Immunohistochemical (IHC) PTK7 scoring protocol and method for assisting cancer treatment

Abstract

In alternative embodiments, immunohistochemical (IHC) methods and kits are provided for reproducibly determining and scoring the expression levels of protein tyrosine-protein kinase-like 7 (PTK7), also known as colon cancer kinase 4 (CCK4); HER2 or receptor tyrosine-protein kinase erbB-2; surface antigen classification 340 (CD340); programmed death-ligand 1 (PD-L1); or surface antigen classification 274 (CD274); B7 homolog 1 (B7-H1); and Ki-67 or MKI67 (proliferation marker Ki-67) in tissue samples. In alternative embodiments, methods and kits are provided for diagnosing or selecting individuals eligible for treatment with cancer or tumor therapies, or for assessing the risk of cancer or tumor recurrence using the IHC methods presented herein. In alternative embodiments, kits are provided comprising components and instructions for carrying out the methods presented herein. This application describes a method for scoring PTK7 expression and using that score for companion or complementary diagnostics, or for assisting in the treatment or improvement of cancer or tumors.

Description

[Technical Field]

[0001] [Related applications] This U.S. utility patent application claims priority under § 119(e) of U.S. Patent Act to U.S. Provisional Patent Application No. 63 / 453,890 (USSN) dated March 22, 2023. The entire above application expressly constitutes part of this specification for all purposes by reference.

[0002] The present invention generally relates to cancer treatment, companion diagnosis or complementary diagnosis, and immunohistochemical methods. In alternative embodiments, an immunohistochemical (IHC) method and kit are provided for reproducibly determining and scoring the degree of expression of protein tyrosine-protein kinase-like 7 (PTK7), also known as colon carcinoma kinase 4 (CCK4); HER2 or receptor tyrosine-protein kinase erbB-2, or cluster of differentiation 340 (CD340); programmed death-ligand 1 (PD-L1), or cluster of differentiation 274 (CD274); B7 homolog 1 (B7-H1); and Ki-67 or MKI67 (Marker of Proliferation Ki-67) in a tissue sample. In alternative embodiments, methods and kits are provided for diagnosing or selecting an individual eligible for treatment with a cancer or tumor treatment method, or for assessing the risk of recurrence of a cancer or tumor using the IHC method presented herein. In alternative embodiments, kits are provided that include components and instructions for performing the methods presented herein. Methods are described in the present application for scoring, or assigning a score, or scoring PTK7 expression and using that score for companion diagnosis or complementary diagnosis, or for assisting in the treatment or amelioration of a cancer or tumor.

Background Art

[0003] Tyrosine-protein kinase-like 7 (PTK7), also known as colon cancer kinase 4 (CCK4), is a receptor tyrosine kinase encoded by the PTK7 gene in humans. PTK7 is expressed in various cancers and tumors, such as lung cancer and non-small cell lung cancer (NSCLC), and has been found to contribute to their progression. PTK7 overexpression has been found to be a candidate biomarker for predicting the presence and prognosis of several types of cancer, including stage I-IV hepatocellular carcinoma (HCC), invasive breast cancer, cervical cancer, colorectal cancer, and thyroid cancer. [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] Manual scoring of qualitative immunohistochemical (IHC) assays is described in pathology scoring manuals for companion diagnostic (CDx) assays. These scoring algorithms rely on subjective ratings of mean IHC intensity and the total percentage of positive tumor staining. These methods cannot accurately apply intensity-based cutoffs, which require a score of the percentage of positive tumors at a specific intensity using a scale of 0 to 3. More objective and accurate methods for scoring PTK7 expression in tissue samples remain needed in this art. [Means for solving the problem]

[0005] In an alternative embodiment, an immunohistochemical (IHC) method for determining and scoring the degree of expression of protein tyrosine-protein kinase-like 7 (PTK7) (also known as colon cancer kinase 4 (CCK4); HER2 or receptor tyrosine-protein kinase erbB-2; surface antigen classification 340 (CD340); programmed death ligand 1 (PD-L1); or surface antigen classification 274 (CD274); B7 homolog 1 (B7-H1); and Ki-67 or MKI67 (proliferation marker Ki-67)) in a tissue sample, (a) Staining the tissue sample with an antibody that specifically binds to PTK7, (b) Determine the total number of evaluable invasive tumor cells or cancer cells stained with anti-PTK7 in at least a portion of the tissue sample, and determine the total number of stained evaluable invasive tumor cells or cancer cells and evaluable invasive tumor cells or cancer cells that were not stained, When invasive tumor cells or cancer cells show cell staining with anti-PTK7 at any intensity exceeding a predetermined threshold, the invasive tumor cells or cancer cells are counted as positively stained with anti-PTK7. (c) To calculate the Tumor Proportion Score (TPS), The Tumor Percentage Score (TPS) is calculated by dividing the number of PTK7-stained evaluable invasive tumor cells or cancer cells found in a tissue sample by the total number of stained and unstained evaluable invasive tumor cells or cancer cells, and then multiplying by 100. A method is provided that includes this.

[0006] In an alternative embodiment of the method, A predetermined threshold is Positive staining intensity of 1+ or higher, evaluated at low magnification. A positive staining intensity of 2+ or higher, as evaluated at medium magnification, The 3+ positive staining intensity to be evaluated at high magnification, Includes, The low magnification is at least approximately 4x, the medium magnification is at least approximately 10x, and the high magnification is at least approximately 20x or at least approximately 40x. This method, At low magnification, the presence of tumor cells or cancer cells stained with anti-PTK7 at any intensity above a predetermined threshold in at least a portion of the tissue sample is evaluated. At medium magnification, determine the positive staining intensity of 1+ or higher among the total number of stained and evaluable tumor cells or cancer cells in the tissue sample, which exceeds a predetermined threshold. It further includes, This method further includes determining a positive staining intensity of 1+ or higher, which exceeds a predetermined threshold, among the total number of stained and evaluable tumor cells or cancer cells in a tissue sample at high magnification. This method further includes, at low magnification, (a) evaluating areas of strong staining of stroma and necrosis by anti-PTK7 in a tissue sample, (b) determining the distribution of evaluable tumor cells, cancer cells, and non-tumorous tissue in the stained tissue sample, or (c) identifying the presence of evaluable tumor cells or cancer cells with a staining intensity of 2+ or higher. The method further includes, at medium or high magnification, (a) distinguishing between positive staining with anti-PTK7 in the cell membrane and positive staining with anti-PTK7 in the cytoplasm of the total number of evaluable tumor cells or cancer cells in a tissue sample, (b) distinguishing between 1+ positive staining intensity, 2+ positive staining intensity, and 3+ positive staining intensity of the total number of evaluable tumor cells or cancer cells in a tissue sample, or (c) distinguishing between 0 staining and 1+ positive cell staining intensity in a tissue sample. The method further includes, at high magnification, (a) confirming the total number of evaluable tumor cells or cancer cells in a tissue sample that have a staining intensity of 2+ or higher and a high level of cytoplasmic staining by comparing positive staining with anti-PTK7 on the cell membrane with positive staining with anti-PTK7 on the cytoplasm, or (b) confirming the total number of evaluable tumor cells or cancer cells in a tissue sample that have a staining intensity of 1+ or higher by comparing cell membrane staining with cytoplasmic staining. The method further includes (a) confirming the distribution of 0-cell staining intensity, 1+-positive cell staining intensity, 2+-positive cell staining intensity, and 3+-positive cell staining intensity in a tissue sample at low magnification, and optionally adjusting the Tumor Proportion Score based on the confirmation. The tissue sample includes tissue sections, and it is determined whether the tissue sections are sufficient to determine and score the expression level of the protein PTK7. The tissue sections are considered sufficient for evaluation if they contain approximately 100 or more evaluable invasive tumor cells or cancer cells. If invasive tumor cells or cancer cells show cell membrane staining with anti-PTK7 at any intensity of 1+ or higher, the invasive tumor cells or cancer cells are counted as positive for anti-PTK7 staining. Prepare sections or portions of the tissue sample on a slide or equivalent, stain the sections or portions of the tissue sample on the slide, The antibody that specifically binds to PTK7 contains a monoclonal mouse anti-PTK7 antibody, or the anti-PTK7 antibody contains monoclonal mouse anti-PTK7 clone 6.60.1, or the anti-PTK7 antibody contains substantially isolated or substantially purified monoclonal mouse anti-PTK7 clone 6.60.1. (a) If the staining signal is clearly brown, or (b) if the staining corresponds to the cell membrane, then staining with any intensity of anti-PTK7 of 1+ or higher is considered to be present. This method further includes excluding the following from the calculation of the Tumor Percentage Score (TPS): tumor cells or cancer cells having only cytoplasmic or nuclear staining; non-invasive neoplastic or carcinoma in situ cells; unassessable or necrotic tumor cells or cancer cells; apoptotic nuclei or nuclear debris; tumor cells or cancer cells in poorly preserved tissue regions; benign epithelial cells; non-tumorous cells and / or lymphocytes with nuclear staining; apoptotic cells; necrotic cells; cells that do not exhibit the intended color; lymphocytes; and stromal cells. This method further includes (i) determining that if the tumor percentage score (TPS) is less than 75%, the tissue sample has diagnostically negative PTK7 expression, and (ii) determining that if the tumor percentage score (TPS) is 75% or higher, the tissue sample has diagnostically positive PTK7 expression. The tissue sample may include formalin-fixed, paraffin-embedded (FFPE) specimens, or the tissue sample section may be prepared by a protocol that includes fixing it in approximately 10% neutral buffered formalin for approximately 6 to 72 hours. The tumor or cancer is non-small cell lung cancer (NSCLC), breast cancer or breast carcinoma, head and neck cancer, colorectal cancer, bladder cancer, lung cancer, gastrointestinal stromal tumor (GIST), prostate cancer, cervical cancer, ovarian cancer, or renal cell carcinoma, and / or Whether the tissue sample is a biopsy sample, or a needle biopsy sample, aspiration sample, cytological specimen, or demineralized bone, or derived from there This is provided herein.

