← Product Code [PRB](/submissions/PA/subpart-b%E2%80%94biological-stains/PRB) · K212176

# CINtec Histology (K212176)

_Ventana Medical Systems, Inc. · PRB · Dec 10, 2021 · Pathology · SESE_

**Canonical URL:** https://fda.innolitics.com/submissions/PA/subpart-b%E2%80%94biological-stains/PRB/K212176

## Device Facts

- **Applicant:** Ventana Medical Systems, Inc.
- **Product Code:** [PRB](/submissions/PA/subpart-b%E2%80%94biological-stains/PRB.md)
- **Decision Date:** Dec 10, 2021
- **Decision:** SESE
- **Submission Type:** Traditional
- **Regulation:** 21 CFR 864.1865
- **Device Class:** Class 2
- **Review Panel:** Pathology

## Indications for Use

CINtec® Histology is a qualitative immunohistochemistry (IHC) test using mouse monoclonal anti-p16 antibody clone E6H4, and is intended for use in the light microscopic assessment of the p16INK4a protein in formalin-fixed, paraffinembedded (FFPE) cervical punch biopsy tissues using OptiView DAB IHC Detection Kit on a VENTANA BenchMark ULTRA instrument. The test is indicated as an adjunct to examination of hematoxylin and eosin (H&E) stained slide(s), to improve consistency in the diagnosis of cervical intraepithelial neoplasia (CIN). Diagnosis of CIN presence or level should be based on H&E stained slide(s) and other clinical and laboratory test information. Intended for in vitro diagnostic (IVD) use. Prescription Use Only.

## Device Story

CINtec Histology is an immunohistochemistry (IHC) assay using recombinant anti-p16INK4a (clone E6H4) antibody. It processes FFPE cervical punch biopsy tissue sections on a VENTANA BenchMark ULTRA instrument using the OptiView DAB IHC Detection Kit. The device detects p16INK4a protein expression via light microscopy. It is used in clinical laboratories by pathologists as an adjunct to H&E staining to assist in diagnosing CIN. The output is a stained slide showing p16 protein distribution, which aids the pathologist in evaluating the presence or grade of CIN, potentially improving diagnostic consistency.

## Clinical Evidence

No clinical trials were performed. Evidence consists of analytical performance testing, including between-lot precision (n=26 cases, 98.7% agreement), immunoreactivity studies across a wide range of normal and neoplastic tissues, and a method comparison study (n=249 cases) comparing recombinant vs. hybridoma antibody formulations. The method comparison showed an Overall Percent Agreement (OPA) of 98.7% (95% CI: 96.4, 99.6).

## Technological Characteristics

IHC assay using recombinant anti-p16 (E6H4) antibody. Reagents include OptiView DAB IHC Detection Kit (biotin-free, indirect system), EZ Prep, Reaction Buffer, ULTRA Cell Conditioning 1 (CC1), Hematoxylin II, and Bluing Reagent. Automated staining on VENTANA BenchMark ULTRA instrument. Recombinant antibody provides high consistency and reproducibility compared to hybridoma-derived antibody.

## Regulatory Identification

A cervical intraepithelial neoplasia (CIN) test system is a device used to detect a biomarker associated with CIN in human tissues. The device is indicated as an adjunct test and not to be used as a stand-alone device. The test results must be interpreted in the context of the patient's clinical history including, but not limited to, prior and current cervical biopsy results, Papanicolaou (Pap) test results, human papillomavirus (HPV) test results, and morphology on hematoxylin and eosin (H&E) stained sections. This device is not intended to detect the presence of HPV.

## Special Controls

*Classification.* Class II (special controls). The special controls for this device are:(1) Premarket notification submissions must include the following information:
(i) The indications for use must specify the biomarker that is intended to be identified and its adjunct use (
*e.g.,* adjunct to examination of H&E stained slides) to improve consistency in the diagnosis of CIN.(ii) Summary of professional society recommendations, as applicable.
(iii) A detailed device description including:
(A) A detailed description of all test components, including all provided reagents and required, but not provided, ancillary reagents.
(B) A detailed description of instrumentation and equipment, including illustrations or photographs of non-standard equipment or manuals.
(C) If applicable, detailed documentation of the device software, including, but not limited to, stand-alone software applications and hardware-based devices that incorporate software.
(D) A detailed description of appropriate positive and negative controls that are recommended or provided.
(E) Detailed specifications for sample collection, processing, and storage.
(F) A detailed description of methodology and assay procedure.
(G) A description of the assay cutoff (the medical decision point between positive and negative) or other relevant criteria that distinguishes positive and negative results, including the rationale for the chosen cutoff or other relevant criteria and results supporting validation of the cutoff.
(H) Detailed specification of the criteria for test results interpretation and reporting.
(iv) Detailed information demonstrating the performance characteristics of the device, including:
(A) Analytical specificity studies such as, but not limited to, antibody characterization (
*e.g.,* Western Blot, peptide inhibition analysis), studies conducted on panels of normal tissues and neoplastic tissues, interference by endogenous and exogenous substances as well as cross-reactivity, as applicable.(B) Device analytical sensitivity data generated by testing an adequate number of samples from individuals with the target condition including limit of blank, limit of detection, and limit of quantification, as applicable.
(C) Device precision/reproducibility data to evaluate within-run, between-run, between-day, between-lot, between-site, between-reader, within-reader and total precision, as applicable, using a panel of samples covering the device measuring range and/or the relevant disease categories (
*e.g.* No CIN, CIN1, CIN2, CIN3, cervical cancer) and testing in replicates across multiple, nonconsecutive days.(D) Device robustness/guardbanding studies to assess the tolerance ranges for various critical test and specimen parameters.
(E) Device stability data, including real-time stability and shipping stability under various storage times, temperatures, and freeze-thaw conditions.
(F) Data from a clinical study demonstrating clinical validity using well-characterized prospectively or retrospectively obtained clinical specimens, as appropriate, representative of the intended use population. The study must evaluate the consistency of the diagnosis of CIN, for example, by comparing the levels of agreements of diagnoses rendered by community pathologists to those rendered by a panel of expert pathologists. Agreement for each CIN diagnostic category (
*e.g.,* No CIN, CIN1, CIN2, CIN3, cancer) and for alternate diagnostic categories (*e.g.,* No CIN, low grade squamous intraepithelial lesion (LSIL)-histology, high grade squamous intraepithelial lesion (HSIL)-histology, cancer) between reference diagnosis by expert pathologist and community pathologist must be evaluated, as applicable. In addition, agreements for CIN binary categories as ≥CIN2 (*i.e.,* CIN2 or CIN3 or cancer) and ≤CIN1 (*i.e.,* No CIN or CIN1) between reference diagnosis by expert pathologist with H&E staining and community pathologist with H&E staining and agreements for alternate CIN binary categories as ≥HSIL-histology (*i.e.,* HSIL-histology or cancer) and ≤LSIL-histology (*i.e.,* No CIN or LSIL-histology) between reference diagnosis by an expert pathologist with H&E + [biomarker specified in paragraph (b)(1)(i) of this section] and a community pathologist with H&E + [biomarker specified in paragraph (b)(1)(i) of this section] must be evaluated and compared, as applicable.(G) The staining performance of the device as determined by the community pathologists during review of the study slides must be evaluated. The staining performance criteria assessed must include overall staining acceptability, background staining acceptability, and morphology acceptability, as applicable.
(H) Appropriate training requirements for users, including interpretation manual, as applicable.
(I) Identification of risk mitigation elements used by the device, including a description of all additional procedures, methods, and practices incorporated into the instructions for use that mitigate risks associated with testing.
(2) The device's 21 CFR 809.10(b) compliant labeling must include a detailed description of the protocol, including the information described in paragraph (b)(1)(ii) of this section, as applicable, and a detailed description of the performance studies performed and the summary of the results, including those that relate to paragraph (b)(1)(ii) of this section, as applicable.

