Abstract A24: Investigation of the spatial heterogeneity of specific immune cell phenotypes in the tumor microenvironment of follicular lymphoma
Authors: Nelson Lilli , Mansfield James , Lloyd Roslyn , Loos Chris van der , Rose Chris , Byers Richard 
Published: May 2015

Abstract
Background. Tumor-infiltrating lymphocytes (TILs) are present in the tumor microenvironment of many cancers, with consequent tumor immunogenicity and association with survival. In particular, increased levels of regulatory T cells (Tregs) are associated with poorer prognosis in some cancers. However, given the complexity of TIL phenotype their visualization in situ is difficult, and impossible without multiplex immunophenotyping. An understanding of both the phenotype and spatial distribution of TILs in situ within the tumor microenvironment would be advantageous to understanding their role in tumour immunobiology, especially given growing interest in tumour immunotherapy. Here we present a multi-marker, computer-aided method for analysing the distributions of CD3/FOXP3 (Treg) and CD3/CD69 (Tact) T cells in follicular lymphoma sections using a multispectral imaging (MSI) and automated analysis approach. An hypothesized interaction distance (HID) analysis was used to determine whether the spatial patterns of Tregs and Tacts were prognostically significant
Design. A single section of a tissue microarray containing triplex follicular lymphoma cores from 40 subjects [24 male, 16 female, age 35 to 75 years at diagnosis, median 55 years , 2- 171 months follow-up] was stained for CD3, FOXP3, CD69 and hematoxylin. Each core was imaged using MSI and the individual staining of each marker separated from each other using spectral unmixing. CD3+ TILs were located using automated image analysis. The FOXP3 and CD69 status of each CD3+ TIL was then determined and the spatial distributions of the CD3/FOXP3 and CD3/CD69 cells were used as input into the HID analysis.
Results. Multiplexed IHC staining, MSI and automated per-cell quantitative analysis was successful. Kaplan-Meier analysis demonstrated favourable outcome with higher numbers of CD3+, CD3+/FOXP3+ and CD3+/CD69+ cells. HID analysis demonstrated the association of favourable outcome with a high entropy , representative of a diffuse spatial pattern, of FOXP3+ and CD69+ positive T cells.
Conclusion. In this study we report that higher Treg cell counts in a diffuse pattern was associated with favorable prognosis. This supports the importance of Tregs in the tumour microenvironment. It is pertinent to mention that contradictory findings are routinely reported from studies investigating the role of Tregs in solid and haematological malignancies. This is due to the complex interactions between pro-/anti- tumour immune factors present in the tumour microenvironment. The resultant effects are due to the summation of the activities of these factors. 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: Lilli Nelson, James Mansfield, Roslyn Lloyd, Chris van der Loos, Chris Rose, Richard Byers. Investigation of the spatial heterogeneity of specific immune cell phenotypes in the tumor microenvironment of follicular lymphoma. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A24. doi:10.1158/1538-7445.CHTME14-A24