CINtec PLUS Cytology

P190024 · Ventana Medical Systems, Inc. · QKF · Mar 10, 2020 · Hematology

Device Facts

• Record ID: P190024
• Device Name: CINtec PLUS Cytology
• Applicant: Ventana Medical Systems, Inc.
• Product Code: QKF · Hematology
• Decision Date: Mar 10, 2020
• Decision: APPR
• Regulation: 21 CFR 864.1860
• Device Class: Class 3

Indications for Use

The CINtec® PLUS Cytology test is a qualitative immunocytochemical assay intended for the simultaneous detection of the p16INK4a and Ki-67 proteins in cervical specimens collected by a clinician using an endocervical brush/spatula or broom collection device and placed in the ThinPrep® Pap Test PreserveCyt® Solution. The CINtec PLUS Cytology test includes a ready-to-use cocktail of primary antibodies which contains a mouse monoclonal antibody directed against human p16INK4a (p16) protein (clone E6H4), and a recombinant rabbit monoclonal antibody directed against human Ki-67 protein (clone 274-11AC3V1) for use on the BenchMark ULTRA instrument with 3,3-diaminobenzidine tetrahydrochloride (DAB) and Fast Red detection systems. The CINtec PLUS Cytology test is indicated: To be used in women 25 - 65 years old with 12 Other High Risk (HR) HPV positive test results using the cobas® 4800 HPV Test in primary HPV screening, to determine the need for referral to colposcopy. To be used in women 25 - 65 years old with HPV16/18 positive test results using the cobas® 4800 HPV Test in primary HPV screening where the CINtec PLUS Cytology test results will be used in conjunction with the physician's assessment of patient screening history, other risk factors, and professional guidelines to guide patient management. To be used in women 30 - 65 years old with NILM (Negative for Intraepithelial Lesion or Malignancy) and 12 Other HR HPV positive test results using the cobas 4800 HPV Test in adjunctive cervical cytology and HR HPV screening, to determine the need for referral to colposcopy. To be used in women 30 - 65 years old with NILM (Negative for Intraepithelial Lesion or Malignancy) and HPV16/18 positive test results using the cobas® 4800 HPV Test in adjunctive cervical cytology and HR HPV screening where the CINtec PLUS Cytology test results will be used in conjunction with the physician's assessment of patient screening history, other risk factors, and professional guidelines to guide patient management. Results from the CINtec PLUS Cytology test should be interpreted by a qualified pathologist.

Device Story

CINtec PLUS Cytology is an immunocytochemical assay for simultaneous detection of p16INK4a and Ki-67 proteins in cervical cytology specimens. Input: cervical samples in PreservCyt solution. Operation: automated staining on BenchMark ULTRA instrument using primary antibody cocktail (mouse anti-p16 clone E6H4; rabbit anti-Ki-67 clone 274-11AC3V1) and dual-detection systems (DAB for p16, Fast Red for Ki-67). Output: stained slides evaluated by cytotechnologist/pathologist via light microscopy. Dual-stained cells (red nucleus, brown cytoplasm) indicate positive result. Used in clinical labs to triage HR HPV-positive women for colposcopy referral or management. Benefits: improved risk stratification for cervical precancer/cancer compared to cytology alone.

Clinical Evidence

Prospective multicenter IMPACT trial (N=35,263). Evaluated CINtec PLUS as triage for HR HPV+ women (25-65y). Primary endpoints: sensitivity/specificity for ≥CIN2 and ≥CIN3 histology (CPR reference). In 12 Other HR HPV+ women (25-65y), sensitivity for ≥CIN3 was 86.2% (95% CI: 77.8-91.7) and specificity 55.6% (95% CI: 53.7-57.4). In NILM/12 Other HR HPV+ women (30-65y), sensitivity for ≥CIN3 was 66.7% (95% CI: 41.7-84.8) and specificity 69.4% (95% CI: 66.8-71.8).

Technological Characteristics

Immunocytochemistry assay; uses mouse monoclonal anti-p16 (E6H4) and rabbit recombinant anti-Ki-67 (274-11AC3V1). Detection: DAB (brown) for p16, Fast Red (red) for Ki-67. Automated on BenchMark ULTRA system. Requires ThinPrep PreservCyt samples. Connectivity: PC-controlled via Ventana System Software (VSS) v12.3.

Indications for Use

Indicated for women 25-65 years old with HR HPV positive test results (cobas 4800) in primary screening or adjunctive cytology/HPV screening to determine colposcopy referral or guide management in conjunction with clinical history.

Regulatory Classification

Identification

Immunohistochemistry test systems (IHC's) are in vitro diagnostic devices consisting of polyclonal or monoclonal antibodies labeled with directions for use and performance claims, which may be packaged with ancillary reagents in kits. Their intended use is to identify, by immunological techniques, antigens in tissues or cytologic specimens. Similar devices intended for use with flow cytometry devices are not considered IHC's.

Special Controls

(2) Class II (special control, guidance document: “FDA Guidance for Submission of Immunohistochemistry Applications to the FDA,” Center for Devices and Radiologic Health, 1998). These IHC's are intended for the detection and/or measurement of certain target analytes in order to provide prognostic or predictive data that are not directly confirmed by routine histopathologic internal and external control specimens. These IHC's provide the pathologist with information that is ordinarily reported as independent diagnostic information to the ordering clinician, and the claims associated with these data are widely accepted and supported by valid scientific evidence. Examples of class II IHC's are those intended for semiquantitative measurement of an analyte, such as hormone receptors in breast cancer.

Reference Devices

• cobas 4800 HPV Test (P100020)
• ThinPrep 2000 Processor (Hologic, Inc.)
• ThinPrep 5000 Processor (Hologic, Inc.)

Submission Summary (Full Text)

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SUMMARY OF SAFETY AND EFFECTIVENESS DATA (SSED)

I. GENERAL INFORMATION

Device Generic Name: CINtec PLUS Cytology

Device Trade Name: CINtec® PLUS Cytology

Device Procode: QKF

Applicant's Name and Address: Ventana Medical Systems, Inc.
1910 E Innovation Park Drive
Tucson, AZ 85755

Date(s) of Panel Recommendation: None

Premarket Approval Application (PMA) Number: P190024

Date of FDA Notice of Approval: March 10, 2020

II. INDICATIONS FOR USE

The CINtec® PLUS Cytology test is a qualitative immunocytochemical assay intended for the simultaneous detection of the p16INK4a and Ki-67 proteins in cervical specimens collected by a clinician using an endocervical brush/spatula or broom collection device and placed in the ThinPrep® Pap Test PreserveCyt® Solution. The CINtec PLUS Cytology test includes a ready-to-use cocktail of primary antibodies which contains a mouse monoclonal antibody directed against human p16INK4a (p16) protein (clone E6H4), and a recombinant rabbit monoclonal antibody directed against human Ki-67 protein (clone 274-11AC3V1) for use on the BenchMark ULTRA instrument with 3,3-diaminobenzidine tetrahydrochloride (DAB) and Fast Red detection systems.

The CINtec PLUS Cytology test is indicated:

- To be used in women 25 - 65 years old with 12 Other High Risk (HR) HPV positive test results using the cobas® 4800 HPV Test in primary HPV screening, to determine the need for referral to colposcopy.

To be used in women 25 - 65 years old with HPV16/18 positive test results using the cobas® 4800 HPV Test in primary HPV screening where the CINtec PLUS Cytology test results will be used in conjunction with the physician's assessment of patient screening history, other risk factors, and professional guidelines to guide patient management.

- To be used in women 30 - 65 years old with NILM (Negative for Intraepithelial Lesion or Malignancy) and 12 Other HR HPV positive test results using the cobas 4800 HPV Test in adjunctive cervical cytology and HR HPV screening, to determine the need for referral to colposcopy.

To be used in women 30 - 65 years old with NILM (Negative for Intraepithelial Lesion or Malignancy) and HPV16/18 positive test results using the cobas® 4800 HPV Test in adjunctive cervical cytology and HR HPV screening where the CINtec PLUS Cytology test results will be used in conjunction with the physician's assessment of patient screening history, other risk factors, and professional guidelines to guide patient management.

Results from the CINtec PLUS Cytology test should be interpreted by a qualified pathologist.

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# III. CONTRAINDICATIONS

There are no known contraindications.

# IV. WARNINGS AND PRECAUTIONS

The warnings and precautions can be found in the CINtec® PLUS Cytology labeling.

# V. DEVICE DESCRIPTION

CINtec PLUS Cytology is an immunocytochemistry test for use on the Benchmark ULTRA staining system for the simultaneous immunocytochemical detection of the p16INK4a (p16) and Ki-67 proteins in cytological specimens obtained from the uterine cervix. The proteins are detected using a ready-to-use cocktail of primary antibodies which contain a mouse monoclonal antibody directed against human p16INK4a a (p16) protein (clone E6H4), and a recombinant rabbit monoclonal antibody directed against human Ki-67 protein (clone 274-11AC3V1).

The CINtec PLUS Cytology test includes 10 FloLock dispensers filled with ready-to-use Reagents in the configuration as shown in the Figure below.

Figure 1: CINtec PLUS Cytology Configuration
As shown in Figure 1, the test kit includes 10 FloLock dispensers filled with ready-to-use reagents as listed in Table 1 below. Materials required but not provided with CINtec PLUS Cytology test kit are listed in Table 2.

