Aggressive Cushing's Disease: Molecular Pathology and Its Therapeutic Approach.

Cushing's disease is a syndromic pathological condition caused by adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas (ACTHomas) mediated by hypercortisolemia. It may have a severe clinical course, including infection, psychiatric disorders, hypercoagulability, and metabolic abnormalities, despite the generally small, nonaggressive nature of the tumors. Up to 20% of ACTHomas show aggressive behavior, which is related to poor surgical outcomes, postsurgical recurrence, serious clinical course, and high mortality. Although several gene variants have been identified in both germline and somatic changes in Cushing's disease, the pathophysiology of aggressive ACTHomas is poorly understood. In this review, we focused on the aggressiveness of ACTHomas, its pathology, the current status of medical therapy, and future prospects. Crooke's cell adenoma (CCA), Nelson syndrome, and corticotroph pituitary carcinoma are representative refractory pituitary tumors that secrete superphysiological ACTH. Although clinically asymptomatic, silent corticotroph adenoma is an aggressive ACTH-producing pituitary adenoma. In this review, we summarize the current understanding of the pathophysiology of aggressive ACTHomas, including these tumors, from a molecular point of view based on genetic, pathological, and experimental evidence. The treatment of aggressive ACTHomas is clinically challenging and usually resistant to standard treatment, including surgery, radiotherapy, and established medical therapy (e.g., pasireotide and cabergoline). Temozolomide is the most prescribed pharmaceutical treatment for these tumors. Reports have shown that several treatments for patients with refractory ACTHomas include chemotherapy, such as cyclohexyl-chloroethyl-nitrosourea combined with 5-fluorouracil, or targeted therapies against several molecules including vascular endothelial growth factor receptor, cytotoxic T lymphocyte antigen 4, programmed cell death protein 1 (PD-1), and ligand for PD-1. Genetic and experimental evidence indicates that some possible therapeutic candidates are expected, such as epidermal growth factor receptor tyrosine kinase inhibitor, cyclin-dependent kinase inhibitor, and BRAF inhibitor. The development of novel treatment options for aggressive ACTHomas is an emerging task.

ACTH increment post total bilateral adrenalectomy for Cushing's disease: a consistent biosignature for predicting Nelson's syndrome.

PURPOSE: Nelson's syndrome (NS) is regarded as an aggressive complication of total bilateral adrenalectomy (TBA) for Cushing's disease (CD). This challenge may be addressed by using clinical criteria to guide frequency of neuroimaging to enable timely management of NS and also avoid unnecessary frequent imaging. METHODS: All patients (n = 43) with CD subjected to TBA over 35 years at a tertiary care centre were included. NS was defined as a newly appearing or expanding (> 2 mm) pituitary adenoma with or without ACTH levels exceeding 500 pg/ml. Pre-and post-TBA parameters like clinical symptomatology, cortisol, ACTH and radiology were analysed for the prediction of NS. RESULTS: NS developed in 39.5% (n = 17) patients with a median follow-up of 7 years. Half of them had new appearance, while rest had an expansion of pre-existing pituitary tumour. Majority (90%) had ACTH above 500 pg/ml. On Cox proportional hazards analysis, frequent discriminatory features of protein catabolism (≥ 4) (HR 1.15, CI 0.18, 7.06), proximal myopathy (HR 8.82, CI 1.12, 69.58) and annual ACTH increment of 113 pg/ml (HR 12.56, CI 1.88, 88.76) predicted NS. First post-operative year ACTH indices predicting NS included ACTH rise of 116 pg/ml and absolute ACTH of 142 pg/ml (sensitivity, specificity exceeding 90%). Annual ACTH increment exceeding 113 pg/ml, ≥ 4 discriminatory features and uncontrolled hypertension had the best overall prediction. CONCLUSION: Patients who developed NS had higher rebound rise of ACTH following TBA and a more severe disease phenotype at baseline. Consistent ACTH increment can be used as a marker for predicting the development of NS.

A high-throughput analysis of the IDH1(R132H) protein expression in pituitary adenomas.

