The molecular mechanisms underlying the development of pancreatic neuroendocrine tumors (PanNETs) have not been well defined. We report here that the genomic region of the PHLDA3 gene undergoes loss of heterozygosity (LOH) at a remarkably high frequency in human PanNETs, and this genetic change is correlated with disease progression and poor prognosis. We also show that the PHLDA3 locus undergoes methylation in addition to LOH, suggesting that a two-hit inactivation of the PHLDA3 gene is required for PanNET development. We demonstrate that PHLDA3 represses Akt activity and Akt-regulated biological processes in pancreatic endocrine tissues, and that PHLDA3-deficient mice develop islet hyperplasia. In addition, we show that the tumor-suppressing pathway mediated by MEN1, a well-known tumor suppressor of PanNETs, is dependent on the pathway mediated by PHLDA3, and inactivation of PHLDA3 and MEN1 cooperatively contribute to PanNET development. Collectively, these results indicate the existence of a novel PHLDA3-mediated pathway of tumor suppression that is important in the development of PanNETs.p53 | PH domain | everolimus | p53 target gene | mTOR N euroendocrine tumors (NETs) arise from cells of the endocrine and nervous systems, and are found in tissues such as lung, pancreas and pituitary (1-3). NETs often produce, store and release biogenic amines and polypeptide hormones, and secretary granules containing these products provide a diagnostic marker for NETs. The mechanisms underlying the development of NETs remain unclear to date, due to the low incidence of these tumors and due to the lack of suitable experimental model systems, including genetically engineered mouse models. Pancreatic NET (PanNET), which is probably the best-studied NET, is the second-most common pancreatic tumor, having an incidence of ∼1 per 100,000 individuals. Patients having late-stage PanNET often harbor tumors that are unresectable or metastatic and face limited treatment options. Accordingly, the prognosis of patients having metastatic PanNET is the worst among the NET subtypes, with a 5-y survival rate of 27-43% (1). Recently, the drug Everolimus has shown promise in the treatment of PanNETs (4), providing a significant improvement in progression-free survival. Everolimus is an inhibitor of mammalian target of rapamycin (mTOR), a downstream mediator of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway. The striking efficacy of Everolimus demonstrates the importance of the PI3K/Akt pathway in the pathology of PanNETs.In agreement with these clinical results, studies on pancreatic endocrine cell lines have identified the PI3K/Akt signaling pathway as a major proliferation and survival pathway in these cells (5). Activated Akt phosphorylates substrates such as mTOR and controls various biological processes, including protein synthesis, proliferation, cell growth, and survival. Regulation of pancreatic islet β-cell proliferation, cell size, and apoptosis by Akt has been demonstrated using various mouse models. For examp...

ResultsWe enrolled 60 consecutive patients and excluded 10 because of severe calcification and large ulcers (Table). The contrast Background and Purpose-Neovascularization associated with plaque vulnerability, particularly in the plaque shoulder, is susceptible to rupture, causing ischemic events. We aimed to use contrast-enhanced ultrasound to evaluate neovessels in carotid plaques quantitatively, focusing on plaque shoulders. Methods-Using contrast-enhanced ultrasound with perflubutane, we analyzed 50 consecutive patients who underwent carotid endarterectomy. We measured enhanced intensity and assessed the correlation between contrast effect and histopathology, comparing symptomatic and asymptomatic plaques. Results-Enhanced intensity of the plaque shoulder was associated with neovessel density (P<0.01; ρ=0.43). Enhanced intensity of the plaque shoulder was higher in plaques with rupture than in those without (P<0.05), and in symptomatic plaques (n=31) than in asymptomatic ones (n=19; P<0.01). Conclusions-Quantitative evaluation of the contrast effect using contrast-enhanced ultrasound enabled the assessment of neovascularization of plaque shoulders in vivo real time, which may help stratify plaque vulnerability. of plaque shoulders was greater than that of cores (EI S versus EI C , 9.6±3.6 versus 1.1±1.6 dB, respectively; P<0.0001), consistent with histological findings that cores contained lipid and hemorrhage with few neovessels, mainly localized in plaque shoulders ( Figure 2). EI S associated with neovessel density in the plaque shoulder (P=0.0017; ρ=0.43; Figure I in the onlineonly Data Supplement). EI S was higher in plaques with rupture than in those without rupture (EI S , 10.1±3.5 versus 6.9±3.2 dB, respectively; P=0.018). EI S tended to be higher in plaques with intraplaque hemorrhage than in those without rupture (EI S , 9.9±3.6 versus 7.6±2.5 dB, respectively; P=0.095).The contrast effect of the plaque shoulder of symptomatic plaques was significantly greater than that of asymptomatic plaques (EI S , 10.8±3.7 versus 7.7±2.4 dB, respectively; P=0.0016). Among symptomatic plaques, EI S values of plaques that had a time interval from the onset of the last event to carotid endarterectomy of ≤6 months were higher than those of plaques with an interval of >6 months, but the difference was not statistically significant (EI S , 11.3±3.7 versus 9.2±3.7 dB, respectively; P=0.34; Figure II in the online-only Data Supplement). DiscussionThe histological characteristics of vulnerable plaques, including intraplaque hemorrhage, thrombus, inflammatory cells, thin fibrous caps, and neovascularization, were reviewed.2 Neovessels are immature and fragile, particularly in plaque shoulders, because local inflammatory damage and shear stress from the arterial lumen lead to collapse, causing intraplaque hemorrhage and plaque rupture, 1 consistent with the present results. Although MRI 3 and computed tomographic angiography 4 using contrast agents yield highly reproducible evaluations of carotid artery neovascularization...

