KIAA1549-BRAF Fusion Research Articles

Oncogenic Fusions in Gliomas: An Institutional Experience

Gliomas are primary malignancies of the central nervous system (CNS). High-grade gliomas are associated with poor prognosis and modest survival rates despite intensive multimodal treatment strategies. Targeting gene fusions is an emerging therapeutic approach for gliomas that allows application of personalized medicine principles. The aim of this study was to identify detectable fusion oncogenes that could serve as predictors of currently available or newly developed targeted therapeutics in cross-sectional samples from glioma patients using next-generation sequencing (NGS). A total of 637 patients with glial and glioneuronal tumours of the CNS who underwent tumour resection between 2017 and 2020 were enrolled. Detection of fusion transcripts in FFPE tumour tissue was performed by a TruSight Tumour 170 assay and two FusionPlex kits, Solid Tumour and Comprehensive Thyroid and Lung. Oncogene fusions were identified in 33 patients. The most common fusion was the KIAA1549-BRAF fusion, detected in 13 patients, followed by FGFR fusions (FGFR1-TACC1, FGFR2-CTNNA3, FGFR3-TACC3, FGFR3-CKAP5, FGFR3-AMBRA1), identified in 10 patients. Other oncogene fusions were also infrequently diagnosed, including MET fusions (SRPK2-MET and PTPRZ1-MET) in 2 patients, C11orf95-RELA fusions in 2 patients, EGFR-SEPT14 fusion in 2 patients, and individual cases of SRGAP3-BRAF, RAF1-TRIM2, EWSR1-PALGL1 and TERT-ALK fusions. The introduction of NGS techniques provides additional information about tumour molecular alterations that can aid the multimodal management of glioma patients. Patients with gliomas positive for particular targetable gene fusions may benefit from experimental therapeutics, enhancing their quality of life and prolonging survival rates.

Analysis of genetic aberrations in pediatric low-grade gliomas: the experience of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology

Low grade gliomas (LGGs) are the most common brain tumors in children. Our retrospective-prospective study of biological characteristics of sporadic LGGs (not associated with neurofibromatosis type I) included 233 patients aged 0 to 18 years who had been diagnosed and/or treated at the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology, and Immunology in the period from 2009 to 2021. The study was approved by the Independent Ethics Committee and the Scientific Council of the D. Rogachev NMRCPHOI. The median age at the diagnosis was 5 years 4 months (2 months – 17 years). Among the LGGs, the following histological variants were identified: pilocytic astrocytoma (n = 191; 82%), pleomorphic xanthoastrocytoma (n = 16; 7%), ganglioglioma (n = 7; 3%), desmoplastic infantile ganglioglioma (n = 4; 2%), diffuse leptomeningeal glioneuronal tumor (n = 5; 2%), dysembryoplastic neuroepithelial tumor (n = 2, 1%), and diffuse astrocytoma (n = 1; 0,5%). The tumors were located in: the suprasellar region (n = 98; 42%), the brainstem (n = 40; 17%), the cerebellum (n = 35; 15%), the hemispheres (n = 34; 15%) etc. The KIAA1549-BRAF fusion was the most common molecular genetic alteration (n = 107; 46%). The second most frequent genetic aberration was the BRAF V600E mutation (n = 44; 19%). Rare molecular genetic events leading to the activation of the MAPK signaling pathway were detected in 13 (6%) patients. The H3 K27M mutation associated with an aggressive clinical course was identified in three patients with brainstem LGGs (1%). These findings point to the importance of molecular profiling of pediatric LGGs for the selection of an effective strategy for molecular diagnosis and optimal clinical care.

Transcriptomic Portraits and Molecular Pathway Activation Features of Adult Spinal Intramedullary Astrocytomas.

In this study, we report 31 spinal intramedullary astrocytoma (SIA) RNA sequencing (RNA-seq) profiles for 25 adult patients with documented clinical annotations. To our knowledge, this is the first clinically annotated RNA-seq dataset of spinal astrocytomas derived from the intradural intramedullary compartment. We compared these tumor profiles with the previous healthy central nervous system (CNS) RNA-seq data for spinal cord and brain and identified SIA-specific gene sets and molecular pathways. Our findings suggest a trend for SIA-upregulated pathways governing interactions with the immune cells and downregulated pathways for the neuronal functioning in the context of normal CNS activity. In two patient tumor biosamples, we identified diagnostic KIAA1549-BRAF fusion oncogenes, and we also found 16 new SIA-associated fusion transcripts. In addition, we bioinformatically simulated activities of targeted cancer drugs in SIA samples and predicted that several tyrosine kinase inhibitory drugs and thalidomide analogs could be potentially effective as second-line treatment agents to aid in the prevention of SIA recurrence and progression.

