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Genome-wide association studies (GWAS) have mapped risk alleles for at least 10 distinct cancers to a small region of 63 000 bp on chromosome 5p15.33. This region harbors the TERT and CLPTM1L genes; the former encodes the catalytic subunit of telomerase reverse transcriptase and the latter may play a role in apoptosis. To investigate further the genetic architecture of common susceptibility alleles in this region, we conducted an agnostic subset-based meta-analysis (association analysis based on subsets) across six distinct cancers in 34 248 cases and 45 036 controls. Based on sequential conditional analysis, we identified as many as six independent risk loci marked by common single-nucleotide polymorphisms: five in the TERT gene (Region 1: rs7726159, P = 2.10 x 10(-39); Region 3: rs2853677, P = 3.30 x 10(-36) and PConditional = 2.36 x 10(-8); Region 4: rs2736098, P = 3.87 x 10(-12) and PConditional = 5.19 x 10(-6), Region 5: rs13172201, P = 0.041 and PConditional = 2.04 x 10(-6); and Region 6: rs10069690, P = 7.49 x 10(-15) and PConditional = 5.35 x 10(-7)) and one in the neighboring CLPTM1L gene (Region 2: rs451360; P = 1.90 x 10(-18) and PConditional = 7.06 x 10(-16)). Between three and five cancers mapped to each independent locus with both risk-enhancing and protective effects. Allele-specific effects on DNA methylation were seen for a subset of risk loci, indicating that methylation and subsequent effects on gene expression may contribute to the biology of risk variants on 5p15.33. Our results provide strong support for extensive pleiotropy across this region of 5p15.33, to an extent not previously observed in other cancer susceptibility loci.

BACKGROUND Previous studies have established several meningioma genomic subgroups, defined by the mutually exclusive genomic drivers. However, we distinguished a small subset of meningiomas simultaneously harboring multiple drivers from different genomic subgroups, thus referred to as “multiple-driver meningiomas”, that has not been previously investigated. We aimed to characterize the genomic landscape and clinical features of multiple-driver meningiomas. METHODS We identified 42 cases of multiple-driver meningiomas from a cohort of 3,016 cases screened for genomic drivers via molecular inversion probe sequencing (MIPS) and/or whole-exome sequencing (WES) previously in our laboratory. All driver mutations were validated via Sanger sequencing and chromosome 22 loss was accessed with qPCR. Clinical information was collected and genome-to-clinical correlations were statistically analyzed. We selected 10 multiple-driver meningioma cases for tumor-normal WES for further characterization of genomic architecture. RESULTS Multiple-driver meningiomas were significantly enriched for NF2 alteration (p< 0.001), had significantly higher WHO grade (p= 0.005) and proportion of recurrent tumors (p= 0.007) when compared with “single-driver meningiomas”, i.e. harboring driver alterations from a single genomic subgroup. Among female cases, those with multiple drivers were nearly four times more likely to be high-grade (II or III) compared to single-driver female samples (48.6% vs. 12.6%, p< 0.001). Among tumors harboring NF2 alteration, multiple-driver meningiomas demonstrated skull base predilection compared with single-driver meningiomas (50.0% vs. 27.9%, p= 0.005). WES analysis of 10 multiple-driver meningiomas identified 1p and/or 14q loss in 60% of cases. Clonality analysis revealed the presence of sub-clonal cell populations in 9 cases with all driver mutations clustered into the founding clone in 7 cases, suggesting their acquisition in the early phase of tumor development. CONCLUSION Multiple-driver meningiomas demonstrate distinct genomic and clinical features that distinguish them as clinically aggressive. They frequently occur in surgically challenging skull base locations, and are more prevalent among high-grade and recurrent cases.

The prognosis for glioma remains dismal, and little is known about the final disease phase. To obtain information about this period, we surveyed caregivers of patients who were registered in the German Glioma Network and who died from the disease.A questionnaire with 15 items, focusing on medical, logistic, and mental health support and symptom control during the final 4 weeks, was sent to caregivers. For some of the questions, a scale from 1 (inadequate) to 10 (excellent) was used.Of 1655 questionnaires, 605 were returned (36.6%) and evaluated. We found that 67.9% of the patients were taken care of at home for the last 4 weeks; 47.7% died at home, 22.6% died in hospitals, and 19.3% died in hospice facilities. Medical support was provided by general practitioners in 72.3% of cases, by physicians affiliated with a nursing home or hospice in 29.9%, and by general oncologists in 17%. Specialized neuro-oncologists were involved in 6%. The caregivers ranked the medical support with a mean of 7.2 (using a 10-point scale), nursing service with 8.1, and mental health support with 5.5. In 22.9% of cases, no support for the caregivers themselves was offered by medical institutions.Although these data reflect the caregivers' subjective views, they are useful in understanding and improving current patterns of care. While patients and their caregivers are supported mainly by neuro-oncologists for most of the disease phase, the end-of-life phase is managed predominantly by general practitioners and specialists in palliative care. Close cooperation between these specialties is necessary to meet the specific needs of glioma patients.

