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Halioseek™ PD-L1/CD8 stainingD. Halioseek™ PD-L1/CD8 virtual slides of four NSCLC samples x20
Correlation between Halioseek™ PD-L1/CD8 and commercial PD-L1 testsA. Virtual slides (x20) of 5 NSCLC samples stained with Dako 22C3 (top), Halioseek™ PD-L1/CD8 dual stain (middle) or VentanaSP263 (bottom).
Analytical comparison of three assays measuring % PD-L1+ TC. B.
C. % PD-L1+ TC with Halioseek™ PD-L1/CD8 compared with 22C3 (Dako) and SP263 (Ventana) commercial tests.
CD8+ cells density assessed by digital pathology
BackgroundAnti-PD-1/PD-L1 are now established agents in the clinical management of advanced NSCLC patients. Although PD-L1positivity enriches for populations with clinical benefit, the selection of patients can be further improved (Topalian, S. L.et al. Nat. Rev. Cancer 2016). In particular, the presence of tumor-infiltrating lymphocytes (TILs), known to play a crucialrole in the immune response to cancer, could also be predictive of the response to Immune Checkpoint Inhibitors (ICI).In addition to their co-presence, the proximity between PD-L1+ and CD8+ cells in the tumor microenvironment iscorrelated to the response to ICI treatment in melanoma (Tumeh, P. C. et al. Nature, ; Teng, M. W. L. et al. CancerRes., 2015). Determination of this relative proximity, as evidence of a physical interaction between PD-L1+ and CD8+cells, might provide complementary information to stratify NSCLC patients and adapt therapeutic strategy.
Florence Monville1, Emmanuel Prestat1, Nadia Yessaad2, Marine Villard1, Luciana Batista1, Julien Adam3, Jérôme Galon , Jacques Fieschi1. 1- HalioDx, Marseille, France | 2- MI-mAbs CIML, Marseille, France | 3- Institut Gustave Roussy, Villejuif, France | - Centre de Recherche des Cordeliers - Inserm, Paris, France
ConclusionsHalioseek™ PD-L1/CD8 is highly correlated to existingcommercial PD-L1 assays across a wide range of PD-L1positive tumors.
High overall agreement, sensitivity and specificity with bothSP263 and 22C3 commercial assays suggest equivalentpredictive/prognostic value at 1% and 50% cut off values.
The detection and quantification of CD8-positive cell densityon the same slide may improve the stratification of NSCLCpatients.
CD8/PD-L1 proximity assessment may be useful to furtherrefine that stratification.
In summary:Halioseek™ PD-L1/CD8 test combines on a single slide:
The accurate PD-L1expression assessment
The quantification of CD8+ cells and the assessment of their clustering
A proximity index between PD-L1+ and CD8+ cells
A new standardized CD8 and PD-L1 dual assay
References1. Mechanism-driven biomarkers to guide immune
checkpoint blockade in cancer therapy. Topalian, S. L.,Taube, J. M., Anders, R. A. & Pardoll, D. M. Nat. Rev. Cancer16, 275–287 (2016)
2. PD-1 blockade induces responses by inhibiting adaptiveimmune resistance. Tumeh, P. C. et al. Nature 515, 568–571 ( )
3. Classifying Cancers Based on T-cell and PD-L1.Teng, M. W. L., Ngiow, S. F., Ribas, A. & Smyth, M. J. CancerRes. 75, 2139– (2015)
AACR 2017 – Poster ID: 590
MethodPD-L1 classical approach: A total of NSCLC tumors were analyzed with three PD-L1 IHC commercial assays: dualstaining with Halioseek™ PD-L1/CD8 kit* (HalioDx), single staining with 22C3 (Dako) and SP263 (Ventana). An experttrained in interpreting PD-L1 staining according to recommendations of each manufacturer estimated the percentagesof PD-L1+ tumor cells (TC).
Digital pathology analysis: 52 whole Halioseek™ PD-L1/CD8 dual stained virtual slides were analyzed with a proprietarysoftware for automated recognition and localization of tissue, anthracosis, PD-L1 and CD8 staining and quantification ofCD8-positive cells.
Three outputs are generated for each sample: CD8+ cell density, Proximity index between CD8+ cells and PD-L1staining, CD8+ cells clustering index.
Proximity index between CD8-positive cells and PD-L1 stainingHalioseek™ digital pathology software establishes a proximity index based on the measure of the distance between CD8+ cells and PD-L1signal.
H. Comparison of CD8+ cell quantification.
CD8+ cells were counted by Halioseek™ digital pathology software and a trained
representative of tissue and cell density heterogeneity. No bias was observed by Bland & Altman test (data not shown).