Table 1: Components Included with CINtec PLUS Cytology

| Component* | Description/Composition |
| --- | --- |
| Primary Antibody Cocktail (p16/Ki-67) | p16INK4a (E6H4) Mouse Monoclonal Primary Antibody, 4.0 μg/mL (0.004 mg/mL); Ki-67 (274-11AC3V1-IgG) Rabbit Recombinant Primary Antibody, 0.3 μg/mL (0.0003 mg/mL); Avidin Diluent with B5 blocker |
| Brown Detection System for p16 | |
| HQ (hapten)-labeled Goat Anti-mouse IgG | Goat anti-mouse HQ Conjugate for Cytology, 35 μg/mL (0.035 mg/mL); Avidin Diluent with B5 blocker |
| Mouse monoclonal anti-HQ-labeled HRP Tertiary Antibody | Mouse anti HQ-HRP Conjugate for Cytology 7.5 μg/mL (0.0075 mg/mL); Avidin Diluent with B5 blocker |

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Table 2: Materials Required but not Provided with CINtec PLUS Cytology

| Component | Description |
| --- | --- |
| Reaction Buffer 10x | Tris based buffer solution (pH 7.6 ± 0.2) used to rinse slides between staining steps and provide a stable aqueous environment for reactions carried out on the BenchMark ULTRA instrument |
| ULTRA Liquid CoverSlip (LCS), predilute | Pre-diluted coverslip solution used as a barrier between aqueous reagents and air |
| ULTRA CC1 Cell Conditioning Solution (CC1) | Pre-diluted solution used as a pretreatment step in the processing of tissue samples on the BenchMark ULTRA instrument |
| Hematoxylin | Modified Mayer's hematoxylin used for staining cellular nuclei on slides containing cells from frozen tissue, formalin fixed and paraffin embedded (FFPE) tissue, or cytologic preparations |
| Bluing Reagent | Aqueous solution of buffered lithium carbonate used for bluing hematoxylin stained sections on glass slides |
| CC/Mount™ Aqueous mounting media (Diagnostic Biosystems; DBS) | Aqueous mounting medium with very high refractive index. When applied to the stained tissue sections, specimens can be permanently mounted without chromogens fading |

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# Device Instrument and Software

The CINtec PLUS Cytology test is performed on the automated BenchMark ULTRA Advanced Staining System. It consists of four main modular components that work together as a system: 1) a stainer subassembly where all slide processing operations are performed and which contains a reagent carousel, dispenser mechanism, barcode readers, heating elements and other components; 2) an automated fluidics subassembly (AFS) 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 (version 12.3). The VSS provides slide information such as patient and doctor identification, protocol, and dates. It also provides reagent information such as reagent name and number, expiration date, and tracks the number of dispensers remaining in the reagent packaging. The immunocytochemistry staining process is fully automated and the staining protocol is specific for the CINtec PLUS Cytology device.

# Specimen Preparation

The CINtec PLUS Cytology test is intended for use on cervical cytology specimens collected by a clinician using an endocervical brush/spatula or broom collection device and placed in ThinPrep® Pap Test™ PreservCyt® Solution (Hologic, Inc.). Slides are prepared from these specimens using the FDA approved ThinPrep® 2000 or ThinPrep® 5000 automated slide processor (Hologic, Inc.) according to the labeling.

Cytologic samples in PreservCyt Solution (PC) intended for immunocytochemistry staining using CINtec PLUS Cytology test can be stored at room temperature (15°C to 30°C) for 6 weeks followed by 12 additional weeks refrigerated at 2°C to 8°C.

Dried (i.e., air-dried) slides can be stored at room temperature protected from light and should be stained with CINtec PLUS Cytology test within seven days after slide preparation.

# Test Controls

Each staining run will include one control slide prepared from control material known to have both dual-stain positive and negative elements such as a high-grade intraepithelial lesion (HSIL) slide that has dual-stain positive epithelial cells. The superficial cells of the squamous epithelium which is known to be negative for the expression of both p16 and Ki-67 will serve as the negative control.

# Principles of Procedure

CINtec PLUS Cytology staining will be performed by laboratory personnel trained in the use of the CINtec PLUS Cytology test and in operation of the BenchMark ULTRA instrument. The CINtec PLUS Cytology test requires one slide per case and one control slide per staining run. Following cell conditioning, inhibition of endogenous peroxidase activity and incubation with the primary antibody cocktail, the assay uses two ready-to-use detection systems optimized for use on cervical cytology specimens:

- a goat anti-mouse secondary antibody covalently attached to HQ haptens (proprietary hapten) and an anti-HQ hapten, horseradish peroxidase (HRP)-conjugated tertiary antibody, optimized for the detection of the monoclonal mouse antibody clone E6H4;
- a goat anti-rabbit secondary antibody covalently attached to NP haptens (proprietary hapten) and an anti-NP hapten, alkaline-phosphatase (AP)-conjugated tertiary antibody, optimized for the detection of the rabbit recombinant antibody clone 274-11AC3V1.

The chromogenic reactions are based on the HRP-mediated conversion of DAB resulting in a brown precipitate at the p16INK4a antigen site and the AP-mediated conversion of Fast Red with Naphthol Phosphate resulting in a red precipitate at the Ki-67 antigen site.

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Hematoxylin counterstains the cytoplasm and nuclei of all cells with a blue color. The cytoplasm and nuclei of the superficial squamous cells that do not stain with p16 and/or Ki-67 can be used as a reference to compare staining intensity of other cells with specific p16 and Ki-67 staining. After automated counterstaining (i.e. using hematoxylin and bluing reagent), a two-step mounting procedure is followed. First, the slide is mounted using an aqueous mounting medium. Subsequently, the slide is coverslipped using a permanent mounting medium. The staining results are evaluated by a cytotechnologist and/or a pathologist by light microscopy. See device package insert (PI) for additional details. The staining protocol is provided in Table 3 below.

Table 3: Staining Protocol for CINtec PLUS Cytology on the BenchMark ULTRA Instrument

| Protocol Step | Incubation Time |
| --- | --- |
| Baking | Not applicable |
| Deparaffinization | Not applicable |
| Cell Conditioning – CC1 | 16 minutes |
| Primary Antibody Cocktail (p16/Ki-67) | 16 minutes |
| DAB anti-Mouse HQ Linker | 12 minutes |
| DAB HRP Multimer | 8 minutes |
| DAB detection | 8 minutes |
| Red anti-rabbit NP Linker | 8 minutes |
| Red AP Multimer | 8 minutes |
| Red detection | 16 minutes |
| Hematoxylin | 8 minutes |
| Bluing | 4 minutes |

## Interpretation of CINtec PLUS Cytology Staining

### Control Slide

After a CINtec PLUS Cytology staining run is complete and before case slides are evaluated, a cytotechnologist or pathologist will assess the run control slide to determine whether it is valid, as defined in Table 4 below.

Table 4: Assessment of Run Control Slides, CINtec PLUS Cytology Staining

| Control | Valid | Invalid |
| --- | --- | --- |
| Positive Elements | At least one cell has both specific red nuclear staining and specific brown cytoplasmic staining | No cells have specific red nuclear staining and specific brown cytoplasmic staining |
| Negative Elements | Cell types known to be negative for expression of p16 and of Ki-67 (such as superficial squamous epithelial cells) show neither non-specific red nuclear staining nor non-specific brown cytoplasmic staining that interferes with interpretation | Cell types known to be negative for expression of p16 and of Ki-67 (such as superficial squamous epithelial cells) show non-specific red nuclear staining and/or non-specific brown cytoplasmic staining that interferes with interpretation |

If the control slide is valid, then case slides stained on that run can be evaluated. If the control slide fails to demonstrate appropriate positive staining elements, but the individual case slides have internal positive cells showing specific red Ki-67 and/or brown p16 staining, that case slide will be considered valid for evaluation. If the control slide fails to demonstrate appropriate positive staining elements, individual case slides that do not contain internal positive cells shall be re-tested to confirm a negative result. If the

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control slide shows unacceptable non-specific staining of negative elements, then troubleshooting of certain staining factors such as instrument performance should be investigated. Individual case slides contain internal negative elements which allow for assessment of appropriate staining. Individual case slides should be evaluated and those with unacceptable staining of negative elements that interferes with interpretation should be re-tested.

## Definition of a Satisfactory/Unsatisfactory Slide

Similar to screening Pap cytology slides, CINtec PLUS Cytology slides should be assessed for specimen adequacy similar to the criteria described in The Bethesda System for Reporting Cervical Cytology. This assessment should be done in the context of definitions of satisfactory and unsatisfactory slides specific for CINtec PLUS Cytology, which are given in Table 5 below.

Table 5: Definitions of Satisfactory and Unsatisfactory CINtec PLUS Cytology Slides

| Satisfactory Slide | Unsatisfactory Slide |
| --- | --- |
| All of the following will apply:
1. Slides exhibit satisfactory squamous cellularity* OR a dual-stained cell is present on the slide AND
2. Less than or equal to 75% of squamous cells are obscured (e.g., bacteria, mucus, etc. interferes with interpretation) OR a dual-stained cell is present on the slide AND
3. Background is graded as acceptable | Any of the following apply:
1. Slide does not exhibit satisfactory squamous cellularity* AND no dual-stained cell has been identified OR
2. More than 75% of squamous cells are obscured (e.g., bacteria, mucus, etc. interferes with interpretation) AND no dual-stained cell has been identified OR
3. Background is graded as unacceptable |

* Adequate CINtec PLUS Cytology slides should have an estimated minimum of 5,000 total well-visualized/ well-preserved squamous epithelial cells

## Identification of Dual-Stained Cells

CINtec PLUS Cytology-stained slides with acceptable background will then be assigned a CINtec PLUS Cytology test result of positive, negative, or unsatisfactory according to the criteria defined in Table 6 below.

Table 6: Criteria to Assess p16/Ki-67 Dual-Staining Using CINtec PLUS Cytology

| CINtec PLUS Cytology Test Result | Staining Description |
| --- | --- |
| Positive | Presence of at least one dual-stained cervical epithelial cell
Dual-stained cervical epithelial cells may be present in sheets, overlapping clusters, or isolated single cells |
| Negative | Sample meets squamous cellularity criteria*
-AND-
Cervical epithelial cells staining:
1. Only brown for p16 (nuclear and/or cytoplasmic), or
2. Only red for Ki-67 (nuclear)
3. Only blue counterstain;
but do not show dual p16 and Ki-67 staining in the same cell |

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| CINtec PLUS Cytology Test Result | Staining Description |
| --- | --- |
| Unsatisfactory | Sample does not meet squamous cellularity criteria* and no dual-stained cells are identifiable
-OR-
More than 75% of squamous cells are obscured (e.g., bacteria, mucus, etc. interferes with interpretation) and no dual-stained cells are identifiable
-OR-
Sample has unacceptable background that interferes with staining interpretation |

* Adequate CINtec PLUS Cytology slides should have an estimated minimum of 5,000 total well-visualized/ well-preserved squamous epithelial cells. See CINtec PLUS Cytology Interpretation guide for additional information

VI. ALTERNATIVE PRACTICES AND PROCEDURES

There is currently no alternative FDA-cleared or approved immunocytochemistry assay available for the simultaneous detection of the p16 and Ki-67 proteins in alcohol-fixed cervical cytology specimens.

According to current interim guidelines for primary HPV (Human Papilloma virus) screening for cervical cancer in the US and the FDA-approved use of the cobas HPV Test (P100020) in primary HPV screening, an alternative approach for the triage of women ≥25 years with positive HPV test results is referral of HPV Type 16 genotype positive (HPV16+) or HPV Type 18 genotype positive (HPV18+) women to colposcopy, with Pap cytology triage of women positive for 12 Other HR HPV genotypes.

