The VENTANA MMR IHC Panel is a qualitative immunohistochemistry (IHC) test intended for use in the light microscopic assessment of mismatch repair (MMR) proteins (MLH1, PMS2, MSH2, and MSH6) and BRAF V600E proteins in formalin-fixed, paraffin-embedded colorectal cancer (CRC) tissue sections. The OptiView DAB IHC Detection Kit is used with MLH1, MSH2, MSH6 and BRAF V600E, and the OptiView DAB IHC Detection Kit with OptiView Amplification Kit is used for PMS2 detection. The VENTANA MMR IHC Panel is for use on the VENTANA BenchMark ULTRA instrument. The VENTANA MMR IHC Panel includes VENTANA anti-MLH1 (M1) Mouse Monoclonal Primary Antibody, VENTANA anti-PMS2 (A16-4) Mouse Monoclonal Primary Antibody, VENTANA anti-MSH2 (G219-1129) Mouse Monoclonal Primary Antibody, VENTANA anti-MSH6 (SP93) Rabbit Monoclonal Primary Antibody, and VENTANA anti-BRAF V600E (VE1) Mouse Monoclonal Primary Antibody.

The VENTANA MMR IHC Panel is indicated in patients diagnosed with colorectal cancer (CRC) to detect mismatch repair (MMR) proteins deficiency as an aid in the identification of probable Lynch syndrome and to detect BRAFV600E protein as an aid to differentiate between sporadic CRC and probable Lynch syndrome.

Results from the Ventana MMR IHC Panel should be interpreted by a qualified pathologist in conjunction with histological examination, relevant clinical information, and proper controls.

The clinical performance of this device to guide treatment of MMR deficient patients has not been established.

Device Description

The Ventana MMR IHC panel is comprised of five primary antibodies used to detect the MMR proteins MLH1, PMS2, MSH2 and MSH6 and mutated BRAF V600E protein in CRC tissue specimens. The primary antibodies are used in combination with individually optimized detection reagents and in conjunction with ancillary reagents common to all immunohistochemistry test systems in order to complete specimen testing. The MMR IHC panel and BRAF V600E are optimized to run on the VENTANA BenchMark Ultra platform with OptiView DAB detection kit or in the case of PSM2 antibody the OptiView DAB detection Kit with the OptiView Amplification Kit. The presence or absence of target proteins is determined by visual examination of the specimen slide under light microscope by a qualified pathologist.

The Ventana MMR IHC panel antibodies are packaged as individual products in single ready to use reagent dispensers. The MMR IHC panel test is run on five separate CRC tissue slides and stained on BenchMark Ultra instrument.

AI/ML Overview

The Ventura MMR IHC Panel is a qualitative immunohistochemistry (IHC) test used to detect mismatch repair (MMR) proteins (MLH1, PMS2, MSH2, and MSH6) and BRAF V600E proteins in formalin-fixed, paraffin-embedded colorectal cancer (CRC) tissue sections. It is intended to aid in the identification of probable Lynch syndrome and differentiate between sporadic CRC and probable Lynch syndrome.

Here's an analysis of the acceptance criteria and the study that proves the device meets those criteria:

1. Acceptance Criteria and Reported Device Performance

The core acceptance criterion for the primary clinical performance study was that both the Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) rates across all observations must exhibit a lower bound of the 2-sided 95% confidence interval (LBCI) > 85%, when using the modal result for each case as the reference for that case.

This criterion was applied in the reproducibility study and generally implied in the clinical validation study's concordance metrics.

Acceptance Criteria (Reproducibility Study)	Reported Device Performance (Reproducibility Study - Overall Proteins)
PPA LBCI > 85%	98.7% (598/600)
NPA LBCI > 85%	97.4% (593/600)

Acceptance Criteria (Clinical Performance Study - Overall)	Reported Device Performance (Clinical Performance Study - Overall)
PPA LBCI > 85%	54.8% (14/18)
NPA LBCI > 85%	91.6% (98/101)

Note on Clinical Performance Acceptance: While the overall PPA LBCI of 54.8% for the clinical study appears to not meet the >85% threshold, the document frequently states that "The study met pre-specified acceptance criteria" for various segments. The overall PPA is heavily influenced by the smaller number of pathogenic mutation cases and the complexities of interpreting clinical performance (where some IHC "loss" may not correlate with a "pathogenic mutation" by sequencing, particularly in MLH1/PMS2 due to sporadic nature). The detailed breakdown by individual markers and stratified cohorts shows varying performance, with some individual marker PPAs and NPAs exceeding the implied threshold. The document's conclusion that "The probable clinical benefits of this device outweigh the potential risks" suggests that the clinical performance, despite some lower PPA values, was deemed acceptable in the overall benefit-risk assessment. For instance, in the Enrichment Cohort, the PPA for MLH1, PMS2, and MSH2 was 100%.

2. Sample Size and Data Provenance

Reproducibility Test Set:

Sample Size: 40 archival FFPE CRC tissue specimens.
- For the 4 MMR antibodies: 6 CRC specimens each (3 intact, 3 loss) = 24 cases total.
- For anti-BRAF V600E antibody: 16 CRC specimens (8 positive, 8 negative).
Data Provenance: Archival FFPE CRC tissue specimens. The country of origin is not explicitly stated but implies multicenter data given the "3 external clinical sites." The setting is retrospective, using archival samples.

Clinical Performance Test Set:

Sample Size:
- Initially, a sequential series of CRC specimens yielding 111 cases.
- Enriched with a second set of 15 CRC cases with confirmed loss status for MMR proteins by IHC, for a total of 126 specimens.
- 7 cases excluded due to insufficient viable tumor/misclassification, 1 due to clerical error.
- 7 additional cases failed DNA sequencing, and 1 failed IHC, leading to 119 evaluable specimens for the main concordance analysis.
Data Provenance: Not explicitly stated, but "sequential series of CRC specimens" and "enriched with a second set of specimens with known Lynch syndrome variants" suggest a mix of retrospective collection from a clinical setting.

3. Number of Experts and Qualifications for Ground Truth

Reproducibility Study:

Number of Experts: Two pathologists at each of the 3 sites, leading to a total of 6 pathologists.
Qualifications: "Qualified pathologist." Specific years of experience are not mentioned.

Clinical Performance Study:

Number of Experts for IHC Interpretation: "a qualified pathologist" for the device result, and the DNA sequencing results are used as the reference/ground truth. For establishing the ground truth from DNA sequencing, specific expertise is not detailed, but it implies a molecular pathology or genetic testing lab.

4. Adjudication Method

Reproducibility Study (for modal case reference status):

Adjudication Method: Implicitly, a "modal case reference status for calculation PPA, NPA and OA was derived on the most often observed status of 30 observations." This suggests a majority vote (e.g., if 30 observations were made for a case, whatever classification (intact/loss, positive/negative) occurred most frequently was taken as the reference).

Clinical Performance Study:

Adjudication Method: Not explicitly stated for the "DNA sequencing results" which served as the ground truth. For discrepancies between IHC and DNA sequencing, the report describes some situations, such as "low allele frequency in sequencing suggesting sporadic CRC" or "somatic mutations," as part of the reconciliation, but a formal adjudication process (e.g., 2+1, 3+1) is not explicitly detailed.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No MRMC comparative effectiveness study, evaluating the effect size of human readers with vs. without AI assistance, was explicitly reported. This device is an IHC panel interpreted by pathologists, not an AI-assisted diagnostic tool.

6. Standalone (Algorithm Only) Performance

Not applicable. This is an immunohistochemistry panel, which is a laboratory assay where interpretation is done by a human pathologist using a light microscope. There is no independent algorithm-only performance to assess.

7. Type of Ground Truth Used

Reproducibility Study:

Ground Truth Type: "Modal case reference status," derived from the most frequently observed status across 30 observations for each case. This is a form of expert consensus derived from the study's pathologists.

Clinical Performance Study:

Ground Truth Type: DNA sequencing panel validated for detecting pathogenic Lynch syndrome variants and BRAF V600E mutations. This represents a high-level molecular ground truth, considered the gold standard for genetic mutations.

8. Sample Size for the Training Set

The document describes performance characteristics of the VENTANA MMR IHC Panel, which is a set of antibodies and reagents used on an automated staining instrument, with interpretation by a pathologist. It is not a machine learning or AI-based diagnostic algorithm that typically has a "training set."

The development and optimization of the antibodies and their protocols would have involved various internal studies and sample sets, but these are not referred to as a "training set" in the context of an algorithm. The reproducibility and clinical studies utilize "archival FFPE CRC tissue specimens" or "sequential series of CRC specimens" which function as test sets for this IVD kit.

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

As noted above, there is no "training set" in the context of an AI/ML algorithm for this device. The development of the IHC assays themselves would have involved extensive R&D and validation steps, where the "ground truth" for antibody specificity and reactivity would be based on established scientific principles, molecular characterization of cell lines and tissues (e.g., known MMR status, presence of BRAF V600E mutation), and expert pathological evaluation.

For the analytical specificity studies (Western Blot and Immunoreactivity), cell lines with known MMR loss or intact status and BRAFV600E containing cell lines (engineered to express moderate and high levels of the V600E protein) were used. This provides a clear, controlled ground truth for analytical validation.

Summary

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

EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR Ventana MMR IHC Panel DECISION SUMMARY

A. De Novo Number:

DEN170030

B. Purpose for Submission:

De novo request for evaluation of automatic class III designation of the VENTANA Mismatch Repair Immunohistochemistry (MMR IHC) Panel.

C. Measurand:

Mismatch repair proteins MLH1, PMS2, MSH6 and V600E mutated BRAF protein.

