Pediatric Pilocytic Astrocytoma Research Articles

Proteomic Profiling of Cerebrospinal Fluid and Its Extracellular Vesicles from Extraventricular Drainage in Pediatric Pilocytic Astrocytoma, towards Precision Oncology.

Pediatric pilocytic astrocytoma (PA) is the most common brain tumor in children. Complete resection provides a favorable prognosis, except for unresectable PA forms. There is an incomplete understanding of the molecular and cellular pathogenesis of PA. Potential biomarkers for PA patients, especially the non-BRAF-mutated ones are needed. Cerebrospinal fluid (CSF) is a valuable source of brain tumor biomarkers. Extracellular vesicles (EVs), circulating in CSF, express valuable disease targets. These can be isolated from CSF from waste extraventricular drainage (EVD). We analyzed the proteome of EVD CSF from PA, congenital hydrocephalus (CH, non-tumor control), or medulloblastoma (MB, unrelated tumoral control) patients. A total of 3072 proteins were identified, 47.1%, 65.6%, and 86.2% of which were expressed in the unprocessed total and in its large-EV (LEV), and small-EV (SEV) fractions. Bioinformatics identified 50 statistically significant proteins in the comparison between PA and HC, and PA and MB patients, in the same fractions. Kinase enrichment analysis predicted five enriched kinases involved in signaling. Among these, only Cyclin-dependent kinase 2 (CDK2) kinase was overexpressed in PA samples. PLS-DA highlighted the inactive carboxypeptidase-like protein X2 (CPXM2) and aquaporin-4 (AQP4) as statistically significant in all the comparisons, with CPXM2 being overexpressed (validated by ELISA and Western blot) and AQP4 downregulated in PA. These proteins were considered the most promising potential biomarkers for discriminating among pilocytic astrocytoma and unrelated tumoral (MB) or non-tumoral conditions in all the fractions examined, and are proposed to be prospectively validated in the plasma for translational medicine applications.

Generation of patient-derived pediatric pilocytic astrocytoma in-vitro models using SV40 large T: evaluation of a modeling workflow

PurposeAlthough pediatric low-grade gliomas (pLGG) are the most common pediatric brain tumors, patient-derived cell lines reflecting pLGG biology in culture are scarce. This also applies to the most common pLGG subtype pilocytic astrocytoma (PA). Conventional cell culture approaches adapted from higher-grade tumors fail in PA due to oncogene-induced senescence (OIS) driving tumor cells into arrest. Here, we describe a PA modeling workflow using the Simian Virus large T antigen (SV40-TAg) to circumvent OIS.Methods18 pLGG tissue samples (17 (94%) histological and/or molecular diagnosis PA) were mechanically dissociated. Tumor cell positive-selection using A2B5 was perfomed in 8/18 (44%) cases. All primary cell suspensions were seeded in Neural Stem Cell Medium (NSM) and Astrocyte Basal Medium (ABM). Resulting short-term cultures were infected with SV40-TAg lentivirus. Detection of tumor specific alterations (BRAF-duplication and BRAF V600E-mutation) by digital droplet PCR (ddPCR) at defined time points allowed for determination of tumor cell fraction (TCF) and evaluation of the workflow. DNA-methylation profiling and gene-panel sequencing were used for molecular profiling of primary samples.ResultsPrimary cell suspensions had a mean TCF of 55% (+/− 23% (SD)). No sample in NSM (0/18) and ten samples in ABM (10/18) were successfully transduced. Three of these ten (30%) converted into long-term pLGG cell lines (TCF 100%), while TCF declined to 0% (outgrowth of microenvironmental cells) in 7/10 (70%) cultures. Young patient age was associated with successful model establishment.ConclusionA subset of primary PA cultures can be converted into long-term cell lines using SV40-TAg depending on sample intrinsic (patient age) and extrinsic workflow-related (e.g. type of medium, successful transduction) parameters. Careful monitoring of sample-intrinsic and extrinsic factors optimizes the process.

