Radiographic Characteristics of Adrenal Masses Preceding the Diagnosis of Adrenocortical Cancer.

Incidentally discovered adrenal masses are common and the clinical evaluation and surveillance aims to diagnose hormone excess and malignancy. Adrenocortical cancer (ACC) is a very rare malignancy. This study aims to define the imaging characteristics of adrenal tumors preceding the diagnosis of ACC. Patients with prior (>5 months) adrenal tumors (<6 cm) subsequently diagnosed with ACC were identified in a large registry at a tertiary referral center. Retrospective chart and image review for patient characteristics and initial, interval, and diagnostic imaging characteristics (size, homogeneity, borders, density, growth rate, etc.) was conducted. Twenty patients with a diagnosis of ACC and a prior adrenal tumor were identified among 422 patients with ACC. Of these, 17 patients were initially imaged with CT and 3 with MR. Only 2 of the 20 patients had initial imaging characteristics suggestive of a benign lesion. Of initial tumors, 25% were <2 cm in size. Surveillance led to the diagnosis of ACC within 24 months in 50% of patients. The growth pattern was variable with some lesions showing long-term stability (up to 8 years) in size. In conclusion, antecedent lesions in patients with a diagnosis of ACC are often indeterminate by imaging criteria and can be small. Surveillance over 2 years detected only 50% of ACCs. Current practice and guidelines are insufficient in diagnosing ACCs. Given the rarity of ACC, the increased risk and health care costs of additional evaluation may not be warranted.

Laparoscopic resection is inappropriate in patients with known or suspected adrenocortical carcinoma.

BACKGROUND: Complete surgical resection is the mainstay of treatment for patients with adrenocortical cancer (ACC). Use of laparoscopy has been questioned in patients with ACC. This study compares the outcomes of patients undergoing laparoscopic versus open resection (OR) for ACC. METHODS: A retrospective review (2003-2008) of patients with ACC was performed. Data were collected for demographics, operative and pathologic data, adjuvant therapy, and outcome. Chi-square analysis was performed. RESULTS: Eighty-eight patients (66% women; median age, 47 (range, 18-81) years) were identified. Seventeen patients underwent laparoscopic adrenalectomy (LA). Median tumor size of those who underwent LA was 7.0 (range, 4-14) cm versus 12.3 (range, 5-27) cm for OR. Recurrent disease in the laparoscopic group occurred in 63% versus 65% in the open group. Mean time to first recurrence for those who underwent LA was 9.6 months (+/-14) versus 19.2 months (+/-37.5) in the open group (p < 0.005). Fifty percent of patients who underwent LA had positive margins or notation of intraoperative tumor spill versus 18% of those who underwent OR (p = 0.01). Local recurrence occurred in 25% of the laparoscopic group versus 20% in the open group (p = 0.23). Mean follow-up was 36.5 months (+/-43.6). CONCLUSIONS: ACC continues to be a deadly disease, and little to no progress has been made from a treatment standpoint in the past 20 years. Careful and complete surgical resection is of the utmost importance. Although feasible in many cases and tempting, laparoscopic resection should not be attempted in patients with tumors suspicious for or known to be adrenocortical carcinoma.

Management of patients with adrenal cancer: recommendations of an international consensus conference.