Ductal and acinar components of mixed prostatic adenocarcinoma frequently have a common clonal origin.

BackgroundDuctal adenocarcinoma (DA) is an aggressive subtype of prostate cancer. It is most commonly seen in mixed tumors together with conventional acinar adenocarcinoma (AA). The genetic profile of DA and its clonal origin is not fully characterized.ObjectiveTo investigate whether DA represents a distinct genetic subtype and to investigate the somatic relationship between the ductal and acinar components of mixed cancers.Design, Setting, and ParticipantsIn 17 radical prostatectomy specimens ductal and acinar tumor components from the same tumor foci were dissected. DNA was extracted and genomic sequencing performed. After exclusion of two cases with low cell yield, 15 paired samples remained for analysis.ResultsIn 12 of 15 cases a common somatic denominator was identified, while three cases had clonally separate components. In DA, TMPRSS2‐ERG gene fusions were detected in 47% (7/15), clonal FOXA1 alterations in 33% (5/15) and SPOP alterations in 27% (4/15) of cases. In one case KIAA1549−BRAF fusion was identified. Genome doubling events, resulting in an increased ploidy, were identified in the DA in 53% (8/15) of cases, but not seen in any AA. PTEN and CTNNB1 alterations were enriched in DA (6/15) but not seen in any AA. No cancers showed microsatellite instability or high tumor mutation burden.ConclusionsDuctal and acinar prostate adenocarcinoma components of mixed tumors most often share the same origin and are clonally related. DA components in mixed tumor often exhibit genome doubling events resulting in aneuploidy, consistent with the aggressive nature of high grade prostate cancer.

Clinical and molecular features of disseminated pediatric low-grade glioma and glioneuronal tumors: a systematic review and survival analysis.

Disseminated pediatric low-grade gliomas and glioneuronal tumors (dpLGG/GNTs) are associated with a poorer prognosis than nondisseminated pLGG/GNTs. To date there is no comprehensive report characterizing the genome profile of dpLGG/GNTs and their relative survival. This systematic review aims to identify the pattern of genetic alterations and long-term outcomes described for dpLGG/GNT. A systematic review of the literature was performed to identify relevant articles. A quality and risk of bias assessment of articles was done using the GRADE framework and ROBINS-I tool, respectively. Fifty studies published from 1994 to 2020 were included in this review with 366 cases reported. There was sporadic reporting of genetic alterations. The most common molecular alterations observed among subjects were 1p deletion (75%) and BRAF-KIAA1549 fusion (55%). BRAF p.V600E mutation was found in 7% of subjects. A higher proportion of subjects demonstrated primary dissemination compared to secondary dissemination (65% vs 25%). First-line chemotherapy consisted of an alkylation-based regimen and vinca alkaloids. Surgical intervention ranged from biopsy alone (59%) to surgical resection (41%) and CSF diversion (28%). Overall, 73% of cases were alive at last follow-up. Survival did not vary by tumor type or timing of dissemination. All studies reviewed either ranked low or moderate for both quality and risk of bias assessments. Chromosome 1p deletion and BRAF-KIAA1549 fusion were the most common alterations identified in dpLGG/GNT cases reviewed. The relative molecular heterogeneity between DLGG and DLGNT, however, deserves further exploration and ultimately correlation with their biologic behavior to better understand the pathogenesis of dpLGG/GNT.

CTNI-06. TRAM-01: A PHASE 2 STUDY OF TRAMETINIB FOR PATIENTS WITH PEDIATRIC GLIOMA WITH ACTIVATION OF THE MAPK/ERK PATHWAY