TMIC-13. EXHAUSTED CD8+ T CELLS MAINTAIN LOW-GRADE GLIOMAS IN NF1-MUTANT MICE

Abstract T cells are minor cellular constituents of the brain tumor microenvironment, but have recently been shown to play critical roles in glioma pathobiology and treatment. Previous studies from our laboratory have shown that T cells are required for the formation and growth of low-grade gliomas (LGGs) in murine models of the Neurofibromatosis type 1 (NF1) tumor predisposition syndrome. To better characterize the immune landscape of these pediatric low-grade tumors, we leveraged single cell RNA sequencing of human pediatric pilocytic astrocytomas and genetically engineered mouse LGG (Nf1-optic glioma) models. First, we showed that both human and murine LGGs contain a CD8+ T cell population expressing PD-1 and TIGIT, similar to conventional exhausted T cell populations. Second, these PD-1+/TIGIT+ CD8+ T cells are actively recruited to the tumor site by Ccl2 where they dictate tumorigenesis through the elaboration of Ccl4, a cytokine necessary for microglia Ccl5-mediated support of Nf1-OPG growth. As such, Ccl4 expression was enriched in PD-1+/TIGIT+ CD8+ T cells relative to all other T cell populations within these tumors. Third, this “exhausted” CD8+ T cell population was exclusively present in the optic nerves, but not in the blood, dura, or deep cervical nodes of optic glioma-bearing mice. Fourth, targeting this PD1+/TIGIT+ exhausted CD8+ T cell population with immune checkpoint monoclonal antibody inhibitors reduced CD8+ T cell tumoral recruitment, Ccl4 production, and optic glioma growth. Collectively, these findings establish an obligate role for PD-1+/TIGIT+ CD8+ T cells in Nf1-optic glioma control and suggest that immune checkpoint modulation may be useful for the treatment of LGGs in children.

Magnetic resonance imaging characteristics of pediatric pilocytic astrocytoma.

MRI has an important role in diagnosing pilocytic astrocytoma and post-surgical follow-up since the surgical approach has a leading role in its treatment. The purpose of our study is to provide an overview of the typical and atypical MRI findings in a series of pediatric patients with isolated-not NF1-related-pilocytic astrocytomas and to correlate specific MRI patterns with clinical variables. This is a cross-sectional retrospective study providing the analysis of several clinical and neuroradiological findings from a cohort of pediatric pilocytic astrocytoma, starting from the data collected in the Fondazione IRCCS Istituto Neurologico Carlo Besta (FINCB) internal Cancer Registry during an 11-year time period (January 2008-January 2019). Fifty-six patients were included in the study. Median age at diagnosis was 9.4 years; a slight female prevalence was noticed (m/f ratio 44.6%/55.4%). The majority of pPAs had well-defined contours: 51 (91.1%), 47 (88.7%) were hypointense on T1-wi, all of them were hyperintense on T2-wi, 46 (90.2%) were hyperintense on FLAIR, and 48 (85.7%) were heterogeneous on T1-wi and T2-wi sequences. We found positive correlation between pPAs location and age (r = 0.017), and small degree of connection between pPAs location and gender (Cramer's V = 0.268). We presented typical and atypical pPAs MRI findings. Age and tumor location were positevely correlated, while degree of connection between gender and pPAs location was small. All of this may aid clinicians, most of all neuroradiologists, neurosurgeons, and neurologists in proper diagnoses and follow-up of these specific patient population.

LGG-27. Molecular implications of mitogen-activated protein kinase pathway inhibition by the MEK inhibitor trametinib in BRAF-fusion-driven pediatric pilocytic astrocytoma

