An Update on Silent Corticotroph Adenomas: Diagnosis, Mechanisms, Clinical Features, and Management

With the introduction of 2017 World Health Organization (WHO) classification of endocrine tumors, T-PIT can serve as a complementary tool for identification of silent corticotroph adenomas (SCAs) in some cases if the tumor is not classifiable by pituitary hormone expression in pathological tissue samples. An increase of the proportion of SCAs among the non-functioning pituitary adenomas (NFPAs) has been witnessed under the new rule with the detection of T-PIT-positive ACTH-negative SCAs. Studies of molecular mechanisms related to SCA pathogenesis will provide new directions for the diagnosis and management of SCAs. A precise pathological diagnosis can help clinicians better identify SCAs. Understanding clinical features in the context of the pathophysiology of SCAs is critical for optimal management. It could provide information on appropriate follow-up time and aid in early recognition and treatment of potentially aggressive forms. Management approaches include surgical, radiation, and/or medical therapies.

Transsphenoidal Surgery of Invasive Corticotroph Adenomas: Results in A Series of 86 Consecutive Patients

Objective: Surgery is first-line treatment for corticotroph adenomas. Although most of corticotroph adenomas are noninvasive microadenomas that show expansive growth to surrounding tissues, a small subset of them is locally invasive and difficult to manage. The aim of this study was to evaluate surgical outcome of invasive corticotroph adenomas from a single-center. Patients and Methods: The clinical features and outcomes of CD patients who underwent transsphenoidal surgery (TSS) between January 2000 and September 2019 at Peking Union Medical College Hospital were collected from medical records. The clinical, endocrinological, radiological, histopathological, surgical outcomes and a minimum 12-month follow-up of 86 consecutive CD patients with invasive corticotroph adenomas were retrospectively reviewed. Results: Eighty-six patients with invasive corticotroph adenomas were included in the study. The average age at TSS was 37.7 years (range, 12 to 67 years), with a female-to-male ratio of 3.1:1 (65/21). The median duration of symptoms was 52.6 months (range, 1.0 to 264 months). The average of maximum diameter of tumor was 17.6 mm (range, 4.5–70 mm). All 86 patients with invasive corticotroph adenomas were performed TSS by microscopic or endoscopic approach. Gross-total resection was achieved in 63 patients (73.3%), subtotal resection in 18 (20.9%), and partial resection in 5 (5.8%). After surgery, the overall postoperative immediate remission rate was 48.8% (42/86), 51.2 % (44/86) of patients maintained persistent hypercortisolism. In 42 patients with initial remission, 16.7 % (7/42) of them experienced a recurrence. In these patients with persistent disease and recurrent CD, data about further treatment was available for 30 patients. The radiotherapy was used for 15 patients, and 4 (26.7%) of them achieved biochemical remission. Repeat TSS was performed in 5 patients, and none achieved remission. Medication was administrated in 4 patients, and one of them obtained disease control. Adrenalectomy was performed in 6 patients, and 5 (83.3 %) achieved biochemical remission. At last follow-up, (33.3%) 10 of 30 patients were in remission, and 20 patients still had persistent disease. The remission rate in patients with invasive corticotroph adenomas who underwent gross-total resection and first TSS were significantly higher than that in patients undergoing subtotal resection, partial resection, and a second TSS (all P＜0.05). However, there was no significant difference in the remission rate between patient with different tumor size, Knosp Grade and surgical approaches (P＞0.05).Conclusion: The management of invasive corticotroph adenomas remain a therapeutic challenge due to incomplete resection of invasive and/or a large adenoma. With application of multiple techniques assistance, approximately half of the patients could achieve gross-total resection and biochemical remission via TSS by experienced neurosurgeons. The extent of tumor resection and number of operations were associated with surgical remission rate in invasive corticotroph adenomas. If the remission was not achieved by surgery, other treatments including radiotherapy, medical therapy, and even bilateral adrenalectomy are required.

