In Vitro Characterization of VPS34 Lipid Kinase Inhibition by Small Molecules.

VPS34 is a class III phosphoinositide 3-kinase that acts on vesicle trafficking. This kinase has recently attracted significant attention because of the function it plays in the machinery involved in the early steps of autophagy. Moreover, because significant progress had been made in the optimization of specific kinase inhibitors, its potential to be targeted by catalytic inhibitors has been investigated by different groups. The aim of this review is to present the key in vitro assays necessary for characterizing inhibitors of the catalytic activity of VPS34. The review covers catalytic (IC50 on purified recombinant protein) and binding assays (KD, ka, kd on purified recombinant protein), and a cell-based assay (IC50 in GFP-FYVE expressing cell line). The methodology for crystallization of VPS34 protein is also presented as it can provide guidance for the design by medicinal chemistry of small molecular mass kinase inhibitor.

Evaluating the impact of summer vacation on the visual acuity of AMD patients treated with ranibizumab.

PURPOSE: To evaluate the impact of traditional French summer vacation on visual acuity and spectral domain-optical coherence tomography (SD-OCT) of Wet AMD patients being treated with intravitreal Ranibizumab. METHODS: This was a consecutive, comparative, single-centre, prospective analysis. All patients who were being treated with intravitreal injection of 0.5 mg ranibizumab at Cergy Pontoise Hospital, Department of Ophthalmology between July 2013 and September 2014 were included. Patients were divided into two groups: (A) patients who skipped one ranibizumab intravitreal injection during holidays, and (B) patients who received injection during their holidays. Evaluations occurred prior to traditional holiday (baseline) and 2 months later, consisting of BCVA using ETDRS, and a complete ophthalmic examination that included slit-lamp biomicroscopy, fundus examination, fluorescein angiography (FA), indocyanine green angiography (ICGA), and spectral domain-optical coherence tomography (SD-OCT). All patients were being treated with PRN anti-VEGF regimen and criteria for reinjection included a visual acuity loss >5 ETDRS letters and/or an increase of central retinal thickness, presence of subretinal fluid, intraretinal fluid, or pigment epithelium detachment. If reinjection criteria were not met, patients were advised to return in 4 weeks. RESULTS: The mean visual acuity change was -0.071 ± 0.149 (LogMAR) in group A and + 0.003 ± 0.178 in group B (P = 0.041). At the second visit (2 months after preholidays visit), 61.8% of patients in group A had SRF and/or intraretinal cysts, and only 27.6% of patients in group B. There was a significant difference in the persistence of fluid between the two groups (P = 0.007, χ(2)-test). CONCLUSION: This cases series demonstrated the detrimental impact of holidays on visual acuity in patients treated with ranibizumab for AMD, which, in spite of their treatment regimen, still leave in vacation. Therefore, it is important to convey the message of treatment adherence to patients, despite their need of holidays.

Identifying Small Molecules which Inhibit Autophagy: a Phenotypic Screen Using Image-Based High-Content Cell Analysis.

Autophagy plays an important role in cancer and it has been suggested that it functions not only as a tumor suppressor pathway to prevent tumor initiation, but also as a pro-survival pathway that helps tumor cells endure metabolic stress and resist death triggered by chemotherapeutic agents, including acquired resistance. We aimed to identify small-molecule autophagy inhibitors using a HTS/HCA approach through a phenotypic, cell image-based assay, in order to screen multiple biological targets simultaneously and to screen compounds in a physiologically relevant environment. LC3 is a component of the autophagosome, which undergoes a cytoplasmic redistribution from diffuse to punctate dots during autophagy. We employed HeLa cells stably expressing EGFP-LC3 in a primary phenotypic screen. As a first step, a "Validation Library" of about 8,000 pre-selected compounds, about 25% of which had known biological activity and the others representing a range of chemical structures, was run in duplicate both to assess screening suitability and likely hit rate, and to give a valuable preview of possible active structures or biological targets. The primary screen of about 0.25 million compounds yielded around 10,500 positive compounds. These were tested in a suite of further cellular assays designed to eliminate unwanted positives, together with the application of chemi- and bioinformatics to pick out compounds with known biological activity. These processes enabled the selection of compounds that were the most promisingly active and specific. The screening "tree" identified, amongst others with as yet unidentified targets, chemical series active against autophagy-relevant biological targets ULK or Vsp34, validating the phenotypic screening methods selected. Finally, about 400 compounds were fully qualified after following this triage. The development of the assays, compound screening process and the compound triage is described.

