Immunohistochemical (IHC) PTK7 scoring regimens and methods for adjunctive cancer therapy

Abstract

In alternative embodiments, provided are immunohistochemical (IHC) methods and kits 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), in a tissue sample; hER2 or a receptor tyrosine protein kinase erbB-2, or a cluster of differentiation 340 (CD340); a programmed death ligand 1 (PD-L1) or a differentiation cluster 274 (CD274); a B7 homolog 1 (B7-H1); and Ki-67 or MKI67 (a proliferation marker Ki-67), as well as a use thereof. In alternative embodiments, methods and kits are provided for diagnosing or selecting an individual eligible for treatment with a cancer or tumor therapeutic agent, or assessing the risk of cancer or tumor recurrence using an IHC method as provided herein. In alternative embodiments, kits comprising components and instructions for performing the methods as provided herein are provided. Described herein are methods for scoring PTK7 expression and utilizing the score as a companion or supplemental diagnosis to help treat or ameliorate cancer or tumor.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This U.S. Patent Application claims priority benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application Serial Number (USSN) 63 / 453,890, filed March 22, 2023. The aforementioned application is expressly incorporated by reference herein in its entirety and for all purposes. TECHNICAL FIELD

[0003] The present invention relates generally to cancer treatment, companion or complementary diagnostics, and immunohistochemical methods. In alternative embodiments, immunohistochemical (IHC) methods and kits are provided for reproducibly determining the extent of expression of, and scoring, 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 (proliferation marker Ki-67) in a tissue sample. In alternative embodiments, methods and kits are provided for diagnosing or selecting individuals eligible for treatment with a cancer or tumor therapeutic, or assessing the risk of cancer or tumor recurrence using the IHC methods as provided herein. In alternative embodiments, kits comprising components and instructions for carrying out the methods as provided herein are provided. The present application describes methods for scoring PTK7 expression and utilizing the score as a companion or complementary diagnostic to aid in the treatment or amelioration of cancer or tumors. BACKGROUND

[0004] Protein tyrosine-protein kinase-like 7 (PTK7) (also known as Colon carcinoma kinase 4 (CCK4)) is a receptor tyrosine kinase encoded in humans by the PTK7 gene. PTK7 is expressed in a variety of cancers and tumors; for example, in lung cancer and non-small cell lung cancer (NSCLC), and has been found to contribute to the progression of the cancers and tumors. PTK7 overexpression has been found to be a candidate biomarker for predicting the occurrence and prognosis of several types of cancer, including stages I-IV hepatocellular carcinoma (HCC), invasive breast cancer, cervical cancer, colorectal cancer, and thyroid cancer.

[0005] Manual scoring of qualitative immunohistochemistry (IHC) assays is described in the pathology scoring manual for the companion diagnostic (CDx) assay. These scoring algorithms rely on subjective assessment of both the average IHC intensity and the total percentage of positive tumor staining. These methods cannot be accurately applied to intensity-based cutoffs, which require a score of positive tumor percentage at a specific intensity on a scale of 0-3. There remains a need in the art for more objective and accurate scoring methods to assess PTK7 expression in tissue samples. SUMMARY

[0006] In alternative embodiments, there are provided immunohistochemistry (IHC) methods for determining the degree of expression of and scoring in a tissue sample: protein tyrosine

[0007] (a) staining the tissue sample with an antibody that specifically binds PTK7; and

[0008] (b) determining the total number of viable invasive tumor or cancer cells with anti-PTK7 staining and determining the total number of stained and unstained viable invasive tumor or cancer cells in at least a portion of the tissue sample,

[0009] wherein the invasive tumor or cancer cells are counted as anti-PTK7 positive staining if they show anti-PTK7 cell staining at any intensity above a defined threshold, and

[0010] (c) determining a tumor proportion score (TPS),

[0011] wherein the tumor proportion score (TPS) is the number of PTK7 stained viable invasive tumor or cancer cells found in the tissue sample divided by the total number of stained and unstained viable invasive tumor or cancer cells, multiplied by 100.

[0012] In alternative embodiments of the methods provided herein:

[0013] - the defined threshold comprises:

[0014] a positive staining intensity of 1+ or greater evaluated at low magnification;

[0015] a positive staining intensity of 2+ or greater evaluated at medium magnification; or

[0016] 3+ positive staining intensity evaluated at high magnification;

[0017] - 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;

[0018] - the method further comprises:

[0019] evaluating the presence of tumor or cancer cells with any intensity above the defined threshold of anti-PTK7 staining in at least a portion of the tissue sample at the low magnification; and

[0020] determining 1+ or greater positive staining intensity of the total number of stained viable tumor or cancer cells in the tissue sample above the defined threshold at the medium magnification;

[0021] - the method further comprises determining 1+ or greater positive staining intensity of the total number of stained viable tumor or cancer cells in the tissue sample above the defined threshold at the high magnification;

[0022] - the method further comprises at the low magnification: (a) evaluating intense anti-PTK7 staining regions of stroma and necrosis in the tissue sample, (b) determining the distribution of stained viable tumor cells, cancer cells, and non-tumor tissue in the tissue sample; or (c) identifying the presence of viable tumor or cancer cells with 2+ or greater staining intensity;

[0023] - the method further comprises at the medium magnification or the high magnification: (a) distinguishing cell membrane positive anti-PTK7 staining from cytoplasmic anti-PTK7 positive staining in the total number of viable tumor or cancer cells in the tissue sample; (b) distinguishing 1+, 2+, or 3+ positive staining intensity in the total number of viable tumor or cancer cells in the tissue sample; or (c) distinguishing 0 staining from 1+ positive cell staining intensity in the tissue sample;

[0024] - the method further comprises at the high magnification: (a) confirming cell membrane positive anti-PTK7 staining from cytoplasmic positive anti-PTK7 staining for the total number of viable tumor or cancer cells in the tissue sample with 2+ or greater staining intensity and a significant level of cytoplasmic staining; or (b) confirming cell membrane staining from cytoplasmic staining for the total number of viable tumor or cancer cells in the tissue sample with 1+ or greater staining intensity;

[0025] - the method further comprises: (a) confirming the distribution of 0, 1+, 2+, and 3+ positive cell staining intensities in the tissue sample at the low magnification; optionally, adjusting the tumor proportion score based on the confirmation;

[0026] - the tissue sample comprises a tissue section, and confirming whether the tissue section is sufficient for determining the amount of expression of the protein PTK7 and scoring it, and the tissue section is considered sufficient for evaluation if there are about 100 or more viable invasive tumor or cancer cells;

[0027] - if an invasive tumor or cancer cell shows anti-PTK7 cell membrane staining at any intensity of 1+ or greater, the invasive tumor or cancer cell is counted as anti-PTK7 positive staining;

[0028] preparing a section or portion of the tissue sample on a slide or equivalent, and staining the section or portion of the tissue sample on the slide;

[0029] - the antibody that specifically binds PTK7 comprises a monoclonal mouse anti-PTK7 antibody; or the anti-PTK7 antibody comprises monoclonal mouse anti-PTK7 clone 6.60.1; or the anti-PTK7 antibody comprises a substantially isolated or substantially purified monoclonal mouse anti-PTK7 clone 6.60.1;

[0030] - there is anti-PTK7 staining at any intensity of 1+ or greater if: (a) the staining signal is definitively brown, or (b) the staining corresponds to a cell membrane;

