step end-point ELISA assay involving a cascade of enzymatic reactions and one antibody binding reaction. During the DiviTum®TKa test, a serum sample is combined with a reaction mixture containing the substrate bromodeoxyuridine (BrdU). Since BrdU is a substrate analog to thymidine, TK from the serum sample phosphorylates the BrdU to its monophosphate, BrdUMP. The BrdUMP is then phosphorylated to BrdUTP and incorporated into a DNA/RNA hybrid, bound to the 96 well microplate solid surface, using a reverse transcriptase DNA polymerase. After washing, an alkaline phosphatase conjugated anti-BrdU antibody is added to the reaction well. The amount of phosphatase conjugate bound to the DNA is determined by a colorimetric reaction, turning the substrate color from colorless to yellow, and the optical density readings (OD) hence indicate the TKa in the sample. Calibrators with predetermined nominal values are included in the kit. These are used to generate a standard curve by which the OD readings from the patient samples are converted to TKa expressed as DiviTum®TKa Units (DuA).

M. PERFORMANCE CHARACTERISTICS:

1. ANALYTICAL PERFORMANCE

Precision | Reproducibility a.
The precision evaluation study for DiviTum®TKa was designed in accordance with CLSI guideline EP05-A3 to test reproducibility, repeatability, and other possible sources of variation for the assay: Operator, reagent lot and laboratory site. The study was divided into four parts: Two single site studies, a multi-site study and a lot-to-lot study.

The 1st single site precision evaluation study was performed at one site with five samples in five replicates per assay run, by two operators for 10 days per operator, and with three kit lots. The 2nd single site precision evaluation study was performed with six samples in two replicates per assay run for 20 days with two assay runs per day. The study was conducted by two operators at one site. The multisite precision evaluation study was conducted at three individual study sites (of which two are external CLIA certified laboratories) with five samples in five replicates per assay run for five days per site for a total of fifteen days. The lot-to-lot precision evaluation study was performed at one site by two operators using 3 unique (regarding critical raw materials) kit lots. Three kit lots were assayed per day for 5 days. The study was conducted with 5 replicates per run and per kit lot.

The results from the 1st single site precision evaluation study, the multisite precision evaluation study, the 2nd single site precision evaluation study, and the lot-to-lot precision evaluation study fulfilled the pre-defined acceptance criteria for acceptable variation between measurements using DiviTum®TKa. The results of site-to-site reproducibility and within laboratory precision are presented below in Tables 4 (multisite precision evaluation study) and 5 (2nd single site precision evaluation study).

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Traditional 510(k) Submission. DiviTum®TKa A.6 510(k) Summary DiviTum®TKa

Table 4 - Results for the multion study. Samples onsisted of pooled breast cancer serum and beathy bload donor servan respectively.

Sample	Meanactivity(DuA)	N	Within-assay(Repeatability)		Between-assay		Between-Site		Reproducibility
VS5	156	75	SD	%CV	SD	%CV	SD	%CV	SD	%CV
VS7	243	75	9	5.4%	17	10.6%	0	0%	19	11.9%
VS9	590	75	12	5.1%	19	7.9%	18	7.5%	29	12.0%
VS10	703	75	28	4.7%	31	5.3%	84	14.2%	94	15.9%
VS12	1710	74*	30	4.3%	33	4.7%	103	14.6%	112	16.0%

*One outlier was found; one replicate was therefore removed.

Table 5 - Results for the 2m single site presison evaluation. Samples conver and healthy blood donor serm.

Sample	Mean activity(DuA)	N	Within-Assay(Repeatability)		Between-Assay		Between-Day		Within-Laboratory
			SD	%CV	SD	%CV	SD	%CV	SD	%CV
1	172	80	9	5.4%	20	11.4%	0	0.0%	22	12.6%
2	255	79*	12	4.8%	20	8.0%	6	2.3%	25	9.6%
3	552	80	28	5.0%	45	8.2%	0	0.0%	53	9.6%
4	990	80	46	4.7%	81	8.1%	0	0.0%	93	9.4%
5	1698	80	106	6.2%	153	9.0%	0	0.0%	186	11.0%

*One outlier was removed.

b. Linearity/ Assay Reportable Range

i. Linearity.
The assay linearity interval was defined as 100 – 2000 DuA. Sample pools "HIGH" and "LOW" were prepared using native serum samples. The HIGH sample pool had a DuA value (as measured by DiviTum®TKa) higher than the upper limit of the linearity interval (ULLI). The LOW sample pool had a DuA value lower than the lower limit of the linearity interval (LLL). The two sample pools (HIGH and LOW) were mixed to obtain 10 intermediate concentrations with TK activity distributed throughout the whole AMI and near the assay clinical decision point at 250 DuA.

