ATRX Mutations Research Articles

BIOM-18. CLINICOGENOMIC PREDICTORS FOR THE PATTERN OF FAILURE IN HIGH-GRADE GLIOMA

Abstract Background High-grade gliomas, the most common primary brain tumors, are highly lethal and treatment options remain limited. Despite advances in genomic technologies, there are few molecular biomarkers to guide precision medicine for high-grade glioma. Here, we aimed to identify the clinicogenomic features associated with its prognosis and recurrence patterns. MATERIALS/METHODS Our single-institution retrospective analysis included 182 patients diagnosed with high-grade gliomas who underwent next-generation sequencing targeting 82 brain tumor-relevant genes. Clinicopathological status, treatment characteristics, survival, and genomic profiles were analyzed. RESULTS At a median follow-up of 23 months (range, 2-229 months), 151 patients (83%) had progression or recurrences. Local and distant recurrences were observed in 132 (72.5%) and 101 (54.9%) patients, respectively. The most common genomic variants in high-grade gliomas were TP53 (42.9%), IDH1/2 (23.1%), TERT promoter (38.5%), ATRX (13.2%), H3F3A (7.1%), and SETD2 (6.0%) mutation. Regarding copy number variants, amplification of EGFR (20.9%), PDGFRA (9.9%), MYCN (2.2%) and loss of CDKN2A/2B (49.5%), PTEN (37.9%), RB1 (17.6%), and 1p19q codeletion(9.3%) were the most common copy number aberrations. On multivariate cox regression anlalysis, MYCN amplification (HR 6.08 95% CI 1.91-19.35, p = 0.002), and SETD2 mutation (HR 0.19 95% CI 0.06-0.62, p =0.06) were independent predictors of overall survival, in relation to previously established prognostic factors including age, Eastern Cooperative Oncology Group Performance Status (ECOG PS) scale, extent of resection, MGMT promoter methylation, and IDH1/2 mutation. Interestingly, MYCN amplification (HR 5.24 95% CI 1.69-16.27, p = 0.004), SETD2 mutation (HR 0.35 95% CI 0.12-0.99, p =0.048) were independent predictors of failure from distant recurrences. CONCLUSION The assessment of genomic characteristics in conjunction with pattern of failure for high-grade glioma aids to identify patients who are likely to benefit from personalized medicine. We identified SETD2 mutation, and MYCN as a prognostic biomarkers with potential therapeutic implications in patients with high-grade glioma.

Expanded analysis of high-grade astrocytoma with piloid features identifies an epigenetically and clinically distinct subtype associated with neurofibromatosis type 1.

High-grade astrocytoma with piloid features (HGAP) is a recently recognized glioma type whose classification is dependent on its global epigenetic signature. HGAP is characterized by alterations in the mitogen-activated protein kinase (MAPK) pathway, often co-occurring with CDKN2A/B homozygous deletion and/or ATRX mutation. Experience with HGAP is limited and to better understand this tumor type, we evaluated an expanded cohort of patients (n = 144) with these tumors, as defined by DNA methylation array testing, with a subset additionally evaluated by next-generation sequencing (NGS). Among evaluable cases, we confirmed the high prevalence CDKN2A/B homozygous deletion, and/or ATRX mutations/loss in this tumor type, along with a subset showing NF1 alterations. Five of 93 (5.4%) cases sequenced harbored TP53 mutations and RNA fusion analysis identified a single tumor containing an NTRK2 gene fusion, neither of which have been previously reported in HGAP. Clustering analysis revealed the presence of three distinct HGAP subtypes (or groups = g) based on whole-genome DNA methylation patterns, which we provisionally designated as gNF1 (n = 18), g1 (n = 72), and g2 (n = 54) (median ages 43.5years, 47years, and 32years, respectively). Subtype gNF1 is notable for enrichment with patients with Neurofibromatosis Type 1 (33.3%, p = 0.0008), confinement to the posterior fossa, hypermethylation in the NF1 enhancer region, a trend towards decreased progression-free survival (p = 0.0579), RNA processing pathway dysregulation, and elevated non-neoplastic glia and neuron cell content (p < 0.0001 and p < 0.0001, respectively). Overall, our expanded cohort broadens the genetic, epigenetic, and clinical phenotype of HGAP and provides evidence for distinct epigenetic subtypes in this tumor type.

