Immunohistochemistry (IHC) MAGE-A4 Scoring Protocols and Methods to Aid Cancer Treatment

Abstract

In another embodiment, an immunohistochemistry (IHC) method is provided for reproducibly determining and scoring the level of expression of the protein Melanoma Associated Antigen Gene-A4 (MAGE-A4) in tissue samples. In another embodiment, a method is provided for diagnosing, treating, or ameliorating cancer or tumor, or assessing the risk of recurrence, using an IHC method as provided herein. In another embodiment, a kit is provided that includes components and instructions for carrying out a method as provided herein. The present application describes methods for scoring MAGE-A4 expression and using the score as a companion or complementary diagnostic, or for treating or ameliorating cancer or tumor.

Description

[Technical Field]

[0001] Related Applications This Patent Cooperation Treaty (PCT) international patent application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application (USSN) No. 63 / 442,381, filed January 31, 2023. The foregoing application is incorporated herein by reference for all purposes.

[0002] This invention generally relates to cancer treatments, companion or complementary diagnostics, and immunohistochemical methods. In another embodiment, an immunohistochemistry (IHC) method is provided for reproducibly determining and scoring the level of expression of the protein Melanoma Associated Antigen Gene-A4 (MAGE-A4) in tissue samples. In another embodiment, a method is provided for diagnosing, treating, or ameliorating cancer or tumor, or assessing the risk of recurrence, using an IHC method as provided herein. In another embodiment, a kit is provided that includes components and instructions for carrying out the method as provided herein. This application describes methods for scoring MAGE-A4 expression and using the score as a companion or complementary diagnostic, or for treating or ameliorating cancer or tumor. [Background technology]

[0003] The melanoma-associated antigen gene-A4 (MAGE-A4) is located at chromosome location Xq28 and has been linked to several genetic disorders, including dyskeratosis congenita. At least four variants of this gene have been found that encode the same protein.

[0004] MAGE-A4 is expressed in many cancers and tumors, including synovial sarcoma, myxoid / round cell liposarcoma, and lung cancer. Targeted treatment using genetically modified autologous T cells directed against MAGE-A4 is in clinical trials.

[0005] Due to the heterogeneity of tumor morphology in some cancers, it can be confusing how to apply scoring guidelines. There remains a need in the art for simpler and more efficient, as well as robust, reproducible, and accurate scoring methods for assessing MAGE-A4 expression in tissue samples. Summary of the Invention

[0006] In another embodiment, (a) staining a tissue sample with an antibody that specifically binds to MAGE-A4; (b) determining the total number of viable tumor or cancer cells with MAGE-A4 staining in at least a portion of the tissue sample, and determining the total number of stained and unstained viable tumor or cancer cells, wherein a tumor or cancer cell is counted as positively stained with an anti-MAGE-A4 antibody if there is cytoplasmic and / or nuclear MAGE-A4 staining at any intensity above a specified threshold; and (c) determining a MAGE-A4 Tumor Intensity Percentage Score (MAGE-A4 TIPS), wherein MAGE-A4 TIPS is the number of MAGE-A4 stained viable tumor or cancer cells found in the tissue sample divided by the total number of stained and unstained viable tumor or cancer cells multiplied by 100; An immunohistochemistry (IHC) method for determining and scoring the degree of cellular expression of melanoma-associated antigen gene-A4 (MAGE-A4) in a tissue sample is provided, comprising:

[0007] In another embodiment of the IHC method as provided herein, - said specified threshold is 1+ positive staining intensity assessed at high magnification; 2+ positive staining intensity assessed at medium magnification; or 3+ positive staining intensity assessed at low magnification Contains; - said low magnification is at least about 4x magnification; said medium magnification is at least about 10x magnification; and said high magnification is at least about 20x magnification or at least about 40x magnification; - the MAGE-A4 TIPS comprise the number of viable MAGE-A4 tumor or cancer cells that stain 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; - the MAGE-A4 TIPS comprise the number of viable MAGE-A4 tumor or cancer cells that stain with a positive staining intensity of 1+ or greater, divided by the total number of stained and unstained viable tumor or cancer cells, multiplied by 100; - about 70% or more of the MAGE-A4 TIPS indicate a positive diagnostic status of the tissue sample; or the MAGE-A4 TIPS is about 75% or more, about 80% or more, or about 90% or more; - about 5% or more of the MAGE-A4 TIPS indicate a positive diagnostic status of the tissue sample; or about 5% or more of the MAGE-A4 TIPS comprise a number of MAGE-A4 viable tumor or cancer cells that stain with a positive staining intensity of 1+ or greater; or about 10% or more of the MAGE-A4 TIPS comprise a number of MAGE-A4 viable tumor or cancer cells that stain with a positive staining intensity of 2+ or greater; - a section or portion of the tissue sample is prepared on a slide, microscope slide, or the like, and the section or portion of the tissue sample is stained on the slide; - the antibody comprises a monoclonal mouse anti-MAGE-A4 antibody, optionally wherein the monoclonal mouse anti-MAGE-A4 antibody is - monoclonal mouse anti-MAGE-A4 clone OTI1F9 antibody (generated using the full-length human recombinant protein of human MAGE-A4 (NP_001011550) produced in HEK293T cells (Agilent Technologies) as an immunogen), or an antibody with substantially similar affinity for MAGE-A4; - for example, the clone OTI1F9 antibody (Origene, Abcam), or an antibody with a substantially similar affinity for MAGE-A4; - monoclonal mouse anti-MAGE-A4 clone 57B antibody, or an antibody with substantially similar affinity for MAGE-A4; - monoclonal mouse anti-MAGE-A4 clone 6C1 antibody, or an antibody with substantially similar affinity for MAGE-A4; - monoclonal mouse anti-MAGE-A4 clone CPTC-MAGEA4-1 antibody (Developmental Studies Hybridoma Bank), or an antibody with substantially similar affinity for MAGE-A4; - a monoclonal mouse anti-MAGE-A4 clone CPTC-MAGEA4-2 antibody, or an antibody with substantially similar affinity for MAGE-A4; - monoclonal mouse anti-MAGE-A4 clone 8BA20 antibody (Creative Biolabs), or an antibody with substantially similar affinity for MAGE-A4; - monoclonal mouse anti-MAGE-A4 clone 8BA21 antibody (Creative Biolabs), or an antibody with substantially similar affinity for MAGE-A4; - monoclonal mouse anti-MAGE-A4 clone CBFYM-1312 antibody (Creative Biolabs), or an antibody with substantially similar affinity for MAGE-A4; or - monoclonal mouse anti-MAGE-A4 clone 9A7 antibody (Creative Biolabs), or an antibody with substantially similar affinity for MAGE-A4; or - any combination thereof Contains; - the tissue sample comprises a formalin-fixed, paraffin-embedded (FFPE) specimen, and optionally, the FFPE specimen comprises a cancer specimen stained on an automated IHC platform; - the sections of the tissue sample are prepared by a protocol comprising fixation in about 10% neutral buffered formalin for between about 6 hours and about 72 hours; - the tumor or cancer is synovial sarcoma, myxoid / round cell liposarcoma, high-grade myxoid liposarcoma, low-grade myxoid liposarcoma, head and neck cancer, melanoma, esophageal cancer, gastric cancer, colorectal cancer, lung cancer, non-small cell lung cancer, colon cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, salivary gland cancer, prostate cancer, liver cancer, or urothelial cancer; - the tissue sample is derived from a needle biopsy sample, fine needle aspirate, cytology specimen, or bone demineralization; - positive staining is determined using bright-field light microscopy, a microscope objective, a computer monitor and imaging software, or a combination thereof; and / or - said imaging software comprises whole slide imaging software.

[0008] In another embodiment, a method is provided for diagnosing tumors or cancers by determining whether a tissue sample is positive for expression of melanoma-associated antigen gene-A4 (MAGE-A4), comprising the step of determining a MAGE-A4 positive diagnostic status in the tissue sample by a method as provided herein, wherein MAGE-A4 TIPS of approximately 70% or more of the tumor or cancer cells having MAGE-A4 nuclear and / or cytoplasmic staining at an intensity of 2+ or higher is diagnostically positive.

