ATRX Mutations Research Articles

891 Next Generation Sequencing Mutational Profiles of Gliomas Presenting With and Without Seizure

INTRODUCTION: Seizures are among the most common presentation of gliomas in adults, but much remains unknown regarding the relationship between underlying tumor biology and epileptic activity. METHODS: We conducted a single-center retrospective review of 140 adult patients who underwent surgical management of glioma with next generation sequencing of tumor samples obtained 2016-2020. Univariate odds ratios were computed for relevant cohort characteristics. RESULTS: Among patients with documented seizure history, 71 (51.1%) presented with seizures while 68 (48.9%) did not. At time of diagnosis, patients presenting with seizure were younger (47 vs 60.5, p = 0.0001) and had more recurrent disease (60.6% vs 16.7%, p < 0.0001). Patients presenting with seizure had fewer glioblastomas (OR: 0.2 [0.09-0.41], p < 0.0001) but more astrocytomas (OR: 4.78 [1.68-13.65], p = 0.0038) and oligodendrogliomas (OR: 3.47 [1.07-11.27], p = 0.0557). Of the top 15 most common mutations in our cohort, patients presenting with seizure had fewer instances of IDH1 (OR: 5.26 [2.44-11.32], p<0.0001), polysomy 7/monosomy 10 (OR: 0.48 [0.24-0.95], p = 0.0523), and PTEN mutation (OR: 0.46 [0.22-0.98], p = 0.0646), but more instances of 1p/19q codeletion (OR: 3.47[1.07-11.27], p = 0.0557), ATRX (OR: 2.54 [1.15-5.59], p = 0.0315) and CIC (OR: 13.63 [1.72-107.97], p = 0.0046) mutations. Notably, mutations in NF1 (OR: 0.73 [0.28-1.88)], p = 0.6753), TP53 (OR: 1.1 [0.56-2.15], p = 0.9194), TERT (OR: 0.63 [0.31-1.28], p = 0.2679), CDKN2A/B (OR: 0.61 [0.31-1.21], p = 0.2154), and EGFR (OR: 0.49 [0.21-1.13], p = 0.1396) were not associated with seizure presentation. CONCLUSIONS: ATRX and CIC mutations are positively associated with seizure presentation while polysomy 7/monosomy 10 and PTEN mutations are negatively associated. Future analysis of this cohort is needed to better characterize the influence of tumor mutational profile on preoperative as well as postoperative seizure activity.

Artificial-intelligence-based molecular classification of diffuse gliomas using rapid, label-free optical imaging.

Molecular classification has transformed the management of brain tumors by enabling more accurate prognostication and personalized treatment. However, timely molecular diagnostic testing for patients with brain tumors is limited, complicating surgical and adjuvant treatment and obstructing clinical trial enrollment. In this study, we developed DeepGlioma, a rapid (<90 seconds), artificial-intelligence-based diagnostic screening system to streamline the molecular diagnosis of diffuse gliomas. DeepGlioma is trained using a multimodal dataset that includes stimulated Raman histology (SRH); a rapid, label-free, non-consumptive, optical imaging method; and large-scale, public genomic data. In a prospective, multicenter, international testing cohort of patients with diffuse glioma (n = 153) who underwent real-time SRH imaging, we demonstrate that DeepGlioma can predict the molecular alterations used by the World Health Organization to define the adult-type diffuse glioma taxonomy (IDH mutation, 1p19q co-deletion and ATRX mutation), achieving a mean molecular classification accuracy of 93.3 ± 1.6%. Our results represent how artificial intelligence and optical histology can be used to provide a rapid and scalable adjunct to wet lab methods for the molecular screening of patients with diffuse glioma.

Molecular correlates of invasion pattern in HPV-associated endocervical adenocarcinoma: emergence of two distinct risk-stratified tiers.

