Abstract PerkinElmer, Inc., 68 Elm Street, Hopkinton, MA USA (800) 762-4000 or (+ 1) 203 925-4602 www.perkinelmer.com Conclusions • Multispectral imaging enabled the quantitation of three immunostains (CD3, CD69 & FOXP3) in intra- and extra-follicular compartments in follicular lymphoma. • FOXP3 + “Tregs” and CD69+ “Tacts” were automatically counted and used in Kaplan-Meier analysis, each associated with good outcome. • The enumeration of FOXP3+ and CD69+ T cells in these clinical samples was effective and easy to perform. • Need to test whether CD4+ /CD25+ /FOXP3+ T cells have same correlation • This panel of immune markers can easily be changed to a panel of whichever markers are of interest (CD4, CD8, CD20, CD68, Ki67, etc) Phenotyping TILs in situ: Automated Enumeration of Tregs and Tacts in Solid Tumors J.R. Mansfield, 1 C.M. van der Loos, 2 , L.S. Nelson, 3 and R.J. Byers 3 1) PerkinElmer, Hopkinton, MA; 2) Academic Medical Center, Amsterdam, Netherlands; 3) University of Manchester, Manchester, UK Morphologic and cellular segmentation RGB representation of spectral cube With cancer mask inForm™ breast cancer ER/PR co-expression assay With cancer mask and nuclear segmentation Red = cancer mask Green = cancer nuclei Blue = background Field #15 vector red dab 10 0 10 1 10 2 10 3 10 0 10 1 10 2 10 3 0.46% 78.31% 3.83% 17.29% PR - Red ER - DAB Scatter plot of ER vs PR expression 1) Automated, user-trained morphologic segmentation 2) Cellular segmentation (nuclear, cytoplasmic or membrane) Vectra™ Multispectral Imaging Systems • Images at different wavelengths • Assemble the images into a data “cube” • Spectrum at every (x,y) pixel Multispectral imaging technology Once unmixed, stains can be measured accurately. Unmixed Hematoxylin Component Unmixed Red Component Spectra of pure chromogens collected from single- stained sections Unmixed DAB Component 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+ activated T-cells (Tacts) 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). 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. Multispectral imaging of quadruplex-stained follicular lymphoma FOXP3 CD3 RGB representation of multispectral dataset Automated tissue and cellular segmentation FOXP3 Analysis: CD3 positivity, identifying T-cells, was used to identify CD3 rich and poor areas, approximating to extra- follicular (green) and intra-follicular (pink) areas, respectively. Thresholding of CD3 (membrane) and FOXP3 (nuclear) was used to identify double FOXP3+/CD3+ Treg cells (shown as yellow cells), FOXP3-/CD3+ cells (green) and other cells (blue) in both compartments. The CD69 analysis was performed similarly. Sample 1 Sample 2 Sample 1 Sample 2 CD69 Counterstain Sample 1 Extra-follicular cells:1473 CD3+/FOXP3-: 13.9% CD3+/FOXP3+: 12.3% Survival: 171+ months Sample 2 Extra-follicular cells: 1917 CD3+/FOXP3-: 19.66% CD3+/FOXP3+: 0.66% Survival: 54 months Clinical correlation of results 53 samples from 40 patients were automatically analyzed using this methodology and the number of FOXP3+/CD3+ Treg cells in each determined, in both T-cell (CD3+) rich and poor areas. The number of Tregs cells were used in Kaplan-Meier survival analysis, demonstrating association of higher numbers of Tregs with favourable outcome in both T-cell rich (extra-follicular) and poor (intra-follicular) areas (data shown with data split at 25th percentile, median & 75th percentiles for CD3+/FOXP3+ Treg score). This meant patients were divided into groups determined by their Treg numbers using these three statistics as a threshold; . Kaplan-Meier demonstrated that patients with Treg numbers in the top 75%, 50% and 25% all had significant survival advantages over those with lower numbers when divided into two groups based on these proportions Sample 1 Extra-follicular cells:1473 CD3+/FOXP3-: 13.9% CD3+/FOXP3+: 12.3% Survival: 171+ months Sample 2 Extra-follicular cells: 1917 CD3+/FOXP3-: 19.66% CD3+/FOXP3+: 0.66% Survival: 54 months Kaplan Meier survival curves for the activated T-cells (i.e., CD3+ve/CD69+ve), for CD3 rich (extra-follicular) and poor (follicular) areas. RGB Representation of Spectral Cube Spectrum from nucleus with both hematoxylin and DAB Spectrum from membrane with just red stain Spectrum from stroma with just hematoxylin