PMC7427000_01
(A) Five consecutive declines in the patient's calcitonin levels while on the immune checkpoint inhibitor, a > 40% decline. (B) Robustly positive PD-L1 staining after surgical resection of a neck lymph node after vaccine (higher power on the right).
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F1
He then had five consecutive declines in his calcitonin to 5,732 pg/ml and CEA levels remained overall stable at 22.0 ng/mL while on the immune checkpoint inhibitor avelumab, a > 40% decline not previously seen in his NCI clinical course (Figure 1A). A subsequent analysis of the patient's lymph node resected post-vaccination revealed that the tumor was PD-L1 positive (Figure 1B).
Cross sectional imaging studies with computed tomography of the neck (A) prior to PD-L1 administration and (B) after a 40% decrease in calcitonin, showing stable thyroid bed recurrence.
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F2
Response assessment by RECIST v1.1 reported stable disease (Figure 2).
Induction of CEA-specific T cells and changes in peripheral immune cell subsets. (A) Schema showing the timing of sequential immunotherapies and immune assays. (B) CEA-specific T cells were identified in PBMCs by intracellular cytokine staining following a period of in vitro stimulation with overlapping 15-mer peptide pools encoding for the tumor-associated antigen CEA or the negative control peptide pool HLA. Dot plots of IFNgamma and TNF production by CD4+ T cells showing induction of multifunctional CEA-specific T cells (producing >1 cytokine) at 3 months. (C-G) PBMCs were assessed for the frequency of 123 immune cell subsets over the course of immunotherapy. The most notable fluctuations were observed after initiation of avelumab (indicated by black arrow). The frequency over time of Tregs (C), cDC (D), pDC (E), MDSC (F), and B cells (G), indicated as a percentage of total PBMCs.
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T cell responses before vaccination, and after six and seven vaccinations with yeast-CEA, corresponding to 3 and 4 months, respectively; PBMCs were also examined 15 days following one cycle (administered every 2 weeks for 30 days) of avelumab (Figure 3A). As visualized in the dot plots of Figure 3B, the CD4+ CEA-specific cells included multifunctional cells, or cells producing >1 cytokine. PBMC subsets was also evaluated in this patient over his course of treatment at the National Cancer Institute using 11-color flow cytometry on cryopreserved PBMC as previously described (Supplemental Table 1). PBMCs were assayed prior to vaccination, 3 and 4 months following yeast-CEA vaccine, as well as at time points pre and post (15 and 42 days) avelumab (Figure 3A). Using 50% change as a cutoff, the first fluctuation in immune cell subsets was observed 4 months following vaccination with yeast-CEA, and included an increase in regulatory T-cells (Tregs) (51%), an inhibitory immune subset, compared to pre-vaccine levels (Figure 3C). Tregs (Figure 3C) and 57% more conventional dendritic cells (cDC) (Figure 3D), a subset that is involved in antigen presentation, compared to pre-vaccine levels. The most dramatic fluctuations in immune subsets were noted at the time point after 6 weeks of avelumab, and included decreases in Tregs (Figure 3C), cDC (Figure 3D), and plasmacytoid DC (pDC, Figure 3E) compared to pre-avelumab therapy levels. pDC are tolerogenic DC, exhibiting poor immunostimulatory ability, and their interaction with T cells often favors the generation of Tregs. Increases in myeloid derived suppressor cells (MDSCs) (Figure 3F), another immune suppressive subset, and B cells (Figure 3G) were also noted after avelumab, compared to pre-avelumab levels.