Low-grade Oligodendrogliomas Research Articles

The prognostic impact of CDKN2A/B hemizygous deletions in IDH-mutant glioma.

Homozygous deletions of CDKN2A/B are known to predict poor prognosis in gliomas, but the impact of hemizygous deletions is less clear. This study aimed to evaluate the prognostic significance of hemizygous CDKN2A/B deletions in IDH-mutant low-grade astrocytomas and oligodendrogliomas. Tissue samples diagnosed as astrocytoma, IDH-mutant and oligodendroglioma, IDH-mutant, 1p/19q co-deleted CNS WHO grade 2 and 3 were collected from the archives of the Institute of Neuropathology in Heidelberg. DNA methylation analysis was performed on formalin-fixed paraffin-embedded (FFPE) samples. Evaluation of the CDKN2A/B locus was performed by visual inspection of copy-number plots derived from methylation-array data for each case. Hemizygous and homozygous losses were assessed in relation to whole chromosomal or larger segmental losses and gains in the chromosomal profile. Survival probabilities were assessed using the Kaplan-Meier method. A total of 334 low-grade glioma cases were identified, including 173 astrocytomas and 161 oligodendrogliomas. Hemizygous deletions in CDKN2A/B (37/173 in astrocytomas, 15/161 in oligodendrogliomas) did not confer significantly worse survival outcomes compared to CDKN2A/B wildtype cases in neither low grade astrocytoma (log-rank p= 0.2556; HR 2.29, 95% CI [0.76; 6.40], p= 0.135) nor oligodendroglioma (log-rank p= 0.2760; HR 0.17; 95% CI [0.01; 5.05]; p= 0.305), regardless of CNS WHO grade, which was further demonstrated on a subgroup of astrocytoma, IDH mutant CNS WHO 4 cases (log-rank p= 0.1680; HR 4.55, 95% CI [0.88; 24.51], p= 0.0689). Hemizygous CDKN2A/B deletions do not significantly worsen OS or PFS in IDH-mutant astrocytomas and oligodendrogliomas, CNS WHO grade 2 and 3.

BIOM-30. NOVEL HISTOLOGIC AND MOLECULAR BIOMARKERS FOR OLIGODENDROGLIOMA

Abstract Oligodendrogliomas are progressive, infiltrative gliomas. While new molecular criteria were introduced in 2016 for diagnosing oligodendroglioma, the grading criteria remains largely unchanged. We evaluated histologic and molecular features for new biomarkers of aggressive behavior, using a cohort of 184 specimens diagnosed as low grade oligodendroglioma within the Duke Brain Tumor Center Biorepository. To identify tumors meeting current molecular criteria for oligodendroglioma, IDH1/2 status was confirmed by whole exome sequencing and 1p/19q codeletion confirmed using inferred copy number variation. First, we assessed for a histologic feature called hypercellular nodules (HCN), which are foci of increased cellularity in regions of otherwise lower cellularity tumor. In patients with newly diagnosed grade 2 oligodendroglioma, 16% (15/92) had HCN. While the median overall survival (OS) was 24.1 years without HCN, the median OS with HCN was only 13.6 years (p<0.02, Mantel-Cox test). We then assessed for homozygous deletion of CDKN2A. Cases with potential CDKN2A homozygous deletion by inferred copy number variation were validated by fluorescence in situ hybridization. In patients with newly diagnosed grade 2 oligodendroglioma, 2% had homozygous CDKN2A deletion (2/107). While the median OS with intact or hemizygous CDKN2A loss was 21 years, it decreased to 3.8 years with homozygous CDKN2A deletion (p<0.0001, Mantel-Cox test), although interpretation is limited given the rarity of homozygous CDKN2A loss at initial diagnosis. For recurrent grade 2 oligodendroglioma, 6% had homozygous deletion (5/77). While the median OS with intact or hemizygous CDKN2A loss in recurrent oligodendroglioma was 25.4 years, it decreased to 6.7 years with homozygous CDKN2A deletion (p< 0.04, Mantel-Cox test). These studies provide support for using HCN and CDKN2A homozygous deletion as grading criteria for oligodendroglioma. Further studies are ongoing, including covariate analysis, expanded cohorts, and ongoing analysis of whole exome sequencing data to identify additional genetic alterations correlated with overall survival.

