REST promotes ETS1-dependent vascular growth in medulloblastoma.

Expression of the RE1-silencing transcription factor (REST), a master regulator of neurogenesis, is elevated in medulloblastoma (MB) tumors. A cell-intrinsic function for REST in MB tumorigenesis is known. However, a role for REST in the regulation of MB tumor microenvironment has not been investigated. Here, we implicate REST in remodeling of the MB vasculature and describe underlying mechanisms. Using RESTTG mice, we demonstrate that elevated REST expression in cerebellar granule cell progenitors, the cells of origin of sonic hedgehog (SHH) MBs, increased vascular growth. This was recapitulated in MB xenograft models and validated by transcriptomic analyses of human MB samples. REST upregulation was associated with enhanced secretion of proangiogenic factors. Surprisingly, a REST-dependent increase in the expression of the proangiogenic transcription factor E26 oncogene homolog 1, and its target gene encoding the vascular endothelial growth factor receptor-1, was observed in MB cells, which coincided with their localization at the tumor vasculature. These observations were confirmed by RNA-Seq and microarray analyses of MB cells and SHH-MB tumors. Thus, our data suggest that REST elevation promotes vascular growth by autocrine and paracrine mechanisms.

Hemangioblastoma of the Cerebellopontine Angle Evaluated with Pseudocontinuous Arterial Spin Labeling.

Hemangioblastomas of the cerebellopontine angle (CPA) that emerge extra-axially from the peripheral nervous system are extremely rare. We report a case of hemangioblastoma of the CPA evaluated by pseudocontinuous arterial spin labeling (pCASL). The high rate of tumor blood flow determined using pCASL provided additional useful information for the differential diagnosis of the CPA tumors in this patient.

Posterior Petrosal Transotic Approach for Cerebellopontine Angle Hemangioblastoma: Technical Case Report.

BACKGROUND AND IMPORTANCE: Although posterior petrosal approaches are utilized less frequently in many practices today, they continue to provide distinct surgical advantages in carefully selected cases. Here, we report a case of a recurrent cerebellopontine angle (CPA) hemangioblastoma that had failed a prior, more conservative, surgical approach. We provide cadaveric dissections of variations of posterior petrosal approaches to illustrate the advantages of the selected approach. CLINICAL PRESENTATION: A 70-yr-old female presented with a growing left CPA hemangioblastoma. The lesion had undergone a prior subtotal resection from a retrosigmoid approach and subsequent adjuvant radiation treatment. The patient had worsening left facial strength, progressive balance difficulty, and absent left auditory function. Preoperative angiogram demonstrated arterial blood supply from the left anterior inferior cerebellar artery (AICA) that was deemed unsafe for embolization due to significant arteriovenous shunting. A posterior petrosal transotic approach was performed in order to optimize the working angle to the anterior brainstem and afford the ability to occlude the vascular supply from AICA prior to surgical resection of the lesion. CONCLUSION: The posterior petrosal transotic approach offers an improved surgical working angle to the anterior brainstem compared to the translabyrinthine approach. This advantage can be particularly important with vascular tumors that receive blood supply anteriorly, as in this case from AICA, and can improve the safety of the resection.

Added Value of Arterial Spin-Labeling MR Imaging for the Differentiation of Cerebellar Hemangioblastoma from Metastasis.

BACKGROUND AND PURPOSE: In adults with only cerebellar masses, hemangioblastoma and metastasis are the 2 most important differential diagnoses. Our aim was to investigate the added value of arterial spin-labeling MR imaging for differentiating hemangioblastoma from metastasis in patients with only cerebellar masses. MATERIALS AND METHODS: This retrospective study included a homogeneous cohort comprising patients with only cerebellar masses, including 16 hemangioblastomas and 14 metastases. All patients underwent enhanced MR imaging, including arterial spin-labeling. First, the presence or absence of a hyperperfused mass was determined. Next, in the hyperperfused mass, relative tumor blood flow (mean blood flow in the tumor divided by blood flow measured in normal-appearing cerebellar tissue) and the size ratio (size in the arterial spin-labeling images divided by size in the postcontrast T1WI) were measured. To validate the arterial spin-labeling findings, 2 observers independently evaluated the conventional MR images and the combined set of arterial spin-labeling images. RESULTS: All patients with hemangioblastomas and half of the patients with metastases presented with a hyperperfused mass (P < .001). The size ratio and relative tumor blood flow were significantly larger for hemangioblastomas than for metastases (P < .001 and P = .039, respectively). The size ratio revealed excellent diagnostic power (area under the curve = 0.991), and the relative tumor blood flow demonstrated moderate diagnostic power (area under the curve = 0.777). The diagnostic accuracy of both observers was significantly improved after the addition of arterial spin-labeling; the area under the curve improved from 0.574 to 0.969 (P < .001) for observer 2 and from 0.683 to 1 (P < .001) for observer 2. CONCLUSIONS: Arterial spin-labeling imaging can aid in distinguishing hemangioblastoma from metastasis in patients with only cerebellar masses.

