|Year : 2021 | Volume
| Issue : 2 | Page : 72-79
Award E Poster
|Date of Web Publication||21-Apr-2022|
Source of Support: None, Conflict of Interest: None
|How to cite this article:|
. Award E Poster. Int J Neurooncol 2021;4:72-9
Brain stem tumors in children: Experience of 42 cases
Rahul Saha, Sandip Chatterjee; VIMS and Park Clinic
Aim: To evaluate our results of surgery of brain stem tumors in children and teenagers based on our experience of operating on 42 children with this condition.
Materials and Methods: Of the patients we have operated upon, three had PNET, nine had malignant gliomas, 10 had gangliogliomas, and 20 had low-grade gliomas.
Results: Of these 42 children, 28 had a reasonable quality of life. Since the diagnosis was a slow-growing tumor in the majority of cases, it seemed appropriate to attempt a total removal.
Conclusion: The adjuncts used include intraoperative neuromonitoring of brainstem, brainstem mapping, ultrasonic aspirator, and neuronavigation, and these will be illustrated in the presentation.
Corpus callosum glioma: An institutional experience
Pawan Kumar Verma, Awadhesh Jaiswal, Kuntal Kanti Das, Sanjay Behari; SGPGIMS
Introduction: The corpus callosum is the largest interhemispheric commissure providing a connection between homologous cortical areas. Lesion involving these fibers may produce a variety of deficits in the form of headache, seizure, personality changes, and neurocognitive changes. These fibers also act as a medium for migration of lesions from one lobe to contralateral lobes responsible for rapid progression or recurrence of disease. Herein, we present our experience of management of corpus callosum lesions and the lessons we learnt.
Materials and Methods: The authors documented retrospective data of histologically proven corpus callosum glioma and their surgical management. The preoperative symptoms, extent of tumor excision, operative nuances, postoperative complications, and histological subtypes of glioma were analyzed.
Results: We treated 25 patients (mean age: 40.8 years, male:female—15:10). The common presenting complains were seizure, headache, behavioral changes, vomiting, hemiparesis, incontinence, and memory loss. The extent of excision achieved was gross total (n=12), near total (n=4), and subtotal including biopsy (n=9). The most common histological pathology was World Health Organization (WHO) grade II (N=14) followed by WHO grade IV (N=10). The major postoperative complications noted were neurocognitive dysfunction, behavioral changes, decreased verbal output, hemiparesis, and wound bulge.
Conclusions: The complete surgical excision of corpus callosum glioma is not feasible on every occasion. While attempting to remove the total tumor, resection may produce a significant morbidity in the form of neurocognitive deficit, limb weakness, memory loss, and other behavioral changes. The comprehensive management with a good functional outcome can be achieved by maximal safe tumor resection followed by radiotherapy and chemotherapy.
Classification of pediatric brain tumors using radiomic features of T2-weighted images
Abhilasha Indoria, Nivedditta Veeraraghavan, Vani Santosh, Arivazhagan Arimappamagan, Shilpa Rao, Nishanth Sadashiva, Karthik Kulanthaivelu, Jitender Saini; NIMHANS, Bangalore, Karnataka, India
Objective: About half of the major pediatric tumors (medulloblastoma, ependymoma, and pilocytic astrocytoma) originate in the posterior fossa. In cases of medulloblastoma and ependymoma, gross total resection is desirable and whole-brain spinal axis radiotherapy is part of the management paradigm, whereas in cases of pilocytic astrocytoma, surgery is the mainstay of primary management. The long-term outcome is favorable with pilocytic astrocytoma, Therefore, knowing the tumor type preoperatively is desirable. The objective of this study was to explore the machine learning classification model using radiomic features of T2-weighted (T2W) images, which are routinely acquired for discriminating between major pediatric posterior fossa brain tumors (ependymoma, medulloblastoma, and pilocytic astrocytoma). This discrimination using conventional magnetic resonance imaging (MRI) is difficult due to overlapping radiological characteristics, but it can be improved by the inclusion of advanced techniques such as MRS and DWI.
Materials and Methods: MRI data of 87 age-matched subjects (32 medulloblastoma, 30 ependymoma, and 25 pilocytic astrocytoma) was retrieved from PACS. Region of interest (ROI) was manually drawn on the preprocessed T2W images to delineate the tumor using three-dimensional slicer version 4.10.2. T2W images’ z-score normalized for feature extraction. In all, 18 first-order statistical features and 23 GLCM features were extracted by using PyRadiomics library. Dataset was split into training and testing data. Feature importance was calculated from the training dataset using randomized search cross-validation and correlation matrix. Important features were selected based on the importance score. Fourfold cross-validation was carried out on the training dataset for selected features to obtain the cross-validation score (accuracy). Random forest classifier along with OneVsRest classifier was trained on a reduced feature set after hyperparameter tuning. Classier performance was assessed. The average intensity histogram for the ROIs was also calculated for the three groups.
