Jürgen Honegger

Targeting hyperactivation of the AKT survival pathway to overcome therapy resistance of melanoma brain metastases

Brain metastases are the most common cause of death in patients with metastatic melanoma, and the RAF‐MEK‐ERK and PI3K‐AKT signaling pathways are key players in melanoma progression and drug resistance. The BRAF inhibitor vemurafenib significantly improved overall survival. However, brain metastases still limit the effectiveness of this therapy. In a series of patients, we observed that treatment with vemurafenib resulted in substantial regression of extracerebral metastases, but brain metastases developed. This study aimed to identify factors that contribute to treatment resistance in brain metastases. Matched brain and extracerebral metastases from melanoma patients had identical ERK, p‐ERK, and AKT immunohistochemistry staining patterns, but there was hyperactivation of AKT (p‐AKT) and loss of PTEN expression in the brain metastases. Mutation analysis revealed no differences in BRAF, NRAS, or KIT mutation status in matched brain and extracerebral metastases. In contrast, AKT, p‐AKT, and PTEN expression was identical in monolayer cultures derived from melanoma brain and extracerebral metastases. Furthermore, melanoma cells stimulated by astrocyte‐conditioned medium showed higher AKT activation and invasiveness than melanoma cells stimulated by fibroblast‐conditioned medium. Inhibition of PI3K‐AKT signaling resensitized melanoma cells isolated from a vemurafenib‐resistant brain metastasis to vemurafenib. Brain‐derived factors appear to induce hyperactivation of the AKT survival pathway and to promote the survival and drug resistance of melanoma cells in the brain. Thus, inhibition of PI3K‐AKT signaling shows potential for enhancing and/or prolonging the antitumor effect of BRAF inhibitors or other anticancer agents in melanoma brain metastases.

Objective criteria for successful transsphenoidal removal of suprasellar nonfunctioning pituitary adenomas. A prospective study.

SummaryBackground. Despite ample experience with transsphenoidal surgery, objective data on which suprasellar tumour expansion and growth pattern allows for radical adenoma resection are still sparse. Hence, we have performed a prospective study to establish the predictive value of tumour dimension and shape for the intra-operative descent of the diaphragma, the completeness of tumour resection and the outcome of patients harbouring pituitary adenomas with suprasellar extension.Method. Included in the study were 105 patients with nonfunctioning pituitary adenomas and suprasellar extension who underwent primary transsphenoidal surgery between January 1998 and December 2005. The precise suprasellar extension, the degree of dumbbell-shape, the configuration of the adenomas and the depth of the pituitary fossa were evaluated. Completeness of resection was assessed by MRI at 3 months postoperatively.Findings. The mean cranio-caudal diameter of the tumours was 28.0 mm (range 9.2–57.8 mm). On average, the suprasellar extension measured 11.9 mm (range 2.1–25.8 mm). Total removal of the suprasellar tumour was accomplished in 83% (87 of 105) of the patients. A second operation for residual adenoma was only indicated in 2 cases. The vertical intracranial extension was the strongest independent predictor of subtotal resection (p < 0.001). Irregular and multilobular configuration was a second highly-significant and independent predictor for incomplete resection (p < 0.003). In contrast, dumbbell-shape and shallow pituitary fossa were not independent predictive factors for incomplete tumour resection. The complication rate was very low. None of our patients suffered postoperative rhinorrhea, meningitis or visual deterioration.Conclusions. One-stage transsphenoidal surgery allows total or near-total resection of most suprasellar pituitary adenomas with low surgical morbidity. Quantitative assessment of tumour dimension and configuration contributes to establishing guidelines for the selection of the appropriate approach and prediction of surgical outcome.

Diagnosis of Primary Hypophysitis in Germany.