[0009] In another embodiment of the methods for diagnosing a tumor or cancer as provided herein, the tumor or cancer is synovial sarcoma, myxoid / round cell liposarcoma, head and neck cancer, melanoma, esophageal cancer, gastric cancer, colorectal cancer, lung cancer, non-small cell lung cancer, colon cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, salivary gland cancer, prostate cancer, liver cancer, or urothelial cancer.

[0010] In another embodiment, a method is provided for treating or ameliorating a tumor or cancer in a patient, comprising determining and scoring the amount of MAGE-A4 in a tissue sample from the patient using a method as provided herein, and if the tissue sample is determined or scored as having high or diagnostically positive MAGE-A4 TIPS, treating the patient with a cancer therapy to which the patient is likely to respond favorably.

[0011] In another embodiment of the method for treating or ameliorating a tumor or cancer in a patient, the method comprises: - the tumor or cancer is synovial sarcoma, myxoid / round cell liposarcoma, head and neck cancer, melanoma, esophageal cancer, gastric cancer, colorectal cancer, lung cancer, non-small cell lung cancer, colon cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, salivary gland cancer, prostate cancer, liver cancer, or urothelial cancer; - the cancer therapy comprises administering to the patient an anti-cancer drug or treatment; and / or - the anti-cancer treatment comprises immunotherapy, monoclonal antibody therapy, adoptive cell therapy (ACT), T cell receptor (TCR) therapy, or chimeric antigen receptor (CAR) T cell therapy.

[0012] In another embodiment, a kit is provided comprising an antibody that specifically binds to MAGE-A4 and MAGE-A4 scoring guidelines as provided in the methods provided herein or as provided in the methods for diagnosing tumors or cancers as provided herein.

[0013] In another embodiment, contacting a sample or a portion thereof comprising cancer or tumor cells from an individual with an antibody or a portion thereof that specifically binds to MAGE-A4; and determining a MAGE-A4 Tumor Intensity Ratio Score (MAGE-A4 TIPS) by dividing the number of MAGE-A4 stained viable 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 viable cancer or tumor cells and multiplying the result by 100, thereby obtaining a MAGE-A4 TIPS; A method for assessing the degree of MAGE-A4 expression is provided, comprising:

[0014] 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.

[0015] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. [Brief explanation of the drawings]

[0016] [Figure 1] Figure 1 shows a synovial sarcoma tissue sample (Block ID: SB00046226) stained with MAGE-A4 IHC (SK032). [Figure 2] Figure 2 shows a squamous non-small cell lung cancer (case ID: 1408330B) tissue sample stained with MAGE-A4 IHC (SK032). [Figure 3] Figure 3 shows the match test procedure.

[0017] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee. The drawings presented herein illustrate exemplary embodiments provided herein and are not intended to limit the scope of the invention as encompassed by the claims. The figures are described in detail herein. Like reference symbols in the various drawings indicate like elements. DETAILED DESCRIPTION OF THE INVENTION

[0018] In another embodiment, an immunohistochemistry (IHC) method is provided for determining and scoring the degree of cellular expression of the protein Melanoma Associated Antigen Gene-A4 (MAGE-A4) in a tissue sample. In another embodiment, a scoring method is provided for assessing MAGE-A4 expression in a tumor or cancer. In another embodiment, a method is provided for diagnosing and / or treating a tumor or cancer, comprising using a scoring method as provided herein to assess MAGE-A4 expression and determine whether a high or diagnostically positive MAGE-A4 Tumor Intensity Percentage Score (MAGE-A4 TIPS) is present. In certain embodiments, if the tissue sample is determined or scored as having a high or diagnostically positive MAGE-A4 TIPS, the patient is treated with a cancer therapy to which the patient is likely to respond favorably.

[0019] Synovial sarcoma (SS) is a rare malignant tumor with a poor prognosis and low response rates to various classes of therapeutic agents. This necessitates the development of targeted biomarkers for patient selection for novel adoptive T cell therapy (ACT) in SS with evidence of clinical efficacy. The cancer-testis antigen, melanoma-associated antigen gene-A4 (MAGE-A4), is highly expressed in SS and correlates with clinical stage. The restricted expression pattern of MAGE-A4 in normal tissues makes it an attractive target for the development of diagnostic assays and ACT.

[0020]

[0003] Embodiments herein are directed to immunohistochemistry methods for determining and scoring the degree of nuclear and / or cytoplasmic expression of MAGE-A4 in a tissue sample. In various embodiments, the methods include staining the tissue sample with an antibody that specifically binds to MAGE-A4; determining the total number of viable tumor or cancer cells having MAGE-A4 nuclear and / or cytoplasmic staining in at least a portion of the tissue sample, and determining the total number of stained and unstained viable tumor or cancer cells, wherein tumor or cancer cells are counted as positively stained with the anti-MAGE-A4 antibody if there is MAGE-A4 cytoplasmic and / or nuclear staining at any intensity above a specified threshold; and determining a MAGE-A4 Tumor Intensity Ratio Score (MAGE-A4 TIPS), wherein MAGE-A4 TIPS is the number of MAGE-A4-stained viable tumor or cancer cells found in the tissue sample divided by the total number of stained and unstained viable tumor or cancer cells, multiplied by 100. Such embodiments may provide a robust, reliable, reproducible and accurate method of scoring MAGE-A4 as well as a simpler and more efficient scoring method, which may provide significant advantages as a reliable and efficient diagnostic assessment.

[0021] In certain embodiments, the tissue sample contains at least about 50 viable tumor or cancer cells. In certain embodiments, the tissue sample contains at least about 100 viable tumor or cancer cells. In certain embodiments, the tissue sample contains at least about 200 viable tumor or cancer cells. In certain embodiments, the tissue sample contains at least about 500 viable tumor or cancer cells.

[0022] In some embodiments, tumor or cancer cells counted as positively stained exclude staining of normal or non-neoplastic structures, staining of non-viable tumor or cancer cells, necrotic cells, cell debris, stromal staining, staining of immune cells, staining of benign cells, or edge artifact staining on the periphery of the tissue sample. In certain embodiments, tumor or cancer cells counted as positively stained exclude tumor or cancer cells that stain with a positive staining intensity of less than 2+. Such embodiments can provide the advantage of increasing the accuracy and reproducibility of the MAGE-A4 scoring method by excluding staining cells with certain structures or characteristics from the MAGE-A4 TIPS determination.

[0023] In certain embodiments, the defined threshold comprises a positive staining intensity of 1+ assessed at high magnification. In certain embodiments, the defined threshold comprises a positive staining intensity of 2+ or greater assessed at medium magnification. In certain embodiments, the defined threshold comprises a positive staining intensity of 3+ assessed at low magnification (see Figures 1 and 2). In some aspects, low magnification is at least about 4x magnification; medium magnification is at least about 10x magnification; or high magnification is at least about 20x magnification or at least about 40x magnification.

[0024] In certain embodiments, MAGE-A4 TIPS comprise the number of viable MAGE-A4 tumor or cancer cells stained with a positive staining intensity of 2+ or greater, divided by the total number of viable stained and unstained tumor or cancer cells, multiplied by 100. In other embodiments, MAGE-A4 TIPS comprise the number of viable MAGE-A4 tumor or cancer cells stained with a positive staining intensity of 1+ or greater, divided by the total number of viable stained and unstained tumor or cancer cells, multiplied by 100. Such embodiments can provide the advantage of versatility in staining intensity at a defined threshold.

[0025] In certain embodiments, about 70% or more MAGE-A4 TIPS indicate a positive diagnostic status for the tissue sample. In certain embodiments, the MAGE-A4 TIPS are about 75% or more, about 80% or more, or about 90% or more. In certain embodiments, about 5% or more MAGE-A4 TIPS indicate a positive diagnostic status for the tissue sample. In certain embodiments, about 5% or more MAGE-A4 TIPS include the number of MAGE-A4 viable tumor or cancer cells staining with a positive staining intensity of 1+ or more; or about 10% or more MAGE-A4 TIPS include the number of MAGE-A4 viable tumor or cancer cells staining with a positive staining intensity of 2+ or more. Such embodiments can provide the advantage of accuracy in determining diagnostic status related to MAGE-A4 expression.