Abstract BACKGROUND Pediatric low-grade gliomas (PLGG) are the most frequent brain tumors in children. It is known that the majority of PLGG have activation of the MAPK/ERK pathway. METHODS This ongoing multicenter phase II trial includes three progressing/refractory PLGG groups: NF1 patients, KIAA1549-BRAF fusion patients and patients with other activation of the MAPK/ERK pathway (excluding V600E). The primary objective was to evaluate the overall response rate based on RANO criteria after daily oral trametinib administration for 18 cycles, lasting 28 days each. Secondary objectives include the assessment of progression-free survival, tolerability of trametinib, serum levels of trametinib and quality of life evaluation during treatment. RESULTS As of February 12 2021, 50 patients have been enrolled (NF1: n=10; KIAA1549-BRAF fusion: n=31; other: n=9 including 5 patients with FGFR1 alterations). Median age is 8.8 years (range 2.4-25.5). Median follow-up is 17.5 months (range 4.7-28.5). Forty-three patients are evaluable. The overall response includes: 4 partial response (PR) (9%), 18 minor response (MR) (42%), 17 stable disease (40%), 4 progressive disease (9%). Median time to response is 5.5 months (range 2.4-13.8). Median duration of response is 6.1 months (range 0.6-26.5). Progression free survival at 12 months is 79.9% (95% CI 68.5-93.6%) and median progression free survival has not yet been reached. Treatment was discontinued for 30 patients: 16 after completing 18 cycles as planned, 5 for progressive disease, 5 for adverse events, 4 for other reasons. A total of 8 patients progressed after discontinuation of treatment including 6 patients (37.5%) that completed 18 cycles. Five of these patients had achieved minor response prior to discontinuation. CONCLUSION Trametinib is a potentially effective targeted therapy for patients with recurrent/refractory PLGG. Treatment was overall well tolerated. This ongoing trial will continue to gather data on response rate, duration of response and safety of trametinib for PLGG.

BIOM-25. ADULT GANGLIOGLIOMA: A 4-PATIENT CASE SERIES WITH MOLECULAR PROFILING

Abstract Accounting for only 2% of all primary brain tumors, ganglioglioma is a rare neoplasm that most frequently arises in the temporal lobe in children or young adults. Ganglioglioma is the most common epilepsy-associated neoplasm and is typically composed of dysplastic ganglion cells and neoplastic glial cells. Recent publications have revealed that the activating p.V600E mutation in the BRAF oncogene is seen in 20-60% of gangliogliomas. However, the genetic landscape of ganglioglioma requires further elucidation, especially in gangliogliomas that arise from outside of the temporal lobe in adults. In this case series, we describe the genetic mutations of adult gangliogliomas originating outside of the temporal lobe in four patients aged 21 to 56 years. Targeted next generation sequencing via the MSK-IMPACT panel covering ~ 500 actionable mutations was used in all cases. None of the cases had the BRAF p.V600E mutation. Case 1 describes a left cerebellar cystic ganglioglioma with NTRK2 fusion mutation (NTRK2-DST) and a tumor mutation burden (TMB) = 0 mutation/megabase (mut/Mb). Case 2 describes a right parietal lobe cystic ganglioglioma with a BRAF-CLEC2Linversion mutation and TMB = 0 mut/Mb. Case 3 describes a cerebellar vermis ganglioglioma with a BRAF fusion mutation (BRAF-KIAA1549) and TMB = 0 mut/Mb. The last case describes a right cerebellopontine angle ganglioglioma with a MAP2K1 splicing mutation and TMB = 0.9 mut/Mb. To the best of our knowledge, the mutations identified in this case series have not been previously described in adult brain gangliogliomas. The BRAF-KIAA1549 fusion mutation has been found in spinal cord gangliogliomas, but not in brain gangliogliomas. MAP2K1 splicing mutations, unlike MAP2K1 in-frame deletions, have not been found in gangliogliomas. Finally, a different NTRK2 mutation (NTRK2-TLE4) has been described in a pediatric ganglioglioma, but NTRK2-DST has not been found in any adult gangliogliomas.

Gliomas in children and adolescents: investigation of molecular alterations with a potential prognostic and therapeutic impact.

Gliomas represent the most frequent central nervous system (CNS) tumors in children and adolescents. However, therapeutic strategies for these patients, based on tumor molecular profile, are still limited compared to the wide range of treatment options for the adult population. We investigated molecular alterations, with a potential prognostic marker and therapeutic target in gliomas of childhood and adolescence using the next-generation sequencing (NGS) strategy. We selected 95 samples with initial diagnosis of glioma from patients treated at Pediatric Oncology Institute-GRAACC/UNIFESP. All samples were categorized according to the 2021 World Health Organization Classification of Tumors of the CNS, which included 39 low-grade gliomas (LGGs) and 56 high-grade gliomas (HGGs). Four HGG samples were classified as congenital glioblastoma (cGBM). NGS was performed to identify somatic genetic variants in tumor samples using the Oncomine Childhood Cancer Research Assay® (OCCRA®) panel, fromThermo Fisher Scientific®. Genetic variants were identified in 76 of 95 (80%) tumors. In HGGs, the most common molecular alteration detected was H3F3A c.83A > T variant (H3.3 K27M) and co-occurring mutations in ATRX, TP53, PDGFRA, MET, and MYC genes were also frequently observed. One HGG sample was reclassified as supratentorial ependymoma ZFTA-fusion positive after NGS was performed. In LGGs, four KIAA1549-BRAF fusion transcripts were detected and this alteration was the most recurrent genetic event and favorable prognostic factor identified. Additionally, genetic variants in ALK and NTRK genes, which provide potential targets for therapy with Food and Drug Administration-approved drugs, were identified in two different cases of cGBM that were classified as infant-type hemispheric glioma, a newly recognized subgroup of pediatric HGG. Molecular profiling by the OCCRA® panel comprehensively addressed the most relevant genetic variants in gliomas of childhood and adolescence, as these tumors have specific patterns of molecular alterations, outcomes, and effectiveness to therapies.