PURPOSE: Inactivating mutations of isocitrate dehydrogenase (IDH) 1 and 2, mitochondrial enzymes participating in the Krebs tricarboxylic acid cycle play a role in the tumorigenesis of gliomas and also less frequently in acute myeloid leukemia and other malignancies. Inhibitors of mutant IDH1 and IDH2 may potentially be effective in the treatment of the IDH mutation driven tumors. Mutations in the succinate dehydrogenase, the other enzyme complex participating in the Krebs cycle and electron transfer of oxidative phosphorylation occur in the paragangliomas, gastrointestinal stromal tumors, and occasionally in the pituitary adenomas. We aimed to determine whether the IDH1(R132H) mutation, the most frequent IDH mutation in human malignancies, occurs in pituitary adenomas. METHODS: We performed immunohistochemical analysis by using a monoclonal anti-IDH1(R132H) antibody on the tissue microarrays containing specimens from the pituitary adenomas of different hormonal types from 246 patients. In positive samples, the status of the IDH1 gene was further examined by molecular genetic analyses. RESULTS: In all but one patient, there was no expression of mutated IDH1(R132H) protein in the tumor cells by immunohistochemistry. Only one patient with a recurring clinically non-functioning gonadotroph adenoma demonstrated IDH1(R132H)-immunostaining in both the primary tumor and the recurrence. However, no mutation in the IDH1 gene was detected using different molecular genetic analyses. CONCLUSION: IDH1(R132H) mutation occurs only exceptionally in pituitary adenomas and does not play a role in their pathogenesis. Patients with pituitary adenomas do not seem to be candidates for treatment with the inhibitors of mutant IDH1.

Nelson Syndrome: Update on Therapeutic Approaches.

OBJECTIVE: To review the pathophysiology and therapeutic modalities availble for Nelson syndrome. METHODS: We reviewed the current literature including managment for Nelson syndrome. RESULTS: For patients with NS, surgical intervention is often the first-line therapy. With refractory NS or tumors with extrasellar involvement, radiosurgery offers an important alternative or adjuvant option. Pharmacologic interventions have demonstrated limited usefulness, although recent evidence supports the feasibility of a novel somatostatin analog for patients with NS. Modern neuroimaging, improved surgical techniques, and the advent of stereotactic radiotherapy have transformed the management of NS. CONCLUSIONS: An up-to-date understanding of the pathophysiology underlying Nelson Syndrome and evidence-based management is imperative. Early detection may allow for more successful therapy in patients with Nelson Syndrome. Improved radiotherapeutic interventions and rapidly evolving pharmacologic therapies offer an opportunity to create targeted, multifocal treatment regiments for patients with Nelson Syndrome.

Low immunohistochemical expression of MGMT in ACTH secreting pituitary tumors of patients with Nelson syndrome.

MGMT expression in tumors has been correlated with response to treatment with temozolomide therapy. Few medical therapies are available for Nelson syndrome, and the efficacy of such therapeutics remains limited. The aim of the present study was to assess immunohistochemical expression of MGMT in ACTH-secreting pituitary adenomas of patients with Nelson syndrome. Our material consisted of eight specimens from ACTH-secreting pituitary adenomas of patients with Nelson syndrome. Immunohistochemical staining for MGMT was performed using the streptavidin-biotin-peroxidase complex method. MGMT immunoreactivity was assessed microscopically and recorded as an estimated percentage of nuclear MGMT immunostaining (0 = none, 1=<10%, 2=<25%, 3=<50%, 4=>50%). Five of the eight specimens (65%) exhibited no MGMT immunoreactivity, with two out of eight cases (25%) showing slight MGMT staining (<10%) and one out of eight cases (12%) demonstrating moderate MGMT positivity (<25%). Patient male/female ratio was 3:5, with average patient age being 62.4 (range 57­66). Our findings suggest that temozolomide therapy may be of potential use in patients with Nelson syndrome, as these tumors express absent/low levels of MGMT. Absent or low MGMT staining in brain and other neoplasms has been shown to correlate with successful treatment with temozolomide, and recent reports of aggressive pituitary adenomas suggest similar outcomes.

The PPAR-gamma activator rosiglitazone fails to lower plasma ACTH levels in patients with Nelson's syndrome.