[0031] - the method further comprises excluding from the calculation of the tumor proportion score (TPS): tumor or cancer cells with cytoplasmic or nuclear staining only; non-invasive tumor or carcinoma cells, non-viable or necrotic tumor or cancer cells, apoptotic nuclei or nuclear debris, tumor or cancer cells in poorly preserved tissue areas, benign epithelial cells, non-tumor cells with nuclear staining and / or lymphocytes, apoptotic cells, necrotic cells, cells that do not exhibit the expected color, lymphocytes, and stromal cells;

[0032] - the method further comprises (i) if the tumor proportion score (TPS) is less than (<) 75%, the tissue sample is determined to have diagnostic negative PTK7 expression; and (ii) if the tumor proportion score (TPS) is greater than or equal to (>) 75%, the tissue sample is determined to have diagnostic positive PTK7 expression;

[0033] - the tissue sample comprises a formalin-fixed, paraffin-embedded (FFPE) specimen; or the section of the tissue sample is prepared by a protocol comprising fixation in about 10% neutral buffered formalin for about 6 to 72 hours;

[0034] - 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

[0035] - the tissue sample is a biopsy sample, or is or is derived from a punch biopsy sample, fine needle aspirate, cytological specimen, or bone decalcification.

[0036] In alternative embodiments, there is provided a method for diagnosing or selecting an individual eligible for treatment with a cancer or tumor therapeutic, the method comprising determining the amount of nuclear protein PTK7 (also known as colon carcinoma kinase 4 (CCK4)) in a tissue sample from the individual using an IHC method as provided herein and scoring it, wherein if the tissue sample is determined or scored as having high or diagnostic positive PTK7 expression, the individual is eligible for treatment with a cancer therapeutic to which the individual is likely to respond well.

[0037] In alternative embodiments of the methods herein, 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, or renal cell carcinoma.

[0038] In alternative embodiments, there is provided a method for diagnosing or selecting an individual eligible for treatment with a cancer or tumor therapeutic, the method comprising: (a) determining whether cells in a tissue sample from an individual in need thereof have a low or high, or diagnostic negative or diagnostic positive PTK7 expression score (%), as determined by a protocol comprising use of an immunohistochemistry (IHC) method as set forth in accordance with the embodiments herein; and (b) diagnosing or selecting the individual as eligible for treatment with the cancer or tumor therapeutic if the tissue sample is found to have a high or diagnostic positive PTK7 expression score (TPS).

[0039] In alternative embodiments of the methods for diagnosing or selecting an individual eligible for treatment with a cancer or tumor therapeutic:

[0040] - the cancer or tumor therapeutic is a non-small cell lung cancer (NSCLC) therapeutic or a lung cancer therapeutic;

[0041] - the cancer or tumor therapeutic is a pharmaceutical formulation comprising an antibody drug conjugate, a radionuclide, a gallium-68 radionuclide, or a fluorine-18 radionuclide;

[0042] - the individual is a patient having lung cancer or non-small cell lung cancer;

[0043] - the method comprises determining the number of PTK7-positive live tumor or cancer cells, the method comprising determining the number of live tumor or cancer cells having PTK7 staining in the cell membrane;

[0044] - the method comprises determining the number of PTK7-positive live tumor or cancer cells, the method comprising determining the number of live tumor or cancer cells having PTK7 staining in the cell membrane that is of higher intensity than cytoplasmic PTK7 staining;

[0045] - the method further comprises determining that the individual is likely to respond well to treatment with a cancer therapeutic if the tumor proportion score (TPS) is above a threshold value, and optionally the threshold value is about 50 TPS or more, about 75% or more, or about any number between 50% and 90%; and / or

[0046] - the method further comprises selecting the individual for administration of the cancer therapeutic if the tumor proportion score (TPS) is above the threshold value.

[0047] In alternative embodiments, there is provided a kit comprising an antibody that specifically binds PTK7 and a scoring guide comprising a method as provided herein or for use in a method as provided herein, e.g., for use in a method as provided herein for treating or ameliorating a cancer or tumor in an individual in need thereof.

[0048] In alternative embodiments, there is provided a kit comprising a scoring guide comprising a method as provided herein or for use in a method as provided herein, e.g., as used in a method as provided herein for diagnosing or selecting an individual eligible for treatment with a cancer or tumor therapeutic.

[0049] In alternative embodiments of a kit as provided herein, the kit further comprises images indicative of a plurality of PTK7 staining levels.

[0050] In alternative embodiments, methods for assessing the degree of PTK7 expression are provided, the methods comprising: contacting a sample or portion thereof from an individual comprising cancer or tumor cells with an antibody or portion thereof that specifically binds PTK7; and determining a tumor proportion score (TPS) by dividing the number of viable tumor or cancer cells stained for PTK7 by the antibody in the sample or portion thereof by the total number of stained and unstained viable cancer or tumor cells and multiplying the result by 100, thereby obtaining the tumor proportion score (TPS).

[0051] The details of one or more exemplary embodiments of the application are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the application will be apparent from the description and drawings, and from the claims.

[0052] All publications, patents, patent applications cited herein are hereby expressly incorporated by reference in their entirety for all purposes. BRIEF DESCRIPTION OF DRAWINGS

[0053] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0054] The drawings described herein are illustrative of exemplary embodiments provided herein and are not intended to limit the scope of the application as encompassed by the claims.

[0055] FIG. 1 illustrates images of 0-3+ intensity examples from non-small cell lung cancer (NSCLC), tumor tissue stained with PTK7 showing membrane staining, as described in further detail in Example 1 below.

[0056] FIG. 2 provides exemplary images of low, medium, and high examples at each staining intensity. These images are intended as a guide and are not intended to represent the full range of staining variation within each intensity category (0-3+), as described in further detail in Example 1 below.

[0057] Figure 3 A T&T (training and testing) inter-observer variability plot is illustrated: the x-axis illustrates 30 tissue blocks scored by four observers or pathologists, as described in further detail in Example 1 below.

[0058] FIG. 4A illustrates a visual summary of the various FOVs used in this exemplary protocol for use in IHC review, and FIG. 4B illustrates exemplary FOVs as overlaid on images of tissue sections, as described in further detail in Example 1 below.

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

[0060] In an alternative embodiment, immunohistochemistry (IHC) methods and kits are provided for reproducibly determining the extent of expression and scoring of the following in a tissue sample: protein tyrosine kinase-like 7 (PTK7), also known as colon cancer kinase 4 (CCK4); HER2 or receptor tyrosine 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 (proliferation marker Ki-67). In an alternative embodiment, scoring methods are provided to assess PTK7 expression in tumors or cancers. Certain aspects include using the scoring methods provided herein to assess PTK7 expression and determine whether a high or diagnostically positive tumor proportion score is present.

[0061] Alternative embodiments provide methods and kits for diagnosing or selecting individuals eligible for treatment with cancer or tumor therapeutics. In certain embodiments, if a tissue sample is determined or scored as having a high or diagnostic positive tumor proportion score, the patient is diagnosed or selected for treatment with a cancer therapeutic to which the patient is likely to respond well.

[0062] In alternative embodiments, methods and kits are provided for diagnosing, treating, or ameliorating or assessing the risk of cancer or tumor recurrence using the IHC methods as provided herein.

