bluuebunny/crossref_metadata_2025 huggingface dataset:
P04.14 Loss of oligodendroglial features at recurrence in five diffuse low-grade glioma patients treated with repeated surgery
Authors: Darlix A , Duffau H , Rigau V , Gozé C
Published: September 2019

BACKGROUND

Diffuse low-grade gliomas (DLGG) are characterized by a continuous growth and an unavoidable anaplastic transformation. IDH mutation and 1p19q codeletion have been integrated to the 2016 WHO classification to define the oligodendroglioma entity. Whenever feasible, neurosurgical resection is the first treatment option. At recurrence, a second surgical resection is proposed in selected cases. The consistency of molecular patterns (IDH mutation, 1p19q codeletion) at recurrence has been poorly studied in DLGG.

MATERIAL AND METHODS

We conducted a retrospective study on consecutive DLGG patients treated at our institution with repeated surgery (2006–2017). Clinical and biological data were collected for both the initial and subsequent surgery. Additional immunohistochemistry (including tumor morphology, ATRX and p53) and genetic analyses (TERT promoter mutation, CIC mutation, CGHa) were also performed on tumors with joint loss of IDH mutation and of 1p19q codeletion at recurrence.

RESULTS

A total of 71 patients were identified. Analyses were carried out on 56 patients (molecular data missing: n=15). Nine patients (16.1%) presented with a loss of their IDH mutation at second surgery. Five of them (8.9%) had an additional loss of their 1p19q codeletion. These five cases (3 men, median age 36.6 years) were all treated with surgery as the first oncological treatment. The first surgery revealed in all cases tumors with morphological oligodendroglial features, IDH mutation and 1p19q codeletion. Further molecular analysis strengthened the diagnosis of oligodendroglioma (TERT promoter and CIC mutations, no ATRX loss, no expression of p53). Four patients were followed-up after the first surgery; one patient received Temozolomide 14 months later due to FLAIR tumor volume growth. Because of the regrowth of the residual FLAIR tumor volume, a second surgery was performed in all patients, after a median time of 38.9 months. The morphological oligodendroglial features were lost, and the genetic analyses revealed in all cases no IDH mutation, no 1p19q codeletion, no ATRX loss and no expression of p53. No evidence of anaplasia was found histologically or by CGHa analysis in these recurrent tumors.

CONCLUSION

We describe five DLGG patients with a shared histo-molecular evolution characterized by the loss of the initial IDH mutation and of oligodendroglial features at second surgery. While rare, this possible evolution must be acknowledged as it can impact the subsequent therapeutic strategy. This observation is the first of a loss of founder alterations in DLGG genesis (i.e. IDH mutation and 1p19q codeletion); the involved mechanism likely differs from the previously described oligoclonal selection caused by spontaneous tumor genetic drift and/or pressure of chemotherapy, and could be linked with the Darwinian selection of a subpopulation of tumor cells after the first surgery.
P04.16 TP53 mutations in codon 273 is predictive of overall survival in astrocytoma patients
Authors: Noor H , Rapkins R , McDonald K
Published: September 2019

BACKGROUND

Tumour Protein 53 (TP53) is a tumour suppressor gene that is mutated in at least 50% of human malignancies. The prevalence of TP53 mutation is much higher in astrocytomas with reports of up to 75% TP53 mutant cases. Rare cases of TP53 mutation also exist in oligodendroglial tumours (10–13%). P53 pathway is therefore an important factor in low-grade glioma tumorigenesis. Although the prognostic impact of TP53 mutations has been studied previously, no concrete concordance were reached between the studies. In this study, we investigated the prognostic effects of TP53 mutation in astrocytoma and oligodendroglioma.

MATERIAL AND METHODS

A cohort of 65 matched primary and recurrent fresh frozen tumours were sequenced to identify hotspot exons of TP53 mutation. Exons 1 to 10 were sequenced and pathogenic mutations were mostly predominant between Exons 4 and 8. The cohort was further expanded with 78 low grade glioma fresh frozen tissues and hotspot exons were sequenced. Selecting only the primary tumour from 65 matched tumours, a total of 50 Astrocytoma cases and 51 oligodendroglioma cases were analysed for prognostic effects of TP53. Only pathogenic TP53 mutations confirmed through COSMIC and NCBI databases were included in the over survival and progression-free survival analysis.

RESULTS

62% (31/50) of astrocytomas and 16% (8/51) of oligodendrogliomas harboured pathogenic TP53 mutations. Pathogenic hotspot mutations in codon 273 (c.817 C>T and c.818 G>A) was prevalent in astrocytoma with 58% (18/31) of tumours with these mutations. TP53 mutation status was maintained between primary and recurrent tumours in 93% of cases. In astrocytoma, overall survival of TP53 mutant patients was longer compared to TP53 wild-type patients (p<0.01) but was not significant after adjusting for age, gender, grade and IDH1 mutation status. In contrast, astrocytoma patients with specific TP53 mutation in codon 273 showed significantly better survival compared to other TP53 mutant and TP53 wild-type patients combined (p<0.01) in our multivariate analysis. Time to first recurrence (progression-free survival) of TP53 mutant patients was significantly longer than TP53 wild-type patients (p<0.01) after adjustments were made, while TP53 mutation in codon 273 was not prognostic for progression-free survival. In oligodendroglioma patients, TP53 mutations did not significantly affect overall survival and progression-free survival.

