Immunohistochemical assay for detecting expression of programmed death ligand 1 (pd-l1) in tumor tissue

Abstract

The present disclosure provides processes for describing and quantifying the expression of human programmed death ligand-1 (PD-L1) in tumor tissue sections as detected by immunohistochemical assay using an antibody that specifically binds to PD-L1. The results generated using these processes have a variety of experimental, diagnostic and prognostic applications.

1 . A process for scoring the expression of a programmed death ligand 1 (PD-L1) protein in
 a tumor sample removed from an animal, comprising:
 (a) obtaining a tissue section from the tumor sample that has been stained in an immunohistochemical (IHC) assay with an antibody that specifically binds to the PD-L1 protein (anti-PD-L1 Ab); 
 (b) examining each tumor nest in the tissue section for staining; and 
 (c) assigning one or both of a modified H score (MHS) and a modified proportion score (MPS) to the tissue section, 
 wherein assigning the MHS comprises (i) estimating, across all of the viable tumor cells and stained mononuclear inflammatory cells in all of the examined tumor nests, four separate percentages for cells that have no staining, weak staining (+1), moderate staining (+2) and strong staining (+3), wherein a cell must have at least partial membrane staining to be included in the weak, moderate or strong staining percentages, and wherein the sum of all four percentages equals 100; and (ii) inputting the estimated percentages into the formula of 1×(percent of weak staining cells)+2×(percent of moderate staining cells)+3×(percent of strong staining cells), and assigning the result of the formula to the tissue section as the MHS; 
 wherein assigning the MPS comprises estimating, across all of the viable tumor cells and mononuclear inflammatory cells in all of the examined tumor nests, the percentage of cells that have at least partial membrane staining of any intensity, and assigning the resulting percentage to the tissue section as the MPS; and 
 wherein if both the MHS and MPS are assigned, the assignments may be made in either order or simultaneously. 
   
     
     
         2 . The process of  claim 1 , wherein the tumor sample is from a cancer selected from the group consisting of non-small cell lung carcinoma (NSCLC), head and neck squamous carcinoma, and transitional bladder carcinoma, and the process further comprises designating the tumor sample as positive or negative for PD-L1 expression, wherein
 the tumor sample is designated as positive for PD-L1 expression if either of the MHS or the MPS is greater than zero, and   the tumor sample is designated as negative for PD-L1 expression if the MHS is zero or the MPS is zero.   
     
     
         3 . The process of  claim 1 , further comprising:
 examining the stroma surrounding each tumor nest for the presence or absence within the stroma of a band of membrane-stained cells, and   assigning to the tissue section a stroma score of positive if the band is detected in the stroma surrounding at least one tumor nest, or   assigning to the tissue section a stroma score of negative if the band is not detected in the tissue section.   
     
     
         4 . The process of  claim 3 , wherein the tumor sample is from a cancer selected from the group consisting of non-small cell lung carcinoma (NSCLC), head and neck squamous carcinoma, and transitional bladder carcinoma, and the process further comprises designating the tumor sample as positive or negative for PD-L1 expression,
 wherein the tumor sample is designated as positive for PD-L1 expression if the tissue section has any one or more of the following score assignments:
 (i) the stroma score is positive, 
 (ii) the MHS greater than zero, and 
 (iii) the MPS is greater than zero, and 
   wherein the tumor sample is designated as negative for PD-L1 expression if the tissue section has any one or more of the following score assignments:
 (iv) the stroma score is negative and the MHS is zero; 
 (v) the stroma score is negative and the MPS is zero; and 
 (vi) the stroma score is negative, the MHS is zero and the MPS is zero. 
   
     
     
         5 . The process of  claim 3 , wherein the tumor sample is from a cancer selected from the group consisting of melanoma and breast cancer, and the process further comprises:
 examining each tumor nest for the presence or absence of a lattice pattern of membrane-stained dendriform cells within the tumor nest, and   assigning to the tissue section a dendriform pattern score (DS) of positive if the lattice pattern is detected in at least one of the tumor nests, or   assigning a DS of negative if the lattice pattern is not detected in any of the tumor nests.   
     
