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 p16thK4a 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.

Device Description

The CINtec Histology test is a single dispenser 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 primary antibodies and is comprised of 6 dispensers packaged together in one box.

AI/ML Overview

Here's a breakdown of the acceptance criteria and study details for the CINtec Histology device:

1. Acceptance Criteria and Reported Device Performance

The general acceptance criteria for this device type are outlined in 21 CFR 864.1865, specifically under "Special Controls." The clinical study data demonstrated a statistically significant improvement in the consistency of diagnoses by Community Pathologists (CPs) when using CINtec Histology staining. The reported performance relates to the improvement in agreement rates.

Acceptance Criteria Category (Derived from Special Controls)	Specific Metric Referenced in Document	Reported Device Performance (H&E + CINtec Histology)	Baseline Performance (H&E Only)	Difference / Improvement
Clinical Validity (Consistency of CIN Diagnosis)	Positive Percent Agreement (PPA) for ≥HSIL-histology (LAST Cases)	95.9% (165/172)	66.4% (85/128)	29.5% (95% CI: 21.2%; 37.7%) (Statistically Significant)
Clinical Validity (Consistency of CIN Diagnosis)	Negative Percent Agreement (NPA) for ≤LSIL-histology (LAST Cases)	49.6% (131/264)	42.5% (131/308)	7.1% (95% CI: 1.3%; 13.1%) (Statistically Significant)
Clinical Validity (Consistency of CIN Diagnosis)	Positive Percent Agreement (PPA) for ≥HSIL-histology (ALL Cases)	94.1% (206/219)	71.3% (119/167)	22.8% (95% CI: 15.5%; 30.1%) (Statistically Significant)
Clinical Validity (Consistency of CIN Diagnosis)	Negative Percent Agreement (NPA) for ≤LSIL-histology (ALL Cases)	64.4% (567/881)	56.5% (527/933)	7.9% (95% CI: 4.9%; 10.8%) (Statistically Significant)
Staining Performance	Overall Staining Acceptability	99.09% (19,074/19,250)	N/A (criteria for CINtec Histology staining)	N/A
Staining Performance	Morphology Acceptability	99.99% (19,249/19,250)	N/A (criteria for CINtec Histology staining)	N/A
Staining Performance	Background Acceptability	99.99% (19,249/19,250)	N/A (criteria for CINtec Histology staining)	N/A
Precision/Reproducibility	Within-Day (Repeatability) Precision for CINtec Histology Status and CIN Category	100%	N/A	N/A
Precision/Reproducibility	Day-to-Day Precision for CINtec Histology Status and CIN Category	100%	N/A	N/A
Precision/Reproducibility	Instrument-to-Instrument Precision for CINtec Histology Status and CIN Category	100%	N/A	N/A
Precision/Reproducibility	Lot-to-Lot Precision for CINtec Histology Status and CIN Category	100% (CINtec Histology Status), 98.6% (CIN Category)	N/A	N/A
Precision/Reproducibility	Within-Reader Agreement for CINtec Histology Status	98.7%	N/A	N/A
Precision/Reproducibility	Reader-to-Reader Agreement for CINtec Histology Status (Precision Study)	98.7%	N/A	N/A
Reproducibility (Inter-laboratory)	Reader-to-Reader Agreement of CINtec Histology results (Positive: 95.5%, Negative: 92.9%)	N/A	N/A	N/A
Reproducibility (Inter-laboratory)	Day-to-Day Agreement of CINtec Histology results (Positive: 98.2%, Negative: 97.1%)	N/A	N/A	N/A
Reproducibility (Inter-laboratory)	Site-to-Site Agreement of CINtec Histology results (Positive: 96.2%, Negative: 93.9%)	N/A	N/A	N/A
Analytical Specificity	Western Blot, Peptide Inhibition Study, Immunoreactivity (detailed in section M.1.e)	Demonstrated specificity for p16INK4a protein and no unexpected staining	N/A	N/A
Robustness	Tissue Thickness (3, 4, 5, 6, 7 microns), Fixation (10% NBF, zinc formalin, Z-Fix for 6-72 hrs), Staining Options (various combinations)	All demonstrated appropriate specific staining and background levels.	N/A	N/A
Stability	Assay Reagent (24 months at 2-8 °C), Cut-Slide (24 weeks at 2-8°C or 30°C), Shipping Conditions (heated & freeze/thaw)	Acceptable stability confirmed for all conditions.	N/A	N/A

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

The primary clinical study evaluating diagnostic consistency used 1,100 retrospectively collected FFPE cervical punch biopsy specimens.

The data provenance is described as: "retrospectively collected FFPE cervical punch biopsy specimens, which represent a colposcopy referral population." The study involved Board Certified CPs from across the United States.

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

Number of Experts: Three (3) Expert Pathologists (XPs).
Qualifications of Experts: Not explicitly stated beyond "Expert pathologists" and "Board Certified CPs" (for community pathologists). However, the role and processes imply their expertise in cervical pathology.

4. Adjudication Method for the Test Set

The ground truth (Expert-derived Reference Diagnosis) for the test set was established using a 3+1 adjudication method:

Two XPs initially established independent diagnoses based on H&E-stained slides.
Discordant cases were evaluated by a third XP.
Cases for which a "2 out of 3 majority diagnosis" was not achieved were reviewed during an adjudication review meeting that included all three XPs.
"Majority (or consensus) results established the Expert-derived Reference Diagnosis for each case."

This process was repeated for establishing the reference diagnosis based on H&E and CINtec Histology stained slides.

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

Yes, an MRMC comparative effectiveness study was done. This study compared the diagnostic consistency of community pathologists (CPs) with and without the aid of CINtec Histology staining.

The "effect size" can be quantified by the observed differences in Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) between the "H&E Only" and "H&E and CINtec Histology" conditions. The study demonstrated a statistically significant improvement in consistency.

PPA Improvement for ≥HSIL-histology (LAST Cases): 29.5% (from 66.4% to 95.9%)
NPA Improvement for ≤LSIL-histology (LAST Cases): 7.1% (from 42.5% to 49.6%)
PPA Improvement for ≥HSIL-histology (ALL Cases): 22.8% (from 71.3% to 94.1%)
NPA Improvement for ≤LSIL-histology (ALL Cases): 7.9% (from 56.5% to 64.4%)

These values represent the improvement in agreement rates of CPs with the expert reference diagnosis when using CINtec Histology as an adjunct to H&E.

6. If a Standalone (i.e., Algorithm Only Without Human-in-the-Loop Performance) Was Done

No, a standalone (algorithm only) performance study was not done or described. The CINtec Histology device is a qualitative immunohistochemistry test where results are "interpreted using a light microscope by a pathologist." Its indication for use is "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)."

7. The Type of Ground Truth Used

The primary ground truth used for the clinical study was an Expert Consensus Diagnosis (referred to as "Expert-derived Reference Diagnosis"). This was established by a panel of three expert pathologists through initial independent readings and subsequent adjudication (2 out of 3 majority, followed by a review meeting for persistent discordance). This consensus was established twice: once based on H&E alone, and once based on H&E + CINtec Histology.