CONCLUSION

In agreement with others, TP53 mutation is more prevalent in Astrocytoma and mutations in codon 273 are significantly associated with longer survival.
P05.05 Safety and feasibility of temporary blood-brain barrier disruption with the SonoCloud-1/3 implantable ultrasound device in recurrent glioblastoma
Authors: Idbaih A , Canney M , Bouchoux G , Desseaux C , Vignot A , Lafon C , Chapelon J , Delattre J , Belin L , Carpentier A
Published: September 2019

BACKGROUND

The blood-brain barrier (BBB) limits penetration of drug therapies to the brain and may account for the limited effectiveness of chemotherapies in patients with primary brain cancer. An implantable ultrasound (US) device, SonoCloud, was developed to temporarily disrupt the BBB in patients with recurrent glioblastoma (rGBM) prior to carboplatin chemotherapy administration to enhance brain drug concentrations. BBB disruption was investigated with a single emitter device, SonoCloud-1 (SC1) and a three-emitter device, SonoCloud-3 (SC3) in a safety and feasibility study in rGBM patients at the Hôpitaux Universitaires La Pitié Salpêtrière in Paris, France (NCT02253212).

MATERIAL AND METHODS

The SC1 implant consisted of a 1 MHz, 10-mm diameter ultrasound transducer encapsulated in a biocompatible housing while the SC3 consisted of (3) separate SC1 emitters implanted in a triangular pattern to disrupt the BBB over a 3x larger volume. Patients were implanted with the SonoCloud device during tumor debulking or in a dedicated surgical procedure. The devices were activated monthly by connecting the device to an external generator via a transdermal needle. Intravenous injection of SonoVue® microbubbles was performed with device activation to temporarily disrupt the BBB. Magnetic resonance imaging (MRI) was performed after sonications with gadolinium contrast to verify BBB disruption followed by carboplatin infusion at AUC4-6. Patients received treatments until disease progression.

RESULTS

Between 2014 and 2016, 21 patients were registered for the study and implanted with the SC1; 19 patients received at least one sonication. Six additional patients were implanted and received sonications with the SC3. A total of 89 US sessions were performed to disrupt the BBB - 65 with the SC1 and 24 with the SC3. Treatment-related adverse events observed were transient and manageable. No carboplatin-related neurotoxicity was observed. SC1 patients with no or poor BBB disruption (n=8) visible on MRI had a median progression-free survival (PFS) of 2.73 months, and a median overall survival (OS) of 8.64 months. SC1 patients with clear BBB disruption (n=11) had a median PFS of 4.11 months, and a median OS of 12.94 months. The SC3 device was as well-tolerated as the SC1 device, with SC3 patients receiving between 1–12 monthly sonications.

CONCLUSION

These results provide the first safety data on the effects of disrupting the BBB in rGBM patients prior to carboplatin chemotherapy using an implantable low intensity pulsed ultrasound device on enlarged areas of brain. This study has now been completed. A new safety study with a larger device, SonoCloud-9, that covers the tumor and surrounding infiltrative regions, has started in France in early 2019 (NCT03744026). Work supported by CarThera and APHP.
P06.01 Delta24-ACT oncolytic adenovirus as a therapeutic approach for DIPG
Authors: Laspidea V , Puigdelloses M , García-Moure M , Iñigo-Marco I , Gallego J , Patiño-García A , Fueyo J , Gomez-Manzano C , Alonso M M
Published: September 2019

BACKGROUND

Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain tumor, being the leading cause of pediatric death caused by cancer. We previously showed that administration of the oncolytic virus Delta-24-RGD to DIPG murine models was safe and led to an increase in the median survival of these animals. However, not all the animals responded, underscoring the need to improve this therapy. In order to increase the antitumoral effect of the virus, we have engineered Delta-24-RGD with the costimulatory ligand 4-1BBL (Delta24-ACT). 4-1BB is a costimulatory receptor that promotes the survival and expansion of activated T cells, and the generation and maintenance of memory CD8+ T cells. In this project, we evaluated the oncolytic effect of Delta24-ACT and the antitumor immune response in DIPG murine models.

MATERIALS AND METHODS

We use the NP53 and XFM murine DIPG cell lines. Flow cytometry was used to assess cell infectivity and ligand expression. We analyzed viral replication using a method based in hexon detection, and viral cytotoxic effect using the MTS assay. For immunogenic cell death analysis, we measured ATP secretion by a luminometric assay and calreticulin location by flow cytometry and immunofluorescence. For in vivo studies, cells and virus were injected in the pons of the mice, using the screw-guided system.

RESULTS

In vitro, Delta24-ACT was able to infect and induce cell death in a dose-dependent manner in murine DIPG cell lines. In addition, Delta24-ACT was able to replicate in these tumor cells and to express viral proteins. Moreover, infected cells expressed 41BBL in their membranes. Delta24-ACT could induce immunogenic cell death due to an increased secretion of ATP and calreticulin translocation to the membrane of infected cells (in no-infected cells it located in the ER), DAMPs that can trigger the immune response activation. In vivo, Delta24-ACT demonstrated to be safe in all the tested doses and was able to induce a significant increase in the median survival of the treated animals. Moreover, long-term survivors display immunological memory.

