MMR IHC Panel pharmDx (Dako Omnis)

{0} SUMMARY OF SAFETY AND EFFECTIVENESS DATA (SSED) I. GENERAL INFORMATION Device Generic Name: Immunohistochemistry test, DNA mismatch repair (MMR) protein assay Device Trade Name: MMR IHC Panel pharmDx (Dako Omnis) Device Procode: QNH Applicant's Name and Address: Agilent Technologies, Inc. 6392 Via Real Carpinteria, CA 93013 USA Date(s) of Panel Recommendation: None Premarket Approval Application (PMA) Number: P250004 Date of FDA Notice of Approval: August 15, 2025 II. INDICATIONS FOR USE For In Vitro Diagnostic Use. MMR IHC Panel pharmDx (Dako Omnis) is a qualitative immunohistochemical (IHC) assay intended for use in the assessment of mismatch repair (MMR) proteins (MLH1, PMS2, MSH2, and MSH6) in formalin-fixed, paraffin-embedded (FFPE) colorectal cancer (CRC) tissue using EnVision FLEX visualization system on Dako Omnis automated staining instrument. MMR IHC Panel pharmDx (Dako Omnis) consists of MLH1 IHC pharmDx (Dako Omnis), PMS2 IHC pharmDx (Dako Omnis), MSH2 IHC pharmDx (Dako Omnis), and MSH6 IHC pharmDx (Dako Omnis), which must be used together to identify MMR deficient CRC patients. MMR IHC Panel pharmDx (Dako Omnis) is indicated as an aid to identify MMR deficient CRC patients eligible for treatment with OPDIVO® (nivolumab) alone or OPDIVO (nivolumab) in combination with YERVOY® (ipilimumab). III. CONTRAINDICATIONS There are no known contraindications. PMA P250004: FDA Summary of Safety and Effectiveness Data {1} PMA P250004: FDA Summary of Safety and Effectiveness Data # IV. WARNINGS AND PRECAUTIONS The warnings and precautions can be found in the MMR IHC Panel pharmDx (Dako Omnis) labeling: a. MLH1 IHC pharmDx (Dako Omnis (Code GE079) b. MSH2 IHC pharmDx (Dako Omnis) (Code GE085) c. PMS2 IHC pharmDx (Dako Omnis) (Code GE087) d. MSH6 IHC pharmDx (Dako Omnis) (Code GE086) e. MMR Negative Control Reagent, Mouse (Dako Omnis) (Code GE101) f. MMR Negative Control Reagent, Rabbit (Dako Omnis) (Code GE102). # V. DEVICE DESCRIPTION MMR IHC Panel pharmDx (Dako Omnis) is designed to be run on the Dako Omnis automated staining system with Dako Omnis Solution software. The MMR panel primary antibodies and negative control reagents (NCRs) are sold separately, each with adequate reagent for 60 tests. Each primary monoclonal antibody is provided in a ready-to-use 12 mL volume, in liquid form, in a buffer containing stabilizing protein and 0.015 mol/L sodium azide. MMR panel antibodies are listed in Table 1. Table 1: MMR Panel Primary Antibodies | Product Name | Clone | Isotype | | --- | --- | --- | | MLH1 IHC pharmDx (Dako Omnis) | ES05 | Mouse IgG1 kappa | | MSH2 IHC pharmDx (Dako Omnis) | FE11 | Mouse IgG1 kappa | | MSH6 IHC pharmDx (Dako Omnis) | EP49 | Rabbit IgG | | PMS2 IHC pharmDx (Dako Omnis) | EP51 | Rabbit IgG | The isotype monoclonal mouse and rabbit NCRs are provided in a ready-to-use 12 mL volume, in liquid form, containing stabilizing protein and 0.015 mol/L sodium azide. The isotype NCRs are not directed against any known human antigen. They are used to detect nonspecific staining in CRC tissues stained with MMR IHC Panel pharmDx (Dako Omnis) rabbit or mouse antibodies. MMR NCRs are listed in Table 2. Table 2: Negative Control Reagents | Description | Clone | Isotype | | --- | --- | --- | | MMR Negative Control Reagent, Mouse (Dako Omnis) | NA | Mouse IgG1 kappa | | MMR Negative Control Reagent, Rabbit (Dako Omnis) | NA | Rabbit IgG | 2 of 77 {2} Reagents and equipment required for performing testing, but not supplied as part of MMR IHC Panel pharmDx (Dako Omnis), are listed below: - Dako Omnis (Code GI100) - Target Retrieval Solution, pH 9 (50x) (Dako Omnis) (Code GC309) - EnVision FLEX, High pH (Dako Omnis) (Code GV800 or GV823), containing: - EnVision FLEX DAB+ Chromogen (Dako Omnis) - EnVision FLEX Peroxidase-Blocking Reagent (Dako Omnis) - EnVision FLEX Substrate Buffer (Dako Omnis) - EnVision FLEX Visualization Reagent (Dako Omnis) - EnVision FLEX+ Mouse LINKER (Dako Omnis) (Code GV821) - EnVision FLEX+ Rabbit LINKER (Dako Omnis) (Code GV809) - Wash Buffer (20x) (Dako Omnis) (Code GC807) - Sulfuric Acid, 0.3 M (Code GC203) - Hematoxylin (Dako Omnis) (Code GC808) or equivalent - Clearify™ clearing agent (Code GC810) - Distilled or de-ionized water (reagent-grade water) - Drying oven, capable of maintaining 60 °C or less - Ethanol, absolute and 95% - Xylene, or xylene substitute - Bright field microscope (4–20x objective magnification) - Coverslips - Nonaqueous, permanent mounting medium and ancillary reagents required for mounting coverslips - Microscope slides: FLEX IHC Microscope Slides (Code K8020) or Superfrost Plus slides - Tissues to use as process controls - pH meter All instrumentation should be maintained and calibrated per manufacturer’s recommendation. ## Device Instrument and Software MMR IHC Panel pharmDx (Dako Omnis) is designed to be run on the Dako Omnis automated staining system with Dako Omnis Instrument Software (OIS) and the Dako Link Omnis Workstation and Server software (WSS). The Dako Omnis system is designed to process slides on a continuous basis and can run different staining protocols for individual slides at the same time to optimize capacity utilization and patient case management. ## Specimen Preparation Specimens must be handled to preserve the tissue for IHC staining. Standard methods of tissue processing should be used for all specimens. PMA P250004: FDA Summary of Safety and Effectiveness Data 3 of 77 {3} # Paraffin-embedded tissue Formalin-Fixed Paraffin-Embedded (FFPE) tissues are suitable for use with MMR IHC Panel pharmDx (Dako Omnis). Recommended handling and processing conditions are: ≤ 1 hour ischemia time prior to immersion in fixative, and 6 to 48 hours fixation time in 10% neutral buffered formalin (NBF). Alternative fixatives have not been validated and may give erroneous results. Reduced staining was observed with 10% unbuffered formalin, Bouin's fixative, and acetic formalin alcohol (AFA), so they are not acceptable for use with this assay. Specimens should be blocked into a thickness of 3 or 4 mm, fixed in 10% NBF, and dehydrated and cleared in a series of alcohols and xylene, followed by infiltration with melted paraffin. The paraffin temperature should not exceed 60 °C. Handling and processing outside of the recommended conditions should be validated by the user. # Tissue sections FFPE tissue specimens should be cut into sections of 4 µm. After sectioning, tissues should be mounted on FLEX IHC Microscope Slides (Code K8020) or Superfrost Plus microscope slides and then placed in a 58 ± 2 °C calibrated oven for 1 hour. The tissue specimens must be positioned on the glass within the defined slide staining area per the Dako Omnis User Guide. To preserve antigenicity, tissue sections mounted on slides should be stained within 2 months of sectioning when held in the dark at 2–8 °C (preferred), or at room temperature up to 25 °C. Slide storage and handling conditions should not exceed 25 °C at any point after mounting to ensure tissue integrity and antigenicity. # Reagent Preparation Target Retrieval Solution, pH 9 (50x) (Code GC309) and Wash Buffer (20x) (Code GC807) must be prepared according to their respective instructions for use (IFU). Refer to the GC309 and GC807 IFU for proper reagent preparation and storage information. Note the color of the Target Retrieval Solution, pH 9 (50x) is blue. Reagents do not need to be equilibrated to room temperature before loading into the instrument. However, they should be loaded into the instrument before starting the staining procedure, which allows sufficient time for equilibration. # Test Controls ## System level controls System-level controls are intended to ensure the validity of the staining procedure, including reagents, tissue processing and instrument performance. If controls are not fixed in the same way as the test specimen, then the control tissue may only be used as a staining control. PMA P250004: FDA Summary of Safety and Effectiveness Data {4} Negative control tissue (lab-supplied) with known expression should be run for each staining procedure. The negative control should be prescreened CRC tissue with loss of biomarker expression in malignant cells compared to moderate to strong nuclear staining in adjacent internal positive controls. It is recommended that negative control tissue is stained on the same slide as the patient tissue. The positive control should be tissue with positive biomarker expression. Positive nonmalignant elements (lymphocytes, stromal cells, and normal epithelium) present in the patient tissue should be used, where possible, as internal positive controls instead of a separate positive control tissue. In rare cases where nonmalignant elements may have loss of biomarker expression, nonmalignant elements of the negative control tissue may be used to qualify the staining procedure. ## Negative control reagent MMR Negative Control Reagent, Mouse (Dako Omnis) (Code GE101) and MMR Negative Control Reagent, Rabbit (Dako Omnis) (Code GE102) should each be used in place of the respective species-matched primary antibody with a section of each patient specimen to evaluate nonspecific staining and allow correct interpretation of specific staining at the antigen site. Use the Dako Omnis protocol “MMR NCR Mo GE101” for slides stained with the mouse negative control reagent (NCR) and “MMR NCR Rb GE102” for slides stained with the rabbit NCR. Refer to the MMR Negative Control Reagent, Mouse (Dako Omnis) (Code GE101) and MMR Negative Control Reagent, Rabbit (Dako Omnis) (GE102) instructions for use for details. ## Principle of Operation MMR IHC Panel pharmDx (Dako Omnis) primary antibodies and NCRs are used in combination with detection reagents and ancillary