Dynamic expression of CEACAM7 in precursor lesions of gastric carcinoma and its prognostic value in combination with CEA

The study included 345 patients, including 145 patients with gastric carcinoma who underwent primary surgical resection between 2006 and 2009 at the Xijing Hospital of Forth Military Medical University (Xi'an, China), 100 patients with intraepithelial neoplasms (55 low-grade and 45 high-grade), 50 patients with chronic atrophic gastritis, and 50 patients with normal gastric mucosa. The 200 patients with nonneoplastic and precancerous lesions had received a gastric endoscopic check and biopsy between 2008 and 2010 at the Xijing Hospital. All diagnoses, including differentiation status, were made based on the Pathology and Genetics Tumors of Digestive System by three pathologists [22]. GIN is categorized as either high- or low-grade according to both the Vienna [4] classifications. Follow-up was performed on the 145 patients with gastric carcinoma for survival analysis. These patients underwent subtotal gastrectomy with D2 lymph node dissection at Xijing Hospital from 2006 to 2009. The mean age was 56.4 years (range, 32-74 years). All patients received postoperative chemotherapy using a fluorouracil-based regimen. No patients received preoperative chemotherapy or underwent radiotherapy. Follow-up was performed on patients from the date of surgery until either the date of death or March 30, 2011, resulting in follow-up periods ranging from 5 to 67 months (mean, 31 months). Those cases lost to follow-up or those who died of a cause other than gastric cancer were regarded as censored data for the analysis of survival rates. In all cases, informed consent was obtained for the use of resected tumor specimens. This study was approved by the Moral and Ethical Committee of the Xijing Hospital of Forth Military Medical University.

Tissues of gastric carcinoma or precancerous lesions were made into tissue micro-arrays by Tissue Microarrayer (Beecher Instruments, Silver Spring, USA ™), according to the technique of Kononen et al [23]. Briefly, core tissue biopsies (2 mm in diameter) were taken from individual paraffin-embedded tissues (donor blocks) and arranged in a new recipient paraffin block (tissue array block) using a trephine apparatus. The staining results of the different areas in these tissue array blocks showed excellent agreement. One core was chosen from each case for analysis. We defined an adequate case as a tumor that occupied 10% of the core area.

Mouse anti-human CEACAM7 antibody [BAC2] (ab26281) and Rabbit Anti-human CEA antibody (ab-15157) was all purchased from Abcam plc. (Cambridge, UK).

Immunohistochemistry was performed on 4-lm-thick, routinely processed, paraffin sections in series. Briefly, after baking on a panel at 60°C for an hour, the paraffin embedded, formalin fixed tissue samples were deparaffinized with xylene and rehydrated through gradient ethanol immersion. Endogenous peroxidase activity was quenched by 3% (vol/vol) hydrogen peroxide in methanol for 10 minutes, followed by three 3-minute washes with phosphate buffered saline (PBS). This was followed by a step of antigen retrieval. The slides were immersed in 0.01 mol/l citrate buffer solution (pH 6.0) and placed in a microwave oven for 30 min. After a wash in 0.01 mol/l of phosphate-buffered saline (PBS, pH 7.4), The sections were then blocked with 10% (vol/vol) normal goat serum in PBS for 30 minutes followed by incubation in a moist chamber at 4 overnight with the primary antibody to CEACAM7 (1:100, ab26281, Abcam) or CEA 1:10, ab-15157, Abcam) diluted in PBS containing 1% (wt/vol) bovine serum albumin (BSA). Negative controls were performed by replacing the primary antibody with pre-immune mouse serum. After three 3-minute washes with PBS, the sections were treated with second anti-mouse antibody (PV-6002, Santa Cruz) for 30 minutes at room temperature followed by additional three 3-minute washes with PBS. Reaction product was visualized with diaminobenzidine (DAB, ZLI-9032, ZSGB. Beijing, China) at room temperature for 2 min. After being counterstained with Harris hematoxylin (ZLI-9039, ZSGB. Beijing, China) for 3 min, and rinsed with tap water, the sections were immediately dehydrated by sequential immersion in gradient ethanol and xylene, and mounted with pernount and cover slips. Images were obtained under a light microscope (Olympus BX51, Olympus, Japan) equipped with a DP70 digital camera.

For evaluation of cell staining, sections were examined by two independent pathologists without prior knowledge of the clinicopathological status of the specimens. Expression of CEACAM7 and CEA was evaluated according to the ratio of positive cells per specimen (R) and staining intensity (I). The ratio of positive cells per specimen was scored 0 for staining of < 1%, 1 for staining of 2 to 25%, 2 for staining of 26 to 50%, 3 for staining of 51 to 75%, and 4 for staining > 75% of the cells examined. Intensity was graded as following: 0, no signal; 1, weak; 2, moderate; and 3, strong staining. A total score (R × I) of 0 to 12 was finally calculated and graded as negative (-score: 0-2) and positive (+, 3-12).