Cellular heterogeneity, for example, the intratumoral coexistence of cancer cells with and without stem cell characteristics, represents a potential root of therapeutic resistance and a significant challenge for modern drug development in glioblastoma (GBM). We propose here that activation of the innate immune system by stimulation of innate immune receptors involved in antiviral and antitumor responses can similarly target different malignant populations of glioma cells. We used short-term expanded patient-specific primary human GBM cells to study the stimulation of the cytosolic nucleic acid receptors melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene I (RIG-I). Specifically, we analyzed cells from the tumor core versus “residual GBM cells” derived from the tumor resection margin as well as stem cell-enriched primary cultures versus specimens without stem cell properties. A portfolio of human, nontumor neural cells was used as a control for these studies. The expression of RIG-I and MDA5 could be induced in all of these cells. Receptor stimulation with their respective ligands, p(I:C) and 3pRNA, led to in vitro evidence for an effective activation of the innate immune system. Most intriguingly, all investigated cancer cell populations additionally responded with a pronounced induction of apoptotic signaling cascades revealing a second, direct mechanism of antitumor activity. By contrast, p(I:C) and 3pRNA induced only little toxicity in human nonmalignant neural cells. Granted that the challenge of effective central nervous system (CNS) delivery can be overcome, targeting of RIG-I and MDA5 could thus become a quintessential strategy to encounter heterogeneous cancers in the sophisticated environments of the brain.

Two phase III trials of photodynamic therapy (PDT) with BF-200 ALA, a recently approved nanoemulsion formulation of 5-aminolaevulinic acid (ALA) demonstrated high clearance rates in mild-to-moderate actinic keratosis (AK). The comparison to a registered methyl aminolaevulinate (MAL) cream demonstrated significantly superior total patient clearance rates.To evaluate long-term efficacy and safety of PDT for AK 6 and 12 months after the last PDT with BF-200 ALA, MAL or placebo.The follow-up phase (FUP) was performed with patients of two phase III studies. Both studies compared BF-200 ALA with placebo, one of the studies additionally with MAL. Overall recurrence rates and various subgroups (light source, lesion severity, lesion location, complete responders after first PDT) were assessed 6 and 12 months after the last PDT.Recurrence rates were similar for BF-200 ALA and MAL, with a tendency to lower recurrence rates for BF-200 ALA. The proportion of patients who were fully cleared during PDT and remained completely clear for at least 12 months after PDT were 47% for BF-200 ALA (both studies) and 36% for MAL treatment. The subgroup that was illuminated with narrow wavelength LED lamps reached 69% and 53% for BF-200 ALA (both studies, respectively) and 41% for MAL. No safety concerns were reported.The FUP data confirmed the high efficacy and safety of PDT with BF-200 ALA. The slightly lower recurrence rates after BF-200 ALA treatment compared with MAL treatment enhanced the better treatment outcome due to the significantly superior efficacy.