reagents (sold separately) to complete an IHC staining procedure on the Dako Omnis automated staining system. Vials are labeled with a bar code that can be recognized by Dako Omnis Solution software. For each antibody, an optimized staining protocol is provided in the software. Following incubation with one of the four primary antibodies or either of the NCRs, specimens are sequentially incubated with peroxidase block, sequential linker antibodies, and a visualization reagent consisting of secondary antibody molecules and horseradish peroxidase (HRP) molecules coupled to a dextran polymer backbone. The enzymatic conversion of the subsequently added diaminobenzidine (DAB) chromogen results in precipitation of a visible reaction product at the antigen site. The specimen may then be counterstained and coverslipped. Deparaffinization, rehydration, target retrieval, staining and counterstaining procedures are automatically performed on the Dako Omnis system. Coverslipping can be manual or automated, so capabilities for this are also required, but not supplied. PMA P250004: FDA Summary of Safety and Effectiveness Data {5} The presence or absence of target proteins is determined by visual examination of the specimen slide under a light microscope by a qualified pathologist. ## Staining Protocol When processing slides for staining with the MMR IHC Panel pharmDx (Dako Omnis) assay, the Dako Omnis automated platform executes the following protocols: Table 3: Dako Omnis Staining Protocols for MMR IHC Panel pharmDx (Dako Omnis) | Protocol Parameter | Reagent | MLH1 (GE079)* or msNCR (GE101)* | MSH2 (GE085)* | PMS2 (GE087)* or MSH6 (GE086)* or rbNCR (GE102)* | | --- | --- | --- | --- | --- | | | | Temperature and incubation times/cycles | | | | Dewax | Clarify Cleaning Agent | 25 °C, 10 s incubation top, 1 min incubation bottom, 1 cycle | 25 °C, 10 s incubation top, 1 min incubation bottom, 1 cycle | 25 °C, 10 s incubation top, 1 min incubation bottom, 1 cycle | | | DI water | 5 s incubation, 1 cycle | 5 s incubation, 1 cycle | 5 s incubation, 1 cycle | | Target Retrieval | TRS, pH 9 | 97 °C, 30 min incubation | 97 °C, 30 min incubation | 97 °C, 30 min incubation | | | DI water | N/A, cooling fluid | N/A, cooling fluid | N/A, cooling fluid | | Staining | Wash buffer | 2:40 min incubation, 2 cycles | 2:40 min incubation, 2 cycles | 2:40 min incubation, 2 cycles | | | Primary Antibody | 25 min incubation (GE079 or GE101) | 20 min incubation (GE085) | 20 min incubation (GE087, GE086, or GE102) | | | Wash Buffer | 2 min incubation, 10 cycles | 2 min incubation, 10 cycles | 2 min incubation, 10 cycles | | | EnV FLEX Peroxidase-Blocking Reagent | 3 min incubation | 3 min incubation | 3 min incubation | | | Wash Buffer | 2 min incubation, 10 cycles | 2 min incubation, 10 cycles | 2 min incubation, 10 cycles | | | EnV FLEX+ LINKER (Rabbit or Mouse) | 10 min incubation (Mouse) | 10 min incubation (Mouse) | 10 min incubation (Rabbit) | | | Wash Buffer | 2 min incubation, 10 cycles | 2 min incubation, 10 cycles | 2 min incubation, 10 cycles | | | EnV FLEX+ LINKER | 10 min incubation (Rabbit) | 10 min incubation (Rabbit) | 10 min incubation (Mouse) | PMA P250004: FDA Summary of Safety and Effectiveness Data 6 of 77 {6} | Protocol Parameter | Reagent | MLH1 (GE079)* or msNCR (GE101)* | MSH2 (GE085)* | PMS2 (GE087)* or MSH6 (GE086)* or rbNCR (GE102)* | | --- | --- | --- | --- | --- | | | | Temperature and incubation times/cycles | | | | | (Rabbit or Mouse) | | | | | | Wash Buffer | 2 min incubation, 10 cycles | 2 min incubation, 10 cycles | 2 min incubation, 10 cycles | | | EnV FLEX/HRP | 20 min incubation | 20 min incubation | 20 min incubation | | | Wash Buffer | 2 min incubation, 10 cycles | 2 min incubation, 10 cycles | 2 min incubation, 10 cycles | | | Wash Buffer | 2 min incubation, 10 cycles | 2 min incubation, 10 cycles | 2 min incubation, 10 cycles | | | DI water | 31 s incubation, 1 cycle | 31 s incubation, 1 cycle | 31 s incubation, 1 cycle | | | Wash Buffer | 2 min incubation, 10 cycles | 2 min incubation, 10 cycles | 2 min incubation, 10 cycles | | | EnV FLEX Substrate Working Solution** | 5 min incubation | 5 min incubation | 5 min incubation | | | Wash Buffer | 2 min incubation, 10 cycles | 2 min incubation, 10 cycles | 2 min incubation, 10 cycles | | | DI water | 31 s incubation, 1 cycle | 31 s incubation, 1 cycle | 31 s incubation, 1 cycle | | | Wash Buffer | 2 min incubation, 10 cycles | 2 min incubation, 10 cycles | 2 min incubation, 10 cycles | | Counterstaining | Hematoxylin | 3 min incubation | 3 min incubation | 3 min incubation | | | DI Water | 2 min incubation, 10 cycles | 2 min incubation, 10 cycles | 2 min incubation, 10 cycles | | | Wash Buffer | 2 min incubation, 10 cycles | 2 min incubation, 10 cycles | 2 min incubation, 10 cycles | *The staining protocol names for each of the MMR products are as follows: MMR MLH1 IHC pDx GE079, MMR MSH2 IHC pDx GE085, MMR MSH6 IHC pDx GE086, MMR PMS2 IHC pDx GE087, MMR NCR Mouse GE101, and MMR NCR Rabbit GE102. **EnV FLEX Substrate Working Solution consists of two components: EnVision FLEX DAB+ Chromogen and EnVision FLEX Substrate Buffer. Timely mixing of these two components is automatically performed onboard Dako Omnis. PMA P250004: FDA Summary of Safety and Effectiveness Data {7} # Interpretation of MMR IHC Panel pharmDx (Dako Omnis) Results of the MMR IHC Panel pharmDx (Dako Omnis) should be interpreted by a qualified pathologist within the context of clinical presentation, morphology, and other histopathological criteria and complemented by proper controls. A Hematoxylin and Eosin (H&E) stain of the patient tissue is evaluated first to assess tissue histology and preservation quality. MMR IHC Panel pharmDx (Dako Omnis) and the H&E staining should be performed on serial sections from the same paraffin block of the specimen to confirm: 1. The histological diagnosis of colorectal cancer. 2. The specimen contains a minimum of 50 viable malignant cells. 3. The specimen has been properly fixed and prepared for IHC analysis. Only well-preserved and well-stained areas of the specimen should be used to make a diagnostic status determination. The specific staining pattern of MMR IHC Panel pharmDx (Dako Omnis) is nuclear and is evaluated using the following rules: 1. Only nuclear staining is considered; cytoplasmic staining should be ignored. 2. Brown DAB signal must be unequivocal. 3. The staining must cover the entire nucleus. NCR slides must exhibit no or weak staining in malignant cells. If weak staining is present in tumor nuclei it should be used as a baseline to evaluate the species-matched primary antibody slides. Staining at the same intensity or lower that may occur in a species-matched primary antibody slide should be disregarded upon interpretation. NCR slides with moderate or strong staining in malignant cells are invalid and the species-matched antibody slides are considered nonevaluable and must be retested. For example, if the rabbit NCR slide is valid and the mouse NCR slide is invalid, only the mouse NCR and two mouse antibodies, MLH1 and MSH2, must be retested. System level controls are intended to ensure the validity of the staining procedure, including reagents, tissue processing and instrument performance. If controls are not fixed in the same way as the test specimen then the tissue may only be used as a staining control. Nonspecific cytoplasmic staining may be present in some tissues stained with MMR IHC Panel pharmDx (Dako Omnis). As long as cytoplasmic staining does not interfere with the evaluation of biomarker status, then the slide is considered acceptable. If cytoplasmic staining does interfere with the evaluation of biomarker status, then repeat staining for the affected test. PMA P250004: FDA Summary of Safety and Effectiveness Data {8} Components of tumor areas that frequently demonstrate positive staining with MMR proteins, but are excluded from scoring are: 1. Normal cells such as lymphocytes, stromal cells, epithelial cells 2. Edge effects 3. Necrotic areas 4. Areas with adenoma component 5. Areas with obvious fixation artifacts should not be scored or scored with caution Protein status of Intact or Loss is determined for MLH1, PMS2, MSH2, and MSH6, separately, using the following guidelines: Table 4. Guidelines for Determining Protein Status of Intact or Loss | Intact | Nuclear staining in viable malignant cells must be unequivocal, with at least the same overall staining intensity as in adjacent internal positive controls. If focal staining is present, the tissue is considered intact if: 1) continuous in multiple glands/nests and 2) equal or stronger in intensity than internal positive controls. | | --- | --- | | Loss | No or equivocal nuclear staining in viable malignant cells compared to moderate or strong nuclear staining in adjacent internal positive controls. If focal staining is present, the tissue is considered loss if: 1) continuous in only a single gland/nest, 2) discontinuous in multiple glands/nests, or 3) weaker in intensity than internal positive controls. | Only unequivocal brown DAB staining that covers the entire nucleus of tumor cells and exhibits at least the same overall staining intensity as in adjacent internal positive controls should be considered intact MMR biomarker expression. Punctate nuclear staining of tumor cells, along with other incomplete nuclear staining patterns, should be considered loss of MMR biomarker expression. Internal positive control elements must also be assessed when evaluating for MMR biomarker status. Cells with intact nuclear staining must have at least the same overall staining intensity as in adjacent internal positive controls. Cells with loss of nuclear PMA P250004: FDA Summary of Safety and