Local detection of microvessels in IDH-wildtype glioblastoma using relative cerebral blood volume: an imaging marker useful for astrocytoma grade 4 classification

Background The microvessels area (MVA), derived from microvascular proliferation, is a biomarker useful for high-grade glioma classification. Nevertheless, its measurement is costly, labor-intense, and invasive. Finding radiologic correlations with MVA could provide a complementary non-invasive approach without an extra cost and labor intensity and from the first stage. This study aims to correlate imaging markers, such as relative cerebral blood volume (rCBV), and local MVA in IDH-wildtype glioblastoma, and to propose this imaging marker as useful for astrocytoma grade 4 classification. Methods Data from 73 tissue blocks belonging to 17 IDH-wildtype glioblastomas and 7 blocks from 2 IDH-mutant astrocytomas were compiled from the Ivy GAP database. MRI processing and rCBV quantification were carried out using ONCOhabitats methodology. Histologic and MRI co-registration was done manually with experts’ supervision, achieving an accuracy of 88.8% of overlay. Spearman’s correlation was used to analyze the association between rCBV and microvessel area. Mann-Whitney test was used to study differences of rCBV between blocks with presence or absence of microvessels in IDH-wildtype glioblastoma, as well as to find differences with IDH-mutant astrocytoma samples. Results Significant positive correlations were found between rCBV and microvessel area in the IDH-wildtype blocks (p < 0.001), as well as significant differences in rCBV were found between blocks with microvascular proliferation and blocks without it (p < 0.0001). In addition, significant differences in rCBV were found between IDH-wildtype glioblastoma and IDH-mutant astrocytoma samples, being 2–2.5 times higher rCBV values in IDH-wildtype glioblastoma samples. Conclusions The proposed rCBV marker, calculated from diagnostic MRIs, can detect in IDH-wildtype glioblastoma those regions with microvessels from those without it, and it is significantly correlated with local microvessels area. In addition, the proposed rCBV marker can differentiate the IDH mutation status, providing a complementary non-invasive method for high-grade glioma classification.

Microvascular proliferation in the clots: The key finding of acute subdural hematoma transforming into chronic subdural hematoma?

Background: Despite extensive investigations, the exact etiology of chronic subdural hematoma (CSDH) remains elusive. Organized CSDHs are a distinct but less-understood type of CSDH. Case Description: A 50-year-old hypertensive woman experienced headache without any previous head injury. At presentation, the patient showed no focal neurological deficits. Cranial computed tomography (CT) revealed a slightly compressive subdural hematoma that spontaneously regressed and no intracranial vascular lesions. Cerebral magnetic resonance imaging identified a non-enhancing nodular lesion in the subdural hematoma. After the patient presented disorientation and aphasia on post hospitalization day 14, CT showed a considerable enlargement of the subdural hematoma. Partial removal of the bi-layered hematoma was performed through a parietal craniotomy. Histological examination revealed microvascular proliferation in both the outer membrane and the nodular lesion. On postoperative day 35, CT demonstrated a remarkable resolution of the residual hematoma. Conclusion: Development of microvascular proliferation in the clots of an acute subdural hematoma may lead to its rapid enlargement as an organized CSDH. Organized CSDH can be managed by partial removal of the outer membrane and hematoma through a craniotomy.

