Application Of E-cadherin/β-catenin And Ki-67 In Prognosis For Hepatocellular Carcinoma Treated With Orthotopic Liver Transplantation

BackgroundHepatocellular carcinoma(HCC) is one of the most common deadly malignancy, which is the third cause of death by malignant tumor in the world. As a radical effective option, orthotopic liver transplantation(OLT) is praised highly by many liver disease centers. However, as for the recurrence of disease(ROD) is easy and aggressive, especially for the current organ shortage and the expensive cost for the OLT therapy, selection of candidates for OLT become more and more important. Traditionally, the tumor size and number, presence of vascular invasion on which are focused and thought to be closely related to ROD risk. Aside from these macroscopic tumor characters, are there any other microcosmic tumor characters that can be valuable prognosis factors? Here we performed a study on three biological markerswhich were E-cadherin, β-catenin and Ki67, to evaluate their prognostic value.PATIENT AND METHODSBetween Apr. 2002 and Nov. 2005, fifties patients(48 men, 2 women; mean age, 47 years; range, 19-64 years; 44 patients suffered hepatitis B virus infection and 48 ones suffered hepatic cirrhosis; 34 patients' tumour diameter exceeded 5cm; 33 ones had multiple lesions; 27 ones'preoperative α-fetoprotein/AFP serum levels exceeded 400μg/L; 42 ones staging in III-IV phases of TNM) who underwent OLT in our institution were involved in this retrospective study, all cases were diagnosed as HCC by postoperative pathological examination and died as a result of ROD. None of the patients received postoperative adjuvant antineoplastic therapy. 12 months were as a minimum post-transplantation follow-up for survivors.ROD was monitored by means of α-fetoprotein(AFP) serum levels, ultrasound, and computed tomography. Suspicious lesions were biopsied for histological confirmation of recurrence.Four μm tissue section were cut, placed on polylysine treated slides, deparaffinized with xylene, rehydrated through graded alcohols, and incubated in 3% H₂O₂ for 10 min. Antigen retrieval was performed by electrical cooker heating in 10mM sodium citrate buffer(PH 6.0) at the temperature around 95℃ for 15 min. After cooled at room temperature, slides were then incubated at 4℃ overnight with following monoclonal antibodies, anti- E-cadherin, anti-β-catenin, anti-Ki67, all diluted 1:50. The reaction was developed with PowerVision^TMKit. Staining was performed with DAB (All the above agents were supplied by the the Pekin Zhongshanjinqiao Biotechnology Limited Company). After all, slides were counterstained with Mayer's hematoxylin and mounted.E-cadherin and β-catenin membranous signs were semiquantitatively assessed comparing the expression in neoplastic and non-neoplastic liver to categorizedhigh-expressers and low-expressers. Presence or absence of nuclear β-catenin accumulation was assessed in the same samples regardless of the percentage of positive nuclei. Proliferation index was calculated as the ratio of Ki67 positive nuclei with respect to the total number of neoplastic cells: tumors were scored as either low proliferation (<10% positive neoplastic cells) or high proliferation (>10% positive neoplastic cells).We considered overall significance, calculated using the Kaplan-Meier method, the Log-rank test being used for univariate analysis with the other variables, and Cox logistic regression was used for the multivariate analysis. All of the calculations were performed using SPSS 13.0 statistical software package, and the results were considered significant at P < 0.05.ResultsAt univariate analysis, both stage III ,IV of TNM and presence of portal vein tumor embolism (PVTE) correlated with ROD and overall survival/OS (P<0.05), while at multivariate analysis, presence of PVTE showed a trend toward correlation with OS, the same as stage III, IV with OS ; Preoperative serum AFP has statistic significance (P<0.05 ) with ROD only at multivariate analysis.It was found that all of the normal hepatocytes showed weak to moderate membrane positivity for E-cadherin and β-catenin expression. Regarding low membrane expression of E-cadherin, 22 (88%) cases had ROD and 18(72%) cases died, while nuclear β-catenin positive, 6 cases suffered recurrence then died. Regarding high Ki67 proliferation index, 12 cases(92.3%) recurred and 10(76.9%) died. All of these three molecular parameters showed significance in the Kaplan-Meier curves and correlation with ROD or OS whither at univariate or multivariable analysis. But the statistic results didn't show any correlation between the membrane expression of β-catenin with ROD or OS.Conclusions1. As for a small sample study, these three biological markers are able to predict recurrence of HCC after OLT, and perhaps better than the clinical-pathological variables, it'll be helpful to stratify candidate with HCC for OLT.2. The molecular parameters can be assessed on tissues obtained by preoperative biopsies under ultrasound monitor. Especially for nuclear β-catenin and Ki67 proliferation index which needless to compare the expression with non-neoplastic tissue, they can be assessed easily on tumour tissues with immunohistochemistry.3. To validate the prognostic value of these three biological markers, it's indispensable to enlarge samples further.