Immunohistochemical (IHC) protocols and methods for diagnosing and treating cancer-claudin 18.2

Abstract

Alternative embodiments provide an immunohistochemistry (IHC) method for reproducibly determining and scoring the degree of expression of the protein claudin 18.2 in a tissue sample. Alternative embodiments also provide a method for diagnosing, treating, or improving cancer or tumors or assessing the risk of their recurrence using an IHC method such as that provided herein. Alternative embodiments also provide a kit comprising components and instructions for use for performing a method such as that provided herein. This application describes a method for scoring claudin 18.2 expression and utilizing the score as a companion or complementary diagnosis or for treating or improving cancer or tumors.

Description

Technical Field

[0001] [Cross - Reference to Related Applications] This application claims priority to U.S. Provisional Patent Application No. 63 / 464,795, filed May 8, 2023, which is hereby incorporated by reference in its entirety.

[0002] The present invention generally relates to cancer treatment, companion or complementary diagnostics, and immunohistochemical methods. In alternative embodiments, an immunohistochemical (IHC) method for reproducibly determining and scoring the degree of expression of the protein Claudin 18.2 (also known as "CLDN18.2") in tissue samples is provided herein. In alternative embodiments, methods for diagnosing, treating, or improving cancer or a tumor or assessing the risk of its recurrence using an IHC method as provided herein are provided. In alternative embodiments, kits comprising components and instructions for performing a method as provided herein are provided. This application describes methods for scoring Claudin 18.2 expression and using the score as a companion or complementary diagnostic or for treating or improving cancer or a tumor.

Background Art

[0003] Cancer is the second leading cause of death globally, and the global burden is projected to increase to 29 million new cancer cases by 2040. Tight junction proteins are often dysfunctional or modified in several types of cancer cells, resulting in a loss of their regulated paracellular permeability and polarity in tumor cells. Tight junction proteins modulate cytoskeletal elements and signaling molecules bound to these proteins, leading to a loss of regulated cell migration and proliferation. Claudin 18.2 (UniProt number: P56856-2; SEQ ID NO: 1) is a 261-amino acid isoform of claudin 18 and is a member of the tight junction protein family. It is a highly selective marker protein expressed only in differentiated gastric mucosal epithelial cells, with extremely limited expression in other normal healthy tissues, and not expressed in undifferentiated gastric stem cells. There is evidence of abnormal claudin 18.2 expression across the emergence and growth of several primary and metastatic malignancies, such as gastric cancer (GC), gastric junction (GEJ) cancer, esophageal cancer, stomach cancer, gastric cancer metastases, colorectal cancer, colitis-associated colorectal adenocarcinoma, colon cancer, non-squamous non-small cell lung cancer (nsNSCLC), non-small cell lung cancer (NSCLC), adenocarcinoma, cholangiocarcinoma, intraductal neoplasm, pancreatic cancer, pancreatic adenocarcinoma, pancreatic cancer metastases, pancreaticobiliary adenocarcinoma, urogenital tract cancer, biliary carcinoma, and biliary tract cancer. Claudin 18.2 with a distinct expression pattern has emerged as a specific molecule for targeted therapy for several cancers, particularly gastric cancer ("GC"), which is currently the third most common, deadly, and prognostic cancer with a high rate of recurrence and metastasis worldwide. [Overview of the Initiative]

[0004] Claudin 18.2 is known to play a crucial role in carcinogenesis, and there remains a widespread need for methods to score claudin 18.2 expression and utilize the score as a companion or complementary diagnostic tool, or to treat or improve cancer or tumors. Therefore, this disclosure addresses various needs in the art by providing a method for scoring claudin 18.2 expression and utilizing the score as a companion or complementary diagnostic tool, or to treat or improve cancer or tumors.

[0005] In a first general embodiment, the present disclosure provides an immunohistochemistry (IHC) method for determining and scoring the degree of claudin 18.2 cell membrane expression in a tissue sample. In various embodiments, such a method includes the steps of (a) staining the tissue sample with an antibody that specifically binds to claudin 18.2, and (b) determining the total number of viable neoplasms or cancer cells having claudin 18.2 cell membrane staining, and determining the total number of stained and unstained viable neoplasms or cancer cells in at least a portion of the tissue sample. Here, neoplasms or cancer cells are counted as positively stained with anti-claudin 18.2 antibody if they have claudin 18.2 cell membrane staining of any intensity exceeding a specified threshold, and (c) a step to determine the claudin 18.2 score (%). Here, the Claudin 18.2 score (%) is calculated by dividing the number of Claudin 18.2-stained viable neoplasms or cancer cells found in the tissue sample by the total number of stained and unstained viable neoplasms or cancer cells, and multiplying by 100, including the step.

[0006] In some embodiments, such a method further includes a step of excluding cells having pseudo-luminal, pseudo-apical, or secretory staining, so that only neoplasms or cancer cells exhibiting membrane staining are considered positive for claudin 18.2.

[0007] In some embodiments, the tissue sample contains at least about 100 cells.

[0008] In some embodiments, staining of normal or non-neoplastic structures, staining of unviable neoplasms or cancer cells, necrotic cells, cellular debris, stromal staining, or edge artifact staining at the periphery of the tissue sample are excluded from the count of neoplasms or cancer cells as positive.

[0009] In some embodiments, the specified intensity threshold includes the positive intensity of cell staining.

[0010] In some embodiments, the Claudin 18.2 score includes the number of Claudin 18.2 viable neoplasms or cancer cells with a 1+, 2+, or 3+ positive staining intensity, divided by the total number of stained and unstained viable neoplasms or cancer cells, and multiplied by 100.

[0011] In some embodiments, a Claudin 18.2 score of approximately 50% or higher indicates a positive or negative interpretation of a tissue sample.

[0012] In some embodiments, the Claudin 18.2 positive score is approximately 5%, 10%, 20%, 30%, 40%, ≥50%, ≥60%, ≥80%, or ≥90%.

[0013] In some embodiments, a Claudin 18.2 score of approximately 5% or higher indicates a positive interpretation of the tissue sample.

[0014] In some embodiments, a Claudin 18.2 score of approximately 5% or more includes the number of Claudin 18.2 viable neoplasms or cancer cells stained with a 1+, 2+, or 3+ positive staining intensity, or a Claudin 18.2 score of approximately 20% or more includes the number of Claudin 18.2 viable neoplasms or cancer cells stained with a 1+, 2+, or 3+ positive staining intensity.

[0015] In some embodiments, sections or portions of the tissue sample are prepared on a slide, microscope slide or equivalent, and the sections or portions of the tissue sample are stained on the slide.

[0016] In some embodiments, the antibody comprises a monoclonal mouse anti-claudin 18.2 antibody or a monoclonal rabbit anti-claudin 18.2 antibody.

[0017] In some embodiments, the monoclonal rabbit anti-claudin 18.2 antibody includes monoclonal rabbit anti-claudin 18.2 clone EPR19202 or an antibody having substantially equivalent affinity to claudin 18.2 as the clone EPR19202 antibody.

[0018] In some embodiments, the tissue sample includes formalin-fixed paraffin-embedded (FFPE) specimens.

[0019] In some embodiments, the FFPE specimen includes a cancer specimen stained on an automated IHC platform.

[0020] In some embodiments, tissue sample sections are prepared by a protocol that includes fixation in approximately 10% neutral buffered formalin for a period of approximately 6 to 72 hours. In some embodiments, the period is approximately 12 to 60 hours, or approximately 24 to 48 hours.

[0021] In some embodiments, tumors or cancers include gastric cancer, gastric cancer, gastric cancer metastasis, gastroesophageal junction (GEJ) cancer, esophageal cancer, colorectal cancer, colitis-associated colorectal adenocarcinoma, colon cancer, non-squamous non-small cell lung cancer (nsNSCLC), non-small cell lung cancer (NSCLC), adenocarcinoma, cholangiocarcinoma, intraductal neoplasm, pancreatic cancer, pancreatic adenocarcinoma, pancreatic cancer metastasis, pancreaticobiliary adenocarcinoma, urogenital tract cancer, biliary tract cancer, and biliary tract cancer.

[0022] In some embodiments, the tissue sample is derived from a needle biopsy sample, fine-needle aspiration, cytological specimen, or decalcified bone.

[0023] In some embodiments, positive staining is determined using brightfield light microscopy, a microscope objective lens, a computer monitor, and imaging software or a combination thereof. In some embodiments, the imaging software includes whole slide imaging software.

