CLIPPERS among patients diagnosed with non-specific CNS neuroinflammatory diseases.

Chronic Lymphocytic Inflammation with Pontine Perivascular Enhancement Responsive to Steroids (CLIPPERS) is an inflammatory CNS disorder characterized by 1) subacute onset of cerebellar and brainstem symptoms, 2) peripontine contrast-enhancing perivascular lesions with a "salt-and-pepper" appearance on MRI, and 3) angiocentric, predominantly T-lymphocytic infiltration as revealed by brain biopsy. Inflammatory diseases including neuroinfections, CNS lymphoma and neurosarcoidosis must be excluded. Since CLIPPERS was described in 2010, many patients might have been misdiagnosed in the past. We therefore searched medical records from a large tertiary neurological center, the Department of Neurology at Rigshospitalet, Copenhagen University Hospital, for patients discharged between 1999 and 2013 with a diagnosis of "sarcoidosis with other localization", "other acute disseminating demyelination", "other demyelinating disease in the CNS" or "encephalitis, myelitis or encephalomyelitis". Of 206 identified patients, 24 had been examined by brain biopsy and were included for further evaluation. Following clinical, neuroradiological and neuropathological review, 3 patients (12.5%) were reclassified as having CLIPPERS. Median long-term follow-up was 75 months. The present results suggest that clinical re-evaluation of patients previously diagnosed with unspecified inflammatory demyelinating CNS disease or atypical neurosarcoidosis may increase the detection rate of CLIPPERS. Further, potentially severe neurological deficits and progressive parenchymal atrophy on MRI may suggest neurodegenerative features, which emphasizes the need for early immunomodulatory treatment.

Quantitative estimates of vascularity in solid tumors by non-invasive near-infrared spectroscopy.

We examined the relationship between non-invasive estimates of the tumor hemoglobin concentration by near-infrared spectroscopy (NIRS) and histological scores of tumor vascularity by Chalkley counts in seven tumor lines in nude mice [malignant gliomas: U87, U118, U373; small cell lung cancers (SCLC): 54A, 54B, DMS79; prostate cancer: MatLyLu (MLL)]. We also evaluated the effect of continuous anti-angiogenic treatment with TNP-470 on tumor hemoglobin concentration and tumor vascularity in U87 and MLL tumors. Non-invasive NIRS recordings were performed with a custom-built flash near-infrared spectrometer using light guide-coupled reflectance measurements at 800+/-10 nm. Chalkley counts were obtained from CD31-immunostained cryosections. The NIRS recordings in arbitrary absorbance units increased with tumor size in the individual tumors until a plateau was reached at approximately 150 mm(3). This plateau was relatively tumor line-specific. NIRS recordings at the plateau phase were strongly correlated (P<.001, n=71) to the histological vessel score (Chalkley count) of the same individual tumors excised immediately after the NIRS was performed. Non-invasive NIRS recordings of the highly vascularized gliomas (U87, U118, and U373) plus the MatLyLu tumor line were significantly higher than the three less vascularized SCLC tumor lines (P<.001). Continuous treatment with the anti-angiogenic compound TNP-470, an endothelial cell inhibitor, significantly retarded tumor growth in both U87 and MLL tumors, but all tumors eventually grew. When comparing treated and untreated tumors of similar size, both NIRS recordings and Chalkley counts were significantly lower in TNP-470-treated tumors (P<.05). In conclusion, the NIRS technique provides a non-invasive measure of the degree of vascularization in untreated tumors and the NIRS technique can measure modifications in tumor vascularization by anti-angiogenic therapy.

Coregulation of glucose uptake and vascular endothelial growth factor (VEGF) in two small-cell lung cancer (SCLC) sublines in vivo and in vitro.

We examined the relationship between (18)F- labeled 2-fluro-2-deoxy-d-glucose (FDG) uptake, and expression of glucose transporters (GLUTs) in two human small-cell lung cancer (SCLC) lines CPH 54A and CPH 54B. Changes in the expression of GLUTs and vascular endothelial growth factor (VEGF) during 12-, 18-, and 24 hours of severe hypoxia in vivo (xenografts) and in vitro (cell cultures) were recorded for both tumor lines. The two SCLC lines are subpopulations of the same patient tumor. In spite of their common genomic origin they represent consistently different metabolic and microenvironmental phenotypes as well as treatment sensitivities. There were higher levels of Glut-1 protein in 54B and a correspondingly higher FDG uptake in this tumor line (P<.001). During hypoxia a significant upregulation of in VEGF mRNA, GLUT-1 mRNA, and Glut-1 and -3 protein occurred with a distinctly different time course in the two cell lines. A similar co-upregulation of GLUT and VEGF was seen in hypoxic tumors of both lines. There were no significant changes of HIF-1alpha mRNA during hypoxia in either of the cell lines. A more detailed understanding of such correlations between glucose metabolism, angiogenesis, and microenvironmental phenotype of tumors, by positron emission tomography (PET) and molecular techniques might further sophisticate our interpretation of glycolytic predominance in tumors as seen by 18FFDG PET.

Relationship between vessel density and expression of vascular endothelial growth factor and basic fibroblast growth factor in small cell lung cancer in vivo and in vitro.