[0026] In another embodiment, the methods and kits provided herein are used for in vitro diagnostic applications. In another embodiment, the MAGE-A4 IHC provided herein is an immunohistochemistry (IHC) assay using an anti-MAGE-A4 antibody, such as the monoclonal mouse anti-MAGE-A4 clone OTI1F9 antibody (generated using the full-length human recombinant protein of human MAGE-A4 (NP_001011550) produced in HEK293T cells (Agilent Technologies) as an immunogen) or an antibody with substantially similar affinity for MAGE-A4, in detecting MAGE-A4 protein in FFPE tissue samples. In another embodiment, the AUTOSTAINER LINK 48™ automated staining system is used, utilizing FFPE tissue sections.

[0027] In another embodiment, the MAGE-A4 IHC method as provided herein is used to aid in identifying patients with a tumor or cancer, for example, synovial sarcoma, myxoid / round cell liposarcoma, high-grade myxoid liposarcoma, low-grade myxoid liposarcoma, head and neck cancer, melanoma, esophageal cancer, gastric cancer, colorectal cancer, lung cancer, non-small cell lung cancer, colon cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, salivary gland cancer, prostate cancer, liver cancer, or urothelial cancer.

[0028] In another embodiment, a method for assessing the degree of MAGE-A4 expression includes the steps of contacting a sample or a portion thereof containing cancer or tumor cells from an individual with an antibody or a portion thereof that specifically binds to MAGE-A4; and determining a MAGE-A4 Tumor Intensity Ratio Score (MAGE-A4 TIPS) by dividing the number of MAGE-A4-stained viable 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 viable cancer or tumor cells and multiplying the result by 100, thereby obtaining a MAGE-A4 TIPS.

[0029] In certain embodiments, a section or portion of a tissue sample is prepared on a slide, microscope slide, or equivalent, and the section or portion of the tissue sample is stained on the slide. In certain embodiments, the tissue sample comprises an FFPE specimen. In certain embodiments, the FFPE specimen comprises a cancer specimen stained on an automated IHC platform. In certain embodiments, the tissue sample section is prepared by a protocol comprising fixation in about 10% neutral buffered formalin for about 6 hours to about 72 hours. In certain embodiments, the tissue sample is derived from a needle biopsy sample, fine needle aspirate, cytology specimen, or demineralized bone.

[0030] In certain embodiments, the antibody comprises a monoclonal mouse anti-MAGE-A4 antibody or a monoclonal rabbit anti-MAGE-A4 antibody. In some embodiments, the monoclonal mouse anti-MAGE-A4 antibody is selected from the group consisting of monoclonal mouse anti-MAGE-A4 clone OTI1F9 antibody, or an antibody having an affinity for MAGE-A4 substantially similar to clone OTI1F9 antibody; monoclonal mouse anti-MAGE-A4 clone 57B antibody, or an antibody having a substantially similar affinity for MAGE-A4; monoclonal mouse anti-MAGE-A4 clone 6C1 antibody, or an antibody having a substantially similar affinity for MAGE-A4; monoclonal mouse anti-MAGE-A4 clone CPTC-MAGEA4-1 antibody, or an antibody having a substantially similar affinity for MAGE-A4; monoclonal mouse anti-MAGE-A4 clone CPTC-MAGEA4-2 antibody, or an antibody having a substantially similar affinity for MAGE-A4; monoclonal mouse anti-MAGE-A4 clone 8BA20 antibody, or an antibody having a substantially similar affinity for MAGE-A4; clone 8BA21 antibody, or an antibody with substantially similar affinity for MAGE-A4; monoclonal mouse anti-MAGE-A4 clone CBFYM-1312 antibody, or an antibody with substantially similar affinity for MAGE-A4; or monoclonal mouse anti-MAGE-A4 clone 9A7 antibody, or an antibody with substantially similar affinity for MAGE-A4; or any combination thereof.

[0031] In certain embodiments, the tumor or cancer is synovial sarcoma, myxoid / round cell liposarcoma, high-grade myxoid liposarcoma, low-grade myxoid liposarcoma, head and neck cancer, melanoma, esophageal cancer, gastric cancer, colorectal cancer, lung cancer, non-small cell lung cancer, colon cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, salivary gland cancer, prostate cancer, liver cancer, or urothelial cancer.

[0032] 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.

[0033] Embodiments of a method for diagnosing a tumor or cancer

[0010] Embodiments herein provide methods for diagnosing tumors or cancers by determining whether a tissue sample is positive for cellular expression of MAGE-A4. In various aspects, the method includes determining MAGE-A4 diagnostic status in the tissue sample by an IHC method for determining the degree of cytoplasmic and / or nuclear expression of MAGE-A4 as provided herein, wherein about 70% or more of the tumor or cancer cells are diagnostically positive for MAGE-A4 TIPS, having MAGE-A4 nuclear and / or cytoplasmic staining at an intensity of 2+ or higher. In certain embodiments, the tumor or cancer is synovial sarcoma, myxoid / round cell liposarcoma, high-grade myxoid liposarcoma, low-grade myxoid liposarcoma, head and neck cancer, melanoma, esophageal cancer, gastric cancer, colorectal cancer, lung cancer, non-small cell lung cancer, colon cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, salivary gland cancer, prostate cancer, liver cancer, or urothelial cancer.

[0034] Method embodiments for treating cancer and tumors

[0010] Methods for treating or ameliorating a tumor or cancer in a patient are provided, comprising determining and scoring the amount of MAGE-A4 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 MAGE-A4 score, the patient is treated with a cancer or anti-cancer therapy to which the patient is likely to respond favorably. The tumor or cancer may be synovial sarcoma, myxoid / round cell liposarcoma, high-grade myxoid liposarcoma, low-grade myxoid liposarcoma, head and neck cancer, melanoma, esophageal cancer, gastric cancer, colorectal cancer, lung cancer, non-small cell lung cancer, colon cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, salivary gland cancer, prostate cancer, liver cancer, or urothelial cancer.

[0035] In certain embodiments, the cancer therapy comprises administering an anti-cancer drug or anti-cancer therapy to the patient. In other embodiments, the anti-cancer 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 radiotherapy.

[0036] In certain embodiments, the anti-cancer therapy comprises an antibody-drug conjugate, small molecule therapy, immunotherapy, monoclonal antibody therapy, adoptive cell therapy (ACT), T cell receptor (TCR) therapy, or chimeric antigen receptor (CAR) T cell therapy. In another embodiment, the anti-cancer treatment or therapy comprises a tyrosine kinase inhibitor (optionally erlotinib (or TARCEVA™), gefitinib (or IRESSA™), afatinib (or GILOTRIF™), or osimertinib (TAGRISSO™); necitumumab (or PORTRAZZA™), pembrolizumab (or KEYTRUDA™), nivolumab (or OPDIVO™), ipilimumab (YERVOY™), cetuximab (or ERBITUX™), or rituximab (or rituximab). trademark), cisplatin (or PLATINOL™), or carboplatin (or PARAPLATIN™); gemcitabine (or GEMZAR™), docetaxel (or TAXOTERE™), olaratumab (or LARTRUVO™), doxorubicin (or ADRIAMYCIN™, or RUBEX™), pazopanib (or VOTRIENT™), ifosfamide (or IFEX™), trabectedin (or YONDELIS™).

[0037] In another embodiment, the anti-cancer treatment or therapy comprises the use of an anti-cancer agent which may comprise an antibody that specifically or substantially binds to a cancer or tumor, said antibody being conjugated to a cytotoxic agent, optionally wherein said cytotoxic agent comprises a radionuclide (optionally yttrium-90, iodine-131, lutetium-177, radium-223 chloride, strontium-89 chloride, or samarium-153 EDTMP), diphtheria toxin, pseudomonas exotoxin A, denileukin diftitox, moxetumomab pasudotox, calicheamicin or N-acetyl-gamma-calicheamicin, emtansine or DM1, maytansine or a derivative thereof, SN-38 (or 7-ethyl-10-hydroxycamptothecin), or auristatin or monomethylauristatin E (MMAE).

[0038] immunohistochemistry In another embodiment, the immunohistochemistry methodology and / or reagents used with the methods and articles of manufacture or kits as provided herein can include, or include or involve the use of, any IHC protocol, IHC medical equipment, device, and / or image or data analysis system for performing IHC, or IHC reagents known in the art.