Pilocytic astrocytomas (PA) are the most common pediatric brain tumors. They are characterized by driving alterations in the mitogen-activated protein kinase (MAPK) pathway, leading to its constitutive activation and modulating the balance between cell proliferation and oncogene-induced senescence (OIS) sustained by senescence-associated secretory phenotype (SASP) factors. This makes PA susceptible to MAPK inhibitor (MAPKi) therapies, which show encouraging results in phase 1/2 clinical trials. However, little is known about the molecular implications of MAPK inhibition in PA. The DKFZ-BT66 cell line, derived from a primary KIAA:BRAF-fusion positive PA, was used as a model system. DKFZ-BT66 were treated with the MEKi trametinib for different durations in both proliferative and senescent states. Gene expression was analyzed by gene expression profiling and protein expression/phospho-regulation by data-dependent mass spectrometry followed by label-free quantitative analysis. A time course analysis based on differentially expressed genes and phosphorylated proteins was performed, followed by a single-sample gene set enrichment analysis (ssGSEA) and kinase substrate enrichment analysis, respectively. Differential gene expression analysis revealed that MEK inhibition led to the inhibition of the OIS/SASP gene programs in senescent DKFZ-BT66, with downregulation of key OIS/SASP partners such as IL1B on the protein level. This functionally translated into a de-sensitization of these cells towards the senolytic agent navitoclax. ssGSEA showed that most MAPK-related signatures were downregulated upon MEKi treatment, while pathways related to upstream MAPK activators (including FGFR, NTRK and TGFB pathways) were upregulated, in both proliferating and senescent DKFZ-BT66. This data indicates that MAPKi reverses OIS in senescent PA cells, while inducing the activation of MAPK upstream regulators in proliferating and senescent PA cells, identifying putative co-targets that could help increase treatment’s efficacy. Validation of these targets by post-translational modification enrichment analysis of the phospho-proteomics dataset is ongoing.

OTHR-10. Pilocytic astrocytoma with respect to treatment

AIM: To describe the sizes of pilocytic astrocytoma with respect to treatment Methodology Pediatric pilocytic astrocytomas cases from 2001 to 2021 were retrospectively reviewed in this Institutional Review Board approved study. Imaging reports, location of tumour, maximum dimension of tumour at diagnosis, treatment given (operation/chemotherapy/ radiotherapy), degree of tumor excision were captured. RESULTS: Imaging was available in 33 with 23 centered in the posterior fossa (1 extending into thalamus), 4 in suprasellar region, 2 in cerebral hemisphere, 2 in thalamus, 1 in pineal thalamic region and 1 in cervicomedullary spine, Tumor dimension at presentation was 5.40 cm ± 2.34 cm. Tumor size at presentation did not show significant correlation with age. 30 patients underwent operation with tumours completely excised in 15 and partially excised in 14 and no postoperative information for 1. Three patients, where tumour involved the thalamus, did not have operation and were given radiotherapy, average size of tumour being 3.47 + 1.15 cm. compared to the 5.59 + 2.34 size of tumours that underwent operation (p=0.06). Completely excised tumours measured 6.29 ± 2.04 cm at presentation while incompletely excised ones measured 4.76 ± 2.53 cm, not significantly different (p=0.09). Unoperated tumours are statistically smaller than those completely excised (p=0.02). One of the completetly excised tumours was located in the parietal cerebral hemisphere with the rest of the 15 in the posterior fossa. Seven of the incompletely excised tumours were located in the posterior fossa with 4 in suprasellar region, 1 in thalamus, 1 in spine and 1 in cerebral hemisphere. 3 patients with uncompletely excised tumours (1 cerebral, 1 post fossa, 1 spine) had post-operative radiation while 2 suprasellar tumours were given post-operative chemotherapy. CONCLUSION: Completely excised tumours are mainly located in posterior fossa. Tumours not operated on are located in thalamus and significantly smaller than tumours which are completely excised.

Genetic and histopathological associations with outcome in pediatric pilocytic astrocytoma.

Pilocytic astrocytomas (PAs) have a generally favorable prognosis; however, progression or recurrence after resection is possible. The prognostic value of histopathological qualifiers (defined below) or BRAF alterations is not well understood. The aim of this study was to identify the prognostic value of genetic and histopathological features of pediatric PAs. Patients treated for a WHO grade I PA at a single institution were analyzed for histopathological and genetic features and outcomes. "Histopathological qualifier" refers to designations such as "WHO grade I PA with increased proliferative index." BRAF alterations include gene fusions and point mutations. Patients with neurofibromatosis type 1 were excluded. A total of 222 patients were analyzed (51% female, mean age 9.6 years). Tumors were located in the cerebellum/fourth ventricle (51%), optic pathway/hypothalamus (15%), brainstem (12%), and cerebral cortex (11%). BRAF alterations were screened for in 77 patients and identified in 56 (73%). Histopathological qualifiers were present in 27 patients (14%). Resection was performed in 197 patients (89%), 41 (21%) of whom displayed tumor progression or recurrence after resection. Tumor progression or recurrence was not associated with histopathologic qualifiers (p = 0.36) or BRAF alterations (p = 0.77). Ki-67 proliferative indices were not predictive of progression or recurrence (p = 0.94). BRAF alterations, specifically KIAA1549 fusions, were associated with cerebellar/fourth ventricular tumor location (p < 0.0001) and younger patient age (p = 0.03). Patients in whom gross-total resection was achieved had lower rates of progression and recurrence (p < 0.0001). Histopathological features/qualifiers and BRAF alterations were not associated with tumor recurrence/progression in pediatric PAs. The extent of resection was the only factor analyzed that predicted outcome.