PATH-32. THE PROGNOSTIC IMPACT OF CDKN2A/B HETEROZYGOUS DELETIONS IN IDH-MUTANT GLIOMA

Abstract BACKGROUND Homozygous deletions of CDKN2A/B have been shown to be a prognosticator for an unfavorable prognosis in gliomas, but the role of heterozygous CDKN2A/B deletions remains less well understood. The aim of this study was to assess the prognostic impact of CDKN2A/B heterozygous deletions in IDH-mutant low-grade astrocytoma and oligodendroglioma. METHODS Tissue samples diagnosed as astrocytoma, IDH-mutant and oligodendroglioma, IDH-mutant, 1p/19q co-deleted CNS WHO grade 2 and 3 were collected from the archive of the Institute of Neuropathology in Heidelberg. DNA methylation analysis was performed on formalin-fixed paraffin-embedded (FFPE) samples. Evaluation of the CDKN2A/B locus was performed by visual inspection of copy-number plots derived from methylation-array data for each case. Heterozygous and homozygous losses were assessed in relation to whole chromosomal or larger segmental losses and gains in the chromosomal profile. Survival probabilities were assessed using the Kaplan–Meier method. RESULTS A total of 334 low-grade glioma cases were identified. Those cases included 173 astrocytomas and 161 oligodendrogliomas. Heterozygous deletions in CDKN2A/B (37/173 in astrocytomas, 15/161 in oligodendrogliomas) did not confer worse survival outcomes compared to CDKN2A/B wildtype cases in neither low grade astrocytoma (p= 0.2556) nor oligodendroglioma (p= 0.2760), regardless of CNS WHO grade, which was further validated on a subgroup of astrocytoma, IDH mutant CNS WHO 4 cases (p= 0.1680). CONCLUSION In summary, our study suggests that heterozygous CDKN2A/B deletions do not confer worse survival rates with respect to OS and PFS in astrocytoma, IDH-mutant and oligodendroglioma, IDH-mutant, 1p/19q co-deleted CNS WHO grade 2 and 3.

Using a three-dimensional-printed device with the patient's maxillary dental impression allows to perform minimally invasive brain biopsies in dogs and cats: a preliminary study.

To develop an innovative process for stereotactic brain biopsies in dogs and cats that would provide a definitive diagnosis and optimize the management of patients with brain lesions. 4 dogs and 1 cat diagnosed with 1 or more brain lesion(s) underwent brain biopsies between March 24, 2023, and October 25, 2023. Based on trajectories selected on images of MRI and CT scan performed on each patient, a computerized software program was used to design a 3-D-printed patient-specific device with maxillary dental impression located on a baseplate to secure the patient's head and with insertion ports for the biopsy instrumentations located on a C-arm. As proof of concept, the device was successfully used in 2 cadavers before being used on clinical patients. All biopsy samples were submitted for histopathological examination. Histological diagnosis was obtained in 80% (4/5) of the cases (choroid plexus tumor, astrocytoma, meningioma, and chronic meningoencephalitis of unknown origin). In 1 patient, the results of biopsy were nondiagnostic; postmortem diagnosis was consistent with a low-grade oligodendroglioma. All the patients were discharged within 24 hours after the procedure without complications. This novel stereotactic system allows the surgeon to perform safe, easy-to-use, inexpensive, and minimally invasive precise brain biopsies in dogs and cats, without complications. This unique technique could be applied to any size and type of skull and for any type of brain lesions and would provide diagnostic information that would be valuable for future treatment planning and prognosis.

Atypical Presentation of a Pilocytic Astrocytoma at the Regional Hospital of Ségou in Mali country

Introduction: Pilocytic astrocytoma is the most common cerebral glioma in pediatric age, preferentially located in the posterior cerebral fossa. Atypical presentations have rarely been described in the literature. Clinical Case: We report a case of pilocytic astrocytoma whose appearance on CT scan is unusual. It concerns a young boy of 9 years old, who presented convulsive seizures since the age of 6 years old, followed by general practitioners with irregular treatment based on gardenal. 2 months ago the seizures became more frequent and this prompted a specialist consultation with the neurologist, after a brain scan he referred the patient to us. The neurological examination was unremarkable today. Brain CT with contrast showed a right parietal lesion. It was a lesion with a double cystic and fleshy component associated with calcifications with heterogeneous enhancement which amputated the posterior horn of the lateral ventricle. The radiological appearance was in favor of a low-grade oligodendroglioma. The patient underwent surgery with complete macroscopic excision of the lesion. Histological examination revealed a pilocytic astrocytoma (grade I). Conclusion: The usual radiological appearance of pilocytic astrocytoma combines a cystic part and a highly contrasting mural nodule. It rarely presents in the form of a supratentorial lesion with calcifications. This form of presentation is rare and can lead to confusion with other gliomas. We can achieve healing for the patient with total excision.

Synchronous contralateral low-grade oligodendroglioma and high-grade IDH-mutant astrocytoma.

