Phenotyping TILs in situ: automated enumeration of intra- and extra-follicular FOXP3+ regulatory T...
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Transcript of Phenotyping TILs in situ: automated enumeration of intra- and extra-follicular FOXP3+ regulatory T...
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. Byers3 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
Su
rviv
al p
ro
bab
ilit
y (
%)
FOXP3 Tregs in CD3 poor areas
less than 25% centile
=/> 25% centile
P=0.0431
Outcome
0 50 100 150 200
0
20
40
60
80
100
Time
Su
rviv
al p
ro
bab
ilit
y (
%)
FOXP3 Tregs in CD3 poor areas
less than median
=/> median
P=0.0036
Outcome
0 50 100 150 200
10
20
30
40
50
60
70
80
90
100
Time
Su
rviv
al p
ro
bab
ilit
y (
%)
FOXP3 Tregs in CD3 poor areas
less than 75% centile
=/> 75% centile
P=0.0173
Outcome
0 50 100 150 200
0
20
40
60
80
100
Time
Su
rviv
al p
ro
bab
ilit
y (
%)
FOXP3 Tregs in CD3 rich areas
less than 25% centile
=/> than 25% centile
P=0.2179
Outcome
0 50 100 150 200
0
20
40
60
80
100
Time
Su
rviv
al p
ro
bab
ilit
y (
%)
FOXP3 Tregs in CD3 rich areas
less than median
=/> than median
P=0.0034
Outcome
0 50 100 150 200
10
20
30
40
50
60
70
80
90
100
Time
Su
rviv
al p
ro
bab
ilit
y (
%)
FOXP3 Tregs in CD3 rich areas
less than 75% centile
=/> than 75% centile
P=0.0343
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