[0007] In an alternative embodiment, a method is provided for diagnosing or selecting an individual who is eligible for treatment with a cancer or tumor therapy, comprising determining and scoring the amount of nuclear protein PTK7 (also known as colon cancer kinase 4 (CCK4)) in a tissue sample derived from the individual using the IHC method presented herein, wherein if the tissue sample is determined to have high or diagnostically positive PTK7 expression, the individual is eligible for treatment with a cancer therapy to which the individual is likely to respond favorably.

[0008] In alternative embodiments of the methods described herein, the tumor or cancer is non-small cell lung cancer (NSCLC), breast cancer or mammary carcinoma, head and neck cancer, colorectal cancer, bladder cancer, lung cancer, gastrointestinal stromal tumor (GIST), prostate cancer, cervical cancer, or renal cell carcinoma.

[0009] In an alternative embodiment, a method is provided for diagnosing or selecting an individual who is eligible for treatment with a cancer or tumor therapy, comprising: (a) determining whether the PTK7 expression score (%) of cells in a tissue sample from the individual in need is low or high, or diagnostically negative or diagnostically positive, as determined by a protocol including the use of an immunohistochemical (IHC) method described according to the embodiments herein; and (b) diagnosing or selecting the individual as eligible for treatment with a tumor or cancer therapy if the tissue sample is found to have a high or diagnostically positive PTK7 expression score (TPS).

[0010] In an alternative embodiment of a method for diagnosing or selecting individuals who are eligible for treatment using cancer or tumor therapies, Cancer or tumor treatments include non-small cell lung cancer (NSCLC) treatments or lung cancer treatments. Cancer or tumor treatments are pharmaceutical formulations containing antibody-drug conjugates, radionuclides, gallium-68 radionuclides, or fluorine-18 radionuclides. The individual is a patient with lung cancer or non-small cell lung cancer. This method includes determining the number of evaluable tumor cells or cancer cells that are PTK7 positive, which includes determining the number of evaluable tumor cells or cancer cells that have PTK7 staining in the cell membrane. This method includes determining the number of evaluable tumor or cancer cells that are PTK7 positive, which includes determining the number of evaluable tumor or cancer cells that have cell membrane PTK7 staining of higher intensity than cytoplasmic PTK7 staining. This method further includes determining that if the tumor percentage score (TPS) is above a threshold, the individual is likely to respond favorably to treatment with cancer therapy, and optionally the threshold is approximately 50 TPS or higher, approximately 75% or higher, or any number between approximately 50% and 90%, and / or This method further includes one or more steps of selecting an individual to receive cancer treatment if their tumor percentage score (TPS) exceeds a threshold.

[0011] In an alternative embodiment, provided is a kit comprising an antibody that specifically binds to PTK7 and a scoring guideline that is included in or used in the methods presented herein, for example, in a method of treating or ameliorating cancer or a tumor in an individual that requires it as presented herein.

[0012] In an alternative embodiment, provided is a kit comprising a scoring guideline that is included in or used in the methods presented herein, for example, in a method of diagnosing or selecting an individual eligible for treatment with a cancer or tumor treatment method as presented herein.

[0013] In an alternative embodiment of the kits presented herein, the kit further comprises an image showing multiple PTK7 staining levels.

[0014] In an alternative embodiment, provided is a method for assessing the degree of PTK7 expression, comprising contacting a sample containing cancer cells or tumor cells derived from an individual or a part thereof with an antibody that specifically binds to PTK7 or a part thereof, and dividing the number of PTK7-stained evaluable tumor cells or cancer cells in the sample or part thereof to which the antibody specifically binds by the total number of stained evaluable cancer cells or tumor cells and unstained evaluable cancer cells or tumor cells, and multiplying the result by 100 to obtain a tumor proportion score (TPS).

[0015] Details of one or more exemplary embodiments of the present invention are described in the accompanying drawings and the following description. Other features, objects, and advantages of the present invention will become apparent from the description and drawings, as well as from the claims.

[0016] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes as being clearly a part of this specification.

[0017] The patent or application file contains at least one color drawing. Copies of the patent or patent application publication, along with the color drawing(s), may be provided by the Japan Patent Office upon request and payment of the required fees.

[0018] The figures described herein illustrate exemplary embodiments presented herein and are not intended to limit the scope of the invention as defined in the claims. [Brief explanation of the drawing]

[0019] [Figure 1] As described in more detail below in Example 1, these are images of non-small cell lung cancer (NSCLC) tumor tissue stained with PTK7, exhibiting membrane staining, with an intensity of 0 to 3+. [Figure 2] This figure shows example images of low, moderate, and high staining intensity at each level. As described in more detail below in Example 1, these images are intended as guidelines and do not represent the entire range of staining variation within each intensity category (0 to 3+). [Figure 3] This is a T&T (Training and Testing) between-observer variability plot. The x-axis shows 30 tissue blocks scored by four observers or pathologists, as described in more detail below in Example 1. [Figure 4A] As described in more detail below in Example 1, this figure illustrates a visual summary of the various FOVs used for IHC testing in the exemplary protocol. [Figure 4B] As described in more detail below in Example 1, this is a diagram illustrating an exemplary FOV superimposed on an image of a tissue section. [Modes for carrying out the invention]

[0020] Similar reference numerals in various figures indicate the same elements.

