Introduction
Hepatocellular carcinoma (HCC) is one of the most frequent human malignancy worldwide. Especially in China, it has become a major cause of cancer-related death
The L1 cell adhesion molecule (L1CAM, also named as CD171), as a member of the immunoglobulin superfamily, is a 220 kDa type I transmembrane glycoprotein
Materials and methods
Patients and tissue samples
The study was approved by the Research Ethics Committee of 302nd Hospital of PLA, Beijing, China. Informed consent was obtained from all of the patients. All specimens were handled and made anonymous according to the ethical and legal standards.
A total of 130 patients with primary HCC who underwent a curative liver resection at the 302nd Hospital of PLA, Beijing, China, were included in this retrospective study. Tissues used in the study were retrieved from the tissue bank of the Department of Pathology in the 302nd Hospital of PLA. These patients were diagnosed as HCC between 2001 and 2006. None of the patients recruited in this study had chemotherapy or radiotherapy before the surgery. HCC diagnosis was based on World Health Organization (WHO) criteria. Tumor differentiation was defined according to the Edmondson grading system. Liver function was assessed using the Child-Pugh scoring system. Tumor staging was determined according to the sixth edition of the tumor-node-metastasis (TNM) classification of the International Union against Cancer. No distant metastasis was present at the time of surgery. The clinicopathological features of 130 patients are summarized in Table
The median follow-up period was 8.6 years. Postoperative surveillance included routine clinical and laboratory examinations every third month, computed tomography scans of the abdomen, and radiographs of the chest every third month. After 5 years, the examination interval was extended to 12 months.
Immunohistochemistry analysis
Immunohistochemical staining was carried out following the protocol of our previous study
Following a hematoxylin counterstaining, immunostaining was scored by two independent experienced pathologists, who were blinded to the clinicopathological parameters and clinical outcomes of the patients. The scores of the two pathologists were compared and any discrepant scores were trained through re-examining the stainings by both pathologists to achieve a consensus score. The number of positive-staining cells showing immunoreactivity on the membrane for L1CAM in ten representative microscopic fields was counted and the percentage of positive cells was calculated. The percentage scoring of immunoreactive tumor cells was as follows: 0 (0%), 1 (1-10%), 2 (11-50%) and 3 (>50%). The staining intensity was visually scored and stratified as follows: 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). A final score was obtained for each case by multiplying the percentage and the intensity score. Therefore, tumors with a multiplied score exceeding 4 (median of total scores for L1CAM) were deemed to be low expressions of L1CAM; all other scores were considered to be high expressions of L1CAM.
Western blot
The Western blot protocol and semiquantitative analysis were carried out following the protocol of Xu et al.
Quantitative RT-PCR
To measure the mRNA expression levels of L1CAM, total RNA was extracted from frozen liver tissues using TriZol reagent (Invitrogen) following the manufacturer’s instructions. Two micrograms of total RNA was subjected to reverse transcription to synthesize cDNA using the ProtoScript M-MuLV Taq RT-PCR Kit (New England Biolabs), according to the manufacture’s instruction, followed by real-time PCR using the TransStart Green qPCR SuperMix (TransGen Biotech). The primer sequences of L1CAM were forward primer, 5′- ACG AGG GAT GGT GTC CAC TTC AAA-3′, reverse primer, 5′- TTA TTG CTG GCA AAG CAG CGG TAG-3′. The transcription of GAPDH was used as an internal control for normalization. L1CAM expression levels were calculated relative to GAPDH using the delta-delta computed tomography method
Statistical analysis
The software of SPSS version13.0 for Windows (SPSS Inc, IL, USA) and SAS 9.1 (SAS Institute, Cary, NC) was used for statistical analysis. Fisher’s exact test and the X2 test were performed to assess associations between L1CAM expression and clinicopathological parameters. The Kaplan-Meier method was used for survival analysis, and differences in survival were estimated using the log-rank test. A multivariate survival analysis was performed for all parameters that were significant in the univariate analyses using the Cox regression model. Differences were considered statistically significant when
Results
Expression of L1CAM protein and mRNA in HCC
To analyze the clinical value of L1CAM in HCC, we first evaluated its expression at protein and mRNA levels by immunohistochemical analysis, western blot analysis and quantitative RT-PCR.
As the results, the immunostaining was homogeneous throughout the tumor. L1CAM immunostaining was mainly localized on the membrane of tumor cells of HCC tissues (Figure
Immunohistochemical staining of L1CAM expression in hepatocellular carcinoma (HCC) and adjacent nonneoplastic liver tissues (Original magnification × 400)
Immunohistochemical staining of L1CAM expression in hepatocellular carcinoma (HCC) and adjacent nonneoplastic liver tissues (Original magnification × 400).A, L1CAM positive staining was indicated by numerous yellowish granules in the membrane of HCC cells; B, L1CAM negative staining was seen in adjacent nonneoplastic liver tissues.