8. The Sample Size for the Training Set

The document does not explicitly state a separate "training set" for the CINtec Histology product itself. As an immunohistochemistry (IHC) assay for detecting a protein marker, it is a laboratory test with defined reagents and protocols, rather than a machine learning algorithm that requires a dedicated training set. The various analytical and precision studies describe the evaluation of the assay's performance characteristics.

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

As noted above, there is no explicit mention of a "training set" for the device in the context of an algorithm. The development and optimization of the IHC assay would have involved standard laboratory practices, including using known positive and negative control tissues. For instance, the analytical performance section mentions using "cervical carcinoma or CIN2/3 cervical tissue positive for CINtec Histology staining" as a positive control and "normal cervical tissue with negative staining" as a negative control. These are used to confirm assay performance rather than to "train" an algorithm.

Summary

{0}------------------------------------------------

EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR CINtec Histology

DECISION SUMMARY

A. DEN Number:

DEN160019

B. Purpose for Submission:

De Novo request for evaluation of automatic class III designation of the CINtec Histology device

C. Measurand:

p16™K4a protein

D. Type of Test:

Immunohistochemistry, qualitative

E. Applicant:

Ventana Medical Systems, Inc.

F. Proprietary and Established Names:

CINtec Histology

G. Regulatory Information:

1. Regulation section: 21 CFR § 864.1865
1. Classification: Class II (special controls)
1. Product code:

PRB

1. Panel:

{1}------------------------------------------------

H. Indications for use:

1. Indications for use:

2. Special conditions for use statement(s):

For in vitro diagnostic (IVD) use only

For prescription use only

3. Special instrument requirements:

VENTANA BenchMark ULTRA instrument

I. Device Description:

Component	Content
OptiView PeroxidaseInhibitor	3.0% hydrogen peroxide solution.
OptiView HQ UniversalLinker	Cocktail of HQ-labeled antibodies (goat anti-mouse IgG, goat anti-mouse IgM and goat anti-rabbit) (<50µg/mL) in a buffer containing protein with ProClin 300, a preservative.

Table 1: OptiView DAB Detection Kit Components

{2}------------------------------------------------

OptiView HRP Multimer	Mouse monoclonal anti-HQ HRP labeled tertiary antibody(<40µg/mL) in a buffer containing protein with ProClin 300.
OptiView DAB	0.2% 3, 3'-diaminobenzidine tetrahydrochloride (DAB) instabilizer 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 table below lists the ancillary reagents required to perform the CINtec Histology assay.

Table 2: Ancillary Reagents

Reagent	Format Provided	Contents	Purpose
EZ Prep	Bulk / 10XConcentrate /2 liter	Detergent	Removes paraffin from thetissue specimen.
Reaction Buffer	Bulk / 10XConcentrate /2 liter	Tris based buffersolution withdetergent andpreservative	Provides stable environment forantibody-antigen interactions andenzyme reactions. Also used as arinse solution to remove reagentsbetween assay steps.
ULTRA HighTemperatureLiquidCoverslip (LCS)	Bulk / 2 liter	Low densityparaffinichydrocarbon andother oils	Functions as a barrier betweenaqueous solutions and air (i.e.,prevents evaporation of reagentsduring incubation periods on theslide).
ULTRA CellConditioning 1Solution (CC1)	Bulk / Prediluted /1 liter	Tris based buffersolution withdetergent andpreservative	Disrupts covalent bonds at hightemperatures formed by formalin intissue. Increases antibodyaccessibility.
Hematoxylin IICounterstain	Dispenser /Prediluted / 25 mL	Hematoxylin(≤60%); containsglycol and aceticacid stabilizingsolution	Stains cellular nuclei. This is amodified Mayer's hematoxylin.
Bluing Reagent	Dispenser /Prediluted / 25 mL	Solution of 0.1 Mlithium carbonate in0.5 M sodiumcarbonate	Changes the hue of thehematoxylin to a blue color.Applied after hematoxylin.

{3}------------------------------------------------

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 with negative staining 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 for 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 is part of the assay kit to evaluate nonspecific staining. The 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 incubation period for the negative reagent control antibody should be the same as that for the primary antibody.

J. Standard/Guidance Document Referenced:

CLSI I/LA28-A2: Quality Assurance for Design Control and Implementation of Immunohistochemistry Assays; Approved Guideline - Second Edition

Guidance for Submission of Immunohistochemistry Applications to the FDA. 1998

K. Test Principle:

CINtec Histology is an immunohistochemistry device used to stain FFPE cervical punch biopsy tissue slides for the visualization of the p16 Was procein. 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 p1641442 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.

L. 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.

A positive result is defined as "diffuse," when there 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

{4}------------------------------------------------

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.

A negative result is defined as either focal staining of the cervical epithelium or absence of 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.

M. Performance Characteristics:

1. Analytical performance:

a. Precision/Reproducibility:
- i. Within-Day (Repeatability) and Day-to-Day Precision

Within-Day (repeatability) and Day-to-Day precision were evaluated in a study of 24 cervical punch biopsy FFPE tissue specimens [3 cervical squamous cell carcinoma (SCC), 6 CIN3, 6 CIN2, 6 CIN1, and 3 normal cervix cases]. Two replicate slides from each specimen were stained with the CINtec Histology on a single BenchMark ULTRA instrument on each of 5 non-consecutive days. Appropriate control tissue slides were also stained in each run. Each CINtec Histology slide was paired with an H&E slide from an adjacent section for evaluation. All paired slides were randomized, and then evaluated by a single pathologist blinded to the case diagnosis. CINtec Histology status (positive or negative) was determined based on the CINtec Histology slide, and CIN categories (No CIN, LSIL-histology, HSIL-histology, Cancer) were determined based on adjunctive interpretation of the H&E and CINtec Histology slides.

For Within-Day precision (Repeatability). CINtec Histology status for each specimen was compared between duplicates on a single run, with data pooled over the 5 days. The estimate of Within-Day precision was 100%. For Day-to-Day precision. CINtec Histology status of slides from each specimen was compared across all days, using pooled data of all possible pairings. The estimate of Day-to-Day precision was 100%. Results of the study are presented in Table 3.