[0063] PTK7 has been found to be expressed in and contribute to the progression of various cancers and tumors. Such cancers include malignancies with a poor prognosis, including lung cancer and non-small cell lung cancer (NSCLC). PTK7 overexpression has been found to be a candidate biomarker for predicting the development and prognosis of several types of cancer, not only lung cancer and NSCLC, but also stage I-IV hepatocellular carcinoma (HCC), invasive breast cancer, cervical cancer, colorectal cancer, and thyroid cancer. The expression pattern of PTK7 in cancers and tumors compared to normal tissues makes it an attractive target for the development of diagnostic assays.

[0064] Currently available CDx scoring guidelines contain formulas for interpreting IHC staining in tissue samples. These formulas are written as equations, such as CPS (number of PTK7-positive cells (tumor cells and immune cells) / number of total tumor cells). However, they do not describe how to use the numerator and denominator to determine the CPS score for the entire slide. Pathologists often ask what method they should follow to obtain the correct score, but there is no structured approach to addressing this question.

[0065] Attempts to obtain correct IHC scores include using IHC stain color and / or thickness to assess intensity; however, drawbacks of such methods include: (1) color (i.e., dark brown, brown, light brown) is highly subjective, depends on scoring method (i.e., glass slide visual scoring versus digital scoring), and is only applicable to non-intensity-based scoring algorithms; and (2) thickness is typically specific to membrane staining and is not applicable to all cancer indications and biomarkers.

[0066] There is a need in the art not only for robust, reliable, reproducible, objective, and accurate PTK7 scoring methods, but also for scoring methods with greater simplicity and efficiency. Embodiments of the immunohistochemistry (IHC) methods herein can provide considerable advantages for reliable and effective diagnostic evaluation of the extent of PTK7 expression in a tissue sample.

[0067] Embodiments of immunohistochemistry (IHC) methods

[0068] Embodiments herein relate to immunohistochemistry methods for determining and scoring the extent of protein PTK7 expression in a tissue sample. In various embodiments, the methods comprise: staining a tissue sample with an antibody that specifically binds PTK7; determining the total number of live invasive tumor or cancer cells with anti-PTK7 staining, determining the total number of stained and unstained live invasive tumor or cancer cells in at least a portion of the tissue sample, wherein if an invasive tumor or cancer cell exhibits anti-PTK7 cell staining at any intensity above a defined threshold, the invasive tumor or cancer cell is counted as anti-PTK7 positively stained; and determining a tumor proportion score (TPS), wherein the tumor proportion score (TPS) is the number of PTK7 stained live invasive tumor or cancer cells found in the tissue sample divided by the total number of stained and unstained live invasive tumor or cancer cells, multiplied by 100. Such embodiments can not only provide robust, reliable, reproducible, and accurate PTK7 scoring methods, but also scoring methods with greater objectivity, simplicity, and efficiency. Such embodiments can provide considerable advantages for reliable and effective diagnostic evaluation.

[0069] In certain embodiments, 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.

[0070] Various embodiments provide a method of systematic microscopy for obtaining correct biomarker scores using fields of view and changing magnification. Guidance based on magnification is an objective method of assessing IHC intensity. Standardization of magnification of the microscope allows the observer and / or pathologist to follow a specific and unique sequence of steps to ensure that they accurately evaluate the staining intensity of 0-3. Such embodiments provide a method for microscopic scoring that can have the advantage of being highly reproducible.

[0071] In certain embodiments, defining the threshold comprises: a positive staining intensity of 1+ or greater evaluated at a low magnification; a positive staining intensity of 2+ or greater evaluated at a medium magnification; or a 3+ positive staining intensity evaluated at a high magnification. In some embodiments, 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.

[0072] In certain embodiments, the positive staining is determined using a brightfield 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.

[0073] In certain embodiments, the method further comprises: evaluating the presence of tumor or cancer cells with any intensity above a defined threshold staining for anti-PTK7 in at least a portion of the tissue sample at a low magnification; and determining a positive staining intensity of 1+ or greater above a defined threshold of the total number of stained viable tumor or cancer cells in the tissue sample at a medium magnification. In some embodiments, the method comprises determining a positive staining intensity of 2+ or greater at a medium magnification. In certain embodiments, the method further comprises: determining a positive staining intensity of 1+ or greater above a defined threshold of the total number of stained viable tumor or cancer cells in the tissue sample at a high magnification. In some embodiments, the method comprises determining a positive staining intensity of 2+ or greater at a high magnification.

[0074] In certain embodiments, the method further comprises: evaluating areas of intense anti-PTK7 staining of stroma and necrosis in the tissue sample at a low magnification; determining the distribution of viable tumor cells, cancer cells, and non-tumor tissue in the stained tissue sample; or identifying the presence of viable tumor or cancer cells with a staining intensity of 2+ or greater at a low magnification.

[0075] Certain embodiments further comprise distinguishing between cell membrane positive anti-PTK7 staining and cytoplasmic anti-PTK7 positive staining in the total number of viable tumor or cancer cells in the tissue sample at medium or high magnification. Such embodiments can provide the advantage of increasing the accuracy and reproducibility of the PTK7 scoring method by distinguishing between cells with different structural staining patterns or excluding stained cells with certain structures or features from the tumor proportion score determination.

[0076] Other embodiments of the methods herein further comprise distinguishing between 1+, 2+, or 3+ positive staining intensities in the total number of viable tumor or cancer cells in the tissue sample; or distinguishing between 0 staining and 1+ positive cell staining intensities in the tissue sample. Such embodiments can provide the benefit of multifunctionality in defining threshold staining intensities.

[0077] In certain embodiments, the methods further comprise confirming cell membrane and cytoplasmic positive anti-PTK7 staining for the total number of viable tumor or cancer cells in the tissue sample having a 2+ or greater staining intensity and a significant level of cytoplasmic staining at high magnification. Certain embodiments further comprise confirming cell membrane staining and cytoplasmic staining for the total number of viable tumor or cancer cells in the tissue sample having a 1+ or greater staining intensity. In some embodiments, a significant level of cytoplasmic staining comprises a cytoplasmic PTK7 staining level equal to or greater than the membrane PTK7 staining level.

[0078] In certain embodiments, the methods further comprise confirming the distribution of 0, 1+, 2+, and 3+ positive cell staining intensities in the tissue sample at low magnification. Certain embodiments optionally comprise adjusting the tumor proportion score based on the confirmation.

[0079] In certain embodiments, invasive tumor or cancer cells are counted as anti-PTK7 positive staining if they show anti-PTK7 cell membrane staining at any intensity of 1+ or higher.

[0080] In certain embodiments, anti-PTK7 staining at any intensity of 1+ and higher is determined when the staining signal is unequivocally brown. In certain embodiments, anti-PTK7 staining at any intensity of 1+ and higher is determined when the staining corresponds to the cell membrane.

[0081] In certain embodiments, the method further comprises excluding from the calculation of the tumor proportion score (TPS): tumor or cancer cells with only cytoplasmic or nuclear staining; non-invasive tumor or carcinoma cells, non-viable or necrotic tumor or carcinoma cells, apoptotic nuclei or nuclear debris, tumor or carcinoma cells in poorly preserved tissue areas, benign epithelial cells, non-tumor cells with nuclear staining and / or lymphocytes, apoptotic cells, necrotic cells, cells that do not exhibit the expected color, lymphocytes, and stromal cells. Such embodiments can provide the advantage of increasing the accuracy and reproducibility of the PTK7 scoring method by excluding stained cells with certain structures or characteristics from the tumor proportion score determination.

