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.

NI-6 Preoperative differential diagnosis of grade II and grade III in cases with astrocytoma, IDH mutant

Purpose: We attempted to differentiate between IDH-mutant astrocytoma Grade II and grade III by using methionine (MET) positron emission tomography (PET) and magnetic resonance spectroscopy (MRS). Subjects and Methods: We retrospectively analyzed 41 adult supratentorial glioma cases with confirmed histological diagnosis and IDH status from June 2015 to June 2020. These included 21 males, with an average age of 38.5 years (19–59 years), including seven astrocytoma grade II (A-II) and 34 grade III (A-III) cases. We determined the accumulation value rate of the maximum tumor to normal cortex accumulation value (T/N ratio) in MET-PET. We obtained the peak ratios of N-acetyl aspartate (NAA)/ creatine (Cr), choline (Cho)/Cr, and Cho/NAA. We investigated the correlation between the T/N ratios and MRS parameters and examined the contrast effects on MRI. Results: There were no significant differences in the T/N ratio and MRS parameters between A-IIs and A-IIIs. Only Cho/NAA ratios were significantly correlated with the T/N ratios (r = 0.443, P = 0.0037). We divided the distribution map into four areas with the highest T/N ratio of AII (1.59) and the highest Cho/NAA ratio (3.66). That is, 1) T/N ratio ≤ 1.59 & Cho/NAA ≤ 3.66, 2) &gt;1.59 & ≤ 3.66, 3) ≤1.59 & &gt; 3.66, 4) &gt 1.59 & &gt 3.66. The diagnostic rates for A-III were 1) 61.1% (11/18), 2) 100% (7/7), 3) 100% (9/9), and 4) 100% (7/7). We found the contrast effects in only 7 cases (20.6%) of A-III, which were distributed in areas 2) to 4). Conclusion: A-IIs and A-IIIs distributed in area 1) were difficult to distinguish, and they need careful observation as a step before the transition to areas 2)-4). Meanwhile, A-IIIs reaching widespread distribution to areas 2)-4) because of their wide range of malignancies require clinically aggressive treatment. The method might be beneficial in grade analysis of IDH-mutant astrocytomas.

Plasma-Derived Extracellular Vesicles Reveal Galectin-3 Binding Protein as Potential Biomarker for Early Detection of Glioma

Gliomas are the most common type of the malignant brain tumor, which arise from glial cells. They make up about 40% of all primary brain tumors and around 70% of all primary malignant brain tumors. They can occur anywhere in the central nervous system (CNS) and have a poor prognosis. The average survival of glioma patients is approximately 6–15 months with poor aspects of life. In this edge, identification of proteins secreted by cancer cells is of special interest because it may provide a better understanding of tumor progression and provide early diagnosis of the diseases. Extracellular vesicles (EVs) were isolated from pooled plasma of healthy controls (n=03) and patients with different grades of glioma (Grade I or II or III, n=03 each). Nanoparticle tracking analysis, western blot, and flow cytometry were performed to determine the size, morphology, the concentration of glioma-derived vesicles and EV marker, CD63. Further, iTRAQ-based LC-MS/MS analysis of EV protein was performed to determine the differential protein abundance in extracellular vesicles across different glioma grades. We further verified galectin-3 binding protein (LGALS3BP) by ELISA in individual blood plasma and plasma-derived vesicles from control and glioma patients (n=40 each). Analysis by Max Quant identified 123 proteins from the pooled patient exosomes, out of which 34, 21, and 14 proteins were found to be differentially abundant by more than 1.3-fold in the different grades of glioma grade I, pilocytic astrocytoma; grade II, diffuse astrocytoma; grade III, anaplastic astrocytoma, respectively, in comparison with the control samples. A total of seven proteins—namely, CRP, SAA2, SERPINA3, SAA1, C4A, LV211, and KV112—showed differential abundance in all the three grades. LGALS3BP was seen to be upregulated across the different grades, and ELISA analysis from individual blood plasma and plasma-derived extracellular vesicles confirmed the increased expression of LGALS3BP in glioma patients (p&lt;0.001). The present study provides LGALS3BP as a potential biomarker for early detection of glioma and improve survival outcome of the patient. The present study further provides the information of progression and monitoring the tumor grades (grade 1, grade II, grade III).

Hypermethylation of the Promoter of miR-338-5p Mediates Aberrant Expression of ETS-1 and Is Correlated With Disease Severity Of Astrocytoma Patients

The pro-oncogene ETS-1 (E26 transformation-specific sequence 1) is a key regulator of the proliferation and invasion of cancer cells. The present work examined the correlation of the aberrant expression of ETS-1 with histological or clinical classification of astrocytoma: grade I (pilocytic astrocytoma), grade II (diffuse astrocytoma), grade III (anaplastic astrocytoma), and grade IV (glioblastoma multiforme). MicroRNA, miR-338-5p, was predicted by an online tool (miRDB) to potentially target the 3’ untranslated region of ETS-1; this was confirmed by multi-assays, including western blot experiments or the point mutation of the targeting sites of miR-338-5p in ETS-1’s 3’untralation region (3’UTR). The expression of miR-338-5p was negatively associated with that of ETS-1 in astrocytoma, and deficiency of miR-338-5p would mediate aberrant expression of ETS-1 in astrocytoma. Mechanistically, hypermethylation of miR-338-5p by DNA methyltransferase 1 (DNMT1) resulted in repression of miR-338-5p expression and the aberrant expression of ETS-1. Knockdown or deactivation of DNMT1 decreased the methylation rate of the miR-338-5p promoter, increased the expression of miR-338-5p, and repressed the expression of ETS-1 in astrocytoma cell lines U251 and U87. These results indicate that hypermethylation of the miR-338-5p promoter by DNMT1 mediates the aberrant expression of ETS-1 related to disease severity of patients with astrocytoma.

