Immunohistochemical (IHC) MAGE-A4 scoring regimen and methods for assisting cancer therapy

Abstract

In alternative embodiments, there is provided an immunohistochemical (IHC) method for determining and reproducibly scoring the degree of expression of protein melanoma associated antigen gene-A4 (MAGE-A4) in a tissue sample. In alternative embodiments, methods are provided for diagnosing, treating, or ameliorating a cancer or tumor, or assessing the risk of recurrence of the cancer or tumor, using the IHC methods as provided herein. In alternative embodiments, kits are provided that include components and instructions for use in practicing the methods as provided herein. Described herein are methods for scoring MAGE-A4 expression and utilizing the score as a companion or supplemental diagnosis or treatment or amelioration of cancer or tumor.

Description

[0001] Related applications

[0002] This Patent Cooperation Treaty (PCT) international patent application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application Serial No. (USSN) 63 / 442,381, filed January 31, 2023. The entire contents of the above application are expressly incorporated herein by reference for all purposes. Technical Field

[0003] The present invention generally relates to cancer treatment, companion or supplemental diagnostics, and immunohistochemical methods. In an alternative embodiment, an immunohistochemical (IHC) method is provided for determining and reproducibly scoring the expression level of the protein Melanoma Associated Antigen Gene-A4 (MAGE-A4) in a tissue sample. In an alternative embodiment, a method is provided for diagnosing, treating, or ameliorating a cancer or tumor, or assessing the risk of recurrence of the cancer or tumor, using the IHC method as provided herein. In an alternative embodiment, a kit is provided, comprising components and instructions for practicing the method as provided herein. This application describes methods for scoring MAGE-A4 expression and using the score as a companion or supplemental diagnostic, therapeutic, or ameliorative method for cancer or tumors. Background Art

[0004] The melanoma-associated antigen gene-A4 (MAGE-A4) is located at chromosome position Xq28 and has been linked to several inherited disorders, such as dyskeratosis congenita. At least four variants of this gene have been found that encode the same protein.

[0005] MAGE-A4 is expressed in many cancers and tumors, for example, synovial sarcoma, myxoid / round cell liposarcoma, and lung cancer. Targeted therapy using autologous T cells genetically modified to target MAGE-A4 is currently under clinical trials.

[0006] Because tumor morphology in some cancers is heterogeneous, how to apply scoring guidelines can be confusing.There remains a need in the art for simpler and more efficient, as well as robust, reproducible, and accurate scoring methods to assess MAGE-A4 expression in tissue samples. Summary of the Invention

[0007] In an alternative embodiment, an immunohistochemistry (IHC) method is provided for determining and scoring the extent of cellular expression of melanoma associated antigen gene-A4 (MAGE-A4) in a tissue sample, the method comprising:

[0008] (a) Tissue samples were stained with antibodies that specifically bind to MAGE-A4.

[0009] (b) determining the total number of viable tumor or cancer cells that have MAGE-A4 staining, and determining the total number of stained and unstained viable tumor or cancer cells in at least a portion of the tissue sample,

[0010] wherein the tumor or cancer cells are counted as positively stained with the anti-MAGE-A4 antibody if there is any intensity of cytoplasmic and / or nuclear MAGE-A4 staining above a defined threshold; and

[0011] (c) determining the MAGE-A4 tumor intensity ratio score (MAGE-A4 TIPS),

[0012] 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, and then multiplied by 100.

[0013] In an alternative embodiment of the IHC method as provided herein:

[0014] - The defined thresholds include:

[0015] 1+ positive staining intensity assessed at high magnification;

[0016] 2+ positive staining intensity assessed at medium magnification; or

[0017] 3+ positive staining intensity assessed at low magnification;

[0018] - low magnification is at least about 4X magnification; medium magnification is at least about 10X magnification; and high magnification is at least about 20X magnification or at least about 40X magnification;

[0019] -MAGE-A4 TIPS consisted of the number of MAGE-A4 viable tumor or cancer cells stained with a positive staining intensity of 2+ or greater divided by the total number of stained and unstained viable tumor or cancer cells, multiplied by 100;

[0020] -MAGE-A4 TIPS consisted of the number of MAGE-A4 viable tumor or cancer cells stained 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;

[0021] - About 70% or greater of the MAGE-A4 TIPS indicate a positive diagnostic status for the tissue sample; alternatively, the MAGE-A4 TIPS are about 75% or greater, about 80% or greater, or about 90% or greater;

[0022] - about 5% or greater of the MAGE-A4 TIPS indicate a positive diagnostic status for the tissue sample; or, about 5% or greater of the MAGE-A4 TIPS include 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 greater of the MAGE-A4 TIPS include a number of MAGE-A4 viable tumor or cancer cells that stain with a positive staining intensity of 2+ or greater;

[0023] - preparing a section or portion of a tissue sample on a glass slide, microscope slide or equivalent and staining said section or portion of said tissue sample on said slide;

[0024] - the antibody comprises a monoclonal mouse anti-MAGE-A4 antibody, and optionally, the monoclonal mouse anti-MAGE-A4 antibody comprises:

[0025] - monoclonal mouse anti-MAGE-A4 clone OTI1F9 antibody (generated using human MAGE-A4 full-length human recombinant protein (NP_001011550) produced in HEK293T cells as an immunogen) (Agilent Technologies), or an antibody with substantially similar affinity for MAGE-A4;

[0026] - as the clone OTI1F9 antibody (Origene, Abcam), or an antibody with substantially similar affinity for MAGE-A4;

[0027] - monoclonal mouse anti-MAGE-A4 clone 57B antibody, or an antibody with substantially similar affinity for MAGE-A4;

[0028] - monoclonal mouse anti-MAGE-A4 clone 6C1 antibody, or an antibody with substantially similar affinity for MAGE-A4;

[0029] - monoclonal mouse anti-MAGE-A4 clone CPTC-MAGEA4-1 antibody (Developmental Studies Hybridoma Bank), or an antibody with substantially similar affinity for MAGE-A4;

[0030] - the monoclonal mouse anti-MAGE-A4 clone CPTC-MAGEA4-2 antibody, or an antibody with substantially similar affinity for MAGE-A4;

[0031] - Monoclonal mouse anti-MAGE-A4 clone 8BA20 antibody (Creative Biolabs), or an antibody with substantially similar affinity for MAGE-A4;

[0032] - Monoclonal mouse anti-MAGE-A4 clone 8BA21 antibody (Creative Biolabs), or an antibody with substantially similar affinity for MAGE-A4;

[0033] - monoclonal mouse anti-MAGE-A4 clone CBFYM-1312 antibody (Creative Biolabs), or an antibody with substantially similar affinity for MAGE-A4; or

[0034] - monoclonal mouse anti-MAGE-A4 clone 9A7 antibody (Creative Biolabs), or an antibody with substantially similar affinity for MAGE-A4; or

[0035] - any combination thereof;

[0036] - 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;

[0037] - preparing sections of tissue samples by a protocol comprising fixation in about 10% neutral buffered formalin for a period of about 6 hours to about 72 hours;

[0038] - 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 carcinoma;

[0039] - Tissue samples were obtained from needle biopsy specimens, fine needle aspirates, cytology specimens, or bone decalcification;

[0040] - positive staining is determined using a brightfield light microscope, a microscope objective, a computer monitor and imaging software, or a combination thereof; and / or

[0041] - Imaging software includes whole slide imaging software.

