Immunohistochemistry (IHC) protocols and methods for diagnosing and treating cancer

Abstract

In an alternative embodiment, an immunohistochemistry (IHC) method is provided for reproducibly determining and scoring the degree of expression of the protein carcinoembryonic antigen-related cell adhesion molecule 5 or CEACAM5 in a tissue sample. In an alternative embodiment, a method is provided for diagnosing, treating or improving a cancer or tumor or assessing the risk of its recurrence using an IHC method as provided herein. In an alternative embodiment, a kit is provided that includes components and instructions for performing a method as provided herein. This application describes methods for scoring CEACAM5 expression and using the score as a companion or complementary diagnosis or for treating or improving a cancer or tumor.

Description

Technical Field

[0001] The present invention generally relates to cancer treatment, companion or complementary diagnostics, and immunohistochemical methods. In alternative embodiments, an immunohistochemical (IHC) method is provided for reproducibly determining and scoring the degree of expression of the protein carcinoembryonic antigen-related cell adhesion molecule 5 or CEACAM5 (also known as CD66e (surface antigen classification 66e)) in tissue samples. In alternative embodiments, methods are provided for diagnosing, treating, or improving cancer or tumor or for assessing the risk of its recurrence using an IHC method as provided herein. In alternative embodiments, kits are provided that include components and instructions for performing a method as provided herein. This application describes methods for scoring CEACAM5 expression and using the score as a companion or complementary diagnostic or for treating or improving cancer or tumor.

Background Art

[0002] Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), also known as CD66e (surface antigen classification 66e), is a member of the carcinoembryonic antigen (CEA) gene family. There is evidence that high expression of CEACAM5 is strongly associated with CD133-positive colorectal cancer stem cells. CEACAM5 is used as a clinical biomarker for gastrointestinal cancer and may promote tumorigenesis by its role as a cell adhesion molecule. Furthermore, CEACAM5 can regulate differentiation, apoptosis, and cell polarity.

[0003] Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is a cell surface glycoprotein that is overexpressed in several tumor types including the digestive tract, urogenital system, breast cancer, and non-squamous NSCLC. CEACAM5 is a member of the CEA family of proteins that play important roles in cell migration, cell invasion, and cell adhesion.

Summary of the Invention

[0004] In an alternative embodiment, an immunohistochemistry (IHC) method for determining and scoring the degree of cell membrane expression of carcinoembryonic antigen-related cell adhesion molecule 5 or CEACAM5 (also known as CD66e (surface antigen classification 66e)) in a tissue sample, (a) staining the tissue sample with an antibody that specifically binds to CEACAM5; (b) determining the total number of viable tumor or cancer cells having CEACAM5 cell membrane staining and determining the total number of stained and non-stained viable tumor or cancer cells in at least a portion of the tissue sample; wherein a tumor or cancer cell is counted as being positively stained with the anti-CEACAM5 antibody if there is CEACAM5 cell membrane staining of any intensity above a defined threshold; (c) determining a Tumor Intensity Proportion Score (TIPS); wherein the tumor intensity proportion score is the number of CEACAM5-stained viable tumor or cancer cells found in the tissue sample divided by the total number of stained and non-stained viable tumor or cancer cells, multiplied by 100, is provided.

[0005] In an alternative embodiment of the IHC method as provided herein: - CEACAM5 cell membrane staining includes whole membrane staining, discontinuous membrane staining, partial membrane staining, complete membrane staining, punctate membrane staining, linear membrane staining, luminal membrane staining, apical membrane staining, basal membrane staining, lateral membrane staining, lateral basal membrane staining, high cytoplasmic staining of 2+ or more positive staining intensities, high cytoplasmic staining of 3+ positive staining intensities, or combinations thereof; - the total number of viable tumor or cancer cells includes luminal cells, acinar cells, intraluminal cells, multiple layers of cells, cells having a signet ring morphology, cells with high cytoplasmic staining, cells with an intracellular lumen, or combinations thereof; - the total number of viable tumor or cancer cells counted as being positively stained includes all acinar cells having a cell membrane associated with a positively stained lumen. - The tissue sample contains at least about 100 cells. - Tumor or cancer cells that are counted as positively stained do not include tumor or cancer cells with cytoplasmic staining, staining of normal or non-neoplastic structures, staining of non-viable tumors or cancer cells, necrotic cells, cell fragments, stromal staining, or edge artifacts staining at the periphery of the tissue sample. - Cytoplasmic staining includes cytoplasmic staining with a positive staining intensity of 2+ or less, or the established threshold includes a positive staining intensity of 2+ or more, or the established threshold includes a 2+ or 3+ positive staining intensity evaluated at a magnification of about 4× to about 10×, or further includes the step of confirming the positive staining intensity at a magnification of about 20× to about 40×. - The Tumor Intensity Percentage Score (TIPS) includes multiplying the number of CEACAM5-positive viable tumor or cancer cell stains with a positive staining intensity of 2+ or more by 100 and dividing by the total number of stained and non-stained viable tumors or cancer cells, or a TIPS of about 40% or more indicates the diagnostic status of the tissue sample, or the TIPS is about 50% or more, about 60% or more, about 80% or more, or about 90% or more. - A TIPS of about 5% or more indicates the diagnostic status of the tissue sample, or a TIPS of about 5% or more includes the number of CEACAM5-positive viable tumor or cancer cell stains with a positive staining intensity of 2+ or more, or a TIPS of about 10% or more includes the number of CEACAM5-positive viable tumor or cancer cell stains with a positive staining intensity of 2+ or more. - Sections or portions of the tissue sample are prepared on slides, microscope slides, or equivalents, and the sections or portions of the tissue sample are stained on slides. - The antibody includes a monoclonal mouse anti-CEACAM5 antibody or a monoclonal rabbit anti-CEACAM5 antibody, or the monoclonal mouse anti-CEACAM5 antibody includes a monoclonal mouse anti-CEACAM5 clone 769 or an antibody having substantially the same affinity as the clone 769 antibody for CEACAM5. - The tissue sample includes a formalin-fixed paraffin-embedded (FFPE) specimen, or the FFPE specimen includes a cancer specimen stained on an automated IHC platform. - Sections of tissue samples are prepared by a protocol that includes fixation in about 10% neutral buffered formalin for a period of about 6 hours to about 72 hours. - The tumor or cancer is non-squamous non-small cell lung cancer (nsNSCLC), non-small cell lung cancer (NSCLC), adenocarcinoma, synovial sarcoma, myxoid / round cell liposarcoma, high-grade myxoid liposarcoma, low-grade myxoid liposarcoma, head and neck cancer, melanoma, esophageal cancer, gastric cancer, colorectal cancer, lung cancer, colon cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, prostate cancer or urothelial cancer. - The tissue sample is derived from a needle biopsy sample, a fine needle aspirate, a cytology specimen or decalcified bone, and / or - Positive staining is determined using a brightfield light microscope, microscope objective lens, computer monitor and imaging software or a combination thereof, and optionally, the imaging software includes whole slide imaging software.

[0006] In an alternative embodiment, a method for diagnosing a tumor or cancer by determining whether a tissue sample is positive for the expression of carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), the method provided herein, for example, by a method as described above, includes determining the CEACAM5 diagnostic status in the tissue sample, and a tumor intensity ratio score of about 50% or more of tumor cancer cells having a CEACAM5 cell membrane staining of intensity 2+ or more is diagnostically positive. In an alternative embodiment, the tumor or cancer is non-squamous non-small cell lung cancer (nsNSCLC), non-small cell lung cancer (NSCLC), adenocarcinoma, synovial sarcoma, myxoid / round cell liposarcoma, head and neck cancer, melanoma, esophageal cancer, gastric cancer, colorectal cancer, lung cancer, colon cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, prostate cancer or urothelial cancer.

[0007] In an alternative embodiment, a method for treating or ameliorating a tumor or cancer in a patient, comprising determining and scoring the amount of CEACAM5 in a tissue sample derived from the patient, such as provided herein, e.g., as described above, and if the tissue sample is determined or scored to have a CEACAM5 score that is high or diagnostically positive, the patient is treated with a cancer therapeutic agent to which the patient is likely to respond favorably, is provided.

[0008] In an alternative embodiment of the method provided herein, - the tumor or cancer is non-squamous non-small cell lung cancer (nsNSCLC), non-small cell lung cancer (NSCLC), adenocarcinoma, synovial sarcoma, myxoid / round cell liposarcoma, head and neck cancer, melanoma, esophageal cancer, gastric cancer, colorectal cancer, lung cancer, colon cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, prostate cancer or urothelial cancer, - the cancer therapeutic agent comprises administration of an anti-cancer drug or anti-cancer therapy to the patient, and / or - the anti-cancer therapy comprises an antibody-drug conjugate, small molecule therapy, immunotherapy, monoclonal antibody therapy, adoptive cell therapy, T cell receptor therapy or chimeric antigen receptor (CAR) T cell therapy.

[0009] In an alternative embodiment, a kit is provided comprising an antibody that specifically binds to CEACAM5 and CEACAM5 scoring guidelines as provided herein, e.g., as described above, comprising a method as provided herein.

[0010] In an alternative embodiment, a method for assessing the degree of CEACAM5 expression, comprising contacting a sample or a portion thereof comprising cancer or tumor cells derived from an individual with an antibody or a portion thereof that specifically binds to CEACAM5, and Determining a tumor intensity percentage score (TIPS) by dividing the number of CEACAM5-stained viable tumor or cancer cells in a sample or a portion thereof specifically bound by an antibody by the total number of stained and non-stained viable cancer or tumor cells, and multiplying the result by 100, thereby obtaining a tumor intensity percentage score A method is provided that includes

[0011] Details of one or more exemplary embodiments of the invention are set forth in the accompanying drawings and the following description. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims

[0012] All publications, patents, and patent applications cited herein are hereby expressly incorporated by reference in their entirety for all purposes

[0013] [Description of Drawings] The patent or application document contains at least one drawing created in color. Copies of this patent or patent application publication with color drawings will be provided by the Patent Office upon request and payment of the necessary fee

[0014] The drawings presented herein illustrate the exemplary embodiments provided herein and are not meant to limit the scope of the invention as encompassed by the claims

[0015] The drawings are described in detail herein

[0016] Like reference symbols in the various drawings indicate like elements [Modes for Carrying Out the Invention]

[0017] In an alternative embodiment, an immunohistochemistry (IHC) method is provided for determining and scoring the degree of expression of the protein carcinoembryonic antigen-related cell adhesion molecule 5 or CEACAM5 (also known as CD66e (surface antigen classification 66e)) in a tissue sample. In an alternative embodiment, a scoring method is provided for assessing CEACAM5 expression in tumors or cancers such as non-squamous non-small cell lung cancer (nsNSCLC).

[0018] Alternative embodiments of the IHC method herein provide a robust, reliable, standardized, reproducible, and harmonized method for assessing the degree of cell membrane expression of CEACAM5, thus providing greater inter-facility and inter-assay comparability and enabling earlier and proper application of CEACAM5 in diagnostics. Embodiments of the IHC method herein provide high-quality staining and reliable diagnostic assessment.

[0019] The current immunohistochemical scoring method for CEACAM5 measures the percentage of cells in a tissue sample that show total membrane staining and luminal (polarized) membrane staining. These percentages of total staining versus luminal staining are placed into separate scoring categories. The percentages are further broken down into staining intensity bins. Only staining intensities of 2+ and 3+ are considered in determining the final CEACAM5 score in relation to the cut-off. Dividing the membrane expression level scoring into multiple categories and staining intensity bins complicates the scoring process. Such greater complexity of the scoring method then adds to the time required to reach a diagnostic evaluation. Pathologists may also vary their individual determinations regarding the presence or relative staining intensity of the membrane and luminal staining patterns among the various cells in the tissue sample, making the reproducibility of the scoring method difficult. Further, in some cancers including non-small cell lung cancer (nsNSCLC) with non-squamous epithelium, the tumor morphology is heterogeneous, so there may be confusion regarding how to apply the scoring guidelines. There remains a need in the art for a simpler, more efficient, and robust, reproducible, accurate scoring method for evaluating CEACAM5 expression in tissue samples.