Results: An 87% mean accuracy score was achieved after cross-validation. The classifier also performed well on the test dataset with an accuracy of 83.14%. Calculation of average intensity histogram revealed the intensity overlap between the three groups. A higher feature importance score was assigned to second-order features as compared to first-order features by a randomized search algorithm. The top three features in the analysis included only the second-order features, i.e., “glcm IMC1,” “glcm jointenergy,” and “glcm InverseVariance.” First-order features included in the analysis are “skewness,” “energy,” and “minimum.”
Conclusion: We conclude that second-order statistical features derived from T2W images are most useful for discriminating between the three major pediatric brain tumors, i.e., pilocytic astrocytoma, ependymoma, and medulloblastoma, and can be used to predict the tumor type.
Transcriptome profiling data of anaplastic astrocytomas identify a set of novel genes associated with poor prognosis in isocitrate dehydrogenase-mutant tumors
Harsha S. Sugur, P. Sravya, Shilpa Rao, K. Thennarasu, Bhupesh Mehta, A. Arivazhagan, Vani Santosh; National Institute of Mental Health and Neurosciences, Bangalore, Karnataka, India
Background and Objectives: Anaplastic astrocytoma (AA), isocitrate dehydrogenase mutant (IDHmt), and isocitrate dehydrogenase wild type (IDHwt) are heterogeneous in their behavior due to other co-occurring genetic alterations, such as CDKN2A/B deletions and GBM-like molecular alterations in AA, IDHmt, and IDHwt, respectively. However, these genetic alterations alone may not be enough to explain the differences in clinical outcome and a comprehensive study on other molecular alterations obtained from high-throughput approaches is essential. We aimed to study the transcriptome profiling data of AA and validate the shortlisted genes in our cohort for clinical significance.
Materials and Methods: We analyzed the transcriptome data of AA (n=167) where isocitrate dehydrogenase (IDH) status, 1p/19q status, and follow-up were available from the TCGA database. AAs were divided into IDHmt and IDHwt subgroups. The mRNA sequencing data of the subgroups were separately compared with the normals to derive differentially regulated genes using the DESeq2 R package. Various methods such as Venn, pathway (Enrichr), connectivity (STRING), and survival analysis were performed to shortlist robust genes. The mRNA levels of shortlisted genes were validated using the NanoString nCounter technique on our cohort of AA, IDHmt (n=81), and IDHwt (n=7) tumors. Survival analysis was carried out on uniformly treated AA, IDHmt patients with available follow-up details. CDKN2A/B status was evaluated in this cohort by the FISH technique. The mRNA expression was correlated with survival of patients to identify genes that are clinically relevant.
Results: From the TCGA dataset, subgroup analysis of AA, IDHmt (n=100) and AA, IDHwt (n=67) vs. normal (n=5) identified 96 and 136 differentially regulated genes, respectively. Of these, 34 genes (17%) were commonly regulated in both the subgroups. A total of 21 genes were finalized (eight in AA, IDHmt; seven in AA, IDHwt; and six common to both the subgroups) and validated in our cohort of AA (n=88). Except three genes, all others validated robustly. In all, 7.4% of AA, IDHmt showed CDKN2A/B homozygous deletion. Survival analysis on AA, IDHmt cases identified novel genes, such as IRX1 (hazard tatio [HR]=1.002, p=0.002), MYBL2 (HR=1.003, p=0.005), FOXM1 (HR=1.003, p=0.002), and CDKN2A/B deletion (HR=3.99, p=0.014), to be associated with poor prognosis.
Conclusions: Our study reiterates the prognostic significance of CDKN2A/B in AA, IDHmt. On transcriptome profiling, we identified novel genes that are differentially regulated in AA, IDHmt, and IDHwt tumors. Among them, IRX1, MYBL2, and FOXM1 emerged as robust biomarkers associated with poor prognosis in AA, IDHmt patients. This set of novel genes would serve as potential prognostic markers in the routine clinical setting.
Characterization of molecular differences in ependymoma arising in different intracranial and spinal locations
Dheeraj Chinnam, Kirti Gupta, Tanvi Kiran, Aastha Saraswati, Pravin Salunke, Narendra Kumar, Bishan D. Radotra; PGIMER, Chandigarh, India
Background and Objectives: Our understanding of ependymoma (EPN) has greatly evolved in the past decade. Nine consensus molecular subgroups were discovered arising at different locations within brain and spinal cord. As these subgroups carry prognostic relevance, such molecular information must be included within the histological classification. We sought to identify molecular subgroups of EPN arising at intracranial and spinal locations using immunohistochemistry for L1CAM, cyclin D1, H3K27M, and H3K27me3 and correlate their expression with clinical, histological parameters and overall survival.
Materials and Methods: Our cohort consisted of 125 cases of EPN arising across all the major central nervous system compartments. Immunohistochemistry for L1CAM and cyclin D1 was assessed in supratentorial (ST) tumors, whereas expression of H3K27M and H3K27me3 was assessed in posterior fossa (PF) tumors. Spinal tumors were also assessed for these markers.