Adrenocortical carcinomas are rare, highly malignant tumors that account for only 0.2% of deaths due to cancer. Given the limited number of patients seen in most medical centers with this diagnosis, series usually reported are small and clinical trials not randomized or blinded. In an attempt to answer important questions concerning the management of patients with adrenal cancer, a consensus conference was organized and held at the University of Michigan in Ann Arbor, MI, 11-13 September 2003, with the participation of an international group of physicians who had reported on the largest series of patients with this disease and who had recognized basic and clinical research expertise in adrenal cortical cancer. Totally 43 questions were addressed by the presenters and recommendations discussed in plenary and breakout sessions. Evidence for the recommendations of this conference was at the 2-4+ level and based on available literature and participants' experience. In addition to setting up guidelines in specific areas of the diagnosis and treatment of adrenal cancer, the conference recommended and initiated the planning of an international prospective trial for treatment of patients with adrenal cancer in stages III and IV. In terms of new therapies, first trials of dendritic cell therapy in human subjects with adrenal cancer have been started, but it is too early to comment on efficacy. Different strategies of immunotherapy, including DNA vaccination are currently being tried in animal models. There are no clinical gene therapy trials for human adrenal cortical cancer. The adrenals are a preferred target for adenovirus and the results of gene therapy in preclinical studies are promising. In addition, there is evidence that histone deacetylase inhibitors can further enhance the rate of adenoviral infectivity in human adrenal cancer cells. Testing of retroviral vectors, non-viral vectors, small interfering RNA technology, and combined approaches could be performed in various laboratories. Anti-angiogenic substances have only been applied in preclinical studies. The use of these and other agents in the treatment of adrenal cancer should be hypothesis-driven and based on a thorough analysis of tumor biology.

Role of the inhibin/activin system and luteinizing hormone in adrenocortical tumorigenesis.

Adrenal masses are one of the most common endocrine tumors diagnosed. Although most adrenal tumors are inactive adenomas, a considerable proportion is associated with hormonal hyperfunction and/or malignancy. The adrenocortical carcinoma (ACC) is a rare but highly malignant tumor. Most ACCs in adults are diagnosed in an advanced tumor stage limiting therapeutic options. Accordingly, despite some progress in diagnostic and therapeutic approaches, the overall survival rate of patients with ACC remains poor. However, the prerequisite for the development of new diagnostic tools and therapeutic options in the management of patients with ACC is the elucidation of the molecular pathogenesis of adrenal tumorigenesis. Although our understanding of adrenal tumor biology has increased substantially over the last decades, the regulation of many molecular pathways involved in adrenocortical growth and differentiation awaits further elucidation. Luteinizing hormone (LH) and activin have only recently emerged as hormones likely to play opposite roles in adrenocortical hormone secretion and cellular proliferation. Recent evidence from studies on human surgical tumor sample expression and detailed characterization of murine adrenal tumor models suggests stimulatory effects of LH on adrenocortical growth and function. On the contrary, activin, which plays a critical role as a paracrine and autocrine factor regulating cellular growth and differentiation, has been demonstrated to induce apoptosis and suppress proliferation in the human and murine adrenal cortex. In this review, we will summarize molecular and functional aspects of adrenal tumorigenesis and highlight some prospects for future clinical applications.

SF-1, DAX-1, and acd: molecular determinants of adrenocortical growth and steroidogenesis.

The formation of the adrenal cortex in humans is notable for the presence of two discrete zones, the fetal zone (FZ) which regresses soon after birth and the definitive zone (DZ) which gives rise to the classic steroidogenic zones of the adult cortex. Mice possess an analogous structure to the FZ referred to as the X-zone (XZ) which regresses at puberty in the male and during the first pregnancy in the female. Similar to the human FZ in X-linked Congenital Adrenal Hypoplasia caused by loss of function mutations in DAX-1 (Dosage-sensitive sex reversal-Adrenal hypoplasia congenita critical region on the X chromosome), the mouse XZ does not regress when DAX-1 is mutated. Only in humans with DAX-1 mutations, however, is the DZ small and hypofunctional. Patients and mice with SF-1 mutations have complete adrenal aplasia with absence of both the DZ and FZ/XZ. Lastly, the phenotype of the Autosomal Recessive Adrenocortical Dysplasia (acd) mouse is strikingly similar to human Miniature Adult Congenital Adrenal Hypoplasia, lacking an XZ/FZ and possessing a dysfunctional DZ. Current work has addressed the regulation of SF-1 and DAX-1 dependent adrenocortical growth and steroidogenesis in vivo utilizing mouse models of simple and combined SF-1 and DAX-1 deficiency. In addition, the model of compensatory adrenal growth in SF-1 haplo-insufficient mice has been applied to evaluate the potential role of SF-1 in adrenocortical proliferation. Additional efforts aim to positionally clone the acd gene, predicated on the hypothesis that it is a critical component of the adrenal developmental cascade.