[0020] Embodiments herein are directed to immunohistochemical methods for determining and scoring the degree of cell membrane expression of carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) in tissue samples. In various embodiments, the method comprises staining a tissue sample with an antibody that specifically binds to CEACAM5, determining the total number of viable tumor or cancer cells having CEACAM5 cell membrane staining, and determining the total number of stained and non-stained viable tumor or cancer cells in at least a portion of the tissue sample, wherein the tumor or cancer cells are counted as being positively stained with the anti-CEACAM5 antibody if there is any intensity of CEACAM5 cell membrane staining above a defined threshold, as well as determining a tumor intensity percentage score (TIPS), wherein TIPS is the number of CEACAM5 stained viable tumor or cancer cells found in the tissue sample divided by the total number of stained and non-stained viable tumor or cancer cells, multiplied by 100. In various embodiments herein, the tumor intensity percentage score (TIPS) is also referred to as the CEACAM5 positive score. Such embodiments can provide not only a robust, reliable, reproducible, and accurate CEACAM5 scoring method, but also a scoring method with greater simplicity and efficiency. Such embodiments can provide considerable advantages for reliable and efficient diagnostic evaluation.

[0021] In certain embodiments, CEACAM5 cell membrane staining includes total membrane staining, discontinuous membrane staining, partial membrane staining, complete membrane staining, punctate membrane staining, linear membrane staining, luminal membrane staining, apical membrane staining, basal membrane staining, lateral membrane staining, lateral basal membrane staining, high staining of cytoplasm with a positive staining intensity of 2+ or more, high staining of cytoplasm with a positive staining intensity of 3+, or combinations thereof. In certain embodiments, the total number of viable tumors or cancer cells includes luminal cells, glandular luminal cells, intraluminal cells, multiple layers of cells, cells having a signet ring morphology, cells with high cytoplasmic staining, cells having an intracellular lumen, or combinations thereof. In certain embodiments, the total number of viable tumors or cancer cells counted as being positively stained includes all glandular luminal cells having a cell membrane associated with a positively stained lumen. Such embodiments can provide the advantage of including cell membrane staining, cytoplasmic staining, and all observed patterns and levels of all types of viable tumors or cancer cells in the determination of a tumor intensity ratio score. Such embodiments can provide the benefit of a simplified IHC scoring method for scoring the cell membrane expression of CEACAM5 in tissue samples.

[0022] In certain embodiments, the tissue sample includes at least about 100 cells. In certain embodiments, the tissue sample includes at least about 200 cells. In certain embodiments, the tissue sample includes at least about 500 cells.

[0023] In some embodiments, the tumors or cancer cells counted as being positively stained do not include tumor or cancer cells having cytoplasmic staining, staining of normal or non-neoplastic structures, staining of non-viable tumors or cancer cells, necrotic cells, cell fragments, stromal staining, or edge artifacts staining at the periphery of the tissue sample. In certain embodiments, the cytoplasmic staining includes cytoplasmic staining with a positive staining intensity of 2+ or less. Such embodiments can provide the advantage of enhancing the accuracy and reproducibility of the CEACAM5 scoring method by excluding stained cells having certain structures or features from the tumor intensity ratio score (TIPS) determination.

[0024] In certain embodiments, the defined threshold includes a positive staining intensity of 2+ or greater. In certain embodiments, the defined threshold includes a 2+ or 3+ positive staining intensity evaluated at a magnification of about 4× to about 10×. In certain embodiments, the method further includes the step of confirming the positive staining intensity at a magnification of about 20× to about 40×. In certain embodiments, the tumor intensity ratio score includes the number of CEACAM5 positive-stained viable tumors or cancer cells with a positive staining intensity of 2+ or greater, divided by the total number of stained and non-stained viable tumors or cancer cells, and multiplied by 100. Such embodiments can provide the benefit of versatility at the defined threshold staining intensity.

[0025] In certain embodiments, a tumor intensity ratio score (TIPS) of about 40% or greater indicates the diagnostic status of the tissue sample. In certain embodiments, the TIPS is about 50% or greater, about 60% or greater, about 80% or greater, or about 90% or greater. In certain embodiments, a TIPS of about 5% or greater indicates the diagnostic status of the tissue sample. In certain embodiments, a TIPS of about 1% or greater indicates the diagnostic status of the tissue sample. In certain embodiments, a TIPS of about 5% or greater includes the number of CEACAM5 positive-stained viable tumors or cancer cells with a positive staining intensity of 2+ or greater, or about 10% or greater of the TIPS includes the number of CEACAM5 positive-stained viable tumors or cancer cells with a positive staining intensity of 2+ or greater. Such embodiments can provide the benefit of accuracy in determining the diagnostic status associated with CEACAM5 expression.

[0026] In an alternative embodiment, an IHC method is provided that includes a simplified scoring system for membrane staining with a threshold of ≧2+ intensity, and guidance is provided for addressing different morphological patterns of luminal membrane staining. Include luminal tumor cells in the CEACAM5 evaluation when viability and tumor cell identification can be confirmed. Guidance for evaluating tumor cells with strong (3+) cytoplasmic staining that can obscure the membrane staining when tumor cells with clear membrane staining are present within the same 10× field of view.

[0027] In alternative embodiments, the methods and kits as provided herein are used for in vitro diagnostic use. In alternative embodiments, CEACAM5 IHC as provided herein is an immunohistochemical (IHC) assay that uses an anti-CEACAM5 antibody, such as monoclonal mouse anti-CEACAM5 clone 769, in the detection of CEACAM5 protein in formalin-fixed paraffin-embedded (FFPE) tissue samples, such as lung cancer tumor tissue samples. In alternative embodiments, the EnVision FLEX™ visualization system on Dako OMNIS™ is used.

[0028] In alternative embodiments, CEACAM5 IHC as provided herein is used to assist in the identification of patients having a tumor or cancer, such as non-squamous non-small cell lung cancer (nsNSCLC), non-small cell lung cancer (NSCLC), adenocarcinoma, synovial sarcoma, myxoid / round cell liposarcoma, high-grade myxoid liposarcoma, low-grade myxoid liposarcoma, head and neck cancer, melanoma, esophageal cancer, gastric cancer, colorectal cancer, lung cancer, colon cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, prostate cancer or urothelial cancer.

[0029] In alternative embodiments, a method for assessing the degree of CEACAM5 expression comprises contacting a sample or a portion thereof comprising cancer or tumor cells from an individual with an antibody or a portion thereof that specifically binds to CEACAM5, and dividing the number of CEACAM5-stained viable tumor or cancer cells in the sample or the portion thereof specifically bound by the antibody by the total number of stained and non-stained viable cancer or tumor cells, and multiplying the result by 100 to determine a tumor intensity percentage score, thereby obtaining a tumor intensity percentage score.

[0030] In certain embodiments, a section or portion of a tissue sample is prepared on a slide, microscope slide, or equivalent, and the section or portion of the tissue sample is stained on the slide. In certain embodiments, the tissue sample includes a formalin-fixed paraffin-embedded (FFPE) specimen. In certain embodiments, the FFPE specimen includes a cancer specimen stained on an automated IHC platform. In certain embodiments, a section of the tissue sample is prepared by a protocol that includes fixation in about 10% neutral buffered formalin for a period of about 6 hours to about 72 hours. In certain embodiments, the tissue sample is derived from a needle biopsy sample, fine needle aspirate, cytology specimen, or decalcified bone.

[0031] In certain embodiments, the antibody includes a monoclonal mouse anti-CEACAM5 antibody or a monoclonal rabbit anti-CEACAM5 antibody. In some embodiments, the monoclonal mouse anti-CEACAM5 antibody includes monoclonal mouse anti-CEACAM5 clone 769, or an antibody having an affinity substantially equivalent to that of clone 769 antibody against CEACAM5.

[0032] In certain embodiments, the tumor or cancer is non-squamous non-small cell lung cancer (nsNSCLC), non-small cell lung cancer (NSCLC), adenocarcinoma, synovial sarcoma, myxoid / round cell liposarcoma, high-grade myxoid liposarcoma, low-grade myxoid liposarcoma, head and neck cancer, melanoma, esophageal cancer, gastric cancer, stomach cancer, colorectal cancer, lung cancer, colon cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, prostate cancer, or urothelial cancer.

[0033] In certain embodiments, positive staining is determined using a brightfield microscope, microscope objective lens, computer monitor, and imaging software, or a combination thereof. In some aspects, the imaging software includes whole slide imaging software.

[0034] <Embodiments of a method for diagnosing a tumor or cancer> Embodiments herein provide a method for diagnosing a tumor or cancer by determining whether a tissue sample is positive for the expression of carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5). In various aspects, the method includes determining the CEACAM5 diagnostic status in a tissue sample by an IHC method for determining the degree of cell membrane expression of CEACAM5 as provided herein, and a tumor intensity ratio score of about 50% or more of tumor cancer cells having a CEACAM5 cell membrane expression of intensity 2+ or more is diagnostically positive. In certain embodiments, the tumor or cancer is non-squamous non-small cell lung cancer (nsNSCLC), non-small cell lung cancer (NSCLC), adenocarcinoma, synovial sarcoma, myxoid / round cell liposarcoma, head and neck cancer, melanoma, esophageal cancer, gastric cancer, colorectal cancer, lung cancer, colon cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, prostate cancer or urothelial cancer.

[0035] <Embodiments of methods for treating cancer and tumors> A method for treating or ameliorating a tumor or cancer in a patient, the method comprising determining and scoring the amount of CEACAM5 in a patient-derived tissue sample using a method as provided herein, and if the tissue sample is determined or scored to have a CEACAM5 score that is high or diagnostically positive, the patient is treated with a cancer therapeutic agent or anti-cancer therapy to which the patient is likely to respond favorably. The tumor or cancer can be non-squamous non-small cell lung cancer (nsNSCLC), non-small cell lung cancer (NSCLC), adenocarcinoma, synovial sarcoma, myxoid / round cell liposarcoma, head and neck cancer, melanoma, esophageal cancer, gastric cancer, colorectal cancer, lung cancer, colon cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, prostate cancer or urothelial cancer.

[0036] In certain embodiments, the cancer therapeutic agent comprises the administration of an anti-cancer drug or anti-cancer therapy to a patient. In alternative embodiments, the anti-cancer treatment or therapy includes surgery such as cyberknife therapy, chemoembolization, ablation techniques such as radiofrequency ablation (RFA), cryoablation necrosis treatment and / or microwave ablation, and / or radiation therapy such as stereotactic body radiotherapy.

[0037] In certain embodiments, the anti-cancer therapy includes antibody-drug conjugates, small molecule therapies, immunotherapies, monoclonal antibody therapies, adoptive cell therapies, T cell receptor therapies or chimeric antigen receptor (CAR) T cell therapies. In alternative embodiments, the anti-cancer treatment or therapy includes tyrosine kinase inhibitors (optionally, erlotinib (or TARCEVA (trademark)), gefitinib (or IRESSA (trademark)), afatinib (or GILOTRIF (trademark)) or osimertinib (TAGRISSO (trademark))), necitumumab (or PORTRAZZA (trademark)), pembrolizumab (or KEYTRUDA (trademark)), nivolumab (or OPDIVO (trademark)), ipilimumab (YERVOY (trademark)), cetuximab (or ERBITUX (trademark)), cisplatin (or PLATINOL (trademark)) or carboplatin (or PARAPLATIN (trademark))).