After baking on a panel at 60°C for an hour, the sections were deparaffinized with xylene and rehydrated through gradient ethanol immersion. This was followed by a step of antigen retrieval. The slides were immersed in 0.01 mol/l citrate buffer solution (pH 6.0) and placed in a microwave oven for 30 min. After a wash in 0.01 mol/l of phosphate-buffered saline (PBS, pH 7.4), samples were incubated with blocking buffer (5% normal goat serum, 1% bovine serum albumin and 0.1% triton X-100) for one hour at room temperature. Samples were then incubated with primary antibodies diluted in the blocking buffer at 4°C overnight, followed by 3 washes in PBS for 10 minutes each. Finally, samples were stained with secondary antibodies diluted in blocking buffer for 30 minutes followed by 3 washes in PBS. Coverslips were mounted on slides with Prolong Antifade (Invitrogen). Antibodies used for immunostaining include mouse anti-CEACAM7 (1:100, ab26281, Abcam), rabbit anti-CEA (1:10, ab-15157, Abcam), secondary antibodies used were goat anti-mouse IgG Alexa 549 (1:300, Invitrogen), goat anti-rabbit IgG Alexa 488 (1:300, Invitrogen), DAPI (1:1000, Molecular Probes). Adjacent normal tissues were used as negative controls (20 mm away from the cancer tissue). Immunostained tissues were visualized and images were captured by using FlUOVIEW (FVLIOi, OLYMPUS.

All statistical analyses were performed using IBM SPSS 19.0 software. Measurement data were analyzed using Student's t or one-way ANOVA test, while categorical data were studied using theχ2 or nonparametric test. Survival curves were estimated using the Kaplan-Meier method, and the log-rank test was used to calculate differences between the curves. Multivariate analysis using the Cox proportional hazards regression model was performed to assess the prognostic values of protein expression. Correlation coefficient between expression of CEACAM7 and CEA was estimated using the Spearman correlation method. Statistical significance was set at P < 0.05.

Immunohistochemistry was performed on consecutive sections of 145 gastric carcinomas using either anti-CEACAM7 or anti-CEA antibody (Figure 1). Immunohistochemical analysis shows that 100 of 145 (69.0%) samples were categorized as CEACAM7-negative (Figure 1A), and 45 of 145 (31.0%) samples were categorized as CEACAM7-positive (Figure 1C, 1E). CEACAM7 expression was concordant with CEA expression in 80.0% (116 of 145) of gastric carcinoma cases (Spearman R = 0.605, P < 0.001). CEACAM7 and CEA double-positivity was observed in 40 cases, double-negativity was observed in 76 cases, and 29 cases were found to be either CEACAM7-positive or CEA-positive only. The expression correlation coefficient was estimated using the Spearman Rank Correlation method. The Spearman R value was 0.605 (P < 0.001), indicating a close correlation between CEACAM7 and CEA expression in gastric carcinomas. As it is inconvenient and unnecessary to check the localization of the two antigens by immunofluorescence in all the 40 carcinoma tissues that are positive for both CEACAM7 and CEA, we investigated their localization in seven tissue sections that were randomly selected from the 40 carcinoma tissues sections. Laser confocal microscopy analysis showed that they co-localized in the membrane and cytoplasm of cancerous cells (Figure 2A,B,C,D).

Relationship between CEACAM7 and CEA expression and various clinicopathological features of gastric carcinomas is summarized in Table 1. CEACAM7 was more frequently expressed in poorly differentiated tumors than in well and moderately differentiated gastric carcinomas (41.3% vs. 20.0%, P = 0.006). CEACAM7 positivity was significantly higher in diffuse-type gastric carcinomas than in intestinal-type gastric carcinomas, (39.7% vs. 22.2%, P = 0.023). CEA expression significantly correlated with lymph node metastasis (P = 0.031).

The results of CEACAM7 and CEA expression in normal mucosa and precancerous lesions were summarized in Table 2. Positivity for CEACAM7 expression was found in chronic atrophic gastritis (12%; 6/50), low-grade GIN (29.1%; 16/55), and high-grade GIN (28.9%; 13/45). CEACAM7 expression was not found in normal mucosa (0/50). Positivity for CEACAM7 expression in low grade GINs was significantly higher than for chronic atrophic gastritis (P = 0.032), and CEACAM7 was more frequently expressed in atrophic chronic gastritis than in normal gastric mucosa (P = 0.012), but there was no significant difference in CEACAM7 expression between low-grade GIN and high-grade GIN (P = 0.982). CEA positivity was found in normal mucosa (6%; 3/50), chronic atrophic gastritis (8%; 4/50), low-grade GIN (57.1%; 13/55), and high-grade GIN (60%; 20/45). The CEA positivity for low-grade GIN was significantly higher than for chronic atrophic gastritis (P = 0.030), but was lower than that for high grade GIN (P = 0.028). There was no significant difference in CEA positivity between normal mucosa and chronic atrophic gastritis (P = 0.500). CEACAM7 expression was localized to the luminal surface of chronic atrophic gastritis and GINs (Figure 3C, E, and 3G), no immunoreaction was detected in normal mucosa (Figure 3A). CEA was localized on the luminal surface of normal mucosa, chronic atrophic gastritis, low-grade GIN, and high-grade GIN (Figure 3B, D, F, and 3H), whereas both CEA and CEACAM7 expression was localized to the entire surface of gastric cancerous cells (Figure 3I and 3J)

One-year and three-year survival rates were 92.0% and 54.1%, respectively, for CEACAM7 and CEA double-negative patients, 98.3% and 45.1%, respectively, for CEACAM 7 and CEA single-positive (either CEACAM7 positive or CEA positive) patients, and 64.0% and 8.5% for CEACAM7 and CEA double-positive patients. Significant difference was observed between the survival rate of double-negative and double-positive patients (Figure 4, P = 0.001). However, no significant difference was found between double-negative and single-positive patients (Figure 4, P = 0.391). Multivariate analysis demonstrated that double-positivity for CEACAM7 and CEA expression (P = 0.004) and tumor stage (P = 0.001) were factors independently associated with poorer patient prognosis (Table 3).