Central neurocytoma exhibits radial glial cell signatures with FGFR3 hypomethylation and overexpression.

We explored the genomic events underlying central neurocytoma (CN), a rare neoplasm of the central nervous system, via multiomics approaches, including whole-exome sequencing, bulk and single-nuclei RNA sequencing, and methylation sequencing. We identified FGFR3 hypomethylation leading to FGFR3 overexpression as a major event in the ontogeny of CN that affects crucial downstream events, such as aberrant PI3K-AKT activity and neuronal development pathways. Furthermore, we found similarities between CN and radial glial cells based on analyses of gene markers and CN tumor cells and postulate that CN tumorigenesis is due to dysregulation of radial glial cell differentiation into neurons. Our data demonstrate the potential role of FGFR3 as one of the leading drivers of tumorigenesis in CN.

A Clinicopathological and Molecular Analysis of Sellar/Suprasellar Neurocytoma Mimicking Pituitary Adenoma.

Objective: To investigate the clinicopathological characteristics, molecular genetic characteristics and prognosis of extraventricular neurocytoma located in the sellar/suprasellar region. Methods: Seven archived tumor samples derived from 4 patients with neurocytoma in the sellar/suprasellar region were collected from the First Affiliated Hospital of Fujian Medical University and the Affiliated Hospital of Qingdao University and retrospectively analyzed for clinical manifestations, imaging features, and histopathological features. Neuronal and pituitary biomarkers and molecular features were detected in these tumor tissues by immunohistochemistry and FISH or Sanger sequencing. The related literature was reviewed. Results: Three patients were female, while 1 was male, with an average age of 35.5 years (range: 27 to 45 years). The initial manifestations were mainly headache and blurred vision in both eyes. The first MRI examination showed marginally enhancing masses in the intrasellar or intra- to suprasellar region. The diagnosis of pituitary adenomas was based on imaging features. The levels of pituitary hormones were normal. Histologically, the tumor cells were arranged in a sheet-like, monotonous architecture and were uniform in size and shape with round to oval, exquisite and hyperchromatic nuclei, which densely packed close to one another and were separated only by a delicate neuropil background. There was no evident mitosis, necrosis or microvascular proliferation. The three cases of recurrent tumors were highly cellular and showed increased mitotic activity. Immunohistochemically, the tumor cells were positive for syn, CR, CgA, and vasopressin and were focally positive for NeuN, TTF-1, NF, CK8, vimentin, and S100 proteins. Other markers, including IDH1, BRAF VE1, Olig-2, and EMA, were negative. Pituitary transcription factors and anterior pituitary hormones were negative. Molecular genetic testing showed that the tumor cells lacked IDH gene mutations, LOH of 1p/19q, MYCN amplification, and EGFR alteration. With a median follow-up of 74.5 months (range 23 to 137 months), 3 patients relapsed at 11, 50, and 118 months after the initial surgery. Conclusion: The morphological features and immunophenotypes of neurocytoma in the sellar/suprasellar region are similar to those of classic central neurocytoma. The prognosis is relatively good. Gross-subtotal resection and atypical subtype may be related to tumor recurrence.

Metabolic, immunohistochemical, and genetic profiling of a cerebellar liponeurocytoma with spinal dissemination: a case report and review of the literature.

Cerebellar liponeurocytoma (cLNC), categorized as a World Health Organization grade II tumor, is a rare neoplasm characterized by advanced neuronal/neurocytic differentiation and focal lipid accumulation in neuroepithelial tumor cells. However, the expression and genetic profiling of cLNC have been poorly studied. A 44-year-old woman with a three-year history of cerebellar ataxia and numbness in lower extremities underwent radiological examination revealing multiple contrast-enhancing tumors at the floor of the fourth ventricle and in the lower vermis, and spinal dissemination. The high uptake of 11 C-methionine in positron emission tomography (Met-PET) supported the preoperative cLNC diagnosis. Subtotal removal of the tumor around the obex and inferior vermis was performed. Histologically, the tumor was composed of small, uniform cells with round nuclei in a sheet-like fashion. Tumor cells were diffusely reactive for the neuronal markers synaptophysin and neurofilament. Vacuolate cells with a displacement of nuclei suggested the accumulation of lipid, which was further supported by immunohistochemical staining of S-100. These findings confirmed the diagnosis of cLNC. Next-generation sequencing of tumoral DNA detected a splice site mutation in the ATRX gene. Further reports of cLNC cases with detailed expression and genetic profiles are essential for precise diagnosis and clarifying the oncogenic pathway in cLNC.