[0038] In an alternative embodiment, the anti-cancer treatment or therapy may include the use of an anti-cancer agent that includes an antibody that specifically binds, or substantially binds, to a cancer or tumor, where the antibody is conjugated to a cytotoxic agent, and optionally, the cytotoxic agent includes a radionuclide (optionally, yttrium-90, iodine-131, lutetium-177, radium-223 chloride, strontium-89 chloride or samarium-153 EDTMP), diphtheria toxin, Pseudomonas exotoxin A, denileukin diftitox, moxetumomab pasudotox, calicheamicin or N-acetyl-γ-calicheamicin, emtansine or DM1, maytansine or a derivative thereof, SN-38 (or 7-ethyl-10-hydroxycamptothecin) or an auristatin or monomethyl auristatin E (MMAE).

[0039] <Immunohistochemistry> In alternative embodiments, immunohistochemistry methodologies and / or reagents used with the methods and manufactured products or kits as provided herein are those known in the art, for example, U.S. Patent No. (USPN) 10,634,590 (describing a slide holder assembly fixture for use in IHC); U.S. Patent No. 10,565,479 (describing a method for identifying unclear regions in digital images of stained tissue); U.S. Patent No. 10,564,076 (describing a system for analytical (or IHC) sample preparation); No. 10,551,395 (describing an automated histological staining system); No. 10,551,378 (describing a tissue staining method); No. 10,504,224 (describing a digital tissue image analysis system for IHC); No. 10,501,777 (describing simultaneous, multiplex detection and quantification of protein expression in IHC); No. 10,488,340 (describing a method for extracting images of target fluorophores in biological materials); No. 10,453,195 (describing a method for detecting a tissue region of interest using digital pathology imaging); No. 10,438,381 (describing a device, system and method for creating digital images of tissue sections); No. 10,430,943 (describing a method and program for automated nuclear region / number estimation for IHC image analysis); No. 10,416,176 (describing a method for processing specimens in an automated histological staining system); No. 10,393,633 (describing a method for processing and inhibiting the degradation of IHC samples); No. 10,217,011 (describing the handling of IHC slides); No. 10,209,165 (describing an automated or semi-automated method for evaluating the quality of staining of specimens containing cells); No. 10,126,216 (describing a method for fixing tissue samples for IHC); No. 9,423,322; No. 8,515,683 (describing a method and system for automated detection of immunohistochemical (IHC) patterns); USPN No. 10,816,443 (describing an automated batch staining device for staining biological specimens on microscope slides);Or U.S. Patent Application Publication No.: US2019 / 0178867 A1 (describing the detection of specific tissue targets in thin sections of tissue samples imaged in a brightfield microscope without using a chromogenic stain specific for those tissue targets); US2019 / 0156510 A1 (describing an image analysis method for IHC tissue sample analysis); US2019 / 0293637 A1 (methods and systems for quantitative immunohistochemistry (IHC) of target protein molecules); US2019 / 0080450 A1 (describing an automated determination of the staining quality of IHC-stained biological samples); or US2020 / 0316589 A1 (describing a multiwell solid-phase support container for treating and testing fixed biological materials). It may include, or consist of, or consist of the use of any IHC protocol, IHC medical equipment, device and / or image or data analysis system, or IHC reagent for performing IHC as described therein.

[0040] In an alternative embodiment, the antibodies, antigen-binding fragments thereof or monomeric or dimeric antigen-binding proteins (e.g., including synthetic or recombinant forms) as provided herein and used in an IHC protocol or kit as provided herein are substantially purified or isolated or are in the form of an unpurified or partially purified culture supernatant.

[0041] In an alternative embodiment, the methods as provided herein can use or include reagents for detecting or visualizing antibody-antigen interactions using any product or method known in the art, such as an IHC protocol or reagent.

[0042] In an alternative embodiment, the methods as provided herein include the use of chromogenic immunohistochemistry (CIH), where the primary antibody (e.g., a recombinant antibody (Ab) as provided herein or an antigen-binding fragment or monomeric or dimeric antigen-binding protein thereof) or the secondary antibody (e.g., the secondary antibody binds to a primary antibody or recombinant antibody (Ab) or an antigen-binding fragment or monomeric or dimeric antigen-binding protein thereof as provided herein) is conjugated to an enzyme capable of catalyzing a chromogenic reaction, e.g., peroxidase, e.g., immunoperoxidase), e.g., horseradish peroxidase (HRP). In an alternative embodiment, the chromogenic moieties used in the methods as provided herein are coumarin, rhodamine, 2,3,6,7-tetrahydro-11-oxo-1H,5H,11H-[1]benzopyrano[6,7,8-ij]quinolizin-1-0-carboxylic acid, 7-(diethylamino)coumarin-3-carboxylic acid, coumarin derivatives, rhodamine derivatives, tetramethylrhodamine, diarylrhodamine derivatives, QSY 7, QSY 9, QSY 21, diazo chromophores, DABSYL, tartrazine, triarylmethane compounds, fast red, fast blue, fuchsine, cascade blue acetyl, Dapoxyl sulfonic acid / carboxylic acid succinimidyl ester, DY-405, Alexa Fluor 405 succinimidyl ester, cascade yellow succinimidyl ester, pyridyloxazole succinimidyl ester (PyMPO), pacific blue succinimidyl ester, DY-415, 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester, DYQ-425, 6-FAM phosphoramidite, lucifer yellow, iodoacetamide, Alexa Fluor 430 succinimidyl ester, Dabcyl succinimidyl ester, NBD chloride / fluoride, QSY 35 succinimidyl ester, DY-485XL, Cy2 succinimidyl ester, DY-490, Oregon Green 488 carboxylic acid succinimidyl ester, Alexa Fluor 488 succinimidyl ester, BODIPY 493 / 503C3 succinimidyl ester, DY-480XL, BODIPY FL C3 succinimidyl ester, BODIPY FL C5 succinimidyl ester, BODIPY FL-X succinimidyl ester, DYQ-505, Oregon Green 514 carboxylic acid succinimidyl ester, DY-510XL, DY-481XL, 6-carboxy-4’,5’-dichloro-2’,7’-dimethoxyfluorescein succinimidyl ester (JOE), DY-520XL, DY-521XL, BODIPY R6G C3 succinimidyl ester, erythrosin isothiocyanate, 5-carboxy-2’,4’,5’,7’-tetrabromosulfonefluorescein succinimidyl ester, Alexa Fluor 532 succinimidyl ester, 6-carboxy-2’,4,4’,5’7,7’-hexachlorofluorescein succinimidyl ester (HEX), BODIPY 530 / 550 C3 succinimidyl ester, DY-530, BODIPY TMR-X succinimidyl ester, DY-555, DYQ-1, DY-556, Cy3 succinimidyl ester, DY-547, DY-549, DY-550, Alexa Fluor 555 succinimidyl ester, Alexa Fluor 546 succinimidyl ester, DY-548, BODIPY 558 / 568 C3 succinimidyl ester, rhodamine red-X succinimidyl ester, QSY 7 succinimidyl ester, BODIPY 564 / 570 C3 succinimidyl ester, BODIPY 576 / 589 C3 succinimidyl ester, carboxy-X-rhodamine (ROX), succinimidyl ester, Alexa Fluor 568 succinimidyl ester, DY-590, BODIPY 581 / 591 C3 succinimidyl ester, DY-591, BODIPY TR-X succinimidyl ester, Alexa Fluor 594 succinimidyl ester, DY-594, carboxynaphthofluorescein succinimidyl ester, DY-605, DY-610, Alexa Fluor 610 succinimidyl ester, DY-615, BODIPY 630 / 650-X succinimidyl ester, erioglaucine, AlexaFluor 633 succinimidyl ester, Alexa Fluor 635 succinimidyl ester, DY-634, DY-630, DY-631, DY-632, DY-633, DYQ-2, DY-636, BODIPY 650 / 665-X succinimidyl ester, DY-635, Cy5 succinimidyl ester, Alexa Fluor 647 succinimidyl ester, DY-647, DY-648, DY-650, DY-654, DY-652, DY-649, DY-651, DYQ-660, DYQ-661, Alexa Fluor 660 succinimidyl ester, Cy5.5 succinimidyl ester, DY-677, DY-675, DY-676, DY-678, Alexa Fluor 680 succinimidyl ester, DY-679, DY-680, DY-682, DY-681, DYQ-3, DYQ-700, Alexa Fluor 700 succinimidyl ester, DY-703, DY-701, DY-704, DY-700, DY-730, DY-731, DY-732, DY-734, DY-750, Cy7 succinimidyl ester, DY-749, DYQ-4, Cy7.5 succinimidyl ester, 7-diethylaminocoumarin-3-carboxylic acid, succinimidyl ester, dabsylsulfonyl chloride, fluorescein isothiocyanate (FITC) carboxysuccinimidyl ester (DY-495), rhodamine green carboxylic acid succinimidyl ester (DY-505), eosin isothiocyanate (EITC), 6-carboxy-2’,4,7,7’-tetrachlorofluorescein succinimidyl ester (TET), carboxyrhodamine 6G succinimidyl ester, carboxytetramethylrhodamine succinimidyl ester (TMR, TAMRA) (DY-554), QSY 9 succinimidyl ester, sulforhodamine B sulfonyl chloride (DY-560), Texas Red (sulforhodamine 101), galocyanine, fast green FCF, malachite green or QSY 21 succinimidyl ester, or comprising the same.

[0043] In alternative embodiments, the methods as provided herein include the use of immunofluorescence, and the primary or secondary antibody is tagged with a fluorophore, such as fluorescein or fluorescein isothiocyanate (FITC), a triarylmethane dye, such as rhodamine or a rhodamine derivative (e.g., tetramethylrhodamine (TRITC), rhodamine 6G, rhodamine 123, rhodamine B, carboxytetramethylrhodamine (TAMRA), tetramethylrhodamine (TMR), sulforhodamine 101), aminomethylcoumarin acetate (AMCA), ALEXA™ or DYLIGHT™ fluor, or a fluorophore or dye as described in U.S. Patent Application No. US2019 / 0018018 A1. 3,3'-Diaminobenzidine (DAB) can also be used.

[0044] In alternative embodiments, the methods as provided herein include the use of a direct or one-step staining method, where the primary antibody (e.g., an antibody (Ab) as provided herein or an antigen-binding fragment or monomeric or dimeric antigen-binding protein thereof (including synthetic or recombinant forms)) is labeled and reacts directly with the antigen, e.g., in a tissue section. This technique utilizes only one antibody and is thus simple and rapid but may be less sensitive due to limited signal amplification.

[0045] In alternative embodiments, the methods as provided herein include the use of an indirect method where an unlabeled primary antibody (first layer) binds to a target antigen (e.g., CEACAM5) in, e.g., a tissue or organ, and then a labeled secondary antibody (second layer) reacts with the primary antibody. The secondary antibody can be specific for the isotype of the animal species from which the primary antibody is derived, e.g., IgG. This method can be more sensitive than direct detection strategies due to signal amplification by the binding of several secondary antibodies to each primary antibody when the secondary antibody is conjugated to a detection agent such as a fluorophore or enzyme reporter.

[0046] In an alternative embodiment, further amplification is achieved when the secondary antibody is conjugated to a biotin molecule that can recruit several detection molecules, such as complexes of avidin-, streptavidin- or NEUTRAVIDIN™ protein-binding enzymes.

