Chromatin texture analysis of cortical adrenal gland adenomas, including incidentalomas, and adjacent normal-appearing cortical tissue

OBJECTIVE: To describe, by morphometric and chromatin texture analysis, a series of adrenal gland lesions, including Cushing's and Conn's adenomas and incidentalomas. STUDY DESIGN: The material for the study consisted of five consecutive cases of incidentaloma, three cases of Conn's adenoma and three cases of Cushing's adenoma. Also included were five cases of adrenal carcinoma. Sections were stained according to the Feulgen procedure. Measurements were taken from the nodules and from two different zones, identified as outer and inner parts, of the normal-appearing adrenal cortex adjacent to the tumor. Data on approximately 50 nuclei were recorded for each of these three sites (tumor and outer and inner normal-appearing adrenal cortex). The nuclei were subjected to feature extraction and were analyzed by identification procedures--i.e., establishing nuclear and lesion signatures. RESULTS: The total optical density (OD) distributions of the nuclei from the normal-appearing adrenal cortex pointed to their diploid or near-diploid nature. In incidentalomas there was a very small increase in the number of nuclei, with increased total OD. In Conn's adenoma there was a noticeable but modest extension of the total OD distribution into the higher OD range. This trend continued for Cushing's adenoma. The pixel OD histograms for nuclei from normal-appearing tissue and from incidentalomas were hardly distinguishable. Starting with nuclei from Conn's adenoma, a shift toward lower pixel OD values began. The trend continued for nuclei from Cushing's adenoma and was very pronounced for nuclei from carcinoma. The nuclear signatures showed no appreciable difference between nuclei from normal-appearing cortex and from incidentaloma. Nuclei from Conn's adenoma were more similar to those from normal tissue in their signatures than nuclei from Cushing's adenoma. In fact, the nuclear signatures from Cushing's adenoma were almost identical to those of carcinoma. The lesion signatures for normal tissue, incidentaloma and Conn's adenoma confirmed the results seen in the nuclear signatures. There was a very modest increase in the number of nuclei with greater deviation from normal in incidentalomas, and the trend was more obvious in Conn's adenoma. However, in Cushing's adenoma there was a very substantial increase in the number of nuclei, with large deviations of their nuclear chromatin texture from normal. CONCLUSION: Computer-assisted analysis of nuclear characteristics proved useful in identifying and describing differences between groups of tumors arising in the adrenal cortex and highlighted the similarity between incidentalomas and adjacent normal-appearing cortex and between Cushing's adenoma and adrenal carcinoma.     

The effect of sampling on quantitative nuclear image features in histologic sections of lung carcinomas

A feasibility study showed that quantitative nuclear image (QNI) analysis, in which the morphology of the nucleus is described by a number of mathematical parameters, can be used to make the therapeutically and prognostically important distinction between small cell lung carcinoma (SCLC) and non-SCLC, which can be difficult to make with subjective histologic typing. In the present study, the effects of sample size and sample site on the QNI features were investigated. For all sample sites in a given tumor, comparison was made between the histologic classification, the ultrastructural findings and the classification based on the QNI features. Using a running mean, it was found that a sample size of 25 nuclei is sufficiently large. Histologic and quantitative classifications of samples from different sites of the same tumors were in agreement with regard to the separation of SCLC and non-SCLC in 19 of 20 sections. In the case in which disagreement occurred in one section, the ultrastructural findings supported the quantitative classification. These data indicate that sampling from different sites has no essential influence on the QNI classification of lung carcinomas.     

The first insight into the trace element status of human adrenal gland accompanied by elemental alterations in adrenal adenomas

BackgroundThe baseline status of trace metals in adrenal tissue is unresolved, while the elemental profile for any adrenal pathology has not been examined so far. This study aimed to determine the baseline status of important toxic (Ni, As, Cd, Pb, Th, U) and essential trace elements (Mn, Co, Cu, Zn, Se) in healthy adrenal tissues (HATs) as well as to examine whether there are alterations in the elemental composition of adenomatous adrenal tissues (AATs). Furthermore, this study aimed to find potential trace metals that could play a role in the pathogenesis of adrenal adenoma (AA).MethodsThe study included 45 patients diagnosed with AA. Impacts of relevant parameters such as gender, age, smoking habits and nodular sizes were considered. All samples were subjected to microwave digestion and the trace elements were determined by inductively coupled plasma mass spectrometry (ICP-MS).ResultsThis is the first study that provided an insight into the elemental status of HATs. It was also shown that AATs had altered trace metal contents. Compared to HATs, the most significant findings were related to the high content of essential (Cu, Mn, Se, Zn) and Pb as a non-essential metal. Although gender, age and smoking habits had a modest effect on metal profiles, the most significant alterations were related to the nodular diameter above 4 cm, indicating that the growth of benign tumor could influence changes in elemental composition.ConclusionFor the first time the baseline contents of essential and toxic trace metals in HATs were determined. The results of this study may highlight the role of toxic and essential trace metals in AAs and could provide new insights into the molecular basis of pathophysiological changes caused by the hazardous effects of trace metals on adrenal structure and function.     

