Glioblastoma, IDH-Wildtype With Epithelioid Morphology and a BCR::NTRK2 Fusion.

Glioblastoma, IDH-wildtype (GBM) is a high-grade astrocytic glioma that accounts for the majority of malignant brain tumors in adults. Within this entity, epithelioid GBM represents a histological subtype characterized by a loosely cohesive aggregate of large cells with abundant cytoplasm, and vesicular nuclei that usually harbors the BRAF V600E mutation. Molecular alterations in GBMs are frequent and play an important role in the diagnosis of this entity. Among the many genetic alterations reported, NTRK fusions are rare and account for <2% of gliomas. Furthermore, NTRK2 fusions are most seen in pediatric populations. Recent approval of the TRK inhibitor larotrectinib by the Food and Drug Administration (FDA) has brought interest in the study and recognition of NTRK fusions in multiple types of tumors. Trials that assess the response to this drug in cancers carrying NTRK fusions have yielded favorable results. We discuss a rare presentation of an adult-type GBM with epithelioid morphology and a BCR::NTRK2 gene fusion.

Inhibition of USP7 upregulates USP22 and activates its downstream cancer-related signaling pathways in human cancer cells.

Deubiquitinases (DUBs) play important roles in various human cancers and targeting DUBs is considered as a novel anticancer therapeutic strategy. Overexpression of ubiquitin specific protease 7 and 22 (USP7 and USP22) are associated with malignancy, therapy resistance, and poor prognosis in many cancers. Although both DUBs are involved in the regulation of similar genes and signaling pathways, such as histone H2B monoubiquitination (H2Bub1), c-Myc, FOXP3, and p53, the interdependence of USP22 and USP7 expression has never been described. In the study, we found that targeting USP7 via either siRNA-mediated knockdown or pharmaceutical inhibitors dramatically upregulates USP22 in cancer cells. Mechanistically, the elevated USP22 occurs through a transcriptional pathway, possibly due to desuppression of the transcriptional activity of SP1 via promoting its degradation upon USP7 inhibition. Importantly, increased USP22 expression leads to significant activation of downstream signal pathways including H2Bub1 and c-Myc, which may potentially enhance cancer malignancy and counteract the anticancer efficacy of USP7 inhibition. Importantly, targeting USP7 further suppresses the in vitro proliferation of USP22-knockout (USP22-Ko) A549 and H1299 lung cancer cells and induces a stronger activation of p53 tumor suppressor signaling pathway. In addition, USP22-Ko cancer cells are more sensitive to a combination of cisplatin and USP7 inhibitor. USP7 inhibitor treatment further suppresses in vivo angiogenesis and tumor growth and induced more apoptosis in USP22-Ko cancer xenografts. Taken together, our findings demonstrate that USP7 inhibition can dramatically upregulate USP22 in cancer cells; and targeting USP7 and USP22 may represent a more effective approach for targeted cancer therapy, which warrants further study. Video Abstract.

Expansion of endogenous T cells in CSF of pediatric CNS tumor patients undergoing locoregional delivery of IL13R〿2-targeting CAR T cells: an interim analysis.

Outcomes for pediatric brain tumor patients remain poor, and there is optimism that chimeric antigen receptor (CAR) T cell therapy can improve prognosis. Here, we present interim results from the first six pediatric patients treated on an ongoing phase I clinical trial (NCT04510051) of IL13BBζ-CAR T cells delivered weekly into the lateral cerebral ventricles, identifying clonal expansion of endogenous CAR-negative CD8+ T cells in the cerebrospinal fluid (CSF) over time. Additionally, of the five patients evaluable for disease response, three experienced transient radiographic and/or clinical benefit not meeting protocol criteria for response. The first three patients received CAR T cells alone; later patients received lymphodepletion before the first infusion. There were no dose limiting toxicities (DLTs). Aside from expected cytopenias in patients receiving lymphodepletion, serious adverse events possibly attributed to CAR T cell infusion were limited to one episode of headache and one of liver enzyme elevation. One patient withdrew from treatment during the DLT period due to a Grade 3 catheter-related infection and was not evaluable for disease response, although this was not attributed to CAR T cell infusion. Importantly, scRNA- and scTCR-sequence analyses provided insights into CAR T cell interaction with the endogenous immune system. In particular, clonally expanded endogenous CAR- T cells were recovered from the CSF, but not the peripheral blood, of patients who received intraventricular IL13BBζ-CAR T cell therapy. Additionally, although immune infiltrates in CSF and post-therapy tumor did not generally correlate, a fraction of expanded T cell receptors (TCRs) was seen to overlap between CSF and tumor. This has important implications for what samples are collected on these trials and how they are analyzed. These initial findings provide support for continued investigation into locoregionally-delivered IL13BBζ-CAR T cells for children with brain tumors.

