PMC6079623_01
(A) CT scan of the patient neck with IV contrast. Irregular infiltrative mass in the left side of the neck adjacent to the base of the tongue, invading the oropharynx and extending caudally to supraglottic and glottic larynx was shown both before and after the fifth cycle of anti-programmed cell death protein-1 (PD-1) treatment [(B,D) respectively]. It shows mild increase in size measuring about 5.1 cm x 4.6 cm 10 days after the fifth cycle (C) compared to 4.5 cm x 4.3 cm before anti-PD-1 treatment (A). (B) PET CT carried out at day 239 after fifth cycle (7 months, 25 days) of anti-PD-1 treatment showing progression of the disease (F) compared tp PET CT obtained at 10 days after the fifth cycle (E).
fimmu-09-01769-g001.jpg
PMC6079623_01_fimmu-09-01769-g001.jpg
F1
After the fifth cycle of nivolumab treatment, the patient's bleeding from the tumor site at the neck stopped and CT scan follow-up showed stable disease, no progression, or distant metastasis (Figure 1A). It showed a mild increase in size, measuring about 5.1 cm x 4.6 cm, 10 days after the fifth cycle (Figures 1A-C) compared to 4.5 cm x 4.3 cm before nivolumab treatment (Figures 1A-A) which suggests of pseudo-progression. PET CT scan was carried out at day 239 after the fifth cycle (7 months, 25 days) and the patient was found to have progressed (Figures 1B-F).
Antibody response to the NY-ESO-1 antigen as measured in the plasma by enzyme-linked immunosorbent assay (ELISA). (A) The results are expressed as the mean OD value and error bars indicate the SD for the triplicate values in each dilution. Out of the four different plasma dilutions tested (1:100, 1:400, 1:1,600, and 1:6,400), 1:100 and 1:400 were found to be the optimum dilutions to differentiate the anti-NY-ESO-1 antibody level before and after nivolumab treatment. (B) Bar graph represents the mean OD values were measured at 1:400 dilution. Each ELISA experiment was repeated six times and the shown data corresponds to one representative experiment. (C) Enzyme-linked immunospot (ELISPOT) assay for interferon-gamma production to investigate T cell response to the NY-ESO-1 antigen in patient's peripheral blood mononuclear cells against NY-ESO-1 overlapping peptides (PepMix). The assay was repeated three times and the shown data corresponds to one representative experiment. Statistical analysis for ELISA and ELISPOT were performed using non-parametric unpaired ANOVA followed by multiple comparison Dunnet's test and p values <0.05 were considered statistically significant.
fimmu-09-01769-g002.jpg
PMC6079623_01_fimmu-09-01769-g002.jpg
F2
ELISA results showed that out of the four different plasma dilutions tested (1:100, 1:400, 1:1,600, and 1:6,400), 1:100 and 1:400 were found to be the optimum dilutions to differentiate the anti-NY-ESO-1 antibody level before and after nivolumab treatment (Figure 2A). ELISA results showed that the NY-ESO-1 antibody levels at 1:400 plasma dilution were significantly higher before nivolumab treatment compared to samples taken 11 days after the third cycle (third cycle-11 days, ****p < 0.0001), 8 days after the fifth cycle (fifth cycle-8 days, ****p < 0.0001), and at progression stage (fifth cycle-226 days, ****p < 0.0001) (Figure 2B). IFN-gamma secretion was demonstrated against a pool of 43 peptides representing the whole length of the NY-ESO-1 protein (PepMix) (Figure 2C). IFN-gamma secretion was slightly increased in T cells tested 11 days after the third cycle (third cycle-11 days) and was significantly higher 8 days after the fifth cycle (fifth cycle-8 days, **p = 0.002) of nivolumab treatment compared to control before treatment. Interestingly, there was a significant decrease in IFN-gamma secretion by the patient T cells collected at progression (fifth cycle-226 days, **p = 0.0028) (Figure 2C).
Flow cytometry was used to determine the expression of programmed cell death protein-1 (PD-1) in the patient CD3+, CD4+, and CD8+ T cells before nivolumab treatment. Panels (A,B) are dot plots for isotype control and for PD-1 staining in CD3+, respectively. Isotype control and PD-1 staining in CD4+ and CD8+ cells are represented in panels (C,D).
fimmu-09-01769-g003.jpg
PMC6079623_01_fimmu-09-01769-g003.jpg
F3
Nivolumab treatment demonstrated a 15-fold decrease in the expression of PD-1 by the CD3+ T cells when compared to the value obtained before treatment (Figure 3B). T cells expressed the PD-1 molecule, its expression was dominant in the CD4+ T cells population before treatment (Figure 3D). The expression of PD-1 in the CD4+ and CD8+ T cells population was below detection limits after nivolumab treatment (data not shown).
Multiplex analysis of cytokines/chemokines in patient plasma before and after nivolumab treatment, and after progression. (A,B) Significant downregulation of the immune activation biomarkers (IL-10 and CX3CL-1 also known as Fractalkine) at progression (fifth cycle-226 days). (C,D) Significant upregulation of the immune inhibition biomarkers (IL-6 and IL-8) at progression (fifth cycle-226 days). The assay was repeated three times and the shown data corresponds to one representative experiment. Statistical analysis was performed using non-parametric unpaired ANOVA followed by multiple comparison Dunnet's test and p values <0.05 were considered statistically significant.
fimmu-09-01769-g004.jpg
PMC6079623_01_fimmu-09-01769-g004.jpg
F4
Two biomarkers (IL-10 and CX3CL-1 also known as Fractalkine), important for immune activation, were significantly reduced at progression (fifth cycle-226 days, Figures 4A,B). Moreover, two biomarkers (IL-6 and IL-8), important for immune inhibition, were significantly upregulated at progression (fifth cycle-226 days, Figures 4C,D).