Adenomas In Cushing's Disease Research Articles

1233 A Benchmark for Early Biochemical Success and Long-term Remission After Resection of Corticotroph Adenomas in Cushing Disease

INTRODUCTION: In Cushing disease, the definition of success after resection of corticotroph adenomas remains unclear given inconsistent pathology and high recurrence rates despite initial biochemical remission. METHODS: We collected 12 years of in-hospital cortisol data after the removal of pathology-confirmed corticotroph adenomas in patients with Cushing disease. A novel cortisol halving time analysis was performed using exponential decay modeling, results were dichotomized a priori, and then applied to an external cohort of patients. Halving time was compared to first post-operative cortisol and nadir cortisol variables by estimating long-term remission and time-to-recurrence using Cox regression modeling and the Kaplan-Meier method. RESULTS: A total of 320 patients met inclusion/exclusion criteria for the final analysis, and 26 of those patients developed recurrent disease. Median follow-up time was 2.1 years, although 62 patients had five years or longer follow-up time. Higher first post-operative cortisol and higher nadir were associated with increased risk of recurrence. Patients who had a first post-operative cortisol = 50 µd/dL were 4.1 times more likely to recur than those with a first post-operative cortisol < 50 µd/dL (HR 4.1, 1.8-9.2; p = 0.0003). Halving time was not associated with recurrence (HR 1.7, 0.8-3.8, p = 0.18). Patients with a nadir cortisol =2 µg/dL were 6.6 times more likely to recur than those with a nadir cortisol of < 2 µg/dL (HR 6.6, 2.6-16.6, p < 0.0001). CONCLUSIONS: Post-operative nadir serum cortisol is the most important cortisol variable associated with recurrence and time-to-recurrence. Compared to first post-operative cortisol and cortisol halving time, a nadir < 2 µg/dL occurring within the first three post-operative days showed the strongest association with long-term remission.

Diurnal Range and Intra-patient Variability of ACTH Is Restored With Remission in Cushing's Disease.

Single ACTH measurements have limited ability to distinguish patients with Cushing's disease (CD) from those in remission or with other conditions. To investigate the changes in ACTH levels before and after transsphenoidal surgery (TSS) to identify trends that could confirm remission from CD and help establish ACTH cutoffs for targeted clinical trials in CD. Retrospective analysis of CD patients who underwent TSS from 2005 to -2019. Referral center. CD patients (n = 253) with ACTH measurements before and after TSS. TSS for CD. Remission after TSS. Remission was observed in 223 patients after TSS. Those in remission had higher ACTH variability at AM (P = .02) and PM (P < .001) time points compared to nonremission. The nonremission group had a significantly narrower diurnal range compared to the remission group (P = <.0001). A decrease in plasma ACTH of ≥50% from mean preoperative levels predicted CD remission after TSS, especially when using PM values. The absolute plasma ACTH concentration and ratio of preoperative to postoperative values were significantly associated with nonremission after multivariable logistic regression (adj P < .001 and .001, respectively). Our findings suggest that ACTH variability is suppressed in CD, and remission from CD is associated with the restoration of this variability. Furthermore, a decrease in plasma ACTH by 50% or more may serve as a predictor of remission post-TSS. These insights could guide clinicians in developing rational outcome measures for interventions targeting CD adenomas.

MRI for Cushing Disease: A Systematic Review.