Primary pilocytic astrocytoma of the cerebellopontine angle in pediatric patient with neurofibromatosis type 1: a case report

BackgroundCerebellopontine angle tumor (CPA) in pediatrics is rare as compared to adults. We describe a case of pediatric pilocytic astrocytoma presented as a right CPA mass with a concurrent clinical diagnosis of neurofibromatosis type 1 (NF1).Case presentationA 14-year-old boy with a newly diagnosed hypertension presented with a short history of headache and blurring vision. Neurological examination revealed bilateral papilloedema, partial right third nerve palsy and mild sensorineuronal hearing deficits. Skin examination identified multiple café au lait spots with cutaneous neurofibromas. Preoperative neuroimaging suggested the diagnosis of an extraaxial CPA mass consistent with meningioma, with obstructive hydrocephalus. A left ventriculoperitoneal shunt was inserted and the child was subjected for a suboccipital retrosigmoid approach for tumor resection. The histopathological features, however, were typical for pilocytic astrocytoma.ConclusionsA careful evaluation of the clinical presentation and radiological images of CPA lesions is necessary prior to surgical embarkment. To the best of our knowledge, this case is the first report of pilocytic astrocytoma in the CPA in pediatric with NF1.

Comparison of adult and pediatric pilocytic astrocytomas using competing risk analysis: A population-based study

BackgroundPilocytic astrocytoma(PA) is a relatively benign tumor occurring primarily in the pediatric population. Comparison of characteristics and survival of this tumor between adult and pediatric patients in a single, population-based study is yet to be performed. ObjectiveWe aimed to directly compare the characteristics and survival of pilocytic astrocytoma between pediatric and adult patients in a single, population-based study. MethodsWe utilized the SEER database using data from 1983 to 2016. All patients with histologically confirmed, intracranial pilocytic astrocytoma were included and divided into a pediatric(age<18 years) or adult group. Due to lower risk of tumor-specific-mortality, we utilized a competing risk analysis to account for mortality from other causes. Univariable and multivariable competing risk analysis(CRA) was performed, and sub-distribution hazard ratio(SHR) or adjusted SHR(aSHR) was reported. ResultsA total of 4357 patients comprised the final cohort, with 3014(69.2%) pediatric patients. As compared to the pediatric group, adult patients were predominantly White(p < 0.01), with PA less likely fully resected(p = 0.01), smaller tumor size(p < 0.01), and were less often located in the cerebellum(p < 0.01). Multivariable CRA revealed a worse prognosis for the adult group(p < 0.01), regional or distal extension(p < 0.01), and non-cerebellar locations including frontal(p < 0.01), parietal(p < 0.01), ventricular(p < 0.01) or brainstem(p < 0.01). Improved prognosis was seen with more recent year-of-diagnosis(2003–2016, p = 0.03), gross-total/total resection(p < 0.01), and biopsy only patients(p = 0.02). ConclusionsPilocytic astrocytomas in adult patients have a worse prognosis than pediatric patients. Cumulative incidence of cancer-specific-mortality is higher in adults when adjusted for other factors. PAs with regional or distal extension and non-cerebellar locations carry worse outcomes. Surgery remains an effective treatment and GTR/TR should be achieved when possible.

LGG-07. IS BRAF ALTERATION OR A HISTOLOGIC ‘QUALIFIER’ A PREDICTOR OF OUTCOME IN PEDIATRIC PILOCYTIC ASTROCYTOMA?