The pattern-based (Silva) classification of invasive human papilloma virus (HPV)-associated endocervical adenocarcinomas (HPVA) is an established and reproducible method to predict outcomes for this otherwise stage-dependent group of tumours. Previous studies utilising targeted sequencing have shown a correlation between mutational profiles and an invasive pattern. However, such correlation has not been explored using comprehensive molecular testing. Clinicopathologic data including invasive pattern (Silva groups A, B, and C) was collected for a cohort of invasive HPVA, which previously underwent massive parallel sequencing using a panel covering 447 genes. Pathogenic alterations, molecular signatures, tumour mutational burden (TMB), and copy number alterations (CNA) were correlated with pattern of invasion. Forty five HPVA (11 pattern A, 17 pattern B, and 17 pattern C tumours) were included. Patients with pattern A presented at stage I with no involved lymph nodes or evidence of recurrence (in those with >2 months of follow-up). Patterns B and C patients also mostly presented at stage I with negative lymph nodes, but had a greater frequency of recurrence; 3/17 pattern B and 1/17 pattern C HPVAs harboured lymphovascular space invasion (LVI). An APOBEC mutational signature was detected only in Silva pattern C tumours (5/17), and pathogenic PIK3CA changes were detected only in destructively invasive HPVA (patterns B and C). When cases were grouped as low-risk (pattern A and pattern B without LVI) and high-risk (pattern B with LVI and pattern C), high-risk tumours were enriched in mutations in PIK3CA, ATRX, and ERBB2. There was a statistically significant difference in TMB between low-risk and high-risk pattern tumours (P= 0.006), as well as between Pattern C tumours with and without an APOBEC signature (P= 0.002). CNA burden increased from pattern A to C. Our findings further indicate that key molecular events in HPVA correlate with the morphologic invasive properties of the tumour and their aggressiveness. Pattern B tumours with LVI clustered with pattern C tumours, whereas pattern B tumours without LVI approached pattern A genotypically. Our study provides a biologic foundation for consolidating the Silva system into low-risk (pattern A + B without LVI) and high-risk (pattern B with LVI and pattern C) categories.

A Clinicopathologic and Molecular Characterization of Uterine Sarcomas Classified as Malignant PEComa.

Perivascular epithelioid cell tumors (PEComas) are a distinctive group of mesenchymal neoplasms that demonstrate features of smooth muscle and melanocytic differentiation. Here, we present the clinicopathologic, immunohistochemical, and molecular features of 15 uterine sarcomas diagnosed as malignant PEComa. The median patient age was 56 years (range: 27 to 86y). The median tumor size was 8.0cm (range: 5.0 to 14.0cm). All tumors were classified as malignant based on the presence of mitoses (15/15; 100%), necrosis (15/15; 100%), lymphovascular invasion (8/15; 53%), and high nuclear grade (13/15; 87%). Molecular analysis revealed the mammalian target of rapamycin pathway gene mutations in 7 cases (47%), including mutually exclusive variants in TSC1 (27%) and TSC2 (20%). Recurrent alterations were also identified in TP53 (53%), RB1 (30%), ATRX (33%), and BRCA2 (13%). Tumors with inactivating ATRX mutations all demonstrated loss of ATRX expression by immunohistochemistry. Loss of expression was also observed in 2 tumors without demonstrable ATRX alterations. Clinical follow-up was available for 14 patients (range: 5 to 92mo; median: 15mo). Five patients developed local recurrence and 9 developed metastases; 2 patients died of their disease. Our series expands the spectrum of molecular events in tumors diagnosed as malignant PEComa and further highlights the important role of targeted sequencing in tumors with focal melanocytic marker expression.

Aggressive Pituitary Tumors and Pituitary Carcinomas: From Pathology to Treatment.

Aggressive pituitary tumors (APTs) and pituitary carcinomas (PCs) are heterogeneous with regard to clinical presentation, proliferative markers, clinical course, and response to therapy. Half of them show an aggressive course only many years after the first apparently benign presentation. APTs and PCs share several properties, but a Ki67 index greater than or equal to 10% and extensive p53 expression are more prevalent in PCs. Mutations in TP53 and ATRX are the most common genetic alterations; their detection might be of value for early identification of aggressiveness. Treatment requires a multimodal approach including surgery, radiotherapy, and drugs. Temozolomide is the recommended first-line chemotherapy, with response rates of about 40%. Immune checkpoint inhibitors have emerged as second-line treatment in PCs, with currently no evidence for a superior effect of dual therapy compared to monotherapy with PD-1 blockers. Bevacizumab has resulted in partial response (PR) in few patients; tyrosine kinase inhibitors and everolimus have generally not been useful. The effect of peptide receptor radionuclide therapy is limited as well. Management of APT/PC is challenging and should be discussed within an expert team with consideration of clinical and pathological findings, age, and general condition of the patient. Considering that APT/PCs are rare, new therapies should preferably be evaluated in shared standardized protocols. Prognostic and predictive markers to guide treatment decisions are needed and are the scope of ongoing research.

Genomic profiling and prognostic factors of H3 K27M-mutant spinal cord diffuse glioma.

