NIRVANA is an ongoing multicentric phase 2 non-randomized prospective trial (ClinicalTrials.gov number NCT02799901) exploring the association of nivolumab (240 mg every 2 weeks until disease progression, intolerable toxicity, or withdrawal of consent) with multisite hypofractionned radiotherapy (performed on week 2 and week 4) in adults with previously untreated advanced melanoma (stage III unresectable or IV) without brain metastases. Written informed consent was obtained from all patients. The primary outcome is the overall survival. Biomarker assessment is a prespecified exploratory outcome of the trial and safety is one of the secondary endpoints.

Herein, blood specimens for correlative studies were planned at baseline (day 0), week 2 and week 6 from 17 patients included from the Department of Dermatology in Nice University Hospital. The cytokine profile was analyzed before and after non-specific stimulation of innate and adaptive immune system and then correlated with tumor response rate, progression-free survival (PFS) and incidence of irAEs. Baseline lymphocyte and monocyte counts were gathered to adjust the baseline cytokines levels on the respective amount of producing cells.

The results in melanoma patients were compared with those of a cohort of 9 age-matched healthy donors (2:1).

PFS was defined as the time from the start of nivolumab to the date of disease progression or death, whichever occurred first. Patients who were alive without disease progression were censored at the date of their last disease assessment. Overall survival was defined as the time from the start of nivolumab to death from any cause.

In terms of tumor response, patients were classified as either responders (complete response, partial response, stabilization) or non-responders.

Disease progression and tumor response were evaluated using the response evaluation criteria in solid tumors (V.1.1) by radiologists at each participating institution.21 In case of tumor progression, in order to exclude pseudoprogression, a second CT scan was performed 4 weeks after the last evaluation.

Data on irAE incidence were collected and graded according to the US National Cancer Institute common toxicity criteria for adverse events (V.4.0).

Blood samples were taken to assess cytokine levels on day 0, week 2, week 6, and within 2 weeks of any progression. The 1 mL of whole blood was collected and stimulated with immune ligands (anti-CD3 as T-cells stimulant, and R848 as TLR 7/8 agonist to stimulate Natural Killer cells and foster T-cells’ response) on single lyophilized spheres (LyoSphere, Qiagen) within 8 hours from blood collection. Stimulated blood samples were incubated for 16 to 24 hours at 37°C and then centrifuged at 2000 to 3000 × g for 15 min to harvest the stimulated serum. Stimulated serum was stored at −20°C until the analysis and freeze-thaw cycles were minimized to preserve the quality of the samples. Serum levels of cytokines were measured either using the ProcartaPlex Immunoassay Kit (Luminex, ThermoFisher) or with custom-designed cartridges Ella (ProteinSimple). We measured 11 cytokines (IL-17A, IL-6, IL-1β, IFN-, IL-12p70, tumor necrosis factor-α (TNF-α), IL-10, IL-5, IL-4, IL-13 and GM-CSF) following the manufacturers’ instructions.22

For descriptive statistics, data are presented as mean and SD for continuous variables with Gaussian distribution, as median and IQR for continuous variables with non-Gaussian distribution, and as counts and percentages for categorical variables. The D’Agostino-Pearson normality test was used to determine if a variable had a Gaussian distribution or not. Groups of continuous values were compared by the Mann-Whitney test. Categorical variables were compared using χ² test. Median follow-up was estimated with Schemper method. Event-time distributions were estimated with the use of the Kaplan-Meier method. Log-rank test was used to compare survival data. Comparisons were two tailed and statistical significance was set at p<0.05. To take into account the multiple testing problems, independent variables were sought with Spearman’s correlation method. Bonferroni correction was then applied to quantitative independent variables to adjust the statistical significance level for the comparisons of cytokines levels. Statistical analyses were performed using SPSS V.16.0 on Windows and GraphPad Prism V.7.0 (GraphPad Software).