CML-363 Recent Perspective of Next-Generation Sequencing (NGS) in Mechanisms of Resistance in Patients With Chronic Myeloid Leukemia

Although tyrosine kinase inhibitors (TKIs) have successfully been used to treat CML, some patients develop therapy resistance. Most resistance is due to mutations in the tyrosine kinase domain of BCR::ABL. However, there are still many cases with unknown causes of resistance to TKIs. Determine BCR::ABL-independent biomarkers of resistance to TKIs in patients with CML using NGS. This study included 24 patients (12 men and 12 women) with a median age of 43 years (IQR 26-72 years) with CML resistant to TKI therapy. The control group was 5 patients (3 men and 2 women) with a median age of 54 years (IQR 33-72 years) with CML and optimal response (MMR) to TKI. A myeloid panel of 116 genes with an average reading depth of 200x or 1000x on MiSeq (Illumina) was used. The clinical significance of mutations was evaluated using COSMIC, ClinVar, and VarSome. All studied patients were found to have genetic abnormalities. However, more mutations were detected in the group of patients with resistance than in the control group (average of 5 and 1 mutations, respectively). Multiple mutations were detected in 63% of patients with resistance, although 92% of these mutations had unclear clinical significance. The most common abnormalities were found in tumor suppressor NF1 (10/24), transcription regulators TET2 (10/24) and ATRX (7/24), cohesin complex member STAG2 (6/24), and epigenetic regulator ASXL1 (6/24). Mutations were also found in the intron region of BCR (2/24). Two patients had pathogenic mutations in ASXL1 (p. G646Wfs∗12), which could cause TKI resistance. Despite mutations in NF1 and ATRX being more common in treatment-resistant patients, in our study, these mutations were also noted in optimal responders. The detection of multiple mutations in patients with CML may explain resistance to TKI therapy; however, to better understand the pathogenesis of CML, it is important to know not only the presence of certain mutations but also their level of expression and to study the effects of various regulatory elements, such as transcription factors and miRNAs.

ATRX status in patients with gliomas: Radiomics analysis.

The aim of this study was to develop a noninvasive radiomics analysis model based on preoperative multiparameter MRI to predict the status of the biomarker alpha thalassemia/mental retardation X-linked syndrome (ATRX) in glioma noninvasively.Material and methods:A cohort of 123 patients diagnosed with gliomas (World Health Organization grades II–IV) who underwent surgery and was treated at our center between January 2016 and July 2020, was enrolled in this retrospective study. Radiomics features were extracted from MR T1WI, T2WI, T2FLAIR, CE-T1WI, and ADC images. Patients were randomly split into training and validation sets at a ratio of 4:1. A radiomics signature was constructed using the least absolute shrinkage and selection operator (LASSO) to train the SVM model using the training set. The prediction accuracy and area under curve and other evaluation indexes were used to explore the performance of the model established in this study for predicting the ATRX mutation state.Results:Fifteen radiomic features were selected to generate an ATRX-associated radiomic signature using the LASSO logistic regression model. The area under curve for ATRX mutation (ATRX(−)) on training set was 0.93 (95% confidence interval [CI]: 0.87–1.0), with the sensitivity, specificity and accuracy being 0.91, 0.82 and 0.88, while on the validation set were 0.84 (95% CI: 0.63–0.91), with the sensitivity, specificity and accuracy of 0.73, 0.86, and 0.79, respectively.Conclusions:These results indicate that radiomic features derived from preoperative MRI facilitat efficient prediction of ATRX status in gliomas, thus providing a novel evaluation method for noninvasive imaging biomarkers.

P02.01.B The telomere maintenance mechanism spectrum and its dynamics in gliomas

Abstract The activation of the telomere maintenance mechanism (TMM) is one of the critical drivers of cancer cell immortality. In gliomas, TERT expression and TERT promoter mutation are considered to reliably indicate telomerase activation, while ATRX mutation indicates alternative lengthening of telomeres (ALT). However, these relationships have not been extensively validated in tumor tissues. Here, we show through the direct measurement of telomerase activity and ALT in a large set of glioma samples that the TMM in glioma cannot be defined in the dichotomy of telomerase activity and ALT, regardless of TERT expression, TERT promoter mutation and ATRX mutation. Moreover, we observed that a considerable proportion of gliomas lack both telomerase activity and ALT (Neither group). And this Neither group exhibited evidence of slow growth potential. From a set of longitudinal samples from a separate cohort of glioma patients, we discovered that the TMM is not fixed but changes with glioma progression. Collectively, these results suggest that the TMM is a dynamic entity and that reflects the plasticity of the oncogenic biological status of tumor cells and that the TMM should be defined by the direct measurement of telomerase enzyme activity and evidence of ALT.

ATRX histone binding and helicase activities have distinct roles in neuronal differentiation.