H3 K27-altered diffuse midline glioma is a highly lethal pediatric-type tumor without efficacious treatments. Recent findings have highlighted the heterogeneity among diffuse midline gliomas with different locations and ages. Compared to tumors located in the brain stem and thalamus, the molecular and clinicopathological features of H3 K27-altered spinal cord glioma are still largely elusive, thus hindering the accurate management of patients. Here we aimed to characterize the clinicopathological and molecular features of H3 K27M-mutant spinal cord glioma in 77 consecutive cases. We found that the H3 K27M-mutant spinal cord glioma, with a median age of 35 years old, had a significantly better prognosis than H3 K27M-mutant brain tumors. We noticed a molecular heterogeneity of H3 K27M-mutant spinal cord astrocytoma via targeted sequencing with 34 cases. TP53 mutation which occurred in 58.8% of cases is mutually exclusive with PPM1D (26%) and NF1 (44%) mutations. The TP53-mutant cases had a significantly higher number of copy number variants (CNV) and a marginally higher proportion of pediatric patients (age at diagnosis <18 years old, p=0.056). Cox regression and Kaplan-Meier curve analysis showed that the higher number of CNV events (≥3), chromosome (Chr) 9p deletion, Chr 10p deletion, ATRX mutation, CDK6 amplification, and retinoblastoma protein(RB)pathway alteration are associated with worse survival. Cox regression analysis with clinicopathological features showed that glioblastoma histological type and a high Ki-67 index (>10%) are associated with a worse prognosis. Interestingly, the histological type, an independent prognostic factor in multivariate Cox regression, can also stratify molecular features of H3 K27M-mutant spinal cord glioma, including the RBpathway, KRAS/PI3K pathway, and chromosome arms CNV. In conclusion, although all H3 K27M-mutant spinal cord diffuse glioma were diagnosed as WHO Grade 4, the histological type, molecular features representing chromatin instability, and molecular alterations associated with accelerated cell proliferative activity should not be ignored in clinical management.

A mysterious appearance at zone 1

We present the case of a 76-year-old male with history of Type II diabetes presenting for routine HbA1c testing. The HbA1c profile via capillary electrophoresis (CE) revealed the presence of an unknown haemoglobin peak of 19.3% in Zone 1. Blood film showed microcytosis, hypochromia, poikilocytosis and red cell dimorphism. The unknown haemoglobin migrated in the HbH position on the Sebia Capillarys3 Hemoglobin(E) program and on High Pressure Liquid Chromatography (HPLC). Many HbH bodies were detected on Brilliant Creysl Blue (BCB) staining and biochemistry was consistent with haemolysis. A presumptive diagnosis of acquired HbH disease, or alpha thalassaemia-MDS (AT-MDS), was determined. The acquired nature was determined by previously normal red cell indices and HbA1c chromatogram. AT-MDS is an acquired disorder that most commonly occurs due to rare somatic mutations in the ATRX gene, or less commonly chromosome 16p. There is a male predominance (6:1) and the median diagnosis age is 68. These mutations occur in 0.2–‘.8% of MDS cases and produce a more severe alpha thalassaemia phenotype when compared to germ line mutations of ATRX. This case highlights a novel diagnostic pathway for a rare disease and emphasises the importance of investigating ‘incidental’ haemoglobin variants detected during HbA1c testing.

Radiomic features of contralateral and ipsilateral hemispheres for prediction of glioma genetic markers

Purpose: Radiomic features of gliomas are often used to predict genetic markers from radiological studies. Radiomic features were extracted from the contralateral brain to test if tumor texture is driving the predictive power of machine learning models. Ideally, these contralateral models would be a negative control for tumor radiomics models, since many studies use contralateral normal appearing white matter for normalization. This study used those features to attempt to predict IDH mutation status, MGMT promoter methylation, TERT promoter mutation, and ATRX mutation status with random forests.Methods: Radiomic features were extracted from the tumor region, a mirrored contralateral region, a spherical region within the tumor, a spherical region on the contralateral, and a spherical sample of the ipsilateral side. These features were used independently to predict IDH, MGMT, TERT, and ATRX using random forests.Main Findings: Contralateral features alone were as predictive of IDH mutation status as tumor features and had predictive power for several genetic markers.Conclusion: Normalization with contralateral brain should be done carefully, and further investigation of potential radiological changes to the contralateral is warranted.

