PD-L1 IHC 22C3 pharmDx

{0} # SUMMARY OF SAFETY AND EFFECTIVENESS DATA (SSED) ## I. GENERAL INFORMATION | Device Generic Name: | In vitro diagnostic immunohistochemistry (IHC) test for detection of PD-L1 in formalin-fixed, paraffin-embedded (FFPE) human tissue sections | | --- | --- | | Device Trade Name: | PD-L1 IHC 22C3 pharmDx | | Device Procode: | PLS | | Applicant's Name and Address: | Agilent Technologies, Inc. 5301 Stevens Creek Blvd. Carpinteria, CA 95051 | | Date(s) of Panel Recommendation: | None | | Premarket Approval Application (PMA) Number: | P150013/S033 | | Date of FDA Notice of Approval: | February 10, 2026 | The original PD-L1 IHC 22C3 pharmDx PMA (P150013) was approved on October 2, 2015, for the qualitative detection of PD-L1 protein in formalin-fixed, paraffin-embedded (FFPE) non-small cell lung cancer (NSCLC) tissue using EnVision FLEX visualization system on the Autostainer Link 48. PD-L1 protein expression is determined by using Tumor Proportion Score (TPS), which is the percentage of viable tumor cells showing partial or complete membrane staining. The indication for use of the device as approved in P150013 is to aid in identifying patients with NSCLC whose tumors are considered PD-L1 positive if $TPS \geq 50\%$ and for whom safety and efficacy of KEYTRUDA® (pembrolizumab) have been established. The SSEDs to support the previously approved indications are available on the CDRH website and are incorporated by reference here. The current supplement was submitted to expand the indication for the PD-L1 IHC 22C3 pharmDx to include patients with epithelial ovarian, fallopian tube, or primary peritoneal carcinoma (EOC) for treatment with KEYTRUDA® (pembrolizumab). PD-L1 protein expression is determined by using Combined Positive Score (CPS), which is the number of PD-L1 staining cells (tumor cells, lymphocytes, macrophages) divided by the total number of viable tumor cells, multiplied by 100. The specimen should be considered to have PD-L1 expression if $CPS \geq 1$. ---PMA P150013/S033: FDA Summary of Safety and Effectiveness Data 1 of 34 {1} ## II. INDICATIONS FOR USE For in vitro diagnostic use. PD-L1 IHC 22C3 pharmDx is a qualitative immunohistochemical assay using monoclonal mouse anti-PD-L1, Clone 22C3 intended for use in the detection of PD-L1 protein in formalin-fixed, paraffin embedded (FFPE) non-small cell lung cancer (NSCLC), esophageal squamous cell carcinoma (ESCC), cervical cancer, head and neck squamous cell carcinoma (HNSCC), triple-negative breast cancer (TNBC), gastric or gastroesophageal junction (GEJ) adenocarcinoma, and epithelial ovarian, fallopian tube, or primary peritoneal carcinoma (EOC) tissues using EnVision FLEX visualization system on Autostainer Link 48. PD-L1 protein expression in NSCLC is determined by using Tumor Proportion Score (TPS), which is the percentage of viable tumor cells showing partial or complete membrane staining at any intensity. PD-L1 protein expression in ESCC, cervical cancer, HNSCC, TNBC, gastric or GEJ adenocarcinoma, and EOC is determined by using Combined Positive Score (CPS), which is the number of PD-L1 staining cells (tumor cells, lymphocytes, macrophages) divided by the total number of viable tumor cells, multiplied by 100. PD-L1 IHC 22C3 pharmDx is indicated as an aid in identifying patients for treatment with the therapies for the indications listed in Table 1. Table 1. PD-L1 IHC 22C3 pharmDx companion diagnostic indications, PD-L1 expression levels, and therapies | Tumor Indication | PD-L1 Expression Level | Therapy | | --- | --- | --- | | NSCLC | TPS ≥ 1% | KEYTRUDA® (pembrolizumab)* | | ESCC | CPS ≥ 10 | | | Cervical Cancer | CPS ≥ 1 | | | HNSCC | CPS ≥ 1 | | | TNBC | CPS ≥ 10 | | | Gastric or GEJ adenocarcinoma | CPS ≥ 1 | | | Epithelial ovarian, fallopian tube, or primary peritoneal carcinoma (EOC) | CPS ≥ 1 | | | NSCLC | TPS ≥ 50% | LIBTAYO® (cemiplimab)** | *See the KEYTRUDA® product label for specific clinical circumstances guiding PD-L1 testing. **See the LIBTAYO® product label for specific clinical circumstances guiding PD-L1 testing. PMA P150013/S033: FDA Summary of Safety and Effectiveness Data 2 of 34 {2} ### III. CONTRAINDICATIONS There are no known contraindications. ### IV. WARNINGS AND PRECAUTIONS The warnings and precautions can be found in the PD-L1 IHC 22C3 pharmDx labeling. ### V. DEVICE DESCRIPTION #### Device Kit Components PD-L1 IHC 22C3 pharmDx contains optimized reagents required to complete an immunohistochemical (IHC) staining procedure for formalin-fixed and paraffin-embedded (FFPE) specimens using the EnVision FLEX visualization system and the Dako Autostainer Link 48 with DakoLink software version 4.2. The principal component of the kit is the mouse monoclonal anti PD-L1 clone 22C3 antibody that binds to PD-L1 protein expressed on FFPE tissue. Each kit includes 19.5 mL of PD-L1 primary antibody (approximately 3μg/mL protein concentration) and reagents shown in Table 2 necessary to perform 50 tests in up to 15 individual runs. Wash buffer and hematoxylin are required for the assay but not included in the kit. PT Link Pre-Treatment Module is required for deparaffinization, rehydration, and target retrieval of the tissues. Cover-slipping is required but can be performed by either manual or automated methods. Device kit components are shown in Table 2 below. Table 2: Device Kit Components | Reagent | Description | Qty x Vol | | --- | --- | --- | | Peroxidase-Blocking Reagent | Buffered solution containing hydrogen peroxide, detergent and 0.015 mol/L sodium azide. | 1 x 34.5 mL | | Monoclonal Mouse anti-PD-L1, Clone 22C3 | Monoclonal mouse anti-PD-L1 antibody in a buffered solution, containing stabilizing protein (3 μg/mL protein concentration), and 0.015 mol/L sodium azide. | 1 x 19.5 mL | | Negative Control Reagent | Monoclonal mouse control IgG antibody in a buffered solution, containing stabilizing protein, and 0.015 mol/L sodium azide. | 1 x 15 mL | | Linker, Anti-Mouse | Rabbit secondary antibody against mouse immunoglobulins in a buffered solution containing stabilizing protein and 0.015 mol/L sodium azide. | 1x 34.5 mL | | Visualization Reagent-Horseradish Peroxidase (HRP) | Dextran coupled with peroxidase molecules and goat secondary antibody molecules against rabbit and mouse immunoglobulins in a buffered solution containing stabilizing protein and an antimicrobial agent. | 1 x 34.5 mL | | DAB+ Buffered Substrate | Buffered solution, containing hydrogen peroxide and an antimicrobial agent. | 15 x 7.2 mL | PMA P150013/S033: FDA Summary of Safety and Effectiveness Data 3 of 34 {3} | Reagent | Description | Qty x Vol | | --- | --- | --- | | DAB+ Chromogen | 3,3'-diaminobenzidine tetrahydrochloride (DAB) in an organic solvent. | 1 x 5 mL | | DAB Enhancer | Cupric sulfate in water. | 1 x 34.5 mL | | Target Retrieval Solution Low pH (50X) | Buffered solution, pH 6.1, containing detergent and an antimicrobial agent. | 6 x 30 mL | | Cell Line Control Slides | Each slide contains sections of two pelleted, formalin-fixed paraffin-embedded cell lines: NCI-H226 with moderate PD-L1 protein expression (positive control), and MCF-7 with negative PD-L1 protein expression (negative control). | 15 slides | PD-L1 IHC 22C3 pharmDx comes with individually labeled reagent components that are recognized together. The kit components can only be used on the specified instrument with the PD-L1 IHC 22C3 pharmDx protocol. The DakoLink software has been designed to recognize and group PD-L1 IHC 22C3 pharmDx reagents, mandating that the reagents are run together. ### Device Instrument and Software PD-L1 IHC 22C3 pharmDx assay is performed on the Dako Autostainer Link 48 automated staining system using the DakoLink software version 4.2. The Autostainer system is designed to mimic the staining steps performed manually by a lab technician. The PD-L1 IHC 22C3 pharmDx protocol is assay specific. The DakoLink software has been designed to recognize and group PD-L1 IHC 22C3 pharmDx reagents, requiring that all system reagents are used together. Deparaffinization, rehydration, and target retrieval (3-in-1) procedures are performed in the PT Link Pre-treatment module (PT100/200 modules; deparaffinization performed separately in PT100). ### Specimen Preparation Epithelial ovarian, fallopian tube, or primary peritoneal carcinoma specimens must be handled appropriately to preserve the tissue for IHC staining. Standard methods of tissue processing should be used for all specimens. FFPE tissues are suitable for use. Fixation time for 12-72 hours in 10% neutral buffered formalin (NBF) is recommended. Alternative fixatives have not been validated and may give erroneous results. Fixation times of ≤ 3 hours may result in variable PD-L1 detection. Specimens should be blocked into a thickness of 3 or 4 mm, fixed in 10% NBF. Specimens are then processed through a tissue processor where they are dehydrated and cleared in a series of alcohols and xylene, followed by infiltration with melted paraffin. The paraffin temperature should not exceed 60 °C. After specimen processing, tissue specimens should be cut into sections of 4-5 μm using a tissue microtome, mounted on charged microscope slides. Slides are then placed in a 58 ± 2°C oven for 1 hour as an initial step for deparaffinization. To preserve antigenicity, tissue sections, once mounted on slides, should be stored in the dark at 2-8 °C (preferred), or at room temperature up to 25°C. Tissue sections should be PMA P150013/S033: FDA Summary of Safety and Effectiveness Data 4 of 34 {4} stained within 7.5 months of sectioning when stored at 2-8°C, or within 4 months when stored at 25 °C. Slide storage and handling conditions should not exceed 25°C at any point post-mounting to ensure tissue integrity and antigenicity. ## Test Controls Run controls are included in each staining run to establish the validity of the test results. In the device labeling, Dako recommends the following controls to be run with the assay: 1) Control cell line slides provided as part of the kit should be used to verify the staining procedure. One Control Slide should be stained with the primary antibody to PD-L1 in each staining run. Each slide contains sections of two pelleted, FFPE cell lines: one with moderate PD-L1 protein expression and one that is negative for PD-L1 protein expression. The evaluation of the Control Slide supplied in the kit indicates the validity of the staining run. The Control Slides should not be used as an aid in interpretation of patient results. 