[0042] In an alternative embodiment, a method of diagnosing a tumor or cancer by determining whether a tissue sample is positive for expression of melanoma associated antigen gene-A4 (MAGE-A4) is provided, the method comprising:

[0043] MAGE-A4 positive diagnostic status in a tissue sample is determined by the methods as provided herein, wherein about 70% or greater of tumor cancer cells having MAGE-A4 TIPS with an intensity of 2+ or greater MAGE-A4 nuclear and / or cytoplasmic staining are diagnostically positive.

[0044] In alternative embodiments 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 carcinoma.

[0045] In alternative embodiments, methods are provided for treating or ameliorating a tumor or cancer in a patient, the methods 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 high or diagnostically positive MAGE-A4 TIPS, the patient is treated with a cancer therapy to which the patient is likely to respond favorably.

[0046] In an alternative embodiment of the method for treating or ameliorating a tumor or cancer in a patient:

[0047] - 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 carcinoma;

[0048] - the cancer treatment comprises administering an anti-cancer drug or anti-cancer therapy to the patient; and / or

[0049] - The anti-cancer therapy includes immunotherapy, monoclonal antibody therapy, adoptive cell therapy (ACT), T cell receptor (TCR) therapy or chimeric antigen receptor (CAR) T cell therapy.

[0050] In alternative embodiments, kits are provided, comprising an antibody that specifically binds to MAGE-A4 and a MAGE-A4 scoring guide as described in the methods provided herein or as described in the methods for diagnosing a tumor or cancer as provided herein.

[0051] In an alternative embodiment, a method for assessing the extent of MAGE-A4 expression is provided, the method comprising:

[0052] contacting a sample from the individual comprising cancer or tumor cells, or a portion thereof, with an antibody, or a portion thereof, that specifically binds MAGE-A4; and

[0053] The MAGE-A4 tumor intensity ratio score (MAGE-A4TIPS) is determined by dividing the number of viable tumor or cancer cells stained by MAGE-A4 specifically bound by the antibody in the specimen or a portion thereof by the total number of stained and unstained viable tumor or cancer cells and multiplying the result by 100 to obtain MAGE-A4 TIPS.

[0054] 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.

[0055] All publications, patents, and patent applications cited herein are expressly incorporated by reference in their entirety for all purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The patent or application file contains at least one drawing drawn in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0057] The drawings set forth herein are illustrative of exemplary embodiments provided herein and are not meant to limit the scope of the invention encompassed by the claims.

[0058] The accompanying drawings are described in detail herein.

[0059] Like reference symbols in the various drawings denote like elements. DETAILED DESCRIPTION

[0060] In an alternative 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 an alternative embodiment, a scoring method for assessing MAGE-A4 expression in a tumor or cancer is provided. In an alternative embodiment, a method for diagnosing and / or treating a tumor or cancer is provided, the method comprising using a scoring method as provided herein to assess MAGE-A4 expression and determining whether there is a high or diagnostically positive MAGE-A4 tumor intensity ratio score (MAGE-A4TIPS). In certain embodiments, if a tissue sample is determined or scored as having a high or diagnostically positive MAGE-A4TIPS, the patient is treated with a cancer treatment to which the patient may respond favorably.

[0061] Synovial sarcoma (SS) is a rare malignancy with a poor prognosis and low response rate to multiple classes of therapeutic agents. This necessitates the development of targeted biomarkers for patient selection for novel adoptive T cell therapies (ACTs), which have demonstrated clinical efficacy in SS. Melanoma-associated antigen gene-A4 (MAGE-A4), a cancer-testis antigen, is highly expressed in SS and correlates with advanced clinical stages. The restricted expression pattern of MAGE-A4 in normal tissues makes it an attractive target for the development of diagnostic assays and ACTs.

[0062] Embodiments herein are directed to immunohistochemical methods for determining and scoring the extent of cellular and / or cytoplasmic expression of MAGE-A4 in a tissue sample. In various embodiments, the methods comprise: staining the tissue sample with an antibody that specifically binds to MAGE-A4; determining the total number of viable tumor or cancer cells with nuclear and / or cytoplasmic staining of MAGE-A4, and determining the total number of stained and unstained viable tumor or cancer cells in at least a portion of the tissue sample, wherein a tumor or cancer cell is counted as positively stained with an anti-MAGE-A4 antibody if cytoplasmic and / or nuclear MAGE-A4 staining is present at any intensity above a defined threshold; and determining a MAGE-A4 Tumor Intensity Proportional Score (MAGE-A4 TIPS), wherein the MAGE-A4 TIPS is the number of viable tumor or cancer cells stained with MAGE-A4 present in the tissue sample divided by the total number of viable tumor or cancer cells stained and unstained, multiplied by 100. Such embodiments may not only provide a robust, reliable, reproducible, and accurate MAGE-A4 scoring method, but may also provide a scoring method with greater simplicity and efficiency. Such embodiments may provide considerable advantages for reliable and effective diagnostic assessments.

[0063] In certain embodiments, the tissue sample comprises at least about 50 viable tumor or cancer cells. In certain embodiments, the tissue sample comprises at least about 100 viable tumor or cancer cells. In certain embodiments, the tissue sample comprises at least about 200 viable tumor or cancer cells. In certain embodiments, the tissue sample comprises at least about 500 viable tumor or cancer cells.

[0064] In some embodiments, the count of positively stained tumor or cancer cells excludes staining of normal or non-neoplastic structures, staining of non-viable tumor or cancer cells, necrotic cells, cell debris, stained stroma, stained immune cells, stained benign cells, or edge artifact staining on the periphery of the tissue sample. In certain embodiments, the count of positively stained tumor or cancer cells excludes 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 stained cells with specific structures or characteristics from the MAGE-A4 TIPS determination.

[0065] In certain embodiments, a defined threshold comprises a 1+ positive staining intensity assessed at high magnification. In certain embodiments, a defined threshold comprises a 2+ or greater positive staining intensity assessed at medium magnification. In certain embodiments, a defined threshold comprises a 3+ positive staining intensity assessed at low magnification (see Figure 1 and Figure 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.

[0066] In certain embodiments, MAGE-A4 TIPS comprise the number of MAGE-A4 viable tumor or cancer cells stained with a positive staining intensity of 2+ or greater divided by the total number of stained and unstained viable tumor or cancer cells, multiplied by 100. In other embodiments, MAGE-A4 TIPS comprise the number of MAGE-A4 viable tumor or cancer cells stained 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. Such embodiments can provide the benefit of diversity in defining threshold staining intensities.

[0067] In certain embodiments, about 70% or greater of the MAGE-A4 TIPS indicate a positive diagnostic status for a tissue sample. In certain embodiments, the MAGE-A4 TIPS are about 75% or greater, about 80% or greater, or about 90% or greater. In certain embodiments, about 5% or greater of the MAGE-A4 TIPS indicate a positive diagnostic status for a tissue sample. In certain embodiments of MAGE-A4 TIPS, about 5% or greater of the MAGE-A4 TIPS include the number of MAGE-A4 viable tumor or cancer cells that stain with a positive staining intensity of 1+ or greater; or about 10% or greater of the MAGE-A4 TIPS include the number of MAGE-A4 viable tumor or cancer cells that stain with a positive staining intensity of 2+ or greater. Such embodiments may provide a benefit in the accuracy of diagnostic status determinations related to MAGE-A4 expression.

