Unmet needs in the international neuroendocrine tumor (NET) community: Assessment of major gaps from the perspective of patients, patient advocates and NET health care professionals.

Due to the increasing incidence and prevalence of neuroendocrine tumors (NETs), there is a need to assess any gaps in awareness and care. A survey was undertaken in 2017 to identify perceived unmet needs from the perspectives of patients/families, patient advocates and health care professionals (HCPs). The survey consisted of 33-37 questions (depending on type of respondent) across four areas: information, care, treatments and research. In total, 443 participants from 26 countries responded: 338 patients/families, 35 advocates and 70 HCPs. Perceived unmet needs regarding provision of information at diagnosis differed between groups. While 59% of HCPs believed they provided sufficient information, informational needs were mostly/fully met for only 30% of patients and 18% of advocates. Additionally, 91% of patients and 97% of advocates felt that patients had to search for information themselves. Availability of Gallium-68-Dotatate PET/CT scan was limited for the majority of patients (patients: 73%; advocates: 85%; HCP: 86%), as was access to treatments, particularly peptide receptor radionuclide therapy (patients: 42%; advocates: 95%; HCPs: 77%). All groups felt that standards of care, including psychological needs and diagnosis of mental health, were not fully met. Although about two-thirds of patients were managed by a multidisciplinary team, 14% of patients reportedly did not have enough contact. All groups supported more patient involvement in research; patients and advocates prioritized improvement in diagnosis and HCPs focused on clinical trials. This survey revealed significant unmet needs but differing perceptions regarding these among the groups. There is a need for investigation and collaboration to improve standards of care for NET patients.

Consensus-Derived Quality Performance Indicators for Neuroendocrine Tumour Care.

Quality performance indicators (QPIs) are used to monitor the delivery of cancer care. Neuroendocrine tumours (NETs) are a family of individually uncommon cancers that derive from neuroendocrine cells or their precursors, and can occur in most organs. There are currently no QPIs available for NETs and their heterogeneity makes QPI development difficult. CommNETs is a collaboration between NET clinicians, researchers and advocates in Canada, Australia and New Zealand. We created QPIs for NETs using a three-step consensus process. First, a multidisciplinary team used the nominal group technique to create candidates (n = 133) which were then curated into appropriateness statements (62 statements, 44 sub-statements). A two-stage modified RAND/UCLA Delphi consensus process was conducted: an online survey rated the statement appropriateness then the top-ranked statements (n = 20) were assessed in a face-to-face meeting. Finally, 10 QPIs met consensus criteria; documentation of primary site, proliferative index, differentiation, tumour board review, use of a structured pathology report, presence of distant metastasis, 5- and 10-year disease-free and overall survival. These NET QPIs will be trialed as a method to monitor and improve care for people with NETs and to facilitate international comparison.

IL-8 associates with a pro-angiogenic and mesenchymal subtype in glioblastoma.

Glioblastoma (GBM) is a highly aggressive brain tumor characterized by a high rate of vascularization. However, therapeutic targeting of the vasculature through anti-vascular endothelial growth factor (VEGF) treatment has been disappointing, for which Angiopoietin-2 (Ang-2) upregulation has partly been held accountable. In this study we therefore explored the interplay of Ang-2 and VEGFA and their effect on angiogenesis in GBM, especially in the context of molecular subclasses. In a large patient cohort we identified that especially combined high expression of Ang-2 and VEGFA predicted poor overall survival of GBM patients. The high expression of both factors was also associated with increased IL-8 expression in GBM tissues, but in vitro stimulation with Ang-2 and/or VEGFA did not indicate tumor or endothelial cell-specific IL-8 responses. Glioblastoma stem cells (GSCs) of the mesenchymal (MES) subtype showed dramatically higher expression of IL8 when compared to proneural (PN) GSCs. Secreted IL-8 derived from MES GSCs induced endothelial proliferation and tube formation, and the MES GBMs had increased counts of proliferating endothelial cells. Our results highlight a critical pro-angiogenic role of IL-8 in MES GBMs.

Identification of Two Protein-Signaling States Delineating Transcriptionally Heterogeneous Human Medulloblastoma.