Acquired resistance to KRAS G12C small-molecule inhibitors via genetic/nongenetic mechanisms in lung cancer.

Inherent or acquired resistance to sotorasib poses a substantialt challenge for NSCLC treatment. Here, we demonstrate that acquired resistance to sotorasib in isogenic cells correlated with increased expression of integrin ß4 (ITGB4), a component of the focal adhesion complex. Silencing ITGB4 in tolerant cells improved sotorasib sensitivity, while overexpressing ITGB4 enhanced tolerance to sotorasib by supporting AKT-mTOR bypass signaling. Chronic treatment with sotorasib induced WNT expression and activated the WNT/ß-catenin signaling pathway. Thus, silencing both ITGB4 and ß-catenin significantly improved sotorasib sensitivity in tolerant, acquired, and inherently resistant cells. In addition, the proteasome inhibitor carfilzomib (CFZ) exhibited synergism with sotorasib by down-regulating ITGB4 and ß-catenin expression. Furthermore, adagrasib phenocopies the combination effect of sotorasib and CFZ by suppressing KRAS activity and inhibiting cell cycle progression in inherently resistant cells. Overall, our findings unveil previously unrecognized nongenetic mechanisms underlying resistance to sotorasib and propose a promising treatment strategy to overcome resistance.

Metastatic Testicular Sex Cord Tumor Harboring a EWSR1::ATF1 Gene Fusion-A Case Report of a Novel Neoplasm: "Inflammatory and Nested Testicular Sex Cord Tumor".

We present a case report of a 54-year-old male with a metastatic testicular sex cord tumor harboring a EWSR1::ATF1 gene fusion. The tumor displayed a solid and nested architecture with sclerotic stroma and variable inflammatory infiltrate, and was positive for SF-1, inhibin, EMA, CD30, and WT1 expression. Further genetic analysis identified a EWSR1::ATF1 gene fusion. Overall findings were consistent with an "inflammatory and nested testicular sex cord tumor," a recently described testicular neoplasm characterized by EWSR1::ATF1 gene fusion and aggressive clinical behavior. Due to the aggressive nature of this entity and the limited response to current treatment options available, identification of potential biomarkers for early diagnosis and targeted therapies are critical. This case report provides important insights into the genomic landscape of testicular sex cord-stromal tumors, especially within the CTNNB1-negative subset of patients with an aggressive clinical course, and further supports the distinction of "inflammatory and nested testicular sex cord tumor" as a separate entity from Sertoli cell tumors due to its characteristic morphological, immunohistochemical and molecular, features and clinical behavior.

Targeting ITGB4/SOX2-driven lung cancer stem cells using proteasome inhibitors.

This study investigates the role of integrin ß4 (ITGB4) and stemness-associated factor SOX2 in platinum resistance in lung squamous cell carcinoma (LUSC). The expression of SOX2 and ITGB4 is found to be high in all LUSC subtypes, but the impact of ITGB4 expression on overall patient survival varies by subtype. Cancer stem cells (CSCs) isolated from LUSC patients were found to be resistant to cisplatin, but knocking down ITGB4 or SOX2 sensitized them to cisplatin. Carfilzomib (CFZ) synergized with cisplatin and suppressed CSC growth by inhibiting ITGB4 and SOX2 expression. Additionally, CFZ was found to inhibit SOX2 expression epigenetically by inhibiting histone acetylation at the SOX2 promoter site. CFZ also suppressed the growth of SOX2-dependent small cell lung cancer cells in vitro and in vivo. The study highlights the unique function of CFZ as a transcriptional suppressor of SOX2, independent of its proteasome inhibitory function.

Predicting survival of NSCLC patients treated with immune checkpoint inhibitors: Impact and timing of immune-related adverse events and prior tyrosine kinase inhibitor therapy.

