Pituitary neuroendocrine tumors with PIT1/SF1 co-expression show distinct clinicopathological and molecular features.

Pituitary neuroendocrine tumors (PitNETs) are classified according to cell lineage, which requires immunohistochemistry for adenohypophyseal hormones and the transcription factors (TFs) PIT1, SF1, and TPIT. According to the current WHO 2022 classification, PitNETs with co-expression of multiple TFs are termed "plurihormonal". Previously, PIT1/SF1 co-expression was prevailingly reported in PitNETs, which otherwise correspond to the somatotroph lineage. However, little is known about such tumors and the WHO classification has not recognized their significance. We compiled an in-house case series of 100 tumors, previously diagnosed as somatotroph PitNETs. Following TF staining, histopathological features associated with PIT1/SF1 co-expression were assessed. Integration of in-house and publicly available sample data allowed for a meta-analysis of SF1-associated clinicopathological and molecular features across a total of 270 somatotroph PitNETs. The majority (74%, 52/70) of our densely granulated somatotroph PitNETs (DGST) unequivocally co-expressed PIT1 and SF1 (DGST-PIT1/SF1). None (0%, 0/30) of our sparsely granulated somatotroph PitNETs (SGST) stained positive for SF1 (SGST-PIT1). Among DGST, PIT1/SF1 co-expression was significantly associated with scarce FSH/LH expression and fewer fibrous bodies compared to DGST-PIT1. Integrated molecular analyses including publicly available samples confirmed that DGST-PIT1/SF1, DGST-PIT1 and SGST-PIT1 represent distinct tumor subtypes. Clinicopathological meta-analyses indicated that DGST-PIT1 respond more favorably towards treatment with somatostatin analogs compared to DGST-PIT1/SF1, while both these subtypes show an overall less aggressive clinical course than SGST-PIT1. In this study, we spotlight that DGST with co-expression of PIT1 and SF1 represent a common, yet underrecognized, distinct PitNET subtype. Our study questions the rationale of generally classifying such tumors as "plurihormonal", and calls for a refinement of the WHO classification. We propose the term "somatogonadotroph PitNET".

Sphenoid Sinus Mucosal Flap after Transsphenoidal Surgery-A Systematic Review.

Background and Objectives: Skull base reconstruction is a crucial step during transsphenoidal surgery. Sphenoid mucosa is a mucosal membrane located in the sphenoid sinus. Preservation and lateral shifting of sphenoid mucosa as sphenoid mucosal flap (SMF) during the transsphenoidal exposure of the sella may be important for later closure. This is the first systematic review to evaluate the utility of sphenoid mucosal flap for sellar reconstruction after transsphenoidal surgery. Materials and Methods: A systematic literature search was performed in January 2023: Cochrane, EMBASE, PubMed, Scopus, and Web of Science. The following keywords and their combinations were used: "sphenoid mucosa", "sphenoid sinus mucosa", "sphenoid mucosal flap", "sphenoid sinus mucosal flap". From a total number of 749 records, 10 articles involving 1671 patients were included in our systematic review. Results: Sphenoid sinus mucosa used to be applied for sellar reconstruction as either a vascularized pedicled flap or as a free flap. Three different types of mucosal flaps, an intersinus septal flap, a superiorly based flap and an inferiorly based flap, were described in the literature. Total SMF covering compared to partial or no SMF covering in sellar floor reconstruction resulted in fewer postoperative CSF leaks (p = 0.008) and a shorter duration of the postoperative lumbar drain (p = 0.003), if applied. Total or partial SMF resulted in fewer local complications (p = 0.012), such as fat graft necrosis, bone graft necrosis, sinusitis or fungal infection, in contrast to no SMF implementation. Conclusions: SMF seems to be an effective technique for skull base reconstruction after transsphenoidal surgery, as it can reduce the usage of avascular grafts such as fat along with the incidence of local complications, such as fat graft necrosis, bone graft necrosis, sinusitis and fungal infection, or it may improve the sinonasal quality of life by maintaining favorable wound healing through vascular flap and promote the normalization of the sphenoid sinus posterior wall. Further clinical studies evaluating sphenoid mucosal flap preservation and application in combination with other techniques, particularly for higher-grade CSF leaks, are required.

What to report in sellar tumor MRI? A nationwide survey among German pituitary surgeons, radiation oncologists, and endocrinologists.

