Carcinoembryonic antigen-related cell adhesion molecules (CEACAM) 1, 5 and 6 as biomarkers in pancreatic cancer

Abstract

Background: Aim of this study was to assess the biological function in tumor progression and metastatic process carcinoembryonic antigen-related cell adhesion molecules (CEACAM) 1, 5 and 6 in pancreatic adenocarcinoma (PDAC).

Experimental design: CEACAM knock down cells were established and assessed in vitro and in a subcutaneous and intraperitoneal mouse xenograft model. Tissue and serum expression of patients with PDAC were assessed by immunohistochemistry (IHC) and by enzyme linked immunosorbent assays.

Results: Presence of lymph node metastasis was correlated with CEACAM 5 and 6 expression (determined by IHC) and tumor recurrence exclusively with CEACAM 6. Patients with CEACAM 5 and 6 expression showed a significantly shortened OS in Kaplan-Meier survival analyses. Elevated CEACAM6 serum values showed a correlation with distant metastasis and. Survival analysis revealed a prolonged OS for patients with low serum CEACAM 1 values. In vitro proliferation and migration capacity was increased in CEACAM knock down PDAC cells, however, mice inoculated with CEACAM knock down cells showed a prolonged overall-survival (OS). The number of spontaneous pulmonary metastasis was increased in the CEACAM knock down group.

Conclusion: The effects mediated by CEACAM expression in PDAC are complex, though overexpression is correlated with loco-regional aggressive tumor growth. However, loss of CEACAM can be considered as a part of epithelial-mesenchymal transition and is therefore of rather importance in the process of distant metastasis.

Figure 1. CEACAM levels decreased to <5% for CEACAM 1, 5 and 6 after shRNA knock down as shown in flow cytometry.

(A). Basal CEACAM expression varied depending on the CEACAM subtype, highest levels were found for CEACAM 1, intermediate for CEACAM 6 and lowest levels for CEACAM 5. As seen in proliferation (B) and migration assays (C), CEACAM knock down cells showed a higher proliferative and migrative potential compared to control cells. Differences in adhesion on stimulated (TNFα) endothelial cells were not detectable between CEACAM kd and control cells. Neither the average number (D) nor the average adhesion time to the endothelial cells was different (E). Murine xenograft tumors were highly positive for CEACAM 1, 5 and 6. Immunohistochemical staining in control cells showed complete absence of CEACAM expression in immunohistochemistry in knock down cells (F).

Figure 2. Subcutaneous murine xenograft model showed prolonged survival in CEACAM knock down group compared to the control group (A).

Intraperitoneally, no differences in the peritoneal carcinomatosis index (PCI) were observed at time of scarification (80 days after injection). CEACAM knock down showed more DNA copies of human DNA in the left lung (P = 0.021) (C) which is correlated with a higher metastatic load in the lung, however, no difference was observed for circulating tumor cells in the blood (D).

Figure 3. TMA immunohistochemical staining of CEACAM 1 negative (A) and positive (B), CEACAM 5 negative (C) and positive (D), CEACAM 6 negative (E) and positive (F).

Figure 4. Kaplan-Meier survival analysis (log-rank test) for correlation between overall-survival (OS) and CEACAM expression on the TMA.

(A–C). CEACAM 1 positive patients showed a median OS of 17.0 months (14.0–20.1 months), CEACAM 1 negative 23.0 months (9.5–36.5 months, P = .279). OS of CEACAM 5 positive patients was 16.0 months (12.8–19.2 months) vs. 22.0 months (4.1–47.9 months) (p = .025). CEACAM 6 positive patients showed an OS of 14.0 months (7.9–20.0) vs. 22.0 months (5.6–38.4 months, P = .010). Survival curves for correlation between overall-survival and CEACAM serum expression (D–F). CEACAM 1 positive patients showed an OS of 11.8 months (2.4–25.2) vs. 18.3 months (10.0–26.2 months, P = .022). Median OS for patients positive for CEACAM 5 was 15.7 months (2.4–32.3 months) vs. 18.6 months (8.7–28.6 months, P = .651), and median OS for patients with CEACAM 6 positivity was 18.8 months (9.5–28.1 months) vs. 12.8 months (3.3–22.3 months, P = .187). Boxplots for CEACAM 1 serum expression (G), CEACAM 5 (H) and CEACAM 6 (I) compared pancreatic ductal adenocarcinoma (PDAC), chronic pancreatic (CP) and healthy blood donors (BD). Receiver operating curve (ROC) for CEACAM serum expression (J).