## Predicate Devices

- CINtec® Histology ([DEN160019](/device/DEN160019.md))

## Submission Summary (Full Text)

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>
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FDA U.S. FOOD & DRUG ADMINISTRATION

# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY
ASSAY AND INSTRUMENT

## I Background Information:

A 510(k) Number
K212176

B Applicant
Ventana Medical Systems, Inc.

C Proprietary and Established Names
CINtec Histology

D Regulatory Information
|  Product Code(s) | Classification | Regulation Section | Panel  |
| --- | --- | --- | --- |
|  PRB | Class II | 21 CFR 864.1865 - Cervical Intraepithelial Neoplasia (CIN) Test System | PA - Pathology  |

## II Submission/Device Overview:

A Purpose for Submission:
Change in the CINtec® Histology device from the hybridoma anti-p16 (E6H4) antibody to recombinant anti-p16 (E6H4) antibody.

B Measurand:
p16<sup>INK4a</sup> protein

C Type of Test:
Immunohistochemistry, qualitative

Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.gov

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K212176 - Page 2 of 17

## III Intended Use/Indications for Use:

### A Intended Use(s):

CINtec® Histology is a qualitative immunohistochemistry (IHC) test using mouse monoclonal anti-p16 antibody clone E6H4, and is intended for use in the light microscopic assessment of the p16$^{\text{INK4a}}$ protein in formalin-fixed, paraffin-embedded (FFPE) cervical punch biopsy tissues using OptiView DAB IHC Detection Kit on a VENTANA BenchMark ULTRA instrument. The test is indicated as an adjunct to examination of hematoxylin and eosin (H&amp;E) stained slide(s), to improve consistency in the diagnosis of cervical intraepithelial neoplasia (CIN). Diagnosis of CIN presence or level should be based on H&amp;E stained slide(s) and other clinical and laboratory test information.

### B Indication(s) for Use:

Same as above

### C Special Conditions for Use Statement(s):

For *in vitro* diagnostic (IVD) use only
Rx - For Prescription Use Only

### D Special Instrument Requirements:

VENTANA BenchMark ULTRA instrument

## IV Device/System Characteristics:

### A Device Description:

CINtec® Histology is an immunohistochemical (IHC) assay system comprised of an anti-p16 primary antibody optimized for use with the BenchMark ULTRA automated slide staining instrument and the OptiView DAB IHC Detection Kit. The antibody is diluted in a Tris-HCl buffer containing carrier protein and 0.1% ProClin 300 as a preservative, and provided as a ready-to-use liquid in a FloLock dispenser. CINtec Histology is available in a 50 test size and a 250 test size.

The OptiView DAB IHC Detection Kit (OptiView) is an indirect, biotin-free system for detecting mouse IgG, mouse IgM, and rabbit IgG primary antibodies and is comprised of 6 dispensers packaged together in one box. The components of the OptiView DAB IHC Detection Kit are provided in Table 1.

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Table 1: OptiView DAB IHC Detection Kit Components

|  Component | Content  |
| --- | --- |
|  OptiView Peroxidase Inhibitor | 3.0% hydrogen peroxide solution  |
|  OptiView HQ Universal Linker | Cocktail of HQ-labeled antibodies (goat anti-mouse IgG, goat anti-mouse IgM, and goat anti-rabbit IgG) (<50 μg/mL) in a buffer containing protein with ProClin 300, a preservative  |
|  OptiView HRP Multimer | Mouse monoclonal anti-HQ HRP labeled tertiary antibody (<40 μg/mL) in a buffer containing protein with ProClin 300  |
|  OptiView DAB | 0.2% 3, 3'-diaminobenzidine tetrahydrochloride (DAB) in a stabilizer solution in preservative  |
|  OptiView H2O2 | 0.04% hydrogen peroxide in a phosphate buffer solution  |
|  OptiView Copper | Copper sulfate (5.0 g/L) in an acetate buffer in preservative  |

The ancillary reagents required to perform the CINtec Histology assay are provided in Table 2.