There is an urgent need for more effective therapies in glioblastoma (GBM). Data from the single arm UKT-03 trial (Glas et al., J Clin Oncol 27, 1257, 2009) suggested that combined lomustine/temozolomide (CCNU/TMZ) therapy might have superior activity in MGMT-methylated GBM. The phase III CeTeG/NOA-09 trial was set up to test this hypothesis in a randomized setting. Patients with MGMT-methylated GBM were randomized (1:1) for standard therapy with daily TMZ (75 mg/m2) during local radiotherapy (RT, 30 x 2 Gy) followed by 6 courses of TMZ (150–200 mg/m2/day for 5 days q4w) or experimental therapy with CCNU/TMZ in addition to local RT. Six 6-week courses of CCNU/TMZ (CCNU 100 mg/m2 d1, TMZ 100 mg/m2 d2-6) were applied with the first course starting on the first day of RT while continuous concomitant TMZ was not applied. The TMZ dose was adapted in further courses. The primary endpoint was overall survival (OS) in the modified intention-to-treat (mITT) population of patients who had received at least one dose of study medication. For confirmatory analysis, differences in OS were analyzed using the logrank test stratified for center and recursive partitioning analysis group. The trial randomized 141 patients; 129 patients (63 TMZ, 66 CCNU/TMZ) belonged to the mITT population. OS was superior in the CCNU/TMZ arm as compared to the TMZ arm (p=0.049). Significant OS differences were also found in the as-randomized population (p=0.043) and in the subgroup of 103 patients with an IDHwt GBM (p=0.039). In the mITT population, median OS was prolonged from 31.4 months (95%CI 27.0–44.8 months) with TMZ to 37.9 months (95% CI 29.2–51.4 months) with CCNU/TMZ (hazard ratio for death 0.60, 95% CI 0.35–1.03, p=0.064). In summary, CCNU/TMZ combination chemotherapy showed improved efficacy as compared to TMZ standard therapy in patients with newly diagnosed MGMT promotor-methylated GBM.

Summary Background Photodynamic therapy (PDT) with 5-aminolaevulinic acid (ALA) or its methylester [methyl-5-aminolaevulinate (MAL) or 5-amino-4-oxopentanoate] was recently ranked as first-line therapy for the treatment of actinic keratosis (AK) and is an accepted therapeutic option for the treatment of neoplastic skin diseases. BF-200 ALA (Biofrontera Bioscience GmbH, Leverkusen, Germany) is a gel formulation of ALA with nanoemulsion for the treatment of AK which overcomes previous problems of ALA instability and improves skin penetration. Objectives To evaluate the efficacy and safety of PDT of AKs with BF-200 ALA in comparison with a registered MAL cream and with placebo. Methods The study was performed as a randomized, multicentre, observer-blind, placebo-controlled, interindividual trial with BF-200 ALA, a registered MAL cream and placebo in a ratio of 3 : 3 : 1. Six hundred patients, each with four to eight mild to moderate AK lesions on the face and/or the bald scalp, were enrolled in 26 study centres in Germany, Austria and Switzerland. Patients received one PDT. If residual lesions remained at 3 months after treatment, PDT was repeated. Results PDT with BF-200 ALA was superior to placebo PDT with respect to patient complete clearance rate (78·2% vs. 17·1%; P

Although clinically aggressive meningiomas (MGMs) are rare tumors, more effective therapies are urgently needed. As a prerequisite for successful immunotherapy we determined the infiltration and activation status of T cells in primary (p-) and recurrent (r-) MGMs and their impact on survival. Presence of tumor-infiltrating lymphocytes (TILs) was assessed in a large, clinically well-annotated study sample of 202 cases (n=123 pMGMs, and n=79 rMGMs) with a substantial proportion of higher-grade MGMs (n=43 WHO°I, n=97 WHO°II, n=62 WHO°III). TIL quantification was performed by a semi-automated analysis on whole tissue sections stained by multi-color immunofluorescence for CD3, CD8, FOXP3, and PD-1. Ranging from 0.01–24.88 %, median T cell infiltration accounted for 0.59 TILs per total cell count (TCC). Although there were no significant changes for the proportion of helper and cytotoxic T cells in pMGM of different WHO grades, higher numbers of cytotoxic T cells were associated with an improved progression-free survival (PFS) independent of prognostic confounders. rMGM were characterized by significantly lower numbers of TILs in general (median: 0.33 % per TCC), helper and cytotoxic T cells and a significant increase of regulatory T cells. As for the activation of TILs, about one third expressed the immune checkpoint molecule PD-1 and predominantly were CD8-positive. We observed a significant WHO-dependent decrease of PD-1(+)/CD8(+) TILs which in univariate and multivariate analyses were associated with a poorer PFS. In line with these findings proportions of PD-1(+)/CD8(+) TILs were significantly lower in rMGM arguing for PD-1 as an activation rather than an exhaustion marker. In summary, we were able to identify intratumoral cytotoxic TILs as well as of PD-1-expressing cytotoxic TILs as novel and easy applicable biomarkers for a better survival, which might facilitate the selection of patients who could benefit from immunotherapeutic approaches and mandates for an intervention in primary rather than recurrent tumors.