Endothelial cells by inactivation of VHL gene direct angiogenesis, not vasculogenesis via Twist1 accumulation associated with hemangioblastoma neovascularization.

Inactivation of the VHL tumour suppressor gene is a highly frequent genetic event in the carcinogenesis of central nervous system-(CNS) hemangioblastomas (HBs). The patterning of the similar embryonic vasculogenesis is an increasing concern in HB-neovascularization, and the classic vascular endothelial growth factor (VEGF)-mediated angiogenesis driven by VHL loss-of-function from human endothelium have been questioned. With this regard, we identify a distinct, VHL silencing-driven mechanism in which human vascular endothelial cells by means of increasing cell proliferation and decreasing cell apoptosis, is concomitant with facilitating accumulation of Twist1 protein in vascular endothelial cells in vitro. Importantly, this molecular mechanism is also pinpointed in CNS-HBs, and associated with the process of HB-neovascularization. In contrast with recent studies of HB-neovascularization, these modified cells did not endow with the typical features of vasculogenesis, indicating that this is a common angiogenesis implementing the formation of the vascular network. Taken together, these findings suggest that vasculogenesis and angiogenesis may constitute complementary mechanisms for HB-neovascularization, and could provide a rational recognition of single anti-angiogenic intervention including targeting to the Twist1 signalling for HBs.

Neuroprotective effects respond to cerebral ischemia without susceptibility to HB-tumorigenesis in VHL heterozygous knockout mice.

The von Hippel-Lindau (VHL) tumor suppressor gene plays a prominent role in the development of hemangioblastomas (HBs) within specific regions of the human' central nervous system (CNS). Alterations in VHL gene are rarely observed in the more common features of human VHL-related tumors in animal models, and VHL heterozygous knockout (VHL+/-) mice do not develop HBs. We tested whether VHL heterozygous knockout mice exhibited genetic predisposition to the development of HBs and conferred a selective advantage involving growth of blood vessels to its carrier. No differences were observed between wild-type and VHL+/- mice in development ad reproduction. The heterozygous VHL+/- mice did not develop higher genetic susceptibility to CNS-HBs over their lifetime. Furthermore, this recessive VHL gene heterozygosity is relatively stable. Interestingly, we found these heterozygous VHL+/- mice gained an advantage conferring to angiogenic ability in a particular environment, compared with wild-type mice. The heterozygous VHL+/- mice obviously enhanced hypoxia inducible factor-1 (HIF)-dependent and Twist1 angiogenic mechanism in response to acute cerebral ischemia, resulting in decreased cerebral tissue damage and neuroprotective response through neovascularization. Our findings provide evidence of partial loss function of VHL as a novel precise therapeutic target in acute cerebral ischemia.

Cerebellar hemangioblastoma with perivascular pseudorosette formation and glial differentiation: A case report.

Hemangioblastoma is a well-circumscribed, highly vascular, lipid-rich and low-grade tumor of uncertain histogenesis. Its histopathological features have been well established. Herein, we present a case of cerebellar hemangioblastoma in a 43-year-old woman. Histologically, the tumor was predominantly composed of cellular areas showing eosinophilic or vacuolated stromal cells arranged in nests and sheets. Focally, conventional reticular areas could be seen. Additionally, in some areas, the stromal cells were arranged radially around blood vessels, exhibiting perivascular pseudorosette structures, which were similar mostly to those of ependymomas. Immunohistochemically, the stromal cells markedly showed diffused staining for Vimentin, S-100, CD56, NSE, inhibin-a, podoplanin, alpha-Thalassemia/mental retardation syndrome X and carbonic anhydrase IX, and were negative for cytokeratin, epithelial membrane antigen, oligodendrocyte transcription factor 2, neuronal nuclear antigen, synaptophysin, isocitrate dehydrogenase 1 (R132H), P53 and CD34. Interestingly, the reticular and cellular areas either showed no or individual scattering of the GFAP-positive cells, respectively, while, the perivascular pseudorosette areas strongly and diffusely expressed GFAP. Nuclear mitosis and necrosis were not observed. The MIB-1 antibody labeling index was especially low (about 3%). Based on these findings, the patient was diagnosed with cerebellar hemangioblastoma. In the present case, we documented a distinctive histological appearance of perivascular pseudorosettes in hemangioblastoma and provided the evidence for stromal cells with glial differentiation.