Pediatric Basal Ganglia Region Tumors: Clinical and Radiologic Features Correlated with Histopathologic Findings.

PURPOSE: To summarize the clinical and radiologic features of pediatric basal ganglia region tumors (PBGRT) in correlation with their histopathologic findings to reduce inappropriate surgery and identify tumors that can benefit from maximal safe resection. METHODS: The records of 35 children with PBGRT treated in our hospital from December 2011 to December 2015 were analyzed retrospectively. The clinical and radiologic features of these tumors were summarized and correlated with their histopathologic diagnosis. RESULTS: Our series included 15 astrocytomas and 11 germ cell tumors (GCTs). Basal ganglia astrocytomas were characterized by various clinical presentations and an ill-circumscribed mass with the involvement of surrounding structures on neuroimaging and mostly occurred in the first decade of life (n = 10; 66.7%). Basal ganglia GCT mostly occurred in the second decade of life (n = 8; 72.7%) with hemiparesis as the most common symptom (n = 9; 81.8%). The tumors were located predominantly in the caput of caudate nucleus (n = 8; 72.7%) with hemiatrophy as the typical sign (n = 8; 72.7%). Occasionally, other tumors also could occur in this region, including primitive neuroectodermal tumor (n = 1), atypical teratoid/rhabdoid tumor (n = 1), anaplastic ependymoma (n = 1), lymphoma (n = 1), extraventricular neurocytoma (n = 1), gangliogliomas (n = 2), oligodendroglioma (n = 1), and dysembryoplastic neuroepithelial tumor (n = 1). CONCLUSIONS: Astrocytoma and GCT are the most common PBGRTs. Low-grade astrocytomas could benefit from maximal surgical resection, whereas GCTs merit neoadjuvant chemoradiation therapy followed by second-look surgery. We advocate routine testing of tumor markers and analysis of their clinical and radiologic features to optimize the therapeutic strategy.

A clinicopathologic study of extraventricular neurocytoma.

In 2007, extraventricular neurocytoma was classified as a separate entity among glioneuronal tumors. However, extraventricular neurocytoma is not fully understood and may be misdiagnosed. Here, we describe the clinical and pathological features, prognoses, and treatments of 13 extraventricular neurocytoma cases, and compare their immunophenotypes with those of oligodendroglioma, diffuse astrocytoma, and ependymoma. Six typical and 7 atypical cases comprised the 13 extraventricular neurocytoma cases. Histological features included oligodendroglioma-like perinuclear halo, neuropil-like matrix, ganglion or ganglioid cells, perivascular pseudorosettes, vessel hyalinization, calcifications, and myxoid degeneration. Atypical histological features included increased mitotic figures, focal necrosis, endothelial cell proliferation, and/or a Ki-67 index of >2%. All lesions expressed synaptophysin and microtubule-associated protein-2, which distinguished them from other similar tumors. Two patients with atypical extraventricular neurocytoma had tumor recurrence, one of whom had cerebrospinal fluid dissemination, suggesting that atypical histological features might represent adverse prognostic factors. In conclusion, the present study identified morphological and immunohistochemical features that would aid the differential diagnosis of extraventricular neurocytoma. In addition, radiotherapy with subtotal resection could be considered an effective treatment for extraventricular neurocytoma, but because a pediatric patient died of intracranial hemorrhage during radiotherapy, radiotherapy-related side effects should be considered, especially when treating children. Additional cases with long-term follow-up are needed to develop optimal management protocols for extraventricular neurocytoma.

Central, Extraventricular and Atypical Neurocytomas: a Clinicopathologic Study of 35 Cases from Pakistan Plus a Detailed Review of the Published Literature.