[0082] In certain embodiments of the methods herein, a tissue sample is determined to have diagnostic negative PTK7 expression if the tumor proportion score (TPS) is less than (<) 75%. In certain embodiments, a tumor proportion score of less than 70% indicates diagnostic negative PTK7 expression. In certain embodiments, a tumor proportion score of less than 60% indicates diagnostic negative PTK7 expression. In certain embodiments, a tissue sample is determined to have diagnostic positive PTK7 expression if the tumor proportion score (TPS) is greater than or equal to (>) 75%. In certain embodiments, a tumor proportion score of greater than 75% indicates diagnostic positive PTK7 expression. In certain embodiments, a tumor proportion score of greater than 80% indicates diagnostic positive PTK7 expression. In certain embodiments, a tumor proportion score of greater than 85% indicates diagnostic positive PTK7 expression.

[0083] In certain embodiments, the antibody that specifically binds PTK7 comprises a monoclonal mouse anti-PTK7 antibody. In certain embodiments, the anti-PTK7 antibody comprises monoclonal mouse anti-PTK7 clone 6.60.1. In certain embodiments, the anti-PTK7 antibody comprises a substantially isolated or substantially purified monoclonal mouse anti-PTK7 clone 6.60.1.

[0084] In certain aspects of the methods herein, the tissue sample comprises a tissue section; in certain embodiments, the tissue section is determined to be sufficient for determining the amount of expression of the protein PTK7 and scoring it. In certain embodiments, a tissue section is considered sufficient for evaluation if there are about 100 or more viable invasive tumor or carcinoma cells. In certain embodiments, a tissue section is considered sufficient for evaluation if there are about 200 or more viable invasive tumor or carcinoma cells. In certain embodiments, a tissue section is considered sufficient for evaluation if there are about 500 or more viable invasive tumor or carcinoma cells.

[0085] In various embodiments, a section or portion of a tissue sample is prepared on a slide or equivalent and the section or portion of the tissue sample is stained on the slide. In certain embodiments, a section or portion of a tissue sample is prepared on a slide, microscope slide, or equivalent and the section or portion of the tissue sample is stained on the slide. In certain embodiments, the tissue sample comprises a formalin-fixed, paraffin-embedded (FFPE) specimen. In certain embodiments, the FFPE specimen comprises a cancer specimen that is stained on an automated IHC platform. In certain embodiments, the section of the tissue sample is prepared by a protocol comprising fixation in about 10% neutral buffered formalin for a period of about 6 to 72 hours. In certain embodiments, the tissue sample is a biopsy sample, a needle biopsy sample, a sample derived from a needle biopsy sample, a fine needle aspirate, a cytological specimen, or a decalcified bone.

[0086] Embodiments of methods for diagnosing tumors or cancers

[0087] Embodiments herein provide methods of diagnosing a tumor or cancer by determining whether a tissue sample is positive for expression of PTK7. In various aspects, the methods comprise determining a PTK7 diagnostic status in a tissue sample by using an IHC method for determining the degree of cell membrane expression of PTK7 as provided herein, wherein the method can comprise determining whether PTK7 staining is at a defined threshold, which can comprise: a positive staining intensity of 1+ or greater assessed at low magnification; a positive staining intensity of 2+ or greater assessed at medium magnification; or a 3+ positive staining intensity assessed at high magnification. Such embodiments can provide the benefit of versatility in the aspect of staining intensity of the defined threshold. In alternative embodiments, a tissue sample is determined to have a diagnostic negative PTK7 expression if the tumor proportion score (TPS) is less than (<) 75%, and a tissue sample is determined to have a diagnostic positive PTK7 expression if the tumor proportion score (TPS) is greater than or equal to (>) 75%. Such embodiments can provide the benefit of accuracy in the determination of diagnostic status related to PTK7 expression.

[0088] In certain embodiments, 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, or renal cell carcinoma.

[0089] Embodiments herein provide methods for diagnosing or selecting an individual eligible for treatment with a cancer or tumor therapeutic. In various embodiments, such a method comprises determining the amount of nuclear protein PTK7 (also known as colon carcinoma kinase 4 (CCK4)) in a tissue sample from an individual using an IHC method according to the embodiments provided herein and scoring it, wherein if the tissue sample is determined or scored as having high or diagnostic positive PTK7 expression, the individual is diagnosed or selected for treatment with a cancer therapeutic to which the individual is likely to respond well. In certain embodiments, 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, or renal cell carcinoma.

[0090] Embodiments herein provide methods for diagnosing or selecting an individual eligible for treatment with a cancer or tumor therapeutic. In various embodiments, such a method comprises: (a) determining whether cells in a tissue sample from an individual have a low or high, or diagnostic negative or diagnostic positive PTK7 expression score (TPS) as determined by a protocol comprising use of an immunohistochemistry (IHC) method as shown in various embodiments herein; and (b) diagnosing or selecting the individual as eligible for treatment with a cancer or tumor therapeutic if the tissue sample is found to have a high or diagnostic positive PTK7 expression score (TPS). In certain embodiments, the cancer or tumor therapeutic is a non-small cell lung cancer (NSCLC) therapeutic or a lung cancer therapeutic. In certain embodiments, the cancer or tumor therapeutic 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 having lung cancer or non-small cell lung cancer.

[0091] In certain embodiments, determining the number of live tumor or cancer cells that are PTK7 positive comprises determining the number of live tumor or cancer cells having PTK7 staining in the cell membrane. In certain embodiments, determining the number of live tumor or cancer cells that are PTK7 positive comprises determining the number of live tumor or cancer cells having PTK7 staining in the cell membrane that is of higher intensity than cytoplasmic PTK7 staining.

[0092] In certain embodiments, such a method further comprises determining that the individual is likely to respond well to treatment with a cancer therapeutic if the tumor proportion score (TPS) is above a threshold value. In certain embodiments, the threshold value is about 50% or more, about 75% or more, or about any number between 50% and 90%. In some aspects, the method further comprises selecting the individual for administration of a cancer therapeutic if the tumor proportion score (TPS) is above a threshold value.

[0093] Embodiments of methods for treating cancers and tumors

[0094] Provided are methods for treating or ameliorating a tumor or cancer in a patient, the methods comprising determining the amount of PTK7 in a tissue sample from the patient using a method as provided herein and scoring it, wherein if the tissue sample is determined or scored as having a high or diagnostic positive tumor proportion score, the patient is treated with a cancer therapeutic or anti-cancer therapy to which the patient is likely to respond well.

[0095] In certain embodiments, 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, or renal cell carcinoma.

[0096] In certain embodiments, the cancer therapeutic 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; ablation techniques, such as radiofrequency ablation (RFA), cryoablation, and / or microwave ablation; and / or radiation therapy, such as stereotactic body radiation therapy.

[0097] In certain embodiments, the anti-cancer therapy comprises an antibody drug conjugate, a small molecule therapy, an immunotherapy, a monoclonal antibody therapy, an adoptive cell therapy, a T cell receptor therapy, or a chimeric antigen receptor (CAR) T cell therapy. In alternative embodiments, the anti-cancer treatment or therapy comprises: a tyrosine kinase inhibitor (optionally erlotinib (or TARCEVA™), gefitinib (or IRESSA™), afatinib (or GILOTRIF™), or osimertinib (TAGRISSO™)); nectinumumab (or PORTRAZZA™), pembrolizumab (or KEYTRUDA™), nivolumab (or OPDIVO™), ipilimumab (YERVOY™), cetuximab (or ERBITUX™), cisplatin (or PLATINOL™), or carboplatin (or PARAPLATIN™).