Table 2: Ancillary Reagents

|  Reagent | Format Provided | Contents | Purpose  |
| --- | --- | --- | --- |
|  EZ Prep | Bulk / 10X Concentrate / 2 liter | Detergent | Removes paraffin from the tissue specimen  |
|  Reaction Buffer | Bulk / 10X Concentrate / 2 liter | Tris based buffer solution with detergent and preservative | Provides stable environment for antibody-antigen interactions and enzyme reactions. Also used as a rinse solution to remove reagents between assay steps  |
|  ULTRA High Temperature Liquid Coverslip (LCS) | Bulk / 2 liter | Low density paraffinic hydrocarbon and other oils | Functions as a barrier between aqueous solutions and air (i.e., prevents evaporation of reagents during incubation periods on the slide)  |
|  ULTRA Cell Conditioning 1 Solution (CC1) | Bulk / Prediluted / 1 liter | Tris based buffer solution with detergent and preservative | Disrupts covalent bonds at high temperatures formed by formalin in tissue Increases antibody accessibility.  |

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|  Reagent | Format Provided | Contents | Purpose  |
| --- | --- | --- | --- |
|  Hematoxylin II Counterstain | Dispenser / Prediluted / 25 mL | Hematoxylin (≤60%); contains glycol and acetic acid stabilizing solution | A modified Mayer's hematoxylin used for staining cellular nuclei.  |
|  Bluing Reagent | Dispenser / Prediluted / 25 mL | Solution of 0.1 M lithium carbonate in 0.5 M sodium carbonate | Applied after hematoxylin and changes the hue of the hematoxylin to a blue color.  |

## Controls:

Positive and negative tissue controls that are fixed and processed in the same manner as the test specimens should be used when performing this test. Positive and negative control tissue is used to confirm that the assay performed as expected. For optimal quality control, cervical carcinoma or CIN2/3 cervical tissue positive for CINtec Histology staining is suitable for use as a positive tissue control, and normal cervical tissue is suitable for use as a negative tissue control. Normal human tonsil tissue is also suitable for use as a tissue control, as tonsil contains both positive and negative staining elements when stained with CINtec Histology. Within normal tonsil tissue, there is nuclear and/or cytoplasmic staining of scattered squamous epithelial cells primarily in crypt epithelium and scattered follicular dendritic cells in germinal centers and absence of staining in the majority of lymphocytes.

A negative reagent control mouse monoclonal antibody shall be used to evaluate nonspecific staining. This negative reagent control should be used to stain an adjacent section of the patient specimen tissue on a separate slide from the CINtec Histology slide. The staining protocol for the negative reagent control antibody should be the same as that for the primary antibody.

## B Principle of Operation:

CINtec Histology is an immunohistochemistry device used to stain FFPE cervical punch biopsy tissue slides for the visualization of the p16$^{\text{INK4a}}$ protein. The test process involves the sequential application of antibodies and a chromogen, with interposed washing steps. The assay steps are as follows: 1) the anti-p16 antibody specifically binds to an epitope in the p16$^{\text{INK4a}}$ protein; 2) a HQ-labeled secondary antibody binds to the primary antibody (HQ is a proprietary hapten covalently linked to the secondary antibody); 3) a tertiary horseradish peroxidase (HRP)-labeled antibody directed against HQ binds to the HQ-labeled secondary antibody; and 4) the resulting complex is visualized with hydrogen peroxide and DAB, due to the formation of a visible brown precipitate at the antigen site. The specimen slide is then counterstained with hematoxylin and cover slipped. Results are interpreted using a light microscope by a pathologist.

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Table 3: CINtec Histology, Staining Protocol

|  Staining Procedure: CINtec Histology  |   |
| --- | --- |
|  Procedure Type | Parameter Input  |
|  Deparaffinization | Selected
Not modifiable  |
|  Cell Conditioning
(Antigen Unmasking) | ULTRA CC1,
48 minutes  |
|  Pre-Primary
Peroxidase Inhibitor | Selected
Not modifiable  |
|  Antibody (Primary) | CINtec Histology
OR
negative reagent control  |
|  Antibody (Primary)
Incubation | 12 minutes, 36°C  |
|  OptiView HQ Linker | 8 minutes
Not modifiable  |
|  OptiView HRP
Multimer | 8 minutes
Not modifiable  |
|  Counterstain | Hematoxylin II, 4 minutes
Not modifiable  |
|  Post Counterstain | Bluing, 4 minutes
Not modifiable  |

## Interpretation of Results

CINtec Histology is a qualitative test. The results are interpreted as either positive or negative based on the p16 staining pattern in the FFPE cervical tissue section.

**Positive Result:** A positive result is defined as “diffuse” staining which is continuous staining of cells in the basal and parabasal cell layers of the cervical squamous epithelium, with or without staining of the intermediate or intermediate to superficial cell layers. Diffuse staining of any intensity is considered to be positive for CINtec Histology status. In some specimens, nuclear expression may be faint or undetectable, but nuclear p16 staining is not required to interpret the CINtec Histology status. Cellular p16 staining for CINtec Histology may be nuclear and/or cytoplasmic.

**Negative Result:** A negative result is defined as either focal staining of the cervical epithelium or absence of the p16 staining in the cervical epithelium. Focal staining is defined as staining of isolated cells or small cell clusters, i.e., a non-continuous staining pattern, and particularly not in the basal and parabasal cells.

