LTBK-03 MULTICENTER RANDOMIZED PLACEBO CONTROLLED TRIAL OF AUTOLOGOUS FORMALIN FIXED TUMOR VACCINE FOR NEWLY DIAGNOSED GLIOBLASTOMAS

Abstract

BACKGROUND

Unlike immune checkpoint inhibitors, various vaccines have failed to demonstrate sufficient evidence in phase III studies due to the multiple antigenicity of neoplasms. Autologous formalin-fixed tumor vaccine (AFTV) from paraffin-embedded tissue is stable, and contains multiple tumor peptides, which are designed to induce killer lymphocytes in vivo (Nat Med 1995, 1996). We conducted three AFTV trials of Phase Ib or IIa for recurrent and newly-diagnosed glioblastoma (ndGBM), which demonstrate promising results. (Clin Cancer Res 2004; J Neurosurg 2011, 2013). In addition, a multicenter double-blind Phase IIb/III trial has been initiated.

METHODS

Patients from 16 to 75 years of age with supratentorial ndGBM, extensive resection, KPS of at least 60, and no prolonged steroids, were eligible. Planned patient enrollment was 120. AFTV or placebo, including for adjuvant treatment, was administered intradermally 3 times preceding (1) temozolomide chemoradiotherapy, (2) first and (3) second courses of adjuvant temozolomide.

RESULTS

In 30 months, 63 patients were registered for phase IIb and 61 cases were analyzed (mean age, 61 years). Median resection rate was 95%, median KPS was 80. In the whole cohort, median OS and progression-free survival (PFS) was 31.5 and 14.5 months, respectively. Comparison of the AFTV group (median OS, 25.6 months; 3-year OS rate, 38%) and the placebo group (median OS, 31.5 months; 3-year OS rate, 41%) did not reveal a significant difference in OS (HR, 1.19; 95% CI, 0.57 - 2.47; P = 0.64). Median PFS was 13.5 months in both groups (P = 0.98). According to a subgroup analysis of tumors with negative p53 immunostaining, 3-year OS of AFTV and placebo was 79% and 43%, respectively (P = 0.072). Surprisingly, for patients undergoing total resection of tumors, 3-year PFS of AFTV and placebo was 81% and 46%, respectively (P = 0.067). There were no severe adverse effects.

CONCLUSIONS

Phase IIb of AFTV trial for ndGBM did not demonstrate obvious survival impact in the whole cohort. However, subgroup analyses of tumors with negative p53 immunostaining or after total resection revealed possible better outcomes after AFTV therapy. These results will be investigated further in the Phase III part of the study.

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LTBK-04 FIRST RESULTS OF THE RANDOMIZED PHASE II STUDY ON DEPATUX –M ALONE, DEPATUX-M IN COMBINATION WITH TEMOZOLOMIDE AND EITHER TEMOZOLOMIDE OR LOMUSTINE IN RECURRENT EGFR AMPLIFIED GLIOBLASTOMA: FIRST REPORT FROM INTELLANCE 2/EORTC TRIAL 1410

Abstract

BACKGROUND

Depatux-m is a tumor-specific antibody-drug-conjugate consisting of antibody (ABT-806) bound to the toxin monomethylauristatin-F. We investigated depatux-m in EGFR-amplified recurrent glioblastoma (40–50% of glioblastoma).

METHODS

EORTC-1410-BTG (NCT02343406) is a randomized open label phase II study. Eligible were patients with centrally confirmed EGFR-amplified glioblastoma at first recurrence after temozolomide chemo-irradiation, occurring ≥3 months after radiotherapy. After stratification for WHO status and time of relapse (<16 or ≥16 weeks after the first day of the last temozolomide cycle), patients were randomized to either a) treatment with depatux-m 1.0 mg/kg every 2 weeks intravenously, or b) the same treatment combined with temozolomide 150–200 mg/m2 day 1–5 every 4 weeks, or c) either lomustine or temozolomide (TMZ/LOM) depending on the time of relapse. Primary endpoint was overall survival (OS); 240 patients/170 events were needed to detect a HR reduction of 0.54 in the depatux-m arms.

RESULTS

Between March 2015 and July 2016 260 patients were randomized. With 199 events observed, for the primary comparison of depatux-m in combination with TMZ versus TMZ/LOM a HR of 0.71 was observed (95%CI [0.50, 1.02]; p = 0.031 one-sided; median OS 9.6 months versus 8.2 months, 1-year OS rate 39.7% versus 28.2%). No OS difference was observed between depatux-m monotherapy (median OS 7.9 months) and TMZ/LOM (HR 1.04; 95%CI [0.73, 1.48]. In patients with relapse ≥16 weeks after end of 1stline TMZ treatment, combined depatux-m/TMZ was associated with improved OS (HR 0.45, 95%CI [0.23, 0.87]; median OS 14.9 months versus 9.5 months). The main toxicity observed in depatux-m treated patients was ocular (grade 3: 27.9%, grade 4: 1%).

CONCLUSION

Although the primary endpoint was not met, depatux-m in combination with TMZ might improve OS in EGFR amplified recurrent glioblastoma. Outcome for depatux-m monotherapy was similar to LOM/TMZ control. Ocular events observed were consistent with earlier studies.