According to current consensus guidelines for managing abnormal cervical cancer screening tests, there are two alternative management approaches for women ≥30 years with normal Pap cytology and positive HPV test results in the Pap/HPV co-testing setting: (i) repeat co-testing at 12 months, or (ii) HPV DNA genotyping with referral of HPV16+ or HPV18+ women directly to colposcopy and repeat co-testing at 12 months in 12 Other HR HPV+ women. Both are considered acceptable patient management approaches.

The patient's age, medical history (including past HPV and Pap cytology status), and thorough physical examination will provide further information on a patient's risk of cervical disease, as well as the need for referral to colposcopy. The CINtec PLUS Cytology test should only be used in conjunction with this clinical information.

VII. MARKETING HISTORY

The product is currently distributed/marketed in fifty nine countries. The product has not been withdrawn to date from the market in any country for reasons relating to safety and effectiveness of the device.

VIII. POTENTIAL ADVERSE EFFECTS OF THE DEVICE ON HEALTH

Failure of the device to perform as expected or failure to correctly interpret test results may lead to incorrect test results, and subsequently improper patient management decisions in cervical cancer.

For the specific adverse events that occurred in the clinical study, please see Section X below.

IX. SUMMARY OF NONCLINICAL STUDIES

A. Laboratory Studies

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# 1. Analytical Specificity

The CINtec PLUS Cytology consists of the p16INK4a (E6H4) mouse monoclonal primary antibody which targets amino acids 144 to 151 of human p16 protein and the Ki-67 (274-11AC3V1-IgG) rabbit recombinant primary antibody which targets the epitope -LAGFKELF- of human Ki-67 protein.

The following studies were conducted with the p16INK4a (E6H4) and the Ki-67 (274-11AC3V1-IgG) antibodies to establish antibody specificity.

## a. Western Blot

**p16 Primary Antibody**: Western blot analysis was performed on whole cell lysates from four cell lines representing a range of staining intensities when stained with anti- p16INK4a, and purified recombinant p16INK4a and Trefoil Factor 3 (TFF3) proteins (negative control). The cell lines were as follows: High expression cell lines: HeLa (4+) and SK Mel 28 (3+); Moderate expression cell line: DU145 (2+); Negative expression cell line: MDA MB 231 (0+). The Western blot assay tested the ability of the antibody to specifically bind denatured p16INK4a recombinant and endogenous proteins immobilized on a Polyvinylidene difluoride (PVDF) membrane.

**Anti- p16INK4a** antibody detected one ~17 kD band corresponding to the expected molecular weight for endogenous p16INK4a protein in all three immunohistochemistry (IHC) positive cell lines. The intensity of the ~17 kD band correlated with the IHC staining score, showing the strongest signal strength in HeLa (4+), decreasing in SK Mel 28 (3+), and the lowest in DU 145 (2+). No band was observed in the IHC negative cell line MDA MB 231. The antibody bound specifically to purified recombinant p16INK4a protein and not to an equivalent amount of unrelated recombinant protein TFF3.

**Ki-67 Primary Antibody**: Western blot analysis was performed on whole cell lysates prepared from L428 (a Hodgkin's lymphoma positive for Ki-67 antigen, DSMZ ATCC 197) and LNCaP (a prostate carcinoma cell line with low expression of Ki-67 protein, ATCC CRL-1740) cell lines, a purified recombinant TFF3 protein (negative control) and a Ki-67 protein fragment.

The Ki-67 primary antibody detected a ~350kD band corresponding to endogenously expressed Ki-67 protein in the Ki-67 high-expressing L428 cell lysate. No band in this size range was detected by Western blot in the Ki-67 low-expressing cell line LNCaP. A single band of ~95kD was detected in the lane loaded with recombinant Ki-67 protein fragment when probed with Ki-67 primary antibody consistent with the expected molecular weight for the recombinant Ki-67 protein fragment. No band was observed in the TFF3 lane.

## b. Peptide inhibition studies

Analytical specificity of CINtec PLUS Cytology test for its corresponding epitope-specific peptides was evaluated to demonstrate decreased staining of p16INK4a in the presence of a p16 epitope-specific peptide and decreased staining of Ki-67 in the presence of a Ki-67 epitope-specific peptide when compared to a no-peptide control. The primary antibody cocktail was diluted at a 1:1 volume ratio with p16-specific and Ki-67 specific peptide solutions with concentrations of molar ratios of approximately 1-fold, 10-fold, 100-fold, 1,000-fold and 10,000-fold molar excess of peptide compared to the final concentration of anti-p16 antibody in the solution (2.6×10⁻⁸ M) and anti-Ki-67 antibody in the solution (2×10⁻⁹ M). Two control solutions were used in the study. Undiluted primary antibody cocktail (optimal) and primary antibody cocktail diluted at a ratio 1:1 with the antibody diluent, because all peptide solutions contained only 50% of the primary antibody cocktail.

Fifty-two (52) slides were stained using the CINtec PLUS Cytology test kit on a BenchMark ULTRA instrument as follows:

- One slide from each specimen (three cervical cytology specimen pools and one CaSki cells) was stained with each solution (ten specific peptide solutions, two control solutions).

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- One slide from each specimen was incubated with diluent only, which served as a negative control. The slides were evaluated for stain intensity and background by a qualified reader. The p16 and Ki-67 staining intensities determined for the cervical cytology specimens and CaSki slides stained with diluted primary antibody cocktail solution were used as a reference, since this solution contains the same amount of both antibodies as the solutions containing p16-specific or Ki-67-specific peptides (50%).

The specificity of the anti-p16 antibody for the p16 protein and the Ki-67 antibody for the Ki-67 protein was demonstrated by decreased p16 and Ki-67 staining intensities when the slides were stained with primary antibody cocktail containing p16-specific and Ki-67-specific peptides. Complete inhibition of the p16 or Ki-67 staining was observed when the corresponding peptides were present in the solution. No inhibition of the p16 or Ki-67 staining was seen when the unrelated peptides were present in the solution.

## 2. Robustness

### a. Guardbanding studies

Studies were performed to evaluate the robustness (guardbanding) of the CINtec PLUS Cytology device to variations in the staining protocol, instrument settings, and concentrations of manufactured reagents. Study sample characteristics are provided in Table 7 below.

Table 7: Guardbanding Studies - Sample Characteristics

| Sample Category | Pap Category | Number of Samples for Low testing | Number of Samples for High Testing |
| --- | --- | --- | --- |
| Intermediate | NILM | 10 | 10 |
| | ASCUS | 10 | 10 |
| | LSIL | 10 | 10 |
| Positive | HSIL | 30 | 30 |

One reader team was used for evaluation of guardbanding and robustness slides. A cytotechnologist completed a primary read including adequate cellularity, signal intensity, background, CINtec PLUS Cytology status, and whether a positive slide had <10 dual-stained cells. A pathologist completed the confirmation read by confirming all entries. Any responses that the confirmation reader disagreed with included a comment and a consensus discussion between the readers.

The conditions and parameters tested are provided in Table 8 below.

Table 8: Guardbanding Studies - Conditions and Parameters Tested

| Test Category | Variable | Low Condition | Reference Condition | High Condition |
| --- | --- | --- | --- | --- |
| Staining Protocol | Cell conditioning time | 16 minutes | 16 minutes | 24 minutes |
| | Primary antibody time | 12 minutes | 16 minutes | 20 minutes |

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| Test Category | Variable | Low Condition | Reference Condition | High Condition |
| --- | --- | --- | --- | --- |
| | Hematoxylin incubation time | 4 minutes | 8 minutes | 12 minutes |
| Instrument Specifications | Reaction buffer (RB) volume* | 300 μL | 270 μL | 240 μL |
| | Mixing flow rate | 450 mL/min | 480 mL/min | 510 mL/min |
| Reagent Concentration | Reagent formulations | -2% of target concentrations | Target concentrations | +2% of target concentrations |
| Potential impact to functional staining | Reduced signal intensity (possible loss in sensitivity) | n/a | Increased signal intensity | |
| | | | Weak counterstain leading to perceived high background staining | Decreased specificity (increase in background) Nucleus appears black in color and poor staining quality of Ki-67 (red nuclear staining) |

*Reaction buffer volume refers to the amount of reaction buffer applied to the slide prior to the reagent (in dispenser) application. The reagent is applied directly into this reaction buffer “puddle” and therefore is diluted based on the volume of reaction buffer in this specification. For this reason, a larger reaction buffer volume will give a small effective concentration of the reagent being dispensed and therefore is included in the “low” robustness setting.

Study results are provided in Tables 9 and 10 below.

Table 9: Pass Rates for HSIL Sample Category

| Test Condition | Test Group | Metric | No. of Passing Slides | No. of Svaluable Slides | Pass Rate (CI) |
| --- | --- | --- | --- | --- | --- |
| Protocol Setting | Low test condition | p16 signal intensity | 31 | 31 | 100 (89.0, 100) |
| | | DAB background | 31 | 35 | 88.6 (74.0, 95.5) |
| | | Ki-67 signal intensity | 31 | 31 | 100 (89.0, 100) |
| | | Red background | 31 | 31 | 100 (89.0, 100) |
| | | Status | 30 | 30 | 100 (88.6, 100) |
| | High test condition | p16 signal intensity | 35 | 35 | 100 (90.1, 100) |
| | | DAB background | 35 | 35 | 100 (90.1, 100) |
| | | Ki-67 signal intensity | 35 | 35 | 100 (90.1, 100) |

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Table 10: Pass Rates for Intermediate Sample Category

| Test Condition | Test Group | Metric | No. of Passing Slides | No. of Svaluable Slides | Pass Rate (CI) |
| --- | --- | --- | --- | --- | --- |
| Protocol setting | Low test condition | p16 signal intensity | 24 | 24 | 100 (86.2, 100) |
| DAB background | 24 | 29 | 82.8 (65.5, 92.4) |
| Ki-67 signal intensity | 24 | 24 | 100 (86.2, 100) |
| Red background | 24 | 24 | 100 (86.2, 100) |
| Status | 20 | 23 | 87.0 (67.9, 95.5) |
| High test condition | p16 signal intensity | 31 | 31 | 100 (89.0, 100) |
| DAB background | 31 | 31 | 100 (89.0, 100) |
| Ki-67 signal intensity | 31 | 31 | 100 (89.0, 100) |
| Red background | 31 | 31 | 100 (89.0, 100) |
| Status | 29 | 31 | 93.5 (79.3, 98.2) |
| Instrument Setting | Low test condition | p16 signal intensity | 28 | 28 | 100 (87.9, 100) |
| DAB background | 28 | 29 | 96.6 (82.8, 99.4) |
| Ki-67 signal intensity | 28 | 28 | 100 (87.9, 100) |
| Red background | 28 | 29 | 96.6 (82.8, 99.4) |
| Status | 23 | 27 | 85.2 (67.5, 94.1) |
| | p16 signal intensity | 30 | 30 | 100 (88.6, 100) |