Effectiveness Data {9} staining must have no or equivocal staining compared to adjacent internal positive controls. If the specimen demonstrates equivocal internal positive control staining and a protein status for the biomarker cannot be determined, it is recommended to first evaluate all biomarkers together. If the MMR status cannot be determined using all biomarkers, retesting of equivocal staining should be performed. Specimen qualities that may make a case difficult or challenging to interpret include: focal staining in loss of expression tissue, focal staining in intact tissue, heterogeneous staining; necrosis, stromal cell staining, signet ring cell adenocarcinoma, mucinous colorectal cancer, areas without IPC staining, nonspecific/background staining tissue/staining artifacts (e.g., edge effect, tissue folding, etc.), and punctate staining. After a protein status of Intact or Loss is assigned to each biomarker for a given specimen, a diagnostic status of MMR proficient or MMR deficient is given using the following definitions: Table 5. Definition for MMR Diagnostic Status | MMR Proficient (pMMR) | MMR Deficient (dMMR) | | --- | --- | | Intact for all four biomarkers | Loss of one or more biomarkers | For additional guidance on MMR staining interpretation, refer to the MMR IHC Panel pharmDx (Dako Omnis) Interpretation Manual. VI. ALTERNATIVE PRACTICES AND PROCEDURES There are no other FDA-cleared or approved alternative class III immunohistochemistry assays available for detection of MMR in formalin-fixed, paraffin-embedded (FFPE) colorectal cancer (CRC) specimens to aid in identify MMR deficient CRC patients eligible for treatment with OPDIVO (nivolumab) alone or in combination with YERVOY (ipilimumab). VII. MARKETING HISTORY MMR IHC Panel pharmDx (Dako Omnis) has not been marketed in the United States or any foreign country. VIII. POTENTIAL ADVERSE EFFECTS OF THE DEVICE ON HEALTH MMR IHC Panel pharmDx (Dako Omnis) is intended for in vitro diagnostic (IVD) use only. As with any IVD test, the potential risks are associated with an incorrect test result or incorrect interpretation of results. Failure of the device to perform as expected or failure to correctly interpret test results may lead to improper patient management decisions. PMA P250004: FDA Summary of Safety and Effectiveness Data 10 of 77 {10} For the specific adverse events that occurred in the OPDIVO (nivolumab) alone or OPDIVO (nivolumab) in combination with YERVOY (ipilimumab) clinical studies, please see the FDA approved package inserts, which are available at Drugs@FDA. ## IX. SUMMARY OF NON-CLINICAL STUDIES Non-clinical studies were performed using the MMR IHC Panel pharmDx (Dako Omnis) to establish analytical performance of the device in CRC patients. These studies were conducted to characterize the MMR IHC Panel pharmDx (Dako Omnis), demonstrate the impact of pre-analytical variables on assay performance, evaluate assay precision and robustness, and establish assay stability. The study results detailed below establish sensitivity, specificity, precision, robustness, stability, external reproducibility, and other performance characteristics of the device. ## A. Laboratory Studies ### 1. Analytical Sensitivity A set of 171 unique CRC specimens that were not preselected by mismatch repair (MMR) status was tested to assess if the MMR IHC Panel pharmDx (Dako Omnis) antibodies can detect the presence or absence of the target proteins (MLH1, MSH2, MSH6, and PMS2). The prevalence of loss of staining at the individual MMR biomarker level and the prevalence of dMMR status at the MMR panel level were calculated. The prevalence of dMMR panel status was 8.8%. The prevalence of loss of staining for individual MMR biomarkers was between 0% and 8.8%. Given the observed 0% MSH2 loss status in the 171 specimens included in the analytical sensitivity study, a supplemental MSH2 sensitivity study was initiated to assess further the prevalence of loss of MSH2 biomarker expression in a set of intended use samples that were not preselected based on MMR diagnostic status. An additional set of 98 unique cases was combined with the previously tested 171 cases for a total of 269 unique cases stained with MMR IHC MSH2 pharmDx (Dako Omnis). The prevalence of loss of staining of MSH2 was 2.6%. Table 6 summarizes the results for all biomarkers. Table 6. MMR Panel dMMR/pMMR and MMR Biomarker Intact/Loss Results | Biomarker | N Specimens Total | Result | N Specimens (% of Total) | | --- | --- | --- | --- | | MLH1 | 171 | Intact | 157 (91.8%) | | | | Loss | 14 (8.2%) | | PMS2 | 171 | Intact | 156 (91.2%) | PMA P250004: FDA Summary of Safety and Effectiveness Data {11} | Biomarker | N Specimens Total | Result | N Specimens (% of Total) | | --- | --- | --- | --- | | | | Loss | 15 (8.8%) | | MSH2 | 269 | Intact | 262 (97.4%) | | | | Loss | 7 (2.6%) | | MSH6 | 171 | Intact | 169 (98.8%) | | | | Loss | 2 (1.2%) | | MMR Panel* | 171 | pMMR | 156 (91.2%) | | | | dMMR | 15 (8.8%) | *MMR panel results do not include the additional cases tested for MSH2, as only MSH2 was supplemented. ## 2. Analytical Specificity Specificity testing was conducted to demonstrate that MMR IHC Panel pharmDx (Dako Omnis) will detect the target antigens in the appropriate tissue elements and cellular compartment. Tests included testing of immunoreactivity on normal tissues and neoplastic tissues (tour of tumor); Western blot; and peptide inhibition. ### i. Western Blot Western blots analyses were conducted to demonstrate that the antibodies specifically detect the proteins of predicted molecular weight for each of the 4 MMR pharmDx antibodies using cell lines with known MMR loss or intact status. The MLH1, MSH2, PMS2, and MSH6 antibodies demonstrated the ability to detect the presence or absence of their target antigens in cell lines known to have positive or negative expression, respectively. The Western blot study supports the conclusion that each of the MMR antibodies is specific for their respective antigens. Additionally, IHC testing confirmed alignment between Western blot and IHC results in the tested cell lines. Further studies investigated the specificity using RNA-sequencing and BLAST to evaluate the expression of the target proteins and potential cross-reacting proteins in the cell lines used for Western blot testing. The results confirmed that the MMR antibodies are specific for their respective antigens. ### ii. Epitope Mapping and Peptide Inhibition PMA P250004: FDA Summary of Safety and Effectiveness Data {12} Epitope mapping was conducted to elucidate the epitope of each antibody, and this data was used to synthesize epitope-containing peptides for use in the inhibition study. The UniProt BLAST and ALIGN tools were used to identify the proposed epitope sequence within the context of each of the target proteins and to identify potential cross-reactivity with any non-target proteins. A peptide inhibition study was conducted to confirm the specificity of the MMR IHC Panel pharmDx (Dako Omnis) antibodies (MLH1, MSH2, MSH6 and PMS2) for their respective protein targets as identified in the epitope mapping study. For each biomarker, sections from five CRC specimens (four intact and one loss) were stained with one of the MMR antibodies in the presence or absence of a peptide containing the corresponding epitope. Solutions of various peptide-to-antibody (peptide:antibody) molar ratios (0.5:1 [MSH6 only], 2:1 [MSH6 only], 4:1, 10:1, 50:1, 100:1, 200:1 [PMS2 only], and 300:1 [PMS2 only]) were prepared for use in place of the uninhibited Ready-to-use (RTU) antibody in the primary antibody step of the staining protocol. Slides were also stained with standard RTU antibody as the reference condition. To verify specificity of inhibition, specimens were also stained with the MMR antibody mixed with a negative control peptide and tested at the highest peptide:antibody molar ratio. Each negative control peptide contained the same amino acids as the corresponding epitope-containing peptide in a scrambled order. For MSH2, the peptides (test and negative control) were not soluble in water and had to be reconstituted in 3% ammonium hydroxide, so an ammonium hydroxide diluent in RTU antibody as a control was also included. IHC staining by the MMR antibodies was inhibited in the presence of epitope-containing peptides, confirming specificity to the target antigen. iii. Immunoreactivity in Normal Tissues Immunohistochemistry based specificity testing was conducted to demonstrate that MMR IHC Panel pharmDx (Dako Omnis) will detect the target substance in the appropriate tissue elements and cellular compartment in a variety of normal tissues. The expectation of which structures should be positive and negative was established using peer-reviewed scientific literature. There was no non-specific staining observed in tissue types tested. All tissues were FFPE and stained with MMR IHC Panel pharmDx (Dako Omnis) according to the instructions in the package insert. MMR IHC Panel pharmDx (Dako Omnis) detected expression of the MMR proteins (MLH1, MSH2, MSH6 and PMS2) in all 31 normal tissue types tested. Because the MMR proteins are required for normal DNA PMA P250004: FDA Summary of Safety and Effectiveness Data 13 of 77 {13} replication, it is expected that they were expressed in all normal cells. There were no unexpected results observed in cell types or tissue types tested. The observed staining was consistent with the reported literature for MMR expression in normal tissues. Table 7 summarizes MMR immunoreactivity on the recommended panel of normal tissues. Table 7: Specificity of MMR IHC Panel pharmDx (Dako Omnis) in Normal Tissue | Reactivity Tissue | # positive / total cases | | | | | --- | --- | --- | --- | --- | | | MLH1 | PMS2 | MSH2 | MSH6 | | Salivary Gland | 3/3 | 3/3 | 3/3 | 3/3 | | Bone Marrow | 2/3a | 2/3a | 3/3a | 2/3a | | Pituitary Gland | 3/3 | 3/3 | 3/3 | 1/3 | | Cerebrum | 2/3 | 3/3 | 1/3 | 1/3 | | Adrenal Gland | 2/3 | 2/3 | 3/3 | 2/3 | | Cerebellum | 3/3 | 3/3 | 3/3 | 1/3 | | Uterus | 3/3 | 3/3 | 3/3 | 3/3 | | Ovary | 3/3 | 3/3 | 3/3 | 3/3 | | Cervix | 3/3 | 3/3 | 3/3 | 3/3 | | Breast | 2/3 | 2/3 | 2/3 | 2/3 | | Nerve Peripheral | 3/3 | 3/3 | 3/3 | 1/3 | | Prostate | 3/3 | 3/3 | 3/3 | 3/3 | | Skin | 3/3 | 3/3 | 3/3 | 3/3 | | Testis | 3/3 | 3/3 | 3/3 | 3/3 | | Mesothelial Cells | 3/3 | 3/3 | 3/3 | 3/3 | | Skeletal Muscle | 3/3 | 3/3 | 3/3 | 3/3 | | Lung | 3/3 | 3/3 | 3/3 | 3/3 | | Heart | 3/3 | 3/3a | 3/3a | 2/3 | | Stomach | 3/3 | 3/3 | 3/3 | 2/3 | | Small Intestine | 3/3 | 3/3 | 3/3 | 3/3 | PMA P250004: FDA Summary of Safety and Effectiveness Data 14 of 77 {14} | Reactivity Tissue | # positive / total cases | | | | | --- | --- | --- | --- | --- | | | MLH1 | PMS2 | MSH2 | MSH6 | | Esophagus | 3/3 | 3/3 | 3/3 | 3/3 | | Colon | 3/3 | 3/3 | 3/3 | 3/3 | | Kidney | 3/3 | 3/3 | 3/3 | 3/3 | | Liver | 2/3 | 3/3^{a} | 3/3^{a} | 3/3^{a} | | Pancreas | 3/3 | 3/3 | 3/3 | 3/3 | | Spleen | 3/3 | 3/3 | 3/3 | 2/3 | | Tonsil | 3/3 | 3/3 | 3/3 | 3/3 | | Thyroid | 3/3 | 3/3 | 3/3 | 2/3 | | Thymus | 3/3^{b} | 3/3 | 3/3^{b} | 3/3^{b} | | Parathyroid | 3/3 | 3/3 | 3/3 | 3/3 | | Bladder | 3/3 | 3/3 | 3/3 | 3/3 | a cytoplasmic staining pattern for at least one case b cytoplasmic and extracellular staining pattern for at least one case iv. Immunoreactivity in Neoplastic Tissues Neoplastic tissue specimens were evaluated using various neoplastic tumors in cores from a tissue microarray. The expected staining pattern was established through review of peer-reviewed scientific literature. MMR IHC Panel pharmDx (Dako Omnis) identifies MMR expression in multiple cancer types, and findings of no expression of one or more biomarker in breast and colon samples are consistent with reported dMMR prevalence in the literature. All tissues were FFPE and stained with MMR IHC Panel pharmDx (Dako Omnis) according to the instructions in the package insert. Expression of one or more of the MMR proteins was identified in 42 tumor types. There were no unexpected results observed in the tumor specimens tested. The observed staining was consistent with the reported literature for MMR expression in neoplastic tissues. Table 8 summarizes MMR immunoreactivity on a panel of neoplastic tissues. PMA P250004: FDA Summary of Safety and Effectiveness Data {15} Table 8: Specificity of MMR IHC Panel pharmDx (Dako Omnis) in Neoplastic Tissue | Tissue Type (# tested) | # positive/total cases evaluateda | | | | | --- | --- | --- | --- | --- | | | MLH1 | PMS2 | MSH2 | MSH6 | | Bladder Carcinoma (2) | 2/2 | 2/2 | 2/2 | 2/2 | | Breast Carcinoma (6) | 5/5 | 3/4 | 5/5 | 4/5 | | Cholangiocarcinoma (1) | 1/1 | 1/1 | 1/1 | 1/1 | | Colon Adenocarcinoma (1) | 1/1 | 0/1 | 1/1 | 1/1 | | Endometrial Sarcoma (1) | 1/1 | 1/1 | 1/1 | 1/1 | | Ewing's Sarcoma (1) | 1/1 | 1/1 | 1/1 | 1/1 | | Gastric Adenocarcinoma (2) | 2/2 | 2/2 | 2/2 | 2/2 | | Hepatoma (1) | NE | NE | 1/1 | NE | | Islet cell tumor of pancreas (1) | NE | NE | 1/1 | NE | | Kidney Transitional Cell Carcinoma (1) | 1/1 | 1/1 | 1/1 | NE | | Liver cell adenoma (1) | NE | NE | 1/1 | NE | | Lung Carcinoma (4) | 3/3 | 2/2 | 4/4 | 3/3 | | Lymphoma of Cecum (1) | 1/1 | 1/1 | 1/1 | 1/1 | | Melanoma (3) | 3/3 | 3/3 | 3/3 | 3/3 | | Merkel Cell Tumor (1) | 1/1 | 1/1 | 1/1 | 1/1 | | Ovarian Carcinoma (2) | 2/2 | 2/2 | 2/2 | 2/2 | | Ovarian Dysgerminoma (1) | 1/1 | 1/1 | 1/1 | 1/1 | | Ovarian Granulosa Cell Tumor (1) | 1/1 | 1/1 | 1/1 | 1/1 | | Pancreatic adenocarcinoma (1) | NE | NE | NE | 1/1 | | Pancreatic glucagonoma (1) | NE | NE | 1/1 | 1/1 | | Papillary Serous carcinoma (1) | NE | NE | 1/1 | NE | | Pleomorphic Rhabdomyosarcoma (1) | 1/1 | 1/1 | 1/1 | 1/1 | | PNET Scrotum (1) | 1/1 | NE | 1/1 | 1/1 | | Prostate Adenocarcinoma (2) | 1/1 | 1/1 | 2/2 | 1/1 | | Prostate Benign Prostatic Hyperplasia (1) | 1/1 | 1/1 | 1/1 | 1/1 | | Renal Cell Carcinoma (1) | 1/1 | 1/1 | 1/1 | NE | | Squamous Carcinoma of Ear (1) | 1/1 | 1/1 | 1/1 | 1/1 | | Testicular Embryonal Carcinoma (1) | 1/1 | 1/1 | 1/1 | 1/1 | | Testicular Yolk Sac Tumor (1) | 1/1 | 1/1 | 1/1 | 1/1 | | Thymic carcinoid tumor (1) | NE | NE | 1/1 | 1/1 | | Thymoma (1) | 1/1 | 1/1 | 1/1 | 1/1 | | Thyroid Carcinoma (2) | 1/1 | 1/1 | 2/2 | 2/2 | PMA P250004: FDA Summary of Safety and Effectiveness Data 16 of 77 {16} | Tissue Type (# tested) | # positive/total cases evaluateda | | | | | --- | --- | --- | --- | --- | | | MLH1 | PMS2 | MSH2 | MSH6 | | Uterine Adenomatoid Tumor (1) | 1/1 | 1/1 | 1/1 | 1/1 | a Number evaluated may be less than total number tested due to non-evaluable (NE) internal positive controls for a single biomarker NE = Non-evaluable # 3. Precision The precision of MLH1 IHC pharmDx (Dako Omnis), MSH2 IHC pharmDx (Dako Omnis), MSH6 IHC pharmDx (Dako Omnis), and PMS2 IHC pharmDx (Dako Omnis) was evaluated at one (1) internal laboratory. Mismatch Repair NCRs (mouse and rabbit) were used in the generation of the precision study and did not show staining on any of the tissues. Diagnostic status was recorded as 'Intact' or 'Loss' for biomarker-level analysis and was recorded as 'pMMR' or 'dMMR' for panel-level analysis. Percent agreement of loss (LPA), percent agreement of intact (IPA) and overall percent agreement (OPA), using comparisons to the consensus diagnostic status as reference, were computed with corresponding two-sided $95\%$ percentile bootstrap confidence intervals (CIs). The Wilson score limits were used to calculate confidence intervals for agreement parameters with point estimates equal to $100\%$ . The sample sets varied between each biomarker, so only biomarker results (intact/loss) were determined. The sample sets included 18 challenging cases for MLH1, 5 challenging cases for PMS2, 18 challenging cases for MSH2, and 13 challenging cases for MSH6. # i. Intermediate Precision and Repeatability Table 9. Intermediate Precision of MLH1 | Precision Study | Study Design | % Agreement (95% CI) | | --- | --- | --- | | Intra-rack | Each of 24 CRC specimens (12 loss, 12 intact) was tested on a single Dako Omnis instrument within the same rack/staining module. Intra-rack analysis was performed between 4 replicates stained within the same rack/staining module on a total of 96 comparisons to consensus. | LPA 100.0 (92.6, 100.0) IPA 100.0 (92.6, 100.0) OPA 100.0 (96.2, 100.0) | | Inter-rack | Each of 24 CRC specimens (12 loss, 12 intact) was tested on a single Dako Omnis instrument on different racks/staining modules. Inter-rack analysis was performed between 4 racks/staining modules on a total of 95 comparisons to consensus. | LPA 97.9 (93.8, 100.0) IPA 100.0 (92.4, 100.0) OPA 98.9 (96.8, 100.0) | | Inter-instrument | Each of 24 CRC specimens (12 loss, 12 intact) was tested across 3 different | LPA 100.0 (94.9, 100.0) IPA 100.0 (94.9, 100.0) | | | racks/staining modules on a total of 95 comparisons to consensus. | | PMA P250004: FDA Summary of Safety and Effectiveness Data {17} PMA P250004: FDA Summary of Safety and Effectiveness Data 18 of 77 | Precision Study | Study Design | % Agreement (95% CI) | | --- | --- | --- | | | Dako Omnis instruments. Inter-instrument analysis was performed between 3 different Dako Omnis instruments on a total of 144 comparisons to consensus. | OPA 100.0 (97.4, 100.0) | | Inter-day | Each of 24 CRC specimens (12 loss, 12 intact) was tested on a single Dako Omnis instrument over 5 nonconsecutive days. Inter-day analysis was performed between 5 nonconsecutive days on a total of 120 comparisons to consensus. | LPA 98.3 (95.0, 100.0) IPA 100.0 (94.0, 100.0) OPA 99.2 (97.5, 100.0) | | Inter-lot | Each of 24 CRC specimens (11 loss, 13 intact) was tested on a single Dako Omnis instrument using 3 unique lots of reagents. Inter-lot analysis was performed between 3 unique lots of reagents on a total of 143 comparisons to consensus. | LPA 100.0 (94.4, 100.0) IPA 100.0 (95.3, 100.0) OPA 100.0 (97.4, 100.0) | LPA=Percent Agreement of Loss; IPA=Percent Agreement of Intact; OPA=Overall Percent Agreement Table 10. Percent Loss Results Per Sample for Intermediate Precision of MLH1 | Case ID | Inter-Day Percent Loss Results, % (n/N) | Inter-Instrument Percent Loss Results, % (n/N) | Inter-Lot Percent Loss Results, % (n/N) | Inter-Rack Percent Loss Results, % (n/N) | Intra-Rack Percent Loss Results, % (n/N) | | --- | --- | --- | --- | --- | --- | | 1 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 2 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 3 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 4 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 5 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 6 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 7 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/3) | 0% (0/4) | | 8 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 9 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 10 | 0% (0/5) | 0% (0/6) | 0% (0/6) | N/A | N/A | | 11 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 12 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 13 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | {18} | Case ID | Inter-Day Percent Loss Results, % (n/N) | Inter-Instrument Percent Loss Results, % (n/N) | Inter-Lot Percent Loss Results, % (n/N) | Inter-Rack Percent Loss Results, % (n/N) | Intra-Rack Percent Loss Results, % (n/N) | | --- | --- | --- | --- | --- | --- | | 14 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 15 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 16 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 17 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 