[0021] In alternative embodiments, immunohistochemical (IHC) methods and kits are provided for reproducibly determining and scoring the degree of expression of protein tyrosine-protein kinase-like 7 (PTK7), also known as colon cancer kinase 4 (CCK4); HER2 or receptor tyrosine-protein kinase erbB-2; surface antigen classification 340 (CD340); programmed death-ligand 1 (PD-L1); or surface antigen classification 274 (CD274); B7 homolog 1 (B7-H1); and Ki-67 or MKI67 (proliferation marker Ki-67) in tissue samples. In alternative embodiments, scoring methods for assessing PTK7 expression in tumors or cancers are provided. Certain embodiments include using the scoring methods presented herein to assess PTK7 expression and determine whether there is a high or diagnostically positive tumor percentage score.

[0022] Alternative embodiments provide methods and kits for diagnosing or selecting individuals who are eligible for treatment with cancer or tumor therapies. In certain embodiments, if a tissue sample is determined to have a high or diagnostically positive tumor percentage score, the patient is diagnosed or selected as a candidate for treatment with a cancer therapy that is likely to favorably respond to the patient.

[0023] In an alternative embodiment, methods and kits are provided for diagnosing cancer or tumors, treating cancer or tumors, improving cancer or tumors, or assessing the risk of cancer or tumor recurrence, using the IHC methods presented herein.

[0024] PTK7 is expressed in various cancers and tumors and has been found to contribute to the progression of various cancers and tumors. Such cancers include malignant tumors with poor prognosis, including lung cancer and non-small cell lung cancer (NSCLC). PTK7 overexpression has been found to be a promising candidate biomarker for predicting the presence and prognosis of several types of cancer, including not only lung cancer and NSCLC, but also stage I-IV hepatocellular carcinoma (HCC), invasive breast cancer, cervical cancer, colorectal cancer, and thyroid cancer. By comparing PTK7 expression patterns in cancers and tumors with those in normal tissues, PTK7 is an attractive target for the development of diagnostic assays.

[0025] Currently available CDx scoring guidelines include formulas for interpreting IHC staining in tissue samples. Such formulas are written as equations such as CPS(number of PTK7-positive cells (tumor cells and immune cells) / total number of tumor cells), but they do not describe how to use this numerator and denominator to determine the CPS score for the entire slide. Pathologists often ask what method to follow to obtain the correct score, but there has been no systematic answer to this question.

[0026] Attempts to obtain accurate IHC scores include assessing intensity using the color and / or thickness of IHC staining. However, such methods have drawbacks: (1) color (i.e., dark brown, brown, light brown) is highly subjective, dependent on the scoring method (i.e., glass or digital), and only suitable for scoring algorithms that are not intensity-based; and (2) thickness is generally specific to membrane staining and is not applicable to all cancer indicators and biomarkers.

[0027] There is still a need in the art for not only robust, reliable, reproducible, objective, and accurate PTK7 scoring methods, but also simpler and more efficient scoring methods. Embodiments of immunohistochemical (IHC) methods described herein may offer considerable advantages for reliable and efficient diagnostic assessment of the degree of PTK7 expression in tissue samples.

[0028] [Embodiment of the immunohistochemical (IHC) method] Embodiments herein relate to an immunohistochemical method for determining and scoring the degree of protein PTK7 expression in a tissue sample. In various embodiments, the method includes staining the tissue sample with an antibody that specifically binds to PTK7; determining the total number of evaluable invasive tumor cells or cancer cells stained with anti-PTK7 in at least a portion of the tissue sample, and determining the total number of stained and unstained evaluable invasive tumor cells or cancer cells, wherein if the invasive tumor cells or cancer cells show cell staining with anti-PTK7 at any intensity above a predetermined threshold, the invasive tumor cells or cancer cells are counted as positively stained with anti-PTK7; and determining a tumor percentage score (TPS), wherein the tumor percentage score (TPS) is obtained by dividing the number of PTK7-stained evaluable invasive tumor cells or cancer cells found in the tissue sample by the total number of stained and unstained evaluable invasive tumor cells or cancer cells, and multiplying by 100. Such embodiments may provide not only a robust, reliable, reproducible, and accurate PTK7 scoring method, but also a more objective, simpler, and more efficient scoring method. Such embodiments may offer significant advantages for reliable and efficient diagnostic assessment.

[0029] In certain embodiments, the tumor or cancer is non-small cell lung cancer (NSCLC), breast cancer or mammary carcinoma, head and neck cancer, colorectal cancer, bladder cancer, lung cancer, gastrointestinal stromal tumor (GIST), prostate cancer, cervical cancer, ovarian cancer, or renal cell carcinoma.

[0030] Various embodiments provide a systematic microscopy method that uses multiple fields of view and varies magnification to obtain the correct biomarker score. Magnification-based guidance is an objective method for assessing IHC intensity. Standardizing magnification across microscopes ensures that observers and / or pathologists accurately assess stain intensity from 0 to 3 according to a specific, unique set of steps. Such embodiments provide a microscopy-based scoring method that may have the advantage of high reproducibility.

[0031] In certain embodiments, a predetermined threshold includes a positive staining intensity of 1+ or higher at low magnification, a positive staining intensity of 2+ or higher at medium magnification, or a positive staining intensity of 3+ at high magnification. In some embodiments, low magnification is at least about 4x, medium magnification is at least about 10x, and high magnification is at least about 20x or at least about 40x.

[0032] In certain embodiments, positive staining is determined using a bright-field optical microscope, a microscope objective lens, a computer monitor, and imaging software, or a combination thereof. In some embodiments, the imaging software includes whole-slide imaging software.

[0033] In certain embodiments, the method further includes evaluating, at low magnification, the presence of tumor cells or cancer cells stained with anti-PTK7 at any intensity above a predetermined threshold in at least a portion of the tissue sample; and, at medium magnification, determining a positive staining intensity of 1+ or higher above a predetermined threshold among the total number of stained, evaluable tumor cells or cancer cells in the tissue sample. In some embodiments, the method includes determining a positive staining intensity of 2+ or higher at medium magnification. In certain embodiments, the method further includes determining a positive staining intensity of 1+ or higher above a predetermined threshold among the total number of stained, evaluable tumor cells or cancer cells in the tissue sample at high magnification. In some embodiments, the method includes determining a positive staining intensity of 2+ or higher at high magnification.

[0034] In a particular embodiment, the method further includes evaluating areas of strong staining of stroma and necrosis by anti-PTK7 in a tissue sample at low magnification, determining the distribution of evaluable tumor cells, cancer cells, and non-tumorous tissue in the stained tissue sample, or identifying the presence of evaluable tumor cells or cancer cells having a staining intensity of 2+ or higher.

[0035] Certain embodiments further include distinguishing between positive staining with anti-PTK7 in the cell membrane and positive staining with anti-PTK7 in the cytoplasm of the total number of evaluable tumor or cancer cells in a tissue sample at medium or high magnification. Such embodiments may offer the advantage of increased accuracy and reproducibility of the PTK7 scoring method by distinguishing between cells having different structural staining patterns, or by excluding stained cells having certain structures or characteristics from the determination of the tumor percentage score.

[0036] Other embodiments of the methods described herein further include distinguishing between 1+ positive staining intensity, 2+ positive staining intensity, or 3+ positive staining intensity among the total number of evaluable tumor or cancer cells in a tissue sample, or distinguishing between 0 staining and 1+ positive cell staining intensity in a tissue sample. Such embodiments may offer the benefit of a predetermined threshold flexibility with respect to staining intensity.