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CMET-26. DIAGNOSTIC VALUE OF FDG-PET/CT FOR PATIENTS WITH BRAIN METASTASIS FROM UNKNOWN PRIMARY SITE

Abstract

OBJECTIVE

In 30% of patients with brain metastasis (BM), BM are the first clinical manifestation of systemic malignancy, referred to as BM from cancer of unknown primary site (BM-CUPS). The value of 18F-fluordesoxyglucose positron emission tomography (FDG-PET)/CT in the work-up of BM-CUPS patients remains to be defined.

METHODS

We screened 566 patients operated for BM at the University Hospital Zurich between 2004 and 2014 and identified 127 BM-CUPS patients. A validation cohort from two independent centers (n=100 and 120 patients) was available.

RESULTS

FDG-PET/CT was not superior to CT in localizing the primary lesion (FDG-PET/CT: 73/78, 93.6%; CT: n=70/78, 89.7%; p=0.25, McNemar's test). Thirty-six of 64 patients (56.3%) showed the same result in spotting the primary tumor. FDG-PET/CT identified additional lesions suspicious for extracranial metastases in 28 patients (43.7%). The graded prognostic assessment (GPA) score was determined post-hoc to objectify clinical relevance of additional findings. Information from CT only or FDG-PET/CT was used to assess extracranial metastases. Median GPA was 3 for CT vs. 2.5 for PET/CT (p= 3.8x10-5, McNemar's test), resulting in a predicted survival of 5.3 vs. 3.8 months (p= 6.1x10-5; Wilcoxon's test). Sensitivity of CT and FDG-PET/CT and staging capabilities were comparable in all cohorts.

CONCLUSION

FDG-PET/CT shows similar sensitivity to detect the primary tumor in BM-CUPS patients as CT, but may improve the accuracy of staging by detecting of more metastases. GPA scores and predicted survival differ significantly when calculated based on CT versus FDG-PET/CT. FDG-PET/CT should be prioritized for planning the diagnostic algorithm of BM-CUPS patients and redundant CT imaging should be avoided. Further, randomized trials on BM patients should standardize the methods when stratifying for GPA.

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ACTR-01. RETROSPECTIVE ANALYSIS OF OLIGODENDROGLIOMA TREATMENT AT A SINGLE INSTITUTION

Abstract

BACKGROUND

Recently published results of randomized prospective clinical trials have demonstrated benefit from sequential radiation and chemotherapy after surgery for oligodendroglial tumors (RTOG 9802, 9402; EORTC 26951). We aimed to investigate whether such a benefit could be demonstrated in a retrospective cohort treated at our institution.

METHODS

Patients were identified from the quality control database in the Section of Neuro-Oncology, Yale Cancer Center. Histologic, clinical, imaging, treatment, and outcome data was reviewed. Patients who were 18 to 39 years old and had undergone gross total resection of a WHO II lesion were deemed low-risk (LR). High-risk patients were those ≥40 years old or who had undergone an incomplete resection or those with anaplastic tumors. Primary outcome measure is progression-free survival (PFS). Secondary outcome measure is overall survival (OS). PFS and OS are analyzed in high-risk patients who at initial diagnosis received sequential therapy (HR-SEQ) compared to individuals who only underwent surgery alone or combined with one adjuvant treatment modality (HR-DEL). Survival estimates are based on Kaplan Meier method and survival distributions are compared using the log-rank test.

RESULTS

We identified 150 consecutive biopsy-confirmed oligodendroglioma cases who were treated at Yale Cancer Center between 2002 and 2016. Forty-one patients (27%) are deceased. Thirty patients were considered low risk (1p/19q intact, n=14; co-deletion, n=14; 1p-deleted, n=2). Amongst the high-risk patients, 99 were in the SEQ group (1p/19q intact, n=46; co-deletion, n=47; 1p-deleted, n=2; 19q-deleted, n=4) and 21 in the DEL group (1p/19q intact, n=9; co-deletion, n=8; 1p-deleted, n=3; 19q-deleted, n=1). Median OS was 15.1 years for LR, 6.75 years for HR-SEQ, and 16.95 years for HR-DEL.

CONCLUSIONS

PFS and subgroup analysis based on molecular markers is ongoing. We reserve our conclusions until the analysis is complete. The cohort is likely too small for detailed subgroup analysis.

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TMOD-48. SNAIL1 REGULATES GLIOMAGENESIS INDEPENDENT OF ITS MESENCHYMAL TRANSFORM FUNCTION

Abstract

BACKGROUND

Master mesenchymal factors such as Snail1 have long been implicated in local invasion and metastasis of various cancers including malignant brain tumors such as glioblastoma (GBM). However, mesenchymal factors are expressed early in tumorigenesis, well before first evidence of metastatic cells, suggesting that these factors have other critical functions independent of their mesenchymal regulation.

METHODS

We used a highly penetrant, retrovirally-delivered, PDGFB-induced mouse model of gliomagenesis in a conditional PTEN and/or P53 knockout background to produce GBM tumors of the proneural subtype, in which Snail1 expression is much lower compared to the mesenchymal subtype. We then examined the effect of conditional KO of Snail1 in glial cells when PDGFB overexpression occurred, using in vitro and in vivo loss-of-function experiments augmented by RNAseq expression profiling and computational network analysis.

