Histopathological and genotypic characterization of metastatic colorectal carcinoma with PD-L1 (CD274)-expression: Possible roles of tumour micro environmental factors for CD274 expression

Abstract

Aberrant PD-L1 (CD274) expression has been described in different types of tumour and linked to tumour aggressiveness and a poor prognosis. In primary colorectal carcinomas (CRCs), CD274 expression was reported to be associated with mismatch repair (MMR)-deficiency, BRAF mutation, and "stem-like" immunophenotype defined by down-regulation of homeobox protein CDX2 and membranous expression of activated leukocyte cell adhesion molecule (ALCAM). However, the immunophenotype and genotype of CD274-positive metastatic CRC have not been extensively analysed. In this study, 189 CRC metastases were evaluated immunohistochemically for CD274, MMR proteins, CDX2, and ALCAM expression. Immunostaining for CD4, CD8, and FOXP3 was also performed to characterize tumour-associated immune cells. In addition, 34 arbitrarily selected lesions were genotyped using Sanger- and next-generation sequencing. Univariate analyses showed no clear association between CD274 expression and clinicopathological parameters including MMR-deficiency or "stem-like" immunophenotype after adjustment for multiple testing. Comparison of the clinicopathological profiles of CD274-positive primary and metastatic tumours revealed in the latter younger age of occurrence (60.9 ± 13.3 versus 72.6 ± 13.1 years, p = 0.001), cytoplasm-dominant CD274 expression (p < 0.001), infrequent MMR-deficiency (p < 0.001), and common KRAS mutations (54%, p < 0.001). In five cultured colon cancer cell lines, CD274 was expressed and modulated after exogenous exposure to IFNγ and TGF-β1. Thus, CD274 regulation mechanisms might include tumour micro environmental factors. Based on significantly different characteristics in CD274-positive metastatic and primary CRCs, evaluation of metastases should also be considered when planning immune checkpoint inhibitor therapy.

Keywords: ALCAM (CD166); BRAF; CD274 (PD‐L1); CDX2; IFNγ; KRAS; TGF‐β1; immunohistochemistry; metastatic colorectal carcinoma.

Figure 1

Histology and CD274 expression of metastatic CRCs. Case 1, a case of liver metastasis. Tumour tissue contained highly atypical tumour cells with CD274 expression on the cell membrane and in the cytoplasm (arrow heads). KRAS p.G12D (GGT to GAT) mutation was identified by gene mutation analysis. Case 2, a case of ovarian metastasis. Tumour cells are invading into desmoplastic stroma. Tumour cells showed cytoplasm‐dominant expression of CD274. In this case, BRAF p.V600E mutation was identified.

Figure 2

Basal CD274 expression and other characteristics of colon cancer cells under cultured conditions. (A) Immunoblot analysis for CD274 in colon cancer cells. (B) FACS analyses for cell surface CD274 in colon cancer cells. (C) Immunoblot analyses for the characterization of colon cancer cells. Note that VIM expression uniquely showed inverse correlation to CD274.

Figure 3

IFNγ up‐regulates CD274 through the JAK‐STAT pathway. (A) FACS analyses for cell surface CD274 in IFNγ‐stimulated colon cancer cells. The experiments were performed in triplicate. Columns, mean values; bars, SD. **p < 0.01; *p < 0.05. (B) Representative results of FACS analyses for cell surface CD274 in COLO205 and HCT116 stimulated with IFNγ (100 U/ml) for 24 h. (C) Immunoblot analyses of SW480 and CW‐2 cells with or without IFNγ and JAK inhibitor I treatment. Note that phospho‐STAT2 (Tyr690), phospho‐STAT3 (Tyr705), phospho‐STAT5 (Tyr694), and phospho‐STAT6 (Tyr641) were expressed at under‐detectable levels (data not shown). (D) Immunoblot analyses of COLO205 and CW‐2 cells with or without JAK inhibitor I blockade.

Figure 4

TGF‐β1 modulates CD274 regardless of EMT status. (A) FACS analyses for cell surface CD274 on colon cancer cells with 48 h of TGF‐β1 stimulation. The experiments were performed in triplicate. Columns, mean values; bars, SD. **p < 0.01; *p < 0.05. (B) Immunoblot analyses of colon cancer cells stimulated with TGF‐β1 for 48 h. Note that SW480 uniquely showed mild EMT. However, no correlation between EMT and CD274 expression was detected.