BACKGROUND: Central neurocytomas are rare neuronal neoplasms with a favorable prognosis. They are typically located in the lateral ventricles of the brain and mostly histologically correspond to WHO grade II with a Mib 1 labelling index of <2%. Similar tumors located in the cerebral hemispheres and spinal cord, for example, are called "extraventricular neurocytomas". A few tumors histologically show atypia, mitoses, vascular proliferation and/or necrosis and a Mib 1 index >2 % and are designated as "atypical neurocytomas. AIM: The aim of our study was to describe the common as well as unusual morphologic features and the role of various immunohistochemical stains in the diagnosis of these rare tumors. MATERIALS AND METHODS: We retrieved and reviewed 35 cases diagnosed between 2001 and 2015. RESULTS: Sixty percent of patients were males, and the mean age was 26 years. 31 cases (88.6%) were intraventricular and 4(11.4%) were extraventricular. Histologically, 6 cases (17.1%) were compatible with "atypical neurocytomas". All cases showed the classic morphology comprising nests and sheets of uniform, round cells with uniform round to oval nuclei with finely speckled chromatin and perinuclear cytoplasmic clearing (halos). All cases also showed delicate, fibrillary, neuropil-like matrices. Other common histologic features included capillary-sized blood vessels in a branching pattern in 57.1%, foci of calcification in 34.3% and perivascular pseudorosettes in 20%. Rare findings included Homer- Wright or true rosettes in 8.6% and ganglioid cells in 2.9%. Synaptophysin was the most consistent and valuable marker, being positive in almost all cases. GFAP positivity in tumor cells was seen in 25.7% of cases. Follow up was available in 13 patients. Of these 9 had histologically typical and 4 had atypical tumors. Only 1 (with an atypical neurocytoma) died, probably due to complications of surgery within one month, while 12 (including 3 with atypical neurocytomas) remained alive. Recurrence developed in 1 of these 12 patients (histologically consistent with typical morphology) almost 9 years after surgery. Only 4 patients, including 2 with atypical tumors, received postoperative radiotherapy, all with surgery in 2010 or later. Overall, prognosis was excellent with prolonged, recurrence free survival and most patients, even without receiving radiation therapy, were alive and well for many years, even a decade or more after surgery, without developing any recurrence, indicating the benign nature of these neoplasms.

[THE STUDY OF THE ORIGIN OF HUMAN GLIOMA BASED ON CELL LINES AND TUMOR SPECIMENS].

The present study is dedicated to the search of human glioma "cells of origin". Specimens of the tumor tissue have been analyzed via the RT-PCR method to potentially reveal the expression of molecular markers characteristic of various cells of nerve tissue (NeuN, MOG, MBP, NG2, Olig2, Vimentin, GFAP, AldhlL1), as well as markers of stem (Oct4, c-Kit) and cancerous stem (CD133) cells. We have shown that the expression profiles of these markers for different types of gliomas intersect, which does not allow to determine the type of "cells of origin". So, in order to study the origin of glioma using cell lines derived from primary cultures, we need a more sophisticated culture conditions, rather than the commonly used serum-based media.

Atypical extraventricular neurocytoma:A report of two cases.

Central neurocytomas are tumors with neuronal differentiation, generally arising in the lateral ventricles in the region of foramen of Monro. Whenever these tumors arise in the brain parenchyma they are called "extraventricular neurocytomas". We present two unusual cases of extraventricular atypical neurocytomas at uncommon locations with a very high Ki-67 index. The WHO grading of this tumor is yet to be answered.

Clinical course of central neurocytoma with malignant transformation-an indication for craniospinal irradiation.

Central neurocytoma is generally considered to be a benign tumor and the literature suggests that a cure may be attained by surgery ± adjuvant focal irradiation. However, there is a need for change in the therapeutic strategy for the subgroup of patients with aggressive central neurocytoma. An example case is presented and the literature on central neurocytoma cases with malignant features and dissemination via the cerebrospinal fluid is reviewed and the radiotherapeutic strategies available for central neurocytoma treatment is discussed. Nineteen cases including the present report with a malignant course and cerebrospinal fluid dissemination have been described to date, most of them involving an elevated MIB-1 labeling index. Our case exhibited atypical central neurocytoma with an initially elevated MIB-1 labeling index (25-30 %). The primary treatment included surgery and focal radiotherapy. Three years later the disease had disseminated throughout the craniospinal axis. A good tumor response and symptom relief were achieved with repeated radiation and temozolomide chemotherapy. Central neurocytoma with an initially high proliferation activity has a high tendency to spread via the cerebrospinal fluid. The chemo- and radiosensitivity of the tumor suggest a more aggressive adjuvant therapy approach. Cases with a potential for malignant transformation should be identified and treated appropriately, including irradiation of the entire neuroaxis and adjuvant chemotherapy may be considered.