RESULTS

Snail1 expression peaked early in gliomagenesis although its expression is downregulated once PDGFB-induced proneural tumors are formed. In an immune intact background, Snail1-null GBM developed at a much slower pace. Snail1 cooperated with oncogene activity to promote higher transformative capacity and to influence the cancer stem cell compartment.

CONCLUSION

Our data confirms a dual role for Snail1 in GBM progression, in which it cooperates with oncogenes to promote early transformation while independently also regulating mesenchymal changes at later stages of tumor development. Our platform combining RNAseq expression profiling and computational network analysis with biological validation provides a potentially powerful tool to study mechanistic events as gliomagenesis and proneural subtype GBM transformed into mesenchymal subtype GBM.

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SURG-11. LASER INTERSTITIAL THERMOTHERAPY (LITT) FOR NEWLY DIAGNOSED AND RECURRENT GLIOBLASTOMA: ASSOCIATION BETWEEN TIME TO INITIATION OF CHEMOTHERAPY POST-PROCEDURE AND OUTCOME

Abstract

BACKGROUND

LITT is used for cytoreduction of unresectable glioblastoma and is associated with blood brain barrier disruption with an increased permeability peak at 2 weeks. We investigated if time to initiation of chemotherapy post-LITT was associated with progression free survival (PFS) and overall survival (OS).

METHODS

Records of glioblastoma patients who underwent LITT at our institution between 2013-2017 were reviewed. Patients with inadequate follow-up or no further treatment after LITT were excluded. A time-to-event analysis was performed to investigate the association between PFS, OS and the time to initiation of chemotherapy after LITT.

RESULTS

The study included 21 patients; 17 recurrent glioblastoma (rGBM) (6 secondary glioblastoma), 4 newly diagnosed glioblastoma (nGBM). Median age was 53.6 (19.8-64.9) years. Three patients (14%) had isocitrate dehydrogenase (IDH)-1 mutation by immunohistrochemistry and two patients had unknown IDH status. Pre- and post-operative median KPS were 90 (60-100) and 80 (40-100) respectively. Eleven patients had difficulty weaning steroids (4 patients initiated steroids peri-operatively, 7 patients prior to surgery). For rGBM post-LITT median PFS was 3.36 months (95% CI (0.21, 0.51)) and median OS was 18.48 months (95% CI (0.66,NA)) with 5 deaths. Median PFS and OS for nGBM has not been reached. Eighteen patients (86%) received post-LITT chemotherapy of which eight initiated treatment >3 weeks post-LITTdue to poor functionality (6), pregnancy (1), and patient choice (1). Among the patients receiving chemotherapy, time to initiation of chemotherapy was not associated with PFS or OS. Chemotherapy in rGBM cohort included lomustine (6), temozolomide (5), bevacizumab (3), bevacizumab + lomustine (1), lapatinib (1), and Novo-TTF (1). Median time to initiation ofi bevacizumab (4 patients) after LITT was 30.5 (17-45) days, without complications.

CONCLUSIONS

LITT may be an effective cytoreductive treatment for glioblastoma. Timing of onset of chemotherapy after LITT for glioblastoma is not associated with PFS or OS.

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EPID-13. A SINGLE INSTITUTION ANALYSIS: DETERMINING GENETIC VARIATION AND SURVIVAL OUTCOMES AMONG ASIAN AND NON-ASIAN NEUROSURGERY PATIENTS

Abstract

BACKGROUND

Globally, brain cancer compromises 2% of all total emerging cancers, yet has one of the lowest survival five-year survival rates. Differences in clinical outcomes between brain tumor patients of different races remains poorly understood.

METHODS

A retrospective chart review was performed on brain tumor resection patients 18 years old. Demographics, treatment variables, and survival outcomes were collected. Primary outcomes were recurrence rate, progression free survival (PFS), and overall survival (OS).

RESULTS

A total of 666 patients were included in final analysis. Females and males had nearly a 1:1 ratio (n = 345 and n = 321, respectively). Mean age was 51.3 (± 17.2) years. Females composed 64.3% (n = 54) of Asians patients; males constituted 35.7% (n = 30). Mean age of the Asians was 53.5 (± 16.5) years. Tumor pathologies included meningioma (n = 210 total) and glioblastoma (n = 192 total). There were 210 meningioma patients, of which Non-Asian patients comprised 81.43% of the group (n = 171) and Asian patients composed 18.57% (n = 39). We identified 192 glioblastoma patients in total. Non-Asian patients made up 92.7% of the glioblastoma cohort (n = 178) with the remaining 7.3% (n = 14) composed of Asian patients. There were no statistically significant differences between these groups in both cohorts in recurrence (P = .9052 and P = .7894, respectively), PFS (P = .6953 and P = .845, respectively), or OS (P = 1.0 and P = .9126, respectively).

CONCLUSION

Studies evaluating the survival between patients of different racial backgrounds against several tumor varieties are rare. Patients of certain racial backgrounds may need additional care consideration despite the same mutational composition as their counterparts. Repeated studies using national databases, such as the SEER database, may yield more conclusive results.