[0039] In another embodiment, 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 in the form of unpurified or partially purified culture supernatants.

[0040] In another embodiment, the methods as provided herein can use or include reagents for detecting or visualizing antibody-antigen interactions using any product or method known in the art, e.g., IHC protocols or reagents.

[0041] In another embodiment, the methods as provided herein include the use of chromogenic immunohistochemistry (CIH), wherein a primary antibody (e.g., a recombinant antibody (Ab) or antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein as provided herein) or a secondary antibody (e.g., a secondary antibody that binds to the primary antibody, i.e., a recombinant antibody (Ab) or antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein as provided herein) is conjugated to an enzyme, such as a peroxidase, e.g., an immunoperoxidase, e.g., horseradish peroxidase (HRP), that can catalyze a color-developing reaction. In another embodiment, the chromogenic moiety used in the methods as provided herein is selected from the group consisting of coumarin; rhodamine; 2,3,6,7-tetrahydro-11-oxo-1H,5H,11H-[1]benzopyrano[6,7,8-ij]quinolizine-10-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; fuchsin; Cascade Blue acetyl; dapoxyl sulfonic acid / carboxylic acid succinimidyl ester; DY-405; Alexa Fluor 405 succinimidyl ester; Cascade Yellow succinimidyl ester; pyridyloxazole succinimidyl ester (PyMPO); Pacific Blue succinimidyl ester; DY-415; 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester; DYQ-425; 6-FAM phosphoramidite; Lucifer Yellow; iodoacetamide; Alexa Fluor 430 succinimidyl ester; Dabcyl succinimidyl ester; NBD chloride / fluoride; QSY 35 succinimidyl ester; DY-485XL; Cy2 succinimidyl ester; DY-490; Oregon Green 488 carboxylic acid succinimidyl ester; Alexa Fluor 488 succinimidyl ester;BODIPY 493 / 503 C3 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'-tetrabromosulfonefluorescein succinimidyl ester; Alexa Fluor 532 succinimidyl ester; 6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein succinimidyl ester (HEX); BODIPY 530 / 550 C3 succinimidyl ester; DY-530; BODIPY TMR-X succinimidyl ester; DY-555; DYQ-1; DY-556; Cy3 succinimidyl ester; DY-547; DY-549; DY-550; Alexa Fluor 555 succinimidyl ester; Alexa Fluor 546 succinimidyl ester; DY-548; BODIPY 558 / 568 C3 succinimidyl ester; Rhodamine Red-X succinimidyl ester; QSY 7 succinimidyl ester; BODIPY 564 / 570 C3 succinimidyl ester; BODIPY 576 / 589 C3 succinimidyl ester; Carboxy-X-rhodamine (ROX); succinimidyl ester; Alexa Fluor 568 succinimidyl ester; DY-590; BODIPY 581 / 591 C3 succinimidyl ester; DY-591; BODIPY TR-X succinimidyl ester; Alexa Fluor 594 succinimidyl ester; DY-594; Carboxynaphthofluorescein succinimidyl ester; DY-605; DY-610; Alexa Fluor 610 succinimidyl ester; DY-615;BODIPY 630 / 650-X succinimidyl ester; Erioglaucine; 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 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; Dabsyl sulfonyl chloride; Fluorescein isothiocyanate (FITC) carboxysuccinimidyl ester (DY-495); Rhodamine Green carboxylic acid succinimidyl ester (DY-505); eosin isothiocyanate (EITC); 6-carboxy-2',4,7,7'-tetrachlorofluorescein succinimidyl ester (TET); carboxyrhodamine 6G succinimidyl ester; carboxytetramethylrhodamine succinimidyl ester (TMR, TAMRA) (DY-554); QSY 9 succinimidyl ester; sulforhodamine B sulfonyl chloride (DY-560); Texas Red (sulforhodamine 101); gallocyanine; Fast Green FCF; Malachite Green; or QSY 21 succinimidyl ester.

[0042] In another embodiment, the methods provided herein include the use of immunofluorescence, wherein a primary or secondary antibody is tagged with a fluorophore, such as fluorescein or fluorescein isothiocyanate (FITC), a triarylmethane dye, such as rhodamine or a rhodamine derivative (e.g., tetramethylrhodamine (TRITC), rhodamine 6G, rhodamine 123, rhodamine B, carboxytetramethylrhodamine (TAMRA), tetramethylrhodamine (TMR), sulforhodamine 101), aminomethylcoumarin acetate (AMCA), or ALEXA™ or DYLIGHT™ fluor, a fluorophore, or a dye. 3,3'-Diaminobenzidine (DAB) can also be used.

[0043] In another embodiment, the methods as provided herein involve the use of a direct or one-step staining method in which a primary antibody (e.g., an antibody (Ab) as provided herein, an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein (e.g., including synthetic or recombinant forms)) is labeled and reacts directly with the antigen, e.g., in a tissue section. This technique utilizes only one antibody and is therefore simple and rapid, although sensitivity may be lower due to little signal amplification.

[0044] In another embodiment, the method as provided herein involves the use of an indirect method, in which an unlabeled primary antibody (first layer) binds to a target antigen (e.g., MAGE-A4 protein), e.g., in a tissue or organ, and then a labeled secondary antibody (second layer) reacts with the primary antibody. The secondary antibody can be directed against the isotype of the animal species from which the primary antibody is derived, e.g., IgG. This method can be more sensitive than direct detection strategies when the secondary antibodies are conjugated to a detectable substance such as a fluorescent or enzyme reporter, due to signal amplification resulting from the binding of several secondary antibodies to each primary antibody.

[0045] In another embodiment, further amplification is achieved when the secondary antibody is conjugated to several detection molecules, for example, a biotin molecule that can recruit avidin-linked, streptavidin-linked, or NEUTRAVIDIN™ protein-linked enzyme complexes.

[0046] In another embodiment, IHC is performed on tissue sections or tissue biopsies, e.g., paraformaldehyde (PFA)-fixed tissues or organs, or FFPE tissues. In another embodiment, the tissues are sectioned or sliced, or used whole. Prior to sectioning, tissue samples can be embedded in media, e.g., paraffin wax or cryomedia. Tissue sections can be sectioned or sliced ​​using a variety of instruments, most commonly a microtome, cryostat, or vibratome. Specimens can be sectioned or sliced ​​to a thickness of approximately 3 μm to 5 μm. Sections or slices can be mounted on slides, dehydrated using increasing concentrations of alcohol washes (e.g., 50%, 75%, 90%, 95%, 100%), and cleaned with detergents such as xylene before being imaged or evaluated under a microscope.

[0047] Depending on the method of fixation and tissue preservation, samples may require additional steps, such as deparaffinization and antigen retrieval, to make MAGE-A4 epitopes available for antibody binding. For formalin-fixed, paraffin-embedded tissues, antigen retrieval is often required and may involve pre-treating sections with heat or proteases.

[0048] In another embodiment, IHC is performed using the ENVISION DUOFLEX DOUBLESTAIN SYSTEM™ (Agilent, San Jose, CA), which allows staining of two or more markers on a single slide. In another embodiment, 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 another embodiment, IHC is performed using the EnVision FLEX Mini Kit, High pH, ​​a highly sensitive visualization system intended for use with IHC in conjunction with the Dako AUTOSTAINER™ instrument; this dual-link system detects primary mouse and rabbit antibodies, and the reaction is visualized with 3,3'-diaminobenzidine (DAB) chromogen (DAB forms a water-insoluble brown precipitate when oxidized, e.g., by peroxidase).

[0049] Products and Kits Articles of manufacture and kits for carrying out the methods as provided herein are provided, which comprise, for example, at least one anti-MAGE-A4 antibody, e.g., a monoclonal antibody, e.g., the monoclonal mouse anti-MAGE-A4 clone OTI1F9 antibody (generated using full-length human recombinant protein of human MAGE-A4 produced in HEK293T cells (Agilent Technologies) as an immunogen) or an antibody having a substantially similar affinity for MAGE-A4, and / or reagents for performing IHC, e.g., reagents as described herein (see Example 1 below), and optionally the articles of manufacture and kits further comprise instructions for carrying out the methods as provided herein.