ATRX is a chromatin remodeler, which is mutated in ATRX syndrome, a neurodevelopmental disorder. ATRX mutations that alter histone binding or chromatin remodeling activities cluster in the PHD finger or the helicase domain respectively. Using engineered mouse embryonic stem cells that exclusively express ATRX protein with mutations in the PHD finger (PHDmut) or helicase domains (K1584R), we examine how specific ATRX mutations affect neurodifferentiation. ATRX PHDmut and K1584R proteins interact with the DAXX histone chaperone but show reduced localization to pericentromeres. Neurodifferentiation is both delayed and compromised in PHDmut and K1584R, and manifest differently from complete ATRX loss. We observe reduced enrichment of PHDmut protein to ATRX targets, while K1584R accumulates at these sites. Interestingly, ATRX mutations have distinct effects on the genome-wide localization of the polycomb repressive complex 2 (PRC2), with PHDmut and ATRX knockout showing reduced PRC2 binding at polycomb targets and K1584R showing loss at some sites and gains at others. Notably, each mutation associated with unique gene signatures, suggesting distinct pathways leading to impaired neurodifferentiation. Our results indicate that the histone binding and chromatin remodeling functions of ATRX play non-redundant roles in neurodevelopment, and when mutated lead to ATRX syndrome through separate regulatory pathways.

The telomere maintenance mechanism spectrum and its dynamics in gliomas

BackgroundThe activation of the telomere maintenance mechanism (TMM) is one of the critical drivers of cancer cell immortality. In gliomas, TERT expression and TERT promoter mutation are considered to reliably indicate telomerase activation, while ATRX mutation and/or loss indicates an alternative lengthening of telomeres (ALT). However, these relationships have not been extensively validated in tumor tissues.MethodsTelomerase repeated amplification protocol (TRAP) and C-circle assays were used to profile and characterize the TMM cross-sectionally (n = 412) and temporally (n = 133) across glioma samples. WES, RNA-seq, and NanoString analyses were performed to identify and validate the genetic characteristics of the TMM groups.ResultsWe show through the direct measurement of telomerase activity and ALT in a large set of glioma samples that the TMM in glioma cannot be defined solely by the combination of telomerase activity and ALT, regardless of TERT expression, TERT promoter mutation, and ATRX loss. Moreover, we observed that a considerable proportion of gliomas lacked both telomerase activity and ALT. This telomerase activation-negative and ALT negative group exhibited evidence of slow growth potential. By analyzing a set of longitudinal samples from a separate cohort of glioma patients, we discovered that the TMM is not fixed and can change with glioma progression.ConclusionsThis study suggests that the TMM is dynamic and reflects the plasticity and oncogenicity of tumor cells. Direct measurement of telomerase enzyme activity and evidence of ALT should be considered when defining TMM. An accurate understanding of the TMM in glioma is expected to provide important information for establishing cancer management strategies.

Multiplatform molecular analyses refine classification of gliomas arising in patients with neurofibromatosis type 1

Gliomas arising in the setting of neurofibromatosis type 1 (NF1) are heterogeneous, occurring from childhood through adulthood, can be histologically low-grade or high-grade, and follow an indolent or aggressive clinical course. Comprehensive profiling of genetic alterations beyond NF1 inactivation and epigenetic classification of these tumors remain limited. Through next-generation sequencing, copy number analysis, and DNA methylation profiling of gliomas from 47 NF1 patients, we identified 2 molecular subgroups of NF1-associated gliomas. The first harbored biallelic NF1 inactivation only, occurred primarily during childhood, followed a more indolent clinical course, and had a unique epigenetic signature for which we propose the terminology “pilocytic astrocytoma, arising in the setting of NF1”. The second subgroup harbored additional oncogenic alterations including CDKN2A homozygous deletion and ATRX mutation, occurred primarily during adulthood, followed a more aggressive clinical course, and was epigenetically diverse, with most tumors aligning with either high-grade astrocytoma with piloid features or various subclasses of IDH-wildtype glioblastoma. Several patients were treated with small molecule MEK inhibitors that resulted in stable disease or tumor regression when used as a single agent, but only in the context of those tumors with NF1 inactivation lacking additional oncogenic alterations. Together, these findings highlight recurrently altered pathways in NF1-associated gliomas and help inform targeted therapeutic strategies for this patient population.

Pervasive conditional selection of driver mutations and modular epistasis networks in cancer.

Cancer driver mutations often display mutual exclusion or co-occurrence, underscoring the key role of epistasis in carcinogenesis. However, estimating the magnitude of epistasis and quantifying its effect on tumor evolution remains a challenge. We develop a method (Coselens) to quantify conditional selection on the excess of nonsynonymous substitutions in cancer genes. Coselens infers the number of drivers per gene in different partitions of a cancer genomics dataset using covariance-based mutation models and determines whether coding mutations in a gene affect selection for drivers in any other gene. Using Coselens, we identify 296 conditionally selected gene pairs across 16 cancer types in the TCGA dataset. Conditional selection affects 25%-50% of driver substitutions in tumors with >2 drivers. Conditionally co-selected genes form modular networks, whose structures challenge the traditional interpretation of within-pathway mutual exclusivity and across-pathway synergy, suggesting a more complex scenario where gene-specific across-pathway epistasis shapes differentiated cancer subtypes.