CONTEXT Representative data on diagnostic findings in primary hypophysitis (PrHy) are scarce. OBJECTIVE The objective of the study was to collate consistent data on clinical features in a large series of patients with PrHy. Another objective was to gain information on current practice in a diagnostic work-up. DESIGN The Pituitary Working Group of the German Society of Endocrinology conducted a nationwide retrospective cross-sectional cohort study in Germany. PATIENTS Seventy-six patients with PrHy were identified. MAIN OUTCOME MEASURES Clinical and endocrinological features were assessed. RESULTS Headache (50%) and increase in body mass (18%) were the most frequent nonendocrine symptoms. Hypophysitis was associated with pregnancy in only 11% of the female patients. Diabetes insipidus was found in 54% of the patients at presentation. Hypogonadotropic hypogonadism was the most frequent endocrine failure (62%), whereas GH deficiency was the least frequent (37%). With 86%, thickening of the pituitary stalk was the prevailing neuroradiological sign. Compared with surgical cases, the cases without histological confirmation presented more often with suprasellar lesions and had less severe nonendocrine symptoms. Granulomatous hypophysitis was associated with more severe clinical symptoms than lymphocytic hypophysitis. Examination of cerebrospinal fluid was predominantly performed in participating neurosurgical centers, whereas thyroid antibodies were almost exclusively assessed in endocrinological centers. CONCLUSION In contrast to the literature, hypogonadism was found to be the most frequent endocrine failure in PrHy. Weight gain was identified as a clinical sign of PrHy. In the majority of patients, PrHy can be reliably identified by characteristic clinical signs and symptoms, obviating histological confirmation. The diagnostic approach should be standardized in PrHy.

Rathke's cleft cyst rupture as potential initial event of a secondary perifocal lymphocytic hypophysitis: proposal of an unusual pathogenetic event and review of the literature.

Herein, we report on a lymphocytic hypophysitis related to a ruptured Rathke’s cleft cyst which is not associated with pregnancy. A 45-year-old woman initially presented with headache and temporary double vision followed by amenorrhea. Preoperative imaging showed an intra- and suprasellar cystic mass. Complete resection of the tumor mass was performed via a transnasal, transseptal approach. Pathological examination displayed lymphocytic infiltrates within fibrotic tissue and residual pituitary cells accompanied by epithelial tissue of a Rathke’s cleft cyst. The strongest inflammatory reaction was observed at the site of disrupture of the cyst integrity, suggesting that high protein levels from ruptured Rathke’s cleft cyst might have triggered a lymphocytic hypophysitis. Our review of the literature provides further insights regarding the clinical behavior and different histological types of the lesions as well as the inflammatory changes that can occur in Rathke’s cleft cysts.

Treatment of Primary Hypophysitis in Germany.

CONTEXT The best treatment of primary hypophysitis (PrHy) is a matter of debate. OBJECTIVE Our main objective was to analyze the treatment practice for PrHy in Germany and to compare the outcome of the main treatment options. DESIGN The Pituitary Working Group of the German Society of Endocrinology conducted a nationwide retrospective cross-sectional cohort study. PATIENTS Seventy-six patients with PrHy were eligible for the study. MAIN OUTCOME MEASURES Clinical and endocrinological outcomes, side effects and complications of therapy, initial response, and recurrence rates were assessed. Outcome depending on the treatment modality was evaluated. RESULTS For mere observation, regression of space-occupying lesions was observed in 46%, unchanged size in 27%, and progression reported in 27%. Pituitary function improved in 27% of patients during observation. Deterioration of pituitary function was only found in patients with progressive lesions. The initial response to glucocorticoid pulse therapy was most favorable, with early failure in only 3%. However, the overall failure and recurrence rate was 41%. Recurrence rate was not related to duration of steroid administration. Side effects of steroids occurred in 63%. The surgical approach was transsphenoidal in 94%. The histological subtype was lymphocytic hypophysitis in 70% and granulomatous hypophysitis in 30%. Progression or recurrence was observed in 25% after surgical treatment. CONCLUSION Glucocorticoid pulse therapy is associated with a high recurrence rate. Evidence suggests that surgery is not able to prevent recurrence. Considering the favorable results of observation, conservative management is recommended in PrHy unless symptoms are severe or progressive.