Synchronous contralateral low-grade oligodendroglioma and high-grade IDH-mutant astrocytoma.

OS07.6.A INVESTIGATING REGIONAL HETEROGENEITY AND PROGRESSION-ASSOCIATED MARKERS IN OLIGODENDROGLIOMAS

Abstract BACKGROUND Oligodendrogliomas are defined by IDH-mutation, 1p/19q-codeletion and TERT promoter mutation. Molecular stratification of oligodendrogliomas has so far proved difficult, despite recent progress in other brain tumours. Loss of H3K27 trimethylation (H3K27me3) is seen in many, but not all oligodendrogliomas, and we have observed that this trimethylation mark is lost predominantly in low-grade oligodendrogliomas, and more often retained in high-grade tumours. The key aims of this project are to establish the downstream effects of H3K27me3 regulation in oligodendrogliomas to identify progression-associated biomarkers. MATERIAL AND METHODS 78 oligodendrogliomas (CNS WHO grade 2: n=38, and grade 3: n=40) were tested for H3K27me3 expression. These tumours showed either down-regulation (H3K27me3-), retention of expression (H3K27me3+) or intratumoural heterogeneity (H3K27me3Het). 65 regions across these three types were micro-dissected for DNA methylation profiling and RNA sequencing. RESULTS Of the H3K27me3- tumours 85% (35/41) were grade 2, of the H3K27me3+ tumours 100% (10/10) were grade 3, and of the H3K27me3Het tumours 87% (20/23) were grade 3. With DNA methylation arrays and derived copy number data we have shown a different breakpoint of the 1p chromosome in H3K27me3+ samples or areas, and in 15 paired samples, 6 gains and 12 losses in H3K27me3+ regions, and overall 24 copy number differences between H3K27me3+ and me3- areas within the same tumours, indicative of spatial genetic heterogeneity. RNA sequencing suggests distinct expression profiles between H3K27me3+ and H3K27me3- areas. CONCLUSION We have shown a close correlation between areas with high-grade features and H3K27me3 expression both between and within tumours. We have shown that H3K27me3+ and me3- areas within the same tumours show copy number changes and have differential gene expression. Using pathway analysis we have identified pathways and genes involved in tumour progression and mediation of histone modification. We are currently using these targets for functional studies and protein level validation with the overall aim of developing immunohistochemistry biomarkers.

EVALUATION OF THE EFFECTIVENESS OF STEREOTACTIC BIOPSY OF SUPRATENTORIAL TUMORS AT MILITARY HOSPITAL 103

Objectives: To evaluate the histological diagnostic results using stereotactic biopsy of supratentorial tumors at Military Hospital 103. Subjects and methods: A retrospective cross-sectional study on 9 stereotactic biopsies of supratentorial tumors at Military Hospital 103 from 6/2021 - 9/2022. Results: 9 patients in the study included 5 men and 4 women, aged from 29 to 79 years old. The clinical features were dominated by headache (77%), hemiplegia (22.2%), and epilepsy (11.1%). The deep lesions (capsula interna lesions and thalamic lesions) were 44.4%. The histological diagnostic accuracy rate was 88.9%. Histological types include low-grade astrocytoma (44.4%), high-grade astrocytoma (11.1%), oligodendroglioma (11.1%), lymphoma (22.2%), metastatic carcinomas (0%), and brain tissue (11.1%). The mortality rate was 0%. Conclusion: Stereotactic biopsy of supratentorial tumors is a safe procedure that provides a very accurate histological diagnostic result with a low incidence of complications and mortality rate.

IL-3 DISCONNECTING BRAIN TUMOR NETWORKS FOR IMPROVED THERAPIES

Abstract Integration into communicating tumor cell networks is a way how the entire brain tumor organism can progress in the brain, and resist therapies. It started with our discovery of ultra-long membrane protrusions, tumor microtube (TM), in tumor cells from glioblastomas and other incurable adult and pediatric glioma entities (Osswald et al., Nature 2015). These TMs are used for very effective brain invasion, and later interconnect single tumor cells to communicating and resistant multicellular networks, but also glioma cells to neurons in the brain, with neuron-glioma synapses stimulating glioma growth(Venkataramani et al., Nature 2019) and invasion (Venkataramani et al., Cell 2022). Increasing formation of these homotypic and heterotypic networks is seen with increasing glioma malignancy: lowest in low-grade oligodendrogliomas, and highest in glioblastomas and K27M mutated gliomas. The genetic reduction of GAP-43, a crucial neurodevelopmental factor that drives TM formation and function, not only deprived glioblastoma cells from the ability to form functional tumor networks, but also made radiotherapy much more effective, by and large eradicating experimental tumors from the mouse brain, which is typically not possible with radiotherapy alone. Consistent with the concept of tumor cell resilience by network integration, radiotherapy and also chemotherapy (Weil et al., Neuro-Oncology 2017) preferentially killed the unconnected glioblastoma cells, leaving the tumor cell networks largely intact. Damage to the tumor network by surgical measures induced a self-healing response. Taken together, integration into multicellular, highly functional, communicating and resilient networks is not only a feature of the nervous system, but also of incurable, most aggressive brain tumors. In this talk, clinical concepts that originate from these discoveries are presented, including a network disconnection strategy currently tested in the German MecMeth trial (EudraCT2021-000708-39), and the AMPAR inhibitor perampanel as a potential inhibitor of neuron-glioma synapses. Furthermore, new basic and translational developments in the field of brain tumor networks are presented and discussed.