18 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 19 | 100% (5/5) | 100% (6/6) | 0% (0/6) | 75% (3/4) | 100% (4/4) | | 20 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 21 | 100% (5/5) | 100% (6/6) | 100% (5/5) | 100% (4/4) | 100% (4/4) | | 22 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 23 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 24 | 80% (4/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 25 | N/A | N/A | N/A | 0% (0/4) | 0% (0/4) | Note: n/N denotes the number of loss replicates/the total number of replicates. Table 11. Intermediate Precision of MSH2 | Precision Study | Study Design | % Agreement (95% CI) | | --- | --- | --- | | Intra-rack | Each of 24 CRC specimens (12 loss, 12 intact) was tested on a single Dako Omnis instrument within the same rack/staining module. Intra-rack analysis was performed between 4 replicates stained within the same rack/staining module on a total of 96 comparisons to consensus. | LPA 100.0 (92.6, 100.0) IPA 100.0 (92.6, 100.0) OPA 100.0 (96.2, 100.0) | | Inter-rack | Each of 24 CRC specimens (12 loss, 12 intact) was tested on a single Dako Omnis instrument on different racks/staining modules. Inter-rack analysis was performed between 4 racks/staining modules on a total of 96 comparisons to consensus. | LPA 100.0 (92.6, 100.0) IPA 100.0 (92.6, 100.0) OPA 100.0 (96.2, 100.0) | | Inter-instrument | Each of 24 CRC specimens (12 loss, 12 intact) was tested across 3 different Dako Omnis instruments. Inter-instrument analysis was performed between 3 different | LPA 100.0 (94.9, 100.0) IPA 100.0 (94.9, 100.0) OPA 100.0 (97.4, 100.0) | PMA P250004: FDA Summary of Safety and Effectiveness Data {19} | Precision Study | Study Design | % Agreement (95% CI) | | --- | --- | --- | | | Dako Omnis instruments on a total of 144 comparisons to consensus. | | | Inter-day | Each of 24 CRC specimens (12 loss, 12 intact) was tested on a single Dako Omnis instrument over 5 nonconsecutive days. Inter-day analysis was performed between 5 nonconsecutive days on a total of 120 comparisons to consensus. | LPA 100.0 (94.0, 100.0) IPA 100.0 (94.0, 100.0) OPA 100.0 (96.9, 100.0) | | Inter-lot | Each of 24 CRC specimens (12 loss, 12 intact) was tested on a single Dako Omnis instrument using 3 unique lots of reagents. Inter-lot analysis was performed between 3 unique lots of reagents on a total of 144 comparisons to consensus. | LPA 98.6 (95.8, 100.0) IPA 98.6 (95.8, 100.0) OPA 98.6 (96.5, 100.0) | LPA=Percent Agreement of Loss; IPA=Percent Agreement of Intact; OPA=Overall Percent Agreement Table 12. Percent Loss Results Per Sample in Intermediate Precision of MSH2 PMA P250004: FDA Summary of Safety and Effectiveness Data 20 of 77 {20} | Case ID | Inter-Day Percent Loss Results, % (n/N) | Inter-Instrument Percent Loss Results, % (n/N) | Inter-Lot Percent Loss Results, % (n/N) | Inter-Rack Percent Loss Results, % (n/N) | Intra-Rack Percent Loss Results, % (n/N) | | --- | --- | --- | --- | --- | --- | | 1 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 2 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 3 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 4 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 5 | 0% (0/5) | 0% (0/6) | 16.7% (1/6) | 0% (0/4) | 0% (0/4) | | 6 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 7 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 8 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 9 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 10 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 11 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 12 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 13 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 14 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 15 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 16 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 17 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 18 | 100% (5/5) | 100% (6/6) | 83.3% (5/6) | 100% (4/4) | 100% (4/4) | | 19 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 20 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 21 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 22 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 23 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 24 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | Note: n/N denotes the number of loss replicates/the total number of replicates. Table 13. Intermediate Precision of MSH6 | Precision Study | Study Design | % Agreement (95% CI) | | --- | --- | --- | | Intra-rack | Each of 24 CRC specimens (12 loss, 12 intact) was tested on a single Dako Omnis instrument within the same rack/staining module. Intra-rack analysis was performed between 4 replicates stained within the same rack/staining module on a total of 96 comparisons to consensus. | LPA 100.0 (92.6, 100.0)IPA 100.0 (92.6, 100.0)OPA 100.0 (96.2, 100.0) | PMA P250004: FDA Summary of Safety and Effectiveness Data {21} | Precision Study | Study Design | % Agreement (95% CI) | | --- | --- | --- | | Inter-rack | Each of 23 CRC specimens (11 loss, 12 intact) was tested on a single Dako Omnis instrument on different racks/staining modules. Inter-rack analysis was performed between 4 racks/staining modules on a total of 92 comparisons to consensus. | LPA 100.0 (92.0, 100.0) IPA 100.0 (92.6, 100.0) OPA 100.0 (96.0, 100.0) | | Inter-instrument | Each of 24 CRC specimens (12 loss, 12 intact) was tested across 3 different Dako Omnis instruments. Inter-instrument analysis was performed between 3 different Dako Omnis instruments on a total of 144 comparisons to consensus. | LPA 100.0 (94.9, 100.0) IPA 100.0 (94.9, 100.0) OPA 100.0 (97.4, 100.0) | | Inter-day | Each of 24 CRC specimens (10 loss, 14 intact) was tested on a single Dako Omnis instrument over 5 nonconsecutive days. Inter-day analysis was performed between 5 nonconsecutive days on a total of 120 comparisons to consensus. | LPA 100.0 (92.9, 100.0) IPA 98.6 (95.7, 100.0) OPA 99.2 (97.5, 100.0) | | Inter-lot | Each of 24 CRC specimens (12 loss, 12 intact) was tested on a single Dako Omnis instrument using 3 unique lots of reagents. Inter-lot analysis was performed between 3 unique lots of reagents on a total of 144 comparisons to consensus. | LPA 100.0 (94.9, 100.0) IPA 100.0 (94.9, 100.0) OPA 100.0 (97.4, 100.0) | LPA=Percent Agreement of Loss; IPA=Percent Agreement of Intact; OPA=Overall Percent Agreement Table 14. Percent Loss Results Per Sample in Intermediate Precision of MSH6 PMA P250004: FDA Summary of Safety and Effectiveness Data {22} | Case ID | Inter-Day Percent Loss Results, % (n/N) | Inter-Instrument Percent Loss Results, % (n/N) | Inter-Lot Percent Loss Results, % (n/N) | Inter-Rack Percent Loss Results, % (n/N) | Intra-Rack Percent Loss Results, % (n/N) | | --- | --- | --- | --- | --- | --- | | 1 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 2 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 3 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 4 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 5 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 6 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 7 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 8 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 9 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 10 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 11 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 12 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 13 | 0% (0/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 14 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 15 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 16 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 17 | 100% (5/5) | 100% (6/6) | 100% (6/6) | N/A | N/A | | 18 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 19 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 20 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 21 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 22 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 23 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 24 | 20% (1/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 25 | N/A | N/A | N/A | N/A | 100% (4/4) | Note: n/N denotes the number of loss replicates/the total number of replicates. Table 15. Intermediate Precision of PMS2 PMA P250004: FDA Summary of Safety and Effectiveness Data {23} | Precision Study | Study Design | % Agreement (95% CI) | | --- | --- | --- | | Intra-rack | Each of 24 CRC specimens (13 loss, 11 intact) was tested on a single Dako Omnis instrument within the same rack/staining module. Intra-rack analysis was performed between 4 replicates stained within the same rack/staining module on a total of 96 comparisons to consensus. | LPA 100.0 (93.1, 100.0) IPA 100.0 (92.0, 100.0) OPA 100.0 (96.2, 100.0) | | Inter-rack | Each of 23 CRC specimens (13 loss, 11 positive) was tested on a single Dako Omnis instrument on different racks/staining modules. Inter-rack analysis was performed between 4 racks/staining modules on a total of 96 comparisons to consensus. | LPA 100.0 (93.1, 100.0) IPA 100.0 (92.0, 100.0) OPA 100.0 (96.2, 100.0) | | Inter-instrument | Each of 24 CRC specimens (12 loss, 12 intact) was tested across 3 different Dako Omnis instruments. Inter-instrument analysis was performed between 3 different Dako Omnis instruments on a total of 144 comparisons to consensus. | LPA 100.0 (94.9, 100.0) IPA 98.6 (95.8, 100.0) OPA 99.3 (97.9, 100.0) | | Inter-day | Each of 24 CRC specimens (12 loss, 12 intact) was tested on a single Dako Omnis instrument over 5 nonconsecutive days. Inter-day analysis was performed between 5 nonconsecutive days on a total of 120 comparisons