Prostate Cancer Case Study
Quantification of Infiltrating Lymphocytes Post T-Cell Antibody Treatment for Prostate Cancer
Collaborator Challenge: Understand T-cell and cytokine response in patients treated with antibody therapy meant to induce an immune response.
Aureon Objective: Establish quantitative biometrics from neighboring inflammatory infiltrates using tissue specimens and clinical history derived from a clinical trial in prostate cancer. To achieve this, Aureon utilized a modified systems pathology approach and integrated prostate-derived morphometric image analysis features, molecular measurements of tumor and non-tumor elements using immunofluorescent multiplexing, nascent RNA peT-FISH analyses, as well as clinical data.
Results: A number of multiplexed immunofluorescent assays were developed to quantify T-cell subsets (CD4, 8, 25, 69, 86) in formalin-fixed, paraffin-embedded tissue samples from 6 patients with hormone refractory prostate cancer treated with an antibody to a known T-cell receptor. In addition, a nascent RNA peT-FISH, semi-quantitative assay was performed to evaluate IFNg transcript profiles within lymphocyte populations from the same patient group. There was considerable antigen heterogeneity reflected in part by the degree of lymphocytic infiltration and the distribution of lymphocyte subsets within the diffuse and aggregated foci. Across all 6 samples, CD8 > CD4 and CD86 > CD69 with an approximate ratio of 2-5:1. In addition, three cases contained CD25 positive lymphocytes. Together the results suggest that the majority of lymphocytes were CD8 and CD86 (+) with limited CD4 (+) cells and rare Tregs (CD4+/25+). The predominance of CD8 expressing cells and the presence of IFNg transcripts both suggest an active, immune-mediated mechanism, possibly against a tumor antigen or as a result of antibody-based therapies designed to promote T-cell-mediated immunity. Finally, although clinical information was not initially provided, the quantitative feature data from each of the domains correlated with the observed histopathology and was related in part to dosing levels within the limited cohort.
Next Steps: Obtain tissue samples from a control arm to assess effect of neoadjuvant hormonal therapy on the immune response and comparison with evaluated treatment group. Develop enhanced multiplex assays to further characterize the infiltrative lymphocyte population and employ on both groups for final analysis. Early results suggest that a predictive model would allow quantitative biologic features to be linked with outcome, and potentially support the development of a ‘directed’ therapeutic plan.
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Figure 1. Triplex and duplex ‘multiplex’ assays are utilized to localize CD4, CD25, CD69, CD8, and CD86 in formalin-fixed, paraffin-embedded sections of prostate tissue. Individual fluorochromes exhibiting discrete spectral profiles are used to extract quantitative data used for feature generation with respect to marker distribution.
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Figure 2. Spleen control tissue samples are employed to demonstrate co-localization of CD4 and either CD69 or CD25. As illustrated, the majority of lymphocytes express both CD4 and CD69 with a reduced number of CD4 and CD25 positive cells noted.
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Figure 3. Hematoxylin and Eosin (H&E)-stained prostate tissue samples (post-treatment) exhibit both a diffuse and nodular lymphocytic infiltrate (3A and 3B). Using a quantitative histologic imaging software tool on a standard H&E image (3C), cellular elements are segmented and classified to yield quantitative data including features associated with nodular / clustered (yellow) and diffuse / isolated (black) lymphocytes (3D).