CONCLUSIONS

Delta24-ACT treatment led to antitumoral effect in DIPG murine cell lines in vitro. Of significance, we have demonstrated that in vivo administration of Delta24-ACT is safe and results in an enhanced antitumor effect. Future in vivo studies will explore the underlying immune mechanism of the virus.
P08.03 Separation and divorce after the diagnosis of child’s brain tumor
Authors: Sardi I , Censullo M , Rousseau M , Guidi M , Giordano F , Fonte C , Farina S , Carra F , Genitori L
Published: September 2019

BACKGROUND

The diagnosis of a child’s brain tumor is a terrible situation for every member of the family. Numerous are the case of separations and divorces in Italy after a diagnosis of a child’s cancer. In particular, it happens with parents of children affected with brain tumor, being the most frequent solid tumor and the first cause of a tumor child’s death. The crisis related to the discovery of a tumor consists of four phases: shock, reaction, processing and re-orientation. It can happen that the diagnosis, experienced as a traumatic experience, can unite the family members as well as separate them. If there is already a process of family disintegration, a trauma can be a cause for breakup. The aim of our study was to investigate the possible correlation between brain tumor diagnosis in children and parental separations/divorces.

MATERIAL AND METHODS

We considered 427 patients afferent from 2012 to 2018 to the Neuro-Oncology Unit of the Meyer Children’s Hospital. Brain tumors are the 55–60% of all the tumors of our hospital, with an extra-regionality greater than 65%. The data analysis was conducted through information obtained directly from the families during follow-up visits or by telephone interviews.

RESULTS

Consistent with literature data in our series, the most frequent brain tumors were low-grade gliomas medulloblastomas, high-grade gliomas, ependymomas, midline diffuse gliomas, craniopharyngiomas, germ cell tumors and other rare pediatric tumors. The population was divided in 16 females and 18 males from different Italian regions: 65% from Central Italy, 23% from the South and Islands, 12% from the North. Data analysis showed 34 cases of separation and/or divorce, equal to 7% of our whole population, during treatment and more frequently at the end of treatment or after death. The median age of the 34 patients at the diagnosis of brain tumor was 9.5 years (range 1–19 years), with a higher percentage of cases of separations (41%) for parents of patients aged 10 years-14 years; 7 were the cases of separation and/or divorce when the diagnosis of brain tumor was made around 12–48 months after the child birth.

CONCLUSION

The diagnosis of a child’s brain tumor can generate stress in the family leading to different reactions, such as conflicts between parents or a real family crisis. The results of our study suggest a possible correlation between the diagnosis of a child’s brain tumor and the cases of separation and/or divorce. High risk medulloblastomas and high-grade gliomas that are likely to have a shorter path due to the unfavorable prognosis of the disease, appear to be the pathologies more often related to situations of family disputes. However, further investigations are necessary to verify the trend emerged from our study respect to the normal population.
P08.06 Clinical value scores for treatment of newly diagnosed glioblastoma with TTFields
Authors: Kelly J , Proescholdt C
Published: September 2019

BACKGROUND

High quality and value of recommended treatments is of specific importance in cancer care. ESMO, ASCO and NCCN have developed tools intended to help assessing the clinical value of cancer treatments in a standardised way, allowing for a comparative discussion. Tumor treating fields (TTFields) is a novel, device based cancer treatment, that was recently demonstrated to be effective in newly diagnosed glioblastoma (GBM). This new modality augments the treatments discussed with glioblastoma patients today.

MATERIAL AND METHODS

ESMO and ASCO frameworks each calculate a score for the clinical value of a cancer treatment, called Magnitude of Clinical Benefit Scale (MCBS) by ESMO and the Net Health Benefit (NHB) by ASCO. NCCN self reports “evidence blocks” which are assessed by clinician panels and were recently published for the first line treatment of newly diagnosed GBM with TTFields. We apply and compare the ESMO, ASCO and NCCN tools for TTFields treatment of newly diagnosed GBM.

RESULTS

The resulting ASCO NHB score for TTFields treatment of newly diagnosed GBM is 56. ESMO MCBS scores for TTFields in GBM are resulting in A/5, these being the highest achievable scores for this framework. All frameworks value the increase in overall survival by TTFields and the moderate toxicity profile. ESMO additionally values quality of life, while ASCO values palliation and treatment free intervals. NCCN’s specific focus is on the quality and consistency of the evidence. NCCN evidence blocks also contain an affordability score.

CONCLUSION

All three frameworks consider the clinical efficacy of a treatment and it’s toxicity profile in their clinical value assessment. Beyond that, their respective focus is on slightly different aspects and their definition of clinical value therefore varies in detail. However, all value scores suggest that TTFields treatment of newly diagnosed GBM provides a substantial clinical benefit. The high ESMO and ASCO scores are based on the significantly extended progression free and overall survival for TTFields treated patients, without adding systemic toxicities. The NCCN evidence blocks strongly support the NCCN category 1 recommendation for the use of TTFields in newly diagnosed GBM.
P09.05 177Lu-DOTATATE therapy in progressive meningioma
Authors: Müther M , Roll W , Zinnhardt B , Schäfers M , Rahbar K , Brokinkel B , Stummer W
Published: September 2019

BACKGROUND

Progressive meningioma is a challenging condition with a decreasing number of treatment options over the course of disease. Neurovascular involvement and multifocal disease often complicate surgical management. In addition, repeated radiotherapy carriers a risk of side effects. In somatostatin receptor expressing meningioma, peptide receptor radiotherapy (PPRT) with 177Lu-DOTATATE poses a promising alternative. However, current evidence is scarce. Hereby we present our single center experience of (PPRT) with 177Lu-DOTATATE in progressive meningioma.

METHODS

Eight patients (median age: 71 years; range 56–77) with progressive meningioma underwent PRRT using 177Lu-DOTATATE were included in this retrospective analysis. Response to therapy was assessed by interim and post-therapy 68Ga-DOTATATE-PET-CT and MRI. 177Lu-DOTATATE scintigraphies 48h p.i. were evaluated according to Krenning scale. Additionally, clinical outcome and follow up imaging were analyzed for progression free interval times.