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| Test Condition | Test Group | Metric | No. of Passing Slides | No. of Svaluable Slides | Pass Rate (CI) |
| --- | --- | --- | --- | --- | --- |
| | High test condition | DAB background | 30 | 32 | 93.8 (79.9, 98.3) |
| | | Ki-67 signal intensity | 30 | 30 | 100 (88.6, 100) |
| | | Red background | 30 | 31 | 96.8 (83.8, 99.4) |
| | | Status | 26 | 30 | 86.7 (70.3, 94.7) |
| | | P16 signal intensity | 30 | 30 | 100 (88.6, 100) |
| Reagent Formulation | Low test Condition | DAB background | 30 | 30 | 100 (88.6, 100) |
| | | Ki-67 signal intensity | 30 | 30 | 100 (88.6, 100) |
| | | Red background | 30 | 30 | 100 (88.6, 100) |
| | | Status | 26 | 29 | 89.7 (73.6, 96.4) |
| | High test condition | p16 signal intensity | 32 | 32 | 100 (89.3, 100) |
| | | DAB background | 32 | 32 | 100 (89.3, 100) |
| | | Ki-67 signal intensity | 32 | 32 | 100 (89.3, 100) |
| | | Red background | 32 | 33 | 97.0 (84.7, 99.5) |
| | | Status | 25 | 32 | 78.1 (61.2, 89.0) |

## b. Slide Preparation and Coverslipping Robustness Studies

This study was performed to demonstrate the following:

- Ability of CINtec PLUS Cytology to achieve equivalent performance on at least two slide types (Arcless ThinPrep slides and Superfrost PLUS slides).
- Acceptable performance of various pre-staining drying times (60 minutes [recommended], 45 min and 30 min) for test slide that is prepared before performing the CINtec PLUS Cytology test.
- The robustness of post-processing aqueous mounting medium drying time post CINtec PLUS Cytology test (60 min [recommended], 50 min, 40 min and 30 min) to provide acceptable performance.

Stained ThinPrep slides were assessed for signal intensity, cellularity adequacy, background, and CINtec PLUS Cytology test status (positive or negative), by a single cytotechnologist reader. The sample size and study characteristics are provided in Table 11 below.

Table 11: Number of Cases and Slides for Each Test Condition

| Number of Cases | Test condition | Number of slides |
| --- | --- | --- |
| 20 | Slide type assessment (ThinPrep Arcless) | 20 |
| | Slide type assessment (Superfrost PLUS) | 20 |
| | Cover-slipping robustness for 60 minutes pre-staining | 20 |
| | Cover-slipping robustness for 45 minutes pre-staining | 20 |
| | Cover-slipping robustness for 30 minutes pre-staining | 20 |
| 20 | Cover-slipping robustness for 60 minutes post-processing | 20 |
| | Cover-slipping robustness for 50 minutes post-processing | 20 |
| | Cover-slipping robustness for 40 minutes post-processing | 20 |
| | Cover-slipping robustness for 30 minutes post-processing | 20 |

Results are as follows:

Recommended Condition Pass Rate for ThinPrep Arcless Slides:

The recommended condition of drying the ThinPrep Arcless slide for 60 minutes at room temperature after fixation in ethanol provided a passing rate of 100% (20/20). The post-process method of coverslipping using CC/Mount™ aqueous mounting medium (CC/Mount) and then drying for 60 minutes at 60°C in the oven provided a passing rate of 90.0%

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(18/20). The two slides that failed at the recommended condition were due to higher DAB background which is not an anticipated or plausible failure mode for the conditions being tested.

Superfrost PLUS Slides Pass Rate:

The coverslipping robustness pass rate for the Superfrost PLUS Slides was 95.0% (19/20). The one failed slide that failed was due to DAB background, which is not an anticipated or plausible failure mode for the conditions being tested.

Pre-processing Slide Equivalency for ThinPrep Arcless Slides:

Drying the slide for 30 minutes or 45 minutes at room temperature afterfixation in ethanol were the two test conditions for pre-processing. There were 20 ThinPrep Arcless slides prepared and stained for each of the two testing conditions. Both test conditions had an equivalency rate of 95.0% (19/20).

Post-processing Slide Equivalency for ThinPrep Arcless Slides:

Three testing conditions post-processing were considered as follows: (1) dry the slide for 30 minutes, (2) 40 minutes, and, (3) 50 minutes at 60°C after processed with CC/Mount. The 30-minute post-processing had an equivalency rate of 88.9% (16/18) and did not meet the passing endpoint of 90%, 40-minute post-processing equivalency rate was 94.4% (17/18), and 50-minute post-processing equivalency rate was 100% (18/18).

CINtec PLUS Result Concordance Between the Recommended and Test Conditions: From all the testing conditions, 30-minute pre-staining had a concordance of 100% (20/20) for CINtec PLUS result. The 40 and 50-minute poststaining concordance were 100% (20/20). The 45-minute pre-staining dry time was 95% (19/20) and the 30-minute post-staining was 99.4% (17/18) for CINtec PLUS result concordance.

Since the 30-minute dry time failure, it is recommended that the pre- and post-staining drying conditions remain at 60 minutes in a 60°C oven pre-staining (or overnight at room temperature), and 60 minutes at room temperature for post-staining.

c. Effectiveness of Recommended Control Sample

This study was performed to evaluate the effectiveness of control samples to detect assay-related failures. The control samples are ThinPrep patient samples that are known to be positive for CINtec PLUS Cytology and have acceptable background. The ThinPrep samples may be used as controls either as individual cases or in a sample pool where multiple patient cases are pooled together for the purpose of creating more slides per sample. Twenty (20) control sample pools created from qualified individual patient samples were used in this study. The test condition levels and parameters assessed are shown in Table 12 below:

Table 12: Test Condition Levels, Settings and Number of Pools

| Category | Sub-category | Low Severe (10 pools) | Low Moderate (20 pools) | Target (20 pools) | High Moderate (20 pools) | High Severe (10 pools) |
| --- | --- | --- | --- | --- | --- | --- |
| Instrument | Rate of Mixing | 7 mL/min | 225 mL/min | 480 mL/min | 805 mL/min | 1100 mL/min |
| | Puddle Volume | 0.500g | 0.400g | 0.270g | 0.190g | 0.190g |

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| Category | Sub-category | Low Severe (10 pools) | Low Moderate (20 pools) | Target (20 pools) | High Moderate (20 pools) | High Severe (10 pools) |
| --- | --- | --- | --- | --- | --- | --- |
| Instrument | Reaction buffer (RB) formulation change | Not Tested | Not Tested | Bulk RB formulate to design | Half water and Half Reaction Buffer | Water only (in place of RB) |
| Reagent Dispense | Dispense | Missed dispense (0 μL) | Partial dispense (50 μL) | 1 complete dispense (100 μL) | 2 dispenses (200 uL) | 4 dispenses (400 uL) |

- Five severe conditions were tested using slides prepared from 10 pools, five moderate conditions were tested using slides prepared from 20 pools.
- One test slide made from each pool was evaluated using each condition. In addition, one slide from each pool was stained using recommended target conditions (reference slide). The results showed that three of the five severe conditions caused assay failure and the other two conditions were not severe enough to drive the assay to failing results despite being at the uppermost limits of the instrument.
- All 10 pools tested under the instrument category for the high/severe reaction buffer condition (RB) had a failing rate of 100% (10/10).
- All 10 pools tested under the instrument category for the mixers/puddle volume at high/severe conditions had a failing rate of 100% (10/10).
- All 10 pools tested under the reagent dispense category for low/severe conditions had a failing rate of 100% (10/10).
- For two out of the five severe conditions, the 10 pools passed and had appropriate positivity of CINtec PLUS Cytology stain and background acceptability. These included high/severe conditions for mixers/puddle volume and high/severe conditions for reagent dispense.

All 20 pools tested under target and moderate conditions had a passing rate of 100% (20/20).

## 3. Interference

The effect of exogenous and endogenous substances on the performance of the CINtec PLUS Cytology device was evaluated. One hundred twenty (120) ThinPrep (TP) Pap test samples qualified for this study (qualified as HSIL, satisfactory for cellularity, acceptable for background, and acceptable for obscuring elements) were treated with different potentially interfering substances and/or re-processed with glacial acetic acid (GAA) in order to assess the effect on CINtec PLUS Cytology test results. From the 120 individual patient samples included in the study, a total of 60 cases were selected for testing with whole blood and 10 cases were selected for testing with each of the other 6 interfering substances specified in Table 13 below. Each case was tested with CINtec PLUS cytology prior to the addition of the interfering substance as a reference for assay/sample performance. Once each case had a reference slide prepared and stained with CINtec PLUS Cytology device, its respective interfering substance was added to the sample and a slide was then prepared from each vial using the TP 2000 processor and stained with the CINtec PLUS Cytology device.

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Table 13: Interfering Substances Sample Set

| Pap Cytology | Interfering Substance (amount added to vial) | Number of Sample Cases | Number of slides (includes 1 test slide and 1 Reference slide per sample) | Number of Reprocessed Slides | Total Number of slides |
| --- | --- | --- | --- | --- | --- |
| HSIL | Whole blood (1% of vial volume) | 60 | 120 | 60 | 180 |
| HSIL | Mucus (250μL) | 10 | 20 | 10 | 30 |
| HSIL | Leukocytes (1% of vial volume) | 10 | 20 | NA | 20 |
| HSIL | Vaginal Douche (75μL) | 10 | 20 | NA | 20 |
| HSIL | anti-Fungal Cream (18mg-22mg) | 10 | 20 | NA | 20 |
| HSIL | Vaginal Lubricant (30mg-35mg) | 10 | 20 | NA | 20 |
| HSIL | Vaginal Deodorant (30mg-35mg) | 10 | 20 | NA | 20 |

Results are provided in Table 14 below.