Background: Approximately one quarter of the acute ischemic stroke patients notice the event at awakening. Such patients with stroke at awakening are usually excluded from thrombolysis, since the time of stroke onset cannot be definitely identified. We compared the hyperacute CT findings of awakening stroke patients with those of stroke patients with known onset to assess whether the time of stroke onset is shortly before awakening. Methods: Subjects were cardioembolic stroke patients who were consecutively admitted to our department within 3 h after the recognition of stroke during the period between January 2000 and March 2003. The patients were classified into three groups: group A with stroke of known onset, group B with stroke at awakening, and group C with stroke of unknown onset due to lack of a witness. The clinical and CT findings in each group were compared. Results: A total of 81 patients fulfilled the study criteria. There were 46 patients in group A, 17 patients in group B, and 18 patients in group C. There was no significant difference in CT findings between groups A and B. In group C, however, definite hypodense areas were more commonly found than in group A (56 vs. 0%; p < 0.001) or in group B (56 vs. 11%; p = 0.012). Conclusion: Based on our CT findings, stroke at awakening seems to be developing shortly before in a large subset of patients, making them potential candidates for acute stroke therapies.

Background and Purpose-To establish the diagnostic criteria for the site of occlusion in the vertebral arteries (VAs) using duplex color-coded ultrasonography. Methods-In 128 consecutive patients who underwent conventional cerebral angiography, we prospectively measured the diameter, mean flow velocity (MV), peak systolic flow velocity, and end-diastolic flow velocity of both VAs. The diameter-ratio (diameter of contralateral VA divided by that of target VA) and MV-ratio (MV of contralateral VA divided by that of target VA) were determined. Based on the angiographic findings, we classified the VAs into 4 types (5 groups) as follows: (1) the origin of VA occlusion (Origin group: nϭ9); (2) VA occlusion before branching into the posterior inferior cerebellar artery (PICA) (Before group: nϭ10); (3A) symptomatic VA occlusion after branching into the PICA (After group: nϭ12); (3B) asymptomatic or hypoplastic occlusive VA after branching into the PICA (PICA end group: nϭ15); and (4) no significant occlusive lesions in the VA (Control group: nϭ194). Results-No flow signals in the VAs apparently indicated the Origin group. Preserved peak systolic flow velocity but end-diastolic flow velocity of zero cm/s indicated the Before group. MV Ͻ18 cm/s and MV-ratio Ն1.4 indicated the PICA end group or After group. Furthermore, these groups could be distinguished as follows: a diameter-ratio Ͻ1.4 indicated the After group. A diameter-ratio Ն1.4 indicated the PICA end group. Either MV Ն18 cm/s or MV Ͻ18 cm/s in combination with MV-ratio Ͻ1.4 indicated the Control group. Conclusion-Duplex color-coded ultrasonography can accurately diagnose the site of VA occlusion.

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