Christine M. Betts

Disruptive mitochondrial DNA mutations in complex I subunits are markers of oncocytic phenotype in thyroid tumors

Oncocytic tumors are a distinctive class of proliferative lesions composed of cells with a striking degree of mitochondrial hyperplasia that are particularly frequent in the thyroid gland. To understand whether specific mitochondrial DNA (mtDNA) mutations are associated with the accumulation of mitochondria, we sequenced the entire mtDNA in 50 oncocytic lesions (45 thyroid tumors of epithelial cell derivation and 5 mitochondrion-rich breast tumors) and 52 control cases (21 nononcocytic thyroid tumors, 15 breast carcinomas, and 16 gliomas) by using recently developed technology that allows specific and reliable amplification of the whole mtDNA with quick mutation scanning. Thirteen oncocytic lesions (26%) presented disruptive mutations (nonsense or frameshift), whereas only two samples (3.8%) presented such mutations in the nononcocytic control group. In one case with multiple thyroid nodules analyzed separately, a disruptive mutation was found in the only nodule with oncocytic features. In one of the five mitochondrion-rich breast tumors, a disruptive mutation was identified. All disruptive mutations were found in complex I subunit genes, and the association between these mutations and the oncocytic phenotype was statistically significant (P = 0.001). To study the pathogenicity of these mitochondrial mutations, primary cultures from oncocytic tumors and corresponding normal tissues were established. Electron microscopy and biochemical and molecular analyses showed that primary cultures derived from tumors bearing disruptive mutations failed to maintain the mutations and the oncocytic phenotype. We conclude that disruptive mutations in complex I subunits are markers of thyroid oncocytic tumors.

Toker cells are probably precursors of Paget cell carcinoma: a morphological and ultrastructural description

The present paper documents an investigation of the morphology, immunohistochemistry, and ultrastructure of Toker cells (TC), aiming for a better definition of these elements and better understanding of their histogenesis. We studied 12 nipples removed for nipple adenoma from twelve patients and a case of supernumerary nipple. In addition four cases of Pagets carcinoma (PC) restricted to the nipple without underlying tumor were studied for comparison. All cases were stained with hematoxylin and eosin (H&E), Alcian blue pH 2.5 and periodic acid-Schiff (PAS) preceded by diastase digestion and with immunohistochemistry using antisera anti cytokeratin 7, cytokeratin 20, protein S100, GCDFP-15, c-Erb-B2, CAM 5.2, and epithelial membrane antigen (EMA). Two cases from the nipple adenoma series were studied by electron microscopy. In seven cases within the series of 12 nipple adenomas as well as in the case of supernumerary nipple, keratin 7 antibody highlighted numerous cells located within the nipple epidermis which in three cases showed dendritic processes. These same elements were also positive with CAM 5.2. All these same elements were negative with Alcian Blue (AB), PAS and the other antisera employed. Ultrastructural examination demonstrated that these cells differed from keratinocytes while they presented the same features as the glandular cells seen in the related nipple adenoma. The cells constituting Pagets carcinoma showed more irregular nuclei and were more easily seen in the context of the epidermis. The immunocytochemical profile of the cancer cells was similar to that of TC, but in addition the neoplastic cells were c-Erb-B2 and EMA positive in all cases, and one case also displayed numerous cells immunoreactive with anti GCDFP-15 antibody. Keratin 7 highlighted dendritic cells in two cases and AB, PAS was negative in all patients. The immunocytochemical profile and the ultrastructural features of TC are similar to those of the glandular cells constituting the ducts and the adenoma. These findings together with the localization of TC near or around the openings of the lactiferous sinuses indicate that TC might be ductal cells with a dendritic aspect and migrate through the galactophorous ostia. PC cells not related to ductal carcinomas have a similar but not superimposable immunohistochemical profile to TC, and in two cases the neoplastic elements were also dendritic which suggests that these same cells are likely to be the neoplastic counterpart of TC.

Microglandular adenosis, apocrine adenosis, and tubular carcinoma of the breast. An immunohistochemical comparison.