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TMIC-36. CROSSTALK OF GLIOMA STEM CELLS WITH VASCULAR ENDOTHELIAL CELLS PERSISTS THEIR PRONEURAL PHENOTYPE AND THERAPY RESISTANCE VIA ENDOCAN-CD11A INTERACTION

Abstract

GBM cells tend to invade into adjacent normal brain tissues and cannot be completely resected surgically. These remaining tumor cells are the "seeds" to escape post-surgical therapies, hence contributing to tumor recurrence. The exact mechanisms by which these seeds confer to therapy resistance remains elusive. Several studies have shown the functional contribution of tumor microenvironment to glioma stemness and highlighted the importance of vascular endothelial(VE) cells in tumor initiation and progression. Here, we aim to determine the role of crosstalk of VE cells with glioma stem cells (GSCs) to develop GBM recurrence. We first assessed the effect of VE conditioned media (VE-CM) on patient-derived GBM neurospheres in vitro and in vivo. Culturing GBM neurospheres with VE-CM or their coculturing with VE cells promoted growth and migration of GBM neurospheres. Mice coinjected with VE cells and GBM neurospheres resulted in an increase of tumor growth compared to the injection of GBM neurospheres alone. To understand the mechanism of action, we performed RNA sequencing with CD31+ VE cells and A2B5+ GSCs directly isolated from patients with therapy-refractory epilepsy or GBM to identify factors secreted by these cells. We identified that Endocan is upregulated in VE cells, whereas CD11a – a receptor for Endocan, is upregulated in GSCs. Preferential expression of CD11a in perivascular region of GBM tissues was observed by immunohistochemistry. Irradiating VE cells substantially increased ESM1, by which VE cells provide extrinsic signals for elevated malignancy of GSCs after radiation therapy. Functionally, adding VE-CM or recombinant ESM1 protected GBM neurospheres from undergoing irradiation induced apoptosis and mesenchymal transition. We are currently investigating exact mechanism by which Endocan secretion from VE cells promotes GSC malignancy after radiation. Taking these data together, targeting ESM1 in VE cells or CD11a in GBM cells may provide a novel and effective strategy for preventing GBM recurrence.

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ATIM-20. CLINICAL OUTCOMES WITH IPILIMUMAB IN COMBINATION WITH BEVACIZUMAB IN PATIENTS WITH RECENTLY DIAGNOSED GLIOBLASTOMA - A RETROSPECTIVE COHORT REVIEW

Abstract

BACKGROUND

Median survival for patients with glioblastoma remains under a year. Whilst there is accumulating interest in the role of checkpoint inhibitors in newly diagnosed glioblastoma, the results of clinical trials are awaited to establish clinical efficacy. We have previously presented clinical outcomes in patients with relapsed glioblastoma treated with the anti-CTLA-4 monoclonal antibody ipilimumab in combination with the anti-VEGF monoclonal antibody bevacizumab.

METHODS

We retrospectively identified patients with newly diagnosed WHO grade IV glioma who received treatment with ipilimumab and bevacizumab at our centre between March 2015 and March 2017. Baseline demographics, tumour characteristics, concurrent therapy, radiological responses, and survival data were analysed.

RESULTS

Nineteen patients were identified, 18 with glioblastoma and one with a glioneuronal tumour (Grade IV). Median age was 52 years (range 22–85) and 79% were male. 5% (1/19) had an IDH mutation, and 38% (6/16) had MGMT promotor methylation. Ipilimumab (3mg/kg, 3 weekly, 4 cycles) and bevacizumab (10mg/kg 2 weekly), given with concurrent G-CSF or GM-CSF were commenced after radiotherapy (except in one patient who did not receive radiotherapy). 58% of patients had prior surgical debulking (42% biopsy only), 79% had prior radical radiotherapy with concomitant temozolomide, 16% had short course radiotherapy, and 5% did not receive radiotherapy. 84% of patients received adjuvant temozolomide, and 89% received concurrent valganciclovir. In those with visible disease on pre-treatment MRI, 62% (8/13) had a radiological response. At time of analysis, 63% of patients remained alive, and 58% were alive and progression free. Median follow up was 15 months. Median survival in patients who had had debulking surgery was 23 months, and median survival in those who had a biopsy only was 16 months.

CONCLUSION

This combination requires prospective evaluation in clinical trials to formally determine efficacy. Data on this cohort continues to be collected and will be updated.

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CSIG-41. UPREGULATED EXPRESSION OF THE ARYL HYDROCARBON RECEPTOR PATHWAY IN BRAIN METASTASES FROM MALIGNANT MELANOMA

Abstract

OBJECTIVE

The Aryl-hydrocarbon-receptor (AHR) is a ligand activated transcription factor linked to exogenic carcinogenic agents such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In 2011, the tryptophan (TRP) catabolite kynurenine (KYN) was identified as an endogenous ligand for AHR. The AHR is involved in several crucial processes such as cell migration, tumorigenesis, and immune function. Dysregulation of AHR, its nuclear translocator (ARNT), its repressor AHRR and the KYN building Enzyme TRP-2,3-dioxygenase 2 (TDO) has been linked to poor survival in patients with Glioblastoma. We aimed to evaluate the AHR-pathway in brain metastases.