PURPOSE: While MRI has become the imaging modality of choice in the diagnosis of sellar tumors, no systematic attempt has yet been made to align radiological reporting of findings with the information needed by the various medical disciplines dealing with these patients. Therefore, we aimed to determine the prevailing preferences in this regard through a nationwide expert survey. METHODS: First, an interdisciplinary literature-based catalog of potential reporting elements for sellar tumor MRI examinations was created. Subsequently, a web-based survey regarding the clinical relevance of these items was conducted among board certified members of the German Society of Neurosurgery, German Society of Radiation Oncology, and the Pituitary Working Group of the German Society of Endocrinology. RESULTS: A total of 95 experts (40 neurosurgeons, 28 radiation oncologists, and 27 endocrinologists) completed the survey. The description of the exact tumor location, size, and involvement of the anatomic structures adjacent to the sella turcica (optic chiasm, cavernous sinus, and skull base), occlusive hydrocephalus, relationship to the pituitary gland and infundibulum, and certain structural characteristics of the mass (cyst formation, hemorrhage, and necrosis) was rated most important (> 75% agreement). In contrast, the characterization of anatomic features of the nasal cavity and sphenoid sinus as well as the findings of advanced MRI techniques (e.g., perfusion and diffusion imaging) was considered relevant by less than 50% of respondents. CONCLUSION: To optimally address the information needs of the interdisciplinary treatment team, MRI reports of sellar masses should primarily focus on the accurate description of tumor location, size, internal structure, and involvement of adjacent anatomic compartments.

Exoscopic vs. microscopic transsphenoidal surgery for Cushing's disease: a retrospective single-center study on 388 patients.

Microscopic and endoscopic transsphenoidal surgeries represent the standard treatment for Cushing's disease (CD). At our institution a new exoscopic approach was implemented. After proof of the general use for transsphenoidal pituitary surgery, the aim of this study was to compare the exoscopic 4K3D video microscope with the microscopic transsphenoidal surgery for patients with CD. We conducted a retrospective analysis on 388 patients with CD treated in our medical center via microscopic transsphenoidal surgery (MTS) between January 2008 and July 2019 or via exoscopic transsphenoidal surgery (ExTS) between May 2019 and May 2021. Parameters investigated included histology, pre- and postoperative MRI with tumor size, pre- and postoperative ACTH and cortisol levels, duration of surgery, perioperative and postoperative complications as well as clinical outcome. Patients who underwent ExTS in CD experienced a lower incidence of SIADH/diabetes insipidus (p = 0.0164), a higher rate of remission (p = 0.0422), and a shorter duration of surgery (p < 0.0001), compared to MTS. However, there was no significant difference regarding new postoperative pituitary insufficiency and intraoperative CSF space opening. We found that ExTS had multiple benefits compared to MTS for tumor resection in case of CD. These results are in line with our previous publication on the general applicability of an exoscope in pituitary surgery. To our knowledge, this is the first clinical study proving the superiority of ExTS in CD. These results are promising, nevertheless further studies comparing exoscopic with the endoscopic approach are necessary to finally evaluate the utility of the new technique.

A Copeptin-Based Approach in the Diagnosis of Diabetes Insipidus.

BACKGROUND: The indirect water-deprivation test is the current reference standard for the diagnosis of diabetes insipidus. However, it is technically cumbersome to administer, and the results are often inaccurate. The current study compared the indirect water-deprivation test with direct detection of plasma copeptin, a precursor-derived surrogate of arginine vasopressin. METHODS: From 2013 to 2017, we recruited 156 patients with hypotonic polyuria at 11 medical centers to undergo both water-deprivation and hypertonic saline infusion tests. In the latter test, plasma copeptin was measured when the plasma sodium level had increased to at least 150 mmol per liter after infusion of hypertonic saline. The primary outcome was the overall diagnostic accuracy of each test as compared with the final reference diagnosis, which was determined on the basis of medical history, test results, and treatment response, with copeptin levels masked. RESULTS: A total of 144 patients underwent both tests. The final diagnosis was primary polydipsia in 82 patients (57%), central diabetes insipidus in 59 (41%), and nephrogenic diabetes insipidus in 3 (2%). Overall, among the 141 patients included in the analysis, the indirect water-deprivation test determined the correct diagnosis in 108 patients (diagnostic accuracy, 76.6%; 95% confidence interval [CI], 68.9 to 83.2), and the hypertonic saline infusion test (with a copeptin cutoff level of >4.9 pmol per liter) determined the correct diagnosis in 136 patients (96.5%; 95% CI, 92.1 to 98.6; P<0.001). The indirect water-deprivation test correctly distinguished primary polydipsia from partial central diabetes insipidus in 77 of 105 patients (73.3%; 95% CI, 63.9 to 81.2), and the hypertonic saline infusion test distinguished between the two conditions in 99 of 104 patients (95.2%; 95% CI, 89.4 to 98.1; adjusted P<0.001). One serious adverse event (desmopressin-induced hyponatremia that resulted in hospitalization) occurred during the water-deprivation test. CONCLUSIONS: The direct measurement of hypertonic saline-stimulated plasma copeptin had greater diagnostic accuracy than the water-deprivation test in patients with hypotonic polyuria. (Funded by the Swiss National Foundation and others; ClinicalTrials.gov number, NCT01940614 .).