Table 3: Within-Day (Repeatability) and Day-to-Day Precision: Number of Slides Agreeing with Modal CINtec Histology Status and Modal CIN Category

	Modal CINtec Histology Status
Modal CIN Category		Positive	Negative	Total
No CIN	# of cases	N=0	N=3	N=3
		CINtec HistologyStatus		29/29[1](100.0%)	29/29[1](100.0%)
		CINCategory		29/29[1](100.0%)	29/29[1](100.0%)
LSIL[2]- Histology		# of cases		N = 4	N = 6
		CINtec HistologyStatus	20/20(100.0%)	40/40(100.0%)	60/60(100.0%)
		CINCategory	20/20(100.0%)	40/40(100.0%)	60/60(100.0%)
HSIL[3]- Histology		# of cases	N = 12	N = 0	N = 12
		CINtec HistologyStatus	120/120(100.0%)		120/120(100.0%)
		CINCategory	120/120(100.0%)		120/120(100.0%)
Cancer		# of cases	N = 3	N = 0	N = 3
		CINtec HistologyStatus	30/30(100.0%)		30/30(100.0%)
		CINCategory	30/30(100.0%)		30/30(100.0%)
Total		# of cases	N = 17	N = 7	N = 24
		CINtec HistologyStatus	170/170(100.0%)	69/69[1](100.0%)	239/239[1](100.0%)
		CINCategory	170/170(100.0%)	69/69[1](100.0%)	239/239[1](100.0%)

{5}------------------------------------------------

[ ^ CINtec Histology staining not evaluable for one study sample due to background staining [2]LSIL - Low Grade Squamous Intraepithelial Lesion

13JHSIL - High Grade Squamous Intraepithelial Lesion

ii. Instrument-to-Instrument Precision

Precision of the CINtec Histology assay across 3 BenchMark ULTRA instruments was determined by staining 3 replicate slides of 28 cervical punch biopsy cases (8 normal cervix, 6 CIN1, 6 CIN2, 4 CIN3, and 4 cervical carcinoma cases) using the OptiView DAB IHC Detection kit. Appropriate control tissue slides were also stained in each run. All slides were randomized, and then evaluated by a single pathologist, who was blinded to the case diagnosis, for positive or negative

{6}------------------------------------------------

CINtec Histology status. Each CINtec Histology slide was then paired with an H&E slide from the same case. After randomization of the paired slides, a single pathologist evaluated the CIN categories (No CIN, LSIL-histology, HSILhistology, Cancer) based on the H&E and CINtec Histology slides.

For Instrument-to-Instrument precision, CINtec Histology status of slides for each specimen was compared between instruments by pairwise comparisons. The estimate of Instrument-to-Instrument precision was 100%. Results of the study are presented in Table 4.

		Modal CINtec Histology Status
Modal CIN Category		Positive	Negative	Total
No CIN	# of cases	N = 0	N = 8	N = 8
	CINtec HistologyStatus	72/72(100.0%)	72/72(100.0%)	72/72(100.0%)
	CIN Category	72/72(100.0%)	72/72(100.0%)	72/72(100.0%)
LSIL- Histology	# of cases	N = 4	N = 3	N = 7
	CINtec HistologyStatus	36/36(100.0%)	27/27(100.0%)	63/63(100.0%)
	CINCategory	36/36(100.0%)	27/27(100.0%)	63/63(100.0%)
HSIL- Histology	# of cases	N = 9	N = 0	N = 9
	CINtec HistologyStatus	81/81(100.0%)	81/81(100.0%)	81/81(100.0%)
	CINCategory	81/81(100.0%)	81/81(100.0%)	81/81(100.0%)
Cancer	# of cases	N = 4	N = 0	N = 4
	CINtec HistologyStatus	36/36(100.0%)	36/36(100.0%)	36/36(100.0%)
	CINCategory	36/36(100.0%)	36/36(100.0%)	36/36(100.0%)
Total	# of cases	N = 17	N = 11	N = 28

Table 4: Instrument-to-Instrument Precision: Number of Slides Agreeing with Modal CINtec Histology Status and Modal CIN Category

{7}------------------------------------------------

CINtec HistologyStatus	153/153(100.0%)	99/99(100.0%)	252/252(100.0%)
CINCategory	153/153(100.0%)	99/99(100.0%)	252/252(100.0%)

iii. Lot-to-Lot Precision

Lot-to-Lot precision of the CINtec Histology was evaluated by testing 3 lots of the CINtec Histology primary antibody on duplicate slides of 18 cervical punch biopsy tissue specimens (4 normal cervix, 4 CIN1. 4 CIN2. 4 CIN3. and 2 cervical carcinoma cases) on one BenchMark ULTRA instrument using the OptiView DAB IHC Detection kit. Appropriate control tissue slides were also stained in each run.

Each CINtec Histology slide was paired with an adjacent H&E slide from the same case. Slide sets were randomized, and evaluated by a single pathologist blinded to the case diagnosis and lot number. CINtec Histology status (positive or negative) was determined based on the CINtec Histology slide, and CIN categories [No CIN, LSL-histology, HSIL-histology, Cancer] were determined based on adjunctive interpretation of H&E and CINtec Histology slide.

For Lot-to-Lot precision, CINtec Histology status of slides for each specimen was compared between lots and slide replicates by pairwise comparisons. The estimate of Lot-to Lot precision was 100%. Results of the study are presented in Table 3.

		Modal CINtec Histology Status
Modal CINCategory		Positive	Negative	Total
No CIN	# of cases	N = 0	N = 6	N = 6
	CINtec HistologyStatus		36/36(100.0%)	36/36(100.0%)
	CINCategory		34/36(94.4%)	34/36(94.4%)
LSIL- Histology	# of cases	N = 3	N = 2	N = 5
	CINtec HistologyStatus	18/18(100.0%)	12/12(100.0%)	30/30(100.0%)

Table 5: Lot-to-Lot Precision: Number of Slides Agreeing with Modal CINtec Histology Status and Modal CIN Category

{8}------------------------------------------------

	CIN Category	18/18(100.0%)	12/12(100.0%)	30/30(100.0%)
HSIL- Histology	# of cases	N = 10	N = 0	N = 10
	CINtec Histology Status	60/60(100.0%)		60/60(100.0%)
	CIN Category	60/60(100.0%)		60/60(100.0%)
Cancer	# of cases	N = 3	N = 0	N = 3
	CINtec Histology Status	18/18(100.0%)		18/18(100.0%)
	CIN Category	18/18(100.0%)		18/18(100.0%)
Total	# of cases	N = 16	N = 8	N = 24
	CINtec Histology Status	96/96(100.0%)	48/48(100.0%)	144/144(100.0%)
	CIN Category	96/96(100.0%)	46/48(95.8%)	142/144(98.6%)

iv. Reader Precision

Within- and Between-Reader precision were evaluated on 50 cervical cases (16 normal cervix, 12 CIN1, 12 CIN2, 6 CIN3, and 4 cervical carcinoma cases) stained with the CINtec Histology and the OptiView DAB IHC Detection kit. All slides were randomized, and subsequently evaluated by 3 pathologists for CINtec Histology status. Pathologists were blinded to the case diagnosis. The CINtec Histology slides were re-randomized for a second evaluation of the CINtec Histology status by each of the 3 pathologists following a 4-week washout period. Additionally, each CINtec Histology slide was paired with an H&E slide from the same case and the paired slide sets were randomized. CIN category (No CIN, LSL-histology, HSIL-histology, Cancer) was evaluated by 3 pathologists based on adjunctive interpretation of the H&E and CINtec Histology slides. Following a washout period of at least 4 weeks, slide pairs were re-randomized, and a second evaluation of the CIN category by each of the 3 pathologists was performed.

