Changes in serum interleukin-8 (IL-8) levels reflect and predict response to anti-PD-1 treatment in melanoma and non-small-cell lung cancer patients

Abstract

Background: Surrogate biomarkers of efficacy are needed for anti-PD1/PD-L1 therapy, given the existence of delayed responses and pseudo-progressions. We evaluated changes in serum IL-8 levels as a biomarker of response to anti-PD-1 blockade in melanoma and non-small-cell lung cancer (NSCLC) patients.

Patients and methods: Metastatic melanoma and NSCLC patients treated with nivolumab or pembrolizumab alone or nivolumab plus ipilimumab were studied. Serum was collected at baseline; at 2-4 weeks after the first dose; and at the time-points of response evaluation. Serum IL-8 levels were determined by sandwich ELISA. Changes in serum IL-8 levels were compared with the Wilcoxon test and their strength of association with response was assessed with the Mann-Whitney test. Accuracy of changes in IL-8 levels to predict response was estimated using receiver operation characteristics curves.

Results: Twenty-nine melanoma patients treated with nivolumab or pembrolizumab were studied. In responding patients, serum IL-8 levels significantly decreased between baseline and best response (P <0.001), and significantly increased upon progression (P = 0.004). In non-responders, IL-8 levels significantly increased between baseline and progression (P = 0.013). Early changes in serum IL-8 levels (2-4 weeks after treatment initiation) were strongly associated with response (P <0.001). These observations were validated in 19 NSCLC patients treated with nivolumab or pembrolizumab (P = 0.001), and in 15 melanoma patients treated with nivolumab plus ipilimumab (P <0.001). Early decreases in serum IL-8 levels were associated with longer overall survival in melanoma (P = 0.001) and NSCLC (P = 0.015) patients. Serum IL-8 levels also correctly reflected true response in three cancer patients presenting pseudoprogression.

Conclusions: Changes in serum IL-8 levels could be used to monitor and predict clinical benefit from immune checkpoint blockade in melanoma and NSCLC patients.

Keywords: IL-8; NSCLC; anti-CTLA-4 mAbs; anti-PD-1 mAbs; melanoma; serum biomarkers.

Figure 1

Changes in serum IL-8 levels reflect tumor response in metastatic melanoma and NSCLC patients during treatment with anti-PD-1 mAbs. Metastatic melanoma (n = 29) and NSCLC (n = 19) patients were treated with anti-PD-1 mAbs and serum IL-8 levels were assessed at baseline (BL), at the moment of best response (BR), and at the moment of progressive disease (PD). (A) Results of IL-8 levels are plotted in the different time-points for metastatic melanoma responders (n = 14) (left) and non-responders (n = 15) (right) according to RECIST 1.1. (B) Results of IL-8 levels are plotted in the different time-points for metastatic NSCLC (n = 12) (left) and non-responders (n = 7) (right) according to RECIST 1.1. Insets show the progression of IL-8 values in each individual patient at the different time-points. Statistical comparisons across median IL-8 levels in different time-points were made by nonparametric Mann–Whitney U tests. *P <0.05, **P <0.01, ***P <0.001.

Figure 2

Early changes in serum IL-8 levels are associated with response in melanoma and NSCLC patients treated with single-agent anti-PD-1 mAbs or in melanoma patients treated with the combination of anti-PD-1 plus anti-CTLA-4 mAbs. (A) Percentages of change in serum IL-8 levels in melanoma patients treated with single agent anti-PD-1 mAbs (left panel); NSCLC patients treated with single agent anti-PD-1 mAbs (middle panel); and melanoma patients treated with anti-CTLA-4 (ipilimumab) and anti-PD-1 (nivolumab) mAbs (right panel), from baseline levels to 2–3 weeks after start of treatment. Statistical comparisons across median IL-8 levels in responders versus non-responders were made by nonparametric Wilcoxon test. (B) ROC curves for the correlation of early changes in serum IL-8 levels with response to treatment in the identification melanoma cohort (left panel); the independent NSCLC cohort (central panel); and the independent melanoma cohort (right panel). The cut-off point of ≥9.2% increase in serum IL-8 levels is designated by a larger circle. AUC and null hypothesis P-value are indicated inside the graph in each case. **P <0.01; ****P <0.0001.

Figure 3

In situ IL-8 mRNA measurement using quantitative fluorescence. (A) IL-8 mRNA transcripts were detected using multiplexed in situ hybridization in FFPE preparations containing human colon cancer HT-29 cells as a positive control and HEK-293 cells as negative control. The signal in each preparation was measured using quantitative fluorescence. (B) Representative fluorescence captions showing the IL-8 mRNA signal (red channel) in HT-29 cells (upper panels) and HEK-293 cells (lower panels). Nuclei were highlighted with DAPI (blue channel). (C) Level of IL-8 mRNA in the tumor (cytokeratin-positive) and stromal (cytokeratin-negative) compartment of NSCLC samples with high and low levels of serum IL-8. (D) Representative fluorescence caption showing the localized IL-8 mRNA signal (red channel), cytokeratin positivity (green channel) and DAPI (blue channel) staining. The number of independent 20× fields of view analyzed is indicated within each bar and scores are expressed as arbitrary units (AU). Error bars represent SEM. *P <0.05, **P <0.01, ***P <0.001.

Figure 4

Changes in serum IL-8 levels are associated with overall survival in melanoma and NSCLC patients treated with anti-PD-1 mAbs. Kaplan–Meier curves of overall survival (OS) stratified by early changes in serum IL-8 levels in (A) melanoma and (B) NSCLC patients treated with single-agent anti-PD1 mAbs. Data were analyzed using the log-rank (Mantel–Cox) test.