Pilocytic Astrocytoma Patients Research Articles

PATH-14. UTILITY OF DNA METHYLATION ANALYSIS IN HISTOLOGICALLY-CONFIRMED PA PATIENTS WITH POOR PROGNOSIS

Abstract INTRODUCTION Pilocytic astrocytoma (PA) is a circumscribed low-grade astrocytic glioma, generally considered to be associated with a good prognosis. However, some PA patients experience unfavorable outcomes; advanced age was known as an independent poor prognostic factor. This study retrospectively reviewed PA patients and conducted molecular analyses for elderly patients. METHODS We analyzed data from 29 histologically confirmed PA patients treated at a single center from 2002 to 2021 and conducted integrated molecular analyses including DNA methylation profiling for elderly PA patients. RESULTS The median age at diagnosis was 14 years (range 3-82 years), with 4 patients (14%) being elderly (patients≥60years old). Elderly patients had significantly worse progression-free survival and overall survival. DNA methylation analysis was performed on 2 of the 4 elderly patients. Despite histological diagnoses of PA, methylation profiling classified one case as high-grade astrocytoma with piloid features (methylation class scores below 0.3 in both v11b4 and v12.5) and the other as glioblastoma, IDH-wildtype (score over 0.5 in both v11b4 and v12.5), using classifiers from the German Cancer Research Center and t-SNE analysis. CONCLUSION Elderly patients with PA morphology showed unfavorable outcomes in this cohort. In those patients, DNA methylation profiling revealed the possibility of high-grade astrocytic tumors, including newly defined entities.

Efficacy of various extent of resection on survival rates of patients with pilocytic astrocytoma: based on a large population

Pilocytic astrocytoma (PA) is classified as a Grade I benign neuroglial tumor. The extent of surgical resection is a critical factor influencing the prognosis for patients with PA. In prior researches of PA, the extent of surgical resection is generally categorized into GTR, STR and biopsy. In some researches on brain tumor surgeries, the extent of resection also includes GTL. There is no existing research specifically comparing the efficacy of GTR versus GTL in PA treatment. In this study, the data we used are from the SEER database. We categorized the extent of resection into GTL, GTR, STL, STR, biopsy, and no surgery based on SEER classification of surgical procedures, to investigate the impact of extent of resection on PA patient survival. A multivariate logistic regression model was utilized to acquire odds ratios (OR) for different extent of resection. Survival outcomes across different extent of resection (GTL, GTR, STL, STR, biopsy, no surgery) were assessed using Kaplan–Meier survival curve analysis, with curve comparisons conducted via log-rank tests. The impact of various risk factors on survival was assessed using the Cox proportional hazards model. The hazard ratio (HR) was employed to quantify the influence of one or more factors on overall survival throughout the follow-up period. Multivariate Cox analysis revealed that age, tumor location, extent of resection, as well as the application of radiotherapy and chemotherapy, all significantly impacted prognosis. Compared to GTL, GTR did not significantly increase the risk of mortality (HR 1.17; 95% CI 0.73–1.86, p = 0.5). Furthermore, there was no statistically significant difference between the Kaplan–Meier survival curves of the two groups (p = 0.18). We employed propensity score matching (PSM) to balance the differences in baseline characteristics of patients receiving chemotherapy or radiotherapy. A total of 4429 patients were included in this study. Age, diagnosis period, race, tumor size, and tumor location as influential on the extent of resection. Age, tumor location, extent of resection, and application of radiotherapy and chemotherapy influenced the survival of PA patients. The Kaplan–Meier survival curves revealed that the long-term survival rate for GTR is slightly higher than that for GTL. The PSM analysis revealed that the application of radiotherapy and chemotherapy was associated with the reduction of overall survival in PA patients. In conclusion, there was no significant difference in survival between GTR and GTL, so GTR with less damage was preferred. The application of radiotherapy and chemotherapy can reduce overall survival of patients with PA.

A novel radiological classification of midbrain pilocytic astrocytomas and its implication for surgical management: a single-institution experience of 57 cases.

