Luiz Eduardo Wildemberg

The Gene of the Ubiquitin-Specific Protease 8 Is Frequently Mutated in Adenomas Causing Cushing's Disease

CONTEXT We have recently reported somatic mutations in the ubiquitin-specific protease USP8 gene in a small series of adenomas of patients with Cushings disease. OBJECTIVE To determine the prevalence of USP8 mutations and the genotype-phenotype correlation in a large series of patients diagnosed with Cushings disease. DESIGN We performed a retrospective, multicentric, genetic analysis of 134 functioning and 11 silent corticotroph adenomas using Sanger sequencing. Biochemical and clinical features were collected and examined within the context of the mutational status of USP8, and new mutations were characterized by functional studies. PATIENTS A total of 145 patients who underwent surgery for an ACTH-producing pituitary adenoma. MAIN OUTCOMES MEASURES Mutational status of USP8. Biochemical and clinical features included sex, age at diagnosis, tumor size, preoperative and postoperative hormonal levels, and comorbidities. RESULTS We found somatic mutations in USP8 in 48 (36%) pituitary adenomas from patients with Cushings disease but in none of 11 silent corticotropinomas. The prevalence was higher in adults than in pediatric cases (41 vs 17%) and in females than in males (43 vs 17%). Adults having USP8-mutated adenomas were diagnosed at an earlier age than those with wild-type lesions (36 vs 44 y). Mutations were primarily found in adenomas of 10 ± 7 mm and were inversely associated with the development of postoperative adrenal insufficiency. All the mutations affected the residues Ser718 or Pro720, including five new identified alterations. Mutations reduced the interaction between USP8 and 14-3-3 and enhanced USP8 activity. USP8 mutants diminished epidermal growth factor receptor ubiquitination and induced Pomc promoter activity in immortalized AtT-20 corticotropinoma cells. CONCLUSIONS USP8 is frequently mutated in adenomas causing Cushings disease, especially in those from female adult patients diagnosed at a younger age.

AIP expression in sporadic somatotropinomas is a predictor of the response to octreotide LAR therapy independent of SSTR2 expression.

mutation and/or a history of medical treatment with SRL before surgery. Biochemical assessment was performed 3 months after surgery by oral glucose tolerance test (OGTT) and evaluations of IGF1 levels. Patients were considered non-cured based on nonsuppressible GH levels on OGTT and plasma IGF1 levels higher than those of age-matched normal subjects. Medical therapy with OCT-LAR was started at a dose of 20 mg every 4 weeks, and the dose was increased to 30 mg in patients with non-controlled disease after 3 months of therapy. The efficacy of medical therapy was evaluated during the last patient visit. Patients were considered to have non-controlled disease if they had plasma IGF1 levels greater than those of age-matched normal subjects or basal GH levels O1.0 ng/ml after at least 6 months of treatment with OCT-LAR at a dosage of 30 mg. The AIP and SSTR2 expressions were analyzed through immunohistochemistry in paraffin-embedded

Low Aryl Hydrocarbon Receptor-Interacting Protein Expression Is a Better Marker of Invasiveness in Somatotropinomas than Ki-67 and p53

Background: Some pituitary adenomas exhibit fast growth and invade surrounding structures. To date, there is no robust marker to predict invasiveness. Aim: To evaluate Ki-67, p53 and aryl hydrocarbon receptor-interacting protein (AIP) expression and compare these between invasive and noninvasive somatotropinomas and nonfunctioning pituitary adenomas (NFPAs). Methods: Protein expression was determined by immunohistochemistry. Tumors were classified according to percentage of immunolabeled nuclei for Ki-67 and p53. AIP immunopositivity was graded according to a score encompassing pattern and intensity. Invasiveness was defined according to radiological and surgical criteria. Results: Thirty-eight sporadic somatotropinomas were studied. Median Ki-67 labeling index in invasive and noninvasive tumors was 1.6 (range 0–20.6) and 0.26 (0–2.2), respectively (p = 0.01). With a 2.3% cut-off point obtained by ROC curve analysis, invasive adenomas were distinguished with 100% specificity, 39% sensitivity, and 63% accuracy. Low AIP expression was also correlated with tumor invasiveness (p = 0.001), with sensitivity, specificity and accuracy of 78, 80, and 79%, respectively. Expression of p53 was not different among tumors. Twenty-nine NFPAs were studied, with no significant difference between Ki-67, p53 and AIP expression in invasive and noninvasive tumors. High AIP expression was more frequent in NFPAs, with Ki-67 >3% (p = 0.051), especially when only gonadotrope cell adenomas (n = 25) were considered (p = 0.012). Conclusions: These data suggest, for the first time, that AIP is a better marker of invasiveness in somatotropinomas than Ki-67 and p53. In addition, low AIP expression is observed in invasive somatotropinomas, in contrast with high AIP expression in NFPAs (mainly gonadotrope cell tumors) with high proliferative indices.