Four replicates per linearity panel member were analysed using DiviTum®TKa. Expected values were calculated using the measured value of sample HIGH and LOW together with proportional data obtained from the mixing of samples.

Measured values were plotted on the vertical (y-) axis and the expected values on the horizontal (x-) axis. Weighted least squares (WLS) linear regression analysis with an intercept was applied on the dataset. The results from WLS linear regression is presented in Table 6. The curve is described by the following equation (curve-equation): Measured value=4.157+0.96 x Expected value

Table 6. WLS linear regression results. Curve-equation: y = 4.157 + 0.96x.

Range (DuA)	Slope (95% CI)	Intercept (95% CI)	R2
61 - 2249	0.96 (0.9123 to 1.008)	4.157 (-59.70 to 68.01)	0.996

ii. High Dose Hook Effect

A dilution linearity test was performed in accordance with CLSI guideline EP34 1st edition to prove the absence of a Hook effect within the expected range of

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Traditional 510(k) Submission. DiviTum®TKa A.6 510(k) Summary DiviTum®TKa

DiviTum®TKa in patient samples with high TKa.: No Hook effect was observed within a TK-concentration range that reaches up to approximately 100,000 DuA, which is almost 50 times higher than the ULOO in DiviTum®TKa.

C. Traceability, Stability, Expected values (controls, calibrators, or methods)
- Traceability and Calibrators i.

DiviTum®TKa is a complex multi-step end-point assay involving five enzymatic reactions and one antibody binding reaction. It is not possible to characterize the measured TK activity in this assay using SI units. According to ISO 17511:2020, in IVD assays using units that are non-SI traceable, the calibration hierarchy shall be defined in a way that enables consistent realization of the corresponding (non-SI) units of measure. Biovica therefore established in-house measurement procedures and calibrators to support values assigned to the product calibrators. The definition of 1 DuA (DiviTum unit of Activity) is the TK activity of 1 pg/mL of recombinant TK1, MyBiosource. It equals the reaction rate catalyzed by 1 pg/mL of recombinant TK under the standard DiviTum®TKa assay conditions.

ii. Reagent Stability
DiviTum®TKa is stable for 12 months in storage at +2 to +8°C. The expiry date stated on the labeling will be 12 months in storage at +2 to +8°C. The FDA recognized consensus standard CLSI EP25A was used as guideline for stability studies.
iii. Stability during assay run
To verify in use stability, assay runs were performed at different temperatures using a minishaker chamber to control/maintain the temperature during thawing (30min), mixing of reagents (10min), and incubation (1.5h). The temperatures tested were +18℃, +23°C, and +28°C. The %CV between the three assays run at each temperature was <15% for all temperatures, and hence the stability of the kit is not significantly affected by variations in lab temperature in the range of normal room temperature (+18-28°C.)
Detection Limit d.
A detection capability study has been performed in accordance with CLSI guideline EP17-A2 to characterize measurement accuracy in the low-end region of the assay.
i. Limit of Blank (LOB):
LOB was calculated to 33 DuA, using the non-parametric option.
ii. Limit of Detection (LOD): LOD was calculated to 47 DuA.
iii. Limit of Quantitation (LOO): The estimated LoQ of DiviTum®TKa is 80 DuA.
iv. Analytical Measuring Interval (AMI)

The Analytical Measuring Interval (AMI) for DiviTum®TKa is set to 100 DuA to 2000 DuA. The AMI is established from the LoQ, Linearity and Precision studies. Samples below 100 DuA will be reported as <100 DuA and samples above 2000 DuA will be reported as >2000 DuA.