Surgery for midbrain pilocytic astrocytoma (PA) remains a formidable challenge. To facilitate decision-making and achieve a better outcome in the management of patients with midbrain PA, the authors have proposed a novel radiological classification of midbrain PAs with long-term follow-up. Fifty-seven midbrain PA patients who underwent surgery at Beijing Tiantan Hospital, Capital Medical University, from January 2008 to June 2021, were reviewed. Based on tumor location and the topological anatomical change identified on MRI, midbrain PAs were categorized into four types: crural (12/57, 21.1%), tegmental (25/57, 43.9%), aqueductal (5/57, 8.8%), and tectal (15/57, 26.3%) PAs. The relevant clinical, radiological, and pathological data; surgical procedures and results; and long-term outcomes were collected and analyzed. The 1-, 3-, and 5-year survival rates reached 98%, 96%, and 96%, respectively, with gross-total resection achieved in 66.7% of cases, followed by near-total resection in 17.5% cases. The clinical and radiological features, selection of surgical approaches, and long-term postoperative deficits were distinct among each type. Crural PAs were associated with younger age (median 9 years, IQR 5.0-12.8 years); the largest tumor volume (median 31.9 cm3, IQR 17.2-42.6 cm3); the lowest preoperative Karnofsky Performance Scale (KPS) score (median 65, IQR 50-70); the most frequent preoperative motor deficit (91.7%); a mixed solid-cystic component (75%); occupation of the crural cistern; elevation and rotation of the thalamus (medial and/or lateral); displacement of the anterior third ventricle, uncus, and anterior commissure; the most diverse surgical approaches; more frequent use of multimodality image-guided surgery (58.3%); and the most remarkable improvement in KPS score at long-term follow-up. Tegmental PAs were associated with adolescents and young adults (median age 21 years, IQR 8-33 years); tumor volume (median 13.9 cm3, IQR 9.5-20.5 cm3); a good preoperative KPS score (median 80, IQR 70-80); a mixed solid-cystic component (72%); occupation of the ambient cistern and cerebellomesencephalic fissure; a close relationship with the dorsal pons, superior cerebellar peduncle, and posterior inferior third ventricle; and a higher probability of permanent postoperative sensory deficits (40%). Aqueductal and tectal PAs were associated with small tumor volume (median 9.14 cm3, IQR 5.1-17.4 cm3 and median 11.84 cm3, IQR 5.7-18.3 cm3, respectively), a higher percentage of hydrocephalus (80% and 86.7%, respectively), and a straightforward selection of limited surgical approaches. A novel and comprehensive radiological classification of midbrain PAs was established, which will serve as a valuable tool in patient management and promote uniform communication and comparison across different studies and publications.

SURG-15. RIGHT FRONTAL-PARIETAL PILOCYTIC ASTROCYTOMA WHO GRADE I ASSOCIATED WITH HEMORRHAGIC COMPONENT AND ESSENTIAL HYPERTENSION IN AN ADULT

Abstract Pilocytic astrocytoma (PA) is a primary glial neoplasm and low-grade tumor. After total resection, the prognosis is very good. A poor prognosis results from leptomeningeal spreading and bleeding, both of which are extremely uncommon in Pilocytic Astrocytoma patients. Case presentation We report the case of a 43-year-old male that presented with a 4 days’ history of right sided frontal headache, non-radiating and mostly felt in the morning. Not associated with vomiting and fever. In addition, there was a sudden decline in level of consciousness with no associated stool or urine incontinence. The patient also developed three episodes of tonic clonic convulsions, lasting less than 3 minutes associated with urine incontinence and frothing from the mouth. Two days later, there was a sudden onset of left sided weakness involving upper and lower limbs. However, there is no history of trauma or hypertension. He was identified as having a hemorrhagic right frontal-parietal PA evidence on MRI associated with essential hypertension. The patient had an intervention combination of medical treatment and craniotomy via fronto-temporal approach plus excision of lesion. Histopathological examination of specimen determined that it was PA WHO grade I. He was treated with oral anti-epileptic therapy (Phenytoin 300 mg once a day), folic acid supplementation (400 mcg once a day), anti-hypertensive therapy (Amlodipine 10 mg and Losartan 50 mg once daily) and he showed marked improvement and was discharged home after 1 month and followed up by primary neurosurgeon, oncologist and physician. CONCLUSION: This case report raises awareness of Pilocytic Astrocytoma among adults and emphasizes the value of early diagnosis and management of patients with Pilocytic Astrocytoma associated with essential hypertension can prevent complications, reduce morbidity and mortality.

GLIOMA-13 RIGHT FRONTO-PARIETAL PILOCYTIC ASTROCYTOMA WHO GRADE I ASSOCIATED WITH HEMORRHAGIC COMPONENT AND ESSENTIAL HYPERTENSION IN AN ADULT: A CASE REPORT