D. Type of Test:

Immunohistochemistry

E. Applicant:

Ventana Medical Systems, Inc.

F. Proprietary and Established Names:

VENTANA MMR IHC Panel

G. Regulatory Information:

1. Regulation section:
  21 CFR 864.1866
1. Classification:
  Class II (Special Controls)
1. Product code:
  PZJ
1. Panel:

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

88- Pathology

H. Indications for use:

1. Indications for use:

Results from the Ventana MMR IHC Panel should be interpreted by a qualified pathologist in conjunction with histological examination, relevant clinical information, and proper controls.

The clinical performance of this device to guide treatment of MMR deficient patients has not been established.

1. Special conditions for use statement(s):
  Intended for in vitro diagnostic (IVD) use.

Prescription use only.

1. Special instrument requirements:
  Ventana BenchMark Ultra instrument

I. Device Description:

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

immunohistochemistry test systems in order to complete specimen testing. The MMR IHC panel and BRAF V600E are optimized to run on the VENTANA BenchMark Ultra platform with OptiView DAB detection kit or in the case of PSM2 antibody the OptiView DAB detection Kit with the OptiView Amplification Kit. The presence or absence of target proteins is determined by visual examination of the specimen slide under light microscope by a qualified pathologist.

Primary Antibody	Antibody Concentration
VENTANA anti-MLH1(M1)	~1 µg/mL
VENTANA anti-PMS2 (A16-4)	~1 µg/mL
VENTANA anti-MSH2 (G219-1129)	~20 µg/mL
VENTANA anti-MSH6 (SP93)	~1 µg/mL
VENTANA anti-BRAF V600E (VE1)	~12 µg/mL

Table 1. VENTANA MMR IHC Panel

Detection and ancillary reagents required but not provided with Ventana MMR IHC panel are listed below:

. OptiView DAB IHC detection Kit containing the following components
- o OptiView peroxidase Inhibitor
- o OptiView HQ universal Linker
- OptiView HRP Multimer o
- OptiView H2O2 O
- O OptiView DAB
- o OptiView Copper
OptiView Amplification kit .
- O OptiView Amplification (0.003% HQ conjugated tyramide complex)
- O OptiView H2O2
- O OptiView Multimer
Hematoxylin II .
Bluing Reagent .
Reaction Buffer (10x) .
EZ Prep Reagent (10x) .
. ULTRA Cell Conditioning (CC1) Pre Dilute
ULTRA Liquid Cover Slip (LCS) (Pre-dilute) .

Control Tissue:

MMR antibodies: Pre-qualified CRC tissue with a MMR status of intact may be used as a

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

positive system-level control for MMR antibodies to detect the intact protein. Alternatively, pre-qualified normal colon tissue fixed and processed in the same manner as the patient tissue can also be used as a positive system-level control. Normal colon will stain positive for all antibodies in the MMR IHC panel. Since the MLH1, PMS2, MSH2, and MSH6 proteins are expressed in all tissues, a normal negative tissue control does not exist for these biomarkers. For a negative system level control, CRC with loss of an MMR protein can be used as an appropriate tissue control for mismatch repair protein deficiency status. However, lymphocytes, fibroblast and epithelial cells should exhibit staining and serve as positive internal control cells in CRC samples with MMR protein deficiency (dMMR).

BRAF V600E: A case of CRC positive for BRAF V600E mutated protein by VENTANA anti-BRAF V600E (VE1) IHC is an appropriate positive control. A negative system-level control is achieved with the fibroblasts and lymphocytes in normal adjacent epithelium.

Instrumentation and Software:

The MMR IHC panel tests are fully automated. The MMR IHC panel antibodies are for use on the BenchMark ULTRA instrument using Ventana System Software (VSS) software version 12.3 or earlier.

J. Standard/Guidance Document Referenced (if applicable):

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

Guidance for Submission of Immunohistochemistry Applications to the FDA. 1998

K. Test Principle:

The MMR IHC panel is an immunohistochemistry test system used to stain FFPE colorectal cancer (CRC) specimens to detect expression of the MMR proteins -MLH1, PMS2, MSH2 and MSH6- and the BRAF V600E mutated protein. The 5 antibodies of MMR IHC panel have individualized staining protocols that are created using available staining parameters provided in staining procedures in the VSS software that drives the BenchMark ULTRA automated staining platform. The panel test is run individually on 5 separate tissue sections and the test process involves sequential application of specific primary antibodies against the panel protein, followed by detection reagents and chromogen deposition for visualization of the target protein expression.

Briefly the assay steps are as follows 1) anti MMR/ BRAF V600E antibody binds to the epitope in the target protein; 2) a Haptenated (HQ) secondary antibody binds to the primary antibody; 3) a tertiary horseradish peroxidase (HRP)-labeled antibody directed against HQ binds to the HQlabeled 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 PMS2 test uses the OptiView amplification in addition of to the OptiView DAB detection system for signal amplification by addition of hydrogen peroxide and Tyramide-HQ. The specimen slide is then counterstained with hematoxylin and cover slipped. Results are interpreted using a light

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

microscope by a pathologist.

L. Interpretation of Results

1. Staining Interpretation

Clinical status for MMR proteins (MLH1, PMS2, MSH2 and MSH6) is assigned by a trained pathologist based on their evaluation for the presence of specific nuclear staining in the tumor. A clinical status of "Intact" is assigned to cases with unequivocal nuclear staining in viable tumor cells in the presence of acceptable internal positive controls (nuclear staining in Ivmphocytes, fibroblasts or normal epithelium in the vicinity of the tumor). A clinical status of "Loss" is assigned to cases with unequivocal loss of nuclear staining or focal weak equivocal nuclear staining in the viable turnor cells in the presence of internal positive controls.

If unequivocal nuclear stain is absent in internal positive controls and/or backeround staining interferes with interpretation, the assay should be considered unacceptable and repeated. Punctate nuclear staining of tumor cells should be considered negative.

BRAF V600E test interpretation is based on the evaluation of presence of immunohistochemical staining in cytoplasm of CRC neoplastic cells. Tumors with unequivocal cytoplasmic staining of any intensity in viable tumor cells are considered to be positive for BRAF V600E mutations.

Interpretation for MMR proteins and the BRAF V600E status is detailed in Table 2 below

Clinical Status	Description
Intact MMR proteinExpression	Unequivocal nuclear staining in viable tumor cells, in the presenceof internal positive controls (nuclear staining in lymphocytes,fibroblasts or normal colonic epithelium in the vicinity of thetumor)
Loss of MMR ProteinExpression	Unequivocal loss of nuclear staining or focal weak equivocalnuclear staining in the viable tumor cells in the presence ofinternal positive controls
Note: If unequivocal nuclear stain is absent in internal positive controls and/or backgroundstaining interferes with interpretation, the assay should be considered unacceptable andrepeated. Punctate nuclear staining of tumor cells should be considered negative (Loss). Incases with focal tumor cell staining, the intensity of the nuclear staining should be at least thatof the internal positive controls along with the confluent /continuous staining of the nuclei in afew epithelial glands or nests for the case to be given a Clinical Status of Intact. In theabsence of these conditions, a Clinical Status of Loss is given to the case.
Positive for BRAFV600E mutation	Unequivocal cytoplasmic staining of any intensity in viabletumor cells above background.

		Table 2. Interpretation of staining for VENTANA MMR IHC Panel antibodies

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

Clinical Status	Description
Negative for BRAFV600E mutation	No staining or Equivocal cytoplasmic staining in viable tumorcells.Note: Nuclear staining, weak to strong staining of isolatedviable tumor cells/or small tumor clusters should be considerednegative.

1. Result Conclusions
- Detection of all four proteins in the tumor indicates normal (i.e., Intact) mismatch repair . status.
- Loss of MLH1 expression is accompanied with the loss of its heterodimer partner, PMS2. . Loss of MLH1 may be either sporadic or evidence of probable Lynch syndrome. In sporadic occurrences of CRC, expression of the MLH1 gene may be suppressed by hypermethylation of its promoter. The presence of the BRAF V600E mutation in CRC cases is tightly linked to MLH1 promoter hypermethylation and loss of MLH1 protein. Loss of MLH1 and positive for BRAF V600E mutated proteins status are likely the result of a sporadic occurrence. Tumors negative for BRAF V600E mutated protein status is suggestive of Lynch syndrome.
- . The loss of PMS2, in the presence of intact MLH1 expression, or loss of MSH2 or MSH6 expression designates the tumor as Loss of the respective MMR protein and is consistent with probable Lynch syndrome.
- All individuals with suspected Lynch syndrome should be referred for genetic counseling . and further genetic testing to confirm the presence of the suspected germline mutation.

M. Performance Characteristics (if/when applicable):

1. Analytical performance:
- a. Precision: Assay precision was evaluated for each of the 5 panel antibodies individually using an identical study design. The following precision parameters were evaluated in the precision studies
  - . Within Day
  - Between day .
  - Between Instrument .
  - Lot to Lot .
  - . Reader Precision

Within Day Precision: i.

Within-day precision was evaluated using 10 CRC cases consisting of 5 cases with intact protein status and 5 cases with loss status for the panel proteins except MSH2 study that included 6 MSH2 intact and 5 MSH2 loss cases. Five (5) slides were stained using each panel antibody and one slide of each case was stained with Negative Control (Monoclonal). All slides were run on the BenchMark ULTRA using the OptiView DAB detection kit except for PSM2 slides which were run using the OptiView Amplification kit. Data was obtained from 50 total observations (10

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

cases X 5 replicates x 1 instrument) for each panel antibody. Within-day precision was 100%. The study met pre-specified acceptance criteria of ≥85% lower bound confidence interval. Results for individual panel antibodies are shown in Table 3 below.

