Intratumor morphologic and molecular genetic heterogeneity in astrocytomas of different grade of malignancy in the material from the first operation

Introduction. Intratumor heterogeneity is one of the key reasons for unfavourable prognosis in malignant tumors. Astrocytic tumors are known to develop therapy resistance inevitably during the course of disease. One of possible reason is tumor heterogeneity. Purpose. The aim of this work was to assess the intratumor morphologic and molecular heterogeneity in diffuse astrocytoma, anaplastic astrocytomas and primary glioblastomas. Material and methods. We conducted morphologic (n=22) and molecular-genetic (n=8) analysis of surgical specimens obtained from primarily operated glioblastoma giv (gb), anaplastic astrocytomas giii (aa) and diffuse astrocytoma gii (da) patients aged 18 years and older in whom total or subtotal tumor resection was performed. Tissue sampling for the analysis was performed from 5 equidistant areas of each tumor. Morphologic diagnosis was established according to who classification of central nervous system tumors (2007/2016). Mgmt, c-kit, top2a, pdgfr-α, ercc1, vegf genes mrnaexpression was assessed by rt-pcr. Idh1 and idh2 mutational status was evaluated by allele-specific pcr. Results. Morphologic heterogeneity was evident in 72,7 % tumors (16/22) overall. Heterogeneity was observed in 68,8 % (11/16) of gb, 80 % (4/5) of aa and in the only case of da. In 50 % of cases at least 3 different morphologic variants were seen in different areas of the tumor. This morphologic heterogeneity presented as the combination of different grades of anaplasia (gii – giv) in one tumor. Molecular profile was assessed in 48 expression analysis of genes: mgmt, c-kit, top2a, pdgfr-α, ercc1, vegf from 8 patients. Intratumoral molecular heterogeneity was revealed in 41,7 % of cases (20/48). Conclusion. The presence of intratumoral heterogeneity should be taken into account during surgery for adequate tumor sampling for histologic and molecular analysis which is critical for proper assessment of prognosis and following treatment planning.

Frequent FGFR1 hotspot alterations in driver-unknown low-grade glioma and mixed neuronal-glial tumors

Purpose Low-grade gliomas (LGG) and mixed neuronal-glial tumors (MNGT) show frequent MAPK pathway alterations. Oncogenic fibroblast growth factor receptor 1 (FGFR1) tyrosinase kinase domain has been reported in brain tumors of various histologies. We sought to determine the frequency of FGFR1 hotspot mutations N546 and K656 in driver-unknown LGG/MNGT and examined FGFR1 immunohistochemistry as a potential tool to detect those alterations. Methods We analyzed 476 LGG/MNGT tumors for KIAA-1549-BRAF fusion, IDH1/2, TERT promotor, NF1, H3F3A and the remaining cases for FGFR1 mutation frequency and correlated FGFR1 immunohistochemistry in 106 cases. Results 368 of 476 LGG/MNGT tumors contained non-FGFR1 alterations. We identified 9 FGFR1 p.N546K and 4 FGFR1 p.K656E mutations among the 108 remaining driver-unknown samples. Five tumors were classified as dysembryoplastic neuroepithelial tumor (DNT), 4 as pilocytic astrocytoma (PA) and 3 as rosette-forming glioneuronal tumor (RGNT). FGFR1 mutations were associated with oligodendroglia-like cells, but not with age or tumor location. FGFR1 immunohistochemical expression was observed in 92 cases. FGFR1 immunoreactivity score was higher in PA and DNT compared to diffuse astrocytoma, but no correlation between FGFR1 mutation in tumors and FGFR1 expression level was observed. Conclusion FGFR1 hotspot mutations are the fifth most prevailing alteration in LGG/MNGT. Performing FGFR1 sequencing analysis in driver-unknown low-grade brain tumors could yield up to 12% FGFR1 N546/K656 mutant cases.