2) Run controls are to be provided by the end-user laboratory. Positive and negative run controls should be fresh biopsy/surgical specimens of the same tumor indication as the patient specimen, fixed, processed and embedded as soon as possible in the same manner as the patient sample(s). The positive control tissue should include weak to moderate positive staining for PD-L1 to detect subtle changes in assay sensitivity. Negative control tissue is required to detect unintended antibody cross reactivity to tissue and is expected to be negative for PD-L1 expression. 3) The kit includes a Negative Control Reagent that is used in parallel with the PD-L1 clone 22C3 primary antibody on patient tissue. The matched negative control aids the reader in differentiating a true signal from tissue-specific background staining that may occur from reaction with detection chemistry and not the anti PD-L1 primary antibody. Additional information about the use of controls is available in the product labeling. ## Principle of Procedure PD-L1 IHC 22C3 pharmDx contains optimized reagents required to complete an IHC staining procedure on FFPE specimens using the Autostainer Link 48. Following deparaffinization of the tissue sections, rehydration and target retrieval, the slides are incubated with the primary monoclonal antibody to PD-L1 (clone 22C3) or the Negative Control Reagent. The slides are then incubated with an anti-mouse Linker antibody, which is specific to the host species of the primary antibody. Following this, the slides are incubated with a ready-to-use Visualization Reagent consisting of secondary antibody molecules and HRP molecules coupled to a dextran polymer backbone. The enzymatic conversion of the subsequently added DAB+ Chromogen results in precipitation of a visible reaction product at the antigen sites. The color of the chromogenic reaction is modified by a chromogen enhancement reagent, DAB Enhancer. The specimen is then counterstained with hematoxylin and cover slipped. PMA P150013/S033: FDA Summary of Safety and Effectiveness Data 5 of 34 {5} # **Staining Protocol** PD-L1 IHC 22C3 pharmDx is designed to be run on the Autostainer Link 48 with DakoLink software. The staining protocol on the Autostainer Link 48 is as follows: | Peroxidase-Blocking Reagent (2 drop zones x150μL): | 5 minutes (± 1 minute) | | --- | --- | | Rinse in buffer | | | Monoclonal Mouse anti-PD-L1 (or Negative Control Reagent) (2 drop zones x150μL): | 30 minutes (± 1 minute) | | Rinse in buffer | | | Linker, anti-Mouse Ig (2 drop zones x150μL): | 30 minutes (± 1 minute) | | Rinse in buffer | | | Visualization Reagent (2 drop zones x150μL): | 30 minutes (± 1 minute) | | Rinse in buffer: | 5 minutes | | DAB+ solution (2 drop zones x150μL): | 2 x 5 minutes (± 1 minute) | | Rinse in buffer | | | DAB+ Enhancer (2 drop zones x150μL): | 5 minutes (± 1 minute) | | Rinse in buffer | | | Hematoxylin (2x150μL): | 5 minutes (± 1 minute) | | Rinse in deionized water | | | Rinse in buffer: | 5 minutes | | Rinse in deionized water | | Remove slides from autostainer and place in bath of reagent water. Slides may then be permanently mounted using non-aqueous, permanent mounting medium. # **Interpretation of PD-L1 Staining** Interpretation of stained slides should be performed by a pathologist using a light microscope with an objective of 20x magnification. All viable tumor cells on the entire slide must be evaluated and included in the PD-L1 scoring assessment together with tumor associated PD-L1 positive lymphocytes and macrophages. A minimum of 100 viable tumor cells must be present in the PD-L1 stained slide for the specimen to be considered adequate for evaluation. For specimens with less than 100 viable tumor cells, tissue from a deeper level of the block or potentially another block, could present sufficient tumor cells for PD-L1 evaluation. PD-L1 expression in epithelial ovarian, fallopian tube, or primary peritoneal carcinoma is determined by CPS, which is the number of PD-L1 staining cells (tumor cells, lymphocytes, macrophages) divided by the total number of viable tumor cells, multiplied by 100. Distinction of viable tumor cells, lymphocytes, and macrophages is essential for accurate denominator estimation. Although the result of the calculation can exceed 100, the maximum score is defined as CPS 100. CPS is defined as follows: PMA P150013/S033: FDA Summary of Safety and Effectiveness Data 6 of 34 {6} $$\mathrm{CPS} = \frac {\# \mathrm{PD-L1~staining~cells~(tumor~cells,~lymphocytes,~macrophages)}}{\mathrm{Total~\#~of~viable~tumor~cells}} \times 100$$ By definition, PD-L1 staining cells are: - Tumor cells with convincing partial or complete linear membrane staining (at any intensity) that is perceived distinct from cytoplasmic staining and - Lymphocytes and macrophages (mononuclear inflammatory cells, MICs) within the tumor nests and/or adjacent supporting stroma with convincing membrane and/or cytoplasmic staining (at any intensity). MICs must be directly associated with the response against the tumor. For each staining run, slides should be examined in the order recommended in the labeling to determine the validity of the staining run and enable assessment of the staining of the sample tissue. Patient specimens stained with PD-L1 and the negative control reagent (NCR) from PD-L1 IHC 22C3 pharmDx should be examined when evaluating PD-L1 expression. Specimens stained with NCR must have 0 specific staining and ≤ 1+ nonspecific staining. Due to the histological characteristics of epithelial ovarian, fallopian tube and primary peritoneal carcinoma they are grouped together as epithelial ovarian cancer (EOC) in this document. The tables below provide details about which tissue elements are included in and excluded from the CPS numerator and denominator in EOC. Table 3. CPS Numerator Inclusion/Exclusion Criteria for EOC | Tissue Elements | Included in the Numerator | Excluded from the Numerator | | --- | --- | --- | | **Tumor cells** | • Convincing partial or complete linear membrane staining (at any intensity) of viable invasive tumor cells | • Nonstaining tumor cells • Tumor cells with only cytoplasmic staining • Noninvasive neoplasia (Serous tubal intraepithelial carcinoma) | | **Immune cells** | • Membrane and/or cytoplasmic* staining (at any intensity) of mononuclear inflammatory cells (MICs) within tumor nests and adjacent supporting stroma**: • Lymphocytes (including lymphocyte aggregates) • Macrophages*** (including intraluminal and intracystic regions) • Only MICs directly associated with the response to the tumor are scored. | • Nonstaining MICs • MICs (including lymphoid aggregates) not directly associated with tumor • MICs associated with noninvasive neoplasia (Serous tubal intraepithelial carcinoma) • MICs associated with benign structures • Neutrophils, eosinophils, and plasma cells | PMA P150013/S033: FDA Summary of Safety and Effectiveness Data 7 of 34 {7} | **Other Cells** | - Not Included | - Benign epithelial cells - Stromal cells (fibroblasts, endothelial cells) - Necrotic cells and/or cellular debris, cystic fluid | | --- | --- | --- | *In MICs, membrane and cytoplasmic staining are often indistinguishable due to high nuclear to cytoplasmic ratio. Therefore, membrane and/or cytoplasmic staining of MICs is included in the score. **Adjacent MICs are defined as being within the same 20x field as the tumor. However, MICs that are NOT directly associated with the response to the tumor should be excluded. ***Macrophages and histiocytes are considered the same cells. **Table 4. CPS Denominator Inclusion/Exclusion Criteria for EOC** | **Tissue Elements** | **Included in the Denominator** | **Excluded from the Denominator** | | --- | --- | --- | | **Tumor Cells** | All viable invasive tumor cells | - Nonviable tumor cells - Noninvasive neoplasia (Serous tubal intraepithelial carcinoma) | | **Immune Cells** | Not included | - All immune cells | | **Other Cells** | Not included | - Benign epithelial cells - Stromal cells (fibroblasts) - Necrotic cells and/or cellular debris | The specimen should be considered to have PD-L1 expression if CPS ≥ 1. ## **VI. ALTERNATIVE PRACTICES AND PROCEDURES** There is currently no alternative FDA-cleared or approved immunohistochemistry assay available for use as an aid in identifying patients with PD-L1 expression in EOC tissues for treatment with KEYTRUDA® (pembrolizumab). ## **VII. MARKETING HISTORY** PD-L1 IHC 22C3 pharmDx has been marketed in the United States since approval of P150013 on October 