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Context Mechanisms underlying pituitary corticotroph adenoma adrenocorticotropin (ACTH) production are poorly understood, yet circulating ACTH levels closely correlate with adenoma phenotype and clinical outcomes. Objective We characterized the 5′ ends of proopiomelanocortin (POMC) gene transcripts, which encode the precursor polypeptide for ACTH, in order to investigate additional regulatory mechanisms of POMC gene transcription and ACTH production. Methods We examined 11 normal human pituitary tissues, 32 ACTH-secreting tumors, as well as 6 silent corticotroph adenomas (SCAs) that immunostain for but do not secrete ACTH. Results We identified a novel regulatory region located near the intron 2/exon 3 junction in the human POMC gene, which functions as a second promoter and an enhancer. In vitro experiments demonstrated that CREB binds the second promoter and regulates its transcriptional activity. The second promoter is highly methylated in SCAs, partially demethylated in normal pituitary tissue, and highly demethylated in pituitary and ectopic ACTH-secreting tumors. In contrast, the first promoter is demethylated in all POMC-expressing cells and is highly demethylated only in pituitary ACTH-secreting tumors harboring the ubiquitin-specific protease 8 (USP8) mutation. Demethylation patterns of the second promoter correlate with clinical phenotypes of Cushing disease. Conclusion We identified a second POMC promoter regulated by methylation status in ACTH-secreting pituitary tumors. Our findings open new avenues for elucidating subcellular regulation of the hypothalamic-pituitary-adrenal axis and suggest the second POMC promoter may be a target for therapeutic intervention to suppress excess ACTH production.

Background Corticotrophs are a well-known tissue susceptible to cytotoxicity by lymphocytes, as seen in autoimmune hypophysitis, suggesting that immunological approach may be one of the therapeutic strategies for ACTH-producing adenomas. However, the efficacy of immune therapy for general ACTH-producing adenomas remains unclear. And cortisol levels expected to affect the tumor microenvironment (TME). Objectives To clarify whether ACTH-producing adenomas are sensitive to immunotherapy and if this approach can be a potential treatment for these challenging tumors. Methods TME was examined by immunostaining using pituitary adenomas specimens. This study consists of two phases: In phase 1, TME of ACTH-producing adenomas (n=29) was analyzed by comparing with GH-producing pituitary adenomas (GHomas) (n=10) and nonfunctioning pituitary adenomas (NPAs) derived from gonadotrophs origin (Gnomas) (n=10). In phase 2, corticotroph adenomas were divided into three groups that were considered to be at different cortisol exposure; naïve ACTH-producing adenomas (Naïve, n=29), treatment with metyrapone (Met, n=13), and silent corticotroph adenomas (SCAs) (n=12). These TME were analyzed and compared. To detect T-lymphocytes and M2 macrophages, antibodies to CD3, 4, or 8, and CD163 were used, respectively. The association between TME and patients’ background data including urinary free cortisol levels were analyzed. Results Phase 1: Tumor infiltrating lymphocytes (TILs) evaluated by CD3 + cells inACTH-producing adenomas were less than those in the GHomas (p Conclusions We revealed for the first time that low TILs are common features among corticotroph adenomas regardless cortisol levels. Moreover, the CD4 + cells but not CD8 + cells were higher in tumors with low cortisol status, suggesting induction of CD8 + cells are required for the immune therapy to corticotroph adenomas. Regarding with CD163 + cells, no association with tumor behavior was shown rather related to cortisol exposure status. Presentation: No date and time listed

Objectives There is a little evidence for the TRH test criteria in the diagnosis of Central Hypothyroidism (CeH). Therefore, we investigated the significance of the TRH test for Central Hypothyroidism due to Non-Functional Pituitary Adenoma. Methods 107 cases of Non-functional pituitary adenoma (NFPA) in Gunma University Hospital Neurosurgery and Toranomon Hospital Intercerebral Pituitary Surgery, between 2007 to 2020 are studied. Subjects divided into CeH group (n = 19) with FT4 below the reference value and a normal group (n = 88). Serum TSH level was determined before (basal-TSH value) and 30, 60, 120 minutes after TRH administration. Peak-TSH value, delayed and prolonged responses were analyzed. A peak-TSH occurring at 60 minutes or later was considered as a delayed response. If 120-minutes TSH value to peak-TSH value ratio is equal to or higher than 0.6 was considered as a prolonged response. Results The basal-TSH was higher in the CeH group than in the normal group (median 2.7 vs. 1.5μIU/mL) (p Conclusion In central hypothyroidism due to Non-Functional Pituitary Adenoma, the basal-TSH value does not usually decrease, but rather be higher value within the reference range. 30-minute value or peak value was not useful in diagnosis, yet the prolonged response was more significant. Central Hypothyroidism in Non-Functional Pituitary Adenoma, can be diagnosed with sensitivity of 79% and specificity of 78% when the 120-minute value to peak value ratio is equal to or lower than 0.65. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.

