Phase 2 trial of perioperative chemo-immunotherapy for gastro-esophageal adenocarcinoma: The role of M2 macrophage landscape in predicting response

Abstract

We present the clinical results of a phase 2 trial combining neoadjuvant docetaxel, cisplatin, 5 Flourouracil, and the PD-L1 inhibitor avelumab in locally advanced gastro-esophageal adenocarcinoma (GEA). Fifty-one patients receive neoadjuvant therapy with 50 proceeding to surgery. Grade 3-4 adverse events occur in 40%; complete/major pathological response is found in 7/50 (14%) and 9/50 (18%), with 2-year disease-free survival of 67.5%. There is no correlation between tumor regression and PD-L1 or mismatch repair (MMR) status. Multiplex immunohistochemistry and longitudinal single-cell transcriptomic profiling reveal alterations in certain innate immune cell populations, particularly noting an M2-tumor-associated macrophage (M2-TAM) proliferation in non-responding tumors. These findings describe the effective nature of this treatment regimen for GEA and reveal associated features of the inflammatory milieux associated with response to chemo-immunotherapy. The specific character of the inflammatory environment in non-responders may, in the future, help personalize treatment. This study was registered at ClinicalTrials.gov (NCT03288350).

Keywords: chemotherapy; gastroesophageal adenocarcinoma; immune microenvironment; immunotherapy; pathologic response; single cell transcriptomics; spatial proteomics; surgery; tumor associated macrophage.

Graphical abstract

Figure 1

A CONSORT diagram showing patient inclusion in the study Fifty-one patients were enrolled of which 50 underwent surgery. There were 37 patients who continued to receive adjuvant therapy.

Figure 2

Stratified radiological and survival outcomes of the cohort (A) A waterfall plot showing the change in the maximum standardized uptake values (SUV_max) correlated with pathological response, PD-L1 status, MMR status, and grade of differentiation for the entire cohort (n = 50). (B) (i) Kaplan-Meier plot of DFS for the cohort as a whole (median DFS not met) and (ii) stratified by pCR status until (median DFS not met, log rank [Mantel-Cox] = 0.091) 36 months after histological confirmation. Similar plots were created for OS for the cohort as a whole (iii) (median OS not met) and stratified by pCR status (median OS not met, log rank [Mantel-Cox] = 0.175).

Figure 3

Multiplex imaging density and proximity analyses stratified by response to neoadjuvant therapy (A) Seven-colored composite microphotographs (20X, scale: 50 μm) of representative paired treated and untreated sections of MPR, TRG2, and TRG3 responders (left to right column). (B) (i) The results of a spatially resolved analysis of mIHC-defined immune markers using Halo software. Density of M2-TAM (CD68⁺CD163⁺), potential regulatory T cells (TREG Foxp3⁺), CD8⁺T cells, and CTLs (CD8⁺granzymeB⁺) was measured stratified by TRG (TRG0/1 n = 7, TRG 2 n = 11, and TRG3 n = 18). A significant reduction in the percentage change in M2-TAM density was associated with MPR (two-way ANOVA, Tukey’s correction for multiple comparison, p = 0.038). Though non-significant, increment in mean changes in CTLs and CD8⁺T cells densities was correlated with favorable outcome. All boxplots are shown with the horizontal line denoting the median, vertical line denoting the 95% confidence interval, and “+” denoting the mean. (B) (ii) Spatial analysis of the measured immune cells indicated a predictive role of intra-tumoral M2-TAM content in pre-treatment gastro-esophageal cancer tissues stratified by TRG (TRG0/1 n = 7, TRG 2 n = 13, and TRG3 n = 21). The mean density of pre-existing, intra-tumoral (infiltrated) M2-TAM among patients with MPR was significantly higher compared to that of poor responders (TRG3) (unpaired t test with Welch’s correction, p = 0.024). (B) (iii) Calculation of the average distance of M2-TAM from the tumor core stratified by TRG (TRG0/1 n = 7, TRG 2 n = 13, and TRG3 n = 21). M2-TAMs were found to be significantly further from tumor cells present in TRG0/1 patients’ specimen compared to that of TRG3 patients (Welch’s unpaired two tailed t test, p = 0.005. (C) Forty-two formalin-fixed paraffin-embedded (FFPE) specimens from pre-treatment biopsies were available. Patients were arranged (left to right) in a heatmap based on their pathological responses. Clinical and post-treatment TNM stages, tumor location, objective response to treatment, and survival were also stratified. High or low subgroups of M2-TAM, TREG, and CD8+T cells were determined using cohort medians. Analysis revealed that 22 out of 42 patients harbor high pre-existing M2-TAM (>median = 390.8 cells/mm²). In total, 63% of these patients had ≥500 M2-TAM/mm², and 93% of such patients were pathological non-responders (TRG2 and TRG3). Similar analysis for potential TREG and CD8+T cells was conducted and also displayed in the heatmap (last 3 rows).

Figure 4

Single-cell analysis highlighting differential gene expression and cellular distribution among specific clusters of interest (A) A series of UMAPs showing the spatial reduction of cells included from the 20 samples in the scRNA-seq analysis including (i) stratified by cell types, (ii) time in the treatment cycle, (iii) sample number, (iv) response to neoadjuvant therapy, and (v) cell type allocation: CTL (CD8+GZMB+), TREG (CD4+FOXP3+), and M2-TAM (CD68⁺CD163+/CD206+) cells from all collected samples (n = 20). (B) A heatmap showing the top 10 genes (weighted by lowest adjusted p value) per cluster of M2-TAM, TREG, and CTL cell clusters among all samples (n = 20). (C) A bar plot showing the percentage distribution of M2-TAM, TREG, and CTL cells stratified by sample, response to treatment, and stage in the treatment cycle (treatment pre n = 10, treatment mid n = 2, and treatment post n = 5) with the corresponding number of cells per sample. (D) A transposition of the same data showing the combined cell distribution across all samples stratified by cell type and response to therapy.

Figure 5

Exploring single-cell characteristics of M2-TAMs among poor responders to neoadjuvant therapy (A) For patients with a TRG3, the number of cells allocated to the M2-TAM, TREG, and CTL clusters was summated and the percentage distribution calculated at each time point in the treatment cycle (treatment pre, n = 6, treatment mid, n = 3, and treatment post n = 5). (B) A volcano plot of an unmatched comparison of differentially expressed genes among M2-TAM (CD68⁺CD163+/CD206+) cells of patients with TRG3 lesions between the treatment pre (n = 6) and treatment post (n = 5) samples using a LogFold change cutoff of +/−1.5 and p value of <0.05. (C) A heatmap of differentially expressed genes of M2-TAM (CD68⁺CD163+/CD206+) among patients with TRG3 lesions, comparing treatment pre (n = 6) and treatment mid (n = 3) with treatment post (n = 5) with a LogFold change cutoff of +/−1.5 and p value of <0.05. (D) Functional enrichment pathways of differentially expressed genes of treatment-naive versus post-treatment TRG3 samples with upregulated (blue) and downregulated (red) genes. (E) A plot demonstrating ligand-receptor differential gene expression between M2-TAM and other TIME cell lineages among TRG3 samples (n = 14). (F) A dot plot comparing (i) MIF and (ii) CD68 expression among M2-TAMs stratified by TRG status (MPR n = 1, TRG 2 n = 2, and TRG 3 n = 3).