BOND MMR Antibody Panel

K213348 · Leica Biosystems Newcastle, Ltd. · PZJ · Feb 21, 2023 · Immunology

Device Facts

• Record ID: K213348
• Device Name: BOND MMR Antibody Panel
• Applicant: Leica Biosystems Newcastle, Ltd.
• Product Code: PZJ · Immunology
• Decision Date: Feb 21, 2023
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 864.1866
• Device Class: Class 2

Indications for Use

The BOND MMR Antibody Panel is intended to be used for the qualitative identification by light microscopy of human mismatch repair (MMR) proteins MLH1, MSH2, MSH6 and PMS2 in formalin-fixed, paraffin-embedded (FFPE) colorectal cancer (CRC) tissue sections by immunohistochemical staining. The BOND MMR Antibody Panel includes BOND Ready-to-Use Primary Antibody MLH1 (Mismatch Repair Protein) (ES05), BOND Ready-to-Use Primary Antibody MSH2 (Mismatch Repair Protein) (79H11), BOND Ready-to-Use Primary Antibody MSH6 (Mismatch Repair Protein) (EP49) and BOND Ready-to-Use Primary Antibody PMS2 (Mismatch Repair Protein) (EP51). The BOND MMR Antibody Panel is intended for use on the BOND-III or BOND-MAX fully automated systems with BOND Polymer Refine Detection. The BOND MMR Antibody Panel is indicated for the detection of MMR protein deficiency as an aid in the identification of potential hereditary nonpolyposis colorectal cancer (HNPCC)/Lynch Syndrome in patients diagnosed with CRC. Patients with “MMR Loss” results should receive additional diagnostic testing consistent with clinical practice guidelines for diagnosis of Lynch syndrome. The BOND MMR Antibody Panel is not intended for use in indications other than CRC. This test should not be used for diagnosis of CRC. The clinical interpretation of any staining or its absence when using the BOND MMR Antibody Panel should be complemented by morphological studies and proper controls and should be evaluated within the context of the patient’s clinical history and other diagnostic tests by a qualified pathologist. The clinical performance of this device to guide treatment of MMR deficient patients has not been established.

Device Story

The BOND MMR Antibody Panel is an immunohistochemistry (IHC) test for qualitative identification of MLH1, MSH2, MSH6, and PMS2 proteins in FFPE colorectal cancer tissue. The device uses four ready-to-use monoclonal antibodies applied to tissue sections on BOND-III or BOND-MAX automated staining platforms. The system performs automated deparaffinization, antigen retrieval, and staining using BOND Polymer Refine Detection (poly-HRP anti-mouse/rabbit IgG and DAB chromogen). A pathologist evaluates the stained slides via light microscopy to determine protein expression status (intact or loss). The output aids in identifying patients who may require further genetic testing for Lynch syndrome. The device is intended for use by pathologists in clinical laboratory settings.

Clinical Evidence

Clinical validity was established by comparing BOND MMR Antibody Panel results to a validated DNA sequencing panel for pathogenic mutations in 133 evaluable FFPE CRC cases (combined sequential and enrichment cohorts). The combined cohort showed 93.3% PPA, 95.9% NPA, and 94.7% OPA. Analytical performance studies (precision, reproducibility, stability) were conducted across multiple sites, days, and instruments, meeting pre-specified acceptance criteria.

Technological Characteristics

IHC-based protein expression assay. Components: Mouse/Rabbit monoclonal antibodies (ES05, 79H11, EP49, EP51) in Tris buffered saline with ProClin 950. Detection: Biotin-free, polymeric HRP-linker (BOND Polymer Refine Detection). Platform: BOND-III/BOND-MAX automated slide stainers. Connectivity: Standalone instrument. Software: Embedded instrument/processing module software.

Indications for Use

Indicated for detection of MMR protein deficiency in patients diagnosed with colorectal cancer (CRC) as an aid in identifying potential hereditary nonpolyposis colorectal cancer (HNPCC)/Lynch Syndrome. Not for CRC diagnosis or treatment guidance.

Regulatory Classification

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.

Special Controls

*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.

Predicate Devices

• VENTANA Mismatch Repair Immunohistochemistry (MMR IHC) Panel (DEN170030)

Submission Summary (Full Text)

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510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION
DECISION SUMMARY

I Background Information:

A. 510(k) Number:
K213348

B. Applicant
Leica Biosystems Newcastle, Ltd.

C. Proprietary and Established Names:
BOND MMR Antibody Panel

D. Regulatory Information
| Product Code | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| PZJ | Class II | 21 CFR 864.1866 - Lynch Syndrome Test Systems | 88 Pathology |

II Submission/Device Overview:

E. Purpose for Submission:
New device

F. Measurand:
Mismatch repair proteins MLH1, MSH2, MSH6 and PMS2.

G. Type of Test:
Immunohistochemistry

III Intended Use/Indications for Use:

H. Indications for Use:
The BOND MMR Antibody Panel is intended to be used for the qualitative identification by light microscopy of human mismatch repair (MMR) proteins MLH1, MSH2, MSH6 and PMS2 in formalin-fixed, paraffin-embedded (FFPE) colorectal cancer (CRC) tissue sections by immunohistochemical staining. The BOND MMR Antibody Panel includes BOND Ready-to-Use Primary Antibody MLH1 (Mismatch Repair Protein) (ES05), BOND Ready-to-Use Primary Antibody MSH2 (Mismatch Repair Protein) (79H11), BOND Ready-to-Use Primary Antibody MSH6 (Mismatch Repair Protein) (EP49) and

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BOND Ready-to-Use Primary Antibody PMS2 (Mismatch Repair Protein) (EP51). The BOND MMR Antibody Panel is intended for use on the BOND-III or BOND-MAX fully automated systems with BOND Polymer Refine Detection.

The BOND MMR Antibody Panel is indicated for the detection of MMR protein deficiency as an aid in the identification of potential hereditary nonpolyposis colorectal cancer (HNPCC)/Lynch Syndrome in patients diagnosed with CRC. Patients with “MMR Loss” results should receive additional diagnostic testing consistent with clinical practice guidelines for diagnosis of Lynch syndrome. The BOND MMR Antibody Panel is not intended for use in indications other than CRC. This test should not be used for diagnosis of CRC.

The clinical interpretation of any staining or its absence when using the BOND MMR Antibody Panel should be complemented by morphological studies and proper controls and should be evaluated within the context of the patient’s clinical history and other diagnostic tests by a qualified pathologist.

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

## I. Special conditions for use statement(s):

Rx - For prescription use.

For in vitro diagnostic use.

## J. Special instrument requirements:

BOND-III automated system or BOND-MAX automated system.

## IV Device/System Characteristics:

## K. Device Description:

The BOND MMR Antibody Panel consists of the following BOND Ready-to-Use (RTU) Primary Antibody (PA) products:

- MLH1 (Mismatch Repair Protein) (ES05) (PA0988-U)
- MSH2 (Mismatch Repair Protein) (79H11) (PA0989-U)
- MSH6 (Mismatch Repair Protein) (EP49) (PA0990-U)
- PMS2 (Mismatch Repair Protein) (EP51) (PA0991-U)

MLH1 (Mismatch Repair Protein) (ES05) is a mouse anti-human monoclonal antibody produced as a tissue culture supernatant, and supplied in Tris buffered saline with carrier protein, containing 0.35 % ProClin 950 as a preservative and in a total volume of 7 mL. The antibody is optimally diluted for use on the automated BOND-MAX or BOND-III instrument staining platforms in combination with BOND Polymer Refine Detection.

MSH2 (Mismatch Repair Protein) (79H11) is a mouse anti-human monoclonal antibody produced as a tissue culture supernatant, and supplied in Tris buffered saline with carrier protein, containing 0.35 % ProClin 950 as a preservative and in a total volume of 7ml. The antibody is optimally diluted for use on the automated BOND-MAX or BOND-III instrument staining platforms in combination with BOND Polymer Refine Detection.

MSH6 (Mismatch Repair Protein) (EP49) is a rabbit anti-human monoclonal antibody produced as an affinity-purified tissue culture supernatant, and supplied in Tris buffered saline with carrier protein,

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containing 0.35 % ProClin 950 as a preservative and in a total volume of 7ml.. The antibody is optimally diluted for use on the automated BOND-MAX or BOND-III instrument staining platforms in combination with BOND Polymer Refine Detection.

PMS2 (Mismatch Repair Protein) (EP51) is a rabbit anti-human monoclonal antibody produced as an affinity purified tissue culture supernatant, and supplied in Tris buffered saline with carrier protein, containing 0.35 % ProClin 950 as a preservative and in a total volume of 7ml. The antibody is optimally diluted for use on the automated BOND-MAX or BOND-III instrument staining platforms in combination with BOND Polymer Refine Detection.

BOND Ready-To-Use Reagents use containers that fit into the reagent trays. These reagents are provided in concentrations optimized for the BOND System.

Reagents required but not provided with the BOND MMR Antibody Panel are listed below:

- BOND Ready-to-Use Negative Control Negative (Mouse) (Catalog No. PA0996)
- BOND Ready-to-Use Negative Control Negative (Rabbit) (Catalog No. PA0777)
- BOND Polymer Refine Detection (Catalog No. DS9800)
- BOND Dewax Solution (Catalog No. AR9222)
- BOND Epitope Retrieval Solution 1 (Catalog No. AR9961)
- BOND Epitope Retrieval Solution 2 (Catalog No. AR9640)
- BOND Wash Solution 10X Concentrate (Catalog No. AR9590)

The Test Components/Accessories of the BOND MMR Antibody Panel System are summarized below:

| Components of the BOND MMR Antibody Panel System | | | |
| --- | --- | --- | --- |
| BOND Ready-to-Use (RTU) Primary Antibodies | BOND Detection System | BOND Ancillary Reagents/BOND Consumables | BOND Platforms |
| • BOND MMR Antibody Panel
○ MLH1 (Mismatch Repair Protein) (ES05) (PA0988-U)
○ MSH2 (Mismatch Repair Protein) (79H11) (PA0989-U)
○ MSH6 (Mismatch Repair Protein) (EP49) (PA0990-U)
○ PMS2 (Mismatch Repair Protein) (EP51) (PA0991-U)
• Negative (Mouse) BOND Ready-to-Use Negative Control (PA0996)
• Negative (Rabbit) BOND Ready-to-Use Negative Control (PA0777) | • BOND Polymer Refine Detection (DS9800) | • BOND Wash Solution 10X (AR9590) Concentrate
• BOND Dewax Solution (AR9222)
• BOND Epitope Retrieval Solution 1 (AR9961)
• BOND Epitope Retrieval Solution 2 (AR9640)
• BOND Universal Covertile (S21.4611 & S21.2001) | • BOND-MAX Instrument
• BOND-III Instrument |

## Quality Controls

Internal Positive Tissue Control: Normal epithelial cells in the vicinity of the tumor or infiltrating lymphocytic cells, fibroblasts, nerves, and muscle integral to the patient specimen can serve as internal positive tissue controls for each of the primary antibodies, as they should exhibit expression of the mismatch repair proteins (MLH1, MSH2, MSH6 and PMS2) even if the tumor in the patient specimen is

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deficient for the protein. The presence of staining elicited by the primary antibody in an internal positive tissue control indicates the following:

1. The tissue of the patient specimen has been subjected to suitable processing and fixation to allow the epitope of the target protein to be detected via IHC.
2. The staining procedure has been performed correctly on the slide mounted with the patient specimen.
3. The primary antibody is performing as intended and staining the corresponding protein.