Abstract Pilocytic astrocytoma (PA) is a primary glial neoplasm and low-grade tumor. After total resection, the prognosis is very good. A poor prognosis results from leptomeningeal spreading and bleeding, both of which are extremely uncommon in Pilocytic Astrocytoma patients. CASE PRESENTATION: We report the case of a 43-year-old male that presented with a 4 days’ history of right sided frontal headache, non-radiating and mostly felt in the morning. Not associated with vomiting and fever. In addition, there was a sudden decline in level of consciousness with no associated stool or urine incontinence. The patient also developed three episodes of tonic clonic convulsions, lasting less than 3 minutes associated with urine incontinence and frothing from the mouth. Two days later, there was a sudden onset of left sided weakness involving upper and lower limbs. However, there is no history of trauma or hypertension. He was identified as having a hemorrhagic right fronto-parietal PA evidence on MRI associated with essential hypertension. The patient had an intervention combination of medical treatment and craniotomy via fronto-temporal approach plus excision of lesion. Histopathological examination of specimen determined that it was PA WHO grade I. He was treated with oral anti-epileptic therapy (Phenytoin 300 mg once a day), folic acid supplementation (400 mcg once a day), anti-hypertensive therapy (Amlodipine 10 mg and Losartan 50 mg once daily) and he showed marked improvement and was discharged home after 1 month and followed up by primary neurosurgeon, oncologist and physician. CONCLUSION: This case report raises awareness of Pilocytic Astrocytoma among adults and emphasizes the value of early diagnosis and management of patients with Pilocytic Astrocytoma associated with essential hypertension can prevent complications, reduce morbidity and mortality.

Prognostic Values of Core Genes in Pilocytic Astrocytom

Pilocytic astrocytoma (PA) is the most common primary brain tumor in children and adolescents. Treatment strategy largely depends on its key genes and molecular mutations. This study aimed to identify potential biomarkers of PA closely related to its prognosis. The gene expression profiles (series numbers GSE50161, GSE66354, and GSE86574) of PA and normal brain tissues were downloaded from the Gene Expression Omnibus database. The Gene Expression Omnibus2R was used to identify differentially expressed genes. The overlapping differentially expressed genes were subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) database. A protein-protein interaction network was constructed using Search Tool for the Retrieval of Interacting Genes (STRING) and Cytoscape. The Gene Expression Profiling Interactive Analysis 2 (GEPIA2) tool analyzed the impact of hub genes on PA prognosis based on the Kaplan-Meier curves. Compared with normal brain tissues (n= 36), a total of 37 upregulated and 144 downregulated genes were identified in PA (n= 40). In the protein-protein interaction network construction and GEPIA2 survival analysis, 2 of the top 10 hub genes were significantly associated with decreased overall survival of PA patients, namely Gamma-aminobutyric acid A receptor alpha 2 (hazard ratio = 2.8, P < 0.01) and regulating synaptic membrane exocytosis protein 1) (hazard ratio= 3.2, P < 0.01). This bioinformatics analysis reveals that low expression of Gamma-aminobutyric acid A receptor alpha 2 and regulating synaptic membrane exocytosis protein 1 is associated with a favorable prognosis for PA patients. These 2 hub genes could be novel biomarkers for prognosis assessment, furthermore a key element for treatment decisions in the future.

Placement of EVD in pediatric posterior fossa tumors: safe and efficient or old-fashioned? The Vienna experience.

The perioperative treatment of hydrocephalus in pediatric posterior fossa tumors with an external ventricular drain (EVD) is the treatment of choice in our center. We analyzed our experience in using EVD concerning safety and effectivity. This is a single-center retrospective cohort study of 100 consecutive pediatric patients who underwent resection for a newly diagnosed tumor in the posterior fossa between 2011 and 2022. Of the 100 patients with posterior fossa tumors, 80 patients (80%) had radiological signs of hydrocephalus at presentation, 49 patients (49%) of whom underwent placement of an EVD. In 40 patients, the EVD was inserted at a mean of 2.25days prior to the tumor resection; 9 had the EVD inserted during tumor resection (frontal trajectory in 7 patients, occipital trajectory in 2 patients). Histology revealed pilocytic astrocytoma in 48 patients, medulloblastoma in 32, ependymoma in 11, and other histologic entities in 9 patients. Gross total/near-total resection was achieved in 46 (95.83%) of the 48 pilocytic astrocytomas, 30 (93.75%) of the 32 medulloblastomas, and 11 (100%) of the 11 ependymomas. The mean number of total days with the EVD in place was 8.61 ± 3.82 (range 2-16days). The mean number of days with an EVD after tumor resection was 6.35 ± 3.8 (range 0-16days). EVD-associated complications were seen in 6 patients (12.24%) including one infection. None of these resulted in a worse clinical course or any long-term sequelae. Permanent CSF diversion at 6months after surgery was necessary in 13 patients (13%), including two VP shunt, two SD-shunt, six endoscopic third ventriculostomy (ETV), and three combined VP shunt and ETV procedures. Patients with a medulloblastoma or ependymoma had a higher rate of permanent CSF diversion needed than the group of pilocytic astrocytoma patients (27.9% versus 2.13%, p < 0.001). In patients with metastatic disease, 7 of 17 patients (41.18%) needed a permanent CSF diversion, compared to 6 of 83 patients (7.23%) in the group without metastasis (p = 0.001). The treatment of hydrocephalus in pediatric posterior fossa tumors with an EVD as a temporary measure is safe and effective, provided that a multi-professional understanding for its handling is given and there is no need for a long transport of the children.