MPC-5 Characteristics of H3 G34-mutant gliomas

Introduction: Diffuse hemispheric gliomas, H3 G34-mutant (DHG H3G34-mutant) are newly recognized infiltrating gliomas of the cerebral hemispheres of pediatric and young adult patients. We experienced 6 DHG H3G34-mutant cases. In this study, we describe the clinical, radiological and pathological characteristics of these cases. Result: Mean age at diagnosis was 16.8 years (range:10–26). Three patients were male. Among six cases, tumors located in cerebral cortex in five cases and multiple sites including basal ganglia and cortex in a case. All tumors showed no or only a faint contrast-enhancement and harbored restriction of diffusion. One patient underwent total resection, four underwent partial resection and one underwent biopsy. Pathological diagnosis were CNS embryonal tumors (n=3/6), glioblastoma, IDH-wildtype (n=2/6) and anaplastic astrocytoma, IDH-wildtype (n=1/5). All cases were negative for Olig2 and positive for GFAP in immunohistochemistry. Mean Ki-67 index was 38% (range: 10–60%). All cases revealed at least one of mitosis, necrosis or microvascular proliferation. Especially, mitosis was the most frequently found (n=5/6). The H3F3A mutations were G34R mutations in all cases. One case revealed a characteristic mutation pattern, therefore now we are performing further examination. Adjuvant chemoradiotherapies were performed for all cases. Mean progression free survival was 10.1 months (range: 1.6–33.1). Discussion: As published literatures reported, all cases exhibited restriction of diffusion and negative for Olig2. For a cerebral hemispheric tumor of pediatric or young adult patient which shows restriction of diffusion and no contrast-enhancement, and of which pathological findings is malignant and olig2 is negative, genetic analysis of H3F3A gene might be essential.

CS-5 A case of anaplastic pleomorphic xanthoastrocytoma in the pineal region

Tumors in the pineal gland are rare tumors that account for about 0.3% of all brain tumors and have various histological types of tumors develop with germinoma, pineocytoma, and pineoblastoma in that order. On the other hand, pleomorphic xanthoastrocytoma (PXA) is a rare tumor of less than 0.2% and frequently occurs in supratentorial cerebral surface of children and young adults.A case was a 61-year-old man whose pineal tumor was found due to visual disturbance. MRI showed a 23 mm-sized lesion with cysts and inhomogeneous enhancement in the pineal gland. Partial calcification was observed, but there was no non-communicating hydrocephalus, and no increase in HCG-β and AFP with blood sampling. A midline suboccipital craniotomy was performed in the sitting position, a head-up surgery was performed using a 4K / 3D video microscope system (ORBEYE exoscope, Olympus) by the infratentorial supracerebellar approach (ITSCA). The bridging veins and precentral cerebellar vein were dissected to expose the posterior surface of the tumor, and internal decompression was performed. For the complication of air embolism, artificial cerebrospinal fluid was sprayed, and the bilateral internal jugular veins were compressed to confirm the inflow point. There was strong adhesion to the Rt vein of Rosenthal, and the site was removed intracapsularly. Finally, subtotal resection was performed with remaining the upper part of the tumor, a blind lesion behind the Vein of Galen. Vertical gaze palsy occurred after this operation, but it gradually improved over time. Tumor showed pathologically remarkable polymorphism, poor microvascular proliferation and necrosis, but mitotic figures 4–5 / 10HPF, MIB-1 index 10%, GFAP positive, no BRAF V600E mutation. There are few reports of PXA occurring in the pineal gland, and this case is the sixth case. It is also the first report for pineal tumors using ORBEYE through ITSCA in the sitting position.

Uncovering a Key Role of ETS1 on Vascular Abnormality in Glioblastoma

Glioblastoma (GBM) is the most aggressive type of brain tumor. Microvascular proliferation and abnormal vasculature are the hallmarks of the GBM, aggravating disease progression and increasing patient morbidity. Here, we uncovered a key role of ETS1 on vascular abnormality in glioblastoma. ETS1 was upregulated in endothelial cells from human tumors compared to endothelial cells from paired control brain tissue. Knockdown of Ets1 in mouse brain endothelial cells inhibited cell migration and proliferation, and suppressed expression of genes associated with vascular abnormality in GBM. ETS1 upregulation in tumor ECs was dependent on TGFβ signaling, and targeting TGFβ signaling by inhibitor decreased tumor angiogenesis and vascular abnormality in CT-2A glioma model. Our results identified ETS1 as a key factor regulating tumor angiogenesis, and suggested that TGFβ inhibition may suppress the vascular abnormality driven by ETS1.