Do somatic USP8, USP48 and BRAF mutations differ in their genotype-phenotype correlation in Asian Indian patients with Cushing’s disease?

Purpose To estimate the prevalence of USP8, USP48 and BRAF mutations in patients with Cushing’s disease (CD) from the Indian subcontinent, and determine their genotype-phenotype correlation. Methods We prospectively recruited 46 patients with CD who underwent surgery between September 2015 and July 2019 at our institute. Fresh frozen tumour tissue was obtained in all patients. Using Sanger sequencing, the presence of somatic USP8 mutations was documented and the frequency of USP48 and BRAF mutations in USP8 wild-type corticotroph adenomas was determined. Clinical, hormonal and surgical data were then compared between USP8-, USP48- and BRAF-variant carriers and patients with wild-type tumours. Results Signature USP8 mutations were detected in 17 (37%) patients. Of the 29 USP8 wild-type adenomas, 4 (13.8%) harboured USP48 mutations, one of them being a splice-site mutation that has previously not been described. BRAF mutations were not found in any of the 29 patients. Corticotroph adenomas with USP8 mutations had a higher incidence of Crooke’s hyaline change than wild-type tumours (70.6% vs. 37.9%, p = 0.032). Adenomas with USP48 mutations had a higher rate of cavernous sinus invasion than their wild-type counterparts (50% vs. 4%, p = 0.042). No other significant phenotypic difference could be established between mutant and wild-type tumours. Conclusions The prevalence of USP8 mutations in our series of patients with CD was 37%. The prevalence of USP48 mutations in USP8 wild-type adenomas was 13.8%, including a novel splice-site mutation. BRAF mutations were not found in any USP8 wild-type tumour. USP8-mutants showed significantly more Crooke’s hyaline change and USP48-mutants were more likely to demonstrate cavernous sinus invasion.

Clinical, Biological, Radiological Pathological and Immediate Post-Operative Remission of Sparsely and Densely Granulated Corticotroph Pituitary Tumors: A Retrospective Study of a Cohort of 277 Patients With Cushing’s Disease

PurposeCushing’s disease is the most common cause of endogenous hypercortisolemia due to a corticotroph pituitary tumor. Up-to-date there is no reliable biomarker of invasiveness among corticotroph tumors, while it is well established in the literature that sparsely granulated somatotroph tumors are characterized by poorer prognosis. The aim of the study was to correlate multiple data including clinical, biochemical, radiological, and pathological findings (including granulation pattern) as well as immediate post-operative remission status among patients operated on due to corticotroph tumors.MethodsWe enrolled all patients consecutively operated on for planned transsphenoidal neurosurgery due to corticotroph PitNETs in years 2010–2018. We excluded from analysis silent corticotroph tumors, plurihormonal PitNETs, and the Crooke’s cell adenomas.ResultsWe recorded 348 hormonally active corticotroph PitNETs. The results of the analysis showed the female predominance 79.88% (n = 278), with the mean age of Cushing’s disease occurrence 43.27 years of age. The mean time from the first signs and symptoms to the operation was 2 years. The women were diagnosed earlier (20–40 years of age vs. 50–60 years of age among men). We performed a detailed analysis of 277 cases classified by granularity pattern as DG or SG corticotroph PitNETs. Densely granulated tumors (DG) occurred four times more frequently than sparsely granulated (SG) (n = 225 vs. n = 52), at similar age (mean 42.94; median 40 vs. mean 45.46; median 45.5; p = 0.3896), but were characterized by lower Knosp’s scale grades (p = 0.0147*), smaller preoperative tumors’ volumes measured at MRI, and more commonly exhibited lower Ki-67 labeling index (<3%) (p = 0.0168*). What is more, DG adenomas more frequently achieved an immediate remission status (measured as postoperative cortisol concentration <2 µg/dl; p = 0.0180*), and the mean postoperative cortisol concentration in DG group was lower than in SG group (mean 5.375 µg/dl vs. 10.47 µg/dl; median 2.49 µg/dl vs. 6.52 µg/dl; p = 0.0028**).ConclusionsOur study indicates that DG corticotroph adenomas occurred at younger age, more commonly were microadenomas as compared to SG tumors, less frequently had invasive features in comparison to SG corticotroph adenomas (p = 0.0019**), and more commonly achieved an immediate postsurgical hormonal remission (p = 0.0180*). We highlight the need for an accurate differentiation of DG and SG subtypes in the pathomorphological diagnosis of corticotropic tumors, especially in invasive PitNETs.