[0024] In a second general aspect, the present disclosure provides a method for treating or ameliorating a tumor or cancer in a patient, the method comprising determining the degree of cell membrane expression of Claudin 18.2 in a tissue sample herein and determining the Claudin 18.2 interpretation state in a patient-derived tissue sample using any of the various embodiments of the method for scoring, and treating the patient with a cancer therapeutic agent to which the patient is likely to respond favorably based on the Claudin 18.2 interpretation state.

[0025] In some embodiments, the tissue sample has a Claudin 18.2 score of about 20% or more of neoplastic or cancer cells having Claudin 18.2 cell membrane staining of intensity 2+ or greater.

[0026] In some embodiments, the tumor or cancer is gastric cancer, gastric carcinoma, gastric cancer metastasis, esophageal-gastric junction (GEJ) cancer, esophageal cancer, colorectal cancer, colitis-associated colorectal adenocarcinoma, colon cancer, non-squamous non-small cell lung cancer (nsNSCLC), non-small cell lung cancer (NSCLC), adenocarcinoma, cholangiocarcinoma, intraductal papillary neoplasm, pancreatic cancer, pancreatic adenocarcinoma, pancreatic cancer metastasis, pancreatobiliary adenocarcinoma, genitourinary cancer, biliary tract cancer, and cholangiocarcinoma.

[0027] In some embodiments, the cancer therapeutic agent includes administration of an anti-cancer drug or anti-cancer therapy to the patient.

[0028] In some embodiments, the anti-cancer therapy includes an antibody-drug conjugate, small molecule therapy, immunotherapy, monoclonal antibody therapy, adoptive cell therapy, T cell receptor therapy, or chimeric antigen receptor (CAR) T cell therapy.

[0029] In a third general aspect, the present disclosure provides a method for assessing the degree of Claudin 18.2 expression in a tissue sample derived from an individual, the method comprising contacting a tissue sample or a portion thereof comprising cancer or neoplastic cells with an antibody or a portion thereof that specifically binds to Claudin 18.2; determining the total number of viable neoplastic or cancer cells having Claudin 18.2 cell membrane staining; determining the total number of stained and non-stained viable neoplastic or cancer cells; and determining the Claudin 18.2 score (%) by dividing the number of Claudin 18.2 stained viable neoplastic or cancer cells in the sample or a portion thereof specifically bound by the antibody by the total number of stained and non-stained viable cancer or neoplastic cells and multiplying the result by 100, thereby obtaining the Claudin 18.2 score (%).

[0030] In a fourth general aspect, the present disclosure provides a kit comprising an antibody that specifically binds to Claudin 18.2 and Claudin 18.2 scoring guidelines as shown in various embodiments of the method for determining and scoring the degree of cell membrane expression of Claudin 18.2 in a tissue sample herein.

[0031] The patent or application documents contain at least one drawing created in color. Copies of this patent or patent application publication with color drawings will be provided by the Patent Office upon request and payment of the necessary fee.

Brief Description of the Drawings

[0032] [Figure 1] Exemplary images of Claudin 18.2 staining in gastric cancer using the Claudin 18.2 IHC (antibody clone) EPR19202 assay are illustrated as described herein. [Figure 2]Representative images of positive staining are shown: confident partial membrane staining (center, red arrow) and complete linear membrane staining (black arrow or center upper right arrow) are considered positive; 20× images are shown here for detailed illustration, but scoring and calculation of Claudin 18.2 positive membrane staining % can also be measured using various objective lenses, including 4×, 10×, 20×, and 40×. [Figure 3] Exemplary examples of benign tissue staining: Exemplary examples of staining of normal (non-neoplastic) tissue, as shown herein, are excluded from the Claudin 18.2 scoring protocol as provided herein. [Figure 4A-4B] Images illustrating that mucin can be stained with high intensity (H&E staining, Figure 4A and Claudin 18.2 staining, Figure 4B) are provided, which are not considered nonspecific background staining and are excluded from the Claudin 18.2 percent score using the Claudin 18.2 scoring protocol provided herein. [Figures 5A-5F] Positive control tissue images: Gastric cancer tissue is illustrated in 10× (Figures 5A, 5D), 20× (Figures 5B, 5E), and 40× (Figures 5C, 5F), with Figures 5A, 5B, and 5C stained with a negative control reagent, and Figures 5D, 5E, and 5F stained with an anti-claudin 18.2 antibody, demonstrating membrane staining in a range of weak (red arrows) to moderate (black arrows) intensity using a claudin 18.2 scoring protocol as provided herein. [Figures 6A-6D] Examples of negative control tissue images are shown: Figure 6A: H&E staining of gastric cancer; Figure 6B: Using a low-magnification objective lens, the tissue is initially scanned to observe any Claudin 18.2 staining; Figure 6C: Using a high-magnification 10× or 20× lens, it is possible to confirm whether any staining is present; and Figure 6D: Using a 20× or 40× objective lens, it is possible to visualize whether any specific membrane staining of 1+ intensity is present. In this example, there is no Claudin 18.2 staining, and this specimen can serve as a good negative control tissue. [Figure 7]An example image of a tissue specimen stained with H&E is shown, which is first evaluated by H&E to assess histology and preservation quality. [Figure 8] A negative control reagent slide is provided as an example, which is used to evaluate nonspecific staining and enable interpretation of Claudin 18.2 stained slides. [Modes for carrying out the invention]

[0033] The drawings provided herein illustrate exemplary embodiments and are not intended to limit the scope of the invention as encompassed by the claims.

[0034] The drawings are described in detail herein.

[0035] Similar reference symbols in various drawings indicate the same elements.

[0036] In alternative embodiments, an IHC method for determining and scoring the degree of expression of the protein claudin 18.2 (also known as SEQ ID NO: 1, CLDN18.2) in tissue samples is provided herein. In alternative embodiments, a scoring method for evaluating claudin 18.2 expression in neoplastic tissue, tumors or cancers, such as gastric cancer ("GC"), gastric cancer, gastroesophageal junction ("GEJ") cancer, esophageal cancer, gastric cancer metastasis, colorectal cancer, colon cancer, colitis-associated colorectal adenocarcinoma, non-squamous non-small cell lung cancer ("nsNSCLC"), non-small cell lung cancer ("NSCLC"), adenocarcinoma, cholangiocarcinoma, intraductal neoplasm, pancreatic cancer, pancreatic adenocarcinoma, pancreatic cancer metastasis, pancreaticobiliary adenocarcinoma, urogenital tract cancer, biliary tract cancer and biliary tract cancer is provided herein.

[0037] The alternative embodiments of the IHC method described herein provide a robust, reliable, standardized, reproducible, and harmonized method for evaluating the degree of cell membrane expression of claudin 18.2, thus resulting in greater inter-institutional and inter-test comparability and enabling earlier and more appropriate application of claudin 18.2 in clinical practice. The embodiments of the IHC method described herein provide high-quality staining and reliable diagnostic evaluation.

[0038] Current immunohistochemical scoring methods for claudin 18.2 measure the percentage of cells in a tissue sample that exhibit partial or full membrane staining. These percentages are further divided into staining intensity bins. Only 2+ and 3+ intensity stainings are considered to determine the final claudin 18.2 diagnostic score in relation to the cutoff. Dividing membrane expression level scoring into multiple staining intensity bins complicates the scoring process. This greater complexity in such scoring methods, in turn, adds to the time required to reach a diagnostic assessment. Pathologists may also vary their individual decisions regarding the presence or relative staining intensity of membrane staining patterns among various cells in a tissue sample, making the reproducibility of the scoring method difficult. Furthermore, in some cancers, including gastric cancer, the heterogeneous tumor morphology can lead to confusion regarding how to apply scoring guidelines. A simpler, more efficient, and robust, reproducible, and accurate scoring method for assessing claudin 18.2 expression in tissue samples remains needed in the art.

[0039] Embodiments herein relate to immunohistochemical methods for determining and scoring the degree of claudin 18.2 cell membrane expression in tissue samples. In various embodiments, the method includes the steps of (a) staining a tissue sample with an antibody that specifically binds to claudin 18.2; (b) determining the total number of viable neoplasms or cancer cells having claudin 18.2 cell membrane staining, and determining the total number of stained and unstained viable neoplasms or cancer cells in at least a portion of the tissue sample, wherein a neoplasm or cancer cell is counted as positively stained with an anti-claudin 18.2 antibody if it has any intensity of claudin 18.2 cell membrane staining exceeding a specified threshold; and (c) determining a claudin 18.2 score (%), wherein the claudin 18.2 score (%) is obtained by dividing the number of claudin 18.2-stained viable neoplasms or cancer cells found in the tissue sample by the total number of stained and unstained viable neoplasms or cancer cells and multiplying by 100.