Introduction: Immune checkpoint inhibitors (ICIs) produce a broad spectrum of immune-related adverse events (irAEs) affecting various organ systems. While ICIs are established as a therapeutic option in non-small cell lung cancer (NSCLC) treatment, most patients receiving ICI relapse. Additionally, the role of ICIs on survival in patients receiving prior targeted tyrosine kinase inhibitor (TKI) therapy has not been well-defined. Objective: To investigate the impact of irAEs, the relative time of occurrence, and prior TKI therapy to predict clinical outcomes in NSCLC patients treated with ICIs. Methods: A single center retrospective cohort study identified 354 adult patients with NSCLC receiving ICI therapy between 2014 and 2018. Survival analysis utilized overall survival (OS) and real-world progression free survival (rwPFS) outcomes. Model performance matrices for predicting 1-year OS and 6-month rwPFS using linear regression baseline, optimal, and machine learning modeling approaches. Results: Patients experiencing an irAE were found to have a significantly longer OS and rwPFS compared to patients who did not (median OS 25.1 vs. 11.1 months; hazard ratio [HR] 0.51, confidence interval [CI] 0.39- 0.68, P-value <0.001, median rwPFS 5.7 months vs. 2.3; HR 0.52, CI 0.41- 0.66, P-value <0.001, respectively). Patients who received TKI therapy before initiation of ICI experienced significantly shorter OS than patients without prior TKI therapy (median OS 7.6 months vs. 18.5 months; P-value < 0.01). After adjusting for other variables, irAEs and prior TKI therapy significantly impacted OS and rwPFS. Lastly, the performances of models implementing logistic regression and machine learning approaches were comparable in predicting 1-year OS and 6-month rwPFS. Conclusion: The occurrence of irAEs, the timing of the events, and prior TKI therapy were significant predictors of survival in NSCLC patients on ICI therapy. Therefore, our study supports future prospective studies to investigate the impact of irAEs, and sequence of therapy on the survival of NSCLC patients taking ICIs.

Clinical and Molecular Features of KRAS-Mutated Lung Cancer Patients Treated with Immune Checkpoint Inhibitors.

Background: The molecular and clinical features of KRAS-mutated lung cancer patients treated with immunotherapy have yet to be characterized, which could guide the development of therapeutics targeting KRAS with potential immuno-oncology treatment combinations. Research Question: Do KRAS-mutated patients with different subtypes and comutations have different clinical responses and overall survival (OS) to checkpoint inhibitors? Study Design and Methods: 87 patients with NSCLC at the City of Hope who received immune checkpoint inhibitors were identified and analyzed retrospectively. Tumor genomic alterations were extracted from the clinical data with next-generation sequencing using various platforms. Demographic, clinical, molecular, and pathological information was collected with the approval of the institutional review board of the City of Hope. OS was calculated if it was available at the study time point, and responses were determined according to the RECIST v1.1. Results: Among 87 patients, 32 had a KRAS G12C mutation (36.8%), 19 had G12V (21.9%), 18 had G12D (20.7%), 6 had G12A (6.9%), 3 had G12R (3.45%), and 10 had amplification (11.49%) and other uncommon mutations. G12D had a statistically significant Odds Ratio (OR) between patients who had responses and progression of the disease (OR (95% CI) = 0.31 (0.09−0.95), p < 0.05), with 5 G12D-mutated patients having responses and 11 G12D-mutated patients having progression of the disease. In the univariate analysis with OS, there was a trend of better OS in the G12D-mutated patients, with no statistically significant difference in terms of OS between the patients who had G12D mutation and the patients who had other KRAS mutations (HR (95% CI) = 0.53 (0.21−1.36), p = 0.185). The median OS was significantly worse with KRAS comutation CDKN2A/B loss (4.2 vs. 16.9 months, HR = 3.07 (1.09−8.69), p < 0.05) and MET (3.4 vs. 17 months, HR = 3.80 (1.44−10.05), p < 0.01), which were included for the multivariate analysis. The OS with other KRAS comutations was not statistically significant, including STK11 and KEAP1. Conclusion: KRAS mutation subtypes such as G12D and comutations such as CDKN2/A and MET may modulate the immunotherapy responses and outcomes in lung cancer.

Transcription factor AP2 enhances malignancy of non-small cell lung cancer through upregulation of USP22 gene expression.