Results: Age ranged from 11 months to 70 years with male:female ratio of 1:1.2. PF-EPN constituted the majority (47.2%), followed by ST-EPN (32%) and spinal (20.8%) tumors. Amongst ST tumors, positivity of L1CAM and cyclin D1 was observed in 47.5% of cases. The follow-up ranged from 0.5 to 51.6 months. There was a significant correlation of L1CAM expression with younger age (age<20 years), higher grade, and lesser overall survival in comparison to tumors negative for these markers. Amongst PF tumors, tumors with loss of H3K27me3 (PF-Group A) comprised 69.4% cases, whereas PF-Group B constituted 29.6%. PF-A tumors dominated in less than 5 years, whereas PF-B tumors were more common in above 20 years. The follow-up ranged from 0.17 to 50.5 months. Loss of H3K27me3 expression (PF-A) was associated with lesser mean overall survival. Only a single case of PF-EPN harbored H3K27M mutation. Among the spinal tumors, the follow-up ranged from 0.46 to 45.4 months; none of these tumors showed expression of L1CAM, cyclin D1, H3K27M, or H3K27me3.
Conclusion: Expression for two RELA-associated markers, i.e., L1CAM and cyclin D1 in ST tumors and H3K27M and H3K27me3 within PF location, can be used to identify tumors with adverse clinical outcome.
Aggressiveness of recurrent glioblastoma multiforme depends on integrin beta 5 and paxillin, a novel molecular target
Anagha Acharekar, Jacinth Rajendra, Shilpee Dutt; Shilpee Dutt Laboratory, Tata Memorial Centre, Advanced Centre for Treatment Research and Education in Cancer, Navi Mumbai, Maharashtra, India
Objective: Therapy refractory recurrent glioblastoma multiforme (GBM) is a clinical challenge mainly because of its highly invasive nature. Therefore, better therapeutics of recurrent GBM relies on a better understanding of its molecular characteristics.
Materials and Methods: Paired primary and recurrent GBM cells were captured from the cellular model developed in the laboratory. Parent and recurrent cells were subjected to whole transcriptome and proteome analysis to identify candidate molecular targets. Functional studies were done by clonogenic, migration, and invasion assays. Mechanistic studies were performed by molecular biology methods like quantitative polymerase chain reaction, western blots, immunofluorescence, immunohistochemistry (IHC), and genetic knockdowns of genes. Results were validated in primary cultures from patient samples.
Results: Integrated analysis of iTRAQ proteomics and RNA seq transcriptomics revealed deregulation of focal adhesion pathway, which also corroborated with the aggressive phenotypes of recurrent population such as enhanced proliferation, anchorage independent growth, invasion, and migration. Further, among the focal adhesion proteins, integrin beta 5 (ITGB5) was significantly upregulated at the mRNA and protein levels in recurrent cells of two cell lines and three primary patient samples. Moreover, IHC of six primary and recurrent patient biopsies showed higher expression of ITGB5 in recurrent patient samples, thus suggesting a role of ITGB5 in aggressiveness of recurrent GBM. ITGB5 knockdown reduced migration and invasion of recurrent cells but enhanced cell–cell adhesion. At the molecular level, paxillin (PXN), a key downstream adaptor protein, remained stabilized at the protein level and at the focal adhesions in spite of genetic perturbation of ITGB5. The presence of PXN in the absence of ITGB5 in recurrent cells explains the direct influence of PXN on enhanced cell–cell adhesion in recurrent cells. This was proved by the reduction in cell–cell adhesion upon PXN knockdown. Moreover, in the recurrent population, the cell survival was affected after PXN knockdown and not ITGB5. This result also supports the unpromising clinical outcome of cilengitide, an integrin inhibitor. ITGB5 inhibition does not abrogate the PXN location at FAs due to which the cell survival is not hampered on ITGB5 targeting. Thus, PXN is a better therapeutic target, in order to efficiently target recurrent GBM.
Conclusion: Taken together, here we identified that PXN is responsible for the aggressive nature of recurrent GBM and can be a potential novel molecular target.
Clinical predictive score for survival in spinal metastasis
Tamajyoti Ghosh, S. K. Sengupta, Subir Dey, A. S. Carvalho, Sanjay Kumar; AFMC
Objectives: To validate the NESMS, Revised Tokuhashi, and Tomita score in predicting the 6-month survival outcomes among the spinal metastasis cases treated at our institute. We also tried to determine other variable that may correlate with poor survival in spinal metastasis.
Materials and Methods: Observational study of all spinal metastasis cases treated at AFMC between the period October 1, 2019, and November 30, 2020. Inclusion criteria were all cases of spinal metastasis (operative + nonoperative) and age group 30–80 years. Exclusion criteria were patients <30 years of age and >80 years old and patients who lost follow-up. Patients with spinal metastasis were clinically examined and evaluated with laboratory investigations and neuroimaging and PET scan. NESMS, Revised Tokuhashi, and Tomita scores were collected along with all other clinical data. Patients were followed up over a period of 6 months for two end points: death or survival at 6 months. We tried to correlate the 6 months mortality with the different spinal metastasis scores and other independent variables. The 6-month survival outcome along with different spinal metastasis scores was visually assessed using Kaplan–Meir curves.