[0068] In alternative embodiments, the methods and kits as provided herein are used for in vitro diagnostic purposes. In alternative embodiments, MAGE-A4 IHC as provided herein is an immunohistochemistry (IHC) assay using an anti-MAGE-A4 antibody (e.g., monoclonal mouse anti-MAGE-A4 clone OTI1F9 antibody (generated using human MAGE-A4 full-length human recombinant protein (NP_001011550) produced in HEK293T cells as an immunogen) (Agilent Technologies) or an antibody with substantially similar affinity for MAGE-A4) in the detection of MAGE-A4 protein in FFPE tissue samples. In alternative embodiments, the methods and kits as provided herein are used for in vitro diagnostic purposes. In alternative embodiments, the MAGE-A4 IHC as provided herein is an immunohistochemistry (IHC) assay using an anti-MAGE-A4 antibody (e.g., monoclonal mouse anti-MAGE-A4 clone OTI1F9 antibody (generated using human MAGE-A4 full-length human recombinant protein (NP_001011550) produced in HEK293T cells as an immunogen) (Agilent Technologies) or an antibody with substantially similar affinity for MAGE-A4). TM Automated dyeing system.

[0069] In alternative embodiments, the MAGE-A4 IHC method as provided herein is used as an aid in identifying patients with a tumor or cancer (e.g., 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).

[0070] In an alternative embodiment, a method for assessing the extent of MAGE-A4 expression comprises: contacting a sample or portion thereof containing cancer or tumor cells from an individual with an antibody or portion thereof that specifically binds MAGE-A4; and determining a MAGE-A4 Tumor Intensity Ratio score (MAGE-A4 TIPS) by dividing the number of viable tumor or cancer cells stained with MAGE-A4 specifically bound by the antibody in the sample or portion thereof by the total number of viable stained and unstained tumor or cancer cells and multiplying the result by 100, thereby obtaining the MAGE-A4 TIPS.

[0071] In certain embodiments, sections or portions of tissue samples are prepared on slides, microscope slides, or the like, and the sections or portions of the tissue samples are stained on the slides. In certain embodiments, the tissue samples comprise FFPE specimens. In certain embodiments, the FFPE specimens comprise cancer specimens stained on automated IHC platforms. In certain embodiments, sections of tissue samples are prepared by a protocol comprising fixation in about 10% neutral buffered formalin for a period of about 6 hours to about 72 hours. In certain embodiments, the tissue samples are derived from needle biopsy specimens, fine needle aspirates, cytology specimens, or bone decalcification.

[0072] 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 comprises: a monoclonal mouse anti-MAGE-A4 clone OTI1F9 antibody or an antibody having a substantially similar affinity for MAGE-A4 as clone OTI1F9 antibody; a monoclonal mouse anti-MAGE-A4 clone 57B antibody or an antibody having a substantially similar affinity for MAGE-A4; a monoclonal mouse anti-MAGE-A4 clone 6C1 antibody or an antibody having a substantially similar affinity for MAGE-A4; a monoclonal mouse anti-MAGE-A4 clone CPTC-MAGEA4-1 antibody or an antibody having a substantially similar affinity for MAGE-A4; a monoclonal mouse anti-MAGE-A4 clone 57B antibody or an antibody having a substantially similar affinity for MAGE-A4; a monoclonal mouse anti-MAGE-A4 clone 6C1 antibody or an antibody having a substantially similar affinity for MAGE-A4; a monoclonal mouse anti-MAGE-A4 clone CPTC-MAGEA4-1 antibody or an antibody having a substantially similar affinity for MAGE-A4; The invention further comprises the following: a monoclonal mouse anti-MAGE-A4 clone 8BA20 antibody or an antibody with a substantially similar affinity for MAGE-A4; a monoclonal mouse anti-MAGE-A4 clone 8BA21 antibody or an antibody with a substantially similar affinity for MAGE-A4; a monoclonal mouse anti-MAGE-A4 clone CBFYM-1312 antibody or an antibody with a substantially similar affinity for MAGE-A4; or a monoclonal mouse anti-MAGE-A4 clone 9A7 antibody or an antibody with a substantially similar affinity for MAGE-A4; or any combination thereof.

[0073] 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 carcinoma.

[0074] 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.

[0075] Embodiments of methods for diagnosing tumors or cancer

[0076] Embodiments herein provide methods for diagnosing a tumor or cancer by determining whether a tissue sample is positive for cellular expression of MAGE-A4. In various aspects, the methods include determining the MAGE-A4 diagnostic status of a tissue sample using an IHC method for determining the extent of cytoplasmic and / or nuclear expression of MAGE-A4 as provided herein, wherein approximately 70% or greater of tumor cancer cells having MAGE-A4 nuclear and / or cytoplasmic staining of 2+ or greater intensity are diagnostically positive. 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.

[0077] Embodiments of methods for treating cancer and tumors

[0078] Provided are methods for treating or ameliorating a tumor or cancer in a patient, the methods 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 treatment or anti-cancer therapy to which the patient is likely to respond favorably. The tumor or cancer can 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.

[0079] In certain embodiments, the cancer treatment comprises administering an anti-cancer drug or anti-cancer therapy to the patient. In alternative embodiments, the anti-cancer treatment or therapy comprises surgery, such as CyberKnife therapy; chemoembolization; ablative techniques, such as radiofrequency ablation (RFA), cryoablation, and / or microwave ablation; and / or radiation therapy, such as stereotactic body radiation therapy.

[0080] In certain embodiments, the anticancer therapy comprises an antibody drug conjugate, a small molecule therapy, an immunotherapy, a monoclonal antibody therapy, adoptive cell therapy (ACT), a T cell receptor (TCR) therapy, or a chimeric antigen receptor (CAR) T cell therapy. In alternative embodiments, the anticancer treatment or therapy comprises: a tyrosine kinase inhibitor (optionally erlotinib (or TARCEVA TM ), gefitinib (or IRESSA TM ), afatinib (or GILOTRIF TM ), or osimertinib (TAGRISSO TM )); Nexituzumab (or PORTRAZZA TM ), pembrolizumab (or KEYTRUDA TM ), nivolumab (or OPDIVO TM ), ipilimumab (YERVOY TM ), Cetuximab (or ERBITUX TM ), cisplatin (or PLATINOL TM ) or carboplatin (or paraplatin TM ); gemcitabine (or GEMZAR TM ), docetaxel (or TAXOTERE TM ), Olaratumab (or LARTRUVO TM ), doxorubicin (or ADRIAMYCIN TM or RUBEX TM ), pazopanib (or VOTRIENT TM ), ifosfamide (or IFEX TM ), Trabectedin (or YONDELIS TM ).

[0081] In alternative embodiments, the anti-cancer treatment or therapy comprises the use of an anti-cancer drug, which may comprise an antibody that specifically or substantially binds to the cancer or tumor, wherein the antibody is conjugated to a cytotoxic agent, and optionally the cytotoxic agent comprises a radionuclide (optionally yttrium-90, iodine-131, lutetium-177, radium-223 chloride, strontium-89 chloride, or samarium-153EDTMP), diphtheria toxin, Pseudomonas aeruginosa exotoxin A, denileukin, moxetumomabpasudotox, calicheamicin or N-acetyl-γ-calicheamicin, emtansine or DM1, maytansine or a derivative thereof, SN-38 (or 7-ethyl-10-hydroxycamptothecin), or auristatin or monomethylauristatin E (MMAE).