Co-expression of intermediate filaments glial fibrillary acidic protein and cytokeratin in pituitary adenoma.

PURPOSE: To analyze the co-expression of the intermediate filaments GFAP and cytokeratin in 326 pituitary adenomas with regard to the distribution pattern, the subtype of the adenoma and clinical prognostic data. METHODS: Tissue from 326 pituitary adenomas and 13 normal anterior pituitaries collected in the Institute of Neuropathology, University Medical Center Hamburg-Eppendorf, between 2006 and 2009 was investigated by immunohistochemistry, immunofluorescence and electron microscopy. RESULTS: Co-expression of intermediate filaments GFAP and cytokeratin was associated with hormone expression in 62/278 cases (22%), but only found in 2/48 (4%) of null cell adenomas (p < 0.01). Simultaneous co-expression of GFAP and cytokeratin in the same cells was demonstrated in 26 out of 326 pituitary adenomas and in all 13 pituitaries. In pituitary intermediate filaments were demonstrated in a larger area of the cytoplasm than in adenoma (p < 0.01), however, overlapping expression was seen in 2.6% of the total area in both, pituitary and adenoma. Congenially, cells with overlapping expression were found near vessels and in follicles. Furthermore, adenomas with cellular co-expression of GFAP and cytokeratin were associated with a lower recurrence rate (7.7%) compared to adenomas without co-expression of intermediate filaments (17.8%). CONCLUSIONS: Cellular co-expression of the intermediate filaments GFAP and cytokeratin in pituitary adenomas and the pituitary was demonstrated and shown to be associated with hormone expression and low recurrence rate. The results are discussed with regard to the biology of folliculostellate cells, neural transformation and tumor stem cells. This study may complement the understanding of pituitary adenoma biology.

Double adenomas of the pituitary reveal distinct lineage markers, copy number alterations, and epigenetic profiles.

PURPOSE: Pituitary adenoma (PA) constitutes the third most common intracranial neoplasm. The mostly benign endocrine lesions express no hormone (null cell PA) or the pituitary hormone(s) of the cell lineage of origin. In 0.5-1.5% of surgical specimens and in up to 10% of autopsy cases, two or three seemingly separate PA may coincide. These multiple adenomas may express different hormones, but whether or not expression of lineage-restricted transcription factors and molecular features are distinct within multiple lesions remains unknown. METHODS: Searching the data bank of the German Pituitary Tumor Registry 12 double pituitary adenomas with diverse lineage were identified among 3654 adenomas and 6 hypophyseal carcinomas diagnosed between 2012 and 2020. The double adenomas were investigated immunohistochemically for expression of hormones and lineage markers. In addition, chromosomal gains and losses as well as global DNA methylation profiles were assessed, whenever sufficient material was available (n = 8 PA). RESULTS: In accordance with the literature, combinations of GH/prolactin/TSH-FSH/LH adenoma (4/12), GH/prolactin/TSH-ACTH adenoma (3/12), and ACTH-FSH/LH adenoma (3/12) were observed. Further, two out of 12 cases showed a combination of a GH/prolactin/TSH adenoma with a null-cell adenoma. Different expression pattern of hormones were confirmed by different expression of transcription factors in 11/12 patients. Finally, multiple lesions that were molecularly analysed in 4 patients displayed distinct copy number changes and global methylation pattern. CONCLUSION: Our data confirm and extend the knowledge on multiple PA and suggest that such lesions may origin from distinct cell types.