Role of phosphorylation, gene dosage and Dax-1 in SF-1 mediated steroidogenesis.

The mechanisms by which SF-1 (Steroidogenic Factor-1) and Dax-1 (Dosage-sensitive sex reversal-Adrenal hypoplasia congenita critical region on the X chromosome) dictate adrenal-specific transcriptional programs are the focus of this laboratory. SF-1-mediated transcription is upregulated by phosphorylation of serine 203 located in the hinge region of SF-1. An SF-1S203A mutant attenuates SF-1 activation, while substitution of S203 with a charged aspartate (SF-1S203D) results in a dose dependent increase in SF-1 mediated transcription. Ser203 serves as a substrate for Erk2 in vitro and is critical for activation of SF-1 by multiple components of the MAPK pathway. Isoelectric focusing demonstrates multiple immuno-reactive SF-1 species in mouse adrenal and NCI-H295A cell extracts. We propose that differential phosphorylation of SF-1 by various mitogens serves to couple extracellular signals to adrenal-specific transcriptional programs. Mouse studies utilizing SF-1 heterozygous mice explore the in vivo role of SF-1 levels, SF-1 phosphorylation and SF-1 interaction with Dax-1 in adrenal steroidogenesis. SF-1 heterozygous mice exhibit a marked decrease in baseline and post-stress corticosterone with a concomitant increase in ACTH. The role of Dax-1 in these SF-1 dependent processes is explored in compound SF-1 (+/-)/Dax-1 KO mice that exhibit an increase in basal corticosterone and a decrease in basal ACTH compared to simple SF-1 (+/-) mice. These finding are consistent with an inhibitory role for Dax-1 in SF-1 mediated transcription. Mice that express epitope tagged SF-1 (wild type, SF-1S203A and SF-1S203D) are being used to rescue the heterozygous adrenal phenotype and to determine the in vivo role of SF-1 phosphorylation in adrenal function.

Steroidogenic factor-1: its role in endocrine organ development and differentiation.

The cloning of the first steroid hormone receptor over a decade ago provided vital insight into the mechanisms by which steroid hormones activate gene transcription. When bound by hormone, these receptors function as ligand-dependent transcription factors by binding to unique response elements in the promoter of specific target genes. Over 60 receptors have now been characterized in this superfamily of steroid receptors. Many receptors known as orphan receptors have been cloned by homology and have no known ligands but appear to be mediators of endocrine function in the adult and in many cases are essential developmental regulators in endocrine organogenesis. One such receptor is steroidogenic factor-1 (SF-1). While initially cloned as a transcriptional regulator of the various steroidogenic enzyme genes in the adrenal and gonad, it has become clear through genetic ablation experiments in mice that SF-1 is an essential factor in adrenal and gonadal development and for the proper functioning of the hypothalamic-pituitary-gonadal axis. In addition, these studies have revealed that SF-1 is necessary for the formation of the ventromedial nucleus of the hypothalamus. While we have learned much since the initial cloning of SF-1, the mechanisms by which SF-1 regulates these various developmental programs remain elusive. This article focuses on the characterization of SF-1 and its emerging role in endocrine homeostasis. Specific attention is placed on the mechanisms of action of this unique member of the nuclear receptor superfamily.

Phosphorylation of the nuclear receptor SF-1 modulates cofactor recruitment: integration of hormone signaling in reproduction and stress.