K212176 - Page 5 of 17

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C Instrument Description Information:

1. Instrument Name:

VENTANA BenchMark ULTRA instrument which fully automates the processes of baking, deparaffinization, and staining. The BenchMark ULTRA instrument is composed of four main modular components that work together as a system: 1) a stainer subassembly where all slide processing operations are performed containing a reagent carousel, dispenser mechanism, barcode readers, heating elements and other components; 2) an automated fluidics subassembly that provides the compressed air and bulk fluids required by the stainer subassembly; 3) a waste bottle subassembly that collects the waste generated by the system during staining operations; and, 4) a personal computer (PC) running on a Microsoft Windows platform that controls and monitors the system through the Ventana System Software (VSS) host operating software.

2. Specimen Identification:

Specimen identification is performed through the VSS host operating software. Barcodes reflecting the specific protocols for the CINtec Histology assay are printed by the user and affixed to the appropriate patient specimen slides. Prior to starting the assay run, the barcodes on the slides and dispensers are scanned and qualified by the host software to ensure all elements are in place to complete the staining run. The specific automated staining procedure is performed for each slide, based on the protocol associated to the key code from the slide barcode.

3. Specimen Sampling and Handling:

Cervical punch biopsy specimens are collected by the clinician and placed in 10% neutral buffered formalin. Tissue sections are prepared from formalin-fixed, paraffin-embedded (FFPE) cervical punch biopsy specimens and mounted on glass slides. Slides are placed on the VENTANA BenchMark ULTRA instrument for staining with the CINtec Histology device.

4. Calibration:

Not applicable. See section titled Controls above.

5. Quality Control:

See Controls in section IV.A above.

V Substantial Equivalence Information:

A Predicate Device Name(s):

CINtec® Histology

K212176 - Page 6 of 17

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# B Predicate 510(k) Number(s):

DEN160019

|  Device Name | CINtec® Histology  |
| --- | --- |
|  Regulation Number | 21 CFR 864.1865  |
|  Regulation Name | A cervical intraepithelial neoplasia (CIN) test system  |
|  Regulatory Classification | Class II  |
|  Product Code | PRB  |
|  DEN# | DEN160019  |

C Table 4: Comparison with Predicate(s):

|  Device & Predicate Device(s): | K212176 | DEN160019  |
| --- | --- | --- |
|  Device Trade Name | CINtec® Histology | CINtec® Histology  |
|  General Device Characteristic Similarities  |   |   |
|  Intended Use/Indications For Use | Same | CINtec® Histology is a qualitative immunohistochemistry (IHC) test using mouse monoclonal anti-p16 antibody clone E6H4, and is intended for use in the light microscopic assessment of the p16INK4a protein in formalin-fixed, paraffin-embedded (FFPE) cervical punch biopsy tissues using OptiView DAB IHC Detection Kit on a VENTANA BenchMark ULTRA instrument. The test is indicated as an adjunct to examination of hematoxylin and eosin (H&E) stained slide(s), to improve consistency in the diagnosis of cervical intraepithelial neoplasia (CIN). Diagnosis of CIN presence or level should be based on H&E stained slide(s) and other clinical and laboratory test information.Intended for in vitro diagnostic (IVD) use.Prescription Use Only.  |
|  Antibody | Same | anti-p16  |
|  Clone | Same | E6H4  |
|  Differences  |   |   |
|  Method of antibody production | Recombinant technology | Hybridoma technology  |

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|  Cell line | Chinese hamster ovary cells (CHO) | Murine hybridoma cell line (clone E6H4)  |
| --- | --- | --- |

## VI Standards/Guidance Documents Referenced:

The subject and predicate device (CINtec Histology) have special controls in the device-specific classification regulation 21 CFR 864.1865. The special controls as outlined in the predicate device De Novo Decision Summary (DEN160019) are unchanged except the results of the performance testing that continues to meet current acceptance criteria. For a detailed description of the performance testing, refer to Sections 13: Labeling and Section 18: Performance Testing of this Traditional 510(k) Notification.

## VII Performance Characteristics (if/when applicable):

A summary of performance testing is provided below. The subject recombinant CINtec Histology performs equivalently to the predicate hybridoma CINtec Histology and both meet current acceptance criteria.

### A Analytical Performance:

1. Precision/Reproducibility:
i. Between-Lot precision

Three production lots of the recombinant CINtec® Histology formulated with three independent recombinant anti-p16 antibody raw material lots were used in this study to stain each of the 26 cervical tissue cases (6 No CIN, 6 CIN1, 6 CIN2, 6 CIN3, and 2 cervical squamous cell carcinomas (SCC). These cases were qualified based on H&amp;E staining. Eighteen sections were cut from each tissue block. For each lot, duplicate slides from each case were stained with H&amp;E, CINtec® Histology and a Negative Control (Monoclonal). The H&amp;E stained slides were adjacent to the CINtec® Histology slides. All CINtec® Histology and Negative Control (Monoclonal) stained slides were run on the same BenchMark ULTRA instrument. Appropriate control tissue slides were also stained in each run.

Each CINtec® Histology stained slide was than paired with its adjacent H&amp;E and Negative Control (Monoclonal) slides. The order of the 26 slide sets was randomized. The sections stained with recombinant CINtec® Histology were evaluated by a study pathologist for p16 staining intensities (0-4), p16 staining pattern (diffuse, focal, negative) and background (acceptable, unacceptable) and assigned a CINtec® Histology status (positive, negative) according to scoring criteria described in the section titled "Interpretation of Results" above. Slide sets were also assessed for histological diagnosis (No CIN, CIN1, CIN2, CIN3 or invasive carcinoma) based on conjunctive evaluation of the H&amp;E and recombinant CINtec® Histology stained slides and assigned to CIN category (CIN2+ vs. CIN1-). Agreement across lots based on CINtec® Histology status (positive/negative) and by CIN status categories (CIN1-/CIN2+) were evaluated.