Giant cell glioblastoma (gcGB), a subtype of GB, is characterized by the presence of numerous multinucleated giant cells. The prognosis for gcGB is poor, but it may have a better clinical outcome compared with classic GB. The molecular alterations that lead to the multinucleated cell phenotype of gcGB have not been elucidated. Giant cell GB has a higher frequency of the tumor suppressor protein p53 mutations than GB, however, and a role for the mitotic Aurora B kinase has been suggested. We analyzed Aurora B expression in gcGB (n = 28) and GB (n = 54) patient tumor samples by immunohistochemistry; 17 gcGB and 22 GB samples were analyzed at the DNA and mRNA levels. No mutations in the Aurora B gene (AURKB) were found, but its mRNA and protein levels were significantly higher in gcGB than in GB. Fifty-nine percent of gcGB samples but only 18% of the GB samples showed p53 mutations. Ectopic overexpression of Aurora B induced a significant increase inthe proportion of multinucleated cells in p53 mutant U373-MG, but not in p53 wild-type U87-MG, glioma cells. RNAi of p53 in U87-MG cells led to an increase in the fraction of multinucleated cells that was further augmented by ectopic overexpression of Aurora B. These results suggest that loss of p53 function and dysregulated Aurora B protein levels might represent factors that drive the development of multinucleated cells in gcGB.

Focal cortical dysplasia type IIb is characterized by epilepsy-associated malformations that are often composed of balloon cells and dysplastic neurons. There are many histopathologic similarities between focal cortical dysplasia type IIb and cortical tubers in tuberous sclerosis complex (TSC), an autosomal-dominant phakomatosis caused by mutations in the TSC1 or TSC2 genes that encode hamartin and tuberin. We previously found that an allelic variant of TSC1 (hamartin) is increased in focal cortical dysplasia type IIb. Here, we investigated the subcellular localization of hamartin and its interaction with tuberin in vitro. Coimmunoprecipitation assays with tuberin revealed reduced tuberin binding of hamartin compared with wild-type hamartin. Tuberin binding was also reduced for 2 TSC1 stop mutants (hamartin and hamartin) that are present in brain lesions of TSC patients. Colocalization assays of hamartin and tuberin were performed in HEK293T cells, and the subcellular localization of the hamartin variants were studied using immunocytochemistry. There was an impairment of tuberin binding of hamartin and aberrant nuclear distribution of hamartin in these cells, whereas hamartin and hamartin were, like wild-type tuberin, localized in the cytoplasm. These data suggest a fundamental functional impairment of hamartin and the 2 TSC1 stop mutants hamartin and hamartin in vitro. Future studies will be needed to characterize the roles of these TSC1 sequence variants in the genesis of dysplastic epileptogenic developmental brain lesions.

The median survival of glioblastoma patients is approximately 12 months. However, 3-5% of the patients survives for more than 3 years and are referred to as long-term survivors. The clinical and molecular factors that contribute to long-term survival are still unknown. To identify specific parameters that might be associated with this phenomenon, we performed a detailed clinical and molecular analysis of 55 primary glioblastoma long-term survivors recruited at the six clinical centres of the German Glioma Network and one associated centre. An evaluation form was developed and used to document demographic, clinical and treatment-associated parameters. In addition, environmental risk factors, associated diseases and occupational risks were assessed. These patients were characterized by young age at diagnosis and a good initial Karnofsky performance score (KPS). None of the evaluated socioeconomic, environmental and occupational factors were associated with long-term survival. Molecular analyses revealed MGMT hypermethylation in 28 of 36 tumours (74%) investigated. TP53 mutations were found in 9 of 31 tumours (29%) and EGFR amplification in 10 of 38 tumours (26%). Only 2 of 32 tumours (6%) carried combined 1p and 19q deletions. Comparison of these data with results from an independent series of 141 consecutive unselected glioblastoma patients registered in the German Glioma Network revealed significantly more frequent MGMT hypermethylation in the long-term survivor group. Taken together, our findings underline the association of glioblastoma long-term survival with prognostically favourable clinical factors, in particular young age and good initial performance score, as well as MGMT promoter hypermethylation.