Anomalous vascularization in a Wnt medulloblastoma: a case report.

BACKGROUND: Medulloblastoma is the most common malignant brain tumor in children. To date only few cases of medulloblastoma with hemorrhages have been reported in the literature. Although some studies speculate on the pathogenesis of this anomalous increased vascularization in medulloblastoma, the specific mechanism is still far from clearly understood. A correlation between molecular medulloblastoma subgroups and hemorrhagic features has not been reported, although recent preliminary studies described that WNT-subtype tumors display increased vascularization and hemorrhaging. CASE PRESENTATION: Herein, we describe a child with a Wnt-medulloblastoma presenting as cerebellar-vermian hemorrhagic lesion. Brain magnetic resonance imaging (MRI) showed the presence of a midline posterior fossa mass with a cystic hemorrhagic component. The differential diagnosis based on imaging included cavernous hemangioma, arteriovenous malformation and traumatic lesion. At surgery, the tumor appeared richly vascularized as documented by the preoperative angiography. CONCLUSIONS: The case we present showed that Wnt medulloblastoma may be associated with anomalous vascularization. Further studies are needed to elucidate if there is a link between the hypervascularization and the Wnt/ß-catenin signaling activation and if this abnormal vasculature might influence drug penetration contributing to good prognosis of this medulloblastoma subgroup.

Medulloblastoma Genotype Dictates Blood Brain Barrier Phenotype.

The childhood brain tumor, medulloblastoma, includes four subtypes with very different prognoses. Here, we show that paracrine signals driven by mutant ß-catenin in WNT-medulloblastoma, an essentially curable form of the disease, induce an aberrant fenestrated vasculature that permits the accumulation of high levels of intra-tumoral chemotherapy and a robust therapeutic response. In contrast, SHH-medulloblastoma, a less curable disease subtype, contains an intact blood brain barrier, rendering this tumor impermeable and resistant to chemotherapy. The medulloblastoma-endothelial cell paracrine axis can be manipulated in vivo, altering chemotherapy permeability and clinical response. Thus, medulloblastoma genotype dictates tumor vessel phenotype, explaining in part the disparate prognoses among medulloblastoma subtypes and suggesting an approach to enhance the chemoresponsiveness of other brain tumors.

CD41 and CD45 expression marks the angioformative initiation of neovascularisation in human haemangioblastoma.

The initiation and formation of haemangioblastoma (HB) neovascularisation remain unknown, with concomitant controversy on its cytological origin. We detected HB-derived specific haematopoietic progenitors identified by surface expression of CD41 and CD45, which are similar to human embryonic vasculogenesis. CD41/CD45 cells expressed mesodermal markers, including SCL, Flk1 and c-kit. CD41 also seemed to appear before CD45 on haematopoietic progenitors. In vitro analysis showed that the CD41(+)/CD45 subpopulation gave rise to occasional primitive erythroid activity and endothelial marker expression. Meanwhile, kinetic investigation of the CD41(+)/CD45(+) subpopulation showed that some molecules, including SCL, Flk1 and c-kit, were involved in vascular formation. The CD45(+)/c-kit(+) population that lacked primitive haematopoiesis came from CD41(+) cells. Acquisition of CD45 expression by the haematopoietic progenitors was associated with advanced differentiation towards the vascular cell lineage. Taken together, the present data suggested that CD41 and CD45 expression marked the onset of HB neovascularisation and the stepwise development of the angioformative period. Our findings provide new insights into the mechanisms of HB neovascularisation and the underlying therapeutic targets of anti-vascular treatment.

Coagulation and angiogenic gene expression profiles are defined by molecular subgroups of medulloblastoma: evidence for growth factor-thrombin cross-talk.

