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Variable impact of chemotherapy +/- cetuximab on immune modulation
in a prospective cohort of 163 cancer patients
AACR 2015
Philadelphia
Abstract # 1327
Cristiana Lo Nigro 1, Martino Monteverde 1, Marie-Christine Etienne-Grimaldi 2, Giuliana Strola 3, Laura Lattanzio 1, Daniela Vivenza 1, Federica Tonissi1,
Annalisa Ghiglia 1, Marco Merlano 4 and Gérard Milano 2
of Cancer Genetics and Translational Oncology, Santa Croce General Hospital, Cuneo, Italy; 2 Oncopharmacology department, Centre Antoine Lacassagne, Nice, France ;
3 Laboratory Department, Santa Croce General Hospital, Cuneo, Italy; 4 Medical Oncology Department, Santa Croce General Hospital, Cuneo, Italy.
Figure 2
Elimination, inactivation or reprogramming of tumor-induced
Tregs and MDSCs by conventional chemotherapeutic drugs
Mechanisms through which targeted anticancer agents
affect the immune system
Figure 4
INTRODUCTION
Effect of 5FU-cisplatin ± Cetuximab (head&neck cancer cohort)
Immunomodulation by anticancer agents, either conventional chemotherapies or targeted therapies, is
currently of major interest. Interestingly, previous studies have shown that chemotherapeutic agents alone
(Figure 1) [1,2] and anti-EGFR therapies alone (Figure 2) [3] can induce more or less pronounced changes in
the immunological cell profile. In particular, monoclonal antibodies (mAb) may directly activate or inhibit
molecules of the immune system [4]. We thus prospectively examined in 163 advanced cancer patients the
impact of conventional 5FU-based chemotherapy, combined or not with cetuximab, on immune cell profile. This
study is a companion study of a greater ongoing project (joint French-Italian) aimed at examining the individual
patient capacity to produce ADCC during anti-EGFR antibody treatment with cetuximab [5].
RESULTS
Basal cell counts of T cells, NK, NKT and invNKT cells were similar between the four patient groups.
PATIENTS AND METHODS
Patients
This prospective study was conducted on 163 patients included at the Santa Croce Hospital (Cuneo, Italy) between 2009 and
2014. There were 71 metastatic colorectal cancer patients receiving FOLFIRI, associated (N=60) or not (N=11) with Cetuximab
(Cetux), and 92 head&neck squamous cell carcinoma patients receiving 5FU+platinum-based chemotherapy, associated (N=69)
or not (N=23) with Cetux (Table 1).
Flow cytometry analysis
For each patient, 12 ml blood samples were taken early in the morning at baseline and after 2-month treatment. Phenotyping of
peripheral-blood lymphocytes was performed on whole-blood by flow cytometry analysis. Lymphocytes from whole blood were
isolated by physical parameters (SSC vs FSC) and absolute numbers were calculated (WBC count). WBC (95% CD3- CD56+) at
0.3 to 1 x 106/ml were incubated for 20 min at room temperature with TCR Va24 FITC labelled mAb, TCR Vb11 PE labelled mAb,
D56-specific APC labelled mAb and CD3-specific PC7 labelled mAb. After lysis with NH4Cl, samples were washed with PBS and
cells were analyzed with a Beckman Coulter Fc500 flow cytometer. Data were analyzed using the CXP 2.2 software (Beckman
Coulter, Fullerton, CA).
The following mAb were used for cell characterization: APC-labeled antihuman CD56 (cloneN901(NKH-1) from Immunotech
Marseille, France), PC7-labeled antihuman CD3 (cloneUCH123 from Immunotech Marseille, France), FITC-labeled antihuman
TCR Va24 (clone C15 from Immunotech Marseille, France), PE-labeled antihuman TCR Vb11 (clone C21 from Immunotech
Marseille, France) [6]. T cells were defined as CD3+ (irrespective of CD56), NK cells as CD3- CD56+, NKT cells as CD3+ CD56+,
and invariant CD1d-restricted natural killer T (invNKT) cells were characterized by coexpression of T-cell antigen receptor-Va24
and -Vb11 along with CD3+ and CD56+ (Figure 3).
Statistics
Non-parametric tests were applied. Wilcoxon paired-test was used for analysis of intra-patient evolution of cell count at 2-month
relative to baseline. Mann-Whitney test was used for comparing basal cell counts between patient groups as well as for
comparing intra-patient cell count change between Cetuximab group and No-Cetuximab group. All tests were two-sided and p
values below 0.05 were considered significant. Statistics were performed on SPSS software (version 15.0).
Figure 5
5FU-Cisplatin chemotherapy in Head&Neck cancer
Analysis of intra-patient cell count evolution at 2-month relative to baseline showed that 5FU+platinum alone
negatively significantly modulated both T, NK, NKT and invNKT cells, with a median decrease of 44% to 66%
relative to baseline (p values comprised between <0.001 and 0.20, Table 2A, Figure 4). In the presence of
Cetuximab, the above negative modulation of cell count observed with 5FU+platinum was significantly
attenuated for all immune cells ; this phenomena was highly significant for T cells (Intergroup statistics p =
0.002, Table 2A, Figure 4) and a similar trend was observed in NKT cells.
