Immunohistochemical (IHC) regimens and methods for diagnosis and treatment of cancer

Abstract

In alternative embodiments, there is provided an immunohistochemical (IHC) method for reproducibly determining and scoring the degree of expression of a protein carcino-embryonic antigen associated cell adhesion molecule 5 or CEACAM5 in a tissue sample. In alternative embodiments, methods are provided for diagnosing, treating, or ameliorating a cancer or tumor, or assessing the risk of recurrence of the cancer or tumor, using the IHC methods as provided herein. In alternative embodiments, kits are provided that include components and instructions for use in practicing the methods as provided herein. Described herein are methods for scoring CEACAM5 expression and utilizing the score as a companion or supplemental diagnosis or treatment or amelioration of cancer or tumor.

Description

Technical Field

[0001] The present invention generally relates to cancer treatment, concomitant or supplementary diagnosis and immunohistochemical methods. In an alternative embodiment, an immunohistochemistry (IHC) method is provided for reproducibly determining and scoring the expression level of the protein carcinoembryonic antigen-related cell adhesion molecule 5 or CEACAM5 (also known as CD66e (differentiation cluster 66e)) in a tissue sample. In an alternative embodiment, a method for diagnosing, treating or improving cancer or tumors or assessing the risk of recurrence of the cancer or tumor using the IHC method as provided herein is provided. In an alternative embodiment, a kit is provided, the kit comprising components and instructions for use for practicing the method as provided herein. The application describes a method for scoring CEACAM5 expression and using the score as a concomitant or supplementary diagnosis or treatment or improvement of cancer or tumors. Background Art

[0002] Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), also known as CD66e (cluster of differentiation 66e), is a member of the carcinoembryonic antigen (CEA) gene family. Evidence suggests that high CEACAM5 expression is closely associated with CD133-positive colorectal cancer stem cells. CEACAM5 is used as a clinical biomarker for gastrointestinal cancer and may promote tumor development through its role as a cell adhesion molecule. In addition, CEACAM5 may 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 those of the gastrointestinal tract, genitourinary system, breast cancer, and non-squamous non-small cell lung cancer (NSCLC). CEACAM5 is a member of the CEA protein family and plays a key role in cell migration, cell invasion, and cell adhesion. Summary of the invention

[0004] In an alternative embodiment, an immunohistochemistry (IHC) method is provided for determining and scoring the extent of cell membrane expression of carcinoembryonic antigen-related cell adhesion molecule 5 or CEACAM5 (also known as CD66e (cluster of differentiation 66e)) in a tissue sample, the method comprising:

[0005] (a) Tissue samples were stained with antibodies that specifically bind to CEACAM5.

[0006] (b) determining the total number of viable tumor or cancer cells having CEACAM5 cell membrane staining, and determining the total number of stained and unstained viable tumor or cancer cells in at least a portion of the tissue sample,

[0007] wherein a tumor or cancer cell is counted as positively stained with an anti-CEACAM5 antibody if there is any intensity of CEACAM5 cell membrane staining above a defined threshold; and

[0008] (c) Determine the Tumor Intensity Proportion Score (TIPS),

[0009] The tumor intensity ratio score is the number of CEACAM5-stained viable tumor or cancer cells present in the tissue sample divided by the total number of stained and unstained viable tumor or cancer cells, multiplied by 100.

[0010] In an alternative embodiment of the IHC method as provided herein:

[0011] - 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, basal membrane staining, lateral membrane staining, basolateral membrane staining, high cytoplasmic staining with a positive staining intensity of 2+ or greater, high cytoplasmic staining with a positive staining intensity of 3+, or a combination thereof;

[0012] - the total number of viable tumor or cancer cells includes luminal cells, glandular luminal cells, intraluminal cells, multi-layered cells, cells with signet ring morphology, cells with high cytoplasmic staining, cells with intracytoplasmic lumens, or combinations thereof;

[0013] - The total number of viable tumor or cancer cells counted as positively stained includes all glandular luminal cells with cell membranes associated with the positively stained lumen;

[0014] - the tissue sample comprises at least about 100 cells;

[0015] Tumor or cancer cells counted as positively stained excluded tumor or cancer cells with cytoplasmic staining, staining of normal or non-neoplastic structures, staining of non-viable tumor or cancer cells, necrotic cells, cell debris, stromal staining, or edge artifact staining on the periphery of the tissue specimen;

[0016] - Cytoplasmic staining comprises a cytoplasmic staining of 2+ or less positive staining intensity; or, the defined threshold comprises a positive staining intensity of 2+ or greater; or, the defined threshold comprises a 2+ positive or 3+ positive staining intensity assessed at a magnification of about 4X to about 10X; or further comprises confirming a positive staining intensity at a magnification of about 20X to about 40X;

[0017] - the Tumor Intensity Ratio Score (TIPS) comprises the number of CEACAM5 viable tumor or cancer cells stained with a positive staining intensity of 2+ or greater divided by the total number of stained and unstained viable tumor or cancer cells, multiplied by 100; or, a TIPS of about 40% or greater is indicative of a diagnostic status of the tissue sample; or, a TIPS of about 50% or greater, about 60% or greater, about 80% or greater, or about 90% or greater;

[0018] - about 5% or greater of the TIPS are indicative of the diagnostic status of the tissue sample; or, about 5% or greater of the TIPS include the number of CEACAM5 viable tumor or cancer cells stained with a positive staining intensity of 2+ or greater; or about 10% or greater of the TIPS include the number of CEACAM5 viable tumor or cancer cells stained with a positive staining intensity of 2+ or greater;

[0019] - preparing a section or portion of a tissue sample on a glass slide, a microscope slide or equivalent and staining said section or portion of said tissue sample on said slide;

[0020] - the antibody comprises a monoclonal mouse anti-CEACAM5 antibody or a monoclonal rabbit anti-CEACAM5 antibody; or, the monoclonal mouse anti-CEACAM5 antibody comprises a monoclonal mouse anti-CEACAM5 clone 769, or an antibody having a substantially similar affinity for CEACAM5 as the clone 769 antibody;

[0021] - The tissue sample comprises a formalin-fixed, paraffin-embedded (FFPE) sample; or, the FFPE sample comprises a cancer sample stained on an automated IHC platform;

[0022] - preparing sections of tissue samples by a protocol comprising fixation in about 10% neutral buffered formalin for a period of about 6 hours to about 72 hours;

[0023] - 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 carcinoma;

[0024] - tissue sample from a needle biopsy, fine needle aspirate, cytology specimen or bone decalcification; and / or

[0025] - Positive staining is determined using a bright field light microscope, a microscope objective, a computer monitor and imaging software, or a combination thereof; and optionally, the imaging software comprises whole slide imaging software.

[0026] In an alternative embodiment, a method for diagnosing a tumor or cancer by determining whether a tissue sample is positive for expression of carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is provided, the method comprising: determining the diagnostic status of CEACAM5 in a tissue sample by a method provided herein, e.g., as described above, wherein about 50% or more of the tumor cancer cells having a 2+ or greater intensity of CEACAM5 cell membrane staining have a tumor intensity ratio score that 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.

[0027] In an alternative embodiment, a method for treating or ameliorating a tumor or cancer in a patient is provided, the method comprising determining and scoring the amount of CEACAM5 in a tissue sample from the patient using a method as provided herein, e.g., as described above, wherein if the tissue sample is determined or scored as having a high or diagnostically positive CEACAM5 score, the patient is treated with a cancer therapeutic agent to which the patient may respond favorably.

[0028] In an alternative embodiment of the method as provided herein:

[0029] - 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 carcinoma;

[0030] - the cancer treatment comprises administering an anti-cancer drug or anti-cancer therapy to the patient; and / or

[0031] - The anti-cancer therapy comprises antibody drug conjugates, small molecule therapy, immunotherapy, monoclonal antibody therapy, adoptive cell therapy, T cell receptor therapy, or chimeric antigen receptor (CAR) T cell therapy.

[0032] In alternative embodiments, kits are provided, comprising an antibody that specifically binds CEACAM5 and a guide for scoring CEACAM5 as provided herein, e.g., comprising a method as provided herein, e.g., as described above.

[0033] In an alternative embodiment, a method for assessing the extent of CEACAM5 expression is provided, the method comprising:

[0034] contacting a sample or a portion thereof from the individual comprising cancer or tumor cells with an antibody or a portion thereof that specifically binds CEACAM5; and

[0035] The tumor intensity ratio score (TIPS) is determined by dividing the number of viable tumor or cancer cells stained by antibody-specifically bound CEACAM5 in the sample or portion thereof by the total number of stained and unstained viable tumor or cancer cells and multiplying the result by 100 to obtain the tumor intensity ratio score.

[0036] The details of one or more exemplary embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0037] All publications, patents, and patent applications cited herein are expressly incorporated by reference in their entirety for all purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The patent or application file contains at least one drawing drawn in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0039] The drawings set forth herein are illustrative of exemplary embodiments provided herein and are not meant to limit the scope of the present invention as encompassed by the claims.

[0040] The drawings are described in detail herein.

[0041] Like reference symbols in the various drawings denote like elements. DETAILED DESCRIPTION

[0042] In an alternative embodiment, an immunohistochemistry (IHC) method is provided for determining and scoring the extent of expression of the protein carcinoembryonic antigen-related cell adhesion molecule 5 or CEACAM5, also known as CD66e (cluster of differentiation 66e) in a tissue sample. In an alternative embodiment, a scoring method for assessing CEACAM5 expression in a tumor or cancer, such as non-squamous non-small cell lung cancer (nsNSCLC), is provided.

[0043] Alternative embodiments of the IHC methods herein provide a robust, reliable, standardized, reproducible, and harmonized method for assessing the extent of cell membrane expression of CEACAM5, thereby creating greater inter-laboratory and inter-study comparability and allowing for early and effective application of CEACAM5 in clinical practice. Embodiments of the IHC methods herein provide high-quality staining and reliable diagnostic assessment.

[0044] The current immunohistochemical scoring method for CEACAM5 measures the percentage of cells that exhibit full membrane staining and luminal (polarized) membrane staining in a tissue sample. The percentages of these full staining versus luminal staining are placed in separate scoring categories. The percentages are further divided into staining intensity boxes. In order to determine the final CEACAM5 score relative to the cutoff value, only staining with 2+ and 3+ intensities is considered. Scoring membrane expression levels into multiple categories and staining intensity boxes increases the complexity of the scoring process. The greater complexity of this scoring method then increases the time required to reach a diagnostic assessment. The individual determination of the presence or relative staining intensity of membrane and luminal staining patterns between various cells in a tissue sample by a pathologist may also be different, thereby adding difficulty to the reproducibility of the scoring method. In addition, how to apply the scoring guidelines may be confusing because some cancers, including tumor morphology in non-squamous non-small cell lung cancer (NSCLC), are heterogeneous. There is still a need in the art for simpler and more effective, as well as robust, reproducible and accurate scoring methods to assess CEACAM5 expression in tissue samples.