IntroductionPediatric pilocytic astrocytomas (PA) are the most common pediatric central nervous system tumor. Surgical resection is the primary treatment for PA with five-year survival rates up to 95%. Despite a favorable prognosis, our understanding about the prognostic value of histopathological findings, such as histopathologic qualifier* or BRAF alterations is evolving. MethodsPatients treated for a WHO grade 1 PA at Washington University in St. Louis/St. Louis Children’s Hospital were analyzed for clinical details, including pathology diagnosis (*histopathologic qualifier refers to designations in the diagnosis such as “WHO Grade I pilocytic astrocytoma with increased proliferative index”). BRAF alterations include gene fusions and point mutations. Results224 patients were analyzed (51% female, mean age 9.6 years). Tumors were located in the cerebellum/fourth ventricle (50%), optic pathway/hypothalamus (15%), brainstem (12%), and cerebral cortex (11%). BRAF alterations were identified in 55/77 patients (71.4%) and additional histopathologic qualifiers were present in 27/220 patients (12.3%). 196 patients (87.5%) underwent surgical treatment and 22 (9.8%) had biopsy alone. 45 patients (22%) displayed tumor progression or recurrence after resection. The presence of a histopathologic ‘qualifier’ in the topline or BRAF alteration was not associated with tumor progression or recurrence (p=0.36, p=0.77). Ki-67 proliferative indices were not predictive of progression or recurrence (p=0.94), including when controlling for extent of resection and adjuvant therapy. BRAF alterations, specifically KIAA1549 fusions, were associated with cerebellar/fourth ventricular tumor location (p<0.001) and younger patient age (p=0.03). Extent of resection was the only predictor of outcome identified in this study; patients with gross total resection had significantly lower rates of progression and recurrence (p<0.0001). Conclusion BRAF alterations and histopathologic qualifiers were not associated with tumor progression or recurrence in pediatric PA, although BRAF fusions were more common in tumors located in the cerebellum/fourth ventricle and in younger patients.

HTERT Transduction Extends the Lifespan of Primary Pediatric Low-Grade Glioma Cells While Preserving the Biological Response to NGF.

The neurotrophin nerve growth factor (NGF) modulates the growth of human gliomas and is able to induce cell differentiation through the engagement of tropomyosin receptor kinase A (TrkA) receptor, although the role played in controlling glioma survival has proved controversial. Unfortunately, the slow growth rate of low-grade gliomas (LGG) has made it difficult to investigate NGF effects on these tumors in preclinical models. In fact, patient-derived low-grade human astrocytoma cells duplicate only a limited number of times in culture before undergoing senescence. Nevertheless, replicative senescence can be counteracted by overexpression of hTERT, the catalytic subunit of telomerase, which potentially increases the proliferative potential of human cells without inducing cancer-associated changes. We have extended, by hTERT transduction, the proliferative in vitro potential of a human LGG cell line derived from a pediatric pilocytic astrocytoma (PA) surgical sample. Remarkably, the hTERT-transduced LGG cells showed a behavior similar to that of the parental line in terms of biological responses to NGF treatment, including molecular events associated with induction of NGF-related differentiation. Therefore, transduction of LGG cells with hTERT can provide a valid approach to increase the in vitro life-span of patient-derived astrocytoma primary cultures, characterized by a finite proliferative potential.

The age of adult pilocytic astrocytoma cells

Adult pilocytic astrocytomas (PAs) have been regarded as indistinguishable from pediatric PAs in terms of genome-wide expression and methylation patterns. It has been unclear whether adult PAs arise early in life and remain asymptomatic until adulthood, or whether they develop during adulthood. We sought to determine the age and origin of adult human PAs using two types of “marks” in the genomic DNA. First, we analyzed the DNA methylation patterns of adult and pediatric PAs to distinguish between PAs of different anatomic locations (n = 257 PA and control brain tissues). Second, we measured the concentration of nuclear bomb test-derived 14C in genomic DNA (n = 14 cases), which indicates the time point of the formation of human cell populations. Our data suggest that adult and pediatric PAs developing in the infratentorial brain are closely related and potentially develop from precursor cells early in life, whereas supratentorial PAs might show age and location-specific differences.

Immune Checkpoint-Associated Locations of Diffuse Gliomas Comparing Pediatric With Adult Patients Based on Voxel-Wise Analysis.