2, 2015. PD-L1 IHC 22C3 pharmDx has also been marketed in Albania, Algeria, Argentina, Australia, Austria, Azerbaijan, Belgium, Bahrain, Bosnia-Herzegovina, Botswana, Brazil, Brunei, Bulgaria, Canada, Chile, China, Colombia, Costa Rica, Croatia, Cyprus, Czech Republic, Denmark, Ecuador, Egypt, Estonia, Ethiopia, Finland, France, Germany, Greece, Hong Kong, Hungary, Iceland, India, Indonesia, Iraq, Ireland, Israel, Italy, Japan, Jordan, Kazakhstan, Kosovo, Kuwait, Latvia, Lebanon, Liechtenstein, Lithuania, Luxembourg, Macau, Macedonia, Malaysia, Malta, Mexico, Montenegro, Morocco, Netherlands, New Zealand, Norway, Oman, Panama, Paraguay, Peru, Philippines, Poland, Portugal, Puerto Rico, Qatar, Romania, Russia, Saudi Arabia, Serbia, Singapore, Slovakia, Slovenia, South Africa, South Korea, Spain, Sweden, Switzerland, Taiwan, Thailand, Turkey, Ukraine, United Arab Emirates, United Kingdom, Uruguay and Vietnam. PMA P150013/S033: FDA Summary of Safety and Effectiveness Data 8 of 34 {8} ## **VIII. POTENTIAL ADVERSE EFFECTS OF THE DEVICE ON HEALTH** Failure of the device to perform as expected or failure to correctly interpret test results may lead to incorrect PD-L1 test results and subsequently improper assignment of treatment with KEYTRUDA®. Patients with a false negative assay result may not be considered for treatment with KEYTRUDA (pembrolizumab). Patients with a false positive assay result may receive treatment with KEYTRUDA (pembrolizumab) for which there is no expectation of benefit and exposure to potential toxicity. There is also a risk of delayed results, which may lead to delay in treatment. For the specific adverse events that occurred in the clinical study, please see the KEYTRUDA® (pembrolizumab) FDA approved package insert which is available at Drugs@FDA. ## **IX. SUMMARY OF NONCLINICAL STUDIES** ### **A. Laboratory Studies** Non-clinical studies were performed using PD-L1 IHC 22C3 pharmDx to establish analytical performance of the device. Antibody characterization studies for clone 22C3, including specificity and tour of body/ tour of tumor, control cell line validation, kit stability and preanalytical variables were submitted and reviewed in the original PMA (P150013) for this device. There have been no changes to the device design including reagent formulation or kit configuration since approval of the original PMA (P150013). Results from studies performed to support the ovarian carcinoma indication at CPS ≥ 1 are summarized in the sections below. #### **1. Analytical Sensitivity** Analytical sensitivity was assessed by calculating prevalence of PD-L1 positivity in participants with platinum-resistant EOC from the KEYNOTE-B96 study. A total of 643 participants were randomly assigned in a 1:1 ratio to pembrolizumab + chemotherapy (322 participants) or placebo + chemotherapy (321 participants). All 643 participants had a tumor tissue sample tested with PD-L1 IHC 22C3 pharmDx. Most participants (72.5%, n=466) had a tumor tissue PD-L1 expression score of CPS ≥1, and 27.5% of participants had a tumor tissue PD-L1 expression score of CPS < 1. While 201 of 466 (31.3%) participants with CPS ≥1 had a tumor tissue PD-L1 expression score of CPS ≥10. #### **2. Analytical Specificity** ##### **a. Western Blot** See Summary of Safety and Effectiveness Data for P150013. ##### **b. Immunoreactivity in Human Tissues** See Summary of Safety and Effectiveness Data for P150013. ---PMA P150013/S033: FDA Summary of Safety and Effectiveness Data 9 of 34 {9} ### 3. Precision Precision of the PD-L1 IHC 22C3 pharmDx was evaluated at Agilent Technologies, Inc. to demonstrate that the assay produces consistent results in normal day-to-day testing of FFPE EOC specimens. Testing included repeatability, combined precision, and intra-/inter-reader precision: - Repeatability was measured within a run - Combined precision was measured between Autostainer Link 48 instruments, operators, testing days, and lots - Precision was measured within and between readers Intermediate Precision and Repeatability Studies (Combined Precision): Thirty-eight EOC FFPE tissue blocks, each representing a unique study sample (19 PD-L1 positive and 19 PD-L1 negative, including 13 near cutoff samples at CPS ≥1) that encompassed PD-L1 IHC staining status range of positive and negative were used in this study. This study was performed using the following design factors on Autostainer Link 48 instruments: - Three kit lots of PD-L1 IHC 22C3 pharmDx - Three operators - Three Autostainer Link 48 instruments - Across three days - One pathologist reader - 3 replicates per condition - Across all intermediate precision conditions (within-staining run) All slides were blinded and randomized and evaluated using the PD-L1 IHC 22C3 pharmDx scoring algorithm. Each sample had 3 results and a majority PD-L1 status was assigned based on 3 results. A total of 38 unique test samples were included for assessment at the CPS ≥1 cutoff. For each sample, median CPS and range of CPS of 3 results were calculated. In addition, percent positive (CPS ≥1, "Positive" with regard to PD-L1 therapy) results were calculated. Results are presented in Table 5, below: Table 5. Per-Specimen Agreement Results: Combined Precision: Results per Specimen (Days) | Specimen | Consensus* Result | Number of Results | Median (CPS) | Range (CPS) | Between Day (SD) | Percent Positive (n/N) ** | Percent Agreement with Consensus (n/N) | 95% CI*** | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 1 | Negative | 3 | 0 | 0, 0 | 0.000 | 0% (0/3) | 100% (3/3) | 43.9-100% | | 2 | Negative | 3 | 0 | 0, 0 | 0.000 | 0% (0/3) | 100% (3/3) | 43.9-100% | | 3 | Negative | 3 | 0 | 0, 0 | 0.000 | 0% (0/3) | 100% (3/3) | 43.9-100% | | 4 | Negative | 3 | 0 | 0, 0 | 0.000 | 0% (0/3) | 100% (3/3) | 43.9-100% | | 5 | Negative | 3 | 0 | 0, 0 | 0.000 | 0% (0/3) | 100% (3/3) | 43.9-100% | | 6 | Negative | 3 | 0 | 0, <1 | 0.289 | 0% (0/3) | 100% (3/3) | 43.9-100% | PMA P150013/S033: FDA Summary of Safety and Effectiveness Data 10 of 34 {10} | Specimen | Consensus* Result | Number of Results | Median (CPS) | Range (CPS) | Between Day (SD) | Percent Positive (n/N) ** | Percent Agreement with Consensus (n/N) | 95% CI*** | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 7 | Negative | 3 | 0 | 0, <1 | 0.289 | 0% (0/3) | 100% (3/3) | 43.9-100% | | 8 | Negative | 3 | 0 | 0, 0 | 0.000 | 0% (0/3) | 100% (3/3) | 43.9-100% | | 9 | Negative | 3 | 0 | 0, <1 | 0.289 | 0% (0/3) | 100% (3/3) | 43.9-100% | | 10 | Negative | 3 | 0 | 0, <1 | 0.289 | 0% (0/3) | 100% (3/3) | 43.9-100% | | 11 | Negative | 3 | 0 | 0, <1 | 0.289 | 0% (0/3) | 100% (3/3) | 43.9-100% | | 12 | Negative | 3 | 0.5 | 0, <1 | 0.289 | 0% (0/3) | 100% (3/3) | 43.9-100% | | 13 | Negative | 3 | 0.5 | 0, <1 | 0.289 | 0% (0/3) | 100% (3/3) | 43.9-100% | | 14 | Negative | 3 | 0.5 | 0, <1 | 0.289 | 0% (0/3) | 100% (3/3) | 43.9-100% | | 15 | Negative | 3 | 0.5 | 0, <1 | 0.289 | 0% (0/3) | 100% (3/3) | 43.9-100% | | 16 | Negative | 3 | 0.5 | <1, <1 | 0.000 | 0% (0/3) | 100% (3/3) | 43.9-100% | | 17 | Negative | 3 | 0.5 | <1, <1 | 0.000 | 0% (0/3) | 100% (3/3) | 43.9-100% | | 18 | Negative | 3 | 0.5 | <1, <1 | 0.000 | 0% (0/3) | 100% (3/3) | 43.9-100% | | 19 | Negative | 3 | 0.5 | <1, 1 | 0.289 | 33.3% (1/3) | 66.7% (2/3) | 20.8-93.9% | | 20 | Positive | 3 | 1 | 1, 1 | 0.000 | 100% (3/3) | 100% (3/3) | 43.9-100% | | 21 | Positive | 3 | 4 | 3, 8 | 2.646 | 100% (3/3) | 100% (3/3) | 43.9-100% | | 22 | Positive | 3 | 7 | 3, 9 | 3.055 | 100% (3/3) | 100% (3/3) | 43.9-100% | | 23 | Positive | 3 | 8 | 4, 13 | 4.509 | 100% (3/3) | 100% (3/3) | 43.9-100% | | 24 | Positive | 3 | 10 | 8, 20 | 6.429 | 100% (3/3) | 100% (3/3) | 43.9-100% | | 25 | Positive | 3 | 12 | 12, 25 | 7.506 | 100% (3/3) | 100% (3/3) | 43.9-100% | | 26 | Positive | 3 | 13 | 10, 30 | 10.786 | 100% (3/3) | 100% (3/3) | 43.9-100% | | 27 | Positive | 3 | 15 | 5, 30 | 12.583 | 100% (3/3) | 100% (3/3) | 43.9-100% | | 28 | Positive | 3 | 15 | 7, 20 | 6.557 | 100% (3/3) | 100% (3/3) | 43.9-100% | | 29 | Positive | 3 | 20 | 15, 30 | 7.638 | 100% (3/3) | 100% (3/3) | 43.9-100% | | 30 | Positive | 3 | 20 | 20, 30 | 5.774 | 100% (3/3) | 100% (3/3) | 43.9-100% | | 31 | Positive | 3 | 25 | 18, 30 | 6.028 | 100% (3/3) | 100% (3/3) | 43.9-100% | | 32 | Positive | 3 | 25 | 18, 25 | 4.041 | 100% (3/3) | 100% (3/3) | 43.9-100% | | 33 | Positive | 3 | 25 | 20, 35 | 7.638 | 100% (3/3) | 100% (3/3) | 43.9-100% | | 34 | Positive | 3 | 25 | 20, 25 | 2.887 | 100% (3/3) | 100% (3/3) | 43.9-100% | | 35 | Positive | 3 | 30 | 15, 45 | 15.000 | 100% (3/3) | 100% (3/3) | 43.9-100% | | 36 | Positive | 3 | 30 | 20, 30 | 5.774 | 100% (3/3) | 100% (3/3) | 43.9-100% | | 37 | Positive | 3 | 30 | 30, 35 | 2.887 | 100% (3/3) | 100% (3/3) | 43.9-100% | | 38 | Positive | 3 | 40 | 30, 60 | 15.275 | 100% (3/3) | 100% (3/3) | 43.9-100% | SD = Standard Deviation; CI = Confidence Interval; CPS = Combined Positive Score; n = number of results; N = Total number of results *Consensus result was determined based on the majority call across all testing sites and days **Percent positive represents the proportion of specimens scoring positive (CPS ≥ 1) out of total number of results ***95% confidence intervals were calculated using the Wilson score method PMA P150013/S033: FDA Summary of Safety and Effectiveness Data 11 of 34 {11} # Intra-Run Repeatability Five replicate sections from each EOC specimen were stained with PD-L1 IHC 22C3 pharmDx within the same run on the Autostainer Link 48. A total of 38 unique test specimens were included