SYST-04. TRAM-01: A PHASE 2 STUDY OF TRAMETINIB FOR PATIENTS WITH PEDIATRIC GLIOMA WITH ACTIVATION OF THE MAPK/ERK PATHWAY

Abstract BACKGROUND Pediatric low-grade gliomas (PLGG) are the most frequent brain tumors in children. It is known that the majority of PLGG have activation of the MAPK/ERK pathway. METHODS This ongoing multicenter phase II trial includes three progressing/refractory PLGG groups: NF1 patients, KIAA1549-BRAF fusion patients and patients with other activation of the MAPK/ERK pathway (excluding V600E). The primary objective was to evaluate the overall response rate based on RANO criteria after daily oral trametinib administration for 18 cycles, lasting 28 days each. Secondary objectives include the assessment of progression-free survival, tolerability of trametinib, serum levels of trametinib and quality of life evaluation during treatment. RESULTS As of February 12 2021, 50 patients have been enrolled (NF1: n=10; KIAA1549-BRAF fusion: n=31; other: n=9 including 5 patients with FGFR1 alterations). Median age is 8.8 years (range 2.4-25.5). Median follow-up is 17.5 months (range 4.7-28.5). Forty-three patients are evaluable. The overall response includes: 4 partial response (PR) (9%), 18 minor response (MR) (42%), 17 stable disease (40%), 4 progressive disease (9%). Median time to response is 5.5 months (range 2.4-13.8). Median duration of response is 6.1 months (range 0.6-26.5). Progression free survival at 12 months is 79.9% (95% CI 68.5-93.6%) and median progression free survival has not yet been reached. Treatment was discontinued for 30 patients: 16 after completing 18 cycles as planned, 5 for progressive disease, 5 for adverse events, 4 for other reasons. A total of 8 patients progressed after discontinuation of treatment including 6 patients (37.5%) that completed 18 cycles. Five of these patients had achieved minor response prior to discontinuation. CONCLUSION Trametinib is a potentially effective targeted therapy for patients with recurrent/refractory PLGG. Treatment was overall well tolerated. This ongoing trial will continue to gather data on response rate, duration of response and safety of trametinib for PLGG.

Polymorphous Low-Grade Neuroepithelial Tumor of the Young (PLNTY): Molecular Profiling Confirms Frequent MAPK Pathway Activation.

Polymorphous low-grade neuroepithelial tumor of the young (PLNTY) is a recently described epileptogenic tumor characterized by oligodendroglioma-like components, aberrant CD34 expression, and frequent mitogen-activated protein kinase (MAPK) pathway activation. We molecularly profiled 13 cases with diagnostic histopathological features of PLNTY (10 female; median age, 16 years; range, 5-52). Patients frequently presented with seizures (9 of 12 with available history) and temporal lobe tumors (9 of 13). MAPK pathway activating alterations were identified in all 13 cases. Fusions were present in the 7 youngest patients: FGFR2-CTNNA3 (n = 2), FGFR2-KIAA1598 (FGFR2-SHTN1) (n = 1), FGFR2-INA (n = 1), FGFR2-MPRIP (n = 1), QKI-NTRK2 (n = 1), and KIAA1549-BRAF (n = 1). BRAF V600E mutation was present in 6 patients (17 years or older). Two fusion-positive cases additionally harbored TP53/RB1 abnormalities suggesting biallelic inactivation. Copy number changes predominantly involving whole chromosomes were observed in all 10 evaluated cases, with losses of chromosome 10q occurring with FGFR2-KIAA1598 (SHTN1)/CTNNA3 fusions. The KIAA1549-BRAF and QKI-NTRK2 fusions were associated respectively with a 7q34 deletion and 9q21 duplication. This study shows that despite its name, PLNTY also occurs in older adults, who frequently show BRAF V600E mutation. It also expands the spectrum of the MAPK pathway activating alterations associated with PLNTY and demonstrates recurrent chromosomal copy number changes consistent with chromosomal instability.