[0040] In various embodiments, a method for evaluating the degree of claudin 18.2 expression in an individual-derived tissue sample includes the steps of: contacting a tissue sample or portion thereof containing cancer or neoplastic cells with an antibody or portion thereof that specifically binds to claudin 18.2; determining the total number of viable neoplasms or cancer cells having claudin 18.2 cell membrane staining; determining the total number of stained and unstained viable neoplasms or cancer cells; and determining a claudin 18.2 score (%) by dividing the number of claudin 18.2-stained viable neoplasms or cancer cells in the sample or portion thereof that are specifically bound by the antibody by the total number of stained and unstained viable cancer or neoplastic cells, and multiplying the result by 100, thereby obtaining a claudin 18.2 score (%). Such embodiments can provide a robust, reliable, reproducible, and accurate claudin 18.2 scoring method, as well as a scoring method with greater simplicity and efficiency. Such embodiments can provide considerable advantages for reliable and efficient diagnostic evaluation.

[0041] In certain embodiments, claudin 18.2 cell membrane staining includes whole membrane staining, discontinuous membrane staining, partial membrane staining, complete membrane staining, linear membrane staining, or a combination thereof. In certain embodiments, the total number of viable neoplasms, tumors, or cancer cells includes multilayer cells, cells with signet ring morphology, or a combination thereof. Such embodiments can offer the advantage of including cell membrane staining and all observed patterns and levels of all types of viable neoplasms, tumors, or cancer cells in determining the claudin 18.2 positive score. Such embodiments can offer the advantage of a simplified IHC scoring method for scoring claudin 18.2 cell membrane expression in tissue samples.

[0042] In certain embodiments, the tissue sample contains at least about 100 cells. In certain embodiments, the tissue sample contains at least about 200 cells. In certain embodiments, the tissue sample contains at least about 500 cells.

[0043] In some embodiments, neoplasms, tumors, or cancer cells counted as positively stained do not include neoplasms, tumors, or cancer cells having cytoplasmic staining, staining of normal or non-neoplasmic structures, staining of unviable neoplasms, tumors, or cancer cells, necrotic cells, cell fragments, stromal staining, or edge artifact staining at the periphery of the tissue sample. In certain embodiments, cytoplasmic staining includes cytoplasmic staining of any (1+, 2+, 3+) positive staining intensity. Such embodiments can offer the advantage of improving the accuracy and reproducibility of the Claudin 18.2 scoring method by excluding stained cells with certain structures or features from the Claudin 18.2 positive score determination.

[0044] In certain embodiments, the defined threshold includes a positive staining intensity of 1+ or greater. In certain embodiments, the defined threshold includes a positive staining intensity of 2+ or greater. In certain embodiments, the defined threshold includes a 2+ positive or 3+ positive staining intensity. In certain embodiments, the claudin 18.2 positive score includes the number of claudin 18.2 viable neoplasms, tumors, or cancer cells stained with a positive staining intensity of 2+ or greater, divided by the total number of stained and unstained viable neoplasms, tumors, or cancer cells, and multiplied by 100. Such embodiments can offer the benefit of versatility in the staining intensity of the defined threshold.

[0045] In certain embodiments, a Claudin 18.2 positive score of approximately 20% or higher indicates the diagnostic status of the tissue sample. In certain embodiments, a Claudin 18.2 positive score of approximately 40% or higher indicates the diagnostic status of the tissue sample. In certain embodiments, the Claudin 18.2 positive score is approximately 50% or higher, approximately 60% or higher, approximately 80% or higher, or approximately 90% or higher. In certain embodiments, a Claudin 18.2 positive score of approximately 5% or higher indicates the diagnostic status of the tissue sample. In certain embodiments, a Claudin 18.2 positive score of approximately 1% or higher indicates the diagnostic status of the tissue sample. In certain embodiments, a Claudin 18.2 positive score of approximately 5% or higher includes a number of Claudin 18.2 viable neoplasms, tumors, or cancer cells with a positive staining intensity of 2+ or higher, or a Claudin 18.2 positive score of approximately 10% or higher includes a number of Claudin 18.2 viable neoplasms, tumors, or cancer cells with a positive staining intensity of 2+ or higher. In certain embodiments, a claudin 18.2 positive score of approximately 20% or more includes the number of claudin 18.2 viable neoplasms, tumors, or cancer cells stained with a positive staining intensity of 2+ or higher. Such embodiments can provide an advantage in accuracy in determining the diagnostic status associated with claudin 18.2 expression.

[0046] In alternative embodiments, an IHC method is provided that includes a simplified scoring system for membrane staining at a specific intensity threshold, providing guidance for addressing different morphological patterns of membrane staining. Partial membrane staining of neoplasms or tumor cells should be included in the Claudin 18.2 assessment when viability and neoplasm or tumor cell identification can be confirmed.

[0047] In alternative embodiments, methods and kits as provided herein are used for in vitro diagnostic use. In alternative embodiments, the claudin 18.2 IHC provided herein is an IHC assay that uses an anti-claudin 18.2 antibody, such as monoclonal rabbit anti-claudin 18.2 clone EPR19202 (available from Abcam), or an antibody having substantially equivalent affinity to the clone EPR19202 antibody for claudin 18.2, in the detection of claudin 18.2 protein in formalin-fixed paraffin-embedded (FFPE) tissue samples, e.g., lung cancer tissue samples. In alternative embodiments, the EnVision FLEX® visualization system is used with Dako OMNIS®.

[0048] In alternative embodiments, Claudine 18.2 IHC as provided herein may be used to aid in the identification of patients with tumors or cancers, such as gastric cancer (GC), gastric junction (GEJ) cancer, esophageal cancer, gastric cancer, gastric cancer metastases, colorectal cancer, colitis-associated colorectal adenocarcinoma, colon cancer, non-squamous non-small cell lung cancer (nsNSCLC), non-small cell lung cancer (NSCLC), adenocarcinoma, cholangiocarcinoma, intraductal neoplasm, pancreatic cancer, pancreatic adenocarcinoma, pancreatic cancer metastases, pancreaticobiliary adenocarcinoma, urogenital tract cancer, biliary tract cancer, and biliary tract cancer.

[0049] In an alternative embodiment, a method for evaluating the degree of claudin 18.2 expression includes the steps of: contacting a sample or portion thereof containing cancer or tumor cells of an individual with an antibody or portion thereof that specifically binds to claudin 18.2; and determining a claudin 18.2 positivity score (%) by dividing the number of claudin 18.2-stained surviving tumor or cancer cells in the sample or portion thereof that are specifically bound by the antibody by the total number of stained and unstained surviving cancer or tumor cells, and multiplying the result by 100, thereby obtaining a claudin 18.2 positivity score (%).

[0050] In certain embodiments, sections or portions of tissue samples are prepared on slides, microscope slides or equivalents, and the sections or portions of tissue samples are stained on the slides. In certain embodiments, the tissue sample includes formalin-fixed paraffin-embedded (FFPE) specimens. In certain embodiments, the FFPE specimen includes cancer specimens stained on an automated IHC platform. In certain embodiments, sections of tissue samples are prepared by a protocol that includes fixation in about 10% neutral buffered formalin for a period of about 6 to about 72 hours. In certain embodiments, the tissue sample is derived from needle biopsy samples, fine-needle aspirations, cytological specimens or demineralized bone.

[0051] In certain embodiments, the antibody comprises monoclonal rabbit anti-claudin 18.2 antibody or monoclonal rabbit anti-claudin 18.2 antibody. In some embodiments, the monoclonal rabbit anti-claudin 18.2 antibody comprises monoclonal rabbit anti-claudin 18.2 clone EPR19202, or an antibody having substantially equivalent affinity to clon 769EPR19202 antibody for claudin 18.2.

[0052] In certain embodiments, tumors or cancers include gastric cancer (GC), gastric esophagogastric junction (GEJ) cancer, esophageal cancer, gastric cancer, gastric cancer metastasis, colorectal cancer, colitis-associated colorectal adenocarcinoma, colon cancer, non-squamous non-small cell lung cancer (nsNSCLC), non-small cell lung cancer (NSCLC), adenocarcinoma, cholangiocarcinoma, intraductal neoplasm, pancreatic cancer, pancreatic adenocarcinoma, pancreatic cancer metastasis, pancreaticobiliary adenocarcinoma, urogenital tract cancer, biliary tract cancer, and biliary tract cancer.

[0053] In certain embodiments, positive staining is determined using a bright-field microscope, a microscope objective lens, a computer monitor, and imaging software, or a combination thereof. In some embodiments, the imaging software includes whole-slide imaging software.