4K 3-dimensional video microscope system (orbeye) for transsphenoidal pituitary surgery.

BACKGROUND: In previous reports on experiences with an exoscope, this new technology was not found to be applicable for transsphenoidal pituitary surgery. As a specialized center for pituitary surgery, we were using a 4K 3D video microscope (Orbeye, Olympus) to evaluate the system for its use in transsphenoidal pituitary surgery in comparison to conventional microscopy. METHOD: We report on 296 cases performed with the Orbeye at a single institution. An observational study was conducted with standardized subjective evaluation by the surgeons after each procedure. An objective measurement was added to compare the exoscopic and microscopic methods, involving surgery time and the initial postoperative remission rate in matched cohorts. RESULTS: The patients presented with a wide range of pathologies. No serious events or minor complications occurred based on the usage of the 4K 3D exoscope. There was no need for switching back to the microscope in any of the cases. Compared to our microsurgically operated collective, there was no significant difference regarding duration of surgery, complications, or extent of resection. The surgeons rated the Orbeye beneficial in regard to instrument size, positioning, surgeon's ergonomics, learning curve, image resolution, and high magnification. CONCLUSIONS: The Orbeye exoscope presents with optical and digital zoom options as well as a 4K image resolution and 3D visualization resulting in better depth perception and flexibility in comparison to the microscope. Split screen mode offers the complementary benefit of the endoscope which may increase the possibilities of lateral view but has to be evaluated in comparison to endoscopic transsphenoidal procedures in the next step.

Real-life analysis of 280 patients with surgically treated acromegaly: a single-center experience from 2008 to 2015.

OBJECTIVE: The purpose of this study was to analyze the clinical and biochemical outcome of consecutive patients with acromegaly after microscopic transsphenoidal surgery (MTS) at a single center over an 8-year period. METHODS: A retrospective analysis of patients with acromegaly treated via MTS between 2008 and 2015 at the authors' center was performed. The mean follow-up was 29 months (range 1-120 months). Parameters investigated included tumor size, pre- and postoperative insulin-like growth factor-I, growth hormone levels, pretreatment, perioperative complications, and clinical outcome. RESULTS: A total of 280 patients with acromegaly were treated surgically at the authors' center over the abovementioned time frame and were included in analyses. For 231 of these patients, complete follow-up data were available for evaluation. One hundred eighty-eight patients (81%) showed remission initially according to current criteria. So far, 23 of these patients relapsed in the further course, so that on follow-up 165 patients (71%) demonstrated full remission by surgery alone. Most patients in whom remission after surgery failed were treated with somatostatin receptor ligands and/or dopamine agonists as second-line treatment. The main postoperative complications included transient hyponatremia and diabetes insipidus (13/280; 4.6%). CSF leakage only occurred in 2 cases (2/280; 0.7%). No surgery-related death occurred. CONCLUSIONS: The data underline the effectiveness of MTS in acromegaly. Many patients with recurrent disease or incomplete tumor resection can be successfully managed pharmacologically.

The USP8 mutational status may predict long-term remission in patients with Cushing's disease.

OBJECTIVE: Almost half of the cases of Cushing's disease (CD) tumours carry recurrent activating somatic mutations in the ubiquitin-specific protease eight gene (USP8). The USP8 mutational status could predict remission in patients with CD, so our objective was to correlate the presence of somatic USP8 mutations with the rate of recurrence after transsphenoidal surgery (TSS) retrospectively. DESIGN: Biochemical, radiological and clinical data were retrospectively assessed in 48 patients. USP8 mutational status was determined from corticotroph tumour samples. Association between USP8 mutational status, remission and recurrence was investigated. PATIENTS: Patients with Cushing's disease from a single-centre cohort who underwent TSS between 1991 and 2012. MEASUREMENTS: Long-term remission and recurrence rate after TSS with at least 6 months follow-up. Biochemical, radiological and clinical data, including sex, age at diagnosis, tumour size and pre-operative hormonal levels. USP8 mutational status. RESULTS: Patients with USP8 mutant corticotroph tumours (18 of 48; 37%) were diagnosed significantly earlier (mean ± SD 46 ± 10 years vs 53 ± 11 years; P = 0.028) and presented with higher pre-operative 24-hour urinary-free cortisol levels (median IQR µg/24 hours 1174.0, 1184.5 vs 480.0, 405.3; P = 0.045). The incidence of recurrence in a 10-year follow-up was significantly higher in patients with USP8 mutant tumours after the initial remission (58% vs 18% P = 0.026). Recurrence appeared significantly earlier in these patients (months 70, 44-97 95% CI vs 102, 86-119 95% CI; P = 0.019). CONCLUSION: Recurrence appears to be more frequent and earlier after TSS in patients with USP8 mutant corticotroph tumours.