A case of oligodendroglioma with prominent neuronal differentiation.

We report a case of oligodendroglioma showing marked neuronal differentiation, which arose in the right frontal lobe of a 46-year-old woman. The resected tumor was composed of a mixture of oligodendroglioma, gangliocytoma, and neurocytoma areas with predominance of gangliocytoma-like areas. The oligodendroglioma areas showed immunoreactivity for Olig2 and mutant isocitrate dehydrogenase 1 protein, whereas the gangliocytoma and neurocytoma areas were positive for synaptophysin and NeuN. Ki-67 labeling index was approximately 5% to 10% in the oligodendroglioma areas. Molecular cytogenetic analyses demonstrated chromosomal losses of 1p and 19q and a mutation of isocitrate dehydrogenase 1 (G395A, R132H) in both the oligodendroglioma and gangliocytoma areas. These data suggest that this tumor is an oligodendroglioma associated with prominent neuronal differentiation. There seems to be a close relationship between oligodendroglial progenitor cells and neuronal cells.

Clinicopathological and genetic characteristics of extraventricular neurocytomas.

Extraventricular neurocytoma (EVN) shares histological features with central neurocytoma, but has a wide morphological spectrum. Little is known regarding its clinicopathologic nature, biological behavior and genetic abnormalities. The aim of this study is to examine the diagnostic criteria, genetic abnormalities and biological behavior of EVN. Clinicopathological and molecular genetic studies were performed in seven EVNs. Among them, three cases showed atypical histology. Immunohistochemically, synaptophysin was robustly positive, but neuronal muclear antigen was positive in only half the cases (4/7cases). Isocitrate dehydrogenase enzyme isoform 1 (IDH1) (H09 immunostaining), α-internexin and p53 were negative in all cases. One case was positive for galectin-3. None of the cases showed IDH1 R132 and IDH2 R172 mutation by direct sequencing. One case showed high polysomy of the epidermal growth factor receptor (EGFR) gene; however, O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation and 1p/19q co-deletion were not detected. Array-based comparative genomic hybridization (CGH) study was performed in two cases, revealing different profiles, with loss and gain of multiple chromosomal loci. Two children (18%) had tumor recurrence after initial surgery, and one of them showed worse histology at recurrence and EGFR high polysomy. One patient died from the disease at 18.5 months after surgery. From our study, we concluded that EVNs were characterized by the absence of p53 overexpression, α-internexin positivity, MGMT promotor methylation and IDH1/IDH2 mutation. Oligodendrocyte transcription factor 2 expression was seen in a scattered positive pattern but quite large numbers of tumor cells were negative. EVN is a WHO grade II tumor but some cases (2/7 cases in our series) can show late recurrence but mortality is low (1/7 cases in our series). CGH study suggested genetic heterogeneity of EVNs and unknown subclassification, which requires verification in more cases.

MIB-1 labeling index predicts recurrence in intraventricular central neurocytomas.

Despite the relatively low-grade of most central neurocytomas (CN), evidence suggests the existence of an aggressive subset with a propensity for recurrence. Recent studies have found the MIB-1 labeling index to be a prognostic indicator in CN. Here we review our experience with CN to analyze the relationships between extent of resection, adjuvant therapy, tumor histology, and clinical outcomes based on aggressive histology, as defined by MIB-1 labeling. A retrospective review was performed on histologically proven CN surgically resected from 1993 to 2009 at the University of California at San Francisco. Recurrence rates were analyzed using the Kaplan-Meier method with respect to MIB-1 labeling and extent of resection. All MIB-1 labeling indices were analyzed. A total of 18 patients were identified with a mean age of 30 years (range 17-58 years) and median follow-up of 40 months (5-173 months). The treatments were: gross total resection (GTR) alone (17% of patients), subtotal resection (STR) alone (50% of patients), STR plus radiotherapy (XRT: external beam or stereotactic radiosurgery: 28% of patients), or STR plus chemotherapy (5% of patients). The extent of resection and a MIB-1 labeling index >4% was predictive of recurrence (p<0.01). In the 33% of the patients in whom the tumor recurred, all had STR with MIB-1 labeling >4% with median time to recurrence of 23.5 months. The 2-year and 4-year recurrence rates in patients with MIB-1 labeling >4% were 50% and 75% respectively. No patient with a MIB-1 labeling index <4% who received STR alone had a recurrence. Thus, in patients with CN who were treated with STR, histology demonstrating a MIB-1 labeling index >4% can be a clinically useful prognostic indicator and can help guide adjuvant treatment.