The utility of DNA methylation analysis in elderly patients with pilocytic astrocytoma morphology.

Pilocytic astrocytoma (PA) is a circumscribed low-grade astrocytic glioma, generally considered to be associated with a good prognosis. However, a subset of PA patients shows unfavorable outcomes. In this study, we retrospectively reviewed PA patients and performed further molecular analysis, such as DNA methylation profiling, to identify prognostic factors. We analyzed 29 histologically-confirmed PA patients from a single center from 2002 to 2021 and conducted integrated molecular analyses among elderly PA patients since age was an independent prognostic factor for poor outcomes. The median age at diagnosis was 14years (range 3-82years) and 4 patients (14%) were elderly (patients ≥ 60years old). Age over 60 was associated with poor progression-free survival and overall survival. We performed DNA methylation analysis on 2 of the 4 elderly patients. Both cases were histologically diagnosed as PA, but DNA methylation profiling revealed one as high-grade astrocytoma with piloid features (all methylation class scores were below 0.3 in both v11b4 and v12.5) and the other as glioblastoma, IDH-wildtype (score was over 0.5 in both v11b4 and v12.5), using the German Cancer Research Center methylation profiling classifiers and t-SNE analysis. Elderly patients with PA morphology showed unfavorable outcomes in this cohort. In those patients, further molecular analysis and DNA methylation profiling revealed the possibility of high-grade astrocytic tumors, including newly defined entities.

EGFL7 expression profile in IDH-wildtype glioblastomas is associated with poor patient outcome.

BackgroundDespite the advances in glioblastoma (GBM) treatment, the average life span of patients is 14 months. Therefore, it is urgent to identity biomarkers of prognosis, treatment response, or development of novel treatment strategies. We previously described the association of high epidermal growth factor-like domain multiple 7 (EGFL7) expression and unfavorable outcome of pilocytic astrocytoma patients. The present study aims to analyze the prognostic potential of EGFL7 in GBM isocitrate dehydrogenase (IDH)-wildtype, using immunohistochemistry and in silico approaches.MethodsSpearman’s correlation analysis of The Cancer Genome Atlas RNA sequencing data was performed. The genes strongly correlated to EGFL7 expression were submitted to enrichment gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Additionally, EGFL7 expression was associated with patient overall survival. The expression of EGFL7 was analyzed through immunohistochemistry in 74 GBM IDH-wildtype patients’ samples, and was associated with clinicopathological data and overall survival.ResultsIn silico analysis found 78 genes strongly correlated to EGFL7 expression. These genes were enriched in 40 biological processes and eight KEGG pathways, including angiogenesis/vasculogenesis, cell adhesion, and phosphoinositide 3-kinase–Akt, Notch, and Rap1 signaling pathways. The immunostaining showed high EGFL7 expression in 39 cases (52.7%). High immunolabelling was significantly associated with low Karnofsky Performance Status and poor overall survival. Cox analysis showed that GBMs IDH-wildtype with high EGFL7 expression presented a higher risk of death compared to low expression (hazard ratio, 1.645; 95% confidence interval, 1.021 to 2.650; p = .041).ConclusionsThis study gives insights regarding the genes that are correlated with EGFL7, as well as biological processes and signaling pathways, which should be further investigated in order to elucidate their role in glioblastoma biology.

Clinical factors and conventional MRI may independently predict progression-free survival and overall survival in adult pilocytic astrocytomas.

Pilocytic astrocytoma (PA) is rare in adults, and only limited knowledge on the clinical course and prognosis has been available. The combination of clinical information and comprehensive imaging parameters could be used for accurate prognostic stratification in adult PA patients. This study was conducted to predict the prognostic factors from clinical information and conventional magnetic resonance imaging (MRI) features in adult PAs. A total of 56 adult PA patients were enrolled in the institutional cohort. Clinical characteristics including age, sex, anaplastic PA, presence of neurofibromatosis type 1, Karnofsky performance status, extent of resection, and postoperative treatment were collected. MRI characteristics including major axis length, tumor location, presence of the typical 'cystic mass with enhancing mural nodule appearance', proportion of enhancing tumor, the proportion of edema, conspicuity of the nonenhancing margin, and presence of a cyst were evaluated. Univariable and multivariable Cox proportional hazard modeling were performed. The 5-year progression-free survival (PFS) and overall survival (OS) rates were 83.9% and 91.l%, respectively. On univariable analysis, older age, larger proportion of edema, and poor definition of nonenhancing margin were predictors of shorter PFS and OS, respectively (all Ps < .05). On multivariable analysis, older age (hazard ratio [HR] = 1.04, P = .014; HR = 1.14, P = .030) and poor definition of nonenhancing margin (HR = 3.66, P = .027; HR = 24.30, P = .024) were independent variables for shorter PFS and OS, respectively. Age and the margin of the nonenhancing part of the tumor may be useful biomarkers for predicting the outcome in adultPAs.