Low concordance between surgical and radiological assessment of degree of resection and treatment-related hypothalamic damage: results of KRANIOPHARYNGEOM 2007.

BACKGROUND: Assessment of presurgical hypothalamic involvement (psHI) and treatment-related hypothalamic damage (trHD) is relevant for the decision on risk-adapted treatment and rehabilitation strategies in craniopharyngioma. PATIENTS AND METHODS: 129 surgical reports of childhood-onset craniopharyngioma patients recruited 2007-2014 in KRANIOPHARYNGEOM 2007 were analyzed. Data on psHI were available based on surgeon's (63%), reference neuroradiologist's (95%), and local radiologist's (23%) assessment. The surgical degree of resection (DoR) was assessed by neurosurgeon (95%), reference neuroradiologist (73%), and local radiologist (61%). TrHD was assessed by neurosurgeon (33%), by reference neuroradiologist (95%), and by local radiologist (2%). Neurosurgical center size was categorized based on patient load. RESULTS: Surgical assessments on psHI (n = 78), DoR (n = 89) and trHD (n = 42) as documented in surgical reports could be compared with the assessment of respective parameters by reference neuroradiologist. Differences with regard to DoR (p = 0.0001) and trHD (p < 0.0001) were detectable between surgeon's and reference neuroradiologist's assessment, whereas psHI was assessed similarly. Concordance for DoR and trHD was observed in 48 and 62%, respectively. Surgeons estimated a higher rate of complete resections and a lower rate of trHD. Neuroradiological reference assessment of trHD had higher predictive value for hypothalamic sequelae then surgical assessment. Observed differences were not related to neurosurgical center size. CONCLUSIONS: Observed differences between surgical and neuroradiological estimation of risk factors in craniopharyngioma support the necessity of neuroradiological reference review to assure standards of quality. This could be established by central internet-based neuroradiological review in KRANIOPHARYNGEOM 2007. Standardization of surgical reports including specific assessment of tumor/damage location is recommended.

Pituitary adenomas in children and young adults.

PURPOSE: Pituitary adenomas are rare in the pediatric population. We present a recent cohort of children operated transsphenoidally on pituitary adenoma from a single center. METHODS: Inclusion criteria were age < 21 years and histopathological diagnosis of adenoma after transsphenoidal surgery. The electronic file was used for prospective and retrospective data collection on symptoms, pituitary function before/after surgical intervention, and surgical complications. Surgical reports were used for assessment of resection grade. Follow-up data were collected from outpatient clinical visit and/or correspondence concerning pituitary function and MRI. RESULTS: Among 962 consecutive patients who underwent transsphenoidal surgery at the University Medical Center Hamburg-Eppendorf (Germany) between April 2013-December 2016, 50 patients (5.2%) were 21 years old or younger (range 6-21 years, mean 16.5). Twenty-five (50%) patients had pituitary adenomas; M:F = 13:12. Eight were macroadenomas (> 1cm3). Time from onset of symptoms to first operation had a range of 0-48 months (mean 18.5 months). Nine (36%) patients overall complained of headaches preoperatively. One patient had objective (hemianopsia) and four subjective visual disturbances preoperatively. Histopathological diagnosis revealed 13 (52%) ACTH adenomas, 5 (20%) prolactinomas, 3 (12%) mixed GH-prolactinoma, 1 (4%) GH adenoma, 2 (8%) TSH-producing adenomas, and 1 (4%) non-functioning adenoma. There were no surgical complications. On clinical follow-up (range 1-34.5 months, mean 11.7 months), 21/24 (88%) patients with hormonally active tumors were in biochemical remission by surgery alone; 2 patients with Cushing disease and 1 with GH-hypersecretion required further treatment. CONCLUSIONS: Our recent series confirms the effectiveness and safety of transsphenoidal surgery, especially in the pediatric patient population.