Evaluation of BRAF Gene Status in Gliomas

Background: Development of different molecular markers has given a new insight in the glioma management. KIAA1549-BRAF gene fusion has a diagnostic and prognostic significance. Aim: The aim of this study was to determine the KIAA1549-BRAF gene fusion in glioma and their correlation with various clinical parameters. Material and Methods: Forty cases of glioma were studied for KIAA1549-BRAF gene fusion by reverse transcription-PCR (RT-PCR). Results: Overall, KIAA1549-BRAF gene fusion was found in 22% (9/40) cases of glioma. Children had higher KIAA1549-BRAF fusion (72%; 8/11) as compared to adults (10%; 3/29) and this difference was statistically significant. Cerebellar location of tumor was significantly associated with KIAA1549-BRAF fusion. KIAA1549-BRAF fusion was highest in pilocytic astrocytoma (89%), and this difference was statistically significant. Statistically significant difference was noted between KIAA1549-BRAF fusion expression and WHO grade I glioma. Conclusion: Overall, KIAA1549-BRAF gene fusion was found in 22% (9/40) cases of glioma. Childhood age, pilocytic astrocytoma histology, cerebellar location and WHO grade I tumor were significantly associated with KIAA1549-BRAF gene fusion.

Integrated genotype–phenotype analysis of long‐term epilepsy‐associated ganglioglioma

The BRAF p.V600E mutation is the most common genetic alteration in ganglioglioma (GG). Herein, we collected a consecutive series of 30 GG specimens from Xuanwu Hospital in order to corroborate the genetic landscape and genotype–phenotype correlation of this enigmatic and often difficult‐to‐classify epilepsy‐associated brain tumor entity. All specimens with histopathologically confirmed lesions were submitted to targeted next‐generation sequencing using a panel of 131 genes. Genetic alterations in three cases with histologically distinct tumor components, that is, GG plus pleomorphic xanthoastrocytoma (PXA), dysembryoplastic neuroepithelial tumor (DNT), or an oligodendroglioma (ODG)‐like tumor component, were separately studied. A mean post‐surgical follow‐up time‐period of 23 months was available in 24 patients. Seventy seven percent of GG in our series can be explained by genetic alterations, with BRAF p.V600E mutations being most prevalent (n = 20). Three additional cases showed KRAS p.Q22R and KRAS p.G13R, IRS2 copy number gain (CNG) and a KIAA1549‐BRAF fusion. When genetically studying different histopathology patterns from the same tumor we identified composite features with BRAF p.V600E plus CDKN2A/B homozygous deletion in a GG with PXA features, IRS2 CNG in a GG with DNT features, and a BRAF p.V600E plus CNG of chromosome 7 in a GG with ODG‐like features. Follow‐up revealed no malignant tumor progression but nine patients had seizure recurrence. Eight of these nine GG were immunoreactive for CD34, six patients were male, five were BRAF wildtype, and atypical histopathology features were encountered in four patients, that is, ki‐67 proliferation index above 5% or with PXA component. Our results strongly point to activation of the MAP kinase pathway in the vast majority of GG and their molecular‐genetic differentiation from the cohort of low‐grade pediatric type diffuse glioma remains, however, to be further clarified. In addition, histopathologically distinct tumor components accumulated different genetic alterations suggesting collision or composite glio‐neuronal GG variants.