Gene fusions are frequent in ACTH-secreting neuroendocrine neoplasms of the pancreas, but not in their non-pancreatic counterparts

Ectopic Cushing syndrome is a rare clinical disorder resulting from excessive adrenocorticotrophic hormone (ACTH) produced by non-pituitary neoplasms, mainly neuroendocrine neoplasms (NENs) of the lung, pancreas, and gastrointestinal tract, and other less common sites. The genetic background of ACTH-producing NENs has not been well studied. Inspired by an index case of ACTH-producing pancreatic NEN carrying a gene fusion, we postulated that ACTH-producing NENs might be enriched for gene fusions. We herein examined 21 ACTH-secreting NENs of the pancreas (10), lung (9), thymus (1), and kidney (1) using targeted RNA sequencing. The tumors were classified according to the most recent WHO classification as NET-G1/typical carcinoid (n = 4), NETG-2/atypical carcinoid (n = 14), and NET-G3 (n = 3). Overall, targeted RNA sequencing was successful in 11 cases (4 of 10 pancreatic tumors, 5 of 9 pulmonary tumors, and in the one renal and one thymic tumor). All four successfully tested pancreatic tumors revealed a gene fusion: two had a EWSR1::BEND2 and one case each had a KMT2A::BCOR and a TFG::ADGRG7 fusion, respectively. EWSR1 rearrangements were confirmed in both tumors with a EWSR1::BEND2 by FISH. Gene fusions were mutually exclusive with ATRX, DAXX, and MEN1 mutations (the most frequently mutated genes in NETs) in all four cases. Using RNA-based variant assessment (n = 16) or via the TSO500 panel (n = 5), no pathogenic BCOR mutations were detected in any of the cases. Taken together, gene fusions were detected in 4/4 (100%) pancreatic versus 0/7 (0%) non-pancreatic tumors, respectively. These results suggest a potential role for gene fusions in triggering the ACTH production in pancreatic NENs presenting with ectopic Cushing syndrome. While the exact mechanisms responsible for the ectopic ACTH secretion are beyond the scope of this study, overexpressed fusion proteins might be involved in promoter-mediated overexpression of pre-ACTH precursors in analogy to the mechanisms postulated for EWSR1::CREB1-mediated paraneoplastic phenomena in certain mesenchymal neoplasms. The genetic background of the ACTH-producing non-pancreatic NENs remains to be further studied.

Cancer-associated SMARCAL1 loss-of-function mutations promote alternative lengthening of telomeres and tumorigenesis in telomerase-negative glioblastoma cells.

Telomere maintenance mechanisms are required to enable the replicative immortality of malignant cells. While most cancers activate the enzyme telomerase, a subset of cancers uses telomerase-independent mechanisms termed alternative lengthening of telomeres (ALT). ALT occurs via homology-directed-repair mechanisms and is frequently associated with ATRX mutations. We previously showed that a subset of adult glioblastoma (GBM) patients with ATRX-expressing ALT-positive tumors harbored loss-of-function mutations in the SMARCAL1 gene, which encodes an annealing helicase involved in replication fork remodeling and the resolution of replication stress. However, the causative relationship between SMARCAL1 deficiency, tumorigenesis, and de novo telomere synthesis is not understood. We used a patient-derived ALT-positive GBM cell line with native SMARCAL1 deficiency to investigate the role of SMARCAL1 in ALT-mediated de novo telomere synthesis, replication stress, and gliomagenesis in vivo. Inducible rescue of SMARCAL1 expression suppresses ALT indicators and inhibits de novo telomere synthesis in GBM and osteosarcoma cells, suggesting that SMARCAL1 deficiency plays a functional role in ALT induction in cancers that natively lack SMARCAL1 function. SMARCAL1-deficient ALT-positive cells can be serially propagated in vivo in the absence of detectable telomerase activity, demonstrating that the SMARCAL1-deficient ALT phenotype maintains telomeres in a manner that promotes tumorigenesis. SMARCAL1 deficiency is permissive to ALT and promotes gliomagenesis. Inducible rescue of SMARCAL1 in ALT-positive cell lines permits the dynamic modulation of ALT activity, which will be valuable for future studies aimed at understanding the mechanisms of ALT and identifying novel anticancer therapeutics that target the ALT phenotype.

Alternative lengthening of telomeres (ALT) in pediatric high-grade gliomas can occur without ATRX mutation and is enriched in patients with pathogenic germline mismatch repair (MMR) variants.