RESULTS

Eight patients were included: Six patients with grade II and two patients with grade I meningiomas, according to the 2016 WHO classification. Six of eight patients harbored multifocal disease. One patients suffered systemic metastatic disease. Patients received a median of three cycles (range: 1 - 5) of PRRT with 177Lu-DOTATATE (mean injected dose 7.1 GBq) between 1/2015 and 1/2019. Tumor uptake in 48h p.i. 177Lu-Scintigraphies was heterogeneous (Krenning scale; median: 3; range: 1–4). Post-therapy imaging scheduled eight weeks after completion of therapy showed progressive disease in five patients, three patients had stable disease. Median follow-up post therapy response evaluation was 24 months in patients with stable disease. Median time to progression was 10 months.

CONCLUSION

In this cohort of eight progressive meningioma patients 177Lu-DOTATATE therapy showed heterogeneous efficacy.
P10.02 Differentiating microglia and tumour associated macrophages in high grade glioma
Authors: Woolf Z , Swanson M , Park T , Brooks A , Dragunow M
Published: September 2019

BACKGROUND

Glioblastoma multiforme (GBM) is the most common primary brain tumour that affects adults. This aggressive tumour is invariably fatal, carrying a rapid progression and a dismal median survival period of only 15 months despite multimodal treatment approaches. Central to GBM pathogenesis is the immunosuppressive profile of these tumours. The two cell types that are highly abundant in these tumours and play critical roles in the immunosuppressive niche are the brain’s resident microglia and their peripheral counterparts - tumour associated macrophages (TAMs). Despite microglia and TAMs being ontogenetically distinct, these cells have largely been grouped together in research owing to the previous lack of cell-specific markers. Recent evidence has suggested that although TAMs may hold a predominantly pro-tumoral role, microglia may adopt a more anti-tumoral phenotype. Therefore, the differentiation of these two cell types is critical in elucidating the potentially characteristic roles of these two cell types in GBM pathogenesis.

MATERIAL AND METHODS

Tissue sections from resected low- and high-grade glioma tumours, along with epilepsy tissue (control), were used for immunohistochemistry (IHC) staining of macrophage pan-makers (Iba1, CD45, PU.1) and microglial-specific markers (TMEM119, P2RY12). Marker co-localisation was then used to differentiate microglia from TAMs. We further investigated a wider subset of cell-specific markers using multicolour flow cytometry and immunocytochemical staining of isolated cells from patient tissue samples.

RESULTS

Immunofluorescent staining of glioma and epilepsy tissue revealed two clear populations of cells; one population displayed long processes and co-labelling for both pan- and microglial-specific markers, whilst the other population displayed an amoeboid phenotype with only pan-maker staining. Preliminary analysis comparing microglia/TAM populations in low-grade, high-grade and epilepsy tissue suggests a clear difference in the proportions of these cells.

CONCLUSION

Our work complements RNA-Seq studies, showing that TMEM119 and P2RY12, alongside other markers, can indeed identify two distinct myeloid cell populations within glioma tissue. This provides a strong basis for further study where we aim to elucidate the respective roles of microglia and TAMs within tumours. Ultimately, this may hold the potential for differential targeting of these cells using immunotherapies.
P10.04 Incidence and characteristics of neurological adverse events secondary to immunotherapy with checkpoint inhibitors
Authors: Velasco R , Argyriou A , Bruna J , Anastopoulou G G , Alemany M , Simó M , Piulats J M , Nadal E , Kalofonos H P
Published: September 2019

BACKGROUND

To explore the incidence and clinical phenotype of serious neurological adverse events (NAEs) in cancer patients who received immunotherapy with checkpoint inhibitors (ICIs) at two institutions.

MATERIAL AND METHODS

We reviewed files of cancer patients who were treated with ICIs from 2010 to 2018 and then searched for ICIs-related NAEs.

RESULTS

We identified 1185 ICIs-treated patients. Males and females were 63.7% and 36.3%, respectively, with a mean age of 63.4±7.3 years-old. Nivolumab was given to 536 (45.2%) patients, Pembrolizumab to 301 (25.4%) patients, Atezolizumab to 135 (11.4%), Ipilimumab to 104 (8.8%), Durvalumab-Tremelimumab to 77 (6.5%), and other ICIs to 32 (2.8%). Of those patients, 24 (2%) developed a ICI-related NAE. No differences were identified in age, sex, tumor type and class of ICIs between patients who developed neurotoxicity compared to those without neurologic adverse events. The distribution of NAE by agent was: Nivolumab (2.1%), Pembrolizumab (2%), Atezolizumab (1.5%), Ipilimumab (1%), Durvalumab-Tremelimumab (3.9%), and others (2.8%). The median number of cycles received before NAEs onset were 4.5 (1–10), and the median time was 110 days. PNS involvement was evident in 14 patients (58.3%) and CNS involvement in 41.7%, while 2 patients with aseptic meningitis also presented polyradicular involvement. Among PNS complications, there were 5 cases (20.8%) with axonal sensory neuropathies, 4 (16.7%) with Guillain-Barre-like syndromes, 4 (16.7%) with muscle involvement (myositis and myasthenias), and one with other syndromes. Seven patients (50%) with PNS-related NAEs were treated with steroids, 2 (14.3%) with IVIG, and 5 (35.7%) remained untreated. In general the outcome was good after ICIs discontinuation, with about half of patients improving or completely resolving NAEs.

