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The fifth edition of the World Health Organization (WHO) classification of tumors of the central nervous system, published in 2021, contains substantial updates in the classification of tumor types. Many of these changes are relevant to radiologists, including "big picture" changes to tumor diagnosis methods, nomenclature, and grading, which apply broadly to many or all central nervous system tumor types, as well as the addition, elimination, and renaming of multiple specific tumor types. Radiologists are integral in interpreting brain tumor imaging studies and have a considerable impact on patient care. Thus, radiologists must be aware of pertinent changes in the field. Staying updated with the most current guidelines allows radiologists to be informed and effective at multidisciplinary tumor boards and in interactions with colleagues in neuro-oncology, neurosurgery, radiation oncology, and neuropathology. This review represents the first of a two-installment review series on the most recent changes to the WHO brain tumor classification system. This first installment focuses on the changes to the classification of adult and pediatric gliomas of greatest relevance for radiologists.

Fig. S1: MDM2 transcript expression. RNA extracted from flank tumor xenografts from 51 PDX models was assessed by qRT-PCR for MDM2 expression in pooled triplicate samples and compared to expression in normal brain (NB). Fig. S2: MDM2 Amplification in PDX lines. Copy-number profiles were generated from whole exome sequencing of flank xenografts. MDM2 exons are highlighted in red. GBM10, GBM12, and GBM102 show normal MDM2 copy number. GBM108 and GBM143 demonstrate high-level amplification of MDM2. Fig. S3: Apoptosis by TUNEL assay in GBM108. Established flank tumor xenografts were randomized and treated for 5 days with placebo (n=5 mice) versus 50 mg/kg SAR405838 (n=5 mice). Tumor samples were then processed for TUNEL staining. TUNEL-positive cells were quantitated in six high-powered fields (HPF) for each sample. Results are plotted as mean and standard error of the mean. * P

Controversy exists surrounding whether heterogeneous disruption of the blood–brain barrier (BBB), as seen in glioblastoma (GBM), leads to adequate drug delivery sufficient for efficacy in GBM. This question is especially important when using potent, targeted agents that have a poor penetration across an intact BBB. Efficacy of the murine double minute-2 (MDM2) inhibitor SAR405838 was tested in patient-derived xenograft (PDX) models of GBM. In vitro efficacy of SAR405838 was evaluated in PDX models with varying MDM2 expression and those with high (GBM108) and low (GBM102) expression were evaluated for flank and orthotopic efficacy. BBB permeability, evaluated using TexasRed-3 kDa dextran, was significantly increased in GBM108 through VEGFA overexpression. Drug delivery, MRI, and orthotopic survival were compared between BBB-intact (GBM108-vector) and BBB-disrupted (GBM108-VEGFA) models. MDM2-amplified PDX lines with high MDM2 expression were sensitive to SAR405838 in comparison with MDM2 control lines in both in vitro and heterotopic models. In contrast with profound efficacy observed in flank xenografts, SAR405838 was ineffective in orthotopic tumors. Although both GBM108-vector and GBM108-VEGFA readily imaged on MRI following gadolinium contrast administration, GBM108-VEGFA tumors had a significantly enhanced drug and gadolinium accumulation, as determined by MALDI-MSI. Enhanced drug delivery in GBM108-VEGFA translated into a marked improvement in orthotopic efficacy. This study clearly shows that limited drug distribution across a partially intact BBB may limit the efficacy of targeted agents in GBM. Brain penetration of targeted agents is a critical consideration in any precision medicine strategy for GBM. Mol Cancer Ther; 17(9); 1893–901. ©2018 AACR.

Genetics of recurrent PDX

Purpose:Glioblastoma is the most frequent and lethal primary brain tumor. Development of novel therapies relies on the availability of relevant preclinical models. We have established a panel of 96 glioblastoma patient-derived xenografts (PDX) and undertaken its genomic and phenotypic characterization.Experimental Design:PDXs were established from glioblastoma, IDH-wildtype (n = 93), glioblastoma, IDH-mutant (n = 2), diffuse midline glioma, H3 K27M-mutant (n = 1), and both primary (n = 60) and recurrent (n = 34) tumors. Tumor growth rates, histopathology, and treatment response were characterized. Integrated molecular profiling was performed by whole-exome sequencing (WES, n = 83), RNA-sequencing (n = 68), and genome-wide methylation profiling (n = 76). WES data from 24 patient tumors was compared with derivative models.Results:PDXs recapitulate many key phenotypic and molecular features of patient tumors. Orthotopic PDXs show characteristic tumor morphology and invasion patterns, but largely lack microvascular proliferation and necrosis. PDXs capture common and rare molecular drivers, including alterations of TERT, EGFR, PTEN, TP53, BRAF, and IDH1, most at frequencies comparable with human glioblastoma. However, PDGFRA amplification was absent. RNA-sequencing and genome-wide methylation profiling demonstrated broad representation of glioblastoma molecular subtypes. MGMT promoter methylation correlated with increased survival in response to temozolomide. WES of 24 matched patient tumors showed preservation of most genetic driver alterations, including EGFR amplification. However, in four patient–PDX pairs, driver alterations were gained or lost on engraftment, consistent with clonal selection.Conclusions:Our PDX panel captures the molecular heterogeneity of glioblastoma and recapitulates many salient genetic and phenotypic features. All models and genomic data are openly available to investigators.