5FU-Irinotecan chemotherapy in colorectal cancer
Analysis of intra-patient cell count evolution at 2-month relative to baseline showed that FOLFIRI alone had no
significant impact on both T, NK, NKT and invNKT cells. In contrast, the combination of Cetuximab with
FOLFIRI induced a significant decrease in NK cells and invNKT cells (Table 2B, Figure 5).
140
Illustration of flow cytometry plots of invariant CD1d-restricted natural killer T (invNKT) cells in two patients
Plots
from
two
patients
representative of the two patient’s
categories, i.e. with invNKT cells/µl
lower or upper the median value
(0.280 invNKT cells/µl) are shown:
(A)
(B)
invNKT High (1196 cells/µl)
invNKT Low (0.179 cells/µl).
5FU-Irinotecan + Cetuximab
120
120
100
80
60
40
20
0
-20
*
**
-40
**
T cells
*
*
**
*
-60
NK
NKT
100
80
60
40
0
-20
-40
-60
T cells
NK
NKT
invNKT
Cell count change at 2 months relative to baseline: * p <0.001 ; ** 0.001 ≤ p <0.05 (Wilcoxon paired-test).
Table 1
Table 2
Patient characteristics
Head&Neck cancer cohort
*
**
20
invNKT
Cell count change at 2 months relative to baseline: * p <0.001 ; ** 0.001 ≤ p <0.05 (Wilcoxon paired-test).
Intra-patient cell count change (%) at 2 months relative to baseline
Metastatic colorectal cancer cohort
2A - Head&neck cancer cohort
5FU-Pt
group
5FU-Pt + Cetuximab
group
FOLFIRI
group
FOLFIRI + Cetuximab
group
23
69
11
60
50
57
23-75
60
61
39-87
65
64
48-74
67
67
48-83
15
7
57
12
8
4
41
19
3
2
0.6-14
5
2
0.2-30
6
6
1,1-11
7
5
1,6-39
5FU-cisplatin group
N
Sex
mean
median
extremes
men
women
Treatment duration
(months) mean
median
extremes
The frequency of invNKT cells was
identified by coexpression of the Tcell antigen receptor (TCR)-Va24
chain and TCR-Vb11 chain (lower
panels) after gating on CD3+ T cells
(upper panels).
5FU-Irinotecan
140
**
5FU-cisplatin + Cetuximab
Age
Figure 3
Effect of FOLFIRI ± Cetuximab (colorectal cancer cohort)
//
5FU-cisplatin
Mean Intra-patient cell count change at 2 months
(%)
Figure 1
Mean Intra-patient cell count change at 2 months
(%)
1 Laboratory
T cells
NK
NKT
invNKT
5FU-cisplatin + Cetux group
Median
Q1/Q3
% of cases
with decrease
-66
-65
-52
-44
-80/-48
-83/-32
-66/-8
-74/-8
96%
87%
82%
78%
Median
Q1/Q3
% of cases
with decrease
-36
-56
-30
-38
-63/-2
-71/-22
-50/+32
-67/0
76%
89%
61%
72%
Stat*
p<.001
p=.002
p=.020
p=.004
Inter-group
Statistics**
Stat*
p<.001
p<.001
ns
p<.001
p=.002
ns
p=.089
ns
p value of Wilcoxon paired-test* or Mann-Whitney test**. ns means not significant.
2B - Metastatic colorectal cancer cohort
5FU-irinotecan group
CONCLUSION
Present results reveal an opposite modulation of peripheral
immune cells by cetuximab according to the associated
chemotherapy protocol: as compared with chemotherapy alone,
cetuximab in combination with 5FU-platinum attenuates the
negative modulation of immune cells, whereas in combination with
5FU-irinotecan, cetuximab induces a negative modulation of NK
and invNKT cells. Such data draw attention on the origin of the
underlying molecular mechanisms and would merit to be further
explored. These striking results may be of importance in the
context of current and future settings of associations between
chemotherapy-antibody combination and immunotherapy.
T cells
NK
NKT
invNKT
Median
Q1/Q3
-15
-17
+18
-32
-24/-2
-34/+20
-17/+43
-57/+28
5FU-irinotecan + Cetux group
% of cases
with decrease
Stat*
73%
55%
27%
55%
ns
ns
ns
ns
Median
Q1/Q3
% of cases
with decrease Stat*
-3
-43
-1
-18
-23/+21
-60/-11
-23/+46
-54/+37
56% ns
80% p<.001
51% ns
55% p=.008
Inter-group
Statistics**
ns
p=.031
ns
ns
p value of Wilcoxon paired-test* or Mann-Whitney test**. ns means not significant.
REFERENCES
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10.1158/0008-5472
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4- Levy EM et al. J Biomed Biotechnol 2011; Doi: 10.1155/2011/676198
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doi: 10.1016/j.critrevonc
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