The ROCOCO performance scoring system translates dosimetric differences into clinically relevant endpoints: Comparing IMPT to VMAT in an example pilocytic astrocytoma dataset

Background and purposeProton therapy is expected to outperform photon-based treatment regarding organs at risk (OAR) sparing but to date there is no method to practically measure clinical benefit. Here, we introduce the novel ROCOCO Performance Scoring System (RPSS) translating dose differences into clinically relevant endpoints and apply this to a treatment plan comparison of volumetric modulated arc therapy (VMAT) and intensity modulated proton therapy (IMPT) in 20 pilocytic astrocytoma patients. Material and methodsThe RPSS was developed on the basis of expert-based weighting factors and toxicity scores per OAR. The imaging datasets of 20 pilocytic astrocytoma patients having undergone radiotherapy were included in this in silico dosimetric comparison trial as proof of principle. For each of these patients, treatment plans to a total dose of 54 Gy (RBE) were generated for VMAT and IMPT and these were compared regarding radiation dose to the clinical target volume (CTV) and OARs. The RPSS was calculated for each treatment plan comparing VMAT and IMPT. ResultsIn 40 analysed treatment plans, the average and low dose volumes to various OARs were significantly reduced when using IMPT compared to VMAT (p < 0.05). Using the RPSS, a significant difference between both treatment modalities was found, with 85% of the patients having a lower RPSS in favour of the IMPT plan. ConclusionThere are dosimetric differences between IMPT and VMAT in pilocytic astrocytoma patients. In absence of clinically validated NTCP models we introduce the RPSS model in order to objectively compare treatment modalities by translating dosimetric differences in potential clinical differences.

LGG-54. DETECTION OF THE KIAA1549-BRAF FUSION GENE IN CELLS FORMING MICROVASCULAR PROLIFERATIONS IN PILOCYTIC ASTROCYTOMA

Microvascular proliferation (MVP), an aberrant vascular structure is a histopathological hallmark of glioblastoma multiforme (GBM). Although MVP tends to be associated with high-grade glioma, it has also been detected in WHO grade I pilocytic astrocytoma (PA). However, little is known about the mechanism underlying its formation. Using TP53 point mutations as a marker for tumor-derived cells, we earlier reported that MVP was partially converted from tumor cells via mesenchymal transition. In the current study we used the KIAA1549-BRAF fusion gene as a marker to assess whether MVPs in PA contained tumor-derived cells and/or phenotypically distinct tumor cells expressing vascular markers. Samples from three PA patients harbored the KIAA1549 exon 15, BRAF exon 9 fusion gene. In two patient samples with abundant MVP, RT-PCR assay detected strong bands arising from the KIAA1549-BRAF fusion gene in both tumor cells and cellular components of MVP. Digital PCR showed that vis-à-vis tumor tissue, its relative expression in cellular components of MVP was 42% in one- and 76% in another sample. FISH revealed amplified signals in both tumor cells and cellular components of MVP indicative of tandem duplication. Our findings suggest that in patients with PA, some cellular components of MVP contained tumor derived cell and/or phenotypically distinct tumor cells expressing vascular markers.

Evaluation of the prognostic potential of EGFL7 in pilocytic astrocytomas.

Pilocytic astrocytoma (PA) is the most frequent solid neoplasm in childhood. It has a good 5-year overall survival (90% in childhood and 52% in adults). However, up to 20% of patients experience residual tumor growth, recurrence, and death. Although the main genetic alteration of PAs, including KIAA1549:BRAF fusion, involves chromosome 7q34, we previously found frequent loss in chr9q34.3 locus in a small subset of these tumors. Among the genes present in this locus, EGFL7 is related to poor prognosis in several tumor types. In this study, we aimed to assess EGFL7 expression through immunohistochemistry, and to evaluate its prognostic value in a series of 64 clinically and molecularly well-characterized pilocytic astrocytomas. We found high expression of EGFL7 in 71.9% of patients. Low EGFL7 expression was associated with older patients, the mean age mainly older than 11 years (P = 0.027). EGFL7 expression was not associated with presence of KIAA1549:BRAF fusion, BRAF mutation, FGFR1 mutation, nor FGFR1 duplication. Moreover, high EGFL7 expression was associated with high FGFR1 (P = 0.037) and 5'-deoxy-5'-methyltioadenosine phosphorylase (MTAP) (P = 0.005) expression, and with unfavorable outcome of patients (P = 0.047). Multivariate analysis revealed low EGFL7 expression related to older patients and high EGFL7 expression related to retained expression of MTAP. In addition, we found a borderline significance of unfavorable outcome and high EGFL7 expression. Finally, EGFL7 expression was not associated with overall or event-free survival of PA patients. Our findings point to EGFL7 expression as a novel candidate prognostic marker in PA, which should be further investigated.