Molecular landscape of pediatric type IDH wildtype, H3 wildtype hemispheric glioblastomas

The WHO (2021) Classification classified a group of pediatric-type high-grade gliomas as IDH wildtype, H3 wildtype but as of currently, they are characterized only by negative molecular features of IDH and H3. We recruited 35 cases of pediatric IDH wildtype and H3 wildtype hemispheric glioblastomas. We evaluated them with genome-wide methylation profiling, targeted sequencing, RNAseq, TERT promoter sequencing, and FISH. The median survival of the cohort was 27.6 months. With Capper et al.'s36 methylation groups as a map, the cases were found to be epigenetically heterogeneous and were clustered in proximity or overlay of methylation groups PXA-like (n = 8), LGG-like (n = 10), GBM_MYCN (n = 9), GBM_midline (n = 5), and GBM_RTKIII (n = 3). Histology of the tumors in these groups was not different from regular glioblastomas. Methylation groups were not associated with OS. We were unable to identify groups specifically characterized by EGFR or PDGFRA amplification as proposed by other authors. EGFR, PDGFRA, and MYCN amplifications were not correlated with OS. 4/9 cases of the GBM_MYCN cluster did not show MYCN amplification; the group was also enriched for EGFR amplification (4/9 cases) and the two biomarkers overlapped in two cases. Overall, PDGFRA amplification was found in only four cases and they were not restricted to any groups. Cases in proximity to GBM_midline were all hemispheric and showed loss of H3K27me3 staining. Fusion genes ALK/NTRK/ROS1/MET characteristic of infantile glioblastomas were not identified in 17 cases successfully sequenced. BRAF V600E was only found in the PXA group but CDKN2A deletion could be found in other methylation groups. PXA-like cases did not show PXA histological features similar to findings by other authors. No case showed TERT promoter mutation. Mutations of mismatch repair (MMR) genes were poor prognosticators in single (p ≤ 0.001) but not in multivariate analyses (p = 0.229). MGMT had no survival significance in this cohort. Of the other common biomarkers, only TP53 and ATRX mutations were significant poor prognosticators and only TP53 mutation was significant after multivariate analyses (p = 0.024). We conclude that IDH wildtype, H3 wildtype pediatric hemispheric glioblastomas are molecularly heterogeneous and in routine practice, TP53, ATRX, and MMR status could profitably be screened for risk stratification in laboratories without ready access to methylation profiling.

Characterization of Different Subtypes of Immune Cell Infiltration in Glioblastoma to Aid Immunotherapy.

Glioblastoma multiforme (GBM) has been identified as a frequently occurring adult primary brain cancer that is highly aggressive. Currently, the prognostic outcome for GBM patients is dismal, even with intensive treatment, and the median overall survival (OS) is 14.6 months. Immunotherapy, which is specific at the cellular level and can generate persistent immunosurveillance, is now becoming a promising tool to treat diverse cancers. However, the complicated nature of the tumor microenvironment (TME) makes it challenging to develop anti-GBM immunotherapy because several cell types, cytokines, and signaling pathways are involved in generating the immunosuppressive environment. Novel immunotherapies can illustrate novel tumor-induced immunosuppressive mechanisms. Here, we used unsupervised clustering analysis to identify different subtypes of immune cell infiltration that actuated different prognoses, biological actions, and immunotherapy responses. Gene cluster A, with a hot immune cell infiltration phenotype, had high levels of immune-related genes (IRGs), which were associated with immune pathways including the interferon-gamma response and interferon-alpha response, and had low IDH1 and ATRX mutation frequencies. Gene cluster B, a cold immune cell infiltration subtype, exhibited a high expression of the KCNIP2, SCRT1, CPLX2, JPH3, UNC13A, GABRB3, ARPP21, DLGAP1, NRXN1, DLL3, CA10, MAP2, SEZ6L, GRIA2, and GRIA4 genes and a low expression of immune-related genes, i.e., low levels of immune reactivity. Our study highlighted the complex interplay between immune cell infiltration and genetic mutation in the establishment of the tumor immune phenotype. Gene cluster A was identified as an important subtype with a better prognosis and improved immunotherapy response.