Four cases of microglandular adenosis (MA), together with four cases of apocrine adenosis (AA) and 10 cases of tubular carcinoma (TC) of the breast were studied at the light and immunohistochemical level. One case of MA was studied with electron microscopy. MA is characterized by an absence of myoepithelial cells (ME), epithelial membrane antigen (EMA), and gross cystic disease fluid protein (GCDFP-15). The absence of EM A in MA makes it unique among benign glandular hyperplasias of the breast. AA contains myoepithelial cells and a distinct basal lamina. It is characterized by the presence of GCDFP-15, the specific apocrine marker, which is not present in MA. TC lacks both myoepithelial cells and a basal lamina. It is negative for GCDFP-15. Periductal and vascular elastosis are common and usually prominent, whereas they are not found in either MA and AA. Other stromal changes further distinguish the three lesions. These three distinct entities can be separated objectively and unequivocally and it is essential that this be done so as to prevent confusion.

A Mutation Threshold Distinguishes the Antitumorigenic Effects of the Mitochondrial Gene MTND1, an Oncojanus Function

The oncogenic versus suppressor roles of mitochondrial genes have long been debated. Peculiar features of mitochondrial genetics such as hetero/homoplasmy and mutation threshold are seldom taken into account in this debate. Mitochondrial DNA (mtDNA) mutations generally have been claimed to be protumorigenic, but they are also hallmarks of mostly benign oncocytic tumors wherein they help reduce adaptation to hypoxia by destabilizing hypoxia-inducible factor-1α (HIF1α). To determine the influence of a disassembling mtDNA mutation and its hetero/homoplasmy on tumorigenic and metastatic potential, we injected mice with tumor cells harboring different loads of the gene MTND1 m.3571insC. Cell cultures obtained from tumor xenografts were then analyzed to correlate energetic competence, apoptosis, α-ketoglutarate (α-KG)/succinate (SA) ratio, and HIF1α stabilization with the mutation load. A threshold level for the antitumorigenic effect of MTND1 m.3571insC mutation was defined, above which tumor growth and invasiveness were reduced significantly. Notably, HIF1α destabilization and downregulation of HIF1α-dependent genes occurred in cells and tumors lacking complex I (CI), where there was an associated imbalance of α-KG/SA despite the presence of an actual hypoxic environment. These results strongly implicate mtDNA mutations as a cause of oncocytic transformation. Thus, the antitumorigenic and antimetastatic effects of high loads of MTND1 m.3571insC, following CI disassembly, define a novel threshold-regulated class of cancer genes. We suggest these genes be termed oncojanus genes to recognize their ability to contribute either oncogenic or suppressive functions in mitochondrial settings during tumorigenesis.

Primary Oat-Cell Carcinoma of the Larynx

A primary oat-cell carcinoma of the larynx in a 63-year-old man is reported. The oat-cell pattern appeared intermingled at one edge with a squamous carcinoma. The origin of this mixed tumour is considered together with the literature of extrapulmonary carcinomas having an oat-cell pattern.

Respiratory complex I is essential to induce a Warburg profile in mitochondria-defective tumor cells.

BackgroundAerobic glycolysis, namely the Warburg effect, is the main hallmark of cancer cells. Mitochondrial respiratory dysfunction has been proposed to be one of the major causes for such glycolytic shift. This hypothesis has been revisited as tumors appear to undergo waves of gene regulation during progression, some of which rely on functional mitochondria. In this framework, the role of mitochondrial complex I is still debated, in particular with respect to the effect of mitochondrial DNA mutations in cancer metabolism. The aim of this work is to provide the proof of concept that functional complex I is necessary to sustain tumor progression.MethodsComplex I-null osteosarcoma cells were complemented with allotopically expressed complex I subunit 1 (MT-ND1). Complex I re-assembly and function recovery, also in terms of NADH consumption, were assessed. Clones were tested for their ability to grow in soft agar and to generate tumor masses in nude mice. Hypoxia levels were evaluated via pimonidazole staining and hypoxia-inducible factor-1α (HIF-1α) immunoblotting and histochemical staining. 454-pyrosequencing was implemented to obtain global transcriptomic profiling of allotopic and non-allotopic xenografts.ResultsComplementation of a truncative mutation in the gene encoding MT-ND1, showed that a functional enzyme was required to perform the glycolytic shift during the hypoxia response and to induce a Warburg profile in vitro and in vivo, fostering cancer progression. Such trigger was mediated by HIF-1α, whose stabilization was regulated after recovery of the balance between α-ketoglutarate and succinate due to a recuperation of NADH consumption that followed complex I rescue.ConclusionRespiratory complex I is essential for the induction of Warburg effect and adaptation to hypoxia of cancer cells, allowing them to sustain tumor growth. Differently from other mitochondrial tumor suppressor genes, therefore, a complex I severe mutation such as the one here reported may confer anti-tumorigenic properties, highlighting the prognostic values of such genetic markers in cancer.