Negative Reagent Control: For each patient specimen, a negative reagent control must be used to stain an additional slide-mounted tissue section to that stained with the primary antibody. This will determine whether the primary antibody is reacting with the patient tissue in a non-specific manner. BOND Ready-to-Use Negative Control Negative (Mouse) is recommended as the negative reagent control for use in conjunction with MLH1 (Mismatch Repair Protein) (ES05) and MSH2 (Mismatch Repair Protein) (79H11). BOND Ready-to-Use Negative Control Negative (Rabbit) is recommended as the negative reagent control for use in conjunction with MSH6 (Mismatch Repair Protein) (EP49) and PMS2 (Mismatch Repair Protein) (EP51).

## Instrumentation and Software:

The BOND MMR Antibody Panel is intended for use on the BOND-III or BOND-MAX fully automated systems. The BOND-MAX and BOND-III instruments are fully automated slide stainers that perform automated deparaffinization (dewaxing), antigen retrieval, immunohistochemistry (IHC) staining/in situ hybridization (ISH) staining, and counterstaining. The major components of the BOND staining platforms are the processing module, computer (BOND controller), handheld ID scanner, and slide label printer. The BOND staining platforms are composed of a number of discrete software components including the BOND application software, BOND instrument/processing module software, BOND service software, and Laboratory interface system - integration package (LIS-IP).

## L. Test Principle:

Immunohistochemical techniques can be used to demonstrate the presence of antigens in tissue and cells. MLH1 (Mismatch Repair Protein) (ES05), MSH2 (Mismatch Repair Protein) (79H11), MSH6 (Mismatch Repair Protein) (EP49) and PMS2 (Mismatch Repair Protein) (EP51) primary antibodies are Ready-to-Use products that have been specifically optimized for use on the automated BOND-MAX or BOND-III systems in combination with BOND Polymer Refine Detection.

The BOND Polymer Refine Detection Process is as follows:

- The specimen is incubated with hydrogen peroxide to quench endogenous peroxidase activity.
- The BOND Ready-to-Use Primary Antibody is applied.
- A post primary antibody solution enhances penetration of the subsequent polymer reagent.
- A poly-HRP anti-mouse/rabbit IgG reagent localizes the primary antibody.
- The substrate chromogen, 3,3'-diaminobenzidine (DAB), visualizes the complex via a brown precipitate.
- Hematoxylin (blue) counterstaining allows the visualization of cell nuclei.

## M. Interpretation of Results

Hematoxylin and Eosin stained slides are used to assess the overall quality of the sample for each case. An assessment should be made of the quality and quantity of the tumor content of the CRC sample. Cases should be voided if there is insufficient tumor (less than 50 viable tumor cells) available to score or if the sample is poorly fixed or otherwise of a poor enough quality that renders interpretation of the case impossible. The MMR protein status for each IHC marker shall be assigned following the decision

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summary illustrated in the flowchart below.

## Decision Summary Flowchart - MMR Protein Status for each IHC Marker


*Any staining seen in the Negative Isotype Controls should be taken into account when interpreting the MMR antibody stained test sections. If any staining is judged to be of sufficient intensity to adversely affect the interpretation of the test sections then this should be classed as unacceptable and the case voided.

** Weaker than internal positive control cells

To obtain the BOND MMR Antibody Panel results, each individual marker (MLH1, MSH2, MSH6 and PMS2) will be scored as protein loss or protein intact according to the device's prespecified scoring criteria. The loss of one or more of these proteins within one specimen will be considered an MMR loss. To obtain an intact panel result, all four proteins must be scored as intact.

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V Substantial Equivalence Information:

N. Predicate Device Name:
Ventana MMR IHC panel

O. Predicate 510(k) Number:
DEN170030

P. Comparison with Predicate:

| | Leica Biosystems BOND MMR Antibody Panel (K213348) [Subject Device] | VENTANA Mismatch Repair Immunohistochemistry (MMR IHC) Panel (DEN170030) [Predicate Device] |
| --- | --- | --- |
| Similarities | | |
| Indications for Use | For In Vitro Diagnostic Use The BOND MMR Antibody Panel is intended to be used for the qualitative identification by light microscopy of human mismatch repair (MMR) proteins MLH1, MSH2, MSH6 and PMS2 in formalin-fixed, paraffin-embedded (FFPE) colorectal cancer (CRC) tissue sections by immunohistochemical staining. The BOND MMR Antibody Panel includes BOND Ready-to-Use Primary Antibody MLH1 (Mismatch Repair Protein) (ES05), BOND Ready-to-Use Primary Antibody MSH2 (Mismatch Repair Protein) (79H11), BOND Ready-to-Use Primary Antibody MSH6 (Mismatch Repair Protein) (EP49) and BOND Ready-to-Use Primary Antibody PMS2 (Mismatch Repair Protein) (EP51). The BOND MMR Antibody Panel is intended for use on the BOND-III or BOND-MAX fully automated systems with BOND Polymer Refine Detection. The BOND MMR Antibody Panel is indicated for the detection of MMR protein deficiency as an aid in the identification of potential hereditary nonpolyposis colorectal cancer (HNPCC)/Lynch Syndrome in patients diagnosed with CRC. Patients with “MMR Loss” results should receive additional diagnostic testing consistent with clinical practice guidelines for diagnosis of Lynch syndrome. The BOND MMR Antibody Panel is not intended for use in indications other than CRC. This test should not be used for diagnosis of CRC. The clinical interpretation of any staining or its absence when using the BOND MMR Antibody Panel should be complemented by morphological studies and proper controls and should be evaluated within the context of the patient’s clinical history and other diagnostic tests by a qualified pathologist. | For In Vitro Diagnostic Use 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 and MSH6, 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 |

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VI Standard/Guidance Document Referenced (if applicable):

Guidance for Submission of Immunohistochemistry Applications to the FDA. 1998

| | The clinical performance of this device to guide treatment of MMR deficient patients has not been established. | established |
| --- | --- | --- |
| Target Population | Patients diagnosed with CRC | Same |
| Tissue Type | Formalin-Fixed Paraffin-Embedded Colorectal Cancer Tissue Sections | Same |
| Technology | Immunohistochemistry based protein expression | Same |
| Read/Review Equipment | Light Microscopy | Same |
| Controls | Internal positive tissue controls and negative reagent controls | External and internal positive tissue controls and negative reagent controls |
| Result Interpretation | Pathologist assessment | Same |
| Differences | | |
| Supplemental BRAFV600E testing | Not applicable | As an aid to differentiate between sporadic CRC and probable Lynch syndrome |
| Assay Target | 4 Mismatch repair (MMR) proteins MLH1, MSH2, MSH6 and PMS2 | 4 Mismatch repair (MMR) proteins MLH1, MSH2, MSH6 and PMS2
BRAF V600E protein |
| Detection Kit | BOND Polymer Refine Detection (DS9800): Biotin-free, polymeric HRP-linker antibody conjugate, for MLH1, MSH2, MSH6 and PMS2. | OptiView DAB IHC Detection Kit: Biotin-free, Multimeric HRP-linker antibody conjugate for MLH1, MSH2, MSH6 and BRAF V600E.
OptiView DAB IHC Detection Kit with OptiView Amplification Kit: Biotin-free, Multimeric HRP-linker antibody conjugate, with tyramide- based amplification for PMS2. |
| Staining Instruments | LBS BOND -MAX and BOND-III Instruments | VENTANA BenchMark ULTRA Instrument |

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Performance Characteristics:

# Q. Analytical performance

# a. Precision

Three studies were performed to evaluate the precision of the BOND MMR Antibody Panel.

# i. Precision across Lots and Days on the BOND-III and BOND-MAX Instruments

This study was conducted at 1 site using 3 lots of BOND Ready To Use MMR Primary Antibody for each of the 4 MMR proteins. Slides from 40 FFPE CRC tissue cases, 10 cases per MMR protein, with 5 cases being protein deficient and 5 cases expressing intact protein were tested. Each case was stained 18 times [3 Slide Staining Assembly (SSAs) x 6 days] on 1 BOND-III and 1 BOND-MAX instrument. Testing was performed over 6 nonconsecutive days following a randomized testing order. Therefore, a total of 1440 slides (4 antibodies x 10 samples x 3 SSAs x 2 instruments x 6 days) were evaluated. A single pathologist read and scored all stained slides.

Positive percent agreement (PPA), negative percent agreement (NPA) and overall percent agreement (OPA) were calculated, comparing the antibody status of each processed slide determined by the pathologist to the majority score. Point estimates along with 2-sided $95\%$ confidence intervals were calculated separately by instrument and by antibody. All slides from one case for MSH2 staining were excluded from BOND-III data analysis $(n = 18)$ and from BOND-MAX data analysis $(n = 18)$ because the case had insufficient tumor.