Objective: Pediatric diffuse gliomas (pDGs) are relatively rare and molecularly distinct from pediatric pilocytic astrocytoma and adult DGs. Immunotherapy is a promising therapeutic strategy, requiring a deep understanding of tumor immune profiles. The spatial locations of brain tumors might be related to the molecular profiles. We aimed to analyze the relationship between the immune checkpoint molecules with the locations of DGs comparing pediatric with adult patients.Method: We studied 20 pDGs patients (age ≤ 21 years old), and 20 paired adult patients according to gender and histological types selected from 641 adult patients with DGs. Immune checkpoint molecules including B7-H3, CD47, and PD-L1, as well as tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs), were manifested by immunohistochemical staining. Expression difference analyses and Spearman's correlation were performed. MRI data were voxel-wise normalized, segmented, and analyzed by Fisher's exact test to construct the tumor frequency and p value heatmaps. Survival analyses were conducted by Log-rank tests.Result: The median age of pediatric patients was 16 years. 55% and 30% of patients were WHO II and III grades, respectively. The left frontal lobe and right cerebellum were the statistically significant locations for pDGs, while the anterior horn of ventricles for adult DGs. A potential association between the expression of PD-L1 and TAMs was found in pDGs (p = 0.002, R = 0.670). The right posterior external capsule and the lateral side of the anterior horn of the left ventricle were predominant locations for the adult patients with high expression of B7-H3 and low expression of PD-L1 compared to pediatric ones, respectively. Pediatric patients showed significantly improved overall survival compared with adults. The prognostic roles of immune checkpoint molecules and TILs/TAMs were not significantly different between the two groups.Conclusion: Immune checkpoint-associated locations of diffuse gliomas comparing pediatric with adult patients could be helpful for the immunotherapy decisions and design of clinical trials.

Cytokines in Pediatric Pilocytic Astrocytomas: A Clinico-Pathological Study

Pilocytic astrocytomas (PCA) are WHO Grade I tumors with a favorable prognosis. Surgical resection is usually curative. Nonetheless, progressive and/or metastatic disease occurs in 20% of patients. For these patients, treatment options are limited. The role of the immune system in PCA has not previously been reported. We hypothesize that the circulating cytokines contribute to tumorigenicity in PCA. This is an exploratory study with a focus on the identification of circulating cerebrospinal (CSF) cytokines associated with PCA. The primary objective is to demonstrate that CSF cytokines will be differentially expressed in the subset of PCAs that are difficult to treat in comparison to their surgically amendable counterparts. This is a single-institution, retrospective study of prospectively collected data. Patients with a confirmed histological diagnosis of PCA who have simultaneous intraoperative CSF sampling are included. Cerebrospinal fluid samples are subjected to multiplex cytokine profiling. Patient-derived PCA lines from selected patients in the same study cohort are cultured. Their cell culture supernatants are collected and interrogated using the sample multiplex platform as the CSF. A total of 8 patients are recruited. There were two patients with surgically difficult tumors associated with leptomeningeal involvement. Multiplex profiling of the cohort’s CSF samples showed elevated expressions of IFN-γ, IL-2, IL-12p70, IL-1β, IL-4, and TNF-α in these two patients in comparison to the remaining cohort. Next, primary cell lines derived from the same PCA patients demonstrated a similar trend of differential cytokine expression in their cell culture supernatant in vitro. Although our findings are preliminary at this stage, this is the first study in pediatric PCAs that show cytokine expression differences between the two groups of PCA with different clinical behaviors.

Adult pilocytic astrocytoma in the molecular era: a comprehensive review.

Adult pilocytic astrocytoma (PA) is less prevalent than pediatric PA and is associated with a worse prognosis. In a literature review, we found that 88.3% of the molecular alterations in adult PA are associated with MAPK pathway dysregulation. The most common alterations are fusions of BRAF. Understanding of the mechanisms underlying this pathway has evolved substantially, heralding advancements in specific targeted therapy. Here, we review clinical and molecular features of adult PA, characteristics predicting aggressive behavior and approaches to standard and investigational therapies. We highlight epigenetic profiling and integrated diagnosis as an essential component of classifying PA.