ContextThe most frequent cause of central hypothyroidism (CeH) is pituitary adenomas, but the mechanisms remain unclear.ObjectiveWe investigated serum thyroid levels and GH/IGF-1 in central hypothyroidism in untreated patients with pituitary nonfunctioning and GH-secreting adenomas.DesignThis was a retrospective cross-sectional study of cases collected from Gunma University and Toranomon Hospitals between 2007 and 2016.PatientsOne-hundred thirty-nine cases of nonfunctioning pituitary adenoma (NFPA) and 150 cases of GH-secreting pituitary adenoma (GHPA) were analyzed.Main Outcome MeasuresThe correlations between thyroid levels, several clinicopathological parameters, and GH/IGF-1 were examined.ResultsTwenty-four percent of NFPA patients had CeH. The severity did not correlate with tumor size, age, or sex, and all cases had normal TSH levels. In contrast, only 8.7% of GHPA patients had CeH; approximately half had normal TSH levels and approximately half had low TSH levels. Serum TSH levels in GHPA patients were significantly lower and free T4 (FT4) and free T3 levels were higher than those in patients with NFPA. Furthermore, approximately one-fourth of GHPA patients had normal FT4 and low TSH levels. In addition, serum FT4 levels and serum TSH levels were positively and negatively correlated, respectively, with serum IGF-1 levels. Furthermore, IGF-1 levels in patients with GHPA decreased with age.Conclusions(i) NFPA patients with CeH had TSH levels within a normal range. (ii) GHPA patients had a low incidence of CeH, which may be a result of stimulated thyroid function by GH/IGF-1. (iii) We found an age-dependent decrease in serum IGF-1 levels in patients with GHPA.

Context Paradoxical increases in serum cortisol in the dexamethasone suppression test (DST) have been rarely observed in Cushing disease (CD). Its pathophysiology and prevalence remain unclear. Case Description A 62-year-old woman with suspected CD showed paradoxical increases in cortisol after both 1-mg and 8-mg DST (1.95-fold and 2.52-fold, respectively). The initiation of metyrapone paradoxically decreased plasma adrenocorticotropic hormone (ACTH) levels and suppressed cortisol levels. Moreover, the pituitary tumor considerably shrank during metyrapone treatment. Ex Vivo Experiments The resected tumor tissue was enzymatically digested, dispersed, and embedded into Matrigel as 3D cultured cells. ACTH levels in the media were measured. In this tumor culture, ACTH levels increased 1.3-fold after dexamethasone treatment (P < 0.01) while control tumor cultures exhibited no increase in ACTH levels, but rather a 20% to 40% suppression (P < 0.05). Clinical Study A cross-sectional, retrospective, multicenter study that included 92 patients with CD who underwent both low-dose and high-dose DST from 2014 to 2020 was performed. Eight cases (8.7%) showed an increase in serum cortisol after both low-dose and high-dose DST. Conclusion This is the first report of a patient with glucocorticoid (GC)-driven positive feedback CD who showed both ACTH suppression and tumor shrinkage by metyrapone. Our cohort study revealed that 8.7% of patients with CD patients possibly possess GC-driven positive-feedback systems, thereby suggesting the presence of a new subtype of CD that is different from the majority of CD cases. The mechanisms exhibiting GC positive feedback in CD and the therapeutic approach for these patients remain to be investigated.