METHODS

Using qRT-PCR, gene expression was analyzed in 19 brain-metastases of melanoma patients, 10 control tissues from peritumoral brain and 10 glioblastoma (GBM) samples. Differences in the expression of AHR, ARNT, AHRR, TDO as well as the AHR/AHRR ratio between tumor tissue and control were analyzed. Results are depicted in mean values with standard deviation (SD) and in Arbitrary Units (AU). p<.05 was considered significant.

RESULTS

Both, AHR and ARNT were massively overexpressed in metastatic tissues (p<.01) (6- and 4.5-fold) in comparison to control (AHR: 3.64 ± 2.32 vs. .58 ±.50; p<0.01 and ARNT: 3.81 ± 2.25 vs. .83 ±.48, p<.01). AHRR and TDO where not significantly upregulated (.73 ±.88 vs. .41 ±.35; p=.31 and .37 ±.61 vs. .14 ±.09; p=.46). The ratio of AHR and its repressor AHRR (AHRR/AHR) was significantly different between metastases and control, hinting at a distinct regulatory imbalance (.55 ± 1.09 vs. .18 ± 1.60 p<.01). The difference of AHR expression in metastases and GBM did not quite reach significance (3.64 ± 2.32 vs. 1.27 ± 1.89; p=.055). ARNT was significantly elevated in metastases compared to GBM (3.81 ± 2.25 vs. 1.7 ±.1.15, p<.01).

CONCLUSION

The AHR pathway is significantly upregulated in melanoma brain metastases and might play a pathophysiological role in growth and progression of brain metastases.

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EXTH-59. THE IDH1 MUTANT INHIBITOR AG-120 SHOWS STRONG INHIBITION OF 2-HG PRODUCTION IN AN ORTHOTOPIC IDH1 MUTANT GLIOMA MODEL IN VIVO

Abstract

Mutations in isocitrate dehydrogenase (IDH) 1 and 2 result in accumulation of the oncometabolite 2-hydroxyglutarate (2-HG), which drives multiple oncogenic processes, including increased histone and DNA methylation, leading to a block in cellular differentiation. IDH1/2 mutations occur in >70% of diffuse low-grade gliomas (LGG). Standard of care treatment for patients with diffuse LGG involves combined modality approaches including surgery, radiation, and chemotherapy. Here we present preclinical data from studies using AG-120, a potent, orally available inhibitor of the mIDH1 protein currently in clinical trials. In an orthotopic mouse xenograft model of a human mIDH1-R132H glioma, strong inhibition of 2-HG production in brain tumor samples (>77% inhibition) was observed when AG-120 was dosed orally at 150 mg/kg twice daily. Pharmacokinetic analysis revealed that AG-120 was detectable in the brain and brain-tumor tissues of the mice, although at much lower exposures than in the plasma, indicating that AG-120 is highly potent against the mIDH1-R132H protein in vivo. Results from ongoing preclinical studies testing the potential activity of AG-120 combined with radiation therapy in an orthotopic human mIDH1-R132H glioma model will also be shared.

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STEM-21. CONTEXT-SPECIFIC TUMOR SUPPRESSIVE FUNCTION OF THE CANONICAL Wnt PATHWAY IN PEDIATRIC MEDULLOBLASTOMA HIGHLIGHTS A THERAPEUTIC STRATEGY FOR TREATMENT-REFRACTORY SUBGROUPS

Abstract

Current molecular subgroups of childhood medulloblastoma (MB) recognize distinct disease entities of which activated Wnt signaling is associated with a distinct subgroup and the best overall outcome. In contrast, non-Wnt MBs are characterized by metastatic disease, increased rate of recurrence, and poor overall survivorship. Given the excellent clinical outcome in Wnt-driven MB, we aimed to convert treatment-resistant MB subgroups 3 and 4 into an ostensibly benign tumor. Activated Wnt signaling by way of Wnt agonists decreased in vitro self-renewal of primary MB cells. Comparative RNA-sequencing of control and transgenic lines containing a stabilized beta-catenin mutant demonstrated a reduction in self-renewal genes following beta-catenin overexpression, including Sox2 and Bmi1. In order to validate the therapy-sensitive nature of Wnt-activated cells, we developed stable human Group 3 and 4 patient-derived lines containing a 7XTOPFlash reporter to determine the presence of endogenous Wnt signaling. Rare subclonal Wnt-active cells demonstrated a reduced self-renewal and tumor-initiating capacity through in vivo limiting dilution assays when compared to bulk Wnt-inactive cells from Group 3 and 4 MBs. The therapeutic relevance of these findings were demonstrated with an in vivo survival advantage in mice with orthotopic injections of cells containing stabilized beta-catenin overexpression or endogenous Wnt-active cells. Resulting xenograft tumors were smaller in size, maintained a lower rate of proliferation, and reduction in MB self-renewal genes. To develop a rationale clinical therapeutic, we used a novel substrate-competitive peptide inhibitor for GSK. Treatment with our peptide inhibitor showed a significant reduction in tumor burden and metastatic disease with a corresponding increase in survival of patient-derived Group 3 and 4 tumors that were otherwise treatment-resistant. Our work establishes activated Wnt signaling as a novel treatment paradigm in childhood MB, identifies a rationale therapeutic approach for recurrent MB, and provides evidence for the context-specific tumor suppressive function of the canonical Wnt pathway.