[0045] Embodiments herein relate to immunohistochemical methods for determining and scoring the degree of cell membrane expression of carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) in a tissue sample. In various embodiments, the method comprises: staining the tissue sample with an antibody that specifically binds to CEACAM5; determining the total number of viable tumors or cancer cells with CEACAM5 cell membrane staining, and determining the total number of stained and unstained viable tumors or cancer cells in at least a portion of the tissue sample, wherein if there is any intensity of CEACAM5 cell membrane staining above a defined threshold, the tumor or cancer cell is counted as positively stained with an anti-CEACAM5 antibody; and determining a tumor intensity ratio score (TIPS), wherein TIPS is the number of CEACAM5-stained viable tumors or cancer cells present in the tissue sample divided by the total number of stained and unstained viable tumors or cancer cells, multiplied by 100. In various embodiments herein, the tumor intensity ratio score (TIPS) may also be referred to as a CEACAM5 positive score. Such embodiments may not only provide a robust, reliable, reproducible and accurate CEACAM5 scoring method, but may also provide a scoring method with greater simplicity and efficiency. Such implementations may provide considerable advantages for reliable and efficient diagnostic assessments.

[0046] In certain embodiments, the CEACAM5 cell membrane staining includes full membrane staining, discontinuous membrane staining, partial membrane staining, complete membrane staining, dot membrane staining, linear membrane staining, luminal membrane staining, apical membrane staining, basement membrane staining, lateral membrane staining, basolateral membrane staining, high cytoplasmic staining of 2+ or greater positive staining intensity, high cytoplasmic staining of 3+ positive staining intensity, or a combination thereof. In certain embodiments, the total number of viable tumor or cancer cells includes luminal cells, glandular luminal cells, intraluminal cells, multilayered cells, cells with signet ring morphology, cells with high cytoplasmic staining, cells with intracytoplasmic lumens, or a combination thereof. In certain embodiments, the total number of viable tumor or cancer cells counted as positive staining includes all glandular luminal cells with cell membranes associated with positively stained lumens. Such embodiments can provide the advantage of including all observed modes and levels of cell membrane staining, cytoplasmic staining, and all types of viable tumor or cancer cells in the determination of tumor intensity ratio scores. Such embodiments can provide the benefit of a simplified IHC scoring method for scoring cell membrane expression of CEACAM5 in tissue samples.

[0047] In some embodiments, the tissue sample comprises at least about 100 cells. In some embodiments, the tissue sample comprises at least about 200 cells. In some embodiments, the tissue sample comprises at least about 500 cells.

[0048] In some embodiments, tumors or cancer cells counted as positive staining exclude tumors or cancer cells with cytoplasmic staining, staining of normal or non-tumor structures, staining of non-viable tumors or cancer cells, necrotic cells, cell debris, matrix staining, or edge artifact staining on the periphery of the tissue sample. In certain embodiments, cytoplasmic staining includes cytoplasmic staining of 2+ or less positive staining intensity. Such embodiments can provide the advantage of increasing the accuracy and reproducibility of the CEACAM5 scoring method by excluding stained cells with specific structures or characteristics from the determination of the tumor intensity ratio score (TIPS).

[0049] In certain embodiments, the defined threshold comprises a positive staining intensity of 2+ or greater. In certain embodiments, the defined threshold comprises a 2+ positive or 3+ positive staining intensity assessed at a magnification of about 4X to about 10X. In certain embodiments, the method further comprises confirming the positive staining intensity at a magnification of about 20X to about 40X. In certain embodiments, the tumor intensity ratio score comprises the number of CEACAM5 viable tumors or cancer cells stained with a positive staining intensity of 2+ or greater divided by the total number of stained and unstained viable tumors or cancer cells, multiplied by 100. Such embodiments can provide the benefit of diversity in terms of staining intensity for defining thresholds.

[0050] In certain embodiments, a tumor intensity ratio score (TIPS) of about 40% or greater indicates the diagnostic status of a 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 a tissue sample. In certain embodiments, a TIPS of about 1% or greater indicates the diagnostic status of a tissue sample. In certain embodiments, a TIPS of about 5% or greater includes the number of CEACAM5 viable tumors or cancer cells stained with a positive staining intensity of 2+ or greater; or a TIPS of about 10% or greater includes the number of CEACAM5 viable tumors or cancer cells stained with a positive staining intensity of 2+ or greater. Such embodiments may provide benefits in terms of the accuracy of the determination of the diagnostic status associated with CEACAM5 expression.

[0051] In an alternative embodiment, an IHC method is provided that includes a simplified scoring system for membrane staining of ≥2+ intensity thresholds and provides guidelines for handling different morphological patterns of luminal membrane staining. When viability and tumor cell identity can be confirmed, luminal tumor cells are included in the CEACAM5 assessment. When tumor cells with clear membrane staining are presented in the same 10X field of view, guidelines for evaluating tumor cells with strong (3+) cytoplasmic staining that may mask membrane staining are used.

[0052] In alternative embodiments, the methods and kits as provided herein are used for in vitro diagnostic purposes. In alternative embodiments, CEACAM5 IHC as provided herein is an immunohistochemistry (IHC) assay using an anti-CEACAM5 antibody (e.g., monoclonal mouse anti-CEACAM5 clone 769) in the detection of formalin-fixed, paraffin-embedded (FFPE) tissue samples (e.g., lung cancer tissue samples). In alternative embodiments, the Dako Omnis TM EnVision FLEX TM Visualization system.

[0053] In alternative embodiments, CEACAM5 IHC as provided herein is used as an aid in identifying patients with a tumor or cancer, e.g., 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 carcinoma.

[0054] In an alternative embodiment, a method for assessing the extent of CEACAM5 expression comprises: contacting a sample or portion thereof containing cancer or tumor cells from an individual with an antibody or portion thereof that specifically binds to CEACAM5; and determining a tumor intensity ratio score by dividing the number of viable tumor or cancer cells stained with CEACAM5 specifically bound by the antibody in the sample or portion thereof by the total number of stained and unstained viable tumor or cancer cells and multiplying the result by 100, thereby obtaining the tumor intensity ratio score.

[0055] In some embodiments, a section or portion of a tissue sample is prepared on a slide, a microscope slide, or an equivalent, and the section or portion of the tissue sample is stained on the slide. In some embodiments, the tissue sample comprises a formalin-fixed, paraffin-embedded (FFPE) specimen. In some embodiments, the FFPE specimen comprises a cancer specimen stained on an automated IHC platform. In some embodiments, a section of a tissue sample is prepared by a scheme including fixation in about 10% neutral buffered formalin for a period of about 6 hours to about 72 hours. In some embodiments, the tissue sample is derived from a needle biopsy specimen, a fine needle aspirate, a cytology specimen, or bone decalcification.

[0056] In certain embodiments, the antibody comprises a monoclonal mouse anti-CEACAM5 antibody or a monoclonal rabbit anti-CEACAM5 antibody. In some embodiments, the monoclonal mouse anti-CEACAM5 antibody comprises monoclonal mouse anti-CEACAM5 clone 769, or an antibody having substantially similar affinity for CEACAM5 as the clone 769 antibody.

[0057] 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 carcinoma.

[0058] In certain embodiments, positive staining is determined using a bright field light microscope, a microscope objective, a computer monitor, and imaging software, or a combination thereof. In some aspects, the imaging software comprises whole slide imaging software.

[0059] Embodiments of methods for diagnosing tumors or cancer

[0060] Embodiments herein provide methods for diagnosing a tumor or cancer by determining whether a tissue sample is positive for expression of carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5). In various aspects, the method comprises: determining the diagnostic status of CEACAM5 in a tissue sample by an IHC method as provided herein for determining the extent of cell membrane expression of CEACAM5, wherein about 50% or more of the tumor cancer cells having a cell membrane expression of CEACAM5 of 2+ or greater intensity have a tumor intensity ratio score that 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.

[0061] Embodiments of methods for treating cancer and tumors

[0062] Provided are methods for treating or ameliorating a tumor or cancer in a patient, the method comprising determining and scoring the amount of CEACAM5 in a tissue sample from the patient using a method as provided herein, wherein if the tissue sample is determined or scored as having a high or diagnostically positive CEACAM5 score, the patient is treated with a cancer treatment or anticancer therapy to which the patient may respond favorably. The tumor or cancer may 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.

[0063] In certain embodiments, the cancer treatment comprises administering an anticancer drug or anticancer therapy to the patient. In alternative embodiments, the anticancer treatment or therapy comprises: surgery, such as CyberKnife therapy; chemoembolization; ablation techniques, such as radiofrequency ablation (RFA), cryoablation and / or microwave ablation; and / or radiation therapy, such as stereotactic body radiation therapy.

[0064] In certain embodiments, the anticancer therapy comprises an antibody drug conjugate, a small molecule therapy, an immunotherapy, a monoclonal antibody therapy, an adoptive cell therapy, a T cell receptor therapy, or a chimeric antigen receptor (CAR) T cell therapy. In alternative embodiments, the anticancer therapy or therapy comprises: a tyrosine kinase inhibitor (optionally erlotinib (or TARCEVA TM ), gefitinib (or IRESSA TM ), afatinib (or GILOTRIF TM ), or osimertinib (TAGRISSO TM )); Nexituzumab (or PORTRAZZA TM ), pembrolizumab (or KEYTRUDA TM ), nivolumab (or OPDIVO TM ), ipilimumab (YERVOY TM ), cetuximab (or ERBITUX TM ), cisplatin (or PLATINOL TM ) or carboplatin (or paraplatin TM ).

[0065] In alternative embodiments, the anti-cancer treatment or therapy comprises the use of an anti-cancer drug, which may comprise an antibody that specifically or substantially binds to a cancer or tumor, wherein the antibody is conjugated to a cytotoxic agent, and optionally the cytotoxic agent comprises a radionuclide (optionally yttrium-90, iodine-131, lutetium-177, radium-223 chloride, strontium-89 chloride, or samarium-153EDTMP), diphtheria toxin, Pseudomonas aeruginosa exotoxin A, denileukin, moxetumomabpasudotox, calicheamicin or N-acetyl-γ-calicheamicin, emtansine or DM1, maytansine or a derivative thereof, SN-38 (or 7-ethyl-10-hydroxycamptothecin), or auristatin or monomethyl auristatin E (MMAE).

[0066] Immunohistochemistry

[0067] In alternative embodiments, the immunohistochemical methods and / or reagents used with the manufacturing methods and manufacturing products or kits as provided herein may include or comprise the use of any IHC protocol, IHC medical device, apparatus and / or image or data analysis system to practice IHC or IHC reagents known in the art, such as, for example, as described in U.S. Patent Nos. (USPN) 10,634,590 (describing a slide holder assembly fixture for IHC); 10,565,479 (describing a method for identifying blurred areas in a digital image of stained tissue); 10,564,076 (describing a system for analyzing (or IHC) sample preparation); 10,551,395 (describing an automated histological staining system); 10,551,378 (describing a tissue staining method); 10,504,224 (describing a digital tissue image analysis system for IHC); 10,501,777 (describing simultaneous, multiplexed detection and quantification of protein expression in IHC); 10,488,340 (describing a method for extracting 10,453,195 (describing methods for detecting tissue regions of interest using digital pathology imaging); 10,438,381 (describing apparatus, systems, and methods for generating digital images of tissue sections); 10,430,943 (describing methods and procedures for automated nuclear area / number estimation for IHC image analysis); 10,416,176 (describing methods for processing specimens in an automated tissue staining system); 10,393,63 3 (describing methods for processing and inhibiting degradation of IHC samples); 10,217,011 (describing treatment of IHC slides); 10,209,165 (describing automated or semi-automated methods for assessing staining quality of a specimen containing cells); 10,126,216 (describing methods for fixing tissue samples for IHC); 9,423,322; 8,515,683 (describing methods and systems for automatically detecting immunohistochemistry (IHC) patterns); USPN 10,816,443 (describing an automated batch stainer for staining biological specimens on microscope slides); or U.S. Patent Application Publication No.: US2019 / 0178867A1 (describing the detection of a specific tissue object within a thin section of a tissue sample as imaged in a bright field microscope without the use of a chromogenic stain specific to the specific tissue object within the thin section of the tissue sample); US2019 / 0156510 A1 (describing an image analysis method for analyzing IHC tissue samples); US2019 / 0293637 A1 (methods and systems for quantitative immunohistochemistry (IHC) of target protein molecules); US2019 / 0080450 A1 (describing automated determination of staining quality of biological samples stained by IHC);Or as described in US2020 / 0316589 A1 (describing a porous solid support container for handling and testing fixed biological materials). ;

[0068] In alternative embodiments, the antibodies, antigen-binding fragments thereof, or monomeric or dimeric antigen-binding proteins (including, e.g., synthetic or recombinant forms) as provided herein used in the IHC protocols or kits as provided herein are substantially purified or isolated, or are in the form of unpurified or partially purified culture supernatants.