Assessment of Therapeutic Outcome and Role of Reirradiation in Patients With Radiation-induced Glioma

Background: We retrospectively reviewed the clinical characteristics and treatment outcomes to clarify the optimal therapeutic strategy, especially the role of reirradiation in patients with radiation-induced glioma (RIG). Methods: We identified patients with high-grade glioma who satisfied Cahan’s criteria for RIG in our database during 2001–2021 and analyzed the outcomes.Results: We identified 11 patients with RIG. The primary diseases included germinomas (n=2), acute lymphoblastic lymphomas (n=2), medulloblastomas (n=3), diffuse astrocytoma with isocitrate dehydrogenase (IDH) 2 mutant (n=1), pilocytic astrocytoma (n=1), pituitary adenoma (n=1), and a metastatic tumor from lung cancer (n=1). The median latency period was 17 years (range: 9–30 years). The RIGs included glioblastoma with IDH 1/2 wild-type (n=7), glioblastoma not otherwise specified (n=2), anaplastic astrocytoma with IDH1/2 wild-type (n=1), and anaplastic astrocytoma not otherwise specified (n=1). All patients underwent tumor removal or biopsy, 5 patients postoperatively received reirradiation combined with chemotherapy, and 6 patients were treated with chemotherapy alone. The median progression-free and overall survival times were 11.3 and 28.3 months, respectively. The median progression-free survival time of patients treated with reirradiation and chemotherapy (n=5) tended to be longer than that of patients that received chemotherapy alone (n=6) (17.0 vs 8.1 months; p=0.45); the median survival time was similar (29.6 vs 27.4 months; p=0.28). Local recurrences were observed less frequently in patients who received reirradiation combined with chemotherapy (50%) than in those who received chemotherapy alone (100%; p=0.046). None of the patients developed radiation necrosis. In one case, the different IDH2 mutational states between the primary and secondary tumors were useful for diagnosing the secondary tumor as RIG. Conclusions: RIG can occur more than 20 years after successful treatment of the primary disease using radiotherapy; thus, follow-up times should be extended to 30 years. Reirradiation combined with chemotherapy appears to be feasible and have favorable outcomes. Identifying the IDH1/2 mutational status can have a diagnostic effect on establishing RIG in recurrent gliomas.

Histomorphological spectrum of central nervous system lesions in a tertiary care hospital

Background: Central nervous system (CNS) lesions show considerable geographic and racial variations with respect to the incidence and their pattern of distribution. CNS neoplasm account for 2% of all cancers. There has been a rapid increase in incidence of CNS tumors. Many non-neoplastic CNS lesions can clinically and radiologically mimic brain tumors and to differentiate them histopathological examination is necessary.Methods: This was a descriptive cross-sectional study of 30 cases, carried out from March 2020 to February 2021, in a tertiary health care hospital. The cases were diagnosed histopathologically and categorized according to the WHO 2021 classification.Results: The study included 30 cases. 3 (10%) of them were non-neoplastic while 27 (90%) were neoplastic with glioblastoma and diffuse astrocytoma being the most common. Overall, tumors of neuroepithelial tissue (60%) was the most common entity. Frontal lobe was the most commonly observed site in the neoplastic lesion. Headache was the most common presentation. Age range varied from 7 years to 66 years with the mean age of 32.7 years.Conclusions: The spectrum of neoplastic lesions were astrocytoma, glioblastoma, ependymoma, hemagioblastoma, medulloblastoma, meningioma, non-hodgkins lymphoma, oligodendroglioma, pitutary adenoma, schwannoma and non-neoplastic were A-V malformation, brain abscess, subdural empyema.

MPC-2 Clinical course and prognosis of lower-grade glioma, IDH wildtype and pTERT mutant

Background and Purpose: In the cIMPACT-Now update 3, it was proposed that grade 2 astrocytic gliomas without IDH-mutations and grade 3 astrocytic gliomas with TERT promoter mutations should be designated as diffuse IDH wildtype astrocytic glioma with molecular features of WHO grade IV glioblastoma. Therefore, we investigated whether this group of tumors actually corresponds to grade IV prognostically in cases that we encountered ourselves. Cases and Methods: Among the 65 patients having primary astrocytic glioma who were operated in our hospital from January 2016 to March 2021, the prognostic values of seven patients with lower-grade glioma, IDH wildtype, and pTERT mutant were investigated. Results: Among the seven patients, the median age was 59 years (50–66 years). Four of them had anaplastic astrocytoma, two had diffuse astrocytoma, and no tumor lesion could be identified upon histological examination for one patient. The male-to-female ratio was 1:6. MGMT methylation was observed in two patients (29%). The median survival was 20 months, with a significantly worse prognosis when compared with lower-grade glioma without the TERT promoter mutation (13 patients: median survival 40 months), but a better prognosis when compared with glioblastoma (45 patients: median survival 13 months) (Log-rank p = 0.0051). Conclusion: Although EGFR amplification, combined whole chromosome 7 gain, and whole chromosome 10 loss were not examined, the prognostic value of lower-grade glioma, IDH wildtype, and pTERT mutant was not as poor as that of glioblastoma. Further investigation is required to confirm whether these groups of tumors should be treated in the same way as grade IV glioblastoma.