[0047] In an alternative embodiment, IHC is performed on tissue sections or tissue biopsies, such as paraformaldehyde (PFA)-fixed tissues or organs, or formalin-fixed paraffin-embedded tissues. In an alternative embodiment, the tissue is sectioned, sliced, or used whole. Prior to sectioning, the tissue sample can be embedded in a medium, such as paraffin wax or cryomedia. Tissue sections can be sectioned or sliced using various instruments, most commonly a microtome, cryostat or vibratome. The specimen can be sectioned or sliced in the range of about 3 μm to 5 μm. The sections or slices are mounted on slides, dehydrated using a gradient of alcohol washes (e.g., 50%, 75%, 90%, 95%, 100%), cleared using a clearing agent such as xylene, and then imaged under a microscope.

[0048] Depending on the method of fixation and tissue preservation, the sample may require additional steps including deparaffinization and antigen retrieval to make the CEACAM5 epitope available for antibody binding. In formalin-fixed paraffin-embedded tissues, antigen retrieval is often required and may include pretreatment of the sections with heat or protease.

[0049] In an alternative embodiment, IHC is performed using the ENVISION DUOFLEX DOUBLESTAIN SYSTEM™ (EnVision DuoFLEX Doublestain System) (Agilent, San Jose, CA) which enables staining of two or more markers on a single slide. In an alternative embodiment, IHC is performed using the EnVision FLEX HRP Magenta, High pH (Dako Omnis) system, and the binding can be visualized by the EnVision FLEX HRP magenta chromogen. In an alternative embodiment, IHC is performed using the EnVision FLEX Mini Kit, High pH, a high-sensitivity visualization system intended for use in IHC together with a Dako AUTOSTAINER™ instrument, and this dual-link system detects primary mouse and rabbit antibodies and the reaction is visualized by the 3,3'-diaminobenzidine (DAB) chromogen (DAB forms a water-insoluble brown precipitate when oxidized, for example, by peroxidase).

[0050] <Manufactured products and kits> Manufactured products and kits for carrying out methods as provided herein and / or reagents for performing IHC, including, for example, monoclonal mouse anti-CEACAM5 antibodies, such as clone 769 anti-CEACAM5 antibody, see Example 1 below, are provided, and optionally, the manufactured products and kits may further include instructions for use for carrying out methods as provided herein.

[0051] Any of the above aspects and embodiments can be combined with any other aspect or embodiment as disclosed herein in the summary, drawings, and / or detailed description sections.

[0052] As used in this specification and the claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0053] Unless explicitly stated otherwise or otherwise apparent from the context, as used in this specification, the term "or" is to be understood as inclusive and applies to both "or" and "and".

[0054] Unless explicitly stated otherwise or otherwise apparent from the context, in this specification, the term "about" is understood to be within the normal tolerance ranges in the art, for example, within two standard deviations of the mean. About (the use of the term "about") can be understood to be within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the indicated value. Unless clear from the context, all numerical values provided in this specification are modified by the term "about".

[0055] Unless explicitly stated otherwise or otherwise apparent from the context, as used in this specification, the terms "substantially all", "substantially most", "substantially all of" or "a majority of" include at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% or more of the recited amount of the composition.

[0056] The entire contents of each patent, patent application, publication, and document referred to herein are hereby incorporated by reference into this specification. The citation of such patents, patent applications, publications, and documents does not constitute an admission that any of the foregoing is relevant prior art, nor does it constitute any admission as to the content or date of these publications or documents. The incorporation by reference of these documents should not be construed as an assertion or admission that any part of the content of any document is essential material for meeting the statutory disclosure requirements of any country or region for a patent application. Nevertheless, the right to rely on any of such documents, as necessary, to provide material that is considered essential to the claimed subject matter by a reviewing authority or court is reserved.

[0057] Modifications may be made to the foregoing without departing from the basic aspects of the present invention. Although the present invention has been described in considerable detail with respect to one or more specific embodiments, those skilled in the art will recognize that changes may be made to the embodiments specifically disclosed herein, and that these modifications and improvements are within the scope and spirit of the present invention. The invention illustrated herein may, as appropriate, be practiced in the absence of any element(s) (which may be plural) not specifically disclosed herein. Thus, for example, in each example herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms. Accordingly, the terms and expressions used are used as terms of description and not of limitation, and equivalents or portions thereof of the features shown and described are not excluded, and it is recognized that various modifications may be within the scope of the present invention. Embodiments of the present invention are set forth in the following claims.

[0058] The present invention will be further described in connection with the embodiments described herein, but it should be understood that the present invention is not limited to such embodiments.

Examples

[0059] Unless otherwise specified in the examples, all recombinant DNA techniques are carried out according to standard protocols, for example, as described in Sambrook et al. (2012) Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, NY as well as Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA, Volumes 1 and 2. Other references for standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK).

Brief Description of Drawings

[0060]

Figure 1

Figure 2

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Figure 9A - B

Figure 10A - B

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[0061] [Example 1: CEACAM5 IHC] This example demonstrates the use of the CEACAM5 staining protocol as provided herein.

[0062] Exemplary scoring guidelines are provided herein for the evaluation of CEACAM5 expression in non-squamous non-small cell lung cancer (non-squamous NSCLC) specimens stained with a mouse monoclonal CEACAM5 antibody called mouse mAb clone 769. This antibody is provided in the CEACAM5 IHC 769 assay for the identification of CEACAM5 expression in formalin-fixed paraffin-embedded (FFPE) non-squamous NSCLC specimens using the Dako Omnis automated staining system.

[0063] Evaluation of the CEACAM5 IHC staining pattern in non-squamous non-small cell lung cancer (non-squamous NSCLC) FFPE tissue. Complete the protocol-specific Pathology Report Form (PRF) for each specimen evaluated (including "non-evaluable" and "indeterminate" as described below).

[0064] <CEACAM5 Scoring Summary> <Definition of Positive Staining> CEACAM5 positive staining is defined as any partial or complete tumor cell membrane staining of ≧2+ intensity. Membrane staining can be linear or punctate. Staining may appear as complete or partial staining including the basal, outer, or basolateral aspects of the membrane, or as apical staining of the luminal aspect of the cell.

[0065] See Figures 1 - 19, representative staining examples are described below.

[0066] The CEACAM5 positive percent score is determined by the following formula:

[0067]

Number

[0068] To determine the percentage of stained cells, at least 100 evaluable viable tumor cells must be present in the CEACAM5 stained slide.

[0069] The overall CEACAM5 diagnostic status is determined according to the following criteria: · If ≧X% of the tumor cells in the specimen show ≧2+ intensity CEACAM5 positive staining, where X is a predetermined cut-off, the specimen is considered diagnostically positive. · If <X% of the tumor cells in the specimen show ≧2+ intensity CEACAM5 positive staining, where X is a predetermined cut-off, the specimen is considered diagnostically negative.

[0070] For adenocarcinomas showing glandular lumens, CEACAM5 partial cell membrane staining may appear as apical membrane staining on the luminal side of the cells. In the case of ≧2+ intensity luminal staining, all cells associated with a structure that may have a membrane associated with the positive lumen are considered positive.

[0071] <Definition of negative staining> Negative staining is defined as no membrane staining or any membrane staining of tumor cells with <2+ intensity.

[0072] Scoring includes the following: · ≧2+ intensity, partial or complete, punctate membrane staining and linear membrane staining are considered positive. · All viable tumor cells on the slide are evaluated for both the intensity and frequency of positive staining.

[0073] Cell types and staining patterns to be excluded from staining scoring: ·Cytoplasmic staining is generally not considered positive staining, but positive cytoplasmic staining may be inferred for tumor cells with intense cytoplasmic staining (3+) when positive membrane staining is identified in other tumor cells present within the 10× objective field of view of highly stained cells in the cytoplasm. Further details and examples are provided in Section 2.1 and Figure 4. ·Staining of normal, non-neoplastic structures (Figure 5) ·Detached non-viable tumor cells ·Necrotic cells and cell debris (Figure 6) ·Stromal staining (Figure 7) ·Edge artifact staining at the periphery of tissue specimens

[0074] <Scoring of non-squamous NSCLC> Staining intensity is scored using the following scale (Table 1):

[0075] JPEG2025522755000003.jpg49170

[0076] Examples of CEACAM5 staining at each intensity are shown in Figure 8.

[0077] CEACAM5-positive tumor staining of ≥2+ is scored using a 4× to 10× microscope objective lens. Using a 20× objective lens, 1+ and punctate and / or delicate 2+ membrane staining intensities and 2+ intracellular lumens that may not be apparent with 4× and 10× objective lenses can be identified and / or confirmed. Using a 20× to 40× objective lens, membrane staining localization can be confirmed as needed. A 40× objective lens should not always be used for scoring (i.e., for assessing intensity and percent positive).

[0078] This assay has been evaluated on non-squamous NSCLC tissue samples. To allow for proper confirmation of the presence of the tumor and histological diagnosis, to evaluate tumor viability, and to allow for discrimination from non-tumor portions of the slides that should be excluded from the evaluation, each CEACAM5 stained sample should be accompanied by an H&E stained slide. H&E and NCR stained slides can also assist in the identification of endogenous pigments (e.g., due to endogenous anthracosis, hemosiderin pigment, etc., see for example Figure 9).

[0079] CEACAM5 expression patterns observed in non-squamous NSCLC: · Luminal staining: In lung adenocarcinoma, two morphological patterns of structures showing luminal (apical membrane) staining can be observed: nested and alveolar patterns. If ≥2+ intensity luminal staining is observed in either pattern, all cells within structures that may have a membrane associated with the positive lumen should be considered positive.

[0080] · Nested pattern: Solid nests separated by stroma and containing multiple layers of cells and small concentric luminal spaces. Tumors with solid tumor nests containing glandular lumens may show CEACAM5 apical membrane staining of the lumen. In the case of ≥2+ intensity luminal staining, it is assumed that all cells within the nest may have a membrane association with the lumen, and thus all cells within the nest should be considered positive (Figures 10 and 11).

[0081] · Alveolar pattern: Glandular structures (single or multiple layers) containing irregularly shaped luminal spaces of various sizes. Cells showing ≥2+ intensity apical membrane staining on the luminal surface are considered positive. The cell layers behind the cells facing those lumens may also have a membrane associated with the positive lumen and should thus be considered positive as well. Further details and examples are provided in the attached Figures 3 and 12.

[0082] Cells with signet ring morphology: Cells with signet ring cell morphology may show intense staining in the vacuolar lumen and cytoplasm. In these cases, membrane staining may be difficult to distinguish due to the intensity of cytoplasmic staining. For cells with signet ring cell morphology, highly stained tumor cells are considered positive for membrane staining and scored for cytoplasmic intensity. For example, if the cytoplasmic intensity is ≧2+ in cells with signet ring morphology, the cells are considered positive for CEACAM5 (Figure 13).

[0083] Highly stained cytoplasmic cells: Cytoplasmic staining without obvious membrane staining is generally not considered positive staining. However, if positive membrane staining is identified in tumor cells within the 10× objective field of highly stained cytoplasmic cells, positivity can be inferred for highly stained cytoplasmic tumor cells (3+ intensity) (Figure 4).

[0084] Viable cells in the lumen: Viable tumor cells in the lumen and alveolar space should be included in the scoring if the membrane staining is ≧2+ intensity and viability can be confirmed. Corresponding H&E slides may be required to determine viability. Care must be taken to distinguish tumor cells from macrophages that should not be scored. If the cells are non-viable, or viability or tumor identity cannot be confirmed, the lumen cells should be excluded (Figure 14).

[0085] Intralumens within the cytoplasm: Cells with intralumens within the cytoplasm having an intralumenal staining of ≧2+ intensity are considered positive (Figure 15). H&E may be used to determine whether the specimen contains this expression pattern.