[0069] In alternative embodiments, methods as provided herein may use or comprise reagents for detecting or visualizing antibody-antigen interactions using, for example, any product or method, and IHC protocols or reagents known in the art.

[0070] In alternative embodiments, the methods as provided herein include the use of chromogenic immunohistochemistry (CIH), wherein a primary antibody (e.g., a recombinant antibody (Ab) or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein as provided herein) or a secondary antibody (e.g., wherein the secondary antibody binds to a primary antibody as provided herein, or a recombinant antibody (Ab) or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein) is conjugated to an enzyme that can catalyze a reaction that produces a color, such as a peroxidase (e.g., an immunoperoxidase), such as horseradish peroxidase (HRP). In alternative embodiments, the chromogenic moiety used in the methods as provided herein is or comprises coumarin; rhodamine; 2,3,6,7-tetrahydro-11-oxo-1H,5H,11H-[1]benzopyrano[6,7,8-ij]quinoline-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; basic fuchsin; cascade blue acetyl; Dapoxyl sulfonic acid / carboxylic acid succinimidyl ester; DY-405; Alexa Fluor 405 succinimidyl ester; Cascade Yellow succinimidyl ester; Pyridyl oxazole succinimidyl ester (PyMPO); Pacific Blue succinimidyl ester; DY-415; 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester; DYQ-425; 6-FAM phosphoramidite; Fluorescent 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'-tetrabromosulfone fluorescein succinimidyl ester; Alexa Fluor532 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 / 591C3 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; Wool poppy red; Alexa Fluor 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 succinimide ester; DY-703; DY-701; DY-704; DY-700; DY-730; DY-731; DY-732; DY-734; DY-750; Cy7 succinimide ester; DY-749; DYQ-4; Cy7.5 succinimide ester; 7-diethylaminocoumarin-3-carboxylic acid; succinimide ester; Dabsyl sulfonyl chloride; fluorescein isothiocyanate (FITC) carboxysuccinimide 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); gallic blue; fast green FCF; malachite green; or QSY 21 succinimidyl ester. ;

[0071] In alternative embodiments, the methods as provided herein include the use of immunofluorescence, wherein the primary or secondary antibody is labeled with a fluorophore (e.g., 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 Fluor®, TM or Dylight TM Fluorescent dyes, or fluorophores or dyes as described in US Patent Application No. US2019 / 0018018 A1. 3,3'-Diaminobenzidine (DAB) may also be used.

[0072] In alternative embodiments, methods as provided herein include the use of a direct method or a one-step staining method, wherein, for example, in a tissue section, a primary antibody (e.g., an antibody (Ab) or an antigen-binding fragment thereof as provided herein, or a monomeric or dimeric antigen-binding protein (including, for example, a synthetic or recombinant form)) is labeled and reacts directly with the antigen. Although this technique utilizes only one antibody and is therefore simple and rapid, the sensitivity may be low because the signal amplification is minimal.

[0073] In alternative embodiments, methods as provided herein include the use of an indirect approach in which an unlabeled primary antibody (first layer) binds to a target antigen (e.g., CEACAM5) in, for example, a tissue or organ, and then a labeled secondary antibody (second layer) reacts with the primary antibody. The secondary antibody can be directed against an isotype, e.g., IgG of the animal species from which the primary antibody is derived. This approach may be more sensitive than direct detection strategies because if several secondary antibodies are conjugated to detection reagents such as fluorescent or enzyme reporters, the signal amplification caused by the binding of the secondary antibodies to each primary antibody.

[0074] In an alternative embodiment, if the secondary antibody is conjugated to several detection molecules, such as a biotin molecule, the biotin molecule can recruit avidin-, streptavidin- or NeutrAvidin TMThe protein-binding enzyme complex achieves further amplification.

[0075] In alternative embodiments, IHC is performed on tissue sections or tissue biopsy sections (e.g., paraformaldehyde (PFA) fixed tissue or organ, or formalin fixed paraffin embedded tissue). In alternative embodiments, the tissue is sliced ​​or used as a whole. Before slicing, the tissue sample can be embedded in a medium, such as paraffin or freezing medium. Tissue sections can be sliced ​​on a variety of instruments, most commonly microtomes, cryostat slicers, or vibrating slicers. The specimen can be sliced ​​in the range of about 3 μm to 5 μm. The sections can be mounted on slides, dehydrated using increasing concentrations of alcohol washes (e.g., 50%, 75%, 90%, 95%, 100%), and cleaned with a detergent such as xylene, and then imaged under a microscope.

[0076] Depending on the method of fixation and tissue preservation, samples may require additional steps to make the CEACAM5 epitope available for antibody binding, including deparaffinization and antigen retrieval. For formalin-fixed paraffin-embedded tissues, antigen retrieval is often necessary and may include pre-treatment of sections with heat or proteases.

[0077] In an alternative embodiment, the EnVision DuoFLEX Doublestain System is used TM IHC is performed using the EnVisionDuoFLEX Dual Staining System (Agilent, San Jose, CA), which allows 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 (DakoOmnis) system, and binding can be visualized by the EnVision FLEX HRP Magenta chromogen. In an alternative embodiment, IHC is performed using the EnVision FLEX Micro Kit, High pH, ​​which is a high sensitivity visualization system designed to be used with the Dako Autostainer TM The ANALYZER® instruments are used together in IHC; this dual-link system detects mouse and rabbit primary antibodies, and the reactions are visualized by 3,3'-diaminobenzidine (DAB) chromogen (DAB forms a water-insoluble brown precipitate when oxidized, for example, by peroxidase).

[0078] Manufacturing products and kits

[0079] Articles of manufacture and kits for practicing the methods provided herein are provided, comprising, for example, a monoclonal mouse anti-CEACAM5 antibody (e.g., a clone 769 anti-CEACAM5 antibody), and / or reagents for practicing IHC, including, for example, reagents as described herein, see Example 1 below; and optionally, the articles of manufacture and kits can further include instructions for practicing the methods described herein.

[0080] Any of the above aspects and embodiments may be combined with any other aspects or embodiments disclosed herein in the Summary, Figures, and / or Detailed Description sections.

[0081] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0082] Unless otherwise stated or obvious from the context, as used herein, the term "or" is construed as inclusive and encompasses both "or" and "and."

[0083] Unless otherwise specified or apparent from the context, as used herein, the term "about" is understood to be within the normal tolerance range of the art, such as within 2 standard deviations of the mean value. Approximately (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 stated value. Unless otherwise clearly stated from the context, all numerical values ​​provided herein are modified by the term "about".

[0084] Unless specifically stated or obvious from the context, as used herein, the terms "substantially all," "substantially a majority," "substantially all," or "majority" encompass at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or more of a reference amount of a composition.

[0085] The entire contents of each patent, patent application, publication and document cited herein are hereby incorporated by reference. The reference to the above patents, patent applications, publications and documents does not admit that any of the above is relevant prior art, nor does it constitute any admission of the contents or dates of these publications or documents. The reference to these documents alone should not be construed as an assertion or admission that any part of the contents of any document is considered to be necessary material to satisfy the statutory disclosure requirements of patent applications in any country or region. Nevertheless, the right to rely on any such document to provide materials that the examining body or court considers to be essential to the subject matter claimed is reserved where appropriate.

[0086] Without departing from the basic aspects of the present invention, the foregoing may be modified. Although the present invention has been described in considerable detail with reference to one or more specific embodiments, it will be appreciated by those of ordinary skill in the art that changes may be made to the embodiments specifically disclosed in this application, and these modifications and improvements are still within the scope and spirit of the present invention. The present invention illustratively described herein may be properly practiced in the absence of any elements not specifically disclosed herein. Thus, for example, in each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced by any of the other two terms. Therefore, the terms and expressions that have been used are used as terms of description rather than limitation, and equivalents of the features shown and described or parts thereof are not excluded, and it should be recognized that various modifications are possible within the scope of the present invention. Embodiments of the present invention are set forth in the following claims.

[0087] The present invention will be further described with reference to the examples described herein; however, it should be understood that the invention is not limited to such examples.

[0088] Example

[0089] Unless otherwise indicated in the examples, all recombinant DNA techniques were performed 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 and in Ausubel et al., (1994) Current Protocols in Molecular Biology, Current Protocols, USA, Volumes 1 and 2. Other references to standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, NY; Brown (1998) Molecular Biology Lab Fax, 2nd edition, Academic Press (UK), Volumes I and II.

[0090] Figure 1 Representative images of CEACAM5 staining in non-squamous NSCLC using the CEACAM5 IHC (antibody clone) 769 assay are shown, as described in further detail in Example 1 below.

[0091] Figure 2 Representative images of examples of positive staining are shown: punctate (red arrows, or arrows at the left edge of the image) and linear (black arrows, or arrows in the middle of the image) membrane staining of intensity ≥2+ are considered positive; the 40x image shown here is for illustrative purposes, as described in further detail in Example 1 below.

[0092] Figure 3 Partial and complete membrane staining: arrow "a" indicates complete membrane staining; arrow "b" indicates partial, basolateral and apical (luminal) membrane staining; and arrow "c" indicates partial, apical (luminal) membrane staining (alveolar pattern), as described in further detail in Example 1 below.

[0093] Figure 4 CEACAM5 scoring is shown: red (or longer) arrows indicate areas where high cytoplasmic staining without distinguishable membrane staining was observed, and cells were scored as CEACAM5 positive because tumor cells within the 10X objective field of view (black (or shorter) arrows) had discernible membrane staining and indicated that the area was highly CEACAM5 positive, as further described in detail in Example 1 below.

[0094] Figure 5 An exemplary benign tissue staining example is shown: staining of normal (non-neoplastic) tissue, as shown here, as described in further detail in Example 1 below.

[0095] Figure 6 Images are shown which demonstrate that staining may occur in necrotic tissue and at a range of intensities, as described in further detail in Example 1 below.

[0096] Figure 7 An image is shown demonstrating that only tumor cells (black (or longer) arrows) were scored as CEACAM5 positive, and any surrounding stroma (red (shorter) arrows) showing CEACAM5 staining was excluded from the percentage score of the CEACAM5 staining protocol as provided herein and was not considered nonspecific background staining, as described in further detail in Example 1 below.