to consensus. | LPA 100.0 (94.0, 100.0) IPA 100.0 (94.0, 100.0) OPA 100.0 (96.9, 100.0) | | Inter-lot | Each of 24 CRC specimens (14 loss, 10 intact) was tested on a single Dako Omnis instrument using 3 unique lots of reagents. Inter-lot analysis was performed between 3 unique lots of reagents on a total of 144 comparisons to consensus. | LPA 100.0 (95.6, 100.0) IPA 100.0 (94.0, 100.0) OPA 100.0 (97.4, 100.0) | LPA=Percent Agreement of Loss; IPA=Percent Agreement of Intact; OPA=Overall Percent Agreement Table 16. Percent Loss Results Per Sample in Intermediate Precision of PMS2 PMA P250004: FDA Summary of Safety and Effectiveness Data {24} | Case ID | Inter-Day Percent Loss Results, % (n/N) | Inter-Instrument Percent Loss Results, % (n/N) | Inter-Lot Percent Loss Results, % (n/N) | Inter-Rack Percent Loss Results, % (n/N) | Intra-Rack Percent Loss Results, % (n/N) | | --- | --- | --- | --- | --- | --- | | 1 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 2 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 3 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 4 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 5 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 6 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 7 | 0% (0/5) | 16.7% (1/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 8 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 9* | 0% (0/5) | 0% (0/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 10 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 11 | 0% (0/5) | 0% (0/6) | 0% (0/6) | 0% (0/4) | 0% (0/4) | | 12 | 0% (0/5) | 0% (0/6) | 100% (6/6) | 0% (0/4) | 0% (0/4) | | 13 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 14 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 15 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 16 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 17 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 18 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 19 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 20 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 21 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 22 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 23 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | | 24 | 100% (5/5) | 100% (6/6) | 100% (6/6) | 100% (4/4) | 100% (4/4) | *Inter-observer variability observed in biomarker status for this case across the different sub-studies was attributed to the challenging quality of the case (i.e., focal staining). The variability had no impact on the intermediate precision results, as the intra-observer scoring was consistent, and the sub studies are analyzed separately. Note: n/N denotes the number of loss replicates/the total number of replicates. # ii. Internal Inter-Reader Precision The between-reader precision of MLH1 IHC pharmDx (Dako Omnis), MSH2 IHC pharmDx (Dako Omnis), MSH6 IHC pharmDx (Dako Omnis), and PMS2 IHC pharmDx (Dako Omnis) was evaluated at one (1) internal laboratory. The sample set included 12 challenging cases. Mismatch Repair NCRs (mouse and rabbit) were used in the generation of the precision study and did not show staining on any of the tissues. PMA P250004: FDA Summary of Safety and Effectiveness Data {25} Diagnostic status was recorded as 'Loss' or 'Intact' for biomarker-level analysis and was recorded as 'dMMR' or 'pMMR' for panel-level analysis. Loss percent agreement (LPA), intact percent agreement (IPA) and overall percent agreement (OPA), using comparisons to the consensus diagnostic status as reference, were computed with corresponding two-sided 95% percentile bootstrap confidence intervals (CIs) for the biomarker-level analysis. dMMR percent agreement (dMPA), pMMR percent agreement (pMPA), and OPA were computed with corresponding two-sided 95% percentile bootstrap CIs for the panel-level analysis. The Wilson score limits were used to calculate confidence intervals for agreement parameters with point estimates equal to 100%. Table 17. Inter-Observer Precision at one site | Biomarker | Study Design | % Agreement (95% CI) | | --- | --- | --- | | Panel | One set of 58 stained specimens (31 dMMR, 27 pMMR) was evaluated in turn by each of 3 observers at a single site. Inter-observer analysis was performed between 3 observers on a total of 172 comparisons to consensus. | dMPA 95.7 (91.3, 98.9) pMPA 98.8 (96.2, 100.0) OPA 97.1 (94.7, 99.4) | | MLH1 | One set of 58 CRC stained specimens (28 loss, 30 intact) was evaluated in turn by each of 3 observers at a single site. Inter-observer analysis was performed between 3 observers on a total of 172 comparisons to consensus. | LPA 94.0 (89.3, 98.8) IPA 97.7 (94.2, 100.0) OPA 95.9 (93.0, 98.3) | | MSH2 | One set of 58 CRC stained specimens (4 loss, 54 intact) was evaluated in turn by each of 3 observers at a single site. Inter-observer analysis was performed between 3 observers on a total of 172 comparisons to consensus. | LPA 90.9 (75.0, 100.0) IPA 98.8 (96.9, 100.0) OPA 98.3 (96.0, 100.0) | | MSH6 | One set of 58 CRC stained specimens (6 loss, 52 intact) was evaluated in turn by each of 3 observers at a single site. Inter-observer analysis was performed between 3 observers on a total of 172 comparisons to consensus. | LPA 82.4 (70.6, 94.4) IPA 99.4 (98.1, 100.0) OPA 97.7 (95.9, 99.4) | | PMS2 | One set of 58 CRC stained specimens (28 loss, 30 intact) was evaluated in turn by each of 3 observers at a single site. Inter-observer analysis was performed between 3 observers on a total of 172 comparisons to consensus. | LPA 95.2 (90.5, 98.8) IPA 97.7 (94.2, 100.0) OPA 96.5 (93.6, 98.8) | dMPA=dMMR Percent Agreement; pMPA=pMMR Percent Agreement; OPA=Overall Percent Agreement; LPA=Percent Agreement of Loss; IPA=Percent Agreement of Intact PMA P250004: FDA Summary of Safety and Effectiveness Data 26 of 77 {26} Table 18. Biomarker-level and Panel-level Percent Loss Results Per Sample in Inter-Observer Precision at One Site | Case ID | MLH1 Percent Loss Results, % (n/N) | MSH2 Percent Loss Results, % (n/N) | PMS2 Percent Loss Results, % (n/N) | MSH6 Percent Loss Results, % (n/N) | Panel Percent dMMR, % (n/N) | | --- | --- | --- | --- | --- | --- | | 1 | 0% (0/2) | 0% (0/2) | 0% (0/2) | 0% (0/2) | 0% (0/2) | | 2 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 3 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 4 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 5 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 6 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 7 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 8 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 9 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 10 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 11 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 12 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 13 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 14 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 15 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 16 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 17 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 18 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 19 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 20 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 21 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 22 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 23 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 24 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 25 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 26 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 27 | 50% (1/2) | 100% (2/2) | 50% (1/2) | 100% (2/2) | 100% (2/2) | | 28 | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3) | 100% (3/3) | | 29 | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3) | 100% (3/3) | | 30 | 100% (3/3) | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3) | | 31 | 100% (3/3) | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3) | | 32 | 100% (3/3) | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3) | PMA P250004: FDA Summary of Safety and Effectiveness Data {27} | Case ID | MLH1 Percent Loss Results, % (n/N) | MSH2 Percent Loss Results, % (n/N) | PMS2 Percent Loss Results, % (n/N) | MSH6 Percent Loss Results, % (n/N) | Panel Percent dMMR, % (n/N) | | --- | --- | --- | --- | --- | --- | | 33 | 100% (3/3) | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3) | | 34 | 100% (3/3) | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3) | | 35 | 100% (3/3) | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3) | | 36 | 100% (3/3) | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3) | | 37 | 100% (3/3) | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3) | | 38 | 100% (3/3) | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3) | | 39 | 100% (3/3) | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3) | | 40 | 100% (3/3) | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3) | | 41 | 100% (3/3) | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3) | | 42 | 100% (3/3) | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3) | | 43 | 100% (3/3) | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3) | | 44 | 100% (3/3) | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3) | | 45 | 100% (3/3) | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3) | | 46 | 100% (3/3) | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3) | | 47 | 100% (3/3) | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3) | | 48 | 100% (3/3) | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3) | | 49 | 100% (3/3) | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3) | | 50 | 100% (3/3) | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3) | | 