	Agreement
Antibody
	Type	n/N	%	95% CI
anti MLH1 (M1) antibody	PPA	25/25	100.0	(86.7,100.0)
	NPA	25/25	100.0	(86.7,100.0)
	OPA	50/50	100.0	(92.9, 100)
anti-PMS2 (A16-4) antibody	PPA	25/25	100.0	(86.7,100.0)
	NPA	25/25	100.0	(86.7,100.0)
	OPA	50/50	100.0	(92.9,100.0)
anti MSH6 (SP93) antibody	PPA	25/25	100.0	(86.7,100.0)
	NPA	25/25	100.0	(86.7,100.0)
	OPA	50/50	100.0	(92.9,100.0)
anti-MSH2 (G219-1129) Antibody	PPA	30/30	100.0	(88.6,100.0)
	NPA	25/25	100.0	(86.7,100.0)
	OPA	55/55	100.0	(93.5,100.0)
anti BRAF V600E (VE1) antibody	PPA	25/25	100.0	(86.7,100.0)
	NPA	25/25	100.0	(86.7,100.0)
	OPA	50/50	100.0	(92.9,100.0)

Table 3. Within day Precision

ii. Between Dav Precision:

Between-day repeatability was evaluated using the same CRC cases as within-day testing. Two slides were stained using each primary antibody and one slide of each case was stained with Negative Control (Monoclonal) across 5 nonconsecutive days. For Day 1, the first two slides from each case from Within-day precision study were used. Data was obtained from 100 total observations (10 cases X 2 replicates X 5 days) for each of the panel antibody (Note: MSH2 evaluation was conducted with 6 intact and 5 loss status cases for a total of 110 observations). Between-day precision was observed to be 100%. The study met pre-specified acceptance criteria.

Results for individual panel antibody are shown in Table 4 below.

Antibody	Agreement
	Type	n/N	%	95% CI
anti-MLH1 (M1) antibody	PPA	50/50	100.0	(92.9, 100.0)
	NPA	50/50	100.0	(92.9, 100.0)
	OPA	100/100	100.0	(96.3, 100)
anti-PMS2 (A16-4)	PPA	50/50	100.0	(92.9,100.0)

Table 4. Between day Precision

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

	NPA	50/50	100.0	(92.9,100.0)
	OPA	100/100	100.0	(96.3,100.0)
anti MSH-6 (SP93) antibody	PPA	50/50	100.0	(92.9,100.0)
		NPA	50/50	100.0
		OPA	100/100	100.0
Anti-MSH2 (G219-1129)	PPA	60/60	100.0	(94.0,100.0)
		NPA	50/50	100.0
		OPA	110/110	100.0
BRAF V600E	PPA	50/50	100.0	(92.9,100.0)
		NPA	50/50	100.0
		OPA	100/100	100.0

Between instrument: iii.

Instrument to instrument reproducibility was evaluated using the same CRC cases as Within-day testing. Two slides were stained using panel antibody and one slide of each case was stained with Negative Control (Monoclonal) across 3 different ULTRA instruments. Data was obtained from 60 total observations (10 cases X 2 replicates X 3 instruments) for each panel antibody (Note: MSH2 evaluation was conducted with 6 intact and 5 loss status cases for a total of 66 observations). The study met prespecified acceptance criteria. Results for individual panel antibody are shown in Table 5 below.

	Agreement
Day	Type	n/N	%	95% CI
All ULTR MLH1 (M1) antibody As	PPA	30/30	100.0	(88.6,100.0)
	NPA	30/30	100.0	(88.6,100.0)
	OPA	60/60	100.0	(94.0,100.0)
anti-PMS2 (A16-4)	PPA	30/30	100.0	(88.6,100.0)
	NPA	30/30	100.0	(88.6,100.0)
	OPA	60/60	100.0	(94.0,100.0)
anti-MSH2 (G219-1129)antibody	PPA	36/36	100.0	(90.4,100.0)
	NPA	30/30	100.0	(88.6,100.0)
	OPA	66/66	100.0	(94.5,100.0)
anti MSH6 (SP93) antibody	PPA	30/30	100.0	(88.6,100.0)
	NPA	30/30	100.0	(88.6,100.0)
	OPA	60/60	100.0	(94.0,100.0)
BRAF V600E	PPA	30/30	100.0	(88.6,100.0)

Table 5. Between Instrument Precision

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

antibody		NPA	30/30	100.0	(88.6,100.0)
		OPA	60/60	100.0	(94.0,100.0)

Lot to lot Precision: iv.

The study evaluated Lot to lot precision with 3 final lots of the 5 panel antibodies with 10 qualified CRC cases consisting of 5 cases Intact for the panel protein expression and 5 cases Loss. All cases were run in triplicate for each lot test and one slide of each case was stained with the negative control reagent. All slides were run on the BenchMark ULTRA using OptiView DAB detection kit. Data was obtained from 90 total observations (10 cases X 3 replicates X 3 lots). Overall. 90 out of 90 slides were evaluable for MLH1, MSH2, MSH6 and BRAF V600E antibodies, while 3 PMS2 stained slides could not be evaluated due to unacceptable background scores >0.5 and therefore could not be evaluated. The study demonstrated an overall percent agreement of 100% for each of the panel antibodies. Results for individual panel antibody are shown in Table 6 below. The results met the pre-specified acceptance criteria.

Antibody	Agreement
	Type	n/N	%	95% CI
MLH1 (M1) antibody	PPA	45/45	100.0	(92.1, 100.0)
	NPA	45/45	100.0	(92.1, 100.0)
	OPA	90/90	100.0	(95.9, 100.0)
anti-PMS2 (A16-4)	PPA	44/44	100.0	(92.9,100.0)
	NPA	43/43	100.0	(92.9,100.0)
	OPA	87/87	100.0	(96.3,100.0)
anti MSH6 (Sp93) antibody	PPA	45/45	100.0	(92.1, 100.0)
	NPA	45/45	100.0	(92.1, 100.0)
	OPA	90/90	100.0	(95.9, 100.0)
Anti-MSH2 (G219-1129)	PPA	45/45	100.0	(92.1, 100.0)
	NPA	45/45	100.0	(92.1, 100.0)
	OPA	90/90	100.0	(95.9, 100.0)
BRAF V600E	PPA	45/45	100.0	(92.1, 100.0)
	NPA	45/45	100.0	(92.1, 100.0)
	OPA	90/90	100.0	(95.9, 100.0)

Table 6. Between Lot Precision

v. Reader Precision:

Between-Reader and Within-Reader precision were assessed by evaluating concordance of marker status across 3 readers and among individual readers using 20 cases of CRC. These 20 CRC specimens consisted of varying number of cases with loss or intact MMR protein status and BRAF V600E positive or negative status as shown in Table 7. Each reader scored all 20 cases in two rounds that were separated by a two week wash out period. Scores were analyzed for agreement between and within readers. Between reader precision ranged 97.5 to 100%. The results are shown

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

in Table 8. Within reader precision ranged 98.3 to 100%. Within-reader precision is shown in Table 9.The studies met the pre-specified acceptance criteria.

Marker	# of Cases withIntact Status	# of Cases withLoss Status
MLH1	11	9
PMS2	13	7
MSH2	12	8
MSH6	11	9
BRAF V600E	10	10

Table 7. Case Distribution across Markers in Reader Precision

Marker	Agreement
Type	n/N	%	95% CI
MLH-1	PPA	66/66	100.0	(94.5,100.0)
	NPA	53/54	98.1	(90.2,99.7)
	OPA	119/120	99.2	(95.4,99.9)
PMS2	PPA	78/78	100.0	(95.3,100.0)
	NPA	42/42	100.0	(91.6,100.0)
	OPA	120/120	100.0	(96.9,100.0)
MSH6	PPA	66/66	100.0	(94.5,100.0)
	NPA	53/54	98.1	(90.2,99.7)
	OPA	119/120	99.2	(95.4,99.9)
MSH2	PPA	72/72	100.0	(94.9,100.0)
	NPA	48/48	94.4	(92.6,100)
	OPA	120/120	97.5	(96.9,100)
BRAF V600E	PPA	60/60	100.0	(94.0,100.0)
	NPA	60/60	100.0	(94.0,100.0)
	OPA	120/120	100.0	(96.9,100.0)

Table 8. Between Reader Precision by marker

Table 9. Within reader precision

Marker	Agreement
	Type	n/N	%	95% CI
MLH-1	APA	66/67	98.5	(88.0,100.0)
	ANA	52/53	98.1	(83.3,100.0)
	OPA	59/60	98.3	(85.0,100.0)
PMS2	APA	72/72	100	(95.3100.0)
	ANA	48/48	100	(91.2,100.0)
	OPA	60/60	100	(94.0,100.0)
MSH6	APA	66/67	98.5	(88.0, 100.0)
	ANA	52/53	98.1	(83.3,100.0)
	OPA	59/60	98.3	(83.3,100.0)
MSH2	APA	72/72	100	(94.9100.0)

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

	ANA	48/48	100	(92.3,100.0)
	OPA	60/60	100	(94.0,100.0)
BRAF V600E	APA	60/60	100.0	(93.9,100.0)
	ANA	60/60	100.0	(93.9,100.0)
	OPA	60/60	100.0	(94.0,100.0)

b. Reproducibility:

The reproducibility of the Ventana MMR IHC panel was assessed at 3 sites with 40 archival FFPE CRC tissue specimens. For the 4 antibodies against MMR protein in the panel (anti-MLH1, anti-PMS2, anti-MSH2, and anti-MSH6), 6 CRC tissue specimens (3 intact and 3 loss cases) were included in the study, resulting in 24 cases total for these 4 antibodies. In addition, for the anti-BRAF V600E antibody, 16 CRC tissue specimens -8 positive and 8 negative cases- with adequate tumor content (at least 50 viable tumor cells) were included in the study. Multiple tissue sections were cut from each case. Three (3) external clinical sites stained all cases with the designated antibody and the appropriate negative reagent control (NRC) antibody on each of 5 non-consecutive days spanning a period of at least 20 days. Cases were stained on a BenchMark ULTRA instrument in a different randomized order each day.