Immunoprofiling of glial tumours of the neurohypophysis suggests a common pituicytic origin of neoplastic cells.

PURPOSE: To analyse the antigen expression profiles of 27 cases of pituicytoma, spindle cell oncocytoma, and granular cell tumour of the sellar region concerning a common pituicytic origin of neoplastic cells. METHODS: Material from 12 female and 15 male patients (13 granular cell tumours of the sellar region, 10 pituicytomas, four spindle cell oncocytomas) collected in the German Registry of Pituitary Tumours between 1993 and 2015 was re-evaluated according to the current WHO classification of tumours of the central nervous system and supplementary immunohistochemistry including S100-protein, CD56, CD68, thyroid transcription factor-1 (TTF-1), and Ki-67 was performed. RESULTS: S100-protein was detected in all 27 tumours and TTF-1 in all 16 tumours that were assessed. Vimentin was expressed in all 13 cases investigated whereas broad spectrum cytokeratin was not detected in any of 14 evaluated cases. GFAP was observed in nine out of 21 cases. 15 out of 17 investigated lesions showed some CD68 expression and five out of 14 cases were labelled with CD56 antibodies. Proliferative activity did not differ significantly between the three tumour subgroups although one primary and one recurrent pituicytoma showed exceptionally high Ki-67-proliferation indices of 15.3 and 12.7 %, respectively (means: granular cell tumour of the sellar region 2.0 %, pituicytoma 2.8 %, spindle cell oncocytoma 2.7 %). CONCLUSIONS: The study confirms and expands earlier data and is in line with the notion that the three tumour types are variants of pituicytoma.

Surgical treatment and outcome of TSH-producing pituitary adenomas.

BACKGROUND: Thyrotropin-producing adenomas (TSHomas) account for approximately 1-2% of all pituitary tumors. Recently, there has been debate on primary treatment as some studies suggest a high rate of hypopituitarism after transsphenoidal surgery and therefore suggest primary use of somatostatin analogs (SSA) instead. We would like to challenge this assumption by presenting our single-center experience with transsphenoidal surgery for TSHoma. METHODS: Fifteen patients treated consecutively between May 2010 and December 2016 were analyzed for long-term postoperative remission and pituitary function. RESULTS: Data on the development of TSH, fT3 and fT4 were available for 12 of 15 patients (mean follow-up was 18 months, 8 patients >12 months), showing mean TSH levels of 1.289 mU/l (0.02-2.04, SD 0.66), mean fT3 levels of 3.76 pmol/l (0.5-6.16, SD 1.8) and mean fT4 levels of 16.5 pmol/l (11.7-21.9, SD 3.66). Six of those patients were substituted with a mean of 85.4 µg L-thyroxine after a median follow-up of 20.7 months. The other six patients did not receive L-thyroxine at a median follow-up of 15.5 months. One patient with a known tumor remnant on MRI stayed euthyreotic with cabergoline at the timepoint of follow-up 22 months after the operation. Control of the corticotrop axis was also available in 12 of 15 with no patient showing a corticotroph insufficiency in the long term. CONCLUSIONS: We argue that transsphenoidal surgery for TSHoma should be considered as the treatment of choice as remission following surgery is highly probable and postoperative hypopituitarism is very unlikely if patients are referred to centers with high pituitary surgery case loads.

Drug priming enhances radiosensitivity of adamantinomatous craniopharyngioma via downregulation of survivin.