Additionally, Western blot analysis as an independent method was performed to confirm L1CAM protein expression. The distinct overexpression of L1CAM protein in HCC tissues compared with adjacent nonneoplastic liver tissues was also detected (P <0.01, Figure
Increased expression levels of L1CAM protein and mRNA in hepatocellular carcinoma (HCC) and adjacent nonneoplastic liver tissues
Increased expression levels of L1CAM protein and mRNA in hepatocellular carcinoma (HCC) and adjacent nonneoplastic liver tissues. (A) Representative Western blotting of L1CAM protein levels in HCC tissues and adjacent nonneoplastic liver tissues. (B) Semiquantitative Western blotting showed significantly increased L1CAM protein level in HCC tissues compared with adjacent nonneoplastic liver tissues. GAPDH was used as internal control. Means, standard deviation (SD), and P values were given (T test). (C) Significantly increased L1CAM mRNA level (P <0.01, Mann–Whitney test) in HCC tissues compared with adjacent nonneoplastic liver was detected by quantitative RT-PCR. GAPDH was used as internal control.
Association of L1CAM expression with the clinicopathological features of HCC
We next evaluated whether L1CAM protein expression was associated with clinicopathological features of patients with HCC by correlating immunohistochemical L1CAM staining results with T stage, tumor grade, presence of cirrhosis, underlying liver disease including alcohol abuse, viral hepatitis B and C, sex, and age (Table
Prognostic values of L1CAM expression in HCC
To further investigate the clinical usefulness of L1CAM expression in HCC, we compared five-year overall survival and five-year disease-free survival according to various clinicopathologic factors including the expression level of L1CAM. Five-year disease-free survival was observed in 30 (23.08%) patients, whereas in 100 (76.92%) patients, disease recurred, and 88 (67.69%) even died during a 5-year follow-up period. We observed a trend that 5-year disease-free survival in the group with high L1CAM expression was significantly poorer than that in the group with low L1CAM expression (P <0.01, log-rank test; Figure
Kaplan-Meier survival curves for L1CAM expression in the HCC patients
Kaplan-Meier survival curves for L1CAM expression in the HCC patients. The HCC patients with high L1CAM expression showed significantly shorter disease-free survival (P <0.01, A) and overall survival (P <0.01, B) rates than those with low L1CAM expression.
Discussion
In this study, we first dmonstrate that L1CAM protein and mRNA expression in human HCC tissue was significantly associated with tumor progression and clinicopathologic features. Immunohistochemical analysis of a large set of HCCs revealed that 63.08% of HCC were high expression for L1CAM. Notably, L1CAM immunoreactivity was distinctly increased in a substantial proportion of HCC cases compared with their adjacent nonneoplastic liver tissue, which was further confirmed by Western bloting analysis and Q-PCR analysis. Then, the expression of L1CAM in HCC tissues with advanced tumor stage and grade was significantly higher than that in early tumor stage and low tumor grade HCC, suggesting that L1CAM expression might be of clinical relevance in the aggressiveness of HCC. The impact of L1CAM expression on clinical outcome was assessed by Kaplan-Meier analyses. High L1CAM expression was associated with a significant trend toward both poorer disease-free and overall survival. Univariate and multivariate analyses clearly demonstrated that L1CAM expression was an independent risk factor predicting overall survival and disease-free survival of patients with HCC. The statistically significant impact of L1CAM expression for overall survival (P = 0.008) was more significant than the tumor stage (P = 0.009) that is widely used at present, suggesting that L1CAM expression could be a useful marker to predict patient survival.
It is necessary to identify biological markers associated with the advancement of tumor progression for early diagnosis of patients with aggressive tumors and poor prognosis, and for the development of new therapeutic strategies and the selection of the appropriate treatment. The cell dhesion molecule families, such as integrins, cadherins, immunoglobulin-like CAMs and selectins, are often aberrantly regulated in human malignancies, leading to the tumor progression
The function of L1CAM determines its contribution to the tumorigensis. At first, L1CAM is a target gene of β-catenin-TCF signaling, which is an important cancer-related pathway. Many β-catenin target genes including metalloproteases, cell-extracellular matrix components, transcription factors, and cell adhesion molecules have been demonstrated to be involved in later stages of tumorigenesis that can confer invasive and metastatic capacities
In conclusion, our data suggest that L1CAM is overexpressed in HCC tissues compared with their benign counterparts. To the best our knowledge, this is the first study evaluating the expression levels of L1CAM mRNA and protein in HCC tissues and its association with clinicopathologic parameters. Especially, the most important finding of this study is that L1CAM also is a novel and potential factor for predicting the poorer prognosis of HCC patients after surgery.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
XDG and LX carried out the experimental studies and drafted the manuscript. LZ, TS, JZ, and HWL carried out part of the experimental studies. RYP and JMZ designed the experiments and modified the manuscript. All authors read and approved the final manuscript.