Histochemistry sections and mutations. A, Eosin staining (original magnification ×100). B, Staining of complex V with an antibody anti-ATP5B subunit (original magnification ×100). C, The MTND6 heteroplasmic insertion of C in position 14249 (arrow) of the mitochondrial genome in tumor and in the same patient's nonneoplastic tissue. D, The homoplasmic MT-TS m.12242A>G mutation (arrow) in tumor and in control nonneoplastic tissue (not of the same patient). E, Fainter staining of MTND6 in oncocytic cells (red circle) compared with normal conjunctival cells (black circle) (original magnification ×100). F, Staining with Ki67 (original magnification ×100).
Bartoletti-Stella A, Salfi NCM, Ceccarelli C, Attimonelli M, Romeo G, Gasparre G. Mitochondrial DNA Mutations in Oncocytic Adnexal Lacrimal Glands of the Conjunctiva. Arch Ophthalmol. 2011;129(5):664-676. doi:10.1001/archophthalmol.2011.95
Oncocytic neoplasms are characterized by a marked mitochondrial hyperplasia. They are of epithelial derivation and usually occur in endocrine and exocrine organs such as the thyroid, parathyroids, kidney, pituitary, and salivary glands.1 Oncocytic lesions of the ophthalmic regions have been reported, especially as oncocytic adenomas of the caruncle.2,3 Similar lesions occur rarely in the adnexal lacrimal glands of the conjunctiva. Truncating mitochondrial DNA (mtDNA) mutations in respiratory complex I (CI) subunits have been suggested to be the genetic hallmark of the oncocytic phenotype.4- 6 The occurrence of such genetic lesions strongly impairs cell respiration and contributes to maintaining the tumor in a low-proliferating, benign state.6 We describe an oncocytoma of the adnexal lacrimal glands of the conjunctiva harboring mtDNA mutations that cause CI disassembly and associate with a low proliferation index.
An oncocytic tumor of the adnexal lacrimal glands of the conjunctiva was collected anonymously. The tumor was partially cystic and composed of cylindrical oncocytic cells with small, ovoid nuclei. It was arranged in a glandular pattern and characterized by an abundant inflammatory stroma with nontumor cells and few lymphocytes, displaying some similarities with Warthin tumor.
Paraffin-embedded sections showed intense cytoplasmic eosin staining of neoplastic cells compared with normal cells (Figure). Staining with a specific antibody against complex V6 confirmed mitochondria abundance in the neoplastic cells (Figure). Association between the oncocytic phenotype and the presence of truncating mtDNA mutations is generally accepted.4- 6 To prove that oncocytic transformation in the ophthalmic region may be unequivocally defined by pathogenic mtDNA mutations, the entire mtDNA sequence was analyzed.4- 6
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).
To our knowledge, clearly pathogenic mtDNA alterations have never been reported in tumors of the ophthalmic region. The frameshift mutation described here was shown to at least partially hamper CI assembly, likely causing an energetic impairment in oncocytic cells. The occurrence of CI-truncating mutations has been extensively reported in benign oncocytic tumors, where the mutations associate with lack of aggressive behavior.4,6 In fact, such mutations render cells unable to stabilize hypoxia-inducible factor 1-α, which is one of the key players in mediating progression to malignancy.6 The CI structural impairment may hence explain the benign nature of the case described.
Correspondence: Dr Romeo, Dipartimento di Scienze Ginecologiche, Ostetriche e Pediatriche, Unità Operativa di Genetica Medica, Policlinico S. Orsola-Malpighi, via Massarenti 9, 40138 Bologna, Italy (firstname.lastname@example.org).
Author Contributions: Dr Gasparre had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Financial Disclosure: None reported.
Funding/Support: This work was supported by grant 8810 from the Associazione Italiana per la Ricerca sul Cancro–Milano (Dr Romeo) and Fondo per gli Investimenti della Ricerca di Base “Futuro in Ricerca” grant J31J10000040001 from the Italian Ministry of University and Research (Dr Gasparre).
Role of the Sponsors: The funding agencies had no role in the design or conduct of this research.