Background: Although paradoxical response of cortisol (F) during high dose dexamethasone (Dex) suppression test (DST) is observed in a part of ectopic ACTH syndrome, few reports have been shown in Cushing’s disease (CD). It suggests a presence of glucocorticoid (GC)-driven positive-feedback loop. However, the underlying mechanism remains elusive. Here we present a case of CD showing clear clinical and pathophysiological evidences of GC positive-feedback using ex vivo 3-dimensional (D) culture method. Case: A 62-year-old woman manifested typical Cushing’s symptoms, including moon face, central obesity, hypertension, hypokalemia, and vertebral fractures. Endocrinological data were consistent with a diagnosis of CD; morning plasma ACTH 299 pg/mL, serum F 28 μg/dL, midnight serum F 43 μg/dL, and 24hr urinary free cortisol 988 μg/day. CRH test showed a slight increase in plasma ACTH levels (1.4 fold), and pituitary MRI revealed a 14 mm macroadenoma invading into the left cavernous sinus. Interestingly, both 1 mg and 8 mg DST showed a paradoxical increase in serum cortisol levels (27.7→64.7μg/dL and 17.17→35.68μg/dL, respectively). These data indicated a presence of positive-feedback response to GC in the tumor. Indeed, after the initiation of metyrapone (1,000 mg/day) administration for the treatment of hypercortisolemia, plasma ACTH levels were decreased to 147.5 pg/mL accompanied with the decrease in serum F levels to 4.12 μg/dL. Moreover, pituitary tumor obviously shrank during the metyrapone treatment. Thereafter, we undertook transsphenoidal surgery and plasma ACTH and serum F levels decreased to 35.8 pg/mL and 7.6μg/dL, respectively. ex vivo studies: To prove the presence of the positive feedback response and explore the underlying mechanisms, we performed a primary culture experiment using the tumor and applied 3D culture method. The resected tumor tissue was enzymatically digested, dispersed and embedded into the Matrigel. Then cells were treated with Dex (0.1-10 nM), and ACTH concentrations were measured after 72 hrs. Interestingly, ACTH levels significantly increased by 10 nM Dex treatment at 72 h (129 %, p Conclusions: To our knowledge, this is the first case of CD who showed clinically obvious GC positive-feedback that was proved by ex vivo 3D primary culture cell models. Notably, tumor shrinkage was observed during the metyrapone treatment, suggesting that GC positive-feedback mechanisms also involve tumor proliferation. Further investigation is required for elucidating the underlying mechanisms.

Temozolomide (TMZ) is an alkylating agent and was a first-line chemotherapeutic agent for malignant gliomas. Recently, TMZ has been documented to be effective against atypical pituitary adenomas (APAs) and pituitary carcinomas (PCs).The clinical and pathological characteristics of APAs and PCs treated with TMZ in Japan were surveyed and analyzed retrospectively.Members of the Japan Society of Hypothalamic and Pituitary Tumors were surveyed regarding the clinical characteristics of APAs and PCs treated with TMZ. Stored tumor samples were gathered from the responders and were assessed by the immunohistochemistry of Ki-67, O(6)-methyl-guanine-DNA methyltransferase, p53, MSH6, and anterior pituitary hormones. Responses to TMZ treatment were defined as complete response (CR), partial response (PR), progressive disease (PD), and stable disease (SD) according to RECIST (Response Evaluation Criteria in Solid Tumors) version 2.0.Three samples from 3 subjects with APA and 11 samples from 10 subjects with PC were available.The 13 subjects had APAs and PCs consisting of 5 prolactin-producing tumors, 5 ACTH-producing tumors, and 3 null cell adenomas. The clinical response to TMZ treatment was as follows: 4 cases of CR and PR (31%), 2 cases of SD (15%), 6 cases of recurrence after CR and PR (46%), and 1 case of PD (8%). However, considerable subjects had recurrent disease after a response to TMZ. The immunohistochemical findings of Ki-67, O(6)-methyl-guanine-DNA methyltransferase, and p53 did not show any significant correlation with the efficacy of TMZ. However, the immunopositivity of MSH6 was positively correlated with TMZ response (P = .015, Fisher's exact test).This study showed that preserving MSH6 function was contributory to the effectiveness of TMZ in malignant pituitary neoplasms. It is necessary to survey more cases and evaluate multifactor analyses.