Immunohistochemical study of neural stem cell lineage markers in canine brains, gliomas, and a glioma cell line.

Neural stem cells (NSCs) produce neuron intermediate progenitor cells (nIPC), oligodendrocyte precursor cells (OPCs), and immature astrocytes. To confirm NSC lineages in the normal canine brain and the association of these cells with gliomas, an immunohistochemical study was conducted on fetal and adult canine brains, gliomas, and a glioma cell line. In fetal brains, glial fibrillary acidic protein (GFAP)- and nestin-immunolabeled NSC were observed in the ventricular zone, β-3 tubulin- and/or neuronal nuclei (NeuN)-immunolabeled nIPC in the subventricular zone (SVZ), and platelet-derived growth factor receptor-α (PDGFR-α)- and OLIG2-immunolabeled OPC and GFAP- and OLIG2-immunolabeled immature astrocytes in the SVZ and intermediate zone. Ki-67 immunohistochemistry revealed that nIPC exhibited high proliferative activity. Quiescent nIPC and OPC were observed in adult brains. Among 58 glioma cases including 4 low-grade oligodendrogliomas (LGOGs), 48 high-grade oligodendrogliomas (HGOGs), 1 low-grade astrocytoma, and 5 high-grade astrocytomas (HGACs), immunohistochemical analyses revealed that oligodendrogliomas expressed PDGFR-α and OLIG2, whereas astrocytomas expressed GFAP and OLIG2. HGOG showed significantly higher immunohistochemical scores for NeuN and β-3 tubulin than LGOG. The Ki-67 labeling index was high in PDGFR-α and NeuN-immunolabeled tumor cells, and low in β-3 tubulin- and synaptophysin-immunolabeled cells. A HGOG cell line possessed the same immunohistochemical characteristics as HGOG. In this study, glioma cells with the OPC and IPC immunophenotypes had a higher Ki-67 labeling index, indicating their high proliferative activity. Furthermore, high-grade gliomas showed the characteristics of nIPC and neurons, which may suggest the pluripotent NSC lineage nature of these tumors.

Double dose of 5-aminolevulinic acid and its effect on protoporphyrin IX accumulation in low-grade glioma.

Administration of 5-aminolevulinic acid (5-ALA) does not regularly elicit fluorescence in low-grade glioma (LGG) at currently established doses and timing of administration. One explanation may be differences in blood-brain barrier (BBB) integrity compared to high-grade glioma. The authors hypothesized that for a BBB semipermeable to 5-ALA there might be a relationship between plasma 5-ALA concentration and its movement into the brain. A higher dose would elicit more 5-ALA conversion into protoporphyrin IX (PPIX). The authors present a case series of patients harboring LGG who received higher doses of 5-ALA. Patients undergoing surgery for indeterminate glioma later diagnosed as LGG were included in this study. 5-ALA was administered at a standard dose of 20 mg/kg body weight (bw) 4 hours prior to induction of anesthesia. A subgroup of patients received a higher dose of 40 mg/kg bw. Fluorescence was evaluated visually and PPIX concentration (cPPIX) was determined ex vivo by hyperspectral measurements in freshly extracted tissue. All adverse events were recorded. A total of 23 patients harboring diffuse low-grade astrocytomas (n = 19) and oligodendrogliomas (n = 4) were analyzed. Thirteen patients received 20 mg/kg bw, and 10 patients received 40 mg/kg bw of 5-ALA. In the 20 mg/kg group, 30.8% (4 of 13) of tumors harbored areas of visible fluorescence, compared to 60% of cases (n = 6 of 10) with 40 mg/kg bw. The threshold to visibility was 1 μg/ml in both groups. Measured over all biopsies, the mean cPPIX was significantly higher in the double-dose group (1.8 vs 0.45 μg/ml; p < 0.001). In non-visibly fluorescent tissue the mean cPPIX was 0.146 μg/ml in the 20 mg/kg and 0.347 μg/ml in the 40 mg/kg group, indicating an increase of 138% (p < 0.001). These observations demonstrate different regions with different levels of PPIX accumulation in LGG. With higher 5-ALA doses cPPIX increases, leading to more regions surpassing the visibility threshold of 1 μg/ml. These observations can be explained by the fact that the BBB in LGG is semipermeable to 5-ALA. Higher 5-ALA doses result in more PPIX conversion, an observation with implications for future dosing in LGG.

