Background: Colorectal cancer is highly prevalent. The stromal tumour microenvironment significantly influences tumour behaviour, and cancer-associated fibroblasts (CAFs), as major component of tumour stroma, are increasingly studied. Specifically, CAF-marker fibroblast activation protein (FAP) is gaining interest as tracer for imaging using radiolabelled FAP-inhibitor (FAPI). We describe patterns of FAP-expression, and associations to intratumoural stroma amount, establishing a biological background and potential future reference to pathology assessment.

Materials and methods: Archival histological material from 125 stage-II/III CRC patients was collected. Haematoxylin-and-eosin staining was performed to determine the tumour-stroma ratio (TSR), indicating stromal percentages in primary tumours, lymph nodes (LNs) and biopsies. On immunohistochemistry stains, FAP-expression was semiquantitatively scored as little-no, heterogeneous, or moderate-high expression. Correlation of TSR and FAP-expression with Chi-square testing was assessed. Other patterns were also described, e.g. tumour epithelial FAP-expression.

Results: In total, 93 patients (40 stage-III colon [CC], 53 stage-II/III RC) were included. Correlation between 41 (44 %) stroma-high CRC (18/40 CC, 45 %; 23/53 RC, 43 %) with high FAP-expression was not significant (P = 0.428 CRC; P = 0.470 CC; P = 0.615 RC). The majority of CRC had any FAP-expression (78 CRC, 84 %), mostly the invasive front, and in most associated LN metastases (87 % CRC tumour-positive LN). However, FAP-expression was often heterogeneous, even staining healthy colon and lymphoid tissue.

Conclusions: CRCs and LN metastases generally express FAP, but levels vary significantly between and within tumours and have no direct correlation with TSR. Care has to be taken translating FAPI-PET/CT results with e.g. disease extent and activity, emphasizing the importance of multidisciplinary approach.

Purpose
Tumor-targeted positron emission tomography (PET) and fluorescence-guided surgery (FGS) could address current challenges in pre- and intraoperative imaging of gastric cancer. Adequate selection of molecular imaging targets remains crucial for successful tumor visualization. This study evaluated the potential of integrin αvβ6, carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM) and human epidermal growth factor receptor-2 (HER2) for molecular imaging of primary gastric cancer, as well as lymph node and distant metastases.

Methods
Expression of αvβ6, CEACAM5, EGFR, EpCAM and HER2 was determined using immunohistochemistry in human tissue specimens of primary gastric adenocarcinoma, healthy surrounding stomach, esophageal and duodenal tissue, tumor-positive and tumor-negative lymph nodes, and distant metastases, followed by quantification using the total immunostaining score (TIS).

Results
Positive biomarker expression in primary gastric tumors was observed in 86% for αvβ6, 72% for CEACAM5, 77% for EGFR, 93% for EpCAM and 71% for HER2. Tumor expression of CEACAM5, EGFR and EpCAM was higher compared to healthy stomach tissue expression, while this was not the case for αvβ6 and HER2. Tumor-positive lymph nodes could be distinguished from tumor-negative lymph nodes, with accuracy ranging from 82 to 93% between biomarkers. CEACAM5, EGFR and EpCAM were abundantly expressed on distant metastases, with expression in 88–95% of tissue specimens.

Conclusion
Our findings show that CEACAM5, EGFR and EpCAM are promising targets for molecular imaging of primary gastric cancer, as well as visualization of both lymph node and distant metastases. Further clinical evaluation of PET and FGS tracers targeting these antigens is warranted.