[0050] Any of the above aspects and embodiments may be combined with any other aspect or embodiment, such as those disclosed in the Summary, Figures, and / or Detailed Description sections of this specification.

[0051] As used in this specification and the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0052] As used herein, unless otherwise expressly stated or apparent from the context, the term "or" is understood to be inclusive, covering both "or" and "and."

[0053] As used herein, unless otherwise specified or clear from the context, the term "about" is understood to be within the normal tolerance in the art, for example, within two standard deviations from the mean value.About (use of the term "about") is 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 clear from the context, all numerical values ​​provided herein are modified by the term "about".

[0054] As used herein, unless otherwise specified or clear from the context, the terms "substantially all," "a substantial majority of," "substantially all of," 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 referenced amount of the composition.

[0055] The entirety of each patent, patent application, publication, and document referred to herein is incorporated herein by reference. The citation of any patent, patent application, publication, and document is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of such publications or documents. The incorporation by reference of any of these documents independently should not be construed as a representation or admission that any portion of the contents of any document is deemed essential material for satisfying any national or local statutory disclosure requirements for patent applications. The right is nevertheless reserved to rely on any such documents, as necessary, to provide material determined by an examining authority or court to be essential to the claimed subject matter.

[0056] Modifications may be made thereto without departing from the essential aspects of the invention. While the present invention has been described in sufficient detail with reference to one or more specific embodiments, those skilled in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, and that these modifications and improvements will still be within the scope and spirit of the invention. The invention illustratively described herein may suitably be practiced in the absence of any element not specifically disclosed herein. Thus, for example, in each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be substituted for either of the other two terms. Accordingly, the terms and expressions used are used as terms of description, rather than as terms of limitation, and are not intended to exclude equivalents of the features shown and described, or portions thereof, recognizing that various modifications are possible within the scope of the invention. Embodiments of the invention are set forth in the following claims.

[0057] The present invention will be further described with reference to the examples set forth herein; however, it should be understood that the present invention is not limited to such examples. [Example]

[0058] Unless otherwise specified in the examples, all recombinant DNA techniques are carried out according to standard protocols, such as those described in Sambrook et al. (2012) Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, NY, and Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. 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, and Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK).

[0059] [Example 1] Exemplary IHC Protocol for Detecting MAGE-A4 Protein This example describes an exemplary IHC method as provided herein and demonstrates the effectiveness of the MAGE-A4 detection IHC protocol as provided herein.

[0060] Results are presented for activities performed as needed to establish inter- and intra-observer scoring accuracy for FFPE synovial sarcoma (SS), myxoid round cell liposarcoma (MRCLS), and squamous cell non-small cell lung cancer (sqNSCLC) specimens stained with the MAGE-A4 IHC (SK032) assay in AUTOSTAINER LINK 48™. Specimens were evaluated using the MAGE-A4 IHC scoring guidelines.

[0061] Inter- and intraobserver precision was tested using the MAGE-A4 IHC scoring guidelines on SS, MRCLS, and sqNSCLC specimens. Specimens were stained with the MAGE-A4 IHC assay. The results reported here provide information on the reproducibility of assay scoring on stained SS, MRCLS, and sqNSCLC specimens when scored multiple times by each of multiple observers.

[0062] reagent MAGE-A4 IHC (SK032) contains optimized reagents along with the protocol required to complete the IHC staining procedure on FFPE specimens using the PT Link Pre-Treatment Module™ (https: / / www.agilent.com / en / product / pt-link-for-pre-treatment / pt-link-accessories / pt-link-pre-treatment-module-for-tissue-specimens-76929) and AUTOSTAINER LINK 48™. First, the specimen is incubated with a peroxidase blocking reagent. After incubation with a primary monoclonal antibody against MAGE-A4 or a negative control reagent (NCR), the specimen is incubated with the primary antibody and then with a ready-to-use (RTU) visualization reagent consisting of a secondary antibody molecule linked to a dextran polymer backbone and a horseradish peroxidase molecule. Subsequent enzymatic conversion of the added chromogen results in the precipitation of a visible reaction product at the antigen site. Specimens may then be counterstained and coverslipped. Results are interpreted using brightfield microscopy.

[0063] JPEG2026505760000002.jpg137170 · EnVision FLEX Hematoxylin (Dako K8008) Automated coverslipper or ClearMount (American MasterTech Scientific, MMCLE126) or equivalent mounting medium Alcohol Reagent, Ethanol: Absolute (Dako 100188) and 95% Xylene (Dako 100941) or a xylene substitute, i.e., Histoclear (Dako 100791) EnVision FLEX Wash Buffer 20x (Dako K8007) Distilled or deionized water (reagent grade water) or Dako Reagent Water (Dako PD00004)

[0064] Reagent preparation Equilibrate kit reagents, buffers, and unstained slides to room temperature, 20-25°C. Prepare 1x wash buffer by diluting 20x wash buffer in distilled or deionized water (reagent grade) and mixing. If the appearance is cloudy, discard the wash buffer. Prepare 1x High pH Target Retrieval Solution (TRS) by diluting 50x High pH Target Retrieval Solution in reagent-grade water and mixing. Prepare a sufficient amount of high pH 1x target retrieval solution by diluting Target Retrieval Solution, High pH (50x) 1:50 with distilled or deionized water (reagent grade). Diluting one 30 mL bottle of Target Retrieval Solution, High pH (50x) 1:50 will yield 1.5 L of 1x reagent, enough to fill one PT Link tank, which can process up to 24 slides per use. A TRS pH test should be performed before use to ensure the 1x TRS is within the reagent specification (pH 9.0 ± 0.2). Prepare DAB+ chromogen immediately before starting the AUTOSTAINER LINK 48™ run: Add 1 drop of liquid DAB+ chromogen per mL of substrate buffer. If using a 7.2mL bottle of DAB+ substrate buffer, add 9 drops of DAB+ chromogen. Although the label says 7.2mL, this is the usable volume and does not include the "dead volume" in the bottle (1.8mL). The color of the Liquid DAB+ Chromogen in the bottle may vary from clear to lavender brown. This will not affect the performance of this product. Dilute according to the guidelines above. Adding too much Liquid DAB+ Chromogen to the DAB+ Substrate Buffer will result in a weakened positive signal.

[0065] Sample preparation All specimens were fixed in neutral buffered formalin and embedded in paraffin blocks. FFPE blocks were sectioned at 4 μm, mounted on Dako FLEX™ IHC microscope slides, and placed at 58 (±2)°C for approximately 1 hour within 18 hours of sectioning, then cooled to room temperature (RT). Cut sections were kept at 2-8°C until use.

[0066] Staining procedure Deparaffinization / Hydrophilizaton / Target Retrieval Procedure:PT Link All specimens were processed using a PT Link with a preheat and cool temperature set to 65° C. All specimens were then processed using a PT Link with a heating temperature set to 97° C. for 20 minutes. Fill the PT Link tanks with 1.5 L per tank of Target Retrieval Solution, High pH, ​​1x Working Solution to cover the tissue sections. Preheat 1x target activation solution to 65°C. Immerse the AUTOSTAINER LINK 48™ rack containing the mounted FFPE tissue sections into preheated Target Retrieval Solution, High pH (1x Working Solution) in the PT Link tank. Incubate at 97°C for 20 minutes. Once the target retrieval incubation period is complete and the temperature has cooled to 65°C, remove each AUTOSTAINER LINK 48™ slide rack with slides from the PT Link tank and immediately place the AUTOSTAINER LINK 48™ rack with slides into a tank (e.g., PT Link Rinse Station, code PT109) containing 1x, room temperature EnVision FLEX™ Wash Buffer (code K8007). · Immerse the slides in diluted room temperature 1x wash buffer for 5 minutes.

[0067] After deparaffinization, hydrophilization, and target retrieval (3-in-1) procedures (specimen pretreatment), the Autostainer rack with slides was placed in the AUTOSTAINER LINK 48™. The AUTOSTAINER LINK 48™ instrument automatically performed the staining process using a pre-programmed protocol by applying the appropriate reagents, monitoring incubation times, and rinsing the slides between reagents.