51 | 100% (3/3) | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3) | | 52 | 100% (3/3) | 0% (0/3) | 100% (3/3) | 33.3% (1/3) | 100% (3/3) | | 53 | 66.7% (2/3) | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3) | | 54 | 33.3% (1/3) | 0% (0/3) | 33.3% (1/3) | 0% (0/3) | 33.3% (1/3) | | 55 | 66.7% (2/3) | 0% (0/3) | 66.7% (2/3) | 0% (0/3) | 66.7% (2/3) | | 56 | 66.7% (2/3) | 33.3% (1/3) | 66.7% (2/3) | 66.7% (2/3) | 66.7% (2/3) | | 57 | 66.7% (2/3) | 33.3% (1/3) | 66.7% (2/3) | 66.7% (2/3) | 66.7% (2/3) | | 58 | 66.7% (2/3) | 66.7% (2/3) | 66.7% (2/3) | 66.7% (2/3) | 66.7% (2/3) | Note: n/N denotes the number of loss or dMMR replicates/the total number of replicates. # iii. Inter-Laboratory (External Reproducibility) Study The reproducibility of the panel (MMR IHC pharmDx Panel (Dako Omnis)) was evaluated at three external testing sites. The inter-/intra-site sample set included 2 challenging cases. MMR diagnostic status was recorded as 'Proficient' or 'Deficient'. dMMR percent agreement (dMPA), PMA P250004: FDA Summary of Safety and Effectiveness Data {28} pMMR percent agreement (pMPA), and overall percent agreement (OPA) were computed with corresponding two-sided 95% percentile bootstrap confidence intervals (Table 19). In the same study, the reproducibility of the individual biomarkers MLH1 IHC pharmDx (Dako Omnis), MSH2 IHC pharmDx (Dako Omnis), MSH6 IHC pharmDx (Dako Omnis), and PMS2 IHC pharmDx (Dako Omnis) were analyzed. Individual MMR antibody status was recorded as 'Intact' or 'Loss'. Mismatch Repair NCRs (mouse and rabbit) were used in the generation of the reproducibility study and did not show staining on any of the tissues. LPA, IPA, and OPA were computed with corresponding two-sided 95% percentile bootstrap confidence intervals for each of the individual MMR antibodies. No acceptance criteria were applied to the statistical analysis of each individual MMR antibody. The Wilson score limits were used to calculate confidence intervals for agreement parameters with point estimates equal to 100%. Table 19. External Reproducibility of MMR IHC Panel pharmDx (Dako Omnis) | Reproducibility Study | Study Design | % Agreement (95% CI) | | --- | --- | --- | | Inter-site | Each of 32 CRC specimens (16 dMMR, 16 pMMR) was tested on 5 nonconsecutive days at each of 3 study sites. Inter-site analysis was performed between 3 sites on a total of 286 comparisons to consensus. | dMPA 98.6 (95.7, 100.0) pMPA 99.3 (97.9, 100.0) OPA 99.0 (97.2, 100.0) | | Intra-site | Each of 32 CRC specimens (16 dMMR, 16 pMMR) was tested on 5 nonconsecutive days at each of 3 study sites. Intra-site analysis was performed for 3 sites on a total of 286 comparisons to consensus. | dMPA 100.0 (97.3, 100.0) pMPA 99.3 (97.9, 100.0) OPA 99.7 (98.9, 100.0) | | Inter-observer | One set of 60 stained specimens (30 dMMR, 30 pMMR) was rotated across 3 sites and evaluated 3 times by the same pathologist at each site. Inter-observer analysis was performed between 3 sites on a total of 540 comparisons to consensus. | dMPA 99.6 (98.9, 100.0) pMPA 100.0 (98.6, 100.0) OPA 99.8 (99.4, 100.0) | PMA P250004: FDA Summary of Safety and Effectiveness Data 29 of 77 {29} | Reproducibility Study | Study Design | % Agreement (95% CI) | | --- | --- | --- | | Intra-observer | One set of 60 stained specimens (30 dMMR, 30 pMMR) was rotated across 3 sites and evaluated 3 times by the same pathologist at each site. Intra-observer analysis was performed for 3 sites on a total of 540 comparisons to consensus. | dMPA 99.6 (98.9, 100.0) pMPA 100.0 (98.6, 100.0) OPA 99.8 (99.4, 100.0) | dMPA=dMMR Percent Agreement; pMPA=pMMR Percent Agreement; OPA=Overall Percent Agreement Table 20. Panel-level Per Site Percent dMMR Results Per Sample in External Reproducibility of MMR IHC Panel pharmDx (Dako Omnis) | Case ID | All Sites Percent dMMR Results, % (n/N) | Site 1 Percent dMMR Results, % (n/N) | Site 2 Percent dMMR Results, % (n/N) | Site 3 Percent dMMR Results, % (n/N) | | --- | --- | --- | --- | --- | | 1 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 2 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 3 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 4 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 5 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 6 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 7 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 8 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 9 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 10 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 11 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 12 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 13 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 14 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 15 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 16 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 17 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 18 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 19 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 20 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 21 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 22 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 23 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 24 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | PMA P250004: FDA Summary of Safety and Effectiveness Data {30} | Case ID | All Sites Percent dMMR Results, % (n/N) | Site 1 Percent dMMR Results, % (n/N) | Site 2 Percent dMMR Results, % (n/N) | Site 3 Percent dMMR Results, % (n/N) | | --- | --- | --- | --- | --- | | 25 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 26 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 27 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 28 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 29 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 30 | 77.8% (7/9) | 100% (3/3) | 33.3% (1/3) | 100% (3/3) | | 31 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 32 | 11.1% (1/9) | 0% (0/3) | 33.3% (1/3) | 0% (0/3) | Note: n/N denotes the number of dMMR replicates/the total number of replicates. Table 21. Panel-level Per Reader Percent dMMR Results Per Sample in External Reproducibility of MMR IHC Panel pharmDx (Dako Omnis) | Case ID | All Readers Percent dMMR Results, % (n/N) | Reader 1 Percent dMMR Results, % (n/N) | Reader 2 Percent dMMR Results, % (n/N) | Reader 3 Percent dMMR Results, % (n/N) | | --- | --- | --- | --- | --- | | 1 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 2 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 3 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 4 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 5 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 6 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 7 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 8 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 9 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 10 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 11 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 12 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 13 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 14 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 15 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 16 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 17 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 18 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | PMA P250004: FDA Summary of Safety and Effectiveness Data {31} | Case ID | All Readers Percent dMMR Results, % (n/N) | Reader 1 Percent dMMR Results, % (n/N) | Reader 2 Percent dMMR Results, % (n/N) | Reader 3 Percent dMMR Results, % (n/N) | | --- | --- | --- | --- | --- | | 19 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 20 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 21 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 22 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 23 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 24 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 25 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 26 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 27 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 28 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 29 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 30 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 31 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 32 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 33 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 34 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 35 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 36 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 37 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 38 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 39 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 40 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 41 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 42 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 43 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 44 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 45 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 46 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 47 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 48 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 49 | 88.9% (8/9) | 66.7% (2/3) | 100% (3/3) | 100% (3/3) | | 50 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 51 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 52 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 53 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | PMA