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Traditional 510(k) Submission, DiviTum®TKa A.6 510(k) Summary DiviTum®TKa

Analytical Specificity l.

The effects of 55 potential interfering substances (Table 7) were tested according to the CLSI guidelines EP07 3rd edition and EP37 1* edition. When tested within normal reference values, none of the 55 tested substances showed any significant interference with DiviTum®TKa. However, interference was observed for serum samples with high lipemia (>427 mg/dl) or with abnormally elevated Bilirubin (at ≥18 mg/dL) or using Cisplatin or with abnormally elevated ALP levels (>980 U/l), the DiviTum®TKa results shall be interpreted with caution.

Table 7. List of the 52 tested substances not interfering with DiviTum®TKa

Exogenous substances

5-fluorouracil
Abemaciclib
Acetylcysteine
Acetylsalycilic acid
Aminoglutethimidine
Ampicillin-Na
Anastrozole
Ascorbic acid
Brivudine
Capecitabine
Carboplatin
Cyclophosphamide
Cyclosporine
Docetaxel
Doxorubicin
Doxycycline
Epirubicin
Eribulin
Everolimus
Exemestane
Foscarnet
Fulvestrant
Goserelin
Heparin
Ibuprofen
Lapatinib
Letrozole
Leuoprorelin
Levodopa
Loperamid
Megestrol Acetate
Methotrexate
Metoklopramid
Metronidazole
Mitomycin C
Morphine
Omeprazole
Paclitaxel
Palbociclib
Paracetamol
Ribociclib

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Traditional 510(k) Submission. DiviTum®TKa A.6 510(k) Summary DiviTum®TKa Tamoxifen Theophylline Toremifene Trastuzumab Zidovudine Endogenous substances Hemoglobin Bilirubin, unconjugated Albumin and y-globulin Alkaline phosphatase HAMA

f. Clinical reference value ("cut-off") See clinical reference value below.

2. COMPARISON STUDIES

Method comparison with predicate device a. Not applicable. The predicate measures a different analyte. Extensive analytical and clinical studies are presented to demonstrate a high safety and efficacy profile.
Matrix comparison b. Not applicable. Serum samples are the only matrix used for analysis.

3. CLINICAL STUDIES

The effectiveness of the DiviTum®TKa as an aid in monitoring disease progression in previously diagnosed postmenopausal HR+ metastatic breast cancer patients undergoing treatment, was determined through a clinical validation study using left-over banked serum samples from metastatic breast cancer patients undergoing treatment. Multiple time-point measurements of TKa in serum samples from patients were linked to clinical data on disease progression. The clinical validation study included 454 patients (subjects) with a total of 1598 DiviTum®TKa serum samples/test results. 52 serum samples/test results obtained after the patient had progressed were excluded before the statistical analysis. These 52 serum samples were distributed over the 454 patients. The number of serum samples/test results - included in all the statistical analyses, and still representing 454 patients, hence constituted 1546 test results of which 1164 test results (samples) were captured during treatment, but still representing all 454 patients. The remaining 382 test results (samples) were from baseline (i.e., pre-treatment). The multiple time-point test results were divided into time intervals based on the original sampling day (phlebotomy) - i.e., actual visit (sampling) day at physician after registration. Patient treatment was initiated according to patient information - i.e., after baseline (>21 days) sampling.

The number of DiviTum®TKa measurements per patient (N=454) and TKa mean, median, minimum, and maximum values per time interval are listed in Table 6.

Table 6 - Number of Divilland TKa measurements (N=454) and TKa mean, median, minimum, and massiman values per time internal

Variable	Statistic	Patients(%)
Number of TKa measurements perpatient	1n1 (%)	55 (12.1%)
	2n2 (%)	50 (11.0%)
	3n3 (%)	100 (22.0%)
	4n4 (%)	102 (22.4%)
	5n5 (%)	147 (32.3%)

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Traditional 510(k) Submission. DiviTum®TKa A.6 510(k) Summary DiviTum®TKa

		Measurement (DuA)
TKa baseline (<21 day)	Mean (SD)	585 (1395)
	Median	227
	Min; Max	33; 15 288
TKa (21-70 days; cycle 2)	Mean (SD)	414 (1226)
	Median	160
	Min; Max	30; 14 439
TKa (71-98 days; cycle 3)	Mean (SD)	239 (408)
	Median	153
	Min; Max	32; 3961
TKa (99-154 days; cycle 4)	Mean (SD)	241 (410)
	Median	144
	Min; Max	30; 3699
TKa (>154 days; cycle 7)	Mean (SD)	243 (966)
	Median	142
	Min; Max	25; 14 674

Elevated serum TKa was defined as above or equal to 250 DuA (clinical reference value).