Acceptance Criteria: Stain intensity shall not vary more than 0.5 point from the median score on a 0-4 scale of each sample on greater than or equal to 85% between lots; all samples shall show equal to or higher than 90% positive/negative agreement for CINtec® Histology status; the

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antibody shall demonstrate background/cross-reactivity less than or equal to 0.5 points on a 0-4 scale in 90% or greater of the tissue samples stained.

Results of the study are presented in Tables 5 and 6 below.

Table 5: Between-Lot Precision for CINtec® Histology

|   |   | Modal CINtec Histology Status  |   |   |
| --- | --- | --- | --- | --- |
|  Modal CIN Category |  | Positive | Negative | Total  |
|  No CIN | # of cases | N = 0 | N = 8 | N = 8  |
|   |  CINtec Histology Status |  | 48/48 (100.0%) | 48/48 (100.0%)  |
|   |  CIN Category |  | 48/48 (100.0%) | 48/48 (100.0%)  |
|  LSIL-Histology | # of cases | N = 1 | N = 3 | N = 4  |
|   |  CINtec Histology Status | 6/6 (100.0%) | 18/18 (100.0%) | 24/24 (100.0%)  |
|   |  CIN Category | 6/6 (100.0%) | 18/18 (100.0%) | 24/24 (100.0%)  |
|  HSIL-Histology | # of cases | N = 12 | N = 0 | N = 12  |
|   |  CINtec Histology Status | 71/71 (100.0%) |  | 71/71 (100.0%)  |
|   |  CIN Category | 69/71 (97.2%) |  | 69/71 (97.2%)  |
|  Cancer | # of cases | N = 2 | N = 0 | N = 2  |
|   |  CINtec Histology Status | 12/12 (100.0%) |  | 12/12 (100.0%)  |
|   |  CIN Category | 12/12 (100.0%) |  | 12/12 (100.0%)  |
|  Total | # of cases | N = 15 | N = 11 | N = 26  |
|   |  CINtec Histology Status | 89/89 (100.0%) | 66/66 (100.0%) | 155/155 (100.0%)  |
|   |  CIN Category | 87/89 (97.8%) | 66/66 (100.0%) | 153/155 (98.7%)  |

Note: A single observation with unevaluable CINtec Histology status for Lot 3 was excluded from analysis.

K212176 - Page 9 of 17

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Table 6. Comparisons of CIN categories across Three Lots

|   |   |   | CIN category-Comparison 2  |   |   |
| --- | --- | --- | --- | --- | --- |
|  Lot Pair | Replicate Pair | CIN category-Comparison 1 | CIN2+ | CIN1- | Total  |
|  All lot pairs | All replicate pairs | CIN 2+ | 157 | 5 | 162  |
|   |   |  CIN 1- | 1 | 145 | 146  |
|   |   |  Total | 158 | 150 | 308  |
|   |   |  Overall Percent Agreement: 98.1%  |   |   |   |
|   |   |  Average Positive Agreement: 98.1%  |   |   |   |
|   |   |  Average Negative Agreement: 98.0%  |   |   |   |

# ii. Immunoreactivity

This study evaluated the immunoreactivity of recombinant CINtec Histology in various normal [Tour of Body (TOB)] and malignant tissues [Tour of Tumor (TOT)]. Additionally, the study evaluated if the positive and negative staining of these same tissue types with recombinant CINtec Histology was consistent with the staining of hybridoma CINtec Histology. Background/cross-reactivity and morphology was also evaluated. The sections from TOB and TOT tissue microarrays and 20 additional individual cases were evaluated in this study. One section from each array or case was stained with recombinant CINtec Histology and hybridoma CINtec Histology and Negative Control (Monoclonal) on BenchMark ULTRA using OptiView DAB IHC detection kit. Furthermore, sections from one prequalified tonsil case were stained with hybridoma CINtec Histology and Negative Control (Monoclonal) were used as a run control. The stained slides were evaluated by pathologist for staining intensities (0-4), background and morphology (acceptable/ unacceptable).

Acceptance Criteria: Background/cross-reactivity less than or equal to 0.5 points on a 0-4 scale in  $90\%$  or greater of the tissue samples stained. The recommended staining protocol shall preserve tissue morphology as noted by the qualified reader in a minimum of  $90\%$  of interpretable samples stained.

Table 7: CINtec Histology staining in FFPE normal tissues (TOB)

|  Tissue types | Recombinant CINtec Histology (# of positive cases/total cases) | Hybridoma CINtec Histology (# of positive cases/total cases) | CINtec Histology (type of positive cells in at least 1 of 3 cases)  |
| --- | --- | --- | --- |
|  Adrenal gland | 3/3 | 3/3 | Adrenocortical epithelial cells  |
|  Appendix | 0/3 | 0/3 | NA  |
|  Bladder | 3/3 | 3/3 | Urothelial cells  |
|  Bone marrow | 2/3 | 2/3 | NA  |
|  Breast | 3/3 | 3/3 | Myoepithelial cells, luminal epithelial cells, stromal cells  |
|  Cerebellum | 3/3 | 3/3 | Purkinje cells  |
|  Cerebrum | 4/4 | 4/4 | Glial cells  |
|  Cervix | 3/3 | 3/3 | Squamous epithelial cells, endocervical cells, stromal cells  |