LGG-13. PILOCYTIC ASTROCYTOMAS WITH NOVEL BRAF FUSIONS DEMONSTRATE MAPK PATHWAY ACTIVATION

BackgroundPilocytic astrocytomas (PAs) are the most common pediatric low-grade glioma subtype. Oftentimes, PAs demonstrate somatic genetic alterations, the most common being the BRAF-KIAA1549 fusion, which results in constitutive activation of the MAPK pathway. Better understanding of the effects of other RAF fusions is necessary to determine the potential utility of MAPK-targeting therapies. MethodsThree patients presented to Children’s Hospital Colorado and were ultimately diagnosed with PAs harboring previously unreported gene fusions identified as FYCO-RAF1, CCTTNNBP2-BRAF, and SLC44A1-BRAF. Utilizing immunohistochemistry, we stained novel samples and controls for ERK and pERK (phosphorylated ERK) to assess the activation of the MAPK pathway. PAs with known BRAF-KIAA1549 fusions (4 samples) and normal brain tissue (5 samples) were used as positive and negative controls, respectively. We additionally performed RNA sequencing to better understand expression changes associated with these fusions, utilizing Metascape and GSEA (Gene Set Enrichment Analysis) for analysis. ResultsImmunohistochemistry of negative control samples demonstrated less p-ERK than ERK (ratios of 0.6–0.9, mean 0.8). All samples with novel fusions demonstrated statistically significantly higher p-ERK expression compared to negative controls (ratios of 1.3–1.7, mean 1.4). These experimental samples also all fell within the p-ERK to ERK expression range of the positive control samples, which demonstrated the widest range of expression (ratios of 1.1–4.5, mean 2.2). Our molecular analysis further confirmed these results, with GSEA demonstrating positively upregulated MAPK and ERK pathways in 2 positive controls and 1 novel fusion sample. Metascape analysis emphasized overall similar gene expression between these samples, demonstrating many shared genes and functional pathways. ConclusionsWe identified 3 previously unreported RAF fusions in PA that demonstrate activation of the MAPK pathway, although not as extensively as seen in some positive control samples with BRAF-KIAA1549 fusions. MEK inhibition may be a useful therapeutic strategy in these tumors if targeted therapy is indicated.

LGG-16. TO BE PRECISE - KNOW YOUR TARGETS: INSTITUTIONAL EXPERIENCE WITH TUMOR TARGETED THERAPY FOR RECURRENT/PROGRESSIVE PEDIATRIC LOW-GRADE GLIOMA AND PLEXIFORM NEUROFIBROMA

IntroductionThe oncogenic drivers of pediatric CNS tumors are rapidly being identified with the implementation of high throughput genetic screening. Precision medicine approaches to treatment have shown promising results, but data remains limited in the community oncology setting. We aim to describe our institutional experience using targeted therapies for plexiform neurofibroma and recurrent/progressive pediatric low-grade glioma (pLGG). MethodsWe performed a retrospective chart review of all patients treated with tumor targeted therapies for recurrent/progressive pLGG and plexiform neurofibroma over the past 5 years. ResultsTen patients treated with tumor targeted therapies were identified. Regimens included combination Dabrafenib and Trametinib (n=3), Trametinib monotherapy (n=2), Selumetinib (n=3), Vemurafenib (n=1), and Larotrectinib (n=1). Median age at therapy initiation was 11.5 years (range 1.1 - 18 years). Tumor molecular status included BRAFV600E mutation (n=4), NF1 mutation (n=2), KIAA1549-BRAF fusion (n=1), NACC-NTRK fusion (n=1), and FGFR1 mutation (n=1). Patients trialed an average of 2 treatment regimens prior to targeted therapy initiation (range 0–5). Mean duration of therapy was 14.5 months (range .5–33 months) with 8 patients remaining on treatment. Based on modified RANO criteria, responses included partial (n=1), stable disease (n=8), and progressive disease (n=1). Progressive disease was noted after 4 months of treatment with Dabrafenib and Trametinib combination therapy, but rate of tumor growth was decreased. Subjective functional improvement was seen in 50% of patients. The most common toxicities included rash (n=5) and pyrexia (n=2). Trametinib was discontinued in one patient due to intra-tumoral hemorrhage of unclear etiology. ConclusionTreatment of pediatric CNS tumors with targeted agents appears to be feasible and efficacious in the community oncology setting. Multi-institutional clinical trials are currently ongoing for each of these therapies. There remains a need for community oncology institutional data regarding their use.