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TMIC-06. IMMUNE MICROENVIRONMENT IN CYSTIC GBM

Abstract

The study objective was to characterize the immune microenvironment in cystic glioblastoma (GBM). We retrospectively reviewed the records of 298 consecutive newly diagnosed and recurrent adult GBM patients treated at our hospital from Jan 2011 to Jun 2017. 23 (7.7%) had cystic tumors and 17 had cystic tumor components. We were able to analyze 23 of these cystic GBM (cGBM) cases. We observed that overall survival in cGBM group is longer than in noncystic counterparts. In 17 samples, 16 were PTEN+, and all of these had TILS CD3, CD4, CD8 within the tumor and in surrounding vessels, 7/17 cGBM were PDL-1+ (>1%). T cells (CD8+, CD4+, and CD8+CD4+), B cells and NK cells were detected in the cystic fluid of cGBM cases. T memory cells (CD4+CD25-CD127+) but not Treg cells were also detected in the cystic fluid. The proportion of active T cells (CD69+) in the cystic fluid was higher than in control PBMC. Immunosuppressive cells Treg and MDSC populations were lower in the cystic fluid than in PBMC. The ProcartaPlex Human Cytokine Panel (34 plex) and IPA data analysis a variety of chemokines were detected in the GBM cyst fluid. IL-6, GRO-alpha, IL-8, IP-10, MCP-1, TNF-a, IL-4, IL-27 were higher in the peripheral blood serum cGBM patients versus non-cystic GBM patients; however, the inhibitory cytokine IL-10 is lower in cyst fluid or PB serum. These results suggest that the mechanism of cystic formation in cGBM could be due to activation of the host immune system. Higher levels of chemokines in the cystic fluid of cGBM patients could enhance recruitment of lymphocytes into the tumor. Intact PTEN status as observed in 16/17 cGBM patients in our study, is critical for anti-glioma immune function and may have played a role in improved survival in our study.

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TMOD-19. SOMATIC GENOME EDITING WITH THE RCAS-CRISPR/Cas9 SYSTEM FOR PRECISION GLIOMA MODELING

Abstract

It has been gradually established that the vast majority of human tumors are extraordinarily heterogeneous at a genetic level. To accurately recapitulate this complexity, it is now evident that in vivo animal models of cancers will require to recreate not just a handful of simple genetic alterations, but possibly dozens and increasingly intricate. Here we have combined the RCAS/tv-a system with the CIRSPR/Cas9 genome editing tools to somatically target neural stem cells (NSCs) for precise modeling of human glioma. We show that deletion, both in pups and adult mice, of a variety of known tumor suppressor genes (Trp53, Cdkn2a and Pten), in combination with the expression of an oncogene driver, leads to high-grade glioma formation. Moreover, by simultaneously delivery into NSCs of pairs of gRNAs we show for the first time that the Bcan-Ntrk1 gene fusions, is able to induce high-grade gliomas. We further established that cells derived from Bcan-Ntrk1 tumors are remarkably sensitive to a Pan-Ntrk inhibitor. Lastly, using homology directed repair (HDR), we generated the Braf V600E mutation into NSCs and we demonstrated that it's sufficient to induce glioma tumor formation. In summary, we have developed an extremely powerful and versatile mouse model for in vivo somatic genome editing. Our system will elicit the generation of more accurate glioma models, particularly suitable for preclinical testing.

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SCDT-30. SURGICAL IMPLANTATION OF AN OSMOTIC PUMP FOR CONVECTION ENHANCED DELIVERY INTO DIPG XENOGRAFT MURINE MODELS

Abstract

INTRODUCTION

Diffuse intrinsic pontine gliomas (DIPG) are fatal, high-grade tumors arising in the pons of children. Convection-enhanced delivery (CED) is a method for directly delivering agents into the tumor, bypassing the blood brain barrier. CED can achieve effective long-term continuous administration when connected to an osmotic pump filled with chemotherapeutic agents. Our objective is to optimize the technique for placement of the CED cannula carrying an osmotic pump into the tumor of preclinical DIPG mouse models.

METHODS

Using agarose gel, Evans blue dye (EBD) was injected at various rates to determine area of distribution. Then, an osmotic pump with a capacity to infuse 100ml over 28 days was used in two cohorts of mice. A cannula was stereotactically implanted into the pons in both cohorts and connected to a subcutaneously placed osmotic pump loaded with EBD or DII. The first cohort assessed the feasibility of implantation into the pons. The second cohort was transplanted with murine DIPG cells and allowed to develop pontine tumors before cannula implantation. The EBD/DII signal was evaluated and compared to tumor cell location to assess delivery to targeted site and distribution.

RESULTS

We have established a methodology for surgical implantation of a cannula attached to an osmotic pump into mouse pons, and assessed the distribution of EBD/DII in healthy as well as tumor bearing murine models of DIPG. We also show that CED cannula installation does not affect the overall survival of the mouse.

CONCLUSION

We show that precise pontine delivery of cargo (dye) has optimal distribution within the pons, tissue integrity is not compromised, and more importantly, does not alter overall survival. This technique will serve as a platform for rapid assessment of tumor response to therapeutic agents. An effective agent can then be translated in clinical setting through upcoming clinical trials.