From the 1164 DiviTum®TKa monitoring tests (during treatment), disease progression within 30 days from testing was seen in 63 cases. Thirty-three (52.4%) of the 63 samples with disease progression had TKa above the clinical reference value (≥250 DuA). Eight hundred and eightyeight (80.7%) of the 1101 samples with TKa <250 DuA were not associated with disease progression within the next 30 days. Table 9 presents the data in a 2 x 2 format and also includes data on progression within 60 days of testing.

			Table 9 - Number of patient samples showing progression within 30- and 60 days after blood sampling
--	--	--	-----------------------------------------------------------------------------------------------------	--	--	--	--

Time interval fromTKa measurement(days)	30 days		60 days
	Progression(samples)	No progression(samples)	Progression(samples)	No progression(samples)
≥250 DuA	33	213	57	189
<250 DuA	30	888	60	858
Total	63	1101	117	1047

Clinical Sensitivity and Specificity: a.

The diagnostical performance is presented in Table 710. At a clinical reference value at 250 DuA, a DiviTum®TKa value ≥ 250 DuA is 52.4% sensitive for disease progression within the next 30 days (48.7% for disease progression within 60 days). A value below the clinical reference value (250 DuA) is 80.7% specific that disease will not progress within the next 30 days (81.9% specific for no-progression within the next 60 days). Sensitivity is represented as the proportion of patients with disease progression that had elevated DiviTum®TKa. Specificity is represented as the proportion of patients without disease progression that did not have elevated DiviTum®TKa. Positive Likelihood Ratio (PLR) is represented as the fold increase of a patient's likelihood of disease progression when having elevated DiviTum®TKa. Negative Likelihood Ratio (NPR) is represented as the fold decrease of a patient's likelihood of disease progression without elevated DiviTum®TKa.

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Traditional 510(k) Submission. DiviTum®TKa A.6 510(k) Summary DiviTum®TKa

Table 7 - Overview Diagnostical Performance

Time intervalProgression within 30 days post testing		Progression within 60 days posttesting
TKa measurement: 21-154 days; cycles 2-7 incl.	Value	95% CI	Value	95% CI
n (samples)	1164	NA	1164	NA
Cases (patients)	63	NA	117	NA
Sensitivity	52.4% (33/63)	42.6 - 61.9	48.7% (57/117)	40.5 - 57.1
Specificity	80.7% (888/1101)	78.1 - 83.1	81.9% (858/1047)	79.3 - 84.6
Concordance	79.1% (921/1164)	76.6 - 81.8	78.6% (915/1164)	74.0 - 81.1
Risk of progression	5.4% (63/1164)	NA	10.1% (117/1164)	NA
Risk of progression forTKa≥250 DuA	13.4% (33/246)	NA	23.2% (57/246)	NA
Risk of progression forTKa<250 DuA	3.3% (30/918)	NA	6.5% (60/918)	NA
Probability of"No progression"	96.7% (888/918)	NA	93.5% (858/918)	NA
Positive LR	2.71	NA	2.70	NA
Negative LR	0.59	NA	0.63	NA

4. Clinical reference value ("cut off")

To determine the clinical reference value, the intended patient population, and the intended purpose of DiviTum®TKa was considered. As TK activity is an indicator for cell proliferation, it was important to determine which values can be expected in a healthy population and a population with other clinical conditions leading to cell proliferation. This was done to get a first indication for a suitable clinical reference value. The clinical reference value was determined by analyzing 123 serum samples from apparently healthy post-menopausal females in a reference study. The clinical reference value was based on the 95th percentile of the distribution in this study. The sample size and statistical method evaluation were done according to the CLSI guideline EP28-A3. Based on the study results the assay clinical reference value for DiviTum®TKa was set to 250 DuA. This clinical reference value/cut-off was verified by published data (Larsson et.al. "Serial evaluation of serum thymidine kinase activity is prognostic in women with newly diagnosed metastatic breast cancer" vol 10, no. 4484, 2020). To assess sensitivity and specificity at various clinical reference values, an ROC analysis was also performed. Results are shown in Figure 1.