Ultrastructural organization of nucleoli in benign naevi and malignant melanomas

SummaryWe have studied the ultrastructural organization of nucleoli in benign naevi and malignant melanomas. In benign naevus cells the nucleoli displayed a compact ribonucleoprotein distribution, with one or two large fibrillar centres. In malignant melanoma cells the nucleoli were large with an irregular, nucleolonema-like ribonucleoprotein distribution and they exhibited numerous, small fibrillar centres. Statistical evaluation of the size of fibrillar centres indicated a mean value of 0.482 μm2±0.136 SD for naevi and 0.221 μm2±0.128 SD for malignant melanomas. These features, together with the more dispersed chromatin pattern of malignant melanoma nuclei compared with those of benign naevus cells, are proposed as diagnostic parameters which differentiate benign naevi from malignant melanomas at the ultrastructural level.

Tubular carcinoma: a variant of secretory breast carcinoma.

Fifteen cases of tubular carcinoma of the breast have been studied using histochemical methods for mucosubstances, immunocytochemical methods for casein and actin and conventional electron microscopy. Mucosubstances and casein were demonstrated lying freely in the lumina of the tubules. Occasionally, mucosubstances and casein assumed the form of target‐like intracytoplasmic ‘inclusions’ like those characteristically seen in lobular carcinoma. The neoplastic cells did not react with antisera specific against actin. Even at ultrastructural level no myoepithelial cells were observed, whilst villi were revealed along the tubular luminal surface. It appears that, in addition to distinctive biological, histological and ultrastructural features, tubular carcinoma has an almost constant histochemical pattern. This suggests a differentiation towards epithelial secretory cells engaged in intensive milk protein production which has also been shown to be a feature of lobular carcinoma. It is concluded that though lobular carcinoma and tubular carcinoma of the breast have been traditionally regarded as two distinct entities, they have certain similar functional characteristics and it is postulated that these two tumours could represent the extreme variants of the same entity: the infiltrative lobular carcinoma being the most undifferentiated and tubular carcinoma the most highly differentiated.

Diagnostic Value of Silver-Stained Interphasic Nucleolar Organizer Regions in Breast Tumors

Seventy-six specimens of normal breast tissue and benign and malignant breast lesions were studied to assess the mean area occupied by silver-stained proteins of the nucleolar organizer regions (MNORA) of the nucleolus. The assessment was performed with a computer-assisted image analyzer. The results indicate that only 30% of malignant lesions have a MNORA value greater than that of normal breast tissue or benign lesions. On the other hand, MNORA values of ductal carcinoma in situ were significantly greater than those of epitheliosis (papillomatosis). MNORA values were also significantly different in grade I and grade III invasive ductal carcinomas, the latter exhibiting the highest MNORA values of all the cases observed. Evaluation of MNORA values may therefore help in differentiating benign epithelial proliferations from ductal carcinomas in situ. Furthermore, because there is evidence that MNORA values are indicative of the cell duplication rate, MNORA values may ultimately be considered an objective prognostic parameter in addition to grading for invasive ductal carcinomas.

Neurofibroma with rhabdomyomatous differentiation: benign 'Triton' tumour of the vagina.

The benign ‘Triton’ tumour is a neural tumour with benign rhabdomyomatous differentiation: it must be distinguished from the better known malignant counterpart. We describe the first case outside the peripheral soft tissues and the first to be studied ultrastructurally. It also represents the second lesion identified in an adult patient.