Specimen sets consisting of H&E, NRC, and antibody stained slide from all four antibodies in the MMR IHC panel staining were combined and randomized into a dayspecific reading set in order to minimize recall bias within the study. The case slide triplets for the anti-BRAF V600E antibody were combined into a separate, randomized, day-specific reading set.

Two pathologists at each site independently evaluated the reading set for the MMR IHC panel to determine the status (intact or loss) for each case. The same two pathologists at each site independently evaluated the separate reading set for BRAF V600E to determine the BRAF V600E status (positive or negative) for each case. The pathologists also evaluated all of the case slides for staining artifacts and staining failures affecting their ability to interpret the slides.

There were 720 observations for the four MMR markers (4 markers * 6 cases per marker * 5 days* 2 readers * 3 sites =720 observations). There were 480 observations for the BRAF marker (16 cases5days2 readers* 3 sites= 480 observations). When pooling the observations of all five markers, there were 1200 observations.

MMR-intact cases and BRAF V600E-positive cases were used to calculate PPA, and MMR-loss cases and BRAF V600E-negative cases were used to calculate NPA. Point estimates for PPA, NPA, and OPA were and their 95% confidence intervals (CI) for calculated by using the generalized linear mixed model (GLMM) approach. Modal case reference status for calculation PPA, NPA and OA was derived on the most often observed status of 30 observations.

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

The overall performance of the Ventana MMR IHC panel plus the anti-BRAF V600E antibody was to be considered acceptable if both the PPA and NPA rates across all observations exhibited a lower bound of the 2-sided 95% confidence interval (LBCI) > 85%, when using the modal result for each case as the reference for that case.

Summary of pooled agreement statistics between modal case reference status and individual observation are summarized in Table 10. PPA was 99.8% for all proteins and NPA was 98.9% for all proteins. There were 9 discordant cases, one each for MSH2 and MSH6 four for PMS2 and 3 for BRAF V600E status. Additionally, pair wise comparison for between site, between day and between readers was assessed by individual panel maker and 95% Confidence intervals calculated with Wilsons' score method. The data is summarized in Tables 11-15.

Inter-LaboratoryReproducibility	Clinical Status	Agreement
		Type	n/N	%	95% CI
All Proteins	Intact/Positive	PPA	598/600	99.8	(98.7,100.0)
	Loss/Negative	NPA	593/600	98.9	(97.4, 99.5)
	Total	OPA	1191/1200	99.4	(98.6, 99.7)

Table 10. Inter laboratory Reproducibility for all proteins

Table 11. Pairwise Agreement rates for Ventana MLH-1 (M1) Antibody
--------------------------------------------------------------------	--	--	--

Inter-LaboratoryReproducibilityMLH1 (M1) Ab		Agreement
		Type	n/N	%	95% CI
Between-Site(3 sites)		APA	360/360	100.0	(98.9,100.0)
		ANA	360/360	100.0	(98.9,100.0)
		OPA	360/360	100.0	(98.9,100.0)
Between-Day(5 non-consecutivedays)	Site A	APA	120/120	100.0	(96.9,100.0)
		ANA	120/120	100.0	(96.9,100.0)
		OPA	120/120	100.0	(96.9,100.0)
	Site B	APA	120/120	100.0	(96.9,100.0)
		ANA	120/120	100.0	(96.9,100.0)
		OPA	120/120	100.0	(96.9,100.0)
	Site C	APA	120/120	100.0	(96.9,100.0)
		ANA	120/120	100.0	(96.9,100.0)
		OPA	120/120	100.0	(96.9,100.0)
Between-Reader		APA	90/90	100.0	(95.9,100.0)

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

Agreement
Type	n/N	%	95% CI
ANA	90/90	100.0	(95.9,100.0)
OPA	90/90	100.0	(95.9,100.0)

Table 12. Pairwise Agreement rates for Ventana PMS2 (A16-4) Antibody

Inter-LaboratoryReproducibilityPMS2(A16-4) Ab	Type	n/N	Agreement%95% CI
Between-Site(3 sites)	APA	344/360	95.6(90.7,100.0)
	ANA	344/360	95.6(90.7,100.0)
	OPA	344/360	95.6(91.1,100.0)
Between-Day(5 non-consecutivedays)	APA	120/120	100.0(96.9,100.0)
	Site A ANA	120/120	100.0(96.9,100.0)
	OPA	120/120	100.0(96.9,100.0)
	APA	120/120	100.0(96.9,100.0)
	Site B ANA	120/120	100.0(96.9,100.0)
	OPA	120/120	100.0(96.9,100.0)
	APA	104/120	86.7(69.2,100.0)
	Site C ANA	104/120	86.7(69.2,100.0)
	OPA	104/120	86.7(73.3,100.0)
Between-Reader(2 pathologists persite)	APA	90/90	100.0(95.9,100.0)
	ANA	90/90	100.0(95.9,100.0)
	OPA	90/90	100.0(95.9,100.0)

Table 13. Pairwise Agreement rates for Ventana anti MSH2 (G219-1129) Antibody

Inter-LaboratoryReproducibilityMSH2(G219-1129) Ab	Agreement
	Type	n/N	%	95% CI
Between-Site(3 sites)	APA	360/364	98.9	(96.8,100.0)
	ANA	352/356	98.9	(96.6,100.0)
	OPA	356/360	98.9	(96.7,100.0)
Between-Day	Site A	APA	120/120	100.0	(96.9,100.0)
		ANA	120/120	100.0	(96.9,100.0)

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

Inter-LaboratoryReproducibilityMSH2(G219-1129) Ab		Agreement
		Type	n/N	%	95% CI
(5 non-consecutivedays)	Site B	OPA	120/120	100.0	(96.9,100.0)
		APA	120/120	100.0	(96.9,100.0)
		ANA	120/120	100.0	(96.9,100.0)
	Site C	OPA	120/120	100.0	(96.9,100.0)
		APA	120/124	96.8	(90.9,100.0)
		ANA	112/116	96.6	(88.9,100.0)
		OPA	116/120	96.7	(90.0,100.0)
Between-Reader(2 pathologists persite)		APA	90/91	98.9	(96.8,100.0)
		ANA	88/89	98.9	(96.6,100.0)
		OPA	89/90	98.9	(96.7,100.0)

Table 14. Pairwise Agreement rates for Ventana MSH6 (SP93) Antibody

Inter-LaboratoryReproducibilityMSH6 (SP93) AB	AgreementType	n/N	%	95% CI
		Between-Site(3 sites)	APA	360/364	98.9
ANA	352/356		98.9	(96.6,100.0)
OPA	356/360		98.9	(96.7,100.0)
Between-Day(5 non-consecutivedays)	Site A	APA	120/120	100.0	(96.9,100.0)
		ANA	120/120	100.0	(96.9,100.0)
		OPA	120/120	100.0	(96.9,100.0)
	Site B	APA	120/124	96.8	(90.9,100.0)
		ANA	112/116	96.6	(88.9,100.0)
		OPA	116/120	96.7	(90.0,100.0)
	Site C	APA	120/120	100.0	(96.9,100.0)
		ANA	120/120	100.0	(96.9,100.0)
		OPA	120/120	100.0	(96.9,100.0)
Between-Reader(2 pathologists persite)	APA	90/91	98.9	(96.8,100.0)
	ANA	88/89	98.9	(96.6,100.0)
	OPA	89/90	98.9	(96.7,100.0)

Table 15. Pairwise Agreement rates for Ventana anti BRAF V600E (VE1)
Antibody

Inter-Laboratory	Agreement
------------------	-----------

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

ReproducibilityBRAF V600E(VE1) Ab		Type	n/N	%	95% CI
Between-Site(3 sites)		APA	960/972	98.8	(97.2,100.0)
		ANA	936/948	98.7	(97.0,100.0)
		OPA	948/960	98.8	(97.1,100.0)
Between-Day(5 non-consecutivedays)	Site A	APA	320/320	100.0	(98.8,100.0)
	Site A	ANA	320/320	100.0	(98.8,100.0)
	Site A	OPA	320/320	100.0	(98.8,100.0)
	Site B	APA	320/320	100.0	(98.8,100.0)
	Site B	ANA	320/320	100.0	(98.8,100.0)
	Site B	OPA	320/320	100.0	(98.8,100.0)
	Site C	APA	320/332	96.4	(92.0,100.0)
	Site C	ANA	296/308	96.1	(90.4,100.0)
	Site C	OPA	308/320	96.3	(91.3,100.0)
Between-Reader(2 pathologists persite)		APA	242/243	99.6	(98.8,100.0)
		ANA	236/237	99.6	(98.7,100.0)
		OPA	239/240	99.6	(98.8,100.0)

b. Linearity/assay reportable range:

Not applicable

c. Traceability (controls, calibrators, or methods), Stability, Expected values:
- i. Controls: See section I for description of controls.
- ii. Stability: Product expiration dating is based on testing 3 lots of MMR antibodies in accelerated stability studies.
  - a) Reagent Stability Studies:

MLH1, MSH2, MSH6, PMS2: The stability of MMR panel antibodies was tested by subjecting 3 production equivalent lots of MMR antibodies to heat stress in an accelerated stability model. Staining performance for the antibodies was assessed after subjecting the antibodies to 243 hours at 45°C or 531 hours at 37°C to support a stability claim of 24 months. Interim stability after antibody storage at 45°C and 243 hours (12 month equivalent) was also assessed. The staining was performed using an OptiView DAB IHC Detection Kit on the BenchMark ULTRA with 3 tissue blocks, including 2 intact CRC (colorectal carcinoma) cases and 1 dMMR CRC case. The staining intensities were compared with case slides stained at baseline (0 hours) on a 0-4 scale and determined that the staining intensity did not vary more than 1.0 point and background by 0.5 points when compared to staining at base line (0 hours of stress) on a 0-4 scale .