for assessment at the CPS ≥1 cutoff. Results are presented in Table 6, below: Table 6. Per-Specimen Agreement Results: Intra-Run Precision: Results per specimen (Replicates) | Specimen | Consensus* | Number of Results | Median (CPS) | Range (CPS) | Within Run (SD) | Percent Positive (n/N) ** | Percent Agreement with Consensus (n/N) | 95% CI*** | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 1 | Negative | 5 | 0 | 0, <1 | 0.27 | 0% (0/5) | 100% (5/5) | 56.6-100% | | 2 | Negative | 5 | 0 | 0, 0 | 0.00 | 0% (0/5) | 100% (5/5) | 56.6-100% | | 3 | Negative | 5 | 0 | 0, <1 | 0.22 | 0% (0/5) | 100% (5/5) | 56.6-100% | | 4 | Negative | 5 | 0 | 0, 0 | 0.00 | 0% (0/5) | 100% (5/5) | 56.6-100% | | 5 | Negative | 5 | 0 | 0, 0 | 0.00 | 0% (0/5) | 100% (5/5) | 56.6-100% | | 6 | Negative | 5 | 0 | 0, 0 | 0.00 | 0% (0/5) | 100% (5/5) | 56.6-100% | | 7 | Negative | 5 | 0 | 0, 0 | 0.00 | 0% (0/5) | 100% (5/5) | 56.6-100% | | 8 | Negative | 5 | 0 | 0, 0 | 0.00 | 0% (0/5) | 100% (5/5) | 56.6-100% | | 9 | Negative | 5 | 0 | 0, 0 | 0.00 | 0% (0/5) | 100% (5/5) | 56.6-100% | | 10 | Negative | 5 | 0 | 0, 0 | 0.00 | 0% (0/5) | 100% (5/5) | 56.6-100% | | 11 | Negative | 5 | 0 | 0, 0 | 0.00 | 0% (0/5) | 100% (5/5) | 56.6-100% | | 12 | Negative | 5 | 0.5 | 0, <1 | 0.27 | 0% (0/5) | 100% (5/5) | 56.6-100% | | 13 | Negative | 5 | 0.5 | 0, <1 | 0.22 | 0% (0/5) | 100% (5/5) | 56.6-100% | | 14 | Negative | 5 | 0.5 | 0, <1 | 0.27 | 0% (0/5) | 100% (5/5) | 56.6-100% | | 15 | Negative | 5 | 0.5 | <1, <1 | 0.00 | 0% (0/5) | 100% (5/5) | 56.6-100% | | 16 | Negative | 5 | 0.5 | <1, <1 | 0.00 | 0% (0/5) | 100% (5/5) | 56.6-100% | | 17 | Negative | 5 | 0.5 | <1, <1 | 0.00 | 0% (0/5) | 100% (5/5) | 56.6-100% | | 18 | Negative | 5 | 0.5 | <1, <1 | 0.00 | 0% (0/5) | 100% (5/5) | 56.6-100% | | 19 | Negative | 5 | 0.5 | <1, <1 | 0.00 | 0% (0/5) | 100% (5/5) | 56.6-100% | | 20 | Positive | 5 | 2 | 1, 4 | 1.14 | 100% (5/5) | 100% (5/5) | 56.6-100% | | 21 | Positive | 5 | 2 | 1, 4 | 1.10 | 100% (5/5) | 100% (5/5) | 56.6-100% | | 22 | Positive | 5 | 4 | 1, 8 | 2.88 | 100% (5/5) | 100% (5/5) | 56.6-100% | | 23 | Positive | 5 | 4 | 4, 5 | 0.45 | 100% (5/5) | 100% (5/5) | 56.6-100% | | 24 | Positive | 5 | 6 | 5, 9 | 1.64 | 100% (5/5) | 100% (5/5) | 56.6-100% | | 25 | Positive | 5 | 10 | 2, 30 | 11.65 | 100% (5/5) | 100% (5/5) | 56.6-100% | | 26 | Positive | 5 | 10 | 10, 10 | 0.00 | 100% (5/5) | 100% (5/5) | 56.6-100% | | 27 | Positive | 5 | 11 | 11, 11 | 0.00 | 100% (5/5) | 100% (5/5) | 56.6-100% | | 28 | Positive | 5 | 12 | 10, 22 | 5.02 | 100% (5/5) | 100% (5/5) | 56.6-100% | | 29 | Positive | 5 | 14 | 14, 18 | 1.79 | 100% (5/5) | 100% (5/5) | 56.6-100% | | 30 | Positive | 5 | 16 | 14, 22 | 3.65 | 100% (5/5) | 100% (5/5) | 56.6-100% | | 31 | Positive | 5 | 21 | 20, 31 | 4.72 | 100% (5/5) | 100% (5/5) | 56.6-100% | PMA P150013/S033: FDA Summary of Safety and Effectiveness Data 12 of 34 {12} | Specimen | Consensus* | Number of Results | Median (CPS) | Range (CPS) | Within Run (SD) | Percent Positive (n/N) ** | Percent Agreement with Consensus (n/N) | 95% CI*** | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 32 | Positive | 5 | 22 | 11, 25 | 6.34 | 100% (5/5) | 100% (5/5) | 56.6-100% | | 33 | Positive | 5 | 28 | 22, 39 | 6.32 | 100% (5/5) | 100% (5/5) | 56.6-100% | | 34 | Positive | 5 | 30 | 23, 42 | 7.12 | 100% (5/5) | 100% (5/5) | 56.6-100% | | 35 | Positive | 5 | 45 | 45, 45 | 0.00 | 100% (5/5) | 100% (5/5) | 56.6-100% | | 36 | Positive | 5 | 60 | 40, 67 | 11.28 | 100% (5/5) | 100% (5/5) | 56.6-100% | | 37 | Positive | 5 | 60 | 50, 70 | 8.94 | 100% (5/5) | 100% (5/5) | 56.6-100% | | 38 | Positive | 5 | 85 | 60, 100 | 17.10 | 100% (5/5) | 100% (5/5) | 56.6-100% | SD = Standard Deviation; CI = Confidence Interval; CPS = Combined Positive Score; n = number of results; N = Total number of results *Consensus was determined based on majority call across all test conditions **Percent positive represents the proportion of specimens scoring positive (CPS ≥ 1) out of total number of results ***95% confidence intervals were calculated using the Wilson score method #### Reader Precision Studies (Inter-Reader and Intra-Reader Precision) In the observer precision studies for PD-L1 IHC 22C3 pharmDx, Within-Reader and Between-Reader components of precision for EOC tissue reads were evaluated. The study included specimens that were stained with PD-L1 IHC 22C3 pharmDx. Specimens were blinded and randomized prior to evaluation for PD-L1 status using the PD-L1 IHC 22C3 pharmDx scoring algorithm. The study included three readers (pathologists). Readers scored all 62 specimens three times each, with a minimum of two-week wash-out period between reads. Results are presented in Table 7, below: **Table 7. Per-Specimen Agreement Results: Observer Precision: Results per Specimen (3 per reader)** | Specimen | Consensus* | Number of Results | Median (CPS) | Range (CPS) | Standard Deviation (SD) | | | Percent Agreement (n/N) ** | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | | | Inter-Read | Inter Observer | Total | Obs 1 | Obs 2 | Obs 3 | All Observers | | 1 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 2 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 3 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 4 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 5 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 6 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 7 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 8 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 9 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 10 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 11 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 12 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 13 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | PMA P150013/S033: FDA Summary of Safety and Effectiveness Data 13 of 34 {13} | Specimen | Consensus* | Number of Results | Median (CPS) | Range (CPS) | Standard Deviation (SD) | | | Percent Agreement (n/N) ** | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | | | Inter-Read | Inter Observer | Total | Obs 1 | Obs 2 | Obs 3 | All Observers | | 14 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 15 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 16 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 17 | Negative | 9 | 0 | 0, <1 | 0.00 | 0.10 | 0.10 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 18 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 19 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 20 | Negative | 7 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/1) | 0% (0/3) | 0% (0/3) | 0% (0/7) | | 21 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 22 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 23 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 24 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 25 | Negative | 8 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/2) | 0% (0/3) | 0% (0/3) | 0% (0/8) | | 26 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 27 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 28 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 29 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 30 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 31 | Positive | 9 | 1 | 0, 2 | 0.00 | 0.63 | 0.63 | 0% (0/3) | 66.7% (2/3) | 100% (3/3) | 55.6% (5/9) | | 32 | Positive | 9 | 1 | 0, 2 | 0.24 | 0.24 | 0.33 | 100% (3/3) | 66.7% (2/3) | 100% (3/3) | 88.9% (8/9) | | 33 | Positive | 9 | 2 | 1, 3 | 0.00 | 0.79 | 0.79 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 34 | Positive | 9 | 3 | 1, 6 | 0.01 | 1.92 | 1.92 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 35 | Positive | 9 | 3 | 1, 10 | 0.00 | 3.04 | 3.04 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 36 | Positive | 9 | 3 | 2, 5 | 0.00 | 0.51 | 0.51 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 37 | Positive | 9 | 5 | 3, 8 | 1.49 | 0.75 | 1.67 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 38 | Positive | 9 | 5 | 3, 10 | 0.00 | 2.01 | 2.01 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 39 | Positive | 9 | 6 | 2, 8 | 0.00 | 0.77 | 0.77 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 40 | Positive | 9 | 8 | 0, 25 | 0.00 | 11.90 | 11.90 | 100% (3/3) | 0% (0/3) | 100% (3/3) | 66.7% (6/9) | | 41 | Positive | 9 | 8 | 2, 12 | 0.00 | 2.18 | 2.18 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 42*** | Positive | 3 | 8 | 5, 8 | 1.73 | N/A | 1.73 | N/A | N/A | 100% (3/3) | 100% (3/3) | | 43 | Positive | 9 | 10 | 4, 10 | 0.00 | 1.25 | 1.25 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 44 | Positive | 9 | 15 | 3, 40 | 0.00 | 16.69 | 16.69 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 45 | Positive | 9 | 15 | 5, 35 | 0.00 | 6.75 | 6.75 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 46 | Positive | 9 | 15 | 6, 30 | 2.11 | 6.06 | 6.41 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 47 | Positive | 9 | 15 | 7, 35 | 1.18 | 4.12 | 4.28 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 48 | Positive | 9 | 15 | 10, 80 | 7.25 | 22.75 | 23.87 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 49 | Positive | 9 | 15 | 10, 40 | 3.54 | 5.89 | 6.87 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 50 | Positive | 9 | 15 | 10, 60 | 0.00 | 10.09 | 10.09 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 51 | Positive | 9 | 15 | 12, 20 | 0.00 | 0.00 | 0.00 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 52 | Positive | 9 | 20 | 10, 75 | 3.12 | 22.02 | 22.24 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 53 | Positive | 9 | 20 | 12, 40 | 0.00 | 8.18 | 8.18 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 54 | Positive | 9 | 25 | 10, 35 | 0.00 | 0.96 | 0.96 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | PMA P150013/S033: FDA Summary of Safety and Effectiveness Data 14 of 34 {14} | Specimen | Consensus* | Number of Results | Median (CPS) | Range (CPS) | Standard Deviation (SD) | | | Percent Agreement (n/N) ** | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | | | Inter-Read | Inter-Observer | Total | Obs 1 | Obs 2 | Obs 3 | All Observers | | 55 | Positive | 9 | 25 | 15, 60 | 0.11 | 11.90 | 11.90 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 56 | Positive | 9 | 30 | 15, 40 | 0.00 | 6.57 | 6.57 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 57 | Positive | 9 | 35 | 25, 60 | 5.77 | 4.08 | 7.07 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 58 | Positive | 9 | 60 | 25, 80 | 11.30 | 6.45 | 13.02 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 59 | Positive | 9 | 60 | 50, 90 | 7.70 | 0.00 | 7.70 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 60 | Positive | 9 | 65 | 25, 100 | 0.00 | 22.11 | 22.11 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 61 | Positive | 9 | 80 | 30, 100 | 14.43 | 15.55 | 21.21 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 62 | Positive | 9 | 90 | 60, 100 | 0.00 | 17.95 | 17.95 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | SD = Standard Deviation; CPS = Combined Positive Score; n = number of agreements; n = number of results; N = Total number of results * Consensus was determined based on majority call across all test conditions * Consensus result was determined based on the majority call across all test conditions ** Percent positive represents the proportion of results scoring positive (CPS ≥ 1) out of the total number of results *** This sample was excluded from analysis due to insufficient washout period ### Precision Agreement Results Summary The overall precision performance across all validation studies is summarized in Table 8, below: **Table 8. Precision of PD-L1 IHC 22C3 pharmDx in epithelial ovarian carcinoma, tested at one site (CPS ≥ 1)** | Precision Study | Diagnostic Cutoff | Study Design | Agreement | | | | --- | --- | --- | --- | --- | --- | | | | | Parameter | n/N | Percent Agreement (95% CI) | | Combined Precision (Inter-Operator, Inter-Instrument, Inter-Day, and Inter-Lot as combined variables) | CPS ≥ 1 | All 38 epithelial ovarian carcinoma specimens (19 PD-L1-negative and 19 PD-L1-positive) with a range of PD-L1 IHC expression were tested by 3 operators, using 3 Autostainer Link 48 instruments, over 3 non-consecutive days, using 3 reagent lots. | NPA | 56/57 | 98.2% (94.7-100.0%) | | | | | PPA | 57/57 | 100.0% (93.7-100.0%)* | | | | | OA | 113/114 | OA 99.1% (97.4-100.0%) | | Intra-run precision (Repeatability) | CPS ≥ 1 | All 38 epithelial ovarian carcinoma specimens (19 PD-L1-negative and 19 PD-L1-positive) with a range of PD-L1 IHC expression were tested with 5 replicates within a run using the Autostainer Link 48 instrument. | NPA | 95/95 | 100.0% (96.1-100.0%) * | | | | | PPA | 95/95 | 100.0% (96.1-100.0%) * | | | | | OA | 190/190 | 100.0% (98.0-100.0%) * | | Inter-observer precision | CPS ≥ 1 | All 61 epithelial ovarian carcinoma specimens (30 PD-L1-negative and 31 PD-L1-positive) with a range of PD-L1 | NPA | 267/267 | 100.0% (98.6-100.0%) * | | | | | PPA | 274/282 | 97.1% (93.2-100.0%) | PMA P150013/S033: FDA Summary of Safety and Effectiveness Data 15 of 34 {15} | Precision Study | Diagnostic Cutoff | Study Design | Agreement | | | | --- | --- | --- | --- | --- | --- | | | | | Parameter | n/N | Percent Agreement (95% CI) | | | | IHC expression, stained with PD-L1 IHC 22C3 pharmDx, were scored by 3 pathologists over 3 non-consecutive days. | OA | 541/549 | 98.5% (96.5-100.0%) | | Intra-observer precision | CPS ≥ 1 | All 62 epithelial ovarian carcinoma specimens (30 PD-L1-negative and 32 PD-L1-positive) with a range of PD-L1 IHC expression, stained with PD-L1 IHC 22C3 pharmDx, were scored by 3 pathologists over 3 non-consecutive days. | NPA | 272/272 | 100.0% (98.6-100.0%) * | | | | | PPA | 274/276 | 99.3% (98.2-100.0%) | | | | | OA | 546/548 | 99.6% (99.1-100.0%) | NPA=Negative Percent Agreement; PPA=Positive Percent Agreement; OA=Overall Percent Agreement; CPS=Combined Positive Score *The percentile bootstrap method cannot compute confidence intervals if 100.0% agreement is observed. Therefore, the Wilson score method was used to compute confidence intervals when the agreement (PPA/NPA/OA) was 100.0%. ### 4. External Reproducibility The external reproducibility study was designed to evaluate the reproducibility of PD-L1 IHC 22C3 pharmDx in determining PD-L1 expression in EOC tissue specimens on the Dako Autostainer Link 48. Reproducibility was assessed in a two-part study: Study Part A assessing Inter- and Intra-Site endpoints and Study Part B assessing Inter- and Intra-Reader endpoints. All IHC tests were interpreted by certified pathologists to determine the positive/negative results based on the CPS ≥ 1 expression level at three external sites. NPA, PPA, and OA with corresponding 95% CI were calculated by using comparisons to the majority call. The results of the reproducibility study for the CPS ≥ 1 cutoff are summarized in tables below. In Part A, reproducibility at the CPS ≥ 1 cutoff was assessed with 40 specimens tested across 3 sites over 5 non-consecutive days. The study included specimens that were pre-qualified at Dako to represent full PD-L1 expression range. The specimen set was randomized and blinded prior to testing at three external sites and assessed for performance with regard to site-to-site and day-to-day reproducibility, or inter- and intra-site reproducibility. Each specimen block was tested at three external sites over five non-consecutive days, yielding 15 total results per specimen (5 results per site). Inter-day variability was assessed within each site, while inter-site variability was assessed across all three sites. Results are presented in Table 9, below: PMA P150013/S033: FDA Summary of Safety and Effectiveness Data 16 of 34 {16} **Table 9. Per-Specimen Agreement Results: Inter-Site External Reproducibility: Results per Specimen (5 per Site)** | Specimen | Consensus* | Number of Results | Median (CPS) | Range (CPS) | Standard Deviation (SD) | | | Percent Positive (n/N) ** | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | | | Inter-Day | Inter-Site | Total | Site 1 | Site 2 | Site 3 | All Sites | | 1 | Negative | 15 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/5) | 0% (0/5) | 0% (0/5) | 0% (0/15) | | 2 | Negative | 15 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/5) | 0% (0/5) | 0% (0/5) | 0% (0/15) | | 3 | Negative | 15 | 0 | 0, <1 | 0.13 | 0.00 | 0.13 | 0% (0/5) | 0% (0/5) | 0% (0/5) | 0% (0/15) | | 4 | Negative | 15 | 0 | 0, <1 | 0.16 | 0.16 | 0.22 | 0% (0/5) | 0% (0/5) | 0% (0/5) | 0% (0/15) | | 5 | Negative | 15 | 0 | 0, <1 | 0.13 | 0.00 | 0.13 | 0% (0/5) | 0% (0/5) | 0% (0/5) | 0% (0/15) | | 6 | Negative | 15 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/5) | 0% (0/5) | 0% (0/5) | 0% (0/15) | | 7 | Negative | 15 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/5) | 0% (0/5) | 0% (0/5) | 0% (0/15) | | 8 | Negative | 15 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/5) | 0% (0/5) | 0% (0/5) | 0% (0/15) | | 9 | Negative | 15 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/5) | 0% (0/5) | 0% (0/5) | 0% (0/15) | | 10 | Negative | 15 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/5) | 0% (0/5) | 0% (0/5) | 0% (0/15) | | 11 | Negative | 15 | 0 | 0, <1 | 0.16 | 0.09 | 0.18 | 0% (0/5) | 0% (0/5) | 0% (0/5) | 0% (0/15) | | 12 | Negative | 15 | 0 | 0, <1 | 0.16 | 0.09 | 0.18 | 0% (0/5) | 0% (0/5) | 0% (0/5) | 0% (0/15) | | 13 | Negative | 15 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/5) | 0% (0/5) | 0% (0/5) | 0% (0/15) | | 14 | Negative | 15 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/5) | 0% (0/5) | 0% (0/5) | 0% (0/15) | | 15 | Negative | 15 | 0 | 0, <1 | 0.13 | 0.22 | 0.26 | 0% (0/5) | 0% (0/5) | 0% (0/5) | 0% (0/15) | | 16 | Negative | 15 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/5) | 0% (0/5) | 0% (0/5) | 0% (0/15) | | 17 | Negative | 15 | 0 | 0, <1 | 0.16 | 0.16 | 0.22 | 0% (0/5) | 0% (0/5) | 0% (0/5) | 0% (0/15) | | 18 | Negative | 15 | 0 | 0, <1 | 0.00 | 0.11 | 0.11 | 0% (0/5) | 0% (0/5) | 0% (0/5) | 0% (0/15) | | 19 | Negative | 15 | 0 | 0, <1 | 0.13 | 0.22 | 0.26 | 0% (0/5) | 0% (0/5) | 0% (0/5) | 0% (0/15) | | 20 | Negative | 15 | 0 | 0, <1 | 0.13 | 0.22 | 0.26 | 0% (0/5) | 0% (0/5) | 0% (0/5) | 0% (0/15) | | 21 | Positive | 15 | 1 | 1, 9 | 2.18 | 1.40 | 2.59 | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (15/15) | | 22 | Positive | 15 | 2 | 0, 5 | 1.10 | 1.19 | 1.62 | 80% (4/5) | 100% (5/5) | 100% (5/5) | 93.3% (14/15) | | 23 | Positive | 15 | 5 | 2, 9 | 1.44 | 1.66 | 2.19 | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (15/15) | | 24 | Positive | 15 | 5 | 3, 20 | 3.58 | 3.15 | 4.77 | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (15/15) | | 25 | Positive | 15 | 8 | 3, 30 | 6.65 | 5.22 | 8.46 | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (15/15) | | 26 | Positive | 15 | 10 | 2, 40 | 7.73 | 10.35 | 12.92 | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (15/15) | | 27 | Positive | 15 | 15 | 0, 50 | 9.09 | 15.99 | 18.39 | 100% (5/5) | 80% (4/5) | 