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 2015Philadelphia

Abstract # 1327

INTRODUCTION

REFERENCES

Table 2Intra-patient cell count change (%) at 2 months relative to baselinePATIENTS AND METHODS

RESULTS

CONCLUSION

Figure 1Elimination, inactivation or reprogramming of tumor-induced Tregs and MDSCs by conventional chemotherapeutic drugs

Table 1Patient characteristics

Figure 4Effect of 5FU-cisplatin ± Cetuximab (head&neck cancer cohort)

Figure 2Mechanisms through which targeted anticancer agents

affect the immune system

Figure 5Effect of FOLFIRI ± Cetuximab (colorectal cancer cohort)

Figure 3Illustration of flow cytometry plots of invariant CD1d-restricted natural killer T (invNKT) cells in two patients

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].

PatientsThis 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 analysisFor 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 V24 FITC labelled mAb, TCR V11 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 V24 (clone C15 from Immunotech Marseille, France), PE-labeled antihuman TCR V11 (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-V24 and -V11 along with CD3+ and CD56+ (Figure 3).  StatisticsNon-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).

Basal cell counts of T cells, NK, NKT and invNKT cells were similar between the four patient groups. 5FU-Cisplatin chemotherapy in Head&Neck cancerAnalysis 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 cancerAnalysis 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).

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.

1- Wolf D et al. Onco Immuno 2014 e275881-92- Alizadeh D and Larmonier N. Cancer Res 2014 May 15;74(10):2663-8. doi: 10.1158/0008-54723- Galluzzi L et al. Nat Rev Drug Discov 2012 ; Feb 3;11(3):215-33. doi: 10.1038/nrd36264- Levy EM et al. J Biomed Biotechnol 2011; Doi: 10.1155/2011/6761985- Monteverde M et al. Crit Rev Oncol Hematol. 2015 Mar 12 pii: S1040-8428(15)00048-7. doi: 10.1016/j.critrevonc6- Molling JW et al. J Clin Oncol 2007; Mar 1;25(7):862-8.

The frequency of invNKT cells was identified by coexpression of the T-cell antigen receptor (TCR)-V24 chain and TCR-V11 chain (lower panels) after gating on CD3+ T cells (upper panels).

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)invNKT High (1196 cells/µl) (B)invNKT Low (0.179 cells/µl).

invNKTNKTNKT cells

Mea

n In

tra-

patie

nt c

ell c

ount

cha

nge

at 2

mon

ths

(%)

140

120

100

80

60

40

20

0

-20

-40

-60

invNKTNKTNKT cells

Mea

n In

tra-

patie

nt c

ell c

ount

cha

nge

at 2

mon

ths

(%)

140

120

100

80

60

40

20

0

-20

-40

-60

Head&Neck cancer cohort

Metastatic colorectal cancer cohort

5FU-Pt

group

5FU-Pt + Cetuximab

group

FOLFIRI

group

FOLFIRI + Cetuximab

group

N

Age mean

median

extremes

Sex men

women

Treatment duration (months) mean

median

extremes

23

50

57

23-75

15

7

3

2

0.6-14

69

60

61

39-87

57

12

5

2

0.2-30

11

65

64

48-74

8

4

6

6

1,1-11

60

67

67

48-83

41

19

7

5

1,6-39

5FU-cisplatin group

5FU-cisplatin + Cetux group

Inter-group Statistics**

Median Q1/Q3 % of cases with decrease Stat*

Median Q1/Q3 % of cases with decrease Stat*

T cells

NK NKT

invNKT

-66

-65 -52

-44

-80/-48

-83/-32 -66/-8

-74/-8

96% p<.001

87% p=.002 82% p=.020

78% p=.004

-36

-56 -30

-38

-63/-2

-71/-22 -50/+32

-67/0

76% p<.001

89% p<.001 61% ns

72% p<.001

p=.002

ns p=.089

ns

2A - Head&neck cancer cohort

2B - Metastatic colorectal cancer cohort

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

5FU-irinotecan group

5FU-irinotecan + Cetux group

Inter-group Statistics**

Median Q1/Q3 % of cases with decrease Stat*

Median Q1/Q3 % of cases with decrease Stat*

T cells NK NKT invNKT

-15 -17 +18 -32

-24/-2 -34/+20 -17/+43 -57/+28

73% ns 55% ns 27% ns 55% ns

-3 -43 -1 -18

-23/+21 -60/-11 -23/+46 -54/+37

56% ns 80% p<.001

51% ns 55% p=.008

ns p=.031

ns ns

5FU-cisplatin 5FU-Irinotecan

5FU-cisplatin + Cetuximab 5FU-Irinotecan + Cetuximab

*

***

*

*

****

//

**

*

**

Cell count change at 2 months relative to baseline: * p <0.001 ; ** 0.001 ≤ p <0.05 (Wilcoxon paired-test).

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

Cell count change at 2 months relative to baseline: * p <0.001 ; ** 0.001 ≤ p <0.05 (Wilcoxon paired-test).