For Within-Reader precision, CINtec Histology status of 2 slides for each specimen was compared between duplicates from the same reader. The estimate of within-reader agreement was 98.7%. For Reader precision, CINtec Histology status of slides from each specimen was compared across 3 pathologists, using pooled data of all possible pairings. The estimate of Reader-to-Reader agreement was 98.7%. The study results are provided in Table 6.

{9}------------------------------------------------

		Modal CINtec Histology Status
Modal CIN Category		Positive	Negative	Total
No CIN	# of cases	N = 0	N = 19	N = 19
	CINtec HistologyStatus		112/113[1](99.1%)	112/113[1](99.1%)
	CINCategory		107/113(94.7%)	107/113(94.7%)
LSIL-Histology	# of cases	N = 5	N = 5	N = 10
	CINtec HistologyStatus	29/30(96.7%)	30/30(100.0%)	59/60(98.3%)
	CINCategory	27/30(90.0%)	18/30(60.0%)	45/60(75.0%)
HSIL-Histology	# of cases	N = 17	N = 0	N = 17
	CINtec HistologyStatus	102/102(100.0%)		102/102(100.0%)
	CINCategory	88/102(86.3%)		88/102(86.3%)
Cancer	# of cases	N = 4	N = 0	N = 4
	CINtec HistologyStatus	24/24(100.0%)		24/24(100.0%)
	CINCategory	23/24(95.8%)		23/24(95.8%)
Total	# of cases	N = 26	N = 24	N = 50
	CINtec HistologyStatus	155/156(99.4%)	142/143[1](99.3%)	297/299[1](99.3%)
	CINCategory	138/156(88.5%)	125/143[1](87.4%)	263/299[1](88.0%)
		Modal CINtec Histology Status
Modal CIN Category		Positive	Negative	Total
No CIN	# of cases	N = 0	N = 10	N = 10
	CINtec HistologyStatus		153/155(98.7%)	153/155(98.7%)
	CINCategory		134/155(86.5%)	134/155(86.5%)
LSIL-	# of cases	N = 2	N = 2	N = 4

Table 6: Reader Precision: Number of Observations Agreeing with Modal CINtec Histology Status and Modal CIN Category

[1] A single observation with unevaluable CINtec Histology status by Reader 2 was excluded

{10}------------------------------------------------

v. Reproducibility:

An inter-laboratory reproducibility study (Laboratory-to-Laboratory precision study) for the CINtec Histology was conducted using 27 cervical cases (10 No CIN. 5 CIN1. 5 CIN2. 5 CIN3. and 2 cervical carcinoma cases) run across 3 BenchMark ULTRA instruments on each of 3 non-consecutive days at 3 external laboratories. The specimens were randomized and evaluated by a total of 6 pathologists (2 pathologists per site) for both CINtec Histology status (positive/negative) and for CIN category (No CIN, LSL-histology, HSILhistology, Cancer) based on adjunctive interpretation of the H&E and CINtec Histology slides. Pathologists were blinded to the case diagnoses. The study results are provided in Table 5.

For Reader-to-Reader precision, CINtec Histology status of 2 slides corresponding to 2 pathologists at each site from each specimen was compared across 3 days and 3 sites and combined for all specimens. The estimates of Reader-to-Reader agreement of CINtec Histology results were 95.5% for positive CINtec Histology results and 92.9% for negative CINtec Histology results.

For Day-to-Day precision, CINtec Histology status of 2 slides corresponding to two different days from each specimen was compared across 3 days and 3 sites using pooled data of all possible pairings. The estimate of Day-to-Day agreement of CINtec Histology results were 98.2% for positive CINtec Histology results and 97.1% for negative CINtec Histology results.

For Site-to-Site precision. CINtec Histology status of 2 slides corresponding to 2 different sites from each specimen was compared across 3 sites using pooled data of all possible pairings. The estimate of Site-to-Site agreement of CINtec Histology results were 96.2% for positive CINtec Histology results and 93.9% for negative CINtec Histology results.

Table 7: Reproducibility: Number of Observations Agreeing with Modal CINtec Histology Status and Modal CIN Category

{11}------------------------------------------------

		Modal CINtec Histology Status
Modal CIN Category		Positive	Negative	Total
Histology	CINtec Histology Status	34/34(100.0%)	22/32(68.8%)	56/66(84.8%)
	CIN Category	28/34(82.4%)	29/32(90.6%)	57/66(86.4%)
	# of cases	N = 11	N = 0	N = 11
HSIL-Histology	CINtec Histology Status	184/186(98.9%)		184/186(98.9%)
	CIN Category	176/186(94.6%)		176/186(94.6%)
	# of cases	N = 2	N = 0	N = 2
Cancer	CINtec Histology Status	36/36(100.0%)		36/36(100.0%)
	CIN Category	31/36(86.1%)		31/36(86.1%)
	# of cases	N = 15	N = 12	N = 27
Total	CINtec Histology Status	254/256(99.2%)	175/187(93.6%)	429/443(96.8%)
	CIN Category	235/256(91.8%)	163/187(87.2%)	398/443(89.8%)

Forty three observations with unevaluable CDNtec Histology status were excluded. Missing data were distributed across all sites and days: (16 from site A, including 2 on day 1, 4 on day 2 and 10 on day 3; 17 from site B, including 3 on day 1, 8 on day 2, and 6 on day 3; and 10 from site C, including 2 on day 1, 5 on day 2, and 3 on day 3.

b. Linearity/assay reportable range:
Not Applicable
Traceability, Stability, Expected values (controls, calibrators, or methods): C.
- i. Assay Reagent stability:

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

Product expiration dating and shipping conditions were established based on testing with three lots of the CINtec Histology reagent. The intended storage condition (2-8 °C) was tested on 3 replicates of two cervical biopsy specimens (CIN2/CIN3) and one tonsil specimen by staining with CINtec Histology device. Additionally one slide from each of the tissues was stained with the negative reagent to assess background staining. The testing time points were as follows: point zero, month 3, 6, 8, 9, 11, 12, 14, 18, 20, 24, and 26 for all three lots. The staining pattern and staining intensity were assessed at each testing time point. The resulting stability data supported expiration dating of 24 months when the product is stored at 2-8 ℃.