To achieve replicative immortality, most cancers develop a telomere maintenance mechanism, such as reactivation of telomerase or alternative lengthening of telomeres (ALT). There are limited data on the prevalence and clinical significance of ALT in pediatric brain tumors, and ALT-directed therapy is not available. We performed C-circle analysis (CCA) on 579 pediatric brain tumors that had corresponding tumor/normal whole genome sequencing through the Open Pediatric Brain Tumor Atlas (OpenPBTA). We detected ALT in 6.9% (n=40/579) of these tumors and completed additional validation by ultrabright telomeric foci in situ on a subset of these tumors. We used CCA to validate TelomereHunter for computational prediction of ALT status and focus subsequent analyses on pediatric high-grade gliomas (pHGGs) Finally, we examined whether ALT is associated with recurrent somatic or germline alterations. ALT is common in pHGGs (n=24/63, 38.1%), but occurs infrequently in other pediatric brain tumors (<3%). Somatic ATRX mutations occur in 50% of ALT+ pHGGs and in 30% of ALT- pHGGs. Rare pathogenic germline variants in mismatch repair (MMR) genes are significantly associated with an increased occurrence of ALT. We demonstrate that ATRX is mutated in only a subset of ALT+ pHGGs, suggesting other mechanisms of ATRX loss of function or alterations in other genes may be associated with the development of ALT in these patients. We show that germline variants in MMR are associated with the development of ALT in patients with pHGG.

Racial/Ethnic and Sex Differences in Somatic Cancer Gene Mutations among Patients with Early-Onset Colorectal Cancer.

NHBs, but not APIs, with early-onset nonhypermutated colorectal cancer had higher adjusted tumor mutation rates versus NHWs. Differences for FLT4, FBXW7, RNF43, LRP1B, APC, PIK3CA, and ATRX mutation rates between racial/ethnic groups and EP300, KRAS, AXIN2, WRN, BRAF, and LRP1B mutation rates by sex were observed in tumors of young patients. See related commentary by Shen et al., p. 530 . This article is highlighted in the In This Issue feature, p. 517.

Elimusertib (BAY1895344), a novel ATR inhibitor, demonstrates in vivo activity in ATRX mutated models of uterine leiomyosarcoma

IntroductionUterine leiomyosarcoma (uLMS) is a rare, highly aggressive malignancy. Recent data suggest 50% of uLMS may harbor alterations in the ATRX gene and such mutations may confer sensitivity to ataxia-telangiectasia-and-Rad3-related (ATR) kinase inhibitors. We sought to investigate the in vivo activity of Elimusertib (BAY1895344), a novel ATR-inhibitor, against ATRX-mutated uLMS patient-derived xenografts (PDXs). MethodsTwo fully characterized uLMS (i.e., LEY-11 and LEY-16) were grafted into female CB-17/SCID mice. Treatments with control vehicle or BAY1895344 (20 mg/kg dosed twice daily 3 days on 4 days off) were given via oral gavage and tumor measurements as well as weights obtained twice weekly. Tumor volume differences were calculated with a two-way ANOVA. Mechanistic studies were performed ex vivo using BAY1895344 treated uLMS tumor samples by western blot analysis. ResultsBoth PDX LEY-11 and PDX LEY-16 harboring ATRX gene mutations demonstrated an aggressive behavior in vivo (i.e., control mice were euthanized on average at day 12.5 for PDX LEY-11 and at day 33 for PDX LEY-16). In both tumor models BAY1895344 20 mg/kg dosed with an intermittent oral schedule was able to induce significant growth inhibition compared to vehicle control treatment (p < 0.001 for both LEY-11 and LEY-16) and prolong median overall survival [PDX LEY-11 (12.5 vs. 42 days, p < 0.001) and PDX LEY-16 (33 vs. 60 days, p < 0.001)]. There were not significant changes in weight between treatment and controls. By western blot assays BAY1895344 exposure decreased phosphorylated-ATR and increased expression of apoptotic molecules in LMS PDXs. ConclusionsBAY1895344 demonstrates promising in vivo activity against biologically aggressive PDX models of uLMS harboring ATRX mutations, with no significant toxicity. Clinical trials of BAY1895344 in uLMS patients are warranted.