Perfusion MR imaging and 1H spectroscopy: their role in the diagnosis of microcystic and lipomatous meningiomas.

Microcystic and lipomatous meningiomas represent two of the recognised rare subtypes of meningiomas. We describe the CT and MR findings in one adult case of each subtype with emphasis on the diagnostic benefits of perfusion-weighted MR imaging (PWI) and/or proton magnetic resonance spectroscopy (MRS).

[Value of perfusion MRI in the study of pilocytic astrocytoma and hemangioblastoma: preliminary findings]. / Intérêt de l'imagerie de perfusion dans l'étude des astrocytomes pilocytiques et des hémangioblastomes : étude préliminaire.

PURPOSE: Pilocytic astrocytomas (PA) and hemangioblastomas (HB) can present the same morphological characteristics on conventional MRI sequences, most usually in the form of a cerebellar cystic mass with a mural nodule that strongly enhances on post-contrast T1 images. We discuss here the value of perfusion MRI in the differentiation of these two tumors, the diagnoses of which have already been histopathologically established. METHOD: Eleven patients with PA and eight with HB underwent first-pass perfusion MRI. The maximum relative cerebral blood volume (rCBV(max)), defined as the ratio between the CBV(max) in tumor tissue and the CBV in healthy, contralateral white matter, is considered to be indicative of the type of tumor. RESULTS: The difference between the rCBV(max) of PA (rCBV(max)=1.19+/-0.71, range 0.6-3.27) compared with that of HB (rCBV(max)=9.37+/-2.37, range 5.38-13) was significant (P<0.001). The first-pass curve crossed the baseline, corresponding to vascular permeability problems in both PA and HB. CONCLUSION: The first-pass method of perfusion MRI is a quick and useful way to differentiate between PA and HB.

[A rare cause of embolic ischemia of the hand: an isolated aneurism of a branch of the axillary artery]. / Une cause rare d'ischémie embolique de la main: un anévrisme isolé d'une branche de l'artère axillaire.

A 48-year-old man was admitted for subacute ischemia of the right hand of sudden onset. The patient, who participated in amateur sports, had an uneventful medical history. Duplex ultrasonography revealed thrombosis of the right radial and ulnar arteries. On heparin, the clinical course was favorable and investigations to search for an embolic source revealed an aneurism of the posterior circumflex artery (arteriography). The etiological work-up was negative as was the search for other aneurismal locations. Surgical excision was carried out. Pathology examination of the surgical specimen revealed a thrombosed aneurism that had developed on an atherosclerotic plaque. Aneurisms of the posterior circumflex artery have been described in professional baseball and volleyball players, but all sports that involve repetitive movements of the arm at extension, external rotation and forced abduction can complicate such damage. Compression of the aneurismal artery by the humeral head leads to extrusion of the thrombus under pressure and to retrograde embolisation towards the leg arteries. Thus, in the same way as for hypothenar hammer syndrome, signs of distal ischemia in an athlete should lead to a search for this type of injury.

[Nontumoral epileptogenic lesions]. / Les lésions épileptogènes non tumorales.

Nontumoral epileptogenic lesions account for the major pathological group of surgical specimens obtained from patients with temporal or extratemporal drug-resistant epilepsy. Hippocampal sclerosis remains the predominant etiology, but cerebral cortical dysplasias actually make up the second major cause of nontumoral epilepsy and are increasingly recognized. The percentage of vascular lesions or glial/glio-mesodermal scars remains stable, but the minor or nonspecific lesion group is decreasing because of imaging investigation technique improvement.

[Incidence and mortality of central nervous system tumors in France: trends over the period 1978-2000 and influence of registration practices on results]. / Incidence et mortalité des tumeurs du système nerveux central en France: évolution de 1978 à 2000 et influence des pratiques d'enregistrement sur les résultats.