Simulated shipping conditions (heated ship stress at 30℃ and 15℃ and freeze/thaw cold ship stress at -20℃) were also tested using 3 lots of the device on 3 replicates of two cervical biopsy specimens (CIN2/CIN3) and one tonsil specimen. Additionally, 1 slide from each of the tissues was stained with the negative reagent to assess background staining. Three lots of the device were held at heated ship stress condition of 30°C for 192 hours, at 15°C for 192 hours and at the freeze/thaw cold ship stress condition of -20 ℃ for 192 hours. The device was then placed at intended storage (2-8°C) for the duration of testing. The testing time points were the same as the assay reagent stability testing time points. Results for all tested simulated shipping conditions were acceptable.

ii. Cut-Slide stability and storage:
The impact of storage time and temperature on tissue section mounted slides prior to staining was assessed. Slides cut from two multi-tissue blocks, each containing an invasive carcinoma (diffuse staining pattern), a CIN1 (focal staining pattern), and a normal cervical epithelium were stained in duplicate on Day 0. Additional slides from each block were then stored at 30°C and 2-8°C and then stained in duplicate. Slides were tested at point zero and at weeks 2, 4, 5, 6, 7, 8, 9, 10, 14, 18, 22, 26. Results for all slides were compared to the Day 0 results. Results showed no change in staining intensity or background staining up to the 26-week testing time point. Cut-slide stability is set at 24 weeks when stored at 2-8°C or 30°C.
d. Detection limit:
Not Applicable
e. Analytical Reactivity:
- i. Western Blot:

Western Blot analysis was conducted to demonstrate that the CINtec Histology anti-p16 primary antibody specifically detects the p16 "NK44 protein. Two different cell lysates were used. The HeLa cell line is known to express p16 ™K4ª and was used as a positive control, while the P693 cell line does not express p16 NK43 and was used as a negative control. It was expected that one single band would be

{13}------------------------------------------------

detected between 15 kD and 20 kD in the HeLa cells, while the P693 cell lysate should have no signal. The lanes containing the HeLa lysate showed a single band ~18 kDa, and, as expected, the lanes containing the negative control lysate showed no detectable signal.

ii. Peptide Inhibition Study:
A peptide inhibition study was conducted to evaluate antibody specificity of the CINtec Histology. The anti-p164x4a (E6H4) antibody was pre-incubated at 1:1 dilutions with three different concentrations (3x106 M, 3x10-7 M and 3x103 M) of p16 ark4a epitope-specific peptides (AGGTRGSNHARIDAAEGPSDIDP, MW 2265 g/mol) and then applied to stain tissue cases that were known to express p16. The peptide concentrations were selected to span a range of molar ratios: approximately a 1000-fold, 100-fold, and 10-fold molar excess of peptide. The primary antibody was also diluted 1:1 with three different concentrations (3x10-6 M, 3x10- M and 3x10-8 M) of a non-specific peptide control (CWQHQPEDRPNFAIILERIEY, MW=2658 g/mol) and 1:1 with diluent only (no peptide control).

All stained slides were evaluated for stain intensity and non-specific background on a 0-4+ scale. Non-specific background was scored as acceptable (≤ 0.5) or unacceptable (> 0.5). The study confirmed specificity of the anti-p16 mK40 (E6H4) primary antibody to only its immunizing peptide.

iii. Immunoreactivity:
Analytical specificity and sensitivity were determined by staining a variety of normal and neoplastic human tissues with the CINtec Histology. For the purposes of this study, any nuclear and/or cytoplasmic staining was considered as positive staining, unless otherwise specified. Many normal tissues demonstrated staining of a few cells or specific cell types as noted. This may be expected due to the role of the p16 mK4a protein in cell cycle regulation. No unexpected staining was observed with the CINtec Histology on the normal and neoplastic tissues.

Tissue	#Positive/ Totalcases	Cell Type
Cerebrum	1/3a,b	Glial cells
Cerebellum	3/3	Purkinje cells
Adrenal gland	3/3a	Adrenocortical epithelial cells
Ovary	2/2a	Stromal cells and endothelial cells
Pancreas	3/3c	Acinar cells
Tissue	#Positive/ Totalcases	Cell Type
Parathyroidgland	1/1	Chief cells
Hypophysis	3/3	Anterior pituitary epithelial cells
Testis	2/3c	Spermatogenic and Leydig cells
Thyroid	2/5c	Follicular and parafollicular cells
Breast	3/3	Myoepithelial cells, luminal epithelialcells, and stromal cells
Spleen	3/3a	Lymphocytes, follicular dendritic cells
Tonsil	3/3a	Squamous epithelial cells,lymphocytes and follicular dendriticcells
Endometrium	2/3a	Stromal cells
Skeletalmuscle	0/3	No specific staining
Nerve (sparse)	1/3a	Schwann cells
Thymus	1/3a	Epithelial reticular cells
Myeloid (bonemarrow)	0/3	No specific staining
Lung	0/4	No specific staining
Heart (cardiacmuscle)	0/3	No specific staining
Esophagus	0/3	No specific staining
Stomach	0/3	No specific staining
Small intestine	3/3a	Lymphocytes
Colon	3/3a	Lymphocytes and plasma cells
Liver	2/3c	Hepatocytes
Salivary gland	2/3a	Striated duct epithelial cells
Kidney	0/3	No specific staining
Prostate	0/3	No specific staining
Cervix	2/3a	Stromal cells and endocervical cells
Tissue	#Positive/ Totalcases	Cell Type
Skin	0/2	No specific staining
Mesothelium	0/2	No specific staining

Table 8: CINtec Histology staining in FFPE normal tissues

{14}------------------------------------------------

{15}------------------------------------------------

4 few cells staining; 8 nuclear staining only; 8 cytoplasmic staining only

	Table 9: CINtec Histology staining in a variety of FFPE neoplastic tissues
	t and and the province of the first of the first of the first of the first of the first of the first of the first of the first of the first of the first of the first of the f

Neoplasm	# Positive /Total cases	Neoplasm	# Positive /Total cases
Glioblastoma	1/1	Hepatocellularcarcinoma	0/1
Atypical meningioma	0/1	Hepatoblastoma	0/1
Malignant ependymoma	1/1	Renal clear cellcarcinoma	0/1
Malignantoligodendroglioma	0/1a	Prostaticadenocarcinoma	1/2
Ovarian Serous papillaryadenocarcinoma	1/1	Leiomyoma	1/1
Ovarian adenocarcinoma	0/1	Uterine endometrialadenocarcinoma	1/1
Islet cell carcinoma	0/1	Uterine endometrialclear cell carcinoma	1/1a
Pancreaticadenocarcinoma	0/1	Cervical squamous cellcarcinoma	2/2
Seminoma	0/1	Embryonalrhabdomyosarcoma	1/1
Embryonal carcinoma	0/1	Malignant melanoma	1/1
Thyroid Medullarycarcinoma	1/1 a,b	Basal cell carcinoma*	1/1
Thyroid Papillarycarcinoma	0/1	Squamous cellcarcinoma	0/1
Breast intraductalcarcinoma	1/1	Neurofibroma	0/1
Breast invasive ductalcarcinoma	2/2	Neuroblastoma	0/1
Neoplasm	# Positive /Total cases	Neoplasm	# Positive /Total cases
Diffuse B-cell lymphoma	1/3	Epithelial malignantmesothelioma	1/1
Lung small cellundifferentiatedcarcinoma	1/1	Hodgkin lymphoma	1/1
Lung squamous cellcarcinoma	0/1	Anaplastic large celllymphoma	1/1
Lung adenocarcinoma	1/1	Bladder transitional cellcarcinoma	0/1
Esophageal squamous cellcarcinoma	0/1	Low gradeleiomyosarcoma	1/1
Esophagealadenocarcinoma	0/1	Osteosarcoma	1/1
Gastric mucinousadenocarcinoma	1/1	Spindle cellrhabdomyosarcoma	1/1
Gastrointestinaladenocarcinoma	3/3	Intermediate gradeleiomyosarcoma	1/1
GIST	3/3

