Identifying T Lymphocytes In Ihc-Stained Tissues Independently Of Cd3+Staining Using Morphometric Features Extracted By Image Analysis

Assessments of leukocyte populations in the context of cancer tissues are typically determined by staining for leukocyte subtype markers in formalin fixed tissues. This requires the identification, categorization, and localization of multiple leukocytes within tissue context utilizing multiple markers. Meeting these demands are challenging due to technical constraints on the number of individual markers which can be visualized or scored in a single slide, and the complexity of staining observed. The use of multispectral imaging with fluorescent multiplexed markers has better enabled the assessment of multiple markers in a single tissue section. However, these wet chemistry and image capture technologies are very complex, and can only be successfully implemented with significant laboratory infrastructure, specialty equipment, and experienced resources. For these reasons, this approach is not widely implementable in the clinical pathology laboratory setting, where such tests are oriented to a companion diagnostic utility. Approaches which rely on widely adopted chromogenic immunohistochemistry (IHC) staining are preferred in the clinical laboratory setting, but the number of assayable markers is limited to 1-3 unique markers in a single tissue. In order to create a useful approach that could be implemented in existing clinical laboratory workflow, Flagship Biosciences has developed an approach for deriving the complex endpoints often necessary in immuno-oncology studies which rely on 1-3 chromagenic stains and computer interpretation of the tissue using only hematoxylin counterstain to identify T-lymphocytes. In a proof-of-concept study, we utilized our Tissue Image Analysis (TIA) tools to identify morphometric parameters which could identify T-lymphocytes, independent of staining for the T-lymphocyte marker CD3. A cohort of non-small cell lung cancer (NSCLC) tissues was stained by CD3 IHC, and both CD3 and isotype-stained tissues were analyzed with Flagship9s CellMap™ software to capture the morphometric and staining features of cells in the tissues. The morphometric features which characterized CD3+ cells were used to approximate the T-lymphocyte population frequency in the isotype-stained tissues. This T-lymphocyte classification scheme was defined based on hematoxylin staining alone, and accuracy of T-lymphocyte classification was verified by CD3 staining. Based on this study, the method described herein could be utilized to reasonably estimate the frequency of T-lymphocyte subsets (e.g. CD4+, CD8+, etc.) or different marker-positive leukocyte (e.g. macrophages) subsets relative to the total T-lymphocyte population without an additional T-lymphocyte marker such as CD3. The approach could, therefore, provide an added dimension of analysis for tissues stained by IHC without adding complexity to the wet assay by necessitating a marker for T-lymphocytes. Citation Format: Nathan T. Martin, Joshua Black, Famke Aeffner, Logan Cerkovnik, Jasmeet Bajwa, Staci J. Kearney, Crystal Pulliam, A.J. Milici, Joseph Krueger. Identifying T lymphocytes in IHC-stained tissues independently of CD3+ staining using morphometric features extracted by image analysis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4162.