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CBIO-29. ACOT7 REGULATES COLONIZATION OF BREAST CANCER CELLS IN THE BRAIN BY REGULATING ENDOPLASMIC RETICULUM STRESS RESPONSE

Abstract

Colonization of cancer cells in a foreign organ is the key for establishment of metastases. To identify the genes that are responsible for colonization of breast cancer cells in the brain, we conducted an in silico analysis using a NKI dataset and identified ACOT-7 to be negatively correlated with patient survival. ACOT-7 is normally expressed in the brain and is responsible for maintaining the cellular pool of coenzyme-A by catalyzing the conversion of acyl-CoA to free fatty acid and CoA. The resulting CoA can be used for anabolic reactions. ACOT-7 was found to be overexpressed in microdissected brain metastases as compared to primary breast cancer tissues, and it was also upregulated in brain metastasis models CN34/CN34BrM2 and BT-474/BT-474BrM3. To understand the role of brain specific protein ACOT-7 in brain metastasis, ACOT-7 knockdown studies were conducted. ACOT7 knockdown in CN34Br cells reduced their ability to grow in the brain, which subsequently resulted in increased median survival by 60% in a preclinical mouse model. To understand the role of ACOT-7, we conducted a whole proteome analysis and found reduced expression of genes responsible for unfolded protein response (UPR)/Endoplasmic reticulum (ER)-stress. The results were confirmed using quantitative RT-PCR and western blotting. ACOT-7 knockdown cells also exhibited increased sensitivity to thapsigargin (an ER stress inducer). In conclusion, ACOT-7 is aberrantly expressed in brain metastases arising from breast cancer, and it regulates the ability of cells to counter ER stress. Hence unfolded protein response/ER stress proteins will be a novel therapeutic target for the treatment of breast cancer brain metastases.

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CSIG-08. MUTANT IDH1 CO-OPERATES WITH ATRX LOSS TO DRIVE THE ALTERNATIVE LENGTHENING OF TELOMERE (ALT) PHENOTYPE IN GLIOMA

Abstract

A subset of human tumors use a recombination-based alternative lengthening of telomere (ALT) pathway to resolve telomeric dysfunction in the absence of TERT activation. In these tumors, which include lower-grade astrocytomas (LGA) and secondary glioblastoma (sGBM), the ALT phenotype is associated with loss-of-function mutations in p53 and ATRX, although these mutations alone are insufficient to drive the process. Because LGA and sGBM also exhibit R132H IDH1 mutations, we examined the possible role of mutant IDH1 in driving ALT-based gliomagenesis. In p53/pRb-deficient human astrocytes, we found that neither expression of mutant IDH1 nor genetic deletion of ATRX was sufficient to drive the ALT phenotype. Combined expression of mutant IDH1 and loss of ATRX, however, resulted in the creation of tumorigenic cells with the ALT phenotype. The tumorigenic IDH1-mutant, ATRX-deficient ALT cells, as well as human IDH1-mutant LGA xenograft cells, consistently downregulated both RAP1 and XRCC1. Furthermore, exogenous re-expression of XRCC1 and/or RAP1 suppressed the ALT phenotype and tumor cell viability. Mechanistically, suppression of RAP1, a component of the telomere-capping shelterin complex, caused telomere dysfunction. Downregulation of XRCC1, a key component of the alternative non-homologous end joining (aNHEJ) pathway, suppressed aNHEJ-mediated lethal fusion of dysfunctional telomeres, and instead allowed IDH1-mutant, ATRX-deficient cells to use homologous recombination and ALT to resolve telomeric dysfunction and escape cell death. These studies show that mutant IDH1 expression initiates telomeric dysfunction and alters DNA repair pathway preference at telomeres, and in doing so co-operates with p53 and ATRX loss to allow cells to overcome a critical bottleneck in gliomagenesis. Agents that alter the linkage between mutant IDH1 and DNA repair pathway preference may therefore be of particular interest in mutant IDH1 driven glioma.

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DRES-05. IDENTIFYING DRIVERS OF CHEMORESISTANCE IN GROUP 3 MEDULLOBLASTOMA THROUGH A GENOME-WIDE CRISPR/Cas9 ACTIVATION SCREEN

Abstract

Medulloblastoma (MB) is the most common malignant brain tumor in children, accounting for nearly 20% of all pediatric brain cancers. Based on transcriptional profiling studies, MB has been characterized into four molecular subgroups--WNT, SHH, Group 3, and Group 4--that have distinct genetics, patient demographics, histology, and clinical outcomes. Group 3 MB carries the worst prognosis, with 5-year survival rates of 50%. Current therapy for MB consists of maximal safe surgical resection, radiation, and chemotherapy, with cisplatin and cyclophosphamide as mainstays in many chemotherapy regimens. However, Group 3 MB commonly develops resistance and becomes refractory to standard therapy. As these mechanisms of resistance remain poorly understood, I used the CRISPR/Cas9-based synergistic activation mediator (SAM) system to conduct a genome-wide, gain-of-function, positive selection screen to identify the specific drivers of chemoresistance in Group 3 MB. I cloned and sequenced a library consisting of 70,290 single-guide RNAs (sgRNAs) targeting each of the 23,430 coding isoforms from the human RefSeq database. I transduced this library into a Group 3 MB patient-derived model stably expressing the other SAM components (dCas9, VP64, MS2, p65, and HSF1) and then selected with cisplatin and 4-hydroperoxycyclophosphamide. Modulators of chemotherapy sensitivity will be prioritized by sgRNAs that have activated genes conferring chemoresistance, and I have performed next-generation sequencing to identify these sgRNAs. I will validate the top hits from our screen and also cross-reference them with recently published whole-genome sequencing data on six pairs of human diagnostic and post-therapy Group 3 MBs. These findings will provide insights into the mechanisms of resistance in Group 3 MB and inform novel therapeutic targets that may sensitize the tumor to chemotherapy and improve future treatment response.