EPCT-13. SINGLE INSTITUTION RETROSPECTIVE ANALYSIS OF TUMOR MUTATIONAL BURDEN AND SURVIVAL IN PEDIATRIC BRAIN TUMORS

Tumor mutational burden (TMB) has been studied across numerous cancer types as a means of risk stratification. To examine the prognostic relevance of TMB to pediatric central nervous system (CNS) tumors, we conducted a retrospective analysis of patients at Albany Medical Center diagnosed from 2012 to present. Patients were <21 at diagnosis, had a primary CNS tumor and available genomic data. Forty-seven patients were included – 22 low-grade gliomas, 10 high-grade gliomas, 5 medulloblastomas, 3 ependymomas, 2 choroid plexus carcinomas, and 5 other CNS tumors, with a median follow up of 36 months, median age at diagnosis 10 (1–19), and 47% female. Median TMB was 1 mutation per megabase (mut/mb); range 0–6. Nine patients did not have available TMB data. Twenty-seven patients had driver mutations and other alterations implicated in cancer development including, including BRAF-KIAA1549 fusion (n=6), NF1 loss (n=5), FGFR1 amplification (n=4), TP53 inactivation (n=4), BRAF V600E mutation (n=3), and H3F3A K28M mutation (n=3). Patients with low TMB (<3 muts/mb; n=24) versus high TMB (≥3 muts/mb; n=14) had a survival of 87% versus 71%, respectively, at last follow-up. Of note, all but one patient in the low TMB cohort had localized disease at diagnosis versus three in the high TMB cohort. High TMB was more prevalent in high- (45%, 9/20) versus low-grade histologies (22%, 4/18). Patients with BRAF alterations had LGGs and low TMB (0–1 muts/mb) with all patients surviving at last follow up. Of the eight deaths observed (median 18 months from diagnosis) TMB was high in 4, low in 3, and unknown in 1; all had high-grade histology. Although limited, our data suggests higher TMB may be associated with worse outcome. This analysis will be expanded via a multi-institutional review of TMB and genomic alterations in pediatric CNS patients to better identify high-risk patients requiring alternative treatment strategies.

Pediatric Glioma

Pediatric gliomas include various types of glioma broadly categorized as low- or hi-grade based on histopathological features. Clinically significant types include cerebellar astrocytomas, optic pathway / hypothalamic pilocytic astrocytomas, and brainstem gliomas. Neurosurgical roles vary for different kinds of pediatric gliomas. Since these representative tumors remain rare, the patients should be directed toward facilities with experienced neurosurgeons. Radiotherapy and chemotherapy are very important as either adjuvant or primary treatment modalities. Recent advancements in molecular biology have revealed unique genetic aberrations in different types of pediatric gliomas. The RAS/MAPK pathway anomalies, including BRAF-KIAA1549 fusion and BRAF V600E mutation, are present in most low-grade gliomas. BRAF/MEK-inhibitors have yielded promising clinical study results. Diffuse midline gliomas, including diffuse intrinsic pontine gliomas, often harbor H3 mutations such as H3K27M. Agents that target these molecular aberrations are unavailable. Because gliomas in infants are sub-categorized by their genetic abnormalities, novel agents targeting ALK, ROS1, or NTRK fusions are promising treatments.

DIFFUSE LEPTOMENINGEAL GLIONEURONAL TUMOR IN CHILDREN: MR CHARACTERISTICS, CLINICAL FEATURES AND OUTCOME. FOUR CLINICAL CASES

Background. Diffuse leptomeningeal glioneuronal tumor (DLGNT) is an extremely rare entity first officially recognized in 2016 WHO classification of tumors of the central nervous system. Magnetic resonance imaging (MRI) of this tumor usually visualizes diffuse meningeal infiltration with contrast enhancement, with the presence of multiple small contrast‑negative cysts, visible mainly in the T2 images. The main molecular markers of DLGNTs include the KIAA1549-BRAF fusion gene, BRAF V600E substitution is less common.The aim of this work is to describe the manifestation of DLGNT, its neuroimaging and molecular genetic characteristics, the experience of using anti‑BRAF and anti‑MEK therapy.Materials and methods. In this article are described four cases of DLGNT. The first patient with the presence of the KIAA1549-BRAF fusion in the tumor tissue received a full course of SIOP‑LGG / 2004 chemotherapy (carbo‑ platin and vincristine), the stabilization of the disease on the MRI remains for 4 years after completion of treatment. Second patient with KIAA1549-BRAF fusion gene in tumour tissue received MEK inhibitor trametinib as first line of treatment with the stabilization of the disease on control MRI which last for 2 years. A third patient with a mutation in the BRAF V600E gene. After disease progression on standard chemotherapy (carboplatin and vincristine) according to the SIOP‑LGG / 2004 protocol, anti‑BRAF therapy with vemurafenib was prescribed. After 10 months on MRI a complete response was recorded, which persists during the drug intake for 2.5 years. In the fourth patient, no molecular genetic aberrations were detected; a refractory / progressive course of the dis‑ ease was noted. To date, the stabilization of the disease is recorded on the fourth line of chemotherapy (everoli‑ mus and temozolomide).Conclusion. Given the rarity of this tumor and the lack of consensus about therapy, despite the limited number of observations, our experience allows us to recommend molecular testing of DLGNT to detect activating events in the BRAF gene, as well as consideration of anti‑BRAF / MEK therapy if either the BRAF V600E mutation is de‑ tected or KIAA1549-BRAF fusion.

