Organ-specific heterogeneity in tumor-infiltrating immune cells and cancer antigen expression in primary and autologous metastatic lung adenocarcinoma

Abstract

Background: Tumor immune microenvironment (TIME) and cancer antigen expression, key factors for the development of immunotherapies, are usually based on the data from primary tumors due to availability of tissue for analysis; data from metastatic sites and their concordance with primary tumor are lacking. Although of the same origin from primary tumor, organ-specific differences in the TIME in metastases may contribute to discordant responses to immune checkpoint inhibitor agents. In immunologically 'cold' tumors, cancer antigen-targeted chimeric antigen receptor (CAR) T-cell therapy can promote tumor-infiltrating lymphocytes; however, data on distribution and intensity of cancer antigen expression in primary tumor and matched metastases are unavailable.

Methods: We performed a retrospective review of a prospectively maintained database of patients who had undergone curative resection of pathological stage I-III primary lung adenocarcinoma from January 1995 to December 2012 followed by metastatic recurrence and resection of metastatic tumor (n=87). We investigated the relationship between the primary tumor and metastasis TIME (ie, tumor-infiltrating lymphocytes, tumor-associated macrophages, and programmed death-ligand 1 (PD-L1)) and cancer antigen expression (ie, mesothelin, CA125, and CEACAM6) using multiplex immunofluorescence.

Results: Brain metastases (n=36) were observed to have fewer tumor-infiltrating lymphocytes and greater PD-L1-negative tumor-associated macrophages compared with the primary tumor (p<0.0001); this relatively inhibitory TIME was not observed in other metastatic sites. In one in three patients, expression of PD-L1 is discordant between primary and metastases. Effector-to-suppressor (E:S) cell ratio, median effector cells (CD20+ and CD3+) to suppressor cells (CD68/CD163+) ratio, in metastases was not significantly different between patients with varying E:S ratios in primary tumors. Cancer antigen distribution was comparable between primary and metastases; among patients with mesothelin, cancer antigen 125, or carcinoembryonic antigen adhesion molecule 6 expression in the primary tumor, the majority (51%-75%) had antigen expression in the metastases; however, antigen-expression intensity was heterogenous.

Conclusions: In patients with lung adenocarcinoma, brain metastases, but not other sites of metastases, exhibited a relatively immune-suppressive TIME; this should be considered in the context of differential response to immunotherapy in brain metastases. Among patients with cancer antigen expression in the primary tumor, the majority had antigen expression in metastases; these data can inform the selection of antigen-targeted CARs to treat patients with metastatic lung adenocarcinoma.

Keywords: antigens; lymphocytes, tumor-infiltrating; programmed cell death 1 receptor; receptors, chimeric antigen; tumor microenvironment.

Figure 1

Metastasis site-specific differences in tumor immune microenvironment. (A) CD20+ (B cells), CD3+ (T cells), CD4+ and CD8+ (T-cell subpopulations), CD68/CD163+ (macrophages), and myeloperoxidase+ (MPO; neutrophils) were compared (as a percentage of total immune cells) between paired primary and metastatic tumors. Among all patients, the primary tumor had higher percentages of B cells, CD3+ and CD4+ T cells, and lower percentages of macrophages. Among patients with brain metastases, percentages of B cells and CD3+, CD4+, and CD8+ T cells were lower, and percentages of macrophages higher compared with primary tumor. Among patients with lung or other sites of metastasis (lymph nodes, pleura, bone, adrenal, kidney, colon, or soft tissue), there was no difference in immune cell populations between the primary tumor and metastases. Median and IQR is displayed. *P<0.01; **p<0.001. (B) Programmed death-ligand 1 (PD-L1) expression on tumor cells (≥1% of tumor cells) differed between the primary tumor and metastatic tumor in 39% of patients; 19% of patients had high (≥1% of tumor cells) PD-L1 expression on the primary tumor but low (<1%) PD-L1 expression on the metastatic tumor, while 20% had low PD-L1 (<1%) PD-L1 expression on the primary tumor but high (≥1%) PD-L1 expression on the metastatic tumor. (C) Tumor-cell PD-L1 expression was stratified into <1%, 1%–49%, and ≥50% of tumor cells. The percentage of primary tumors (n=79) with tumor-cell PD-L1 expression <1% was 53%, 1%–49% was 35%, and ≥50% was 11%. The percentage of metastases (n=79) with PD-L1 expression <1% was 52%, 1%–49% was 41%, and ≥50% was 8%. (D) PD-L1 expression on macrophages (≥27% of macrophages) differed between the primary tumor and the metastatic tumor in 42% of patients; 20% of patients had high (≥27%) macrophage PD-L1 expression on the primary tumor but low (<27%, median value) macrophage PD-L1 expression on the metastatic tumor, while 22% had low macrophage PD-L1 expression on the primary tumor but high macrophage PD-L1 expression on the metastatic tumor. (E) The correlation between tumor cell PD-L1 expression (as a percentage of Pan-CK+ tumor cells) and macrophage PD-L1 expression (as a percentage of CD68/CD163+ macrophages) was assessed within each tumor specimen for both primary and metastatic tumors. The correlation (Spearman’s ρ) between tumor cell PD-L1 expression and macrophage PD-L1 expression was 0.72 within primary tumors, and 0.67 within metastatic tumors. (F) PD-L1 expression on tumor cells and PD-L1 expression on macrophages is shown for 79 paired primary and metastatic tumors, with each horizontal line representing one tumor. The correlation (Spearman’s ρ) between paired primary tumor and metastatic tumors was 0.33 for tumor PD-L1 expression, and 0.29 for macrophage PD-L1 expression. MPO, myeloperoxidase; PD-L1, programmed death-ligand 1.