[0097] Figure 8 Images showing 1+ (upper row), 2+ (middle row), 3+ (lower row) staining intensity according to the CEACAM5 scoring scheme provided herein, as described in further detail in Example 1 below.

[0098] FIG. 9A to FIG. 9B An image is shown which indicates possible pigments present in the sample, such as ankylosing pigment ( Fig.9A arrow) and hemosiderin ( Fig. 9B The pigment of the DAB precipitate (arrow) is distinguishable from the DAB precipitate at medium to high magnification and should not be included in the scoring of CEACAM5 positivity using the CEACAM5 scoring scheme as provided herein, as described in further detail in Example 1 below.

[0099] FIG. 10A to FIG. 10B Image showing luminal staining detail and nested pattern: Fig. 10A 20X H&E (hematoxylin and eosin) staining is shown; and Fig. 10B Shown at 20X is CEACAM5-positive apical membrane staining in the luminal portion of the nests, as described in further detail in Example 1 below.

[0100] Fig.11 Images showing luminal staining details and nested patterns; arrows indicate nests that are either positive or negative for CEACAM5, as described in further detail in Example 1 below.

[0101] FIG. 12A to FIG. 12B Image showing luminal staining detail and alveolar pattern: Fig. 12A : All cells in the outlined alveolar structures were positive; and Fig. 12B: The alveolar structure in the center of this panel contains positive and negative cells, as described in further detail in Example 1 below.

[0102] FIG. 13A to FIG. 13B H&E staining of cells with signet ring morphology is shown ( Fig.13A ) and CEACAM5 staining ( Fig. 13B ) as described in further detail in Example 1 below.

[0103] FIG. 14A to FIG. 14D An image showing viable tumor cells in the intraluminal space ( Fig.14A ; Fig. 14B ), if viability can be confirmed, the viable tumor cells should be scored using the CEACAM5 scoring scheme as provided herein: Fig.14A is the H&E staining of tumor cells, and Fig. 14B is CEACAM5 staining of tumor cells; and FIG. 14C to FIG. 14D : Macrophages and non-viable tumor cells should not be scored. Fig. 14C is H&E stained, and Fig.14D is CEACAM5 staining, as described in further detail in Example 1 below.

[0104] Fig.15 Images showing intracytoplasmic luminal staining (arrows) suggest the possible presence of CEACAM5, as described in further detail in Example 1 below.

[0105] Figure 16A to Fig. 16B Images are shown, which show that mucin can be stained with high intensity (H&E staining is Figure 16A, and CEACAM5 staining is Fig. 16B ); exemplary mucin staining is shown in the boxed area, as described in further detail in Example 1 below.

[0106] Figure 17A to Fig. 17B Positive control tissue images are shown: 10X (Figure 17A) and 20X ( Fig. 17B ) in non-squamous NSCLC tissue, demonstrating a range of weak (1+, red arrow) to moderate (2+, black arrow) intensity membranous staining using the CEACAM5 scoring scheme, as described in further detail in Example 1 below.

[0107] 18A to 18D Negative control tissue images are shown: Fig.18A : H&E staining of non-squamous NSCLC; Fig.18B : Low magnification objectives can be used to initially scan the tissue and observe any CEACAM5 staining; Fig. 18C: A higher magnification of 10X can be used to confirm the presence of any staining; and Fig.18D : A 20X objective can be used to observe whether there is any specific membrane staining at 1+ intensity, as described in further detail in Example 1 below.

[0108] Fig.19 , top panel, shows an H&E (hematoxylin and eosin) stained image of a tissue specimen that was first evaluated by H&E to assess tissue histology and preservation quality;

[0109] Fig.19 , middle panel, shows a negative control reagent slide, which is used to assess non-specific staining and allow interpretation of CEACAM5 stained slides; and

[0110] Fig.19 , lower panel, shows CEACAM5 staining assessed using the CEACAM5 scoring scheme as provided herein, which was considered positive if it was complete and / or partial plasma membrane staining of greater than or equal to (≥) 2+ intensity,

[0111] As described in further detail in Example 1 below.

[0112] Fig. 20 Shown is a table summarizing the results of studies performed using the CEACAM5 staining protocol as provided herein, as described in Example 2 below.

[0113] Example 1: CEACAM5 IHC

[0114] This example demonstrates the use of the CEACAM5 staining protocol as provided herein.

[0115] Provided herein are exemplary scoring guidelines for evaluating CEACAM5 expression in non-squamous non-small cell lung cancer (non-squamous NSCLC) specimens that have been stained with a mouse monoclonal CEACAM5 antibody, designated mouse mAb clone 769. This antibody is provided in the CEACAM5 IHC 769 assay for identification of CEACAM5 expression in formalin-fixed, paraffin-embedded (FFPE) non-squamous NSCLC specimens using the Dako Omnis automated staining system.

[0116] Assessment of CEACAM5 IHC staining pattern in non-squamous non-small cell lung cancer (non-squamous NSCLC) FFPE tissues A protocol-specific Pathology Report Form (PRF) was completed for each specimen evaluated (including "Not Evaluable" and "Indeterminate" as described below).

[0117] CEACAM5 score summary

[0118] Definition of Positive Staining

[0119] CEACAM5 positive staining is defined as any partial or complete tumor cell plasma membrane staining of ≥2+ intensity. Membrane staining can be linear or punctate. The staining can appear as complete or partial staining involving the basal, lateral, or basolateral aspects of the membrane, or as apical staining of the luminal aspect of the cell.

[0120] Representative staining examples are described below, see Figures 1 to 19 。

[0121] The percentage of CEACAM5 positive score is determined by the following formula:

[0122]

[0123] A minimum of 100 evaluable and viable tumor cells must be present on the slide stained for CEACAM5 to determine the percentage of stained cells.

[0124] The overall CEACAM5 diagnostic status is determined according to the following criteria:

[0125] · If ≥X% of the tumor cells exhibit CEACAM5 positive staining of ≥2+ intensity, the specimen is considered diagnostically positive, where X is a predetermined cut-off value.

[0126] · If <X% of the tumor cells exhibit CEACAM5 positive staining of ≥2+ intensity, the specimen is considered diagnostically negative, where X is a predetermined cut-off value.

[0127] For adenocarcinomas showing glandular lumina, CEACAM5 partial plasma membrane staining may appear as apical membrane staining of the luminal aspect of the cell. In the case of luminal staining of ≥2+ intensity, all cells associated with the structure that may have a membrane associated with the positive lumen will be considered positive.

[0128] Definition of Negative Staining

[0129] Negative staining is defined as no tumor cell membrane staining or any membrane staining at <2+ intensity.

[0130] The following are included in the scoring:

[0131] · Partial or complete punctate membrane staining and linear membrane staining at ≥2+

[0132] The intensity is considered positive.

[0133] · Evaluate the intensity and frequency of positive staining of all viable tumor cells on the slide.

[0134] Cell types and staining patterns excluded from staining scoring:

[0135] Cytoplasmic staining is not usually considered positive; however, tumor cells with high cytoplasmic staining (3+) can be inferred to be positive when positive membrane staining is identified in other tumor cells present within the 10x objective field of view of the high cytoplasmic staining cell. More details and examples are provided in Sections 2.1 and Figure 4 Available in.

[0136] Staining of normal, non-neoplastic structures ( Figure 5 )

[0137] · Exfoliated non-viable tumor cells

[0138] Necrotic cells and cell debris ( Figure 6 )

[0139] Matrix staining ( Figure 7 )

[0140] Edge artifact staining on the periphery of the tissue specimen

[0141] Scoring of non-squamous NSCLC

[0142] Staining intensity was scored using the following scale (Table 1):

[0143] Table 1. Staining intensity scale

[0144] Intensity scale Staining intensity What staining intensity is considered CEACAM5 positive? 0 No staining no 1+ weak staining no 2+ Moderate staining yes 3+ Strong staining yes

[0145] Examples of CEACAM5 staining at each intensity are shown in Figure 8 Shown in.

[0146] CEACAM5 positive tumor staining of ≥2+ was scored using a 4X to 10X microscope objective. The 20x objective can be used to identify and / or confirm 1+ and punctate and / or fine 2+ membrane staining intensities and 2+ cytoplasmic lumens that may not be apparent under the 4X and 10X objectives. If necessary, a 20X to 40X objective can be used to confirm membrane staining localization. The 40X objective should not be routinely used for scoring (i.e., assessing intensity and percent positivity).

[0147] Non-squamous NSCLC tissue samples are being evaluated for this assay. An H&E stained slide should accompany each CEACAM5 stained sample to allow for proper confirmation of the presence of tumor and histologic diagnosis, assessment of tumor viability, and to allow for differentiation from non-tumor portions of the slide that should be excluded from the assessment. H&E and NCR stained slides also aid in the identification of intrinsic pigments (due to intrinsic ankylosing pigments, hemosiderin pigments, etc.; see, e.g., FIG. 9 ).

[0148] CEACAM5 expression pattern observed in non-squamous NSCLC:

[0149] - Luminal staining: Two morphological patterns of structures showing luminal (apical membrane) staining can be observed in lung adenocarcinoma: a nested pattern and an alveolar pattern. When luminal staining of intensity ≥2+ is observed in either pattern, all cells within the structure that may have a membrane associated with the positive lumen should be considered positive.

[0150] Nested pattern: solid nests bounded by stroma and containing multiple layers of cells and small concentric luminal spaces. Tumors with solid tumor nests containing glandular lumens may show apical membrane staining of the lumina for CEACAM5. In the case of luminal staining of intensity ≥2+, it is assumed that all cells within the nest are likely to have membrane association with the lumen and therefore all cells within the nest should be considered positive (Figures 10 and Fig.11 ).

[0151] Alveolar pattern: Glandular structures (single or multilayered) containing irregularly shaped luminal spaces of varying sizes. Cells exhibiting apical membrane staining of ≥2+ intensity in the luminal surface were considered positive. Cell layers behind those cells facing the lumen may also have membranes associated with the positive lumen and are therefore considered similarly positive. Further details and examples are provided in the Appendix Figure 3 and Figure 12.

[0152] Cells with signet ring morphology: Cells with signet ring cell morphology can show high intensity staining in the intravacuolar space and cytoplasm. Membrane staining in these cases may be difficult to distinguish due to the intensity of cytoplasmic staining. For cells with signet ring cell morphology, tumor cells with high intensity staining were considered membrane staining positive and scored relative to the cytoplasmic intensity. For example, if the cytoplasmic intensity was ≥2+ in a cell with signet ring morphology, the cell was considered CEACAM5 positive (Figure 13).

[0153] Highly cytoplasmic staining cells: Cytoplasmic staining without clear membrane staining is not usually considered positive staining. However, when positive membrane staining is identified in tumor cells within the 10x objective field of view of highly cytoplasmic staining cells, highly cytoplasmic staining tumor cells (3+ intensity) can be inferred to be positive ( Figure 4 ).

[0154] Viable intraluminal cells: Viable tumor cells within the lumen and alveoli should be included in the score if the membrane stains ≥ 2+ intensity and if viability can be confirmed. Matched H&E slides may be required to determine viability. Care must be taken to distinguish tumor cells from macrophages, which should not be scored. Luminal cells should be excluded if the cells are non-viable or if viability or tumor identity cannot be confirmed (Figure 14).