CONCLUSION

ICIs-related NAE although rare overall, might be severe, and are mostly represented by neuromuscular complications. Early discontinuation of ICIs and possibly treatment with immune-modulating therapies should early be initiated to achieve a favourable neurological outcome
P11.05 Reappraisal of CNS embryonal tumors, not otherwise specified and RELA fusion negative supratentorial ependymomas using immunohistochemistry and sequencing for BCOR
Authors: Nambirajan A , Gurung N , Sashidharan A , Sharma A , Kedia S , Garg A , Suri V , Sarkar C , Sharma M
Published: September 2019

BACKGROUND

BCl2 co-repressor (BCOR) gene internal tandem duplications have recently been identified as the defining molecular alteration in a subset of central nervous system (CNS) high grade neuroepithelial tumors (CNS-HGNET). Their clinicopathological profile is yet to be fully elucidated with available data being largely derived from case reports and small case series. Reported cases presented in young children with a male preponderance, were located predominantly in the posterior fossa, and sometimes but not always showed an ependymoma-like morphology. BCOR protein overexpression and exon 15 internal tandem duplications were characteristic. These tumors associated with a poor overall survival.

MATERIAL AND METHODS

We designed a retrospective study (2002–2019) wherein all cases of CNS embryonal tumors, not otherwise specified (NOS), by definition lacking C19MC locus amplification and Lin28A protein expression, and supratentorial ependymomas (ST-EPN), NOS, lacking RELA and YAP1 fusions and L1CAM protein expression were retrieved and subject to immunohistochemistry for BCOR. Those tumors showing nuclear expression of BCOR were subject to sequencing of exon 15 of BCOR gene.

RESULTS

A total of 19 CNS embryonal tumors, NOS, constituting 86% of all CNS embryonal tumors other than medulloblastomas and atypical teratoid rhabdoid tumors, and 11 ST-EPN, NOS constituting 30% of all ST EPNs, were included. Diffuse nuclear staining for BCOR was seen in 6 tumors, including two patients with CNS embryonal tumor, NOS (10 year old male with spinal cord mass; 5-year-old female with left frontal mass) and 4 patients with ST-EPN-NOS (median age 15 years, ranging from 10–28 years, all females). While the CNS embryonal tumors showed high grade small round cell morphology, the remaining showed ependymoma-like morphology, but with increased cellularity and focal to diffuse membranous expression of epithelial membrane antigen and cytokeratins unlike ependymomas. Among these four patients, one was disease free at last follow-up at 17 months, while the remaining suffered recurrences within a median time duration of 26 months after diagnosis (24 months - 36 months).

CONCLUSION

The clinicopathological spectrum of BCOR-altered CNS tumors appears to be wider than that described in literature. We describe BCOR alterations in a subset of ependymoma-like tumors in older children and young adults with a striking female preponderance. BCOR alterations need to be studied on a broad spectrum of pediatric and adults CNS tumors to aid in better understanding of its true prevalence and biological significance.
P11.07 LAPTM5 functions as a tumor suppressor via CD40 - NFêB pathway inhibition and represents a potential biomarker for temozolomide sensitivity in CD40 proficient glioblastoma
Authors: Berberich A , Bartels F , Tang Z , Pusch S , Hucke N , Kessler T , Dong Z , Wiestler B , Winkler F , Platten M , Wick W , Abdollahi A , Lemke D
Published: September 2019

BACKGROUND

Glioma therapy is challenged by the invasive nature of glioma resulting in tumor recurrence and treatment resistance. Lysosomal protein transmembrane 5 (LAPTM5) was identified to inhibit invasion by screening for invasion-associated genes in glioma. The aim of this study was to decipher the function of LAPTM5 in glioblastoma and its interaction with the CD40 receptor which was shown to be highly expressed in up to 40% of glioblastoma.

METHODS

LAPTM5 expression was correlated with clinical outcome of glioma patients. Knockdown of LAPTM5 was performed in different glioma cell lines to analyze the impact on clonogenicity, invasiveness, sensitivity to temozolomide chemotherapy and tumorigenicity in-vitro and in-vivo in a subcutaneous xenograft mouse model. Expression array was used to elucidate the underlying pathways. CD40 knockdown and overexpression was induced to prove the crosstalk of LAPTM5 and CD40.

RESULTS

LAPTM5 expression correlated with better overall survival in high grade glioma patients and acted as a tumor suppressor in CD40 positive glioblastoma cells. LAPTM5 inhibited CD40-mediated NFκB activation resulting in anti-invasive, anti-clonogenic and temozolomide sensitizing effects in-vitro and in-vivo. Vice-versa, knockdown of LAPTM5 enhanced tumorigenicity by activation of the NFκB pathway which was overcome by NFκB inhibition. Importantly, CD40 expression was required for LAPTM5-mediated tumor suppressive activity.

CONCLUSION

LAPTM5 conveyed tumor suppressive and temozolomide sensitizing effects in CD40-positive glioblastoma by inhibition of CD40-mediated NFκB activation and thereby might provide a reasonable biomarker for sensitivity to temozolomide in CD40-positive glioblastoma.
P11.11 Circulating Pro-Angiogenic Cells and Proteins in Patients with Glioma and Acute Myocardial Infarction: Differences in Neovascularization between Neoplasia and Tissue Regeneration
Authors: Huizer K , Sacchetti A , Dik W , Kros J M , Mustafa D
Published: September 2019

BACKGROUND

Although extensive angiogenesis takes place in glial tumors, anti-angiogenic therapies have remained without the expected success. In the peripheral circulation of glioma patients increased numbers of endothelial precursor cells (EPCs) are present, potentially offering targets for anti-angiogenic therapy (Zheng et al., Ann Neurol, 2007). However, for an anti-angiogenic therapy to be successful, the therapy should specifically target glioma-related EPC subsets and secreted factors. Here we compared the EPC subsets and plasma factors in the peripheral circulation of patients with gliomas to acute myocardial infarctions (representing fysiologic regeneration).