Steroidogenic factor 1 (SF-1) is an orphan nuclear receptor that serves as an essential regulator of many hormone-induced genes in the vertebrate endocrine system. The apparent absence of a SF-1 ligand prompted speculation that this receptor is regulated by alternative mechanisms involving signal transduction pathways. Here we show that maximal SF-1-mediated transcription and interaction with general nuclear receptor cofactors depends on phosphorylation of a single serine residue (Ser-203) located in a major activation domain (AF-1) of the protein. Moreover, phosphorylation-dependent SF-1 activation is likely mediated by the mitogen-activated protein kinase (MAPK) signaling pathway. We propose that this single modification of SF-1 and the subsequent recruitment of nuclear receptor cofactors couple extracellular signals to steroid and peptide hormone synthesis, thereby maintaining dynamic homeostatic responses in stress and reproduction.

Wilms' tumor 1 and Dax-1 modulate the orphan nuclear receptor SF-1 in sex-specific gene expression.

Products of steroidogenic factor 1 (SF-1) and Wilms' tumor 1 (WT1) genes are essential for mammalian gonadogenesis prior to sexual differentiation. In males, SF-1 participates in sexual development by regulating expression of the polypeptide hormone Müllerian inhibiting substance (MIS). Here, we show that WT1 -KTS isoforms associate and synergize with SF-1 to promote MIS expression. In contrast, WT1 missense mutations, associated with male pseudohermaphroditism in Denys-Drash syndrome, fail to synergize with SF-1. Additionally, the X-linked, candidate dosage-sensitive sex-reversal gene, Dax-1, antagonizes synergy between SF-1 and WT1, most likely through a direct interaction with SF-1. We propose that WT1 and Dax-1 functionally oppose each other in testis development by modulating SF-1-mediated transactivation.

Post-translational processing of proopiomelanocortin (POMC) in mouse pituitary melanotroph tumors induced by a POMC-simian virus 40 large T antigen transgene.

Mice harboring a transgene composed of proopiomelanocortin (POMC) gene promoter sequences (nucleotides -706 to +64) ligated to the simian virus (SV) 40 early gene encoding large T antigen developed large POMC-expressing pituitary tumors. Histologically the tumors arose from the intermediate lobe, contained nuclear SV40 T antigen and POMC peptides, but did not express other pituitary hormones. POMC processing in the pituitary tumors was indistinguishable from normal mouse intermediate lobe melanotrophs and was characterized by high proportions of acetylated and carboxyl-terminal shortened beta-endorphins, and amino-terminal acetylated alpha-melanocyte-stimulating hormone, and virtually no adrenocorticotropic hormone (ACTH)(1-39), beta-lipotropin, or POMC. The tumors contained abundant levels of mRNA for the prohormone convertase PC2 and undetectable levels of PC1. Normal mouse neurointermediate lobe also has a high ratio of PC2/PC1 expression that is distinct from the relative abundance of PC1 in anterior lobe and AtT-20 corticotroph cells. In contrast, extracts from tumors transplanted subcutaneously in nude mice contained predominantly nonacetylated forms of beta-endorphin(1-31) and -(1-27), very little ACTH(1-39), almost no corticotropin-like intermediate peptide or alpha-melanocyte-stimulating hormone, and higher proportions of intact POMC. Surprisingly, despite the less efficient proteolytic cleavage, a transplanted tumor expressed both PC1 and PC2. These studies are the first biochemical documentation of a melanotroph pituitary tumor in a rodent species and provide a new model for the investigation of pituitary oncogenesis and the molecular basis of tissue-specific prohormone post-translational processing.

Identification of DNA elements cooperatively activating proopiomelanocortin gene expression in the pituitary glands of transgenic mice.