A Prospective Trial With Ketoconazole Induction Therapy and Octreotide Maintenance Treatment of Cushing’s Disease

Context: The lack of efficacy of octreotide in the medical treatment of Cushing’s disease (CD) may result from suppressive effects of hypercortisolism on somatostatin receptor subtype 2 (SST2) expression by corticotroph adenomas. We previously demonstrated that SST2 mRNA expression levels in corticotroph tumor cells from patients preoperatively treated with cortisol-lowering therapy are significantly higher compared to those from patients with uncontrolled CD at time of surgery. It may be hypothesized that control of cortisol production induced by steroidogenesis inhibitors may reverse SST2 expression and increase efficacy of tumor-directed therapy with octreotide. Objective: To evaluate the efficacy of a sequential strategy of initiation treatment with ketoconazole (KTC) to reduce cortisol levels, followed by octreotide as maintenance therapy in patients with CD. Patients and Design: 14 adult patients with CD were prospectively enrolled. All patients started on KTC (600-800 mg/day), and once cortisol levels were normalized, octreotide 20 mg/4 weeks was initiated which could eventually be increased to 30 mg/4 weeks. After two months of combined therapy, patients were maintained on octreotide monotherapy until the end of the study period (9 months). Treatment success was assessed by the mean of 2 collections of urinary free cortisol (UFC) levels. Results: The mean age of our study population (14 patients) was 48.6 years, 64% (n=8) were female, 85% (n=12) were newly diagnosed and naïve in treatment. Ketoconazole was able to normalize UFC level in 11 (79%) patients. Subsequently, octreotide effectively sustained normal UFC levels in 3 patients (27%) (responders). Four (36%) other patients showed a partial response to octreotide. In 3 patients, normal UFC levels were sustained for one- or two-months following discontinuation of KTC and in the other partial responder, the UFC levels at follow-up decreased by at least 50% of the baseline levels. The remaining 4 (36%) patients developed hypercortisolism as soon as ketoconazole was stopped (non-responders). Responders to octreotide had lower UFC levels at baseline when compared to partial and non-responders (1.40 ± 0.06 vs. 2.05 ± 0.20 ULN, p=0.08). Two of three responders showed improvement in weight, waist circumference, and systolic and diastolic blood pressure during the treatment period. In terms of side effects, one patient discontinued KTC because of gastrointestinal intolerance and 5 patients had a transient increase in liver enzymes. Conclusions: This proof-of-concept study shows that the sequential treatment with ketoconazole to lower cortisol levels followed by octreotide to maintain normal cortisol production seems effective in a subset of patients with mild CD. Ongoing studies aim to evaluate whether this is the result from increased SST2 expression in corticotroph adenomas.

