Abstract 2352: Investigation of the spatial interrelations among multiple types of immune and immune related cells in head and neck squamous cell carcinoma
Authors: Mansfield James Robert , Oguejiofor Kenneth , Galletta Henry , Slater Christian , Byers Richard J. 
Published: August 2015

Abstract
Cancer immunotherapy is rapidly changing the landscape of medical oncology and research. The rise of immunomodulatory therapies such as CTLA-4 inhibitors and the durable effects seen in many tumor types are exciting. Despite huge success in this area, positive effects are only observed in a percentage of treated subjects, and it is still not clear why this is the case or how to differentiate responder from non-responder a priori. To this end, a great deal of research is being directed towards the development of in-situ immune profile analyses of tumor-infiltrating lymphocytes (TILs) in solid tumors that includes investigation of interdistributions of multiple immune cells and other markers important for immunotherapy (e.g., PD-1 and PD-L1). We present a combined method for the simultaneous IHC staining of up to 7 antigens, multispectral imaging (MSI), a per-cell quantitative analysis, cellular phenotyping and multivariate hypothesized interaction distribution (HID) analysis of the spatial interrelations among the cells within sections of standard clinical biopsies to search for patterns correlated with clinical outcome and therapeutic response. Whole sections of FFPE archival head and neck squamous cell carcinoma (HNSCC) biopsies from 130 subjects (96 male, 34 female, median 58 years, median follow up 60 months) were stained simultaneously for CD3, CD8, CD68, FOXP3, PD-1, PD-L1 using tyramide-conjugated fluorophores based on an Opal multiplexed IHC method. Each section was imaged in its entirety at 4x. Using an automated region selection method, approximately 20 multispectral images were acquired from regions of tumor and tumor margin at 20x. Each image was segmented into tumor, stromal and blank compartments. Cellular locations were found using the DAPI staining. The multimarker stain intensities of each cell were used to delineate each cell's phenotype. Patients are stratified based on human papillomavirus (HPV) status (P16 IHC, HPV DNA PCR, HPV DNA ISH). The phenotype, staining intensity pattern and spatial location for each TIL was then used as input into the HID analysis for an exploration of cellular patterns associated with good and poor clinical outcome in HPV positive or negative HNSCC. The complexity of tumor-immune interactions is driving the requirement for highly multiplexed in-situ cellular analyses. It is therefore even more relevant that a method such as exhibited here, capable of defining and measuring the effect on patient outcome of the spatial patterns of multiple cellular phenotypes in the tumor microenvironment is available.
Citation Format: James Robert Mansfield, Kenneth Oguejiofor, Henry Galletta, Christian Slater, Richard J. Byers. Investigation of the spatial interrelations among multiple types of immune and immune related cells in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2352. doi:10.1158/1538-7445.AM2015-2352