Results: Our study included a total of 26 cases of spinal metastasis who were followed up for at least 6 months from the time of diagnosis of spinal metastasis. A total of 15 cases (57.69%) were operated. Total 6-month mortality was 53.38% (14 cases). The mean age of presentation was 57.65 years. Male:female ratio was 38:62. The most common primary tumor was lung carcinoma (38.46%), followed by breast carcinoma (19.23%). NESMS of 0 had 100% (9 cases) 6-month mortality, whereas only one case of NESMS of 2 and above died within 6 months. Similarly 70% (seven cases) 6-month mortality was seen in the Revised Tokuhashi score of 0–8 and 75% 6-month mortality was seen in the Tomita score of more than or equal to 8. Visceral metastasis was commonly found in patients having poor 6-month mortality 71.42% (10 cases). Poor KPS score (mean=57.14) was seen among patients with 6-month mortality. The most common site of visceral metastasis was lung (30.76%), followed by liver (26.92%).
Conclusion: Thus, this study validates the spinal metastasis scoring system of NESMS, Revised Tokuhashi, and Tomita and also independent variables like visceral metastasis and poor KPS in determining poor 6-month mortality among spinal metastasis patients.
Is epithelioid glioblastoma related to pleomorphic xanthoastrocytoma and anaplastic pleomorphic xanthoastrocytoma: Clinicopathological and molecular evaluation of 48 cases
Soutrik Das, B. N. Nandeesh, Subhas Konar, Shilpa Rao, Vani Santosh; NIMHANS, Bangalore, Karnataka, India
Objectives: Pleomorphic xanthoastrocytoma (PXA), anaplastic pleomorphic xanthoastrocytoma (APXA), and epithelioid glioblastoma (EGBM) share some demographic, radiological, histopathological, and molecular similarities. Therefore, it is presumed that these tumors may be closely related entities or represent a spectrum of a single biological entity. However, there is a marked difference in survival and prognosis between these tumors. To the best of our knowledge, there is no Indian study correlating clinical, pathological, and molecular features of these three entities. The aim of this study was to characterize and correlate clinicopathological and molecular profiles of these tumors.
Materials and Methods: In this retrospective study, cases histologically diagnosed as PXA, APXA, and EGBM between January 2011 and March 2019 were evaluated. Demographic details and clinical features of the patients were obtained from the patient case records. Formalin-fixed paraffin-embedded blocks archived in the Neuropathology Department were utilized for histopathological review, immunohistochemistry (IHC), and molecular studies. IHC was carried out using a panel of antibodies against GFAP, synaptophysin, IDH1 R132H, ATRX, p53, MIB1, BRAF V600E, and PDGFRA. Molecular studies comprised CDKN2A/B deletion and EGFR amplification testing by FISH, TERT promoter mutation testing by Sanger sequencing, and MGMT promoter methylation assessment by quantitative (real-time) polymerase chain reaction.
Results: This study included a total of 48 patients (11 PXA, 13 APXA, and 24 EGBM). The mean age for PXA, APXA and EGBM patients were 19.8 years, 28 years, and 34.4 years, respectively. Unlike PXA and APXA patients, EGBM patients consistently lacked eosinophilic granular bodies. BRAF V600E immunopositivity was seen in 73%, 62%, and 79% of PXA, APXA and EGBM patients, respectively, although the intensity of staining was the strongest in EGBM. CDKN2A/B deletion was observed in 73% PXA, 77% APXA, and 80% EGBM patients, with homozygous deletion being more common in all three entities. TERT promoter mutation was more frequent in EGBM (69%) than PXA (50%) and APXA (55%) patients. EGFR amplification was noted only in two cases of EGBM, but none of the PXA and APXA cases.
Conclusions: The present study shows that PXA and APXA share similar histological and molecular alterations, indicating that they belong to one entity with increasing grade of malignancy. EGBM is the most malignant among them with distinct clinical, histological, and molecular features, thus conforming to the group of glioblastomas. We opine that EGBM may not represent the other end of the spectrum of PXA/APXA, although there could exist a subset that shows continuum with PXA/APXA.
Matrix metalloproteinase-14 expression is a potential marker for invasiveness in pituitary adenomas�A study of 156 pituitary resections
Nivetha Ambalavanan, Aruna Nambirajan, Madhu Rajeshwari, Vaishali Suri, Ajay Garg, Sushant Agarwal, Ashish Suri, Chitra Sarkar, Mehar Chand Sharma; NIMHANS, Bangalore, Karnataka, India
Objectives: Pituitary adenomas (PitNETs) represent one of the commonest intracranial tumors in adults. Although majority behave in a benign manner, some tend to be locally invasive with frequent parasellar extension correlating with higher rates of recurrence and treatment resistance. With the aim of identifying potential markers that correlate with invasive behavior, we studied by immunohistochemistry the expression levels of matrix metalloproteinase-14 (MMP14), EZH2, β-catenin, nestin, and micro-vessel density (CD34) in relation to morphofunctional classification of pituitary adenomas, radiological score of invasion (Knosp score), and proliferative index.
