Although the excised tumor mass was very small and mostly composed of tumor cells, microdissection was performed to isolate tumor and normal cells. A clearly pathogenic frameshift mutation (m.14249insC) was detected in the MTND6 subunit of CI (Figure). Because of the physiological polyploidy of the mtDNA, mutations may be homoplasmic or heteroplasmic, meaning that all or only some mtDNA copies may be mutated, respectively. The mutation was heteroplasmic in the tumor, although a slight nontumor cell contamination could not be completely excluded. Also, a novel homoplasmic mutation (Human Mitochondrial Database; http://www.hmtdb.uniba.it) was detected (m.12242A>G) in the transfer RNA gene for serine (MT-TS) (Figure). This mutation was not tumor specific, and positive staining for mitochondria-coded COI (not shown) suggests that the mutation may not affect overall protein translation. Immunohistochemical analysis with an antibody against MTND6 and NDUFB6 subunits of CI showed that MTND6 expression was much fainter in the oncocytic neoplasm compared with nonneoplastic tissue (Figure), in agreement with heteroplasmy of the MTND6 mutation. Although NDUFB6 showed positive staining (not shown), CI assembly is most likely impaired because frameshift mutations in mtDNA-encoded reduced nicotinamide adenine dinucleotide dehydrogenase subunit genes have been shown to result in assembly defects of CI in other oncocytic tumors.4- 6 Finally, to investigate the association between the occurrence of CI-disassembling mtDNA mutations and a low-proliferating state, Ki67 staining was performed. Indeed, the proliferation index was shown to be 1.8% (Figure).