Table 14: Interfering Substance Study Results

| Interfering Substance | Number of Slides | | | | Pass Rate (Two-sided 95% CI) | |
| --- | --- | --- | --- | --- | --- | --- |
| | | Total | Positive | Negative | | Invalid |
| Whole blood | Before GAA treatment | 60 | 19 | 0 | 41 | 31.7% (21.3, 44.2) |
| | After GAA treatment | 60 | 60 | 0 | 0 | 100% (94.0, 100) |
| Mucus | Before GAA treatment | 10 | 0 | 0 | 10 | 0% (0, 27.75) |
| | After GAA treatment | 10 | 4 | 0 | 6 | 40% (16.82, 68.73) |
| Leukocytes | | 10 | 8 | 0 | 2 | 80% (49.02, 94.33) |
| Vaginal Douche | | 10 | 0 | 0 | 0 | 100% (72.25, 100) |

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| Interfering Substance | Number of Slides | | | | Pass Rate (Two-sided 95% CI) |
| --- | --- | --- | --- | --- | --- |
| | Total | Positive | Negative | Invalid | |
| anti-Fungal Cream | 10 | 0 | 0 | 0 | 100% (72.25, 100) |
| Vaginal Lubricant | 10 | 9 | 0 | 1 | 90% (59.58, 98.21) |
| Vaginal Deodorant | 10 | 8 | 0 | 2 | 80% (72.25, 100) |

Based on the above, the presence of leukocytes, mucus or vaginal deodorant in the sample are a limitation to the performance of the CINtec PLUS Cytology device.

## 4. Precision

The precision of the CINtec PLUS Cytology device was evaluated on a cohort of pooled ThinPrep Pap test samples (ThinPrep pools) that represent the possible biological variability that can be observed in clinical practice. The device precision was assessed by testing duplicate slides from each cervical cytology sample pool (within-run), over five non-consecutive days on one BenchMark ULTRA instrument (between-day) and with multiple lots of reagents (between-lot). The resulting slides were evaluated for CINtec PLUS Cytology test status (positive or negative) by three teams (one cytotechnologist and 1 pathologist per team) of readers (between-reader). A subset of these slides were read a second time by the same teams of readers (within-reader precision) after a two week wash out period.

Twelve (12) ThinPrep pools were created (Table 15) to test the precision of the device. Nine (9) ThinPrep pools were created from individual patient samples and 3 pools were created from T24 cell lines prepared in PreservCyt® solution (i.e. ThinPrep Pap test collection media). Six (6) different intermediate pools were created (see Table 15). In order to prepare an adequate volume for multiple replicates (slides) in this study, 8 individual patient samples were pooled together to create each pool in the intermediate and true positive pool categories. Therefore these pools may have additional variability beyond the variability from CINtec PLUS Cytology preparation and reading. The patient samples were selected based on ThinPrep Pap test and cobas® 4800 HPV test status. Specimens that demonstrated adequate cellularity and the absence of obscuring elements on Pap test were included in the study.

Table 15: Sample Pools, Precision Study

| Cell Type/ Pap Cytology Status | HR-HPV (cobas) Status | Pool Category | Number of Pools |
| --- | --- | --- | --- |
| T24 Cell line | Negative | True Negative | 3 |
| NILM | Negative | Intermediate (Patient-specimen Derived Negative) | 3 |
| NILM | Positive | Intermediate | 1 |
| ASC-US | Positive | | 1 |
| LSIL | Positive | | 1 |
| HSIL | Positive | True Positive | 3 |

NILM - Negative for intraepithelial lesion or malignancy; ASC-US - Atypical squamous cells of undetermined significance; LSIL - Low-grade intraepithelial lesion; HSIL - High grade intraepithelial lesion

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A total of 30 slides were prepared across multiple days from each pool with 10 slides allocated to each of the 3 CINtec PLUS Cytology reagent lots. Three runs were performed on each day and each run included 2 replicates stained with one of 3 randomly selected lots.

Each of 30 slides was read by 3 reader teams consisting of 1 cytotechnologist and one pathologist in randomized order. Each cytotechnologist evaluated background acceptability, indicated test result as positive or negative for the presence of p16/Ki-67 dual-stain cell(s) and the number of dual-stained cells present up to a count of 50 cells.

Analysis was performed on each cervical cytology pools by determining the pool-level mode across the sources of variability tested (majority call), then calculating the percent of results same as majority call. Each source of variability was studied independently and total imprecision was also calculated. In addition, background acceptability was calculated across all slides stained and dual-stained (positive) cells were counted in all pools from all categories in order to characterize any relationship between Pap/HPV category and CINtec PLUS Cytology dual-stained cell counts.

Within laboratory precision study results are provided in the Tables 16 through 19 below.

Table 16: Within-Laboratory Precision Study - Overall Precision

| Pool Category | Number of Slides/reads | Mode of CINtec PLUS Cytology Results (majority call) | Percent of Results Same as Majority Call | 95% Confidence Interval |
| --- | --- | --- | --- | --- |
| T24 Cell Line | 270 | Negative | 100% | (98.6, 100) |
| NILM/HPV- | 270 | Negative | 94.1% | (90.7, 97.0) |
| NILM/HPV+ | 90 | Positive | 61.1% | (47.2, 74.4) |
| ASCUS/HPV+ | 90 | Positive | 93.3% | (88.9, 97.8) |
| LSIL/HPV+ | 90 | Positive | 100% | (95.9, 100) |
| HSIL/HPV+ | 270 | Positive | 98.9% | (96.7, 100) |

Table 17: Within-Laboratory Precision Study - Between-Day Precision

| Pool Category | Number of Slides/reads | Mode of CINtec PLUS Cytology Results (majority call) | Percent of Results Same as Majority Call | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | | Day 1 | Day 2 | Day3 | Day 4 | Day 5 |
| T24 Cell Line | 270 | Negative | 100% | 100% | 100% | 100% | 100% |
| NILM/HPV- | 270 | Negative | 88.9% | 90.7% | 98.1% | 98.1% | 94.4% |
| NILM/HPV+ | 90 | Positive | 44.4% | 50.0% | 77.6% | 66.7% | 66.7% |
| ASCUS/HPV+ | 90 | Positive | 94.4% | 88.9% | 88.9% | 94.4% | 100% |
| LSIL/HPV+ | 90 | Positive | 100% | 100% | 100% | 100% | 100% |
| HSIL/HPV+ | 270 | Positive | 100% | 94.4% | 100% | 100% | 100% |

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Table 18: Within-Laboratory Precision Study - Between-Lot Precision

| Pool Category | Number of Slides/reads | Mode of CINtec PLUS Cytology Results (majority call) | Percent of Results Same as Majority Call | | |
| --- | --- | --- | --- | --- | --- |
| | | | Lot 1 | Lot 2 | Lot 3 |
| T24 Cell Line | 270 | Negative | 100% | 100% | 100% |
| NILM/HPV- | 270 | Negative | 92.2% | 93.3% | 96.7% |
| NILM/HPV+ | 90 | Positive | 46.7% | 70.0% | 66.7% |
| ASCUS/HPV+ | 90 | Positive | 90.0% | 96.7% | 93.3% |
| LSIL/HPV+ | 90 | Positive | 100% | 100% | 100% |
| HSIL/HPV+ | 270 | Positive | 100% | 100% | 96.7% |

Table 19: Within-Laboratory Precision Study - Between-Reader Precision

| Pool Category | Number of Slides/reads | Mode of CINtec PLUS Cytology Results (Majority Call) | Percent of Results Same as Majority Call | | |
| --- | --- | --- | --- | --- | --- |
| | | | Reader Team 1 | Reader Team 2 | Reader Team 3 |
| T24 Cell Line | 270 | Negative | 100% | 100% | 100% |
| NILM/HPV- | 270 | Negative | 95.6% | 91.1% | 95.6% |
| NILM/HPV+ | 90 | Positive | 70.0% | 63.3% | 50.0% |
| ASCUS/HPV+ | 90 | Positive | 100% | 90.0% | 90.0% |
| LSIL/HPV+ | 90 | Positive | 100% | 100% | 100% |
| HSIL/HPV+ | 270 | Positive | 98.9% | 98.9% | 98.9% |

# 5. Reproducibility

The primary objective of this study was to evaluate the reproducibility of the CINtec PLUS Cytology test in the detection of p16/Ki-67 dual-staining of cervical epithelial cells in liquid based cytology (LBC) specimens at 3 different sites.

The study used a specimen pooling strategy in which individual, de-identified, residual ThinPrep liquid-based cytology specimens of the same HPV status (positive vs negative) and same Pap cytology result (NILM, ASC-US, LSIL, and HSIL) were combined to make specimen pools that had adequate volume to support the study design. Fourteen (14) different intermediate pools were created. In order to prepare an adequate volume for multiple replicates (slides) in this study, 15 individual patient samples were pooled together to create each pool in the intermediate and true positive categories. Therefore these pools may have additional variability beyond the variability from CINtec PLUS Cytology preparation and reading. The study included 21 specimen pools (Table 20) and 6 distinct cultures of T24 cells (a cell line that is negative for p16/Ki-67 dual-staining), for a total sample size of 27.

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Table 20: Specimen Pool Characteristics

| Sample Type | Dual-Staining Category | Pap Cytology | HPV Status | Anticipated CINtec PLUS Cytology Test Result | Number |
| --- | --- | --- | --- | --- | --- |
| Cell Line | True Negative (T24 cells) | N/A | N/A | Negative | 6 cultures |
| LBC Specimens | Intermediate | NILM | Negative | Positive or Negative | 3 pools |
| | | NILM | Positive | Positive or Negative | 3 pools |
| | | ASC-US | Positive | Positive or Negative | 4 pools |
| | | LSIL | Positive | Positive or Negative | 4 pools |
| LBC Specimens | True Positive | HSIL | Positive | Positive | 7 pools |

Aliquots of sufficient volume of each specimen pool (21 in total), T24 cell culture (6 in total), was created for testing at each site. Two study sites prepared a single slide from each specimen pool, T24 culture (27 slides in total) on a single slide processor (Site 1: ThinPrep 2000 Processor and Site 2: ThinPrep 5000 Processor). At Site 3, one set of slides was prepared on ThinPrep 2000 processor and one set of slides was prepared on ThinPrep 5000 processor. The reproducibility study was conducted for 5 days. On each day, each site stained one set of 27 slides using a BenchMark ULTRA instrument. The 5 staining days were non-consecutive and spanned at least 20 days.

At each site, 2 reader-teams, each consisting of a cytotechnologist and a pathologist, independently evaluated the slides stained at their site for the presence or absence of dual staining and assigned the slide a CINtec PLUS Cytology test result of positive, negative, or invalid. The reader-teams were blinded to any prior determination of HPV status, Pap cytology status, CINtec PLUS Cytology test results from other reader teams, or other clinical information.

Results of the reproducibility studies are provided in the Tables 21 and 22 below.