[0098] In alternative embodiments, the anti-cancer treatment or therapy comprises the use of an anti-cancer drug, which can comprise an antibody that specifically or substantially binds to a cancer or tumor, wherein the antibody is conjugated to a cytotoxic agent, and optionally the cytotoxic agent comprises a radionuclide (optionally yttrium-90, iodine-131, lutetium-177, radium-223 chloride, strontium-89 chloride, or samarium-153 EDTMP), diphtheria toxin, Pseudomonas aeruginosa exotoxin A, denileukin diftitox (or ONTAK™), muromonab-CD3 (or LUMOXITI™), inotuzumab ozogamicin (or BESPONSA™), calicheamicin or N-acetyl-gamma-calicheamicin, maytansinol or DM1, maytansine or a derivative thereof, mertansine, ravtansine, soravtansine, or soravtansinumab, SN-38 (or 7-ethyl-10-hydroxy camptothecin), or auristatin or monomethyl auristatin E (MMAE).

[0099] Immunohistochemistry

[0100] In alternative embodiments, the methods as provided herein can use or comprise reagents for detecting or visualizing antibody-antigen interactions using any products or methods known in the art (e.g., IHC protocols or reagents).

[0101] In alternative embodiments, the immunohistochemical methods and / or reagents used with the methods and articles of manufacture or kits as provided herein can comprise or include or comprise the use of any IHC protocol, IHC medical device, apparatus, and / or image or data analysis system to implement IHC or IHC reagents known in the art.

[0102] In alternative embodiments, the antibodies, antigen-binding fragments thereof, or monomeric or dimeric antigen-binding proteins as provided herein (including, e.g., synthetic or recombinant forms) used in IHC protocols or kits as provided herein are substantially purified or isolated, or in the form of unpurified or partially purified culture supernatants.

[0103] In alternative embodiments, methods as provided herein include the use of chromogenic immunohistochemistry (CIH), in which a first antibody (e.g., a recombinant antibody (Ab) or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein as provided herein) or a second antibody (e.g., in which the second antibody binds to the first antibody or a recombinant antibody (Ab) or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein as provided herein) is conjugated to an enzyme (such as a peroxidase, e.g., an immunoperoxidase, e.g., horseradish peroxidase (HRP)) that can catalyze a chromogenic reaction. In alternative embodiments, a chromogenic moiety used in methods as provided herein is or includes coumarin; rhodamine; 2,3,6,7-tetrahydro-l l-oxo-lH,5H, 11H-[l]benzopyrano[6,7,8-ij]quinoline-l-0-carboxylic acid; 7-(diethylamino)coumarin-3-carboxylic acid; a coumarin derivative; a rhodamine derivative; tetramethylrhodamine; a diaryl rhodamine derivative; QSY 7; QSY 9; QSY 21; a diazonium chromophore; DABSYL; Erioglaucine; a triarylmethane compound; Fast Red; Fast Blue; Fuchsin; Cascade Blue Acetyl; Dapoxyl sulfonic acid / carboxylic acid succinimidyl ester; DY-405; Alexa Fluor 405 succinimidyl ester; Cascade Yellow succinimidyl ester; Pyridyloxyazole succinimidyl ester (PyMPO); Pacific Blue succinimidyl ester; DY-415; 7-hydroxy coumarin-3-carboxylic acid succinimidyl ester; DYQ-425; 6-FAM phosphoramidite; Lucifer Yellow; Iodoacetamide; Alexa Fluor 430 succinimidyl ester; Dabcyl succinimidyl ester; NBD chloride / flouride; QSY 35 succinimidyl ester; DY-485XL; Cy2 succinimidyl ester; DY-490; Oregon Green 488 carboxylic acid succinimidyl ester; Alexa Fluor 488 succinimidyl ester; BODIPY 493 / 503 C3 succinimidyl ester; DY-480XL; BODIPY FL C3 succinimidyl ester; BODIPY FL C5 succinimidyl ester; BODIPY FL-X succinimidyl ester; DYQ-505; Oregon Green 514 carboxylic acid succinimidyl ester; DY-510XL; DY-481XL; 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein succinimidyl ester (JOE); DY-520XL; DY-521XL; BODIPY R6G C3 succinimidyl ester; Erythrosin isothiocyanate; 5-carboxy-2',4',5',7'-tetrabromosulfonfluorescein succinimidyl ester; Alexa Fluor 532 succinimidyl ester;6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein succinimidyl ester (HEX); BODIPY 530 / 550 C3 succinimidyl ester; DY-530; BODIPY TMR-X succinimidyl ester; DY-555; DYQ-1; DY-556; Cy3 succinimidyl ester; DY-547; DY-549; DY-550; Alexa Fluor 555 succinimidyl ester; Alexa Fluor 546 succinimidyl ester; DY-548; BODIPY 558 / 568 C3 succinimidyl ester; Rhodamine Red-X succinimidyl ester; QSY 7 succinimidyl ester; BODIPY 564 / 570 C3 succinimidyl ester; BODIPY 576 / 589 C3 succinimidyl ester; carboxyrhodol (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; carboxynapthofluorescein succinimidyl ester; DY-605; DY-610; Alexa Fluor 610 succinimidyl ester; DY-615; BODIPY 630 / 650-X succinimidyl ester; erythrosin; Alexa Fluor 633 succinimidyl ester; Alexa Fluor 635 succinimidyl ester; DY-634; DY-630; DY-631; DY-632; DY-633; DYQ-2; DY-636; BODIPY 650 / 665-X succinimidyl ester; DY-635; Cy5 succinimidyl ester; Alexa Fluor 647 succinimidyl ester; DY-647; DY-648; DY-650; DY-654; DY-652; DY-649; DY-651; DYQ-660; DYQ-661; Alexa Fluor 660 succinimidyl ester; Cy5.5 succinimidyl ester; DY-677; DY-675; DY-676; DY-678; Alexa Fluor 680 succinimidyl ester; DY-679; DY-680; DY-682; DY-681; DYQ-3; DYQ-700; Alexa Fluor 700 succinimidyl ester; DY-703; DY-701; DY-704; DY-700; DY-730; DY-731; DY-732; DY-734; DY-750; Cy7 succinimidyl ester; DY-749; DYQ-4; Cy7.5 succinimidyl ester; 7-diethylaminocoumarin-3-carboxylic acid; succinimidyl ester; Dabsyl sulfonyl chloride;fluorescein isothiocyanate (FITC) carboxysuccinimidyl ester (DY-495); rhodamine green carboxylic acid succinimidyl ester (DY-505); eosin isothiocyanate (EITC); 6-carboxy-2',4,7,7'-tetrachlorofluorescein carboxylic acid succinimidyl ester (TET); carboxyrhodamine 6G succinimidyl ester; carboxytetramethylrhodamine succinimidyl ester (TMR, TAMRA) (DY-554); QSY 9 succinimidyl ester; sulfo-rhodamine B sulfonyl chloride (DY-560); Texas Red (sulfo-rhodamine 101); gallian blue; fast green FCF; malachite green; or QSY 21 succinimidyl ester.