BACKGROUND: Ubiquitin-specific protease 22 (USP22), a putative cancer stem cell marker, is frequently upregulated in cancers, and USP22 overexpression is associated with aggressive growth, metastasis, and therapy resistance in various human cancers including lung cancer. However, USP22 gene amplification seldom occurs, and the mechanism underlying USP22 upregulation in human cancers remains largely unknown. METHODS: A luciferase reporter driven by a promoter region of USP22 gene was selectively constructed to screen against a customized siRNA library targeting 89 selected transcription factors to identify potential transcription factors (TFs) that regulate USP22 expression in human non-small cell lung cancers (NSCLC). Association of identified TFs with USP22 and potential role of the TFs were validated and explored in NSCLC by biological assays and immunohistochemistry analysis. RESULTS: Luciferase reporter assays revealed that SP1 and activating transcription factor 3 (ATF3) inhibit USP22 transcription, while transcription factor AP-2 Alpha/Beta (TFAP2A/2B) and c-Myc promote USP22 transcription. Binding site-directed mutagenesis and chromosome immunoprecipitation (ChIP) assays validated AP2α and AP2ß are novel TFs of USP22. Furthermore, overexpression of AP2A and AP2B significantly upregulates USP22 expression, and its target: Cyclin D1, concurrently enhances the proliferation, migration, and invasion of NSCLC A549 and H1299 cells in a partially USP22-dependent manner. Moreover, AP2 protein level correlated with USP22 protein in human NSCLC tissues. CONCLUSION: Our findings indicate AP2α and AP2ß are important transcription factors driving USP22 gene expression to promote the progression of NSCLC, and further support USP22 as a potential biomarker and therapeutic target for lung cancer. Video Abstract.

A Case Report of Immunotherapy-Resistant MSI-H Gastric Cancer with Significant Intrapatient Tumoral Heterogeneity Characterized by Histologic Dedifferentiation.

We describe a patient with both gastric adenocarcinoma and metastatic squamous cell carcinoma (SCC) of unknown primary site. The possibility of a single malignant clonal process as opposed to differing primaries was supported by the finding of both histologies exhibiting high microsatellite instability. Despite evidence of tumor microsatellite instability, the patient's disease process did not respond to immune checkpoint inhibition. Our pursuit of whole-exome sequencing and comparing the single-nucleotide variant profiles of both tumors supported a single clonal process with the development of significant intratumoral heterogeneity. High intratumoral heterogeneity has posed a challenge to precision medicine approaches, but we also provide a review of the literature of this phenomenon mediating resistance to immunotherapy strategies.

Small Cell Lung Cancer Transformation following Treatment in EGFR-Mutated Non-Small Cell Lung Cancer.

EGFR-mutated lung adenocarcinoma patients who received tyrosine kinase inhibitors (TKIs) may initially respond to therapy, but over time, resistance eventually occurs. In a small population (5-10%), these patients can have a histological transformation to SCLC. Nine patients with EGFR-mutated lung adenocarcinoma who transformed to SCLC were evaluated at City of Hope. Patient clinical and pathology data, including multiple next-generation sequencing (NGS) results, clinical therapies, histology, and outcomes, were collected across multiple time points. Descriptive statistics were utilized to visualize and interpret the clinical therapeutic timeline and molecular transformation profiles for these patients. All patients received at least one line of EGFR TKI therapies prior to small cell lung cancer transformation, including erlotinib, afatinib, and osimertinib. Two patients also received chemotherapy prior to transformation (one with immunotherapy). The median months to small cell lung cancer transformation was 16 months, ranging from 4-49 months. The median overall survival (OS) was 29 months from diagnosis, with the minimum of 16 months and maximum of 62 months. The majority of patients had EGFR exon 19 deletion (n = 7, 77.8%), and no patients had a change of original oncogenic EGFR mutation over the different time points. Though a TP53 mutation was detected in eight patients (88.9%) either at the first biopsy or the subsequent biopsies, an RB1 alteration was only detected in one patient at presentation, and three patients upon subsequent biopsies (n = 4, 44.4%). Each patient had a unique molecular profile in the subsequent molecular testing post-transformation, but BRAF alterations occurred frequently, including BRAF rearrangement (n = 1), fusion (n = 1), and amplification (n = 1). Our results showed that EGFR-mutated lung adenocarcinoma to SCLC transformation patients have a unique histological, molecular, and clinical profile over multiple time points, with further heterogeneity that is not currently reported in the literature, and we suggest more work is required to better understand the molecular heterogeneity and clinical outcomes over time for this EGFR TKI resistance subtype.