Methods for preoperative diagnosis of prohormone convertase 2 (PC2)-positive ACTH-producing pituitary adenomas (APPAs) have not been established. Also, their characteristics are not evident.This study was designed to understand the meaning of plasma alphaMSH levels and the role of cell proliferation-signaling molecules in PC2-positive APPAs.Nineteen human APPAs (four males and 15 females) were examined for the expression of PC2, phosphorylated ERK1/2, phosphorylated Akt1/2/3 (p-Akt) and receptor tyrosine kinases. alphaMSH was measured in extracted plasma from 17 APPA patients and 30 healthy volunteers.Nine adenomas (47.4%) were immunopositive for PC2 and were large and invasive in nature. In all normal controls and eight PC2-negative cases, plasma alphaMSH was undetectable, whereas in four PC2-positive cases, it was detected at abnormally higher levels. Eight adenomas (42.1%) were immunopositive for both PC2 and p-Akt, and seven others (36.8%) were immunonegative for both, suggesting significant coexpression of PC2 and p-Akt in tumors. Quantitative RT-PCR revealed that PC2 expression is associated with phosphorylation of Akt but not with its gene expression. Most APPAs expressed receptor tyrosine kinases, but membrane-bound receptors could not be identified.Our study suggests that PC2 expression and Akt phosphorylation are related at the molecular level, resulting in a change in cell cycle and an increase in pituitary adenoma size. An elevation of plasma alphaMSH could conjecture the activation of the phosphatidylinositol 3/Akt cascade in PC2-positive APPAs and may become a valuable clinical marker of tumor growth in Cushing's disease.

Among the factors that promote the growth of human pituitary corticotroph adenomas (hPCAs), the proliferative potential of CRH secreted by hPCAs on these tumors is not well known. In this study, the CRH messenger ribonucleic acid (mRNA) transcripts were demonstrated on paraffin sections using the quantitative in situ hybridization method in 37 of 43 hPCAs, including 17 of 22 microadenomas, 15 of 15 macroadenomas, and 5 of 6 locally invasive adenomas according to Hardy's classification of pituitary adenomas. The more important findings were that CRH mRNA signal intensity in pituitary corticotroph adenoma cells was linearly correlated with Ki-67 tumor growth fractions (r = 0.802; P < 0.0001), and in macroadenoma and locally invasive adenoma cells it was significantly higher than in microadenoma cells (P = 0.035). On the other hand, CRH mRNA transcript accumulation was absent or negligible in 10 normal pituitary glands (P = 0.005). This is the first report of the frequent expression of CRH mRNA localized in human pituitary corticotroph adenoma cells. These results indicate that CRH from a local source of corticotroph adenoma cells not only has autocrine/paracrine functions in corticotroph adenomatous tissue, but also is an important factor associated with a proliferative potential of hPCAs.

To investigate the role of tumor suppressor genes in sporadic pituitary adenomas, we first analyzed loss of heterozygosity on 11q13 with microsatellite analysis in 31 tumors. Loss of heterozygosity on 11q13 was detected in 1 mixed GH/PRL adenoma, and the somatic 22-bp deletion of the multiple endocrine neoplasia type 1 (MEN1) gene encoding menin was detected in this tumor. Trisomy 11 suggested by the decreased mean allelic ratios of 66% or 65% for 16 or 13 microsatellite markers, respectively, in 2 of 31 pituitary adenomas was confirmed by interphase fluorescence in situ hybridization. Screening for mutations of the MEN1 gene did not find mutations with PCR-single strand conformation polymorphism analysis in other pituitary adenomas retaining heterozygosity on 11q13. Based on these, it is concluded that inactivation of the MEN1 gene comprises a rare etiology for tumorigenesis of the pituitary gland, and that trisomy 11 or another gene(s) may contribute to the pathogenesis of sporadic pituitary adenomas.

Germ-line mutations of the MEN1 gene were analyzed in five cases of familial and four cases of sporadic multiple endocrine neoplasia type 1 (MEN-1), six cases in three independent pedigrees of familial pituitary adenoma without MEN-1, and three cases of familial isolated primary hyperparathyroidism (FIHP) in Japanese. Eight different types of germ-line mutations in all nine cases of MEN-1 were distributed in exons 2, 3, 7, and 10 and intron 7 of the MEN1 gene. Loss of heterozygosity (LOH) on 11q13 was detected in all nine tumors of these cases with microsatellite analysis. No germ-line mutation of the MEN1 gene was detected in three pedigrees of familial pituitary adenoma and three cases of FIHP. LOH on 11q13 was detected in two cases in one pedigree of familial pituitary adenoma, and one of them showed a heterozygous somatic mutation of the MEN1 gene. No LOH on 11q13 was detected in three cases of FIHP. Based on these, we conclude that the loss of function of menin is etiological for familial or sporadic MEN-1, but not for FIHP or most familial pituitary adenoma without MEN-1.