BACKGROUND: The coagulation system becomes activated during progression and therapy of high-grade brain tumors. Triggering tissue factor (F3/TF) and thrombin receptors (F2R/PAR-1) may influence the vascular tumor microenvironment and angiogenesis irrespective of clinically apparent thrombosis. These processes are poorly understood in medulloblastoma (MB), in which diverse oncogenic pathways define at least four molecular disease subtypes (WNT, SHH, Group 3 and Group 4). We asked whether there is a link between molecular subtype and the network of vascular regulators expressed in MB. METHODS: Using R2 microarray analysis and visualization platform, we mined MB datasets for differential expression of vascular (coagulation and angiogenesis)-related genes, and explored their link to known oncogenic drivers. We evaluated the functional significance of this link in DAOY cells in vitro following growth factor and thrombin stimulation. RESULTS: The coagulome and angiome differ across MB subtypes. F3/TF and F2R/PAR-1 mRNA expression are upregulated in SHH tumors and correlate with higher levels of hepatocyte growth factor receptor (MET). Cultured DAOY (MB) cells exhibit an up-regulation of F3/TF and F2R/PAR-1 following combined SHH and MET ligand (HGF) treatment. These factors cooperate with thrombin, impacting the profile of vascular regulators, including interleukin 1ß (IL1B) and chondromodulin 1 (LECT1). CONCLUSIONS: Coagulation pathway sensors (F3/TF, F2R/PAR-1) are expressed in MB in a subtype-specific manner, and may be functionally linked to SHH and MET circuitry. Thus coagulation system perturbations may elicit subtype/context-specific changes in vascular and cellular responses in MB.

Tumor derived vasculogenesis in von Hippel-Lindau disease-associated tumors.

von Hippel-Lindau disease (VHL) patients develop highly vascular tumors, including central nervous system hemangioblastomas. It has been hypothesized that the vascular nature of these tumors is the product of reactive angiogenesis. However, recent data indicate that VHL-associated hemangioblastoma neoplastic cells originate from embryologically-arrested hemangioblasts capable of blood and endothelial cell differentiation. To determine the origin of tumor vasculature in VHL-associated hemangioblastomas, we analyzed the vascular elements in tumors from VHL patients. We demonstrate that isolated vascular structures and blood vessels within VHL-associated hemangioblastomas are a result of tumor-derived vasculogenesis. Further, similar to hemangioblastomas, we demonstrate that other VHL-associated lesions possess vascular tissue of tumor origin and that tumor-derived endothelial cells emerge within implanted VHL deficient UMRC6 RCC murine xenografts. These findings further establish the embryologic, developmentally arrested, hemangioblast as the tumor cell of origin for VHL-associated hemangioblastomas and indicate that it is also the progenitor cell for other VHL-associated tumors.

Anatomical variation of superior petrosal vein and its management during surgery for cerebellopontine angle meningiomas.

No systematic study is yet available that focuses on the surgical anatomy of the superior petrosal vein and its significance during surgery for cerebellopontine angle meningiomas. The aim of the present study was to examine the variation of the superior petrosal vein via the retrosigmoid suboccipital approach in relation to the tumor attachment of cerebellopontine angle meningiomas as well as postoperative complications related to venous occlusion. Forty-three patients with cerebellopontine angle meningiomas were analyzed retrospectively. Based on the operative findings, the tumors were classified into four subtypes: the petroclival type, tentorial type, anterior petrous type, and posterior petrous type. According to a previous anatomical report, the superior petrosal veins were divided into three groups: Type I which emptied into the superior petrosal sinus above and lateral to the internal acoustic meatus, Type II which emptied between the lateral limit of the trigeminal nerve at Meckel's cave and the medial limit of the facial nerve at the internal acoustic meatus, and Type III which emptied into the superior petrosal sinus above and medial to Meckel's cave. In both the petroclival and anterior petrous types, the most common vein was Type III which is the ideal vein for a retrosigmoid approach. In contrast, the Type II vein which is at high risk of being sacrificed during a suprameatal approach procedure was most frequent in posterior petrous type, in which the superior petrosal vein was not largely an obstacle. Intraoperative sacrificing of veins was associated with a significantly higher rate of venous-related phenomena, while venous complications occurred even in cases where the superior petrosal vein was absent or compressed by the tumor. The variation in the superior petrosal vein appeared to differ among the tumor attachment subtypes, which could permit a satisfactory surgical exposure without dividing the superior petrosal vein. In cases where the superior petrosal vein was previously occluded, other bridging veins could correspond with implications for the crucial venous drainage system, and should thus be identified and protected whenever possible.