MATERIAL AND METHODS

We investigated the five most important EPC subsets and 21 angiogenesis-related plasma factors in peripheral blood samples of 29 patients with glioma, 14 patients with myocardial infarction and 20 healthy people as controls, by an advanced FACS protocol (Huizer et al., PlosOne 2018) and Luminex assay.

RESULTS

In GBM patients all EPC subsets were elevated as compared to healthy subjects. In addition, HPC and KDR+ cell fractions were higher than in MI, while CD133+ and KDR+CD133+ cell fractions were lower. There were differences in relative EPC fractions between the groups: KDR+ cells were the largest fraction in GBM while CD133+ cells were the largest fraction in MI. An increase in glioma malignancy grade coincided with an increase in the KDR+ fraction while the CD133+ cell fraction decreased relatively. Most plasma angiogenic factors were higher in GBM than MI patients. In both MI and GBM, the ratio of CD133+ HPCs correlated significantly with elevated levels of MMP9. In the GBM patients MMP9 correlated strongly with levels of all HPCs.

CONCLUSION

In conclusion, the data demonstrate that EPC traffic in patients with glioma is different from that in normal tissue regeneration. Therefore, the effects of glioma extent beyond the brain, and future therapies aimed at lowering KDR+ cells and HPCs may add to effective treatment.
P11.17 Splicing dysregulation drives glioblastoma malignancy: SRSF3 as a potential therapeutic target to impair glioblastoma progression
Authors: Fuentes-Fayos A C , Vázquez-Borrego M C , Jiménez-Vacas J M , Bejarano L , Blanco-Acevedo C , Sánchez-Sánchez R , Solivera J , Blasco M A , Castaño J P , Luque R M
Published: September 2019

Glioblastomas (GBMs) remain the deadliest human brain tumors, with poor prognosis despite years of research. Currently, standard therapeutic strategies to treat GBM are not efficient and common survival from diagnosis is ~12–16 months. Thus, identification of new diagnostic/prognostic/therapeutic tools to tackle GBMs is crucial. Emerging evidence indicates that the cellular machinery controlling alternative splicing is altered in tumor pathologies, leading to oncogenic splicing events linked to tumor progression. Accordingly, we aimed to determine the expression pattern of the spliceosome components (SCs) and splicing factors (SFs) in high-grade astrocytomas (HGAs), mostly GBMs, and to ascertain the potential consequences of its dysregulation on GBM development. To this end, expression levels of SCs core and selected SFs were measured using a customized-microfluidic qPCR array in a well-characterized cohort of HGAs (n=33). Our results unveiled a profound alteration in the expression of multiple SCs and SFs in HGAs compared to healthy brain control-samples, wherein levels of particular elements (SRSF3/RBM22/PTBP1/RBM3) enabled perfect discrimination between non-pathological vs. tumor human-tissues, and between proneural and mesenchymal-like GBMs vs. control samples in mouse-models. Results were confirmed in an independent validation-cohort (n=49) and available Microarray dataset (Murat), which revealed that the expression of these splicing elements was correlated with relevant tumor markers and with survival. Remarkably, SRSF3/RBM22/PTBP1/RBM3 silencing (using specific siRNAs) decreased several aggressiveness parameters in vitro (e.g. proliferation, migration, tumorsphere formation, VEGFA secretion, etc.) and induced apoptosis, being SRSF3 the most relevant element affecting these parameters. Hence, a preclinical mouse model (U87MG-xenografts) with SRSF3 silencing drastically decreased in vivo tumor development/progression (i.e. tumor size, %MKI67, mitosis number, etc.) likely through a molecular/cellular mechanism involving the regulation of PDGFRB expression and its associated oncogenic signaling pathways. Overall, our results demonstrate that there is a profound dysregulation of the splicing machinery (spliceosome core and SFs) in HGAs/GBMs, which is directly associated to the development/progression of GBMs. Furthermore, this study reveals that SRSF3 can be a novel biomarker of malignancy and a potential therapeutic target to impair GBMs progression.
P11.26 Genome-wide shRNA screen identifies candidate genes driving glioblastoma invasion
Authors: Schuster A , Neirinckx V , Klein E , Nazarov P V , Oudin A , Muller A , Azuaje F , Herold-Mende C , Klink B , Niclou S P
Published: September 2019

BACKGROUND

A major hallmark of glioblastoma (GBM) is its highly invasive capacity, contributing to its aggressive behaviour. Since invasive cells cannot be easily removed by surgery or irradiation, they are left behind and eventually result in lethal recurrence. Therefore, a better understanding of the invasion process and of the key molecular players underlying the invasive capacities of GBM may lead to the identification of new therapeutic targets for GBM patients.

MATERIAL AND METHODS

To identify candidate genes responsible for invasion, a genome-wide shRNA screen was performed in patient-derived GBM sphere cultures. The phenotype of the most promising candidate was validated in in vitro invasion assays, ex vivo brain slice cultures and in vivo orthotopic xenografts in mice. Gene knockdown in invasive GBM cell lines was compared with overexpression in non-invasive cells. RNA sequencing of knockdown cells, along with the generation of deletion constructs were applied to uncover the mechanisms regulating invasion.