Pulmonary adenocarcinoma metastatic to the adrenal gland mimicking normal adrenal cortical epithelium on fine needle aspiration

Fine needle aspiration (FNA) of enlarged adrenal glands detected by computed tomography is a valuable method for extrathoracic staging of pulmonary carcinomas. This paper presents the case of a middle-aged man with pulmonary adenocarcinoma metastatic to the adrenal glands, the FNA sample of which closely resembled normal adrenal cortical epithelium. Through review of the case and comparison with cytologic preparations from normal adrenal glands, the aspiration cytologic features suggesting carcinoma are described.     

Cellular localization of orexin receptors in human adrenal gland, adrenocortical adenomas and pheochromocytomas

Orexin-A and -B are hypothalamic peptides derived from a precursor called prepro-orexin and related with the regulation of the energy balance and arousal. They act on G protein receptors named orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). In the present study, we used immunohistochemical techniques to detect the distribution of OXR in normal human adrenal gland and adrenal tumours (adrenocortical adenomas and pheochromocytomas). OX1R was expressed in the cortex of the normal human adrenal gland (glomerulosa, fasciculata and reticular zones) and OX2R was located in the medulla (epinephrine and norepinephrine cells). By the double immunofluorescence techniques, we demonstrated that virtually all medullar cells (epinephrine and norepinephrine cells) expressed OX2R. As was expected, according to the results obtained in normal tissues, cortical tumours (adrenocortical adenomas) were positive for OX1R but not for OX2R and conversely, medullar tumours (pheochromocytomas) expressed only OX2R.     

Characterization of microtubule-associated proteins isolated from bovine adrenal gland

Following induction of hemopoiesis, poly(A)-rich RNA was prepared from the heart of the tarantula, Eurypelma californicum, and translated in rabbit reticulocyte lysates. In vitro translation products were immunoprecipitated with antiserum against whole dissociated Eurypelma hemocyanin. Analysis of the immunoprecipitate by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed a set of polypeptides comigrating with authentic Eurypelma hemocyanin. The mRNA was transcribed into cDNA, clones were constructed using the pUC9 vector and probed with a synthetic 17-mer oligonucleotide probe complementary to the amino acid sequence of the 'copper A' binding site of chelicerate hemocyanins. One clone, pHC4, contained a 1.62-kb cDNA insert, which was subcloned into phage M13. Sequence analysis by the dideoxynucleotide chain-termination method yielded a nucleotide sequence coding for 526 amino acids of Eurypelma hemocyanin subunit e.     

A dopamine agonist stimulates progesterone secretion from adrenal gland

Administration of either progesterone (P) or a dopamine agonist, Legrotrile mesylate (LM), have been shown to induce the ovulatory release of LH in rats. In order to elucidate the mode of action of dopamine agonists we studied the effects of LM on P secretion by the adrenals. A subcutaneous injection of LM, in doses which induce ovulation, stimulated adrenal P secretion in ovariectomized, estrogen-primed rats and in castrate male rats. Peak plasma P concentrations were found at 2 h with a gradual return to pre-injection levels at 6 hrs following LM injection. These results raise the possibility that P increments following LM administration may be responsible for inducing ovulation in young and old rats.There is considerable evidence to show that modification of hypothalamic monoamine metabolism by pharmacologic agents results in markedly altered pituitary gonadotropin secretion [1]. Lergotrile mesylate (LM), a dopamine agonist, has been shown to advance the ovulatory release of LH on proestrus and induce ovulation and cyclicity in anovulatory aged rats [2,3]. Everett [4] observed that progesterone (P) administration induced ovulation in rats rendered anovulatory under constant light and also advanced ovulation by one day if administered to 5-day cycling rats. A similar effect of LM on ovulation advancement has been noted (Clemens, personal communication). These similarities in action of P and LM on ovulation in young and aged rats, led us to speculate that LM may stimulate P secretion which in turn may elicit the discharge of ovulatory hormones. To test this hypothesis we examined the effects of LM on progesterone secretion in gonadectomized rats.     

RET rearrangements in papillary thyroid carcinomas and adenomas detected by interphase FISH