The brain cancer medulloblastoma consists of different transcriptional subgroups. To characterize medulloblastoma at the phosphoprotein-signaling level, we performed high-throughput peptide phosphorylation profiling on a large cohort of SHH (Sonic Hedgehog), group 3, and group 4 medulloblastomas. We identified two major protein-signaling profiles. One profile was associated with rapid death post-recurrence and resembled MYC-like signaling for which MYC lesions are sufficient but not necessary. The second profile showed enrichment for DNA damage, as well as apoptotic and neuronal signaling. Integrative analysis demonstrated that heterogeneous transcriptional input converges on these protein-signaling profiles: all SHH and a subset of group 3 patients exhibited the MYC-like protein-signaling profile; the majority of the other group 3 subset and group 4 patients displayed the DNA damage/apoptotic/neuronal signaling profile. Functional analysis of enriched pathways highlighted cell-cycle progression and protein synthesis as therapeutic targets for MYC-like medulloblastoma.

GPR56/ADGRG1 Inhibits Mesenchymal Differentiation and Radioresistance in Glioblastoma.

A mesenchymal transition occurs both during the natural evolution of glioblastoma (GBM) and in response to therapy. Here, we report that the adhesion G-protein-coupled receptor, GPR56/ADGRG1, inhibits GBM mesenchymal differentiation and radioresistance. GPR56 is enriched in proneural and classical GBMs and is lost during their transition toward a mesenchymal subtype. GPR56 loss of function promotes mesenchymal differentiation and radioresistance of glioma initiating cells both in vitro and in vivo. Accordingly, a low GPR56-associated signature is prognostic of a poor outcome in GBM patients even within non-G-CIMP GBMs. Mechanistically, we reveal GPR56 as an inhibitor of the nuclear factor kappa B (NF-κB) signaling pathway, thereby providing the rationale by which this receptor prevents mesenchymal differentiation and radioresistance. A pan-cancer analysis suggests that GPR56 might be an inhibitor of the mesenchymal transition across multiple tumor types beyond GBM.

The Impact of the Tumor Microenvironment on the Properties of Glioma Stem-Like Cells.

Glioblastoma is the most common and highly malignant primary brain tumor, and patients affected with this disease exhibit a uniformly dismal prognosis. Glioma stem-like cells (GSCs) are a subset of cells within the bulk tumor that possess self-renewal and multi-lineage differentiation properties similar to somatic stem cells. These cells also are at the apex of the cellular hierarchy and cause tumor initiation and expansion after chemo-radiation. These traits make them an attractive target for therapeutic development. Because GSCs are dependent on the brain microenvironment for their growth, and because non-tumorigenic cell types in the microenvironment can influence GSC phenotypes and treatment response, a better understanding of these cell types is needed. In this review, we provide a focused overview of the contributions from the microenvironment to GSC homing, maintenance, phenotypic plasticity, and tumor initiation. The interaction of GSCs with the vascular compartment, mesenchymal stem cells, immune system, and normal brain cell types are discussed. Studies that provide mechanistic insight into each of these GSC-microenvironment interactions are warranted in the future.

Novel insights into vascularization patterns and angiogenic factors in glioblastoma subclasses.

Glioblastoma (GBM) is a highly vascularized and aggressive type of primary brain tumor in adults with dismal survival. Molecular subtypes of GBM have been identified that are related to clinical outcome and response to therapy. Although the mesenchymal type has been ascribed higher angiogenic activity, extensive characterization of the vascular component in GBM subtypes has not been performed. Therefore, we aimed to investigate the differential vascular status and angiogenic signaling levels in molecular subtypes. GBM tissue samples representing proneural IDH1 mutant, classical-like and mesenchymal-like subtypes were analyzed by morphometry for the number of vessels, vessel size and vessel maturity. Also the expression levels of factors from multiple angiogenic signaling pathways were determined. We found that necrotic and hypoxic areas were relatively larger in mesenchymal-like tumors and these tumors also had larger vessels. However, the number of vessels, basement membrane deposition and pericyte coverage did not vary between the subtypes. Regarding signaling patterns the majority of factors were expressed at similar levels in the subtypes, and only ANGPT2, MMP2, TIMP1, VEGFA and MMP9/TIMP2 were higher expressed in GBMs of the classical-like subtype. In conclusion, although morphological differences were observed between the subtypes, the angiogenic signaling status of GBM subtypes seemed to be rather similar. These results challenge the concept of mesenchymal GBMs being more angiogenic than other subclasses.