BACKGROUND: Peroxisomal proliferator-activated receptors (PPAR)- gamma are expressed abundantly in ACTH-secreting pituitary tumours. The PPAR-gamma activator rosiglitazone has been shown to suppress ACTH secretion in human adrenocorticotroph tumour cells in vitro, and prevent and reduce adrenocorticotroph tumour development in mouse models in vivo. OBJECTIVE: To evaluate the effect of rosiglitazone in patients with persistently elevated plasma ACTH levels postbilateral adrenalectomy for Cushing's disease. PATIENTS: Seven patients were treated with rosiglitazone 8 mg orally per day for 12 weeks. MEASUREMENTS: Plasma ACTH was measured at two hourly intervals from 09:00 h to 17:00 h before and after 6 and 12 weeks of treatment. RESULTS: Plasma ACTH at 09:00 hours immediately before the usual morning hydrocortisone dose was 2599.0 +/- 899.7 ng/l (mean +/- SEM) basally and 1547.6 +/- 515.7 ng/l after 12 weeks of rosiglitazone, whereas levels at 17:00 h were 1433.4 +/- 506.2 ng/l (mean +/- SEM) basally and 1122.3 +/- 460.9 ng/l at 12 weeks (all nonsignificant). CONCLUSION: This study showed no effect of rosiglitazone treatment at maximum approved doses in lowering plasma ACTH levels in patients post bilateral adrenalectomy for Cushing's disease.

Immunocytochemical accumulation of p53 in corticotroph adenomas: relationship with heat shock proteins and apoptosis.

The pathogenesis of corticotroph adenomas is unknown. In a recent study accumulation of p53 protein was detected by immunohistochemistry in a substantial proportion of pituitary corticotroph adenomas, and it has been suggested that it may be causally related to their development. However, other immunohistochemical studies have not confirmed the high incidence of p53 accumulation in this tumor type. Therefore, in the present study, p53 protein accumulation was re-examined in a series of 31 cases of corticotroph adenomas, using different sets of well validated anti-p53 antibodies. Furthermore, in view of the known association of p53 protein with apoptosis, and the known property of p53 to form complexes with heat shock proteins (HSPs), the relationship of p53 accumulation in corticotroph adenomas with apoptosis and HSP-70 was also investigated. Tumor samples from 31 patients with Cushing's disease or Nelson's syndrome were studied. Accumulation of p53 protein was tested by the standard ABC method using two different sets of clone Pab1801 and DO-7 monoclonal antibodies, applied after incubation of sections in a microwave oven. Using the DO-7 antibody, nuclear accumulation of p53 protein was detected in a total of 15 cases, with cytoplasmic staining observed in only 3 tumors. In contrast, using the Pab1801 antibody nuclear staining was observed in only 5 adenomas, with 11 adenomas demonstrating focal cytoplasmic immunoreactivity. Parallel sections of all corticotroph tumors demonstrating cytoplasmic accumulation of p53 protein were tested for the immunohistochemical presence of heat shock protein HSP-70. A striking similar distribution pattern of these two proteins was observed. Apoptosis, identified by the in situ end labeling technique, was detected in a total of 15 out of 28 corticotroph adenomas tested. Calculation of the apoptotic labeling index (ALI) by image analysis showed a significantly lower ALI in those corticotroph adenomas demonstrating nuclear p53 accumulation compared to those with no nuclear p53 immunostaining (p < 0.05). There was no significant difference in the ALI between cytoplasmic p53 positive and negative tumors. It is concluded that depending on the antibody used there is a significant variation of p53 protein detection in corticotroph adenomas. Overall, a significant proportion of corticotroph adenomas studied expressed the p53 protein, which depending on the antibody used, was located either in the nucleus and/or the cytoplasm of tumorous corticotroph cells. Cytoplasmic accumulation of p53, as shown by our colocalization studies with HSP-70, may be due to p53/HSP-70 complex formation. Although such a complex-mediated cytoplasmic exclusion of p53 has no significant effect on apoptosis, nuclear accumulation of p53 protein is associated with a significantly lower apoptotic index indicating a failure of p53 protein to exert its apoptotic action in at least a subset of this tumor type.

Cytokine expression in human anterior pituitary adenomas.