Pituicytoma in a patient with Cushing's disease: case report and review of the literature

Pituicytoma is an exceptionally rare low-grade glioma (WHO grade I) of the neurohypophysis and infundibulum. We are reporting the case of a 48-year-old man who presented with severe Cushing′s syndrome. Endocrinological evaluation unequivocally confirmed pituitary-dependent Cushing’s syndrome (=Cushing’s disease). Cranial MR-imaging displayed a conspicuous area in the dorsal and basal pituitary gland and a minimal bulging of the pituitary gland paramedian of the pituitary stalk on the right side. Transsphenoidal inspection revealed a small tumor in the basal and dorsal pituitary gland. Surprisingly, the definite postoperative histopathological diagnosis of the removed tumor was pituicytoma and not pituitary adenoma. Hence, the microadenoma responsible for Cushing’s disease was not yet removed and persistent hypercortisolism necessitated transsphenoidal re-operation. During re-operation, hemihypophysectomy was performed on the right side. The non-tumorous specimen of the adeno-hypophysis showed signs of Crooke’s hyalinization consistent with Cushing’s disease. Undetectable postoperative ACTH- and cortisol levels provided clear evidence that the underlying ACTH-source was successfully removed during re-operation. Coincidence of pituicytoma and pituitary-dependent Cushing’s disease has not previously been reported.

Tuberculum sellae meningioma symptomatic during pregnancy: pathophysiological considerations

SummaryA 31 year old woman in her second pregnancy presented in the 31st (+4) week of gestation with progressive visual impairment of the right eye. Magnetic resonance imaging (MRI) demonstrated a tuberculum sellae meningioma that was displaced upward by a markedly enlarged pituitary gland. Neuro-ophthalmological follow-up examinations showed a progressive decrease of visual acuity and right temporal field loss. Therefore, a caesarean section was performed in the 34th (+8) week. The meningioma was removed three days after childbirth via a right-sided pterional approach. Post-operatively, visual function was completely restored. Immunohistochemical examination showed positive staining for progesterone receptors (PR) in approximately 50% of tumour cells. Enlargement of the pituitary gland during late pregnancy in conjunction with a preexisting tuberculum sellae meningioma is the most likely pathophysiological factor responsible for visual loss. Enlargement of the PR-positive meningioma in the hormonal milieu of pregnancy might have contributed additionally to visual loss.

Predictive factors for neurocognitive function and Quality of Life after surgical treatment for Cushing’s disease and acromegaly

Background: Cushing’s disease (CD) and acromegaly (AC) are associated with impairment in quality of life (QoL) and neurocognition that can persist after successful treatment. Aim: To investigate the influence of current disease status (remission vs no remission) on neurocognitive function and QoL in treated CD and AC patients and to determine predictive factors (e.g. demographic, clinical, neurosurgical, endocrinological) for post-operative neurocognition and QoL. Subjects and methods: Twenty-four CD and 37 AC patients underwent neuropsychological testing 1 to 10 yr following surgical therapy. Additionally, QoL was assessed. An overnight 2-mg dexamethasone suppression test in CD and IGF-I and GH levels in AC patients were assessed to determine current disease status. The results were compared with 28 sex-, education- and age-matched healthy controls (HC). Results: Impaired QoL was more pronounced than neurocognitive decrease in both pathologies compared to HC. This finding was independent of the current status of disease. In AC, persistent comorbidities were associated with impaired QoL (p<0.05). Older age at operation in AC patients was a significant predictor for adverse effects on psychomotor speed and attentional functions (p<0.05). In CD persistent hypocortisolism, not hypercortisolism, had adverse effects on neurocognition (p<0.01). Conclusions: The current status of disease plays a subordinate role in postoperative outcome concerning QoL and neurocognition in either pathology. A possible explanation might be the considerably improved endocrinopathy after treatment compared to untreated patients, even if no cure is achieved. The lasting impairments might be explained by irreversible changes that have occurred during the active phase of the disease.

Clinical Impact of the Current WHO Classification of Pituitary Adenomas.

