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B cells (CD20+) associated to tumor infiltrating cytotoxic T-cells observed on resected liver
colorectal metastases (LCM) are prognostic.
Abstract Id: 7033 (554P)
M. Van den Eynde (1), B. Mlecnik (2), JP. Machiels (1), D. Debetancourt (1), A. Jouret-Mourin (1), C. Sempoux (1), JF Gigot (1), C. Hubert (1), Y. Humblet (1), N. Haicheur (2), F. Marliot (2), F. Pagès (2), J. Galon (2).
(1) Cliniques universitaires St-Luc, Centre du Cancer, MIRO, BRUSSELS (2) Centre de Recherche des Cordeliers, Université Paris VI, INSERM, PARIS
4. Results 1. Background Numerous data collected from large cohorts of human cancers, demonstrated that the number, type and location of tumor immune infiltrates in primary
colorectal tumors, are prognostic for Disease-Free Survival (DFS) and Overall Survival (OS).
A potential clinical translation of these observations is the establishment of a simple immune score, quantifying the density and location of immune-cells within
the colorectal tumor. This immune score, based on the density of Th1/cytotoxic and memory T cells (CD3/CD8/CD45RO), both in the center and the invasive
margin of the tumors, has important prognosis value that may be superior to the AJCC/UICC TNM –classification (1-3).
Curative surgery of liver colorectal metastases (LCM) is the only hope for cure of metastatic patients. Nevertheless, 70% of resected patients will relapse and
half of them will die from this recurrence.
We previously reported that the immunoscore performed for T-cell infiltration in LCM after curative surgery was strongly related to the patient’s outcome (4-5).
The impact of B cells for prognosis is less characterized.
We report here the prognostic role of B cells infiltration in LCM and the association with cytotoxic T-cells according the validated methodology of the
immunoscore.
A. Demography B. Pa�ent inter-­‐LCM variability Table 1: Patients and disease characteristics
Characteristics
n
Total
88
Sex
Age (diagnosis)
Primary location
Prior treatment lines
for metastatic disease
Liver colorectal
metastases
Preoperative
chemotherapy
To analyse B-cells and the association with cytotoxic T-cells on all liver colorectal metastases resected after preoperative treatment and to validate it as a
potential prognostic marker for the patient.
Preoperative
antibody
Surgical resection
3. Material and Methods CT
CT
CD20
IM
46
42
53%
47%
<50 y-old
≥50-70 y-old
≥70 y-old
13
57
18
15%
65%
20%
CD8IM
colon
rectum
63
25
72%
28%
CD20CT
Mean of all CD8CT
CD20IM
0
1
2
68
11
9
78%
12%
10%
Less of all CD8CT
CD8IM
Synchronous
Metachronous
75
13
85%
15%
294
3,34
FOLFOX
FOLFIRI
Other
38
44
6
43%
50%
7%
Cetuximab
Bevacizumab
None
40
28
20
45%
32%
23%
R0
R1
75
13
85%
15%
Figure 4: Inter-metastatic variability
CD20CT
Each line represents a patient, and each little square a LCM.
The scale of color indicate the mean density value of the 3 most
infiltrated fields of each LCM (blue: low infiltration, red: high
infiltration). The LCM are sorted by CD8 CT in order to compare the
different markers (CD8+, CD20+) in differents regions (CT and IM)
for a same LCM
CD20IM
Most of all
CD8CT
CD8IM
CD20CT
CD20IM
-  Patients with liver colorectal metastases (LCM)
-  Preoperative treatment (chemo +/- targeted therapy)
-  Curative surgery
-  FFPE blocks available for all metastases (+/- primary tumor)
-  Single centre retrospective cohort (Cliniques universitaires St-Luc; UCL)
Survival
HR (High vs Low;95%IC)
Logrank p-­‐value
DFS
OS
DFS
OS
0.44 (0.24-­‐0.81)
0.27 (0.13-­‐0.56)
0.60 (0,34-­‐1.06)
0.54 (0.22-­‐1.32)
0.2
0.0002
0.6
0.2
DFS
OS
DFS
OS
0.63 (0.38-­‐1.06)
0.46 (0.20-­‐1.02)
0.52 (0.25-­‐1.06)
0.60 (0.23-­‐1,59)
0.2
0.05
0.2
0.5
DFS
OS
DFS
OS
0.36 (0.22-­‐0.61)
0.31 (0.15-­‐0.63)
0.38 (0.22-­‐0.67)
0,42 (0.20-­‐0.88)
0.004
0.0006
0.03
0.02
DFS
OS
DFS
OS
0.41 (0.24-­‐0.69)
0.32 (0.16-­‐0.64)
0.68 (0.37-­‐1,23) 0.39 (0.18-­‐0.82)
0.04
0.0008
0.4
0.01
DFS
OS
DFS
OS
0.65 (0.38-­‐1.08)
0,87 (0.44-­‐1.75)
0.69 (0.41-­‐1.16)
0.63 (0.28-­‐1.42)
0.9
0.7
0.5
0.5
DFS
OS
DFS
OS
0.70 (0.41-­‐1.18) 0.60 (0.30-­‐1.22)
0.58 (0.28-­‐1.19)
0.77 (0.29-­‐2.03)
0.4
0.4
0.4
0.6
Figure 6: Kaplan-Meier curves (DFS and OS) for the less infiltrated
LCM per patient for CD20 CT, CD20 IM and CD20 CT/IM.