Serum-Induced Differentiation of Glioblastoma Neurospheres Leads to Enhanced Migration/Invasion Capacity That Is Associated with Increased MMP9.

Glioblastoma (GBM) is a highly infiltrative brain tumor in which cells with properties of stem cells, called glioblastoma stem cells (GSCs), have been identified. In general, the dominant view is that GSCs are responsible for the initiation, progression, invasion and recurrence of this tumor. In this study, we addressed the question whether the differentiation status of GBM cells is associated with their invasive capacity. For this, several primary GBM cell lines were used, cultured either as neurospheres known to enrich for GSCs or in medium supplemented with 10% FCS that promotes differentiation. The differentiation state of the cells was confirmed by determining the expression of stem cell and differentiation markers. The migration/invasion potential of these cells was tested using in vitro assays and intracranial mouse models. Interestingly, we found that serum-induced differentiation enhanced the invasive potential of GBM cells, which was associated with enhanced MMP9 expression. Chemical inhibition of MMP9 significantly reduced the invasive potential of differentiated cells in vitro. Furthermore, the serum-differentiated cells could revert back to an undifferentiated/stem cell state that were able to form neurospheres, although with a reduced efficiency as compared to non-differentiated counterparts. We propose a model in which activation of the differentiation program in GBM cells enhances their infiltrative potential and that depending on microenvironmental cues a significant portion of these cells are able to revert back to an undifferentiated state with enhanced tumorigenic potential. Thus, effective therapy should target both GSCs and differentiated offspring and targeting of differentiation-associated pathways may offer therapeutic opportunities to reduce invasive growth of GBM.

Hypoxia enhances migration and invasion in glioblastoma by promoting a mesenchymal shift mediated by the HIF1α-ZEB1 axis.

Glioblastoma (GBM) is the most common brain tumor in adults and the mesenchymal GBM subtype was reported to be the most malignant, presenting severe hypoxia and necrosis. Here, we investigated the possible role of a hypoxic microenvironment for inducing a mesenchymal and invasive phenotype. The exposure of non-mesenchymal SNB75 and U87 cells to hypoxia induced a strong change in cell morphology that was accompanied by enhanced invasive capacity and the acquisition of mesenchymal marker expression. Further analyses showed the induction of HIF1α and HIF2α by hypoxia and exposure to digoxin, a cardiac glycoside known to inhibit HIF1/2 expression, was able to prevent hypoxia-induced mesenchymal transition. ShRNA-mediated knockdown of HIF1α, and not HIF2α, prevented this transition, as well as the knockdown of the EMT transcription factor ZEB1. We provide further evidence for a hypoxia-induced mesenchymal shift in GBM primary material by showing co-localization of GLUT1, ZEB1 and the mesenchymal marker YKL40 in hypoxic regions of the tumor. Collectively, our results identify a HIF1α-ZEB1 signaling axis that promotes hypoxia induced mesenchymal shift and invasion in GBM in a cell line dependent fashion.

Subclassification of newly diagnosed glioblastomas through an immunohistochemical approach.