NI-13 Prediction of outcome at the time of discontinuing TMZ-adjuvant therapy in IDH-mutant lower grade glioma using 11C-methinine PET

Purpose: This study aimed to clarify whether positron emission tomography with 11C-methyl-L-methionine (11C-met PET) can predict consequential outcomes at the time of discontinuing temozolomide (TMZ)-adjuvant chemotherapy in patients with residual isocitrate dehydrogenase gene (IDH)-mutant lower-grade glioma.Methods: In 30 patients showing residual lesions of IDH-mutant lower grade glioma (16 with diffuse astrocytoma and 14 with anaplastic astrocytoma), we performed 11C-met PET, and calculated the tumor-to-normal brain tissue ratio of standardized uptake values (SUVT/N) at the time of discontinuing TMZ-adjuvant chemotherapy. We determined cutoff values to predict tumor relapse using the receiver operating characteristic curve for various prognostic factors including age, Karnofsky performance scale, number of courses of therapy, residual tumor size, and SUVT/N. The promotor methylation status of O6-methylguanine-DNA methyl-transferase gene (MGMT) was assessed using methylation-specific polymerase chain reaction. Progression-free survival (PFS) was compared between groups divided by cutoff values. Uni- and multivariate analyses were conducted using log-rank testing and Cox regression analysis, respectively.Results: Univariate analysis identified MGMT methylation status (p = 0.04) and an SUVT/N of 1.27 (p = 0.02) as predictors of PFS after TMZ discontinuation. In multivariate analysis, both unmethylated MGMT and SUVT/N ≥ 1.27 remained as strong predictors of unfavorable outcome. Conclusion: The present study suggested that 11C-met PET allows prediction of outcomes comparable to MGMT promotor methylation status at the time of discontinuing TMZ-adjuvant chemotherapy in patients with residual IDH-mutant lower-grade glioma.

TB-9 An attempt to establish a patient-derived brain tumor culture model by organoid culture method

Background: Molecular heterogeneity among and within tumors are one of the reasons for the poor survival rate of brain tumors even with the current standard therapy. However, monolayer culture and neuro-sphere culture (NS) use exogenous growth factors, so may not show the true nature of the tumor. And the culture establishment rate is low, especially low-grade tumors. Therefore, we used the glioblastoma organoid (GBO) culture method showed by Fadi to create culture models of various brain tumors and investigated their characteristics. Methods: We examined the establishment rate in pathological and genotypic types of 56 patients who underwent brain tumor resection at our hospital between January 2020 and June 2021 and were cultured with GBO or NS. If tumor cells are increased visually at 1 month after culture, we defined establishment. Results: There were 15 cases of glioblastoma, 7 cases of anaplastic astrocytoma, 7 cases of diffuse astrocytoma, 3 cases of diffuse midline glioma, 2 cases of anaplastic oligodendroglioma, 5 cases of oligodendroglioma, and 16 cases of others. The establishment rate was 76.5% by the GBO method and 40% by the N S method. By histological type, GBO: 80% in glioblastoma, NS: 58.3% in glioblastoma, GBO: 83.3% in AA, NS: 40% in AA, and GBO: 100% in DA. The IDH mutation and pTERT mutation were investigated in GBO: IDHwt/TERT+ 87.5%, IDHwt/TERT- 64.3%, IDHmt/TERT- 100%, and in NS: IDHwt/TERT+ 75%, IDHwt/TERT- 33.3%, IDHmt/ TERT- 20% in NS. In addition, establishment was observed in GBO 2 case in medulloblastoma, 1 case in ependymoma. Discussion and Conclusion: This suggest that GBO can be used to establish culture models for low-grade tumors. In addition, GBO can establish culture earlier, so it is expected to be applicable to personalized therapies such as preclinical drug efficacy studies tailored to individual patients.