Vasculogenic mimicry and its clinical significance in medulloblastoma.

Vasculogenic mimicry (VM), a process involving the formation of a tubular structure by highly invasive and genetically dysregulated tumor cells, can supplement the function of blood vessels to transport nutrients and oxygen to maintain the growth of tumor cells in many malignant tumors. We aimed to explore the existence of VM and its clinical significance in medulloblastoma in this study. VM was identified in 9 out of 41 (22%) medulloblastoma tissues. Immunohistochemical studies revealed that the presence of VM was associated with the expression of MMP-2, MMP-14, EphA2 and laminin 5γ2. Tumor tissues with VM were associated with lower microvessel density (MVD), which was indirect evidence of the blood supply function of VM. Survival analysis and log-rank tests showed that patients with VM had shorter overall survival time than those without VM. Multivariate analysis and the Cox proportional hazards model identified VM as independent prognostic factor for overall survival. Our results confirmed the existence of VM for the first time and revealed that VM is a strong independent prognostic factor for survival in patients with medulloblastoma.

Hemorrhagic onset of cerebellar pilocytic astrocytoma in an adult: a case report and review of the literature implying a possible relation of degenerative vascular changes to the massive intratumoral hemorrhage.

Pilocytic astrocytoma (PA) is a low-grade astrocytic tumor arising predominantly during the first two decades of life. Hemorrhagic onset of PAs is uncommon, and the etiology of hemorrhage remains unclear. Here we report a case of hemorrhagic onset of cerebellar PA in a 29-year-old man who presented with a week-long history of headache and gait instability. Computed tomography and magnetic resonance imaging revealed a hemorrhagic tumor located in the right cerebellar hemisphere, and total resection was performed. Histological examination showed bipolar glial cell proliferation in a biphasic pattern in a compact area and a loose microcystic area with Rosenthal fibers and eosinophilic granular bodies, indicating PA. Prominent changes in tumor vasculature, including aggregation of sclerotic thick-walled and ectatic thin-walled vessels, was observed, and nodules of thrombi containing complex vascular proliferation suggesting recanalized thrombi formed in partially ruptured vessels were also found. Thus, rupture of these abnormal vessels appeared to be the cause of hemorrhage. Review of the literature revealed that age distribution of patients with hemorrhagic PAs tends to be older than that of patients with general PAs. These findings imply a possibility that degenerative changes in blood vessels in long-standing PAs might be related to the mechanisms of spontaneous intratumoral hemorrhage.

Cerebellar hemangioblastoma supplied by persistent hypoglossal artery.

The authors report a unique case of an intramedullary cerebellar hemangioblastoma that was supplied by a persistent hypoglossal artery. In this case, recognition of the persistent hypoglossal artery through angiographic imaging was crucial in identifying tumor blood supply and in guiding the decision to perform a craniotomy without tumor embolization.

Complete preoperative embolization of hemangioblastoma vessels with Onyx 18.

The authors present a preliminary experience with ethyl-enevinylalcohol copolymer (Onyx) for hemangioblastoma vessel embolization before surgical resection. The patient presented with neck pain, dizziness, blurred vision, vomiting, and loss of balance. Diagnostic imaging revealed a posterior fossa cystic mass with a nodular component. Angiography demonstrated a significant vascular blush with arteriovenous shunting that was characteristic of a hemangioblastoma. Tumor vessels originating off the left posterior inferior cerebellar artery were embolized before surgery using Onyx 18 (ev3, Covidien Vascular Therapies, Mansfield, MA, USA). This resulted in complete obliteration of all tumor vessels, transforming a highly vascular tumor into an avascular mass. A safe and uneventful surgical resection was performed the next day. Onyx is a valuable embolic agent for preoperative hemangioblastoma vessel embolization. Because of its low viscosity, Onyx penetrates deeply into the tumor vasculature and allows complete obliteration of tumor vessels. Risks of the intervention have to be carefully weighed against the benefits. If preoperative embolization is indicated, the use of Onyx should be strongly considered.

Antidepressant hyperforin up-regulates VEGF in CNS tumour cells.