[0104] In alternative embodiments, the methods as provided herein include the use of immunofluorescence, in which the first or second antibody is tagged to a fluorophore such as fluorescein or fluorescein isothiocyanate (FITC), triarylmethane dyes such as rhodamine or rhodamine derivatives (e.g., tetramethylrhodamine (TRITC), rhodamine 6G, rhodamine 123, rhodamine B, carboxytetramethylrhodamine (TAMRA), tetramethylrhodamine (TMR), sulfo-rhodamine 101), aminomethylcoumarin acetate (AMCA), ALEXATM or DYLIGHTTM fluor, or a fluorophore or dye as described in U.S. Patent Application No. US 2019 / 0018018 Al. 3,3'-diaminobenzidine (DAB) can also be used.

[0105] In alternative embodiments, the methods as provided herein include the use of a direct or one-step staining method, in which the first antibody (e.g., an antibody (Ab) or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein as provided herein, including, e.g., synthetic or recombinant forms) is labeled and directly reacted with the antigen, e.g., in a tissue section. While this technique utilizes only one antibody, and is thus simple and fast, sensitivity can be lower due to little signal amplification.

[0106] In alternative embodiments, the methods as provided herein include the use of a direct or one-step staining method, in which the first antibody (e.g., an antibody (Ab) or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein as provided herein, including, e.g., synthetic or recombinant forms) is labeled and directly reacted with the antigen, e.g., in a tissue section. While this technique utilizes only one antibody, and is thus simple and fast, sensitivity can be lower due to little signal amplification.

[0107] In alternative embodiments, methods as provided herein include the use of an indirect approach, in which an unlabeled first antibody (first layer) binds to the target antigen (e.g., PTK7 protein) in, for example, a tissue or organ, and a labeled second antibody (second layer) then reacts with the first antibody. The second antibody can be directed against the isotype of the first antibody's animal species of origin (e.g., IgG). This approach can be more sensitive than a direct detection strategy because if the second antibody is conjugated to a detection agent (such as a fluorescent or enzymatic reporter), signal amplification occurs due to several second antibodies binding to each first antibody.

[0108] In alternative embodiments, further amplification can be achieved if the second antibody is conjugated to several detection molecules (e.g., biotin molecules) that can recruit a complex of avidin, streptavidin, or NEUTRAVIDIN™ protein-bound enzymes.

[0109] In alternative embodiments, IHC is performed on tissue sections or tissue biopsies (e.g., paraformaldehyde (PFA)-fixed tissues or organs, or formalin-fixed paraffin-embedded tissues). In alternative embodiments, tissue sections, or sections, or whole bodies are used. Prior to sectioning, the tissue sample can be embedded in a medium (e.g., paraffin or a cryo-preservation medium). The tissue sections can be sectioned or sectioned on various instruments, most commonly using an ultramicrotome, a cryostat, or a vibratome. The sample can be sectioned or sectioned in a range of about 3 pm to 5 pm. The sections or sections can be mounted on glass slides, dehydrated using increasing concentrations of alcohol (e.g., 50%, 75%, 90%, 95%, 100%), and cleared using a de-pilating agent such as xylene, before being imaged under a microscope.

[0110] Depending on the method of fixation and tissue preservation, additional steps can be required to make the PTK7 epitopes available for antibody binding, including deparaffinization and antigen retrieval. For formalin-fixed paraffin-embedded tissues, antigen retrieval is typically necessary and can include pretreatment of the sections with heat or proteases.

[0111] In alternative embodiments, IHC is performed using the ENVISION DUOFLEX DOUBLESTAIN SYSTEM™ (EnVision DuoFLEX Double Stain System) (Agilent, San Jose, CA), which allows for staining of two or more markers on a single slide. In alternative embodiments, IHC is performed using the EnVision FLEX HRP Magenta (high pH) (Dako Omnis) system and binding can be visualized by EnVision FLEX HRP Magenta Chromogen. In alternative embodiments, IHC is performed using the EnVision FLEX™ MINI™ Kit (high pH), which is a high sensitivity visualization system designed for use with the Dako AUTOSTAINER™ instrument in IHC; this dual-link system detects first mouse and rabbit antibodies and visualizes the reaction by 3,3'-diaminobenzidine (DAB) chromogen (DAB forms a water-insoluble brown precipitate when oxidized by, for example, peroxidase).

[0112] Articles and kits

[0113] Articles of manufacture and kits for carrying out the methods as provided herein are provided; and optionally, the articles of manufacture and kits can further comprise instructions for practicing the methods as provided herein.

[0114] Embodiments of kits herein include an antibody that specifically binds PTK7 and a scoring guide comprising an immunohistochemistry (IHC) method for determining and scoring the degree of expression of protein tyrosine-like kinase 7 (PTK7) in a tissue sample as provided herein. Other embodiments of kits herein include a scoring guide comprising an IHC method as provided herein. In some embodiments, the kits further comprise images indicative of a plurality of PTK7 staining levels.

[0115] Any of the above aspects and embodiments can be combined with any other aspect or embodiment as disclosed herein in the SUMMARY, DETAILED DESCRIPTION, and / or specific embodiments section.

[0116] As used in this specification and claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0117] As used herein, the term “or” shall be understood to be inclusive and to cover both “or” and “and,” unless the context clearly dictates otherwise.

[0118] Unless explicitly stated otherwise or is apparent from context, as used herein, the term "about" is understood to be within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About (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 clear from context, all numerical values provided herein are modified by the term "about."

[0119] Unless explicitly stated otherwise or is apparent from context, as used herein, the term "substantially all," "substantially most," "substantially all," or "most" encompasses at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or more of the referenced amount of the composition.

[0120] The entire contents of each patent, patent application, publication and document referenced herein are hereby incorporated by reference. Citation of the above patents, patent applications, publications, and documents is not admission that any of the foregoing is pertinent prior art, that any of the foregoing is independently prior art at all, or, if pertinent prior art, that any of the foregoing is prior art as to a presently claimed invention. Incorporation by reference of any document herein is not to be construed as an admission that the document is available as prior art to any of the claims or that the document is pertinent to the patentability of any of the claims. To the extent that any meaning or definition of a term in this document conflicts with the meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to the term in this document shall control.

[0121] Modifications can be made to the foregoing without departing from the basic aspects of the application. Although the application has been described in considerable detail with reference to one or more specific embodiments, one skilled in the art will recognize that changes can be made to the embodiments specifically disclosed herein, yet will fall within the scope and spirit of the application. The application illustratively described herein suitably can be practiced in the absence of any element or ingredient not specifically disclosed herein. Thus, for example, in each instance herein, the use of terms such as "containing", "having", "comprising", "consisting of", and "consisting essentially of" should all be construed in an inclusive sense as opposed to an exclusive sense unless the context of the specification clearly dictates otherwise. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof. It is recognized that various modifications are possible within the scope of the application.

[0122] The application will be further described with reference to the examples described herein; however, it is to be understood that the application is not limited to such examples.

[0123] Examples

[0124] Unless otherwise stated in the examples, all recombinant DNA techniques are carried out according to standard protocols, such as those described in Sambrook et al. (2012) Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Further references for standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, NY; Brown (1998) Molecular Biology LabFax, 2nd Edition, Volumes I and II, Academic Press (UK). Standard materials and methods for the polymerase chain reaction can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press and in McPherson et al. (2000) PCR - Basics: From Background to Bench, 1st Edition, Springer Verlag, Germany.

