Mitochondrial D-loop mutations as clonal markers in multicentric hepatocellular carcinoma and plasma.

PURPOSE Hepatocellular carcinoma (HCC) is a highly malignant tumor prone to multicentric occurrence. Differentiation between a true relapse of HCC and a second primary tumor is of clinical importance. We sought to identify mitochondrial mutations in HCC and test their use as clonal markers in this disease. EXPERIMENTAL DESIGN Primary HCC tissue samples were obtained from 19 patients and analyzed for mutations within the mitochondrial displacement loop (D-loop). The discovered mutations were used to determine tumor clonality and provided the basis for detection of tumor DNA in corresponding plasma samples. RESULTS Thirteen of 19 HCC cases (68%) were identified as having D-loop mitochondrial DNA (mtDNA) mutations in at least one tumor. In 3 of these 13 cases, the same mutation was observed in multiple tumors, indicating monoclonal origin. Remarkably, in 8 of 13 mutated cases, we detected deletion/insertion mutations in the C-tract, a recently reported hotspot and potential replication start site of the closed, circular mitochondrial genome. In addition, we detected mutant mtDNA in 8 of 10 tested paired plasma DNA samples using a highly sensitivity molecular assay. CONCLUSIONS mtDNA mutations within the D-loop control region are a frequent event in HCC, providing a molecular tool for the determination of clonality. In addition, detection of tumor-specific mtDNA mutations in plasma DNA needs to be explored further for monitoring patients with primary HCC.