ANGI-05. ORIGIN OF MICROVASCULAR PROLIFERATION IN PILOCYTIC ASTROCYTOMA - MVP IN PILOCYTIC ASTROCYTOMA MIGHT PARTIALLY COMPRISE TUMOR-DERIVED CELLS

Abstract Microvascular proliferation (MVP), an aberrant vascular structure containing multilayered mitotically active endothelial- and smooth-muscle cells/pericytes, is a histopathological hallmark of glioblastoma multiforme (GBM). Although MVP tends to be associated with high-grade glioma, it has also been detected in WHO grade I pilocytic astrocytoma (PA). However, little is known about the mechanism underlying its formation. Using TP53 point mutations as a marker for tumor-derived cells, we earlier reported that MVP was partially converted from tumor cells via mesenchymal transition. In the current study we used the KIAA1549-BRAF fusion gene as a marker to assess whether MVP in PA contained tumor-derived cells. cDNA synthesized from frozen tissue of six PA patients operated at our institute was analyzed to detect the KIAA1549-BRAF fusion gene by reverse transcription polymerase chain reaction (RT-PCR) assay. The breakpoint in the fusion gene was characterized by long and accurate PCR (LA-PCR) and Sanger sequencing of genomic DNA. Distinct tumor cells and cellular components of MVP were obtained by laser microdissection. For the qualitative and quantitative detection of the KIAA1549-BRAF fusion gene we performed genomic and digital PCR assays. Fluorescence in situ hybridization (FISH) was used to assess gene fusion in cellular components of MVP. Samples from three PA patients harbored the KIAA1549 exon 15, BRAF exon 9 fusion gene. In two patient samples with abundant MVP, RT-PCR assay detected strong bands arising from the KIAA1549-BRAF fusion gene in both tumor cells and cellular components of MVP. Digital PCR showed that vis-à-vis tumor tissue, its relative expression in cellular components of MVP was 42% in one- and 76% in another sample. FISH revealed amplified signals in both tumor cells and cellular components of MVP indicative of tandem duplication. Our findings suggest that in patients with PA, some cellular components of MVP may be tumor-derived.

Detection of the KIAA1549-BRAF fusion gene in cells forming microvascular proliferations in pilocytic astrocytoma.

Microvascular proliferation (MVP), an aberrant vascular structure containing multilayered mitotically active endothelial- and smooth-muscle cells/pericytes, is a histopathological hallmark of glioblastoma multiforme (GBM). Although MVP tends to be associated with high-grade glioma, it has also been detected in WHO grade I pilocytic astrocytoma (PA). However, little is known about the mechanism underlying its formation. Using TP53 point mutations as a marker for tumor-derived cells, we earlier reported that MVP was partially converted from tumor cells via mesenchymal transition. In the current study we used the KIAA1549-BRAF fusion gene as a marker to assess whether MVPs in PA contained tumor-derived cells and/or phenotypically distinct tumor cells expressing vascular markers. cDNA synthesized from frozen tissue of six PA patients operated at our institute was analyzed to detect the KIAA1549-BRAF fusion gene by reverse transcription polymerase chain reaction (RT-PCR) assay. The breakpoint in the fusion gene was identified by long and accurate PCR (LA-PCR) and Sanger sequencing of genomic DNA. Distinct tumor cells and cellular components of MVP were obtained by laser microdissection. For the qualitative and quantitative detection of the KIAA1549-BRAF fusion gene we performed genomic and digital PCR assays. Fluorescence in situ hybridization (FISH) was used to assess gene fusion in cellular components of MVP. Samples from three PA patients harbored the KIAA1549 exon 15, BRAF exon 9 fusion gene. In two patient samples with abundant MVP, RT-PCR assay detected strong bands arising from the KIAA1549-BRAF fusion gene in both tumor cells and cellular components of MVP. Digital PCR showed that vis-à-vis tumor tissue, its relative expression in cellular components of MVP was 42% in one- and 76% in another sample. FISH revealed amplified signals in both tumor cells and cellular components of MVP indicative of tandem duplication. Our findings suggest that in patients with PA, some cellular components of MVP contained tumor derived cell and/or phenotypically distinct tumor cells expressing vascular markers.