# Intra-run Repeatability:

Intra-run repeatability was evaluated using 40 FFPE CRC tissue cases, 10 cases per MMR protein, with 5 cases being protein deficient (protein loss) and 5 cases expressing intact protein (intact). Six (6) slides were stained using each panel antibody. Data were obtained using 1 lot of each panel antibody from 60 total observations (10 cases x 3 replicates x 2 days x 1 instrument) for each panel antibody on BOND-III and from 60 total observations (10 cases x 3 replicates x 2 days x 1 instrument) for each panel antibody on BOND-MAX. Intra-run repeatability OPA was $100\%$ for each panel antibody on both instruments except MSH2 on BOND-III (OPA $98.1\%$ , PPA $100\%$ , NPA $95.8\%$ ) and PMS2 on BOND-MAX (OPA $98.3\%$ , PPA $100\%$ , NPA $96.7\%$ ). Results for individual panel antibodies are shown in Table 1 below.

Table 1 Summary of Intra-run Precision

| Antibody | Agreement on BOND-III | | | | Agreement on BOND-MAX | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Type | n/N* | % | 95% CI | Type | n/N* | % | 95% CI |
| Anti-MLH1 (ES05) | PPA | 30/30 | 100 | [88.6% - 100%] | PPA | 30/30 | 100 | [88.6% - 100%] |
| | NPA | 30/30 | 100 | [88.6% - 100%] | NPA | 30/30 | 100 | [88.6% - 100%] |
| | OPA | 60/60 | 100 | [94.0% - 100%] | OPA | 60/60 | 100 | [94.0% - 100%] |
| Anti-MSH2 (79H11) | PPA | 30/30 | 100 | [88.6% - 100%] | PPA | 30/30 | 100 | [88.6% - 100%] |
| | NPA | 23/24 | 95.8 | [79.8% - 99.3%] | NPA | 24/24 | 100 | [86.2% - 100%] |
| | OPA | 53/54 | 98.1 | [90.2% - 99.7%] | OPA | 54/54 | 100 | [93.4% - 100%] |
| Anti-MSH6 (EP49) | PPA | 30/30 | 100 | [88.6% - 100%] | PPA | 30/30 | 100 | [88.6% - 100%] |
| | NPA | 30/30 | 100 | [88.6% - 100%] | NPA | 30/30 | 100 | [88.6% - 100%] |
| | OPA | 60/60 | 100 | [94.0% - 100%] | OPA | 60/60 | 100 | [94.0% - 100%]8 |

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| Anti-PMS2 (EP51) | PPA | 30/30 | 100 | [88.6% - 100%] | PPA | 30/30 | 100 | [88.6% - 100%] |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | NPA | 30/30 | 100 | [88.6% - 100%] | NPA | 29/30 | 96.7 | [83.3% - 99.4%] |
| | OPA | 60/60 | 100 | [94.0% - 100%] | OPA | 59/60 | 98.3 | [91.1% - 99.7%] |

*Number of agreement/Number of pairs

# Between-day Precision:

Between-day repeatability was evaluated using the same CRC cases used for intra-run testing. Three (3) slides were stained using each of the 4 panel antibodies across 6 non-consecutive days on BOND-III and BOND-MAX. For Day 1 and 2, the first six slides from each case from Intra-run precision study were used. Data were obtained from 180 total observations (10 cases x 3 replicates x 6 days x 1 instrument) for each panel antibody on BOND-III and from 180 total observations (10 cases x 3 replicates x 6 days x 1 instrument) for each panel antibody on BOND-MAX [Note: MSH2 evaluation was conducted with 4 intact and 5 loss status cases (9 total cases) for a total of 162 observations per instrument since 1 case was excluded due to insufficient tumor]. Between-day repeatability OPA was $100\%$ for each panel antibody on both instruments except MSH2 on BOND-III (OPA $98.8\%$ , PPA $98.9\%$ , NPA $98.6\%$ ), MSH6 on BOND-III (OPA $99.4\%$ , PPA $98.9\%$ , NPA $100\%$ ) and BOND-MAX (OPA $99.4\%$ , PPA $100\%$ , NPA $98.9\%$ ), PMS2 on BOND-III (OPA $98.9\%$ , PPA $98.9\%$ , NPA $98.9\%$ ) and BOND-MAX (OPA $99.4\%$ , PPA $100\%$ , NPA $98.9\%$ ). The study met pre-specified acceptance criteria of $\geq 85\%$ lower bound confidence interval. Results for individual panel antibodies are shown in Table 2 below.

Table 2 Summary of Between-day Precision

| Antibody | Agreement on BOND-III | | | | Agreement on BOND-MAX | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Type | n/N* | % | 95% CI | Type | n/N* | % | 95% CI |
| Anti-MLH1 (ES05) | PPA | 90/90 | 100 | [95.9% - 100%] | PPA | 90/90 | 100 | [95.9% - 100%] |
| | NPA | 90/90 | 100 | [95.9% - 100%] | NPA | 90/90 | 100 | [95.9% - 100%] |
| | OPA | 180/180 | 100 | [97.9% - 100%] | OPA | 180/180 | 100 | [97.9% - 100%] |
| Anti-MSH2 (79H11) | PPA | 89/90 | 98.9 | [94.0% - 99.8%] | PPA | 90/90 | 100 | [95.9% - 100%] |
| | NPA | 71/72 | 98.6 | [92.5% - 99.8%] | NPA | 72/72 | 100 | [94.9% - 100%] |
| | OPA | 160/162 | 98.8 | [95.6% - 99.7%] | OPA | 162/162 | 100 | [97.7% - 100%] |
| Anti-MSH6 (EP49) | PPA | 89/90 | 98.9 | [94.0% - 99.8%] | PPA | 90/90 | 100 | [95.9% - 100%] |
| | NPA | 90/90 | 100 | [95.9% - 100%] | NPA | 89/90 | 98.9 | [94.0% - 99.8%] |
| | OPA | 179/180 | 99.4 | [96.9% - 99.9%] | OPA | 179/180 | 99.4 | [96.9% - 99.9%] |
| Anti-PMS2 (EP51) | PPA | 89/90 | 98.9 | [94.0% - 99.8%] | PPA | 90/90 | 100 | [95.9% - 100%] |
| | NPA | 89/90 | 98.9 | [94.0% - 99.8%] | NPA | 89/90 | 98.9 | [94.0% - 99.8%] |
| | OPA | 178/180 | 98.9 | [96.0% - 99.7%] | OPA | 179/180 | 99.4 | [96.9% - 99.9%] |

*Number of agreement/Number of pairs

# Between-lot Precision:

The study evaluated lot to lot repeatability with 3 final lots of each of the 4 panel antibodies using the same CRC cases as intra-run testing. Three (3) slides per case were stained using each panel antibody across 6 non-consecutive days (2 days per lot) on BOND-III and BOND-MAX. Data were obtained from 180 total observations (10 cases x 3 replicates x 2 days x 3 lots) for each panel antibody on BOND-III and from 180 total observations (10 cases x 3 replicates x 2 days x 3 lots) for each panel antibody on BOND-MAX. [Note: MSH2 evaluation was conducted with 4 intact and 5 loss $\mathfrak{S}$ taus

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cases (9 cases total) for a total of 162 observations per instrument since 1 case was excluded due to insufficient tumor]. Between-lot repeatability OPA was $100\%$ for each panel antibody on both instruments with the exception of MSH2 on BOND-III (OPA $98.8\%$ , PPA $98.9\%$ , NPA $98.6\%$ ), MSH6 on BOND-III (OPA $99.4\%$ , PPA $98.9\%$ ) and BOND-MAX (OPA $99.4\%$ , NPA $98.9\%$ ). PMS2 on BOND-III (OPA $98.9\%$ , PPA $98.9\%$ , NPA $98.9\%$ ) and BOND-MAX (OPA $99.4\%$ , NPA $98.9\%$ ). The study met pre-specified acceptance criteria of $\geq 85\%$ lower bound confidence interval. Results for individual panel antibodies are shown in Table 3 below.

Table 3 Summary of Between-lot Precision

| Antibody | Agreement on BOND-III | | | | Agreement on BOND-MAX | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Type | n/N* | % | 95% CI | Type | n/N* | % | 95% CI |
| Anti-MLH1 (ES05) | PPA | 90/90 | 100 | [95.9% - 100%] | PPA | 90/90 | 100 | [95.9% - 100%] |
| | NPA | 90/90 | 100 | [95.9% - 100%] | NPA | 90/90 | 100 | [95.9% - 100%] |
| | OPA | 180/180 | 100 | [97.9% - 100%] | OPA | 180/180 | 100 | [97.9% - 100%] |
| Anti-MSH2 (79H11) | PPA | 89/90 | 98.9 | [94.0% - 99.8%] | PPA | 90/90 | 100 | [95.9% - 100%] |
| | NPA | 71/72 | 98.6 | [92.5% - 99.8%] | NPA | 72/72 | 100 | [94.9% - 100%] |
| | OPA | 160/162 | 98.8 | [95.6% - 99.7%] | OPA | 162/162 | 100 | [97.7% - 100%] |
| Anti-MSH6 (EP49) | PPA | 89/90 | 98.9 | [94.0% - 99.8%] | PPA | 90/90 | 100 | [95.9% - 100%] |
| | NPA | 90/90 | 100 | [95.9% - 100%] | NPA | 89/90 | 98.9 | [94.0% - 99.8%] |
| | OPA | 179/180 | 99.4 | [96.9% - 99.9%] | OPA | 179/180 | 99.4 | [96.9% - 99.9%] |
| Anti-PMS2 (EP51) | PPA | 89/90 | 98.9 | [94.0% - 99.8%] | PPA | 90/90 | 100 | [95.9% - 100%] |
| | NPA | 89/90 | 98.9 | [94.0% - 99.8%] | NPA | 89/90 | 98.9 | [94.0% - 99.8%] |
| | OPA | 178/180 | 98.9 | [96.0% - 99.7%] | OPA | 179/180 | 99.4 | [96.9% - 99.9%] |

*Number of agreement/Number of pairs

The results support that the BOND MMR Antibody Panel on BOND-III and on BOND-MAX have acceptable levels of precision (repeatability) across lots and days.

ii. Reproducibility across Laboratories and Pathologists on the BOND-III and BOND-MAX Instruments

This study was conducted at 3 sites using 1 lot of the BOND MMR antibody for each of the 4 MMR proteins. Slides from 30 FFPE CRC tissue cases were tested, including cases being protein deficient and cases expressing intact proteins. Each case was stained on 1 BOND-III and 1 BOND MAX instrument per site, for a total of 6 times (3 sites x 2 instruments). Testing was performed on 6 non-consecutive days over a minimum 20-day testing period, following randomized testing orders. A single pathologist per site read and scored all stained slides from that site. In addition, 3 pathologists read and scored a set of 30 cases stained at 1 site in 3 scoring sessions separated by a washout period of at least 14 days.