[0068] Data interpretation The following information was obtained during the observer reading: MAGE-A4 slides: Percent positive MAGE-A4 scores in each intensity bin (0-3+) Diagnostic results *Positive: ≥2+ staining intensity at MAGE-A4 TIPS ≥75% cutoff *Negative: ≥2+ staining intensity at MAGE-A4 TIPS <75% cutoff ·Average IHC staining intensity NCR Slide: Pass / Fail *Negative control reagent (NCR) stained slides must not exhibit specific staining. Nonspecific staining at ≤1+ staining intensity is acceptable. *Specific staining is defined as nuclear and / or cytoplasmic staining in tumor cells, and nonspecific staining is defined as staining of non-tumor tissue elements. If an NCR specimen exhibits specific staining at any intensity or nonspecific staining at >1+ intensity, do not proceed with evaluation of the MAGE-A4 stained slide. If applicable, comment

[0069] Scoring Guidelines Stained slides for all indicators were scored by a qualified observer.

[0070] specimen Eighty specimens (sqNSCLC, MRCLS, and / or SS) exhibiting variable MAGE-A4 expression were selected for this study. A balanced sample size was ensured per MAGE-A4 TIPS, with a staining intensity of ≥2+ at the ≥75% cutoff, with approximately 20–25% of specimens falling within the range close to the cutoff (65–85%). Stained slides were selected from a previously performed screening run and followed the method described herein. Figure 1 shows a synovial sarcoma tissue sample (Block ID: SB00046226) stained with MAGE-A4 IHC (SK032). Arrows indicate 1+, 2+, or 3+ staining intensity observed at 20x magnification.

[0071] Figure 2 shows a squamous non-small cell lung carcinoma (Case ID: 1408330B) tissue sample stained with MAGE-A4 IHC (SK032). Arrows indicate 1+, 2+, or 3+ staining intensity observed at 10x magnification. Data and biospecimens used in this project were provided by Contract Research Ltd (Charlestown, Nevis) through Azenta Life Sciences with appropriate ethical approval.

[0072] procedure Eighty stained specimens were evaluated by three observers (interobserver) and scored three times by each observer (intraobserver). To minimize recall bias in scoring, there was a washout period of at least 14 days between each observer reading session.

[0073] All slides for all readings were blinded and randomized. A circle label was attached to each slide according to a randomization scheme generated using the RAND function in Excel. Each intra-observer reading had a different randomization scheme. The observer scoring this study was not involved in blinding and randomizing the slides.

[0074] Test Objectives and Procedures The purpose of this study is to determine scoring accuracy among multiple observers.

[0075] Blinded and randomized sets of stained slides were presented in sequence to each of three observers, who each performed three reads. This resulted in nine scores for each case. The scores for each specimen were analyzed to determine the level of diagnostic agreement between observers across multiple reads. The same data set was used for both inter- and intra-observer analyses.

[0076] Statistical Analysis Plan Calculating the Agreement Percentage (NPA, PPA, and OA) A percent agreement calculation was devised to compare each reading from each observer against the consensus, which was defined as the most frequent diagnosis observed.

[0077] Confidence intervals for percentage agreement Two-sided 95% confidence intervals for NPA, PPA, and OA were calculated using bootstrapping, a well-known computational technique that uses random selection to generate a new dataset from the entire original dataset so that statistical uncertainty can be calculated to create statistical confidence intervals. If NPA, PPA, or OA is exactly equal to 100%, the bootstrapping is known to have failed; therefore, Wilson score confidence intervals were calculated.

[0078] Acceptance Criteria The lower limit of the two-sided 95% confidence interval calculated for each concordance parameter (NPA, PPA, and OA) must be ≥ 85%. Any additional analyses performed are not subject to the acceptance criteria.

[0079] Intraobserver precision Test Purpose The purpose of this study was to determine the intra-observer scoring accuracy across multiple readings of the same sample.

[0080] Test Procedure A blinded and randomized set of stained slides was provided to each of three observers in sequence, and each observer performed three reads. This resulted in nine scores for each case. A washout period of at least 14 days was allowed between intraobserver reads. Scores for each specimen were analyzed to determine the level of diagnostic agreement between multiple reads for each observer. Note that the same data set was used for both interobserver and intraobserver analyses.

[0081] Statistical Analysis Plan A percent agreement calculation was devised to compare each reading from each observer to a consensus, which was defined as the most frequent diagnosis observed. Two-sided 95% confidence intervals for NPA, PPA, and OA were calculated using bootstrapping, a well-known computational technique that uses random selection to generate a new dataset from the entire original dataset so that statistical uncertainty can be calculated to create statistical confidence intervals for PPA, NPA, and OA. It is known that if NPA, PPA, or OA is exactly equal to 100%, the bootstrapping has failed; therefore, Wilson score confidence intervals were calculated.

[0082] Acceptance Criteria The study will be accepted once all sub-studies have been completed, discrepancies (if any) have been addressed and resolved, and the final report has been approved.

[0083] result Data from all indicators were combined for analysis and are presented in Tables 2 and 3. Additional analyses per indicator were conducted for informational purposes.

[0084] JPEG2026505760000003.jpg116170

[0085] JPEG2026505760000004.jpg115170

[0086] conclusion Analysis of intraobserver and interobserver precision demonstrated that the lower limits of the two-sided 95% confidence intervals for NPA, PPA, and OA were at least 85% for both interobserver and intraobserver data. Therefore, statistically, both intraobserver and intraobserver precision met the acceptance criteria. NPA, PPA, and OA were calculated using a 75% cutoff for a staining intensity of ≥2+ for MAGE-A4 TIPS. Using the scoring system specified in this report, interobserver and intraobserver precision across multiple readings of the same specimen for SS, MRCLS, and sqNSCLC slides stained with the SK032 MAGE-A4 IHC assay was established. Additional analyses performed on individual index data were not subject to the acceptance criteria.

[0087] Each of the three individual indices showed high agreement (estimates >93%), suggesting that none of the indices biased the results in any favor.

[0088] [Example 2] Exemplary IHC Protocol for Detecting MAGE-A4 Protein This example describes an exemplary IHC method as provided herein and demonstrates the effectiveness of the MAGE-A4 detection IHC protocol as provided herein.

[0089] This study was performed on FFPE myxoid / round cell liposarcoma (MRCLS), synovial sarcoma (SS), and squamous non-small cell lung cancer (sqNSCLC) specimens stained with MAGE-A4 IHC using the EnVision FLEX visualization system on an AUTOSTAINER LINK 48™. WSI scoring was performed by a qualified observer using Aperio and ImageScope software.

[0090] reagent The SK032 MAGE-A4 IHC contains the optimized reagents and protocols required to complete the IHC staining procedure using the AUTOSTAINER LINK 48™ and PT Link Pre-treatment Module™ (https: / / www.agilent.com / en / product / pt-link-for-pre-treatment / pt-link-accessories / pt-link-pre-treatment-module-for-tissue-specimens-76929). After incubation with a primary monoclonal antibody against MAGE-A4 or a negative control reagent (NCR), specimens were incubated with a ready-to-use visualization reagent consisting of a secondary antibody molecule linked to a dextran polymer backbone and a horseradish peroxidase (HRP) molecule. Subsequent enzymatic conversion of the added chromogen results in the precipitation of a visible reaction product at the antigen site. The specimens were then counterstained and coverslipped. Results were interpreted using a light microscope.