TAMI-22. SEGMENTATION OF DISTINCT TUMOR HALLMARKS OF GLIOBLASTOMA ON DIGITAL HISTOPATHOLOGY USING A HIERARCHICAL DEEP LEARNING APPROACH

PURPOSE Glioblastoma is a highly heterogeneous brain tumor. Primary treatment for glioblastoma involves maximally-safe surgical resection. After surgery, resected tissue slides are visually analyzed by neuro-pathologists to identify distinct histological hallmarks characterizing glioblastoma including high cellularity, necrosis, and vascular proliferation. In this work, we present a hierarchical deep learning-based strategy to automatically segment distinct Glioblastoma niches including necrosis, cellular tumor, and hyperplastic blood vessels, on digitized histopathology slides. METHODS We employed the IvyGap cohort for which Hematoxylin and eosin (H&E) slides (digitized at 20X magnification) from n=41 glioblastoma patients were available. Additionally, expert-driven segmentations of cellular tumor, necrosis, and hyperplastic blood vessels (along with other histological attributes) were made available. We randomly employed n=120 slides from 29 patients for training, n=38 slides from 6 cases for validation, and n=30 slides from 6 patients to feed our deep learning model based on Residual Network architecture (ResNet-50). ~2,000 patches of 224x224 pixels were sampled for every slide. Our hierarchical model included first segmenting necrosis from non-necrotic (i.e. cellular tumor) regions, and then from the regions segmented as non-necrotic, identifying hyperplastic blood-vessels from the rest of the cellular tumor. RESULTS Our model achieved a training accuracy of 94%, and a testing accuracy of 88% with an area under the curve (AUC) of 92% in distinguishing necrosis from non-necrotic (i.e. cellular tumor) regions. Similarly, we obtained a training accuracy of 78%, and a testing accuracy of 87% (with an AUC of 94%) in identifying hyperplastic blood vessels from the rest of the cellular tumor. CONCLUSION We developed a reliable hierarchical segmentation model for automatic segmentation of necrotic, cellular tumor, and hyperplastic blood vessels on digitized H&E-stained Glioblastoma tissue images. Future work will involve extension of our model for segmentation of pseudopalisading patterns and microvascular proliferation.

PATH-25. H3 K27M-MUTANT DIFFUSE NON-MIDLINE GLIOMA: REPORT OF A CASE WITH EXTENDED FOLLOW-UP

H3 K27M-mutant diffuse gliomas without midline involvement are extremely rare and their clinical behavior remains elusive due to limited reported follow-up data. We describe an H3 K27M-mutant diffuse non-midline glioma patient with extended follow-up. A 34-year-old woman presented with headache, memory loss, periods of changes in taste and smell, and confusion. Imaging studies revealed an 2.3 cm expansile T2/flair hyperintensity with patchy postcontrast enhancement centered in the left amygdala without associated restricted diffusion and no involvement of the midline structures. The tumor was debulked and consisted of a mitotically active infiltrating astrocytic glioma without tumor necrosis or microvascular proliferation, consistent with anaplastic astrocytoma. Targeted 187-gene neuro-oncology NGS testing detected an H3F3A K27M mutation along with a PTPN11 and a PPM1D mutation. FISH 1p/19q-codeletion testing was negative. MGMT promoter was unmethylated. The patient was treated with chemoradiation with temozolomide for 6 weeks followed by 12 cycles of temozolomide. Four years after initial resection, an area of increased post-contrast enhancement indicating tumor recurrence/progression was noted. Partial resection of the recurrent tumor revealed a mitotically active infiltrating astrocytic glioma with tumor necrosis consistent with glioblastoma. Molecular profiling of the recurrent tumor by the neuro-oncology NGS panel detected similar mutational profile (identical H3F3A K27M and PTPN11 mutation; different PPM1D mutation) with an additional SOS1 mutation. The patient completed 4 cycles of Lomustine and although clinically stable, imaging studies showed slight increase in residual tumor size concerning for tumor progression. Lomustine was discontinued, Bevacizumab therapy was initiated and patient was enrolled in clinical trial (NCT02525692). Despite tumor progression, this patient has had a relatively long disease course (5 years) suggesting that H3 K27M-mutant non-midline diffuse gliomas although molecularly similar, may follow a more favorable clinical course than their midline counterparts possibly due to the hemispheric location, which is more amenable for surgical resection.