Table 4 MMR antibody Case Status

| Marker/Antibody | Positive/Intact | Deficient/Loss | Total |
| --- | --- | --- | --- |
| MLH1 | 25 | 5 | 30 |
| MSH2 | 27 | 3 | |
| MSH6 | 26 | 4 | |
| PMS2 | 24 | 6 | |

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PPA, NPA and OPA were calculated for point estimates along with 2-sided $95\%$ confidence intervals to evaluate intra-pathologist, inter-pathologist, inter-laboratory and inter-instrument precision.

Results of the study are summarized in Tables 5 to 8. The results support that the BOND MMR Antibody Panel on BOND-III and on BOND-MAX have acceptable levels of precision (reproducibility).

# Intra- and Inter-Pathologist Reproducibility:

Intra-pathologist and inter-pathologist reproducibility were assessed by evaluating concordance of marker status across 3 pathologists and within individual pathologists using 30 cases of CRC stained at 1 site on BOND-III and BOND-MAX. These 30 CRC specimens consisted of varying number of cases with loss or intact MMR protein status as shown in Table 4. Each reader scored all 30 cases in three rounds that were separated by a two week wash out period. Data was obtained from 270 total observations (30 cases x 3 replicate x 3 pathologists) for each of the 4 panel antibodies for BOND-III, and from 270 total observations (30 cases x 3 replicate x 3 pathologists) for each panel antibody for BOND-MAX. Intra-pathologist reproducibility OPA ranged from $99.3\%$ to $100\%$ , PPA ranged from $99.2\%$ to $100\%$ . The results are shown below in Table 5. Inter-pathologist reproducibility OPA ranged from $99.3\%$ to $100\%$ , PPA ranged from $99.2\%$ to $100\%$ . The results are shown below in Table 6. The studies met the pre-specified acceptance criteria.

Table 5 Summary of Intra-Pathologist Reproducibility

| Antibody | Agreement on BOND-III | | | | Agreement on BOND-MAX | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Type | n/N* | % | 95% CI | Type | n/N* | % | 95% CI |
| Overall call | PPA | 180/180 | 100 | [97.9% - 100%] | PPA | 180/180 | 100 | [97.9% - 100%] |
| | NPA | 90/90 | 100 | [95.9% - 100%] | NPA | 90/90 | 100 | [95.9% - 100%] |
| | OPA | 270/270 | 100 | [98.6% - 100%] | OPA | 270/270 | 100 | [98.6% - 100%] |
| Anti-MLH1 (ES05) | PPA | 225/225 | 100 | [98.3% - 100%] | PPA | 225/225 | 100 | [98.3% - 100%] |
| | NPA | 45/45 | 100 | [92.1% - 100%] | NPA | 45/45 | 100 | [92.1% - 100%] |
| | OPA | 270/270 | 100 | [98.6% - 100%] | OPA | 270/270 | 100 | [98.6% - 100%] |
| Anti-MSH2 (79H11) | PPA | 252/252 | 100 | [98.5% - 100%] | PPA | 250/252 | 99.2 | [97.2% - 99.8%] |
| | NPA | 18/18 | 100 | [82.4% - 100%] | NPA | 18/18 | 100 | [82.4% - 100%] |
| | OPA | 270/270 | 100 | [98.6% - 100%] | OPA | 268/270 | 99.3 | [97.3% - 99.8%] |
| Anti-MSH6 (EP49) | PPA | 234/234 | 100 | [98.4% - 100%] | PPA | 234/234 | 100 | [98.4% - 100%] |
| | NPA | 36/36 | 100 | [90.4% - 100%] | NPA | 36/36 | 100 | [90.4% - 100%] |
| | OPA | 270/270 | 100 | [98.6% - 100%] | OPA | 270/270 | 100 | [98.6% - 100%] |
| Anti-PMS2 (EP51) | PPA | 216/216 | 100 | [98.3% - 100%] | PPA | 216/216 | 100 | [98.3% - 100%] |
| | NPA | 54/54 | 100 | [93.4% - 100%] | NPA | 54/54 | 100 | [93.4% - 100%] |
| | OPA | 270/270 | 100 | [98.6% - 100%] | OPA | 270/270 | 100 | [98.6% - 100%] |

*Number of agreement/Number of pairs

Table 6 Summary of Inter-Pathologist Reproducibility

| Antibody | Agreement on BOND-III | | | | Agreement on BOND-MAX | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Type | n/N* | % | 95% CI | Type | n/N* | % | 95% CI |
| Overall call | PPA | 180/180 | 100 | [97.9% - 100%] | PPA | 180/180 | 100 | [97.9% - 100%] |
| | NPA | 90/90 | 100 | [95.9% - 100%] | NPA | 90/90 | 100 | [95.9% - 100%] |

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# Inter-Instrument Reproducibility:

Instrument to instrument reproducibility was evaluated by one pathologist using the same cases as Inter-pathologist and Intra-pathologist reproducibility. One slide was stained using each of the 4 panel antibodies, one slide was stained with Negative Control (Mouse), and one slide was stained with Negative Control (Rabbit) for each case across 3 different BOND-III and 3 different BOND-MAX instruments. Data was obtained from 90 total observations (30 cases x 1 replicate x 3 instruments) for each panel antibody for BOND-III, and from 90 total observations (30 cases x 1 replicate x 3 instruments) for each panel antibody for BOND-MAX. Inter-instrument reproducibility OPA ranged from $98.9\%$ to $100\%$ , PPA ranged from $98.8\%$ to $100\%$ , NPA ranged from $93.3\%$ to $100\%$ . The study met pre-specified acceptance criteria. Results for the overall panel and each panel antibody on BOND-III and BOND-MAX are shown in Table 7 below.

Table 7 Summary of Inter-Instrument Reproducibility

| Antibody | Agreement on BOND-III | | | | Agreement on BOND-MAX | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Type | n/N* | % | 95% CI | Type | n/N* | % | 95% CI |
| Overall call | PPA | 60/60 | 100 | [94.0% - 100%] | PPA | 60/60 | 100 | [94.0% - 100%] |
| | NPA | 30/30 | 100 | [88.6% - 100%] | NPA | 29/30 | 96.7 | [83.3% - 99.4%] |
| | OPA | 90/90 | 100 | [95.9% - 100%] | OPA | 89/90 | 98.9 | [94.0% - 99.8%] |
| Anti-MLH1 (ES05) | PPA | 75/75 | 100 | [95.1% - 100%] | PPA | 75/75 | 100 | [95.1% - 100%] |
| | NPA | 15/15 | 100 | [79.6% - 100%] | NPA | 14/15 | 93.3 | [70.2% - 98.8%] |
| | OPA | 90/90 | 100 | [95.9% - 100%] | OPA | 89/90 | 98.9 | [94.0% - 99.8%] |
| Anti-MSH2 (79H11) | PPA | 84/84 | 100 | [95.6% - 100%] | PPA | 83/84 | 98.8 | [93.6% - 99.8%] |
| | NPA | 6/6 | 100 | [61.0% - 100%] | NPA | 6/6 | 100 | [61.0% - 100%] |
| | OPA | 90/90 | 100 | [95.9% - 100%] | OPA | 89/90 | 98.9 | [94.0% - 99.8%] |
| Anti-MSH6 (EP49) | PPA | 78/78 | 100 | [95.3% - 100%] | PPA | 78/78 | 100 | [95.3% - 100%] |
| | NPA | 12/12 | 100 | [75.8% - 100%] | NPA | 12/12 | 100 | [75.8% - 100%] |
| | OPA | 90/90 | 100 | [95.9% - 100%] | OPA | 90/90 | 100 | [95.9% - 100%] |
| Anti-PMS2 | PPA | 72/72 | 100 | [94.9% - 100%] | PPA | 72/72 | 100 | [94.9% - 100%] |
| | NPA | 18/18 | 100 | [82.4% - 100%] | NPA | 17/18 | 94.4 | [74.2% - 99.0%] |
| | OPA | 90/90 | 100 | [95.9% - 100%] | OPA | 89/90 | 98.9 | [94.0% - 99.8%] |

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*Number of agreement/Number of pairs

# Inter-laboratory Reproducibility:

The reproducibility of the BOND MMR Antibody Panel was assessed at 3 sites using the same cases as Inter-pathologist and Intra-pathologist reproducibility. Multiple tissue sections were cut from each case. Three (3) sites (2 external and 1 internal) stained all cases with the designated antibody and the appropriate negative reagent control (NRC) antibody spanning a period of at least 20 days. One pathologist at each site independently evaluated each case to determine the status (intact or loss). Data was obtained from 90 total observations (30 cases x 1 replicate x 3 sites) for each of the 4 panel antibodies for BOND-III, and from 90 total observations (30 cases x 1 replicate x 3 sites) for each panel antibody for BOND-MAX. Reproducibility OPA ranged from $98.9\%$ to $100\%$ , PPA ranged from $98.8\%$ to $100\%$ . The study met pre-specified acceptance criteria. Results for the overall panel and each panel antibody on BOND-III and BOND-MAX are shown in Table 8 below.