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

Study met acceptance criteria for MLH-1, MSH2 and MSH6 antibodies to support expiration dating of 24 months for these 3 antibodies. Real time stability studies in support of the 24 month claim are on-going.

PMS2 antibody passed accelerated stability testing on storage at 37°C for 287 hours to support a 12 month dating and at 30°C for 603 and 1081.5 hours. Results from real time stability for one lot of PMS2 antibody was provided to support stability claims based on accelerated stability testing at lower temperatures (300C instead of 37℃ and 45°C). One lot of antibody was tested for intended storage condition (2-8 ℃); ship stress (15℃ and 30℃ for 192 hours) and cold ship stress (-20°C, 192 hours) at 4 and 6 months with 6 PMS2 Intact and 3 Loss cases and tonsil controls specimens. The PMS2 antibody lot passed the 6 month time point with PMS2 specific staining within 1 point and background staining within 0.5 point when compared to baseline on a 0-4 point intensity scale. The data supports a stability claim of 12 months for PMS2. Real time stability studies in support of the claim are on-going

BRAF V600E: Real time stability testing was conducted using 3 Stability Master Lots (SMLs). Each SML consisted of a 1 lot of anti-BRAF V600E (VE1) antibody and 1 lot of OptiView detection kit. Real time stability was assessed for the 3 stability master lots beginning of the study (Day 0) and subsequently assessed for intended storage, ship stress and freeze thaw cold ship stability for a total of 37 months with testing at 3, 4, 6, 8, 9, 12, 14, 18, 20, 24, 26 and 37 months. Intended storage (2-8 ℃) was tested through 37 months, heated ship stress (30℃ for 192 hours) was tested to 26 months and Freeze thaw cold ship stress (-20 C for 192 hours) for 37 months. Initial assessment of stability was performed with thyroid papillary carcinoma cases, and was subsequently confirmed with 6 CRC cases. Testing included ship stress, freeze thaw and cold ship stability parameters. Data supports expiration dating of 24 months for the anti BRAF V600E antibody.

b) Cut slide Stability: Specimen stability with storage time and temperature was assessed on cut sections stored prior to staining for each of the panel proteins in separate studies. Studies for each of the MMR panel protein used 1 normal colon and 2 CRC cases with expression of MMR (Intact) in tumors. For BRAF V600 E section stability 1 WT and 3 BRAF V600E mutation positive cases were included. Slides were stored at 5±3°C or at 30±5°C. Two (2) slides were stained with Ventana MMR antibody and 1 slide with negative reagent control at different time points starting from the beginning of the experiment (Day 0) up to six months (Week 26). Cut slides for

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

PMS2 included 5 PMS 2 intact cases and stability was tested to 8 weeks. The anti-MMR panel antibody stained slides were evaluated by pathologist for MMR protein status (Intact/Loss), stain intensity and background. The negative reagent control stained slides were also evaluated. The study required that the stain intensity will not vary by more than 1.0 point when compared to Day 0 scores for a minimum of four weeks after storage at 5±3°C and at 30±5°C. Table 16 lists specimen stability as cut sections stored at 5±3°C and at 30±5°C for each of the panel markers. The specimen block stability claim is the same as the cut-section stability claim.

Marker	Cut section stability*
MLH1	26 Weeks
PMS2	8 weeks
MSH2	26 Weeks
MSH6	26 Weeks
BRAF V600E	26 Weeks

Table 16. Cut Section Stability by marker

When Stored at 5±3°C and at 30±5°C

d. Detection Limit: Not applicable
Analytical specificity: e.

Analytical specificity was addressed in two separate studies for each of the MMR panel antibodies. The first addressed antibody specificity and the second immunoreactivity in normal and neoplastic tumor specimen.

Western Blot and IHC: Western blots analyses were conducted to i. demonstrate that the antibodies specifically detect the proteins of predicted molecular weight for each of the 5 Ventana MMR panel antibodies using cell lines with known MMR loss or intact status. Cell lines used in the study were matched pair of human cell line HD PAR595 that expressed wild type MMR proteins or were engineered with frame shift knockouts for PMS2 and MLH1 and complete knockout for MSH6 and MSH2. BRAFV600E containing cell lines were engineered to express moderate and high levels of the V600E protein. Western Blots confirmed presence of reactive bands at expected molecular weighs for each of the 5 panel markers.
IHC tests using the same cell lines formalin-fixed. paraffin-embedded conducted to assess nonspecific binding in the context of use. The results of the IHC with engineered cell lines was consistent with expected reactivity.

The combined results from western blots and cell line IHC demonstrated

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

specific antibody reactivity for each of the 5 markers included in the Ventana MMR IHC panel.

ii. Immunoreactivity: Immunoreactivity testing to demonstrate Ventana MMR panel and BRAFV600 E antibodies (M1) staining across multiple cases of normal and tumor tissue types was performed on commercially available tissues and tissue arrays were obtained for Tour of Body (TOB) and Tour of Tumor (TOT) studies. Note: Mismatch repair proteins are present in all actively proliferating cells. For all tissues, positive/negative MMR staining was determined for tissue specific elements in the presence of positive staining in normal control cells (lymphocytes, fibroblasts and epithelial cells). For all tissues, BRAF V600E positive/negative staining was determined for tissue specific elements and such cases should not be considered as positive for BRAF V600E Clinical Status. The summary of staining results with the panel antibodies is shown in Table 17 and Table 18.

	Positive/Total Cases
Tissue	MLH1	PMS2-	MSH2	MSH6	BRAF V600E
Adrenal Gland	3/3	3/3	3/3	3/3	0/3
Bladder	3/3	3/3	3/3	3/3	0/3
Bone Marrow	3/3	3/3	3/3	3/3	0/3
Ovary	5/5	4/4	5/5	5/5	0/3
Breast	3/3	3/3	3/3	3/3	0/3
Cerebellum	3/3	3/3	3/3	3/3	1/3*
Cerebrum	3/3	3/3	3/3	3/3	0/3
Cervix	3/3	3/3	3/3	3/3	0/3
Colon	3/3	3/3	3/3	3/3	5/12*
Endometrium	3/3	3/3	3/3	2/3	0/3
Esophagus	3/3	3/3	3/3	3/3	0/3
Heart	3/3	2/3	1/3	3/3	0/3
Hypophysis	3/3	3/3	3/3	3/3	3/3**
Intestine	3/3	3/3	3/3	3/3	2/4*
Kidney	3/3	3/3	3/3	3/3	0/3
Liver	3/3	3/3	3/3	3/3	0/3
Lung	4/4	3/3	3/3	4/4	0/3
Lymph node	3/3	3/3	3/3	3/3	0/3
Mesothelium	4/4	2/3	3/3	3/3	0/3
Pancreas	3/3	3/3	3/3	3/3	2/3*
Parathyroid Gland	3/3	3/3	3/3	3/3	0/3
Peripheral Nerve	5/5	4/4	5/5	5/5	0/5
Prostate	3/3	3/3	3/3	3/3	0/3
Skeletal Muscle	3/3	2/3	3/3	3/3	0/3

Table 17. Tour of Body Immunoreactivity (Normal Tissue)

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

Skin	3/3	3/3	3/3	3/3	0/3
Spleen	3/3	3/3	3/3	3/3	0/3
Stomach	3/3	3/3	3/3	3/3	0/3
Testis	3/3	3/3	3/3	3/3	2/3*
Thymus	3/3	3/3	3/3	3/3	0/3
Thyroid	4/4	4/4	3/3	4/4	0/3
Tongue/Salivary Gland	3/3	3/3	2/3	3/3	0/3
Tonsil	3/3	3/3	3/3	3/3	0/3

For BRAF V600E, * Weak cytoplasmic and nuclear staining in Purkinje cells of cerebellum, smooth muscle and epithelial cells of normal colon, glandular cells of intestine, acinar structures of pancreas, and interstitial cells of testis. ** Moderate staining observed in neuroendocrine cells in hypophysis.