Halioseek™ PD-L1/CD8 workflow Digital pathology analysis
Halioseek™ PD-L1/CD8IHC dual staining
+ Visual PD-L1+ TC assessment
Digital pathology workflowDual-stainedvirtual slide
CD8+ cells identification, localization / quantification
PD-L1 staining detection and localization
ROI verification
Final results
CD8+ cell density
Proximity index between CD8+ cells and PD-L1 staining
CD8+ cells clustering index
B. PD-L1+ TC % across NSCLC samples C. Halioseek™ vs SP263 or 22C3
PD-L1 staining detection
0
10
20
30
50
60
LCN
22
LCN
18
LCA0
2
LCN
25
LCN
07
LCN
02
LCN
37
LCN
03
LCN
10
LCN
19
LCN
17
LCA0
7
LCN
36
LCN
33
LCN
16
LCN
21
LCN
06
LCN
39
LCN
01
LCN
09
LCN
05
LCN
11
LCA1
6
LCN
15
LCN
23
LCN
30
LCN
12
LCN
32
LCA1
1
LCN
20
LCA1
3
LCN
35
LCN
31
LCN
13
LCA0
8
LCN
29
LCN
08
LCA0
3
LCA1
2
LCN
27
LCN
38
LCA1
5
LCN
28
LCN
26
LCA0
6
LCA0
5
Prox
imity
Inde
x(a
rbitr
ary
unit)
NSCLC samples
K. The proximity index takes into accountthe localization of CD8+ cells and PD-L1signal within a delimited area (pinkcircles).
L. Distribution of the proximity index across 52 NSCLC samples: proximity indexvalues span between 1 and 52, reflecting several putative classes of NSCLC tumorsdepending on this parameter.
G. CD8 staining detection (red stain). Cells detected by the software are surrounded in yellow.
I. Precision assessment of CD8+ cell density on three NSCLC samples.
Parameters variability:- 2 Halioseek™ lots- 2 revelation lots- 30 slides per sample
1 unstainedNSCLC slide
Stained slide scan Virtual slide
22C3
LCN15 LCN26
Halioseek™
SP263
y = 0,93x - 0,51R² = 0,98
y = 0,95x R² = 0,89
0
10
20
30
50
60
70
80
90
100
0 10 20 30 50 60 70 80 90 100
Hal
iose
ek (P
D-L
1+ T
C %
)
Reference (PD-L1+ TC %)
SP263 22C3 y = x
J. PD-L1 staining detection (Brown stain). Cells detected by the software are surrounded in blue.
Agreement between methods assessed at 1% and 50% cut-offsE. 22C3 vs. Halioseek™
F. SP263 vs. Halioseek™
Discordant samples analysis: Three negative samples (<1%) with 22C3 reported low-positive with Halioseek™ and SP263. One
22C3 and 50% with Halioseek™ and 60% with SP263.
R2 �0.86
0.01
0.10.1
1
10
100
1000
1 10 100Nb.cells: pathologist
Nb.
cells
: DP
CD8: DP vs pathologist (log10)
LCN15 LCN26
LCN32 LCA03
0
10
20
30
50
60
70
80
90
100
LCA0
2
LCN
03
LCN
09LC
N10
LCN
13
LCN
17LC
N21
LCN
23LC
N30
LCN
36LC
N39
LCA1
3LC
N02
LCN
18LC
N20
LCN
22LC
N25
LCA0
9LC
N05
LCN
19LC
N33
LCN
37LC
N31
LCA0
7LC
N29
LCA1
1LC
A10
LCA1
2LC
N26
LCN
27LC
N08
LCA0
8LC
N01
LCA0
5LC
N32
LCA0
3LC
N38
LCA0
6LC
N15
LCN
28
PD-L
1+ T
C %
NSCLC Samples
HDX3 SP263 22C3
Mean Global CV
LCN01 168 cells/mm2 11%
LCN06 185 cells/mm2 12%
LCN13 79 cells/mm2 11%
1 % cut-off
< 1%
Halioseek™ < 1% 15 0
3 28
Overall agreement: 93%
50 % cut-off
< 50%
Halioseek™ < 50% 32 0
1 13
Overall agreement: 98%
1 % cut-off
< 1%
Halioseek™ < 1% 15 0
0 31
Overall agreement: 100%
50 % cut-off
< 50%
Halioseek™ < 50% 31 1
0
Overall agreement: 98%
*For Research Use Only. Not for Use in Diagnostic Procedures.