Abstract 3647: Single cell RNA sequencing reveals mitogenic and progenitor gene programs inBRAF-rearranged pilocytic astrocytomas

Abstract Single-cell RNA sequencing (scRNAseq) has been performed across a range of intermediate to high-grade gliomas that each harbor multiple driver mutations. Pilocytic astrocytoma (PA), the most common childhood brain tumor, is a low-grade glioma. PAs frequently harbor oncogenic KIAA1549-BRAF fusions, and exhibit low rates of other driver mutations. While PAs exhibit a favorable prognosis compared to the higher-grade gliomas, treatment morbidity and tumor recurrence can represent major challenges for some PA patients. We performed scRNAseq of both tumor and non-tumor cells in newly-diagnosed PAs that contained KIAA1549-BRAF rearrangements. To confidently distinguish tumor cells from nontumor cells, we sorted cells by glial progenitor marker A2B5 status and profiled KIAA1549-BRAF fusion status of cells using a sensitive quantitative PCR-based assay. Results were validated using RNA in situ hybridization. When compared to higher-grade gliomas, a higher proportion of the PA tumor cells exhibited a differentiated, astrocyte-like phenotype. A smaller proportion of cells exhibited a progenitor-like phenotype with evidence of proliferation. These progenitor-like tumor cells expressed a mitogen-activated protein kinase (MAPK) program that was absent from higher-grade gliomas. Similar patterns of expression of genes associated with the astrocyte-like and MAPK gene programs were also seen in formalin fixed, paraffin embedded PA tissues using RNA in situ hybridization. Immune cells, especially microglia, comprised almost half of all cells in the PAs and accounted for differences in bulk expression profiles between tumor locations and subtypes. These single cell transcriptional data reveal a transcriptional developmental hierarchy in a pediatric low grade glioma that is skewed towards more mature brain cells compared to higher-grade gliomas. The results indicate that future analyses of bulk PA tissues should attempt to account for considerable infiltration by nontumor cells. Finally, the finding that a MAPK gene program is not uniformly expressed in PA tumor cells has implications for ongoing clinical investigations of therapies directed at the MAPK pathway for the treatment of PA. Citation Format: Zachary J. Reitman, Brenton Paolella, Guillaume Bergthold, Kristine Pelton, Robert Jones, Sarah Becker, Claire E. Sinai, Haley Malkin, Ying Huang, Leslie Grimmett, Zachary T. Herbert, Yu Sun, Jessica Weatherbee, John Alberta, John Daley, Orit Rozenblatt-Rosen, Rosalind Segal, Daphne Haas-Kogan, Mariella G. Filbin, Mario L. Suva, Aviv Regev, Charles Stiles, Mark W. Kieran, Liliana Goumnerova, Keith L. Ligon, Alex K. Shalek, Pratiti Bandopadhayay, Rameen Beroukhim. Single cell RNA sequencing reveals mitogenic and progenitor gene programs inBRAF-rearranged pilocytic astrocytomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3647.

Mathematical skills in children with pilocytic astrocytoma.

Pediatric patients with circumscribed cerebellar pilocytic astrocytoma (PA) tumors generally perform within the normal range on neuropsychological tests after a complete tumor resection. The outcome in academically relevant abilities such as mathematics, which in adults involve some cerebellar functions, is however much less understood. The aim of this study is to retrospectively investigate the neuroplasticity of mathematical skills and associated cognitive functions following cerebellar resection of PA in pediatric patients. Twenty-two children (mean age = 11.2+1.8), including 11 PA patients (females = 6) and 11 healthy controls (females = 6), were administered a battery of mathematical (MaT) and neuropsychological tests. Single-case statistical analyses were carried out (Crawford's t) as well as between-group comparisons (Wilcoxon test). Spearman correlations between MaT and neuropsychological tests were calculated. Thirty-six percent of the patients showed difficulties in some mathematical tasks, 50% of them within a broader cognitive deficit. Verbal working memory was associated with MaT performance both in patients and controls while, crucially, visuospatial memory, and visual-motor integration were associated with MaT in patients only. Among patients, MaT correlated negatively with tumor size and positively with the interval surgery test. The results evince an overall recovery of mathematical abilities despite PA in the majority of patients. This functional reestablishment is supported by visuospatial and visuomotor integration functions that contribute to set up emerging mathematical skills in these patients. Higher levels of compensation are found in more developed tumors as compared to smaller ones.

Circulating serum oncologic miRNA in pediatric juvenile pilocytic astrocytoma patients predicts mural nodule volume.