Materials and Methods: Paraffin blocks of pituitary adenoma (with adequate tissue) from the archives between the period of 2016 and 2019 were included in the study. Preoperative tumor size and invasiveness were reevaluated and assessed using Knosp score in magenetic resonance imaging. The 2017 morphofunctional classification was done with hormonal markers (GH, PRL, ACTH, TSH, FSH, LH, α-subunit) and transcription factors (Pit1, Tpit, SF1, GATA3, ER-α) and LMWCK. Subsequently, the potential markers under investigation (MMP14, EZH2, β-catenin, nestin, CD34) were performed. Intensity and proportion scoring were given to EZH2 and MMP14. Nuclear accumulation of β-catenin was evaluated by immunohistochemistry. Microvessel density was calculated using CD34 immunostain in Image-Pro Plus software in hotspots.
Results: A total of 156 cases were studied. MMP14 expression positively correlated with larger tumor size, higher Knosp score (p-value≤0.0001), and higher MIB1 labeling index (p-value=0.04); however, there was no significant correlation with morphofunctional subtypes. EZH2 protein level expression and microvessel density did not reveal any significant correlation with any of the clinicopathological parameters. Nestin expression in pituitary adenoma was very limited, with focal reticular positivity observed in four cases of gonadotroph adenomas. β-catenin staining was negative (membranous pattern) in all cases with a peculiar loss of membranous staining in Pit1 lineage adenomas.
Conclusions: MMP14 emerges as a potential tissue marker for invasiveness in pituitary adenomas. There appears to be a limited role for EZH2 and Wnt signaling pathway activation in driving invasiveness in pituitary adenomas.
Brain tumors in adolescents and young adults: Experience from a community oncology center
S. P. Shrivastava, V. Bhandari, A. Elhence, S. Singhal, K. p. k. Reddy, J. Elagandula, S. Bansal, P. Jinwala, R. Patidar, V. Asati, P. G. Chitalkar; Department of Medical Oncology, SAIMS, Indore, Madhya Pradesh, India
Objectives: The incidence of cancer in adolescents and young adults (AYAs) 15–39 years has been on the rise. Central nervous system (CNS) tumors account for 10% of AYA cancers and are the most common cause of cancer-related death in this age group. We describe the presentation, treatment, and survival for AYAs diagnosed with CNS tumors from a single institution.
Materials and Methods: Case records (2012–2020) of CNS tumors of AYAs were extracted and analyzed; treatment details and follow-up wherever possible were recorded. Patient status (alive, well, with disease, or dead) was obtained by telephonic interview with caregivers, performed in January–February 2021.
Results: Sixty-three AYA patients (28 low-grade glioma [LGG], 21 high-grade glioma [HGG], three medulloblastoma, five ependymoma, three meningioma, two pituitary adenoma, and one craniopharyngioma) were identified from the hospital records over 108 months from 2012 to 2020. Symptoms were headache 78%, vomiting 63%, seizure 40%, nausea 29%, and visual disturbances 8%, which were present for a median of 3 weeks (1–20) before consulting a health professional. Clinical, imaging, or molecular risk factors were not evaluated. All patients underwent surgery, either maximum safe resection or stereotactic biopsy, to confirm histopathological diagnosis. All patients with LGG had radiotherapy. Among HGG patients, all had radiotherapy and concurrent chemotherapy (temozolomide 75 mg/m2), followed by adjuvant temozolomide. All medulloblastoma patients were managed with surgery followed by craniospinal radiotherapy. Radiotherapy was planned and delivered as per the standard guidelines for the respective histologies. Patients alive without disease; LGG 18/28 (68%) for median 23 months (4–90); among HGG, 4/21(19%) median 6 months (3–21); medulloblastoma 1/3 alive without disease (4–60 months); meningioma 3/3, pituitary adenoma 2/2. However, most patients did not come for regular follow-up.
Conclusions: The treatment of brain tumors in AYAs is challenging. Our retrospective data bring out proportion of histologies similar to older adults. Clinical, histology-molecular, or treatment-related prognostication was not attempted. Other institutions have characterized brain tumors of AYAs as distinct from diseases in children and older adults. This group with its unique sociobiological context will benefit from an age-appropriate, multi-disciplinary approach.
Depletion of mitochondrial DNA induces stemness and treatment resistance in glioblastoma
Sravya Palavalasa, Vidya Prasad Nimbalkar, Nandaki NagKanuri, Harsha Sugur, Brijesh Kumar Verma, Paramita Kundu, Shilpa Rao, Sampath Somanna, Paturu Kondaiah, A. Arivazhagan, Vani Santosh; National Institute of Mental Health and Neurosciences, Bangalore, Karnataka, India
Background: Treatment resistance and cancer stem cells are known to be closely related. Low mitochondrial DNA (mtDNA) content is essential for maintaining stemness of a cell. Recent studies, including our own, have shown that low mtDNA copy number confers poor prognosis in glioblastoma. This study aims to elucidate the biology behind the clinical finding.