INSP-13. Malignant networks in brain tumors: from basic discoveries to clinical implications

Abstract Integration into communicating tumor cell networks is a way how the entire brain tumor organism can progress in the brain, and resist therapies. It started with our discovery of ultra-long membrane protrusions, tumor microtube (TM), in tumor cells from glioblastomas and other incurable adult and pediatric glioma entities (Osswald et al., Nature 2015). These TMs are used for early brain invasion, and later interconnect single tumor cells to communicating and resistant multicellular networks, but also glioma cells to neurons in the brain, with neuron-glioma synapses stimulating glioma progression (Venkataramani et al., Nature 2019). Increasing formation of these homotypic and heterotypic networks is seen with increasing glioma malignancy: lowest in low-grade oligodendrogliomas, and highest in glioblastomas and K27M mutated gliomas. The genetic reduction of GAP-43, a crucial neurodevelopmental factor that drives TM formation and function, not only deprived glioblastoma cells from the ability to form functional tumor networks, but also made radiotherapy much more effective, by and large eradicating experimental tumors from the mouse brain, which is typically not possible with radiotherapy alone. Consistent with the concept of tumor cell resilience by network integration, radiotherapy and also chemotherapy (Weil et al., Neuro-Oncology 2017) preferentially killed the unconnected glioblastoma cells, leaving the tumor cell networks largely intact. Damage to the tumor network by surgical measures induced a self-healing response. Taken together, integration into multicellular, highly functional, communicating and resilient networks is not only a feature of the nervous system, but also of incurable, most aggressive brain tumors. In this talk, clinical concepts that originate from these discoveries are presented, including a network disconnection strategy currently tested in the German MecMeth trial (EudraCT2021-000708-39), and the AMPAR inhibitor perampanel as a potential inhibitor of neuron-glioma synapses. Furthermore, new basic and translational developments in the field of brain tumor networks are presented and discussed.

Distinct metabolic hallmarks of WHO classified adult glioma subtypes.

BackgroundGliomas are complex tumors with several genetic aberrations and diverse metabolic programs contributing to their aggressive phenotypes and poor prognoses. This study defines key metabolic features that can be used to differentiate between glioma subtypes, with potential for improved diagnostics and subtype targeted therapy.MethodsCross-platform global metabolomic profiling coupled with clinical, genetic, and pathological analysis of glioma tissue from 224 tumors—oligodendroglioma (n = 31), astrocytoma (n = 31) and glioblastoma (n = 162)—were performed. Identified metabolic phenotypes were evaluated in accordance with the WHO classification, IDH-mutation, 1p/19q-codeletion, WHO-grading 2–4, and MGMT promoter methylation.ResultsDistinct metabolic phenotypes separate all six analyzed glioma subtypes. IDH-mutated subtypes, expressing 2-hydroxyglutaric acid, were clearly distinguished from IDH-wildtype subtypes. Considerable metabolic heterogeneity outside of the mutated IDH pathway were also evident, with key metabolites being high expression of glycerophosphates, inositols, monosaccharides, and sugar alcohols and low levels of sphingosine and lysoglycerophospholipids in IDH-mutants. Among the IDH-mutated subtypes, we observed high levels of amino acids, especially glycine and 2-aminoadipic acid, in grade 4 glioma, and N-acetyl aspartic acid in low-grade astrocytoma and oligodendroglioma. Both IDH-wildtype and mutated oligodendroglioma and glioblastoma were characterized by high levels of acylcarnitines, likely driven by rapid cell growth and hypoxic features. We found elevated levels of 5-HIAA in gliosarcoma and a subtype of oligodendroglioma not yet defined as a specific entity, indicating a previously not described role for the serotonin pathway linked to glioma with bimorphic tissue.ConclusionKey metabolic differences exist across adult glioma subtypes.