The proopiomelanocortin (POMC) gene is highly expressed in adult mouse pituitary anterior lobe corticotrophs and intermediate lobe melanotrophs. To identify the DNA elements important for this tissue-specific expression, we analyzed a series of POMC reporter genes in transgenic mice. A DNA fragment containing rat POMC 5'-flanking sequences from -323 to -34 recapitulated both basal pituitary cell-specific and hormonally stimulated expression in adult mice when fused to a heterologous thymidine kinase promoter. Developmental onset of the reporter gene expression lagged by 1 day but otherwise closely paralleled the normal ontogeny of murine POMC gene expression, including corticotroph activation at embryonic day 14.5 (E14.5) followed by melanotroph activation at E15.5 to E16.5. AtT20 corticotroph nuclear protein extracts interacted with three specific regions of the functional POMC promoter in DNase I protection assays. The positions of these protected sites were -107 to -160 (site 1), -182 to -218 (site 2), and -249 to -281 (site 3). Individual deletions of these footprinted sites did not alter transgene expression; however, the simultaneous deletion of sites 2 and 3 prevented transgene expression in both corticotrophs and melanotrophs. Electrophoretic mobility shift and Southwestern (DNA-protein) assays demonstrated that multiple AtT20 nuclear proteins bound to these footprinted sites. We conclude that the sequences between -323 and -34 of the rat POMC gene promoter are both necessary and sufficient for correct spatial, temporal, and hormonally regulated expression in the pituitary gland. Our data suggest that the three footprinted sites within the promoter are functionally interchangeable and act in combination with promoter elements between -114 and -34. The inability of any reporter gene construction to dissociate basal and hormonally stimulated expression suggests that these DNA elements are involved in both of these two characteristics of POMC gene expression in vivo.

Pituitary corticotroph hyperplasia in rats implanted with a medullary thyroid carcinoma cell line transfected with a corticotropin-releasing hormone complementary deoxyribonucleic acid expression vector.

CRH stimulates both the synthesis and release of ACTH and other derivatives of POMC by the adenohypophysis. It is uncertain, however, whether it also causes proliferation of corticotrophs. Patients with CRH-producing tumors develop Cushing's syndrome, and some have been reported to have pituitary corticotroph hyperplasia. We now report an animal model that accurately reproduces the human disorder of ectopic production of CRH by a neoplasm. Prolonged CRH secretion by a transplanted medullary thyroid carcinoma cell line stably transfected with a CRH cDNA under transcriptional control of a cytomegalovirus promoter resulted in corticotroph hyperplasia and hypertrophy; the percentage of ACTH-containing cells in animals bearing W2CRH tumors was increased at 9.8 +/- 0.5% (controls, 6.2 +/- 0.3%; W2 implanted tumors, 7.7 +/- 0.4%). Occasional mitotic figures were identified, and the cells were larger, with abundant cytoplasm but generally less intense immunohistochemical staining for ACTH due to relative degranulation compared to controls. Melanotrophs of the intermediate lobe were also increased in number and were larger, with abundant cytoplasm. No corticotroph adenomas were found. Our experiment accurately reproduces the gradually increasing CRH levels in the general circulation produced by a growing tumor, as found in the human ectopic CRH syndrome, and confirms that long term exposure to CRH excess, as produced by a tumor, results in an increased number of corticotrophs in the adenohypophysis.

Ectopic corticotropin-releasing hormone produced by a transfected cell line chronically activates the pituitary-adrenal axis in transkaryotic rats.