MR imaging is key in the diagnostic work-up of Cushing disease. The sensitivity of MR imaging in Cushing disease is not known nor is the prognostic significance of "MR imaging-negative" disease. Our aim was to determine the overall sensitivity and prognostic significance of MR imaging localization of Cushing disease. We performed a systematic review of the MEDLINE and PubMed databases for cohort studies reporting the sensitivity of MR imaging for the detection of adenomas in Cushing disease. This study included 57 studies, comprising 5651 patients. Risk of bias was assessed using the methodological index for non-randomized studies criteria. Meta-analysis of proportions and pooled subgroup analysis were performed. Overall sensitivity was 73.4% (95% CI, 68.8%-77.7%), and the sensitivity for microadenomas was 70.6% (66.2%-74.6%). There was a trend toward greater sensitivity in more recent studies and with the use of higher-field-strength scanners. Thinner-section acquisitions and gadolinium-enhanced imaging, particularly dynamic sequences, also increased the sensitivity. The use of FLAIR and newer 3D spoiled gradient-echo and FSE sequences, such as spoiled gradient-echo sequences and sampling perfection with application-optimized contrasts by using different flip angle evolutions, may further increase the sensitivity but appear complementary to standard 2D spin-echo sequences. MR imaging detection conferred a 2.63-fold (95% CI, 2.06-3.35-fold) increase in remission for microadenomas compared with MR imaging-negative Cushing disease. Pooled analysis is limited by heterogeneity among studies. We could not account for variation in image interpretation and tumor characteristics. Detection on MR imaging improves the chances of curative resection of adenomas in Cushing disease. The evolution of MR imaging technology has improved the sensitivity for adenoma detection. Given the prognostic importance of MR imaging localization, further effort should be made to improve MR imaging protocols for Cushing disease.

508 Syngeneic Pair-wise Transcriptomic/Epigenomic Analysis of Sporadic Cushing’s Adenomas and Adjacent Gland Reveals Novel Tumorigenic Pathways

INTRODUCTION: Sporadic pituitary adenomas are found in more than 10% of the general population and are mutationally bland. The normal human pituitary transcriptome is unknown, precluding syngeneic studies on Cushing's disease (CD) pathogenesis. METHODS: We surgically annotated adenomas and adjacent normal glands and performed single cell RNAseq (scRNAseq; 10X Genomics Chromium 3’) on 28,724 cells from 6 patients (3 CD and 3 non-CD). We performed bulk RNAseq (Illumina Nextseq) in 7 patients (5 CD and 2 non-CD) and DNA methylation analysis (Illumina Infinium MethylEPIC 850k) in 6 patients (3 CD and 3 non-CD). Transcriptomic/epigenomic data were analyzed using R packages and custom Python pipelines for pairwise differential expression analyses. RESULTS: We mapped the post-natal human pituitary gland at single cell resolution and identified canonical resident pituitary cell classes. Corticotrophs were abundant within CD adenomas but rare in the surrounding normal gland, with downregulated POMC expression consistent with repression in the hypercortisolemic state. CD corticotrophs overexpressed signature genes PCSK1, PPP1R17, EGLN, RSPO3 and PMAIP1 compared to surrounding tissues. This gene signature was specific for core adenomas and correlated with anatomic demarcation at the time of surgery. We confirmed overexpression of PCSK1, PPP1R17, RSPO3 and PMAIP1 by independent RNAseq. We identified hypomethylation at the promoters of PMAIP1, PPP1R17 and RSPO3 but not PCSK1 or EGLN1 in CD adenomas. CONCLUSION: We mapped the transcriptome of the adult human pituitary gland, identified novel signature genes for CD and highlighted DNA methylation as an epigenetic mechanism of transcriptional activation in a number of Cushing’s adenoma signature genes. We provide novel, targetable mechanistic pathways for therapeutic intervention in Cushing’s disease.

Clinical, hormonal, radiological and morphological comparison of patients with clinically evident Cushing's disease and patients with silent corticotroph cell adenoma

Purpose: The aim of the study was to review our clinical 7 years experiences with silent corticotroph adenoma (SCA) and Cushing disease (CD) with regard to clinical, radiological features, immunohistochemical and surgical outcomes and compare the results between two groups.&#x0D; Material and Methods: We retrospectively reviewed a series of patients (n=17; SCA 10, CD 7) with corticotroph adenomas and collected biochemical, neuroradiological and pathological data of those during follow-up time. &#x0D; Results: Mean pre-operative ACTH values of the patients with CD and SCA were 60.4±29.5, 45.5±34.6 pg/mL, respectively. Mean preoperative cortisol of the patient with CD (21.2±4.4 vs 11.5±4.0 mcg/dL) was increased than the patients with SCA measurements’. Patients with SCA had larger pituitary mass (20.4±4.5 vs 8.8±4.0 mm) than the patients with CD. And cavernous sinus invasion also was determined in all patients with SCA. Sparsely granulation staining was more common in all patients with SCA (7/10) or CD (4/7).&#x0D; Conclusion: SCAs are totally different from functional CD adenomas related with clinical characteristics and postoperative outcomes. SCA can be thought of as a diverse group of pituitary adenomas.

