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Phenotyping TILs in situ: automated enumeration of intra- and extra-follicular FOXP3+ regulatory T cells in follicular lymphoma
 

Phenotyping TILs in situ: automated enumeration of intra- and extra-follicular FOXP3+ regulatory T cells in follicular lymphoma

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Authors:
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) and R.J. Byers (3).

Affiliations:
1 - PerkinElmer, Hopkinton, MA
2 - Academic Medical Center, Amsterdam, Netherlands
3 - University of Manchester, UK

For further information on the Microscopy Imaging Systems and Software (PerkinElmer) presented in this poster, please visit http://bit.ly/15hJz6D

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    Phenotyping TILs in situ: automated enumeration of intra- and extra-follicular FOXP3+ regulatory T cells in follicular lymphoma Phenotyping TILs in situ: automated enumeration of intra- and extra-follicular FOXP3+ regulatory T cells in follicular lymphoma Presentation Transcript

    • 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. Byers3 1) PerkinElmer, Hopkinton, MA; 2) Academic Medical Center, Amsterdam, Netherlands; 3) University of Manchester, UKSummary Automated tissue and cellular segmentation Clinical correlation of results Outcome Outcome OutcomeIn many cancers, tumor-infiltrating lymphocytes (TILs) indicate levels of tumor CD3 positivity, identifying T-cells, wasimmunogenicity and are a strong predictor of survival. In particular, increased levels of Sample 1 Sample 2 used to identify CD3 rich and poor 100 FOXP3 Tregs in CD3 poor areas 100 100 FOXP3 Tregs in CD3 poor areas FOXP3 Tregs in CD3 poor areasregulatory T cells (Tregs) are associated with poorer prognosis in some cancers. An areas, approximating to extra-follicular 90 less than 25% centile less than median 90 less than 75% centile =/> 25% centile =/> median =/> 75% centileunderstanding of the phenotype and spatial distribution of TILs in situ within tumor regions (green) and intra-follicular (pink) 80 80 80 Survival probability (%) Survival probability (%) Survival probability (%)would be advantageous. However, visual TIL assessment cannot easily determine the type areas, respectively. Thresholding of 70 70of lymphocyte in situ and multimarker quantitation is difficult with standard methods. Here CD3 (membrane) and FOXP3 P=0.04 60 P=0.00we present a multi-marker, computer-aided event-counting method for determining the (nuclear) was used to identify double 60 31 36 60phenotypes of lymphocytes in follicular lymphoma sections using a multispectral imaging FOXP3+/CD3+ Treg cells (shown as 50 50(MSI) and automated tissue segmentation and counting approach. A tissue microarray yellow cells), FOXP3-/CD3+ cells 40 40 P=0.017containing follicular lymphoma cores from 70 patients was stained for CD3, FOXP3 and (green) and other cells (blue) in both 40hematoxylin, of which 40 cores were informative for both triple staining and clinical follow- compartments. 30 3 20 30up. Each core was imaged using MSI and the individual staining of each marker separated 20from each other using spectral unmixing. The images were analyzed using software which 20 10had been trained to recognize the follicular areas based on the tissue morphology, 0 50 100 150 200 0 10specifically based on CD3 rich (extra-follicular) and poor (intra-follicular) areas. The Sample 1 Sample 2 Time Outcome 0 50 100 Outcome 150 200 0 50 100 Outcome 150 200FOXP3 status of each CD3+ TIL was then determined and the number of each Treg Time Time 100 100 100(FOXP3+/CD3+) counted for both the intra- and extra-follicular tissue compartments. FOXP3 Tregs in CD3 rich areas FOXP3 Tregs in CD3 rich areas FOXP3 Tregs in CD3 rich areasResults indicate that machine-learning software can be trained to accurately recognize less than 25% centile less than median 90 less than 75% centile =/> than 25% centile =/> than median =/> than 75% centilefollicular and non-follicular regions within each core, in this instance based on abundance 80 80 80 Survival probability (%) Survival probability (%) Survival probability (%)of CD3 cells. MSI enabled the accurate quantitation of two immunostains in the sample 70without crosstalk. The number of Tregs were determined for each core and used in Kaplan- 60Meier survival analysis, which demonstrated association of FOXP3+/CD3+ Tregs with 60 P=0.21 60favourable outcome in both the intra- and extra-follicular areas. Understanding the number 79 50 40and location (intra- and extra-follicular) of Tregs is an assay with potentially important 40 40clinical prognostic implications. Thus study shows that an automated method for counting P=0.03 P=0.003Tregs can be developed for follicular lymphoma. This multimarker phenotyping and 20 30 43counting approach shows the potential for broad applicability in the enumeration of a wide 20 4 20range of specifically phenotyped TILs in situ in many solid tumors. 0 10 0 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 Time Time Time Sample 1 Sample 2 Extra-follicular cells:1473 Extra-follicular cells: 1917 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 Multispectral imaging technology Morphologic and cellular segmentation CD3+/FOXP3-: 13.9% CD3+/FOXP3-: 19.66% 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 CD3+/FOXP3+: 12.3% CD3+/FOXP3+: 0.66% Spectrum from Survival: 171+ months Survival: 54 months 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 • Images at different nucleus with both hematoxylin and Breast cancer ER/PR 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 DAB wavelengths co-expression assay 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 • Assemble the images into a data “cube” • Spectrum at every (x,y) pixel RGB representation Multispectral imaging of triplex- Conclusions of spectral cube stained follicular lymphoma Spectrum from Spectrum from With cancer mask • Multispectral imaging enabled the quantitation of two immunostains (CD3 Nuance® and Vectra™ membrane with just red stain stroma with just hematoxylin RGB representation of multispectral dataset & FOXP3) in intra- and extra-follicular compartments in follicular Multispectral Imaging FOXP3 lymphoma RGB Representation of Spectral Cube Systems With cancer mask and • FOXP3+ Tregs were automatically counted and used in Kaplan-Meier nuclear segmentation survival analysis, demonstrating association with good outcome Unmixed DAB • Automated multiplexed tissue cytometry analyses are feasible for routine Component 1) Automated user-trained morphologic segmentation clinical studies and work with many multiplexed IHC staining Spectra of pure chromogens using inForm™ Tissue Finder collected from single- stained sections Unmixed Red Component Once unmixed, methodologies. • The enumeration of FOXP3 +’ve T cells in these clinical samples was Unmixed Hematoxylin stains can be 2) Cellular segmentation Component Red = cancer mask measured (nuclear, cytoplasmic or CD3 Green = cancer nuclei accurately. membrane) Blue = background effective and easy to perform. PerkinElmer, Inc., 68 Elm Street, Hopkinton, MA USA (800) 762-4000 or (+1) 203 925-4602 www.perkinelmer.com