P06.01 Pediatric non-pontine diffuse midline glioma H3K27M mutant: a monoinstitutional experience

BACKGROUND The new entity “diffuse-midline-glioma H3K27M-mutant”(DMG) was defined in the 2016 WHO classification. This tumor subtype, regardless of histological features, is associated with an aggressive clinical behavior and poor prognosis, sharing the same outcome as DIPG. MATERIAL AND METHODS We report our experience from 2016 to 2018 in treating patients with radiotherapy and concomitant Nimotuzumab/Vinorelbine, as already published for DIPG’s patients (DOI10.1007/s11060-014-1428-z). All patients’ parents provided written informed consent for treatment. RESULTS Patients were 9: 7/9 females, median age at diagnosis 13 years (range 1–26); 8 with localized disease, 1 with spinal fluid (CSF) dissemination. Five patients had thalamic disease (1 bithalamic), 2 ponto-cerebellar (one with 2 lesions), 1 pineal gland and one with disseminated CSF disease at diagnosis. 3/9 were biopsed in thalamus, the others had partial resection. Central neuropathological review with confirmed histopathological diagnosis of DMG mutated was necessary for inclusion. At the first pathological evaluation tumors had been classified according to the fourth edition of the WHO 2007 as glioblastoma (n=4), anaplastic astrocytoma grade III (n=1), diffuse astrocytoma grade II (n=1), and 1 glial tumor with oligodendroglial features; 2 patients already had DMG at the first pathological evaluation. Immunohistochemical analysis revealed p53 mutation in 3 patients. All 9 patients were evaluated on serial plasma samples with NGS targeted panel (Pan-Cancer) and 6/9 presented p53 mutation in at least one of the samples. 8/9 expressed MAP2K1 gain of function at liquid biopsy. The small number of patients did not allow correlations with the prognosis. Two patients completed the scheduled 2 years of treatment: one had a local relapse at 37 months after diagnosis, the other is alive without evidence of progression at 30 months. With a median follow up of 14.4 months, PFS and OS were 33% and 77% at 1 year; OS was 22% at 2 years, with 2 patients long survivors at 36 and 40 months after diagnosis. CONCLUSION The molecular and phenotypic pattern of DMG allows to include patients in clinical trials/registry shared with patients suffering from DIPG. Our knowledge is still limited about this entity; new insights into molecular profile should help us to study new drugs directed against the effects of the H3K27M mutations and to define new diagnostic/prognostic approach as liquid biopsy able to correlate treatments and prognosis

Immunohistochemical Expression of Epidermal Growth Factor Receptor in Astrocytic Tumors in Iraqi Patients

BACKGROUND: Diffuse astrocytomas constitute the largest group of primary malignant human intracranial tumours. They are classified by the World Health Organization (WHO) into three histological malignancy grades: diffuse astrocytomas (grade II), anaplastic astrocytomas (grade III) and glioblastoma (grade IV) based on histopathological features such as cellular atypia, mitotic activity, necrosis and microvascular proliferation. Epidermal growth factor receptor (EGFR) is a 170-kDa transmembrane tyrosine kinase receptor expressed in a variety of normal and malignant cells regulating critical cellular processes. When activated, epidermal growth factor receptor (EGFR) triggers several signalling cascades leading to increased proliferation and angiogenesis and decreased apoptosis and hence associated with aggressive progression of the tumour. Epidermal growth factor receptor (EGFR) level is known to be a strong indicator associated with the aggressive behaviour of the tumour and acts as a prognostic factor for evaluating the survival rate. AIM: To evaluate the expression of epidermal growth factor receptor (EGFR) in different grades of astrocytoma. MATERIAL AND METHODS: formalin-fixed paraffin-embedded astrocytic tumours of 44 patients were collected from the archival material of pathology department of Ghazi Al Hariri Teaching Hospital during the period from June to December 2018. Hematoxylin and eosin-stained sections were used to characterise the tumours histologically based on cellularity, nuclear hyperchromasia, polymorphism, mitotic activity, vascular proliferation and necrosis with or without pseudopallisading of tumour cells. Diagnosis and grading of astrocytic tumours in this study were made according to WHO criteria (2016). Using a monoclonal antibody to the epidermal growth factor receptor (EGFR) and immunohistochemical analysis, the expression and distribution of epidermal growth factor receptor in astrocytic tumours were examined. RESULTS: The study included 1 case pilocytic astrocytoma (grade I), 20 cases diffuse astrocytoma (grade II), 5 cases anaplastic astrocytoma (grade III) and 18 cases of glioblastoma (grade IV). Expression of EGFR was found in 38.88% of the glioblastoma samples (grade IV). However, none of the astrocytomas of WHO grades I, II and III showed immunoreactivity for EGFR protein. Different patterns of immunoreactive cells and significant intratumor heterogeneity of EGFR expression were observed in glioblastomas. CONCLUSION: The immunohistochemical expression of Epidermal growth factor receptor (EGFR) was restricted only to high-grade astrocytic tumours, namely glioblastoma, thus may use to predict glioblastoma.