{16}------------------------------------------------

ª few cells staining; b nuclear staining only

f. Robustness:

i. Tissue Thickness:
Robustness of staining performance due to tissue thickness was evaluated using 3 unique human cervical cases (cervical carcinoma, CIN1, and normal cervix). Tissues were sectioned and tested in duplicate at 3, 4, 5, 6, and 7 microns. Results for all samples were compared to the recommended 4 micron thickness. All tissue thicknesses demonstrated appropriate specific staining and background levels with CINtec Histology.

ii. Fixation:

The impact of pre-analytical factors (fixation type and fixation time) on the p16 mK4 antigen as detected by the CINtec Histology was assessed using fresh xenograft tumors derived from cancer cell line. Tumors were fixed for 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, 48 hours and 72 hours with each of the following fixatives: 10% neutral buffered formalin (10% NBF), zinc formalin, alcohol formalin, alcohol formalin acetic acid (AFA), Prefer solution and Z-fix. The resulting fixed tissues were paraffin embedded, then sectioned and stained with the CINtec Histology. Slides were read by a single reader and scored for p16 staining intensity and background. Results for all fixation types and times were

{17}------------------------------------------------

compared to those for tissues fixed for 12 hours with 10% NBF. Tissues fixed in 10% NBF, zinc formalin, and Z-Fix across all seven fixation times performed equivalently to tissue fixed in 10% NBF for 12 hours. It is recommended that tissues be fixed with 10% NBF for a minimum of 6 hours before staining with the ClNtec Histology device. Alcohol formalin and Prefer fixatives are not recommended for use with CINtec Histology due to demonstrated weaker or variable staining.

iii. Staining Options:
All user selectable options in the CINtec Histology staining procedure on the BenchMark ULTRA stainer were validated using 6 cervical samples (1 CIN1, 3 CIN2, 2 normal cervix) stained with all possible combinations allowed within the CINtec Histology staining procedure, for a total of 6 staining conditions. Results from each sample were compared against the recommended protocol. All samples tested for all staining conditions yielded equivalent results to the recommended condition with regard to both the CINtec Histology staining pattern and CINtec Histology positive/negative status.
1. Comparison studies:
- a. Method comparison with predicate device:

Not applicable

b. Matrix comparison:
FFPE uterine cervical punch biopsy specimen is the only recommended matrix.
1. Clinical studies:
  To demonstrate that the CINtec Histology results in an improvement in consistency of the diagnosis of cervical intraepithelial neoplasia (CIN), levels of agreement between Community pathologists' (CP) and Expert pathologists' (XP) readings of cervical punch biopsy tissue were evaluated in a clinical study. The clinical study was performed on 1,100 retrospectively collected FFPE cervical punch biopsy specimens, which represent a colposcopy referral population. An XP derived reference diagnosis was established for each study case using the hematoxylin & eosin (H&E) stained slides only and using the H&E and CINtec Histology stained slides. Study slides were assessed by CPs reviewing H&E stained slides only in the first round (Round 1, CP1) and by reviewing H&E and CINtec Histology stained slides in the second round (Round 2, CP2) for each study case by same pathologists after a 4-week washout period. The results were compared to XP diagnosis for each study case to evaluate positive, negative and overall agreements. Two XPs established their independent diagnoses [No CIN, CIN1, CIN2, CIN3, adenocarcinoma in situ (ACIS), or invasive carcinoma] based on the H&E-stained slides for each of the 1,100 cases. The pathologists were also provided with the following clinical information: patient age. Pap cytology result and HPV test result (if available). Discordant cases were evaluated by a third XP. Cases for which a 2 out of 3 majority diagnosis was not achieved were reviewed during an adjudication review meeting that

{18}------------------------------------------------

included all three XPs. Majority (or consensus) results established the Expert-derived Reference Diagnosis for each case evaluated in the study. After a minimum of 4 week washout period, the same XPs evaluated both the H&E and CINtec Histology slides to establish their diagnosis (No CIN, LSIL-histology, HSIL-histology, (ACIS, or invasive carcinoma). The process of establishing the majority diagnoses was that used for establishing the Reference Diagnosis on H&E-stained slides only.

Seventy (70) Board Certified CPs, from across the United States, participated in the study. In the first round (Round 1, CP1), the 1,100 H&E-stained cases were divided into 4 reading sets of 275 cases with comparable distributions of individual diagnostic categories per Reference Diagnosis. The 70 CPs were assigned to 4 groups consisting of either 17 or 18 pathologists per group. For each case within their assigned reading set, the pathologists were provided with the following clinical information: patient age, Pap cytology result and HPV test result (if available). The CPs independently rendered their diagnoses on the H&E-stained slide for each of their assigned cases (No CIN, CIN1, CIN2, CIN3, ACIS, or invasive carcinoma). In addition, CPs were asked during Round 1 reading whether they would request an adjunctive p16 IHC stain (CINtec Histology) in alignment with the following criteria from the LAST recommendations ': 1) the H&E morphologic differential diagnosis is between pre-cancer (CIN2 or CIN3) and a mimic of pre-cancer; 2) the H&E morphologic diagnosis is CIN2; or 3) the H&E morphologic diagnosis is < CIN1 and the biopsy specimen is at high risk for missed high-grade disease, which is defined as prior cytologic interpretation of HSIL, ASC-H (atypical squamous cells, cannot rule out high-grade squamous intraepithelial lesion), ASC-US/HPV16+ (atypical squamous cells of undetermined significance/HPV16+), or AGC-(NOS) (atypical glandular cells- not otherwise specified).

In the second round (Round 2, CP2), the CPs read the H&E-stained slides along with the paired corresponding CINtec Histology-stained slides for the same set of cases within their assigned reading set. After at least a 4-week washout period between Rounds 1 and 2, each pathologist independently rendered their diagnoses (No CIN, LSIL-histology, HSIL-histology, ACIS, or invasive carcinoma). The CPs noted the CINtec Histology status (positive = diffuse CINtec Histology staining; negative = focal or no CINtec Histology staining) along with their histological diagnosis using both the H&E-stained slide along with the CINtec Histology stained slide.