[0082] Immunohistochemistry

[0083] In alternative embodiments, immunohistochemistry methods and / or reagents for use with the methods and articles of manufacture or kits as provided herein can include or comprise the use of any IHC protocol, IHC medical device, apparatus, and / or image or data analysis system for practicing IHC or IHC reagents known in the art.

[0084] In alternative embodiments, the antibodies, antigen-binding fragments thereof, or monomeric or dimeric antigen-binding proteins as provided herein (including, for example, synthetic or recombinant forms) used in the IHC protocols or kits as provided herein are substantially purified or isolated, or are in the form of unpurified or partially purified culture supernatants.

[0085] In alternative embodiments, the methods as provided herein can use or comprise reagents for detecting or visualizing antibody-antigen interactions using any product or method known in the art (eg, IHC protocols or reagents).

[0086] In alternative embodiments, the methods as provided herein comprise the use of chromogenic immunohistochemistry (CIH), wherein a primary antibody (e.g., a recombinant antibody (Ab) or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein as provided herein) or a secondary antibody (e.g., wherein the secondary antibody binds to a primary antibody as provided herein, or a recombinant antibody (Ab) or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein) is conjugated to an enzyme that can catalyze a reaction that produces a color, such as a peroxidase (e.g., an immunoperoxidase), such as horseradish peroxidase (HRP). In alternative embodiments, the chromogenic moiety used in the methods as provided herein is or comprises coumarin; rhodamine; 2,3,6,7-tetrahydro-11-oxo-1H,5H,11H-[1]benzopyrano[6,7,8-ij]quinoline-1-O-carboxylic acid; 7-(diethylamino)coumarin-3-carboxylic acid; a coumarin derivative; a rhodamine derivative; tetramethylrhodamine; a diarylrhodamine derivative; QSY 7; QSY 9; QSY 21; a diazo chromophore; DABSYL; tartrazine; a triarylmethane compound; Fast Red; Fast Blue; Basic Fuchsin; Cascade Blue Acetyl; Dapoxyl Sulfonic Acid / Carboxylic Acid Succinimidyl Ester; DY-405; Alexa Fluor 405 succinimidyl ester; Cascade Yellow succinimidyl ester; Pyridyl oxazole succinimidyl ester (PyMPO); Pacific Blue succinimidyl ester; DY-415; 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester; DYQ-425; 6-FAM phosphoramidite; Fluorescein 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; AlexaFluor 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 / 591C3 succinimidyl ester; DY-591; BODIPY TR-X succinimidyl ester; Alexa Fluor 594 succinimidyl ester; DY-594; carboxynaphthofluorescein succinimidyl ester; DY-605; DY-610; Alexa Fluor 610 succinimidyl ester; DY-615; BODIPY 630 / 650-X succinimidyl ester; Woolpopin; 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; AlexaFluor 660 succinimidyl ester; Cy5.5 succinimidyl ester; DY-677; DY-675; DY-676; DY-678; AlexaFluor 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); gallic blue; Fast Green FCF; malachite green; or QSY 21 succinimidyl ester.

[0087] In alternative embodiments, the methods as provided herein comprise the use of immunofluorescence, wherein the primary or secondary antibody is labeled with a fluorophore (e.g., fluorescein or fluorescein isothiocyanate (FITC)), a triarylmethane dye, such as rhodamine or a rhodamine derivative (e.g., tetramethylrhodamine (TRITC), rhodamine 6G, rhodamine 123, rhodamine B, carboxytetramethylrhodamine (TAMRA), tetramethylrhodamine (TMR), sulforhodamine 101)), aminomethylcoumarin acetate (AMCA), Alexa Fluor®, TM or Dylight TM Fluorescent dyes, or fluorophores or dyes. 3,3'-Diaminobenzidine (DAB) may also be used.

[0088] In alternative embodiments, methods as provided herein include the use of a direct method or a one-step staining method in which, for example, a primary antibody (e.g., an antibody (Ab) or antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein (including, for example, a synthetic or recombinant form) as provided herein) is labeled and reacts directly with the antigen. Although this technique utilizes only one antibody and is therefore simple and rapid, the sensitivity may be low due to minimal signal amplification.

[0089] In an alternative embodiment, the method as provided herein includes the use of an indirect method, wherein an unlabeled primary antibody (first layer) binds to a target antigen (e.g., MAGE-A4 protein) in, for example, a tissue or organ, and then a labeled secondary antibody (second layer) reacts with the primary antibody. The secondary antibody can be directed against an isotype, for example, IgG of the animal species from which the primary antibody is derived. This method may be more sensitive than a direct detection strategy because, if several secondary antibodies are conjugated to a detection reagent, such as a fluorescent or enzyme reporter, the signal resulting from the binding of the secondary antibody to each primary antibody is amplified.

[0090] In an alternative embodiment, if the secondary antibody is conjugated to several detection molecules, for example a biotin molecule, said biotin molecule can recruit avidin-, streptavidin- or NeutrAvidin TM The protein-binding enzyme complex then achieves further amplification.

[0091] In an alternative embodiment, IHC is performed on tissue sections or tissue biopsy sections (e.g., paraformaldehyde (PFA) fixed tissue or organ, or FFPE tissue). In an alternative embodiment, the tissue is sliced ​​or used as a whole. Before slicing, the tissue sample can be embedded in a medium, such as paraffin or freezing medium. Tissue sections can be sliced ​​on a variety of instruments, most commonly microtomes, cryostat microtomes or vibrating microtomes. The specimen can be sliced ​​in the range of about 3 μm to 5 μm. The sections can be mounted on slides, dehydrated using increasing concentrations of alcohol washes (e.g., 50%, 75%, 90%, 95%, 100%), and cleaned with detergents such as xylene, and then imaged or observed under a microscope.

[0092] Depending on the method of fixation and tissue preservation, samples may require additional steps to make the MAGE-A4 epitope available for antibody binding, including deparaffinization and antigen retrieval. For formalin-fixed, paraffin-embedded tissue, antigen retrieval is often necessary and may include pre-treatment of sections with heat or proteases.

[0093] In an alternative embodiment, the EnVision DuoFLEX Doublestain System is used. TM IHC is performed using the EnVision DuoFLEX Dual Staining System (Agilent, San Jose, CA), which allows staining of two or more markers on a single slide. In an alternative embodiment, IHC is performed using the EnVision FLEX HRP Magenta High pH (DakoOmnis) system, and binding can be visualized using the EnVision FLEX HRP Magenta chromogen. In an alternative embodiment, IHC is performed using the EnVision FLEX Micro Kit, High pH, ​​which is a high-sensitivity visualization system designed for use with the Dako Autostainer TM The instruments are used together in IHC; this dual-link system detects mouse and rabbit primary antibodies, and the reactions are visualized by 3,3'-diaminobenzidine (DAB) chromogen (DAB forms a water-insoluble brown precipitate when oxidized, for example, by peroxidase).

[0094] Manufacturing products and kits

[0095] Provided are articles of manufacture and kits for practicing the methods provided herein, comprising, 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 as an immunogen) (Agilent Technologies), or an antibody with substantially similar affinity for MAGE-A4, and / or reagents for practicing IHC, including, e.g., reagents as described herein, see Example 1 below; and optionally, the articles of manufacture and kits can further include instructions for practicing the methods described herein.

[0096] Any of the above aspects and embodiments may be combined with any other aspects or embodiments disclosed herein in the Summary, Drawings, and / or Detailed Description sections.

[0097] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0098] Unless otherwise stated or obvious from the context, as used herein, the term "or" is understood to be inclusive and encompasses both "or" and "and."

