Diapositive 1

1
Variable impact of chemotherapy /- cetuximab on
immune modulation
in a prospective
cohort of 163 cancer patients 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 1 Laboratory 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.
AACR 2015PhiladelphiaAbstract 1327
Figure 4 Effect of 5FU-cisplatin Cetuximab
(headneck cancer cohort)
Figure 5 Effect of FOLFIRI Cetuximab
(colorectal cancer cohort)
Figure 1 Elimination, inactivation or
reprogramming of tumor-induced Tregs and MDSCs
by conventional chemotherapeutic drugs
Figure 2 Mechanisms through which targeted
anticancer agents affect the immune system
INTRODUCTION
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.
//
5FU-cisplatin
5FU-Irinotecan
5FU-cisplatin Cetuximab
5FU-Irinotecan Cetuximab


RESULTS



Basal cell counts of T cells, NK, NKT and invNKT
cells were similar between the four patient
groups.   5FU-Cisplatin chemotherapy in HeadNeck
cancer Analysis of intra-patient cell count
evolution at 2-month relative to baseline showed
that 5FUplatinum 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 lt0.001 and
0.20, Table 2A, Figure 4). In the presence of
Cetuximab, the above negative modulation of cell
count observed with 5FUplatinum 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).




Cell count change at 2 months relative to
baseline p lt0.001 0.001 p lt0.05
(Wilcoxon paired-test).
Cell count change at 2 months relative to
baseline p lt0.001 0.001 p lt0.05
(Wilcoxon paired-test).
Table 1 Patient characteristics
Table 2 Intra-patient cell count change () at 2
months relative to baseline
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 (N60) or not
(N11) with Cetuximab (Cetux), and 92 headneck
squamous cell carcinoma patients receiving
5FUplatinum-based chemotherapy, associated
(N69) or not (N23) 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 V?24 (clone C15 from
Immunotech Marseille, France), PE-labeled
antihuman TCR V?11 (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).
2A - Headneck cancer cohort
Figure 3 Illustration of flow cytometry plots of
invariant CD1d-restricted natural killer T
(invNKT) cells in two patients
p value of Wilcoxon paired-test or Mann-Whitney
test. ns means not significant.
2B - Metastatic colorectal cancer cohort

• The frequency of invNKT cells was identified by
  coexpression of the T-cell antigen receptor
  (TCR)-Va24 chain and TCR-Vb11 chain (lower
  panels) after gating on CD3 T cells (upper
  panels).
• Plots from two patients representative of the two
  patients categories, i.e. with invNKT cells/µl
  lower or upper the median value (0.280 invNKT
  cells/µl) are shown
• invNKT High (1196 cells/µl)
• invNKT Low (0.179 cells/µl).

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.
p value of Wilcoxon paired-test or Mann-Whitney
test. ns means not significant.
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