[0054] <Embodiment of a method for diagnosing tumors or cancer> Embodiments herein provide a method for diagnosing a tumor or cancer by determining whether a tissue sample is positive for claudin 18.2 expression. In various embodiments, the method includes the step of determining the claudin 18.2 diagnostic status in a tissue sample by an IHC method for determining the degree of claudin 18.2 cell membrane expression, as provided herein, wherein approximately 20% or more of tumor cancer cells with a claudin 18.2 positivity score of 2+ or higher are considered diagnostically positive. In certain embodiments, tumors or cancers include gastric cancer (GC), gastric esophagogastric junction (GEJ) cancer, esophageal cancer, gastric cancer, gastric cancer metastasis, colorectal cancer, colitis-associated colorectal adenocarcinoma, colon cancer, non-squamous non-small cell lung cancer (nsNSCLC), non-small cell lung cancer (NSCLC), adenocarcinoma, cholangiocarcinoma, intraductal neoplasm, pancreatic cancer, pancreatic adenocarcinoma, pancreatic cancer metastasis, pancreaticobiliary adenocarcinoma, urogenital tract cancer, biliary tract cancer, and biliary tract cancer.

[0055] <Embodiments of methods for treating cancer and tumors> A method is provided for treating or improving a tumor or cancer in a patient, comprising the step of determining and scoring the amount of claudin 18.2 in a tissue sample derived from the patient using a method such as that provided herein, wherein if the tissue sample is determined or scored to have a high or diagnostically positive claudin 18.2 score, the patient is treated with an anti-cancer drug or anti-cancer therapy in which the patient is likely to respond favorably. The tumor or cancer may be gastric cancer (GC), gastroesophageal junction (GEJ) cancer, esophageal cancer, gastric cancer, gastric cancer metastasis, colorectal cancer, colitis-associated colorectal adenocarcinoma, colon cancer, non-squamous non-small cell lung cancer (nsNSCLC), non-small cell lung cancer (NSCLC), adenocarcinoma, cholangiocarcinoma, intraductal neoplasm, pancreatic cancer, pancreatic adenocarcinoma, pancreatic cancer metastasis, pancreaticobiliary adenocarcinoma, urogenital tract cancer, biliary tract cancer, and biliary tract cancer.

[0056] In certain embodiments, the cancer treatment includes administering an anticancer drug or anticancer therapy to a patient. In alternative embodiments, the anticancer treatment or therapy includes surgical procedures such as CyberKnife therapy and endoscopic mucosal resection, ablation techniques such as chemoembolization, radiofrequency ablation (RFA), cryo-thaw necrosis treatment and / or microwave ablation, and / or radiotherapy such as stereotactic body radiotherapy.

[0057] In certain embodiments, anticancer therapies include antibody-drug conjugates, small molecule therapies, immunotherapies, monoclonal antibody therapies, adoptive cell therapies, T cell receptor therapies, or chimeric antigen receptor ("CAR") T cell therapies. In alternative embodiments, the anticancer treatment or therapy includes the HER2 therapeutic ad-trastuzumab emtansine (or Kadcyla®), pertuzumab (or Perjeta®), trastuzumab (or Herceptin®), enhertu fam-trastuzumab deruxtecan-nxki (or ENHERTU®), tucatinib (or TUKYSA®), pembrolizumab (or Keytruda®), nivolumab (or Opdivo®), ipilimumab (or YERVOY®), ramucirumab (or CYRAMZA®), entrectinib (or ROZLYTREK®), larotrectinib (or VITRAKVI®), or dostallimab-gxly (or JEMPERLI).

[0058] In alternative embodiments, the anticancer treatment or therapy may include the use of an anticancer agent comprising an antibody that specifically binds to or substantially binds to cancer or tumors, wherein the antibody is conjugated with a cytotoxic agent, which optionally includes radionuclides (optionally, yttrium-90, iodine-131, lutetium-177, radium-223 chloride, strontium-89 chloride, or samarium-153 EDTMP), diphtheria toxin, pseudomonas exotoxin A, denileukin diphthitox, moxetumomab pasudotox, calicheamicin or N-acetyl-γ-calicheamicin, emtansine or DM1, mytansine or its derivatives, SN-38 (or 7-ethyl-10-hydroxycamptothecin), or auristatin or monomethyl auristatin E (MMAE).

[0059] <Immunohistochemistry> In alternative embodiments, immunohistochemical methodologies and / or reagents used with methods and manufactured products or kits such as those provided herein are known in the art, for example, U.S. (USPN) Patent No. 10,634,590 (Describes a slide holder assembly fixture for use in IHC); U.S. Patent No. 10,565,479 (Describes a method for identifying blurred areas in digital images of stained tissue); U.S. Patent No. 10,564,076 (System for preparing analytical (or IHC) samples) (Describes a method for automated histological staining); No. 10,551,395 (Describes an automated histological staining system); No. 10,551,378 (Describes a histological staining method); No. 10,504,224 (Describes a digital tissue image analysis system for IHC); No. 10,501,777 (Describes simultaneous, multiplexed detection and quantification of protein expression in IHC); No. 10,488,340 (Describes a method for extracting images of target fluorophores in biological materials); No. 10,453,195 (Describes a method for extracting images of target fluorophores using digital pathological imaging) (Describes methods for detecting tissue regions); No. 10,438,381 (Describes devices, systems, and methods for creating digital images of tissue sections); No. 10,430,943 (Describes methods and programs for automated nuclear region / number estimation for IHC image analysis); No. 10,416,176 (Describes methods for processing specimens in automated histological staining systems); No. 10,393,633 (Describes methods for processing IHC samples and inhibiting their degradation); No. 10,217,011 (IHC slides) (Describes the handling of); No. 10,209,165 (Describes automated or semi-automated methods for evaluating the staining quality of cell-containing specimens); No. 10,126,216 (Describes methods for fixing tissue samples for IHC); No. 9,423,322; No. 8,515,683 (Describes methods and systems for automated detection of immunohistochemical (IHC) patterns); USPN No. 10,816,443 (Describes automated batch staining devices for staining biological specimens on microscope slides);Or, as described in US Patent Application Publication No.: US2019 / 0178867 A1 (Describing the detection of specific tissue targets in thin sections of tissue samples, such as those imaged under a bright-field microscope, without the use of chromogenic stains specific to those tissue targets); US2019 / 0156510 A1 (Describing an image analysis method for IHC tissue sample analysis); US2019 / 0293637 A1 (Method and system for quantitative immunohistochemistry (IHC) of target protein molecules); US2019 / 0080450 A1 (Describing an automated determination of staining quality of IHC-stained biological samples); or US2020 / 0316589 A1 (Describing a multi-well solid-phase support container for treating and testing immobilized biological material), any IHC protocol, IHC medical equipment, devices and / or image or data analysis system, or IHC reagents for performing IHC, which may include, or may consist of, or may consist of, the use thereof.

[0060] In alternative embodiments, the antibodies, antigen-binding fragments thereof, or monomeric or dimeric antigen-binding proteins (including, for example, synthetic or recombinant forms) used in an IHC protocol or kit as provided herein are substantially purified or isolated, or are in the form of unpurified or partially purified culture supernatant.

[0061] In alternative embodiments, methods such as those provided herein may use or include any product or method known in the art, for example, reagents for detecting or visualizing antibody-antigen interactions using IHC protocols or reagents.