WHO classifications should be used for comparing the results from different groups of pathologist and clinicians by standardized histopathological methods. Our present report describes the important parameters of pituitary adenoma pathology as demand of the WHO classification for correlation to endocrine data and prognosis. The combination of HE stain based structures with immunostainings for pituitary hormones allows subclassification of adenomas as the best method not only for correlations to clinical hyperfunctions but also for statements to the sensitivity of drug therapies (somatostatin analogs, dopamine agonists). GH-, PRL- and ACTH-secreting pituitary adenomas are further classified based on the size and number of their secretory granules by electron microscopy, or as is mostly the case nowadays by cytokeratin staining pattern, into densely and sparsely granulated. Granulation pattern may be considered for the prediction of treatment response in patients with GH-secreting adenomas, since the sparsely granulated subtype was shown to be less responsive to somatostatin analog treatment. For prognosis, it is important to identify aggressive adenomas by measurements of the Ki-67 index, of the number of mitoses, and of nuclear expression of p53. Among the criteria for atypical adenomas, high Ki-67 labeling index and invasive character are the most important adverse prognostic factors. Promising molecular markers have been identified that might supplement the currently used proliferation parameters. For defining atypical adenomas in a future histopathological classification system, we propose to provide the proliferative potential and the invasive character separately.

VE1 immunohistochemistry in pituitary adenomas is not associated with BRAF V600E mutation

(6 %) and 1 strong case (1 %); supplemental Figure S1). Twelve of the positive cases were HGH-producing adenomas, two were mixed mammosomatotroph adenomas and the four remaining adenomas expressed ACTH (including the case with score 3). To rule out non-specific enzymatic activity of the avidin–biotin method, the VE1 diaminobenzidine (DAB) staining results were repeated using two different polymer detection systems (Ventana OptiView and Leica BondMax), as recommended by Andrulis et al. [1] to reduce unspecific background staining, and reproducibly confirmed the positive reactions (Fig. 1b). The signal was even stronger than in the initial DAB staining of the same case (Fig. 1a). The cases were also stained with synaptophysin and CD68; possible occasional cross-reactivity was excluded [3]. Ten VE1 positive cases (one score 3, three score 2 and six score 1) were analyzed by clamped probe assay; they showed wild-type BRAF curves (Fig. 1c; for experimental methods see supplemental data). Sanger sequencing of BRAF exon 15 in the same cases confirmed the absence of V600E mutation (Fig. 1d). We minimized sequencing failure because we used only samples with 80 % tumor content and additionally performed clamped probe assays that are able to detect mutations even if the mutation/wild-type ratio is as low as 1 % [9]. Due to their highsequence homology with the BRAF activation segment, we also analyzed the activation segments of ARAF and CRAF by sequencing to rule out VE1 cross-reactivity with a putative homolog ARAF V453E or CRAF V492E mutation, again revealing wild-type sequences for all cases (Fig. 1d; for experimental procedures, see supplemental data). In contrast to many previously investigated cancers, our results indicate that even strong positive immunostaining of VE1 in pituitary adenomas does not indicate the presence of a BRAF V600E mutation, especially if it is restricted to the cells’ membranes. While V600E mutations Activating BRAF mutations are observed in malignant melanomas, pleomorphic xanthoastrocytomas, hairy cell leukemia, papillary thyroid carcinomas, colorectal cancer, ovarian cancer and ganglioglioma [1, 10, 11]. Generation of a monoclonal antibody specific for the BRAF V600E mutation (VE1) was published recently [3]. This antibody has been successfully validated for melanomas [5], lung adenocarcinomas [8] and thyroid carcinomas [2]. Although pituitary adenomas account for up to 10 % of all intracranial tumors [4], there is still a lack of data on VE1 immunostains in these tumors. In three studies in which BRAF in pituitary adenomas was examined by sequencing, a single V600E mutation was found in 145 cases [6, 7, 10]. We screened 78 pituitary adenomas (epidemiological data in supplemental Table 1) with VE1 antibody. Staining was observed in 18 cases (12 weak (15 %); 5 moderate