     
         6 . The process of  claim 5 , further comprising designating the tumor sample as positive or negative for PD-L1 expression,
 wherein the tumor sample is designated as positive for PD-L1 expression if the tissue section has any one or more of the following score assignments:
 (i) the stroma score is positive, 
 (ii) the DS is positive 
 (iii) the MHS greater than zero, and 
 (iv) the MPS is greater than zero; and 
   wherein the tumor sample is designated as negative for PD-L1 expression if the tissue section has any one or more of the following score assignments:
 (v) the stroma score is negative, the DS is negative and the MHS is zero; 
 (vi) the stroma score is negative, the DS is negative and the MPS is zero; and 
 (vi) the stroma score is negative, the DS is negative, the MHS is zero and the MPS is zero. 
   
     
     
         7 . The process of  claim 1 , wherein the tumor sample is from a human and the PD-L1 protein is human PD-L1. 
     
     
         8 . The process of  claim 7 , wherein the anti-PD-L1 Ab is a monoclonal antibody which comprises a light chain mature variable region of SEQ ID NO:6 and a heavy chain mature variable region of SEQ ID NO:14. 
     
     
         9 . The process of  claim 7 , wherein the anti-PD-L1 Ab is a monoclonal antibody which comprises a light chain mature variable region of SEQ ID NO:21 and a heavy chain mature variable region of SEQ ID NO:29. 
     
     
         10 . A process for predicting if a human patient diagnosed with non-small cell lung carcinoma (NSCLC) is likely to respond to treatment with a PD-1 antagonist, comprising:
 (a) obtaining at least one quantitative staining score for the level of expression of programmed death ligand 1 (PD-L1) in a tumor sample removed from the patient; wherein the quantitative staining score is selected from the group consisting of a modified H score (MHS) and a modified P score (MPS), wherein the MHS and MPS are obtained by performing a scoring process that comprises:
 (i) obtaining a tumor tissue section from the sample that has been stained in an immunohistochemical (IHC) assay with an antibody that specifically binds to human PD-L1 (anti-PD-L1 Ab); 
 (ii) examining each tumor nest in the tissue section for staining; and 
 (iii) assigning one or both of a modified H score (MHS) and a modified P score (MPS) to the tissue section, 
 wherein assigning the MHS comprises estimating, across all of the viable tumor cells and stained mononuclear inflammatory cells in all of the examined tumor nests, four separate percentages for cells that have no staining, weak staining, moderate staining and strong staining, wherein a cell must have at least partial membrane staining to be included in the weak, moderate or strong staining percentages, and wherein the sum of all four percentages equals 100; and inputting the estimated percentages into the formula of MHS=1×(percent of weak staining cells)+2×(percent of moderate staining cells)+3×(percent of strong staining cells); and 
 wherein assigning the MPS comprises estimating, across all of the viable tumor cells and mononuclear inflammatory cells in all of the examined tumor nests, the percentage of cells that have at least partial membrane staining of any intensity; and 
 wherein if both the MHS and MPS are assigned, the assignments may be made in either order or simultaneously; and 
   (b) predicting the patient as likely to respond to the treatment if either the MHS or the MPS is at or above a specified threshold or predicting the patient as not likely to respond to the treatment if each of the MHS and the MPS is below the specified threshold.   
     
     
         11 . The process of  claim 10 , wherein the specified threshold for MHS is 190 and the specified threshold for MPS is 90. 
     
     
         12 . The process of  claim 1 , wherein each of the estimated percentages used in calculating one of both of the MHS and the MPS is confined to a discrete estimate selected from the group consisting of 0, 1, 2, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95 and 100. 
     
     
         13 . The process of  claim 1 , wherein the tumor tissue section is a formalin-fixed, paraffin-embedded tissue section. 
     
     
         14 . (canceled) 
     
     
         15 . (canceled) 
     
     
         16 . The process of  claim 1 , wherein the tumor sample is from a human patient who is being evaluated for treatment with a PD-1 antagonist. 
     
     
         17 . The process of  claim 16 , wherein the PD-1 antagonist is an anti-PD-1 monoclonal antibody that comprises a heavy chain and a light chain, and wherein the heavy chain comprises SEQ ID NO:32 and the light chain comprises SEQ ID NO:33. 
     