Vascular endothelial growth factor (VEGF) is a key player in neo-angiogenesis; it sustains the progression of solid neoplasias, brain tumours included. It has recently been demonstrated that the use of antidepressants correlates with increasing VEGF levels in the central nervous system (CNS). In order to elucidate whether the most used natural antidepressant [St. John's wort (SJW) extract] modulates VEGF expression, possible relationship between≤µM hyperforin (Hyp, the bioactive component in SJW) and VEGF in CNS tumours has been now examined in medulloblastoma and glioblastoma cells. Real-time PCR and ELISA revealed that under Hyp VEGF expression increased more than three fold in DAOY medulloblastoma cells; while, U87 glioblastoma cells - constitutively expressing high VEGF levels - showed no significant differences. Moreover, Hyp induced endothelial pro-angiogenic behaviour in a multi-parametric Matrigel colonisation assay, and down-modulation of pro-MMP-2 and pro-MMP-9 activities as measured by gelatin zymography. Should these results be confirmed in vivo for this and other types of CNS tumour, the antidepressant use of SJW extracts must be carefully re-considered, in particular for brain tumour patients.

Downregulation of uPA/uPAR inhibits intermittent hypoxia-induced epithelial-mesenchymal transition (EMT) in DAOY and D283 medulloblastoma cells.

Hypoxia is known to induce overexpression of the urokinase plasminogen activator (uPA) and its receptor (uPAR) and thus overexpression promotes uPAR-mediated survival signaling in various cancers. Moreover, hypoxia/ overexpression of uPAR in cancer cells promote the epithelial-mesenchymal transition (EMT) and thereby invasiveness and metastasis. In this study, we show that intermittent hypoxia has a more pronounced effect than chronic hypoxia and contributes to EMT, invasion and migration in medulloblastoma cells. Intermittent hypoxia induced expression of mesenchymal markers (i.e., SNAIL, Vimentin and N-cadherin) and reduced expression of epithelial markers (i.e., Zo-1, E-cadherin) in medulloblastoma cells. Further, intermittent hypoxia also leads to enhancement in cell invasion, migration and angiogenesis in medulloblastoma cells. Intermittent hypoxia also inhibited expression of pro-anti-apoptotic proteins (Bax and Bad), and induced expression of anti-pro-apoptotic proteins (Bcl2 and Bcl-xL), and activation of ERK in medulloblastoma cells. Transcriptional inactivation of either uPA or uPAR inhibits the intermittent hypoxia-induced invasion and migration, and expression of Vimentin. uPA/ uPAR downregulation also induces E-cadherin expression and inhibits activation of ERK. Thus, transcriptional inactivation of either uPA or uPAR enhances the apoptotic response in medulloblastoma cells exposed to intermittent hypoxia. This study provides evidence of the anti-tumor efficacy of down-regulation of uPA or uPAR in medulloblastoma tumors to target hypoxia-induced cell EMT, invasion and migration, to achieve better therapeutic outcomes in the treatment of malignant medulloblastoma.

The tumour-associated carbonic anhydrases CA II, CA IX and CA XII in a group of medulloblastomas and supratentorial primitive neuroectodermal tumours: an association of CA IX with poor prognosis.

BACKGROUND: Medulloblastomas (MBs) and supratentorial primitive neuroectodermal tumours (PNETs) are the most common highly aggressive paediatric brain tumours. In spite of extensive research on these tumours, there are only few known biomarkers or therapeutic target proteins, and the prognosis of patients with these tumours remains poor. Our aim was to investigate whether carbonic anhydrases (CAs), enzymes commonly overexpressed in various tumours including glioblastomas and oligodendrogliomas, are present in MBs and PNETs, and whether their expression can be correlated with patient prognosis. METHODS: We determined the expression of the tumour-associated carbonic anhydrases CA II, CA IX and CA XII in a series of MB/PNET specimens (n = 39) using immunohistochemistry. RESULTS: Endothelial CA II, cytoplasmic CA II, CA IX and CA XII were expressed in 49%, 73%, 23% and 11% of the tumours, respectively. CA II was detected in the neovessel endothelium and the tumour cell cytoplasm. CA IX was mainly expressed in the tumour cells located in perinecrotic areas. CA XII showed the most homogenous distribution within the tumours. Importantly, CA IX expression predicted poor prognosis in both univariate (p = 0.041) and multivariate analyses (p = 0.016). CONCLUSIONS: We suggest that CA IX should be considered a potential prognostic and therapeutic target in MBs and PNETs.