Treatment of Advanced NSCLC Patients with an Anti-Idiotypic NeuGcGM3-Based Vaccine: Immune Correlates in Long-Term Survivors

Abstract

Background: Racotumomab-alum is an anti-idiotype vaccine targeting the NeuGcGM3 tumor-associated ganglioside. Clinical trials in advanced cancer patients have demonstrated low toxicity, high immunogenicity and clinical benefit. The goal of this study was to identify circulating biomarkers of clinical outcome. Methods: Eighteen patients with stage IIIb/IV non-small-cell lung cancer (NSCLC) were injected with racotumomab-alum as switch maintenance therapy after first-line chemotherapy. Treatment was administered until severe performance status worsening or toxicity. The frequencies of innate and adaptive lymphocytes were assessed by flow cytometry. Circulating factors were measured using multi-analyte flow assay kits. Results: The median overall survival was 16.5 months. Twenty-seven percent of patients were classified as long-term survivors. Patients with lower baseline frequencies of CD4+Tregs and central memory (CM) CD8+T cells displayed longer survival rates. Furthermore, higher baseline frequencies of NKT cells and a high CD8+T/CD4+Treg ratio were associated with longer survival. Interestingly, patients with significantly lower levels of effector memory (EM) CD8+T cells survived longer. The levels of NKT cells and terminal effector memory (EMRA) CD8+T cells were higher in long-term survivors in comparison with short-term survivors in post-immune samples. As expected, the ratio of CD8+T/CD4+Tregs showed significantly higher values during treatment in patients with clinical benefits. Regarding serum factors, pro-tumorigenic cytokines significantly increased during treatment in poor survivors. Conclusions: In advanced NSCLC patients receiving racotumomab-alum vaccine, longer survival could be associated with a unique profile of circulating lymphocyte subsets at baseline and during treatment. Additionally, certain pro-tumor-related cytokines increased in short-term survivors. These results should be confirmed in larger randomized clinical trials. This clinical trial was registered in the Cuban Clinical Trials Register (RPCE00000279).

Keywords: anti-idiotypic cancer vaccine; circulating biomarkers; non-small cell lung cancer.

Figure 1

Immunization and sampling schedule of NSCLC patients treated with racotumomab-alum vaccine. Created with BioRender.com.

Figure 2

Overall survival. (A) Kaplan–Meier curve for OS in months for all patients (n = 18), calculated as the difference between the vaccination onset and the date of death (n = 14), or date of last visit (n = 4). (B) Kaplan–Meier curves for short- and long-term survivors. Differences in survival times were assessed by the log-rank test. Red spots (A) and black spots (B) represent censored patients.

Figure 3

Changes in immune cell populations during racotumomab-alum treatment in long-term (LS) and short-term survivors (SS). (A) gating strategy to analyze NK, NKT and T cell subsets is shown. Lymphocytes were subjected to doublet discrimination. Singlet lymphocytes were separated according CD3 and CD56 expression to identify NK (CD56+CD3−), NKT (CD56+CD3+) and T cells (CD56−CD3+). T cells were plotted using CD4 and CD8 to further divide the population. CD8+T cells were further resolved according to CCR7 and CD45RA expression: CCR7+CD45RA+ as naïve (N), CCR7+CD45RA− as central memory (CM), CCR7−CD45RA− as effector memory (EM) and CCR7−CD45RA+ as terminal effector memory T cells (EMRA). CD4+T cells were plotted for CD25 and CD127 to identify Tregs as CD25+CD127−. (B–D) Behavior of CD8+T cell subpopulations, (E) and (F) CD4+Tregs cells and CD8+T/CD4+Tregs cells ratio and (G) NKT lymphocytes in long- and short-term survivors during racotumomab-alum vaccination. The black triangles and circles show the values for each patient. The asterisks indicate statistically significant differences between the groups (* p < 0.05, Mann–Whitney U test).

Figure 3

Changes in immune cell populations during racotumomab-alum treatment in long-term (LS) and short-term survivors (SS). (A) gating strategy to analyze NK, NKT and T cell subsets is shown. Lymphocytes were subjected to doublet discrimination. Singlet lymphocytes were separated according CD3 and CD56 expression to identify NK (CD56+CD3−), NKT (CD56+CD3+) and T cells (CD56−CD3+). T cells were plotted using CD4 and CD8 to further divide the population. CD8+T cells were further resolved according to CCR7 and CD45RA expression: CCR7+CD45RA+ as naïve (N), CCR7+CD45RA− as central memory (CM), CCR7−CD45RA− as effector memory (EM) and CCR7−CD45RA+ as terminal effector memory T cells (EMRA). CD4+T cells were plotted for CD25 and CD127 to identify Tregs as CD25+CD127−. (B–D) Behavior of CD8+T cell subpopulations, (E) and (F) CD4+Tregs cells and CD8+T/CD4+Tregs cells ratio and (G) NKT lymphocytes in long- and short-term survivors during racotumomab-alum vaccination. The black triangles and circles show the values for each patient. The asterisks indicate statistically significant differences between the groups (* p < 0.05, Mann–Whitney U test).

Figure 4

Changes in serum cytokine levels in long-term (LS) and short-term survivors (SS) at baseline and at post-immune time points (3–6 months). The black triangles and circles show the values for each patient The asterisks indicate statistically significant differences between the groups (* p < 0.05 and ** p < 0.01, Mann–Whitney U test).

Figure 5

Association of immune cell populations at baseline with overall survival. Kaplan–Meier curves representing overall survival vs. immune cell subsets after dichotomizing the data at established cut off. Patients were divided according to the percentage of (A) CD4+Tregs cells, (B) CM CD8+T cells, (C) CD8+T/Tregs cells ratio and (D) NKT cells. Differences in survival times were assessed by the log-rank test.

Figure 5

Association of immune cell populations at baseline with overall survival. Kaplan–Meier curves representing overall survival vs. immune cell subsets after dichotomizing the data at established cut off. Patients were divided according to the percentage of (A) CD4+Tregs cells, (B) CM CD8+T cells, (C) CD8+T/Tregs cells ratio and (D) NKT cells. Differences in survival times were assessed by the log-rank test.