GENE-23. GENOME-WIDE DNA METHYLATION PROFILES DISTINGUISH SILENT FROM NON-SILENT ACTH ADENOMAS

Abstract BACKGROUND Corticotroph adenomas are immunopositive for adrenocorticotrophic hormone (ACTH) associated with elevated blood ACTH levels leading to Cushing disease (CD). Yet silent ACTH adenomas (SCA) immunostain for ACTH but do not cause hypercortisolism. SCA have consistently been shown to have a more aggressive postoperative course, then nonfunctioning and ACTH adenomas. Here we show that genome-wide methylation profiles can be used to distinguish ACTH adenomas from SCA. METHODS16 SCA patients and 19 CD patients that underwent transsphenoidal resection were included. Tumor size was measured by MRI. Tumor histology was proven by immunstain for ACTH and routine histopathology. Sanger sequencing was performed to analyse mutational burden within the ACTH locus (n=3). Genome-wide DNA methylation profiling was performed using a 850k Illumina EPIC array and classified by the DKFZ brain tumor classifier as well as PCA analysis using R (n=17). RESULTS Mean age was 55,4 and 46,2 for SCA and CD patients, respectively. Patients with SCA had significantly larger tumors (SCA: 12,56±3,07ccm; CD: 1,9±1,2ccm p&lt; 0.01). Both SCA and CD tumors showed strong expression of ACTH as proven by immunohistochemistry. Sanger sequencing revealed no mutations within the ACTH gene locus in SCA. Both, SCA and CD tumors classified as ACTH adenomas by genome-wide methylation profiling. Further PCA of DNA methylation profiles allowed subtype tumor classification into SCA and CD adenomas. CONCLUSION SCA show a strong expression of ACTH without causing hypercortisolism. The reason for this is not yet known. Our data suggest that SCA do not harbor specific mutations within the ACTH gene locus. However genome wide methylation profiles allows subgrouping of SCA and CD adenomas.

USP8 Mutations in Pituitary Cushing Adenomas-Targeted Analysis by Next-Generation Sequencing.

Gain-of-function somatic mutations in the ubiquitin specific protease 8 (USP8) gene have recently been reported as a cause of pituitary adenomas in Cushing disease. Molecular diagnostic testing of tumor tissue may aid in the diagnosis of specimens obtained through therapeutic transsphenoidal surgery; however, for small tumors, availability of fresh tissue is limited, and contamination with normal tissue is frequent. We performed molecular testing of DNA isolated from single formalin-fixed and paraffin-embedded (FFPE) tissue sections of 42 pituitary adenomas from patients with Cushing disease (27 female patients and 15 male patients; mean age at surgery, 42.5 years; mean tumor size, 12.2 mm). By Sanger sequencing, we identified previously reported USP8 missense mutations in six tumors. Targeted next-generation sequencing (NGS) revealed known or previously undescribed missense mutations in three additional tumors (two with two different mutations each), with mutant allele frequencies as low as 3%. Of the nine tumors with USP8 mutations (mutation frequency, 21.4%), seven were from female patients (mutation frequency, 25.9%), and two were from male patients (mutation frequency, 13.3%). Mutant tumors were on average 11.4 mm in size, and patients with mutations were on average 43.9 years of age. The overall USP8 mutation frequency in our cohort was lower than in previously described cohorts, and we did not observe USP8 deletions that were frequent in other cohorts. We demonstrate that testing for USP8 variants can be performed from small amounts of FFPE tissue. NGS showed higher sensitivity for USP8 mutation detection than did Sanger sequencing. Assessment for USP8 mutations may complement histopathological diagnosis.

Long-term outcomes of tissue-based ACTH-antibody assay-guided transsphenoidal resection of pituitary adenomas in Cushing disease.