NCOG-40. AWAKE CRANIOTOMY FOR EXCISION OF BRAIN TUMORS; INITIAL 200 CONSECUTIVE CASES FROM AN LMIC

Abstract In recent years, reports from developed countries have shown that awake craniotomy has been shown to improve outcomes of surgical resection of brain tumors. However, no such data is available from low- and middle-income countries. We retrospectively reviewed 200 cases of awake craniotomy performed at our center for excision of brain tumors during last 5 years, and assessed clinical outcomes. Data was collected from patients’ medical records, and included demographics, tumor location/histology, clinical complains, and functional status. We used Karnofsky performance scale (KPS) to assess function. Extent of resection was determined on post-operative MRI. Statistical analysis was done using SPSS version 22. Seven attending surgeons performed these cases; however, 168 (84%) surgeries were performed by a single surgeon who is the senior author (SA Enam). Mean age was 39.3 ± 11.9 years and 79% (158) were male. Left frontal lobe was the most common location for tumors (50; 25%). Although 52% (104) patients had malignant neoplasms, seizures were the most common presenting symptom in 63% (126) cases followed by motor deficits in 29% (58). The most common tumors were low grade oligodendroglioma (58; 29%%) followed by glioblastoma (42; 21%). Mean length of hospital stay was 3.15 days ± 1.7 days. Gross total resection was achieved in 82 (41%) patients. New intraoperative neurological complains were seen in 31 (15.5%) patients, however, 22 (11%) of these had recovered by median follow-up of 1.4 months. KPS at last follow-up improved in 92 (46%), remained stable in 94 (47%) and deteriorated in 14 (7%) patients. Although absence of a control group decreases the strength of this, with our large sample size we can safely conclude that AC allows maximum safe excision of brain tumors, and offers a good chance of preserving patients’ functional status, along with adequate extent of resection.

NIMG-51. DEUTERIUM METABOLIC IMAGING OF BRAIN TUMOR IMMORTALITY USING 2H-PYRUVATE

Abstract Telomere maintenance is essential for tumor immortality and sustained tumor proliferation. Most tumors, including high-grade glioblastomas and low-grade oligodendrogliomas achieve telomere maintenance via reactivation of the expression of telomerase reverse transcriptase (TERT), which is silenced in normal somatic cells. Due to this essential role, TERT is a therapeutic target and TERT inhibitors such as 6-thio-2’-deoxyguanosine are in clinical trials. Non-invasive methods of imaging TERT, therefore, have the potential to provide a readout of tumor proliferation and response to therapy. We previously showed that TERT expression is associated with elevated levels of NADH in gliomas. Since NADH is essential for the conversion of pyruvate to lactate, measuring pyruvate flux to lactate could be useful for imaging TERT expression. In this context, deuterium magnetic resonance spectroscopy (2H-MRS) recently emerged as a novel, clinically translatable method of monitoring metabolic fluxes. The goal of this study was to assess the potential of [U-2H]pyruvate for non-invasive imaging of TERT status in gliomas. Following intravenous injection of [U-2H]pyruvate, lactate production was significantly higher in mice bearing orthotopic oligodendroglioma (SF10417, BT88) or glioblastoma (GBM1, GBM6) tumors relative to tumor-free controls. 2D chemical shift imaging showed localization of lactate production to tumor vs. contralateral normal brain. Importantly, following treatment of mice bearing orthotopic GBM6 or BT88 tumors with the TERT inhibitor 6-thio-2’-deoxyguanosine, lactate production from [U-2H]pyruvate was significantly reduced at early timepoints when alterations in tumor volume could not be detected by anatomical imaging, pointing to the ability of [U-2H]pyruvate to report on pseudoprogression. Collectively, we have, for the first time, demonstrated the utility of [U-2H]pyruvate for metabolic imaging of brain tumor burden and treatment response in vivo. Importantly, since 2H-MRS can be implemented on clinical scanners, our results provide a novel, non-invasive method of integrating information regarding a fundamental tumor hallmark, i.e. TERT, into glioma patient management.

Metabolic imaging detects elevated glucose flux through the pentose phosphate pathway associated with TERT expression in low-grade gliomas.