[0125] Example 1 : Exemplary IHC method

[0126] This example describes an exemplary IHC method as provided herein.

[0127] Figure 1 illustrates images from 0-3+ intensity examples of non-small cell lung cancer (NSCLC) tumor tissue stained with PTK7 showing membrane staining.

[0128] Figure 2 provides exemplary images of low, medium, and high examples at each staining intensity. These images are intended as a guide and are not intended to represent the full range of staining variation within each intensity category (0-3+).

[0129] Membrane staining magnification guide

[0130] The first point of each staining intensity of 1-3 includes a color description (dark brown, golden brown / medium brown, and light brown). These descriptions are typically included in non-intensity based scoring guidelines, where any intensity of 1-3 is considered a positive staining.

[0131] Stepwise approach for assessing intensity

[0132] Table 1

[0133] 1. Scan the sample at 4X

[0134] a. Determine the distribution of tumor and non-tumor tissue.

[0135] b. Assess areas with intense stromal and necrotic staining.

[0136] c. Observe the relative presence or absence of cells staining at 2+ and 3+ within viable tumor areas

[0137] 2. Review the tissue at 10X

[0138] a. Distinguish between 1+, 2+, and 3+

[0139] b. Confirm membrane staining with cytoplasmic staining at all intensities

[0140] c. Record cells staining at 1+, 2+, and 3+ after reviewing all viable tumor

[0141] 3. If necessary, move to 20X

[0142] a. Confirm between 0 and 1+

[0143] b. If there is intense cytoplasmic staining, confirm membrane staining with cytoplasmic staining at 2+ and 3+

[0144] c. Confirm membrane staining with cytoplasmic staining at 1+

[0145] 4. Move back to 4X

[0146] a. Confirm the distribution of 0, 1+, 2+, and 3+ in viable tumor cells (1+ is more difficult to assess at this magnification)

[0147] b. If necessary, adjust the score.

[0148] For intensity-based scoring, positive staining is defined only at certain intensities. This positive staining information is used to determine the diagnostic status of a patient sample. Specific magnification guidelines are included in the following second and third points: ​

[0149] Table 2

[0150]

[0151] Inter-observer variability

[0152] During training, the pathologists were given the above Tables 1 and 2 in the form of a pathology scoring manual. After training, the accuracy of the test pathologist scoring was assessed. The pathologists were provided with a table including the following table (Table 3) to complete for each sample:

[0153] Table 3

[0154]

[0155] Figure 3 A T&T (training and testing) observer variability plot is shown: the x-axis shows the 30 tissue blocks scored by four observers or pathologists. The y-axis shows the score for percentage of tumor cells greater than or equal to 2+. For example, block 140446 received one 25, two 30s, and one 35. This indicates that the evaluation of the blinded slides by four different pathologists, without reference scores, were within 10% of each other for that particular specimen.

[0156] Exemplary protocol

[0157] - Examine the 40X field of view (FOV) and determine the score according to the guidelines as provided herein;

[0158] - Change the objective to 20X. Examine the FOV. Determine the score, especially by evaluating the outer FOV. Weak or unstained areas of cells can be evaluated at higher magnification for confirmation. Ignore non-cellular areas. Re-evaluate / adjust the score.

[0159] - Change the objective to 10X. Repeat the above.

[0160] - Change the objective to 4X. Repeat the above.

[0161] - The result is the score at 4X, which can be repeated for other 4X FOVs if the tissue is large enough. The 4X FOVs can be added together and divided by the number of fields to obtain an average.

[0162] Figure 4A shows a visual summary of the various FOVs used in the IHC examination in this exemplary protocol, and Figure 4B shows exemplary FOVs as superimposed on an image of a tissue section.

[0163] Exemplary embodiments

[0164] Embodiment 1. A method for treating or ameliorating a cancer or tumor in an individual in need thereof, the method comprising:

[0165] (a) determining whether cells in a tissue sample from the individual in need thereof have a low or high, or diagnostic negative or diagnostic positive, PTK7 expression score (TPS), as determined by a protocol comprising use of an immunohistochemistry (IHC) method according to any one of claims 1 to 24; and

[0166] (b) if the tissue sample is found to have a high or diagnostic positive PTK7 expression score (TPS), administering a tumor or cancer therapeutic to the individual.

[0167] Embodiment 2. The method of Embodiment 1, wherein the cancer or tumor is non-small cell lung cancer (NSCLC) or lung cancer.

[0168] Embodiment 3. The method of Embodiment 1 or 2, wherein the treatment or amelioration of the cancer or tumor comprises administering to the individual in need thereof a pharmaceutical formulation comprising an antibody drug conjugate, a radionuclide, a gallium-68 radionuclide, or a fluorine-18 radionuclide.

[0169] Embodiment 4. The method of any one of Embodiments 1 to 3, wherein the individual in need thereof is a patient having lung cancer or non-small cell lung cancer.

[0170] Embodiment 5. The method of any one of Embodiments 1 to 4, wherein determining the number of PTK7 positive viable tumor or cancer cells comprises determining the number of viable tumor or cancer cells having PTK7 staining in the cell membrane.

[0171] Embodiment 6. The method of any one of Embodiments 1 to 5, wherein determining the number of PTK7 positive viable tumor or cancer cells comprises determining the number of viable tumor or cancer cells having cell membrane PTK7 staining of higher intensity than cytoplasmic PTK7 staining.

[0172] Embodiment 7. The method of any one of Embodiments 1 to 6, wherein the method further comprises determining that a subject is likely to respond well to treatment with a cancer therapeutic if the tumor proportion score (TPS) is above a threshold value.

[0173] Embodiment 8. The method of Embodiment 7, wherein the threshold value is about 50% or more, about 75% or more, or about any number between 50% and 90%.

[0174] Embodiment 9. The method of any one of embodiments 1 to 8, further comprising administering the cancer therapeutic if the tumor proportion score (TPS) is above the threshold.

[0175] Embodiment 10. A method for treating or ameliorating a cancer or tumor in a patient, the method comprising determining the amount of nuclear protein PTK7 (also known as colon carcinoma kinase 4 (CCK4)) in a tissue sample from a patient in need thereof using the IHC method of any one of embodiments 1-9 and scoring it, wherein if the tissue sample is determined or scored as having high or diagnostic positive PTK7 expression, the patient is treated with a cancer therapeutic to which the patient is likely to respond well.

[0176] Embodiment 11. The method of embodiment 10, wherein 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, or renal cell carcinoma.

[0177] A number of embodiments of the application have been described. Nevertheless, it will be understood that various modifications can be made without departing from the spirit and scope of the application. Therefore, other embodiments are within the scope of the following claims.

Claims

1. An immunohistochemistry (IHC) method for determining and scoring the expression level of protein tyrosine kinase-like 7 (PTK7) in a tissue sample, the method comprising: (a) Tissue samples were stained with antibodies that specifically bind to PTK7; as well as (b) determining the total number of live invasive tumor or cancer cells that have anti-PTK7 staining, and determining the total number of stained and unstained live invasive tumor or cancer cells in at least a portion of the tissue sample, wherein an invasive tumor or cancer cell is counted as anti-PTK7 positive staining if the invasive tumor or cancer cell shows anti-PTK7 cell staining at any intensity above a defined threshold, and (c) determine the tumor proportion score (TPS), The tumor proportion score (TPS) is the number of PTK7-stained live invasive tumor or cancer cells found in the tissue sample divided by the total number of stained and unstained live invasive tumor or cancer cells, multiplied by 100.