OBJECTIVE Cushing disease is caused by a pituitary micro- or macroadenoma that hypersecretes adrenocorticotropic hormone (ACTH), resulting in hypercortisolemia. For decades, transsphenoidal resection (TSR) has been an efficacious treatment but with certain limitations, namely precise tumor localization and complete excision. The authors evaluated the novel use of a double-antibody sandwich assay for the real-time quantitation of ACTH in resected pituitary specimens with the goals of augmenting pathological diagnosis and ultimately improving long-term patient outcome. METHODS This study involved a retrospective review of records and an analysis of assay values, pathology slides, and MRI studies of patients with Cushing disease who had undergone TSR in the period from 2009 to 2014 and had at least 1 year of follow-up in coordination with an endocrinologist. In the operating room, biopsy specimens from the patients had been analyzed for tissue ACTH concentration. Additional samples were simultaneously sent for frozen-section pathological analysis. The ACTH assay performance was compared against pathology assessments of surgical tumor samples using receiver operating characteristic (ROC) analysis and against pre- and postoperative MRI studies. RESULTS Fourteen patients underwent TSR with guidance by ACTH-antibody assay and pathological assessment of 127 biopsy samples and were followed up for an average of 3 years. The ACTH threshold for discriminating adenomatous from normal tissue was 290,000 pg/mg of tissue, based on jointly maximized sensitivity (95.0%) and specificity (71.3%). Lateralization discordance between preoperative MRI studies and surgical visualization was noted in 3 patients, confirming the impression that MRI alone may not achieve optimal localization. A majority of the patients (85.7%) attained long-term disease remission based on urinary free cortisol levels, plasma cortisol levels, and long-term corticosteroid therapy. Comparisons of patient-months of remission and treatment failure showed that the remission rate in the study sample statistically exceeds the rate in historical controls (71.9%; p = 0.0007, Fisher's exact test). Long-term unexpected hormonal deficiencies were statistically similar between study patients (29%) and those in a meta-analysis (25%; p = 0.7596, Fisher's exact test). CONCLUSIONS These preliminary findings reflect the promising potential of tissue-based ACTH-antibody-guided assay for improving the cure rates of Cushing disease patients undergoing TSR. Further studies with larger sample sizes, further refinements of assay interpretation, and longer-term follow-ups are needed.

High-resolution(18)F-fluorodeoxyglucose positron emission tomography and magnetic resonance imaging for pituitary adenoma detection in Cushing disease.

OBJECT High-resolution PET (hrPET) performed using a high-resolution research tomograph is reported as having a resolution of 2 mm and could be used to detect corticotroph adenomas through uptake of(18)F-fluorodeoxyglucose ((18)F-FDG). To determine the sensitivity of this imaging modality, the authors compared(18)F-FDG hrPET and MRI detection of pituitary adenomas in Cushing disease (CD). METHODS Consecutive patients with CD who underwent preoperative(18)F-FDG hrPET and MRI (spin echo [SE] and spoiled gradient recalled [SPGR] sequences) were prospectively analyzed. Standardized uptake values (SUVs) were calculated from hrPET and were compared with MRI findings. Imaging findings were correlated to operative and histological findings. RESULTS Ten patients (7 females and 3 males) were included (mean age 30.8 ± 19.3 years; range 11-59 years). MRI revealed a pituitary adenoma in 4 patients (40% of patients) on SE and 7 patients (70%) on SPGR sequences.(18)F-FDG hrPET demonstrated increased(18)F-FDG uptake consistent with an adenoma in 4 patients (40%; adenoma size range 3-14 mm). Maximum SUV was significantly higher for(18)F-FDG hrPET-positive tumors (difference = 5.1, 95% CI 2.1-8.1; p = 0.004) than for(18)F-FDG hrPET-negative tumors.(18)F-FDG hrPET positivity was not associated with tumor volume (p = 0.2) or dural invasion (p = 0.5). Midnight and morning ACTH levels were associated with(18)F-FDG hrPET positivity (p = 0.01 and 0.04, respectively) and correlated with the maximum SUV (R = 0.9; p = 0.001) and average SUV (R = 0.8; p = 0.01). All(18)F-FDG hrPET-positive adenomas had a less than a 180% ACTH increase and(18)F-FDG hrPET-negative adenomas had a greater than 180% ACTH increase after CRH stimulation (p = 0.03). Three adenomas were detected on SPGR MRI sequences that were not detected by(18)F-FDG hrPET imaging. Two adenomas not detected on SE (but no adenomas not detected on SPGR) were detected on(18)F-FDG hrPET. CONCLUSIONS While(18)F-FDG hrPET imaging can detect small functioning corticotroph adenomas and is more sensitive than SE MRI, SPGR MRI is more sensitive than(18)F-FDG hrPET and SE MRI in the detection of CD-associated pituitary adenomas. Response to CRH stimulation can predict(18)F-FDG hrPET-positive adenomas in CD.