Evaluation of Somatic Mutations in Solid Metastatic Pan-Cancer Patients.

Metastasis continues to be the primary cause of all cancer-related deaths despite the recent advancements in cancer treatments. To evaluate the role of mutations in overall survival (OS) and treatment outcomes, we analyzed 957 metastatic patients with seven major cancer types who had available molecular testing results with a FoundationOne CDx® panel. The most prevalent genes with somatic mutations were TP53, KRAS, APC, and LRP1B. In this analysis, these genes had mutation frequencies higher than in publicly available datasets. We identified that the somatic mutations were seven mutually exclusive gene pairs and an additional fifty-two co-occurring gene pairs. Mutations in the mutually exclusive gene pair APC and CDKN2A showed an opposite effect on the overall survival. However, patients with CDKN2A mutations showed significantly shorter OS (HR: 1.72, 95% CI: 1.34-2.21, p < 0.001) after adjusting for cancer type, age at diagnosis, and sex. Five-year post metastatic diagnosis survival analysis showed a significant improvement in OS (median survival 28 and 43 months in pre-2015 and post-2015 metastatic diagnosis, respectively, p = 0.00021) based on the year of metastatic diagnosis. Although the use of targeted therapies after metastatic diagnosis prolonged OS, the benefit was not statistically significant. However, longer five-year progression-free survival (PFS) was significantly associated with targeted therapy use (median 10.9 months (CI: 9.7-11.9 months) compared to 9.1 months (CI: 8.1-10.1 months) for non-targeted therapy, respectively, p = 0.0029). Our results provide a clinically relevant overview of the complex molecular landscape and survival mechanisms in metastatic solid cancers.

Protein Phosphatase 2A as a Therapeutic Target in Small Cell Lung Cancer.

Protein phosphatase 2A (PP2A), a serine/threonine phosphatase involved in the regulation of apoptosis, proliferation, and DNA-damage response, is overexpressed in many cancers, including small cell lung cancer (SCLC). Here we report that LB100, a small molecule inhibitor of PP2A, when combined with platinum-based chemotherapy, synergistically elicited an antitumor response both in vitro and in vivo with no apparent toxicity. Using inductively coupled plasma mass spectrometry, we determined quantitatively that sensitization via LB100 was mediated by increased uptake of carboplatin in SCLC cells. Treatment with LB100 alone or in combination resulted in inhibition of cell viability in two-dimensional culture and three-dimensional spheroid models of SCLC, reduced glucose uptake, and attenuated mitochondrial and glycolytic ATP production. Combining LB100 with atezolizumab increased the capacity of T cells to infiltrate and kill tumor spheroids, and combining LB100 with carboplatin caused hyperphosphorylation of the DNA repair marker γH2AX and enhanced apoptosis while attenuating MET signaling and invasion through an endothelial cell monolayer. Taken together, these data highlight the translational potential of inhibiting PP2A with LB100 in combination with platinum-based chemotherapy and immunotherapy in SCLC.

The O-Linked N-Acetylglucosamine Containing Epitope H (O-GlcNAcH) is Upregulated in the Trophoblastic and Downregulated in the Fibroblastic Cells in Missed Miscarriage Human Chorionic Villi With Simple Hydropic Degeneration.

Epitope H contains an O-linked N-acetylglucosamine (O-GlcNAcH) residue in a specific conformation and/or environment recognized by the mouse monoclonal antibody H. O-GlcNAcH is present in several types of cells and in several polypeptides, including cytokeratin 8 and vimentin, on the latter in cells under stress. In the present work, we examined the expression of the O-GlcNAcH in 60 cases of endometrial curettings from missed miscarriage cases containing normal and simple hydropic degenerated chorionic villi in each case, using monoclonal antibody H and indirect immunoperoxidase and Western blot immunoblot. In all cases examined the expression of the O-GlcNAcH was cytoplasmic as follows: (1) syncytiotrophoblastic cells showed very low expression in chorionic villi (CV) with nonhydropic degeneration (NHD) and high expression in hydropic degenerated (HD) CV; (2) cytotrophoblastic cells showed low expression in CV with NHD and high expression in HD CV; (3) fibroblastic cells showed high expression in CV with NHD and very low expression in HD CV; (4) histiocytes showed very low expression in both types of CV; (5) endothelial cells showed high expression in both types of CV. An immunoblot of CV from one case of a legal abortion from a normal first-trimester pregnancy showed 5 polypeptides with 118.5, 106.3, 85, 53, and 36.7 kD bearing the epitope H and the 53 kD corresponded to cytokeratin 8. The expression of the O-GlcNAcH is upregulated in the trophoblastic cells and downregulated in the fibroblastic cells in the HD CV in comparison to the NHD CV.