Concomitant KIAA1549-BRAF fusion and IDH mutation in Pediatric spinal cord astrocytoma: a case report and literature review

Primary tumors of the spinal cord are rare, accounting for 3-6% of tumors in the central nervous system, particularly in children. KIAA1549-BRAF fusion is more common in pilocytic astrocytoma (PA) and IDH1 R132H mutation is rare in infratentorial tumors. Here, we report a 10-year-old male patient who presented with weakness in lower limbs that progressed to difficulty walking. Magnetic resonance imaging (MRI) revealed an intramedullary solid-cystic lesion from the medulla oblongata to the thoracic spin 4 level, with the expansion of the spinal cord. The lesion exhibited patchy enhancement at C4-T1, indicating a tentative diagnosis of astrocytoma. The patient underwent resection of the lesion in the spinal canal from the cervical 6 level to the thoracic 2 level. Histopathology confirmed diagnosis of astrocytoma, WHO grade 2. Genetic analysis showed both IDH1 R132H mutation and KIAA1549-BRAF fusion. Therefore, our integrated diagnosis was astrocytoma, IDH mutation, WHO grade 2. Its molecular analyses include IDH1 R132H mutation and KIAA1549-BRAF fusion. After the operation, the patient did not receive chemo- or radiotherapy, and underwent an aggressive rehabilitation regiment. Follow up 10months later, symptoms improved. To our best knowledge, this is the first case of concomitant IDH mutation and BRAF fusion in pediatric spinal cord astrocytoma.

SNP-based detection of allelic imbalance: A novel approach for identifying KIAA1549-BRAF fusion in pilocytic astrocytoma using DNA sequencing

Pilocytic astrocytoma (PA) is the most common glioma subtype found in children, and it is a non-malignant tumor type. The majority of PAs is caused by an approximately 2 Mb tandem duplication within 7q34 which creates an in-frame KIAA1549-BRAF fusion gene. The kinase domain of BRAF is fused to the N-terminal of KIAA1549, whereby BRAF is constitutively activated. We here present a novel approach for identifying KIAA1549-BRAF fusion based on single nucleotide polymorphism (SNP) analysis and next generation sequencing (NGS). Highly polymorphic SNPs in the duplicated area and in adjacent areas were selected and a custom targeted amplicon based NGS panel was designed. The panel was tested on DNA extracted from formalin fixed and paraffin embedded tissue from a retrospective cohort, consisting of biopsies from patients with PA, anaplastic astrocytoma, oligodendroglioma and glioblastoma as well as two non-tumor biopsies. The panel could distinguish chromosome 7 gain from BRAF fusion and correctly identified 8/9 PA samples with KIAA1549-BRAF fusion confirmed by RNA sequencing. The one biopsy where no fusion was detected was fresh frozen and from the RNA sequencing expected to have very low tumor content. No allelic imbalance was detected in either oligodendroglioma or in the non-tumor biopsies.

Accurate calling of KIAA1549-BRAF fusions from DNA of human brain tumours using methylation array-based copy number and gene panel sequencing data.

KIAA1549-BRAF fusions occur in certain brain tumours and provide druggable targets due to a constitutive activation of the MAP-kinase pathway. We introduce workflows for calling the KIAA1549-BRAF fusion from DNA methylation array-derived copy number as well as DNA panel sequencing data. Copy number profiles were analysed by automated screening and visual verification of a tandem duplication on chromosome 7q34, indicative of the KIAA1549-BRAF fusion. Pilocytic astrocytomas of the ICGC cohort with known fusion status were used for validation. KIAA1549-BRAF fusions were called from DNA panel sequencing data using the fusion callers Manta, Arriba with modified filtering criteria and deFuse. We screened DNA methylation and panel sequencing data of 7790 specimens from brain tumour and sarcoma entities. We identified the fusion in 337 brain tumours with both DNA methylation and panel sequencing data. Among these, we detected the fusion from copy number data in 84% and from DNA panel sequencing data in more than 90% using Arriba with modified filters. While in 74% the KIAA1549-BRAF fusion was detected from both methylation array-derived copy number and panel sequencing data, in 9% it was detected from copy number data only and in 16% from panel data only. The fusion was almost exclusively found in pilocytic astrocytomas, diffuse leptomeningeal glioneuronal tumours and high-grade astrocytomas with piloid features. The KIAA1549-BRAF fusion can be reliably detected from either DNA methylation array or DNA panel data. The use of both methods is recommended for the most sensitive detection of this diagnostically and therapeutically important marker.