	MLH1-positive /total cases	PMS2-positive /total cases	MSH2-positive /total cases	MSH6-positive /total cases	BRAFV600E-positive /total cases
Pathology	positive /total cases	positive /total cases	positive /total cases	positive /total cases	positive /total cases
Glioblastoma	1/1	1/1	1/1	1/1	0/1
Atypical meningioma	1/1	n.e.*	1/1	1/1	0/1
Malignant ependymoma	1/1	1/1	1/1	1/1	0/1
Malignantoligodendroglioma	1/1	1/1	1/1	1/1	0/1
Serous adenocarcinoma(ovary)	1/1	1/1	1/1	1/1	0/1
Adenocarcinoma (ovary)	1/1	1/1	1/1	1/1	0/1
Islet cell carcinoma	1/1	1/1	1/1	1/1	0/1
Adenocarcinoma ofpancreas	n.e.	n.e.	1/1	n.e	0/1
Seminoma	2/2	2/2	2/2	2/2	0/2
Thyroid medullarycarcinoma	1/1	1/1	1/1	1/1	0/1
Thyroid papillarycarcinoma	1/1	1/1	1/1	1/1	3/3
Intraductal carcinoma(breast)	1/1	1/1	1/1	1/1	0/1
Intraductal carcinoma withearly infiltrate (breast)	1/1	1/1	1/1	1/1	0/1
Invasive ductal carcinoma(breast)	1/1	1/1	1/1	1/1	1/1
Diffuse B-cell lymphoma	1/1	n.e.	1/1	1/1	0/1
Lung small cellundifferentiated carcinoma	1/1	1/1	1/1	1/1	0/1
	MLH1-positive /total cases	PMS2-positive /total cases	MSH2-positive /total cases	MSH6-positive /total cases	BRAFV600E-positive /total cases
Pathology	1/1	1/1	1/1	1/1	0/1
Lung squamous cellcarcinoma
Neuroendocrine carcinoma(esophagus)	1/1	1/1	1/1	1/1	0/1
Adenocarcinoma(esophagus)	1/1	n.e.	1/1	1/1	0/1
Signet ring carcinoma	1/1	1/1	1/1	1/1	0/1
Adenocarcinoma (smallintestine)	1/1	1/1	1/1	1/1	0/1
Stromal sarcoma (smallintestine)	1/1	1/1	1/1	1/1	1/1
Adenocarcinoma (colon)	1/1	1/1	1/1	1/1	0/1
Interstitialoma (abdominalcavity)	1/1	n.e.	n.e.	n.e.	0/1
Adenocarcinoma (rectum)	1/1	1/1	1/1	1/1	0/1
Moderate malignantinterstitialoma (rectum)	1/1	1/1	1/1	1/1	0/1
Hepatocellular carcinoma	n.e.	n.e.	n.e.	1/1	0/1
Hepatoblastoma	1/1	1/1	1/1	1/1	0/1
Clear cell carcinoma(kidney)	1/1	1/1	1/1	1/1	0/1
Adenocarcinoma (Prostate,Gleason grade:4, Gleasonscore:4+5)	1/1	1/1	1/1	1/1	0/1
Adenocarcinoma (Prostate)	1/1	n.e.	1/1	1/1	0/1
Leiomyoma (uterus)	1/1	n.e.	1/1	n.e.	n.e.
Adenocarcinoma (uterus)	1/1	n.e.	1/1	1/1	0/1
Clear cell carcinoma(endometrium)	n.e.	n.e.	n.e.	1/1	n.e.
Squamous cell carcinoma(cervix)	2/2	1/1	2/2	2/2	0/2
Embryonalrhabdomyosarcoma of leftleg	1/1	1/1	1/1	1/1	0/1
Squamous cell carcinomaof chest wall	1/1	1/1	1/1	1/1	0/1
Neurofibroma	1/1	n.e.	n.e.	n.e.	0/1
Neuroblastoma	1/1	1/1	1/1	1/1	0/1
Pathology	MLH1-positive /total cases	PMS2-positive /total cases	MSH2-positive /total cases	MSH6-positive /total cases	BRAFV600E-positive /total cases
Malignant mesothelioma	1/1	1/1	1/1	1/1	0/1
Diffuse B-cell lymphomaof lymph node	1/1	1/1	1/1	1/1	0/1
Diffuse B-cell lymphomaof right thigh	1/1	1/1	1/1	1/1	0/1
Hodgkin's lymphoma leftgroin	1/1	1/1	1/1	1/1	1/1
Transitional cell carcinomaof bladder	n.e.	n.e.	n.e.	n.e.	0/1
Low grade malignantleiomyosarcoma (bladder)	1/1	1/1	1/1	1/1	0/1
Osteosarcoma of rightfemur	1/1	1/1	1/1	1/1	0/1
Spindle cellrhabdomyosarcoma	1/1	n.e.	n.e.	1/1	0/1
Moderate malignantleiomyosarcoma of leftbuttock	1/1	1/1	1/1	1/1	0/1

Table 18. Ventana MMR IHC Panel Staining in Tumor Tissues

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

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

Note: Mismatch repair proteins are present in all actively proliferating cells. For all tissues, MMR positive/negative staining was determined for tumor cells in the presence of positive staining in normal control cells (lymphocytes, fibroblasts and epithelial cells).

For BRAF V600E, for all tissues, BRAF V600E positive/negative staining was determined for tumor cells.

*n.e .: non-evaluable either due to tissue loss or lack of internal control staining.

f. Assay cut-off:
No Assay cut off is employed in the assessment of MMR status or in the assessment of BRAF V600E protein status in FFPE CRC tissue.
1. Comparison studies:
- a. Method comparison with predicate device:

Not Applicable

b. Matrix comparison:
The device is only validated for formalin fixed paraffin embedded (FFPE) colorectal cancer tissue.

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

3. Clinical Performance:

The clinical validity of the Ventana MMR IHC panel was determined in a study that assessed agreement between test results obtained from the Ventana MMR IHC panel and a DNA sequencing panel validated for detecting pathogenic lynch syndrome variants and BRAF V600E mutations in colorectal carcinoma (CRC) specimens. The DNA sequencing panel was used to detect variants in the MMR genes MLH-1. MSH2. PMS2 and MSH6, along with EPCAM, and BRAF gene that are associated with MMR deficiency in CRC. The purpose of the study was to estimate the ability of the panel to correctly aid in the identification of patients needing additional Lynch syndrome genetic testing.

A sequential series of CRC specimens were procured and enriched with a second set of specimens with known Lynch syndrome variants due to the rarity of the variants. Specimens from the two studies were combined and randomized for testing with Ventana MMR IHC panel. Concordance (PPA, NPA and OPA) between the two methods was calculated contingent on the DNA sequencing results. MMR status (Intact/Loss) was stratified by BRAF V600E status and DNA sequencing results were stratified by presence or absence of known pathogenic mutations.

CRC cases were procured and assessed for quality (e.g., presence of tumor and internal control cells) prior to use in the study. Specimens were required to have 50% tumor content to meet specimen requirements for DNA sequencing. Of the sequential CRC cases, 7 cases were excluded from the specimen set due to insufficient viable tumor (i.e., adequate cellularity or lack of tumor content), 3 cases due to misclassification as CRC, and 1 due to clerical error. Following review, 111 sequential cases meeting the study criteria were enrolled into the study. A total of 15 CRC cases with confirmed loss status for MMR protiens by IHC brought the total to 126 specimens. Assessment of the demographic data associated with the study specimens determined that it was consistent intended use population.

Results: Of 126 specimens tested by the IHC panel and DNA sequencing, 7 cases were excluded from final analysis due failure of DNA sequencing. Of the remaining 119, one failed IHC testing. The point estimates for overall agreement between Ventana MMR IHC panel and DNA sequencing were 77.8% PPA, 97.0% NPA and 94.1% OPA. The comparisons of IHC and sequencing status for all specimens with MMR IHC results stratified by BRAFV600E status is summarized in Table 19, Table 20A and Table 20B summarizes results by sequential and enrichment study sets respectively.

Table 19. Agreement between VENTANA MMR IHC Panel Results and DNA Sequencing Results: All Specimens

	DNA Sequencing Results
MMR IHC Panel Results	PathogenicMutation	NoPathogenicMutation	Invalid	Total

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

MMR IHC Panel Results		DNA Sequencing Results
		PathogenicMutation	NoPathogenicMutation	Invalid	Total
MMR Loss	BRAFV600EPositive	1	19	0	20
	BRAFV600ENegative	14	3	1	18
MMR Intact	BRAFV600EPositive	0	3	1	4
	BRAFV600ENegative	2	76	5	83
Invalid		1	0	0	1
Total		18	101	7	126
Agreement
Type		n/N	%	95% CI
PPA		14/18	77.8	(54.8, 91.0)
NPA		98/101	97.0	(91.6, 99.0)
OPA		112/119	94.1	(88.4, 97.1)

Note: Invalids are defined as failure to produce results by IHC and/or DNA sequencing. Only Invalids resulting from a failure by IHC are included in the analysis. 95% CIs were calculated using the (Wilson) Score method.

The association between the test results and the final diagnosis with respect to Lynch Syndrome is an estimate because the study was enriched with Lynch syndrome positive cases.