[0037] In certain embodiments, the method further includes, at high magnification, confirming the total number of evaluable tumor or cancer cells in a tissue sample having a staining intensity of 2+ or higher and a high level of cytoplasmic staining by comparing positive staining with anti-PTK7 at the cell membrane with positive staining with anti-PTK7 in the cytoplasm. In certain embodiments, the method further includes confirming the total number of evaluable tumor or cancer cells in a tissue sample having a staining intensity of 1+ or higher by comparing cell membrane staining with cytoplasmic staining. In some embodiments, high level of cytoplasmic staining includes a level of cytoplasmic PTK7 staining equivalent to or higher than the level of membrane PTK7 staining.

[0038] In certain embodiments, the method further includes confirming the distribution of 0-cell staining intensity, 1+-positive cell staining intensity, 2+-positive cell staining intensity, and 3+-positive cell staining intensity in a tissue sample at low magnification. Certain embodiments optionally include adjusting the tumor percentage score based on the confirmation.

[0039] In a particular embodiment, if invasive tumor cells or cancer cells show cell membrane staining with anti-PTK7 at any intensity of 1+ or higher, the invasive tumor cells or cancer cells are counted as positive for anti-PTK7 staining.

[0040] In certain embodiments, staining is determined to be anti-PTK7 staining at any intensity of 1+ or higher if the staining signal is clearly brown. In certain embodiments, staining is determined to be anti-PTK7 staining at any intensity of 1+ or higher if the staining corresponds to a cell membrane.

[0041] In certain embodiments, the method further includes excluding from the calculation of the Tumor Proportion Score (TPS) the following: tumor cells or cancer cells having only cytoplasmic or nuclear staining; non-invasive neoplastic or carcinoma in situ cells; unassessable or necrotic tumor cells or cancer cells; apoptotic nuclei or nuclear debris; tumor cells or cancer cells in poorly preserved tissue regions; benign epithelial cells; non-tumorous cells and / or lymphocytes with nuclear staining; apoptotic cells; necrotic cells; cells that do not exhibit the intended color; lymphocytes; and stromal cells. Such embodiments may offer the advantage of increased accuracy and reproducibility of the PTK7 scoring method by excluding stained cells having certain structures or characteristics from the determination of the Tumor Proportion Score.

[0042] In certain embodiments of the methods described herein, a tissue sample is determined to have diagnostically negative PTK7 expression if the tumor percentage score (TPS) is less than 75%. In certain embodiments, a tumor percentage score of less than 70% indicates diagnostically negative PTK7 expression. In certain embodiments, a tumor percentage score of less than 60% indicates diagnostically negative PTK7 expression. In certain embodiments, a tissue sample is determined to have diagnostically positive PTK7 expression if the tumor percentage score (TPS) is 75% or higher. In certain embodiments, a tumor percentage score exceeding 75% indicates diagnostically positive PTK7 expression. In certain embodiments, a tumor percentage score exceeding 80% indicates diagnostically positive PTK7 expression. In certain embodiments, a tumor percentage score exceeding 85% indicates diagnostically positive PTK7 expression.

[0043] In certain embodiments, the antibody that specifically binds to PTK7 includes monoclonal mouse anti-PTK7 antibody. In certain embodiments, the anti-PTK7 antibody includes monoclonal mouse anti-PTK7 clone 6.60.1. In certain embodiments, the anti-PTK7 antibody includes substantially isolated or substantially purified monoclonal mouse anti-PTK7 clone 6.60.1.

[0044] In certain embodiments of the methods described herein, the tissue sample comprises a tissue section, and in certain embodiments, it is determined whether the tissue section is sufficient to determine and score the expression level of the protein PTK7. In certain embodiments, the tissue section is considered sufficient for evaluation if it contains about 100 or more evaluable invasive tumor cells or cancer cells. In certain embodiments, the tissue section is considered sufficient for evaluation if it contains about 200 or more evaluable invasive tumor cells or cancer cells. In certain embodiments, the tissue section is considered sufficient for evaluation if it contains about 500 or more evaluable invasive tumor cells or cancer cells.

[0045] In various embodiments, sections or portions of tissue samples are prepared on slides or equivalents, and the sections or portions of tissue samples are stained on the slides. In certain embodiments, sections or portions of tissue samples are prepared on slides, microscope slides, or equivalents, and the sections or portions of tissue samples are stained on the slides. In certain embodiments, the tissue sample includes formalin-fixed paraffin-embedded (FFPE) specimens. In certain embodiments, the FFPE specimens include cancer specimens stained on an automated IHC platform. In certain embodiments, sections of tissue samples are prepared by a protocol that includes fixing them in about 10% neutral buffered formalin for a period of about 6 to about 72 hours. In certain embodiments, the tissue sample is a biopsy specimen, a needle biopsy specimen, a needle biopsy specimen, aspiration, a cytological specimen, or a specimen derived from demineralized bone.

[0046] [Examples of methods for diagnosing tumors or cancer] Embodiments herein provide a method for diagnosing a tumor or cancer by determining whether a tissue sample is positive for PTK7 expression. In various embodiments, the method includes determining the diagnostic PTK7 status in a tissue sample by using an IHC method for determining the degree of cell membrane PTK7 expression presented herein, wherein the method may include determining whether the PTK7 staining is at a predetermined threshold, where the predetermined threshold may include a positive staining intensity of 1+ or higher at low magnification, a positive staining intensity of 2+ or higher at medium magnification, or a positive staining intensity of 3+ at high magnification. Such embodiments may offer the benefit of flexibility in the predetermined threshold for staining intensity. In an alternative embodiment, if the tumor percentage score (TPS) is less than 75%, the tissue sample is determined to have diagnostically negative PTK7 expression, and if the tumor percentage score (TPS) is 75% or higher, the tissue sample is determined to have diagnostically positive PTK7 expression. Such embodiments may offer the benefit of accuracy in determining the diagnostic status with respect to PTK7 expression.

[0047] In certain embodiments, the tumor or cancer is non-small cell lung cancer (NSCLC), breast cancer or mammary carcinoma, head and neck cancer, colorectal cancer, bladder cancer, lung cancer, gastrointestinal stromal tumor (GIST), prostate cancer, cervical cancer, or renal cell carcinoma.

[0048] Embodiments herein provide methods for diagnosing or selecting individuals who are eligible for treatment with cancer or tumor therapies. In various embodiments, such methods include determining and scoring the amount of nuclear protein PTK7 (also known as colon cancer kinase 4 (CCK4)) in a tissue sample derived from an individual using an IHC method according to embodiments presented herein, wherein if the tissue sample is determined to have high or diagnostically positive PTK7 expression, the individual is diagnosed or selected as a target for treatment with a cancer therapy in which the individual is likely to respond favorably. In certain embodiments, the tumor or cancer is non-small cell lung cancer (NSCLC), breast cancer or mammoma, head and neck cancer, colorectal cancer, bladder cancer, lung cancer, gastrointestinal stromal tumor (GIST), prostate cancer, cervical cancer, or renal cell carcinoma.

[0049] Embodiments herein provide methods for diagnosing or selecting individuals who are eligible for treatment with cancer or tumor therapies. In various embodiments, such methods include (a) determining whether cells in an individual-derived tissue sample have a low or high PTK7 expression score (TPS), or whether they are diagnostically negative or diagnostically positive, as determined by a protocol that includes using an immunohistochemical (IHC) method described in various embodiments herein, and (b) diagnosing or selecting an individual who is eligible for treatment with cancer or tumor therapies if the tissue sample is found to have a high or diagnostically positive PTK7 expression score (TPS). In certain embodiments, the cancer or tumor therapy is a non-small cell lung cancer (NSCLC) therapy or lung cancer therapy. In certain embodiments, the cancer or tumor therapy is a pharmaceutical formulation comprising an antibody-drug conjugate, a radionuclide, a gallium-68 radionuclide, or a fluorine-18 radionuclide. In certain embodiments, the individual is a patient with lung cancer or non-small cell lung cancer.