DNAR-08. THE ROLE OF SMARCAL1 AS A SYNTHETIC LETHAL VULNERABILITY IN ATRX-DEFICIENT GLIOMAS

Abstract Mechanisms to maintain telomere length over successive cell divisions are a requirement for cancer cell immortalization. Although many cancers maintain telomere length through the activation of telomerase, ~10-15% of human cancers use telomerase-independent mechanisms of telomere maintenance, termed Alternative Lengthening of Telomeres (ALT). In gliomas, the ALT phenotype is associated with loss-of-function mutations in the ATRX gene in IDH-mutant astrocytomas, adult and pediatric glioblastomas (GBM), high-grade astrocytomas with piloid features, and pleomorphic xanthoastrocytomas. In adult GBM, we previously identified a relatively rare subset of ALT-positive ATRX wildtype tumors that harbored loss-of-function mutations in the SMARCAL1 gene, and we found that loss of SMARCAL1 activity plays a causative role in the onset of ALT in these tumors. SMARCAL1 is an annealing helicase that localizes to sites of DNA damage and replication stress and catalyzes the reversal of stalled replication forks. To better understand the relationship between ATRX mutations, SMARCAL1 activity, and the ALT phenotype, we investigated the localization and function of SMARCAL1 in ALT-positive glioma cells and xenografts with native ATRX deficiency. We found that SMARCAL1 localizes to ALT-associated PML bodies in astrocytoma cell lines with concurrent ATRX and IDH1 mutations (derived from grade 3 and 4 tumors). Our data show that inducible suppression of SMARCAL1 via doxycycline-induced RNAi leads to increased DNA double-strand breaks, increased abundance of extrachromosomal telomeric repeats (c-circles), and increased sensitivity to irinotecan, a topoisomerase I inhibitor. In an orthotopic mouse model, SMARCAL1 depletion exhibited a chemosensitizing effect in combination with irinotecan against a patient-derived xenograft (astrocytoma, IDH-mutant Grade 4). Based on these data, we hypothesize that SMARCAL1 activity is critical for resolving ALT-associated replication stress in ATRX-deficient malignant gliomas. We therefore propose that SMARCAL1 functions as a synthetic lethal vulnerability in ATRX-deficient ALT-positive gliomas and that SMARCAL1 inhibition is a viable therapeutic strategy in these tumors.

PATH-07. MULTIPLATFORM MOLECULAR ANALYSES REFINE CLASSIFICATION AND PROGNOSTICATION OF GLIOMAS ARISING IN PATIENTS WITH NEUROFIBROMATOSIS TYPE 1

Abstract BACKGROUND Gliomas arising in patients with neurofibromatosis type 1 (NF1) are heterogeneous, occurring from childhood through adulthood, can be histologically low-grade (LG) or high-grade (HG), and follow an indolent or aggressive clinical course. Comprehensive profiling of genetic alterations beyond NF1 and epigenetic classification of these tumors remain limited. METHODS Next-generation sequencing and DNA methylation profiling was performed on gliomas from 47 NF1 patients and correlated with clinicopathologic features, treatment, and outcomes. RESULTS 30 tumors demonstrated biallelic inactivation of NF1 without additional oncogenic alterations (“molecular LG subgroup”, median age 14 yrs). The remaining 17 tumors harbored additional oncogenic alterations beyond NF1 (“molecular HG subgroup”, median age 28), most frequently CDKN2A homozygous deletion (n=13), ATRX mutation (n=8), PIK3CA or PIK3R1 mutation (n=4), and TP53 mutation (n=3). Survival analysis showed significant differences in time to progression (137 vs 11 mos, p&amp;lt; 0.0001) and median survival time (undefined vs 37 mos, p &amp;gt; 0.0001) for molecular LG versus HG subgroups. DNA methylation profiles of the molecular LG subgroup resolved into a new epigenetic cluster closest to but divergent from the three existing reference classes of sporadic pilocytic astrocytoma. DNA methylation profiles of the molecular HG subgroup demonstrated most tumors epigenetically align with either HGAP or various subclasses of IDH-wildtype GBM. CONCLUSION NF1-associated gliomas stratify into two molecular subgroups. The “molecular LG subgroup” occurs primarily during childhood, harbors biallelic NF1 inactivation only, follows a more indolent clinical course, and has a unique epigenetic signature for which we propose the terminology “pilocytic astrocytoma, arising in the setting of NF1”. The “molecular HG subgroup” occurs primarily during adulthood, harbors additional oncogenic alterations including CDKN2A homozygous deletion and ATRX mutation, follows a more aggressive clinical course, and is epigenetically diverse. These findings highlight recurrently altered pathways in NF1-associated gliomas and help inform targeted therapeutic strategies for this patient population.