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K212176 - Page 11 of 17
|  Colon | 3/3 | 3/3 | Epithelial cells (few)  |
| --- | --- | --- | --- |
|  Esophagus | 3/3 | 3/3 | Squamous epithelial cells (few)  |
|  Heart | 0/3 | 0/3 | NA  |
|  Kidney | 3/3 | 3/3 | Tubular epithelial cells, glomeruli mesangial cells (few)  |
|  Liver | 0/3 | 1/5 | Hepatocytes  |
|  Lung | 3/3 | 3/3 | Pneumocytes, bronchial epithelial cells  |
|  Lymph node | 3/3 | 3/3 | Lymphocytes, follicular dendritic cells  |
|  Mesothelium | 0/3 | 0/3 | NA  |
|  Ovary | 3/3 | 3/3 | Stromal cells (few)  |
|  Pancreas | 3/3 | 3/3 | Islets of Langerhans, acinar cells  |
|  Parathyroid | 2/3 | 2/3 | Chief cells  |
|  Peripheral nerve | 4/4 | 3/3 | Schwann cells  |
|  Pharynx/Oral cavity | 2/3 | 2/3 | Respiratory epithelial cells, striated duct epithelial cells, mucous acinar cells  |
|  Pituitary | 3/3 | 3/3 | Anterior pituitary epithelial cells  |
|  Prostate | 3/3 | 3/3 | Acinar cells, basal cells  |
|  Salivary gland | 3/3 | 3/3 | Striated duct epithelial cells, serous acinar cells  |
|  Skeletal muscle | 0/3 | 0/3 | NA  |
|  Small intestine | 3/3 | 3/3 | Epithelial cells (few)  |
|  Soft tissue | 3/3 | 3/3 | Endothelial cells, fibroblasts, ductal cells  |
|  Spleen | 3/3 | 3/3 | Lymphocytes, follicular dendritic cells  |
|  Stomach | 3/3 | 3/3 | Epithelial cells, fundic glands  |
|  Testis | 3/3 | 3/3 | Spermatogenic cells, Leydig cells  |
|  Thymus | 3/3 | 3/3 | Epithelial reticular cells, lymphocytes, Hassall's corpuscles  |
|  Thyroid | 3/3 | 3/3 | Follicular cells, parafollicular cells (few)  |
|  Tonsil | 5/5 | 5/5 | Squamous epithelial cells, lymphocytes, follicular dendritic cells  |
|  Uterus | 3/3 | 3/3 | Endometrial glandular cells, stromal cells  |
|  Skin | 0/3 | 0/3 | NA  |

{11}

Table 8: CINtec Histology staining in a variety of FFPE neoplastic tissues (TOT)

|  Organ/Anatomic Site | Tumor Type | Recombinant CINtec Histology (# of positive cases/total cases) | Hybridoma CINtec Histology (# of positive cases/total cases)  |
| --- | --- | --- | --- |
|   | Meningioma | 1/1 | 1/1  |
|  Cervix | Adenocarcinoma (Endocervical) | 1/1 | 1/1  |
|   |  Squamous Cell Carcinoma | 1/1 | 1/1  |
|  Colon | Adenocarcinoma | 1/1 | 1/1  |
|   |  Adenosquamous carcinoma | 1/1 | 1/1  |
|  Esophagus | Adenocarcinoma | 1/1 | 1/1  |
|   |  Squamous Cell Carcinoma | 0/1 | 0/1  |
|  Head and Neck | Adenocarcinoma | 1/1 | 1/1  |
|   |  Squamous Cell Carcinoma | 0/1 | 0/1  |
|  Kidney | Papillary Renal Adenoma | 1/1 | 1/1  |
|   |  Renal Cell Carcinoma | 1/2 | 1/1  |
|  Liver | Cholangiocarcinoma | 0/1 | 0/1  |
|   |  Hepatocellular Carcinoma | 1/1 | 1/1  |
|  Lymph Node | Anaplastic Large Cell Lymphoma | 1/1 | 1/1  |
|   |  Follicular Lymphoma | 1/1 | 1/1  |
|   |  Hodgkin's Lymphoma | 1/1 | 1/1  |
|  Lung | NSCLC-Adenocarcinoma | 1/1 | 1/1  |
|   |  NSCLC-Squamous Cell Carcinoma | 0/1 | 0/1  |
|   |  Small Cell Lung Carcinoma | 1/1 | 1/1  |
|  Mesothelium | Mesothelioma | 1/1 | 1/1  |
|   |  Pleural Solitary Fibrous Tumor | 1/1 | 1/1  |
|  Muscle | Alveolar Rhabdomyosarcoma | 0/1 | 0/1  |
|   |  Myxoma | 1/1 | 1/1  |
|  Ovary | Granulosa Cell Tumor | 1/1 | 1/1  |
|   |  Serous Carcinoma | 1/1 | 1/1  |
|   |  Teratoma | 1/1 | 1/1  |
|  Pancreas | Islet Cell Tumor | 1/1 | 1/1  |
|   |  Pancreatic Ductal Adenocarcinoma | 1/1 | 1/1  |
|  Peripheral Nerve | Neurofibrosarcoma | 1/1 | 1/1  |
|   |  Schwannoma | 1/1 | 1/1  |
|  Prostate | Adenocarcinoma | 2/2 | 2/2  |
|   | Pleomorphic Adenoma | 1/1 | 1/1  |