OBJECTIVE: There is increasing evidence for the role of cytokines in pituitary differentiated function and tumorigenesis, but the spectrum of cytokines found in the pituitary is unknown. Therefore profiles of cytokine expression were determined in different human anterior pituitary adenoma sub-types. DESIGN: The reverse transcriptase-linked polymerase chain reaction (PCR) was used to identify the presence of cytokine mRNA within human pituitary adenomas. PATIENTS: Seventeen pituitary adenoma biopsies removed at transsphenoidal surgery were examined: 4 somatotrophinomas, 7 non-functional adenomas, 4 prolactinomas, one case of Cushing's disease and one case of Nelson's syndrome. MEASUREMENTS: RNA was extracted from each adenoma biopsy and reverse transcribed into cDNA. This was specifically amplified in a PCR using oligonucleotide primers complementary to each cytokine. The cytokines investigated were interleukin (IL)-I alpha, IL-I beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, tumour necrosis factor (TNF)-alpha, TNF-beta and transforming growth factor (TGF)-beta 1, beta 2 and beta 3. The products of each PCR were visualized using agarose gel electrophoresis. RESULTS: All 17 adenomas expressed IL-8 transcripts, but no expression of IL-2, IL-5 or IL-7 was found. IL-6 was expressed in all 4 somatotrophinomas, 3 of 7 non-functional tumours, 2 of 4 prolactinomas and in the single case of Nelson's syndrome. At least one of the 3 isoforms of TGF-beta was found in all but 2 tumours; one prolactinoma and one non-functional adenoma. IL-1 alpha, IL-beta, IL-4, TNF-alpha and TNF-beta were expressed sporadically by individual adenomas. CONCLUSION: These data suggest that whilst IL-8 may be important, the local expression of the cytokines IL-2, IL-5 and IL-7 is not important in human anterior pituitary tumorigenesis.

Multiple forms of bioactive and immunoreactive adrenocorticotropin in human pituitary and blood of patients with Nelson's syndrome.

A pool of human pituitaries obtained from allegedly healthy subjects (traffic victims) and plasma samples from patients with Nelson's syndrome were analyzed by high performance liquid chromatography, and the corticosteroidogenic bioactivity and ACTH immunoreactivity were measured. Three bioactive forms of ACTH were detected in plasma samples and pituitary extract. The major form (peak III) coeluted with human ACTH-(1-39), showed a bioactive to immunoreactive ratio (B/I ratio) of about 1, and represented about 80% of the total bioactivity in both the plasma samples and the pituitary extract. Peak I, with a B/I ratio greater than 1, represented about 5%, and peak II, with a highly variable B/I ratio, represented about 7% of the bioactivity in both the plasma and pituitary extracts. A fraction with a very low B/I ratio was found to coelute with corticotropin-like intermediate lobe peptide. These data suggest that in Nelson's syndrome, ACTH secretion by the pituitary gland does not differ from that in normal subjects, at least qualitatively.

The role of octreotide (Sandostatin) in non-growth hormone-, non-thyroid-stimulating hormone-, and non-prolactin-secreting adenomas.

Octreotide may act on non-growth hormone-, non-thyroid-stimulating hormone, and non-prolactin-secreting adenomas. Its efficacy was reported in some corticotropin-secreting adenomas from Nelson's syndrome and from Cushing's disease. In gonadotropin-secreting adenomas, octreotide was shown to be effective in two of eight cases. In nonfunctioning adenomas, visual improvement was observed with octreotide in 14 of 23 cases in a French multicenter study. Among the 33 patients whose tumor volume was checked, shrinkage occurred in seven, but an increase in tumor volume was observed in another seven patients. Mechanism(s) and prediction of efficacy of octreotide remain to be documented.

Electron microscopical morphometry of well-differentiated and undifferentiated ACTH secreting adenomas in Cushing's disease and Nelson's syndrome.