[0050] In a particular embodiment, determining the number of PTK7-positive evaluable tumor cells or cancer cells includes determining the number of evaluable tumor cells or cancer cells having PTK7 staining at the cell membrane. In a particular embodiment, determining the number of PTK7-positive evaluable tumor cells or cancer cells includes determining the number of evaluable tumor cells or cancer cells having cell membrane PTK7 staining of a higher intensity than cytoplasmic PTK7 staining.

[0051] In certain embodiments, such a method further includes determining that an individual is likely to respond favorably to treatment with a cancer treatment if the tumor percentage score (TPS) is above a threshold. In certain embodiments, the threshold is any number between approximately 50% and above, approximately 75% and above, or roughly between 50% and 90%. In some embodiments, the method further includes selecting an individual to be treated with a cancer treatment if the tumor percentage score (TPS) is above a threshold.

[0052] [Embodiments of methods for treating cancer and tumors] A method is provided for treating or improving a tumor or cancer in a patient, comprising determining and scoring the amount of PTK7 in a patient-derived tissue sample using a method presented herein, wherein if the tissue sample is determined or scored to have a high or diagnostically positive tumor percentage score, the patient is treated with a cancer treatment or anti-cancer treatment that is likely to be favorably responded to.

[0053] In certain embodiments, the tumor or cancer is non-small cell lung cancer (NSCLC), breast cancer or mammary carcinoma, head and neck cancer, colorectal cancer, bladder cancer, lung cancer, gastrointestinal stromal tumor (GIST), prostate cancer, cervical cancer, or renal cell carcinoma.

[0054] In certain embodiments, a cancer treatment method includes administering an anticancer drug or anticancer treatment to a patient. In alternative embodiments, the anticancer procedure or treatment includes surgical procedures, e.g., CyberKnife treatment; chemoembolization; ablation techniques, e.g., radiofrequency ablation (RFA), cryoablation and / or microwave ablation; and / or radiotherapy, e.g., stereotactic body radiotherapy.

[0055] In certain embodiments, anticancer treatment includes antibody-drug conjugates, small molecule therapies, immunotherapies, monoclonal antibody therapies, adoptive cell therapies, T cell receptor therapies, or chimeric antigen receptor (CAR) T cell therapies. In alternative embodiments, the anticancer treatment or therapy comprises a tyrosine kinase inhibitor (optionally, erlotinib (i.e., TARCEVA®), gefitinib (i.e., IRESSA®), afatinib (i.e., GILOTRIF®), or osimertinib (TAGRISSO®)); necitumumab (i.e., PORTRAZZA®), pembrolizumab (i.e., KEYTRUDA®), nivolumab (i.e., OPDIVO®), ipilimumab (YERVOY®), cetuximab (i.e., ERBITUX®), cisplatin (i.e., PLATINOL®), or carboplatin (i.e., PARAPLATIN®).

[0056] In an alternative embodiment, the anticancer treatment or therapy comprises the use of an anticancer agent which may include an antibody that specifically or substantially binds to cancer or tumor, wherein the antibody is conjugated with a cytotoxic agent, which optionally includes a radionuclide (optionally, yttrium-90, iodine-131, lutetium-177, radium-223 chloride, strontium-89 chloride, or samarium-153). This includes EDTMP), diphtheria toxin, Pseudomonas exotoxin A, denileukin diphthitox (i.e., ONTAK®), moxetumomab pasudotox (i.e., Lumoxiti®), inotuzumab ozogamicin (i.e., BESPONSA®), calitiamycin or N-acetyl-γ-calitiamycin, emtansine or DM1, mytansine or its derivatives, meltansine, labtansine, solabtansine or milbetuximab solabtansine, SN-38 (7-ethyl-10-hydroxycamptothecin), or auristatin or monomethyl auristatin E (MMAE).

[0057] [Immunohistochemistry] In alternative embodiments, the methods presented herein may use or include any product or method known in the art, such as an IHC protocol or reagent, for detecting or visualizing antibody-antigen interactions.

[0058] In alternative embodiments, immunohistochemical methodologies and / or reagents used in conjunction with the methods and products or kits presented herein may include, or comprise, any IHC protocols, IHC instruments, devices, and / or image or data analysis systems known in the art for performing IHC or for IHC reagents.

[0059] In alternative embodiments, the antibodies, antigen-binding fragments thereof, or monomeric or dimeric antigen-binding proteins (including, for example, synthetic or recombinant forms) used in the IHC protocols or kits presented herein are substantially purified or isolated, or are in the form of unpurified or partially purified culture supernatant.

[0060] In alternative embodiments, the methods presented herein involve the use of chromogenic immunohistochemistry (CIH). In chromogenic immunohistochemistry, a primary antibody (e.g., a recombinant antibody (Ab) or its antigen-binding fragment, or monomeric or dimeric antigen-binding protein as presented herein) or a secondary antibody (e.g., a primary antibody, recombinant antibody (Ab) or its antigen-binding fragment, or monomeric or dimeric antigen-binding protein as presented herein) is conjugated with an enzyme capable of catalyzing a color reaction, such as an immunoperoxidase, such as horseradish peroxidase (HRP). In alternative embodiments, the chromogenic moieties used in the methods presented herein include coumarin; rhodamine; 2,3,6,7-tetrahydro-11-oxo-1H,5H,11H-[1]benzopyrano[6,7,8-ij]quinolidine-1-0-carboxylic acid; 7-(diethylamino)coumarin-3-carboxylic acid; coumarin derivatives; rhodamine derivatives; tetramethylrhodamine; diarylrhodamine derivatives; QSY7; QSY9; QSY21; diazochromophores; DABSYL; tartrazine; triarylmethane compounds; Fast Red; Fast Blue; fuchsine; Cascade Blue acetyl; dapoxylsulfonic acid / succinimidyl carboxylic acid; 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; QSY35 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'-tetrabromosulfone fluorescein succinimidyl ester; Alexa Fluor 532 succinimidyl ester; 6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein succinimidyl ester (HEX); BODIPY 530 / 550 C3 succinimidyl ester; DY-530; BODIPY TMR-X succinimidyl ester; DY-555; DYQ-1; DY-556; Cy3 succinimidyl ester; DY-547; DY-549; DY-550; Alexa Fluor 555 succinimidyl ester; Alexa Fluor 546 succinimidyl ester; DY-548; BODIPY 558 / 568 C3 succinimidyl ester; Rhodamine red-X succinimidyl ester; QSY7 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: fluorescein Isothiocyanate) Carboxysuccinimidyl ester (DY-495); Rhodamine Green carboxylate 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); QSY9 succinimidyl ester; Sulforodamine B sulfonyl chloride (DY-560); Texas Red (Sulforodamine 101); Gallocyanine; Fast Green FCF;Malachite Green; or QSY21 succinimidyl ester, or containing the same.

[0061] In alternative embodiments, the methods presented herein include the use of immunofluorescence. In immunofluorescence, a primary or secondary antibody is tagged with a fluorophore, e.g., fluorescein or fluorescein isothiocyanate (FITC), a triarylmethane dye, e.g., rhodamine or a rhodamine derivative (e.g., tetramethylrhodamine (TRITC), rhodamine 6G, rhodamine 123, rhodamine B, carboxytetramethylrhodamine (TAMRA), tetramethylrhodamine (TMR), sulforhodamine 101), aminomethylcoumarin acetate (AMCA), ALEXA® or DYLIGHT® fluorophore, or a fluorophore or dye described in U.S. Patent Application No. 2019 / 0018018. 3,3'-diaminobenzidine (DAB) may also be used.