Figure 2

Co-localization of CD3+ T cells and CD68/163+ macrophages and patient stratification by effector:suppressor cell ratio in paired primary and metastatic tumors. (A) Co-localization of CD3+ T cells and CD68/CD163+ macrophages was quantified through the Morisita-Horn (MH) index, based on immune cell populations in the tumorous tissue microarray cores. MH index indicated a high degree of co-localization in both primary (MH=0.83) and metastatic tumors (MH=0.84). There was no difference in CD3 and macrophage cell co-localization between primary and metastatic tumors (p=0.13, Wilcoxon signed rank test). (B) Patients were divided into four groups based on median in primary and metastatic tumor. Tumors with greater than median effector cell (CD20+ and CD3+):suppressor cell (CD68/CD163+) ratio (E:S ratio) were considered ‘hot’, and tumors with less than median E:S ratio were considered ‘cold’. (C) Among ‘cold’ primary tumors, the median E:S ratio in corresponding metastases was 0.3 (IQR 0.1, 1.2), and among ‘hot’ primary tumors, the median metastatic E:S ratio was 0.8 (0.3, 2.4) (p=0.082, Wilcoxon rank sum exact test). (D) Immune cell abundance (cells/core) and site of metastasis (brain, lung, lymph node, and other) is displayed for each primary and metastatic tumor, following stratification of patients into groups based on E:S ratio in primary and metastatic tumor. The data are represented with metastatic tumors increasing E:S ratio (cold to hot). ICD, immunogenic cell death; MPO, myeloperoxidase.

Figure 3

Comparison of cancer antigen surface expression between the primary tumor and metastases. (A) Carcinoembryonic antigen adhesion molecule 6 (CEACAM6), cancer antigen 125 (CA125), and mesothelin (MSLN) surface expression is compared between paired primary and metastatic tumors (cut-off value ≥10% of tumor cells with antigen expression). CEACAM6 expression differed between the primary tumor and the metastatic site in 33% of patients; 20% of patients had high CEACAM6 expression (≥10%) in the primary tumor and low CEACAM6 expression in the metastatic tumor, while 13% of patients had low CEACAM6 expression in the primary tumor and high CEACAM6 expression in the metastatic tumor. Similarly, CA125 expression differed between the primary tumor and the metastatic site in 38% of patients, and CA125 expression differed between the primary tumor and the metastatic site in 42% of patients. (B) Cell surface antigen co-expression was compared between the primary tumor and metastases. (C) Cell surface antigen co-localization was assessed for primary and metastatic tumors (n=87, each bar represents one patient). For each tumor, the percentage of tumor cells positive for a single antigen, two antigens (double), and three antigens (triple) is shown. The median expression of at least one antigen was 62% for primary tumors and 72% for metastatic tumors. (D) In metastatic tumors, the percentage of tumor cells positive for a single antigen (n=87) for CEACAM6, CA125, and MSLN is shown (cell surface expression). Median antigen expression (as a percentage of tumor cells) was 52% for CEACAM6, 21% for CA125, and 6% for MSLN. (E) Waterfall plots demonstrating relative cell-surface antigen intensity for CEACAM6, CA125, and MSLN in primary and metastatic tumors with high (≥10% of tumor cells) antigen expression only are shown. Each bar represents one patient. Relative antigen intensity is calculated as the fold-change from background fluorescence.