EXTH-63. A NOVEL MOUSE MODEL OF DIFFUSE MIDLINE GLIOMA FOR TARGETED IMMUNOTHERAPY

In children, diffuse midline gliomas retain poor outcomes, failing to have a durable response to conventional therapies. Immunotherapies hold promise, with the integration of the host's immune system fundamental to their design. Here, we describe a novel genetically engineered immunocompetent model that incorporates interleukin 13 receptor alpha 2 (IL13Rα2), a tumor-associated antigen, to evaluate the antitumor activity of IL13Rα2-CAR T cell and bispecific T cell engager (BiTE) therapies in preclinical studies. The RCAS-Tva delivery system was utilized to induce gliomagenesis through p53 loss and the constitutive expression of PDGFB and human IL13Rα2 in Nestin-Tva;p53fl/fl mice. Pups were injected with virus-producing DF1 cells, encoding either for RCAS-Cre and PDGFB+IL13Rα2 or RCAS-Cre and PDGFB. Kaplan-Meier survival curves established and compared tumor growth dynamics in both models. Tumor tissue was characterized through immunohistochemistry and H&E staining. Cell lines generated from tumor-bearing tissue were used for orthotopic injection and in vitro studies. Expression of PDGFB and IL13Rα2 was confirmed by flow cytometry and western blot. In both groups, de novo tumors developed without significant difference in median survival between RCAS:PDGFB (n=25, 40 days) and RCAS:PDGB+IL13Rα2 (n=32, 39 days). Tumors demonstrated characteristics of high-grade glioma such as infiltration, pseudopalisading necrosis, microvascular proliferation, high Ki-67 index, heterogenous IL13Rα2 expression, with notable presence of CD11b+ macrophages and low count of CD3+ T cells. Orthotopic tumors from developed cell lines were histologically similar to de novo tumors. Treatment of generated cell lines with IL13Rα2-targeting BiTE protein resulted in a loss of glioma cell viability and target-specific activation of T cells. Engineered de novo tumors possess histopathologic features common to diffused midline gliomas. IL13Rα2-positive cell lines derived from de novo tumors were responsive to targeted treatment, opening the opportunity for preclinical assessment of IL13Rα2-directed immunotherapies, with the potential for clinical translation.

CTNI-43. CXCL12 INHIBITION IN MGMT UNMETHYLATED GLIOBLASTOMA – RESULTS OF AN EARLY PROOF-OF-CONCEPT ASSESSMENT IN THE MULTICENTRIC PHASE I/II GLORIA TRIAL (NCT04121455)

BACKGROUND Preclinical studies showed that CXCL12-mediated influx of highly angiogenic monocytes/macrophages is a key driver of tumor re-vascularization and re-growth after radiotherapy (RT) of glioblastoma (GBM). We report findings from a phase I/II proof-of concept (PoC) study on CXCL12 inhibition during and after RT of GBM. METHODS Patients ≥18years with incompletely or unresected GBM without MGMT promoter hypermethylation and ECOG≤2 were eligible to participate. Patients received continuous (24/7) i.v. infusions of 200mg/week (n=3), 400mg/week (n=3) or 600mg/week (n=3) of the CXCL12 inhibitor olaptesed pegol (OLA) for 26 weeks during and after normo- or hypofractionated RT (60Gy/40.05Gy). The primary endpoint was safety as per the incidence of treatment-related adverse events. The study was accompanied by PoC-research including multiparametric MRI biomarkers (relative cerebral blood volume, rCBV; fractional tumor burden with high perfusion, FTBhigh; apparent diffusion coefficient, ADC) and of multiplexed immunofluorescence imaging (CODEX®) of reference and patient samples. Initial results of these analyses are reported for the first six patients enrolled. RESULTS Five of six (83%) patients assessed with advanced MRI showed response under OLA in rCBV/FTBhigh and ADC. Maximum reduction in perfusion (rCBV) from baseline was 55%, maximum reduction of FTBhigh was 55% and maximum increase in ADC was 77%. Furthermore, five of six (83%) patients analyzed showed reduction of enhancing tissue volumes in at least one scan under OLA therapy. In both one patient and two reference samples CXCL12 co-localized with endothelial cells of the microvascular proliferation zone. In a paired sample (before/during OLA) of one patient, endothelial cells stained positive for CXCL12 before but not during treatment and almost all GBM cells were negative in Ki67 staining in the sample obtained under OLA therapy. CONCLUSIONS Advanced MRI and multiplexed immunofluorescence suggest efficacy of combined radiotherapy and CXCL12 inhibition in unmethylated GBM. Funded by NOXXON Pharma AG; ClinicalTrials.gov number, NCT04121455.