Molecular signatures in Glioblastoma (GBM) have been described that correlate with clinical outcome and response to therapy. The Proneural (PN) and Mesenchymal (MES) signatures have been identified most consistently, but others including Classical (CLAS) have also been reported. The molecular signatures have been detected by array techniques at RNA and DNA level, but these methods are costly and cannot take into account individual contributions of different cells within a tumor. Therefore, the aim of this study was to investigate whether subclasses of newly diagnosed GBMs could be assessed and assigned by application of standard pathology laboratory procedures. 123 newly diagnosed GBMs were analyzed for the tumor cell expression of 23 pre-identified proteins and EGFR amplification, together allowing for the subclassification of 65% of the tumors. Immunohistochemistry (IHC)-based profiling was found to be analogous to transcription-based profiling using a 9-gene transcriptional signature for PN and MES subclasses. Based on these data a novel, minimal IHC-based scheme for subclass assignment for GBMs is proposed. Positive staining for IDH1R132H can be used for PN subclass assignment, high EGFR expression for the CLAS subtype and a combined high expression of PTEN, VIM and/or YKL40 for the MES subclass. The application of the proposed scheme was evaluated in an independent tumor set, which resulted in similar subclass assignment rates as those observed in the training set. The IHC-based subclassification scheme proposed in this study therefore could provide very useful in future studies for stratification of individual patient samples.

Mesenchymal differentiation mediated by NF-κB promotes radiation resistance in glioblastoma.

Despite extensive study, few therapeutic targets have been identified for glioblastoma (GBM). Here we show that patient-derived glioma sphere cultures (GSCs) that resemble either the proneural (PN) or mesenchymal (MES) transcriptomal subtypes differ significantly in their biological characteristics. Moreover, we found that a subset of the PN GSCs undergoes differentiation to a MES state in a TNF-α/NF-κB-dependent manner with an associated enrichment of CD44 subpopulations and radioresistant phenotypes. We present data to suggest that the tumor microenvironment cell types such as macrophages/microglia may play an integral role in this process. We further show that the MES signature, CD44 expression, and NF-κB activation correlate with poor radiation response and shorter survival in patients with GBM.

CLMP is essential for intestinal development, but does not play a key role in cellular processes involved in intestinal epithelial development.

Loss-of-function mutations in CLMP have been found in patients with Congenital Short Bowel Syndrome (CSBS), suggesting that its encoded protein plays a major role in intestinal development. CLMP is a membrane protein that co-localizes with tight junction proteins, but its function is largely unknown. We expressed wild-type (WT)-CLMP and a mutant-CLMP (associated with CSBS) in human intestinal epithelial T84 cells that, as we show here, do not produce endogenous CLMP. We investigated the effects of WT-CLMP and mutant-CLMP proteins on key cellular processes that are important for intestinal epithelial development, including migration, proliferation, viability and transepithelial resistance. Our data showed that expression of WT-CLMP or mutant-CLMP does not affect any of these processes. Moreover, our aggregation assays in CHO cells show that CLMP does not act as a strong adhesion molecule. Thus, our data suggest that, in the in vitro model systems we used, the key processes involved in intestinal epithelial development appear to be unaffected by WT-CLMP or mutant-CLMP. Further research is needed to determine the role of CLMP in the development of the intestine.

Gene-eluting stents: comparison of adenoviral and adeno- associated viral gene delivery to the blood vessel wall in vivo.

Gene-eluting stents are being evaluated in animals as an alternative approach to inhibiting in-stent restenosis. Adeno-associated virus type 2 (AAV2) and adenovirus are commonly used for gene transfer applications. We tested the hypothesis that these vectors can achieve prolonged and localized gene delivery to the vessel wall, using stents as delivery platforms. AdbetaGal (5 x 10(9) plaque-forming units) and AAV2betaGal (5.3 x 10(9) DNase-resistant particles) were used to coat BiodivYsio stents with matrix HI coating (Abbott Vascular Devices, Galway, Ireland). After balloon injury, external iliac arteries of New Zealand White rabbits were stented. The reverse transcription-polymerase chain reaction was used to assess viral spread. Expression of LacZ was demonstrated with both vectors at five time points (3, 7, 14, 21, and 28 days). In the adenovirus group the median percentage of cells expressing the transgene on day 3 was 2.73%, which increased to a median expression of 7.31% at 28 days (p > 0.05). Expression was localized to medial cells on day 3, but was observed predominantly in neointimal cells on day 28. In the AAV group, day 3 expression was 5.78%, which decreased to 2.12% on day 28 (p = 0.05). No systemic dissemination of virus was seen in any group. Adenovirus- and AAV2-coated stents can be used to deliver genes to the blood vessel wall for up to 28 days.