Clinical, Morphological, and Molecular Study of Diffuse WHO Grade II and III Astrocytomas: A Retrospective Analysis from a Single Tertiary Care Institute

Introduction Astrocytomas are the most common gliomas, classified on the basis of grade and IDH mutation status according to the World Health Organization (WHO) 2016 update. IDH mutations are seen in 70 to 80% of diffuse grade II and III astrocytomas and are associated with better outcome. They serve as predictive biomarker in IDH-targeted therapies such as small-molecule inhibitors or vaccines. Objective The aim of this study was to analyze the clinical, morphological, immunohistochemical, and molecular genetic characteristics of diffuse astrocytoma (DA: grades II and III). The IDH mutant and wild-type tumors are compared and contrasted with survival analysis on follow-up. Materials and Methods This was a retrospective study conducted on surgically resected tumor specimens. The hematoxylin and eosin-stained slides were examined for histologic features. Immunohistochemistry (IHC) was performed using IDH1R132H, ATRX, p53, and Ki67. All cases of negative immunohistochemical expression of IDH1R132H were subjected to IDH1 mutation analysis by Sanger sequencing. Overall survival was estimated by the Kaplan-Meier method using the log-rank (Mantel–Cox) test. Results The study included 51 cases of DA in the age of 17 to 66 years, mean ± standard deviation was 35.5 ± 9.7 years, and male:female ratio was 2:1.The IDH1R132H cytoplasmic immunopositivity was seen in 36 cases (70.5%), of which 63.6% were of grade II and 72.5% were of grade III. ATRX showed loss of expression in 50 cases (98%), and p53 showed diffuse strong immunohistochemical expression in all the cases of IDH mutant tumors. The difference in the age at presentation for IDH mutant (32.5 years) and wild type tumors (38 years) was statistically significant. Median survival was 55.3 months and 22.2 months in of IDH mutant and wild type cases, respectively. Conclusion IHC and sequencing for IDH mutations is helpful in making an integrated diagnosis and classifying definite molecular subgroups of astrocytic tumors. Mutations in IDH core-elate with survival. IDH mutant tumors showed longer survival duration and are good prognostic indicators.

PEDT-1 Integrated diagnoses of pediatric gliomas in our institute by cIMPACT-NOW recommendations

Purpose:Since many genetic abnormalities in glioma have been revealed in recent years, Integrated diagnoses are necessary in the updated fourth edition of the WHO Classification of Tumors of the Central Nervous System(CNS) published in 2016. The Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy (cIMPACT-NOW) was established to provide a forum to evaluate and recommend proposed changes to future CNS tumor classification. We retrospectively classified pediatric gliomas in our hospital in accordance with cIMPACT-NOW recommendations. Methods: This study includes 13 consecutive glioma patients under the age of 18 who underwent surgical resection at our hospital from 2000 to 2021. Histopathological diagnoses and molecular status such as IDH, H3F3A and BRAF were analyzed. Results: There were four females and nine males, ranging in age from 0 to 17 years, median 9.0 years. Three pilocytic astrocytomas (PA) and a polymorphous low-grade neuroepithelial tumor of the young (PLNTY) had BRAF fusion. Four cases were classified as Diffuse midline glioma, H3K27M-altered. Two cases were classified as Diffuse pediatric-type high-grade glioma, H3-wildtype and IDH-wildtype. No genetic alteration was observed in two diffuse astrocytoma cases. An anaplastic oligodendroglioma case with PDGFRA amplification could not be classified as any new entity. All three PA cases with BRAF fusion occurred in cerebellum and all four H3K27M altered cases occurred in midline location such as thalamus, brainstem and cervical spinal cord. Six cases which were classified as pediatric-type diffuse high grade gliomas had poor prognosis. Conclusion:Genetic status is associated to tumor location and patients prognosis. Integrated diagnoses are important in pediatric glioma patients.