[0086] <Non-specific background staining> The non-specific background staining of NCR and CEACAM5 stained slides must be ≦1+ average intensity throughout the specimen.

[0087] The following are excluded from the evaluation of non-specific background staining: · The stroma near the highly stained tumor cells may have some staining. Stromal staining is not scored as non-specific background staining (Figure 7). · Necrosis can be highly stained but is not scored as non-specific background staining (Figure 6). · Mucin can be highly stained but is not scored as non-specific background staining (Figure 16).

[0088] Non-specific background staining is evaluated across the entire specimen and is not local (i.e., in regions that are a small proportion of the total area). Local staining of >1+ intensity is offset by regions without staining, such that it is acceptable if the average intensity across the entire specimen is ≦1+.

[0089] <System control> Before examining the IHC-stained tumor specimen(s), confirm that the IHC staining of the control tissue is good.

[0090] Perform positive control tissue and negative control tissue in parallel with the patient tissue. Perform NCR staining on the control tissue to confirm that there is no non-specific staining within the staining procedure. See Figure 17 for a representative image of the positive control tissue and Figure 18 for a representative image of the negative control tissue.

[0091] <Control tumor tissue> The control tumor tissue must be identified from pre-screened non-squamous NSCLC tissue. The control must be a fresh biopsy / surgical non-squamous NSCLC specimen that was fixed, processed, and embedded as soon as possible in the same manner as the test specimen(s).

[0092] <Negative control reagent (NCR)> The negative control reagent contains antibodies with matching non-specific species to CEACAM5. Control tissues stained with NCR should not show specific membrane staining, and the presence of non-specific background staining should have an average intensity score of ≤ 1+ across the entire specimen. If specific staining occurs in the NCR-stained tissue, the results are considered invalid, the "failure" checkbox in the scoring form must be recorded, and staining must be repeated for both control and test specimens.

[0093] <Positive control tissue> · Positive control tissue is evaluated to ensure tissue preparation and good staining technique. One positive control tissue is included in each staining procedure. · The tissue selected to be used as a positive tissue control should yield moderately positive staining (≥ 2+ intensity) to detect slight variations in assay sensitivity. · Non-specific background staining should have an average intensity of ≤ 1+ across the entire specimen (see section 2.2). · Only reagent performance is confirmed by specimens processed differently from the test sample(s), and tissue preparation is not verified. · Known positive tissue controls should only be used to monitor the correct performance of the processed tissue and test reagent and should not be used as an aid in formulating a specific diagnosis of patient samples.

[0094] <Negative control tissue> · Non-squamous NSCLC negative control tissue is evaluated to ensure tissue preparation, good staining technique, verify the specificity of the primary antibody, and provide an indication of non-specific staining. · The various cell types present in most tissue sections provide an internal negative control site (which must be verified by the user). · The proposed negative control tumor tissue for non-squamous NSCLC should not show membrane staining in tumor cells. · Non-specific background staining must have an average intensity of ≤ 1+ across the entire specimen. · Known negative tissue controls should only be used to monitor the correct performance of the processed tissue and test reagents and should not be used as an aid in formulating a specific diagnosis of patient samples.

[0095] <Acceptance criteria for control specimens> Based on the staining results of the control specimens, accept or reject the results of the test specimens according to the following criteria. · Accept the test specimen results when the control meets all the necessary requirements. · If the control tissue slide does not meet the necessary requirements, the test specimen results must be considered invalid and the staining including the tissue control and tissue sample must be repeated. · If the staining results of the negative control reagent (NCR) do not meet the necessary requirements, the test specimen results must be considered invalid and the staining including the tissue control and tissue sample must be repeated.

[0096] Once appropriate staining of the control material is established, the staining is judged to be satisfactory for the evaluation of the test samples.

[0097] <Patient tissue slide stained with NCR> The negative control reagent is used in place of the CEACAM5-specific primary antibody to assist in the interpretation of specific staining at the antigen site.

[0098] The absence of membrane staining verifies the specific labeling of the target antigen by the CEACAM5 primary antibody in patient specimens. Staining with NCR should not show any intensity of membrane staining. Non-specific background staining must have an average intensity of ≤ 1+ across the entire specimen.

[0099] <Slide reading order and evaluation> For each staining run, slides must be tested in the order shown in the following table to determine the validity of the staining run and to enable evaluation of the stained sample tissue.

[0100] JPEG2025522755000004.jpg254170JPEG2025522755000005.jpg255162JPEG2025522755000006.jpg85170

[0101] <NCR-stained specimen> To evaluate non-specific staining and enable interpretation of specific staining, sections of each patient specimen must be tested with a negative control reagent (NCR). Figure 19 illustrates the evaluation of an NSCLC with non-squamous epithelium, first with H&E, then with NCR, and finally with CEACAM5 IHC slides.

[0102] Acceptance criteria: · Staining with NCR should not show any specific membrane staining of any intensity. · Non-specific background staining should have an average intensity of ≤ 1+ throughout the specimen.

[0103] <CEACAM5-stained specimen> Carefully examine the entire tumor specimen and determine the percentage of CEACAM5-positive tumor cells with an intensity of ≥ 2+:

[0104]

Number

[0105] Examples of staining of individual intensities are shown in Figure 8. The estimated number of total viable tumor cells in the entire tissue section is used as the denominator for calculating the score.

[0106] The diagnostic status (positive / negative) of CEACAM5 in NSCLC specimens with non-squamous epithelium is determined by the percentage of viable tumor cells expressing CEACAM5-positive (≥ 2+ intensity) membrane staining.

[0107] · Tumor cells are CEACAM5 positive when they show partial or complete peripheral membrane staining of intensity 2+ and 3+. See Figure 3. · CEACAM5 staining may appear as punctate or linear membrane staining as exemplified in Figure 2. · Re-examine the CEACAM5 expression pattern for important scoring details. · Nonspecific background staining should have an average intensity of ≤1+ throughout the specimen.

[0108] For the evaluation and scoring of CEACAM5 immunohistochemical staining, a 4× objective lens magnification can be used for the initial evaluation of the whole specimen, followed by scoring of tumor cell staining of ≥2+ intensity using 4× - 10× objective lenses. A 20× objective lens can be used to identify and / or confirm tumor cells showing 1+ and punctate and / or delicate 2+ membrane staining as well as 2+ intracytoplasmic lumina. Also, 20× - 40× objective lenses can be utilized to confirm the membrane localization of the staining as needed. A 40× objective lens should not always be used for scoring (i.e., for evaluating intensity and percent positive).

[0109] Perform CEACAM5 positive scoring using the following resolution: In the range of zero to 10%, a positive score in 1% increments, except in the range of zero to 1% where "<1%" is specified. In the range of 10% to 100%, positive can be scored in 5% increments, but smaller increments can be used as needed.

[0110] Elements to exclude from scoring and evaluation of nonspecific background The following are listed as additional tissue elements that may be present and may show clearly genuine CEACAM5 staining: · Mucin (see Figure 16) · Shed intracavitary non-viable cells · Benign epithelial cells (see Figure 5) · Immune cells (e.g., macrophages, see Figure 14).

[0111] Staining of these areas is excluded from the determination of CEACAM5 expression status.

[0112] Examples of endogenous pigments that appear in some specimens and ways to distinguish them from authentic CEACAM5 staining are shown in FIG. 9.

[0113] The CEACAM5 positive state is determined and recorded based on a predetermined cut-off point of CEACAM5 positive staining at a ≥2+ intensity level.

[0114] The term "indeterminate" is used to represent samples in which scoring of tumor cell membrane staining is hampered due to reasons attributable to the biology of the tumor tissue sample, rather than due to inappropriate sample preparation or handling, although the criteria for evaluation are met.

[0115] Staining may also occur in certain areas of tissue specimens, such as stroma, necrotic areas, and edge artifacts in the peripheral areas of the tissue. Stromal staining may occur in specimens showing highly positive CEACAM5 expression, as shown in FIG. 7. Staining of these tissue elements is excluded from the determination of CEACAM5 expression status and from the evaluation of non-specific background staining (see section 2.2). Some examples of these staining artifacts are shown in FIGS. 6 and 7.

[0116] FIG. 1 illustrates a representative image of CEACAM5 staining in non-squamous NSCLC using the CEACAM5 IHC (antibody clone) 769 assay as described herein.

[0117] Figure 2 illustrates representative images of positive staining examples: punctate (red arrow or arrow in the left edge image) and linear (black arrow or arrow in the center of the image) membrane staining with intensity greater than ≧2+ is considered positive, and a 40× image is shown here for detailed illustration, but scoring and calculation of the percentage of CEACAM5 positive membrane staining can also be measured with 4× and / or 10× objective lenses.

[0118] Figure 3 of partial and complete membrane staining: Arrow "a" indicates complete membrane staining, arrow "b" indicates partial, basal outer and apical (luminal) membrane staining, and arrow "c" indicates partial, apical (luminal) membrane staining (alveolar pattern).

[0119] Figure 4 illustrates CEACAM5 scoring as provided herein: cytoplasmic staining is generally not contemplated for CEACAM5 scoring, and only membrane staining is relevant to the calculation of the CEACAM5 positive percentage score. However, for cells with intense cytoplasmic staining, a positive can be inferred if positive membrane staining is identified in tumor cells within the 10× objective field of view of the highly cytoplasmic staining cells. In this case, the red (or longer) arrow indicates the region where intense cytoplasmic staining without distinguishable membrane staining is observed. For these regions, the cells are scored as positive for CEACAM5 because the tumor cells within the 10× objective field of view (black (or shorter) arrow) have distinguishable membrane staining and the region is highly positive for CEACAM5.

[0120] Figure 5 illustrates an exemplary benign tissue staining example, and staining of normal (non-neoplastic) tissue as shown herein is excluded from the CEACAM5 scoring protocol as provided herein.

[0121] Figure 6 illustrates an image demonstrating that staining can occur at various intensities in necrotic tissue. Necrotic regions must be excluded from scoring using the CEACAM5 scoring protocol as provided herein and should not be considered non-specific background staining.

[0122] Figure 7 illustrates an image demonstrating that only tumor cells (black (or longer) arrows) are scored for CEACAM5 positivity, and any surrounding stroma (red (shorter) arrows) showing CEACAM5 staining is excluded from the percent score of the CEACAM5 staining protocol as provided herein and is not considered non-specific background staining.

[0123] Figure 8 illustrates images of staining intensities of 1+ (upper row), 2+ (middle row), 3+ (lower row) using the CEACAM5 scoring protocol as provided herein. 2+ and 3+ membrane staining intensities are considered CEACAM5 positive according to the CEACAM5 scoring protocol as provided herein.

[0124] Figures 9A - B illustrate images demonstrating that pigments that may be present in the specimen, such as anthracosis pigment (arrow in Figure 9A) and hemosiderin (arrow in Figure 9B) at moderate to high magnifications, can be distinguished from DAB precipitates and should not be included in the scoring for CEACAM5 positivity using the CEACAM5 scoring protocol as provided herein.

[0125] Figures 10A - B illustrate images of lumen staining details and nested patterns. Figure 10A shows H&E (hematoxylin and eosin) staining at 20×, and Figure 10B shows CEACAM5 positive apical membrane staining at 20× in the lumen portion of the nest. If the nest has a layer of tumor cells and the lumen is positive, then all tumor cells within the nest are positive. In this case, using the CEACAM5 scoring protocol as provided herein, the cells within the dashed line area are considered positive.

[0126] Figure 11 illustrates images of luminal staining details and nested patterns. The nested expression pattern is a solid tumor nest delimited by stroma and containing multiple layers of cells and small concentric luminal spaces. When a nest shows positive luminal staining, all cells within the nest are considered positive using the CEACAM5 scoring protocol as provided herein. Arrows indicate nests that are positive or negative for CEACAM5.