[0062] In alternative embodiments, methods such as those provided herein involve the use of chromogenic immunohistochemistry (CIH), in which a primary antibody (e.g., a recombinant antibody (Ab) or its antigen-binding fragment or monomeric or dimeric antigen-binding protein, as provided herein) or a secondary antibody (e.g., the secondary antibody is bound to a primary antibody or recombinant antibody (Ab) or its antigen-binding fragment or monomeric or dimeric antigen-binding protein, as provided herein) is conjugated to an enzyme capable of catalyzing the chromogenic reaction, such as a peroxidase (e.g., an immunoperoxidase), such as horseradish peroxidase (HRP). In alternative embodiments, the chromogenic moieties used in methods such as those provided herein include coumarin, rhodamine, 2,3,6,7-tetrahydro-11-oxo-1H,5H,11H-[1]benzopyrano[6,7,8-ij]quinolidine-10-carboxylic acid, 7-(diethylamino)coumarin-3-carboxylic acid, coumarin derivatives, rhodamine derivatives, tetramethylrhodamine, diarylrhodamine derivatives, QSY 7, QSY 9, QSY 21, diazochromophores, DABSYL, tartrazine, triarylmethane compounds, Fast Red, Fast Blue, fuchsin, Cascade Blue acetyl, dapoxyl sulfonic acid / succinimidyl carboxylic acid ester, DY-405, and Alexa Fluor 405 succinimidyl ester, Cascade Yellow succinimidyl ester, pyridyloxazole succinimidyl ester (PyMPO), Pacific Blue succinimidyl ester, DY-415, 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester, DYQ-425, 6-FAM phosphoramidite, Lucifer Yellow, iodoacetamide, Alexa Fluor 430 succinimidyl ester, Dabcyl succinimidyl ester, NBD chloride / fluoride, QSY 35 succinimidyl ester, DY-485XL, Cy2 succinimidyl ester, DY-490, Oregon Green 488 carboxylic acid succinimidyl ester, Alexa Fluor 488 succinimidyl ester, BODIPY 493 / 503C3 succinimidyl ester, DY-480XL, BODIPY FL C3 succinimidyl ester, BODIPY FL C5 succinimidyl ester, BODIPY FL-X succinimidyl ester, DYQ-505, Oregon Green 514 carboxylic acid succinimidyl ester, DY-510XL, DY-481XL, 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein succinimidyl ester (JOE), DY-520XL, DY-521XL, BODIPY R6G C3 succinimidyl ester, erythrosine isothiocyanate, 5-carboxy-2',4',5',7'-tetrabromosulfone fluorescein succinimidyl ester, Alexa Fluor 532 succinimidyl ester, 6-carboxy-2',4,4',5'7,7'-hexachlorofluorescein succinimidyl ester (HEX), BODIPY 530 / 550 C3 succinimidyl ester, DY-530, BODIPY TMR-X succinimidyl ester, DY-555, DYQ-1, DY-556, Cy3 succinimidyl ester, DY-547, DY-549, DY-550, Alexa Fluor 555 succinimidyl ester, Alexa Fluor 546 succinimidyl ester, DY-548, BODIPY 558 / 568 C3 succinimidyl ester, Rhodamine Red-X succinimidyl ester, QSY 7 succinimidyl ester, BODIPY 564 / 570 C3 succinimidyl ester, BODIPY 576 / 589 C3 succinimidyl ester, carboxy-X-rhodamine (ROX), succinimidyl ester, Alexa Fluor 568 succinimidyl ester, DY-590, BODIPY 581 / 591 C3 succinimidyl ester, DY-591, BODIPY TR-X succinimidyl ester, Alexa Fluor 594 succinimidyl ester, DY-594, carboxynaphthofluorescein succinimidyl ester, DY-605, DY-610, Alexa Fluor 610 succinimidyl ester, DY-615, BODIPY 630 / 650-X succinimidyl ester, erioglaucine, AlexaFluor 633 succinimidyl ester, Alexa Fluor 635 succinimidyl ester, DY-634, DY-630, DY-631, DY-632, DY-633, DYQ-2, DY-636, BODIPY 650 / 665-X succinimidyl ester, DY-635, Cy5 succinimidyl ester, Alexa Fluor 647 succinimidyl ester, DY-647, DY-648, DY-650, DY-654, DY-652, DY-649, DY-651, DYQ-660, DYQ-661, Alexa Fluor 660 succinimidyl ester, Cy5.5 succinimidyl ester, DY-677, DY-675, DY-676, DY-678, Alexa Fluor 680 succinimidyl ester, DY-679, DY-680, DY-682, DY-681, DYQ-3, DYQ-700, Alexa Fluor 700 succinimidyl ester, DY-703, DY-701, DY-704, DY-700, DY-730, DY-731, DY-732, DY-734, DY-750, Cy7 succinimidyl ester, DY-749, DYQ-4, Cy7.5 succinimidyl ester, 7-diethylaminocoumarin-3-carboxylic acid, succinimidyl ester, dabusil sulfonyl chloride, fluorescein isothiocyanate (FITC) carboxysuccinimidyl ester (DY-495), rhodamine green carboxylic acid succinimidyl ester (DY-505), eosin The product is or contains 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), gallocyanine, Fast Green FCF, malachite green, or QSY 21 succinimidyl ester.

[0063] In alternative embodiments, methods such as those provided herein involve the use of immunofluorescence, where a primary or secondary antibody is tagged with a fluorophore, e.g., fluorescein or fluorescein isothiocyanate (FITC), a triarylmethane dye, e.g., rhodamine or a rhodamine derivative (e.g., tetramethylrhodamine (TRITC), rhodamine 6G, rhodamine 123, rhodamine B, carboxytetramethylrhodamine (TAMRA), tetramethylrhodamine (TMR), sulforhodamine 101), aminomethylcoumarin acetate (AMCA), ALEXA® or DYLIGHT® Fluor, or a fluorophore or dye as described in U.S. Patent Application No. US2019 / 0018018 A1. 3,3'-diaminobenzidine (DAB) may also be used.

[0064] In alternative embodiments, methods such as those provided herein involve the use of a direct or one-step staining method in which a primary antibody (e.g., an antibody (Ab) or its antigen-binding fragment or monomeric or dimeric antigen-binding protein, e.g., including synthetic or recombinant forms) is labeled and reacts directly with the antigen, for example, in a tissue section. This technique utilizes only one antibody and is therefore simple and rapid, but may have lower sensitivity due to limited signal amplification.

[0065] In alternative embodiments, methods such as those provided herein involve the use of an indirect method in which an unlabeled primary antibody (layer 1) binds to a target antigen (e.g., claudin 18.2) in, for example, a tissue or organ, and then a labeled secondary antibody (layer 2) reacts with the primary antibody. The secondary antibody may be for an isotype of the animal species from which the primary antibody originates, e.g., IgG. This method may be more sensitive than direct detection strategies due to signal amplification by the binding of several secondary antibodies to each primary antibody, if the secondary antibody is conjugated to a detection agent such as a fluorescent or enzyme reporter.

[0066] In alternative embodiments, further amplification can be achieved if the secondary antibody is conjugated to a biotin molecule that can recruit a complex of several detection molecules, such as avidin-, streptavidin-, or NEUTRAVIDIN® protein-binding enzymes.

[0067] In alternative embodiments, IHC is performed on tissue sections or tissue biopsies, e.g., paraformaldehyde (PFA) fixed tissue or organs, or formalin-fixed paraffin-embedded tissue. In alternative embodiments, the tissue is sectioned, sliced, or used whole. Prior to sectioning, the tissue sample can be embedded in a medium, e.g., paraffin wax or cryomedia. Tissue sections can be sectioned or sliced ​​using a variety of instruments, most commonly using a microtome, cryostat, or vibratome. The specimen can be sectioned or sliced ​​in the range of approximately 3 μm to 5 μm. The sections or slices can be mounted on slides, dehydrated using progressively increasing concentrations of alcohol washing (e.g., 50%, 75%, 90%, 95%, 100%), cleared using a surfactant such as xylene, and then imaged under a microscope.

[0068] Depending on the fixation and tissue preservation method, samples may require additional steps, including deparaffinization and antigen retrieval, to make the claudin 18.2 epitope available for antibody binding. In formalin-fixed, paraffin-embedded tissues, antigen retrieval is often required and may involve pretreatment of sections with heat or protease.

[0069] In alternative embodiments, IHC is performed using the ENVISION DUOFLEX DOUBLESTAIN SYSTEM® (EnVision DuoFLEX Doublestain System) (Agilent, San Jose, California), which allows staining of two or more markers on a single slide. In alternative embodiments, IHC is performed using the EnVision FLEX HRP Magenta, High pH (Dako Omnis) system, and binding can be visualized with the EnVision FLEX HRP magenta chromogen. In alternative embodiments, IHC is performed using the EnVision FLEX Mini Kit, High pH, ​​a highly sensitive visualization system intended for use in IHC with the Dako AUTOSTAINER® instrument or the Dako OMNIS instrument. This dual-link system detects primary mouse and rabbit antibodies, and the reaction is visualized with the 3,3'-diaminobenzidine (DAB) chromogen (DAB forms a water-insoluble brown precipitate when oxidized, for example, by peroxidase).

[0070] <Manufactured products and kits> For example, manufacturing products and kits for carrying out methods such as those provided herein, including monoclonal rabbit anti-claudin 18.2 antibody, e.g., clone EPR19202 anti-claudin 18.2 antibody, and / or reagents for carrying out IHC, including, for example, reagents described herein, see Example 1 below, and optionally, the manufacturing products and kits may further include instructions for use for carrying out methods such as those provided herein.

[0071] Any of the above aspects and embodiments may be combined with any other aspects or embodiments disclosed herein in the sections of the summary, drawings, and / or detailed description.

[0072] As used herein and in the claims, the singular forms “a,” “an,” and “the” include plural referents unless the context explicitly indicates otherwise.

