Background. In many cancers, tumor-infiltrating lymphocytes (TILs) indicate levels of tumor immunogenicity and are a strong predictor of survival. In particular, increased levels of regulatory T cells (Tregs) are associated with poorer prognosis in some cancers. However, visual TIL assessment cannot easily determine the phenotype of lymphocyte in situ. An understanding of both the phenotype and spatial distribution of TILs in situ within tumor regions would be advantageous. Here we present a multi-marker, computer-aided prognostication 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 was 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 favorable outcome with higher numbers of CD3+, CD3+/FOXP3+ and CD3+/CD69+ cells. HID analysis demonstrated the association of favorable outcome with a high entropy / diffuse 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 tumor 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- tumor immune factors present in the tumor 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 biological behaviour, in this case patient outcome, of cellular pattern is available, as demonstrated for the HID method in the present study.

Citation Format: James Mansfield, Lilli Nelson, Roslyn Lloyd, Chris van der Loos, Ken Oguejiofor, Lia Menasce, Kim Linton, Chris Rose, Richard J. Byers. Favorable diffuse prognostic pattern of FOXP3+ and CD69+ T cells in follicular lymphoma demonstrated using automated imaging and analysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1647. doi:10.1158/1538-7445.AM2014-1647