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{12}

|  Salivary Gland | Warthin's Tumor | 1/1 | 1/1  |
| --- | --- | --- | --- |
|  Small Intestine | Adenocarcinoma | 0/1 | 0/1  |
|   | Gastrointestinal Stromal Tumor (GIST) | 1/1 | 1/1  |
|  Soft Tissue | Angiosarcoma | 1/1 | 1/1  |
|   |  Liposarcoma | 1/1 | 1/1  |
|  Spleen | Diffuse Large B-Cell Lymphoma | 0/1 | 0/1  |
|  Stomach | Adenocarcinoma | 1/1 | 1/1  |
|   |  Gastrointestinal Stromal Tumor (GIST) | 1/1 | 1/1  |
|  Testis | Embryonal Carcinoma | 1/1 | 1/1  |
|   |  Seminoma | 1/1 | 1/1  |
|  Thyroid | Follicular Thyroid Cancer | 1/1 | 1/1  |
|   |  Papillary Thyroid Cancer | 0/1 | 0/1  |
|  Uterus | Clear Cell Carcinoma | 1/1 | 1/1  |
|   |  Endometrioid or Mucinous carcinoma | 1/1 | 1/1  |
|   |  Leiomyoma | 0/1 | 0/1  |
|   |  Leiomyosarcoma | 1/1 | 1/1  |
|  Skin | Basal Cell Carcinoma | 1/1 | 1/1  |
|   |  Invasive Melanoma | 1/1 | 1/1  |
|   |  Squamous Cell Carcinoma | 0/1 | 0/1  |

The recombinant CINtec Histology and hybridoma CINtec Histology performed equivalently when used to stain a variety of normal and neoplastic tissue types. The tissues stained with hybridoma CINtec Histology exhibited same staining pattern as tissues stained with recombinant CINtec Histology.

2. Linearity:

Not applicable

3. Analytical Specificity/Interference:

a. Western Blot

The primary objective of this study was to confirm the specificity of the recombinant anti-p16 $^{\text{INK4a}}$  antibody to bind p16 $^{\text{INK4a}}$  protein. In addition, the results from the Western blot assay using recombinant antibody were compared with the results using hybridoma Millipore anti-p16 $^{\text{INK4a}}$  (E6H4) antibody. The study also addressed the sensitivity of the recombinant anti-p16 $^{\text{INK4a}}$  antibody by evaluating the lysates from cell lines expressing various amounts of p16 $^{\text{INK4a}}$  protein.

K212176 - Page 13 of 17

{13}

One lot of recombinant anti-p16 antibody and one lot of hybridoma anti-p16 antibody were evaluated. Lysates from four cell lines with various expression levels of p16^{INK4a} protein and purified recombinant p16^{INK4a} protein (positive control) and recombinant GADPH protein (negative control) were used in this study.

Acceptance Criteria: Single band between 15-20 kDa (~16 kDa) must be detected on the Western blot membrane for those lanes loaded with recombinant p16^{INK4a} protein or with lysates from p16^{INK4a} -expressing cell lines and consequently probed with recombinant anti- p16^{INK4a} antibody or hybridoma anti- p16^{INK4a} antibody.

Both hybridoma and recombinant anti- p16^{INK4a} (E6H4) antibody lots performed equivalently in the Western Blot assay demonstrating same sensitivity and specificity for p16^{INK4a} recombinant and endogenous protein, with no significant unexpected off target reactivity observed.

### Peptide Inhibition

The objective of this study is to confirm the specificity of CINtec^{®} Histology formulated with the recombinant anti-p16 antibody for p16 protein in the context of the IHC assay.

One lot of the recombinant CINtec^{®} Histology was used on a BenchMark ULTRA instrument for this study. The anti-p16^{INK4a} (E6H4) antibody was pre-incubated at 1:1 dilution with three different concentrations (3×10^{-6} M, 3×10^{-7} M, and 3×10^{-8} M) of p16 INK4a epitope-specific peptides (AGGTRGSNHARIDAAEGPSDIDP, MW=2265 g/mol). The non-specific peptide used in this study is These three concentrations were selected to span a range of molar ratios: approximately a 1000-fold, 100-fold, and 10-fold molar excess of peptide compared to the final concentration of anti-p16 antibody in the solution (3.3×10^{-9} M). The primary antibody was also diluted 1:1 with three different concentrations (3×10^{-6} M, 3×10^{-7} M, and 3×10^{-8} M) of a non-specific peptide control [Alk-a (amino acid sequence: CWQHQPEDRPNFAIILERIEY), MW=2658 g/mol].

CINtec^{®} Histology/no peptide control solution CINtec^{®} Histology reagent was diluted at a 1:1 ratio with the diluent only. All these solutions contained only 50% of the CINtec^{®} Histology reagent. A multi-tissue block (MTB) containing one case of each CIN3, CIN1, and normal cervical epithelium was used in this study.

Duplicate MTB slides were stained with these prepared solutions and also with undiluted CINtec^{®} Histology reagent. In addition, a single slide was stained with the negative reagent control [Negative Control (Monoclonal)]. All stained slides were scored for p16 staining intensity on a 0-4 scale for each tissue. Note: The p16 staining intensities determined for the cervical tissues stained with CINtec^{®} Histology/no peptide control solution were used as a reference, since this solution contains the same amount of anti-p16 antibody as the solutions containing p16- specific or non-specific peptides (50% only).

Acceptance Criteria: Decreased staining in the tissues stained with recombinant CINtec Histology reagent containing p16 epitope-specific peptide when compared to the tissues stained with recombinant CINtec Histology reagent containing diluent or non-specific peptide.

This study confirmed the specificity of recombinant anti-p16 (E6H4) antibody in the context of the IHC assay. A nearly complete inhibition of the anti-p16 antibody binding to its target protein

{14}

in tissues was detected when the p16 specific peptide was present in the recombinant CINtec® Histology reagent and partial restoration of specific staining was observed when the p16 specific peptide concentration in recombinant CINtec® Histology reagent was reduced. In contrast, no reduction in the signal intensities was observed when unrelated non-specific peptide was present in recombinant CINtec® Histology reagent. No staining was detected when the tissues were stained with Negative Control (Monoclonal) or diluent only.

4. Assay Reportable Range:
Not applicable.

5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):
Not applicable.