[0073] Unless expressly indicated or evident from the context, the term “or” as used herein is understood to be inclusive and applies to both “or” and “and.”

[0074] Unless expressly indicated or evident from the context, the term “about” in this specification is understood to mean within the normal tolerances in the art, e.g., within two standard deviations of the mean. “About” (in the use of the term “about”) may be understood to mean within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the indicated value. Unless evident from the context, all numerical values ​​provided herein are modified by the term “about”.

[0075] Unless expressly indicated or evident from the context, as used herein, the terms “substantially all,” “substantially most,” “substantially all,” or “the majority” encompass at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or more of the composition or the mentioned amount of staining intensity 0, 1+, 2+, or 3+.

[0076] Each patent, patent application, publication, and document referenced herein is incorporated herein by reference in its entirety. The references to the above patents, patent applications, publications, and documents do not constitute an endorsement of any of them as relevant prior art, nor do they constitute any endorsement of the content or date of such publications or documents. The incorporation by reference of these documents should not be construed as independently determining or acknowledging that any part of the content of any document is considered essential material for satisfying the statutory disclosure requirements of any country or region of a patent application. Nevertheless, the right to rely on any of such documents to provide material that is considered essential to the subject matter claimed by an examining authority or court is reserved, as needed.

[0077] Modifications can be made to the above without departing from the basic aspects of the present invention. Although the present invention has been described in considerable detail with respect to one or more specific embodiments, those skilled in the art will recognize that modifications can be made to the embodiments specifically disclosed herein, but these modifications and improvements will remain within the scope and spirit of the present invention. The present invention as illustrated herein can be carried out in the absence of any(s) elements not specifically disclosed herein, as appropriate. Thus, for example, in each example herein, any one of the terms “comprising,” “consisting essentially of,” and “consisting of” can be replaced with any one of the other two terms. Thus, the terms and expressions used herein are used as explanatory terms and not restrictive terms, and it is recognized that equivalents or parts thereof of the features shown and described are not excluded, and various modifications may be within the scope of the present invention. Embodiments of the present invention are shown in the following claims.

[0078] The present invention will be further described in relation to the examples described herein, but it should be understood that the present invention is not limited to such examples. [Examples]

[0079] Unless otherwise stated in the examples, all recombinant DNA techniques are 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 Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA, Volumes 1 and 2. Other references for standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK).

[0080] An exemplary claudin 18.2 staining protocol is described in Example 2 below.

[0081] [Example 1: Claudine 18.2 IHC] This example demonstrates the use of the Claudin 18.2 staining protocol as provided herein.

[0082] Exemplary scoring guidelines for evaluating claudin 18.2 expression in gastric cancer specimens stained with a rabbit monoclonal claudin 18.2 antibody called rabbit mAb clone EPR19202 are provided herein. This antibody is provided in the claudin 18.2 IHC EPR19202 assay for the identification of claudin 18.2 expression in formalin-fixed paraffin-embedded (FFPE) gastric cancer specimens using the Dako Omnis automated staining system.

[0083] Evaluation of the claudin 18.2 IHC staining pattern in gastric cancer FFPE tissue. Complete a protocol-specific Pathology Report Form (PRF) for each evaluated specimen (including those classified as "unevaluable" and "indeterminate" as described below).

[0084] <Claudin 18.2 Scoring Overview: Definition of Positive Staining> Claudin 18.2 positive staining is defined as staining of the cell membrane of any part or complete neoplasm or tumor cell at the intensity threshold. Membrane staining can be partial or complete.

[0085] Typical staining examples are shown below; please refer to Figures 1-8.

[0086] The Claudin 18.2 positive percent score is determined by the following formula:

number

[0087] To determine the percentage of stained cells, a minimum of 100 evaluable viable neoplasms or tumor cells must be present in the Claudin 18.2 stained slide.

[0088] The overall Claudine 18.2 diagnostic status is determined according to the following criteria: · If ≥X% of the specimen is composed of neoplasms or tumor cells showing Claudin 18.2 positive staining at the intensity threshold, and X is a predetermined cut-off, it is considered positive in diagnosis. · If <X% of the specimen is composed of neoplasms or tumor cells showing Claudin 18.2 positive staining at the intensity threshold, and X is a predetermined cut-off, it is considered negative in diagnosis.

[0089] <Claudin 18.2 Scoring Summary: Definition of Negative Staining> Negative staining is defined as the absence of membrane staining.

[0090] Scoring includes the following: · Partial or complete membrane staining of any intensity or intensity threshold is considered positive for Claudin 18.2 expression. · All viable neoplasms or tumor cells on the slide are evaluated for both the intensity and frequency of positive staining.

[0091] Cell types and staining patterns excluded from staining scoring: · Cytoplasmic staining · Staining of normal, non-neoplastic structures · Shed non-viable neoplasms or tumor cells · Necrotic cells and cell debris · Stromal staining · Edge artifact staining at the periphery of the tissue specimen · Intraepithelial cancer

[0092] <Scoring of Gastric Cancer> The staining intensity was scored using the following scale (Table 1):

[0093]

Table 1

[0094] This assay has been evaluated on gastric cancer tissue samples. To enable proper confirmation of tumor presence and histological diagnosis, to assess tumor viability, and to distinguish slides from non-tumor portions that should be excluded from evaluation, each Claudin 18.2 stained sample should be accompanied by an H&E stained slide. H&E and NCR stained slides can also assist in the identification of endogenous pigments (such as endogenous anthrax and hemosiderin dyes).

[0095] Cells with signet ring morphology: Cells with signet ring morphology may show high-intensity staining in the cytoplasm. In these cases, membrane staining may be difficult to distinguish due to the intensity of cytoplasmic staining. For cells with signet ring morphology, if partial or complete membrane staining is distinguishable from cytoplasmic staining, high-intensity stained tumor cells are considered positive for membrane staining.

[0096] Cells with high cytoplasmic staining: Cytoplasmic staining without clear membrane staining is not considered positive. However, in many cases of gastric cells with membrane expression of claudin 18.2, cytoplasmic expression is also shown. Tumor cells are scored as positive for claudin 18.2 if they have membrane staining that is distinguishable from cytoplasmic staining.

[0097] <Non-specific background staining> On slides stained with a negative control reagent (NCR), membrane staining in tumor cells should not be observed.

[0098] Nonspecific staining in tissue components that cannot be scored includes the following: -serum - Plasma cells - Smooth muscle - Extracellular mucin - Necrotic debris -Endogenous or other pigments, including, but not limited to, hemosiderin, iron, melanin, surgical tattoo ink, tissue-oriented ink, or endogenous peroxidase reactivity associated with red blood cells. - Necrosis

[0099] Nonspecific staining, including in tumor areas, should not interfere with the interpretation of membrane staining in tumor cells.

[0100] <System Comparison> Before examining the IHC-stained tumor specimen(s), confirm that the control tissue is well-stained by IHC.

[0101] Positive and negative control tissues are prepared in parallel with patient tissue. NCR staining is performed on the control tissue to ensure that there is no nonspecific staining of the control tissue within the staining procedure. See Figure 5 for representative images of positive control tissue and Figure 6 for representative images of negative control tissue.

[0102] <Control tumor tissue> Control tumor tissue must be identified from pre-screened gastric cancer tissue. The control must be a fresh biopsy / gastric cancer specimen that has been fixed, processed, and embedded as quickly as possible using the same method as the test specimen(s).

[0103] <Negative Control Reagent (NCR)> The negative control reagent contains an antibody that is nonspecifically species-matched to claudin 18.2. Control tissue stained with NCR should not show specific membrane staining, and the presence of nonspecific background staining in tumor cells should not interfere with the interpretation of membrane staining in tumor cells. If specific staining occurs in NCR-stained tissue, the result should be considered invalid, the "failure" checkbox on the scoring form should be recorded, and staining should be repeated on both the control and test specimens.

[0104] <Positive control tissue> • Positive control tissues are evaluated to ensure proper tissue preparation and staining techniques. One positive control tissue is included in each staining procedure. • The tissue selected for use as a positive tissue control must yield moderately positive staining to detect even slight changes in assay sensitivity. • Only reagent performance is confirmed using samples processed differently from the test samples (multiple samples are acceptable), and tissue preparation is not verified. • Known positive tissue controls should only be used to monitor the correct performance of processed tissues and test reagents, and should not be used as an aid in developing specific diagnoses of patient samples.