PATH-17. DISTINCT MOLECULAR SUBCLASSES OF H3F3A-WILDTYPE, EGFR-ALTERED PEDIATRIC-TYPE DIFFUSE MIDLINE GLIOMAS WHO CNS GRADE 4

Abstract OBJECTIVES Histone gene H3K27-altered diffuse midline glioma (DMG) are malignant tumors that occur in all age groups. In this study, we report comprehensive genomic profiling from H3F3A-wildtype, EGFR-altered DMG, as a distinct and newly recognized subset of pediatric-type DMG. METHODS Tumors were profiled in the comprehensive genomic profiling (CGP) program at Foundation Medicine between 2013-2020. Information from pathology reports, histopathology review, and patient clinical data was assessed. RESULTS We collected demographic and genomic data from 39 pediatric patients with H3F3A-wildtype, EGFR-altered HGG (17 females, 22 males; median age: 8.5 years, range 1-18 years). Female patients were younger at first diagnosis compared to male (median age 7 years vs. 10 years). All cases were microsatellite stable (MSS). The EGFR alterations consisted of 30 mutations and 9 amplifications. The mutations were distributed across the entire gene with no clear hotspot location. Our genomic data converged to identify three distinct molecular patterns. The first and most common group contained TP53 mutations (n = 17, 43.5%), showed no association with patient sex (8 females, 9 males), contained ATRX mutations (n = 3) and CDKN2A deletions (n = 5); these tumors did not harbor pathogenic mutations in TERTp, PIK3CA/PIK3R1, BCOR/BCORL1, STAG2, or SETD2. The second group featured TERTp-mutant tumors (n = 10), were more common in males (70%), and often demonstrated additional mutations in PIK3CA/PIK3R1 (n = 4), BCOR/BCORL1 (n = 4), CDKN2A/B (n = 4), and SETD2 (n = 2). The third group (n = 12) lacked TERTp and TP53 mutations and had a heterogeneous spectrum of non-recurrent mutations, including one CDKN2A/B deletion. CONCLUSIONS We have identified three distinct molecular subclasses that defined specific genomic tumor subgroups in pediatric-type EGFR altered DMG. Overall, these data increase our understanding of the pathobiology of this DMG subset and can guide the design of clinical trials.

PATH-04. CURRENT STATUS AND PROSPECTS OF MALIGNANT BRAIN TUMOR TREATMENT IN CANCER GENOMIC MEDICINE

Abstract INTRODUCTION The cancer gene panel test was covered by insurance in June 2019 in Japan. Our institution started the test in May 2020 and has experienced 16 cases. We will report on the current status and future prospects. METHODS The subjects were 16 patients (Average age 48.4) who underwent the cancer gene panel test with FoundationOne CDx. [Results] The cases included 9 glioblastomas, 3 anaplastic astrocytomas, 2 anaplastic oligodendrogliomas, an ependymoma, and a pituitary carcinoma. On average, 11.0 genetic changes were detected totally, oncogene mutations were 4.4, and gene mutations of unknown relevance to cancer were 5.6. Major oncogene mutations were IDH1 mutation in 5 cases, ATRX mutation in 2 cases, TP53 mutation in 9 cases, CDKN2A/2B mutation in 5 cases, and BRAF V600E mutation in 1 case. Based on our results, a 25-year-old man with BRAF V600E mutation and a 47-year-old male with IDH1 mutation could be given molecular-targeted drugs. Furthermore, a 59-year-old male with pituitary carcinoma had more than 10 mut/Mb in tumor mutational burden (TMB), indicating that anti-PD-1 antibody drugs can be administered. The chance of finding an appropriate drug by cancer gene panel test is approximately 10-20%. In cases that are resistant to standard treatment, the benefits can be expected if the drugs associated with the cancer gene panel test can be used. CONCLUSIONS Comprehensive cancer gene panel test has the potential to provide personalized medicine. And we suggest that the active use of cancer gene panel test may contribute to the development of new drugs with high response rates and the improvement of prognosis.

PATH-12. DIFFUSE HEMISPHERIC GLIOMA, H3 G34-MUTANT: A COMPREHENSIVE SEQUENCING AND HIGH-RESOLUTION GENOME-WIDE COPY NUMBER ANALYSIS