Therapeutic Vulnerability to ATR Inhibition in Concurrent NF1 and ATRX-Deficient/ALT-Positive High-Grade Solid Tumors

Simple SummaryTumors of the brain and nerves develop frequently in patients with neurofibromatosis type 1. Many are benign growths, such as pilocytic astrocytomas in the brain and neurofibromas in the nerves. However, in some patients, the tumors become malignant and may cause local damage, disseminate to distant sites and result in death. We studied changes in the levels of chromatin proteins and changes in telomeres, in cells obtained from mouse gliomas that are deficient in neurofibromin as well as excess brain and nerve tumor tissue from patients with neurofibromatosis type 1 or sporadic tumors lacking neurofibromin expression. A decrease in the levels of these proteins in experimental cell lines resulted in susceptibility to a class of specific drugs knowns as ATR inhibitors, which may represent a specific vulnerability of these tumor subgroups. We expect our data to provide the required rationale for the development of more accurate animal models to study neurofibromatosis, as well as specific molecularly based drugs for treatment as alternatives to the current, often devastating approaches of surgery, radiation, and chemotherapy.Subsets of Neurofibromatosis Type 1 (NF1)-associated solid tumors have been shown to display high frequencies of ATRX mutations and the presence of alternative lengthening of telomeres (ALT). We studied the phenotype of combined NF1 and ATRX deficiency in malignant solid tumors. Cell lines derived from NF1-deficient sporadic glioblastomas (U251, SF188), an NF1-associated ATRX mutant glioblastoma cell line (JHH-NF1-GBM1), an NF1-derived sarcoma cell line (JHH-CRC65), and two NF1-deficient MPNST cell lines (ST88-14, NF90.8) were utilized. Cancer cells were treated with ATR inhibitors, with or without a MEK inhibitor or temozolomide. In contrast to the glioma cell line SF188, combined ATRX knockout (KO) and TERC KO led to ALT-like properties and sensitized U251 glioma cells to ATR inhibition in vitro and in vivo. In addition, ATR inhibitors sensitized U251 cells to temozolomide, but not MEK inhibition, irrespective of ATRX level manipulation; whereas, the JHH-NF1-GBM1 cell line demonstrated sensitivity to ATR inhibition, but not temozolomide. Similar effects were noted using the MPNST cell line NF90.8 after combined ATRX knockdown and TERC KO; however, not in ST88-14. Taken together, our study supports the feasibility of targeting the ATR pathway in subsets of NF1-deficient and associated tumors.

The chromatin remodeller ATRX facilitates diverse nuclear processes, in a stochastic manner, in both heterochromatin and euchromatin

The chromatin remodeller ATRX interacts with the histone chaperone DAXX to deposit the histone variant H3.3 at sites of nucleosome turnover. ATRX is known to bind repetitive, heterochromatic regions of the genome including telomeres, ribosomal DNA and pericentric repeats, many of which are putative G-quadruplex forming sequences (PQS). At these sites ATRX plays an ancillary role in a wide range of nuclear processes facilitating replication, chromatin modification and transcription. Here, using an improved protocol for chromatin immunoprecipitation, we show that ATRX also binds active regulatory elements in euchromatin. Mutations in ATRX lead to perturbation of gene expression associated with a reduction in chromatin accessibility, histone modification, transcription factor binding and deposition of H3.3 at the sequences to which it normally binds. In erythroid cells where downregulation of α-globin expression is a hallmark of ATR-X syndrome, perturbation of chromatin accessibility and gene expression occurs in only a subset of cells. The stochastic nature of this process suggests that ATRX acts as a general facilitator of cell specific transcriptional and epigenetic programmes, both in heterochromatin and euchromatin.

Abstract 912: A novel genetically engineered H3.3G34R model reveals cooperation with ATRX loss in upregulation of PRC2 target genes

Abstract Pediatric High Grade Glioma (pHGG) is a collection of molecularly distinct subtypes with different prognoses depending on the genetic drivers. One particularly aggressive subtype is H3.3G34R mutant gliomas, which are currently incurable and in need of improved therapies. Interestingly, H3.3G34R mutant gliomas commonly harbor TP53mutations, ATRX mutations, and alterations in PDGFRA signaling. The mechanism by which H3.3G34R promotes gliomagenesis is still somewhat unclear and additional models are needed to dissect its role in tumorigenesis. We used the RCAS Tv-a system to model H3.3G34R pHGG in mice. Nestin expressing progenitors in the frontal cortex of Nestin-Tva (Ntva); p53 fl/fl; ATRX fl/fl or Ntva; p53 fl/fl mice were infected with H3.3G34R or H3.3WT, PDGFA, and Cre, and monitored for signs and symptoms of tumor formation. RNAseq analysis and immunophenotype were used to characterize the tumors. We observed that H3.3G34R did not significantly impact tumor latency independent of ATRX status. ATRX loss significantly increased tumor latency independent of H3.3G34R (P &amp;lt; 0.01) from 90 days to 143 days. H&amp;E analysis revealed that the majority of tumors in all groups were high grade and that mice with ATRX loss were more likely to develop low grade tumors though this trend did not reach significance (P = 0.075). Expression of Ki67, GFAP and Olig2 was present in all groups, as shown by Immunophenotypic analysis. RNAseq analysis of murine tumor tissue revealed that ATRX loss in the context of G34R led to significant differential expression of 113 genes (78 upregulated and 35 downregulated) including upregulation of PRC2 target genes including HoxA genes. These findings highlight the cooperation between ATRX loss and H3.3G34R in gliomagenesis. Citation Format: Aalaa Abdallah, Herminio J. Cardona, David J. Picketts, Daniel J. Brat, Xiao-Nan Li, Oren J. Becher. A novel genetically engineered H3.3G34R model reveals cooperation with ATRX loss in upregulation of PRC2 target genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 912.