Telomerase reverse transcriptase (TERT) is essential for tumor proliferation, including in low-grade oligodendrogliomas (LGOGs). Since TERT is silenced in normal cells, it is also a therapeutic target. Therefore, noninvasive methods of imaging TERT are needed. Here, we examined the link between TERT expression and metabolism in LGOGs, with the goal of leveraging this information for noninvasive magnetic resonance spectroscopy (MRS)-based metabolic imaging of LGOGs. Immortalized normal human astrocytes with doxycycline-inducible TERT silencing, patient-derived LGOG cells, orthotopic tumors, and LGOG patient biopsies were studied to determine the mechanistic link between TERT expression and glucose metabolism. The ability of hyperpolarized [U-13C, U-2H]-glucose to noninvasively assess TERT expression was tested in live cells and orthotopic tumors. TERT expression was associated with elevated glucose flux through the pentose phosphate pathway (PPP), elevated NADPH, which is a major product of the PPP, and elevated glutathione, which is maintained in a reduced state by NADPH. Importantly, hyperpolarized [U-13C, U-2H]-glucose metabolism via the PPP noninvasively reported on TERT expression and response to TERT inhibition in patient-derived LGOG cells and orthotopic tumors. Mechanistically, TERT acted via the sirtuin SIRT2 to upregulate the glucose transporter GLUT1 and the rate-limiting PPP enzyme glucose-6-phosphate dehydrogenase. We have, for the first time, leveraged a mechanistic understanding of TERT-associated metabolic reprogramming for noninvasive imaging of LGOGs using hyperpolarized [U-13C, U-2H]-glucose. Our findings provide a novel way of imaging a hallmark of tumor immortality and have the potential to improve diagnosis and treatment response assessment for LGOG patients.

BIMG-02. IMAGING IMMORTALITY: TERT EXPRESSION ALTERS GLUCOSE METABOLISM IN LOW-GRADE GLIOMAS IN A MANNER THAT CAN BE LEVERAGED FOR NONINVASIVE METABOLIC IMAGING

Telomerase reverse transcriptase (TERT) is essential for tumor immortality and uncontrolled proliferation, including in low-grade oligodendrogliomas (LGOGs). Since it is silenced in somatic cells, TERT is also a therapeutic target. Non-invasive imaging of TERT can differentiate tumor from normal brain or lesions such as gliosis and allow assessment of response to therapy. The goal of this study was to identify magnetic resonance spectroscopy (MRS)-detectable metabolic alterations associated with TERT that can be leveraged for noninvasive imaging in LGOGs. We examined patient-derived BT54 neurospheres in which TERT expression was silenced by RNA interference. 1H-MRS showed that steady-state levels of NAD(P)/H, glutathione, aspartate and AXP were elevated in BT54TERT+ neurospheres relative to BT54TERT-. Glucose flux through the pentose phosphate pathway (PPP) is essential for generating NADPH, which maintains glutathione homeostasis. 13C-MRS confirmed that [2-13C]-glucose flux through the PPP was elevated in BT54TERT+ neurospheres relative to BT54TERT-, an effect associated with higher activity of the PPP enzyme glucose-6-phosphate dehydrogenase (G6PDH). Hyperpolarized 13C-MRS is a method of increasing the signal to noise ratio of 13C-MRS such that it can monitor metabolic fluxes noninvasively in cells, animals and patients. Consistent with elevated PPP flux and G6PDH activity, hyperpolarized [U-13C]-glucose metabolism via the PPP to 6-phosphogluconate (6-PG) was elevated in BT54TERT+ neurospheres relative to BT54TERT-. Importantly, examination of an additional patient-derived LGOG model, the SF10417 model which readily forms orthotopic tumor xenografts in rats, showed that 6-PG production from hyperpolarized [U-13C]-glucose demarcated tumor from normal brain. Furthermore, LGOG patient biopsies had elevated NAD(P)/H, glutathione, aspartate, AXP and G6PDH activity relative to gliosis biopsies, confirming the clinical validity of our observations. Collectively, we have identified a metabolic signature of TERT expression that can be leveraged via hyperpolarized [U-13C]-glucose to improve diagnosis and treatment response monitoring for LGOG patients.

BIMG-08. DEUTERIUM MAGNETIC RESONANCE SPECTROSCOPY USING 2H-PYRUVATE ALLOWS NON-INVASIVE IN VIVO IMAGING OF TERT EXPRESSION IN BRAIN TUMORS