Adrenocorticotrophic hormone (ACTH)-secreting adenomas of patients with Cushing's disease (undifferentiated and well-differentiated ACTH-cell adenomas) were studied ultrastructurally and analysed morphometrically by a computer-supported quantitative image-analysing system. They were compared with identically prepared ACTH tumours (undifferentiated and well-differentiated ACTH-cell adenomas) of pituitaries from bilateral adrenalectomised patients with Nelson's syndrome. The aim of our study was to look for significant differences in ultrastructure and to evaluate these findings statistically regarding adenoma types and clinical syndromes. Clinical syndromes aside, more secretory granules and larger-sized prosecretory granules were measured in the well-differentiated ACTH-cell adenomas. The undifferentiated adenomas showed a greater content of nucleoli and prosecretory granules. Within the adenoma types, comparison of well-differentiated ACTH-cell adenomas showed that the clinical group of Cushing's disease contained larger areas of cytofilaments, whereas the clinical group of Nelson's syndrome had a larger tumour size and more lysosomes. Comparing the undifferentiated adenomas of both clinical groups the adenomas in Cushing's disease contained larger nuclei and more lysosomes, whereas the adenomas in Nelson's syndrome were larger in tumour size and contained larger prosecretory granules. Comparison of well-differentiated and undifferentiated adenomas in Cushing's disease showed more secretory granules and bigger prosecretory granules in well-differentiated adenomas whereas in undifferentiated adenomas the total area of the nuclei is larger, the nucleoli increase in number and size and the lysosomes are more frequent. Comparison of well-differentiated and undifferentiated adenomas in Nelson's syndrome demonstrated more lysosomes in well-differentiated adenomas and a larger total area of the nuclei in undifferentiated adenomas. The differences between the well-differentiated adenomas (mainly more secretory granules and larger prosecretory granules) and undifferentiated adenomas (mainly more and larger nuclei and nucleoli and more prosecretory granules) prove the clear separability between the adenoma types, not demonstrated in the literature up to now. The significant differences between adenomas in Cushing's disease (mainly more cytofilaments) and Nelson's syndrome (mainly more ribosomes and larger prosecretory granules) may be interpreted as different cell reactions due to the hypercortisolism present in Cushing's disease and lacking in Nelson's syndrome following adrenalectomy. Despite the fact that both clinical syndromes are based on the same adenoma types, indistinguishable by light microscopy, significant morphometrical findings in ultrastructure allow a clear discrimination of both clinical types.

In vitro effects of ketoconazole on corticotrope cell morphology and ACTH secretion of two pituitary adenomas removed from patients with Nelson's syndrome.

The direct effects of ketoconazole on the secretion of ACTH by human pituitary adenoma cells from 2 patient with Nelson's syndrome were studied in vitro. Stereologically quantified, intracellular changes affect the surface density of the endoplasmic reticulum (it decreased by 73%), the volume density of the secretion granules (it decreased by 49%), and the volume density of lysosomes (it decreased by 67%). The hormone released in the culture medium decreased depending on the doses of ketoconazole used; 10 mumol/l decreased ACTH levels by 31%. These data show that ketoconazole induce marked changes on corticotrope morphology and ACTH secretion in pituitary cells obtained from patients with Nelson's syndrome.

Peptides related to alpha-melanocyte-stimulating hormone are commonly produced by human pituitary corticotroph adenomas: no relationship with pars intermedia origin.

The presence of immunoreactive (ir)-alpha-MSH has been investigated by immunocytochemistry in 24 pituitary adenomas and one case of corticotroph hyperplasia causing Cushing's disease, in four adenomas causing Nelson's syndrome, and in ten 'silent' corticotroph adenomas. It was found that a high proportion of these adenomas have a population of cells containing ir-alpha-MSH in addition to ir-ACTH. In some instances, these adenomas were clearly not associated with the residual intermediate lobe of the pituitary. Radioimmunoassay of plasma from patients with Cushing's disease or Nelson's syndrome showed elevated levels of ir-alpha-MSH in the majority of cases. Characterization of the ir-alpha-MSH in adenoma cells by immunocytochemistry, using an antiserum selective for acetylated forms of alpha-MSH, suggested that only the desacetyl form was present in each case examined. High-performance liquid chromatography of adenoma tissue extracts revealed material co-eluting with acetylated forms of alpha-MSH in only one of six cases. These results have been compared with corticotroph adenomas in animal pituitary glands, and it is concluded that the presence of alpha-MSH peptides cannot be used as a marker for intermediate lobe tumours, and that desacetyl alpha-MSH is commonly produced by corticotroph adenomas.