Abstract Diffuse hemispheric glioma, H3 G34-mutant (DHG_H3G34) is a novel tumor type in the 2021 WHO CNS classification. We describe a comprehensive sequencing and high-resolution genome-wide copy number analysis of a series of cases clinically tested by a single laboratory (2018-2022). Cases included tumors from 47 unique patients (1 reportedly recurrent) that had an H3-3A G34 mutation detected using an 187-gene mutation and fusion targeted neuro-oncology NGS panel (n=47). A subset (n=18) of cases was also tested Oncoscan chromosomal microarray. Median age at testing was 20 years (range, 12-50). H3-3A G34 mutations included G34R (n=44; 94%), G34V (n=2) and a novel G34E variant considered to be likely clinically relevant (n=1). All DHG_H3G34 were hemispheric tumors, and one tumor was multifocal with midline involvement. Concurrent mutations recurrently involved ATRX (n=38), TERT promoter (n=3; mutually exclusive with ATRX mutations), TP53 (n=43), PDGFRA (n=26), PTEN (n=5), NF1 (n=4, all patients over 20 years), PIK3CA (n=4) and CDKN2A (n=3). The single recurrent tumor also had an MSH6 mutation, and in addition to 3 other cases, had increased number of sequence variants suggestive of a high tumor mutational burden. A single case also showed an FGFR3::FAM184B (exon 17::exon 2) fusion. All cases with available chromosomal microarray data had unbalanced genomes with multiple chromosomal gains/losses, and regions of copy-neutral loss of heterozygosity. The most frequent recurrent (at least 50% of cases) abnormalities were losses involving 3q, 4q, 10q, 13q and 18q, and 17p copy-neutral loss of heterozygosity encompassing TP53 (all but one case had concomitant TP53 mutation), a pattern reminiscent of IDH-mutant astrocytomas. Oncogene amplifications (PDGFRA and PIK3CA) were detected in 3 (of 18) cases. CDKN2A/B deletion was observed in 8 cases (2 homozygous). In conclusion, we describe additional sequence and copy-number abnormalities in DHG_H3G34, expanding the spectrum of genetic changes associated with this novel tumor type.

INNV-08. CASE REPORT OF AN OLDER GLIOBLASTOMA PATIENT SURVIVING MORE THAN 12 YEARS AFTER TREATMENT WITH CILENGITIDE IN ADDITION TO STANDARD THERAPY

Abstract BACKGROUND Glioblastoma is the most common malignant brain tumor. Less than 1% of patients survive longer than 10 years. Integrins are implicated in tumorigenesis due to their role in cell adhesion and intercellular communication. Cilengitide, a selective αvβ3, and αvβ5 integrin inhibitor, was studied for patients with glioblastoma. RESULTS A 77-year-old woman was found to have a left temporoparietal MGMT methylated glioblastoma after presenting with visual field deficits in 2009. She underwent near gross total resection of the tumor. The tumor had retained expression of PTEN and MAPK. No mutations in IDH or ATRX were detected. Genetic testing revealed mutations in TERT promoter, TP53, SYNE1, NF1, PTEN, FBXW7, POLD1, ERG, and PRKDC. She was enrolled in the CENTRIC trial, a phase III clinical trial studying the addition of cilengitide to standard therapy for patients with newly diagnosed glioblastoma with methylated MGMT promoter. In 2013, the initial results of the study failed to meet the endpoint of prolonged overall survival and progression-free survival, therefore the study was halted. The patient received cilengitide through compassionate use until 2016. Surveillance brain MRI obtained every three shows no radiographic evidence of recurrence. CONCLUSION Although it is known that methylated-MGMT promoter is one of the few positive prognostic factors in GBM, overall survival remains poor in older patients. This case highlights a rare clinical response to treatment in an older female who has survived more than 12 years without disease recurrence after receiving cilengitide in addition to standard therapy. Her outcome may have been influenced by the expression of PTEN and the methylation of MGMT, which are associated with improved outcomes. Although the development of cilengitide was halted for lack of efficacy, the existence of individual responders such as this patient suggests that further study of integrin inhibition for glioblastoma patients may be worth pursuing.

EPCO-09. TRANSCRIPTOMIC LANDSCAPE OF LOWER GRADE GLIOMA BASED ON AGE-RELATED NON-SILENT SOMATIC MUTATIONS

Abstract Glioma accounts for 80% of all malignant brain tumours and is the most common adult primary brain tumour. Age is an important factor affecting the development of cancer, as somatic mutations accumulate with age. Here, we aimed to analyse the significance of age-dependent non-silent somatic mutations in glioma prognosis. Histological tumour grade depends on age at diagnosis in patients with IDH1, TP53, ATRX, and EGFR mutations. Age of patients with wild-type IDH1 and EGFR increased with increase in tumour grade, while the age of patients with IDH1 or EGFR mutation remained constant. However, the age of patients with EGFR mutation was higher than that of patients with IDH1 mutation. The hierarchical clustering of patients was dominantly separated by IDH1 and EGFR mutations. Furthermore, patients with IDH1 mutation were dominantly separated by TP53 and ATRX double mutation and its double wild-type counterpart. The age of patients with ATRX and TP53 mutation was lower than that of patients with wild-type ATRX and TP53. Patients with the double mutation showed poorer prognosis than those with the double wild type genotype. Unlike IDH1 mutant, IDH1 wild-type showed upregulation of expression of epithelial mesenchymal transition associated genes.