[0099] Unless otherwise specified or apparent from the context, as used herein, the term "about" is understood to be within the normal tolerance range of the art, for example, within 2 standard deviations of the mean. About (the use of the term "about") can be understood to be within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clearly stated from the context, all numerical values ​​provided herein are modified by the term "about."

[0100] Unless specifically stated or obvious from the context, as used herein, the terms "substantially all," "substantially a majority," "substantially all," or "majority" encompass at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or more of a reference amount of a composition.

[0101] Each patent, patent application, publication, and document cited herein is hereby incorporated by reference in its entirety. The citation of any patent, patent application, publication, or document does not constitute an admission that any of the aforementioned is relevant prior art, nor does it constitute any admission as to the contents or date of such publication or document. The individual citation of these documents should not be construed as an assertion or admission that any portion of the contents of any document is considered essential material to satisfy the statutory disclosure requirements for patent applications in any country or region. Nevertheless, the right to rely on any such document, where appropriate, to provide material deemed essential to the claimed subject matter by an examining authority or court of law.

[0102] Without departing from the basic aspects of the present invention, the foregoing may be modified. Although the present invention has been described in considerable detail with reference to one or more specific embodiments, it will be appreciated by those skilled in the art that changes may be made to the embodiments specifically disclosed in this application, while these modifications and improvements remain within the scope and spirit of the present invention. The present invention illustratively described herein may be appropriately practiced in the absence of any elements not specifically disclosed herein. Thus, for example, in each instance herein, any one of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced by any one of the other two terms. Therefore, the terms and expressions that have been used are used as terms of description rather than limitation, do not exclude equivalents of the features shown and described, or portions thereof, and it should be appreciated that various modifications are possible within the scope of the present invention. Embodiments of the present invention are set forth in the following claims.

[0103] The present invention will be further described with reference to the examples described herein; however, it will be understood that the invention is not limited to such examples.

[0104] Example

[0105] Unless otherwise indicated in the examples, all recombinant DNA techniques were performed according to standard protocols, for example as described in Sambrook et al., (2012) Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, NY, and in Ausubel et al., (1994) Current Protocols in Molecular Biology, Current Protocols, USA, Volumes 1 and 2. Other references to standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, NY; Brown (1998) Molecular Biology LabFax, 2nd ed., Academic Press (UK), Volumes I and II.

[0106] Example 1: Exemplary IHC protocol for detecting MAGE-A4 protein

[0107] This example describes an exemplary IHC method as provided herein and demonstrates the efficacy of the MAGE-A4 detection IHC protocol as provided herein.

[0108] The results of the activities performed are presented as required to establish the Autostainer LINK 48 TM Inter-observer and intra-observer scoring accuracy was determined using MAGE-A4 IHC (SK032) on FFPE synovial sarcoma (SS), myxoid round cell liposarcoma (MRCLS), and squamous cell non-small cell lung cancer (sqNSCLC) specimens stained on the Specimens Specimens were evaluated using the MAGE-A4 IHC scoring guide.

[0109] The MAGE-A4 IHC scoring guide was used to test the inter- and intraobserver accuracy of SS, MRCLS, and sqNSCLC specimens. Specimens were stained using the MAGE-A4 IHC assay. The results reported here demonstrate the reproducibility of the assay's scoring when multiple observers each scored stained SS, MRCLS, and sqNSCLC specimens multiple times.

[0110] Reagents

[0111] MAGE-A4 IHC (SK032) contains the PT Link Pre-Treatment Module TM (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 TM Complete the optimized reagents and protocols required for the IHC staining procedure for FFPE samples. First, the sample is incubated with a peroxidase blocking agent. After the sample is incubated with a monoclonal primary antibody of MAGE-A4 or a negative control reagent (NCR), the sample is incubated with the primary antibody and then incubated with a ready-to-use (RTU) visualization reagent, which consists of a secondary antibody molecule and a horseradish peroxidase molecule coupled to a dextran polymer backbone. The enzymatic conversion of the subsequently added chromogen causes a visible reaction product to precipitate at the antigenic site. The sample can then be counterstained and coverslipped. Use a bright field microscope to interpret the results.

[0112] Table 1: Assay components for an exemplary MAGE-A4 detection IHC protocol

[0113]

[0114] EnVision FLEX Hematoxylin (Dako K8008)

[0115] ●Automatic coverslipper or ClearMount (American MasterTech Scientific, MMCLE126) or equivalent mounting medium

[0116] ● Alcohol reagent, ethanol: anhydrous (Dako 100188) and 95%

[0117] Xylene (Dako 100941) or a xylene substitute, namely Histoclear (Dako 100791)

[0118] EnVision FLEX Wash Buffer 20x (Dako K8007)

[0119] Distilled or deionized water (reagent grade water), or Dako reagent water (Dako PD00004)

[0120] Reagent preparation

[0121] ●Equilibrate the kit reagents, buffer, and unstained slides to room temperature (20-25°C).

[0122] • Prepare 1x Wash Buffer by diluting 20x Wash Buffer in distilled or deionized water (reagent grade) and mixing.

[0123] • If the wash buffer is cloudy in appearance, discard the wash buffer.

[0124] • Prepare 1x high pH Target Repair Solution (TRS) by diluting 50x high pH Target Repair Solution in reagent grade water and mixing.

[0125] ■ Prepare a sufficient volume of High pH 1x Target Retrieval Solution by diluting High pH Target Retrieval Solution (50x) 1:50 with distilled or deionized water (reagent grade). A 30 mL bottle of High pH Target Retrieval Solution (50x) diluted 1:50 provides 1.5 L of 1x reagent, which is sufficient to fill one PT Link reservoir, which can process up to 24 slides per use. A TRS pH test should be performed before use to ensure that the 1X TRS is within reagent specifications (pH 9.0 ± 0.2).

[0126] ■Starting with Autostainer LINK 48 TM Prepare DAB + Chromogen shortly before the run. Add 1 drop of liquid DAB + Chromogen per 1 mL of substrate buffer.

[0127] ■ If using a 7.2 mL bottle of DAB + substrate buffer, add 9 drops of DAB + chromogen. Although the label states 7.2 mL, this is the usable volume and does not account for the "dead volume" (1.8 mL) in the bottle.

[0128] 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. Follow the dilution guidelines above. Adding excess liquid DAB+ chromogen to the DAB+ substrate buffer will result in a decreased positive signal.

[0129] Sample preparation

[0130] All specimens were fixed in neutral buffered formalin and embedded in paraffin blocks. FFPE blocks were cut into 4 μm sections and mounted on Dako FLEX TM The slides were placed on IHC microscope slides and placed at 58 (± 2) °C for approximately 1 hour within 18 hours of sectioning, then cooled to room temperature (RT). The sections were stored at 2-8 °C until use.

[0131] Dyeing process

[0132] Dewaxing / Rehydration / Target Repair Process: PT Link

[0133] All samples were processed using the PT Link with the preheating and cooling temperature set at 65° C. All samples were then processed using the PT Link heating temperature set at 97° C. for 20 minutes.

[0134] • Fill the PT Link reservoirs with 1.5 L / reservoir of Target Retrieval Solution, high pH 1x working solution, to cover the tissue sections.

[0135] Preheat 1x Target Retrieval Solution to 65°C.

[0136] ● Place the Autostainer LINK 48 containing the mounted FFPE tissue sections TM Immerse the rack in preheated high pH target retrieval solution (1x working solution) in the PT Link reservoir and incubate at 97°C for 20 minutes.