The association of osteopontin and LMX1A expression with World Health Organization grade in meningiomas and gliomas.

AIMS: Osteopontin (OPN) and LIM homeobox transcription factor 1, alpha (LMX1A) are important factors related to tumour progression, invasion and metastasis in human cancers. The aim of this study was to test the hypothesis that expression of OPN and of LMX1A correlate with the World Health Organization (WHO) grading system of primary brain tumours. METHODS AND RESULTS: Immunohistochemical analyses of OPN and LMX1A expression were performed in 139 cases of brain tumour, including 65 meningiomas, 71 gliomas, and three central neurocytomas. More than 90% of WHO grade I meningiomas showed negative or weak staining for OPN and LMX1A. However, among all WHO grade II and III meningiomas, 100% and 66.7% showed moderate or strong staining for OPN and LMX1A, respectively. Similarly, higher percentages of WHO grade I and II gliomas than of WHO grade III and IV gliomas showed negative or weak staining for OPN. A higher intensity of immunoreactivity for LMX1A correlated with more advanced grade in WHO grade I-III gliomas, but not in WHO grade IV tumours. CONCLUSIONS: Higher immunostaining intensity for OPN and LMX1A correlated with WHO grades for meningiomas and some gliomas. Contrary to our expectations, LMX1A staining in WHO grade IV gliomas was shown to be weaker than in WHO grade III tumours.

Hemispheric extra-ventricular glioneurocytoma: a clinicopathological review with detailed immunohistochemical profile.

Glioneuronal tumors have recently been recognized in the WHO Classification of Tumors of the Central Nervous System, 2007 [14]. However, the entities included in this category do not encompass all the glioneuronal tumors encountered during practice. We characterize a new entity called glioneurocytoma (GNC) showing distinct morphology with glial and neuronal differentiation. We reviewed 10 cases of glioneurocytomas diagnosed in our department during 2003 and 2004, with emphasis on clinicopathological features, immunohistochemical profile, genetic aberrations and prognosis. The cases included in the study showed equal gender distribution and age range of 23-40 years and mean age of 34.4 years at the time of initial presentation. Most of the tumors were centered in the frontal lobe. In our study, GFAP was the most sensitive and relatively specific marker for glial differentiation and remains the marker of choice for glial differentiation. CD56 and S100 protein were sensitive but non-specific. Vimentin, CD57 and NF were non-contributory in the immunohistochemical work up of glioneurocytomas. We concluded that the diagnosis of glioneurocytomas requires attention to morphological details and proper immunohistochemical assessment, using a panel of both glial and neuronal markers. Particular attention is recommended to the existence of the intermediate neurocytic cells which may be unique for these tumors. Future implication with full molecular analysis for gene expression profiling is suggested for proper and accurate identifying this entity.

Proton MR spectroscopy of central neurocytoma using short and long echo time: new proofs for the existence of glycine and glutamate.

RATIONALE AND OBJECTIVES: Central neurocytomas (CNCs) are rare benign tumors typically located in the lateral ventricle of the central nervous system. The authors report five patients with CNCs and review 16 previously published studies that included 52 patients with CNCs to explore the magnetic resonance spectroscopic features of CNCs. MATERIALS AND METHODS: Five patients with CNCs were retrospectively reviewed. They were examined using point-resolved spectroscopic series with short and/or long echo times. The integrals of choline, creatine, and the 3.55-ppm peak were determined using Magnetic Resonance User Interface software, and the metabolite ratios relative to creatine were obtained. In two cases, T2 relaxation times of choline and the metabolite resonance at 3.55 ppm were calculated using data points acquired with different echo times and an exponential decay model. RESULTS: Consistent with previously published studies, all five patients showed highly increased choline and reduced N-acetylaspartate and creatine. Four patients in the present study and 35 in published data demonstrated prominent peaks at 3.55 ppm, which were assigned to glycine because of its relaxation pattern and long T2 relaxation time. In addition, increased in vivo glutamate and glutamine was also confirmed in three patients examined with short echo times. Alanine and lactate peaks were observed in three and two patients, respectively. CONCLUSIONS: The present study shows that the 3.55-ppm peak characteristic of CNC should be assigned to glycine according to its T2 relaxation time. Besides increased glycine and choline, the presence of glutamate or glutamine, which appears on series with short echo times, may further confirm the diagnosis of CNC.