Abstract 6228: APR-246, which restores p53 function, is highly active against alternative lengthening of telomere (ALT) cell lines and PDXs

Abstract Introduction: Most cancers proliferate by activating telomerase (TA+) while 10% of cancers utilize alternate lengthening of telomeres (ALT). ALT has been associated with resistance to DNA damaging agents, p53 loss-of-function (p53LOF), ATRX mutations, and very poor survival. ATM kinase, which activates functional p53, is constitutively activated in ALT cancers (Science Translational Medicine 18:eabd5750, 2021). We hypothesized that the constitutive activation of ATM kinase in ALT cancers would confer high sensitivity to pharmacological reactivation of p53 function. Methods: We used patient-derived ALT (telomeric DNA C-circle positive) and TA+ neuroblastoma, sarcoma, colorectal, and breast cancer cell lines and xenografts treated with the clinical-stage p53 reactivator eprenetapopt (APR-246) and irinotecan. In vitro cytotoxicity was assayed by DIMSCAN digital imaging microscopy, ATM activation by immunofluorescence microscopy, and protein expression by western blotting. Results: ALT p53LOF cell lines were significantly (p&amp;lt;0.05) less sensitive to DNA-damaging agents relative to TA+ p53LOF comparators. Constitutive phosphorylation (activation) of ATM kinase and the DNA damage marker 53BP1 were observed at telomeres in ALT but not TA+ cell lines. APR-246 induced p53 targets p21 and NOXA and was significantly more cytotoxic (p&amp;lt;0.001) for ALT relative to TA+ cell lines, regardless of TP53 status. Overexpression of p53 in a TP53-null ALT cell line increased sensitivity (p&amp;lt;0.0001) to APR-246. Knockdown of p53 or ATM kinase in ALT TP53 mutated and wild-type cell lines antagonized (p&amp;lt;0.0001) APR-246 activity. Induction of telomere dysfunction in a TA+ p53LOF neuroblastoma cell line using dominant-negative TRF2 (a shelterin protein that blocks ATM activation at telomeres) activated ATM and sensitized cells to APR-246 (p&amp;lt;0.01). APR-246 enhanced the cytotoxicity of irinotecan (as SN38) in ALT cell lines in vitro to a higher degree than in TA+ p53LOF cell lines (p&amp;lt;0.05). Single-agent APR-246 significantly (p&amp;lt;0.0001) increased event-free survival (EFS) of mice with ALT xenografts relative to controls. APR-246 enhanced (p&amp;lt;0.0001) the activity of irinotecan in 3 neuroblastoma, 2 rhabdomyosarcoma, 1 colorectal, and 1 triple-negative breast ALT xenograft models with most mice (47/56) achieving complete responses and an EFS of &amp;gt;100 days (45/56) compared to no complete responses (median EFS ~ 34 days) in mice treated with only irinotecan (p&amp;lt;0.0001). APR-246 + irinotecan had no significant effect relative to irinotecan alone (p=0.08) on EFS of mice with TA+ p53LOF xenografts (median EFS ~ 40 days). Conclusion: ALT cancers of a variety of histologies are highly resistant to DNA damaging agents, have p53LOF, and constitutive activation of ATM kinase, which confers high sensitivity to p53 reactivation by APR-246. APR-246 warrants clinical testing in patients with ALT cancers. Citation Format: Shawn J. Macha, Balakrishna Koneru, Trevor Burrow, Charles Zhu, Dzmitry Savitski, Jonas Nance, Kristyn McCoy, Cody Eslinger, C Patrick Reynolds. APR-246, which restores p53 function, is highly active against alternative lengthening of telomere (ALT) cell lines and PDXs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6228.