To study the etiologic roles of genes on chromosome 12 for the pituitary tumorigenesis of adenomas, mutations of the p27Kip1 gene and allelic ratios of 18 microsatellite markers on the entire chromosome 12 were studied in 33 pituitary adenomas. The p27Kip1 gene on chromosome 12p12-p13 encoding an inhibitor of complexes between cyclins and cyclin-dependent kinases is supposed to function as the tumor suppressor gene. Among 31 sporadic and 2 familial pituitary adenomas, PCR-single strand conformation polymorphism analysis detected three polymorphic changes but no tumor-specific mutations of the p27Kip1 gene. Genotyping of 18 microsatellite markers on the entire chromosome 12 detected the uniformly decreased allelic ratios ranging from 54-66% in 8 of 33 pituitary adenomas (24%), although no loss of heterozygosity was detected. Fluorescence in situ hybridization confirmed trisomy 12 in all 5 available samples out of these 8 samples. Based on these, we conclude that not mutations of the p27Kip1 gene, but trisomy 12 may be etiologically important in a subgroup of pituitary adenomas.

Two of three brothers (the second and third brothers) and their uncle (their mother’s brother) presented acrogigantism without multiple endocrine neoplasia type 1 (MEN 1). An invasive macroadenoma was found in the second brother, and it was histologically confirmed as a sparsely granulated GH cell adenoma. Two distinct microadenomas were found in the third brother, and these were histologically diagnosed as a mixed GH cell and PRL cell adenoma and a sparsely granulated GH cell adenoma, respectively. The loss of heterozygosity (LOH) was analyzed in two adenomas (GH cell adenoma from the second brother and a mixed GH cell and PRL cell adenoma from the third brother) by determining microsatellite polymorphisms of DNAs from tumors and patients’ leukocytes. The LOH was found on the chromosome 11q13, whereas LOH was not detected on 1p31-36, 2p, 3p, 4, 5, 6p, 7, 8, 9p21-22, 12p, and 19q13 in both pituitary adenomas examined. The haplotype analysis showed that the same haplotype on 11q13 was found in their mother...

Silent somatotroph adenomas are defined as tumors showing morphological features consistent with GH production, but no clinical evidence of GH excess. We report here the case of a 46-yr-old woman with a large pituitary macroadenoma, slightly elevated serum GH levels, high serum insulin-like growth factor-I levels, and abnormal GH dynamics, but no acromegaly. The endocrinological abnormalities receded after transphenoidal surgery despite tumor persistence, as shown by neuroimaging. The reverse hemolytic plaque assay, performed for the first time in a silent GH cell adenoma, demonstrated that the number of GH cells releasing GH and the amount of GH discharged from individual cells were less than those in clinically functioning somatotroph adenomas. Thus, it is conceivable that this tumor secreted only small quantities of GH and for only short periods, providing an explanation for the lack of acromegaly. It appears that silent somatotroph adenomas do not represent a distinct entity. It is more likely that there is a continuous spectrum from clinically functioning, sparsely granulated somatotroph adenomas with high serum GH levels to silent somatotroph adenomas with normal serum GH levels. The cause of the lack of GH oversecretion in silent GH cell adenomas has yet to be elucidated.

The reverse hemolytic plaque assay was used to study hormone release in vitro by seven clinically nonfunctioning human pituitary adenomas associated with no clinical or biochemical evidence of hormone excess. Four of seven tumors were oncocytomas, one a null cell adenoma, and two gonadotroph adenomas based on immunocytochemical and ultrastructural features. In all seven tumors, plaques were formed with antiserum against beta FSH; four produced plaques for beta LH, and five for glycoprotein hormone alpha-subunit. The percentage of plaque-forming cells and the mean size of plaques were smaller than those of clinically functioning adenomas studied for comparison (five GH- and/or PRL-producing adenomas). These results correlated with those of hormone release in tissue culture, immunocytochemistry on paraffin secretions of the tumors, and immunocytochemistry after reverse hemolytic plaque assay. We conclude that clinically nonfunctioning pituitary adenomas release small quantities of hormones, primarily gonadotropins, and that hormone release is attributable to only a small percentage of tumor cells.