[0137] Once the target retrieval incubation time has ended and the temperature has cooled to 65°C, remove each Autostainer LINK 48 with slides from the PT Link reservoir. TM Slide rack and immediately place the Autostainer LINK 48 with slides TM Place the rack into a tank (e.g., PT Link rinse station, code PT109) containing 1 x room temperature EnVision FLEX TM Wash buffer (code K8007).

[0138] ● Immerse the slides in diluted room temperature 1x wash buffer for 5 minutes

[0139] After the dewaxing, rehydration and target retrieval (3 in 1) steps (specimen pretreatment), the Autostainer rack with the slides was placed in the AUTOSTAINER LINK 48 TM AUTOSTAINER LINK 48 TM The instrument automates the staining process using pre-programmed protocols by applying the appropriate reagents, monitoring incubation times, and washing slides between reagents.

[0140] Data interpretation

[0141] The following information is captured during the observer read:

[0142] MAGE-A4 slides: Percent positive MAGE-A4 scores for each intensity bin (0 to 3+)

[0143] Diagnostic outcome

[0144] Positive: ≥2+ staining intensity of MAGE-A4 TIPS ≥75% cut-off value

[0145] Negative: ≥2+ staining intensity of MAGE-A4 TIPS <75% cutoff value

[0146] Average IHC staining intensity

[0147] NCR slides: pass / fail

[0148] Slides stained with the negative control reagent (NCR) must not show specific staining. Nonspecific staining with a staining intensity of ≤1+ is acceptable.

[0149] 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 NCR specimens show any intensity of specific staining, or >1+ intensity of nonspecific staining, do not proceed with evaluation of MAGE-A4 stained slides.

[0150] ●Comments (if applicable)

[0151] Scoring Guide

[0152] All stained slides for the indications were scored by qualified observers

[0153] Sample

[0154] Eighty specimens (sqNSCLC, MRCLS, and / or SS) exhibiting a range of MAGE-A4 expression were selected for the study. Efforts were made to ensure a balanced positive / negative sample set at a cutoff of approximately MAGE-A4 TIPS ≥2+ staining intensity ≥75%, with approximately 20-25% of the samples falling within the range close to the cutoff (65-85%). Stained slides were selected from a previously performed screening round and followed the methods described herein.

[0155] Figure 1 A synovial sarcoma tissue sample (block ID: SB00046226) stained with MAGE-A4 IHC (SK032) is shown. Arrows indicate 1+, 2+, or 3+ staining intensity observed at 20X magnification.

[0156] Figure 2 A squamous non-small cell lung cancer (Case ID: 1408330B) tissue sample stained with MAGE-A4 IHC (SK032) is shown. Arrows indicate 1+, 2+, or 3+ staining intensity observed at 10X magnification. The data and biological samples used in this project were provided by Contract Research Ltd (Charlestown, Nevis) with appropriate ethical approval and through Azenta Life Sciences.

[0157] Process

[0158] Eighty stained specimens were evaluated by three observers (interobserver) and scored three times by each observer (intraobserver). There was a washout period of at least 14 days between each observer reading to minimize score recall bias.

[0159] All slides were blinded and randomized for all reads. Circle labels were applied to each slide according to a randomization scheme generated in Excel using the RAND function. Each intraobserver read had a different randomization scheme. The observers who scored the study were not involved in the blinding and randomization of the slides.

[0160] Testing Objections and Processes

[0161] The purpose of this test is to determine the accuracy of ratings between multiple observers.

[0162] Each of the three observers was provided with a set of blinded and randomized stained slides, and each observer read three times. This resulted in nine scores for each case. The scores for each sample were analyzed to determine the level of diagnostic consistency across multiple observers across multiple readings. The same set of data was used for both inter-observer and intra-observer analyses.

[0163] Statistical analysis plan

[0164] Calculation of percent agreement (NPA, PPA, and OA)

[0165] A percent agreement calculation was designed to compare each reading by each observer to a consensus, defined as the most common diagnostic outcome observed.

[0166] Confidence interval for percent agreement

[0167] Two-sided 95% confidence intervals for NPA, PPA, and OA were calculated using bootstrapping, a well-known computational technique that uses random selection to create a new dataset from the entire original dataset so that statistical uncertainty can be calculated to create statistical confidence intervals. Bootstrapping is known to fail in cases where the NPA, PPA, or OA is exactly equal to 100%, so Wilson score confidence intervals were calculated.

[0168] Acceptance criteria

[0169] The lower limit of the two-sided 95% confidence interval calculated for each agreement parameter (NPA, PPA, and OA) must be ≥ 85%.Any additional analyses performed were not subject to the acceptance criteria.

[0170] Intraobserver accuracy

[0171] Test Objectives

[0172] The purpose of such a test is to determine the scoring accuracy of an individual observer across multiple reads of the same sample.

[0173] Testing process

[0174] Each of the three observers was sequentially provided with a set of blinded and randomized stained slides, and each observer read them three times. This resulted in nine scores for each case. There was a washout period of at least 14 days between each intra-observer reading. The scores for each specimen were analyzed to determine the level of diagnostic agreement between the multiple readings of the individual observers. It should be noted that the same set of data was used for both the inter-observer analysis and the intra-observer analysis.

[0175] Statistical analysis plan

[0176] Percent agreement calculations were designed to compare each reading by each observer to the consensus. Consensus was defined as the most common diagnostic outcome 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 create 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. Bootstrapping is known to fail in cases where NPA, PPA, or OA is exactly equal to 100%, so Wilson score confidence intervals were calculated.

[0177] Acceptance criteria

[0178] A study is accepted once all substudies have been completed, discrepancies (if any) have been addressed and resolved, and the final report has been approved.

[0179] result

[0180] Data from all indications were combined for analysis and are presented in Tables 2 and 3. Additional analyses were performed for each indication for informational purposes.

[0181] Table 2: Summary of interobserver results

[0182]

[0183]

[0184] * If 100% agreement is observed, the bootstrap method cannot calculate confidence limits. Wilson score limits are used to calculate confidence intervals for parameters where the point estimate of agreement equals 100%.

[0185] Table 3: Summary of Intraobserver Results

[0186]

[0187] * If 100% agreement is observed, the bootstrap method cannot calculate confidence limits. Wilson score limits are used to calculate confidence intervals for parameters where the point estimate of agreement equals 100%.

[0188] in conclusion

[0189] Analyses of intra- and interobserver accuracy demonstrated that the lower limits of the two-sided 95% confidence intervals for NPA, PPA, and OA were at least 85% for both intra- and interobserver accuracy. Therefore, both intra- and interobserver accuracy met the acceptance criteria statistically. For MAGE-A4 TIPS, NPA, PPA, and OA were calculated using a 75% cutoff at ≥2+ staining intensity. When using the scoring system specified in this report, inter- and intraobserver accuracy was established between and within individual observers across multiple reads of the same sample for SS, MRCLS, and sqNSCLC slides stained with the SK032 MAGE-A4 IHC assay. Additional analyses performed on individual indication data were not subject to the acceptance criteria.

[0190] Each of the three individual indications showed high agreement (point estimates >93%), indicating that no indication favorably biased the results.

[0191] Example 2: Exemplary IHC protocol for detecting MAGE-A4 protein

[0192] This example describes an exemplary IHC method as provided herein and demonstrates the efficacy of the MAGE-A4 detection IHC protocol as provided herein.