100% (5/5) | 93.3% (14/15) | | 28 | Positive | 15 | 15 | 8, 35 | 4.87 | 6.55 | 8.16 | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (15/15) | | 29 | Positive | 15 | 20 | 12, 80 | 15.77 | 22.94 | 27.84 | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (15/15) | | 30 | Positive | 15 | 25 | 10, 81 | 17.56 | 9.12 | 19.79 | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (15/15) | | 31 | Positive | 15 | 30 | 7, 95 | 17.43 | 28.05 | 33.03 | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (15/15) | | 32 | Positive | 15 | 30 | 10, 100 | 26.13 | 19.81 | 32.79 | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (15/15) | | 33 | Positive | 15 | 35 | 15, 80 | 14.87 | 7.05 | 16.45 | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (15/15) | | 34 | Positive | 15 | 35 | 24, 100 | 19.15 | 19.45 | 27.30 | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (15/15) | | 35 | Positive | 15 | 50 | 12, 100 | 26.85 | 10.29 | 28.76 | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (15/15) | | 36 | Positive | 15 | 52 | 35, 100 | 19.49 | 13.25 | 23.57 | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (15/15) | | 37 | Positive | 15 | 70 | 37, 95 | 10.59 | 21.06 | 23.58 | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (15/15) | | 38 | Positive | 15 | 75 | 16, 100 | 20.79 | 11.69 | 23.85 | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (15/15) | | 39 | Positive | 15 | 75 | 31, 100 | 15.65 | 18.28 | 24.06 | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (15/15) | PMA P150013/S033: FDA Summary of Safety and Effectiveness Data 17 of 34 {17} | Specimen | Consensus* | Number of Results | Median (CPS) | Range (CPS) | Standard Deviation (SD) | | | Percent Positive (n/N) ** | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | | | Inter-Day | Inter-Site | Total | Site 1 | Site 2 | Site 3 | All Sites | | 40 | Positive | 15 | 95 | 81, 100 | 5.76 | 0.00 | 5.76 | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (15/15) | SD = Standard Deviation; CPS = Combined Positive Score; n = number of positive results; N = Total number of results * Consensus result was determined based on the majority call across all test conditions ** Percent positive represents the proportion of results scoring positive (CPS ≥ 1) out of the total number of results. Part B included assessment of reader-to-reader variability with specimens that spanned the PD-L1 expression range. These specimens were stained at Agilent (Dako North America) and shipped to the three external sites for assessment of PD-L1 expression by pathologists for both inter-reader and intra-reader reproducibility. Fifty-nine pre-stained specimens were assessed three times by three readers with a minimum of 2 weeks between reads. Fifty-nine pre-stained specimens were assessed three times by three certified pathologists with a minimum of 2 weeks between reads, yielding 9 total results per specimen (3 results per observer). Inter-read variability was assessed within each observer, while inter-observer variability was assessed across all three observers. Results are presented in Table 10, below: **Table 10. Per-Specimen Agreement Results: Inter-Reader External Reproducibility: Results per Specimen (3 per Reader)** | Specimen | Consensus* | Number of Results | Median (CPS) | Range (CPS) | Standard Deviation (SD) | | | Percent Positive (n/N) ** | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | | | Inter-Read | Inter-Observer | Total | Obs 1 | Obs 2 | Obs 3 | All Observers | | 1 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 2 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 3 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 4 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 5 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 6 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 7 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 8 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 9 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 10 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 11 | Negative | 9 | 0 | 0, <1 | 0.17 | 0.00 | 0.17 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 12 | Negative | 9 | 0 | 0, <1 | 0.24 | 0.10 | 0.25 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 13 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 14 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 15 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 16 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 17 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 18 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 19 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 20 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | PMA P150013/S033: FDA Summary of Safety and Effectiveness Data 18 of 34 {18} | Specimen | Consensus* | Number of Results | Median (CPS) | Range (CPS) | Standard Deviation (SD) | | | Percent Positive (n/N) ** | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | | | Inter-Read | Inter-Observer | Total | Obs 1 | Obs 2 | Obs 3 | All Observers | | 21 | Negative | 9 | 0 | 0, <1 | 0.17 | 0.00 | 0.17 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 22 | Negative | 9 | 0 | 0, <1 | 0.17 | 0.17 | 0.24 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 23 | Negative | 9 | 0 | 0, <1 | 0.17 | 0.24 | 0.29 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 24 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 25 | Negative | 9 | 0 | 0, <1 | 0.24 | 0.10 | 0.25 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 26 | Negative | 9 | 0 | 0, 0 | 0.00 | 0.00 | 0.00 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 27 | Negative | 9 | 0 | 0, <1 | 0.17 | 0.00 | 0.17 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/9) | | 28 | Negative | 9 | 0 | 0, 12 | 4.18 | 0.00 | 4.18 | 33.3% (1/3) | 33.3% (1/3) | 33.3% (1/3) | 33.3% (3/9) | | 29 | Negative | 9 | 0.5 | 0, 1 | 0.37 | 0.14 | 0.40 | 0% (0/3) | 0% (0/3) | 66.7% (2/3) | 22.2% (2/9) | | 30 | Positive | 9 | 3 | 1, 9 | 2.11 | 1.86 | 2.81 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 31 | Positive | 9 | 4 | 2, 9 | 0.82 | 2.87 | 2.99 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 32 | Positive | 9 | 5 | 1, 20 | 5.61 | 0.82 | 5.67 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 33 | Positive | 9 | 6 | 3, 15 | 2.98 | 2.46 | 3.87 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 34 | Positive | 9 | 7 | 2, 18 | 4.03 | 1.93 | 4.47 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 35 | Positive | 9 | 8 | 5, 30 | 5.27 | 7.10 | 8.84 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 36 | Positive | 9 | 10 | 3, 45 | 4.96 | 16.55 | 17.28 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 37 | Positive | 9 | 10 | 5, 75 | 20.11 | 9.22 | 22.12 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 38 | Positive | 9 | 12 | 5, 95 | 19.33 | 30.80 | 36.36 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 39 | Positive | 9 | 15 | 2, 38 | 4.89 | 15.05 | 15.82 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 40 | Positive | 9 | 15 | 2, 60 | 14.68 | 16.20 | 21.86 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 41 | Positive | 9 | 15 | 10, 70 | 5.11 | 26.91 | 27.39 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 42 | Positive | 9 | 15 | 10, 45 | 5.49 | 14.66 | 15.66 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 43 | Positive | 9 | 15 | 10, 60 | 5.61 | 20.40 | 21.16 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 44 | Positive | 9 | 16 | 1, 90 | 14.15 | 31.79 | 34.79 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 45 | Positive | 9 | 16 | 10, 60 | 11.30 | 12.60 | 16.93 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 46 | Positive | 9 | 20 | 13, 100 | 11.73 | 39.75 | 41.45 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 47 | Positive | 9 | 30 | 20, 45 | 7.95 | 0.00 | 7.95 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 48 | Positive | 9 | 35 | 9, 100 | 35.28 | 15.97 | 38.73 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 49 | Positive | 9 | 40 | 15, 100 | 10.32 | 38.41 | 39.77 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 50 | Positive | 9 | 40 | 20, 100 | 7.05 | 32.71 | 33.46 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 51 | Positive | 9 | 45 | 30, 95 | 13.25 | 19.16 | 23.29 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 52 | Positive | 9 | 50 | 19, 100 | 5.53 | 37.11 | 37.52 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 53 | Positive | 9 | 50 | 25, 85 | 14.18 | 11.71 | 18.39 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 54 | Positive | 9 | 50 | 37, 100 | 4.24 | 28.47 | 28.78 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 55 | Positive | 9 | 50 | 37, 100 | 1.94 | 32.17 | 32.23 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 56 | Positive | 9 | 55 | 40, 100 | 8.82 | 29.58 | 30.87 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 57 | Positive | 9 | 65 | 25, 100 | 8.50 | 33.10 | 34.17 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 58 | Positive | 9 | 80 | 50, 100 | 15.18 | 13.26 | 20.16 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | | 59 | Positive | 9 | 95 | 60, 100 | 8.50 | 16.69 | 18.73 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (9/9) | SD = Standard Deviation; CPS = Combined Positive Score; n = number of positive results; N = Total number of results PMA P150013/S033: FDA Summary of Safety and Effectiveness Data 19 of 34 {19} *Consensus result was determined based on the majority call across all test conditions **Percent positive represents the proportion