Neoadjuvant intratumoral influenza vaccine treatment in patients with proficient mismatch repair colorectal cancer leads to increased tumor infiltration of CD8+ T cells and upregulation of PD-L1: a phase 1/2 clinical trial

Abstract

Background: In colorectal cancer, the effects of immune checkpoint inhibitors are mostly limited to patients with deficient mismatch repair tumors, characterized by a high grade infiltration of CD8+T cells. Interventions aimed at increasing intratumoral CD8+T-cell infiltration in proficient mismatch repair tumors are lacking.

Methods: We conducted a proof of concept phase 1/2 clinical trial, where patients with non-metastasizing sigmoid or rectal cancer, scheduled for curative intended surgery, were treated with an endoscopic intratumorally administered neoadjuvant influenza vaccine. Blood and tumor samples were collected before the injection and at the time of surgery. The primary outcome was safety of the intervention. Evaluation of pathological tumor regression grade, immunohistochemistry, flow cytometry of blood, tissue bulk transcriptional analyses, and spatial protein profiling of tumor regions were all secondary outcomes.

Results: A total of 10 patients were included in the trial. Median patient age was 70 years (range 54-78), with 30% women. All patients had proficient mismatch repair Union of International Cancer Control stage I-III tumors. No endoscopic safety events occurred, with all patients undergoing curative surgery as scheduled (median 9 days after intervention). Increased CD8+T-cell tumor infiltration was evident after vaccination (median 73 vs 315 cells/mm², p<0.05), along with significant downregulation of messenger RNA gene expression related to neutrophils and upregulation of transcripts encoding cytotoxic functions. Spatial protein analysis showed significant local upregulation of programmed death-ligand 1 (PD-L1) (adjusted p value<0.05) and downregulation of FOXP3 (adjusted p value<0.05).

Conclusions: Neoadjuvant intratumoral influenza vaccine treatment in this cohort was demonstrated to be safe and feasible, and to induce CD8+T-cell infiltration and upregulation of PD-L1 proficient mismatch repair sigmoid and rectal tumors. Definitive conclusions regarding safety and efficacy can only be made in larger cohorts.

Trial registration number: NCT04591379.

Keywords: Clinical Trials as Topic; Gastrointestinal Neoplasms; Immunomodulation; Lymphocytes, Tumor-Infiltrating; Tumor Microenvironment.

Figure 1

Neoadjuvant intratumoral influenza vaccine treatment increases CD8+T-cell infiltration and TCR alpha chain diversity. (A) Overview of the study design and sample time points. At each time point, blood and tumor samples were taken and a QoR-15 questionnaire was filled. (B) Representative pictures showing the quadrant visualization (yellow arrows, left picture) and intratumoral (IT) injection of the influenza vaccine (right picture). (C) Representative tumor tissue slides from immunohistochemistry (IHC) staining with anti-CD3/cytokeratin for digital analysis of T-cell infiltration. Tumor slides with IHC staining of anti-CD8/cytokeratin are not shown. Left picture shows a representative sample from before vaccination. Right picture shows a sample after the vaccination with the central tumor (pink) and invasive margins (light blue). (D) Comparison of the IHC staining density of CD3+ and CD8+ T cells before (green area) and after vaccination (pink area) samples (n=7). (E) Comparison of alpha, beta, gamma, and delta variable chains, and combined variable chain score between time points (n=6). (D, E) CD3+ and CD8+ T-cell densities and normalized expression of TCR variable chain expression depicted as boxplot showing median, upper and lower quartiles. Whiskers extend into a max of 1.5 times the IQR. CRC, colorectal cancer; pMMR, proficient mismatch repair; QoR-15, quality of recovery 15 questionnaire; TCR, T-cell receptor.

Figure 2

Changes in the tumor microenvironment after intratumoral influenza vaccine treatment. (A) Principal component analysis based on the top 400 most variable genes from nCounter IO360 panel, which includes 750 genes typically associated with tumor microenvironment biology. The statistical significance was tested using a PERMANOVA on the centroid differences between time points (n=7). (B) Heatmap of paired differentially expressed genes compared between time points (n=7, same order of patients before and after). (C, D) Significant pathways identified using functional enrichment score analysis. Here, the normalized expression of all represented genes in a pathway are analyzed via a paired t-test comparing before and after vaccination tumor samples. Normalized expression depicted as boxplot showing median, upper and lower quartiles (n=7) Whiskers extend into a max of 1.5 times the IQR. EMT, epithelial to mesenchymal transition; GO, Gene Ontology; NS, NanoString; PERMANOVA, permutational multivariate analysis of variance using distance matrices; TIL, tumor-infiltrating lymphocytes.

Figure 3

Spatial protein analysis within immune-infiltrated regions of tumors before and after intratumoral influenza vaccine treatment. (A, B) Picture of region of interest (ROI) selection in a patient before (A) and after (B) IT influenza vaccination. ROIs were drawn by a gastrointestinal pathologist and based on infiltration of CD45+ (yellow) and CD8+ (red) cells in areas of Pan-CK (green) and DNA (blue) positive regions. (C) Volcano plot of differentially expressed proteins in ROIs of immune-infiltrated regions of tumors before versus after vaccination (n=7). (D, E) Box plots of differentially expressed proteins upregulated (D), and downregulated (E) after vaccination (n=7). Differential expression of proteins depicted as boxplots showing median, upper and lower quartiles. Whiskers extend into a max of 1.5 times the IQR.FDR: False Discovery Rate; HLA-DRA, Human Leukocyte Antigen DR alpha chain; IT, intratumoral; KRT1, keratin 1; logFC, log2 fold change; MKI67, marker Ki-67; PD-L1, programmed death-ligand 1.

Figure 4

Spatial analysis of high immune-infiltrated versus low immune-infiltrated regions of tumors after vaccination. (A) Pictures of region of interest (ROI) selection in a patient after vaccination. Upper ROI designates a tumor area with high immune-infiltration (ROI-H) while the lower ROI designates a tumor area with low immune-infiltration (ROI-L). (B) Box plots of differentially expressed (DE) proteins upregulated on vaccination (from figure 3 (n=4)). (C) Box plots of the downregulated DE proteins on vaccination (from figure 3 (n=4)). Differential expression of proteins depicted as boxplots showing median, upper and lower quartiles. Whiskers extend into a max of 1.5 times the IQR. HLA-DRA, Human Leukocyte Antigen DR alpha chain; KRT1, keratin 1; MKI67, marker Ki-67; PD-L1, programmed death-ligand 1.

Figure 5

Circulating levels of immune cells and C-reactive protein after vaccination. (A) Overview of general immune cell populations (leukocytes (n=10), lymphocytes (n=8), neutrophils (n=8), neutrophils/lymphocytes ratio (n=8), and thrombocytes (n=8)), and the concentration of C-reactive protein (CRP, n=9). (B) Overview of flow cytometry analyses to determine subpopulations (CD3+T cells, CD4+T cells, CD8+T cells, B cells (CD19+), and natural killer cells (CD56+), all n=10). Concentration of immune cells and CRP depicted as boxplots showing median, upper and lower quartiles. Whiskers extend into a max of 1.5 times the IQR.