[0062] In alternative embodiments, the methods presented herein involve the use of a direct method or a one-step staining method. In the direct method or one-step staining method, a primary antibody (e.g., the antibody (Ab) presented herein or its antigen-binding fragment, or monomeric or dimeric antigen-binding protein (e.g., including synthetic or recombinant forms)) is labeled and reacted directly with the antigen, for example, in a tissue section. This technique utilizes only one antibody and is therefore simple and rapid, but may have low sensitivity due to minimal signal amplification.

[0063] In alternative embodiments, the methods presented herein involve the use of a direct method or a one-step staining method. In the direct method or one-step staining method, a primary antibody (e.g., the antibody (Ab) presented herein or its antigen-binding fragment, or monomeric or dimeric antigen-binding protein (e.g., including synthetic or recombinant forms)) is labeled and reacted directly with the antigen, for example, in a tissue section. This technique utilizes only one antibody and is therefore simple and rapid, but may have low sensitivity due to minimal signal amplification.

[0064] In alternative embodiments, the methods presented herein include the use of an indirect method. In the indirect method, for example, an unlabeled primary antibody (first layer) is conjugated to a target antigen (e.g., PTK7 protein) in a tissue or organ, and then a labeled secondary antibody (second layer) is reacted with the primary antibody. The secondary antibody may be for the isotype of the animal species from which the primary antibody originates, for example, IgG. This method may be more sensitive than direct detection strategies because, when the secondary antibody is conjugated with a detection agent such as a fluorescent or enzyme reporter, the signal is amplified due to several secondary antibodies binding to each primary antibody.

[0065] In an alternative embodiment, when a secondary antibody is conjugated with several detection molecules, such as a biotin molecule, a complex of avidin and enzyme, a complex of streptavidin and enzyme, or a complex of NEUTRAVIDIN® protein and enzyme can be recruited, enabling further amplification.

[0066] In alternative embodiments, IHC is performed on tissue sections or tissue biopsy materials, such as paraformaldehyde (PFA) fixed tissue or organs, or formalin-fixed paraffin-embedded tissue. In alternative embodiments, the tissue is sectioned, sliced, or used whole. Before sectioning, the tissue sample can be embedded in a medium, such as paraffin wax or cryomedia. Sectioning or slicing of tissue sections can be performed using various instruments, most commonly using a microtome, cryostat, or vibratome. The sample can be sectioned or sliced ​​in the range of approximately 3 μm to 5 μm. The sections or slices can be placed on a slide, dehydrated using progressively increasing concentrations of alcohol washing (e.g., 50%, 75%, 90%, 95%, 100%), clarified with a surfactant such as xylene, and then imaged under a microscope.

[0067] Depending on the fixation and tissue protection method, additional steps, including deparaffinization and antigen retrieval, may be required for the sample to make the PTK7 epitope available for antibody binding. For formalin-fixed, paraffin-embedded tissues, antigen retrieval is often required, and this may include pretreatment of sections with heat or protease.

[0068] In an alternative embodiment, IHC is performed using the EnVision DuoFLEX Doublestain System (Agilent, San Jose, California). This system allows for the staining of two or more markers on a single slide. In an alternative embodiment, IHC is performed using the EnVision FLEX HRP Magenta (High pH) (Dako Omnis) system, and binding can be visualized using the EnVision FLEX HRP Magenta Chromogen. In an alternative embodiment, IHC is performed using the EnVision FLEX MINI (High pH) kit. This kit is a highly sensitive visualization system intended for use in IHC with the Dako AUTOSTAINER (registered trademark) instrument. This dual-link system allows for the detection of mouse and rabbit primary antibodies and visualization of the reaction using a 3,3'-diaminobenzidine (DAB) chromogen (DAB forms a water-insoluble brown precipitate when oxidized, for example, by peroxidase).

[0069] [Manufactured products and kits] Products and kits for carrying out the methods presented herein are provided. Optionally, the products and kits may further include instructions for carrying out the methods presented herein.

[0070] Embodiments of the kit described herein include an antibody that specifically binds to PTK7, and scoring guidelines including an immunohistochemical (IHC) method for determining and scoring the degree of expression of protein tyrosine-protein kinase-like 7 (PTK7) in a tissue sample, as presented herein. Other embodiments of the kit described herein include scoring guidelines including an IHC method, as presented herein. In some embodiments, the kit further includes images showing multiple PTK7 staining levels.

[0071] Any of the above aspects and embodiments can be combined with any other aspects or embodiments disclosed herein in the sections on the Outline of the Invention, the Drawings and / or Modes for Carrying Out the Invention.

[0072] As used herein and in the claims, the singular forms "a," "an" (i.e., "one"), and "the" ("it" or "the aforementioned") refer to multiple subjects unless the context clearly indicates otherwise.

[0073] Unless otherwise specified or the context makes clear, the term “or” as used herein is inclusive and shall be understood to encompass both “or” and “and.”

[0074] Unless otherwise specified or the context makes clear, the term “about” as used herein is understood to mean within the normal range of acceptance in the art, for example, within two standard deviations of the mean. “About” (the use of the term “about”) can be understood to mean within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless the context makes clear, all numerical values ​​presented herein are modified by the term “about”.

[0075] Unless otherwise specified or the context makes clear, the terms “substantially all,” “substantially most,” “substantially all,” or “majority” as used herein encompass at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or more of the reference amount of the composition.

[0076] Each of the patents, patent applications, publications, and documents referenced herein shall, by reference, constitute part of this Specified. The foregoing references to the aforementioned patents, patent applications, publications, and documents shall not constitute an admission that any of them is relevant prior art, nor shall they constitute any finding of the content or date of such publications or documents. The fact that these documents shall constitute part of this Specified by individual reference shall not be construed as an assertion or finding that any part of the content of any of these documents is considered essential material for satisfying the disclosure requirements of national or local law relating to the patent application. However, the right to rely on any of such documents to provide material that an examining authority or court deems essential with respect to the claimed subject matter is reserved.

[0077] Modifications can be made to the foregoing without departing from the basic aspects of the present invention. Although the present invention is described in considerable detail with reference to one or more specific embodiments, those skilled in the art will understand that modifications can be made to the embodiments disclosed in detail in this application, and that such modifications and improvements still fall within the scope and spirit of the present invention. The inventions described exemplary herein can be adequately implemented even if any element is not disclosed in detail herein. Accordingly, for example, in each case herein, the terms “including,” “consisting essentially of,” and “consisting of” can each be replaced by any of the other two terms. Accordingly, it should be recognized that the terms and expressions used are not limiting but explanatory terms, and that the features or equivalents of parts thereof shown and described are not excluded, and that various modifications are possible within the scope of the present invention.

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

[0079] Unless otherwise specified, all recombinant DNA techniques in the examples are performed according to standard protocols as described in, for example, 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 on 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). Standard materials and methods for polymerase chain reactions can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and McPherson et al. (2000) PCR-Basics: From Background to Bench, First Edition, Springer Verlag, Germany.

[0080] [Example 1: Exemplary IHC Method] In this embodiment, an exemplary IHC method presented herein is described.

[0081] Figure 1 shows examples of PTK7-stained tumor tissue from non-small cell lung cancer (NSCLC) with staining intensities ranging from 0 to 3+, illustrating membrane staining.

[0082] Figure 2 shows example images of low, moderate, and high staining intensity at each level. These images are intended as guidelines and do not represent the entire range of staining variation within each intensity category (0-3+).

[0083] [Membrane staining magnification guidance] The first bullet point for each staining intensity from 1 to 3 includes a description of the color (dark brown, golden / medium brown, and light brown). These descriptions are typically included in non-intensity-based scoring guidelines where any intensity from 1 to 3 is considered positive staining.