Table 8 Summary of Inter-Laboratory Reproducibility

| Antibody | Agreement on BOND-III | | | | Agreement on BOND-MAX | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Type | n/N* | % | 95% CI | Type | n/N* | % | 95% CI |
| Overall call | PPA | 59/59 | 100 | [93.9% - 100%] | PPA | 60/60 | 100 | [94.0% - 100%] |
| | NPA | 30/30 | 100 | [88.6% - 100%] | NPA | 30/30 | 100 | [88.6% - 100%] |
| | OPA | 89/89 | 100 | [95.9% - 100%] | OPA | 90/90 | 100 | [95.9% - 100%] |
| Anti-MLH1 (ES05) | PPA | 74/74 | 100 | [95.1% - 100%] | PPA | 75/75 | 100 | [95.1% - 100%] |
| | NPA | 15/15 | 100 | [79.6% - 100%] | NPA | 15/15 | 100 | [79.6% - 100%] |
| | OPA | 89/89 | 100 | [95.9% - 100%] | OPA | 90/90 | 100 | [95.9% - 100%] |
| Anti-MSH2 (79H11) | PPA | 83/83 | 100 | [95.6% - 100%] | PPA | 83/ 84 | 98.8 | [93.6% - 99.8%] |
| | NPA | 6/6 | 100 | [61.0% - 100%] | NPA | 6/6 | 100 | [61.0% - 100%] |
| | OPA | 89/89 | 100 | [95.9% - 100%] | OPA | 89/90 | 98.9 | [94.0% - 99.8%] |
| Anti-MSH6 (EP49) | PPA | 77/77 | 100 | [95.2% - 100%] | PPA | 78/78 | 100 | [95.3% - 100%] |
| | NPA | 12/12 | 100 | [75.8% - 100%] | NPA | 12/12 | 100 | [75.8% - 100%] |
| | OPA | 89/89 | 100 | [95.9% - 100%] | OPA | 90/90 | 100 | [95.9% - 100%] |
| Anti-PMS2 (EP51) | PPA | 71/71 | 100 | [94.9% - 100%] | PPA | 72/72 | 100 | [94.9% - 100%] |
| | NPA | 18/18 | 100 | [82.4% - 100%] | NPA | 18/18 | 100 | [82.4% - 100%] |
| | OPA | 89/89 | 100 | [95.9% - 100%] | OPA | 90/90 | 100 | [95.9% - 100%] |

*Number of agreement/Number of pairs

# iii. Reproducibility across Days and Laboratories on BOND-III

To evaluate the inter-day and inter-site reproducibility of the BOND MMR Antibody Panel on the BOND-III Instrument, 24 FFPE CRC tissue cases were tested, including 3 intact and 3 loss cases for each of the 4 MMR proteins, at 3 testing sites. At each site, slides were stained with 1 reagent lot of the antibodies using 1 BOND-III instrument. Two staining runs were performed each day to test slides from the 24 cases on each of 5 nonconsecutive test days at each site. A total of 360 slides (6 cases $\times$ 4 antibodies $\times$ 3 sites $\times$ 5 days) were evaluated. At each site, one pathologist read and scored stained slides stained at their site in accordance with the scoring guidance. The slides stained on the same day were scored together in the same scoring session. Different scoring sessions occurred on nonconsecutive days.

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PPA, NPA and OPA were calculated for point estimates along with 2-sided $95\%$ confidence intervals by comparing the antibody status of each processed slide determined by the pathologist to the majority score. The majority score was determined by combining runs across all sites. At one site, 12 slides failed due to temperature error. New unstained slides were repeat stained and read and scored successfully.

Results of the study are summarized in Table 9 to Table 13. The overall reproducibility OPA ranged from $94.4\%$ to $100\%$ , PPA ranged from $91.7\%$ to $100\%$ , NPA ranged from $97.8\%$ to $100\%$ . The lower bound of the two-sided $95\%$ Confidence Interval for OPA, PPA and NPA for each antibody are $\geq 85\%$ with 1 exception. For MSH6, PPA was $91.7\%$ (95% CI: $81.9\% - 96.4\%$ ). All 5 discordant results were from one case and 4 of the 5 slides were stained at a single site. This case was investigated, and the study pathologists agreed that it was challenging to interpret due to the very focal staining impacting scoring.

The results support that the BOND MMR Antibody Panel on BOND-III have acceptable levels of precision (reproducibility) across days and testing sites.

Table 9: Summary of Inter-Laboratory Reproducibility

| Antibody | Agreement on BOND-III | | | |
| --- | --- | --- | --- | --- |
| | Type | n/N* | % | 95% CI |
| Overall call | PPA | 190/195 | 97.4% | [94.1% - 98.9%] |
| | NPA | 164/165 | 99.4% | [96.6% - 99.9%] |
| | OPA | 354/360 | 98.3% | [96.4% - 99.2%] |
| Anti-MLH1 (ES05) | 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 (79H11) | 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-MSH6 (EP49) | PPA | 55/60 | 91.7% | [81.9% - 96.4%] |
| | NPA | 30/30 | 100.0% | [88.6% - 100.0%] |
| | OPA | 85/90 | 94.4% | [87.6% - 97.6%] |
| Anti-PMS2 (EP51) | PPA | 45/45 | 100.0% | [92.1% - 100.0%] |
| | NPA | 44/45 | 97.8% | [88.4% - 99.6%] |
| | OPA | 89/90 | 98.9% | [94.0% - 99.8%] |

*Number of agreement/Number of pairs

Table 10: Percent Agreements for Overall and by Site--MLH1

| Anti-MLH1 (ES05) | Agreement on BOND-III | | | | |
| --- | --- | --- | --- | --- | --- |
| | | Type | n/N* | % | 95% CI |
| Overall call | PPA | 45/45 | 100% | [92.1% - 100.0%] | |
| | | NPA | 45/45 | 100% | [92.1% - 100.0%] |
| | | OPA | 90/90 | 100% | [95.9% - 100.0%] |
| Between | Site 1 | PPA | 15/15 | 100% | [79.6% - 100.0%] |
| Site 2 | | NPA | 15/15 | 100% | [79.6% - 100.0%] |

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Table 11: Percent Agreements for Overall and by Site--MSH2

| Anti-MSH2 (79H11) | Agreement on BOND-III | | | | |
| --- | --- | --- | --- | --- | --- |
| | | Type | n/N* | % | 95% CI |
| Overall call | PPA | 45/45 | 100% | [92.1% - 100.0%] | |
| | | NPA | 45/45 | 100% | [92.1% - 100.0%] |
| | | OPA | 90/90 | 100% | [95.9% - 100.0%] |
| Between-Day (5 non-consecutive days) | Site 1 | PPA | 15/15 | 100% | [79.6% - 100.0%] |
| | | NPA | 15/15 | 100% | [79.6% - 100.0%] |
| | | OPA | 30/30 | 100% | [88.6% - 100.0%] |
| | Site 2 | PPA | 15/15 | 100% | [79.6% - 100.0%] |
| | | NPA | 15/15 | 100% | [79.6% - 100.0%] |
| | | OPA | 30/30 | 100% | [88.6% - 100.0%] |
| | Site 3 | PPA | 15/15 | 100% | [79.6% - 100.0%] |
| | | NPA | 15/15 | 100% | [79.6% - 100.0%] |
| OPA | | 30/30 | 100% | [88.6% - 100.0%] | |

*Number of agreement/Number of pairs

Table 12: Percent Agreements for Overall and by Site--MSH6

| Anti-MSH6 (EP49) | Agreement on BOND-III | | | | |
| --- | --- | --- | --- | --- | --- |
| | | Type | n/N* | % | 95% CI |
| Overall call | PPA | 55/60 | 91.7% | [81.9% - 96.4%] | |
| | | NPA | 30/30 | 100.0% | [88.6% - 100.0%] |
| | | OPA | 85/90 | 94.4% | [87.6% - 97.6%] |
| Between-Day (5 non-consecutive days) | Site 1 | PPA | 20/20 | 100% | [83.9% - 100.0%] |
| | | NPA | 10/10 | 100.0% | [72.2% - 100.0%] |
| | | OPA | 30/30 | 100.0% | [88.6% - 100.0%] |
| | Site 2 | PPA | 19/20 | 95.0% | [76.4% - 99.1%] |
| | | NPA | 10/10 | 100.0% | [72.2% - 100.0%] |
| | | OPA | 29/30 | 96.7% | [83.3% - 99.4%] |
| | Site 3 | PPA | 16/20 | 80.0% | [58.4% - 91.9%] |
| | | NPA | 10/10 | 100.0% | [72.2% - 100.0%] |
| | | OPA | 26/30 | 86.7% | [70.3% - 94.7%] |

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*Number of agreement/Number of pairs

Table 13: Percent Agreements for Overall and by Site--PMS2

| Anti-PMS2 (EP51) | Agreement on BOND-III | | | | |
| --- | --- | --- | --- | --- | --- |
| | | Type | n/N* | % | 95% CI |
| Overall call | PPA | 45/45 | 100.0% | [92.1% - 100.0%] | |
| | | NPA | 44/45 | 97.8% | [88.4% - 99.6%] |
| | | OPA | 89/90 | 98.9% | [94.0% - 99.8%] |
| Between-Day (5 non-consecutive days) | Site 1 | PPA | 15/15 | 100% | [79.6% - 100.0%] |
| | | NPA | 15/15 | 100% | [79.6% - 100.0%] |
| | | OPA | 30/30 | 100% | [88.6% - 100.0%] |
| | Site 2 | PPA | 15/15 | 100% | [79.6% - 100.0%] |
| | | NPA | 15/15 | 100% | [79.6% - 100.0%] |
| | | OPA | 30/30 | 100% | [88.6% - 100.0%] |
| | Site 3 | PPA | 15/15 | 100.0% | [79.6% - 100.0%] |
| | | NPA | 14/15 | 93.3% | [70.2% - 98.8%] |
| | | OPA | 29/30 | 96.7% | [83.3% - 99.4%] |

*Number of agreement/Number of pairs

b. Linearity/assay reportable range:

Not applicable

c. Traceability (controls, calibrators, or methods), Stability, Expected values:

i. Controls: See section IV for description of controls.

ii. Stability:

The below stability studies were conducted for the BOND MMR Antibody Panel :

- Reagent Stability Studies
- Real-Time Stability Tests
- Accelerated Stability Tests
- Freeze Thaw Stability Tests
- Cut Slide Stability Studies

a) Reagent Stability Studies:

Stability testing of the BOND MMR antibodies was evaluated by performing Accelerated stability testing and Real Time shelf-life testing. The Accelerated stability was evaluated using accelerated shelf-life tests and was followed up by long term stability with Real Time shelf-life tests.

The stability of the BOND MMR antibodies was evaluated using three batches of each of the four antibodies.

Prior to the initiation of both accelerated and real-time stability testing, one batch of each

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antibody underwent transport simulation as preconditioning. The Transport Simulation Batches were stored for a minimum of 24 hours at 2-8°C, the required aliquots were removed from recommended storage conditions and transferred to 27-33°C for 2 days, then returned to recommended storage conditions for at least 1 day, then transferred to 37-43°C for 2 days.