A) Cases for which p16 staining was required according to LAST 2012 recommendations by majority of CP (LAST Cases) There were 436 cases for which p16 staining was required by the majority of CPs per Round 1 questionnaire. For these cases, the levels of agreement between EPs using H&E alone or both H&E and CINtec Histology is shown in Table 10 below.
1 Darragh TM, Colgan TJ, Cox JT, et al. The Lower Anogenital Squamous Terminology Standardization Project for HPV-Associated Lesions: Background and Consensus Recommendations from the College of American Pathologists and the American Society for Colposcopy and Cervical Pathology. Arch Pathol Lab Med - Vol 136. October 2012

{19}------------------------------------------------

		Reference Diagnosis =Majority/Consensus Diagnosis by Expert PanelH&E Only					Total
		NoCIN	CIN1	CIN2	CIN3	ACIS orCancer
ReferenceDiagnosis =Majority/Consensus byExpert PanelH&E and CINtecHistology	No CIN	175	4	4	0	0	183
	LSIL-histology	15	61	4	1	0	81
	HSIL-histology	24	29	79	37	0	169
	ACIS orcancer	0	0	0	0	3	3
Total		214	94	87	38	3	436

Table 10: XP Agreement, H&E only vs. H&E and CINtec Histology (LAST Cases)

The levels of agreement between CPs using H&E alone vs. the Reference Diagnosis by XPs using H&E alone and agreements between CPs with H&E and CINtec Histology vs. the Reference Diagnosis by XPs with H&E and CINtec Histology are presented in Table 11 below.

Table 11: CP Agreement with Reference Diagnosis, H&E only vs. H&E+CINtec histology (LAST Cases)

	H&E Only				H&E and CINtec Histology
	Reference Diagnosis =Majority/Consensus Diagnosis byExpert Panel				Reference Diagnosis =Majority/Consensus Diagnosis by ExpertPanel
	No CIN	CIN1	CIN2	≥CIN3	No CIN	LSIL-histology	HSIL-histology	ACISorCancer
Number of cases	214	94	87	41	183	81	169	3
Percent of cases withCP majoritydiagnosis the same asReference Diagnosis	29.0%(62/214)	73.4%(69/94)	72.4%(63/87)	53.7%(22/41)	39.3%(72/183)	72.8%(59/81)	96.4%(163/169)	66.7%(2/3)
Number of CP withdiagnosis the same asCP majority averagedover all cases	10.5	11.8	10.6	10.1	11.3	12.6	14.8	16.5

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

	H&E OnlyReference Diagnosis =Majority/Consensus Diagnosis byExpert Panel		H&E and CINtec HistologyReference Diagnosis =Majority/Consensus Diagnosis by ExpertPanel
	≤CIN1	≥CIN2	≤LSIL-histology	≥HSIL-histology
Number of cases	308	128	264	172
Percent of cases withCP majoritydiagnosis the same asReference Diagnosis	42.5%(131/308)	66.4%(85/128)	49.6%(131/264)	95.9%(165/172)
Number of CP withdiagnosis the same asCP majority averagedover all cases	11.2	10.5	11.9	14.8

The estimate of positive percent agreement (PPA) was based on the comparison of the agreement between CPs using H&E and CINtec Histology vs. Reference Diagnosis by XPs using H&E and CINtec Histology for cases with reference diagnoses of ≥HSILhistology and the agreement between CPs using H&E vs Reference Diagnosis by XPs using H&E for cases with reference diagnoses of ≥CIN2. The estimate of negative percent agreement (NPA) was based on the comparison of the agreement between CPs using H&E and CINtec Histology vs. Reference Diagnosis by XPs using H&E and CINtec Histology for cases with reference diagnoses of <LSIL-histology and the agreement between CPs using H&E vs. Reference Diagnosis by XPs using H&E for cases with reference diagnoses of <CIN1. The clinical study data demonstrated a statistically significant improvement in consistency of the diagnoses by CPs when using CINtec Histology staining as summarized in Table 12.

Agreement	H&E and CINtecHistology	H&E Only	Difference	95% CI
PPA	95.9%(165/172)	66.4%(85/128)	29.5%	21.2%; 37.7%
NPA	49.6%(131/264)	42.5%(131/308)	7.1%	1.3%; 13.1%

Table 12: Positive and Negative Percent Agreements (LAST Cases)

B) All cases regardless of LAST 2012 recommendations (ALL Cases) There were 1,100 cases in the study. The levels of agreement between EPs using H&E alone or both H&E and CINtec Histology are shown in Table 13 below.

{21}------------------------------------------------

		Reference Diagnosis = Majority/ConsensusDiagnosis by Expert PanelH&E Only					Total
		NoCIN	CIN1	CIN2	CIN3	ACIS orCancer
ReferenceDiagnosis =Majority/ConsensusDiagnosis byExpert PanelH&E +p16(CINtec histology)	No CIN	693	13	4	0	0	710
	LSIL-histology	46	120	4	1	0	171
	HSIL-histology	30	31	83	69	1	214
	ACIS or cancer	0	0	0	0	5	5
Total		769	164	91	70	6	1100

Table 13: EP Agreement, H&E only vs. H&E and CINtec Histology (ALL Cases)

Table 14: CP Agreement with Reference Diagnosis, H&E only vs. H&E+CINtec histology (ALL Cases)

	H&E Only				H&E +p16 (CINtec Histology)
	Reference Diagnosis =Majority/Consensus Diagnosis by ExpertPanel - XP1				Reference Diagnosis = Majority/ConsensusDiagnosis by Expert Panel - XP2
	No CIN	CIN1	CIN2	≥CIN3	No CIN	LSIL-histology	HSIL-histology	ACIS orCancer
Number ofcases	769	164	91	76	710	171	214	5
Percent ofcases with CPmajoritydiagnosis thesame asReferenceDiagnosis	50.8%(391/769)	82.9%(136/164)	69.2%(63/91)	73.7%(56/76)	60.1%(427/710)	81.9%(140/171)	94.4%(202/214)	80.0%(4/5)
Number of CPwith diagnosisthe same as CPmajority	12.6	13.2	10.6	12.9	12.8	13.6	15.2	16.0

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

averaged overall cases	≤CIN1	≥CIN2	≤LSIL-histology	≥HSIL-histology
Number ofcases	933	167	881	219
Percent ofcases with CPmajoritydiagnosis thesame asReferenceDiagnosis	56.5%(527/933)	71.3%(119/167)	64.4%(567/881)	94.1%(206/219)
Number of CPwith diagnosisthe same as CPmajorityaveraged overall cases	12.7	11.7	13.0	15.2

The estimate of PPA was based on the comparison of the agreement between CPs using H&E and CINtec Histology vs. Reference Diagnosis by XPs using H&E and CINtec Histology for cases with reference diagnoses of ≥HSIL-histology and the agreement between CPs using H&E vs Reference Diagnosis XPs using H&E for cases with reference diagnoses of ≥CIN2. The estimate of NPA was based on the comparison of the agreement between CPs using H&E and CINtec Histology vs. Reference Diagnosis by XPs using H&E and CINtec Histology for cases with reference diagnoses of _LSILhistology and the agreement between CPs using H&E vs. Reference Diagnosis by XPs using H&E for cases with reference diagnoses of <CIN1. The clinical study data demonstrated a statistically significant improvement in consistency of the diagnoses by CPs when using CINtec Histology staining as summarized in Table 15.

