PerkinElmer, Inc., 68 Elm Street, Hopkinton, MA USA (800) 762-4000 or (+1) 203 925-4602 www.perkinelmer.com Summary Conclusions • Multispectral imaging enabled the quantitation of two immunostains (CD3 & FOXP3) in intra- and extra-follicular compartments in follicular lymphoma • FOXP3+ Tregs were automatically counted and used in Kaplan-Meier survival analysis, demonstrating association with good outcome • Automated multiplexed tissue cytometry analyses are feasible for routine clinical studies and work with many multiplexed IHC staining methodologies. • The enumeration of FOXP3 +’ve T cells in these clinical samples was effective and easy to perform. Phenotyping TILs in situ: Automated Enumeration of Intra- and Extra-Follicular FOXP3+ Regulatory T Cells in Follicular Lymphoma J.R. Mansfield, 1 C.M. van der Loos, 2 , L.S. Nelson, 3 C. Rose, 3 H.E. Sandison, 3 S. Usher, 3 J.A. Radford, 3 K. M. Linton, 3 R.J. Byers 3 1) PerkinElmer, Hopkinton, MA; 2) Academic Medical Center, Amsterdam, Netherlands; 3) University of Manchester, UK Nuance® and Vectra™ Multispectral Imaging Systems • Images at different wavelengths • Assemble the images into a data “cube” • Spectrum at every (x,y) pixel 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 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 Automated tissue and cellular segmentation 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 Outcome 0 50 100 150 200 10 20 30 40 50 60 70 80 90 100 Time FOXP3 Tregs in CD3 poor areas less than 25% centile =/> 25% centile P=0.04 31 Outcome 0 50 100 150 200 0 20 40 60 80 100 Time FOXP3 Tregs in CD3 poor areas less than median =/> median P=0.00 36 Outcome 0 50 100 150 200 10 20 30 40 50 60 70 80 90 100 Time FOXP3 Tregs in CD3 poor areas less than 75% centile =/> 75% centile P=0.017 3 Outcome 0 50 100 150 200 0 20 40 60 80 100 Time FOXP3 Tregs in CD3 rich areas less than 25% centile =/> than 25% centile P=0.21 79 Outcome 0 50 100 150 200 0 20 40 60 80 100 Time FOXP3 Tregs in CD3 rich areas less than median =/> than median P=0.003 4 Outcome 0 50 100 150 200 10 20 30 40 50 60 70 80 90 100 Time FOXP3 Tregs in CD3 rich areas less than 75% centile =/> than 75% centile P=0.03 43 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. Multispectral imaging of triplex- stained follicular lymphoma FOXP3 CD3 RGB representation of multispectral dataset Sample 1 Sample 2 Sample 1 Sample 2 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. An understanding of the phenotype and spatial distribution of TILs in situ within tumor regions would be advantageous. However, visual TIL assessment cannot easily determine the type of lymphocyte in situ and multimarker quantitation is difficult with standard methods. Here we present a multi-marker, computer-aided event-counting method for determining the phenotypes of lymphocytes in follicular lymphoma sections using a multispectral imaging (MSI) and automated tissue segmentation and counting approach. A tissue microarray containing follicular lymphoma cores from 70 patients was stained for CD3, FOXP3 and hematoxylin, of which 40 cores were informative for both triple 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. The images were analyzed using software which had been trained to recognize the follicular areas based on the tissue morphology, specifically based on CD3 rich (extra-follicular) and poor (intra-follicular) areas. The FOXP3 status of each CD3+ TIL was then determined and the number of each Treg (FOXP3+/CD3+) counted for both the intra- and extra-follicular tissue compartments. Results indicate that machine-learning software can be trained to accurately recognize follicular and non-follicular regions within each core, in this instance based on abundance of CD3 cells. MSI enabled the accurate quantitation of two immunostains in the sample without crosstalk. The number of Tregs were determined for each core and used in Kaplan- Meier survival analysis, which demonstrated association of FOXP3+/CD3+ Tregs with favourable outcome in both the intra- and extra-follicular areas. Understanding the number and location (intra- and extra-follicular) of Tregs is an assay with potentially important clinical prognostic implications. Thus study shows that an automated method for counting Tregs can be developed for follicular lymphoma. This multimarker phenotyping and counting approach shows the potential for broad applicability in the enumeration of a wide range of specifically phenotyped TILs in situ in many solid tumors. Multispectral imaging technology RGB representation of spectral cube With cancer mask Breast cancer ER/PR co-expression assay With cancer mask and nuclear segmentation Red = cancer mask Green = cancer nuclei Blue = background 1) Automated user-trained morphologic segmentation using inForm™ Tissue Finder 2) Cellular segmentation (nuclear, cytoplasmic or membrane) Morphologic and cellular segmentation 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