[0155] Intracytoplasmic lumen: cells with intracytoplasmic lumen with luminal staining ≥2+ intensity were considered positive ( Fig.15 ). H&E can be used to determine whether a sample contains this expression pattern.

[0156] Non-specific background staining:

[0157] Nonspecific background staining for NCR and CEACAM5 stained slides should be ≤ 1 + mean intensity across the specimen.

[0158] Exclude the following from the assessment of nonspecific background staining:

[0159] · Stromal staining adjacent to highly stained tumor cells may have some staining. Stromal staining was not scored as nonspecific background staining ( Figure 7 ).

[0160] Necrosis may stain intensely and is not scored as nonspecific background staining ( Figure 6 ).

[0161] • Mucins stained with high intensity and were not scored as non-specific background staining (Figure 16).

[0162] Nonspecific background staining was assessed over the entire specimen, not focally (i.e., in areas where the amount was a small fraction of the total area). Focal staining of >1+ intensity was acceptable if counterbalanced by areas of no staining such that the average intensity over the entire specimen was ≤1+.

[0163] System comparison

[0164] Before examining IHC-stained tumor specimens, confirm that IHC staining of control tissues is satisfactory.

[0165] Positive control tissue and negative control tissue were run in parallel with the patient tissue. NCR staining was run on the control tissue to confirm that non-specific staining of the control tissue was not present in the staining procedure. Representative images of the positive control tissue are shown in Figure 17, and representative images of the negative control tissue are shown in Figure 18.

[0166] Control tumor tissue:

[0167] Control tumor tissue should be identified from pre-screened non-squamous NSCLC tissues. The control should be a fresh biopsy / surgical non-squamous NSCLC specimen that is fixed, processed and embedded in the same manner as the test specimen as soon as possible.

[0168] Negative Control Reagent (NCR)

[0169] The negative control reagent contains material matched to the nonspecific antibody against CEACAM5. Control tissue stained with NCR must show no specific membrane staining, and the presence of nonspecific background staining should be scored as ≤1 + mean intensity over the entire specimen. If specific staining occurs in NCR-stained tissue, the result is considered invalid, a "failed" checkbox should be recorded on the scoring sheet, and staining should be repeated on both the control and test specimens.

[0170] Positive control tissue

[0171] · Evaluate positive control tissue to ensure tissue preparation and satisfactory staining technique. Include a positive control tissue in every staining procedure.

[0172] • Tissues selected for use as positive tissue controls should exhibit moderate positive staining (≥2+ intensity) to detect subtle changes in assay sensitivity.

[0173] Non-specific background staining over the entire specimen should be ≤ 1 + mean intensity (see section 2.2).

[0174] • Specimens processed in a manner different from the test specimens only validate reagent performance and do not validate tissue preparation.

[0175] Known positive tissue controls should be used only to monitor the correct performance of processed tissues and test reagents and not as an aid in making a specific diagnosis from a patient sample.

[0176] Negative control tissue

[0177] Evaluate nonsquamous NSCLC negative control tissue to ensure tissue preparation, satisfactory staining technique, verify primary antibody specificity, and provide an indication of nonspecific staining.

[0178] • Different cell types present in most tissue sections provide internal negative control sites (this should be verified by the user).

[0179] The recommended negative control tumor tissue for non-squamous NSCLC is tumor tissue that does not show membrane staining in tumor cells.

[0180] Nonspecific background staining over the entire specimen should be ≤ 1 + mean intensity.

[0181] Known negative tissue controls should be used only to monitor the correct performance of processed tissues and test reagents and not as an aid in making a specific diagnosis from patient samples.

[0182] Acceptance criteria for control samples

[0183] Based on the staining results of the control specimens, accept or reject the test specimen results according to the following criteria:

[0184] If the control meets all requirements, accept the test sample results.

[0185] If the control tissue slide does not meet the requirements, the test specimen results should be considered invalid and the staining should be repeated, including the tissue control and tissue specimens.

[0186] If the negative control reagent (NCR) staining results do not meet the requirements, the test specimen results should be considered invalid and the staining should be repeated, including tissue controls and tissue samples.

[0187] Once correct staining of the control material has been established, the staining is judged to be satisfactory for assessment of the test specimens.

[0188] Patient tissue sections stained with NCR

[0189] Negative control reagents were used in place of the CEACAM5-specific primary antibody and to aid interpretation of specific staining at antigenic sites.

[0190] The absence of membrane staining confirms specific labeling of the target antigen by the CEACAM5 primary antibody on the patient specimen. NCR staining must not show any intensity of membrane staining. Nonspecific background staining over the entire specimen should be ≤ 1 + mean intensity.

[0191] Slide reading order and evaluation

[0192] For each staining run, slides should be examined in the order presented in the table below to determine the validity of the staining run and to enable assessment of the stained specimen tissue.

[0193]

[0194]

[0195]

[0196] NCR stained specimens

[0197] A negative control reagent (NCR) should be examined using sections from each patient specimen to assess nonspecific staining and allow interpretation of specific staining. Fig.19 Evaluation of H&E, followed by evaluation of NCR, and finally evaluation of CEACAM5 IHC slides of non-squamous NSCLC are shown.

[0198] Acceptance criteria:

[0199] NCR staining must not show any specific membrane staining at any intensity.

[0200] Nonspecific background staining over the entire specimen should be ≤ 1 + mean intensity.

[0201] CEACAM5 stained specimens

[0202] Carefully examine the entire tumor specimen and determine the percentage of CEACAM5-positive tumor cells with an intensity of ≥2+:

[0203]

[0204] Examples of staining at individual intensities are shown in Figure 8 The estimated number of total viable tumor cells in the entire tissue section will be used as the denominator for calculating the score.

[0205] The CEACAM5 diagnostic status (positive / negative) of non-squamous NSCLC specimens was determined by the percentage of viable tumor cells expressing CEACAM5-positive (≥2+ intensity) membrane staining.

[0206] Tumor cells are positive for CEACAM5 if they show partial or complete circumferential membrane staining at 2+ and 3+ intensities, see Figure 3 .

[0207] CEACAM5 staining may appear as punctate or linear membrane staining, such as Figure 2 shown.

[0208] For important scoring details, see CEACAM5 Expression Pattern.

[0209] Nonspecific background staining over the entire specimen should be ≤ 1 + mean intensity.

[0210] For evaluation of CEACAM5 immunohistochemical staining and scoring, a 4X objective can be used for initial assessment of the entire specimen, followed by scoring of tumor cell staining with ≥2+ intensity using a 4X to 10X objective. A 20X can be used to identify and / or confirm tumor cells that exhibit 1+ and punctate and / or fine 2+ membrane staining and 2+ intracytoplasmic lumen. If necessary, a 20X to 40X objective can also be used to confirm membrane localization of staining. A 40X objective should not be routinely used for scoring (i.e., assessment of intensity and percent positivity).

[0211] CEACAM5 positivity was scored using the following resolution: Within the range from 0 to 10%, positivity was scored in 1% increments, unless "<1%" was specified when within the range from 0 to 1%. Within the range from 10% to 100%, positivity was scored in 5% increments, but smaller increments could be used if necessary.

[0212] Elements to be excluded from scoring and from non-specific background evaluation

[0213] Additional tissue elements that may be present and show apparent true CEACAM5 staining include:

[0214] Mucin (see Figure 16)

[0215] There are no viable cells in the lumen of the detached tubes

[0216] Benign epithelial cells (see Figure 5 )

[0217] • Immune cells (eg, macrophages; see Figure 14).

[0218] Staining in these areas was excluded from determining CEACAM5 expression status.

[0219] Examples of endogenous pigments present in some specimens, and how to distinguish them from true CEACAM5 staining, are shown in Figure 9.

[0220] CEACAM5 positivity status was determined based on a pre-determined cutoff point of CEACAM5-positive staining of ≥2+ intensity level and recorded.

[0221] the term" uncertain ” is used to denote samples that have met the evaluation criteria but for reasons attributable to the biology of the tumor tissue sample, rather than due to inappropriate sample preparation or handling, have prevented scoring of tumor cell membrane staining.

[0222] Staining may also occur in certain areas of the tissue specimen, such as in the stroma, necrotic areas, and edge artifacts in the peripheral areas of the tissue. Stromal staining may occur in specimens that show highly positive CEACAM5 expression, such as Figure 7 Staining of these tissue elements was excluded from the determination of CEACAM5 expression status and assessment of nonspecific background staining (see Section 2.2). Examples of some of these staining artifacts are shown in Figure 6 and Figure 7 Shown in.

[0223] Figure 1Shown are representative images of CEACAM5 staining in non-squamous NSCLC using the CEACAM5 IHC (antibody clone) 769 assay as described herein.

[0224] Figure 2 Representative images of positive staining examples are shown: punctate (red arrows, or arrows at the left edge of the image) and linear (black arrows, or arrows in the middle of the image) membrane staining of intensity ≥2+ are considered positive; 40x images are shown here for illustration, however scoring and calculation of % CEACAM5 positive membrane staining can also be measured under 4X and / or 10X objectives.

[0225] Figure 3 Partial and complete membrane staining: arrow "a" indicates complete membrane staining; arrow "b" indicates partial, basolateral and apical (luminal) membrane staining; and arrow "c" indicates partial, apical (luminal) membrane staining (alveolar pattern).

[0226] Figure 4 The CEACAM5 score as provided herein is shown: CEACAM5 scoring generally does not take into account cytoplasmic staining, and only membrane staining is relevant for calculating the CEACAM5 positive percentage score. However, when positive membrane staining is identified in tumor cells within the 10X objective field of view of cells with high cytoplasmic staining, cells with high cytoplasmic staining can be inferred to be positive. In this case, the red (or longer) arrow indicates an area where high cytoplasmic staining is observed without distinguishable membrane staining. For these areas, the cells are scored as CEACAM5 positive because the tumor cells within the 10X objective field of view (black (or shorter) arrow) have discernible membrane staining and indicate that the area is highly CEACAM5 positive.

[0227] Figure 5 An illustrative benign tissue staining example is shown: staining of normal (non-neoplastic) tissue, as shown here, is excluded from the CEACAM5 scoring scheme as provided herein.

[0228] Figure 6 Images are shown that demonstrate that staining may occur in necrotic tissue and at a range of intensities. Necrotic areas should be excluded from scoring using the CEACAM5 scoring scheme as provided herein and are not considered non-specific background staining.

[0229] Figure 7Images are shown demonstrating that only tumor cells (black (or longer) arrows) were scored as CEACAM5 positive, and any surrounding stroma (red (shorter) arrows) showing CEACAM5 staining was excluded from the percentage score of the CEACAM5 staining protocol as provided herein and was not considered nonspecific background staining.

[0230] Figure 8 Images showing 1+ (upper row), 2+ (middle row), 3+ (lower row) staining intensities of the CEACAM5 scoring scheme as provided herein. According to the CEACAM5 scoring scheme as provided herein, 2+ and 3+ membrane staining intensities are considered CEACAM5 positive.

[0231] FIG. 9A to FIG. 9B An image is shown which indicates possible pigments present in the sample, such as ankylosing pigment ( Fig.9A arrow) and hemosiderin ( Fig. 9B The pigment of the retinal follicle (arrow) is distinguishable from the DAB precipitate at medium to high magnification and should not be included in the scoring of CEACAM5 positivity using the CEACAM5 scoring scheme as provided herein.