A Day 0 test was performed where a minimum of 3 cases were stained and a 0-4 scale used as the reference. Day 0 slides were required to have a staining intensity of at least 2+ in the most intensely-staining tumor region to be included in the study.

## Accelerated Stability

Following transport cycling, the lot of the antibody was stored at 34-40°C and at 24-30°C. Timepoints for testing at 34-40°C were 7 and 10 weeks; timepoints for testing at 24-30°C were 14 and 20 weeks. If a testing failure occurred at either timepoint for 34-40°C then the study reverted to testing units stored at 24-30°C. If units stored at 24-30°C failed then accelerated stability was discontinued and real time data used. At each time point, staining intensities of the slides were compared with the ones stained at Day 0. The intensity had to be at least 2+ for all slides to pass the acceptance criteria.

## Real Time Shelf-Life Tests

Real time stability was evaluated for three conditions: long term stability (Real-time Shelf Life Test), in-use stability (In Use Test), transport simulation (Transport Test). The study was continued up to 545 Days (18-month timepoint including a safety margin)

1. The in-use stability test batch was stored at 2-8°C until required for testing where an 'In Use' aliquot of the antibody for each time points was cycled for 35 cycles by storing at 34-40°C for 7 hours. At the end of each 7 hour period containers were returned to 2-8°C. The units were tested on or after 35 cycles.
2. The transport simulation batches were stored for a minimum of 24 hours at 2-8°C, the required aliquots were removed from recommended storage conditions and transferred to 27-33°C for 2 days, then returned to recommended storage conditions for at least 1 day, then transferred to 37-43°C for 2 days. Units were returned thereafter to their recommended storage conditions for a period of at least 24 hours before testing.
3. The real time stability test for the three batches were performed on Day 0 and then subsequent time points. Tissues stained at each time point were compared to a Day 0 control tissue and a -77 -83°C control.

## Freeze-Thaw Tests

The freeze-thaw batch underwent freeze-thaw preconditioning prior to day 0 testing. Three vials of the batch were stored at -77 -83°C for a minimum of 24 hours. The aliquots were then placed at 2-8°C to thaw overnight and were tested alongside and compared to an aliquot stored only at 2-8°C. The freeze-thaw batch was not subjected to transport simulation.

At each time point, staining intensities of the slides were compared with the ones stained at Day 0. The intensity was required to be at least 2+ for all slides to pass the acceptance criteria. If the test failed, then study either reverted to the lower temperatures or in the case of a real time failure the study would be aborted.

## Results

All four antibodies passed the transport simulation testing and the freeze-thaw testing. The anti-

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MLH1, anti-MSH2 and anti-PMS2 antibodies passed the accelerated stability testing at 7 and 10 weeks, supporting that the maximum shelf life for these antibodies is 12 months. Real time stability studies have passed at 545 days. The antibody of anti-MSH6 passed the accelerated stability testing at 7 weeks but failed at 10 weeks. The antibody also failed the 24-30°C accelerated stability testing at 20 weeks. However, anti-MSH6 passed the real time stability testing at 365 days, supporting that the maximum shelf life for this antibody is 7 months.

## b) Cut slide Stability:

To evaluate the stability of the cut slides, 60 x 3μm sections were cut from each of three FFPE CRC tissue cases for a total of 180 slides. Six slides of each FFPE CRC tissue case were stained on Day 0 on the BOND-III system with one lot of each of the four antibodies along with the negative reagent controls, scored on a 0-4+ scale and used as the reference. All three cases are intact for all four MMR proteins with the staining intensity being at least 2+ in the most intensely-staining tumor region. The remaining slides of each FFPE CRC tissue case were stored at ambient/room temperature for 7, 14, 28, 42, 56, 70 and 77 days. At each timepoint, six slides of each FFPE CRC tissue case were stained the same way as above and compared with the ones stained at baseline. In addition, at each of these timepoints, freshly cut sections of the same three tissue cases were stained with each of the four antibodies as a quality control. The intensity was required to be at least 2+ to pass the acceptance criteria. Results of the study showed that the epitopes recognized by each of the four antibodies in FFPE CRC tissue cases remain stable for a period of time up to and including 11 weeks post sectioning when the tissue sections are mounted onto slides and stored at ambient/room temperature, therefore supporting epitope stability claims in cut sections of up to 10 weeks.

## d. Detection Limit:

Not applicable

## e. Analytical specificity:

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

## i. Western Blot and IHC:

Western blot analyses were conducted to demonstrate that the antibodies specifically detect the proteins of predicted molecular weight for each of the 4 BOND MMR antibodies using cell lines with known MMR loss or intact status. Cell lines used in the study were generated using lysates from the PANC-1 (human epithelial pancreas/duct carcinoma) for MLH1 and MSH2 and lysates from the A431 (human epidermoid carcinoma) for MSH6 and PMS2. Western Blots confirmed presence of reactive bands at expected molecular weighs for each of the 4 MMR proteins.

IHC tests were conducted using formalin-fixed, paraffin-embedded cell lines described in the table below to assess nonspecific binding in the context of use. The results of the IHC with engineered cell lines was consistent with expected reactivity.

| Antibody | Formalin-Fixed, Paraffin-Embedded Cell Lines for IHC Testing |
| --- | --- |
| MMR INTACT | Breast adenocarcinoma cell line that carries no specific MMR mutations |

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The combined results from western blots and cell line IHC demonstrated specific antibody reactivity for each of the 4 MMR proteins included in the BOND MMR antibody panel.

# ii. Immunoreactivity:

Immunoreactivity of the BOND MMR antibodies was tested across multiple cases of normal and tumor tissue types. The summary of staining results with the panel antibodies is shown in Table 14 to 16. Staining of these ubiquitously expressed nuclear proteins was observed in normal and neoplastic tissues as expected.

Table 14 Immunoreactivity in FFPE normal tissues

| Tissue | No. of IHC positive cases / total cases | | | |
| --- | --- | --- | --- | --- |
| | MLH1 | MSH2 | MSH6 | PMS2 |
| Cerebrum, grey matter tissue | 3/3 | 3/3 | 3/3 | 3/3 |
| Cerebrum, white matter tissue | 3/3 | 3/3 | 3/3 | 3/3 |
| Cerebellum | 3/3 | 3/3 | 3/3 | 3/3 |
| Adrenal gland | 3/3 | 3/3 | 2/3 | 3/3 |
| Ovary | 3/3 | 3/3 | 3/3 | 3/3 |
| Pancreas | 3/3 | 3/3 | 3/3 | 3/3 |
| Parathyroid | 3/3 | 3/3 | 3/3 | 3/3 |
| Pituitary | 3/3 | 3/3 | 3/3 | 3/3 |
| Testis | 3/3 | 3/3 | 3/3 | 3/3 |
| Thyroid gland | 3/3 | 3/3 | 3/3 | 3/3 |
| Breast | 3/3 | 3/3 | 3/3 | 3/3 |
| Spleen | 3/3 | 3/3 | 3/3 | 3/3 |
| Tonsil | 3/3 | 3/3 | 3/3 | 3/3 |
| Thymus gland | 3/3 | 3/3 | 3/3 | 3/3 |
| Bone marrow | 3/3 | 3/3 | 3/3 | 3/3 |
| Lung | 3/3 | 3/3 | 3/3 | 3/3 |
| Heart, cardiac muscle | 2/3 | 3/3 | 1/3 | 1/3 |
| Esophagus | 3/3 | 3/3 | 3/3 | 3/3 |
| Stomach | 3/3 | 3/3 | 3/3 | 3/3 |
| Uterus | 3/3 | 3/3 | 3/3 | 3/3 |
| Uterus, liver | 3/3 | 3/3 | 3/3 | 3/3 |
| Uterus, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, liver, | 3/3 | 3/3 | 3/3 | 3/3 |

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Table 15 Immunoreactivity in a variety of FFPE neoplastic tissues

| Tissue | Pathology | No. of IHC positive cases / total cases | | | |
| --- | --- | --- | --- | --- | --- |
| | | MLH1 | MSH2 | MSH6 | PMS2 |
| Bladder, urinary | Transitional cell carcinoma | 2/2 | 2/2 | 2/2 | 2/2 |
| Breast | Fibroadenoma | 2/2 | 2/2 | 2/2 | 2/2 |
| Breast | Invasive ductal carcinoma | 2/2 | 2/2 | 2/2 | 2/2 |
| Bone, tibia | Osteosarcoma | 1/1 | 1/1 | 1/1 | 1/1 |
| Bone, scapula | Chondrosarcoma | 1/1 | 1/1 | 1/1 | 1/1 |
| Brain | Meningioma, fibroblastic | 1/1 | 1/1 | 1/1 | 1/1 |
| Brain | Astrocytoma | 1/1 | 1/1 | 1/1 | 1/1 |
| Esophagus | Squamous cell carcinoma | 2/2 | 2/2 | 2/2 | 2/2 |
| Stomach | Adenocarcinoma | 3/3 | 3/3 | 3/3 | 3/3 |
| Intestine, small intestine | Adenoma | 1/1 | 1/1 | 1/1 | 1/1 |
| Intestine, small | Adenocarcinoma | 1/1 | 1/1 | 1/1 | 1/1 |
| | invasive ductal carcinoma | | | | |
| Liver | Squamous cell carcinoma | 1/1 | 1/1 | 1/1 | 1/1 |
| Liver | Squamous cell carcinoma | 1/1 | 1/1 | 1/1 | 1/1 |
| Liver, small intestine | Squamous cell carcinoma | 1/1 | 1/1 | 1/1 | 1/1 |
| Liver, small | Squamous cell carcinoma | 1/1 | 1/1 | 1/1 | 1/1 |