Telomere shortening constitutes a natural barrier to uncontrolled proliferation and all tumors must find a mechanism of maintaining telomere length. Most human tumors, including high-grade primary glioblastomas (GBMs) and low-grade oligodendrogliomas (LGOGs) achieve telomere maintenance via reactivation of the expression of telomerase reverse transcriptase (TERT), which is silenced in normal somatic cells. TERT expression is, therefore, a driver of tumor proliferation and, due to this essential role, TERT is also a therapeutic target. However, non-invasive methods of imaging TERT are lacking. The goal of this study was to identify magnetic resonance spectroscopy (MRS)-detectable metabolic biomarkers of TERT expression that will enable non-invasive visualization of tumor burden in LGOGs and GBMs. First, we silenced TERT expression by RNA interference in patient-derived LGOG (SF10417, BT88) and GBM (GS2) models. Our results linked TERT silencing to significant reductions in steady-state levels of NADH in all models. NADH is essential for the conversion of pyruvate to lactate, suggesting that measuring pyruvate flux to lactate could be useful for imaging TERT status. Recently, deuterium (2H)-MRS has emerged as a novel, clinically translatable method of monitoring metabolic fluxes in vivo. However, to date, studies have solely examined 2H-glucose and the use of [U-2H]pyruvate for non-invasive 2H-MRS has not been tested. Following intravenous injection of a bolus of [U-2H]pyruvate, lactate production was higher in mice bearing orthotopic LGOG (BT88 and SF10417) and GBM (GS2) tumor xenografts relative to tumor-free mice, suggesting that [U-2H]pyruvate has the potential to monitor TERT expression in vivo. In summary, our study, for the first time, shows the feasibility and utility of [U-2H]pyruvate for in vivo imaging. Importantly, since 2H-MRS can be implemented on clinical scanners, our results provide a novel, non-invasive method of integrating information regarding a fundamental cancer hallmark, i.e. TERT, into glioma patient management.

TBMT-01. HYPERPOLARIZED δ-[1-13C]GLUCONOLACTONE MONITORS TERT-INDUCED ELEVATION IN PENTOSE PHOSPHATE PATHWAY FLUX IN BRAIN TUMORS IN VIVO

Telomerase reverse transcriptase (TERT) expression is essential for tumor proliferation and is an attractive therapeutic target for gliomas. TERT expression has previously been shown to enhance glucose flux via the pentose phosphate pathway (PPP) in low grade gliomas expressing TERT. Hyperpolarized δ-[1-13C]gluconolactone has been used to detect flux via the PPP by monitoring its conversion to 6-phospho-[1-13C]gluconate (6PG) in isolated perfused liver. The goal of our study was to evaluate whether hyperpolarized δ-[1-13C]gluconolactone can be used to monitor elevated PPP flux induced by TERT expression in low grade gliomas, thereby providing a non-invasive method of assessing TERT expression in vivo. Immortalized normal human astrocytes without (NHApre) and with TERT expression (NHApost) were used in cell bioreactor experiments. In vivo experiment with rats bearing orthotopic NHApost or patient-derived low-grade oligodendroglioma (SF10417) tumors were contacted. Dynamic 13C MR spectra were acquired at 14T (cells) or 3T (rats) following injection of hyperpolarized δ-[1-13C]gluconolactone. NHApost cells showed significantly higher flux through the PPP compared to NHApre. This finding was in agreement with previous results indicating that TERT expression elevates PPP flux. In all rats δ-[1-13C]gluconolactone and 6PG were observed indicating that δ-[1-13C]gluconolactone crosses the blood-brain barrier and is rapidly metabolized. Furthermore, both models presented homogeneous distribution of δ-[1-13C]gluconolactone in the brain and higher ratio of 6PG-to-δ-[1-13C]gluconolactone in the tumor area. In summary we show in vivo that hyperpolarized δ-[1-13C]gluconolactone metabolism to 6-phospho-[1-13C]gluconate is significantly higher in tumor compared to contralateral normal brain in TERT-expressing genetically-engineered and patient-derived low-grade oligodendrogliomas. Due to its fundamental role in tumor proliferation, TERT is both a tumor biomarker and a therapeutic target. Monitoring HP δ-[1-13C]gluconolactone metabolism, therefore, has the potential to inform on tumor burden and response to therapy in the clinic.

Non-invasive assessment of telomere maintenance mechanisms in brain tumors

Telomere maintenance is a universal hallmark of cancer. Most tumors including low-grade oligodendrogliomas use telomerase reverse transcriptase (TERT) expression for telomere maintenance while astrocytomas use the alternative lengthening of telomeres (ALT) pathway. Although TERT and ALT are hallmarks of tumor proliferation and attractive therapeutic targets, translational methods of imaging TERT and ALT are lacking. Here we show that TERT and ALT are associated with unique 1H-magnetic resonance spectroscopy (MRS)-detectable metabolic signatures in genetically-engineered and patient-derived glioma models and patient biopsies. Importantly, we have leveraged this information to mechanistically validate hyperpolarized [1-13C]-alanine flux to pyruvate as an imaging biomarker of ALT status and hyperpolarized [1-13C]-alanine flux to lactate as an imaging biomarker of TERT status in low-grade gliomas. Collectively, we have identified metabolic biomarkers of TERT and ALT status that provide a way of integrating critical oncogenic information into non-invasive imaging modalities that can improve tumor diagnosis and treatment response monitoring.