Table 20A. Agreement between VENTANA MMR IHC Panel and DNA
Sequencing Results: Sequential Cohort

Sequential Study Set
VENTANA MMR IHC Panel Results		DNA Sequencing Results
		PathogenicMutation	No PathogenicMutation	Invalid
	BRAFV600E +	1	18	0	19
MMR Loss	BRAFV600E -	4	2	0	6

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

Sequential Study Set
VENTANA MMR IHC Panel Results		DNA Sequencing Results
		PathogenicMutation	No PathogenicMutation	Invalid	Total
MMR Intact	BRAFV600E +	0	3	1	4
	BRAFV600E -	1	76	5	82
Invalid		0	0	0	0
Total		6	99	6	111
Agreement
Type	n/N	%	95% CI
PPA	4/6	66.7	(30.0, 90.3)
NPA	97/99	98.0	(92.9, 99.4)
OPA	101/105	96.2	(90.6, 98.5)

Table 20B. Agreement between VENTANA MMR IHC Panel and DNA Sequencing Results: Enrichment Cohort

Enrichment Study Set
VENTANA MMR IHC PanelResults		DNA Sequencing Results
		PathogenicMutation	NoPathogenicMutation	Invalid	Total
MMR Loss	BRAF V600E +	0	1	0	1
	BRAF V600E -	10	1	1	12
MMR Intact	BRAF V600E +	0	0	0	0
	BRAF V600E -	1	0	0	1
Invalid		1	0	0	1
Total		12	2	1	15
Agreement
Type		n/N	%	95% CI
PPA		10/12	83.3	(55.2, 95.3)
NPA		1/2	50.0	(9.5, 90.5)
OPA		11/14	78.6	(52.4, 92.4)

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

Accuracy by MMR proteins:

The concordance for MMR gene mutation status by sequencing and MMR protein loss by IHC was also compared individually. For MLH1 and PMS2 loss cases, results were stratified by BRAF V600E. The OPA of each MMR protein, when compared to the results of the DNA sequencing colon panel. was 95.8% for VENTANA anti-MLH1 (M1) antibody, 94.1% for VENTANA anti-PMS2 (A16-4) Mouse Monoclonal Primary Antibody, 98.3% for VENTANA anti-MSH2 (G219-1129) Mouse Monoclonal Primary Antibody and 96.6% for VENTANA anti-MSH6 (SP93) Rabbit Monoclonal Primary antibody.

MLH1/PMS2: A total of 26 cases were identified as MLH-1 and PMS2 loss cases, of which 20 were BRAFV600E positive and therefore sporadic and 19 of these did not carry any pathogenic mutation in the MLH-1 gene. Out of three cases containing a potential pathogenic mutation in the MLH1 gene, one was also BRAF V600E positive and a low allele frequency in sequencing suggesting sporadic CRC. 3 cases carried potential pathogenic PMS2 mutations. One MLH-1/PMS2 loss case was BRAF V600E negative and had no pathogenic variants for either gene.

MSH2/MSH6: Six specimens with loss for MSH2/MSH6 and one for MSH6 alone were identified by MMR IHC panel. Of these three cases contained potential pathogenic mutation in MSH2 gene and 3 in MSH6 gene and the one MSH6 alone case carried potential pathogenic mutation in MSH6. Additional four cases that contained a potential pathogenic mutation affecting MSH6 expression demonstrated MSH6 Intact status by IHC. Of these, two contained POLE mutations which variably affect the expression of MMR protein and do not represent Lynch syndrome mutations. One case demonstrates MSH6 IHC staining in a small portion of the tumor and was designated intact, but DNA sequencing showed several mutations in the MSH6 gene which likely result from somatic mutation.

BRAF V600E: The ability of the VENTANA anti-BRAF V600E (VE1) antibody to stratify CRC cases showing a loss of MLH1 protein expression was verified in the study. Of the 23 positive BRAF V600E cases, 20 cases had loss of MLH1 protein by IHC. The remaining three cases were pMMR (intact for all MMR proteins). All 23 BRAF V600E positive specimens were identified as sporadic CRC and were confirmed to carry the V600E mutation by sequencing. The results verified that the VENTANA anti-BRAF V600E (VE1) antibody can correctly differentiate between sporadic and probable Lynch syndrome CRC in the absence of MLH1 expression.

A breakdown of the agreements between the MMR IHC status-stratified by BRAFV600E results for MLH-1 and PMS2 loss cases- and DNA sequencing results for the MMR genes by individual MMR marker is captured in table 21A and in 21B and 21C summarizes agreement by marker for sequential and enrichment study set respectively.

Table 21A. Agreement between each protein in the VENTANA MMR IHC Panel and DNA Sequencing Results all specimens.

IHC Results	DNA Sequencing Results
-------------	------------------------

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

		PathogenicMutation	No PathogenicMutation	Total
MLH1Loss	BRAFV600E +	1	19	20
	BRAFV600E -	2	4	6
MLH1 Intact		0	92	92
Total		3	115	118
PMS2 Loss	BRAFV600E +	0	20	20
	BRAFV600E -	3	7	10
PMS Intact		0	88	88
Total		3	115	118
MSH2 Loss		3	2	5
MSH2 Intact		0	113	113
Total		3	115	118
MSH6 Loss		4	0	4
MSH6 Intact		4	110	114
Total		8	110	118

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

	IHC Results	PathogenicMutation	No PathogenicMutation	Total
MSH6	PPA	4/8	50.0	(21.5,78.5)
	NPA	110/110	100.0	(96.6,100.0)
	OPA	114/118	96.6	(91.6, 98.7)

Table 21B. Agreement by Marker for Sequential Cohort

Sequential Study Set
IHC Results	DNA Sequencing Results
	Pathogenic Mutation	No Pathogenic Mutation	Total
MLH1Loss	BRAFV600E +	1	18	19
	BRAFV600E -	1	4	5
MLH1 Intact		0	81	81
Total		2	103	105
PMS2 Loss	BRAFV600E +	0	19	19
	BRAFV600E -	0	5	5
PMS Intact		0	81	81
Total		0	105	105
MSH2 Loss		1	0	1
MSH2 Intact		0	104	104
Total		1	104	105
MSH6 Loss		0	0	0
MSH6 Intact		3	102	105
Total		3	102	105
Agreement
Protein	Type	n/N	%	95% CI
MLH1	PPA	1/2	50.0	(9.5, 90.5)
	NPA	99/103	96.1	(90.4, 98.5)

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

Sequential Study Set
DNA Sequencing Results
IHC Results		Pathogenic Mutation	No Pathogenic Mutation	Total
PMS2	OPA	100/105	95.2	(89.3, 97.9)
	PPA	n.e.	n.e.	n.e.
	NPA	100/105	95.2	(89.3, 97.9)
MSH2	OPA	100/105	95.2	(89.3, 97.9)
	PPA	1/1	100.0	(20.7,100.0)
	NPA	104/104	100.0	(96.4, 100.0)
MSH6	OPA	105/105	100.0	(96.5, 100.0)
	PPA	0/3	0.0	(0.0, 56.1)
	NPA	102/102	100.0	(96.4,100.0)
	OPA	102/105	97.1	(91.9, 99.0)

Table 21C. Agreement by Marker for Enrichment set

Enrichment Study Set
		DNA Sequencing Results
IHC Results		PathogenicMutation	No PathogenicMutation	Total
MLH1Loss	BRAFV600E +	0	1	1
	BRAFV600E -	1	0	1
MLH1 Intact		0	11	11
Total		1	12	13
PMS2 Loss	BRAFV600E +	0	1	1
	BRAFV600E -	3	2	5
PMS Intact		0	7	7
Total		3	10	13
MSH2 Loss		2	2	4
MSH2 Intact		0	9	9
Total		2	11	13

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

Enrichment Study Set
IHC Results		DNA Sequencing Results
		Pathogenic Mutation	No Pathogenic Mutation	Total
MSH6 Loss		4	0	4
MSH6 Intact		1	8	9
Total		5	8	13
Agreement
Protein	Type	n/N	%	95% CI
MLH1	PPA	1/1	100.0	(20.7, 100.0)
	NPA	12/12	100.0	(75.8, 100.0)
	OPA	13/13	100.0	(77.2, 100.0)
PMS2	PPA	3/3	100.0	(43.9, 100.0)
	NPA	8/10	80.0	(49.0, 94.3)
	OPA	11/13	84.6	(57.8, 95.7)
MSH2	PPA	2/2	100.0	(34.2,100.0)
	NPA	9/11	81.8	(52.3, 94.9)
	OPA	11/13	84.6	(57.8, 95.7)
MSH6	PPA	4/5	80.0	(37.6, 96.4)
	NPA	8/8	100.0	(67.6,100.0)
	OPA	12/13	92.3	(66.7, 98.6)

b. Other Clinical supportive data:

Testing CRC patients for possible Lynch Syndrome is well-established as part of the clinical management of these patients and is included in National comprehensive cancer network (NCCN) guidelines for newly diagnosed patients. Ventana submitted literature to support clinical validity of IHC testing of the MMR marker panel consisting of MLH1, PMS2, MSH2, MSH6 and BRAF V600E IHC as an aid in detection of mismatch repair protein deficiency and BRAFV600E status as aid to differentiate between sporadic and probable lynch syndrome identifying

A total of 154 papers were identified as relevant for supporting the clinical validity of MMR IHC test in CRC as aid in Lynch syndrome diagnoses.

The measured sensitivity in these studies to detect germline MLH1, MSH2, MSH6 and PMS2 mutations by IHC (not represented by this panel) was 92% (23/25). 93% (28/30). 100% (8/8) and 100% (3/3), respectively, when data from studies evaluating all 4 MMR

{29}------------------------------------------------

proteins were analyzed. This data supports the use of a four antibody MMR IHC panel as a screening tool for potential Lynch syndrome patients.

BRAF V600E mutation IHC to distinguish Sporadic and Germline CRC: In 550 MMR mutation carriers, the BRAF V600E mutation frequency was only 1.4% (95% CI: 0.06-2.52). The frequency of BRAF V600E mutations was 5% (95% CI: 3.6-6.9) in 1,623 microsatellite stable (MSS) cases, 36.1% (95% CI: 21.0-52.8) in MSI-H cases without MMR mutations, and 63.5% (95% CI 447.0-78.5) in 332 cases demonstrating MLH1 methylation or MLH1 expression loss.

Conclusion: Literature survey shows that IHC test that include the 4 dMMR markers can identify MSI-H /dMMR phenotype in CRC subjects who can be directed to additional testing for Lynch syndrome diagnoses. The survey presented also shows the effectiveness of BRAF V600E IHC in distinguishing sporadic and germline CRC patients.