RESULTS

Through a whole genome shRNA screen, a zinc-finger containing protein was identified as an invasion essential candidate gene. Knockdown of this gene confirmed a strong decrease in invasion capacity in two highly invasive GBM cell lines. In contrast, gene overexpression switched non-invasive GBM cells to an invasive phenotype. Deletion of either one or both zinc-finger motifs led to decreased invasion indicating that the two zinc-finger motifs are essential for regulating invasion. Mutation of the nuclear localisation signal resulted in retention of the protein in the cytoplasm and loss of the invasion phenotype demonstrating that the protein activity is required in the nucleus. Gene expression analyses revealed that invasion-related genes are significantly regulated by the candidate gene once it is localized in the nucleus.

CONCLUSION

We identified a zinc-finger containing protein as a novel driver of GBM invasion, presumably through a transcription factor activity resulting in the induction of an invasive transcriptional program. This protein and its downstream pathway may represent a novel promising target to overcome invasive capacities in GBM.
P11.29 Development of ex vivo models for deeper insights into the biology and therapeutic targeting of tumor microtube networks in gliomas
Authors: Jung E , Domínguez Azorín D , Hausmann D , Mall M , Koch P , Wick W , Winkler F
Published: September 2019

BACKGROUND

The formation of multicellular networks via thin cellular protrusions named tumor microtubes (TMs) emerged as a novel mechanism of therapy resistance in malignant glioma. TMs are also involved in tumor cell invasion and growth. Within these tumor cell networks, connected tumor cells communicate via intercellular calcium waves (ICWs). Only few molecular drivers of TMs (Gap43, Ttyh1, Connexin 43) have been identified until now. Furthermore, the molecular mechanisms underlying ICWs as well as their specific biological role in glioma remains to be elucidated. A better understanding of the biology and the identification of molecular key drivers is essential for the development of drugs targeting TM formation and function.

MATERIAL AND METHODS

For this purpose, we have developed novel ex vivo models that not only provide insights into TM biology but further allow medium throughput drug screening. As classical response parameters such as the inhibition of cell growth or cytotoxicity do not necessarily correlate with effects on TM formation or function, a morphometrical approach employing laser scanning microscopy and machine-learning based image analysis tools is used. The application of fluorescent probes and genetic fluorescent reporter systems provides novel longitudinal insights into cytoskeletal dynamics, the role and exchange of organelles such as mitochondria, mechanisms of homeostasis within tumor cell networks (e.g. redox homeostasis) and ICWs in live cells. In addition to 2D glioma cell and co-culture models we have developed a fully human and mature brain organoid model. Here, complex 3D tumor cell networks corresponding to the morphology and exhibiting calcium communication patterns observed in our mouse model can be established and studied ex vivo. Furthermore, with these models not only the role of the brain microenvironment on TM formation but also direct interactions of glioma cells with neurons and glial cells as well as drug effects such as cytotoxicity on these brain cells can be investigated ex vivo.

CONCLUSION

In summary, novel tumor models enable further insights into TM biology and hence provide the basis for development of TM- and network disrupting drugs. First results of this screening opportunity will be presented.
P11.35 Fluorine-18-labeled PET radiotracers for imaging tryptophan uptake and metabolism in brain tumors
Authors: Muzik O , John F , Mittal S , Juhasz C
Published: September 2019

BACKGROUND

Abnormal metabolism of tryptophan via the serotonin and kynurenine pathways plays a key role in multiple disease processes including cancer. Upregulation of key enzymes of the kynurenine pathway (such as indoleamine 2,3-dioxygenase [IDO] and tryptophan 2,3-dioxygenase [TDO]) plays an important role in immune resistance in human brain tumors. IDO inhibitors have recently entered in human clinical trials, and their use can benefit from molecular imaging evaluating IDO activity. Imaging tryptophan uptake and metabolism in vivo can be achieved with tryptophan derivative PET radiotracers. Human studies with such tracers showed promise but have been confined to carbon-11-labeled compounds (such as alpha-[11C]methyl-L-tryptophan). Preclinical development of fluorine-18-labeled tryptophan-based radiotracers has surged only in recent years. We performed a systematic review of studies reporting on such tracers and summarized their biological characteristics and their potential for imaging key enzymes of the kynurenine pathway.

MATERIAL AND METHODS

A PubMed search using the key words “tryptophan” and “PET”/”positron emission tomography” was performed. English language original articles including data on the preparation and/or radiochemical or biological characteristics of fluorine-18-labeled tryptophan derivative radiotracers have been reviewed.

RESULTS

Nineteen original papers identified by the search included data on 15 unique fluorine-18-labeled tryptophan-derived radiotracers. Automated synthesis was reported for 1-(2-[18F]fluoroethyl)-L-tryptophan, the most extensively evaluated tracer among the 15. Biodistribution studies showed high uptake in the pancreas, and the L-type amino acid transporter was the dominant transport mechanism for most of the reported radiotracers. Multiple tracers showed accumulation in various tumor cell lines, including glioma cell lines, in vitro and in xenografts in vivo, with favorable tumor-to-background uptake ratios in comparison to clinically used fluorine-18-labeled radiotracers (such as glucose and non-tryptophan amino acid analogs). Five of the 15 tracers showed promise for imaging IDO activity, including a fluorine-18-labeled analog of alpha-[11C]methyl-L-tryptophan. Two of the 15 radiotracers were metabolized by TDO but showed rapid defluorination in vivo.