Table 21: Percent Same Result by Pool Category

| Pool Category | Mode of CINtec PLUS Cytology Results (Majority Call) | Percent Same Result as Majority Call | | |
| --- | --- | --- | --- | --- |
| | | % | (n/N) | 95% |
| T24 Cells (True Negative) | Negative | 100% | 240/240 | (98.4, 100) |
| NILM/HPV- | Negative | 94.2% | 113/120* | (90.0, 100) |
| NILM/HPV+ | Positive | 65.8% | 79/120 | (50.0, 77.5) |
| ASC-US/HPV- | Positive | 83.5% | 132/158 | (71.3, 96.2) |
| LSIL/HPV+ | Positive | 88.1% | 140/159 | (81.9, 96.2) |
| HSIL/HPV+ (True Positive) | Positive | 100% | 280/280 | (98.6, 100.0) |

* After all slides at a site had been assigned a CINtec PLUS Cytology test result, the slides were shipped back to the Sponsor. A Sponsor's reader was provided with all evaluable slides from the 3 NILM/HPV-negative pools stained at the sites. The Sponsor's reader evaluated these slides to record the number of dual-stained cells present on each slide. Four out of 60 slides prepared from the 3 NILM/HPV-negative pools had dual stained cells; Three slides had one dual-stained cell and 1 slide had 2 dual-stained cells.

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Table 22: Between-Site Reproducibility Study Results

| Pool Category | Number of Slides/reads | Mode of CINtec PLUS Cytology Results (majority call) | Percent of Results Same as Majority Call | | |
| --- | --- | --- | --- | --- | --- |
| | | | Site 1 | Site 2 | Site 3 |
| T-24 Cell Line | 270 | Negative | 100% | 100% | 100% |
| NILM/HPV- | 270 | Negative | 100% | 86.7% | 95.0% |
| NILM/HPV+ | 90 | Positive | 60.0% | 60.0% | 71.7% |
| ASCUS/HPV+ | 90 | Positive | 72.5% | 82.5% | 89.7% |
| LSIL/HPV+ | 90 | Positive | 85.0% | 77.5% | 94.8% |
| HSIL/HPV+ | 270 | Positive | 100% | 100% | 100% |

# 6. Stability Studies

# a. Sample stability

This study was performed to evaluate the stability of the epitopes in cervical cytology specimens collected and transported in ThinPrep vials containing PreservCyt media and stored for at least 18 weeks, including 6 weeks at room temperature (15°C to 30°C) followed by 12 additional weeks refrigerated at 2°C to 8°C.

Twenty (20) ThinPrep LBC cervical specimens with adequate cellularity, acceptable backgrounds for both biomarkers and positive CINtec PLUS Cytology test were included in the study. One slide from each specimen was prepared and tested at the following testing time points: day zero, 6-week, 19-week. Stained slides were assessed by a qualified reader to determine acceptable cellularity, acceptable background for both biomarkers, positive CINtec PLUS Cytology test result. Sample stability study results are provided in Table 23 below.

Table 23 : Sample Stability Study Results

| Assessment Parameter | 6-wk Testing Time Point | 19-wk Testing Time Point |
| --- | --- | --- |
| CINtec PLUS result (same as for day 0 slide) | 100% | 100% |
| Background (score within 0.25 of day 0 slide | 100% | 95% |
| Staining intensity (score within 0.5 of day 0 slide | 100% | 100% |
| Adequate cellularity (per Bethesda System 2014) | 100% | 100% |

The study supports sample stability for CINtec PLUS Cytology staining that has been stored for 6 weeks at room temperature (15 to $30^{\circ}\mathrm{C}$ ) followed by 12 weeks at $2 - 8^{\circ}\mathrm{C}$ .

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b. Prepared Slide stability

This study was performed to evaluate the ability of the CINtec PLUS Cytology test to achieve equivalent staining on freshly prepared ThinPrep slides (reference slides) and on ThinPrep slides stored protected from light (in slide folders with flaps closed) at room temperature for 7 days before staining with the CINtec PLUS Cytology device.

Twenty (20) ThinPrep liquid based cytology (LBC) cervical specimens that were categorized as HSIL/HPV+ were included in the study. Three slides were prepared from each specimen on Day 0 using the ThinPrep 2000 processor. Slides were then incubated in 99% ethanol solution for 15 minutes before being dried for 60 minutes at room temperature. Overall, 60 slides were created for the study. The first and third slides made from each specimen were stained on Day 0 with the CINtec PLUS Cytology test on the BenchMark ULTRA instrument. These slides were then evaluated by a qualified reader to determine if they met the following inclusion criteria for the study:

- Acceptable cellularity according to Bethesda-based method, acceptable backgrounds for both biomarkers within 0.25 point of each other
- Staining intensity scores for both biomarkers within 0.5 point of each other
- Positive CINtec PLUS Cytology test result

This approach provided assurance that if the readings for first and last slides made from each specimen are equivalent on Day 0, then any different result obtained for the test slides (stained at Day 8) are likely due to slide aging. The second slide from each specimen was stored in a controlled environment protected from light (in slide folders with flaps closed) at room temperature and stained with CINtec Cytology PLUS on Day 8. This slide was evaluated by the qualified reader and compared with the first slide that served as reference slide.

The results from this study demonstrated the following:

- For all 20 cases, staining performance was equivalent between slides stained after being stored for 7 days compared to reference slides stained on Day 0.
- All 20 slides tested after being stored for 7 days after slide preparation showed the same CINtec PLUS Cytology test result as reference slides, acceptable background within 0.25 point from reference slides, and staining intensities within 0.5 point for both biomarkers compared to reference slides and adequate cellularity.

Dried slides can be stored at room temperature protected from light and must be stained with the CINtec PLUS Cytology test within 7 days of preparation.

c. Reagent stability

Reagent stability study was performed to evaluate the interim reagent shelf life and in-use stability of the CINtec PLUS Cytology device. Testing evaluated staining performance (signal intensity and background) of the test on LBC cervical specimens using 3 final lots of the device which were held at the following storage and ship stress conditions and tested at specified intervals until 26 months or failure, whichever occurred first:

- Intended Storage Condition: Test kits from each of the three lots placed at 2-8°C for the duration of testing
- Ship Stress:
- Hot (Category A): Test kits from each of the three lots held at 33±3°C for 192 hours, then placed at 2-8°C for the duration of the study
- Hot (Category B): Test kits from each of the three lots held at 18±3°C for 192 hours, then placed at 2-8°C for the duration of the study
- Cold (Freeze/Thaw): Test kits from each of the three lots held at -20±5°C for 192 hours, then placed at 2-8°C for the duration of the study

At each specified time point, one reference slide and one test slide is created from each enrolled case. The reference slide is stained using a previously released lot of CINtec PLUS Cytology within expiration dating. Stained reference and test slides are then evaluated by qualified readers for adequate cellularity, signal intensity of both biomarkers (p16 and Ki-67), and background. Real-time stability study results are provided in Table 24 below.

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Table 24: Real-Time Stability Testing Results

| All Conditions | | | |
| --- | --- | --- | --- |
| Time Point | Lot 1 (Pass/Fail/Invalid) | Lot 2 (Pass/Fail/Invalid) | Lot 3 (Pass/Fail/Invalid) |
| Day 0 | Positive (100%) | Positive (100%) | Positive (100%) |
| Month 10 | Positive (100%) | Positive (100%) | Positive (100%) |
| Month 15 | Positive (100%) | Positive (100%) | Positive (100%) |

The study results support interim stability dating of 12 months and current shipping category B for the CINtec PLUS Cytology device.

## B. Animal Studies

None

## C. Additional Studies

### ThinPrep 2000/ThinPrep 5000 Equivalency Study

The objective of this study was to verify the ability of the CINtec PLUS Cytology test to achieve equivalent staining on ThinPrep slides prepared on the ThinPrep 2000 and ThinPrep 5000 Processors. All samples were residual ThinPrep liquid-based cytology (LBC) cervical specimens. Specimens were acquired from previously approved external clinical laboratories. Three hundred forty-eight (348) specimens were enrolled in the study and 271 specimens were used for analysis. Samples were enrolled based on a cobas HPV test and a Pap cytology stain with results of NILM/HPV- or HSIL/HPV+. For half of the specimens tested, slide one was made using a T2000 processor and slide two was made on a T5000 processor. For the other half of specimens tested, slide one was made using a T5000 processor and slide two was made on a T2000 processor. A control slide was included in each CINtec PLUS Cytology run to ensure that the system worked as expected. The control slides were produced from known CINtec PLUS Cytology positive specimen pools and evaluated for a positive test result (positive control elements) and acceptable background (negative control elements) to determine staining run validity.

Study slides were evaluated by a reader team consisting of a cytotechnologist and a pathologist. The following parameters were assessed: slide background (non-specific staining), slide cellularity per the Bethesda System criteria, assessment for the presence of obscuring elements (≤75% or >75%), including mucus, bacteria, and inflammatory cells, which may interfere with the interpretation of the assay and assessment for the CINtec PLUS Cytology result. A CINtec PLUS Cytology test result was positive if a minimum of one p16/Ki-67 dual-stained cell was present. Study results are provided in the Table 25 below:

Table 25: CINtec PLUS Cytology Agreement Between Slide Processors

| T5000 Slide CINtec PLUS PLUS Result | T2000 Slide CINtec PLUS | | | Agreement | | |
| --- | --- | --- | --- | --- | --- | --- |
| | Positive | Negative | Total | Measure | % (n/N) | 95% CI |
| Positive | 133 | 7 | 140 | PPA | 91.1 (133/146) | (85.4, 94.7) |
| Negative | 13 | 118 | 131 | NPA | 94.4 (118/125) | (88.9, 97.3) |
| Total | 146 | 125 | 271 | | | |

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# X. SUMMARY OF PRIMARY CLINICAL STUDY

The applicant performed a clinical study in the US to establish a reasonable assurance of safety and effectiveness of the CINtec PLUS Cytology device for the following indications:

(a) To be used in women 25 - 65 years old with 12 Other high-risk (HR) HPV positive test results using the cobas® 4800 HPV Test in primary HPV screening, to determine the need for referral to colposcopy.

To be used in women 25 - 65 years old with HPV16/18 positive test results using the cobas® 4800 HPV Test in primary HPV screening where the CINtec PLUS Cytology test results will be used in conjunction with the physician’s assessment of patient screening history, other risk factors, and professional guidelines to guide patient management.

(b) To be used in women 30 - 65 years old with NILM (Negative for Intraepithelial Lesion or Malignancy) and 12 Other HR HPV positive test results using the cobas 4800 HPV Test in adjunctive cervical cytology and HR HPV screening, to determine the need for referral to colposcopy.

