High-Density of FcγRIIIA+ (CD16+) Tumor-Associated Neutrophils in Metastases Improves the Therapeutic Response of Cetuximab in Metastatic Colorectal Cancer Patients, Independently of the HLA-E/CD94-NKG2A Axis

Abstract

Antibody-dependent cellular cytotoxicity (ADCC) in the anti-tumor effect of cetuximab in metastatic colorectal cancer (mCRC) is only based on the impact of FcγRIIIA (CD16) polymorphisms as predictive of therapeutic response. However, nature, density and therapeutic impact of FcγRIIIA⁺ (CD16) effector cells in tumor remain poorly documented. Moreover, the inhibition of cetuximab-mediated ADCC induced by NK cells by the engagement of the new inhibitory CD94-NKG2A immune checkpoint has only been demonstrated in vitro. This multicentric study aimed to determine, on paired primary and metastatic tissue samples from a cohort of mCRC patients treated with cetuximab: 1) the nature and density of FcγRIIIA⁺ (CD16) immune cells, 2) the expression profile of HLA-E/β2m by tumor cells as well as the density of CD94⁺ immune cells and 3) their impact on both objective response to cetuximab and survival. We demonstrated that FcγRIIIA⁺ (CD16) intraepithelial immune cells mainly correspond to tumor-associated neutrophils (TAN), and their high density in metastases was significantly associated with a better response to cetuximab, independently of the expression of the CD94/NKG2A inhibitory immune checkpoint. However, HLA-E/β2m, preferentially overexpressed in metastases compared with primary tumors and associated with CD94⁺ tumor infiltrating lymphocytes (TILs), was associated with a poor overall survival. Altogether, these results strongly support the use of bispecific antibodies directed against both EGFR and FcγRIIIA (CD16) in mCRC patients, to boost cetuximab-mediated ADCC in RAS wild-type mCRC patients. The preferential overexpression of HLA-E/β2m in metastases, associated with CD94⁺ TILs and responsible for a poor prognosis, provides convincing arguments to inhibit this new immune checkpoint with monalizumab, a humanized anti-NKG2A antibody, in combination with anti- FcγRIIIA/EGFR bispecific antibodies as a promising therapeutic perspective in RAS wild-type mCRC patients.

Keywords: CD16; HLA-E/NKG2A axis; antibody-dependent cellular cytotoxicity; cetuximab; metastatic colorectal carcinoma; tumor-associated neutrophils.

Figure 1

FcγRIIIA (CD16) expression on tumor-infiltrating immune cells. Flow cytometry gating strategy of FcγRIIIA (CD16) expression on a representative primary tumor sample from a CRC patient (A). FcγRIIIA (CD16) positive cells are gated among the living lymphoid (G1) and myeloid (G2) cells and different immune sub-populations are highlighted according to their phenotype: T cells (CD3⁺TCRαβ⁺ or γδ⁺), NK cells (CD3⁻CD56⁺), monocytes/macrophages (CD15⁻CD11b⁺CD14^−/low) and granulocytes (CD15⁺CD11b⁺CD14⁻). Frequency of FcγRIIIA (CD16) positive infiltrating immune sub-populations among total FcγRIIIA⁺ (CD16) positive living cells from the 4 non metastatic CRC patients included prospectively. The bar charts represent mean +/- standard deviation (B).

Figure 2

Potential intraepithelial ADCC effector immune cells in colorectal tumors assess by immunohistochemistry. Intraepithelial FcγRIIIA⁺ (CD16) lymphocytes (A) or neutrophils (B). CD8⁺ lymphocytes in close contact with tumor cells (C). Scarce Nkp46⁺Nk cells, only observed in the stroma (D). CD163⁺ macrophages only found in the stroma (E).

Figure 3

The density of FcγRIIIA⁺ (CD16) intraepithelial TAN in metastasis is associated with a better overall survival in mCRC patients treated with cetuximab. Kaplan-Meier curves depict overall survival of patients with a mCRC featuring a low (solid curve) or a high (broken curve) density of FcγRIIIA⁺ (CD16) intraepithelial TAN (second quartile).

Figure 4

Expression profile of HLA-E in primary and metastatic tumors, and its association with intraepithelial CD94⁺ lymphocytes. Two representative cases of mCRC with distinct HLA-E overexpression profile. (A, B) A discordant profile of HLA-E/β2m overexpression between primary tumor (negative) and its matched metastasis (positive) and (C, D) a concordant case featuring HLA-E overexpression both in primary tumor and its paired metastasis. (E) CD94⁺ intraepithelial lymphocytes. (F) The density of CD94⁺ intraepithelial lymphocytes is quite similar between primary tumors and metastases (p = 0.1 Mann Whitney test).

Figure 5

HLA-E/β2m overexpression by tumor cells and a high density of CD94⁺ intraepithelial lymphocytes in primary tumors are associated with a poor overall survival in mCRC patients. (A) Kaplan-Meier curves depict overall survival of patients with a mCRC without HLA-E/β2m overexpression by tumor cells (solid curve) or with HLA-E/β2m overexpression by tumor cells (broken curve). (B) Kaplan-Meier curves depict overall survival of patients with a mCRC containing a high (broken curve) or a low (solid line) density of CD94⁺ intraepithelial lymphocytes (second quartile).