Objectives: To deplete mtDNA content in glioblastoma cell lines and study the effect on (1) cell death induced by radiation and chemotherapy, (2) proliferation and cell cycle, and (3) stemness.
Materials and Methods: Malignant glioma cell lines, U87 and LN229, were subjected to mtDNA depletion using ethidium bromide. The degree of mtDNA depletion was assessed using real-time quantitative polymerase chain reaction. The mtDNA-depleted and parent cells were exposed to 2 Gy, 4 Gy, and 6 Gy radiation, followed by clonogenic assay. The survival fractions were compared. Cells were subjected to temozolomide (TMZ) for 72 h, followed by MTT assay, and the IC50 was compared. Proliferation was studied through clonogenic assay and population doubling time. FACS was used to perform cell cycle analysis. Neurosphere generation was studied by culturing the cells in serum-free nutrient medium for 14 days.
Results and discussion: Radiation-induced cell death was lesser in the mtDNA-depleted cells than that in the parent cell lines (p=0.002). IC50 for TMZ was higher in the mtDNA-depleted cells (U87: 300.8 µM; LN229: 140.8 µM) than that in the parent cells (U87: 232.1 µM, LN229: 64.7 µM) (p=0.001). Therefore, mtDNA-depleted cells are more resistant to radiation and TMZ therapy. The mtDNA-depleted cells proliferated slower than the parent cells as shown by the longer population doubling time in the mtDNA-depleted cells (U87: 119±1 h; LN229: 102±2 h) than that in the parent cells (U87:40.8±3 h; LN229: 30±1 h). The plating efficiency of the mtDNA-depleted cells (U87: 3%±0.5%; LN229: 5%±1%) was also lower than that of the parent cells (U87: 20%±2%; LN229: 32%±1%). Cell cycle analysis showed reduced mitotic (G2/M phase) and synthesis (S) phase populations in the mtDNA-depleted cells when compared to those in the parent cells, showing that the mtDNA-depleted cells are more quiescent than the parent cells. The number of neurospheres formed in the mtDNA-depleted cells (U87: 45±3; LN229: 67±5) was higher than that in the parent cells (U87: 22±2; LN229: 39±2) (p<0.001).
Conclusion: Depletion of mtDNA content in glioblastoma cell lines induces stemness and hence resistance to radiation and TMZ therapy, thus elucidating the role of mtDNA content in glioblastoma treatment resistance.
Clinical profile and treatment outcomes of medulloblastoma: 5-year experience of a referral institution in South India
Jyothis P. Jose, Suresh Kumar, S. G. D. Gangadharan; Department of Medical Oncology, Madras Medical College and Research Institute
Objectives: Medulloblastoma is the most common malignant brain tumor of childhood and occurs in the posterior fossa. Treatment consists of a combined-modality approach that includes surgery, radiation therapy, and chemotherapy in most patients. The aim of this study was to determine the epidemiological patterns of medulloblastoma in a tertiary care center in Southern India.
Materials and Methods: We undertook a retrospective review of medulloblastoma cases treated at our center from 2015 to 2020, in which the records of all the clinically diagnosed cases in the last 5 years (2015–2020) were analyzed.
Results: During the 5-year period, 42 cases were reviewed, with the mean age at diagnosis being 8 years. There was a slight predilection for the male sex (64.8%). The initial presenting symptom was mostly related to raised intracranial pressure and cerebellar dysfunction. Multimodality treatments included surgery and concurrent craniospinal irradiation, followed by postoperative chemotherapy. Most of the patients showed only a partial response to treatment, mainly because of large tumors at presentation, which could be attributed to the lack of awareness, delayed medical attention, and poor follow-up.
Conclusions: Early diagnosis and treatment is the key to management of medulloblastoma, which still needs to be achieved. Factors that are associated with worse prognosis include young age, disseminated or metastatic disease at the time of diagnosis, residual disease after resection, and large cell and anaplastic histology. The patient should be risk stratified for proper management and referred to experienced centers that have capabilities for modern multimodality therapy.
Diffuse midline gliomas with H3K27 mutation—Clinicopathological correlates
John Abha, A. G. Chacko, R. Moorthy, B. V. Joseph, M. Bindra, G. Chacko, B. Selvamani, Rikki R. John, Gowri Mahasampath; CMC Vellore
Objectives: H3K27M mutant diffuse midline gliomas are infiltrative, midline high-grade gliomas with a K27M mutation in either H3F3A or HISTIH3B/HIST1H3C and have been reported in literature to have a 2-year survival of <10%. The objectives of this study were to assess the morphology, the H3K27M status, and clinical outcomes in previously diagnosed midline gliomas and compare H3K27M positive and negative cases.