Canine Pituitary Organoids as 3D In Vitro Model for Cushing Disease

Cushing disease (CD) is a serious endocrine disorder that is most often caused by an ACTH-secreting pituitary adenoma. Patients can be treated medically when surgery is not an option or was unsuccessful. However, currently used pituitary-targeting drugs are effective in only 40% of patients. To efficiently identify new pituitary-targeting treatment options, we need an in vitro system that closely mimics in vivo conditions. We therefore aimed to establish organoid cultures of normal anterior pituitary and corticotroph adenomas. Organoids or tumoroids are miniature three-dimensional (3D) structures grown from stem cells, that closely resemble the organ or tumor they originate from. Because CD is a thousand times more prevalent in dogs than in humans, and hypophysectomy is the treatment of choice, we used canine tissues. Normal anterior pituitary glands were collected from three healthy dogs that were euthanized for reasons unrelated to the current study. Corticotroph adenomas were collected from six dogs that underwent transsphenoidal hypophysectomy at our University Clinic. The dogs were diagnosed with CD based on clinical signs, endocrine testing, and CT scan imaging. Normal anterior pituitary and corticotroph adenoma cells were cultured in a 3D matrix (basement membrane extract) with anterior pituitary organoid medium containing specific growth factors and ligands, which was refreshed twice a week. The organoids and tumoroids were characterized with histopathology and RT-qPCR. Structures resembling organoids or tumoroids grew from all nine samples (3 normal, 6 adenoma) that were put in culture. Both cystic and dense structures were observed. The organoids and tumoroids expanded rapidly, and could be passaged once every week. The organoids and tumoroids were successfully cultured up until passage number 10, and were then frozen down. Histopathology showed that the organoid or tumoroid cells morphologically resembled healthy anterior pituitary or corticotroph adenoma cells. All organoids cultures expressed mRNA of pituitary stem cell markers SOX2 and SOX9. This study shows that corticotroph adenomas can be cultured as tumoroids in vitro, something not previously published in any species. Based on the many opportunities in organoid culture (e.g., high-throughput drug screenings, gene editing, studying developmental processes), we expect that this in vitro model will pave the way to efficiently and reliably identify new treatment options for CD. Not only for humans, but also for our best friends: dogs.

Clinical Correlation to E-cadherin and Granulation Patterns in Corticotroph Tumors

Corticotroph adenomas, either secretory or silent, are associated with significant disease persistence and/or recurrence. Surgical resection is the first line treatment; however, recurrence rates range from 30 to 60%. Although several clinical parameters (i.e. postoperative cortisol level, tumor invasion) have been reported to predict tumor recurrence, none have high diagnostic accuracy. Granulation pattern classifies corticotroph tumors as densely granulated (DG) or sparsely granulated (SG) types, with the latter usually larger and more aggressively behaving. E-cadherin, a calcium-dependent adhesion molecule strongly expressed in normal pituitary cells, plays a role in epithelial cell behavior, tissue development, and suppresses epithelial-mesenchymal transition. Since loss of E-cadherin expression in sparsely granulated somatotroph pituitary tumors correlates with a more aggressive disease course, we sought to examine correlation between E-cadherin expression and behavior in densely versus sparsely granulated corticotroph tumors. A retrospective chart review of adult patients with corticotroph adenomas, seen at our institution between January 2012 - 2020 yielded 62 patients: 18 (29%) male and 44 female (71%), with median age at diagnosis of 49 years (range 25-82). Inclusion criteria required sufficient tissue for E- cadherin immunostaining (IHC). Microadenomas were identified in 19/62 (31%) patients, and 38/62 (52%) patients had clinical and biochemical findings consistent with excess cortisol secretion. Pre-operative imaging showed that 22/62 (35%) tumors were invasive into surrounding structures. After further classification as to densely granulated (DG) or sparsely granulated (SG) types by ACTH granulation pattern on IHC, 19/56 (34%) adenomas were SG, 37/56 (66%) were DG and 6 were not classified. E-cadherin staining was absent in 7/62 tumors (11%) and diminished in 5/62 (8%) tumors and staining did not correlate with dense versus sparse corticotroph types. Chi-squared analysis found a significant association between tumor size (greater than or less than 1cm) and secretion, with hormonally active more likely tumors to be microadenomas (p=0.004). Microadenomas were exclusively DG tumors (p<0.001). Further analysis did not find correlation between presence or absence of E-cadherin expression and tumor invasion into adjacent structures, or recurrence. In summary, the data suggests that, unlike somatotroph corticotroph adenomas for recurrence or invasion, nor does it correlate strongly with granulation status.