Juvenile pilocytic astrocytomas represent the largest group of pediatric brain tumors. The ideal management for these tumors is early, total surgical resection. To detect and track treatment response, a screening tool is needed to identify patients for surgical evaluation and assess the quality of treatment. The identification of aberrant miRNA profiles in the sera of juvenile pilocytic astrocytoma patients could provide such a screening tool. The authors reviewed the serum profiles of 84 oncologically relevant miRNAs in pediatric juvenile pilocytic astrocytoma patients via qPCR screening. miR-21, miR-15b, miR-23a, and miR-146b were significantly elevated in the sera of JPA patients as compared to non-oncologic controls, oncologic controls, and post-JPA resection samples (p < 0.001, 0.022, 0.034, 0.044). miR-21 had the highest AUC on ROC analysis (AUC > 0.99, sensitivity 75%, specificity 100%). All four miRNAs also correlated well with tumor mural nodule size, though they only poorly correlated with total tumor size, including cystic components (Spearman's R2: miR-21 91.7 vs 6.9%, miR-15b 86.3 vs 23.1%, miR-23a 85.8 vs 23.0%, miR-146b 59.8 vs 11.9%). In this small pilot study, pediatric juvenile pilocytic astrocytoma patients had significant elevations in serum miR-21, miR-15b, miR-23a, and miR-146b levels that do not appear to be driven by hydrocephalus or local distortion of the intracranial contents. These alterations correlate with solid tumor component volume and reverse with complete tumor resection, suggesting that this serum miRNA profile may delineate biomarkers for screening and tracking juvenile pilocytic astrocytoma patients. Additional studies, with a larger cohort, are needed to verify these results.

DTI fiber tractography of cerebro-cerebellar pathways and clinical evaluation of ataxia in childhood posterior fossa tumor survivors.

Pediatric posterior fossa (PF) tumor survivors experience long-term motor deficits. Specific cerebrocerebellar connections may be involved in incidence and severity of motor dysfunction. We examined the relationship between long-term ataxia as well as fine motor function and alteration of differential cerebellar efferent and afferent pathways using diffusion tensor imaging (DTI) and tractography. DTI-based tractography was performed in 19 patients (10 pilocytic astrocytoma (PA) and 9 medulloblastoma patients (MB)) and 20 healthy peers. Efferent Cerebello-Thalamo-Cerebral (CTC) and afferent Cerebro-Ponto-Cerebellar (CPC) tracts were reconstructed and analyzed concerning fractional anisotropy (FA) and volumetric measurements. Clinical outcome was assessed with the International Cooperative Ataxia Rating Scale (ICARS). Kinematic parameters of fine motor function (speed, automation, variability, and pressure) were obtained by employing a digitizing graphic tablet. ICARS scores were significantly higher in MB patients than in PA patients. Poorer ICARS scores and impaired fine motor function correlated significantly with volume loss of CTC pathway in MB patients, but not in PA patients. Patients with pediatric post-operative cerebellar mutism syndrome showed higher loss of CTC pathway volume and were more atactic. CPC pathway volume was significantly reduced in PA patients, but not in MB patients. Neither relationship was observed between the CPC pathway and ICARS or fine motor function. There was no group difference of FA values between the patients and healthy peers. Reduced CTC pathway volumes in our cohorts were associated with severity of long-term ataxia and impaired fine motor function in survivors of MBs. We suggest that the CTC pathway seems to play a role in extent of ataxia and fine motor dysfunction after childhood cerebellar tumor treatment. DTI may be a useful tool to identify relevant structures of the CTC pathway and possibly avoid surgically induced long-term neurological sequelae.

MRS of pilocytic astrocytoma: The peak at 2 ppm may not be NAA.

To determine whether the chemical shift of residual N-acetylaspartate (NAA) signal in pilocytic astrocytomas (PA) is consistent with the position of the NAA peak in controls. MR spectra from 27 pediatric World Health Organization (WHO) grade I pilocytic astrocytoma patients, fifteen patients with WHO grade II and high-grade (III-IV) astrocytomas, and 36 controls were analyzed. All spectra were acquired with a short echo time (35 ms), single voxel point-resolved spectroscopy sequence on clinical 3 tesla scanners. Fully automated LCModel software was used for processing, which included the fitting of peak positions for NAA and creatine (Cr). The chemical shift difference between the NAA and Cr peaks was significantly smaller (by 0.016 ± 0.005 parts per million, P < 1e-10) in PAs than in controls and was also smaller than what was observed in infiltrative astrocytomas. The chemical shift position of the residual NAA peak in PAs is not consistent with NAA. The signal likely originates from an N-acetyl group of one or more other chemicals such as N-acetylated sugars. Magn Reson Med 78:452-456, 2017. © 2016 International Society for Magnetic Resonance in Medicine.