H3K27M-Altered Diffuse Midline Gliomas Among Adult Patients: A Systematic Review of Clinical Features and Survival Analysis

The objective of the study was to summarize the clinical characteristics, histo-genomic profiles, management strategies, and survival outcomes of H3K27M-altered adult diffuse midline gliomas (aDMGs). PubMed, Scopus, and Cochrane databases were used to identify relevant articles. Papers including H3K27M-altered aDMGs with sufficient clinical outcome data were included. Descriptive clinical characteristics and survival analysis were also conducted. Twenty studies describing 135 patients were included. The median age at diagnosis was 42years, and there was a slight male predominance (N= 60, 54%). In our cohort, 15 (11%) patients experienced headache, 10 had nausea and vomiting (7%), and 10 had ataxia (7%). Within this cohort, histopathologic diagnoses included glioblastoma (N= 22, 40%) and anaplastic astrocytoma (N= 21, 38%), while genetic alterations included ATRX mutation (N= 22, 16%), PTPN11 mutation (N= 9, 7%), and MGMT promoter methylation (N= 9, 7%). Among histo-genetic alterations, only ATRX mutation was associated with survival and correlated with worse prognosis (log-rank test, P= 0.04). Neither surgical resection versus biopsy nor greater extent of resection demonstrated survival benefit in our cohort. Chemotherapy was administered in 98 (73%) cases with radiotherapy administered in 71 (53%) cases. Unlike chemotherapy, radiotherapy demonstrated a significant survival benefit (log-rank test, P= 0.019). The median overall survival and progression-free survival within our patient cohort were 10 and 7months, respectively. H3K27M-altered aDMGs were associated with relatively poor survival. ATRX gene mutation was significantly associated with survival disadvantage, while radiotherapy was associated with survival benefit. Large, prospective studies are needed to establish a standard management strategy and provide reliable prognostic conclusions.

Study of Surrogate Immunohistochemical Markers IDH1, ATRX, BRAF V600E, and p53 Mutation in Astrocytic and Oligodendroglial Tumors

Abstract Introduction In consonance with current the World Health Organization (WHO) classification of the central nervous system (CNS) tumors (2016), histological diagnosis of gliomas should be reinforced by molecular information. This study was performed to determine the frequency of isocitrate dehydrogenase 1 (IDH1), α thalassemia/intellectual disability syndrome X-linked (ATRX), p53, and BRAF V600E mutations in different grade astrocytomas and oligodendrogliomas. Methods Seventy-seven cases of astrocytoma and oligodendroglioma (7 pilocytic astrocytomas, 15 diffuse astrocytomas [DA], 4 anaplastic astrocytomas [AA], 29 glioblastomas [GBM], and 22 oligodendrogliomas) were analyzed using immunohistochemistry for IDH1 mutant protein, ATRX, p53, and BRAF as well as their clinicopathological features assessed. Results All pilocytic astrocytoma and primary glioblastoma cases were negative for an IDH1 mutation. IDH1 mutation was detected in 66.7% (10/15) of DA, 50% (2/4) of AA, 20.7% (6/29) of glioblastomas, and 81.8% (18/22) of oligodendroglioma cases. Loss of nuclear ATRX expression was found in 86.7% (13/15), 75% (3/4), and 34.5% (10/29) of DA, AA, and GBM cases, respectively. All oligodendroglioma cases showed retained ATRX expression. Both markers were found statistically significant in the above tumors (p &lt;0.05). BRAF V600E mutation was detected in a single case of pilocytic astrocytoma and pleomorphic xanthoastrocytoma as well as both cases of epithelioid glioblastoma. Conclusions IDH1 and ATRX mutations are very common in diffuse astrocytoma and anaplastic astrocytoma, while they are rare in pilocytic astrocytoma and glioblastoma. Immunohistochemistry for IDH1 and ATRX can successfully characterize the diffuse gliomas into molecularly defined groups in the majority of the cases. BRAF V600E mutation is rare in astrocytic tumors in the Indian population.

HGG-26. Comprehensive Genomic Profiling of Pediatric and AYA High-Grade Gliomas Reveals a High Prevalence of ATRX and Homologous Recombination Repair (HRR) Mutations

Abstract BACKGROUND: Pediatric high-grade glioma (HGG) is a devastating heterogenous disease with a clear need for more effective treatment options. Advances in high-throughput molecular sequencing have advanced our understanding of these tumors and yet outcomes remain poor as only a very small subset of patients have demonstrated benefit from targeted therapy approaches. METHODS: We compiled genomic data from 980 pediatric (N=400) and AYA (18 to 30 yrs, N=580) HGG cases that underwent targeted, panel-based comprehensive genomic profiling (FoundationOne® CDx). RESULTS: Among pediatric patients, mutations in ATRX or HRR-related genes were among the most prevalent at 24.25% and 19.5%, respectively. Other commonly occurring mutations were seen in TP53 (58.25%), H3F3A (43%), and PIK3CA/PIK3R1 (22%). Among AYA patients, mutations in ATRX and HRR-related genes were seen in 51.72% and 17.24% of cases, respectively. The most common mutations seen in this older population were in TP53 (76.38%), IDH1/2 (50.86%), PI3KCA/PIK3R1 (20.52%), and H3F3A (18.28%). Among patients with diffuse midline glioma (harboring H3K27M mutations), ATRX and HRR mutations were reported in 21.27% and 13.57% of cases, respectively. Among pediatric patients with histone wild type HGG, mutations in ATRX and HRR-related genes occurred in 20.61% and 21.05% of cases, respectively. CONCLUSION: Alterations in ATRX and HRR-related genes are among the most prevalent mutations in pediatric and AYA HGG. Consideration should be made towards the development of subtype-specific treatment protocols using PARP and/or ATR inhibitors aimed at this subgroup of patients.

