P1 Quantification of the inflammatory tumor microenvironment in carcinomas of the larynx

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Introduction Recent studies have shown that inflammatory cells play a major role in tumor development and progression. Tumor infiltrating neutrophils are a driver of tumor progression and angiogenesis in some murine cancer models. In head and neck cancer patients substantial heterogeneity exists in the tumor microenvironment with respect to the number and localization of tumor-associated neutrophils. Our previous data suggested induction of pro-angiogenic factors in neutrophils by tumor cells in vitro. It is the aim of this project to quantitatively analyse the inflammatory microenvironment in laryngeal carcinomas and to use this method for the development of predictive biomarkers. Methods We used automated digital tissue analysis and developed a software based macro to quantify tumor-associated neutrophils and angiogenesis markers. Data were validated and developed by comparison to manual observer counts. Data analysis was separately performed for stromal and intratumoral regions of the malignant tissue. Absolute numbers of neutrophils and CD31-positive vessels were determined. Results After several rounds of validation, good correlation between manual observer assisted counts and digital tissue analysis was achieved. Quantification of tumor-associated neutrophils revealed a clear dominance of stromal over tumor-infiltrating neutrophils. Because of substantial intratumoral heterogeneity in a number of specimens, a minimum number of four digital images per stromal/tumor region was required for analysis. Tumors were grouped as neutrophil “high” or “low” according to a neutrophil count above or below the mean count. In the neutrophil “low” group only 20% of patients showed a dense vascularization with more than 9 capillaries per visual field. In contrast, 38% of patients with highly inflammatory tumors and neutrophil counts above the mean, had densely vascularized tumors. Conclusions We developed an automated digital analysis method to quantify the number of tumor-infiltrating neutrophils. Tumors with a strong neutrophilic infiltrate showed a tendency towards higher vessel density. This method will help to understand the role of neutrophils in the inflammatory head and neck cancer microenvironment and can be used in future immunological biomarker studies.