[0127] Figures 12A - B illustrate images of luminal staining details and alveolar patterns. Structures with this pattern have multiple layers of cells. When the lumen is positive, cells showing apical membrane staining with an intensity of 2+ or greater (≥) on the luminal surface are considered positive, and the cell layers behind the cells facing those lumens are also considered positive using the CEACAM5 scoring protocol as provided herein. Figure 12A: All cells in the outlined alveolar structure are positive. Figure 12B: The alveolar structure in the center of this panel contains positive and negative cells.

[0128] Figures 13A - B illustrate images of H&E staining (Figure 13A) and CEACAM5 staining (Figure 13B) of cells with a signet ring morphology. Cells with a signet ring morphology may show high-intensity staining in the vacuolar inner space and cytoplasm. Membrane staining may be difficult to distinguish due to cytoplasmic staining intensity. In these cases, tumor cells are considered positive for membrane staining and scored regarding cytoplasmic intensity, i.e., when the cytoplasmic intensity is ≥2+ in the signet ring subtype, the cells are considered positive using the CEACAM5 scoring protocol as provided herein.

[0129] Figures 14A - D illustrate images of viable tumor cells in the luminal space (Figures 14A, 14B), and when viability can be confirmed, scoring must be performed using the CEACAM5 scoring protocol as provided herein. Figure 14A is an H&E stain of the tumor cells, and Figure 14B is a CEACAM5 stain of the tumor cells as described herein. H&E may be required for this evaluation. Care must be taken to distinguish tumor cells from macrophages. Figures 14C - D: Macrophages and non-viable tumor cells must not be scored. Figure 14C is an H&E stain as described herein, and Figure 14D is a CEACAM5 stain.

[0130] Figure 15 illustrates an image of intracytoplasmic luminal (arrow) staining demonstrating that CEACAM5 can be present. H&E staining can be used to confirm the presence of the intracytoplasmic lumen. These cells are considered positive when the staining is ≥2+ and must be included in the scoring using the CEACAM5 scoring protocol as provided herein.

[0131] Figures 16A - B illustrate images demonstrating that mucin can be stained with high intensity (H&E stain, Figure 16A and CEACAM5 stain, Figure 16B), which is not considered non-specific background staining and is excluded from the CEACAM5 percent score using the CEACAM5 scoring protocol as provided herein. Example mucin staining shown in the area surrounded by the square.

[0132] Figures 17A - B illustrate positive control tissue images: demonstrating membrane staining of various weak (1+, red arrows or the two arrows at the top center of the image and the second arrow from the top right to bottom of the image) to moderate (2+, black arrows or the two downward arrows at the center and bottom right of the image and the arrow at the top right of the image) intensities using the CEACAM5 scoring protocol as provided herein, in non-squamous NSCLC tissue at 10× (Figure 17A) and 20× (Figure 17B).

[0133] Figures 18A - D illustrate negative control tissue images: Figure 18A: H&E staining of non - squamous NSCLC; Figure 18B: Using a low - magnification objective lens, the tissue is first scanned to observe any CEACAM5 staining; Figure 18C: Using a higher magnification of 10×, it can be confirmed whether any staining is present; and Figure 18D: Using a 20× objective lens, it can be visualized whether any specific membrane staining of 1+ intensity is present. In this example, there is no CEACAM5 staining, and this specimen can serve as a good negative control tissue.

[0134] Figure 19, the top panel illustrates an image of H&E (hematoxylin and eosin) staining of a tissue specimen, which is first evaluated by H&E to assess tissue histology and preservation quality. Figure 19, the middle panel illustrates a negative control reagent slide, which is used to evaluate non - specific staining and enables the interpretation of CEACAM5 staining slides. And Figure 19, the bottom panel illustrates CEACAM5 staining, which is evaluated and considered positive staining when it is complete and / or partial cell membrane staining of intensity 2+ or more (≥) using the CEACAM5 scoring protocol as provided herein.

[0135] [Example 2: External reproducibility study of CEACAM5 IHC 769 in non - squamous non - small cell lung cancer specimens] This example demonstrates the use of the CEACAM5 staining protocol as provided herein.

[0136] The purpose of this study was to evaluate the following: - A device for investigation regarding inter - facility, intra - facility / date, inter - observer, and intra - observer reproducibility in a set of formalin - fixed paraffin - embedded (FFPE) human non - squamous non - small cell lung cancer tissue specimens, CEACAM5 IHC 769, implemented in three external test facilities, and - Inter - and intra - facility / date, inter - and intra - observer reproducibility of CEACAM5 IHC 769 using the Dako Omnis instrument in FFPE non - squamous NSCLC specimens with ≥2+ intensity ≥50% CEACAM5 - positive tumor cells. The results of this study were used to demonstrate assay reproducibility according to applicable regulatory guidelines.

[0137] CEACAM5 IHC 769 is a qualitative immunohistochemical (IHC) assay using monoclonal mouse anti - CEACAM5 clone 769, intended for use in the detection of human CEACAM5 protein in formalin - fixed paraffin - embedded (FFPE) non - squamous non - small cell lung cancer.

[0138] The CEACAM5 IHC 769 assay is used as an adjunct to identify patients who may benefit from participating in clinical trials using Sanofi SAR408701 (Tusamitamab ravtansine), developed as a treatment for patients with advanced non - squamous NSCLC.

[0139] Tusamitamab ravtansine, an anti - CEACAM5 antibody - drug conjugate, is an immune complex consisting of an anti - cancer fetal antigen - related cell adhesion molecule 5 (CEACAM5) conjugated to a cytotoxic agent and has potential anti - neoplastic activity. When administered, the antibody portion of Tusamitamab ravtansine targets CEACAM5 on tumor cells. Upon antibody / antigen binding and internalization, the immune complex releases the cytotoxic agent, which results in tumor cell death.

[0140] The objective of this study was to evaluate the performance of CEACAM5 IHC 769 with respect to inter - and intra - facility, inter - and intra - date, and inter - and intra - observer reproducibility at the assay cut - off of ≥2+ intensity ≥50% CEACAM5 - positive tumor cells.

[0141] The diagnostic endpoint was defined as positive when ≥50% of tumor cells with ≥2+ intensity of CEACAM5 expression were present. Negative was defined as ≥2+ intensity staining in <50% of the tumor cell staining. In this study, the reproducibility of the diagnostic endpoint (positive / negative) evaluation using CEACAM5 IHC 769 according to the CEACAM5 staining protocol as provided herein was evaluated.

[0142] Endpoint: The following study endpoints were evaluated: · Inter-facility reproducibility across 3 facilities and 5 rounds · Intra-facility / date reproducibility across 5 rounds within 3 facilities · Inter-observer reproducibility across 3 pathologists and 3 readings · Intra-observer reproducibility across 3 readings by each of 3 pathologists.

[0143] This study was a 3-facility, blinded, randomized reproducibility study of CEACAM5 IHC 769 in FFPE human non-squamous non-small cell lung cancer (nsNSCLC) tissue specimens.

[0144] The study was divided into two parts: Part A (inter-facility and intra-facility / date endpoints) and Part B (inter-observer and intra-observer endpoints). All slides were re-randomized with respect to the evaluation order between rounds / readings and interpreted by pathologists blinded to specimen identity and previous evaluations. Slide sets were read by facility pathologists trained with respect to the scoring algorithm. The maximum washout period did not exceed 30 days. If exceeded, proficiency testing and / or refresher workshops were given.

[0145] Study Part A: Each of 3 external facilities performed automated staining runs testing 50 FFPE human nsNSCLC specimens. Specimens were validated by Agilent and distributed to 3 facilities, with each facility receiving 5 replicate sets. Staining was performed on 5 discontinuous days over a period of at least 20 days.

[0146] Study part B: A set of 66 pre-stained specimens of nsNSCLC was circulated among all three test facilities such that it was certified by Agilent and evaluated three times by the same pathologist at each facility. Four unique wildcards were included in each read but not analyzed.

[0147] Efforts were made to select specimens representing the full range of biomarker expression with a balanced distribution of positive and negative samples, based on a cut-off of ≥2+ intensity in ≥50% of CEACAM5-positive tumor cells. Approximately 20% of the specimens were selected in the range near the cut-off of ≥2+ intensity from ≥40% to ≤60% of CEACAM5-positive tumor cells.

[0148] Study part A: The targeted enrollment included 50 specimens, consisting of approximately 25 positive samples and 25 negative samples.

[0149] Study part B: A set of 66 specimens consisting of approximately 33 positive samples and 33 negative samples for analysis. Additionally, four wild card specimens (12 in total) were included for each read to reduce the potential for recall bias.

[0150] Statistical analysis: Concordance estimates for all study endpoints: The following methods were used to evaluate the analytical concordance of diagnostic outcomes (positive / negative) for study part A (between and within facilities) and study part B (between and within observers): 1. Evaluation of NPA, PPA, and OA based on the consensus score using bootstrap confidence intervals. The negative percent agreement (NPA), positive percent agreement (PPA), and overall agreement (OA) were calculated for between-facility, within-facility / date, between-observer, and within-observer reproducibility by comparing the diagnostic status (positive / negative) from each slide to the consensus diagnostic outcome. The bootstrap method was used to calculate two-sided 95% confidence intervals for NPA, PPA, and OA. 2. Analysis regarding IHC staining percentage score. The variability (continuous data) of the CEACAM5 staining percentage score was evaluated by analysis of variance (ANOVA) based on a mixed model using facility and round (study part A) and observer and reading (study part B) as random factors. The mean, standard deviation, and coefficient of variation for facility and observer measurements of each specimen were reported.

[0151] Acceptance criteria: The lower bounds of the two-sided 95% confidence intervals for NPA, PPA, and OA must be at least 85% of each reproducibility measurement.

[0152] Results: The results of this external reproducibility study demonstrated high concordance for all diagnostic endpoints and met all predefined acceptance criteria for each of the study endpoints with a ≥2+ intensity ≥50% CEACAM5 positive tumor cell cut-off.

[0153] Conclusion: The results of this study demonstrate that the concordance of diagnostic endpoint determination met the predefined acceptance criteria. The reproducibility results for the ≥2+ intensity ≥50% cut-off of CEACAM5 positive and negative status across three observers met the acceptance criteria of CI lower bound ≥85% for NPA, PPA, and OA reproducibility estimates.

[0154] <Overall study design> This study was designed to evaluate the assay performance of CEACAM5 IHC 769 in three independent CLIA-certified clinical laboratories. The study utilized blinded and randomized FFPE tissue sections from NSCLC specimens of non-squamous epithelium expressing different levels of CEACAM5. All specimens during the study were evaluated by a certified pathologist in the scoring of CEACAM5. The study consisted of part A that evaluated combined staining and scoring reproducibility, and part B that evaluated observer evaluation reproducibility.

[0155] A study was designed to investigate the reproducibility of positive / negative interpretation of CEACAM5 IHC 769 based on 2+ intensity and 50% CEACAM5 positive tumor cell score as predetermined cut-off values where ≧2+ intensity ≧50% is defined as positive and ≧2+ intensity <50% is defined as negative.

[0156] The distribution of samples in Part A and Part B was based on the predefined CEACAM5 positive tumor % score ranges for negative, positive, and near cut-off samples and is described in Table 1:

[0157] JPEG2025522755000008.jpg34170

[0158] Approximately 20% of the specimens were selected in the near cut-off range.