[0232] FIG. 10A to FIG. 10B Image showing luminal staining detail and nested pattern: Fig. 10A 20X H&E (hematoxylin and eosin) staining is shown; and Fig. 10B 20X CEACAM5 positive apical membrane staining in the luminal portion of the nest is shown. When a nest has multiple tumor cell layers and the lumen is positive, then all tumor cells within the nest are positive. In this case, cells within the dotted area are considered positive using the CEACAM5 scoring scheme as provided herein.

[0233] Fig.11 Images showing luminal staining details and nested patterns: The nested expression pattern is a solid tumor nest defined by a stroma containing multiple cell layers and small concentric luminal spaces. If a nest shows positive luminal staining, all cells within the nest are considered positive using the CEACAM5 scoring scheme as provided herein. Arrows indicate nests that are CEACAM5 positive or negative.

[0234] FIG. 12A to FIG. 12B Images showing luminal staining details and alveolar patterns: When structures with this pattern have multiple cell layers and the lumen is positive, using the CEACAM5 scoring scheme provided herein, cells in the luminal surface exhibiting apical membrane staining of greater than or equal to (≥) 2+ intensity are considered positive, and cell layers behind those cells facing the lumen are considered similarly positive. Fig. 12A: All cells in the outlined alveolar structures were positive; and Fig. 12B : The alveolar structure in the center of this panel contains both positive and negative cells.

[0235] FIG. 13A to FIG. 13B H&E staining of cells with signet ring morphology is shown ( Fig.13A ) and CEACAM5 staining ( Fig. 13B ) images. Cells with signet ring morphology may show high intensity staining in the intravacuolar space and cytoplasm. Membrane staining may be difficult to distinguish due to the intensity of cytoplasmic staining. In these cases, tumor cells are considered membrane staining positive and scored relative to cytoplasmic intensity, i.e., if the cytoplasmic intensity is ≥2+ in the signet ring subtype, the cells are considered positive using the CEACAM5 scoring scheme as provided herein.

[0236] FIG. 14A to FIG. 14D An image showing viable tumor cells in the intraluminal space ( Fig.14A ; Fig. 14B ), if viability can be confirmed, the viable tumor cells should be scored using the CEACAM5 scoring scheme as provided herein: Fig.14A is the H&E staining of tumor cells, and Fig. 14B is CEACAM5 staining of tumor cells, as described herein. This assessment may require H&E. Care should be taken to distinguish tumor cells from macrophages. FIG. 14C to FIG. 14D : Macrophages and non-viable tumor cells should not be scored. Fig. 14C is H&E stained, and Fig.14D was CEACAM5 staining as described herein.

[0237] Fig.15 Images showing staining of intracytoplasmic luminal tubules (arrows) indicate the possible presence of CEACAM5. H&E staining can be used to confirm the presence of intracytoplasmic luminal tubules. When staining ≥ 2+, these cells are considered positive and should be included in the scoring using the CEACAM5 scoring scheme as provided herein.

[0238] Figure 16A to Fig. 16B Images are shown, which show that mucin can be stained with high intensity (H&E staining is Figure 16A, and CEACAM5 staining is Fig. 16B ); this is not considered nonspecific background staining and is not excluded from the CEACAM5 percentage score using the CEACAM5 scoring scheme as provided herein. Exemplary mucin staining is shown in the boxed area.

[0239] Figure 17A to Fig. 17BPositive control tissue images are shown: 10X (Figure 17A) and 20X ( Figure 7 B) Non-squamous NSCLC tissue below, demonstrating the range of weak (1+, red arrows, or top two arrows at the center of the image and the second arrow down from the upper right corner of the image) to moderate (2+, black arrow, or two lower arrows at the center and lower right corner of the image and the arrow in the upper right corner of the image) intensity membrane staining using the CEACAM5 scoring scheme as provided herein.

[0240] 18A to 18D Negative control tissue images are shown: Fig.18A : H&E staining of non-squamous NSCLC; Fig.18B : Low magnification objectives can be used to initially scan the tissue and observe any CEACAM5 staining; Fig. 18C : A higher magnification of 10X can be used to confirm the presence of any staining; and Fig.18D : A 20X objective can be used to see if there is any specific membrane staining at 1+ intensity. In this example, there is no CEACAM5 staining, and this specimen can be used as a good negative control tissue.

[0241] Fig.19 , top panel, shows an H&E (hematoxylin and eosin) stained image of a tissue specimen that was first evaluated by H&E to assess tissue histology and preservation quality;

[0242] Fig.19 , middle panel, shows a negative control reagent slide, which is used to assess non-specific staining and allow interpretation of CEACAM5 stained slides; and

[0243] Fig.19 , lower panel, shows CEACAM5 staining assessed using the CEACAM5 scoring scheme as provided herein, which was considered positive if it was complete and / or partial plasma membrane staining of greater than or equal to (≥) 2+ intensity,

[0244] Example 2: External reproducibility study of CEACAM5 IHC 769 on non-squamous non-small cell lung cancer specimens This example demonstrates the use of the CEACAM5 staining protocol as provided herein.

[0245] The aims of this study were to evaluate:

[0246] - Study Unit CEACAM5 IHC 769, regarding inter-site, intra-site / inter-day, inter-observer, and intra-observer reproducibility performed on a panel of formalin-fixed, paraffin-embedded (FFPE) human non-squamous non-small cell lung cancer tissue specimens at three external testing sites; and

[0247] - CEACAM5 IHC 769 Inter-site, intra-site / inter-day, inter-observer and intra-observer reproducibility of FFPE non-squamous NSCLC specimens with ≥2+ CEACAM5-positive tumor cells with ≥50% intensity using the Dako Omnis instrument. The results of this study are used to demonstrate assay reproducibility in accordance with applicable regulatory guidelines.

[0248] CEACAM5 IHC 769 is a qualitative immunohistochemistry (IHC) assay using monoclonal mouse anti-CEACAM5 clone 769 and is intended for the detection of human CEACAM5 protein in formalin-fixed, paraffin-embedded (FFPE) non-squamous non-small cell lung cancer.

[0249] The CEACAM5 IHC 769 assay is used as an aid to identify patients who may benefit from participation in a clinical trial using the developmental SanofiSAR408701 (Tusamitamab ravtansine) as a treatment for patients with advanced non-squamous NSCLC.

[0250] Tusamitamab ravtansine, an anti-CEACAM5 antibody-drug conjugate, is an immunoconjugate consisting of an anti-carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) conjugated to a cytotoxic agent with potential anti-tumorigenic activity. Upon administration of the anti-CEACAM5 antibody-drug conjugate Tusamitamab ravtansine, the antibody portion targets CEACAM5 on tumor cells. Following antibody / antigen binding and internalization, the immunoconjugate releases the cytotoxic agent, which causes tumor cell death.

[0251] The aim of this study was to evaluate the performance of CEACAM5 IHC 769 in terms of inter-site, intra-site / inter-day and inter-observer and intra-observer reproducibility when examined at an assay cutoff of ≥50% CEACAM5-positive tumor cells ≥2+ intensity.

[0252] The diagnostic endpoint was defined as positive if the percentage of tumor cells expressing CEACAM5 with ≥2+ intensity was ≥50%. Negative was defined as staining with ≥2+ intensity in <50% of tumor cells staining. This study assessed the reproducibility of diagnostic endpoint (positive / negative) assessment using CEACAM5 IHC 769 according to the CEACAM5 staining protocol as provided herein.

[0253] end : The following endpoints of the study were assessed:

[0254] Inter-site reproducibility across three sites and five rounds

[0255] Intra-site / inter-day reproducibility over five runs at three sites

[0256] Interobserver reproducibility across three pathologists and three readings

[0257] • Intra-observer reproducibility across three readings by each of three pathologists.

[0258] This study was a three-site, blinded, randomized, reproducible study of CEACAM5 IHC769 in FFPE human non-squamous non-small cell lung cancer (NSCLC) tissue specimens.

[0259] The study was divided into two parts: Part A (inter-site and intra-site / inter-day endpoints) and Part B (inter-observer and intra-observer endpoints). All slides were re-randomized to the order of evaluation between rounds / readings and interpreted by a pathologist who was blinded to the specimen identity and previous evaluation. The slide pool was read by a site pathologist who was trained in the scoring algorithm. The maximum washout period did not exceed 30 days; if exceeded, proficiency testing and / or refresher training were performed.

[0260] Study Part A: Each of three external sites performed an automated staining run, testing 50 FFPE human nsNSCLC specimens. Specimens were qualified at Agilent and distributed to three sites, with each site receiving a set of five replicates. Staining was performed on five non-consecutive days over a period of at least twenty days.

[0261] Study Part B: A pool of 66 NSCLC pre-stained specimens was qualified at Agilent and rotated across three testing sites to be evaluated three times at each site by the same pathologist. Included in the pool but not analyzed were four unique wildcards for each read.

[0262] Based on a cutoff of ≥2+ intensity in ≥50% of CEACAM5-positive tumor cells, an attempt was made to select specimens that represented the full range of biomarker expression with a balanced distribution of positive and negative samples. Approximately 20% of the specimens were selected from the near cutoff range of ≥40% to ≤60% of CEACAM5-positive tumor cells ≥2+ intensity.

[0263] Study Part A: Targeted enrollment includes 50 specimens, consisting of approximately 25 positive samples and 25 negative samples.

[0264] Study Part B: A pool of 66 samples, consisting of approximately 33 positive samples and 33 negative samples, was used for analysis. In addition, 4 wildcard samples per read (12 total) were included to reduce the possibility of recall bias.

[0265] Statistical analysis: Consensus estimates for all study endpoints:

[0266] Analytical agreement for diagnostic outcomes (positive / negative) was assessed for study part A (between and within sites) and study part B (between and within observers) using the following methods:

[0267] 1. Evaluation of NPA, PPA, and OA based on consensus scores with bootstrap confidence intervals.

[0268] The negative percentage agreement (NPA), positive percentage agreement (PPA), and overall percentage agreement (OA) were calculated between sites, within sites / days, between observers, and within observers by comparing the diagnostic status (positive / negative) from each slide with the consensus diagnostic outcome. Two-sided 95% confidence intervals for NPA, PPA, and OA were calculated using the bootstrap method.

[0269] 2. Analysis of IHC staining percentage scores.

[0270] The variability of the CEACAM5 staining percentage score (continuous data) was assessed by analysis of variance (ANOVA) based on a mixed model with site and round (study part A) and observer and reading (study part B) as random factors. The mean, standard deviation, and coefficient of variation of the site and observer measurements for each specimen are reported.

[0271] Acceptance Criteria : For each reproducibility measurement, the lower limit of the two-sided 95% confidence interval for NPA, PPA, and OA must be at least 85%.

[0272] result : The results of this external reproducibility study demonstrated that all diagnostic endpoints were highly consistent at a cutoff of ≥50% of CEACAM5-positive tumor cells ≥2+ intensity and all pre-specified acceptance criteria were met for each of the study endpoints.

[0273] in conclusion : The results of this study showed that the consistency of diagnostic endpoint decision-making met the pre-defined acceptance criteria. The reproducibility results of the cutoff value of ≥2+ intensity ≥50% for both positive and negative CEACAM5 status across three observers met the acceptance criteria of a lower confidence limit (CI) of ≥85% for the reproducibility estimates of NPA, PPA, and OA.