OBJECTIVE In this study, the authors investigated the underlying mechanisms responsible for high tumor recurrence rates of adamantinomatous craniopharyngioma (ACP) after radiotherapy and developed new targeted treatment protocols to minimize recurrence. ACPs are characterized by the activation of the receptor tyrosine kinase epidermal growth factor receptor (EGFR), known to mediate radioresistance in various tumor entities. The impact of tyrosine kinase inhibitors (TKIs) gefitinib or CUDC-101 on radiation-induced cell death and associated regulation of survivin gene expression was evaluated. METHODS The hypothesis that activated EGFR promotes radioresistance in ACP was investigated in vitro using human primary cell cultures of ACP (n = 10). The effects of radiation (12 Gy) and combined radiochemotherapy on radiosensitivity were assessed via cell death analysis using flow cytometry. Changes in target gene expression were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Survivin, identified in qRT-PCR to be involved in radioresistance of ACP, was manipulated by small interfering RNA (siRNA), followed by proliferation and vitality assays to further clarify its role in ACP biology. Immunohistochemically, survivin expression was assessed in patient tumors used for primary cell cultures. RESULTS In primary human ACP cultures, activation of EGFR resulted in significantly reduced cell death levels after radiotherapy. Treatment with TKIs alone and in combination with radiotherapy increased cell death response remarkably, assessed by flow cytometry. CUDC-101 was significantly more effective than gefitinib. The authors identified regulation of survivin expression after therapeutic intervention as the underlying molecular mechanism of radioresistance in ACP. EGFR activation promoting ACP cell survival and proliferation in vitro is consistent with enhanced survivin gene expression shown by qRT-PCR. TKI treatment, as well as the combination with radiotherapy, reduced survivin levels in vitro. Accordingly, ACP showed reduced cell viability and proliferation after survivin downregulation by siRNA. CONCLUSIONS These results indicate an impact of EGFR signaling on radioresistance in ACP. Inhibition of EGFR activity by means of TKI treatment acts as a radiosensitizer on ACP tumor cells, leading to increased cell death. Additionally, the results emphasize the antiapoptotic and pro-proliferative role of survivin in ACP biology and its regulation by EGFR signaling. The suppression of survivin by treatment with TKI and combined radiotherapy represents a new promising treatment strategy that will be further assessed in in vivo models of ACP.

BRAF V600E analysis for the differentiation of papillary craniopharyngiomas and Rathke's cleft cysts.

AIMS: The differential diagnosis of cystic epithelial masses of the sellar region, especially the histopathological differentiation of craniopharyngiomas and Rathke's cleft cysts, poses a challenge even to experienced diagnosticians. Recently, BRAF V600E mutations have been described as a genetic hallmark of papillary craniopharyngiomas. We investigated a series of 33 Rathke's cleft cysts to determine the frequency of BRAF V600E mutations and its suitability as an additional diagnostic marker for the differentiation of cystic lesions of the sellar region. METHODS: Thirty-three Rathke's cleft cysts and 18 papillary craniopharyngiomas were analysed for BRAF mutational status by immunohistochemistry using a monoclonal antibody (VE1) that selectively recognizes the BRAF V600E mutant epitope and additional BRAF pyrosequencing in a subset of samples. RESULTS: Thirty of 33 specimens diagnosed as Rathke's cleft cysts were negative by VE1 immunohistochemistry and pyrosequencing, whereas in three cysts and in all the 18 papillary craniopharyngiomas, a BRAF V600E mutation was detected. Clinical and histological re-evaluation of the three BRAF V600E mutated cases formerly diagnosed as Rathke's cleft cysts revealed unusual presentations. Two of them were rediagnosed as papillary craniopharyngiomas. The patient of the third case had a history of craniopharyngioma operated 14 years before, and reoperation showed a cystic epithelial lesion with unclear histology. CONCLUSIONS: The determination of BRAF mutational status is recommended in any cystic sellar lesion and can in most cases be provided by VE1 immunohistochemistry even in specimens of low cellularity. Confirmation by (pyro-)sequencing should be attempted whenever sufficient epithelium is available due to variable staining results.

Hippocampal and cerebellar atrophy in patients with Cushing's disease.

OBJECT Cushing's disease (CD) may cause atrophy of different regions of the human brain, mostly affecting the hippocampus and the cerebellum. This study evaluates the use of 3-T MRI of newly diagnosed patients with CD to detect atrophic degeneration with voxel-based volumetry. METHODS Subjects with newly diagnosed, untreated CD were included and underwent 3-T MRI. Images were analyzed using a voxelwise statistical test to detect reduction of brain parenchyma. In addition, an atlas-based volumetric study for regions likely to be affected by CD was performed. RESULTS Nineteen patients with a mean disease duration of 24 months were included. Tumor markers included adrenocorticotropic hormone (median 17.5 pmol/L), cortisol (949.4 nmol/L), and dehydroepiandrosterone sulfate (5.4 µmol/L). The following values are expressed as the mean ± SD. The voxelwise statistical test revealed clusters of significantly reduced gray matter in the hippocampus and cerebellum, with volumes of 2.90 ± 0.26 ml (right hippocampus), 2.89 ± 0.28 ml (left hippocampus), 41.95 ± 4.67 ml (right cerebellar hemisphere), and 42.11 ± 4.59 ml (left cerebellar hemisphere). Healthy control volunteers showed volumes of 3.22 ± 0.25 ml for the right hippocampus, 3.23 ± 0.25 ml for the left hippocampus, 50.87 ± 4.23 ml for the right cerebellar hemisphere, and 50.42 ± 3.97 ml for the left cerebellar hemisphere. CONCLUSIONS Patients with untreated CD show significant reduction of gray matter in the cerebellum and hippocampus. These changes can be analyzed and objectified with the quantitative voxel-based method described in this study.