[0105] <Negative control tissue> • Gastric cancer-negative control tissue is evaluated to ensure tissue preparation and good staining techniques, to verify the specificity of the primary antibody, and to provide indications for nonspecific staining. • The various cell types present in most tissue sections provide internal negative control sites (which must be verified by the user). • A proposed negative control tumor tissue for gastric cancer is one in which tumor cells do not show membrane staining. • Known negative tissue controls should only be used to monitor the correct performance of processed tissues and test reagents, and should not be used as an aid in developing specific diagnoses of patient samples.

[0106] <Acceptability Criteria for Control Samples> Based on the staining results of the control sample, the results of the test sample will be accepted or rejected according to the following criteria. • Accept the test sample results only if the control meets all requirements. If the control tissue slide does not meet the requirements, the test sample results must be considered invalid, and staining including the tissue control and tissue sample must be repeated. If the negative control reagent (NCR) staining results do not meet the requirements, the test specimen results must be considered invalid, and staining including tissue controls and tissue samples must be repeated.

[0107] Once appropriate staining of the control material is established, the staining is deemed satisfactory for evaluating the test sample.

[0108] <Patient tissue slide stained with NCR> The negative control reagent is used in place of the Claudin 18.2 specific primary antibody to assist in the interpretation of specific staining at the antigen site.

[0109] The absence of membrane staining verifies the specific labeling of the target antigen by the Claudin 18.2 primary antibody in patient specimens. Staining with NCR should not show any membrane staining of any intensity.

[0110] <Slide reading order and evaluation> For each staining run, slides must be tested in the order shown in the following table to determine the validity of the staining run and enable evaluation of the stained sample tissue.

[0111]

Table 2

[0112] <Specimen stained with NCR> To evaluate non-specific staining and enable interpretation of specific staining, sections of each patient specimen must be tested with the negative control reagent (NCR).

[0113] Acceptance criteria: · Staining with NCR should not show any specific membrane staining of any intensity.

[0114] <Specimen stained with Claudin 18.2> Carefully examine the entire tumor specimen and determine the percentage of Claudin 18.2 positive tumor cells with a ≥2+ intensity:

Number

[0115] The estimated total number of surviving tumor cells in the entire tissue section is used as the denominator for calculating the score.

[0116] The claudin 18.2 diagnostic status (positive / negative) of a gastric cancer specimen is determined by the percentage of surviving tumor cells that express claudin 18.2 positivity (≧2+ intensity) exceeding the intensity threshold of membrane staining. Tumor cells are positive for claudin 18.2 expression if they show partial or complete peripheral membrane staining of any intensity. Claudin 18.2 staining may appear as partial or complete membrane staining in Figure 2. • Re-examine the claudin 18.2 expression pattern for important scoring details.

[0117] Claudin 18.2 positive scores are scored using the following resolutions: in the range of 0 to 10%, positive scores are scored in 1% increments, except in the range of 0 to 1% where "<1%" is specified. In the range of 10% to 100%, positives can be scored in 5% increments, but smaller increments may be used if necessary.

[0118] <Factors to exclude from scoring and from the evaluation of non-specific background> The following are additional tissue elements that may be present and clearly exhibit genuine claudin 18.2 staining: • Mucin (see Figure 4) • Benign epithelial cells (see Figure 3)

[0119] Staining of these regions is excluded from determining the claudin 18.2 expression status.

[0120] Claudin 18.2 positivity is determined and recorded based on a predetermined cutoff point for claudin 18.2 positivity staining at an intensity threshold.

[0121] The term "indeterminate" is used to describe samples that meet the criteria for evaluation, but for which scoring of tumor cell membrane staining is hindered due to reasons attributable to the biology of the tumor tissue sample, rather than due to improper sample preparation or handling.

[0122] Staining may also occur in certain areas of tissue specimens, such as the stroma, necrotic areas, and edge artifacts in the peripheral regions of the tissue. Stromal staining may occur in specimens showing highly positive claudin 18.2 expression. Staining of these tissue elements should be excluded from determining the claudin 18.2 expression status and from evaluating nonspecific background staining.

[0123] [Alternative Embodiments] In an alternative embodiment, an IHC method for determining and scoring the degree of cell membrane expression of claudin 18.2 in a tissue sample, The tissue sample is stained with an antibody that specifically binds to claudin 18.2. The steps include determining the total number of viable neoplasms, tumors, or cancer cells with claudin 18.2 cell membrane staining, and determining the total number of stained and unstained viable neoplasms, tumors, or cancer cells in at least a portion of a tissue sample. Here, neoplasms, tumors, or cancer cells are counted as positively stained with anti-claudin 18.2 antibody if they have claudin 18.2 cell membrane staining of any intensity exceeding a specified threshold, and Steps to determine the Claudin 18.2 positive score (%) Here, the claudin 18.2 positivity score (%) is calculated by dividing the number of claudin 18.2-stained surviving neoplasms, tumors, or cancer cells found in the tissue sample by the total number of stained and unstained surviving neoplasms, tumors, or cancer cells, and multiplying by 100. A method is provided that includes this.

[0124] In alternative embodiments of the IHC method as provided herein: - Claudin 18.2 cell membrane staining includes whole membrane staining, discontinuous membrane staining, partial membrane staining, complete (peripheral) membrane staining, linear membrane staining, or combinations thereof; - The total number of viable neoplasms, tumors, or cancer cells includes all viable neoplasms, tumors, or cancer cells in the specimen, including cells with multiple layers, cells with signet ring morphology, or combinations thereof. - The tissue sample contains at least approximately 100 viable neoplasms, tumors, or cancer cells. Neoplasms, tumors, or cancer cells that are counted as positively stained do not include neoplasms, tumors, or cancer cells that have cytoplasmic staining of normal or non-neoplasmic structures, staining of non-viable neoplasms, tumors, or cancer cells, necrotic cells, cell fragments, stromal staining, or edge artifact staining at the periphery of the tissue sample. - The Claudin 18.2 positivity score includes the number of Claudin 18.2-stained viable neoplasms, tumors, or cancer cells at the intensity staining threshold, divided by the total number of stained and unstained viable neoplasms, tumors, or cancer cells, multiplied by 100, or a Claudin 18.2 positivity score of approximately 40% or higher indicates the diagnostic status of the tissue sample, or a Claudin 18.2 positivity score of approximately 50% or higher, approximately 60% or higher, approximately 80% or higher, or approximately 90% or higher. - A Claudin 18.2 positivity score of approximately 5% or higher indicates the diagnostic status of the tissue sample, or a Claudin 18.2 positivity score of approximately 5% or higher includes the number of Claudin 18.2 viable neoplasms, tumors, or cancer cells with a positive staining intensity of 2+ or higher, or a Claudin 18.2 positivity score of approximately 10% or higher includes the number of Claudin 18.2 viable neoplasms, tumors, or cancer cells with a positive staining intensity of 2+ or higher, or a Claudin 18.2 positivity score of approximately 20% or higher includes the number of Claudin 18.2 viable neoplasms, tumors, neoplasms, or cancer cells with a Claudin 18.2 cell membrane staining intensity of 2+ or higher. - Sections or portions of the tissue sample are prepared on a slide, microscope slide or equivalent, and the sections or portions of the tissue sample are stained on the slide. - The antibody contains monoclonal rabbit anti-claudin 18.2 antibody or monoclonal rabbit anti-claudin 18.2 antibody, or the rabbit anti-claudin 18.2 antibody contains monoclonal rabbit anti-claudin 18.2 clone EPR19202 or an antibody having substantially equivalent affinity to clon EPR19202 antibody for claudin 18.2. -Tissue samples include formalin-fixed paraffin-embedded (FFPE) specimens, or FFPE specimens include cancer specimens stained on an automated IHC platform. -Tissue sample sections are prepared according to a protocol that includes fixation in approximately 10% neutral buffered formalin for a period of approximately 6 to 72 hours. - Tumors or cancers include gastric cancer, gastric cancer, gastric cancer metastasis, gastroesophageal junction (GEJ) cancer, esophageal cancer, colorectal cancer, colitis-associated colorectal adenocarcinoma, colon cancer, non-squamous non-small cell lung cancer (nsNSCLC), non-small cell lung cancer (NSCLC), adenocarcinoma, bile duct cancer, intraepithelial neoplasm of the bile duct, pancreatic cancer, pancreatic adenocarcinoma, pancreatic cancer metastasis, pancreaticobiliary adenocarcinoma, urogenital tract cancer, biliary tract cancer, and biliary tract cancer. -Tissue samples may be derived from surgical excision or biopsy, needle biopsy samples, fine-needle aspiration, cytological specimens or decalcification of bone, and / or Positive staining is determined using a bright-field optical microscope, microscope objective lenses, a computer monitor, and imaging software or a combination thereof, and optionally the imaging software includes whole-slide imaging software.