CONCLUSION

Most fluorine-18-labeled tryptophan derivative PET tracers share common transport mechanisms and biodistribution characteristics. Several of these radiotracers show promise for imaging IDO activity in vivo, and, therefore, could be leading candidates for testing and validation toward human tumor PET imaging applications.
P11.39 Chloroquine as an anti-glioblastoma therapeutic: repurposing of an old drug
Authors: Roy L , Poirier M , Fortin D
Published: September 2019

BACKGROUND

Glioblastoma (GBM) is the most common and aggressive type of primary brain tumour in adults. These tumours depict anarchic proliferation and brain infiltration as well as radio- and chemoresistant profiles. The complete surgical resection is unachievable and responses to standard therapy are transitory. Recurrence is thus inevitable and patient prognosis is generally less than 15 months. Transforming growth factor-beta (TGF-β) holds a substantial role in supporting the GBM phenotype. We showed that TGF-β 1 expression levels correlate with overall and progression-free survival in newly diagnosed GBM patient. We also observed that chloroquine (CQ) can reduce the production of TGF-β together with proliferation, invasion, radioresistance and radio-induced invasion in vitro. Unfortunately, little is known regarding the ability of CQ to penetrate the blood-brain barrier (BBB). Therefore, our objective is to determine whether intravenous (IV) or intra-arterial (IA) infusions of CQ and hydroxychloroquine (HCQ), a pharmacological analog of CQ, can yield therapeutic brain concentrations.

MATERIAL AND METHODS

To assess BBB penetration, the brain, plasma and cerebrospinal fluid (CSF) concentrations of CQ/HCQ were measured by LCMS/MS at different timepoints post-IV or post-IA infusions with 20 mg/kg of CQ/HCQ in tumour-free Wistar rat. For the survival studies, We implanted 10’000 F98 murin glioblastoma cells in the right putamen of Fischer rats. Ten days post-implantation, IA and IV infusion were accomplished through cannulations of the external right carotid and tail vein respectively.

RESULTS

With IV injections, CQ/HCQ brain concentrations 15 minutes post-injection reached 15.76 mg/g (0.18 µM) and 1.67 mg/g (0.078 µM) respectively. However, following IA infusions, we observed a 1.74 and 20.9 fold increase (20 mg/kg HCQ) as well as 7.1 and 84.7-fold-increase (20 mg/kg CQ) in contra- and ipsilateral brain concentrations respectively. Although brain concentrations gradually decreased over time post-IA infusions, the ipsilateral hemisphere CQ concentration was still 82.81 mg/g (34.52 µM) after 6 hours. Whereas plasma concentrations were very similar following IV and IA infusions, both molecules barely accumulated in the CSF and only when using IA infusions. The median survival of the control group (IA phosphate-buffered saline) and the group treated with 20 mg/kg CQ IV were 23.5 days and 24.5 days respectively. However, rats injected with 20 mg/kg CQ IA had a median survival of 28.5 days.

CONCLUSION

These results suggest that IA CQ could be used to abrogate the GBM phenotype. As TGF-β is associated with resistance to both radio- and chemotherapy, we plan to characterize the combination of IA infusions of CQ in combination with radiation or chemotherapy (carboplatin).
P11.41 Comparison of fibroblast activation protein expression and localization in glioblastomas and brain metastases
Authors: Busek P , Zubal M , Chmielova B , Vanickova Z , Hrabal P , Krepela E , Sedo A
Published: September 2019

BACKGROUND

Fibroblast activation protein (FAP) is a transmembrane serine protease that is frequently upregulated in the tumor microenvironment. In several cases, FAP protein itself and/or FAP expressing stromal cells have been shown to contribute to cancer progression and to be associated with more aggressive cancer behaviour and shorter patient survival. The aim of this study was to determine FAP expression in glioblastomas and brain metastases and to identify the cell types that express FAP in the microenvironment of these malignancies.

MATERIAL AND METHODS

FAP enzymatic activity and protein concentration were determined in samples from patients with brain metastases, glioblastomas and pharmacoresistant epilepsy (control non-tumorous brain tissue) by an enzymatic assay using a specific fluorogenic substrate and ELISA, respectively. Immunohistochemical labelling with antibodies against FAP and markers of astroglia, epithelial cancer cells and mesenchymal stromal cells was performed to characterize FAP expressing cells.

RESULTS

FAP was significantly upregulated in the majority of glioblastomas and brain metastases in comparison to non-tumorous brain tissue. In glioblastomas, FAP was localized perivascularly and in mesenchymal cells, and in part of the tumors also in the glioma cells. In brain metastases, FAP positivity was abundantly present in the stroma and predominantly co-localised with markers of mesenchymal stromal cells (TE-7, SMA, PDGFRbeta, NG2), but there was no overlap between FAP and markers of epithelial cancer cells (EpCAM, pancytokeratin).

CONCLUSION

FAP is upregulated in the microenvironment of human glioblastomas and brain metastases compared to non-tumorous brain tissue. In glioblastomas, FAP is expressed in part of the glioma cells, in pericytes and mesenchymal stromal cells, whereas no positivity in cancer cells and more abundant FAP+ stroma was detected in brain metastases. The selective expression of FAP in these brain tumors may be useful for the visualization and possibly therapeutic targeting of their tumor microenvironment.

GRANT SUPPORT

Ministry of Health of the Czech Republic, grant No. 15-31379A, Progres Q28/LF1, 2015064 LM EATRIS and the project,Center for Tumor Ecology - Research of the Cancer Microenvironment Supporting Cancer Growth and Spread” (reg. n. CZ.02.1.01/0.0/0.0/16_019/0000785) supported by the Operational Programme Research, Development and Education.