[0091] JPEG2026505760000005.jpg115170 · EnVision FLEX Hematoxylin (Dako K8008) Automated coverslipper or ClearMount (American MasterTech Scientific, MMCLE126) or equivalent mounting medium Alcohol Reagent, Ethanol: Absolute (Dako 100188) and 95% Xylene (Dako 100941) or a xylene substitute, i.e., Histoclear (Dako 100791) EnVision FLEX Wash Buffer 20x (Dako K8007) Distilled or deionized water (reagent grade water) or Dako reagent water (Dako PD00004)

[0092] Reagent preparation Equilibrate kit reagents, buffers, and unstained slides to room temperature, 20-25°C. Prepare 1x wash buffer by diluting 20x wash buffer in distilled or deionized water (reagent grade) and mixing. If the appearance is cloudy, discard the wash buffer. Prepare 1x High pH Target Retrieval Solution (TRS) by diluting 50x High pH Target Retrieval Solution in reagent-quality water and mixing. Prepare a sufficient amount of high pH 1x target retrieval solution by diluting Target Retrieval Solution, High pH (50x) 1:50 with distilled or deionized water (reagent grade). Diluting one 30 mL bottle of Target Retrieval Solution, High pH (50x) 1:50 will yield 1.5 L of 1x reagent, enough to fill one PT Link tank processing up to 24 slides per use. A TRS pH test should be performed before use to ensure the 1x TRS is within the reagent specification (pH 9.0 ± 0.2). Prepare DAB+ chromogen immediately before starting the AUTOSTAINER LINK 48™ run: Add 1 drop of liquid DAB+ chromogen per mL of substrate buffer. If using a 7.2mL bottle of DAB+ substrate buffer, add 9 drops of DAB+ chromogen. Although the label says 7.2mL, this is the usable volume and does not include the "dead volume" in the bottle (1.8mL). The color of the Liquid DAB+ Chromogen in the bottle may vary from clear to lavender brown. This will not affect the performance of this product. Dilute according to the guidelines above. Adding too much Liquid DAB+ Chromogen to the DAB+ Substrate Buffer will result in a weakened positive signal.

[0093] Sample preparation All specimens were fixed in neutral buffered formalin and embedded in paraffin blocks. FFPE blocks were sectioned at 4 μm, mounted on charged microscope slides, and placed at 58 (±2)°C for approximately 1 hour within 18 hours of sectioning, then cooled to room temperature (RT). Sections were kept at 2-8°C until use.

[0094] Staining procedure Deparaffinization / Hydrophilizaton / Target Retrieval Procedure:PT Link All specimens were processed using a PT Link (codes PT100 / PT101 and / or PT200) with a preheat and cool temperature set to 65°C. All specimens were then processed using a PT Link with a heating temperature set to 97°C for 20 minutes. Fill the PT Link tanks with 1.5 L per tank of Target Retrieval Solution, High pH, ​​1x Working Solution to cover the tissue sections. Preheat the target activation solution to 65°C. Immerse the Autostainer rack containing the mounted FFPE tissue sections into preheated Target Retrieval Solution, High pH (1x Working Solution) in the PT Link tank. Incubate at 97°C for 20 minutes. Once the target retrieval incubation period is complete and the temperature has cooled to 65°C, remove each Autostainer slide rack with slides from the PT Link tank and immediately place the Autostainer rack with slides into a tank (e.g., PT Link Rinse Station, code PT109) containing 1x, room temperature EnVision FLEX Wash Buffer (code K8007). · Immerse the slides in diluted room temperature wash buffer for 5 minutes.

[0095] After deparaffinization, hydrophilization, and target retrieval (3-in-1) procedures (specimen preparation), the Autostainer rack with slides was placed in the AUTOSTAINER LINK 48™. The AUTOSTAINER LINK 48™ instrument automatically performed the staining process using a pre-programmed protocol by applying the appropriate reagents, monitoring incubation times, and rinsing the slides between reagents. Stained slides for all indicators were scored by a qualified observer.

[0096] Data interpretation The following information was obtained during the observer reading: MAGE-A4 slides: Percent positive MAGE-A4 scores in each intensity bin (0-3+) Diagnostic results *Positive: ≥2+ staining intensity at MAGE-A4 TIPS ≥75% cutoff *Negative: ≥2+ staining intensity in MAGE-A4 TIPS <75% cutoff ·Average IHC staining intensity NCR Slide: Pass / Fail *Negative control reagent (NCR) stained slides must not exhibit specific staining. Nonspecific staining at ≤1+ staining intensity is acceptable. *Specific staining is defined as nuclear and / or cytoplasmic staining in tumor cells, and nonspecific staining is defined as staining of non-tumor tissue elements. If an NCR specimen exhibits specific staining at any intensity or nonspecific staining at >1+ intensity, do not proceed with evaluation of the MAGE-A4 stained slide. If applicable, comment

[0097] specimen Eighty specimens (MRCLS and / or SS, sqNSCLC) showing variable MAGE-A4 expression were selected for this study. A balanced sample size was ensured per MAGE-A4 TIPS with a staining intensity of ≥2+ at the ≥75% cutoff, with approximately 20-25% of specimens falling within the range (65-85%) close to the cutoff for staining intensity of ≥2+. Stained slides were selected from a previously performed screening run and followed the procedure described in the protocol.

[0098] Equipment for digital scoring A computer system with a Windows 7 or higher operating system. Per Aperio System requirements, the minimum computer monitor requirement is a 24-inch screen size, supporting 24-bit color depth and displaying a resolution of 1680(h) x 1050(v). The computer monitor's contrast and color management settings must be set to the default factory settings.

[0099] procedure Test Purpose The purpose of this study was to compare the slide diagnostic status and WSI diagnostic status by observers scoring the same set of slides on two platforms: bright-field light microscopy and a high-resolution computer monitor. Slide scanning was performed using the APERIO AT2 Scanner™.

[0100] Initial WSI Requirements Before scanning the slides, APERIO AT2™ operators were trained. Observers viewing the slides in ESLIDE MANAGER™ and IMAGESCOPE™ software were trained to navigate the WSI using the viewing software.

[0101] Scoring Guidelines Stained slides for all indicators were scored by a qualified observer.

[0102] WSI scanning procedure Before scanning the slides, they were inspected for dust and / or residue. If any residue was present, the operator cleaned the slide with a Kimwipe. Scanning of slides using an APERIO AT2 SCANNER™. The operator reviewed the WSI for complete tissue area coverage and image clarity. If the WSI did not appear satisfactory to the operator, the operator rescanned the associated slide before any scoring evaluation by the observer.

[0103] Blinding and randomization To avoid bias during scoring of stained sections, blinding and randomization of the stained sections were performed according to the CAP pathology guidelines (Pantanowitz et al., Arch. Pathol. Lab. Med. 137(12): 1710-1722). Slides were evaluated by each observer in a random order according to a randomization key. To record the results, a score sheet based on the randomized order of the slides was prepared, and qualified observers were instructed to read the slides only in the order in which they were presented. WSI reading was performed using a function in the APERIO IMAGESCOPE™ viewing software that blinded observers to the slide labels.

[0104] Scoring of slides and WSI Scoring of MAGE-A4 slides was performed by three qualified observers, both manually using a light microscope and digitally by viewing whole slide images (WSI) in APERIO IMAGESCOPE™ viewing software.

[0105] Consistency Test Procedure Three observers manually scored 80 blinded, randomized specimens using bright-field light microscopy on glass slides as Read 1. After a minimum 5-day washout, the same observers scored the WSI of the same set of specimens using APERIO IMAGESCOPE™ viewing software as Read 2, as shown in Figure 3. The slide and WSI readings could be performed in reverse order. Readings were separated into multiple scoring sessions as long as the minimum washout period between slide and whole slide image (WSI) readings for a given specimen was met.

[0106] Match analysis Because this study had a reference (results from slide scoring), negative agreement, positive agreement, and overall agreement (NPA, PPA, OA) were calculated by comparing observer- and specimen-matched WSI scores with slide scores, and two-sided 95% confidence intervals were calculated using the bootstrap method. The MAGE-A4 TIPS, with a cutoff of ≥2+ staining intensity ≥75% applied to slide scores, was used to define the reference for agreement analysis. It is known that bootstrap failure occurs when NPA, PPA, or OA is exactly equal to 100%, and therefore, Wilson score confidence intervals were calculated.

[0107] Acceptance Criteria The lower limit of the two-sided 95% confidence interval for NPA, PPA, or OA, respectively, must be at least 85% when all data from the three observers and three indicators are pooled. The acceptance criteria apply to MAGE-A4 TIPS at a staining intensity of ≥2 and a ≥75% cutoff. Additional analyses, including per-observer or per-indicator analyses, were not subject to the acceptance criteria.

[0108] result Data from all indicators were combined for analysis. For exploratory purposes, analyses were conducted for each indicator and are presented in Table 6.

[0109] JPEG2026505760000006.jpg118170

[0110] A continuous score plot was created to compare the MAGE-A4 TIPS scores at ≥2+ staining intensity ≥75% between the paired slide scores and their status for the WSI scores for each observer and index.