[0125] References DOI 10.21037 / tcr-19-1876 and DOI 10.1080 / 21688370.2021.1967080 are associated with claudin 18.2 and gastric cancer.

[0126] Sequence List

[0127] [Table 3]

Claims

1. An immunohistochemistry (IHC) method for determining and scoring the degree of claudin 18.2 cell membrane expression in tissue samples, (a) A step of staining the tissue sample with an antibody that specifically binds to claudin 18.2, (b) A step of determining the total number of viable neoplasms or cancer cells having claudin 18.2 cell membrane staining, and determining the total number of stained and unstained viable neoplasms or cancer cells in at least a portion of the tissue sample. Here, neoplasms or cancer cells are counted as positively stained with anti-claudin 18.2 antibody if they have claudin 18.2 cell membrane staining of any intensity exceeding a specified threshold, and (c) Claudin 18.2 Step to determine score (%) Here, the Claudin 18.2 score (%) is calculated by dividing the number of Claudin 18.2-stained surviving neoplasms or cancer cells found in the tissue sample by the total number of stained and unstained surviving neoplasms or cancer cells, and multiplying by 100. Methods that include...

2. The method according to claim 1, further comprising the step of excluding cells having pseudo-luminal, pseudo-apical, or secretory staining, wherein only neoplasms or cancer cells exhibiting membrane staining are considered positive for claudin 18.

2.

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

4. The method according to claim 1, wherein the neoplasms or cancer cells counted as positive do not include staining of normal or non-neoplasmic structures, staining of unviable neoplasms or cancer cells, necrotic cells, cell fragments, stromal staining, or edge artifact staining at the periphery of the tissue sample.

5. The method according to any one of claims 1 to 3, wherein the specified intensity threshold includes the positive intensity of cell staining.

6. The method according to claim 5, wherein the Claudin 18.2 score includes the number of Claudin 18.2 viable neoplasms or cancer cells stained with a 1+, 2+, or 3+ positive staining intensity, divided by the total number of stained and unstained viable neoplasms or cancer cells, and multiplied by 100.

7. The method according to any one of claims 1 to 6, wherein a Claudin 18.2 score of approximately 5% or more indicates a positive or negative interpretation of the tissue sample.

8. The method according to claim 7, wherein the Claudin 18.2 positive score is approximately 10%, 20%, 30%, 40%, 50% or more, approximately 60% or more, approximately 80% or more, or approximately 90% or more.

9. The method according to any one of claims 1 to 8, wherein a Claudin 18.2 score of approximately 5% or more indicates a positive interpretation of the tissue sample.

10. The method according to claim 9, wherein approximately 5% or more of the claudin 18.2 score includes the number of claudin 18.2 viable neoplasms or cancer cells stained with a 1+, 2+, or 3+ positive staining intensity, or approximately 20% or more of the claudin 18.2 score includes the number of claudin 18.2 viable neoplasms or cancer cells stained with a 1+, 2+, or 3+ positive staining intensity.

11. The method according to any one of claims 1 to 10, wherein a section or portion of the tissue sample is prepared on a slide, microscope slide or equivalent, and the section or portion of the tissue sample is stained on the slide.

12. The method according to any one of claims 1 to 11, wherein the antibody comprises a monoclonal mouse anti-claudin 18.2 antibody or a monoclonal rabbit anti-claudin 18.2 antibody.

13. The method according to claim 12, wherein the monoclonal rabbit anti-claudin 18.2 antibody comprises monoclonal rabbit anti-claudin 18.2 clone EPR19202, or an antibody having substantially equivalent affinity to claudin 18.2 as the clone EPR19202 antibody.

14. The method according to any one of claims 1 to 13, wherein the tissue sample includes a formalin-fixed paraffin-embedded (FFPE) specimen.

15. The method according to claim 14, wherein the FFPE sample includes a cancer sample stained on an automated IHC platform.

16. The method according to claim 14, wherein the section of the tissue sample is prepared by a protocol including fixation in about 10% neutral buffered formalin for a period of about 6 hours to about 72 hours.

17. The method according to claim 16, wherein the period is approximately 12 hours to approximately 60 hours, or approximately 24 hours to approximately 48 hours.

18. The method according to any one of claims 1 to 17, wherein the tumor or cancer is gastric cancer, stomach cancer, gastric cancer metastasis, gastroesophageal junction (GEJ) cancer, esophageal cancer, colorectal cancer, colitis-associated colorectal adenocarcinoma, colon cancer, non-squamous non-small cell lung cancer (nsNSLC), non-small cell lung cancer (NSLC), adenocarcinoma, bile duct cancer, intraepithelial neoplasm of the bile duct, pancreatic cancer, pancreatic adenocarcinoma, pancreatic cancer metastasis, pancreaticobiliary adenocarcinoma, urogenital tract cancer, biliary tract cancer, and biliary tract cancer.

19. The method according to any one of claims 1 to 18, wherein the tissue sample is derived from a needle biopsy sample, a fine-needle aspiration, a cytological specimen, or decalcification of bone.

20. The method according to any one of claims 1 to 19, wherein positive staining is determined using a bright-field optical microscope, a microscope objective lens, a computer monitor and imaging software, or a combination thereof.

21. The method according to claim 20, wherein the imaging software includes whole slide imaging software.

22. A method for diagnosing a tumor or cancer by determining whether a tissue sample is positive for claudin 18.2 expression, comprising the step of determining the interpretability of claudin 18.2 in a tissue sample by the method of any one of claims 1 to 21, wherein about 10% or more of neoplasms or cancer cells having a claudin 18.2 cell membrane staining of intensity 1+ or greater are positive for claudin 18.2 score.

23. The method according to claim 22, wherein the tumor is gastric cancer, gastric cancer, gastric cancer metastasis, gastroesophageal junction (GEJ) cancer, esophageal cancer, colorectal cancer, colitis-associated colorectal adenocarcinoma, colon cancer, non-squamous non-small cell lung cancer (nsNSLC), non-small cell lung cancer (NSLC), adenocarcinoma, bile duct cancer, intraepithelial neoplasm of the bile duct, pancreatic cancer, pancreatic adenocarcinoma, pancreatic cancer metastasis, pancreaticobiliary adenocarcinoma, urogenital tract cancer, biliary tract cancer, and biliary tract cancer.

24. A method for treating or improving a tumor or cancer in a patient, comprising the steps of: determining a claudin 18.2 interpretation status in a tissue sample derived from the patient using the method according to any one of claims 1 to 23; and treating the patient with a cancer drug that is likely to respond favorably to the claudin 18.2 interpretation status.

25. The method according to claim 24, wherein the tissue sample has a claudin 18.2 score of about 20% or more of neoplasms or cancer cells having a claudin 18.2 cell membrane staining intensity of 2+ or higher.

26. The method according to claim 24, wherein the tumor or cancer is gastric cancer, gastric cancer, gastric cancer metastasis, gastroesophageal junction (GEJ) cancer, esophageal cancer, colorectal cancer, colitis-associated colorectal adenocarcinoma, colon cancer, non-squamous non-small cell lung cancer (nsNSLC), non-small cell lung cancer (NSLC), adenocarcinoma, bile duct cancer, intraepithelial neoplasm of the bile duct, pancreatic cancer, pancreatic adenocarcinoma, pancreatic cancer metastasis, pancreaticobiliary adenocarcinoma, urogenital tract cancer, biliary tract cancer, and biliary tract cancer.

27. The method according to claim 24 or 25, wherein the cancer treatment drug comprises administering an anticancer drug or anticancer therapy to the patient.

28. The method according to claim 27, wherein the anticancer therapy comprises an antibody-drug conjugate, small molecule therapy, immunotherapy, monoclonal antibody therapy, adoptive cell therapy, T cell receptor therapy, or chimeric antigen receptor (CAR) T cell therapy.

29. A kit comprising an antibody that specifically binds to claudin 18.2 and a claudin 18.2 scoring guideline as described in any one of claims 1 to 28.

30. A method for evaluating the degree of claudin 18.2 expression in a tissue sample derived from an individual, comprising the steps of: contacting the tissue sample or a portion thereof containing cancer or neoplastic cells with an antibody or a portion thereof that specifically binds to claudin 18.2; determining the total number of viable neoplasms or cancer cells having claudin 18.2 cell membrane staining; determining the total number of stained and unstained viable neoplasms or cancer cells; and determining a claudin 18.2 score (%) by dividing the number of claudin 18.2-stained viable neoplasms or cancer cells in the sample or a portion thereof that are specifically bound by the antibody by the total number of stained and unstained viable cancer or neoplastic cells, and multiplying the result by 100 to obtain the claudin 18.2 score (%).

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