Identification Of The Cell Clonal Origin Of Multinodular Hepatocellular Carcinoma By Analyzing The Mitochondrial DNA D-Loop Region Variations

Objective To explore the feasibility of identifying clonal origin of hepatocellular carcinoma(HCC)by analyzing the mitochondrial DNA D-Loop region variations and the clinicopathologic characteristics.Methods Study group(multinodular HCCs)were 42 patients with a total of 112 HCC nodules who were consequentially hospitalized for radical resection of HCC in the department of hepatobiliary surgery of the first affiliated hospital to Guangxi medical university from April 2004 to August 2007.Controls were 20 HCCs(40 samples)hospitalized in the same period that consisted of two sub-groups:control groupⅠconsisted of 16 single nodular HCC cases that each had two pieces of inconsecutive tumor tissues and control groupⅡconsisted of 4 HCC cases with portal vein tumor embolus whose tumor tissues and portal vein tumor embolus were collected simultaneously.Normal control were 5 patients who were donors for liver transplantation or underwent liver trauma without any liver desease.Polymerase chain reaction(PCR)and direct sequencing were applied to study the mtDNA D-Loop region.The sequences were compared among multinodular lesions in study group,between inconsecutive tumor tissues and between tumor and embolus tissues in the control group with regard to their clinicopathologic characteristics.Results In study group,20 cases were categorized as multicentric occurrence(MO)based on their variant mtDNA D-Loop sequences in each nodule from the same patient.And 22 cases were characterized as intrahepatic metastasis(IM)based on the identical mtDNA D-Loop sequences found in each nodule from the same patient.In all 20 cases in the control group,the inconsecutive tumor tissues or the portal vein tumor embolus and original tumors shared identical mtDNA D-Loop sequences.HBeAg(P=0.008),tumor size(sizes of all nodules)(P=0.029),position of nodules(P=0.041),cirrhosis (P=0.011),portal vein and microscope tumor embolus(P=0.023)and differentiation degree(Edmondson grade)of the main nodule(P=0.026)had the significant differences in the two original HCCs(IM and MO),and were considered to be the important factors in differentiating the cell clonal origin in multinodular HCC.Positive HBeAg,cumulative diameter of all nodules≤7cm, nodules located in different lobes,cirrhosis,without portal vein or microscope tumor embolus and/or well/moderate differentiation of main nodular histopathology were attributed to a high rate of MO.Tumor-free survival of the MO subjects(20.7±4.5 months)was significantly longer than that of the IM subjects(6.3±1.3 months,P=0.022).Similarly,overall survival of the MO subjects(29.1±4.4 months)was longer than that of the IM subjects(10.1±1.5 months,P=0.006).Multivariate analysis revealed that the IM/MO characteristic was an independent factor for either tumor-free survival(P=0.012)or overall survival(P=0.011).Conclusions 1.There is a high rate of changes in mtDNA D-Loop region.And our study speculates a novel discrimination of MO and IM origins among multinodular HCCs using PCR and direct sequencing of the mtDNA D-Loop sequences.2.HBeAg,tumor size(sizes of all nodules),position of nodules,cirrhosis,portal vein and microscope tumor embolus and differentiation degree of the main nodule are the important factors to differentiate IM from MO.Positive HBeAg,cumulative diameter of all nodules ≤7cm,nodules located in different lobes,cirrhosis,without portal vein or microscope tumor embolus and/or well/moderate differentiation of main nodular histopathology are attributed to a high rate of MO.3.MO HCC patients might have a favorable outcome compared with IM patients.