| BackgroundHepatocellular carcinoma (HCC) is the most common primary malignant tumor in liver, which ranks fifth in overall frequency, and around 372,000 new cases of HCC are diagnosed annually. As an effective treatment, LT has been accepted as the important potentially curative option for patients with HCC after 20 years development. However,due to the high incidence of recurrence in patients treated with LT , LT is still a debate. Recurrence of disease is the most predictive clinical prognostic indicator for all HCC patients. So it is urgency to improve predictive power of recurrence and establish optimal recipients selection criteria. Patients have the similar pathogenesis and treatment,but have differentprognosis because of individual diversity. Gene polymorphism is hotspot in individual diversity study. Several works have showed that MDR1 expression were associated with carcinogenesis, and tumor progression. Kurzawski et al. indicated the association with colon cancer. A report by Stanulla et al. suggested a significant reduction in the risk of relapse in the central nervous system in childhood acute lymphoblastic leukemia (ALL) for patients with the T allele at 3435. However, HCC remains with no prognostic classification according to genotype subsets, and there is no research about the polymorphism of MDR1 in HCC patients treated with LT. In Chinese population, the three most frequent SNPs of MDR1 gene are G2677A/T , C1236T and C3435T . In this study, we investigated the three MDR1 gene SNPs in association with the risk of HCC recurrence after LT and determined whether some polymorphisms could provide further discriminative power over current recipients selective systems for recurrence-free prognostication in HCC patients undergoing LT.Materials and methodsFifty HCC patients treated with LT from cadaveric donors between February 2003 and January 2005 in our center were enrolled in this study. All these HCC patients received orthotopic LT. The mean follow-up of the remaining fifty patients was 16.1 months (median 16.3 months; range 1-34.7months), among them, 39 (78%) had been histologically proven HCC with cirrhosis.Immunosuppressive regimens after LT were based on a standardized protocol using prednisone, tacrolimusand and mycophenolate. All the patients did not receive regular prophylactic chemotherapy after LT. The surviving patients were followed up closely at the outpatient clinic, and tumor recurrence or metastasis was monitored by alpha-phetoprotein (AFP), ultrasonography, chest X-ray, andEmission Computed Tomography.Genotype IdentificationGenomic DNAs were isolated from EDTA-anticoagulated whole blood of recipients using the Qiaamp DNA blood mini kit (Qiagen, German). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used for genotype identification in this study. PCR mixture contained 50 ng/μl genomic DNA 4 μl, 10× PCR buffer 5 μl, MgCl2 (1 mmol/L) 4 μl, dNTP (200 μM) 2 μl, each primer (0.2 μM) 2 μl and Taq gold DNA polymerase (1 U/μl ) 0.6 μl in a final volume of 50 μl. Cycling was performed in a GeneAmp PCR system as follows: initial denaturation at 94°C for 10 minutes and 34 cycles of 94 °C for 45 seconds, 54°C (C1236T G2677A,T) or 56°C (C3435T) for 45 seconds, 72°C for 45 seconds, and a final step at 72°C for 10 minutes. PCR product was analyzed by 1.5% agarose gel electrophoresis after ethidium bromide staining. Enzymatic digestion mixture was performed at 37°C overnight in a total volume of 20 μl including 1 U restriction endonuclease, 1 × restriction buffer and 6 ul PCR products. The digestion products were thereafter electrophoresed at 15% PAGE gel followed by silver staining for genotype determination. Statistical AnalysisThe data were analyzed statistically using the software SPSS, version 13.0 (SPSS Inc, Chicago, IL). Chi-square analysis was performed to evaluate categorical variables related to tumor recurrence. The tumor-free survival and overall survival were calculated with the Kaplan-Meier method, and the differences were compared with the log-rank test. Cox's proportional hazard model with the forward maximum likelihood estimation was used in multivariate analysis. P value less than 0.05 was considered statistically significant.ResultsBetween recurrence and recurrence -free patients, there was no significant differences in the distribution of allelic frequency of MDR1 C1236T, G2677T, C3435T. We took AA, AG or AT (carrying at least one variant A allele) as 2677A carrier group and TT, GT or GG as 2677A noncarrier one. The association between recurrence-free and 2677A carrier was significant (P<0.05).In relationships between 2677A carrier polymorphism and clinicopathological data, there were no significant differences in clinicopathological parameters as to age, gender, portal vein tumor thrombi (PVTT), tumor size, tumor number, hepatic cirrhosis background, preoperative alpha-fetoprotein (AFP) level or histopathologic grading after LT between the MDR-1 2677A carrier and 2677A noncarrier groupsBy this time, 20 patients died of the recurrence, 8 patients were still alive with recurrence, and 22 patients were alive without recurrence. Among 12 2677A carriers,9 patients were alive without recurrence. Only 13 patients were alive without recurrence in 38 2677A noncarriers. The corresponding 1-year survival rates were 91.7% and 65.6%, and the 1-year tumor-free survival rates were 83.3% and 36.5%, respectively.2677A carrier group showed significantly higher tumor-free survival rate than 2677A noncarrier group (P<0.05), but no significant difference in overall survival, (P>0.05). Univariate analysis revealed that PVTT, tumor size, tumor number, preoperative AFP level, histopathologic grading and 2677A carrier genotype were significant variables for predicting tumor-free survival. With respect to overall survival, PVTT, tumor size, tumor number, preoperative AFP level and histopathologic grading provided significant predictive values. The multivariate analysis of these variables revealed that pretransplant AFP level, tumor number, PVTT, and 2677A carrier genotype were independent factors for predicting tumor-free survival.Conclusions1 MDR1 polymorphism 2677A carrier genotype were independent factors for predicting tumor-free survival.2 Between recurrence and recurrence -free patients, there was no significant differences in the distribution of allelic frequency of MDR1 polymorphism C1236T, G2677T, C3435T. |
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