LCM/patient
Markers
Survival
HR (IS3-­‐4 vs 0-­‐2;95%IC)
Logrank p-­‐value
Mean of all CD8-­‐CD20
DFS
OS
0.44 (0.22-­‐0.91)
0.29 (0.09-­‐1.00)
0.5
0.03
Less of all CD8-­‐CD20
DFS
OS
0.31 (0.18-­‐0.53)
0.25 (0.12-­‐0.51)
0.0005
0.00003
Most of all
CD8-­‐CD20
DFS
OS
0.92 (0.53-­‐1.56)
0.95 (0.42-­‐2.12)
0.7
0.9
Figure 7: Kaplan-Meier curves (DFS and OS) for the less
infiltrated LCM per patient for IS CD8-CD20
CD8-­‐CD20 (IS3-­‐4 vs IS0-­‐2) CD20 CT HR=0.36 (0.22-­‐0.61), p=0.004 IM
B. Immunoscore (combined markers and regions).
A. Each marker (CD8+/CD20+) in each region (CT/IM)
Male
Female
CD8 CT CT
IM
100%
Figure 5: Kaplan-Meier curves (DFS and OS) for the less infiltrated
LCM per patient for CD8 CT, CD8 IM and CD8 CT/IM.
A. Inclusion criteria:
CD8
Table 2: Univariate Cox regression for DFS and OS
LCM/patient Markers
Total
Mean/patient
2. Aim C. CD20+ and CD8+ prognos�c for DFS and OS HR=0.41 (0.24-­‐0.69), p=0.04 HR=0.31 (0.18-­‐0.53), p=0.0005 B. Methods:
-  Whole-slide immunohistochemistry (on all resected LCM).
HR=0.32 (0.16-­‐0.64), p=0.0008 -  CD8 (Dako®)
HR=0.31 (0.15-­‐0.63), p= 0.0006 -  CD20 (Ventana®)
-  Software analysis (Definiens®):
HR=0.25 (0.12-­‐0.51), p=0.00003 Figure 1: Whole slide LCM immunohistochemistry for CD8
-  Whole slide quantitative assessment
-  Center of the Tumor (CT) and Invasive Margin (IM)
-  Mean value of the 3 most infiltrated fields (0.8 mm²) for each
markers was defined in the CT and IM for all LCM in order to define
an immune score.
CD8-­‐CD20 (IS4 versus others) CD20 IM CD8 IM HR=0.71 (0.61-­‐0.83), p=0.0007 HR=0.38 (0.22-­‐0.67), p=0.03 C. End-points: regarding CD8+ and CD20+ cells infiltration
(Immunoscore)
HR=0.68 (0.37-­‐1.23), p=0.4 -  Disease-Free Survival and Overall Survival analyses
+ Kaplan-Meier estimator
HR=0.59 (0.46-­‐0.76), p=0.0001 + Univariate Cox regression and compared by log-rank tests
(Low versus High Immunoscore).
Figure 2: Whole slide quantitative analysis with the software Definiens®
CD20 High IS
The total number of high densities (Hi, minimal p-value
approach for cut-off values) in CT and IM for each
marker was used to stratify patients for the
immunoscore (IS)
HR=0.42 (0.20-­‐0.88), p=0.02 CD8 combina�on CT/IM (HiHi versus others) CD20 combina�on CT/IM (HiHi versus others) HR=0.6 (0.47-­‐0.77), p=0.001 HR=0.58 (0.42-­‐0.81), p=0.1 The markers were combined in CT and IM (CD8-CD20)
and finally regrouped to an IS of 0-2 Hi (IS0-2:low IS )
or 3-4 Hi (IS3-4:high IS).
CD8 IM
Figure 3: The immunoscore (IS) is defined by the CD8+ and CD20+ cells infiltration
in the center (CT) and the invasive margin (IM) for each LCM.
For patients with multiple LCM, the mean value of all
densities, the less and the most infiltrated LCM per
patient were analyzed
5. Conclusions
5. Conclusions The T and B-cell infiltration shows important inter metastatic variability in a same
patient.
B cells (CD20+) associated with cytotoxic T cells (CD8+) in both tumor regions (CT/IM) of
the least infiltrated LCM per patient are highly prognostic after curative resection.
These results confirm the important role of B-cells (CD20+) and tumor regions for the
tumor immune response.
Low IS
CT
HR=0.39 (0.18-­‐0.82), p=0.01 HR=0.53 (0.35-­‐0.78), p=0.0008 HR=0.36 (0.21-­‐0.59), p=0.00004 6. References 1. Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce-Pagès C, et al. Type, density, and location of immune cells within human colorectal
tumors predict clinical outcome. Science. 2006 Sep 29;313(5795):1960–4.
2. Mlecnik B, Tosolini M, Kirilovsky A, Berger A, Bindea G, Meatchi T, et al. Histopathologic-based prognostic factors of colorectal cancers are associated with the
state of the local immune reaction. J. Clin. Oncol. 2011 Feb 20;29(6):610–8.
3. Pagès F, Berger A, Camus M, Sanchez-Cabo F, Costes A, Molidor R, et al. Effector memory T cells, early metastasis, and survival in colorectal cancer. N.
Engl. J. Med. 2005 Dec 22;353(25):2654–66.
4. Halama N, Michel S, Kloor M, Zoernig I, Benner A, Spille A, et al. Localization and density of immune cells in the invasive margin of human colorectal cancer
liver metastases are prognostic for response to chemotherapy. Cancer Res. 2011 Sep 1;71(17):5670–7
5. Van den Eynde M, Mlecnik B, Machiels JP, Debetancourt D, Mourin A, Gigot JF, et al. T-cell infiltration (TCI) observed on whole liver colorectal metastases
(LCM) resected after preoperative treatment is a prognostic survival factor. Marker in cancer, Joint meeting EORTC-ASCO-NCI. Brussels November 2013.