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| Tissue | Pathology | No. of IHC positive cases / total cases | | | |
| --- | --- | --- | --- | --- | --- |
| | | MLH1 | MSH2 | MSH6 | PMS2 |
| intestine | | | | | |
| Intestine, colon | Adenoma | 1/1 | 1/1 | 1/1 | 1/1 |
| Intestine, colon | Adenocarcinoma | 3/3 | 3/3 | 3/3 | 3/3 |
| Intestine, rectum | Adenocarcinoma | 2/3 | 3/3 | 3/3 | 2/3 |
| Kidney | Clear cell carcinoma | 1/1 | 1/1 | 1/1 | 1/1 |
| Liver | Hepatocellular carcinoma | 1/1 | 1/1 | 1/1 | 1/1 |
| Lung | Adenocarcinoma | 1/1 | 1/1 | 1/1 | 1/1 |
| Lung | Small cell carcinoma | 1/1 | 1/1 | 1/1 | 1/1 |
| Lymph node, neck | Lymphoma, Hodgkin lymphoma | 1/1 | 1/1 | 1/1 | 1/1 |
| Lymph node, neck | Lymphoma, anaplastic large cell lymphoma | 1/1 | 1/1 | 1/1 | 1/1 |
| Head and neck, oral cavity, hard palate | Adenocarcinoma | 1/1 | 1/1 | 1/1 | 1/1 |
| Tongue | Squamous cell carcinoma | 1/1 | 1/1 | 1/1 | 1/1 |
| Head and neck, nasopharynx | Nasopharyngeal carcinoma, NPC | 1/1 | 1/1 | 1/1 | 1/1 |
| Ovary | Granulosa cell tumor | 1/1 | 1/1 | 1/1 | 1/1 |
| Ovary | Adenocarcinoma | 1/1 | 1/1 | 1/1 | 1/1 |
| Ovary | Endometrioid adenocarcinoma | 1/1 | 1/1 | 1/1 | 1/1 |
| Pancreas | Adenocarcinoma | 1/1 | 1/1 | 1/1 | 1/1 |
| Prostate | Adenocarcinoma | 2/2 | 2/2 | 2/2 | 2/2 |
| Skin, trunk | Squamous cell carcinoma | 1/1 | 1/1 | 1/1 | 1/1 |
| Head and neck, nasal cavity | Melanoma | 1/1 | 1/1 | 1/1 | 1/1 |
| Testis | Seminoma | 1/1 | 1/1 | 1/1 | 1/1 |
| Thyroid | Adenoma | 2/2 | 2/2 | 2/2 | 2/2 |
| Thyroid | Follicular carcinoma | 1/1 | 1/1 | 1/1 | 1/1 |

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| Tissue | Pathology | No. of IHC positive cases / total cases | | | |
| --- | --- | --- | --- | --- | --- |
| | | MLH1 | MSH2 | MSH6 | PMS2 |
| Thyroid | Follicular papillary adenocarcinoma | 1/1 | 1/1 | 1/1 | 1/1 |
| Uterus, cervix | Squamous cell carcinoma | 2/2 | 2/2 | 2/2 | 2/2 |
| Uterus, endometrium | Adenocarcinoma | 2/2 | 2/2 | 2/2 | 2/2 |
| Liver | Metastatic colon adenocarcinoma | 1/1 | 1/1 | 1/1 | 1/1 |
| Lung | Metastatic cancers from gastrointestinal site | 1/1 | 1/1 | 1/1 | 1/1 |
| Ovary | Metastatic colon signet ring cell carcinoma | 1/1 | 1/1 | 1/1 | 1/1 |
| Lymph node | Metastatic esophagus squamous cell carcinoma | 1/1 | 1/1 | 1/1 | 1/1 |

Table 16 Immunoreactivity in FFPE colorectal cancer tissues

| Tissue | Pathology | No. of IHC positive cases / total cases | | | |
| --- | --- | --- | --- | --- | --- |
| | | MLH1 | MSH2 | MSH6 | PMS2 |
| Colon | Adenocarcinoma | 63/68 | 67/68 | 66/68 | 63/68 |
| | Partly mucinous adenocarcinoma | 12/12 | 11/12 | 11/12 | 12/12 |
| | Mucinous adenocarcinoma | 8/9 | 9/9 | 8/9 | 8/9 |
| | Squamous carcinoma | 1/1 | 1/1 | 1/1 | 1/1 |
| Rectum | Adenocarcinoma | 7/8 | 8/8 | 8/8 | 7/8 |
| | Partly mucinous adenocarcinoma | 3/3 | 3/3 | 3/3 | 3/3 |
| | Mucinous adenocarcinoma | 0/1 | 1/1 | 1/1 | 0/1 |

f. Assay cut-off:

No assay cut off is employed in the assessment of MMR status in FFPE CRC tissue.

## R. Comparison studies:

a. Method comparison:

See Clinical Performance below

b. Matrix comparison:

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

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S. Clinical Performance:

The clinical validity of the BOND MMR Antibody Panel was determined in a study that assessed agreement between test results obtained from the BOND MMR Antibody Panel and a molecular test based on a DNA sequencing panel validated for detecting presence of pathogenic mutations likely to affect MMR protein expression in CRC. The purpose of the study was to evaluate the ability of the panel to correctly aid in the identification of patients needing additional Lynch syndrome genetic testing.

Eligible remnant FFPE CRC tissues (“cases”) were procured, with at least 100 sequential cases with unknown mismatch repair (MMR) status (also referred to as the Sequential cohort) and at least 48 cases with known MMR protein deficiencies (also referred to as the Enrichment cohort). Specimens from the two cohorts were combined, randomized and processed to create slides evaluation with BOND MMR Antibody Panel testing and the DNA sequencing testing. Staining with the BOND MMR Antibody Panel was performed on BOND-III system in accordance with the instructions for use (IFUs). One pathologist at the testing site read and scored BOND MMR Antibody Panel stained slides in accordance with the scoring guidance.

Inclusion Criteria:
- FFPE CRC tissue block with matched normal blood, saliva, or FFPE tissue and
- (Sequential cohort) With unknown MMR protein status, sequentially obtained from a single US site or
- (Enrichment cohort) With known immunohistochemistry (IHC) MMR protein status obtained from multiple sites, including:
- MLH1 and PMS2 loss
- MSH2 and MSH6 loss
- isolated PMS2 loss
- isolated MSH6 loss

Exclusion Criteria:
- Tissue requirements for comparator DNA sequencing panel were not met
- Lack of sufficient tumor.

There were 155 cases procured for the study. One case was excluded due to not meeting the Enrichment cohort inclusion criteria. Of the remaining 154 cases eligible for the study, One case had insufficient tumor for both DNA sequencing and BOND MMR Antibody Panel testing, three cases had insufficient tumor for DNA sequencing, and seven cases did not have MMR Stained Slides interpreted due to the H&E Slide showing less than 50 viable tumor cells present for evaluation, not meeting the tissue requirements required by the BOND MMR Antibody Panel,. Therefore, of the 154 eligible cases, 143 were tested by both methods (94 sequential cases and 49 enriched cases). Assessment of the demographic data associated with the study specimens determined that it was consistent with the intended use population.

MMR Status of the Enrichment Cohort

| | MLH1 and PMS2 loss | MSH2 and MSH6 loss | PMS2 loss | MSH6 loss |
| --- | --- | --- | --- | --- |
| # Cases | 25 | 14 | 6 | 4 |

BOND MMR Antibody Panel results (MMR loss or MMR intact) were compared to the DNA sequencing panel results for pathogenic mutation(s) in the combined cohort (Sequential and Enrichment cohorts), and in the Sequential cohort and the Enrichment cohort, respectively. PPA, NPA, and OPA along with 2-

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sided 95% Wilson score confidence intervals (CIs) were calculated.

Of the 143 specimens tested by the BOND MMR Antibody Panel and the DNA sequencing panel, ten (10) cases had invalid DNA sequencing panel results due to testing not meeting quality control standards and were excluded from the agreement analysis. The remaining 133 of 143 cases had valid results by both methods and were evaluable. Results of the study are summarized in Tables 17 to 19. The point estimates of agreement between the BOND MMR Antibody Panel and the DNA sequencing panel in the combined cohort were PPA 93.3%, NPA 95.9% and OPA 94.7%.

Table 17 Agreement between BOND MMR Antibody Panel Results and DNA Sequencing Panel Results: Combined Cohort

| Combined Cohort | DNA Sequencing Panel Results | | | | |
| --- | --- | --- | --- | --- | --- |
| | | Pathogenic Mutations(s) | No Pathogenic Mutations(s) | Invalid | Total |
| BOND MMR Antibody Panel Results | MMR Protein Loss | 56 | 3 | 4 | 63 |
| | MMR Protein Intact | 4 | 70 | 6 | 80 |
| | Total | 60 | 73 | 10 | 143 |
| Agreement | | | | | |
| | | Number of Agreements | Number of Pairs | % Agreement | 95% Confidence Interval |
| PPA | | 56 | 60 | 93.3% | [84.1% - 97.4%] |
| NPA | | 70 | 73 | 95.9% | [88.6% - 98.6%] |
| OPA | | 126 | 133 | 94.7% | [89.5% - 97.4%] |

Table 18 Agreement between BOND MMR Antibody Panel Results and DNA Sequencing Panel Results: Sequential Cohort

| Sequential Cohort | DNA Sequencing Panel Results | | | | |
| --- | --- | --- | --- | --- | --- |
| | | Pathogenic Mutations(s) | No Pathogenic Mutations(s) | Invalid | Total |
| BOND MMR Antibody Panel Results | MMR Protein Loss | 15 | 1 | 3 | 19 |
| | MMR Protein Intact | 3 | 66 | 6 | 75 |
| | Total | 18 | 67 | 9 | 94 |
| Agreement | | | | | |
| | | Number of Agreements | Number of Pairs | % Agreement | 95% Confidence Interval |
| PPA | | 15 | 18 | 83.3% | [60.8% - 94.2%] |
| NPA | | 66 | 67 | 98.5% | [92.0% - 99.7%] |
| OPA | | 81 | 85 | 95.3% | [88.5% - 98.2%] |

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Table 19 Agreement between BOND MMR Antibody Panel Results and DNA Sequencing Panel Results: Enrichment Cohort

| Enrichment Cohort | DNA Sequencing Panel Results | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| | | Pathogenic Mutations(s) | No Pathogenic Mutations(s) | Invalid | Total | |
| BOND MMR Antibody Panel Results | MMR Protein Loss | 41 | 2 | 1 | 44 | |
| | MMR Protein Intact | 1 | 4 | 0 | 5 | |
| | Total | 42 | 6 | 1 | 49 | |
| Agreement | | | | | | |
| | | Number of Agreements | Number of Pairs | % Agreement | 95% Confidence Interval | |
| PPA | | 41 | 42 | 97.6% | [87.7% - 99.6%] | |
| NPA | | 4 | 6 | 66.7% | [30.0% - 90.3%] | |
| OPA | | 45 | 48 | 93.8% | [83.2% - 97.9%] | |

Accuracy by MMR proteins: The concordance for MMR gene mutation status by the DNA sequencing panel and MMR protein loss by the BOND MMR Antibody Panel was also compared individually for each antibody. When compared to the results to the DNA sequencing panel, the OPA of each MMR protein ranged from $94.0\%$ to $98.5\%$ , the PPA ranged from $65.0\%$ to $97.5\%$ , the NPA ranged from $95.7\%$ to $99.2\%$ . Results are summarized in Tables 20 to 22.