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Traditional 510(k) Submission, DiviTum®TKa A.6 510(k) Summary DiviTum®TKa

Image /page/14/Figure/2 description: The image contains two ROC curves, one for progression within 30 days and another for progression within 60 days. Both curves plot sensitivity against specificity. The ROC curve for progression within 30 days has a point marked at 250.0 (0.81, 0.52), while the ROC curve for progression within 60 days has a point marked at 250.0 (0.82, 0.49).

Figure 1 - A and B: ROC curves for progression within 30 days (B), with the clinical reference value) ont-off at 250 DuA noted.

5. Expected values/Reference range:

The reference range was established by measuring the TKa in 123 healthy blood donors. The reference interval for healthy serum donors is 55 to 254 DuA. The 95th percentile for healthy serum donors is 254 DuA.

Groups with non-malignant conditions were analyzed to evaluate possible effects on the TKa value, see Table 811. The groups were chosen based on the possibility that the disorder affects the cell cycle and/or cell proliferation or being common in the study population.

Groups with malignant conditions were also analyzed, see Table 12. They were chosen based on the association with breast cancer.

Group	Sample size	Rationale for inclusion
Viral Infection (EBV & HSV)	41	Enhanced cellular proliferation
Parkinson's Disease	43	Common in intended population
Non-alcoholic Steatohepatitis (NASH)	41	Enhanced cellular proliferation
Type 2 Diabetes	41	Enhanced cellular proliferation
Rheumatoid Arthritis	46	Enhanced cellular proliferation
Chronic Kidney Disease (CKD)	40	Common in intended population
Chronic Obstructive Pulmonary Disease (COPD)	45	Common in intended population

Table 8 - Non-malignant diseases or conditions and rationales for inclusion of chosen groups

Table 9 - Malignant diseases and rationales for inclusion of chosen groups. BC: Breast Cancer

Group	Sample size	Rationale for inclusion
Breast cancer stage IV	60	Patient population; intended use
Breast cancer stage III	48	Can develop into BC stage IV

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Traditional 510(k) Submission. DiviTum®TKa A.6 510(k) Summary DiviTum®TKa

Breast cancer stage II	43	Can develop into BC stage IV
Breast cancer stage I	42	Can develop into BC stage IV
Lung cancer (all stages)	43	Common breast cancer metastatic site
Bone cancer (all stages)	59	Common breast cancer metastatic site
Brain cancer (all stages)	42	Common breast cancer metastatic site
Liver cancer (all stages)	26	Common breast cancer metastatic site

To calculate the reference interval the Harrel-David non-parametric bootstrap method was chosen. The method generated a 95th percentile of 254 DuA. The results are summarized in Table 10.

Table 10 - Results for upper reference limit estimation using not parametric Harrell-Davis bootstrap method. Mean names ar presented.

Apparently Healthy Post-Menopausal Female Specimens, Age ≥ 55 < 85; U.S. Collected
N	123
Mean (DuA)	139
SD (DuA)	56
Median (DuA)	125
Max (DuA)	301
95th percentile (DuA)	254
97.5th percentile (DuA)	276

Based on these results, it can be concluded that values below 254 DuA are within the normal range for a healthy population of post-menopausal women (U.S. collected). The TKa was found to be slightly increased in patients with cell cycle affecting viral infections (acute Herpes Simplex and Epstein-Barr viral infections) as well as in certain autoimmune diseases (Rheumatoid Arthritis). Elevated levels of TKa are seen in patients with any type of late stage or aggressive malignant disease (cancer) as TKa is a biomarker for cell proliferation.

N. PROPOSED LABELING:

Labeling has been done in accordance with the requirements of 21 CFR Part 809.10.

O. CONCLUSION:

The submitted information in this premarket notification is complete and supports a substantial equivalence decision.

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.