Low somatostatin receptor subtype 2, but not dopamine receptor subtype 2 expression predicts the lack of biochemical response of somatotropinomas to treatment with somatostatin analogs

Objectives: To evaluate somatostatin receptor 2A (SSTR2A) and dopamine receptor 2 (DR2) protein expression in somatotropinomas and to relate it to response to somatostatin analogues (SA). Design and patients: SSTR2A and DR2 expression was analyzed by immunohistochemistry in 88 somatotropinomas from patients submitted to either pre-surgical or adjuvant SA treatment. Tumors were scored according to percentage of immunostained cells: 0 (<25%), 1 (25–50%), and 2 (>50%). Relation between protein expression and response to SA was performed in 66 patients. Response to SA was assessed by percent IGF-I reduction, being considered as an IGF-I per cent reduction higher than 50%. Disease control was also assessed (GH<1.0 ng/ml and normal IGF-I). Results: SSTR2A and DR2 were expressed in 100% and 98% of tumors, respectively. Biochemical response and disease control rates were 48% and 32%, respectively. Median IGF-I percent reduction after 3 months of SA treatment was lower in the SSTR2A score 0 than in the scores 1 and 2 (p<0.001, both), and after 6 months in the score 0 than in the score 1 (p=0.001) and 2 (p<0.001). Biochemical response and disease control were associated with SSTR2 expression (p<0.001 and p=0.004, respectively). A negative predictive value for biochemical response of 100% was found when a SSTR2A expression <25% of immunostained cells cut-off point was considered. No relation was found between DR2 expression and biochemical response and disease control. Conclusion: SSTR2A and DR2 are highly expressed in somatotropinomas. Low SSTR2A, but not DR2, expression is a negative predictive factor to response to SA.

Dopamine receptor subtype 2 expression profile in nonfunctioning pituitary adenomas and in vivo response to cabergoline therapy

To determine the dopamine receptor subtype 2 (DR2) mRNA levels and protein expression and to evaluate the effect of adjuvant cabergoline therapy on tumour volume (TV) in patients with postoperative residual nonfunctioning pituitary adenoma (NFPA).

The Role of Temozolomide in the Treatment of a Patient with a Pure Silent Pituitary Somatotroph Carcinoma

OBJECTIVE To describe a case of a pure silent somatotroph pituitary carcinoma. METHODS We describe a 54-year-old female with a clinically nonfunctioning pituitary macroadenoma diagnosed 15 years earlier. RESULTS The patient underwent transsphenoidal surgery and no visible tumor remnant was observed for 6 years. A magnetic resonance imaging (MRI) detected the recurrence of a 1.2 × 1.5 cm macroadenoma. The patient was submitted to conventional radiotherapy (4500 cGy), and the tumor volume remained stable for 7 years. Then, an MRI revealed a slight increase in tumor size, and 2 years later, a subsequent MRI detected a very large, invasive pituitary mass. The patient was resubmitted to transsphenoidal surgery, and the histopathological examination showed diffuse positivity for growth hormone (GH). The nadir GH level during an oral glucose tolerance test was 0.06 ng/mL, and the pre- and postoperative insulin like growth factor type I (IGF-I) levels were within the normal range. Abdominal, chest, brain, and spine MRI showed multiple small and hypervascular liver and bone lesions suggestive of metastases. Liver biopsy confirmed metastasis of GH-producing pituitary carcinoma. The patient has been treated with temozolomide and zoledronic acid for 7 months and with octreotide long-acting release (LAR) for 4 months. The primary tumor and metastases are stable. CONCLUSION Despite being an extremely rare event, pituitary carcinoma may develop several years after the successful treatment of even a silent GH-producing pituitary adenoma, which suggests that close long-term follow-up is necessary.

Ki-67 is a predictor of acromegaly control with octreotide LAR independent of SSTR2 status and relates to cytokeratin pattern.

INTRODUCTION Only one study has evaluated Ki-67 as a predictor of the response to somatostatin analog therapy in acromegaly; however, other predictors like somatostatin receptor type 2 (SSTR2) and cytokeratin pattern expressions were not considered. OBJECTIVE To evaluate whether Ki-67 is a predictor of octreotide LAR (OCT-LAR) response in somatotropinomas independent of SSTR2 and cytokeratin expression patterns. METHODS Protein expression was analyzed by immunohistochemistry. The percentage of cell nuclei that were immunolabeled for Ki-67 and the percentage of cells with positive SSTR2 staining were calculated. SSTR2 expression was considered high when ≥25%, and a cutoff of 2.3% was designated for Ki-67. Tumors were classified as densely or sparsely granulated according to the cytokeratin pattern. RESULTS Thirty-one somatotropinomas were studied. Fourteen patients (45.2%) were controlled with OCT-LAR therapy. The median Ki-67 labeling index (LI) was higher in patients not controlled with OCT-LAR than in those controlled (1.63 and 0.15 respectively, P=0.002). Higher SSTR2 expression and densely granulated tumors were correlated with control as well (P=0.04 and 0.038 respectively). There was no difference in Ki-67 levels between patients with high and low SSTR2 expression (P=0.651). After multivariate analysis, both Ki-67 and SSTR2 remained statistically significant as predictors of OCT-LAR response (P=0.017 and 0.012 respectively). The Ki-67 LI was higher in sparsely than in densely granulated tumors (P=0.047). CONCLUSIONS Ki-67 is a predictor of response to OCT-LAR in acromegaly, independent of SSTR2 expression and relates to cytokeratin patterns.