[0193] This study was performed in an Autostainer LINK 48 TM WSI was performed on FFPE myxoid / round cell liposarcoma (MRCLS), synovial sarcoma (SS), and non-small cell lung cancer (sqNSCLC) squamous cell carcinoma specimens stained with MAGE-A4 IHC using the EnVision FLEX visualization system. WSI scoring was performed by qualified observers using Aperio and ImageScope software.

[0194] Reagents

[0195] SK032 MAGE-A4 IHC Contains Complete Autostainer LINK 48 TM and PT Link Pre-treatment Module TM The optimized reagents and protocols required for the IHC staining procedure (https: / / www.agilent.com / en / product / pt-link-for-pre-treatment / pt-link-accessories / pt-link-pre-treatment-module-for-tissue-specimens-76929) are provided. After incubation of the specimen with the primary antibody against MAGE-A4 or the negative control reagent (NCR), the specimen is incubated with a ready-to-use visualization reagent consisting of a secondary antibody molecule and a horseradish peroxidase (HRP) molecule conjugated to a dextran polymer backbone. Enzymatic conversion of the subsequently added chromogen results in the precipitation of a visible reaction product at the antigenic site. The specimen is then counterstained and coverslipped. Results are interpreted using a light microscope.

[0196] Table 4: Assay components for an exemplary MAGE-A4 detection IHC protocol

[0197]

[0198] EnVision FLEX Hematoxylin (Dako K8008)

[0199] ●Automatic coverslipper or ClearMount (American MasterTech Scientific, MMCLE126) or equivalent mounting medium

[0200] ● Alcohol reagent, ethanol: anhydrous (Dako 100188) and 95%

[0201] Xylene (Dako 100941) or a xylene substitute, namely Histoclear (Dako 100791)

[0202] EnVision FLEX Wash Buffer 20x (Dako K8007)

[0203] Distilled or deionized water (reagent grade water), or Dako reagent water (Dako PD00004)

[0204] Reagent preparation

[0205] ●Equilibrate the kit reagents, buffer, and unstained slides to room temperature (20-25°C).

[0206] • Prepare 1x Wash Buffer by diluting 20x Wash Buffer in distilled or deionized water (reagent grade) and mixing.

[0207] • If the wash buffer is cloudy in appearance, discard the wash buffer.

[0208] • Prepare 1x High pH Target Repair Solution (TRS) by diluting 50x High pH Target Repair Solution in reagent quality water and mixing.

[0209] ■ Prepare a sufficient volume of High pH 1x Target Retrieval Solution by diluting High pH Target Retrieval Solution (50x) 1:50 with distilled or deionized water (reagent grade). A 30 mL bottle of High pH Target Retrieval Solution (50x) diluted 1:50 provides 1.5 L of 1x reagent, which is sufficient to fill one PT Link reservoir, which can process up to 24 slides per use. A TRS pH test should be performed before use to ensure that the 1X TRS is within reagent specifications (pH 9.0 ± 0.2).

[0210] ●At the start of AUTOSTAINER LINK 48 TM Prepare DAB + Chromogen shortly before the run. Add 1 drop of liquid DAB + Chromogen per 1 mL of substrate buffer.

[0211] ■ If using a 7.2 mL bottle of DAB + substrate buffer, add 9 drops of DAB + chromogen. Although the label states 7.2 mL, this is the usable volume and does not account for the "dead volume" (1.8 mL) in the bottle.

[0212] 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. Follow the dilution guidelines above. Adding excess liquid DAB+ chromogen to the DAB+ substrate buffer will result in a decreased positive signal.

[0213] Sample preparation

[0214] All specimens were fixed in neutral buffered formalin and embedded in paraffin blocks. FFPE blocks were cut into 4 μm sections, mounted on pre-filled 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 stored at 2-8°C until use.

[0215] Dyeing process

[0216] Dewaxing / Rehydration / Target Repair Process: PT Link

[0217] All samples were processed using a PT Link (code PT100 / PT101 and / or PT200) with preheating and cooling set to 65° C. All samples were then processed using a PT Link heating temperature set to 97° C. for 20 minutes.

[0218] • Fill the PT Link reservoirs with 1.5 L / reservoir of Target Retrieval Solution, high pH 1x working solution, to cover the tissue sections.

[0219] ●Preheat the target repair solution to 65°C.

[0220] Immerse the autostainer rack containing the mounted FFPE tissue sections into the preheated high pH target retrieval solution (1x working solution) in the PT Link reservoir. Incubate at 97°C for 20 minutes.

[0221] ●Once the target retrieval incubation time has concluded and the temperature has cooled to 65°C, remove each autostainer slide rack with slides from the PT Link reservoir and immediately place the autostainer rack with slides into a reservoir (e.g., PT Link Wash Station, code PT109) filled with 1x room temperature EnVision FLEX Wash Buffer (code K8007).

[0222] • Immerse slides in diluted room temperature wash buffer for 5 minutes.

[0223] After the dewaxing, rehydration and target retrieval (3 in 1) steps (specimen pretreatment), the Autostainer rack with the slides was placed in the AUTOSTAINER LINK 48 TM AUTOSTAINER LINK 48 TM The instrument automates the staining process using pre-programmed protocols by applying the appropriate reagents, monitoring incubation times, and washing slides between reagents.

[0224] All stained slides for the indications were scored by qualified observers

[0225] Data interpretation

[0226] The following information is captured during the observer read:

[0227] MAGE-A4 slides: Percent positive MAGE-A4 scores for each intensity bin (0 to 3+)

[0228] Diagnostic outcome

[0229] Positive: ≥2+ staining intensity of MAGE-A4 TIPS ≥75% cut-off value

[0230] Negative: ≥2+ staining intensity of MAGE-A4 TIPS <75% cutoff value

[0231] Average IHC staining intensity

[0232] NCR slides: pass / fail

[0233] Slides stained with the negative control reagent (NCR) must not show specific staining. Nonspecific staining with a staining intensity of ≤1+ is acceptable.

[0234] 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 NCR specimens show any intensity of specific staining, or >1+ intensity of nonspecific staining, do not proceed with evaluation of MAGE-A4 stained slides.

[0235] ●Comments (if applicable)

[0236] Sample

[0237] Eighty specimens (MRCLs and / or SS) exhibiting a range of MAGE-A4 expression were selected for the study. Efforts were made to ensure a balanced positive / negative sample ratio, with a MAGE-A4 TIPS staining intensity of ≥2+ at a cutoff of approximately ≥75%, and approximately 20-25% of the samples falling within the cutoff range (65-85%) with a staining intensity of 2+ or greater. Stained slides were selected from previously performed screening rounds and followed the methods described in the protocol.

[0238] Equipment for digital scoring

[0239] A computer system with Windows 7 or later operating system. According to Aperio system requirements, the minimum computer monitor requirement must support 24-bit color depth and a display resolution of 1680(h)x1050(v) with a screen size of 24 inches. The computer monitor contrast and color control settings must be set to the default factory settings.

[0240] Process

[0241] Test Objectives

[0242] The purpose of this test is to compare the diagnostic status of glass and WSI by having observers score the same set of slides on two platforms: a bright field light microscope and a high-resolution computer monitor. TM Perform a scan of the glass slide.

[0243] Initial WSI Requirements

[0244] Aperio AT2 TM Operators are trained before scanning slides. View eSlide manager TM and ImageScope TM Observers of the slides in the software were trained to navigate the WSI using the viewing software.