Table 15: Positive and Negative Percent Agreements, All Cases

Agreement	H&E and CINtecHistology	H&E Only	Difference	95% CI
PPA	94.1%(206/219)	71.3%(119/167)	22.8%	15.5%; 30.1%
NPA	64.4%(567/881)	56.5%(527/933)	7.9%	4.9%; 10.8%

C) Percent of CINtec Histology positive results by CIN Diagnosis

The association between majority/consensus CINtec Histology status (Positive or Negative) by expert panel and the majority/consensus diagnosis by expert panel using

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

H&E alone is shown in Table 16. CNtec Histology positive results showed an increasing trend with increasing severity of CIN diagnosis.

	Reference Diagnosis =Majority/Consensus Diagnosis by Expert Panel with H&E
	No CIN	CIN1	CIN 2	CIN3	Cancer
Percent CINtecHistologypositive results	7.5%(57/755)	58.3%(95/163)	94.5%(86/91)	98.6%(69/70)	100%(1/1)

Table 16: Percent of CINtec Histology positive results by CIN Diagnosis

Note: Fifteen Observations with unevaluable CINtec Histology status were excluded, 14 were No CIN and 1 was CIN1 by expert panel using H&E only

D) CINtec Histology Staining Performance

A total of 19.250 ClNtec Histology interpretations were rendered during the study by the 70 CPs. The staining performance criteria assessed included overall staining acceptability, background staining acceptability (background does not interfere with the clinical interpretation of the stain) and morphology acceptability (cellular elements of interest are visualized allowing clinical interpretation of the stain). The results are presented in Table 17 below.

Table 17: CINtec Histology Staining Performance

	Number of Interpretationsn/N	Rate
Staining Acceptability	19,074 / 19,250	99.09%
Morphology Acceptability	19,249 / 19,250	99.99%
Background Acceptability	19,249 / 19,250	99.99%

1. Clinical cut-off:
  Refer to Section M - Interpretation of Results.
1. Expected values/Reference range:
  Not applicable

N. System Descriptions:

1. Modes of Operation:

The CINtec Histology assay is performed on the BenchMark ULTRA instrument which includes the staining system with embedded software, host PC with installed VSS software system (version 12.2), and system peripherals (printer, slide labeler, mouse & keyboard). Reagents are loaded onto the instrument to perform the assay. The CINtec Histology assay protocol is assay specific. The system performs all operations required to automatically process slides for IHC staining with the CINtec Histology device.

2. Software:

{24}------------------------------------------------

FDA has reviewed the applicant's Hazard Analysis and software development processes for this line of product types:

Yes X or No ______________________________________________________________________________________________________________________________________________________________

1. Calibration and Quality Controls: See discussion of controls in section I above

O. Proposed Labeling:

The labeling is sufficient and it satisfies the requirements of 21 CFR Parts 801 and 809, as applicable, and the special controls for this device type.

P. Patient Perspectives:

This submission did not include specific information on patient perspectives for this device.

Q. Identified Risks to Health and Identified Mitigations:

Identified Risks to Health	Identified Mitigations
Inaccurate test results, such as false positive orfalse negative results	General controls and special controls (1)and (2)
Failure to correctly interpret test results canlead to false positive or false negative results	General controls and special controls (1)and (2)

R. Benefit/Risk Analysis:

Summary
Summary ofthe Benefit(s)	This is an immunohistochemistry test intended to improve the consistency ofCIN diagnosis by community pathologists in punch biopsies of the cervixuteri, providing a clinically important medical need.
	In unselected specimens, this device provides an improvement in theconsistent diagnosis of cervical pre-cancers when used in combination withH&E, as compared to H&E alone. In cases that are considered by pathologiststo be challenging, difficult, or of critical clinical importance, the device isexpected to provide a larger benefit in diagnosis.
Summary
Summary ofthe Risk(s)	There is minimal potential risk associated with use of this device given thecombination of required general controls and special controls. The primary risksto patients are related to the consequences of clinical decisions based on falsenegative and false positive results due to inaccurate test results or failure tocorrectly interpret test results. A false positive could lead to an additionalcolposcopy and possibly to a cervical loop electrosurgical excision procedure(LEEP). These are relatively low risk procedures. A false negative or no resultcould lead to a delay in diagnosis of a cervical high grade squamousintraepithelial lesion (HSIL). Such a delay, unless it is on the order of years, isunlikely to result in progression of HSIL to invasive carcinoma, and ismitigated by the concomitant use of H&E with the device, and continuedfollow-up of patients with other abnormal tests indicating potential uterinecervical disease.There are additional risks associated with collection of tissue specimens fortesting with the device. The test requires that a colposcopic biopsy of thecervix uteri be obtained. This is a standard procedure in clinical care, and therisk to patients is minimal. Further, the risk to laboratory workers is no greaterthan that for the routine collection and handling of tissue specimens, giventhat the test is for use by laboratory professionals in a clinical laboratorysetting. These risks are mitigated by the appropriate CLIA (ClinicalLaboratory Improvement Amendments) categorization of the device andlabeling.The risks of the test are mitigated since the results from this test are intendedto be used with results from other clinical, cervical cytology and HPV testingresults. The assay is not to be used as a standalone diagnostic and is to be usedin conjunction with other clinical and laboratory findings such as results ofpatient Pap tests and HPV tests. The risks are further mitigated by the specialcontrols established for this device.
Summary ofOther Factors	None
ConclusionsDo theprobablebenefitsoutweigh theprobable risks?	Yes, the probable benefits of this device, which allows evaluation of p16staining by community pathologists for the diagnosis of CIN on a moreconsistent basis, outweigh the probable risks, given the combination ofrequired general controls and special controls established for this device.

{25}------------------------------------------------

S. Conclusion:

The information provided in this de novo submission is sufficient to classify this device into class II under regulation 21 CFR 864.1865.

{26}------------------------------------------------

FDA believes that the stated special controls and applicable general controls, including design controls, provide reasonable assurance of the safety and effectiveness of the device type. The device is classified under the following:

Product Code:	PRB
Device Type:	A cervical intraepithelial neoplasia (CIN) test system
Class:	II (special controls)
Regulation:	21 CFR 864.1865

(a) 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.

(b) Classification. Class II (special controls). A cervical intraepithelial neoplasia (CIN) test system must comply with the following special controls:
- 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, standalone 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 cut-off (the medical decision point between positive and negative) or other relevant criteria that distinguishes

{27}------------------------------------------------

positive and negative results, including the rationale for the chosen cut-off or other relevant criteria and results supporting validation of the cut-off.

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, betweenrun, between-day, between-lot, between-site, between-reader, withinreader 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 wellcharacterized 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, CIN2, 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 expert pathologist

{28}------------------------------------------------

with H&E+[biomarker specified in paragraph (1)(i) of this section] and community pathologist with H&E+[biomarker specified in paragraph (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.
Identification of risk mitigation elements used by the device, including I. a description of all additional procedures, methods, and practices incorporated into the instructions for use that mitigate risks associated with testing.
1. The device's 21 CFR 809.10(b) compliant labeling must include a detailed description of the protocol, including the information described in paragraph (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 (1)(ii) of this section, as applicable.

Regulation Number and Section

§ 864.1865 Cervical intraepithelial neoplasia (CIN) test system.

(a)
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.(b)
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.