Computational Tissue Analysis-Based Quantification of Tumor-Infiltrating Leukocytes Using Morphometrics in Immunohistochemistry-Stained NSCLC Samples

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Quantification of tumor-infiltrating lymphocytes (TILs) is predictive of patient response to immunotherapy regimens. Because of the role of anti-PD-L1 and anti-PD-1 treatments in promoting tumor-specific cytotoxic T-cell responses, the identification and quantification of TILs and PD-L1-positive tumor cells by immunohistochemistry (IHC) may provide important information for disease prognosis. Using Computational Tissue Analysis (cTATM)-based morphometric features instead of an IHC-based lymphocyte assay enables a simpler work ow for TIL assessment and can be combined with additional IHC assays, enabling the identification and classification of immune cell subsets within the complex tumor microenvironment (TME).

Materials and Methods:
Non-small cell lung cancer (NSCLC) samples were stained with IHC assays for CD45, CD3, CD8, and PD-L1. Serial sections were stained with appropriate isotype-negative controls. cTA tools were used to determine the morphometric parameters that identified hematoxylin-stained leukocytes on isotype-stained slides and used to quantify the total immune cell population in tumor nests and surrounding stroma. The same morphometric ruleset was used to quantify total leukocytes in CD45-, CD3-, CD8-, and PD-L1-stained tissues. The results of this analysis were then compared with the results of the biomarker- specific assay to evaluate the frequency of biomarker-positive TILs in the context of total infiltrating leukocytes quantified by the cTA ruleset.

Morphometric parameters were established that determined that the number of hematoxylin-stained leukocytes correlated with the number of leukocytes identified by CD45 in serial sections. The relative populations of CD3-positive and CD8-positive TILs were consistent with available literature findings. The cTA morphometric ruleset separated PD-L1-positive leukocytes from PD-L1-positive tumor staining in the tumor nests and surrounding stromal tissue.

Methods were developed that use morphometric features to identify the total number of infiltrating immune cells in tumor tissues stained with hematoxylin and eosin or by IHC biomarker assays. These methods provide an additional dimension of data without requiring additional biomarker staining. In some IHC assays, like anti-PD-L1 assays, both the immune infiltrate and tumor cells may stain positive, and the accuracy and precision of quantifying one population by visual pathology is hindered by the staining of the second population. The cTA-based methods can be used to aid pathologists in the interpretation of these types of biomarker assays in which both tumor cells and immune infiltrates may stain positive.

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