HGG-50. Specific sensitivity of pediatric high-grade glioma with ATRX inactivation to PARP inhibitor combinations

Pediatric high-grade gliomas (pHGG) account for approximately 12% of pediatric brain tumors. Despite advances in molecular diagnosis and identification of discrete molecular subtypes, pHGG are the leading cause of cancer-related death in children. Thus, current research focuses on identifying novel therapeutic targets. Sequencing analyses across pediatric cancer types identified DNA repair perturbations as potentially targetable events in certain types of pediatric brain tumors. Herein, we investigated the potential of PARP inhibitors (PARPi), impeding the central role of PARP in DNA damage repair, in pHGG. We screened a patient-derived primary pHGG cell line panel (n=7) for their sensitivity towards 6 different PARPi (niraparib, olaparib, pamiparib, rucaparib, talazoparib, veliparib) using cell viability assays. Basal expression of DNA repair related proteins was assessed by immunoblot, and propidium iodide-based flow cytometry was used for cell cycle analysis. All pHGG were resistant towards single compound PARP inhibition. Interestingly, two H3F3A-G34R mutant pHGG models harboring inactivating ATRX mutations were characterized by elevated basal levels of pH2AX, suggesting increased stress resulting from DNA damage. Consequently, simultaneous targeting of PARP and other components of DNA repair in the respective models showed strong synergistic effects on cell viability, which was not observed to a comparable extent in other models such as BRAFV600E/TERT promotor mutant pHGG. Combination of talazoparib and irinotecan resulted in S-phase arrest. Within a precision oncology approach, we treated a 11-year-old child suffering from H3F3A-G34R mutant pHGG with ATRX mutation, that progressed during radiation, with niraparib and topotecan. The patient achieved partial remission and disease stabilization for 1 year. Taken together, PARPi combinations show potential for the treatment of pHGG with ATRX mutations. Currently, all cell models are characterized for DNA repair signatures by DNA sequencing. Further, in depth characterization of DNA damage responses upon concomitant PARP and topoisomerase inhibition in ATRX-mutated pHGG are ongoing.

HGG-34. Upfront Molecular Targeted Therapy for the Treatment of BRAF-mutant Pediatric High-Grade Glioma

Abstract BACKGROUND: The prognosis for pediatric high-grade glioma (pHGG) is poor despite aggressive multi-modal therapy. Objective responses to targeted therapy with BRAF inhibitors have been reported in some patients with recurrent BRAF-mutant pHGG but are rarely sustained. METHODS: We performed a retrospective, multi-institutional review of patients with BRAF-mutant pHGG treated with off-label BRAF +/- MEK inhibitors as part of their initial therapy. RESULTS: Nineteen patients were identified, with a median age of 10.7 years (range: 1.8–20.3). Histologic diagnoses included HGG (n=6), glioblastoma (n=3), anaplastic ganglioglioma (n=4), diffuse midline glioma (n=3), high-grade neuroepithelial tumor (n=1), anaplastic astrocytoma (n=1), and anaplastic astroblastoma (n=1). Recurrent concomitant oncogenic alterations included CDKN2A/B loss, H3 K27M, as well as mutations in ATRX, EGFR and TERT. Eight patients received BRAF inhibitor monotherapy. Eleven patients received combination therapy with BRAF and MEK inhibitors. Most patients tolerated long-term treatment well with no grade 4–5 toxicities. Objective and durable imaging responses were seen in the majority of patients with measurable disease. At a median follow-up of 2.3 years (range,0.3–6.5), three-year progression-free (PFS) and overall survival (OS) for the cohort were 65% and 82%, respectively, and superior to a historical control cohort treated with conventional therapies. CONCLUSIONS: Upfront targeted therapy for patients with BRAF-mutant pHGG is feasible and effective, with superior clinical outcomes observed compared to historical data. This promising treatment paradigm is currently being evaluated prospectively in the Children’s Oncology Group ACNS1723 clinical trial.