Materials and Methods: Seventy-three cases of diffuse midline gliomas of World Health Organization (WHO) grades II–IV over a 9.5-year study period underwent H32K7M immunohistochemical evaluation. Morphological parameters and clinical details such as gender, age, site of the tumor, and outcomes in the form of recurrence-free survival (RFS) and overall survival (OS) were assessed and were correlated with mutational status.
Results: Forty-four of the 73 tumors were positive for the H3K27M mutation. H3K27M-mutant diffuse midline gliomas occurred more commonly in adults and in the thalamus. They corresponded most often to a WHO Grade III histology. These tumors showed a range of histological features and H3K27M mutation positivity could not be predicted by any one parameter. The MIB-1% was significantly higher in the mutant group of tumors (p=0.03). When compared to the wild group, the H3K27M-mutant group showed better OS (p=0.66) and RFS (p=0.37). In the cohort of H3K27M-mutant tumors, children showed worse OS when compared to adults (p=0.07). Spinal cord H3K27M-mutant tumors showed shorter OS and RFS compared to wild-type tumors (p=0.25). RFS in the mutant group was worse in the supratentorial tumors compared to that in the infratentorial tumors (p=0.30).
Conclusions: Although literature states that H3K27M-mutant diffuse midline gliomas are more common in children, these tumors occurred more commonly in adults and in the thalamus in our study group. Although they are considered to have a worse prognosis, there are conflicting reports in literature. In the present study, H3K27M-mutant tumors had a better RFS and OS than the wild-type cases. However, among H3K27M-mutant tumors, children had worse OS than adults. Spinal cord H3K27M-mutant tumors had a shorter RFS and OS when compared to brainstem and thalamus H3K27M-mutant tumors.
HSP70 in the role of a new systemic marker for prognosis of glioblastoma: Its targeting can help downregulate the inflammatory response via IL-6 expression
Richa Shrivastava, Sandeep K. Sorte, Aadesh Shrivastava, Puneet Gandhi; Department of Research, Bhopal Memorial Hospital and Research Centre, Bhopal, Madhya Pradesh, India, Department of Neurosurgery, Bhopal Memorial Hospital and Research Centre, Bhopal, Madhya Pradesh, India, Department of Neurosurgery, All India Institute of Medical Sciences, Bhopal, Madhya Pradesh, India
Objectives: Glioblastoma (GB) is the most malignant type of diffuse glioma with a high degree of heterogeneity and poor prognosis. The heat shock proteins (HSPs) are involved in many different mechanisms that govern GB malignancy, demonstrating their relevance for prognosis and as potential therapeutic targets. Our objective was to look into how HSP70 expression in circulation was associated with patient survival and whether it could serve as an independent prognosticator and plausible target for therapeutic intervention.
Materials and Methods: The cohort comprised 36 isocitrate dehydrogenase-wildtype GB patients with neurological deficit, median age 54 years, compared with 36 healthy subjects. A presurgery blood sample was collected as per institutional ethics approval. Herein, candidate biomarkers HSP70 and interleukin-6 (IL6) were considered for quantitative comparison using plasma/serum samples using the enzyme-linked immunosorbent assay technique. Nonparametric statistical analysis was carried out and a p-value <0.05 was considered.
Results: The systemic concentration (median value) of HSP70 was 37.91 ng/ml in GB and was significantly different from controls (p<0.0001). HSP70 marker was found to be significantly negatively correlated with overall survival (r=−0.46; p=0.006). To establish the diagnostic accuracy, AUC for HSP70 was 0.77 at a cut-off >16.7 with >85% specificity. Using log-rank analysis, based on ROC cut-off of HSP70, the survival curve was constructed with a significant hazard ratio (p=0.001). Cox-regression analysis revealed that a higher level of HSP70 marker was as an indicator of poor outcome. Using multivariate analysis, both markers were interlinked with each other (p=0.0114) but were independent of confounding factors, namely, age, site, therapy, KPS, tumor volume, and extent of resection (p>0.05). The median circulating level of IL6 was 99.681 pg/ml, AUC 0.90 with specificity >85%, and threshold >50.89.
Conclusions: This is the first evidence-based study indicating that the HSP70 can serve as a potential noninvasive prognosticator of GB. Current findings also suggest that it can serve as a direct target for inhibitors like N-amino-ethylamino derivative of colchicine for better clinical outcome and by downregulating the inflammation due to its positive association with IL6.
Keywords: Circulating, glioblastoma, HSP-70, IL-6, molecular markers, prognosis
Acknowledgment Women Scientist Scheme (WOS-A), Department of Science & Technology, New Delhi for fellowship to RS, WOS-A/LS-684/2016.