MLH1: Of the 37 MLH1 mutation positive cases, 33 had MLH1 protein loss when assessed by the BOND MMR Antibody Panel. Of the 4 MLH1 mutation positive cases with discordant BOND MMR Antibody results (i.e., intact), the following was found:

- One case had MLH1 promoter hypermethylation
- One case had a single somatic MLH1 pathogenic mutation with Copy-neutral loss of heterozygosity (CN-LOH)
- One case had 2 MLH1 somatic mutations as well as MLH1 promoter hypermethylation
- One case had a single somatic MLH1 pathogenic mutation and microsatellite instability-high (MSI-H) status.

Three of the cases also showed PMS2 protein loss.

MSH2: Of the 13 MSH2 mutation positive cases, 12 had MSH2 protein loss when assessed by the BOND MMR Antibody Panel. A single MSH2 mutation positive case with discordant BOND MMR Antibody results (i.e., intact) was identified. This case was characterized by a single MSH2 pathogenic mutation combined with MSI-H status; an MSH2 Variant of Unknown Significance (VUS) was also identified. MSH6 protein was also intact.

MSH6: Of the 20 MSH6 mutation positive cases, 13 had MSH6 protein loss when assessed by the BOND MMR Antibody Panel. Of the 7 MSH6 mutation positive cases with discordant BOND MMR Antibody results (ie, MSH6 intact), the cases were characterized as follows:

- A single MSH2 pathogenic mutation combined with MSI-H status; an MSH2 VUS was also identified. MSH2 protein was preserved.
- One pathogenic variant of somatic origin in the MSH2 gene, MSI-H status and MSH2 protein loss.

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- Germline pathogenic mutation in MSH2 with CN-LOH and MSH2 protein loss, as well as a single germline pathogenic mutation in PMS2 and a single somatic pathogenic mutation in MSH6.
- A germline and a somatic mutation in MSH6 as well as multiple VUS.
- Two somatic mutations in MSH6 consistent with biallelic somatic mutations as well as a single germline mutation in PMS2 with PMS2 protein loss.
- Two pathogenic variants of somatic origin in MSH6 gene, and one pathogenic mutation of somatic mutation in MSH2, MSI-High status, and a PMS2 VUS was also identified.
- One pathogenic variant of somatic origin in MSH6 gene with MLH1 and PMS2 protein loss, as well as MSI-High.

Of the 7 MSH6 mutation positive cases with discordant BOND MMR Antibody results (i.e. MSH6 intact), 4 demonstrated IHC loss when stained using the BOND MMRS Antibody Panel and these cases should receive additional diagnostic testing consistent with clinical practice guidelines for diagnosis of Lynch syndrome.

PMS2: Of the 40 PMS2 mutation positive cases, 39 had PMS2 protein loss when assessed by the BOND MMR Antibody Panel. A single PMS2 mutation positive cases with discordant BOND MMR Antibody results (i.e., intact) was identified. This case was characterized by a MLH1 promoter hypermethylation combined with MSI-S status. MLH1 protein staining was also intact.

Table 20 Agreement for Each Protein between BOND MMR Antibody Panel Results and DNA Sequencing Panel Results: Combined Cohort

| Combined Cohort | DNA Sequencing Panel Results | | | | |
| --- | --- | --- | --- | --- | --- |
| | | Pathogenic Mutations(s) | No Pathogenic Mutations(s) | Invalid | Total |
| BOND MMR anti-MLH1 Results | Protein Loss | 33 | 1 | 3 | 37 |
| | Protein Intact | 4 | 95 | 7 | 106 |
| | Total | 37 | 96 | 10 | 143 |
| | | | | | |
| BOND MMR anti-MSH2 Results | Protein Loss | 12 | 1 | 1 | 14 |
| | Protein Intact | 1 | 120 | 8 | 129 |
| | Total | 13 | 121 | 9 | 143 |
| | | | | | |
| BOND MMR anti-MSH6 Results | Protein Loss | 13 | 1 | 1 | 15 |
| | Protein Intact | 7 | 113 | 8 | 128 |
| | Total | 20 | 114 | 9 | 143 |
| | | | | | |
| BOND MMR anti-PMS2 Results | Protein Loss | 39 | 4 | 3 | 46 |
| | Protein Intact | 1 | 89 | 7 | 97 |

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Table 21 Agreement for Each Protein between BOND MMR Antibody Panel Results and DNA Sequencing Panel Results: Sequential Cohort

| Sequential Cohort | DNA Sequencing Panel Results | | | | |
| --- | --- | --- | --- | --- | --- |
| | | Pathogenic Mutations(s) | No Pathogenic Mutations(s) | Invalid | Total |
| BOND MMR anti-MLH1 Results | Protein Loss | 10 | 1 | 3 | 14 |
| | Protein Intact | 2 | 72 | 6 | 80 |
| | Total | 12 | 73 | 9 | 94 |
| | | | | | |
| BOND MMR anti-MSH2 Results | Protein Loss | 3 | 0 | 0 | 3 |
| | Protein Intact | 0 | 83 | 8 | 91 |
| | Total | 3 | 83 | 8 | 94 |
| | | | | | |
| BOND MMR anti-MSH6 Results | Protein Loss | 2 | 0 | 0 | 2 |
| | Protein Intact | 4 | 80 | 8 | 92 |
| | Total | 6 | 80 | 8 | 94 |
| | | | | | |
| BOND MMR anti-PMS2 Results | Protein Loss | 11 | 2 | 3 | 16 |
| | Protein Intact | 1 | 71 | 6 | 78 |
| | Total | 12 | 73 | 9 | 94 |
| Agreement | | | | | |
| | | Number of Agreements | Number of Pairs | % Agreement | 95% Confidence Interval |

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Table 22 Agreement for Each Protein between BOND MMR Antibody Panel Results and DNA Sequencing Panel Results: Enrichment Cohort

| Enrichment Cohort | DNA Sequencing Panel Results | | | | |
| --- | --- | --- | --- | --- | --- |
| | | Pathogenic Mutations(s) | No Pathogenic Mutations(s) | Invalid | Total |
| BOND MMR anti-MLH1 Results | Protein Loss | 23 | 0 | 0 | 23 |
| | Protein Intact | 2 | 23 | 1 | 26 |
| | Total | 25 | 23 | 1 | 49 |
| | | | | | |
| BOND MMR anti-MSH2 Results | Protein Loss | 9 | 1 | 1 | 11 |
| | Protein Intact | 1 | 37 | 0 | 38 |
| | Total | 10 | 38 | 1 | 49 |
| | | | | | |
| BOND MMR anti-MSH6 Results | Protein Loss | 11 | 1 | 1 | 13 |
| | Protein Intact | 3 | 33 | 0 | 36 |
| | Total | 14 | 34 | 1 | 49 |
| | | | | | |
| BOND MMR anti-PMS2 Results | Protein Loss | 28 | 2 | 0 | 30 |
| | Protein Intact | 0 | 18 | 1 | 19 |
| | Total | 28 | 20 | 1 | 49 |
| Agreement | | | | | |
| | | Number of Agreements | Number of Pairs | % Agreement | 95% Confidence Interval |
| Anti-MLH1 | PPA | 23 | 25 | 92.0% | [75.0% - 97.8%] |
| | NPA | 23 | 23 | 100.0% | [85.7% - 100.0%] |
| | OPA | 46 | 48 | 95.8% | [86.0% - 98.8%] |
| Anti-MSH2 | PPA | 9 | 10 | 90.0% | [59.6% - 98.2%] |
| | NPA | 37 | 38 | 97.4% | [86.5% - 99.5%] |

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29

| | OPA | 46 | 48 | 95.8% | [86.0% - 98.8%] |
| --- | --- | --- | --- | --- | --- |
| Anti-MSH6 | PPA | 11 | 14 | 78.6% | [52.4% - 92.4%] |
| | NPA | 33 | 34 | 97.1% | [85.1% - 99.5%] |
| | OPA | 44 | 48 | 91.7% | [80.4% - 96.7%] |
| Anti-PMS2 | PPA | 28 | 28 | 100.0% | [87.9% - 100.0%] |
| | NPA | 18 | 20 | 90.0% | [69.9% - 97.2%] |
| | OPA | 46 | 48 | 95.8% | [86.0% - 98.8%] |

T. Instrument Name

BOND-III automated system or BOND-MAX automated system.

U. System Descriptions:

1. Modes of Operation:

The BOND MMR Antibody Panel consists of the Ready-to-Use MLH1 (Mismatch Repair Protein) (ES05), MSH2 (Mismatch Repair Protein) (79H11), MSH6 (Mismatch Repair Protein) (EP49) and PMS2 (Mismatch Repair Protein) (EP51) primary antibodies and is intended to identify by light microscopy of human mismatch repair (MMR) proteins MLH1, MSH2, MSH6 and PMS2 in formalin-fixed, paraffin-embedded (FFPE) colorectal cancer (CRC) tissue sections by immunohistochemical staining. The primary antibodies are specifically optimized for use on the automated BOND-MAX or BOND-III systems in combination with BOND Polymer Refine Detection.

2. Software:

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

Yes ☐ x or No ☐

3. Level of Concern:

Moderate

4. Specimen Handling:

Refer to Device Description section above.

5. Calibration and Quality Controls:

Calibration is not applicable. Refer to Device Description section above for quality controls.

V. Other Supportive Instrument Performance Characteristics Data Not Covered in the "Performance Characteristics" Section Above:

Not applicable

W. 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.

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X. Patient Perspectives

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

Y. Conclusion:

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

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