Hepatocellular carcinoma (HCC) is one of the most common solid tumors, characterized by progressive disease, a high postsurgical recurrence, and extremely poor prognosis. Curative resection is a typical first-choice treatment for HCC; however, the prognosis remains poor after partial hepatectomy, even for small tumors.1 Nonetheless, many HCC patients with cirrhosis cannot undergo hepatectomy due to their limited liver function and the extent of the tumor.2 Liver transplantation (LT) offers a potential curative treatment for small HCCs. Increasing evidence suggests that LT is the best therapeutic choice for HCC, especially in patients with cirrhosis.3 Unfortunately, nearly 20%-40% of HCC patients who undergo LT experience HCC recurrence within 5 years,4 making HCC recurrence one of the most prevalent causes of poor long-term survival.3 Studies have shown that the number of tumor nodules, tumor size, microscopic/macroscopic vascular invasion, and pre-operative α-fetoprotein (AFP) level are all correlated with the recurrence of HCC after LT.5,6 It is still widely recognized, however, that those factors remain insufficient for recognizing patients at high risk for recurrence and/or selecting those at low risk.2 Reliable prognostic factors for HCC recurrence after LT remain to be identified to be able to reliably select HCC patients with a low risk of recurrence who would benefit from LT.
Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a member of the CEA subfamily and immunoglobulin superfamily.7,8 CEACAM1 has been detected in several types of tumor cells; however, there are different views regarding the prognostic relevance of CEACAM1 expression in different cancers.9–13 Previous studies have shown that CEACAM1 expression can be determined via the presence of either membranous or cytoplasmic staining.14,15 Cruz et al9 indicated that the loss of CEACAM1 expression in HCC specimens reflected aggressive tumor biology and predicted a poor prognosis for patients with HCC.9 Additionally, studies conducted previously by our research group demonstrated that cytoplasmic CEACAM1 and membranous CEACAM1 exert different functions in HCC progression and angiogenesis. Moreover, CEACAM1 cytoplasmic expression is reportedly closely related to poorer relapse-free survival (RFS).16 However, the prognostic value of CEACAM1 for tumor recurrence in HCC patients treated with LT remains unclear.In the present study, CEACAM1 expression in HCC specimens was assessed by immunohistochemistry. The purpose of this study was to investigate the effect of CEACAM1 on RFS in HCC patients that underwent LT.
Patients who underwent cadaveric donor LT for HCC at the Department of General Surgery from the Qilu Hospital of Shandong University between January 2002 and April 2007 were enrolled in this study. Patients were included if (1) HCC was diagnosed prior to transplantation and was confirmed by histopathologic examinations postsurgically; (2) clinicopathologic variables, such as serum concentrations of AFP, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyl transferase (GGT), portal vein tumor thrombi (PVTT), tumor size, tumor number, and histopathologic grading (Edmondson-Steiner grade) were detected before surgery and during follow-up; (3) all patients were hepatitis B virus (HBV)-positive (HBsAg+) or hepatitis C virus (HCV)-positive (anti-HCV+); and (4) no patient received any adjuvant antineoplastic therapy before surgery. Patients were excluded if they were positive for both HBsAg and anti-HCV.
During follow-up, serum AFP, abdominal ultrasonography, and thoracic radiographs were performed once monthly for the first 6 months, then every 3 months until 3 years postoperatively. Thereafter, the same parameters were measured every 6 months. Emission computed tomography (CT) was performed every 3 months for the first year, biannually for the second year, then annually. Metastases were evaluated using either bone scintigraphy or thoracic CT scan. Finally, RFS was defined as the interval between the date of surgery and the date of diagnosis/relapse.
This study was approved by the investigation and Ethical Committee of Qilu Hospital according to the standards of the Declaration of Helsinki.
All specimens were provided by the Department of Pathology of Qilu Hospital, and all specimens were identified as HCC by pathologists. Formalin-fixed, paraffin-embedded sections that were 4 μm thick were created, and immunohistochemical staining for CEACAM1 was performed as previously described.16 A mouse monoclonal antibody against CEACAM1 (Abcam, USA; 29H2, dilution 1:75) was employed according to the manufacturer's instructions. All immunohistochemistry slides were examined using an Envision kit (Dako, Glostrup, Denmark) and streptavidin-peroxidase complex reagent (Dako). Subsequently, 4-μm paraffin-embedded sections were deparaffinized, rehydrated, and treated with 3.0% hydrogen peroxidase for 20 minutes to block endogenous peroxidase activity prior to antigen retrieval. After washing with phosphate-buffered saline (PBS, pH 7.4) three times, the slides were covered with normal serum in a humidity chamber for 30 minutes at room temperature. The sections were then incubated with the primary antibody in a humidity chamber for 60 minutes at 37.0°C. After washing with PBS again, the sections were incubated with the secondary antibody (Envision; Dako) for 40 minutes at 37°C in a humidity chamber before adding streptavidin-peroxidase complex reagent (Dako). Sections were incubated at room temperature for 45 minutes and covered with 3,3′-diaminobenzidine tetrahydrochloride solution. The sections were immersed in running tap water and counterstained with hematoxylin for 1 minute then immersed in a series of alcohol baths of increasing concentrations and xylene. All sections were covered with coverslips. Adjacent normal liver tissue served as a positive control. Negative controls were samples in which the primary antibody was omitted.
The results of immunohistochemical staining were separately assessed by two independent pathologists who were blind to the patients' clinicopathologic characteristics and clinical outcomes. Cases with discrepancies were jointly resolved by both pathologists re-evaluating the immunostained slides until a consensus was reached.