Cavernous sinus sampling in patients with Cushing's disease.

OBJECT Correct diagnosis and precise localization of adenomas in patients with Cushing's disease are essential for avoiding unsuccessful transsphenoidal pituitary exploration. In addition to the well-established inferior petrosal sinus sampling, preoperative cavernous sinus sampling (CSS) was introduced as a potentially improved way to predict adenoma lateralization. The authors present their results with CSS in a consecutive series of patients with Cushing's disease. METHODS During 1999-2014, transsphenoidal surgeries were consecutively performed in 510 patients with Cushing's disease. For most patients, suppression of cortisol in high-dose dexamethasone tests and stimulation of adrenocorticotropic hormone and cortisol after administration of corticotropin-releasing hormone were sufficient to prove the diagnosis of adrenocorticotropic hormone-dependent hypercortisolism. Of the 510 patients, 67 (13%) were referred to the department of neuroradiology for CSS according to the technique of Teramoto. The indications for CSS were unclear endocrine test results or negative MRI results. Data for all patients were retrospectively analyzed. RESULTS A central/peripheral gradient was found in 59 patients; lateralization to the left or right side was found in 51. For 8 patients with a central/peripheral gradient, no left/right gradient could be determined. For another 8 patients with equivocal test results, no central/peripheral gradient was found. No severe CSS-associated complications were encountered. Of the 51 patients who underwent transsphenoidal surgery, the predicted lateralization was proven correct for 42 (82%). CONCLUSIONS As MRI techniques have improved, the number of potential candidates for this invasive method has decreased in the past decade. However, because detecting minute adenomas remains problematic, CSS remains a useful diagnostic tool for patients with Cushing's disease.

Adamantinomatous craniopharyngiomas express tumor stem cell markers in cells with activated Wnt signaling: further evidence for the existence of a tumor stem cell niche?

INTRODUCTION: Early disease onset, clinical manifestation, histomorphology, and increased tendency to relapse distinguish the adamantinomatous craniopharyngioma (adaCP) from the more favorable papillary variant (papCP). A molecular hallmark of adaCP is the activated Wnt signaling pathway indicated by nuclear ß-catenin accumulation in a subset of tumor cells. A mouse model recently illustrated that these cells are the driving force in tumorigenesis of adaCP. This observation and the peculiar growth pattern points to the existence of a specific tumor stem cell (TSC) population in human CP. MATERIALS AND METHODS: To prove this hypothesis, the TSC markers CD133 (Prominin1) and CD44 were examined in papCP (n = 8) and adaCP (n = 25) on mRNA level using quantitative real time PCR of total tumor RNA. Furthermore, we investigated protein expression performing immunohistochemical analyses of formalin-fixed paraffin embedded tumor samples. RESULTS: PapCP revealed a homogenous CD44 expression pattern predominantly at the cell membrane, whereas CD133 labeling was hardly detectable. In adaCP, on the other hand all markers were consistently and predominantly co-expressed in nuclear ß-catenin accumulating cell clusters, which was confirmed by double immunofluorescence staining. Overall expression of CD44 was significantly decreased in adaCP versus papCP, whereas CD133 showed significantly higher protein and mRNA levels in adaCP. CONCLUSIONS: Our results indicate tumor stem cell-like characteristics of ß-catenin accumulating cell clusters in adaCP, which may represent a tumor stem cell niche and might contribute to tumor recurrence. The potential impact of these special cell groups in regard to future CP management, including postoperative follow-up and additional treatment remains to be explored.