BACKGROUND: In France, cancer incidence figures are produced by cancer registries covering only 13.5% to 16% of the whole population of the country. Thus, to produce national figures, estimates have to be computed. Registration disparities between registries concerning tumors of the Central Nervous System (CNS) could have biased these estimates. METHODS: National estimates are based on modelling of the incidence/mortality ratio. The most recent estimations for year 2000 were calculated by the French Cancer Registry Network (FRANCIM) and the department of biostatistics of Lyon University Hospital. Since benign tumors are not recorded in some cancer registries, a new estimate of the incidence of CNS tumors was produced by estimating the number of benign tumors in these registries. RESULTS: In 2000 in France, the number of estimated cases of CNS tumors was 2697 in men and 2602 in women, with incidence rates (World standard) of 7.4 and 6.4 per 100,000 respectively. The incidence increased between 1978 and 2000, on an average by 2.25% per year in men and 3.01% per year in women. However, these estimates do not provide a correct picture of CNS incidence. First of all, pathological diagnoses are not performed in 3.5%-27.5% of the patients with CNS tumors registered in French registries. Second, figures for benign tumors (mainly meningiomas) were provided by only two of nine cancer registries. If benign tumors had been registered by all cancer registries, computed incidence would have increased by 12% for men and 26% for women. CONCLUSION: Incidence of CNS tumors is increasing in France, as in many other countries. To improve comparability with other countries, French cancer registries should also collect data on benign tumors. The discrepancies observed between registries in the proportion of patients without information on histology show differences in diagnostic practices and should be the starting point for a survey on this topic.

[Perfusion-diffusion 1H spectroscopy: role in the diagnosis and follow-up of supratentorial brain tumours in adults]. / Spectroscopie 1H, perfusion, diffusion: quelle place pour ces techniques lors du diagnostic et du suivi des principales tumeurs cérébrales sus-tentorielles de l'adulte?

INTRODUCTION: In a few years, magnetic resonance imaging (MRI) has evolved from a morphology-based examination to one that encompasses metabolism and function. STATE OF ART: MRI is a well-established tool for the initial evaluation of brain tumors, but conventional MR sequences have some limitations. Conventional MRI is unable to distinguish high-grade glioma from metastasis and abscess, to define precisely the histopathological grade of gliomas, to determine exactly the limits of tumor extension, to characterize meningeal tumors. Differentiation of tumor recurrence from treatment-related changes may be difficult with standard MR imaging because the interpretation is essentially based on volume analysis. PERSPECTIVES: 1H Spectroscopy, diffusion and perfusion imaging become possible with the development of MR imagers and can be routinely performed in clinical settings. They give complementary information about tumor metabolism and vascularity and allow a better analysis of post-treatment modifications. Functional and metabolic explorations should be used to characterize brain tumors.

[Chronic expanding hematoma of the soleus: a case report]. / Hématome encapsulé des membres à expansion progressive: à propos d'une localisation au muscle soléaire.

Trauma-induced hematomas of the limbs usually resorb without sequelae. In certain circumstances which are not fully understood, the hematoma may expand progressively, eventually leading to the development of a tumor-like mass in the soft tissues. We report the case of a chronic expanding hematoma observed in the right soleus muscle of a 75-year-old man. The mass grew +9 cm compared with the other side over a period of two to three years with no notion of recent trauma. Surgical biopsy disclosed a thick capsule containing "chocolate pus". Pathology and cytology examination led to the diagnosis of pseudo-tumor calcinosis subsequent to a hematoma which the patient had developed 34 years earlier when as a mountain guide he had experienced a tear of the soleus muscle. Local care required complete resection of the soleus muscle. The patient was able to resume activities without pain. Well described in the literature, encapsulated hematoma of the limbs is not well known in France. This case illustrated the potentially long latency period (34 years in our patient). Pathologically similar to tumor calcinosis, chronic expanding hematoma should be entertained as a possible diagnosis in a patient with a longstanding mass and a history of past trauma. The differential diagnosis with sarcoma is established by magnetic resonance imaging which reveals a peripheral low intensity signal on T1 and T2 sequences.

[Tuberous sclerosis without mental impairment, diagnosed in adulthood]. / Sclérose tubéreuse de Bourneville sans altération intellectuelle, diagnostiquée à l'âge adulte.

INTRODUCTION: Tuberous sclerosis complex (TSC) is an autosomal dominant inherited phakomatosis, usually diagnosed in childhood and characterized by cutaneous and neurological tumors, the latter often leading to epilepsy and mental retardation. EXEGESIS: We report a case of TSC diagnosed in a 33-year-old man, without any known family history of phakomatosis, presenting with facial angiofibromas, hypomelanotic macules, a giant-cell astrocytoma and retinal phakomas without any mental impairment or epilepsy. CONCLUSION: TSC may occur in patients who do not have any family history of phakomatosis because de novo mutations are frequent. TSC may be diagnosed in adulthood since a high phenotypic variability is observed. Facial angiofibromas are highly suggestive of tuberous sclerosis complex. They should lead to brain imaging in search for astrocytoma, subependymal nodules and cortical tubers which number is directly correlated with the risk of seizures and the degree of mental impairment.