FABP4 is a candidate marker of cerebellar liponeurocytomas.

Cerebellar liponeurocytoma (cLPN) is a very rare central nervous system (CNS) tumour recently recognized as a clinical and pathological entity distinct from medulloblastoma (MB), and included in the WHO classification of CNS tumours under the heading "glioneuronal tumours". cLPN typically develop in adult age and have a favourable prognosis compared with MB. In this work, we reviewed the clinical and neuroradiological data of two novel cases of adult cLPN diagnosed at our institution; one patient developed distant metastases. We tried to identify novel molecular markers for this malignancy. We found that the transcription factor NEUROG1 (but not ATOH1) is expressed in cLPN, unlike normal adult cerebellum, and that fatty acid binding protein 4 (FABP4), typically found in adipocytes, is significantly overexpressed compared with both normal adult cerebellum and human MB. These findings suggest cLPN occur as a result of transformation of cerebellar progenitors, which are distinct from cerebellar granule progenitors, and aberrantly differentiate into adipocyte-like tumour cells. They also suggest that analysis of FABP4 expression is of help to differentiate cLPN from MB.

Expression of N-myc downstream regulated gene 1 (NDRG1) in central neurocytoma.

N-myc downstream regulated gene 1 (NDRG1), also known as Cap43, Drg-1, and rit42, is expressed in various normal tissues and cancers, in which it is often associated with a favorable prognosis. It also plays a critical role in central nervous system development, with NDRG1 deficiency resulting in neural defects in mice. Central neurocytoma (CN) is a relatively rare tumor of the neurocytes in the brain, which occurs mainly in young adults. In the present study, we found that tissue samples from four patients with CN had both nuclear and cytoplasmic/membranous expression of NDRG1 protein in highly differentiated CN tumor cells. NDRG1 was also expressed in intratumoral microvessels. Immunohistochemical study of serial sections from the same patients revealed a marked association between the expression pattern of NDRG1 and that of neuron-specific enolase, a tumor differentiation marker. The data presented in this study suggest that NDRG1 could be considered a potential differentiation marker for CN.

Extraventricular neurocytomas: a morphological and histogenetic consideration. A study of six cases.

AIMS: Various molecular markers have been used for diagnosis, management and prognostication of gliomas. Neurocytomas are close morphological mimics of oligodendrogliomas. While combined 1p/19q deletion has been used as a molecular signature of oligodendroglial tumours, it has also been variably reported to occur in neurocytomas, especially those in extraventricular locations (EVN). In recent studies, presence of IDH1 mutation has shown immense prognostic significance in glial tumours including oligodendrogliomas, but its role in neurocytoma pathogenesis remains unexplored. In this study, EVN cases were analysed for histomorphological features, IDH1 mutation using an antibody for specifically detecting mutant IDH1 protein, and 1p/19q deletion by fluorescence in situ hybridisation (FISH) assay. RESULTS: Over a period of 10 years (2000-2009), 60 cases of neurocytoma were diagnosed, of which six were EVN. These six cases were assessed for histomorphology, IDH1 mutation and 1p/19q deletion. Five cases showed atypical histological features. While none showed mIDH1, four of the five atypical cases harboured 1p/19q deletion either in isolation or in combination. The only case which was well-differentiated (typical) did not show 1p/19q loss. CONCLUSIONS: EVNs are more commonly associated with aggressive histological features. IDH1 mutations, although frequent in oligodendrogliomas, are not seen in EVN. However, similar to oligodendrogliomas, 1p/19q deletion is found in these tumours. Thus, a potential histogenetic link between oligodendrogliomas and EVN remains debatable. This molecular alteration may also have prognostic connotations, being associated with atypical morphological features. Due to the rarity of these tumours, multicentric pooling of larger studies is needed to have an insight into the impact of these molecular aberrations on their biological behaviour.