ZAC1 and SSTR2 Are Downregulated in Non-Functioning Pituitary Adenomas but Not in somatotropinomas

Introduction There are few data regarding ZAC1 expression in clinically non-functioning pituitary adenomas (NFPA). Because somatotropinomas and NFPA behave differently with respect to tumor shrinkage during somatostatin analogs (SA) therapy, we sought to compare the ZAC1 and somatostatin receptor (sstr) types 1, 2, 3 and 5 mRNA expression in these two pituitary adenoma subtypes and in normal human pituitaries. Methods ZAC1 and SSTR mRNA expression levels were evaluated using real-time RT-PCR (TaqMan) in 20 NFPA and compared with the expression levels in 23 somatotropinomas and five normal pituitaries. The NFPA invasiveness was evaluated using magnetic resonance imaging with Hardy’s modified criteria. Ki-67 and p53 were evaluated using immunohistochemistry. Results A total of 20 patients with NFPA [6 males, median age 56 years (range: 30-78)], 23 with acromegaly [12 males, median age 43 years (range: 24–57)] and five normal pituitaries [4 males, median age 48 years (range: 36–54)] were included. Four of the patients (20%) had Hardy’s grade 2 tumors; all of the others had Hardy’s grade 3 tumors. The Ki-67 median expression was 2.35 (range: 0.2–9.23), and only four of the tumors (20%) were positive for p53. The ZAC1 mRNA expression was significantly lower in NFPA than in somatotropinomas and in normal pituitaries (p<0.001 for both), as well as the SSTR2 (p=0.001 and 0.01, respectively). The SSTR3 expression was higher in the NFPA than in the somatotropinomas and in the normal pituitaries (p=0.03 and 0.02, respectively). No correlation was found between the ZAC1 mRNA expression and the tumor invasiveness, Ki-67 and p53. Conclusion ZAC1 and SSTR2 are underexpressed and SSTR3 is overexpressed in NFPA compared to those in somatotropinomas and in normal pituitaries, which might explain the lack of tumor shrinkage that is observed in response to commercially available SA therapy in patients with NFPA.

Familial isolated pituitary adenomas experience at a single center: clinical importance of AIP mutation screening

We present four FIPA kindred discussing clinical and molecular data and emphasizing the differences regarding AIP status, as well as the importance of genetic screening. Family 1 consists of five patients harboring somatotropinomas with germline E24X mutation in AIP. In one of the patients, acromegaly was diagnosed through active screening, being cured by surgery. Families 2 and 3 are composed of two patients with non-functioning pituitary adenomas. Family 4 comprises patients harboring a prolactinoma and a somatotropinoma. No mutations in AIP were found in these families. No patient in Family 1 was controlled with octreotide treatment, while the acromegalic patient in Family 4 was controlled with octreotide LAR. In conclusion, FIPA is a heterogeneous condition, which may be associated with AIP mutation. Genomic and clinical screening is recommended in families with two or more members harboring pituitary adenomas, allowing early diagnosis and better outcome.

Somatostatin receptor ligands in the treatment of acromegaly

First-generation somatostatin receptors ligands (SRL) are the mainstay in the medical treatment of acromegaly, however the percentage of patients controlled with these drugs significantly varies in the different studies. Many factors are involved in the resistance to SRL. In this review, we update the physiology of somatostatin and its receptors (sst), the use of SRL in the treatment of acromegaly and the factors involved in the response to these drugs. The SRL act through interaction with the sst, which up to now have been characterized as five subtypes. The first-generation SRL, octreotide and lanreotide, are considered sst2 specific and have biochemical response rates varying from 20 to 70%. Tumor volume reduction can be found in 36–75% of patients. Several factors may determine the response to these drugs, such as sst, AIP, E-cadherin, ZAC1, filamin A and β-arrestin expression in the somatotropinomas. In patients resistant to first-generation SRL, alternative medical treatment options include: SRL high dose regimens, SRL in combination with cabergoline or pegvisomant, or the use of pasireotide. Pasireotide is a next-generation SRL with a broader pattern of interaction with sst. In the light of the recent increase of treatment options in acromegaly and the deeper knowledge of the determinants of response to the current first-line therapy, a shift from a trial-and-error treatment to a personalized one could be possible.