[0274] Overall study design

[0275] This study was designed to evaluate the performance of the CEACAM5 IHC 769 assay in three independent CLIA-certified clinical laboratories. This study utilized blinded and randomized FFPE tissue sections from non-squamous NSCLC specimens expressing varying levels of CEACAM5. All specimens in the study were evaluated by a pathologist certified in the scoring of CEACAM5. The study consisted of Part A, which evaluated the combined reproducibility of staining and scoring, and Part B, which evaluated the reproducibility of observer assessment.

[0276] This 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 pre-determined cutoffs, where ≥50% of tumor cells ≥2+ intensity were defined as positive and <50% of tumor cells ≥2+ intensity were defined as negative.

[0277] The sample distributions for Part A and Part B were based on the predefined CEACAM5-positive tumor score % ranges for negative, positive, and near-cutoff samples and are described in Table 1:

[0278] Table 1. CEACAM5 score ranges for predefined specimen categories

[0279] Sample type CEACAM5 score Negative <50% of CEACAM5-positive tumor cells were at ≥2+ intensity Near cutoff value 40% to 60% of CEACAM5-positive tumor cells are at ≥2+ intensity Positive ≥50% of CEACAM5-positive tumor cells were at ≥2+ intensity

[0280] About 20% of the samples were selected within the near cut-off range.

[0281] For Part A : Staining was performed for a minimum of twenty day period for each site. Each set was evaluated by a qualified pathologist to whom H&E, CEACAM5, and NCR slides were provided for each case. Each evaluation was limited to a maximum of 30 specimens per day to minimize the possibility of scoring fatigue. There was a washout period of at least 14 calendar days, but no more than 30 days, between each of the 3 readings per observer per specimen built into the study. The schedule was coordinated with laboratory personnel to ensure study requirements were met.

[0282] For Part B: Inter-observer and Intra-observer Reproducibility

[0283] This part of the study was conducted using a single set of 66 permanent mounted CEACAM5, NCR and H&E stained slides, prepared at Agilent and provided to each research site (each research site rotated for evaluation three times), thereby ensuring that the scores from each pathologist (to be analyzed) were based on the same set of slides. In order to mitigate recall bias, a set of 4 wildcard specimens (each set is unique to each reading) was added and evaluated, but the set was not included in the data analysis. After a round of evaluation at all sites, designated Agilent personnel incorporated the next unique wildcard set and randomized the order of reading the slide set according to the randomization procedure. There was a washout period of at least 14 calendar days between each of the 3 readings for each observer per specimen built into the study, but no more than 30 days.

[0284] Methods used to avoid bias:

[0285] • The specimens within the evaluation were completely anonymous and the specimen ID was blinded from the testing site (except for the personnel assigned to randomize the H&E specimens).

[0286] • All IHC stained slides were randomized based on the order of evaluation, and the identity of the specimen was blinded to the pathologist.

[0287] Pathologists were instructed not to discuss the scoring results of the specimens with each other.

[0288] • Conduct five daytime tests (rounds) of Part A on five non-consecutive days.

[0289] At each site, there was a washout period of 14 to 30 days between each of the five scoring assessments (rounds) for Part A and between each of the three scoring assessments (readings) for Part B.

[0290] • For Part B, four predetermined wildcard samples were randomized into the test samples, with each of the three reads having four different wildcards.

[0291] Material

[0292] Description of research diagnostic medical device

[0293] CEACAM5 IHC 769 contains the optimized reagents and staining protocol required to complete immunohistochemistry (IHC) staining procedures on FFPE specimens using the Dako Omnis. One kit contains enough reagents to perform 60 tests (D60362).

[0294] Table 2: CEACAM5 IHC 769 Components (1 Assay Kit)

[0295]

[0296] In Part A, only one lot number of the CEACAM5 IHC 769 kit was used by the site

[0297] Device Operation

[0298] CEACAM5 IHC 769 contains the optimized reagents and protocols required to complete the IHC staining procedure of FFPE specimens using the Dako Omnis. After incubation with a monoclonal primary antibody to CEACAM5 or a negative control reagent (NCR), the specimen is incubated with a ready-to-use chromogenic reagent consisting of a secondary antibody molecule and a horseradish peroxidase molecule coupled to a dextran polymer backbone. Enzymatic conversion of the subsequently added chromogen results in the precipitation of a visible reaction product at the antigenic site. The specimen can then be counterstained and mounted. The results are read using a light microscope. CEACAM5 IHC 769 (Product Code GE025) is suitable for automated staining using Dako Omnis instruments.

[0299] The working procedures followed the CEACAM5 IHC 769 Instructions for Use (IFU) for nsNSCLC specimens (D60362).

[0300] Interpretation of staining followed the scoring guidelines in the IFU (D60362) and the detailed scoring method in the CEACAM5 IHC769 Pathology Scoring Manual for nsNSCLC specimens (D55521). CEACAM5-positive staining was defined as any partial or complete tumor cell plasma membrane staining of ≥2+ intensity.

[0301] Tissues for study were screened using CEACAM5 IHC 769 according to the IFU of the device (D60362). Prior to the start of the study, a certified pathologist confirmed that the selected specimens met the tissue inclusion / exclusion criteria. Screening of specimens was performed by a certified reader (pathologist or scientist).

[0302] Sample profile pool design (Part A and Part B)

[0303] Tissue specimens were selected according to the study protocol and external specimen selection and review guidelines. A summary of the nsNSCLC specimens selected in this study, expressed as the number of specimens (n) and the percentage of the total set (%), is shown in Table 3:

[0304] Table 3. Summary of nsNSCLC sample sets from Part A and Part B

[0305]

[0306] Scoring of nsNSCLC specimens

[0307] Scoring was performed using bright field microscopy according to the IFU of the device and the pathology scoring manual. A brief overview of the scoring process is described here.

[0308] This reproducibility study used a cutoff of ≥2+ staining intensity on the tumor cell membrane of ≥50% of CEACAM5 positive tumor cells. Based on this cutoff, samples were classified as positive (≥cutoff) or negative (<cutoff).

[0309] The entire specimen was evaluated. All viable tumor cells on the entire CEACAM5-stained slide were evaluated and included in the CEACAM5 score 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. A minimum of 100 viable tumor cells must be present in the CEACAM5-stained slide to determine the percentage of CEACAM5-stained cells. Positive was defined as any partial or full tumor cell membrane staining greater than or equal to 2+ intensity (≥2+).

[0310] Statistical analysis

[0311] Part A of the study was analyzed using 50 samples, and Part B was analyzed using 66 samples. Based on the diagnostic outcome of the sample screening, the samples were selected so that approximately 50% of the total counts were negative and 50% were positive. In the near cutoff range, an attempt was made to enrich the sample set by approximately 20-25%. The sample sets for Study Part A and Study Part B contained overlapping samples.

[0312] Based on the percentage of positive scores generated during the review and selection process, specimens close to the cutoff were selected, as the percentage of positive scores in the near cutoff range was 40% to 60% at an intensity of 2+ or higher. Specimens reported as near the cutoff at the conclusion of the study were based on whether the median of the study pathologist's continuous score was within the near cutoff range.

[0313] Calculation of percent agreement and confidence intervals

[0314] For inter-site and intra-site (inter-day) and inter-observer and intra-observer reproducibility measures, estimates of agreement and corresponding confidence intervals were calculated by comparison with the consensus to calculate NPA / PPA / OA.

[0315] The percentage of NPA and PPA agreement required for comparison with the reference condition was calculated. For reproducibility parameters such as site, date, observer and reading, there was no reference. The IHC diagnostic status (positive / negative) was compared between each test condition and the common outcome (the most frequently occurring observation). The total number of comparisons per sample was equal to the total score.

[0316] result

[0317] Results reported for Part A and Part B

[0318] The following subsections present the results of the statistical analysis of inter-site, intra-site, inter-observer and intra-observer reproducibility. All line data (i.e., recorded data points for continuous CEACAM5 and data points for negative / positive diagnostic endpoints based on a cutoff of ≥2+ intensity ≥50% for both Part A and Part B) were tabulated.

[0319] The following results are reported in the statistical analysis summary tables (Tables 5 to 8):

[0320] - Sample distribution profile: number (n) and percentage (%) of samples assessed as CEACAM5 positive (P) or negative (N) and close to the ≥2+ intensity ≥50% cutoff (NCO 50%); a sample was described as "near 50% cutoff" if the median CEACAM5 score of the sample fell within 40% to 60%

[0321] - Number of comparisons to consensus: number of consistent negative, consistent positive, inconsistent negative, and inconsistent positive comparisons

[0322] -NPA

[0323] -PPA

[0324] -OA

[0325] - 95% confidence interval for each NPA, PPA and OA

[0326] Overall study results

[0327] Table 4 provides an overall summary of the distribution of specimens based on external pathologist assessment of reproducibility:

[0328] Table 4. Median of external pathologist raw scores for each specimen, ≥2+ intensity ≥50% cutoff Summary of nsNSCLC specimens

[0329]

[0330] Table 5. Inter-site reproducibility analysis, ≥2+ intensity ≥50% cutoff

[0331]

[0332] The estimates of NPA, PPA, and OA for the within-site reproducibility endpoints are presented in Table 6:

[0333] Table 6. Intra-site (inter-day) reproducibility analysis, ≥2+ intensity ≥50% cutoff

[0334]

[0335] For each reproducibility metric, the lower limits of the two-sided 95% confidence intervals for NPA, PPA, and OA were greater than 85%, thus meeting the acceptance criteria for all analyses in Part A.

[0336] Inter-observer and intra-observer reproducibility (Part B) / Summary of experimental design and data recording (Part B)

[0337] The purpose of this study was to demonstrate that Agilent's scoring methodology and interpretation guidelines for the CEACAM5 IHC 769 assay produce consistent results in normal observer-to-observer testing.

[0338] The data were analyzed to determine the consistency of assay performance across the three sites based on a cutoff of ≥2+ intensity ≥50%.The same data were used for inter-observer (Table 7) and intra-observer analyses (Table 8).

[0339] Interobserver reproducibility results

[0340] The estimates of NPA, PPA, and OA for the interobserver reproducibility endpoints are presented in Table 7:

[0341] Table 7. Interobserver reproducibility analysis, ≥2+ intensity ≥50% cutoff

[0342]

[0343] Statistical analysis of intra-observer reproducibility

[0344] NPA / PPA / OA

[0345] The estimates of NPA, PPA, and OA for the intraobserver reproducibility endpoints are presented in Table 8:

[0346] Table 8. Intra-observer reproducibility analysis, ≥2+ intensity ≥50% cutoff

[0347]

[0348]

[0349] For each reproducibility metric, the lower limits of the two-sided 95% confidence intervals for NPA, PPA, and OA were greater than 85%, thus meeting the acceptance criteria for all analyses in Part B.

[0350] Summary presentation of diagnostic assessment values ​​for reproducibility studies

[0351] The results with 95% confidence intervals for the four study objectives (within-site, between-site, within-observer, and between-observer) presented above are summarized in: Fig. 20 ： Summary of results with ≥2+ intensity ≥50% cutoff.

[0352] Variance Component Analysis

[0353] The basic continuous raw CEACAM5 scores in the reproducibility study were also evaluated based on the analysis of variance model (ANOVA). Mean, standard deviation (SD) and coefficient of variation (%CV) values ​​were calculated, and the data from study part A and part B were analyzed and presented in Table 9 and Table 10, respectively. It should be noted that no acceptance criteria were applied to these results. For some samples, some or all variance components were not estimable due to lack of variability in the scores (e.g., all scores were 0).