Activation of the RET protooncogene through somatic rearrangements represents the most common genetic alteration in papillary thyroid carcinoma (PTC). Three main rearranged forms of RET have been described: RET/PTC1 and RET/PTC3, which arise from a paracentric inversion of the long arm of chromosome 10, and RET/PTC2, which originates from a 10;17 translocation. We have developed a dual-color FISH approach to detect RET/PTC rearrangements in interphase nuclei of thyroid lesions. By using a pool of three cosmids encompassing the RET chromosome region and a chromosome 10 centromeric probe, we could discriminate between the presence of an inversion (RET/PTC1 and RET/PTC3) or a translocation (RET/PTC2). We have investigated a series of thyroid tissue samples from Italian and French patients corresponding to a total of 69 PTCs and 22 benign lesions. Among PTCs, 13 (18.8%) showed a RET rearrangement, and 11 (15.9%) of these carried an inversion (RET/PTC1 or RET/PTC3) in more than 10% of the nuclei examined. Activated forms of RET were also observed in three adenomas. RT-PCR analysis on the same samples confirmed the presence and the type of rearrangement predicted using FISH analysis. An interesting difference in the frequency and type of RET rearrangements was detected between the Italian and the French patients. Furthermore, we identified a putative novel type of rearrangement in at least one PTC sample. Several PTCs carried a significant number of cells characterized by a trisomy or a tetrasomy of chromosome 10. Overall, the FISH approach in interphase nuclei represents a powerful tool for detecting, at the single cell level, RET/PTC rearrangements and other anomalies involving the RET chromosome region.     

Distinguishing gorilla mitochondrial sequences from nuclear integrations and PCR recombinants: guidelines for their diagnosis in complex sequence databases

Nuclear integrations of mitochondrial DNA (Numts) are widespread in many taxa and if left undetected can confound phylogeny interpretation and bias estimates of mitochondrial DNA (mtDNA) diversity. This is particularly true in gorillas, where recent studies suggest multiple integrations of the first hypervariable (HV1) domain of the mitochondrial control region. Problems can also arise through the inadvertent incorporation of artifacts produced by in vitro recombination between sequence types during polymerase chain reaction amplification. This issue has attracted little attention yet could potentially exacerbate errors in databases already contaminated by Numts. Using a set of existing diagnostic tools, this study set out to systematically inventory Numts and PCR recombinants in a gorilla HV1 sequence database and address the degree to which existing public databases are contaminated. Phylogenetic analysis revealed three distinct gorilla HV1 Numt groups (I, II, and III) that could be readily differentiated from mtDNA sequences by Numt-specific diagnostic sites and sequence-based motifs. Several instances of genuine recombination were also identified by a suite of detection methods. The location of putative breakpoints was identified by eye and by likelihood analysis. Findings from this study reveal widespread nuclear contamination of gorilla HV1 GenBank databases and underline the importance of recognizing not only Numts but also PCR recombinant artifacts as potential sources of data contamination. Guidelines for the routine identification of Numts and in vitro recombinants are presented and should prove useful in the detection of similar artifacts in other species mtDNA databases.     

Polypeptide hormone receptors in vivo: demonstration of insulin binding to adrenal gland and gastrointestinal epithelium by quantitative radioautography

A tissue-screening survey employing quantitative radioautography was carried out at 2 min after the intravascular injection of 125I-insulin into laboratory rats. The results revealed a substantial binding of insulin to cells forming the proximal convoluted tubule in kidney, hepatocytes of liver, acinar cells of the pancreas, parenchymal cells of the adrenal cortex and medulla, and epithelial cells of the gastrointestinal tract. Control experiments indicated that this binding was due to a specific interaction with the insulin receptor, except in the case of kidney where the binding was shown to be nonspecific. Although the major target for insulin action (liver) clearly demonstrated specific insulin binding, several other classical targets (adipocytes, skeletal, cardiac, and smooth muscle cells) showed no specific 125I-insulin binding and therefore indicated the limits of sensitivity of the in vivo radioautographic method. Nevertheless, the working hypothesis of a direct correlation of insulin receptor density with insulin action points to the hitherto unemphasized targets of pancreas, adrenal gland, and gastrointestinal tract as major sites of insulin action in the body.     

Morphological evidence for a close interaction of chromaffin cells with cortical cells within the adrenal gland

Summary The adrenal medulla appears to exert a regulatory influence on adrenocortical steroidogenesis. We have therefore studied the morphology of rat, porcine and bovine adrenals in order to characterize the contact zones of adrenomedullary and adrenocortical tissues. The distribution of chromaffin cells located within the adrenal cortex and of cortical cells located within the adrenal medulla was investigated. Chromaffin cells were characterized by immunostaining for synaptophysin and chromogranin A, both being considered specific for neuroendocrine cells. Cortical cells were characterized by immunostaining for 17-hydroxylase, an enzyme of the steroid pathway. Cellular contacts of chromaffin cells and cortical cells were examined at the electron microscopical level. In rat and porcine adrenals, rays of chromaffin cells, small cell clusters and single chromaffin cells or small invaginations from the medulla could be detected in all three zones of the cortex. Chromaffin cells often spread in the subcapsular space of the zona glomerulosa. In porcine and bovine adrenals, 17-hydroxylase immunoreactive cells were localized within the medulla. Single cortical cells and small accumulations of cells were spread throughout this region. At the ultrastructural level, the chromaffin cells located within the cortex in pig and rat adrenals formed close cellular contacts with cortical cells in all three zones. Our morphological data provide evidence for a possible paracrine role of chromaffin cells; this may be important for the neuroregulation of the adrenal cortex.