Abstract A069: Combination of immune status and tumor microvascularization provides strong prognostic markers for prostate cancer recurrence prediction
Authors: Harder Nathalie , Athelogou Maria , Hessel Harald , Buchner Alexander , Stief Christian , Kirchner Thomas , Schmidt Günter , Huss Ralf , Tan Tze Heng 
Published: February 2019

Abstract
Introduction: The composition of different immune cell populations in the tumor microenvironment plays an important role for tumor progression in various cancer types. In particular, tumor-associated macrophages (TAMs) and tumor-infiltrating T cells (TILs) are relevant players. On the other hand, structural changes such as neoangiogenesis triggered by cancerous tumors to increase their nutrient supplies have also been associated with tumor progression. In this work we systematically quantified these factors in prostate cancer (PCa) and correlated them with clinical outcome data using a Tissue Phenomics approach. By investigating the prognostic relevance of TAMs (CD68/CD163), TILs (CD3/CD8), and microvessels (CD34) in the tumor, tumor microenvironment (TME), and stroma we identified strong prognostic markers for PCa recurrence prediction in patients after radical prostatectomy.
Methods: In this study, we analyzed a cohort of 90 PCa patients, of whom 40 suffered from tumor progression measured by prostate cancer antigen (PSA) recurrence after prostatectomy. The cohort comprised low- and intermediate-risk PCa patients (Gleason-Score≤7b) since providing a reliable prognosis is particularly difficult for such grades. Tissue sections were immunohistochemically stained using the duplex stains CD68/CD163 for TAMs, CD3/CD8 for TILs, and CK18/p63 to identify and characterize glands as cancerous vs. noncancerous based on their expression level of p63 (in cancerous glands p63 is not expressed). To quantify tumor neoangiogenesis microvessels were stained by CD34. All sections were geometrically aligned per case (virtual multiplexing) to enable coanalysis of stains, and quantified within relevant regions-of-interest (tumor, TME, stroma) using fully automated computational methods (1, 2). In particular, we determined region-specific densities and average distances of TAMs, TILs, and microvessels, as well as ratios of all measures. We systematically analyzed the prognostic power of each measure by optimizing a cutoff with respect to the disease-free survival statistic (log-rank test) using cross-validation to avoid for overfitting.
Results: The top-ranking prognostic markers regarding robustness and prediction performance were related to microvessel density combined with immune cell densities. In particular, we found that within the TME, a coverage of CD8(+) cytotoxic T cells larger than 10% of the coverage of CD34(+) microvessels is correlated with a good prognosis and long-term disease-free survival (cross-validated p &lt;3.1•10-7, accuracy=83%). This corresponds to high densities of CD8(+) cells and/or low microvessel densities, which both have been shown to be associated with good prognosis in prostate cancer. In addition, we found that a larger average distance of CD68(+) M1 macrophages to CD34(+) microvessels above 75.7µm in the tumor region is associated with good prognosis (cross-validated p &lt;2.9•10-8, accuracy=82%). Again, low tumor microvessel density seems to be beneficial as well as high densities of CD68(+) macrophages. The CD68(+) M1-polarized phenotype is associated with tumor-suppressing properties, indicating that a higher density of this population compared to the tumor-promoting M2-polarized phenotype fosters disease-free survival.
Conclusion: Our results indicate a considerable prognostic potential of markers combining microvessel density with measures of TAMs and TILs to predict PSA recurrence in PCa. This application shows that systematic analysis as performed by Tissue Phenomics enables discovery of non-obvious combined prognostic markers characterizing the tumor landscape with high potential to improve patient treatment. In future work we aim to validate our findings on additional data from other clinical sites.
References:
1. Yigitsoy M, et al. Hierarchical patch-based co-registration of differently stained histopathology slides. Proc SPIE 2017. doi:10.1117/12.2254266.
2. Brieu N, et al. Slide specific models for segmentation of differently stained digital histopathology whole slide images. Proc SPIE 2016. doi:10.1117/12.2208620.
Citation Format: Nathalie Harder, Maria Athelogou, Harald Hessel, Alexander Buchner, Christian Stief, Thomas Kirchner, Günter Schmidt, Ralf Huss, Tze Heng Tan. Combination of immune status and tumor microvascularization provides strong prognostic markers for prostate cancer recurrence prediction [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A069.