6. Detection Limit:
Not applicable.

7. Assay Cut-Off:
See Interpretation of Results in section IV.B above.

8. Accuracy (Instrument):
Please see comparison study below.

9. Carry-Over:
Not applicable.

B Comparison Studies:

1. Method Comparison with Predicate Device:
A method comparison study was performed to compare the performance of the CINtec Histology formulated with the recombinant anti-p16 antibody formulation and the CINtec Histology formulated with the hybridoma anti-p16 antibody (predicate device). The study was conducted using 249 cervical cases [114 No CIN, 17 CIN1, 53 CIN2, 51 CIN3, and 14 cervical SCC cases] run on one BenchMark ULTRA instrument. The cases (H&amp;E stained slides) were reviewed and enrolled into the study by an experienced pathologist. Individual slides were stained with the recombinant CINtec Histology and hybridoma CINtec Histology device, randomized and evaluated for CINtec Histology status (positive/negative) by another pathologist (study pathologist) who was not involved in the selection of study cases.

Acceptance Criteria: Overall percent agreement (OPA) shall demonstrate a lower bound for the two-sided 95% confidence interval (LBCI) of greater than or equal to 85%. Background should be (less than or equal to 0.5 points (on a 0-4 scale) in 90% or greater of the tissue samples stained. Tissue morphology should be preserved as noted by the qualified reader in a minimum of 90% of interpretable samples stained.

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{15}

A summary of the study results is provided in Table 9 below.

Table 9. Agreement on CINtec Histology Status for Recombinant and Hybridoma p16 Antibodies by Enrollment Diagnosis

|   |   | Hybridoma CINtec Histology Result [a] |   |   | Agreement  |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  Enrollment Diagnosis | Recombinant CINtec Histology Result [b] | Positive | Negative | Total | Measure[c] | % (n/N) | 95% CI [d]  |
|  Normal | Positive | 4 | 0 | 4 | PPA | 100.0 (4/4) | (51.0, 100.0)  |
|   |  Negative | 0 | 107 | 107 | NPA | 100.0 (107/107) | (96.5, 100.0)  |
|   |  Total | 4 | 107 | 111 | OPA | 100.0 (111/111) | (96.7, 100.0)  |
|  CIN 1 | Positive | 10 | 0 | 10 | PPA | 100.0 (10/10) | (72.2, 100.0)  |
|   |  Negative | 0 | 6 | 6 | NPA | 100.0 (6/6) | (61.0, 100.0)  |
|   |  Total | 10 | 6 | 16 | OPA | 100.0 (16/16) | (80.6, 100.0)  |
|  CIN 2 | Positive | 48 | 0 | 48 | PPA | 98.0 (48/49) | (89.3, 99.6)  |
|   |  Negative | 1 | 1 | 2 | NPA | 100.0 (1/1) | (20.7, 100.0)  |
|   |  Total | 49 | 1 | 50 | OPA | 98.0 (49/50) | (89.5, 99.6)  |
|  CIN 3 | Positive | 45 | 1 | 46 | PPA | 97.8 (45/46) | (88.7, 99.6)  |
|   |  Negative | 1 | 0 | 1 | NPA | 0 (0/1) | (0.0, 79.3)  |
|   |  Total | 46 | 1 | 47 | OPA | 95.7 (45/47) | (85.8, 98.8)  |
|  SCC | Positive | 11 | 0 | 11 | PPA | 100.0 (11/11) | (74.1, 100.0)  |
|   |  Negative | 0 | 3 | 3 | NPA | 100.0 (3/3) | (43.9, 100.0)  |
|   |  Total | 11 | 3 | 14 | OPA | 100.0 (14/14) | (78.5, 100.0)  |

[a] Hybridoma CINtec Histology result is the final status determined for each slide stained using hybridoma p16 antibody.
[b] Recombinant CINtec Histology result is the final status determined for each slide stained using recombinant p16 antibody.
[c] PPA = Positive Percent Agreement; NPA = Negative Percent Agreement; OPA = Overall Percent Agreement.
[d] Two-sided 95% confidence interval calculated using the Wilson score method.
Note: Cases that do not have evaluable status for slides stained with either Hybridoma or Recombinant p16 antibodies were excluded.

Table 10. Comparison of histology status between Recombinant CINtec® Histology and hybridoma CINtec® Histology

|  |   |   |   |
| --- | --- | --- | --- |
|   | Hybridoma CINtec® Histology Status  |   |   |
|  Recombinant CINtec® Histology Status – | Positive | Negative | Total  |
|  Positive | 118 | 1 | 119  |
|  Negative | 2 | 117 | 119  |

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{16}

|  Total | 120 | 118 | 238  |
| --- | --- | --- | --- |
|  Overall Percent Agreement (OPA) 98.7% (235/238) 95% CI (96.4, 99.6)  |   |   |   |
|  Positive Percent Agreement (PPA) 98.3% (118/120) 95% CI (94.1, 99.5)  |   |   |   |
|  Negative Percent Agreement (NPA) 99.2% (117/118) 95% CI (95.4, 99.9)  |   |   |   |

2. **Matrix Comparison:**
Not applicable.

**C Clinical Studies:**

1. **Clinical Sensitivity:**
Not applicable

2. **Clinical Specificity:**
Not applicable

3. **Other Clinical Supportive Data (When 1. and 2. Are Not Applicable):**
Not applicable

**D Clinical Cut-Off:**
See Interpretation of Results in section IV.B above.

**E Expected Values/Reference Range:**
Not applicable

**F Other Supportive Instrument Performance Characteristics Data:**
Not applicable

**VIII Proposed Labeling:**
The labelling supports the finding of substantial equivalence for this device.

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

K212176 - Page 17 of 17

---

**Source:** [https://fda.innolitics.com/submissions/PA/subpart-b%E2%80%94biological-stains/PRB/K212176](https://fda.innolitics.com/submissions/PA/subpart-b%E2%80%94biological-stains/PRB/K212176)

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