[0159] For Part A: Staining was performed over a period of at least 20 days for each facility. Each set was evaluated by a certified pathologist, and the pathologist was provided with H&E, CEACAM5, and NCR slides for each case. Each evaluation session was limited to a maximum of 30 scoring specimens per day to minimize the potential for scoring fatigue. A washout period of at least 14 calendar days, shorter than 30 days, was taken between each of the three readings per observer for each specimen, which was incorporated into the study. The schedule was coordinated with the laboratory staff to ensure that the study requirements were met.

[0160] For Part B: Inter-observer, intra-observer reproducibility This part of the study was conducted using a single set of 66 permanently mounted CEACAM5, NCR, and H&E stained slides prepared by Agilent and provided to the research facilities (spun to be evaluated three times by each facility), ensuring that the scores (to be analyzed) from each pathologist were based on the same set of slides. To reduce recall bias, a set of four wild - card specimens (each set unique to each reading) was added and evaluated but not included in the data analysis. After all facilities had gone through the round of evaluations, the assigned Agilent personnel incorporated the following specific wild - card sets and randomized the slide - set reading order according to a randomization procedure. A wash - out period of at least 14 calendar days and shorter than 30 days was taken between each of the three readings per observer for each specimen, as incorporated into the study.

[0161] Methods to avoid bias: · Specimens within the evaluation were fully anonymized and the specimen numbers from the test facilities were blinded (away from the personnel assigned to randomize the H&E specimens). · All IHC - stained slides were randomized based on the order of evaluation and the identification of the specimens was blinded to the pathologists. · Pathologists were instructed not to discuss the scoring results of the specimens with each other. · A five - day - interval test (round) for Part A was conducted over five discontinuous days. · A wash - out period of 14 - 30 days was continued between each of the five scoring evaluations (rounds) of Part A and between each of the scoring evaluations (readings) of Part B at each facility. · For Part B, four predetermined wild - card specimens were randomized to be test specimens with four different wild - cards for each of the three readings.

[0162] [Materials] <Description of Diagnostic Medical Devices for Investigation> CEACAM5 IHC 769 contains optimized reagents and staining protocols necessary to complete immunohistochemical (IHC) staining procedures for FFPE specimens using Dako Omnis. One kit contains sufficient reagents to perform 60 tests (D60362).

[0163] JPEG2025522755000009.jpg52170

[0164] In Part A, only one lot number of the CEACAM5 IHC 769 kit was used by the facility.

[0165] <Device Operation> CEACAM5 IHC 769 contains optimized reagents and protocols necessary to complete IHC staining procedures for FFPE specimens using Dako Omnis. After incubation with the primary monoclonal antibody against CEACAM5 or the negative control reagent (NCR), the specimen is incubated with a ready-to-use visualization reagent consisting of secondary antibody molecules and horseradish peroxidase molecules coupled to the dextran polymer backbone. As a result of the enzymatic conversion of the subsequently added chromogen, a visible reaction product precipitate occurs at the antigen site. The specimen is then counterstained and can be mounted. The results are read using an optical microscope. CEACAM5 IHC 769 (product code GE025) is applicable to automated staining using the Dako Omnis instrument.

[0166] The operating procedure followed the Instructions for Use (IFU) (D60362) for the use of CEACAM5 IHC 769 for nsNSCLC specimens.

[0167] The interpretation of the staining followed the scoring guidelines in the IFU (D60362) and the detailed scoring method in the Pathology Scoring Manual for CEACAM5 IHC 769 for nsNSCLC specimens (D55521). CEACAM5 positive staining is defined as any partial or complete tumor cell membrane staining of ≧2+ intensity.

[0168] Tissue screening for the study was performed using CEACAM5 IHC 769 according to the device's IFU (D60362). It was confirmed by a certified pathologist that the selected specimens met the tissue inclusion / exclusion criteria prior to the start of the study. Specimen screening was performed by a certified reader (pathologist or scientist).

[0169] Specimen profile set design (Parts A and B) Tissue specimens were selected according to the study protocol and guidance on external specimen selection and review. The summarized profiles of the nsNSCLC specimens selected for this study, presented as the number of specimens (n) and the percentage (%) of the total set, are shown in Table 3:

[0170] JPEG2025522755000010.jpg51170

[0171] <Scoring of nsNSCLC specimens> Scoring was performed using a brightfield microscope according to the device's IFU and the pathologic scoring manual. The outline of the scoring process is described herein.

[0172] In this reproducibility study, a cutoff of ≥ 50% CEACAM5-positive tumor cells with a staining intensity of ≥ 2+ on the tumor cell membrane was used. Specimens were classified as positive (≥ cutoff) or negative (< cutoff) based on this cutoff.

[0173] The entire specimen was evaluated. All viable tumor cells on all CEACAM5-stained slides were evaluated and included in the CEACAM5 scoring assessment. H&E-stained slides were used to determine whether a sufficient number of tumor cells were present in the tissue sections of a given specimen. To determine the percentage of CEACAM5-stained cells, at least 100 viable tumor cells must be present in the CEACAM5-stained slide. Positive was defined as any partial or complete tumor cell membrane staining of 2+ intensity or greater (≧2+).

[0174] <Statistical analysis> In part A of the study, 50 specimens were utilized for analysis, and in part B, 66 specimens were utilized for analysis. The specimens were selected such that approximately 50% of the total number were negative and 50% were positive based on the diagnostic outcomes from specimen screening. An attempt was made to enrich the specimen set using approximately 20 - 25% in the vicinity of the cutoff. The specimen sets for study part A and study part B contained overlapping specimens.

[0175] Specimens near the cutoff were selected based on the positive percent score created during the review and selection process, being within the range of 40% - 60% of intensity 2+ or greater. Specimens reported to be near the cutoff in the study conclusions were based on whether the median of the consecutive scores of the study pathologists was within the cutoff vicinity range.

[0176] <Calculation of percent agreement and confidence intervals> For measures of reproducibility between facilities and within facilities (between dates) and between observers and within observers, the agreement estimates and their respective confidence intervals were calculated by the following method: comparison to consensus for calculating NPA / PPA / OA.

[0177] Calculating the concordant percentages of NPA and PPA required a comparison to a reference condition. There was no reference for reproducibility parameters such as facility, date, observer, and reading. The comparison was made for the IHC diagnostic status (positive / negative) between each test condition and the consensus outcome (the most frequently occurring observation). The total number of comparisons per specimen was equivalent to the total number of scores.

[0178] [Results] <Reported Results of Part A and Part B> The following sections present the results of the statistical analysis for reproducibility between facilities, within facilities, between observers, and within observers. All line data (i.e., reported data points for continuous CEACAM5 and data points for negative / positive diagnostic endpoints based on a ≥2+ intensity ≥50% cutoff for Parts A and B) are tabulated. ·The following results are reported in the summary tables of the statistical analysis (Tables 5 - 8): - Specimen distribution profile: number (n) and percentage (%) of specimens evaluated as CEACAM5 positive (P) or negative (N) and near the ≥2+ intensity ≥50% cutoff (NCO50%); specimens are described as "near 50% cutoff" if the median of their CEACAM5 scores falls within 40% - 60%. - Number of comparisons to the consensus: number of concordant negative, concordant positive, discordant negative, and discordant positive comparisons - NPA - PPA - OA - 95% confidence intervals for each NPA, PPA, and OA

[0179] <Overall Study Results> Table 4 provides an overall summary of the specimen profile distribution based on the external reproducibility pathologist evaluation:

[0180] JPEG2025522755000011.jpg51170

[0181] JPEG2025522755000012.jpg119170

[0182] Estimated values of NPA, PPA, and OA for the in - facility reproducibility endpoints are shown in Table 6:

[0183] JPEG2025522755000013.jpg147170

[0184] The lower bounds of the two - sided 95% confidence intervals for NPA, PPA, and OA were all higher than 85% for each reproducibility measure, and thus the acceptance criteria were met for all analyses in Part A.

[0185] <Inter - observer, intra - observer reproducibility (Part B) / Overview of experimental design and data recording (Part B)> The purpose of this study was to demonstrate that the Agilent scoring method and interpretation guidelines of the CEACAM5 IHC 769 assay yield consistent results in a normal inter - observer test.

[0186] Data were analyzed to determine the assay performance consistency across three facilities based on a ≥2 + intensity ≥50% cut - off. The same data were used for inter - observer (Table 7) and intra - observer analysis (Table 8).

[0187] <Inter - observer reproducibility results> <npa ppa oa> Estimated values of NPA, PPA, and OA for the inter - observer reproducibility endpoint are shown in Table 7:

[0188] JPEG2025522755000014.jpg55170

[0189] <Intra - observer reproducibility total analysis> <npa ppa oa> Estimated values of NPA, PPA, and OA for the within - observer reproducibility endpoints are shown in Table 8:

[0190] JPEG2025522755000015.jpg113170

[0191] Lower bounds of the two - sided 95% confidence intervals for NPA, PPA, and OA were higher than 85% for each reproducibility measure, and thus the acceptance criteria were met for all analyses in Part B.

[0192] <Summary display of diagnostic estimates in the reproducibility study> Results of the four study objectives (intra - facility, inter - facility, within - observer, and between - observer) shown above with 95% confidence intervals are summarized in Figure 20: Summary of results with intensity ≥2+ and cut - off ≥50%

[0193] <Analysis of variance components> The underlying continuous CEACAM5 scores in the reproducibility study were also evaluated based on an analysis of variance model (ANOVA). Values of the mean, standard deviation (SD), and coefficient of variation (%CV) were calculated, and data from both study parts A and B were analyzed and shown in Tables 9 and 10 respectively. It should be noted that the acceptance criteria were not applied to these results. For some specimens, some or all of the variance components could not be estimated due to no variability in the scores (e.g., all scores of 0).

[0194] JPEG2025522755000016.jpg255169JPEG2025522755000017.jpg75170

[0195] JPEG2025522755000018.jpg254170JPEG2025522755000019.jpg70170

[0196] [Discussion / Conclusion] The data in this example provides estimates of CEACAM5 IHC reproducibility parameters with a ≥2+ intensity ≥50% cut-off within the context of the evaluation of nsNSCLC cases. The study met all acceptance criteria. The calculated lower bound of the two-sided 95% confidence interval was greater than 85% for all endpoints of the ≥2+ intensity ≥50% cut-off analysis.

[0197] Some embodiments of the present invention are described. Nevertheless, it can be understood that various modifications can be made without departing from the spirit and scope of the present invention. Accordingly, other embodiments are within the scope of the following claims.

[0198] [Alternative Embodiment] In an alternative embodiment, an immunohistochemistry (IHC) method for determining and scoring the degree of cell membrane expression of carcinoembryonic antigen-related cell adhesion molecule 5 or CEACAM5 (also known as CD66e (surface antigen classification 66e)) in a tissue sample, staining the tissue sample with an antibody that specifically binds to CEACAM5, determining the total number of viable tumor or cancer cells having CEACAM5 cell membrane staining and determining the total number of stained and non-stained viable tumor or cancer cells in at least a portion of the tissue sample, wherein a tumor or cancer cell is counted as being positively stained with the anti-CEACAM5 antibody if there is CEACAM5 cell membrane staining of any intensity above a defined threshold, and determining a tumor intensity ratio score, wherein the tumor intensity ratio score is the number of CEACAM5-stained viable tumor or cancer cells found in the tissue sample divided by the total number of stained and non-stained viable tumor or cancer cells, multiplied by 100, is provided.