[0354] Table 9. ANOVA mixed model for 50 samples from study part A: ANOVA mixed model in 50 nsNSCLC specimens treated at three sites and five days per site

[0355]

[0356]

[0357]

[0358] Table 10. ANOVA mixed model for 66 samples from study part B:

[0359] ANOVA mixed model for 66 nsNSCLC specimens by three observers with three readings per observer

[0360]

[0361]

[0362]

[0363] Discussion / Conclusion

[0364] The data in this example provide estimates of the reproducibility parameters of CEACAM5 IHC 769 at a ≥2+ intensity ≥50% cutoff within the context of evaluating nsNSCLC cases. This study met all acceptance criteria. The calculated lower limit of the two-sided 95% confidence interval was greater than or equal to 85% for all endpoints analyzed at a ≥2+ intensity ≥50% cutoff.

[0365] A number of embodiments of the present invention have been described. However, it will be appreciated that various modifications may be made without departing from the spirit and scope of the present invention. Accordingly, other embodiments are within the scope of the following claims.

[0366] Alternative Implementation

[0367] In an alternative embodiment, an immunohistochemistry (IHC) method is provided for determining and scoring the extent of cell membrane expression of carcinoembryonic antigen-related cell adhesion molecule 5 or CEACAM5 (also known as CD66e (cluster of differentiation 66e)) in a tissue sample, the method comprising:

[0368] Tissue samples were stained with antibodies that specifically bind to CEACAM5;

[0369] determining the total number of viable tumor or cancer cells having CEACAM5 cell membrane staining, and determining the total number of stained and unstained viable tumor or cancer cells in at least a portion of the tissue sample,

[0370] wherein a tumor or cancer cell is counted as positively stained with an anti-CEACAM5 antibody if there is any intensity of CEACAM5 cell membrane staining above a defined threshold; and

[0371] Determine the tumor intensity ratio score,

[0372] The tumor intensity ratio score is the number of CEACAM5-stained viable tumor or cancer cells present in the tissue sample divided by the total number of stained and unstained viable tumor or cancer cells, multiplied by 100.

[0373] In an alternative embodiment of the IHC method as provided herein:

[0374] - CEACAM5 cell membrane staining includes total membrane staining, discontinuous membrane staining, partial membrane staining, complete (circumferential) membrane staining, punctate membrane staining, linear membrane staining, luminal membrane staining, apical membrane staining, basement membrane staining, lateral membrane staining, basolateral membrane staining, high cytoplasmic staining with a positive staining intensity of 2+ or higher in the signet ring subtype, high cytoplasmic staining with a positive staining intensity of 3+ when cells within a 10X field of view have clear membrane staining, or a combination thereof;

[0375] - The total number of viable tumor or cancer cells includes all viable tumor or cancer cells in the sample, including luminal cells, glandular luminal cells, intraluminal cells, multiple cell layers, cells with signet ring morphology, cells with high cytoplasmic staining, cells with intracytoplasmic lumens, or combinations thereof;

[0376] - The total number of viable tumor or cancer cells counted as positively stained includes all glandular luminal cells with cell membranes associated with the positively stained lumen;

[0377] - the tissue sample contains at least about 100 viable tumor or cancer cells;

[0378] Tumor or cancer cells counted as positively stained excluded those with only cytoplasmic staining (with exceptions), staining of normal or non-neoplastic structures, staining of non-viable tumor or cancer cells, necrotic cells, cell debris, stromal staining, or marginal artifact staining on the periphery of the tissue specimen;

[0379] - Cytoplasmic staining comprises a cytoplasmic staining of 2+ or less positive staining intensity; or, the defined threshold comprises a positive staining intensity of 2+ or greater; or, the defined threshold comprises a 2+ positive or 3+ positive staining intensity assessed at a magnification of about 4X to about 10X; or further comprises confirming a positive staining intensity at a magnification of about 20X to about 40X;

[0380] - the Tumor Intensity Ratio Score (TIPS) comprises the number of CEACAM5 viable tumor or cancer cells stained with a positive staining intensity of 2+ or greater divided by the total number of stained and unstained viable tumor or cancer cells, multiplied by 100; or, a TIPS of about 40% or greater is indicative of a diagnostic status of the tissue sample; or, a TIPS of about 50% or greater, about 60% or greater, about 80% or greater, or about 90% or greater;

[0381] - about 5% or greater of the TIPS are indicative of the diagnostic status of the tissue sample; or, about 5% or greater of the TIPS include the number of CEACAM5 viable tumor or cancer cells stained with a positive staining intensity of 2+ or greater; or about 10% or greater of the TIPS include the number of CEACAM5 viable tumor or cancer cells stained with a positive staining intensity of 2+ or greater;

[0382] - preparing a section or portion of a tissue sample on a glass slide, a microscope slide or equivalent and staining said section or portion of said tissue sample on said slide;

[0383] - the antibody comprises a monoclonal mouse anti-CEACAM5 antibody or a monoclonal rabbit anti-CEACAM5 antibody; or, the monoclonal mouse anti-CEACAM5 antibody comprises a monoclonal mouse anti-CEACAM5 clone 769, or an antibody having a substantially similar affinity for CEACAM5 as the clone 769 antibody;

[0384] - The tissue sample comprises a formalin-fixed, paraffin-embedded (FFPE) sample; or, the FFPE sample comprises a cancer sample stained on an automated IHC platform;

[0385] - preparing sections of tissue samples by a protocol comprising fixation in about 10% neutral buffered formalin for a period of about 6 hours to about 72 hours;

[0386] - 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 carcinoma;

[0387] - tissue sample derived from surgical resection or biopsy, needle biopsy specimen, fine needle aspirate, cytology specimen or bone decalcification; and / or

[0388] - Positive staining is determined using a bright field light microscope, a microscope objective, a computer monitor and imaging software, or a combination thereof; and optionally, the imaging software comprises whole slide imaging software.

Claims

1. An immunohistochemistry (IHC) method for measuring and scoring the cell membrane expression of carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) in a tissue sample, the method comprising: (a) Tissue samples were stained with antibodies that specifically bind 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 unstained viable tumor or cancer cells in at least a portion of the tissue sample, wherein a tumor or cancer cell is counted as positively stained with an anti-CEACAM5 antibody if there is any intensity of CEACAM5 cell membrane staining above a defined threshold; and (c) determining a tumor intensity ratio score, The tumor intensity ratio score is the number of CEACAM5-stained viable tumor or cancer cells present in the tissue sample divided by the total number of stained and unstained viable tumor or cancer cells, multiplied by 100.

2. The method of claim 1, wherein the CEACAM5 cell membrane staining comprises 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, basolateral membrane staining, high cytoplasmic staining with a positive staining intensity of 2+ or greater, high cytoplasmic staining with a positive staining intensity of 3+, or a combination thereof.

3. The method of claim 1 or claim 2, wherein the total number of viable tumor or cancer cells comprises luminal cells, glandular luminal cells, intraluminal cells, multi-layered cells, cells with signet ring morphology, cells with high cytoplasmic staining, cells with intracytoplasmic lumens, or a combination thereof.

4. The method of any one of the preceding claims, wherein the total number of viable tumor or cancer cells counted as positively stained includes all glandular luminal cells having a cell membrane associated with the positively stained lumen.

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

6. The method of claim 1, wherein the tumor or cancer cells counted as positively stained exclude tumor or cancer cells with cytoplasmic staining, staining of normal or non-neoplastic structures, staining of non-viable tumor or cancer cells, necrotic cells, cell debris, matrix staining, or edge artifact staining on the periphery of the tissue sample.

7. The method of claim 6, wherein the cytoplasmic staining comprises a cytoplasmic staining of 2+ or less positive staining intensity.

8. The method of any one of the preceding claims, wherein the determined threshold comprises a positive staining intensity of 2+ or greater.

9. The method of claim 8, wherein the determined threshold comprises a 2+ positive or 3+ positive staining intensity assessed at a magnification of about 4X to about 10X; or further comprises confirming the positive staining intensity at a magnification of about 20X to about 40X.

10. The method of claim 8, wherein the tumor intensity ratio score comprises the number of CEACAM5 viable tumor or cancer cells stained with a positive staining intensity of 2+ or greater divided by the total number of stained and unstained viable tumor or cancer cells, multiplied by 100.

11. The method of any of the preceding claims, wherein a tumor intensity ratio score of about 40% or greater is indicative of a diagnostic status of the tissue sample.

12. The method of claim 11, wherein the tumor intensity ratio score is about 50% or greater, about 60% or greater, about 80% or greater, or about 90% or greater.

13. The method of any of the preceding claims, wherein a tumor intensity ratio score of about 5% or greater is indicative of a diagnostic status of the tissue sample.

14. The method of claim 13, wherein a tumor intensity ratio score of about 5% or greater includes the number of CEACAM5 viable tumor or cancer cells stained with a positive staining intensity of 2+ or greater; or a tumor intensity ratio score of about 10% or greater includes the number of CEACAM5 viable tumor or cancer cells stained with a positive staining intensity of 2+ or greater.

15. The method according to any one of the preceding claims, wherein the sections or portions of the tissue sample are prepared on a glass slide, a microscope slide or equivalent and the sections or portions of the tissue sample are stained on the slide.

16. The method of any one of the preceding claims, wherein the antibody comprises a monoclonal mouse anti-CEACAM5 antibody or a monoclonal rabbit anti-CEACAM5 antibody.

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

18. The method of any of the preceding claims, wherein the tissue sample comprises a formalin-fixed, paraffin-embedded (FFPE) specimen.

19. The method of claim 18, wherein the FFPE specimen comprises a cancer specimen stained on an automated IHC platform.

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

21. The method of any of the preceding claims, wherein 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, gastric cancer, colorectal cancer, lung cancer, colon cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, prostate cancer, or urothelial carcinoma.

22. The method of any one of the preceding claims, wherein the tissue sample is derived from a needle biopsy sample, a fine needle aspirate, a cytology specimen, or a bone decalcification.

23. The method of any of the preceding claims, wherein positive staining is determined using a bright field light microscope, a microscope objective, a computer monitor and imaging software, or a combination thereof.

24. The method of claim 23, wherein the imaging software comprises whole slide imaging software.

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

26. The method of claim 25, wherein 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 carcinoma;.

27. 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 tissue sample from the patient using the method of any one of claims 1 to 25, wherein if the tissue sample is determined or scored as having a high or diagnostically positive CEACAM5 score, the patient is treated with a cancer treatment to which the patient is likely to respond favorably.

28. The method of claim 27, wherein 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 carcinoma.

29. The method of claim 27 or 28, wherein the cancer treatment comprises administering an anti-cancer drug or anti-cancer therapy to the patient.

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

31. A kit comprising an antibody that specifically binds to CEACAM5 as described in any of the preceding claims, and a CEACAM5 scoring guide.

32. A method for assessing the expression level of CEACAM5, the method comprising: contacting a sample or a portion thereof from an individual comprising cancer or tumor cells with an antibody or a portion thereof that specifically binds to CEACAM5; and determining the tumor intensity ratio score by dividing the number of viable tumor or cancer cells stained by CEACAM5 specifically bound by the antibody in the sample or a portion thereof by the total number of stained and unstained viable tumor or cancer cells and multiplying the result by 100, thereby obtaining the tumor intensity ratio score.

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