[0084] [Stepwise method for assessing intensity:] [Table 1]

[0085] Regarding intensity-based scoring, positive staining is defined only when it reaches a certain intensity. This positive staining information is used to determine the diagnostic status of the patient sample. Specific guidance regarding magnification is provided in the second and third bullet points:

[0086] [Table 2]

[0087] [Inter-observer variability] During training, pathologists will be provided with Tables 1 and 2 in the form of a pathology scoring manual. After training, pathologists will be tested on their accuracy in scoring. Pathologists will be provided with a form including the following table (Table 3) to be completed for each specimen:

[0088] [Table 3]

[0089] Figure 3 illustrates a Training and Testing (T&T) inter-observer variability plot. The x-axis shows 30 tissue blocks scored by four observers or pathologists. The y-axis shows the score for the percentage of tumor cells with a score of 2+ or higher. For example, block 140446 received one score of 25, two scores of 30, and one score of 35. This demonstrates that the evaluations of all four different pathologists, who assessed the blinded slide without using a reference score, fell within 10% for this particular specimen.

[0090] [Example protocol] - The field of view (FOV) is investigated at 40x magnification, and a score is determined according to the guidelines presented herein. - Change the objective lens to 20x. Investigate the field of view (FOV). Determine the score by evaluating the peripheral FOV in particular. Weakly stained or unstained areas of cells can be evaluated at higher magnification for confirmation. Ignore non-cellular areas. Re-evaluate / readjust the score. - Change the objective lens to 10x. Repeat the above steps. - Change the objective lens to 4x. Repeat the above steps. - As a result, a score is obtained at 4x FOV, and this protocol can be repeated for other 4x FOVs if the tissue is large enough. The average can be obtained by summing the scores for 4x FOV and dividing by the number of fields of view.

[0091] Figure 4A illustrates a visual summary of the various FOVs used for IHC examination in this exemplary protocol, and Figure 4B illustrates exemplary FOVs overlaid on images of tissue sections.

[0092] [Example Embodiments] Embodiment 1. A method for treating or improving cancer or tumors in an individual requiring treatment or improvement of cancer or tumors, (a) Determining whether the PTK7 expression score (TPS) of cells in a tissue sample from an individual requiring treatment or improvement of cancer or tumor is low or high, or diagnostically negative or diagnostically positive, as determined by a protocol including the use of an immunohistochemical (IHC) method as described in any one of Embodiments 1 to 24, (b) If a tissue sample is found to have a high or diagnostically positive PTK7 expression score (TPS), the individual shall be subjected to tumor or cancer treatment. Methods that include...

[0093] Embodiment 2. The method of Embodiment 1, wherein the cancer or tumor is non-small cell lung cancer (NSCLC) or lung cancer.

[0094] Embodiment 3. The method of Embodiment 1 or 2, wherein the treatment or improvement of cancer or tumor is performed by administering a pharmaceutical formulation containing an antibody-drug conjugate, a radionuclide, a gallium-68 radionuclide, or a fluorine-18 radionuclide to an individual in need thereof.

[0095] Embodiment 4. Any of Embodiments 1 to 3, wherein the individual requiring treatment or improvement of cancer or tumor is a patient with lung cancer or non-small cell lung cancer.

[0096] Embodiment 5. A method according to any one of Embodiments 1 to 4, wherein determining the number of evaluable tumor cells or cancer cells that are PTK7 positive includes determining the number of evaluable tumor cells or cancer cells that have PTK7 staining in their cell membranes.

[0097] Embodiment 6. A method according to any one of Embodiments 1 to 5, wherein determining the number of evaluable tumor cells or cancer cells that are PTK7 positive includes determining the number of evaluable tumor cells or cancer cells that have cell membrane PTK7 staining of a higher intensity than cytoplasmic PTK7 staining.

[0098] Embodiment 7. Any method of Embodiments 1 to 6, further comprising determining that if the tumor percentage score (TPS) is above a threshold, the subject is likely to respond favorably to treatment with cancer therapy.

[0099] Embodiment 8. The method of Embodiment 7, wherein the threshold is approximately 50% or more, approximately 75% or more, or any number between approximately 50% and 90%.

[0100] Embodiment 9. Any method of Embodiments 1 to 8, further comprising administering a cancer treatment if the tumor percentage score (TPS) exceeds a threshold.

[0101] Embodiment 10. A method for treating or improving cancer or tumor in a patient, comprising determining and scoring the amount of nuclear protein PTK7 (also known as colon cancer kinase 4 (CCK4)) in a tissue sample from the patient in need, using an IHC method of any of Embodiments 1 to 9, wherein if the tissue sample is determined to have high or diagnostically positive PTK7 expression and is scored, the patient is treated with a cancer treatment that is likely to favorably respond to the patient.

[0102] Embodiment 11. The method of Embodiment 10, wherein the tumor or cancer is non-small cell lung cancer (NSCLC), breast cancer or mammary carcinoma, head and neck cancer, colorectal cancer, bladder cancer, lung cancer, gastrointestinal stromal tumor (GIST), prostate cancer, cervical cancer, or renal cell carcinoma.

[0103] While several embodiments of the present invention are described, it should be understood that various modifications can be made without departing from the spirit and scope of the invention. Therefore, other embodiments also fall within the scope of the appended claims.

Claims

1. An immunohistochemical (IHC) method for determining and scoring the degree of expression of protein tyrosine-protein kinase-like 7 (PTK7) in a tissue sample, (a) A step of staining the tissue sample with an antibody that specifically binds to PTK7, (b) A step of determining the total number of evaluable invasive tumor cells or cancer cells stained with anti-PTK7 in at least a portion of the tissue sample, and determining the total number of stained evaluable invasive tumor cells or cancer cells and evaluable invasive tumor cells or cancer cells that were not stained, wherein if an invasive tumor cell or cancer cell shows cell staining with anti-PTK7 at any intensity exceeding a predetermined threshold, the invasive tumor cell or cancer cell is counted as positively stained with anti-PTK7. (c) A step of determining a tumor percentage score (TPS), wherein the tumor percentage score (TPS) is obtained by dividing the number of PTK7-stained evaluable invasive tumor cells or cancer cells found in the tissue sample by the total number of stained evaluable invasive tumor cells or cancer cells and unstained evaluable invasive tumor cells or cancer cells, and multiplying by 100. A method that includes this.

2. The predetermined threshold is A positive staining intensity of 1+ or higher, as evaluated at low magnification, A positive staining intensity of 2+ or higher, as evaluated at medium magnification, Evaluation of 3+ positive staining intensity at high magnification The method according to claim 1, including the method described in claim 1.

3. The method according to claim 2, wherein the low magnification is at least about 4 times, the medium magnification is at least about 10 times, and the high magnification is at least about 20 times or at least about 40 times.

4. The steps include evaluating the presence of tumor cells or cancer cells stained with anti-PTK7 at an arbitrary intensity exceeding the predetermined threshold in at least a portion of the tissue sample at the low magnification, The step of determining a positive staining intensity of 1 or more, which exceeds the predetermined threshold, among the total number of stained and evaluable tumor cells or cancer cells in the tissue sample at the aforementioned intermediate magnification. The method according to claim 2 or 3, further comprising:

5. The method according to claim 4, further comprising the step of determining, at the high magnification, a positive staining intensity of 1 or more that exceeds the predetermined threshold among the total number of stained and evaluable tumor cells or cancer cells in the tissue sample.

