Tumor-infiltrating M2 macrophage in pretreatment biopsy sample predicts response to chemotherapy and survival in esophageal cancer

Abstract

The association between the tumor microenvironment (TME) and treatment response or survival has been a recent focus in several types of cancer. However, most study materials are resected specimens that were completely modified by prior chemotherapy; therefore, the unmodified host immune condition has not yet been clarified. The aim of the present study was to evaluate the relationship between TME assessed in pre-therapeutic biopsy samples and chemoresistance in esophageal cancer (EC). A total of 86 endoscopic biopsy samples from EC patients who received neoadjuvant chemotherapy (NAC) prior to surgery were evaluated for the number of intratumoral CD4⁺ lymphocytes (with/without Foxp3 expression), CD8⁺ lymphocytes (with/without PD-1 expression), monocytes (CD14⁺ ) and macrophages (CD86⁺ , CD163⁺ and CD206⁺ ) by multiplex immunohistochemistry (IHC). The number of tumor-infiltrating CD206⁺ macrophages I significantly correlated with cT, cM, cStage and neutrophil/lymphocyte ratio (NLR), whereas the number of lymphocytes (including expression of Foxp3 and PD-1) was not associated with clinico-pathological features. The high infiltration of CD163⁺ or CD206⁺ macrophages was significantly associated with poor pathological response to NAC (P = 0.0057 and 0.0196, respectively). Expression of arginase-1 in CD163⁺ macrophages tended to be higher in non-responders (29.4% vs 18.2%, P = 0.17). In addition, patients with high infiltration of M2 macrophages exhibited unfavorable overall survival compared to those without high infiltration of M2 macrophages (5-year overall survival 57.2% vs 71.0%, P = 0.0498). Thus, a comprehensive analysis of TME using multiplex IHC revealed that M2 macrophage infiltration would be useful in predicting the response to NAC and long-term survival in EC patients.

Keywords: M2 macrophage; biopsy; esophageal cancer; multiplex immunohistochemistry; neoadjuvant chemotherapy.

Figure 1

Multiplex fluorescent immunohistochemistry1 of esophageal cancer biopsies (200× fields). A, CD14, CD4, CD8, Foxp3, PD‐1, DAPI and cytokeratin. B, CD86, CD163, CD206, DAPI and cytokeratin. In each panel: top, fluorescence imaging; bottom left, bright color imaging; bottom right, pseudocolor H&E imaging

Figure 2

The association between the histopathological response to chemotherapy and the number of tumor‐infiltrating immune cells. A, CD14, CD4, CD8, CD86, CD163 and CD206 cells in non–responders and responders. B, The ratio of Foxp3⁺CD4⁺ in CD4⁺ lymphocytes and PD‐1⁺CD8⁺ in CD8⁺ lymphocytes as a percentage of the parent populations

Figure 3

Difference in arginase‐1 expression in M2 macrophages among each therapeutic response. A, Representative immunohistochemical images of M2 macrophages. B, The ratio of arginase‐1⁺CD163⁺ and arginase‐1⁺CD206⁺ macrophages for each therapeutic response. P < 0.05 (Mann‐Whitney U test) was considered significant

Figure 4

Kaplan‐Meier curves of overall survival. A, According to the histopathological response to chemotherapy. B, According to the infiltration of M2 macrophages (CD163⁺ or CD206⁺). C, Patients in stage 1/2 and stage 3/4 according to the infiltration of M2 macrophages