To be used in women 30- 65 years old with NILM (Negative for Intraepithelial Lesion or Malignancy) and HPV16/18 positive test results using the cobas® 4800 HPV Test in adjunctive cervical cytology and HR HPV screening where the CINtec PLUS Cytology test results will be used in conjunction with the physician’s assessment of patient screening history, other risk factors, and professional guidelines to guide patient management.

Data from this clinical study were the basis for the PMA approval decision. A summary of the clinical study is presented below.

## A. Study Design

A multicenter, prospective study (IMPACT trial, IMproved Primary Screening And Colposcopy Triage) was conducted to evaluate the performance of the CINtec PLUS Cytology test as a triage test to stratify high-risk (HR) HPV-positive (HPV+) women 25–65 years old in primary HPV cervical cancer screening using the cobas 4800 HPV Test for referral to colposcopy. Furthermore, the study evaluated the performance of the CINtec PLUS Cytology test as a triage test to stratify women 30 - 65 years with NILM (negative for intraepithelial lesion or malignancy) Pap cytology and positive HPV test results by either the cobas 4800 HPV Test in adjunctive cervical cytology and HPV screening for referral to colposcopy.

### Baseline Phase

The study enrolled 35,263 women 25 - 65 years undergoing routine cervical cancer screening in the US from September 2017 to October 2018 at 32 clinical sites in the Baseline Phase. A total of 34,914 women were eligible to participate in the study.

All women had one cervical sample collected in PreservCyt media at Study Visit 1 (SV1). The specimens were collected using a spatula/brush or broom-type device; approximately half of the specimens were collected with each device. Pap cytology and HPV testing by cobas 4800 HPV Test (Roche Molecular Systems, Inc.), were performed on the PreservCyt samples for all subjects at four testing sites. Pap cytology results were classified according to the criteria of The Bethesda System for Reporting Cervical Cytology. CINtec PLUS Cytology testing was performed on all women with cobas 4800 HPV positive results using the residual volume remaining in the PreservCyt cervical sample collected at SV1.

Women 25 - 65 years with positive cobas HPV Test results (positive by the cobas 4800 HPV Test) were invited to undergo colposcopy.

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Colposcopy was performed at Study Visit 2 (SV2) with colposcopists blinded to the CINtec PLUS Cytology results. Colposcopy was conducted following the principles recommended by the American Society for Colposcopy and Cervical Pathology (ASCCP) as follows. Biopsies were obtained on all visible lesions, and a single random cervical biopsy was obtained from the squamocolumnar junction (SCJ) if no lesions were visible. Endocervical curettage was performed in all patients in whom the SCJ was not visualized or only partially visualized. All biopsies were examined by a Central Pathology Review (CPR) process which included up to three expert pathologists. Discordant histology results were adjudicated according to a pre-defined protocol. The slides that were prepared from the biopsies and reviewed by the CPR panel were stained with hematoxylin and eosin (H&E) and CINtec Histology (p16 IHC assay). The expert pathologist first evaluated H&E-stained slides to establish the CPRH&E reference diagnosis, then evaluated both H&E- and p16 histology-stained slides for that case to establish the CPRH&E+p16 reference diagnosis

The clinical performance of the CINtec PLUS Cytology test in HPV+ (cobas 4800 HPV+) women was assessed using CPR histology results as reference diagnoses at the clinical endpoints ≥CIN2 and ≥CIN3

1. Clinical Inclusion and Exclusion Criteria

Enrollment in the IMPACT study was limited to patients who met the following inclusion criteria:

a. Female 25 - 65 years presenting for routine cervical cancer screening
b. Intact cervix
c. Willing and able to undergo colposcopy and biopsy (and possibly ECC) within 12 weeks from the date of the cervical sample collection
d. Willing and able to provide written informed consent
e. Willing and able to participate in the 1-year Follow-up Phase if required

Patients were not permitted to enroll in the IMPACT study if they met any of the following exclusion criteria:

a. Known pregnancy
b. Current or planned participation in another cervical cancer screening study or in a cervical treatment or vaccine study
c. Incomplete informed consent
d. Any medical condition that, in the opinion of the investigator, would result in increased risk of bleeding at biopsy
e. Known history of ablative or excisional therapy (eg, LEEP, cone biopsy) within the past 12 months
f. Known history of hysterectomy (including supracervical)

2. Follow-up Schedule

All women from SV2 who had a diagnosis of <CIN2 by at least one CPR method and did not undergo treatment during the Baseline Phase were invited to participate in the Year 1-Follow-up Phase (N=6,242). Women who proceeded to the Follow-up Phase underwent Pap cytology and cobas HPV testing at Year 1-Visit 1, approximately one year after Baseline SV1. All women with NILM Pap cytology and negative cobas HPV Test results (both cobas 4800 HPV from Year 1-Visit 1 exited the study. Colposcopy and biopsy/ECC were performed only in non-pregnant women who remained in the study from Year 1-Visit 1. Adverse events and complications were recorded at all visits.

3. Clinical Endpoints

With regards to safety, the incidence of serious adverse events and their potential relation to the investigational device were recorded and monitored during the IMPACT clinical study, and are presented later in this section. Potential safety issues regarding sampling and false positive and false negative test results are discussed in Section VIII.

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With regards to effectiveness, the clinical performance of CINtec PLUS Cytology in HR HPV+ women as tested by the cobas 4800 HPV test was assessed using central pathology review (CPR) histology results as reference diagnoses. The analyses were performed for the ≥CIN2 and ≥CIN3 disease thresholds, as determined by CPR.

With regard to an acceptable performance of CINtec PLUS Cytology in the IMPACT clinical study, performances of CINtec PLUS Cytology test was assessed by the risk (1-NPV) at the ≥CIN3 clinical endpoint, separately for women positive for HPV16/18 (HPV16/18+) and 12 Other HR HPV (12 Other HR HPV+).

## B. Accountability of PMA Cohort

Of the 35,263 subjects enrolled in the study, 34,914 proceeded to Study Visit 1 (SV1). Of these subjects, 5,046 had cobas 4800 HPV positive results. These subjects were referred to colposcopy and and 4,201 proceeded to and completed Study Visit 2 (SV2).

The patient accountability chart is provided in Figure 2 below.

Figure 2: Baseline Analysis Population Disposition for cobas 4800 HPV+ Subjects

## C. Study Population Demographics and Baseline Parameters

The demography of the study population is typical for a cervical cancer screening study performed in the US. Demographic characteristics of the intended use population is provided in the Table 26 below.

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Table 26: Demographic Characteristics of Subjects in the Intended Use Populations

| Demographic Characteristics | HPV+ women 25-65 years old | NILM/HPV+ women 30-65 years old |
| --- | --- | --- |
| | cobas 4800 HPV+ (N=5046) | Cobas 4800 HPV+ (N=2212) |
| Age | | |
| Mean (SD) | 36.8 (10.3) | 41.6 (9.4) |
| Median | 34.0 | 39.0 |
| Min, Max | 25.0, 65.0 | 30.0, 65.0 |
| Age Group | | |
| 25-29 | 1557 (30.9) | 0 (0.0) |
| 30-39 | 1869 (37.0) | 1151 (52.0) |
| 40-49 | 859 (17.0) | 552 (25.0) |
| 50-59 | 585 (11.6) | 395 (17.9) |
| 60-65 | 176 (3.5) | 114 (5.2) |
| Ethnicity | | |
| Hispanic or Latino | 1325 (26.3) | 562 (25.4) |
| Not Hispanic or Latino | 3666 (72.7) | 1629 (73.6) |
| Unknown | 18 (0.4) | 7 (0.3) |
| Not Reported | 37 (0.7) | 14 (0.6) |
| Race | | |
| American Indian/Alaskan Native | 30 (0.6) | 16 (0.7) |
| Asian | 95 (1.9) | 42 (1.9) |
| Black/African-American | 1308 (25.9) | 601 (27.2) |
| Native Hawaiian/Pacific Islander | 12 (0.2) | 1 (0.0) |
| White | 3387 (67.1) | 1467 (66.3) |
| Other | 144 (2.9) | 65 (2.9) |
| Unknown/Not Reported | 70 (1.4) | 20 (0.9) |
| Highest Level of Education | | |
| Elementary | 203 (4.0) | 110 (5.0) |
| High School (or GED) | 1399 (27.7) | 640 (28.9) |
| Vocational/Some College | 1302 (25.8) | 535 (24.2) |
| College Degree | 1402 (27.8) | 601 (27.2) |
| Some Graduate Work | 87 (1.7) | 32 (1.4) |
| Graduate Degree (Master's or Higher) | 461 (9.1) | 233 (10.5) |
| Unknown/Not Reported | 192 (3.8) | 61 (2.8) |

## D. Safety and Effectiveness Results

### 1. Safety Results

The analysis of safety was based on the 5,046 patients. The key safety outcomes for this study (percent of false negative and percent of false positive) are presented in Tables below.

**Adverse effects that occurred in the PMA clinical study:**

There were no adverse events in the PMA clinical study.

### 2. Effectiveness Results

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The analysis of effectiveness was based on the 5,046 evaluable patients. Key effectiveness outcomes (sensitivity, specificity, positive and negative likelihood ratios and positive and negative predictive values along with 95% confidence intervals) are presented in Tables 27 through 64 below.

## EXPECTED RESULTS

A total of 35,263 women 25 - 65 years undergoing routine cervical cancer screening across 32 clinical sites were enrolled in the study. Of these, 5046 women had cobas 4800 positive results: 785 women had HPV 16+ results, 287 women had HPV18+ results and 3,974 women had 12 Other HR HPV+ results.