Statistical analyses were performed using SPSS 13.0 for windows (SPSS Inc., USA). The association between CEACAM1 expression and clinicopathologic variables was assessed using either the χ2 test or two-sided Fisher exact test. The correlation of CEACAM1 expression and HCC recurrence was evaluated using Kaplan-Meier method. Multivariate analysis was performed to identify variables that were independent prognostic factors for RFS. Values of P <0.05 (two-tailed) was considered statistically significant.
Correlation between CEACAM1 expression and clinicopathologic variables
CEACAM1 membranous expression was detected in HCC specimens collected from 25 patients. As exemplified in Figure 1, loss of membranous expression was detected in 23 HCC specimens (19 cases with cytoplasmic expression and four with loss of CEACAM1 expression). The association between CEACAM1 expression and clinicopathologic variables were further analyzed. Loss of membranous CEACAM1 expression was significantly associated with larger tumor size (P <0.0001), multiple number (P=0.004), and serum AFP levels (P=0.001), as summarized in Table 1.
Univariate and multivariate survival analyses
To determine whether CEACAM1 expression patterns were related to the recurrence of HCC in patients treated with LT, univariate and multivariate survival analyses were performed. On univariate analysis, patients with loss of membranous CEACAM1 expressions were prone to experiencing earlier HCC recurrence than patients with membranous CEACAM1 expression (median, 21 months versus 41 months respectively, and 43.5% vs. 76.0% respectively; P=0.027 as shown in Table 2 and Figure 2). Furthermore, univariate analysis also revealed that AFP level, tumor size, and tumor number provided significant predictive values for HCC recurrence after LT. In contrast, other clinical indexes, including sex, age, hepatitis type, ALT, AST, GGT, PVTT, and Edmondson-Steiner grade had no prognostic significance for recurrence of HCC (Table 2). Multivariate analysis revealed that loss of membranous CEACAM1 expression was a novel independent prognostic factor for RFS (hazard ratio, 1.153-111.613, P=0.037; Table 3).
LT offers the only curative therapy for patients with HCC; however, HCC recurrence after LT remains as one of the most prevalent causes of poor long-term survival.3
The Milan criteria and the University of California, San Francisco (UCSF) criteria are both widely used to improve the survival of HCC patients undergoing LT.17
Zheng et al18 also proposed Hangzhou criteria according to their Chinese experience. Each of those criteria are based on the number of HCC nodules, maximum tumor diameter, or both. However, histopathologic evaluation of the explanted HCC specimens can give better information regarding the tumor behavior.19 Additionally, molecular markers can also provide additional prognostic value for the risk stratification patients.
In multicellular organisms, cell-to-cell adhesion is vital in the mediation and integration of single cells into functional tissues and organs.20 Furthermore, many cell adhesion molecules are able to suppress cell growth and migration primarily through contact inhibition in tumor tissue; however, loss of cell-to-cell adhesion may promote tumor cell growth as well as cell dissemination. Members of the immunoglobulin superfamily of cell adhesion molecules (IgCAMs), including CEACAM1, constitute a large group of cell surface glycoproteins with ancient roots in the animal kingdom that specialize in cell-to-cell adhesion.20 Different expression patterns of CEACAM1, including membranous and cytoplasmic expressions have been previously defined.14,15
CEACAM1 cytoplasmic expression and loss of CEACAM1 expression in HCC specimens reflected aggressive tumor biology and predicted a poor prognosis for patients with HCC in previous studies.9,16 In the present study, loss of membranous CEACAM1 expression in HCC specimens was significantly correlated with both tumor size and the number of tumor nodules. Furthermore, loss of membranous CEACAM1 may be involved in the recurrence of HCC after LT. Thus, we propose that membranous CEACAM1 expression in HCC cells can promote cell adhesion and mediate the integration of single tumor cells into HCC tissues, which, in turn, is able to suppress HCC cells growth and migration through contact inhibition in HCC tissues. Loss of membranous CEACAM1 expressed in HCC cells may disrupt cell-to-cell adhesion and help tumor cells escape from the primary mass, which might cause the tumor cells to disseminate into the circulation and/or distant organs. In turn, “tumor self-seeding” by circulating HCC cells may occur in the new transplant and/or distant organs.21
In univariate analysis, the RFS in patients with loss of membranous CEACAM1 expression who underwent LT was significantly less than patients with CEACAM1 membranous expression. This result suggests that HCC patients with CEACAM1 membranous expression may be good candidates for LT, whereas patients with loss of membranous CEACAM1 expression may be less suitable. In addition, multivariate analysis revealed that loss of membranous CEACAM1 expression was an independent poor predictor for RFS in HCC patients undergoing LT.
In summary, this study demonstrated that loss of membranous CEACAM1 expression in HCC was closely associated with aggressive tumor biology and poorer RFS in patients undergoing LT. These results suggest that loss of membranous of CEACAM1 in HCC might be a useful predictor of relapsing phenotype in patients undergoing LT and could serve as a novel selection criterion for LT. To the best of our knowledge, this is the first study to investigate the prognostic value of CEACAM1 expression in HCC patients undergoing LT. We speculate that HCC cells with loss of membranous CEACAM1 may lose cellular adhesion to the primary mass and promote tumor cells to disseminate into the circulation. “Tumor self-seeding” may occur in the new transplant and/or distant organs;21 however, the specific mechanisms require further exploration. Additionally, this study was confined to HCC patients who underwent LT at a single center. In order to verify the usefulness of this biomarker, large-scale, population-based prospective studies from different research centers need to be performed.
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