Is it a new culprit? “TERT promoter mutation” in an aggressive pediatric pilocytic astrocytoma

Recent advances in the genomic study have revolutionized the discovery of molecular biomarkers in glioma not only to aid in targeted therapy but also to prognosticate character of tumor and outcome of a patient.The usually benign nature of pilocytic astrocytoma (PA) can now be challenged with the discovery of genomic alterations that could promote more aggressive clinical behavior. CDKN2A deletion and ATRX gene inactivation or mutation have been the most common molecular alterations in worse prognosis.We will discuss a case of pilocytic astrocytoma showing the progressive recurrence with pilomyxoid features and the presence of TERT promoter mutation.

Novel Surgical and Radiologic Risk Factors for Progression or Recurrence of Pediatric Pilocytic Astrocytoma

Introduction: Pilocytic astrocytomas (PA) are a common, benign childhood tumor known for their slow growth rates and excellent prognosis. The aim of our study was to characterize patient, tumor, and imaging-related risk factors for recurrence and progression of disease. Methods: We identified 116 patients with PA who underwent surgery at our institution between 2000 and 2015. Data were collected retrospectively from the clinical charts. Results: The mean age at resection was 7 ± 5 years (range 0.5–31) and mean follow-up was 6 ± 3 years. Initial resection was complete in 33 patients (29%), subtotal in 78 patients (67%), and biopsy in 5 patients (4%). A total of 45/116 (40%) patients experienced either recurrence or progression after initial resection with a mean time to recurrence or progression of 2.2 years. Bivariate analysis identified subtotal resection, tumor location, age at diagnosis, and imaging features (i.e., T2 invasion, exophytic component, hemorrhage, and solid tumors) as factors significantly associated with recurrence or progression (p < 0.05). Conversely, PAs that were completely resected, predominately cystic, and located in the cerebellum were significantly associated with no recurrence or progression (p < 0.05). Multivariate regression analysis narrowed down 4 robust risk factors: extent of resection, T2 invasion, predominantly solid lesions, and presence of an exophytic component (p < 0.05). Conclusion: Total surgical removal of PA has been the most important prognostic factor for the clinical course of PA. Our study reveals additional risk factors for the recurrence or progression of disease: tumor invasion, solid composition, and tumors with an exophytic component.

P04.19 Analysis of tumor immune microenvironment and immune checkpoint modulators across infantile and pediatric pilocytic astrocytomas to elucidate the role of immunotherapy in these tumors

Abstract BACKGROUND Pilocytic astrocytomas are the most common central nervous system tumors in pediatric age-group. Although grade I, some of the cases show recurrence and progression, and few might not be amenable to surgery due to location or size, and hence have a less favorable prognosis. Drugs blocking immune check-point interactions such as those including programmed cell death ligand-1 (PD-L1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) are now in clinical use for certain tumors. We performed this study to understand the potential candidature of pilocytic astrocytomas in infants and children for immunotherapy by analyzing the expression of immune checkpoint proteins and immune infiltrate, and correlating with clinical details, wherever possible. MATERIALS AND METHODS Cases with adequate tissue (2010–2017) diagnosed in pediatric age-group (&lt;18 years) were retreived from the archives of Department of Pathology, AIIMS, New Delhi. Immunohistochemistry for PD-L1 (SP263, Ventana), CTLA-4, CD3, CD8, CD4 and CD68 was performed. Quantification of cytotoxic lymphocytes was done using digital imaging in the core of the tumor. RESULTS A total of 50 pilocytic astrocytomas were included, 14 of them were &lt;3 years (infants), while 36 were of pediatric age-group (3–18 years). Overall, male preponderance was noted. Cerebellum was the most common location, followed by 4th venrticle, optic pathway, hypothalamus, cerebrum and thalamus. Almost all CD3 lymphocytes were cytotoxic T-lymphocyes (CD8 positive, CTLs). Helper T-lymphocyte infiltration was not seen. Median CTL density/mm3 was 13/mm3(Range:1–85/mm3). CTLA-4 was positive in 4 cases, positivity ranged from 1–4 cells/lpf. PD-L1 was found to be positive in 7 cases, and the positivity ranged from 1+ to 2+ in 1 to 5% of tumor cells. A median TAM (tumor associated macrophages) density of 44/hpf (range: 1–98/hpf) was noted. There was no correlation of CTL density with PD-L1 or CTLA-4 expression, and neither with TAM density. On correlation with clinical parameters, a higher density of CTLs and TAMs was noted in infants, and a higher proportion of cases revealed PD-L1 positivity, though not statistically significant. There was no correlation of TILs or TAMs with the tumor location. CONCLUSION Immune check point blockade using PD-(L)1 or CTLA4 inhibitors may not be a potential therapeutic option for unresectable or recurrent pilocytic astrocytomas, as low positivity rate as well as extremely low percentage of tumor/ immune cells found to be positive. However, alternate forms of immunotherapy might be helpful as most of the cases showed immune infiltrates and a high density of tumor-associated macrophages (TAMs). Large scale studies with larger numbers and longer follow-up periods including in-vitro and clinical studies are warranted for decoding the tumor immunogram.