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EXTH-31. RNA NANOPARTICLE-BASED TARGETED GENE THERAPY FOR GLIOBLASTOMA

Abstract

Systemic administration of therapeutic siRNA/microRNA for targeted treatment of glioblastoma, one of the most deadly cancers, requires robust and efficient delivery platform without immunogenicity. Here we report newly emerged multivalent naked RNA nanoparticle (RNP), named FA-pRNA-3WJ, based on pRNA 3-way-junction (3WJ) from bacteriophage phi29 to target glioblastoma cells with folate (FA) ligand and deliver siRNA/microRNA for tumor cell killing. Systemically injected FA-pRNA-3WJ RNPs successfully targeted and delivered siRNA into brain tumor cells in mice, and efficiently reduced luciferase reporter gene expression (4-fold lower than control). The FA-pRNA-3WJ RNP also can target human patient-derived glioblastoma stem cells, thought to be responsible for tumor initiation and deadly recurrence, without accumulation in adjacent normal brain cells, nor other major internal organs. FA-3WJ-LNA-miR21 specifically targeted and delivered anti-miR-21 LNA and knocked down miR-21 expression in glioblastoma cells in vitro and in vivo with favorable biodistribution. Systemically injected FA-3WJ-LNA-miR21 RNP efficiently rescued PTEN and PDCD4, resulting in glioblastoma cell apoptosis and tumor growth regression. This RNA nanotechnology was employed even to regulate the anchoring of nanoparticles on the surface of extracellular vesicles (EVs) for specific cancer targeting and intracellular trafficking. Taking advantage of the RNA aptamer ligand for specific targeting and EVs for efficient membrane fusion, EGFRapt-pRNA-3WJ RNP was constructed and anchored onto EVs loaded with survivin siRNA. In the preliminary animal tests, systemic administration of the EV-siSurvivin coated with EGFRapt-pRNA-3WJ RNP inhibited tumor growth in orthotopic breast cancer xenograft mouse model by successfully downregulating the Survivin expression. The studies are indicative of the clinical benefit of FA-3WJ RNP based tumor targeting and gene therapy for the successful targeted therapy of primary and recurrent glioblastoma.

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GENE-04. COMPREHENSIVE GENOMIC CHARACTERIZATION OF AGGRESSIVE MENINGIOMAS IDENTIFIES MOLECULAR SIGNATURES THAT PREDICT CLINICAL OUTCOMES

Abstract

BACKGROUND

Current pathological grading schemes do not fully predict meningioma behavior, and the molecular basis for meningioma is incompletely understood. Here, we perform whole exome sequencing (WES), DNA methylation arrays, RNA-seq, NanoString (NS), and immunohistochemistry using tissue microarrays (TMAs) on meningiomas to elucidate molecular drivers of aggressive behavior and identify clinically relevant biomarkers.

METHODS

We constructed a database containing clinical data and tissue from 283 patients who underwent resection of meningioma between 1990 and 2015. Histology was re-evaluated using current grading criteria. We conducted WES (n=24), methylation arrays (n=26), and RNA-seq (n=42) in a discovery cohort composed of 14 grade I, 23 grade II and 5 grade III meningiomas. We further carried out NS profiling of 282 cancer-associated transcripts (n=96), and TMAs (n=241) in an independent validation cohort composed of 132 grade I, 87 grade II and 22 grade III meningiomas. WES data was analyzed via the bcbio pipeline with the ensemble method and CNVkit, methylation data was processed in the minfi Bioconductor package, and RNA-seq data was analyzed with the DESeq2 Bioconductor package. Hierarchical clustering and statistics were performed in R. 

RESULTS

WES demonstrated increased somatic mutation burden was associated with decreased overall survival (OS) (p=0.005). Unsupervised hierarchical clustering of methylation arrays segregated meningioma samples into three groups with significant differences in OS (p=0.0465) and age (p=0.0123). RNA-seq showed enrichment of a FOXM1 mediated transcriptional network with an overrepresentation of cell division genes. High FOXM1 mRNA expression was associated with decreased local recurrence free survival (LRFS) (p=0.004). Consistently, high FOXM1 mRNA expression in NS samples was associated with decreased LRFS (p=0.001) and OS (p=0.006), and high FOXM1 protein expression was associated with decreased LRFS in TMA samples (p=0.049).

CONCLUSION

Comprehensive genomic characterization of aggressive meningiomas identifies molecular signatures associated with poor outcomes, suggesting novel prognostic markers and therapeutic targets.

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