Metachronous oligodendroglial tumours with different IDH mutational profile in a young patient

Aims Oligodendroglioma is molecularly defined by mutation of isocitrate dehydrogenase (IDH) and 1p19q codeletion. IDH mutation is an early driver of tumorigenesis, via its oncometabolite 2-hydroxyglutarate, regardless of the exact mutational subtype in homologues IDH1 or IDH2. IDH mutant cells then acquire 1p19q codeletion, with haploinsufficiency likely to contribute to oncogenesis by reduced expression of genes on 1p and 19q, as well as mutations in TERT, FUBP1 (on 1p31.1) in ~30% and CIC (on 19q13.2) in ~&gt;60% of 1p19q-codeleted gliomas. We present a case of a young patient with metachronous oligodendroglial tumours, initially thought to represent contralateral recurrence of the same disease. However, IDH mutation analysis in each tumour revealed distinct types of mutations, involving both IDH1 and IDH2, indicating different cellular lineages of tumorigenesis. We aim to present this unusual combination by illustrating the histology and molecular profile, and review the literature with regards to multifocal but molecularly distinct glioma. Method Case: The patient is a 33 year old man initially presenting with seizures, who was found to have a frontal lobe lesion (hence called tumour 1) with focal radiological enhancement, followed by a contralateral lesion in the parietal lobe 6 months later (hence designated as tumour 2). He underwent separate surgical debulking, and each time, tumour tissue was histologically and genetically examined. Testing included targeted mutation screening by immunohistochemistry and PCR based methods, pyrosequencing for MGMT methylation analysis, FISH for chromosomal LOH analysis of 1p and 19q, immunohistochemistry for mismatch repair enzymes and next generation sequencing. Results Histology of tumour 1 revealed a neoplasm with uniform cells, round nuclei and oligodendroglioma-like clear cell change, without mitoses, microvascular proliferation or necrosis. Immunohistochemistry showed absence of IDH1 R132H mutation, retained expression of ATRX and no altered p53 staining. The ki-67 index reached 6%. Sequencing of IDH1/2 mutations revealed a rare IDH2 mutation (non-/R172K). FISH confirmed codeletion of 1p19q, and the integrated diagnosis was oligodendroglioma, IDH mutant and 1p19q codeleted, WHO grade II. Histology of tumour 2 demonstrated oligodendroglioma morphology in areas, but more cellular and nuclear pleomorphism and focally brisk mitotic activity (7 mitoses in 10 hpf; ki67 index 20%), while both microvascular proliferation and necrosis were absent. Immunohistochemistry showed IDH1 R132H mutation and retained ATRX, while p53 was not expressed. FISH studies confirmed codeletion of 1p19q, and the integrated diagnosis was anaplastic oligodendroglioma, IDH mutant and 1p19q codeleted, WHO-2016 grade III. NGS data and MMR results are compared. Conclusion We present a patient with two histologically similar, but molecularly distinct oligodendroglial tumours affecting both cerebral hemispheres. Apart from the grade, the important difference is the presence of different IDH mutations, 1) a rare IDH2 mutation (non-R172K) and 2) the common IDH1 (R132H) mutation. While both types of IDH mutations identified are known to occur in oligodendroglioma, the difference clearly indicates two distinct lineages of tumorigenesis, especially as IDH mutation is considered an early event in gliomagenesis. IDH2 mutations are often associated with oligodendrogliomas, while IDH1 R132H is recognised to be frequent in both diffuse oligodendroglial and astroglial neoplasms. Multifocal divergent gliomas have been described previously but oligodendrogliomas with differing IDH mutations in the same patient have not knowingly been reported yet. Importantly, though therapeutically irrelevant here, multicentric gliomas do not automatically imply relatedness. However, a common origin or predisposition (here, even predating IDH mutation) may not be ruled out.