Glioblastoma cells survive genotoxic stress via GCN5-mediated modulation of DNA double-strand break repair
Debashmita Sarkar, Saket V. Mishra, Madhura Ketkar, Shilpee Dutt; Shilpee Dutt Laboratory, Tata Memorial Centre, Advanced Centre for Treatment, Research and Education in Cancer, Navi Mumbai Homi Bhabha National Institute, Training School Complex, Mumbai, Maharashtra, India
Objectives: Modulation of the DNA damage response pathways in glioblastoma (GBM) by novel mechanisms poses challenge to the current treatment modalities as reflected by the high mortality rate. We hypothesize that DSBs generated in GBM during radiation therapy are repaired by molecular complexes distinct from genotoxic responses in normal cells. Here we interrogated the mechanism of DSB repair regulation by GCN5, a histone acetyl transferase in order to understand the differential repair response of GBM to radiotherapy.
Materials and Methods: DSBs in GBM cells were induced by ɣ-irradiation and doxorubicin. Functional studies were done by genetic knockdown and pharmacological inhibition of GCN5. Expression studies were done using western blot and quantitative polymerase chain reaction. Immunoprecipitation followed by mass spectrometry was performed to study interactors of GCN5. DSB repair pathways were studied by westerns, immunofluorescence, and in vivo HR–NHEJ reporter assay.
Results: We found that in GBM cells post ɣ-irradiation treatment, GCN5 is recruited to DNA DSBs within 30 min. Recruitment of GCN5 to DSBs post irradiation was further confirmed by co-localization of GCN5 with gamma-H2AX. Accordingly, GCN5 knockdown led to unresolved ɣ-H2AX foci until 24 h post IR as compared to scrambled control, wherein the gamma-H2AX foci resolved in 6 h. SiRNA-mediated knockdown of GCN5 led to significant decrease in the mRNA and protein levels of DNAPK and phosphorylation of ATM, major kinases of NHEJ and HR repair pathways, respectively, whereas the expression of other proteins such as Mre11, Rad50, Rad51, ATR, and Ku80 remained unaffected. To identify binding partners of GCN5 post induction of DSBs, immunoprecipitation of GCN5 followed by mass spectrometric analysis was done, which identified DNAPK as the potential binding partner of GCN5. Physical interaction of GCN5, DNAPK, and ATM was further confirmed by immunoprecipitation of GCN5, followed by western analysis. Pharmacological inhibition of GCN5 by butyrolactone-3 yielded similar results, suggesting the importance of acetyltransferase activity of GCN5 in mediating DSB repair. Accordingly, DNAPK was found to be significantly acetylated post DSB induction. Finally, we demonstrated that in irradiated cells, both HR and NHEJ repair efficiencies as assessed by HR–NHEJ in vivo reporter assays were significantly reduced upon GCN5 knockdown.
Conclusion: Taken together, we identified GCN5 as an atypical protein that is recruited to double-strand breaks and augment survival of GBM cells under genotoxic stress by regulating both HR and NHEJ repair via controlling ATM and DNAPK signaling, respectively. Therefore, this study identifies GCN5 as a novel molecular target in GBM therapeutics.
Clinical audit of long-term outcomes and prognostic factors in adolescent and young adults with medulloblastoma
Madan Maitre, Roshankumar Patil, Archya Dasgupta, Ayushi Sahay, Sridhar Epari, Neelam Shirsat, Abhishek Chatterjee, Rahul Krishnatry, Jayant Sastri Goda, Aliasgar Moiyadi, Vijay Patil, Girish Chinnaswamy, Nazia Bano, Rakesh Jalali, Tejpal Gupta; Tata Memorial Hospital, Mumbai, Maharashtra, India
Purpose: Medulloblastomas comprising 20%–25% of all primary brain tumors in children are much rarer in adulthood. Disease biology varies substantially across different age groups; however, owing to rarity, adolescents and young adults (AYAs) with medulloblastoma are traditionally treated using pediatric protocols. This is a retrospective audit of AYA medulloblastoma treated at a comprehensive cancer center.
Materials and Methods: Data regarding demography, clinical presentation, imaging characteristics, histopathological features, molecular profiling, risk stratification, treatment details, and outcomes were retrieved from medical records. All time-to event outcomes were analyzed using the Kaplan-Meier method and compared with the log-rank test. Univariate and multi-variate analyses of relevant prognostic factors were done, and p-value <0.05 was considered statistically significant.
Results: A total of 162 patients aged ≥15 years with medulloblastoma were included. Median age of the study cohort was 25 years (range: 15–59 years) with leptomeningeal metastases seen in 31 (19%) patients at initial diagnosis. Following surgery, patients were treated with appropriate risk-stratified adjuvant therapy comprising craniospinal irradiation plus boost with or without systemic chemotherapy. At a median follow-up of 50 months, 5-year Kaplan–Meier estimates of progression-free survival and overall survival were 53.5% and 59.5%, respectively, for the study cohort. High-risk disease and anaplastic histology emerged as significant and independent predictors of poor survival on multi-variate analysis.
Conclusions: Medulloblastoma is a rare tumor in the AYA cohort with key differences in disease biology and resultant outcomes compared to that in the pediatric population. Contemporary management of AYA medulloblastoma comprising maximal safe resection followed by appropriate risk-stratified adjuvant therapy provides acceptable long-term outcomes.
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