[0199] In an alternative embodiment of the IHC method as provided herein: - CEACAM5 cell membrane staining includes total membrane staining, discontinuous membrane staining, partial membrane staining, complete (peripheral) membrane staining, punctate membrane staining, linear membrane staining, luminal membrane staining, apical membrane staining, basal membrane staining, lateral membrane staining, lateral basal membrane staining, high cytoplasmic staining with a positive staining intensity of 2+ or more in the signet ring subtype, high cytoplasmic staining with a positive staining intensity of 3+ when cells within a 10× field of view have distinct membrane staining, or combinations thereof. - The total number of viable tumors or cancer cells includes luminal cells, glandular cells, intracavitary cells, multiple layers of cells, cells with a signet ring morphology, cells with high cytoplasmic staining, cells with an intracytoplasmic lumen, or combinations thereof, and includes all viable tumors or cancer cells in the specimen. - The total number of viable tumors or cancer cells counted as positively stained includes all glandular cells having a cell membrane associated with a positively stained lumen. - The tissue sample includes at least about 100 viable tumors or cancer cells. Tumors or cancer cells counted as positively stained do not include tumors or cancer cells having solely cytoplasmic staining (with exceptions), staining of normal or non-neoplastic structures, staining of non-viable tumors or cancer cells, necrotic cells, cell debris, stromal staining, or edge artifacts staining at the periphery of the tissue sample. - Cytoplasmic staining includes cytoplasmic staining with a positive staining intensity of 2+ or less, or the defined threshold includes a positive staining intensity of 2+ or more, or the defined threshold includes a 2+ or 3+ positive staining intensity evaluated at a magnification of about 4× to about 10×, or further includes the step of confirming the positive staining intensity at a magnification of about 20× to about 40×. - The Tumor Intensity Percentage Score (TIPS) includes dividing the number of CEACAM5 viable tumor or cancer cell stainings with a positive staining intensity of 2+ or more by the total number of stained and non-stained viable tumors or cancer cells and multiplying by 100, or a TIPS of about 40% or more indicates the diagnostic status of the tissue sample, or the TIPS is about 50% or more, about 60% or more, about 80% or more, or about 90% or more. - At least about 5% of the TIPS indicate the diagnostic status of the tissue sample, or at least about 5% of the TIPS include the number of CEACAM5-positive viable tumors or cancer cells with a staining intensity of 2+ or more, or at least about 10% of the TIPS include the number of CEACAM5-positive viable tumors or cancer cells with a staining intensity of 2+ or more. - The section or portion of the tissue sample is prepared on a slide, microscope slide, or equivalent, and the section or portion of the tissue sample is stained on the slide. - The antibody includes a monoclonal mouse anti-CEACAM5 antibody or a monoclonal rabbit anti-CEACAM5 antibody, or the monoclonal mouse anti-CEACAM5 antibody includes an antibody having substantially the same affinity as monoclonal mouse anti-CEACAM5 clone 769 or clone 769 antibody against CEACAM5. - The tissue sample includes a formalin-fixed paraffin-embedded (FFPE) specimen, or the FFPE specimen includes a cancer specimen stained on an automated IHC platform. - The section of the tissue sample is prepared by a protocol including fixation in about 10% neutral buffered formalin for a period of about 6 hours to about 72 hours. - The tumor or cancer is non-squamous non-small cell lung cancer (nsNSCLC), non-small cell lung cancer (NSCLC), adenocarcinoma, synovial sarcoma, myxoid / round cell liposarcoma, high-grade myxoid liposarcoma, low-grade myxoid liposarcoma, head and neck cancer, melanoma, esophageal cancer, gastric cancer, stomach cancer, colorectal cancer, lung cancer, colon cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, prostate cancer, or urothelial cancer. - The tissue sample is derived from a surgical resection or biopsy, needle biopsy sample, fine needle aspirate, cytology specimen, or decalcification of bone, and / or - Positive staining is determined using a brightfield light microscope, microscope objective lens, computer monitor, and imaging software, or a combination thereof. Optionally, the imaging software includes whole slide imaging software.< / npa> < / npa>

Claims

1. An immunohistochemistry (IHC) method for determining and scoring the degree of cell membrane expression of carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5) in tissue samples, (a) A step of staining the tissue sample with an antibody that specifically binds to CEACAM5, (b) In at least a portion of the tissue sample, determine the total number of surviving tumor or cancer cells having CEACAM5 cell membrane staining, and determine the total number of stained and unstained surviving tumor or cancer cells. Here, tumor or cancer cells are counted as positively stained with anti-CEACAM5 antibody if they have any intensity of CEACAM5 cell membrane staining exceeding a specified threshold, and (c) Steps to determine the tumor intensity percentage score, Here, the tumor intensity ratio score is calculated by dividing the number of CEACAM5-stained surviving tumors or cancer cells found in the tissue sample by the total number of stained and unstained surviving tumors or cancer cells, and multiplying by 100. Methods that include...

2. The method according to claim 1, wherein the CEACAM5 cell membrane staining includes whole membrane staining, discontinuous membrane staining, partial membrane staining, complete membrane staining, punctate membrane staining, linear membrane staining, luminal membrane staining, apical membrane staining, basement membrane staining, lateral membrane staining, basement membrane staining, high-level cytoplasmic staining with a positive staining intensity of 2+ or higher, high-level cytoplasmic staining with a positive staining intensity of 3+ or a combination thereof.

3. The method according to claim 1, wherein the total number of surviving tumor or cancer cells includes luminal cells, glandular luminal cells, intraluminal cells, multilayered cells, cells having a signet ring morphology, highly stained cytoplasmic cells, cells having intracytoplasmic lumen, or a combination thereof.

4. The method according to claim 1, wherein the total number of surviving tumor or cancer cells counted as positively stained includes all glandular cells having a cell membrane associated with a positively stained lumen.

5. The method according to claim 1, wherein the tissue sample comprises at least about 100 cells.

6. The method according to claim 1, wherein tumor or cancer cells counted as positively stained do not include tumor or cancer cells having cytoplasmic staining, staining of normal or non-neoplastic structures, staining of non-viable tumor or cancer cells, necrotic cells, cell fragments, stromal staining, or tumor or cancer cells having edge artifact staining at the periphery of the tissue sample.

7. The method according to claim 6, wherein the cytoplasmic staining includes cytoplasmic staining with a positive staining intensity of 2+ or less.

8. The method according to claim 1, wherein the specified threshold includes a positive staining intensity of 2+ or higher.

9. The method according to claim 8, wherein the defined threshold includes a 2+ positive or 3+ positive staining intensity evaluated at a magnification of approximately 4× to approximately 10×, or further includes a step of confirming the positive staining intensity at a magnification of approximately 20× to approximately 40×.

10. The method according to claim 8, wherein the tumor intensity ratio score includes the number of CEACAM5 surviving tumors or cancer cells with a positive staining intensity of 2+ or higher, divided by the total number of stained and unstained surviving tumors or cancer cells, and multiplied by 100.

11. The method according to claim 1, wherein a tumor intensity percentage score of approximately 40% or more indicates the diagnostic status of the tissue sample.

12. The method according to claim 11, wherein the tumor intensity percentage score is approximately 50% or more, approximately 60% or more, approximately 80% or more, or approximately 90% or more.

13. The method according to claim 1, wherein a tumor intensity percentage score of approximately 5% or more indicates the diagnostic status of the tissue sample.

14. The method according to claim 13, wherein the tumor intensity percentage score of approximately 5% or more includes the number of CEACAM5 surviving tumors or cancer cells with a positive staining intensity of 2+ or more, or the tumor intensity percentage score of approximately 10% or more includes the number of CEACAM5 surviving tumors or cancer cells with a positive staining intensity of 2+ or more.

15. The method according to claim 1, wherein a section or portion of the tissue sample is prepared on a slide, microscope slide or equivalent, and the section or portion of the tissue sample is stained on the slide.

16. The method according to claim 1, wherein the antibody comprises a monoclonal mouse anti-CEACAM5 antibody or a monoclonal rabbit anti-CEACAM5 antibody.

17. The method according to claim 16, wherein the monoclonal mouse anti-CEACAM5 antibody comprises monoclonal mouse anti-CEACAM5 clone 769, or an antibody having substantially equivalent affinity to the clone 769 antibody for CEACAM5.

18. The method according to claim 1, wherein the tissue sample includes a formalin-fixed paraffin-embedded (FFPE) specimen.

19. The method according to claim 18, wherein the FFPE sample includes a cancer sample stained on an automated IHC platform.

20. The method according to claim 18, wherein sections of the tissue sample are prepared by a protocol including fixation in about 10% neutral buffered formalin for a period of about 6 to about 72 hours.

21. The method according to claim 1, wherein the tumor or cancer is non-squamous non-small cell lung cancer (nsNSLC), non-small cell lung cancer (NSLC), adenocarcinoma, synovial sarcoma, myxoid / round cell liposarcoma, high-grade myxoid liposarcoma, low-grade myxoid liposarcoma, head and neck cancer, melanoma, esophageal cancer, gastric cancer, stomach cancer, colorectal cancer, lung cancer, colon cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, prostate cancer, or urothelial carcinoma.

22. The method according to claim 1, wherein the tissue sample is derived from a needle biopsy sample, a fine-needle aspiration, a cytological specimen, or bone demineralization.

23. The method according to claim 1, wherein positive staining is determined using a bright-field optical microscope, a microscope objective lens, a computer monitor and imaging software, or a combination thereof.

24. The method according to claim 23, wherein the imaging software includes whole-slide imaging software.

25. A method for diagnosing a tumor or cancer by determining whether a tissue sample is positive for the expression of carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5), comprising the step of determining the CEACAM5 diagnostic status in a tissue sample by the method according to any one of claims 1 to 24, wherein about 50% or more of the tumor or cancer cells having a CEACAM5 cell membrane staining of intensity 2+ have a tumor intensity percentage score that is diagnostically positive.

26. The method according to claim 25, wherein the tumor or cancer is non-squamous non-small cell lung cancer (nsNSLC), non-small cell lung cancer (NSLC), adenocarcinoma, synovial sarcoma, myxoid / round cell liposarcoma, head and neck cancer, melanoma, esophageal cancer, gastric cancer, colorectal cancer, lung cancer, colon cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, prostate cancer, or urothelial cancer.

27. A method for treating or improving a tumor or cancer in a patient, comprising the step of determining and scoring the amount of CEACAM5 in a tissue sample derived from the patient using the method according to any one of claims 1 to 24, wherein if the tissue sample is determined or scored to have a high or diagnostically positive CEACAM5 score, the patient is treated with an anti-cancer drug that is likely to elicit a favorable response from the patient.

28. The method according to claim 27, wherein the tumor or cancer is non-squamous non-small cell lung cancer (nsNSLC), non-small cell lung cancer (NSLC), adenocarcinoma, synovial sarcoma, myxoid / round cell liposarcoma, head and neck cancer, melanoma, esophageal cancer, gastric cancer, colorectal cancer, lung cancer, colon cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, prostate cancer, or urothelial cancer.

29. The method according to claim 27, wherein the cancer treatment drug comprises administering an anticancer drug or anticancer therapy to the patient.

30. The method according to claim 29, wherein the anticancer therapy comprises an antibody-drug conjugate, small molecule therapy, immunotherapy, monoclonal antibody therapy, adoptive cell therapy, T cell receptor therapy, or chimeric antigen receptor (CAR) T cell therapy.

31. A kit comprising an antibody that specifically binds to CEACAM5 and a CEACAM5 scoring guideline as described in any one of claims 1 to 24.

32. A method for evaluating the degree of CEACAM5 expression, comprising the steps of: contacting a sample or portion thereof containing cancer or tumor cells of an individual with an antibody or portion thereof that specifically binds to CEACAM5; and determining a tumor intensity percentage score by dividing the number of CEACAM5-stained surviving tumor or cancer cells in the sample or portion thereof that are specifically bound by the antibody by the total number of stained and unstained surviving cancer or tumor cells, and multiplying the result by 100, thereby obtaining the tumor intensity percentage score.

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