Hypothalamic CRH is the primary positive regulatory factor of the pituitary-adrenal axis. The purpose of our study was to analyze the chronic effects of CRH on the production and secretion of POMC peptides from both the anterior lobe (AL) and neurointermediate lobe (NIL) of the pituitary by mimicking the syndrome of ectopic CRH secretion from neuroendocrine tumors. We first generated stably transfected W2 medullary thyroid carcinoma cell lines with a rat CRH expression vector under the transcriptional control of a cytomegalovirus gene promoter. These cell lines constitutively expressed the foreign gene, accurately processed the encoded prepro-CRH, and secreted biologically active CRH with an estimated potency equivalent to that of synthetic CRH-(1-41)NH2. The cell line designated W2CRH-7 was implanted sc in the syngeneic rat strain WAG/Rij and produced tumors that abundantly secreted CRH into the peripheral circulation. Four weeks postimplantation, W2CRH-7, but not wild-type W2, cells caused significant increases in the AL content of beta-endorphin-like immunoreactivity comparable to that caused by adrenalectomy (ADX). Plasma ACTH and serum beta-endorphin-like immunoreactivity were increased to a greater extent by ADX than by W2CRH-7 cell implantation. The NIL of both male and female rats showed either no change or a tendency to decreased beta-endorphin concentrations with no change in the acetylation or carboxy-shortening profiles judged by cation exchange chromatography in response to the ectopic CRH treatment. Rats of both sexes maintained a profound activation of the pituitary adrenal axis up to 16 weeks postimplantation, with normalized adrenal gland weights 5 times that of controls. The chronic secretion of CRH by W2CRH-7 cells resulted in a complete cessation of body growth in all rats up to the maximum time tested of 16 weeks. The lack of growth was partly ameliorated by concomitant ADX, suggesting an important role for adrenal glucocorticoids in these effects. We conclude that 1) the transplantable W2CRH-7 cell line provides a highly effective and reproducible means of sustained CRH treatment that mimics the syndrome of ectopic CRH expression by neuroendocrine tumors; 2) AL corticotrophs respond to chronic CRH by a sustained production and secretion of POMC peptides, leading to a marked adrenal cortical hyperplasia, with no evidence of biologically significant desensitization; 3) chronic CRH tends to decrease the NIL content of beta-endorphin,with remarkably little effect on posttranslational processing; and 4) the syndrome of chronic ectopic CRH in WAG/Rij rats includes a cessation of body growth at least partly due to products of the adrenal glands.

Pituitary-specific and hormonally regulated gene expression directed by the rat proopiomelanocortin promoter in transgenic mice.

All aspects of POMC biosynthesis exhibit tissue-specific regulation. The single copy gene is highly expressed in anterior lobe (AL) corticotrophs and intermediate lobe (IL) melanotrophs of the pituitary gland and in the arcuate nucleus of the hypothalamus. POMC gene transcription in corticotrophs is induced by hypothalamic CRH and vasopressin and inhibited by adrenal glucocorticoids, while in melanotrophs it is predominantly regulated by beta-adrenergic neural input and dopamine. To identify the rat POMC (rPOMC) gene sequences necessary and sufficient to target expression and hormonal regulation in corticotrophs and melanotrophs, we generated 13 transgenic mice carrying rPOMC fusion genes. The genes consisted of 706 or 480 basepairs of rPOMC 5' flanking sequences ligated to either the E. coli LacZ gene encoding beta-galactosidase or the K1 mutant of the SV40 large T-antigen gene. Overall, half of the transgenic lines had reporter gene expression in their AL and IL in a pattern indistinguishable from ACTH immunohistochemistry. In three of these lines, beta-galactosidase or K1 T-antigen was localized by double immunofluorescence exclusively to ACTH-positive corticotrophs and melanotrophs. Transcriptional regulation of the rPOMC-LacZ fusion gene in response to hormonal manipulation was quantified by a fluorescence assay for beta-galactosidase enzyme activity in pituitary extracts. There was a 15-fold increase in AL enzyme activity after adrenalectomy and a 3-fold increase in IL activity after haloperidol treatment. X-gal histochemistry of pituitaries from hormonally treated mice confirmed the cellular specificity of these effects.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of naloxone administered into the periaqueductal gray on shock-elicited freezing behavior in the rat.

Freezing behavior that occurs following footshock was found to increase in rats in which naloxone was injected into the ventrolateral region of the mesencephalic periaqueductal gray (PAG) area of the brain prior to footshock administration. Since naloxone administered into the ventrolateral region of the PAG induced minimal freezing in rats which did not receive footshock, the results suggest that the effect of naloxone on shock-induced freezing is not due to a nonspecific decrease in motor activity. Naloxone had no effect on freezing when injected into the dorsolateral region of the PAG. The data are consistent with the theory that conditioned fear induces opioid mediated analgesia, and that the ventrolateral region of the PAG is an important component of a pain-inhibitory system involved in this analgesia.