A combination of stromal PD-L1 and tumoral nuclear β-catenin expression as an indicator of colorectal carcinoma progression and resistance to chemoradiotherapy in locally advanced rectal carcinoma

Abstract

Programmed cell death-1 (PD-1) and its ligand (PD-L1) are significant mediators of immune suppression in the tumor microenvironment. We focused on the immunological impact of PD-1/PD-L1 signaling during tumor progression in colorectal carcinoma (CRC) and its association with resistance to neoadjuvant chemoradiotherapy (NCRT) in locally advanced rectal carcinoma (LAd-RC). Histopathological and immunohistochemical analyses of 100 CRC cases (including 34 RC) without NCRT and 109 NCRT-treated LAd-RC cases were performed. Membranous tumoral PD-L1 expression was identified in 9 of 100 (9%) CRC cases, including 1 of 34 (2.9%) RC cases, but PD-L1 immunopositivity was not associated with any clinicopathological factors, with the exception of deficient mismatch repair (dMMR) status. In contrast, stromal PD-L1+ immune cells, which frequently exhibited coexpression of PD-1 and CD8 markers, were significantly correlated with tumor vessel invasion, nuclear β-catenin+ tumor budding cancer stem cell (CSC)-like features, and unfavorable prognosis. In the LAd-RC cases, stromal CD8+ (but not PD-L1+) immune cell infiltration in pretreatment-biopsied samples was significantly and positively associated with therapeutic efficacy. After NCRT, tumoral PD-L1 expression was observed in only 2 of 83 (2.4%) tumors, independent of dMMR status, whereas high stromal PD-L1+ and tumoral nuclear β-catenin positivity were significantly linked to a poor response to NCRT and high tumor budding features. In addition, high stromal PD-L1 immunoreactivity was significantly associated with poorer overall survival. In conclusion, a combination of stromal PD-L1+ immune cells and nuclear β-catenin+ tumor budding may contribute to tumor progression in CRC and resistance to NCRT in LAd-RC, through formation of niche-like lesions that exhibit immune resistance and CSC properties.

Keywords: PD-1; PD-L1; cancer stem cell; colorectal carcinoma; neoadjuvant chemoradiotherapy; β-catenin.

Figure 1

High density of infiltrating immune cells in the outer stroma of CRC without NCRT. (A) Staining with hematoxylin and eosin (HE) or IHC for the indicated immune cell‐related markers in CRC without NCRT. Note the high density of infiltrating immune cells expressing the indicated markers in the outer stroma compared to the inner and middle areas. Closed boxes in the upper panels are magnified in the lower panels. Original magnification, ×100 (upper) and ×400 (lower). (B) LIs of immunopositive immune cells for the indicated markers in the inner (In), middle (Md), and outer (Out) stroma in CRC samples. The data shown are means ± SDs. (C) Number of immune cells immunopositive for the indicated markers per high‐power field (HPF) in the In, Md, and Out intratumoral lesions in CRC samples. The data shown are means ± SDs.

Figure 2

Relationship between stromal immune cell infiltrations and nuclear β‐catenin‐positive tumor buds in CRC without NCRT. (A) Staining with hematoxylin and eosin (HE), IHC for β‐catenin (visualized using 3,3′‐diaminobenzidine), and double IHC for β‐catenin (visualized using sodium cobalt) and the indicated immune cell‐related markers (visualized using 3,3′ ‐diaminobenzidine) in CRC samples with a tumor budding score (BD): 3 without NCRT. Note the infiltrating immune cells expressing the indicated markers around the nuclear β‐catenin+ tumor budding cells (indicated by arrows). Closed boxes in the upper panels are magnified in the lower panels. Original magnification, ×100 (upper) and ×400 (lower). (B) Association between LIs of immunopositive immune cells expressing the indicated markers (left) or IHC score of nuclear β‐catenin (Nu‐β‐cat) (right) and BD score in the outer stroma of CRC samples. The data shown are means ± SDs.

Figure 3

Relationship between immune cell‐related markers, BD score, and prognosis in CRC without NCRT. OS (left) and PFS (right) relative to PD‐L1 (A), PD‐1 (B), CD4 (C), CD8 (D), CD68, and BD score (F) in CRC without NCRT. n, number of cases.

Figure 4

Expression of immune cell‐related molecules in resected LAd‐RC after NCRT. (A) Staining with hematoxylin and eosin (HE) and IHC for the indicated immune cell‐related markers and tumoral β‐catenin in samples of LAd‐RC cases that responded poorly to NCRT and had moderate budding features (TE: G1/BD: 2) (upper panels) or responded well and had low budding scores (TE: G2/BD: 1) (lower panels). Note the high density of infiltrating immune cells expressing the indicated markers around the tumor budding cells (indicated by arrows) in the stroma of RC with TE: G1/BD: 2 (upper panels). For two RC cases, the closed boxes in the left panels are magnified in the right panels. Closed boxes in the upper panels are also magnified in the lower panels in each LAd‐RC case. Original magnification, ×40 (left), ×100 (upper), and ×400 (lower). (B, C) Relationship between IHC score for immune cells expressing the indicated molecules, tumoral nuclear β‐catenin (Nu‐β‐cat), TE grade (B), and BD score (C) in samples from LAd‐RC patients treated with NCRT. The data shown are means ± SDs.

Figure 5

Relationship between immune cell‐related markers, BD score, and prognosis in LAd‐RC patients who received NCRT. OS (left) and PFS (right) relative to PD‐L1 (A), PD‐1 (B), CD4 (C), CD8 (D), CD68, and BD score (F) in LAd‐RC after NCRT. n, number of cases.

Figure 6

Schematic representation of the CSC niche‐like lesions in CRC patients and in LAd‐RC patients receiving NCRT. Densely infiltrating immune cells expressing PD‐L1, PD‐1, or CD8 and nuclear β‐catenin+ tumor budding cells are present in the outer stroma adjacent to tumor invasive fronts. Note the nuclear β‐catenin+ tumor cells, along with migration of PD‐L1+/PD‐1+/CD8+ immune cells, into vessels.