of results scoring positive (CPS ≥ 1) out of the total number of results #### External Reproducibility Percent Agreement Results The overall external reproducibility percent agreement results across all validation studies is summarized in Table 11, below: **Table 11. Reproducibility of PD-L1 IHC 22C3 pharmDx in epithelial ovarian carcinoma, tested at three external sites (CPS ≥ 1)** | Reproducibility Study | Diagnostic Cutoff | Study Design | Agreement | | | | --- | --- | --- | --- | --- | --- | | | | | Parameter | n/N | Percent Agreement (95% CI) | | Inter-site | CPS ≥ 1 | All 40 epithelial ovarian carcinoma specimens (20 PD-L1-negative and 20 PD-L1-positive) with a range of PD-L1 IHC expression were tested on 5 non-consecutive days. Inter-site analysis was performed for 3 sites on a total of 600 comparisons to majority call. | NPA | 300/300 | 100.0% (98.7-100.0%)* | | | | | PPA | 298/300 | 99.3% (98.3-100.0%) | | | | | OA | 598/600 | 99.7% (99.2-100.0%) | | Intra-site | CPS ≥ 1 | All 40 epithelial ovarian carcinoma specimens (20 PD-L1-negative and 20 PD-L1-positive) with a range of PD-L1 IHC expression were tested on 5 non-consecutive days at each of 3 study sites. Intra-site analysis was performed for 3 sites on a total of 600 comparisons to majority call. | NPA | 300/300 | 100.0% (98.7-100.0%)* | | | | | PPA | 298/300 | 99.3% (98.3-100.0%) | | | | | OA | 598/600 | 99.7% (99.2-100.0%) | | Inter-observer | CPS ≥ 1 | All 59 epithelial ovarian carcinoma specimens (29 PD-L1-negative and 30 PD-L1-positive) with a range of PD-L1 IHC expression, stained with PD-L1 IHC 22C3 pharmDx, were scored by 3 pathologists, 1 at each of 3 study sites, on 3 non-consecutive days. Inter-observer analysis was performed between 3 sites on a total of 531 comparisons to majority call. | NPA | 256/261 | 98.1% (95.0-100.0%) | | | | | PPA | 270/270 | 100.0% (98.6-100.0%)* | | | | | OA | 526/531 | 99.1% (97.6-100.0%) | PMA P150013/S033: FDA Summary of Safety and Effectiveness Data 20 of 34 {20} | Reproducibility Study | Diagnostic Cutoff | Study Design | Agreement | | | | --- | --- | --- | --- | --- | --- | | | | | Parameter | n/N | Percent Agreement (95% CI) | | Intra-observer | CPS ≥ 1 | All 59 epithelial ovarian carcinoma specimens (29 PD-L1-negative and 30 PD-L1-positive) with a range of PD-L1 IHC expression, stained with PD-L1 IHC 22C3 pharmDx, were scored by 3 pathologists, 1 at each of 3 study sites, on 3 non-consecutive days. Intra-observer analysis was performed for 3 sites on a total of 531 comparisons to majority call. | NPA | 255/258 | NPA 98.8% (96.5-100.0%) | | | | | PPA | 272/273 | PPA 99.6% (98.9-100.0%) | | | | | OA | 527/531 | OA 99.2% (97.9-100.0%) | NPA=Negative Percent Agreement; PPA=Positive Percent Agreement; OA=Overall Percent Agreement; CPS=Combined Positive Score *The percentile bootstrap method cannot compute confidence intervals if 100.0% agreement is observed. Therefore, the Wilson score method was used to compute confidence intervals when the agreement (PPA/NPA/OA) was observed 100%. ### 5. Robustness Robustness of the staining performance of PD-L1 IHC 22C3 pharmDx in EOC was evaluated by testing the performance of the assay when varying the following conditions as described below: - Target retrieval time at three incubation times - 18 minutes - 20 minutes-standard - 22 minutes - Target Retrieval Solution temperature at three incubation temperatures - 95 °C - 97 °C - standard - 99 °C - Target Retrieval Solution pH at three pH levels - pH 5.9 - pH 6.1- standard - pH 6.3 Studies were performed with 38 EOC specimens. Specimens were blinded and randomized prior to evaluation at a predetermined cutoff. Negative percent agreement (NPA), positive percent agreement (PPA), and overall percent agreement (OA) and 95% confidence interval (CI) were calculated for CPS≥1 cutoff by pairwise comparison to the standard condition. No significant difference in results was observed for the experimental conditions above when compared to the standard conditions. PMA P150013/S033: FDA Summary of Safety and Effectiveness Data 21 of 34 {21} ## 6. Stability Studies ### a. PD-L1 IHC 22C3 pharmDx Stability Product Shelf Life: Real time shelf-life stability testing with three reagent lots of PD-L1 IHC 22C3 pharmDx was previously performed for NSCLC and presented in P150013. The protocol included transport simulation, working stability and in-use/on-board stability testing. The shelf life for PD-L1 IHC 22C3 pharmDx remains unchanged and is 9 months when stored at 2-8 °C. Finished Good Shelf Life: 9 months at 2-8 °C In-Use/On-Board Stability Testing Eighteen cycles to room temperature Working/Reconstituted Stability Testing DAB Substrate-Chromogen Solution: 5 Days at 2-8 °C, protected from light. Target Retrieval Solution: 5 days at room temperature in a PT-Link with up to 3 uses for 3-in-1 pretreatment. ### b. FFPE Cut Section Stability A stability study was conducted to evaluate the stability of the antigen in cut tissue sections of EOC FFPE blocks using PD-L1 IHC 22C3 pharmDx when stored in the dark at 2-8 °C or 25 °C. The study included 10 EOC specimens. Based on these studies, stability dating for cut slides in EOC is 3.5 months for cut sections stored at 2-8 °C and 3 months for cut sections stored at 25 °C. ## 7. Impact of Tumor Heterogeneity The objective of this study was to investigate whether tumor heterogeneity affects PD-L1 IHC staining results with PD-L1 IHC 22C3 pharmDx. ### a. Primary vs. Metastatic Tumor Tissues Matched primary versus metastatic blocks were obtained from 11 subjects and evaluated by PD-L1 IHC 22C3 pharmDx and specimens were determined to be positive or negative based on the CPS ≥ 1 cutoff. There were 8 concordant negative pairs, 1 concordant positive pair, and 2 pairs with discordant PD-L1 status. Results may not be representative of all EOC specimens, as tumor heterogeneity is unique for each specimen. ### b. Intra-Case Heterogeneity Two sister FFPE blocks from 24 EOC subjects obtained from the same tumor were evaluated to assess concordance for PD-L1 status. In 20 of 24 cases, the diagnostic outcomes based on the CPS ≥ 1 cutoff were concordant across intra-case (sister) blocks. Four cases showed discordant results across sister blocks. Results may not be PMA P150013/S033: FDA Summary of Safety and Effectiveness Data 22 of 34 {22} representative of all EOC specimens, as tumor heterogeneity is unique for each specimen. # c. Intra-Block Heterogeneity Thirty individual FFPE EOC tissue blocks were divided in 3 portions across a minimum of 200 μm: anterior, middle, and posterior. Of the 30 cases assessed, all 30 cases were concordant when compared to the diagnostic status of front and back sections based on the CPS ≥ 1 cutoff. Results may not be representative of all EOC specimens, as tumor heterogeneity is unique for each specimen. # B. Animal Studies None # C. Additional Studies # 1. Impact on Ischemia/Fixation See SSED for P150013. # 2. Control Cell Line Validation See SSED for P150013. # X. SUMMARY OF PRIMARY CLINICAL STUDY The clinical performance of PD-L1 IHC 22C3 pharmDx as a companion diagnostic for PD-L1 detection (CPS≥1) in EOC patients to determine eligibility for treatment with KEYTRUDA® (pembrolizumab) was demonstrated in the study KEYNOTE-B96. This study was conducted in the US and geographical regions including, Asia, Europe, Australia and the rest of the world (ROW). A summary of the clinical study is presented below. # A. Study Design KEYNOTE-B96, a randomized, double-blind, phase III study evaluated the efficacy of KEYTRUDA in combination with paclitaxel, with or without bevacizumab, in 643 patients with platinum-resistant EOC who had received ≤ 2 prior lines of therapy. Randomization was stratified by bevacizumab use in the study (yes vs. no), region (US vs. EU vs. ROW), and PD-L1 status (CPS <1 vs. CPS 1 to <10 vs. CPS ≥10) as determined using the PD-L1 IHC 22C3 pharmDx kit. Patients were enrolled regardless of tumor PD-L1 expression status. The primary efficacy endpoint was progression-free survival (PFS) per RECIST v1.1 by investigator assessment in participants with CPS ≥1. Key secondary endpoints included overall survival (OS) in participants with CPS ≥1. PMA P150013/S033: FDA Summary of Safety and Effectiveness Data 23 of 34 {23} # 1. Clinical Inclusion and Exclusion Criteria Enrollment in the KEYNOTE-B96 study was limited to female subjects with platinum-resistant EOC, who had received one or two prior lines of systemic therapy, and who were at least 18 years of age. A summary of key additional inclusion criteria are as follows: - Have histologically confirmed epithelial (including high-grade serous or predominantly serous, low-grade serous, any-grade endometrioid, malignant mixed Müllerian tumors [carcinosarcoma], or clear cell) ovarian, fallopian tube, or primary peritoneal carcinoma. - Have received 1 or 2 prior lines of systemic therapy for EOC, including at least 1 prior platinum-based therapy. Note: Participants must have received at least 4 cycles but not more than 9 cycles of platinum-based therapy in first-line treatment. - Have radiographic evidence of disease progression within 6 months (180 days) after the last dose of platinum-based chemotherapy for EOC (i.e., platinum-resistant disease). Note: Participants with secondary platinum-refractory disease who were either platinum-resistant or platinum-sensitive in the first line were eligible. - Have been a candidate for paclitaxel chemotherapy (and bevacizumab, if used). - Have an ECOG performance status of 0 to 1 assessed within 3 days before randomization. - Provided tumor tissue sample for prospective determination of PD-L1 status. # 2. Follow-up Schedule Treatment with KEYTRUDA continued until RECIST v1.1-defined progression of disease, unacceptable toxicity, or a maximum of 24 months. Administration of KEYTRUDA was permitted beyond RECIST-defined disease progression if the patient was clinically stable and considered to be deriving clinical benefit by the investigator. Assessment of tumor status was performed at Week 9 and then every 9 weeks for the first year, followed by every 12 weeks thereafter. # 3. Clinical Endpoints Primary endpoint: 1. Progression-free survival (PFS), defined as the time from randomization to the first documented disease progression as assessed by the investigator according to RECIST v1.1, or death due to any cause, whichever occurred first. Secondary efficacy endpoints: 1. Overall survival (OS), defined as the time from randomization to the time of death due to any cause. PMA P150013/S033: FDA Summary of Safety and Effectiveness Data 24 of 34 {24} 2. PFS by blinded independent central review (BICR), defined as the time from randomization to the first documented disease progression per RECIST v1.1 as assessed by BICR, or death due to any cause, whichever occurs first. 