6. At the aforementioned low magnification, (a) A step of evaluating the areas of strong staining of the interstitial tissue and necrotic tissue by anti-PTK7 in the tissue sample, or (b) A step of determining the distribution of evaluable tumor cells, cancer cells, and non-tumorous tissue in the stained tissue sample, or (c) A step to identify the presence of evaluable tumor cells or cancer cells with a staining intensity of 2+ or higher. The method according to any one of claims 2 to 5, further comprising:

7. In the aforementioned medium magnification or high magnification, (a) A step of distinguishing between positive staining with anti-PTK7 in the cell membrane and positive staining with anti-PTK7 in the cytoplasm of the total number of evaluable tumor cells or cancer cells in the tissue sample, or (b) A step of distinguishing between 1+positive staining intensity, 2+positive staining intensity, and 3+positive staining intensity from the total number of evaluable tumor cells or cancer cells in the tissue sample, or (c) A step of distinguishing between the staining intensity of 0-positive cells and 1+-positive cells in the tissue sample. The method according to any one of claims 2 to 6, further comprising:

8. (a) At the high magnification, (i) confirm the total number of evaluable tumor cells or cancer cells in the tissue sample having a staining intensity of 2+ or higher and a high level of cytoplasmic staining by comparing positive staining with anti-PTK7 in the cell membrane with positive staining with anti-PTK7 in the cytoplasm, or (ii) confirm the total number of evaluable tumor cells or cancer cells in the tissue sample having a staining intensity of 1+ or higher by comparing cell membrane staining with cytoplasmic staining, or (b) The steps of confirming the distribution of 0 cell staining intensity, 1+ positive cell staining intensity, 2+ positive cell staining intensity, and 3+ positive cell staining intensity in the tissue sample at the low magnification, and optionally adjusting the tumor percentage score based on the confirmation. The method according to claim 7, further comprising:

9. (a) The step of determining whether the tissue sample includes a tissue section and whether the tissue section is sufficient to determine and score the expression level of the protein PTK7, and considering the tissue section to be sufficient for evaluation if there are approximately 100 or more evaluable invasive tumor cells or cancer cells present. (b) If invasive tumor cells or cancer cells show cell membrane staining with anti-PTK7 at any intensity of 1+ or higher, the invasive tumor cells or cancer cells are counted as positive for staining with anti-PTK7, (c) The steps of preparing a section or part of the tissue sample on a slide or equivalent, and staining the section or part of the tissue sample on the slide, (d) The antibody that specifically binds to PTK7 comprises a monoclonal mouse anti-PTK7 antibody, optionally comprising monoclonal mouse anti-PTK7 clone 6.60.1, optionally comprising substantially isolated or substantially purified monoclonal mouse anti-PTK7 clone 6.60.1, (e) (i) If the staining signal is clearly brown, or (ii) If the staining corresponds to the cell membrane, the step of considering that there is staining with anti-PTK7 of any intensity of 1+ or higher, (f) The method further includes the step of excluding from the calculation of the tumor percentage score (TPS) tumor cells or cancer cells having only cytoplasmic or nuclear staining, non-invasive neoplastic or carcinoma in situ cells, unassessable or necrotic tumor cells or cancer cells, apoptotic nuclei or nuclear debris, tumor cells or cancer cells in poorly preserved tissue regions, benign epithelial cells, non-tumor cells and / or lymphocytes, apoptotic cells, necrotic cells, cells that do not show the intended color, lymphocytes, and stromal cells, (g) The method further includes the steps of (i) determining that the tissue sample has diagnostically negative PTK7 expression when the tumor percentage score (TPS) is less than 75%, and (ii) determining that the tissue sample has diagnostically positive PTK7 expression when the tumor percentage score (TPS) is 75% or more. (h) The tissue sample comprises a formalin-fixed paraffin-embedded (FFPE) specimen, and optionally includes the step of preparing the tissue sample by a protocol that includes fixing sections of the tissue sample in approximately 10% neutral buffered formalin for approximately 6 to 72 hours. (i) The step in which the tumor or cancer is non-small cell lung cancer (NSCLC), breast cancer or mammary carcinoma, head and neck cancer, colorectal cancer, bladder cancer, lung cancer, gastrointestinal stromal tumor (GIST), prostate cancer, cervical cancer, ovarian cancer, or renal cell carcinoma, (j) The step in which the tissue sample is a biopsy sample, a needle biopsy sample, a sample derived from a needle biopsy sample, aspiration, a cytological specimen, or demineralized bone. The method according to any one of claims 1 to 8, comprising one or more of the above.

10. A method for diagnosing or selecting an individual who is eligible for treatment with cancer or tumor therapy, comprising the step of determining and scoring the amount of nuclear protein PTK7 (also known as colon cancer kinase 4 (CCK4)) in a tissue sample derived from the individual using the IHC method described in any one of claims 1 to 22, wherein if the tissue sample is determined to have high or diagnostically positive PTK7 expression, the individual is diagnosed or selected as a target for treatment with cancer therapy in which the individual is likely to respond favorably.

11. The method according to claim 10, wherein the tumor or cancer is non-small cell lung cancer (NSCLC), breast cancer or mammary carcinoma, head and neck cancer, colorectal cancer, bladder cancer, lung cancer, gastrointestinal stromal tumor (GIST), prostate cancer, cervical cancer, or renal cell carcinoma.

12. A method for diagnosing or selecting individuals who are eligible for treatment using cancer or tumor therapies, (a) The step of determining whether the PTK7 expression score (TPS) of cells in the tissue sample derived from the individual requiring it is low or high, or diagnostically negative or diagnostically positive, so as to be determined by a protocol comprising the use of an immunohistochemical (IHC) method according to any one of claims 1 to 24, (b) If the tissue sample is found to have a high or diagnostically positive PTK7 expression score (TPS), the individual is diagnosed or selected as eligible for treatment with cancer or tumor therapy. A method that includes this.

13. The method according to claim 12, wherein the cancer or tumor treatment method is a non-small cell lung cancer (NSCLC) treatment method or a lung cancer treatment method, or the cancer or tumor treatment method is a pharmaceutical preparation containing an antibody-drug conjugate, a radionuclide, a gallium-68 radionuclide, or a fluorine-18 radionuclide.

14. The method according to claim 12 or 13, wherein the individual is a patient having lung cancer or non-small cell lung cancer.

15. The method according to any one of claims 12 to 14, wherein determining the number of PTK7-positive evaluable tumor cells or cancer cells includes the step of determining the number of evaluable tumor cells or cancer cells having PTK7 staining at the cell membrane, or determining the number of PTK7-positive evaluable tumor cells or cancer cells includes the step of determining the number of evaluable tumor cells or cancer cells having cell membrane PTK7 staining of a higher intensity than cytoplasmic PTK7 staining.

16. (a) A step in which, if the tumor percentage score (TPS) exceeds a threshold, it is determined that the individual is likely to respond favorably to treatment with cancer therapy, wherein the threshold is optionally any number between approximately 50% and 75%, or approximately 50% and 90%, or (b) If the tumor percentage score (TPS) exceeds the threshold, the individual is selected as a target for the cancer treatment. The method according to any one of claims 12 to 15, further comprising:

17. A kit comprising an antibody that specifically binds to PTK7, and a scoring guideline comprising the method according to any one of claims 1 to 16, further comprising optionally an image showing multiple PTK7 staining levels.

18. A kit comprising a scoring guideline comprising the method according to any one of claims 1 to 16, and optionally further comprising images showing multiple PTK7 staining levels.

19. A method for evaluating the degree of PTK7 expression, comprising the steps of: contacting a sample or a portion thereof containing cancer cells or tumor cells derived from an individual with an antibody or a portion thereof that specifically binds to PTK7; and determining a tumor percentage score (TPS) by dividing the number of PTK7-stained evaluable tumor cells or cancer cells in the sample or a portion thereof to which the antibody has specifically bound by the total number of stained evaluable cancer cells or tumor cells and evaluable cancer cells or tumor cells that have not been stained, and multiplying the result by 100.

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