[0111] conclusion When slide and WSI comparisons from all three observers and all three indices were pooled, the lower limits of the two-sided 95% confidence intervals for NPA, PPA, and OA were at least 85%. Using the WSI and scoring systems specified in this report for SS, MRCLS, and squamous NSCLC slides stained with the SK032 MAGE-A4 IHC assay and interpreted in MAGE-A4 TIPS at a cutoff of ≥2+ staining intensity ≥75%, equivalence has been established between light microscopy and digital WSI scoring.

[0112] In addition to performing analyses on all indices pooled, analyses were performed at the per-indicator, per-observer, and per-indicator-per-observer levels for informational purposes only. Based on the per-indicator analyses, all indices showed high agreement in both the negative and positive sample pools, and therefore, none appear to highly bias the pooled indices results.

[0113] Several embodiments of the present invention have been described. However, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

1. (a) staining the tissue sample with an antibody that specifically binds to melanoma-associated antigen gene-A4 (MAGE-A4); (b) determining the total number of viable tumor or cancer cells with MAGE-A4 staining in at least a portion of the tissue sample, and determining the total number of stained and unstained viable tumor or cancer cells, wherein tumor or cancer cells are counted as positively stained with anti-MAGE-A4 antibody if there is cytoplasmic and / or nuclear MAGE-A4 staining at any intensity above a defined threshold; and (c) determining a MAGE-A4 tumor intensity ratio score (MAGE-A4 TIPS), wherein the MAGE-A4 TIPS is the number of MAGE-A4 stained viable tumor or cancer cells found in the tissue sample divided by the total number of stained and unstained viable tumor or cancer cells multiplied by 100. An immunohistochemistry (IHC) method for determining and scoring the degree of cellular expression of melanoma-associated antigen gene-A4 (MAGE-A4) in a tissue sample, comprising:

2. The specified threshold is 1+ positive staining intensity assessed at high magnification; 2+ positive staining intensity assessed at medium magnification; or 3+ positive staining intensity assessed at low magnification The method of claim 1 , comprising:

3. 3. The method of claim 2, wherein the low magnification is at least about 4x magnification; the medium magnification is at least about 10x magnification; and the high magnification is at least about 20x magnification or at least about 40x magnification.

4. 3. The method of claim 2, wherein the MAGE-A4 TIPS comprises the number of viable MAGE-A4 tumor or cancer cells that stain 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.

5. 3. The method of claim 2, wherein the MAGE-A4 TIPS comprises the number of viable MAGE-A4 tumor or cancer cells that stain with a positive staining intensity of 1+ or greater divided by the total number of stained and unstained viable tumor or cancer cells multiplied by 100.

6. The method of any one of claims 1 to 5, wherein about 70% or more of the MAGE-A4 TIPS indicate a positive diagnostic status of the tissue sample.

7. 7. The method of claim 6, wherein the MAGE-A4 TIPS is about 75% or more, about 80% or more, or about 90% or more.

8. The method of any one of claims 1 to 5, wherein about 5% or more of MAGE-A4 TIPS indicate a positive diagnostic status of the tissue sample.

9. The method of claim 8, wherein about 5% or more of the MAGE-A4 TIPS comprise a number of viable MAGE-A4 tumor or cancer cells that stain with a positive staining intensity of 1+ or greater; or about 10% or more of the MAGE-A4 TIPS comprise a number of viable MAGE-A4 tumor or cancer cells that stain with a positive staining intensity of 2+ or greater.

10. 10. The method of any one of claims 1 to 9, 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.

11. The method of any one of claims 1 to 10, wherein the antibody comprises a monoclonal mouse anti-MAGE-A4 antibody.

12. 12. The method of claim 11, wherein the monoclonal mouse anti-MAGE-A4 antibody comprises monoclonal mouse anti-MAGE-A4 clone OTI1F9, an antibody having affinity for MAGE-A4 substantially similar to the clone OTI1F9 antibody, monoclonal mouse anti-MAGE-A4 clone 57B, monoclonal mouse anti-MAGE-A4 clone 6C1, monoclonal mouse anti-MAGE-A4 clone CPTC-MAGEA4-1, monoclonal mouse anti-MAGE-A4 clone CPTC-MAGEA4-2, monoclonal mouse anti-MAGE-A4 clone 8BA20, monoclonal mouse anti-MAGE-A4 clone 8BA21, monoclonal mouse anti-MAGE-A4 clone CBFYM-1312, or monoclonal mouse anti-MAGE-A4 clone 9A7.

13. (a) the tissue sample comprises a formalin-fixed, paraffin-embedded (FFPE) specimen; (b) the FFPE specimen comprises a cancer specimen stained on an automated IHC platform; (c) the sections of the tissue sample are prepared by a protocol comprising fixation in about 10% neutral buffered formalin for between about 6 hours and about 72 hours; (d) the tumor or cancer is synovial sarcoma, myxoid / round cell liposarcoma, high-grade myxoid liposarcoma, low-grade myxoid liposarcoma, head and neck cancer, melanoma, esophageal cancer, gastric cancer, colorectal cancer, lung cancer, non-small cell lung cancer, colon cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, salivary gland cancer, prostate cancer, liver cancer, or urothelial cancer; (e) the tissue sample is derived from a needle biopsy sample, fine needle aspirate, cytology specimen, or bone demineralization; (f) positive staining is determined using bright field light microscopy, a microscope objective, a computer monitor and imaging software, or a combination thereof; and / or (g) the imaging software comprises whole slide imaging software; The method according to any one of claims 1 to 12.

14. Determining melanoma associated antigen gene-A4 (MAGE-A4) diagnostic status in a tissue sample by the method of any one of claims 1 to 13, wherein about 70% or more of the tumor or cancer cells have MAGE-A4 nuclear and / or cytoplasmic staining at an intensity of 2+ or higher and a MAGE-A4 Tumor Intensity Percentage Score (MAGE-A4 TIPS) is diagnostically positive.

1. A method for diagnosing tumors or cancer by determining whether a tissue sample is positive for expression of melanoma associated antigen gene-A4 (MAGE-A4), comprising:

15. 15. The method of claim 14, wherein the tumor or cancer is synovial sarcoma, myxoid / round cell liposarcoma, head and neck cancer, melanoma, esophageal cancer, gastric cancer, colorectal cancer, lung cancer, non-small cell lung cancer, colon cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, salivary gland cancer, prostate cancer, liver cancer, or urothelial cancer.

16. 16. A method for treating or ameliorating a tumor or cancer in a patient, comprising determining and scoring the amount of MAGE-A4 in a tissue sample from the patient using the method of any one of claims 1 to 15, wherein if the tissue sample is determined or scored as having a high or diagnostically positive MAGE-A4 score, then the patient is treated with a cancer therapy to which the patient is likely to respond favorably.

17. 17. The method of claim 16, wherein the tumor or cancer is synovial sarcoma, myxoid / round cell liposarcoma, head and neck cancer, melanoma, esophageal cancer, gastric cancer, colorectal cancer, lung cancer, non-small cell lung cancer, colon cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, salivary gland cancer, prostate cancer, liver cancer, or urothelial cancer.

18. 18. The method of claim 16 or 17, wherein the cancer therapy comprises administration of an anti-cancer drug or treatment to the patient, and optionally the anti-cancer treatment comprises immunotherapy, monoclonal antibody therapy, adoptive cell therapy (ACT), T cell receptor (TCR) therapy, or chimeric antigen receptor (CAR) T cell therapy.

19. A kit comprising an antibody that specifically binds to MAGE-A4 and MAGE-A4 scoring guidelines, as set forth in the method of any one of claims 1 to 18.

20. contacting a sample or a portion thereof containing cancer or tumor cells from an individual with an antibody or a portion thereof that specifically binds to MAGE-A4; and determining a MAGE-A4 Tumor Intensity Ratio Score (MAGE-A4 TIPS) by dividing the number of MAGE-A4 stained viable 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 viable cancer or tumor cells, and multiplying the result by 100, thereby obtaining MAGE-A4 TIPS. A method for assessing the degree of MAGE-A4 expression, comprising:

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