[0245] Scoring Guide :

[0246] All stained slides for the indications were scored by qualified observers

[0247] WSI scanning process

[0248] Before scanning the slide, the slide is inspected for dust and / or residue. If any residue is present, the operator cleans the slide with a Kimwipe.

[0249] Using Aperio AT2 Scanner TM The slides were scanned. The operator inspected 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 glass slide before the observer made any scoring assessments.

[0250] Blinding and randomization

[0251] According to the CAP pathology guidelines (Pantanowitz et al., Arch. Pathol. Lab. Med. 137 (12): 1710-1722), blinding and randomization of the stained sections were performed to avoid bias during the scoring of the stained sections. Each observer evaluated the slides in a random order according to the randomization key. A score sheet based on the randomized slide sequence was prepared to record the results, and certified observers were instructed to read the slides only in the order in which the slides were presented. Using Aperio Image Scope TM A function in the viewing software that blinds the observer to the slide label performs the WSI reading.

[0252] Scoring of glass slides and WSI

[0253] Scoring of MAGE-A4 slides was performed by three qualified observers. Scoring was performed manually using a light microscope and by scanning in an Aperio ImageScope. TM Scoring was performed digitally by viewing the whole slide image (WSI) in the viewing software.

[0254] Conformance testing process

[0255] Three observers manually scored 80 blinded and randomized samples on glass slides using bright-field light microscopy to obtain a reading of 1. After a minimum washout period of 5 days, the same observers scored the samples using an Aperio ImageScope. TM View the software scoring the WSI of the same set of specimens to obtain reading 2, such as Figure 3 As shown. Glass and WSI readings may occur in the reverse order. Readings are divided into multiple scoring periods as long as the minimum washout period between glass and whole slide image (WSI) readings for a given specimen is met.

[0256] Consistency analysis

[0257] Since this study had a reference (results from slide scoring), negative, positive, and overall agreement percentages (NPA, PPA, OA) were calculated by comparing the WSI scores with the glass scores matched by observer and specimen, and two-sided 95% confidence intervals were calculated using bootstrapping. The reference for the agreement analysis was defined using MAGE-A4 TIPS with a staining intensity of ≥2+ at a cutoff of ≥75% applied to the glass scores. In cases where the NPA, PPA, or OA was exactly equal to 100%, the bootstrapping method was known to fail, so the Wilson score confidence interval was calculated.

[0258] Acceptance criteria

[0259] When all data from three observers and indications are pooled, the lower limit of the two-sided 95% confidence interval for NPA, PPA, and OA must each be at least 85%.Acceptance criteria apply to MAGE-A4 TIPS with a staining intensity of ≥2+ at a cutoff of ≥75%.

[0260] Additional analyses, including analyses by observer or by indication, were not subject to the acceptance criteria result

[0261] Data from all indications were combined for analysis. Analyses were performed for each indication for exploratory purposes and are shown in Table 6.

[0262] Table 6. Summary of MRCLS, NSCLC, SS Glass and WSI

[0263]

[0264]

[0265] * If 100% agreement is observed, the bootstrap method cannot calculate confidence limits. Wilson score limits are used to calculate confidence intervals for parameters where the point estimate of agreement equals 100%.

[0266] Continuous score curves were generated to compare MAGE-A4 TIPS with a score of ≥2+ staining intensity in ≥75% between paired glass and WSI scoring conditions for each observer and indication.

[0267] in conclusion

[0268] When glass and WSI comparisons from all three observers and indications were pooled, the lower limits of the two-sided 95% confidence intervals for NPA, PPA, and OA were at least 85%. When the WSI system and scoring system specified in this report were used, equivalence was established between scoring SS, MRCLS, and squamous NSCLC slides stained with the SK032 MAGE-A4 IHC assay using light microscopy and light WSI, interpreted as MAGE-A4 TIPS with ≥2+ staining intensity at a cutoff of ≥75%.

[0269] In addition to the analysis of all indications combined, analyses by indication, by observer, and by indication-observer rating were performed for informational purposes only. Based on the by indication analysis, all indications showed high agreement in both the negative and positive sample pools, so no single indication appeared to significantly bias the combined indication results.

[0270] A number of embodiments of the present invention have been described. However, it will be appreciated that various modifications may be made without departing from the spirit and scope of the present invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

1. 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, the method comprising: (a) Tissue samples were stained with antibodies that specifically bind to MAGE-A4. (b) determining the total number of viable tumor or cancer cells that have MAGE-A4 staining, and determining the total number of stained and unstained viable tumor or cancer cells in at least a portion of the tissue sample, wherein the tumor or cancer cells are counted as positively stained with the anti-MAGE-A4 antibody if there is any intensity of cytoplasmic and / or nuclear MAGE-A4 staining above a defined threshold; as well as (c) determining the MAGE-A4 tumor intensity ratio score (MAGE-A4 TIPS), 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, and then multiplied by 100.

2. The method according to claim 1, wherein defining a threshold comprises: 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.

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

5. The method of claim 2, wherein the MAGE-A4 TIPS comprises the number of MAGE-A4 viable tumor or cancer cells stained 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 of the preceding claims, wherein about 70% or greater of MAGE-A4 TIPS indicate a positive diagnostic status for the tissue sample.

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

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

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

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

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

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 substantially similar affinity for MAGE-A4 as 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 method according to any one of the preceding claims, wherein: (a) The tissue sample includes a formalin-fixed, paraffin-embedded (FFPE) specimen; (b) the FFPE specimens include cancer specimens stained on an automated IHC platform; (c) preparing said sections of said tissue sample by a protocol comprising fixation in about 10% neutral buffered formalin for a period of about 6 hours to 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 carcinoma; (e) The tissue sample is derived from a needle biopsy sample, fine needle aspirate, cytology specimen, or bone decalcification; (f) determining positive staining using a brightfield light microscope, a microscope objective, a computer monitor and imaging software, or a combination thereof; and / or (g) The imaging software includes whole slide imaging software.

14. A method for diagnosing a tumor or cancer by determining whether a tissue sample is positive for expression of melanoma associated antigen gene-A4 (MAGE-A4), the method comprising: The MAGE-A4 diagnostic status in a tissue sample is determined by the method of any one of claims 1 to 13, wherein a tumor intensity proportion score (MAGE-A4 TIPS) of about 70% or greater of tumor cancer cells having MAGE-A4 nuclear and / or cytoplasmic staining of 2+ or greater intensity is diagnostically positive.

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 carcinoma.

16. A method for treating or ameliorating a tumor or cancer in a patient, the method 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, the patient is treated with a cancer treatment to which the patient is likely to respond favorably.

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 carcinoma.

18. The method of claim 16 or 17, wherein the cancer treatment comprises administering an anti-cancer drug or anti-cancer therapy to the patient, and optionally the anti-cancer therapy 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 a MAGE-A4 scoring guide as described in any one of claims 1 to 18.

20. A method for assessing the expression level of MAGE-A4, comprising: contacting a sample or a portion thereof from the individual comprising cancer or tumor cells with an antibody or a portion thereof that specifically binds to MAGE-A4; as well as The MAGE-A4 Tumor Intensity Ratio score (MAGE-A4 TIPS) is determined by dividing the number of viable tumor or cancer cells stained by MAGE-A4 specifically bound by the antibody in the specimen or portion thereof by the total number of stained and unstained viable tumor or cancer cells and multiplying the result by 100 to obtain the MAGE-A4 TIPS.

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