In 21 human small cell lung cancer (SCLC) cell lines, we determined the expression of mRNA and secreted protein levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). The VEGF expression was highly variable between cell lines, with a > 100-fold variation, under identical in vitro conditions. The bFGF expression in cell lines was generally very low. Nine of the cell lines were further analyzed during growth as solid tumor xenografts in nude mice (in vivo). A more uniform VEGF protein expression was present in vivo. Compared with the variable in vitro expression, VEGF was relatively up-regulated in the tumor lines CPH 54A and CPH 54B and down-regulated in GLC 3. One line, DMS 79, had a high VEGF expression in vivo as well as in vitro. The vessel density was determined by Chalkley point counting on CD31 immunostained cryosections of tumors of each of the nine SCLC lines. We found a strong positive correlation between vessel density and tissue VEGF protein expression (r(s) = 0.75; P = 0.02) and a comparatively strong negative correlation (r(s) = -0.80; P = 0.01) between vessel density and tissue bFGF expression. No significant correlation was present between vessel density and in vitro VEGF expression. We conclude that VEGF and bFGF expression is dependent on microenvironmental conditions, as well as cell line-specific factors, and that a strong positive correlation exists between in vivo VEGF expression and vessel density, whereas high tissue levels of bFGF are not correlated with higher vessel densities in SCLC xenografts.

Therapeutic synergy of TNP-470 and ionizing radiation: effects on tumor growth, vessel morphology, and angiogenesis in human glioblastoma multiforme xenografts.

We examined the effect on tumor growth, vessel morphology, and expression of angiogenic factors of combining radiotherapy and antiangiogenesis in the human glioblastoma line U87 grown in the flank or intracranially in the nude mouse. The antiangiogenic agent TNP-470 was given 6.7 mg/kg s.c. daily on day 1-7 starting 1 week after transplantation. Irradiation (IR), 10 Gy x 1, was administered on day 7. A series of tumors were excised 8 and 48 h after the end of treatment. The vascular morphology was evaluated in CD31 immunostained cryosections and by electron microscopy, and the pattern of expression of angiogenic factors (mRNA and protein) was quantitatively analyzed by phosphorimaging of Northern blots and Western blots. Significant inhibition of s.c. flank tumor growth relative to untreated controls was achieved by monotherapy with both TNP-470 (P < 0.001) and IR (P < 0.001). A significant enhancement of this effect was obtained by combining TNP-470 and IR (P < 0.05). We saw no effect of TNP-470 either alone or in addition to the effect of IR on the survival of mice with intracranial tumors. CD31 immunostaining of s.c. tumors showed acute endothelial swelling and luminal protrusion in irradiated tumor vessels but never in tumors pretreated with TNP-470, and not in the untreated controls. The vessel density (Chalkley point counts) was unchanged by TNP-470 therapy. In the TNP-470-treated tumors, we observed a distinct broadening of the endothelial basement membrane by an approximately 400-700-nm-thick electron-dense yet uncharacterized fibrillar material. TNP-470 treated tumors +/- IR also had a significantly increased mRNA expression of angiopoietin-1, whereas angiopoietin-2, vascular endothelial growth factor and basic fibroblast growth factor mRNA were unchanged by the treatments. In conclusion, TNP-470 significantly enhanced the tumor effect of ionizing IR, and our findings strongly indicate that acute microvascular damage after IR is effectively prevented by concurrent TNP-470 treatment. A significant up-regulation of angiopoietin-1 seems to play a role in this protective mechanism, which as yet is not fully elucidated.

Effect of radiation therapy on small-cell lung cancer is reduced by ubiquinone intake.

The effect of oral ubiquinone (Q10) intake on the in vivo response of tumors to single dose radiotherapy was examined. The human small-cell lung cancer (SCLC) line CPH 054A, which is sensitive to relatively low doses of X-radiation, was grown as subcutaneous transplants in the flanks of nude nu/nu mice. When macroscopical growth was established, groups of mice received either 10, 20 or 40 mg/kg Q10 in 30 mL soy oil intragastrically daily on 4 consecutive days. Controls received either 30 mL of pure soy oil or nothing. Three h after the last dose half of the tumors in each group received a single radiation dose of 5 Gy, using a 300 kV therapeutic unit. The macroscopic growth pre- and posttreatment was analyzed according to a transformed Gompertz algorithm using the software program GROWTH. Treatment with Q10 or soy oil alone had no effect on tumor growth compared with untreated controls. Groups of tumors that received Q10 and radiotherapy had a significantly lower specific growth delay (SGD) than the radiotherapy-only groups. This effect was significant at 40 mg/kg and borderline at 20 mg/kg, whereas at 10 mg/kg no radioprotection was seen. We conclude that systemic Q10 reduces the response to single dose tumor irradiation inxenotransplanted human SCLC tumors.

Tumor angiogenesis--a new therapeutic target in gliomas.

Tumor growth is critically dependent on angiogenesis, which is sprouting of new vessels from pre-existing vasculature. This process is regulated by inducers and inhibitors released from tumor cells, endothelial cells, and macrophages. Brain tumors, especially glioblastoma multiforme, have significant angiogenic activity primarily by the expression of the angiogenic factor VEGF Anti-angiogenic therapy represents a new promising therapeutic modality in solid tumors. Several agents are currently under evaluation in clinical trials. The present review describes the principal inducers and inhibitors of angiogenesis in tumors and summarizes what is known about their mechanisms of action in relation to CNS tumors. Potential areas for clinical use are also discussed.