Contribution of dynamic contrast MR imaging to the differentiation between dural metastasis and meningioma.

PURPOSE: To determine the perfusion-sensitive characteristics of cerebral dural metastases and compare them with the data on meningiomas. METHODS: Twenty-two patients presenting with dural tumor underwent conventional and dynamic susceptibility-contrast MR imaging: breast carcinoma metastases, two patients; colorectal carcinoma metastasis, one patient; lung carcinoma metastasis, one patient; Merkel carcinoma metastasis, one patient; lymphoma, one patient; meningiomas, 16 patients. The imaging characteristics were analyzed using conventional MR imaging. The cerebral blood volume (CBV) maps were obtained for each patient and the relative CBV (rCBV) in different areas was calculated using the ratio between the CBV in the pathological area (CBVp) and in the contralateral white matter (CBVn). RESULTS: The differentiation between a meningioma and a dural metastasis can be difficult using conventional MR imaging. The rCBVs of lung carcinoma metastasis (1 case: 1.26), lymphoma (1 case: 1.29), breast carcinoma metastasis (2 cases: 1.50,1.56) and rectal carcinoma metastasis (1 case: 3.34) were significantly lower than that of meningiomas (16 cases: mean rCBV = 8.97+/-4.34, range 4-18). Merkel carcinoma metastasis (1 case: 7.56) showed an elevated rCBV, not different from that of meningiomas. CONCLUSION: Dural metastases are sometimes indistinguishable from meningiomas using conventional MR imaging. rCBV mapping can provide additional information by demonstrating a low rCBV which may suggest the diagnosis of metastasis.

[Magnetic resonance cerebral blood volume maps. Comparison with histologic findings in different types of brain lesions]. / Volume sanguin cérébral déterminé par IRM de perfusion.

Recent developments in magnetic resonance (MR) have made it possible to obtain measurements of the microvasculature within brain lesions. Cerebral blood volume (CBV) maps calculated from dynamic contrast-enhanced MR imaging are particularly sensitive for depicting the microvasculature, and can enable the detection of neovascularization as well as its quantification in relative terms. The purpose of the present work is to compare the results of CBV maps calculated from MR imaging with those from histologic examination of the same region of interest: the biopsy site. Nineteen patients with brain lesions were studied (18 brain tumors and one case of multiple sclerosis). All patients underwent stereotactic biopsy, and calculation of CBV was performed from perfusion MR imaging. Three histopathologic parameters were assessed: the number of vessels (vessel density), the vessel size and the surface area filled by vessels (%). We observed a statistically significant correlation between the vessel density and the CBV, which is consistent with some previous publications. A noninvasive imaging method for characterizing the functional properties, especially hemodynamic activity, of malignant processes seems to be of great benefit to clinical practice.

Dynamic contrast-enhanced MRI: differentiating melanoma and renal carcinoma metastases from high-grade astrocytomas and other metastases.

We performed conventional and dynamic susceptibility-contrast MRI imaging in 38 patients with brain tumours: 20 with metastases (breast carcinoma: two; renal carcinoma: five; colorectal carcinoma: one; lung carcinoma: seven; melanoma: five), and 18 with high-grade astrocytomas. We obtained cerebral blood volume (CBV) maps and calculated the relative CBV (rCBV) in different areas using the ratio between the CBV in the pathological area (CBVp) and in the contralateral white matter (CBVn). We calculated the maximum rCBV (rCBVmax) for each tumour and compared the mean rCBVmax in each group of tumours. The mean rCBV of melanoma metastases (5.35+/-2.32, range 3.14-9.23) and of renal carcinoma metastases (8.17+/-2.39, range 5.41-11.64) were significantly greater than those of high-grade astrocytomas (2.61+/-1.17, range 1.3-5.0) ( P=0.002 and <0.001, respectively) and of lung carcinoma metastases (2.94+/-0.86, range 1.43-4.04) ( P=0.003 and 0.002). There was no statistically significant difference between the mean rCBV of lung metastases and of high-grade astrocytomas ( P=0.59). Large, solitary, necrotic metastases can be indistinguishable from high-grade astrocytomas using conventional MRI. Demonstration of an elevated rCBV which may suggest a hypervascular lesion such as renal carcinoma or melanoma.