Vascular endothelial growth factor (VEGF) or vascular permeability factor (VPF) is a selective mitogen for endothelial cells; it increases microvascular permeability and has been shown to relax isolated canine coronary arteries by an endothelium-dependent mechanism. In many tissues VEGF/VPF is expressed after an appropriate stimulus, mostly hypoxia. In the kidney VEGF/VPF is constitutively expressed in glomerular podocytes and epithelia of collecting duct. Glomerular and peritubular capillary endothelia also constitutively express specific VEGF receptors. The in vivo function of renal VEGF/VPF is unknown.In the present study the effects of human recombinant VEGF165 on renal haemodynamics and glomerular permselectivity was investigated in the isolated perfused kidney of the rat.In kidneys preconstricted by noradrenaline (NA 1.5 x 10(-7) mol/l) VEGF/VPF (155 pmol/l) caused an almost complete return of renal perfusion flow rate to pre-NA values (before NA 113 +/- 4%, after NA 100%, 15 min with VEGF/VPF 111 +/- 4%). Shortly after VEGF/VPF administration VEGF/VPF-induced relaxation commenced, and became significant after 2 min (15 min with VEGF/VPF vs without VEGF/VPF 111 +/- 4% vs 103 +/- 2%; P0.05). In the presence of the NO-synthase inhibitor N(W)-nitro-L-arginine (L-NNA; 5 x 10(-5) mol/l) VEGF/VPF caused only small, transient relaxations (before NNA 109 +/- 5%, after NNA 100%, 15 min with VEGF 95 +/- 2%). The cyclooxygenase inhibitor diclofenac failed to inhibit the relaxing activity of VEGF/VPF (before NA 119 +/- 4%, after NA + diclofenac 100%, 15 min with VEGF/VPF 123 +/- 5%). VEGF demonstrated no significant increase in renal protein excretion rate (after NA pretreatment (= 100%): 12.5 min with VEGF/VPF vs without VEGF/VPF: 119 +/- 10% vs 132 +/- 11%, n.s.) (after NNA pretreatment (= 100%) 12.5 min with VEGF/VPF vs without VEGF/VPF 94 +/- 5% vs 96 +/- 4%; n.s.) or clearance quotient of albumin. Glomerular filtration rate was not influenced by VEGF/VPF in kidneys pretreated with NA (before NA 105 +/- 5%, after NA 100%, 12.5 min with VEGF/VPF 94 +/- 2%) or with NNA (before NNA 107 +/- 6%, after NNA 100%, 12.5 min with VEGF/VPF 96 +/- 2%). Fractional glucose and fractional sodium excretion showed flow-dependent changes.VEGF/VPF can contribute to the relaxing capacity of the renal vasculature. This relaxation is partly mediated by the NO/endothelium-derived relaxing factor (EDRF) pathway. In the isolated perfused rat kidney the glomerular permeability for albumin is not affected by VEGF/VPF.

BACKGROUND: A relevant fraction of patients does not receive salvage therapy at recurrence of glioblastoma initially treated according to current standards of care. Primary treatment may be of higher relevance in this patient group. Risk factors to prospectively determine which patients have a higher probability to receive salvage therapy have not been prospectively studied. METHODS: Patients (n = 1004) with first recurrence of a glioblastoma received no salvage treatment (n = 374; 37.3%, group N) or ≥1 line of postprogression therapy consisting of surgical resection +/- second radio- and/or chemotherapy (n = 630; 62.7%, group Y) in the prospectively assembled cohort of the German Glioma Network. Age, gender, Karnofsky Performance Status (KPS), extent of resection, O6-methylguanine methyltransferase (MGMT) and isocitrate dehydrogenase (IDH) 1/2 status as well as first line therapy were assessed as risk factors not to receive salvage therapy. RESULTS: Median overall survival in the whole cohort was 14.2 (95% confidence interval 13.4-15.0) months. It was 8.7 (95% CI 8.1-9.2) months in group N as opposed to 18.5 (95% CI 17.2-19.8) months in group Y patients. Positive prognostic factors to receive salvage treatment were age ≤ 60 years (odds ratio (OR), 2.9, p < 0.001), complete resection at first-line treatment (OR 1.8, p = 0.011) and KPS >70 (OR 1.7, p = 0.035), but not initial treatment with radiochemotherapy (versus radio- or chemotherapy) (OR 1.6, p = 0.051), gender (OR 1.1, p = 0.718), or MGMT (OR 1.5, p = 0.087). A multivariate analysis with test and validation set will be presented. CONCLUSION: In this analysis, the administration of salvage treatment expectedly was associated with prolonged survival. Older patients with incomplete resections or biopsies with lower KPS may particularly benefit from improved first-line treatment since these patients are less likely to receive salvage therapy. A concept of the EORTC and NOA proposes to assess the addition of bevacizumab to such patients with tumors lacking MGMT promoter methylation.