G9a Promotes Invasion and Metastasis of Non-Small Cell Lung Cancer through Enhancing Focal Adhesion Kinase Activation via NF-κB Signaling Pathway.

Potential roles of euchromatic histone methyltransferase 2 (EHMT2 or G9a) in invasion and metastasis are not well understood in non-small cell lung cancer (NSCLC). Here, we investigated the effect and underlying mechanisms of G9a and therapeutic implications of targeting G9a in the invasion and metastasis of NSCLC. Overexpression of G9a significantly enhanced in vitro proliferation and invasion, while knockdown of G9a drastically suppressed in vivo growth and metastasis of A549 and H1299 NSCLC cells. Knockdown or inhibition of G9a significantly decreased the expression of focal adhesion kinase (FAK) protein and activation of FAK pathway. In addition, defactinib, a potent FAK inhibitor, partially abolished the G9a-enhanced invasion in these NSCLC cells. Furthermore, targeting G9a was found to suppress NF-κB transcriptional activity in NSCLC cells through stabilizing NF-κB inhibitor alpha (IκBα), while an NF-κB inhibitor Parthenilide partially abolished the G9a-enhanced FAK activation, which suggests that G9a-enhanced invasion and activation of FAK is mediated by elevated NF-κB activity. Notably, a strong positive correlation between the IHC staining of G9a and phosphorylated FAK proteins was identified in H1299 xenografts and 159 cases of NSCLC tissues (R = 0.408). IMPLICATIONS: The findings of this study strongly demonstrate that G9a may promote invasion and metastasis of NSCLC cells by enhancing FAK signaling pathway via elevating NF-κB transcriptional activity, indicating potential significance and therapeutic implications of these pathways in the invasion and metastasis of NSCLCs that overexpress G9a protein.

Erratum: A Non-genetic Mechanism Involving the Integrin ß4/Paxillin Axis Contributes to Chemoresistance in Lung Cancer.

[This corrects the article DOI: 10.1016/j.isci.2020.101496.].

Alternative lengthening of telomeres, ATRX loss and H3-K27M mutations in histologically defined pilocytic astrocytoma with anaplasia.

Anaplasia may be identified in a subset of tumors with a presumed pilocytic astrocytoma (PA) component or piloid features, which may be associated with aggressive behavior, but the biologic basis of this change remains unclear. Fifty-seven resections from 36 patients (23 M, 13 F, mean age 32 years, range 3-75) were included. A clinical diagnosis of NF1 was present in 8 (22%). Alternative lengthening of telomeres (ALT) was assessed by telomere-specific FISH and/or CISH. A combination of immunohistochemistry, DNA sequencing and FISH were used to study BRAF, ATRX, CDKN2A/p16, mutant IDH1 p.R132H and H3-K27M proteins. ALT was present in 25 (69%) cases and ATRX loss in 20 (57%), mostly in the expected association of ALT+/ATRX- (20/24, 83%) or ALT-/ATRX+ (11/11, 100%). BRAF duplication was present in 8 (of 26) (31%). H3-K27M was present in 5 of 32 (16%) cases, all with concurrent ATRX loss and ALT. ALT was also present in 9 (of 11) cases in the benign PA precursor, 7 of which also had ATRX loss in both the precursor and the anaplastic tumor. In a single pediatric case, ALT and ATRX loss developed in the anaplastic component only, and in another adult case, ALT was present in the PA-A component only, but ATRX was not tested. Features associated with worse prognosis included subtotal resection, adult vs. pediatric, presence of a PA precursor preceding a diagnosis of anaplasia, necrosis, presence of ALT and ATRX expression loss. ALT and ATRX loss, as well as alterations involving the MAPK pathway, are frequent in PA with anaplasia at the time of development of anaplasia or in their precursors. Additionally, a small subset of PA with anaplasia have H3-K27M mutations. These findings further support the concept that PA with anaplasia is a neoplasm with heterogeneous genetic features and alterations typical of both PA and diffuse gliomas.