Tables 27 through 29 below show percent of positive, negative and invalid results of the CINtec PLUS Cytology test for women 25 - 65 years old stratified by testing site, by age group and by Pap cytology results

Table 27: Percent of CINtec PLUS Cytology Results for Women 25 - 65 Years Old Stratified by Testing Site

| | cobas 4800 12 Other HR HPV+ | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| | CINtec PLUS Cytology Result | | | | | |
| Site | Positive | Negative | | | Invalid | |
| Site 1 | 45.7% (315/690) | 47.5% (328/690) | | | 6.8% (47/690) | |
| Site 2 | 47.4% (439/926) | 47.5% (440/926) | | | 5.1% (47/926) | |
| Site 3 | 45.8% (697/1522) | 49.2% (749/1522) | | | 5.0% (76/1522) | |
| Site 4 | 29.3% (245/836) | 59.8% (500/836) | | | 10.9% (91/836) | |
| Combined | 42.7% (1696/3974) | 50.8% (2017/3974) | | | 6.6% (261/3974) | |
| | cobas 4800 HPV 16+ | | | cobas 4800 HPV 18+ | | |
| | CINtec PLUS Cytology Result | | | CINtec PLUS Cytology Result | | |
| Site | Positive | Negative | Invalid | Positive | Negative | Invalid |
| Site 1 | 61.7% (79/128) | 29.7% (38/128) | 8.6% (11/128) | 51.2% (21/41) | 43.9% (18/41) | 4.9% (2/41) |
| Site 2 | 63.0% (114/181) | 30.9% (56/181) | 6.1% (11/181) | 58.3% (35/60) | 35.0% (21/60) | 6.7% (4/60) |
| Site 3 | 65.7% (209/318) | 30.2% (96/318) | 4.1% (13/318) | 57.7% (79/137) | 38.7% (53/137) | 3.6% (5/137) |
| Site 4 | 59.5% (94/158) | 35.4% (56/158) | 5.1% (8/158) | 28.6% (14/49) | 63.3% (31/49) | 8.2% (4/49) |
| Combined | 63.2% (496/785) | 31.3% (246/785) | 5.5% (43/785) | 51.9% (149/287) | 42.9% (123/287) | 5.2% (15/287) |

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Table 28: Percent of CINtec PLUS Cytology Results for Women 25 - 65 Years Old Stratified by Age Group

| | cobas 4800 12 Other HR HPV+ | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| Age Group (Years) | CINtec PLUS Cytology Result | | | | | |
| | Positive | | Negative | | Invalid | |
| 25-29 | 46.6% (617/1324) | | 46.8% (620/1324) | | 6.6% (87/1324) | |
| 30-39 | 42.5% (606/1427) | | 50.9% (727/1427) | | 6.6% (94/1427) | |
| 40-49 | 36.1% (228/632) | | 58.1% (367/632) | | 5.9% (37/632) | |
| 50-65 | 41.5% (245/591) | | 51.3% (303/591) | | 7.3% (43/591) | |
| Combined | 42.7% (1696/3974) | | 50.8% (2017/3974) | | 6.6% (261/3974) | |
| Age Group (Years) | cobas 4800 HPV 16+ | | | cobas 4800 HPV 18+ | | |
| | CINtec PLUS Cytology Result | | | CINtec PLUS Cytology Result | | |
| | Positive | Negative | Invalid | Positive | Negative | Invalid |
| 25-29 | 72.8% (131/180) | 22.2% (40/180) | 5.0% (9/180) | 47.2% (25/53) | 49.1% (26/53) | 3.8% (2/53) |
| 30-39 | 66.0% (217/329) | 29.2% (96/329) | 4.9% (16/329) | 52.2% (59/113) | 41.6% (47/113) | 6.2% (7/113) |
| 40-49 | 49.4% (81/164) | 42.7% (70/164) | 7.9% (13/164) | 49.2% (31/63) | 46.0% (29/63) | 4.8% (3/63) |
| 50-65 | 59.8% (67/112) | 35.7% (40/112) | 4.5% (5/112) | 58.6% (34/58) | 36.2% (21/58) | 5.2% (3/58) |
| Combined | 63.2% (496/785) | 31.3% (246/785) | 5.5% (43/785) | 51.9% (149/287) | 42.9% (123/287) | 5.2% (15/287) |

Table 29: Percent of CINtec PLUS Cytology Results for Women 25 - 65 Years Old Stratified by Pap Cytology Results

| | cobas 4800 12 Other HR HPV+ | | |
| --- | --- | --- | --- |
| | CINtec PLUS Cytology Result | | |
| Pap Cytology | Positive | Negative | Invalid |
| NILM | 29.9% (775/2590) | 62.3% (1614/2590) | 7.8% (201/2590) |
| ASC-US | 58.6% (372/635) | 39.1% (248/635) | 2.4% (15/635) |
| LSIL | 75.0% (419/559) | 22.5% (126/559) | 2.5% (14/559) |
| ASC-H | 86.2% (50/58) | 13.8% (8/58) | 0.0% (0/58) |
| HSIL | 91.4% (53/58) | 6.9% (4/58) | 1.7% (1/58) |

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| | cobas 4800 12 Other HR HPV+ | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| | CINtec PLUS Cytology Result | | | | | |
| Pap Cytology | Positive | | Negative | | Invalid | |
| AGC or ACIS | 90.9% (10/11) | | 9.1% (1/11) | | 0.0% (0/11) | |
| UNSAT | 25.9% (15/58) | | 22.4% (13/58) | | 51.7% (30/58) | |
| Combined | 42.7% (1694/3969) | | 50.7% (2014/3969) | | 6.6% (261/3969) | |
| Pap Cytology | cobas 4800 HPV 16+ | | | cobas 4800 HPV 18+ | | |
| | CINtec PLUS Cytology Result | | | CINtec PLUS Cytology Result | | |
| | Positive | Negative | Invalid | Positive | Negative | Invalid |
| NILM | 42.8% (167/390) | 50.0% (195/390) | 7.2% (28/390) | 34.4% (54/157) | 61.1% (96/157) | 4.5% (7/157) |
| ASC-US | 73.9% (99/134) | 23.9% (32/134) | 2.2% (3/134) | 56.8% (25/44) | 36.4% (16/44) | 6.8% (3/44) |
| LSIL | 84.9% (118/139) | 11.5% (16/139) | 3.6% (5/139) | 78.6% (44/56) | 17.9% (10/56) | 3.6% (2/56) |
| ASC-H | 93.6% (44/47) | 4.3% (2/47) | 2.1% (1/47) | 85.7% (6/7) | 0.0% (0/7) | 14.3% (1/7) |
| HSIL | 100.0% (51/51) | 0.0% (0/51) | 0.0% (0/51) | 100.0% (9/9) | 0.0% (0/9) | 0.0% (0/9) |
| AGC or ACIS | 100.0% (11/11) | 0.0% (0/11) | 0.0% (0/11) | 100.0% (10/10) | 0.0% (0/10) | 0.0% (0/10) |
| UNSAT | 41.7% (5/12) | 8.3% (1/12) | 50.0% (6/12) | 33.3% (1/3) | 0.0% (0/3) | 66.7% (2/3) |
| Combined | 63.1% (495/784) | 31.4% (246/784) | 5.5% (43/784) | 52.1% (149/286) | 42.7% (122/286) | 5.2% (15/286) |

Population of Women 30 - 65 Years Old with NILM Pap Cytology Results

Tables 30 and 31 below show percent of positive, negative and invalid results of the CINtec PLUS Cytology test for women 30 - 65 years with NILM Pap cytology results stratified by testing site and by age group.

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Table 30: Percent of CINtec PLUS Cytology Results for NILM Women 30 - 65 Years Old Stratified by Testing Site

| | cobas 4800 12 Other HR HPV+ | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| | CINtec PLUS Cytology Result | | | | | |
| Site | Positive | | Negative | | Invalid | |
| Site 1 | 32.2% (99/307) | | 61.2% (188/307) | | 6.5% (20/307) | |
| Site 2 | 34.9% (158/453) | | 59.6% (270/453) | | 5.5% (25/453) | |
| Site 3 | 29.4% (174/592) | | 63.7% (377/592) | | 6.9% (41/592) | |
| Site 4 | 17.4% (73/420) | | 71.0% (298/420) | | 11.7% (49/420) | |
| Combined | 28.4% (504/1772) | | 63.9% (1133/1772) | | 7.6% (135/1772) | |
| | cobas 4800 HPV 16+ | | | cobas 4800 HPV 18+ | | |
| | CINtec PLUS Cytology Result | | | CINtec PLUS Cytology Result | | |
| Site | Positive | Negative | Invalid | Positive | Negative | Invalid |
| Site 1 | 44.3% (27/61) | 41.0% (25/61) | 14.8% (9/61) | 23.8% (5/21) | 66.7% (14/21) | 9.5% (2/21) |
| Site 2 | 42.9% (30/70) | 51.4% (36/70) | 5.7% (4/70) | 43.8% (14/32) | 46.9% (15/32) | 9.4% (3/32) |
| Site 3 | 38.5% (40/104) | 57.7% (60/104) | 3.8% (4/104) | 45.1% (23/51) | 52.9% (27/51) | 2.0% (1/51) |
| Site 4 | 40.5% (30/74) | 54.1% (40/74) | 5.4% (4/74) | 11.1% (3/27) | 88.9% (24/27) | 0.0% (0/27) |
| Combined | 41.1% (127/309) | 52.1% (161/309) | 6.8% (21/309) | 34.4% (45/131) | 61.1% (80/131) | 4.6% (6/131) |

Table 31: Percent of CINtec PLUS Cytology Results for NILM Women 30 - 65 Years Old Stratified by Age Group

| | cobas 4800 12 Other HR HPV+ | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| Age Group (Years) | CINtec PLUS Cytology Result | | | | | |
| | Positive | | Negative | | Invalid | |
| 30-39 | 29.6% (276/934) | | 62.6% (585/934) | | 7.8% (73/934) | |
| 40-49 | 24.4% (104/427) | | 69.3% (296/427) | | 6.3% (27/427) | |
| 50-65 | 30.2% (124/411) | | 61.3% (252/411) | | 8.5% (35/411) | |
| Combined | 28.4% (504/1772) | | 63.9% (1133/1772) | | 7.6% (135/1772) | |
| Age Group (Years) | cobas 4800 HPV 16+ | | | cobas 4800 HPV 18+ | | |
| | CINtec PLUS Cytology Result | | | CINtec PLUS Cytology Result | | |
| | Positive | Negative | Invalid | Positive | Negative | Invalid |
| 30-39 | 45.8% (71/155) | 49.0% (76/155) | 5.2% (8/155) | 27.4% (17/62) | 67.7% (42/62) | 4.8% (3/62) |
| 40-49 | 32.6% (30/92) | 57.6% (53/92) | 9.8% (9/92) | 30.3% (10/33) | 60.6% (20/33) | 9.1% (3/33) |

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| | cobas 4800 12 Other HR HPV+ | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| Age Group (Years) | CINtec PLUS Cytology Result | | | | | |
| | Positive | | Negative | | Invalid | |
| 50-65 | 41.9% (26/62) | 51.6% (32/62) | 6.5% (4/62) | 50.0% (18/36) | 50.0% (18/36) | 0.0% (0/36) |
| Combined | 41.1% (127/309) | 52.1% (161/309) | 6.8% (21/309) | 34.4% (45/131) | 61.1% (80/131) | 4.6% (6/131) |

## CLINICAL PERFORMANCE…