2. The method according to claim 1, wherein the defining threshold comprises: positive staining intensity of 1+ or greater evaluated at low magnification; Positive staining intensity of 2+ or greater evaluated at moderate magnification; or 3+ positive staining intensity evaluated at high 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 according to claim 2 or 3, further comprising: evaluating at said low magnification for the presence of tumor or cancer cells in at least a portion of said tissue sample with anti-PTK7 staining at any intensity above said defined threshold; as well as The total number of stained viable tumor or cancer cells in the tissue sample is determined at the medium magnification to have a positive staining intensity of 1+ or greater above the defined threshold.

5. The method according to claim 4, further comprising: The total number of stained viable tumor or cancer cells in the tissue sample is determined at the high magnification to have a positive staining intensity of 1+ or greater above the defined threshold.

6. The method according to any one of claims 2 to 5, further comprising: At the low magnification, (a) evaluating areas of intense anti-PTK7 staining of stroma and necrosis in said tissue sample, (b) determining the distribution of viable tumor cells, cancer cells, and non-tumor tissue in the stained tissue sample; or (c) Identifying the presence of viable tumor or cancer cells with a staining intensity of 2+ or greater.

7. The method according to any one of claims 2 to 6, further comprising: At the medium magnification or at the high magnification, (a) distinguishing between cell membrane positive anti-PTK7 staining and cytoplasmic anti-PTK7 positive staining in the total number of living tumor or cancer cells in the tissue sample; (b) distinguishing 1+, 2+ or 3+ positive staining intensities among the total number of viable tumor or cancer cells in the tissue sample; or (c) Differentiating the staining intensity of 0 staining and 1+ positive cells in the tissue sample.

8. The method according to claim 7, further comprising: (a) At the low magnification described, (i) confirming the cell membrane positive anti-PTK7 staining and the cytoplasm positive anti-PTK7 staining with respect to the total number of viable tumor or cancer cells in the tissue sample having a staining intensity of 2+ or greater and a significant level of cytoplasm staining; or (ii) confirming cell membrane staining versus cytoplasmic staining with respect to the total number of viable tumor or cancer cells in the tissue sample having a staining intensity of 1+ or greater; or (b) confirming the distribution of staining intensities of 0, 1+, 2+, and 3+ positive cells in the tissue sample at the low magnification; and optionally adjusting said tumor proportion score based on said confirmation.

9. A method according to any one of the preceding claims, or according to any one of claims 1 to 8, wherein (a) the tissue sample comprises a tissue section, and confirming whether the tissue section is sufficient for determining and scoring the expression amount of protein PTK7, and if about 100 or more living invasive tumor or cancer cells are present, the tissue section is considered sufficient for evaluation; (b) if an invasive tumor or cancer cell shows anti-PTK7 cell membrane staining at any intensity of 1+ or higher, the invasive tumor or cancer cell is counted as anti-PTK7 positive staining; (c) preparing a section or portion of said tissue sample on a glass slide or equivalent, and staining said section or portion of said tissue sample on said slide; (d) an antibody that specifically binds to PTK7 comprises a monoclonal mouse anti-PTK7 antibody, and optionally the anti-PTK7 antibody comprises a monoclonal mouse anti-PTK7 clone 6.60.1, and optionally the anti-PTK7 antibody comprises substantially isolated or substantially purified monoclonal mouse anti-PTK7 clone 6.60.1; (e) Anti-PTK7 staining was present at any intensity of 1+ or higher in the following cases: (i) the staining signal is unequivocally brown, or (ii) The staining corresponds to the cell membrane. (f) the method further comprises excluding from calculation of the tumor proportion score (TPS): tumor cells or cancer cells with only cytoplasmic or nuclear staining; non-invasive tumors or cancer cells in situ, inactive or necrotic tumor or cancer cells, apoptotic nuclei or nuclear fragments, tumor or cancer cells in poorly preserved tissue areas, benign epithelial cells, non-tumor cells and / or lymphocytes with nuclear staining, apoptotic cells, necrotic cells, cells that do not exhibit the expected color, lymphocytes, and stromal cells; (g) The method further comprises: (i) if the tumor proportion score (TPS) is less than (<) 75%, the tissue sample is determined to have diagnostic negative PTK7 expression; and (ii) If the tumor proportion score (TPS) is greater than or equal to (≥) 75%, the tissue sample is determined to have diagnostically positive PTK7 expression. (h) the tissue sample comprises a formalin-fixed paraffin-embedded (FFPE) specimen; and optionally, sections of the tissue sample are prepared by a protocol comprising fixation in about 10% neutral buffered formalin for about 6 to 72 hours; (i) 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 (j) The tissue sample is a biopsy sample, a needle biopsy sample, a sample derived from a needle biopsy sample, a fine needle aspirate, a cytology specimen, or a bone decalcification.

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

11. The method of claim 10, wherein 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, or renal cell carcinoma.

12. A method for diagnosing or selecting an individual eligible for treatment with a cancer or tumor therapeutic agent, the method comprising: (a) determining whether cells in a tissue sample from an individual in need thereof have a low or high, or diagnostically negative or diagnostically positive, PTK7 expression score (TPS) as determined by a protocol comprising use of an immunohistochemistry (IHC) method according to any one of claims 1 to 24; and (b) diagnosing or selecting the individual as eligible for treatment with the cancer or tumor therapeutic agent if the tissue sample is found to have a high or diagnostic positive PTK7 expression score (TPS).

13. The method of claim 12, wherein the cancer or tumor therapeutic agent is a non-small cell lung cancer (NSCLC) therapeutic agent or a lung cancer therapeutic agent; or, the cancer or tumor therapeutic agent is a pharmaceutical formulation comprising an antibody drug conjugate, a radionuclide, a gallium-68 radionuclide, or a fluorine-18 radionuclide.

14. The method according to any one of claims 12 to 13, wherein the individual is a patient suffering from lung cancer or non-small cell lung cancer.

15. The method of any one of claims 12 to 14, wherein determining the number of PTK7-positive viable tumor or cancer cells comprises determining the number of viable tumor or cancer cells having PTK7 staining in the cell membrane; or, determining the number of PTK7-positive viable tumor or cancer cells comprises determining the number of viable tumor or cancer cells having cell membrane PTK7 staining with a higher intensity than cytoplasmic PTK7 staining.

16. The method according to any one of claims 12 to 15, further comprising: (a) determining that the individual is likely to respond well to treatment with a cancer therapeutic if the tumor proportion score (TPS) is above a threshold, wherein optionally the threshold is above about 50%, above about 75%, or about any number between 50% and 90%; or (b) selecting the individual for administration of the cancer therapeutic if the tumor proportion score (TPS) is above the threshold.

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

18. A kit comprising a scoring guide comprising the method according to any one of claims 1 to 16, and optionally further comprising an image indicating a plurality of PTK7 staining levels.

19. A method for assessing the degree of PTK7 expression, the method comprising: contacting a sample from the individual comprising cancer cells or tumor cells, or a portion thereof, with an antibody or portion thereof that specifically binds to PTK7; and determining a tumor proportion score (TPS) by dividing the number of live tumor or cancer cells stained by PTK7 specifically bound by the antibody in the sample or a portion thereof by the total number of stained and unstained live cancer cells or tumor cells, and multiplying the result by 100 to obtain the tumor proportion score (TPS).

Images