The Senescence-associated Secretory Phenotype Mediates Oncogene-induced Senescence in Pediatric Pilocytic Astrocytoma.

Pilocytic astrocytoma is the most common childhood brain tumor, characterized by constitutive MAPK activation. MAPK signaling induces oncogene-induced senescence (OIS), which may cause unpredictable growth behavior of pilocytic astrocytomas. The senescence-associated secretory phenotype (SASP) has been shown to regulate OIS, but its role in pilocytic astrocytoma remains unknown.Experimental Design: The patient-derived pilocytic astrocytoma cell culture model, DKFZ-BT66, was used to demonstrate presence of the SASP and analyze its impact on OIS in pilocytic astrocytoma. The model allows for doxycycline-inducible switching between proliferation and OIS. Both states were studied using gene expression profiling (GEP), Western blot, ELISA, and cell viability testing. Primary pilocytic astrocytoma tumors were analyzed by GEP and multiplex assay. SASP factors were upregulated in primary human and murine pilocytic astrocytoma and during OIS in DKFZ-BT66 cells. Conditioned medium induced growth arrest of proliferating pilocytic astrocytoma cells. The SASP factors IL1B and IL6 were upregulated in primary pilocytic astrocytoma, and both pathways were regulated during OIS in DKFZ-BT66. Stimulation with rIL1B but not rIL6 reduced growth of DKFZ-BT66 cells and induced the SASP. Anti-inflammatory treatment with dexamethasone induced regrowth of senescent cells and inhibited the SASP. Senescent DKFZ-BT66 cells responded to senolytic BCL2 inhibitors. High IL1B and SASP expression in pilocytic astrocytoma tumors was associated with favorable progression-free survival. We provide evidence for the SASP regulating OIS in pediatric pilocytic astrocytoma, with IL1B as a relevant mediator. SASP expression could enable prediction of progression in patients with pilocytic astrocytoma. Further investigation of the SASP driving the unpredictable growth of pilocytic astrocytomas, and its possible therapeutic application, is warranted.

MicroRNA profile of pediatric pilocytic astrocytomas identifies two tumor-specific signatures when compared to non-neoplastic white matter.

Pilocytic astrocytoma (PA) is a low-grade neoplasm frequently found in childhood. PA is characterized by slow growth and a relatively good prognosis. Genetic mechanisms such as activation of MAPK, BRAF gene deregulation and neurofibromatosis type 1 (NF1) syndrome have been associated with PA development. Epigenetic signature and miRNA expression profile are providing new insights about different types of tumor, including PAs. In the present study we evaluated global miRNA expression in 16 microdissected pediatric PA specimens, three NF1-associated PAs and 11 cerebral white matter (WM) samples by the microarray method. An additional cohort of 20 PAs was used to validate by qRT-PCR the expression of six miRNAs differentially expressed in the microarray data. Unsupervised hierarchical clustering analysis distinguished one cluster with nine PAs, including all NF1 cases and a second group consisting of the WM samples and seven PAs. Among 88 differentially expressed miRNAs between PAs and WM samples, the most underexpressed ones regulate classical pathways of tumorigenesis, while the most overexpressed miRNAs are related to pathways such as focal adhesion, P53 signaling pathway and gliomagenesis. The PAs/NF1 presented a subset of underexpressed miRNAs, which was also associated with known deregulated pathways in cancer such as cell cycle and hippo pathway. In summary, our data demonstrate that PA harbors at least two distinct miRNA signatures, including a subgroup of patients with NF1/PA lesions.