P250004: FDA Summary of Safety and Effectiveness Data 32 of 77 {32} | Case ID | All Readers Percent dMMR Results, % (n/N) | Reader 1 Percent dMMR Results, % (n/N) | Reader 2 Percent dMMR Results, % (n/N) | Reader 3 Percent dMMR Results, % (n/N) | | --- | --- | --- | --- | --- | | 54 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 55 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 56 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 57 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 58 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 59 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 60 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | Note: n/N denotes the number of loss replicates/the total number of replicates. Table 22. External Reproducibility of MLH1 | Reproducibility Study | Study Design | % Agreement (95% CI) | | --- | --- | --- | | Inter-site | Each of 32 CRC specimens (8 loss, 24 intact) was tested on 5 nonconsecutive days at each of 3 study sites. Inter-site analysis was performed between 3 sites on a total of 286 comparisons to consensus. | LPA 98.6 (95.8, 100.0) IPA 99.5 (98.6, 100.0) OPA 99.3 (98.3, 100.0) | | Intra-site | Each of 32 CRC specimens (8 loss, 24 intact) was tested on 5 nonconsecutive days at each of 3 study sites. Intra-site analysis was performed for 3 sites on a total of 286 comparisons to consensus. | LPA 98.6 (95.8, 100.0) IPA 99.5 (98.6, 100.0) OPA 99.3 (98.3, 100.0) | | Inter-observer | One set of 60 stained specimens (18 loss, 42 intact) was rotated across 3 sites and evaluated 3 times by the same pathologist at each site. Inter-observer analysis was performed between 3 sites on a total of 540 comparisons to consensus. | LPA 100.0 (97.7, 100.0) IPA 100.0 (99.0, 100.0) OPA 100.0 (99.3, 100.0) | | Intra-observer | One set of 60 stained specimens (18 loss, 42 intact) was rotated across 3 sites and evaluated 3 times by the same pathologist at each site. Intra-observer analysis was performed for 3 sites on a total of 540 comparisons to consensus. | LPA 100.0 (97.7, 100.0) IPA 100.0 (99.0, 100.0) OPA 100.0 (99.3, 100.0) | LPA=Percent Agreement of Loss; IPA=Percent Agreement of Intact; OPA=Overall Percent Agreement PMA P250004: FDA Summary of Safety and Effectiveness Data {33} Table 23. Biomarker-level Per Site Percent Loss Results Per Sample in External Reproducibility of MLH1 | Case ID | All Sites Percent Loss Results, % (n/N) | Site 1 Percent Loss Results, % (n/N) | Site 2 Percent Loss Results, % (n/N) | Site 3 Percent Loss Results, % (n/N) | | --- | --- | --- | --- | --- | | 1 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 2 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 3 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 4 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 5 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 6 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 7 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 8 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 9 | 88.9% (8/9) | 100% (3/3) | 100% (3/3) | 66.7% (2/3) | | 10 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 11 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 12 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 13 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 14 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 15 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 16 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 17 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 18 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 19 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 20 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 21 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 22 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 23 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 24 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 25 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 26 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 27 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 28 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 29 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 30 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 31 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 32 | 11.1% (1/9) | 0% (0/3) | 33.3% (1/3) | 0% (0/3) | Note: n/N denotes the number of loss replicates/the total number of replicates. PMA P250004: FDA Summary of Safety and Effectiveness Data {34} Table 24. Biomarker-level Per Reader Percent Loss Results Per Sample in External Reproducibility of MLH1 | Case ID | All Readers Percent Loss Results, % (n/N) | Reader 1 Percent Loss Results, % (n/N) | Reader 2 Percent Loss Results, % (n/N) | Reader 3 Percent Loss Results, % (n/N) | | --- | --- | --- | --- | --- | | 1 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 2 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 3 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 4 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 5 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 6 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 7 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 8 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 9 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 10 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 11 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 12 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 13 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 14 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 15 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 16 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 17 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 18 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 19 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 20 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 21 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 22 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 23 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 24 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 25 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 26 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 27 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 28 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 29 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 30 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 31 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 32 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 33 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | PMA P250004: FDA Summary of Safety and Effectiveness Data 35 of 77 {35} | Case ID | All Readers Percent Loss Results, % (n/N) | Reader 1 Percent Loss Results, % (n/N) | Reader 2 Percent Loss Results, % (n/N) | Reader 3 Percent Loss Results, % (n/N) | | --- | --- | --- | --- | --- | | 34 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 35 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 36 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 37 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 38 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 39 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 40 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 41 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 42 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 43 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 44 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 45 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 46 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 47 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 48 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 49 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 50 | 100% (9/9) | 100% (3/3) | 100% (3/3) | 100% (3/3) | | 51 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 52 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 53 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 54 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 55 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 56 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 57 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 58 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 59 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | | 60 | 0% (0/9) | 0% (0/3) | 0% (0/3) | 0% (0/3) | Note: n/N denotes the number of loss replicates/the total number of replicates. PMA P250004: FDA Summary of Safety and Effectiveness Data 36 of 77 {36} Table 25. External Reproducibility of MSH2 | Reproducibility Study | Study Design | % Agreement (95% CI) | | --- | --- | --- | | Inter-site | Each of 32 CRC specimens (7 loss, 25 intact) was tested on 5 nonconsecutive days at each of 3 study sites. Inter-site analysis was performed between 3 sites on a total of 286 comparisons to consensus. | LPA 96.8 (90.0, 100.0) IPA 99.1 (97.8, 100.0) OPA 98.6 (96.8, 100.0) | | Intra-site | Each of 32 CRC specimens (7 loss, 25 intact) was tested on 5 nonconsecutive days at each of 3 study sites. Intra-site analysis was performed for 3 sites on a total of 286 comparisons to consensus. | LPA 100.0 (94.0, 100.0) IPA 99.1 (97.8, 100.0) OPA 99.3 (98.3, 100.0) | | Inter-observer | One set of 60 stained specimens (10 loss, 50 intact) was rotated across 3 sites and evaluated 3 times by the same pathologist at each site. Inter-observer analysis was performed between 3 sites on a total of 540 comparisons to consensus. | LPA 100.0 (95.9, 100.0) IPA 100.0 (99.2, 100.0) OPA 100.0 (99.3, 100.0) | | Intra-observer | One set o…