Identification of small compound biomarkers of pituitary adenoma: a bilateral inferior petrosal sinus sampling study

Evaluation of the pathogenic mechanisms underlying Cushing disease (CD) is limited partly by the inaccessibility of the pituitary gland for biopsy. We used bilateral inferior petrosal sinus sampling (BIPSS), the...

Ghrelin Immunexpression in Pituitary Adenomas

Ghrelin, an orexigenic hormone occurs mainly in stomach, and in several tumor types. We investigated ghrelin immunopositivity using the streptavidin‐biotin‐peroxidase complex method in the full spectrum of pituitary adenoma subtypes. Quantification was undertaken by analyzing both the percentage of tumor cells stained for ghrelin and the staining intensity.Cytoplasmic ghrelin immunopositivity was identified in several adenoma subtypes. Not every cell was immunopositive; the intensity of staining differed among cells within the given lesion and among the various tumors. The highest scores were noted in GH adenomas exposed to somatostatin analogues. In decreasing order, lower scores were encountered in ACTH‐producing, silent subtype 1 and 2 adenomas in Cushing disease, silent subtype 3 adenomas, untreated GH adenomas, dopamine agonist‐treated PRL adenomas, gonadotroph adenomas, and ACTH adenomas in Nelson syndrome. No immunoreactivity was found in TSH, untreated PRL‐producing, and null cell adenomas. The high immunoexpression of ghrelin in GH adenomas exposed to somatostatin analogues, may be due to increased ghrelin synthesis or to suppression of its release.

Ghrelin immunoexpression in pituitary adenomas

Ghrelin, an orexigenic hormone, is normally produced mainly in stomach. In addition, it has been demonstrated in gastric carcinoid tumors and less often in other neuroendocrine tumors. We investigated ghrelin expression by immunohistochemistry (streptavidin-biotin-peroxidase complex method) in the full spectrum of resected pituitary adenoma subtypes. Quantification of staining considered both the frequency of ghrelin-reactive tumor cells as well as their staining intensity. Cytoplasmic ghrelin immunopositivity was identified in several adenoma subtypes. Cellular staining varied considerably. In addition, the intensity of cell staining differed within the same tumor and between adenoma subtypes. The highest scores were noted in GH producing adenomas exposed to long-acting somatostatin analogs. In decreasing order, lower scores were encountered in ACTH adenomas in Cushing disease, silent subtype 3 adenomas, untreated GH adenomas, silent corticotroph adenomas of subtypes 1 and 2, dopamine agonist-treated PRL adenomas, ACTH adenomas in Nelson syndrome, and gonadotroph adenomas. No significant immunoreactivity was noted in TSH, untreated PRL, and null cell adenomas. The high immunoexpression of ghrelin in GH adenomas exposed to long-acting somatostatin analogs remains unexplained, but may be due to either increased ghrelin production or to suppression of its release. Based on our findings, it appears that ghrelin immunopositivity does not serve as a biomarker of biologic behavior, prognosis and therapeutic responsiveness in pituitary adenomas.

The diagnostic value of fused positron emission tomography/computed tomography in the localization of adrenocorticotropin-secreting pituitary adenoma in Cushing’s disease