3. Change from baseline and time to deterioration (TTD) of the quality of life (QoL) and symptom scores from the Global Health Status/QoL scale (items 29 and 30) of the EORTC QLQ-C30 and the abdominal/GI symptom scale (items 31 to 36) of the EORTC QLQ-OV28, respectively. # **B. Accountability of PMA Cohort** Participants with platinum-resistant EOC in KEYNOTE-B96 comprise the primary participant population for this application. A total of 643 participants were randomly assigned in a 1:1 ratio to pembrolizumab + paclitaxel, with or without bevacizumab (322 participants) or placebo + paclitaxel, with or without bevacizumab (321 participants). All 643 participants had a tumor tissue sample tested with PD-L1 IHC 22C3 pharmDx. Most participants (72.5%, n=466) had a tumor tissue PD-L1 expression score of CPS ≥1, and 27.5% of participants had a tumor tissue PD-L1 expression score of CPS < 1. While, 201 of 466 (31.3%) participants with CPS ≥1 had a tumor tissue PD-L1 expression score of CPS ≥10. The patient/sample accountability is shown in Table 12 below. **Table 12: Accountability of PMA Cohort** | | Number of Study Subjects n (%) | | --- | --- | | Enrolled | 643 (100%) | | Tumor sample collected at baseline | 643 (100%) | | Specimens with insufficient tissue | 0 (0.0%) | | Total evaluable PD-L1 expression | 643 (100%) | | PD-L1 positive CPS ≥ 1 | 466 (72.5%) | | PD-L1 positive CPS ≥ 10 | 201* (31.3) | | PD-L1 negative CPS < 1 | 177 (27.5%) | | Site of Tumor: (N=643) | | | Primary Site | 452 (70.3%) | | Metastatic Site | 188 (29.2%) | | Unknown | 3 (0.5%) | | Specimen type (N=643) | | | Newly Obtained** | 29 (4.5%) | | Archival | 611 (95%) | | Unknown | 3 (0.5%) | | Sample Procedure (N= 643) | | | Biopsy | 128 (19.9%) | | Resection | 512 (79.6%) | | Unknown | 3 (0.5%) | | *CPS ≥10 is a subset of CPS ≥1 population (201 of 466 participants with CPS ≥1 had CPS ≥10). | | | **Specimen type derived based on sample collection date. If the sample was obtained within 42 days of randomization, the sample is categorized as 'Newly | | PMA P150013/S033: FDA Summary of Safety and Effectiveness Data 25 of 34 {25} Obtained'; otherwise, the sample is categorized as 'Archival'. Three subjects had unknown specimen type classification. ### C. Study Population Demographics and Baseline Parameters The baseline characteristics for study subjects with PD-L1 CPS ≥1 is shown in the table 13 below. **Table 13: Patient Characteristics With PD-L1 CPS ≥1** | | Pembrolizumab + paclitaxel, with or without bevacizumab | | Placebo + with paclitaxel, with or without bevacizumab | | Total | | | --- | --- | --- | --- | --- | --- | --- | | | n | (%) | n | (%) | n | (%) | | Participants in population | 234 | | 232 | | 466 | | | **Sex** | | | | | | | | Female | 234 | (100.0) | 232 | (100.0) | 466 | (100.0) | | **Age (Years)** | | | | | | | | < 65 | 145 | (62.0) | 144 | (62.1) | 289 | (62.0) | | ≥65 | 89 | (38.0) | 88 | (37.9) | 177 | (38.0) | | Mean | 61.5 | | 61.3 | | 61.4 | | | SD | 10.2 | | 9.8 | | 10.0 | | | Median | 62.0 | | 61.5 | | 62.0 | | | Range | 37 to 85 | | 37 to 82 | | 37 to 85 | | | **Race** | | | | | | | | Asian | 51 | (21.8) | 40 | (17.2) | 91 | (19.5) | | Black Or African American | 5 | (2.1) | 4 | (1.7) | 9 | (1.9) | | Multiple | 5 | (2.1) | 11 | (4.7) | 16 | (3.4) | | American Indian Or Alaska Native, White | 3 | (1.3) | 7 | (3.0) | 10 | (2.1) | | Black Or African American, White | 1 | (0.4) | 3 | (1.3) | 4 | (0.9) | | White, Asian | 1 | (0.4) | 1 | (0.4) | 2 | (0.4) | | Native Hawaiian Or Other Pacific Islander | 1 | (0.4) | 1 | (0.4) | 2 | (0.4) | | White | 155 | (66.2) | 158 | (68.1) | 313 | (67.2) | | Missing | 17 | (7.3) | 18 | (7.8) | 35 | (7.5) | | **Ethnicity** | | | | | | | | Hispanic Or Latino | 27 | (11.5) | 35 | (15.1) | 62 | (13.3) | | Not Hispanic Or Latino | 195 | (83.3) | 186 | (80.2) | 381 | (81.8) | | Not Reported | 8 | (3.4) | 7 | (3.0) | 15 | (3.2) | | Unknown | 4 | (1.7) | 4 | (1.7) | 8 | (1.7) | | IIIA1 | 7 | (3.0) | 5 | (2.2) | 12 | (2.6) | | IIIA2 | 5 | (2.1) | 7 | (3.0) | 12 | (2.6) | PMA P150013/S033: FDA Summary of Safety and Effectiveness Data 26 of 34 {26} | IIIB | 16 | (6.8) | 14 | (6.0) | 30 | (6.4) | | --- | --- | --- | --- | --- | --- | --- | | IIIC | 96 | (41.0) | 98 | (42.2) | 194 | (41.6) | | IVA | 28 | (12.0) | 19 | (8.2) | 47 | (10.1) | | IVB | 61 | (26.1) | 70 | (30.2) | 131 | (28.1) | | **Histology Subtype** | | | | | | | | High Grade Serous | 206 | (88.0) | 208 | (89.7) | 414 | (88.8) | | Low Grade Serous | 4 | (1.7) | 6 | (2.6) | 10 | (2.1) | | Carcinosarcoma | 3 | (1.3) | 3 | (1.3) | 6 | (1.3) | | Clear Cell | 14 | (6.0) | 13 | (5.6) | 27 | (5.8) | | Endometrioid | 5 | (2.1) | 2 | (0.9) | 7 | (1.5) | | Others (Carcinoma) | 2 | (0.9) | 0 | (0.0) | 2 | (0.4) | | **Prior Lines of Therapy** | | | | | | | | One line | 88 | (37.6) | 81 | (34.9) | 169 | (36.3) | | Two lines | 145 | (62.0) | 151 | (65.1) | 296 | (63.5) | | Three lines | 1 | (0.4) | 0 | (0.0) | 1 | (0.2) | | **Prior Anti-PD-1 or Anti-PD-L1** | | | | | | | | Yes | 5 | (2.1) | 7 | (3.0) | 12 | (2.6) | | No | 229 | (97.9) | 225 | (97.0) | 454 | (97.4) | | **Prior Bevacizumab/bev-biosimilar** | | | | | | | | Yes and bevacizumab treated in study | 68 | (29.1) | 66 | (28.4) | 134 | (28.8) | | Yes and bevacizumab not treated in study | 40 | (17.1) | 42 | (18.1) | 82 | (17.6) | | No | 126 | (53.8) | 124 | (53.4) | 250 | (53.6) | | **Prior PARPi** | | | | | | | | Yes | 85 | (36.3) | 96 | (41.4) | 181 | (38.8) | PMA P150013/S033: FDA Summary of Safety and Effectiveness Data 27 of 34 {27} | | Pembrolizumab + paclitaxel, with or without bevacizumab | | Placebo + paclitaxel, with or without bevacizumab | | Total | | | --- | --- | --- | --- | --- | --- | --- | | | n | (%) | n | (%) | n | (%) | | No | 149 | (63.7) | 136 | (58.6) | 285 | (61.2) | | **Prior Anti-VEGF Therapy** | | | | | | | | Yes, both bevacizumab/bev-biosimilar and other VEGF inhibitors | 0 | (0.0) | 2 | (0.9) | 2 | (0.4) | | Yes, bevacizumab/bev-biosimilar only | 108 | (46.2) | 106 | (45.7) | 214 | (45.9) | | Yes, other VEGF inhibitors only | 1 | (0.4) | 0 | (0.0) | 1 | (0.2) | | No | 125 | (53.4) | 124 | (53.4) | 249 | (53.4) | | **Platinum Free Interval** | | | | | | | | <3 months from last platinum therapy to subsequent progression | 103 | (44.0) | 115 | (49.6) | 218 | (46.8) | | 3-6 months from last platinum therapy to subsequent progression | 130 | (55.6) | 115 | (49.6) | 245 | (52.6) | | >6 months from last platinum therapy to subsequent progression | 1 | (0.4) | 2 | (0.9) | 3 | (0.6) | | SD=Standard deviation. Missing values in race are mainly because this information is not permitted to report in France and Netherlands. Western Europe includes countries in the European Economic Area, United Kingdom, and Switzerland. Database Cutoff Date: 05MAR2025. | | | | | | | PMA P150013/S033: FDA Summary of Safety and Effectiveness Data 28 of 34 {28} # D. Safety and Effectiveness Results # 1. Safety Results The analysis of safety was based on 643 patients. In the trial, there were no device-related adverse events. The results from KEYNOTE-B96 demonstrate that pembrolizumab in combination with paclitaxel, with or without bevacizumab, has a manageable safety profile consistent with the well-established safety profiles of pembrolizumab monotherapy, paclitaxel, and bevacizumab. The overall incidence and nature (severity, outcome) of adverse events of special interest (AEOSI) in the pembrolizumab in combination with paclitaxel, with or without bevacizumab group were generally consistent with pembrolizumab monotherapy. No new safety signals were identified. Please refer to Drugs@FDA for complete safety information on KEYTRUDA® (pembrolizumab). # 2. Effectiveness Results A summary of the efficacy results for the participants with tumors expressing PD-L1 CPS ≥1 is provided in Table 14. The trial demonstrated a statistically significant improvement in PFS in patients with PD-L1 positive tumors (CPS ≥1) randomized to KEYTRUDA in combination with paclitaxel, with or without bevacizumab compared to placebo in combination with paclitaxel, with or without bevacizumab at the first interim analysis (Table 14 and Figure 1). At the second interim analysis, the trial demonst…