     
         18 . A method of treating a human patient diagnosed with non small cell lung carcinoma (NSCLC), the method comprising:
 a. obtaining a diagnostic report that provides one or both of a modified H score (MHS) and a modified P score (MPS) for PD-L1 expression for a tumor sample removed from the patient; and   b. treating the patient with a PD-1 antagonist if one or both of the MHS and the MPS is at or above a specified threshold, or   c. treating the patient with a therapy that does not include a PD-1 antagonist if the MHS and the MPS are below the specified threshold,   wherein the specified threshold is selected from the group consisting of: the MHS is greater than zero; the MPS is greater than zero; and the MHS and the MPS is greater than zero.   wherein the MHS and the MPS were determined by a scoring process comprising:
 (i) staining a tissue section from the tumor sample in an immunohistochemical (IHC) assay with an antibody that specifically binds to human PD-L1 (anti-PD-L1 Ab); 
 (ii) examining each tumor nest in the tissue section for staining; and 
 (iii) assigning one or both of the MHS and MPS to the tissue section, 
   wherein assigning the MHS comprises estimating, across all of the viable tumor cells and stained mononuclear inflammatory cells in all of the examined tumor nests, four separate percentages for cells that have no staining, weak staining, moderate staining and strong staining, wherein a cell must have at least partial membrane staining to be included in the weak, moderate or strong staining percentages, and wherein the sum of all four percentages equals 100; inputting the estimated percentages into the formula of MHS=1×(percent of weak staining cells)+2×(percent of moderate staining cells)+3×(percent of strong staining cells); and   wherein assigning the MPS comprises estimating, across all of the viable tumor cells and mononuclear inflammatory cells in all of the examined tumor nests, the percentage of cells that have at least partial membrane staining of any intensity;   wherein if both the MHS and MPS are assigned, the assignments may be performed in either order or simultaneously.   
     
     
         19 . The method of  claim 18 , wherein the specified threshold is selected from the group consisting of (i) MHS=190, (ii) MPS=90 and (iii) MHS=190 and MPS=90. 
     
     
         20 . The method of  claim 18 , wherein the PD-1 antagonist is BMS-936558 (nivolumab), MPDL3280A, CT-011, ONO-4538, BMS-936559, MEDI4736, AMP-224 or an anti-PD-1 antibody which comprises a heavy chain and a light chain, and wherein the heavy chain comprises SEQ ID NO:32 and the light chain comprises SEQ ID NO:33. 
     
     
         21 . (canceled) 
     
     
         22 . The method of  claim 18 , wherein the anti-PD-L1 antibody is a monoclonal antibody which comprises a light chain mature variable region of SEQ ID NO:21 and a heavy chain mature variable region of SEQ ID NO:29. 
     
     
         23 . A method of designating a tumor sample as PD-L1 positive or PD-L1 negative, which comprises
 (a) obtaining a tissue section from the tumor sample that has been stained with an antibody that binds to the PD-L1 protein (anti-PD-L1 Ab),   (b) examining each tumor nest in the tissue section for staining,   (c) assigning to the tissue section a modified proportion score (MPS),   (d) examining the stroma surrounding each tumor nest for the presence or absence of a band of anti-membrane-stained cells within the stroma, (e) assigning to the tissue section a stroma score (SS) of positive or negative, wherein a positive SS is assigned if the band is detected and a negative SS is assigned to the tissue section if the band is not detected, and designating the tumor sample as PD-L1 positive if the assigned MPS is greater than 0 or if the assigned SS is positive, and designating the tumor sample as PD-L1 negative if the assigned MPS is 0 and the assigned SS is negative.   
     
     
         24 . The method of  claim 23 , wherein the tumor sample is selected from the group consisting of gastric cancer, bladder cancer, melanoma, NSCLC, head and neck cancer, colon or rectal adenocarcinoma, Hodgkins lymphoma, non-Hodgkins lymphoma, multiple myeloma, Myelodysplastic syndrome (MDS), anal canal squamous cell carcinoma, pancreas adenocarcinoma, esophageal squamous cell carcinoma or adenocarcinoma, biliary tract adenocarcinoma (gallbladder and biliary tree but excluding ampulla of vater cancers), carcinoid tumors, neuroendocrine carcinomas, well or moderately differentiated pancreatic neuroendocrine tumor, estrogen receptor (ER) positive HER2 negative breast cancer, HER2 positive breast cancer, triple negative breast cancer, ovarian epithelial, fallopian tube or primary peritoneal carcinoma, endometrial carcinoma, cervical squamous cell cancer, vulvar squamous cell carcinoma, small cell lung cancer, mesothelioma (malignant pleural esothelioma), thyroid cancer, including of papillary or follicular subtype, salivary gland carcinoma, nasopharyngeal carcinoma, glioblastoma multiforme, leiomyosarcoma, prostate adenocarcinoma. 
     
     
         25 . The method of  claim 23 , wherein the tumor sample is melanoma.