Suppression of MCP-1, IFN-γ and IL-6 production of HNSCC ex vivo by pembrolizumab added to docetaxel and cisplatin (TP) exceeding those of TP alone is linked to improved survival

Abstract

Background: Adding pembrolizumab, an anti-PD-1 antibody approved for treatment of head and neck squamous cell carcinoma (HNSCC) to neoadjuvant (induction-) chemotherapy utilizing docetaxel and cisplatin (TP) followed by radiotherapy may improve outcome in larynx organ-preservation (LOP) that is investigated in the European Larynx-Organ preservation Study (ELOS). As biomarkers for response to TP and pembrolizumab +TP are missing but may include cytokines, this work aims on determining cytokines potentially linked to outcome as prognostic markers sufficient to predict and/or monitor response to successful LOP.

Methods: Collagenase IV digests were generated from 47 histopathological confirmed HNSCC tumor samples and seeded in 96-well plates containing pembrolizumab, docetaxel, cisplatin either solely or in binary or ternary combination. According to the FLAVINO protocol, supernatants were collected after 3 days, adherent cells fixed using ethanol, air-dried and pan-cytokeratin positive epithelial cells counted using fluorescence microscopy. The cytokines IL-6, IL-8, IFN-γ, IP-10, MCP-1, TNF-α, and VEGF in the supernatant were quantified by sandwich ELISA.

Results: The mode of interaction between pembrolizumab and TP was assessed and correlated to outcome (overall, disease-specific and progression-free survival of patients). Suppression of MCP-1, IFN-γ and IL-6 production by pembrolizumab + TP exceeding the suppressive effect of TP was detected in the majority of samples and linked to improved survival. Multivariate Cox proportional hazard regression modeling revealed MCP-1, IFN-γ and IL-6 as independent outcome predictors.

Conclusions: Comparing response to TP vs. pembrolizumab vs. TP + pembrolizumab may allow for identification of patients with superior outcome independent from treatment applied.

Keywords: CCL2); PD-1:PD-L1 immune-checkpoint inhibitor (ICI) pembrolizumab; biomarker research; local and locoregional advanced head and neck squamous cell carcinoma (HNSCC); monocyte chemoattractant protein 1 (MCP-1; neoadjuvant (induction) chemotherapy; predictive assay for chemoresponse-evaluation.

Figure 1

Cytokine production of head and neck squamous cell carcinoma of 47 patients treated ex vivo according the FLAVINO protocol with immune-checkpoint blockade (ICB) utilizing the anti-PD-1 antibody pembrolizumab either alone (Pemb; 50 ng/ml pembrolizumab mono), TP or PembTP, Pemb combined with half maximum tolerable plasma concentrations of docetaxel (T; 275 nM) and cisplatin (P; 3.33 µM). Mean concentrations (pg/ml) and normalized mean (% of untreated control) as well as median concentrations (pg/ml) and normalized mean (% of untreated control) inclusive 95% CI and interquartile range (IQR) measured in 72-hours supernatants are shown for (A) interleukin 6 (IL-6); (B) IL-8; (C) interferon gamma (IFN-γ); (D) interferon-induced protein 10 (IP-10; CXCL10); (E) monocyte chemo-attractant protein 1(MCP-1; CCL2); (F) vascular endothelial growth factor A (VEGF); (G) tumor-necrosis-factor alpha (TNF-α); (H) mean and 95% CI (left) and median and IQR (right).

Figure 2

Kaplan-Meier cumulative survival plots for (from left to right) overall, disease-specific, and progression-free survival of 47 patients treated ex vivo with immune-checkpoint inhibitor (ICI) utilizing the anti-PD-1 antibody pembrolizumab either alone (pembrolizumab mono) vs. TP or pembrolizumab combined with docetaxel (T) and cisplatin (P) according to half maximum tolerable plasma concentration (275 nM and 3.33 µM, respectively) were compared. Binary split of the cohort was according to the mode of action, suppression (Supp.) of MCP-1 or no effect or suppression of IFN-γ or IL-6 production by pembrolizumab + TP vs. those with stimulation (Stim.). Numbers for patients at risk are provided for (I) monocyte chemoattractant protein 1 (MCP-1; CCL2); (II) interferon gamma (IFN-γ); (III) interleukin 6 (IL-6). P values shown are from log rank tests (2-sided).

Figure 3

Boxplots demonstrating deviating cytokine production of head and neck squamous cell carcinoma in treatment response groups according to binary classification in Figure 2 .