[Focal cortical dysplasia possibly related to a probable prenatal ischemic injury]. / Dysplasie corticale focale liée à un probable accident ischémique anténatal.

Focal cortical dysplasias are a frequent etiology of partial seizure disorders refractory to medical treatment. We report the case of a patient with focal cortical dysplasia, confirmed by surgery, in association with ischemic cerebral lesions that possibly occurred during the intra-uterine development. This observation reinforces the hypothesis of a possible factor of causality between prenatal ischemia and anomalies of cortical development.

Chordoid glioma of the third ventricle: CT and MRI, including perfusion data.

Chordoid glioma is a homogeneous tumour involving the third ventricular region of middle-aged women, containing a small central cyst or necrosis. Histologically the tumour has a chordoid appearance. We report a new case with a haemodynamic imaging approach which indicates tumour angiogenesis at the capillary level.

Cerebral blood volume mapping by MR imaging in the initial evaluation of brain tumors.

PURPOSE: To assess the contribution of magnetic resonance (MR) cerebral blood volume (CBV) mapping in the initial evaluation of brain tumors. METHODS: 63 patients presenting a brain tumor underwent dynamic susceptibility-contrast MR imaging before surgery or biopsy: 28 high grade gliomas, 8 low grade gliomas, 2 pilocytic astrocytomas, 4 lymphomas, 12 metastases, 9 meningiomas. The CBV maps were obtained for each patient and the relative CBV (rCBV) in different areas was calculated using the ratio between the CBV in the pathological area (CBVp) and in the contralateral normal tissue(CBVn). The maximum rCBV (rCBVmax) for each tumor was determined and the mean values of rCBVmax in each group of tumors were compared using an unpaired Student t test (p=0.05). RESULTS: The rCBVmax for high grade gliomas (mean +/- SD: 2.6 +/- 1,2) was statistically different from low grade gliomas (0.9 +/- 0.4) (p<0.001), lymphomas (0.7 +/- 0.2) (p=0.002), meningiomas (9.1 +/- 4.4) (p<0.001) and kidney metastases (8.9 +/- 2.1) (p<0.001). The two pilocytic astrocytomas had a much lower rCBVmax than high grade gliomas. No statistically significant difference was found between high grade gliomas and lung metastases (2.4 +/- 0.9) (p=0.72). CONCLUSION: CBV mapping provides additional information on the vascularity of the lesions, which is not available with conventional MR imaging. It might be useful for differentiating certain lesions showing contrast enhancement, mainly high grade gliomas from kidney metastases, meningiomas, lymphomas or pilocytic astrocytomas.

Cortical dysplasia: electroclinical, imaging, and neuropathologic study of 13 patients.

PURPOSE: The aim of this study was to correlate the electroclinical and radiologic data with the neuropathologic findings and surgical outcome in epileptic patients with epilepsy and Taylor's focal cortical dysplasia (TFCD) and to characterize further the abnormal intermediate filaments expression in the balloon cell present in the peculiar dysplasia. METHODS: We retrospectively selected 13 TFCD patients who underwent surgery for intractable epilepsy with the aim of removing the magnetic resonance (MR)-detectable lesion and/or the epileptogenic zone defined by stereoelectroencephalographic recordings. The surgical specimens were analyzed by means of routine neuropathologic and immunocytochemical studies. Antisera against different intermediate filaments also were used in serial adjacent sections to evaluate their coexpression in balloon cells. RESULTS: Histopathologic abnormalities typical of TFCD were found not only within the MR-visible lesions but also in most of the epileptogenic zones with no MR signal alterations. Furthermore, the MR-visible lesions contained a high proportion of cells with an abnormal expression of intermediate filament proteins. After a long follow-up, 10 of the patients are now seizure free. CONCLUSIONS: Our findings indicate that highly epileptogenic zones may correspond to tissue alterations not revealed by neuroimaging. Furthermore, the immunocytochemical data show that the dysplastic tissue detected by MR contained high concentrations of cells filled with abnormal intermediate filaments. The detected colocalization of neuronal and glial markers in balloon cells indicates a failure of cellular commitment during development.