Quantitative in-situ hybridization histochemistry of anterior pituitary hormone mRNA species in human pituitary adenomas.

We have examined the anterior pituitary hormone messenger (m) RNA species contained in biopsies of 41 pituitary tumours obtained at hypophysectomy using in-situ hybridization histochemistry. The adenoma were grouped clinically into 12 prolactinomas, 8 somatotrope adenomas, 16 non-functioning, 4 Nelson's syndrome, and 1 thyrotrope adenoma. Of these, 10 contained no detectable anterior pituitary hormone mRNA species and 11 appeared to be expressing the gene responsible for the patients' clinical state in isolation. In a number of cases the accumulation of specific mRNA species was not accompanied by an increase in the circulating levels of the corresponding hormone or subunit. Evidence of activation of more than 1 anterior pituitary hormone gene was present in 16 adenomas of which only 7 showed a pattern of activation or amplification of gene expression which would suggest deregulation of either the inositol phospholipid or cAMP second messenger pathway. It was therefore not possible from these data to postulate that isolated deregulation of a single second messenger transduction pathway is a common etiological factor in pituitary tumour formation.

Alpha-amidated peptides derived from pro-opiomelanocortin in human pituitary tumours.

Human pituitary tumours, obtained at surgery for Cushing's disease and Nelson's syndrome, were extracted and the content and molecular forms of pro-opiomelanocortin (POMC)-derived peptides determined by radioimmunoassay, gel chromatography, reversed-phase high-performance liquid chromatography (HPLC) and sequence analysis. In the tumours from patients with Cushing's disease the mean concentrations of amidated peptides relative to the total amount of POMC were as follows: alpha-MSH, 1.7%; amidated gamma-MSH (gamma 1-MSH), 8.5% and the peptide linking gamma-MSH and ACTH in the precursor (hinge peptide or joining peptide) in its amidated form (HP-N), 17.1%. The same relative concentrations in the tumours from patients with Nelson's syndrome were 8.5% (alpha-MSH), 7.5% (gamma 1-MSH) and 12.2% (HP-N). More than 95% of the ACTH(1-39) immunoreactivity eluted as synthetic ACTH(1-39) by gel chromatography and HPLC. The remaining ACTH(1-39) immunoreactivity eluted as partly glycosylated high molecular weight forms. All the alpha-MSH and its glycine-extended precursor ACTH(1-14) were of low molecular weight, mainly non- or mono-acetylated forms, but significant amounts of diacetylated analogues were also present. gamma 1-MSH and gamma 2-MSH immunoreactivities eluted as high molecular weight forms and were partly glycosylated. No low molecular weight forms of gamma 1-MSH or gamma 2-MSH could be detected in the pituitary tumours. Amidated hinge peptide was mainly of the 30 amino acid form. In conclusion, all the molecular forms of the amidated peptides detected in tumours from patients with Cushing's disease and Nelson's syndrome were similar to the molecular forms found in the normal human pituitary. The main difference between the tumours and the normal pituitary was the greater amount of peptides produced, particularly alpha-MSH and gamma 1-MSH.

Treatment of pituitary macroadenomas secreting PRL, HGH or ACTH with long-acting bromocriptine.

Long-acting im bromocriptine was administered to 7 patients with pituitary macroadenomas (4 acromegalics, 1 Nelson's syndrome and 2 prolactinomas), with good tolerance except during the first 24 h. During a 42-day period hormonal, CT-scan and visual field variations were followed. In acromegalics HGH decrease was not evident, except in some isolated sample. In Nelson's syndrome ACTH showed a 94% fall on day 14, even though a spontaneous oscillation cannot be ruled out, and recovery took place from day 21 on. PRL remained undetectable in both. In prolactinomas, PRL suffered a great decrease (91.8% and 96.3% on days 21 and 28 respectively) and remained well below its initial values up to the end of the study, in spite of partial recovery. In these 2 patients CT-scan evidenced shrinkage of tumor mass, which was not observed in the remaining 5 cases. Visual fields did not improve in the 2 cases initially affected (Nelson's syndrome and 1 prolactinoma). Long-acting bromocriptine seems to have the same therapeutic uses of the oral form with the possible advantage of a better tolerance of full initial doses.