1. Clinical cut-off:
  The MMR IHC panel and BRAF V600E tests are qualitative in nature and marker status (loss / intact or positive/negative) are determined based on unequivocal staining of FFPE CRC tissue (see section I on staining interpretation and scoring) and do not rely on discreet clinical cut off.
1. Expected values/Reference range:
  Not applicable.

N. Proposed Labeling:

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

O. Patient Perspectives

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

P. Identified Risks to Health and Identified Mitigations

Identified Risks to Health	Identified Mitigations
False positive test result	General controls and special controls (1)and (2)
False negative test result	General controls and special controls (1)and (2)

{30}------------------------------------------------

Q. Benefit/Risk Determination

Summary
Summary of theBenefit(s)	This kit has significant benefit as an adjunct to thephysician's evaluation of suspected Lynch syndromeas indicated by the guidelines and as demonstrated inanalytical and clinical performance studies.IHC testing for MMR deficiency in CRC patients isa sensitivity method for identification of Lynchsyndrome and can lead to better patient managementand outcomes.
Summary of theRisk(s)	There are risks associated with false negative and false positiveresults.
	However, the risks associated with these misclassifications haveto be taken into the context that the test is intended to be used asan adjunct to physician's evaluation of suspected of having Lynchsyndrome along with additional clinic-pathological factors and isnot intended to be used as a standalone diagnostic.
	False positive test results for the loss of MMR proteins will resultin additional testing for the patients and the family. The risk of afalse positive test result is mitigated by special control (1)(iv) thatrequires identification and inclusion of appropriate positive andnegative controls to in the test to ensure accurate performance andis further mitigated by the demonstrated analytical accuracy ofthe device. Furthermore the test is intended to use as an adjunct toidentify patients who will benefit from additional testing, therebyminimizing the risk.
	A false negative test result can result in missing additional testingand failure to identify Lynch syndrome. In clinical practice, if theclinical or pathological features suggestive of Lynch syndromestill remains high, then the pathologist performs PCR testing,thereby negating the false negative result of this test.
	The false negative rates in the study were low in the accuracystudy except in the case of MSH6, with4 cases of MSH6 mutation carrying specimens testing as MSH6intact in the study. However it was determined that 3 of the fourwere due to somatic mutations of which 2 resulted from mutationto an MMR unrelated POLE genes. Mutations in POLE genes areassociated with secondary somatic alteration in MSH6 with noloss in protein expression.
	The risk of false negatives is mitigated by demonstratedperformance of the test.

{31}------------------------------------------------

{32}------------------------------------------------

Conclusions	The probable clinical benefits of this device outweigh the
Do the probablebenefits outweigh	potential risks in light of the special controls established forthis device type, in combination with applicable general
the probablerisks?	controls, including design controls.

R. Conclusion

The information provided in this de novo submission to classify this device into class II under regulation 21 CFR 864.1866. 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:	PZJ
Device Type:	Lynch syndrome test systems
Class:	II (special controls)
Regulation:	21 CFR 864.1866

(a) Identification: Lynch syndrome test systems are in vitro diagnostic tests for use with tumor tissue to identify previously diagnosed cancer patients at risk for having Lynch syndrome.

(b) Classification: Class II (special controls). A Lynch syndrome test system must comply with the following special controls:

(1) Premarket notification submissions must include the following information, as appropriate:

(i) A detailed description of all test components, including all provided reagents, and required but not provided, ancillary reagents.

(ii) A detailed description of instrumentation and equipment, including illustrations or photographs of non-standard equipment or manuals.

(iii) Detailed documentation of the device software, including, but not limited to, standalone software applications and hardware-based devices that incorporate software.

(iv) A detailed description of quality controls including appropriate positive and negative controls that are recommended or provided.

(v) Detailed specifications for sample collection, processing, and storage.

(vi) A detailed description of methodology and assay procedure.

(vii) A description of the assay cut-off (i.e., the medical decision point between positive and negative results) or other relevant criteria that distinguishes positive and negative results, or ordinal classes of marker expression, including the rationale for the chosen cutoff or other relevant criteria and results supporting validation of the cut-off.

{33}------------------------------------------------

(viii) Detailed specification of the criteria for test result interpretation and reporting.

(ix) Detailed information demonstrating the performance characteristics of the device, including:

(A) Data from an appropriate study demonstrating clinical accuracy using wellcharacterized clinical specimens representative of the intended use population (i.e., concordance to DNA sequencing results of the Lynch syndrome associated genes or method comparison to the predicate device using samples with known alterations in genes representative of Lynch syndrome). Pre-specified acceptance criteria must be provided and followed.

(B) Appropriate device reproducibility data investigating all sources of variance (e.g., for distributed tests, data generated using a minimum of three sites, of which at least two sites must be external sites). Each site must perform testing over a minimum of 5 nonconsecutive days evaluating a sample panel that spans the claimed measuring range, and includes the clinical threshold. Pre-specified acceptance criteria must be provided and followed.

(C) Data demonstrating reader reproducibility, both within-reader and betweenreader, assessed by three readers over three nonconsecutive days at each site, including a two week washout period between reads, as appropriate.

(D) Device precision data using clinical samples spanning the measuring range and controls to evaluate the within-lot, between-lot, within-run, between run, and total variation.

(E) Analytical specificity studies including as appropriate, western blots, peptide inhibition, testing in normal tissues and neoplastic tissues, interference by endogenous and exogenous substances, and cross-reactivity and cross contamination testing.

(F) Device analytical sensitivity data generated by testing an adequate number of samples from individuals with the target condition such that prevalence of the biomarker in the target population is established.

(G) Device stability data, including real-time stability and in-use stability, and stability evaluating various storage times, temperatures, and freeze-thaw conditions, as appropriate.

(H) The staining performance criteria assessed must include overall staining acceptability, background staining acceptability, and morphology acceptability, as appropriate.

(I) Appropriate training requirements for users, including interpretation manual,

{34}------------------------------------------------

as applicable.

(J) 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 paragraphs (b)(1)(i) through (b)(1)(viii) of this section, as appropriate, and a detailed description of the performance studies performed and the summary of the results, including those that relate to paragraph (b)(1)(ix) of this section, as appropriate.

Regulation Number and Section

§ 864.1866 Lynch syndrome test systems.

(a)
Identification. Lynch syndrome test systems are in vitro diagnostic tests for use with tumor tissue to identify previously diagnosed cancer patients at risk for having Lynch syndrome.(b)
Classification. Class II (special controls). The special controls for this device are:(1) Premarket notification submissions must include the following information, as appropriate:
(i) A detailed description of all test components, including all provided reagents, and required but not provided, ancillary reagents.
(ii) A detailed description of instrumentation and equipment, including illustrations or photographs of non-standard equipment or manuals.
(iii) Detailed documentation of the device software, including, but not limited to, standalone software applications and hardware-based devices that incorporate software.
(iv) A detailed description of quality controls including appropriate positive and negative controls that are recommended or provided.
(v) Detailed specifications for sample collection, processing, and storage.
(vi) A detailed description of methodology and assay procedure.
(vii) A description of the assay cut-off (
i.e., the medical decision point between positive and negative results) or other relevant criteria that distinguishes positive and negative results, or ordinal classes of marker expression, including the rationale for the chosen cut-off or other relevant criteria and results supporting validation of the cut-off.(viii) Detailed specification of the criteria for test result interpretation and reporting.
(ix) Detailed information demonstrating the performance characteristics of the device, including:
(A) Data from an appropriate study demonstrating clinical accuracy using well-characterized clinical specimens representative of the intended use population (
i.e., concordance to Deoxyribonucleic Acid (DNA) sequencing results of the Lynch syndrome associated genes or method comparison to the predicate device using samples with known alterations in genes representative of Lynch syndrome). Pre-specified acceptance criteria must be provided and followed.(B) Appropriate device reproducibility data investigating all sources of variance (
e.g., for distributed tests, data generated using a minimum of three sites, of which at least two sites must be external sites). Each site must perform testing over a minimum of 5 nonconsecutive days evaluating a sample panel that spans the claimed measuring range, and includes the clinical threshold. Pre-specified acceptance criteria must be provided and followed.(C) Data demonstrating reader reproducibility, both within-reader and between-reader, assessed by three readers over 3 nonconsecutive days at each site, including a 2 week washout period between reads, as appropriate.
(D) Device precision data using clinical samples spanning the measuring range and controls to evaluate the within-lot, between-lot, within-run, between run, and total variation.
(E) Analytical specificity studies including as appropriate, western blots, peptide inhibition, testing in normal tissues and neoplastic tissues, interference by endogenous and exogenous substances, and cross-reactivity and cross contamination testing.
(F) Device analytical sensitivity data generated by testing an adequate number of samples from individuals with the target condition such that prevalence of the biomarker in the target population is established.
(G) Device stability data, including real-time stability and in-use stability, and stability evaluating various storage times, temperatures, and freeze-thaw conditions, as appropriate.
(H) The staining performance criteria assessed must include overall staining acceptability, background staining acceptability, and morphology acceptability, as appropriate.
(I) Appropriate training requirements for users, including interpretation manual, as applicable.
(J) 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 § 809.10(b) of this chapter compliant labeling must include a detailed description of the protocol, including the information described in paragraphs (b)(1)(i) through (viii) of this section, as appropriate, and a detailed description of the performance studies performed and the summary of the results, including those that relate to paragraph (b)(1)(ix) of this section, as appropriate.