Abstract C286: Phenotyping TILs in situ: Automated enumeration of Tregs and Tacts in solid tumors.
Authors: Mansfield James , Nelson Lilli , van der Loos Chris , Byers Richard 
Published: August 2014

Abstract
In many cancers, tumor-infiltrating lymphocytes (TILs) indicate levels of tumor immunogenicity and predict survival. In particular, increased levels of regulatory T cells (Tregs) are associated with poorer prognosis, whilst CD69+ T-cells may also be prognostic. Understanding the phenotype and pattern of TILs in situ within tumors would be advantageous. However, visual TIL assessment cannot easily determine the type of lymphocyte in situ and multimarker quantitation is difficult with standard methods. We present a multi-marker, computer-aided event-counting method for determining the phenotypes of lymphocytes in a range of tumor types using a multispectral imaging (MSI) automated tissue segmentation and counting approach. This paper will demonstrate the use of automated methods for counting Tregs, Tacts and other immune cells in follicular lymphoma, melanoma and lymph nodes. Automated, multiplexed tissue cytometric analyses are feasible for routine clinical studies, work with many multiplexed IHC staining methodologies for a range of immune cell types and are of importance for translational cancer studies in general and cancer immunotherapy in particular. A tissue microarray containing follicular lymphoma (FL) cores from 70 patients was chromogenically immunostained for CD3, CD69 and FOXP3, counterstained with hematoxylin, of which 40 cores were informative for both triplex staining and clinical follow-up. Each core was imaged using MSI and the individual staining of each marker separated from each other using spectral unmixing. Images were analyzed using software trained to recognize different tissue compartments based on morphology, specifically based on CD3 rich (extra-follicular) and poor (intra-follicular) areas. The FOXP3 or CD69 status of each CD3+ TIL was then determined and number Treg (FOXP3+/CD3+) and CD69+ T-cells counted in the intra- and extra-follicular areas. The intra-follicular (CD3 poor) and extra-follicular (CD3 rich) regions were accurately recognized within each core, based on abundance of CD3 cells. MSI enabled the accurate quantitation of CD3, CD69 and FOXP3 without crosstalk. The number of FOXP3+/CD3+ Tregs and CD69+ T-cells were counted in each core and used in Kaplan-Meier survival analysis, which demonstrated association of FOXP3+/CD3+ Tregs with favourable outcome in both the intra- (p=0.0173) and extra-follicular (p=0.0173) areas, as well as CD69+ T-cells in intra-follicular (p=0.0175) areas; CD69+ T-cells were not prognostic in extra-follicular areas (p=4509).
Conclusions: This study demonstrates use of an automated method for counting Tregs in follicular lymphoma, showing association of FOXP3+ Tregs with good outcome. Given the generic nature of the method automated multiplexed tissue cytometry analyses are feasible for routine clinical studies and work with many multiplexed IHC staining methodologies, of importance for translational cancer studies in general and cancer immunotherapy in particular.
Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C286.
Citation Format: James Mansfield, Lilli Nelson, Chris van der Loos, Richard Byers. Phenotyping TILs in situ: Automated enumeration of Tregs and Tacts in solid tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C286.