A total of 72 patients were included in the NIRVANA trial between April 2017 and June 2019. Eighteen patients were included in this ancillary study testing immune response, among them one was excluded due to positive hepatitis B virus serology according to the study’s non-inclusion criteria. Therefore, 17 patients were included in the current study. The median patient age was 72 years (IQR, 67.5–78.0), and a majority of the patients were male (n=13, 76.5%). Characteristics of patients are detailed in table 1. Over a median follow-up of 16 months (2.2–28.4), two patients died, yielding a 12-month overall survival rate of 83.3%. The tumor control rate was 64.7% (tumor stabilization (n=6), partial response (n=2) or complete response (n=3)). The remaining six patients (35.3%) presented immediate tumor progression. The 12-month PFS rate was 67.7%. The incidence of any grade of irAEs and grade 3–4 irAEs was 52.9% (n=9 patients) and 11.7% (n=2 patients), respectively.

To validate our conditions, we first test on the seven first inclusions, the cytokine level with and without in vitro stimulation. Without in vitro stimulation of immune cells, the levels of cytokines were mostly undetectable at baseline and did not differ between patients whereas differences could be observed after in vitro stimulation (figure 1).

We then compared inflammatory (IL-1 and IL-6) and Th1 (TNF-α and IFN-γ) baseline cytokine levels after in vitro stimulation from the 17 melanoma patients and 9 age and sex-matched healthy donors. Melanoma patients had significantly lower TNF-α, IFN-γ and IL-6 levels after in vitro stimulation compared with healthy donors (respectively p<0.0001, p=0.002, p=0.002, figure 2).

We first explored the Th1 pathway: IFN-γ was significantly higher in responders compared with non-responders on week 6 (p=0.03, figure 3A), while IL-2 was not statistically different (p=0.13) between responders and non-responders (figure 3B). Moreover, PFS was significantly improved in patients with higher levels of IFN-γ at week 6 (p=0.04, figure 3C).

We then explored Th17 pathway: IL-17A at baseline was significantly higher in responders compared with non-responders (p=0.03), as well as on week 2 (p=0.005) and week 6 (p=0.02, figure 3D), while IL-6 was not significantly different in responders compared with non-at each time point (figure 3E). PFS was significantly improved in patients with higher levels of IL-17A at week 2 (p=0.04, figure 3F). After adjustment on the baseline lymphocyte count, IL-17A was still significantly higher in responders compared with non-responders (p=0.02).

Cytokines from Th2 pathway also exhibited an increase during follow-up: IL-4 and IL-5 were slightly but significantly higher in responders compared with non-responders on week 2 (p=0.049 and p=0.048 respectively, figure 3G,H) and PFS was significantly improved in patients with higher levels of IL-4 at week 2 (p=0.01, figure 3I).

Clinical characteristics at baseline did not correlate with tumor response or PFS (table 1).

To adjust for multiple analysis problems, independence of variables was tested with Spearman’s method. All tested cytokines were correlated with IL-17A levels, whereas age and lactate dehydrogenase were independent (online supplemental file 1). Bonferroni’s correction was then applied to the statistical significance level, which yielded a corrected p at p<0.02 (table 1).

Baseline cytokine levels of IFN-γ, IL-17A and IL-4 did not significantly differ when complete and partial responders were compared with stable patients and non-responders (online supplemental file 2).

IrAEs were significantly more frequent in responders than non-responders (81.8% vs 16.7% respectively, p=0.04), and were also identified as a factor associated with improved prognosis for 1-year PFS (88.9% vs 38.1%, p=0.02). Patients that presented irAEs had significantly higher IL-6 levels before treatment with nivolumab on Day 0 than patients without irAEs (p=0.03), and the levels remained higher during the course of nivolumab treatment on week 2 (p=0.003) and week 6 (p=0.01, figure 4). The same observation was made with IL-17A levels (p=0.009 on day-0; p=0.04 on week 2; p=0.003 on week 6, figure 4), whereas IL-6 levels were comparable between responders and non-responders at each point (data not shown). IFN-γ levels in patients with irAEs were significantly higher on week 2 (p=0.018) and week 6 (p=0.01) but not on day 0 (p=0.25, figure 4).