Despite the high resolution of magnetic resonance imaging (MRI) of the pituitary gland, up to 40% of cases of Cushing's disease (CD) have normal MRI. Fused images of positron emission tomography and computed tomography (PET-CT) may have a potential diagnostic role in CD in general and in such cases in particular. Objective of this study is to explore the diagnostic potential of PET-CT for localization of adrenocorticotropin-secreting pituitary adenomas in CD. PET-CT was performed in 12 cases with de novo (7 cases) or persistent CD (5 cases) that were proven to have CD on biochemical, radiological and/or histopathological findings. These cases had a definite CD confirmed on histopathological and immunostaining examination of the subsequent transphenoidal surgical specimens (10 cases) and/or bilateral inferior petrosal sinus sampling (IPSS, 4 cases). PET-CT was positive in 7 of the 12 cases of CD (58%) showing a focal area of uptake in the pituitary gland. In these seven cases, MRI was positive in six (85.7%) but negative in one case (14.3%). In the other five cases with negative PET-CT, MRI was positive in two and negative in three cases. Of four cases with negative MRI, PET-CT was positive in one case (25%). We conclude that PET-CT is positive in around 60% of the cases of CD. Although the majority of cases with positive PET-CT had positive MRI, PET-CT may detect some cases with negative MRI and thus provides important diagnostic information. If these findings are confirmed in larger studies, PET-CT might become an important diagnostic technique, especially when the more invasive and technically demanding procedure of IPSS is not available or inconclusive.

Immunohistochemical properties of silent corticotroph adenoma and Cushing’s disease

Proopiomelanocortin processing in corticotroph cells is known to be operated by prohormone convertase (PC) 1/3 which is activating several pro-proteins and prohormones by intracellular limited proteolysis processing. In this study, we hypothesized that PC1/3 expression differs between Cushing's disease (CD) and silent corticotroph adenoma (SCA), and investigated whether PC1/3 expression is involved in the adrenocorticotropin (ACTH) silence of SCA. We performed immunohistochemical analysis of pituitary adenoma specimens for six adenohypophysial hormones, PC1/3 and chromogranin A (CgA). Subjects for this study consisted of 12 anterior pituitary adenomas of CD (1 male, 11 female; 14-70 years old) and 31 non-functioning adenomas (23 male, 8 female; 32-71 years old).ACTH immunoreactivity was observed in all of CD and three of 31 non-functioning adenomas. The three cases diagnosed as SCA were also positive for growth hormone and follicle-stimulating hormone. Cushing's adenomas and SCAs were all positive for PC1/3. PC1/3-positive cells did not always colocalize with ACTH but some of them colocalized with CgA in SCAs. Even if PC1/3 is not present in corticotroph cells, PC1/3 immunoreactivity in SCA may originate from CgA-positive cells. We conclude that immunohistochemistry for PC1/3 is not helpful for differential diagnosis between CD and SCA in clinical practice, though the regulation of PC1/3 expression is likely to be an important etiological factor in ACTH silence of SCA. The diversity of immunohistochemical properties of SCA leads us to speculate that it is not a single entity and may be a general diagnostic term for adenomas of varying etiology.

An Assessment of Petrosal Sinus Sampling for Localization of Pituitary Microadenomas in Children with Cushing Disease

Pituitary adenomas in Cushing disease (CD) are usually small and difficult to visualize. Bilateral inferior petrosal venous sampling (BIPSS) before and after ovine CRH stimulation is reserved for patients who have ACTH-dependent Cushing syndrome and negative magnetic resonance imaging (MRI) or positive MRI but inconsistent biochemical data. The objective of the study was to evaluate the usefulness of BIPSS as a tool for localization of a pituitary adenoma in children with CD. The study was a retrospective review of the records of 141 children who were admitted for evaluation of CD from 1982 to 2004. The study was conducted at a tertiary care center. Lateralization of ACTH secretion during BIPSS was compared with MRI and surgical findings for the localization of a microadenoma. A total of 94 patients, 49 males and 45 females with an age range of 5.3 to 18.7 yr (13 +/- 3.2 yr), underwent BIPSS. Localization of a microadenoma by BIPSS agreed with surgical location in only 58% of the cases (95% confidence interval, 43-66). The combined use of information from the MRI and inferior petrosal venous sampling did not predict the location of the tumor more frequently than MRI alone (P > 0.1), which in this study localized a lesion in 39% of the patients (95% confidence interval, 28-50). The procedure was completed successfully in all patients, and no serious complications were recorded. Although BIPSS was safe and well tolerated in an experienced center, lateralization of the ACTH gradient during BIPSS was a poor predictor of the site of the adenoma in children with CD.