Figures S1-S8, Supplementary Methods, Supplementary Bibliography

BackgroundMeningiomas are the most common primary central nervous system (CNS) tumor in adults and CNS World Health Organization grade 2 (atypical) meningiomas show an intermediate risk of recurrence/progression. Molecular parameters are needed to better inform management following gross total resection (GTR).MethodsWe performed comprehensive genomic analysis of tumor tissue from 63 patients who underwent radiologically confirmed GTR of a primary grade 2 meningioma, including a CLIA-certified target next-generation sequencing panel (n = 61), chromosomal microarray (n = 63), genome-wide methylation profiling (n = 62), H3K27me3 immunohistochemistry (n = 62), and RNA-sequencing (n = 19). Genomic features were correlated with long-term clinical outcomes (median follow-up: 10 years) using Cox proportional hazards regression modeling and published molecular prognostic signatures were evaluated.ResultsThe presence of specific copy number variants (CNVs), including -1p, -10q, -7p, and -4p, was the strongest predictor of decreased recurrence-free survival (RFS) within our cohort (P < .05). NF2 mutations were frequent (51%) but did not show a significant association with RFS. DNA methylation-based classification assigned tumors to DKFZ Heidelberg benign (52%) or intermediate (47%) meningioma subclasses and was not associated with RFS. H3K27 trimethylation (H3K27me3) was unequivocally lost in 4 tumors, insufficient for RFS analysis. Application of published integrated histologic/molecular grading systems did not improve prediction of recurrence risk over the presence of -1p or -10q alone.ConclusionsCNVs are strong predictors of RFS in grade 2 meningiomas following GTR. Our study supports incorporation of CNV profiling into clinical evaluation to better guide postoperative patient management, which can be readily implemented using existing, clinically validated technologies.

Background EGFR targeting antibody-drug conjugates (ADCs) are highly effective against EGFR-amplified tumors, but poor distribution across the blood–brain barrier (BBB) limits their efficacy in glioblastoma (GBM) when administered systemically. We studied whether convection-enhanced delivery (CED) can be used to safely infuse ADCs into orthotopic patient-derived xenograft (PDX) models of EGFRvIII mutant GBM. Methods The efficacy of the EGFR-targeted ADCs depatuxizumab mafodotin (Depatux-M) and Serclutamab talirine (Ser-T) was evaluated in vitro and in vivo. CED was performed in nontumor and tumor-bearing mice. Immunostaining was used to evaluate ADC distribution, pharmacodynamic effects, and normal cell toxicity. Results Dose-finding studies in orthotopic GBM6 identified single infusion of 2 μg Ser-T and 60 μg Depatux-M as safe and effective associated with extended survival prolongation (>300 days and 95 days, respectively). However, with serial infusions every 21 days, four Ser-T doses controlled tumor growth but was associated with lethal toxicity approximately 7 days after the final infusion. Limiting dosing to two infusions in GBM108 provided profound median survival extension of over 200 days. In contrast, four Depatux-M CED doses were well tolerated and significantly extended survival in both GBM6 (158 days) and GBM108 (310 days). In a toxicity analysis, Ser-T resulted in a profound loss in NeuN+ cells and markedly elevated GFAP staining, while Depatux-M was associated only with modest elevation in GFAP staining. Conclusion CED of Depatux-M is well tolerated and results in extended survival in orthotopic GBM PDXs. In contrast, CED of Ser-T was associated with a much narrower therapeutic window.

Background Glioblastoma (GBM) is an incurable disease with few approved therapeutic interventions. Radiation therapy (RT) and temozolomide (TMZ) remain the standards of care. The efficacy and optimal deployment schedule of the orally bioavailable small-molecule tumor checkpoint controller lisavanbulin alone, and in combination with, standards of care were assessed using a panel of IDH-wildtype GBM patient-derived xenografts. Methods Mice bearing intracranial tumors received lisavanbulin +/−RT +/−TMZ and followed for survival. Lisavanbulin concentrations in plasma and brain were determined by liquid chromatography with tandem mass spectrometry, while flow cytometry was used for cell cycle analysis. Results Lisavanbulin monotherapy showed significant benefit (P < .01) in 9 of 14 PDXs tested (median survival extension 9%-84%) and brain-to-plasma ratios of 1.3 and 1.6 at 2- and 6-hours postdose, respectively, validating previous data suggesting significant exposure in the brain. Prolonged lisavanbulin dosing from RT start until moribund was required for maximal benefit (GBM6: median survival lisavanbulin/RT 90 vs. RT alone 69 days, P = .0001; GBM150: lisavanbulin/RT 143 days vs. RT alone 73 days, P = .06). Similar observations were seen with RT/TMZ combinations (GBM39: RT/TMZ/lisavanbulin 502 days vs. RT/TMZ 249 days, P = .0001; GBM26: RT/TMZ/lisavanbulin 172 days vs. RT/TMZ 121 days, P = .04). Immunohistochemical analyses showed a significant increase in phospho-histone H3 with lisavanbulin treatment (P = .01). Conclusions Lisavanbulin demonstrated excellent brain penetration, significant extension of survival alone or in RT or RT/TMZ combinations, and was associated with mitotic arrest. These data provide a strong clinical rationale for testing lisavanbulin in combination with RT or RT/TMZ in GBM patients.

Introduction Neurenteric cysts (NECs) are rare, congenital lesions lined by endodermal cell-derived columnar or cuboidal epithelium. Based on previous studies, gross total removal of the capsule has been presumed to be the ideal surgical goal. Objective This series was undertaken to further understand the risk of recurrence based on the extent of capsule resection. Methods Records were retrospectively reviewed for all patients with radiographic or pathological evidence of intracranial NEC from 1996 to 2021. Results A total of eight patients were identified; four of eight (50%) presented with headache, and four had signs of one or more cranial nerve syndromes. One patient (13%) presented with third nerve palsy, one (13%) had sixth nerve palsy, and two (25%) with hemifacial spasm. One patient (13%) presented with signs of obstructive hydrocephalus. Magnetic resonance imaging demonstrated T2 hyper- or isointense lesions. Diffusion-weighted imaging was negative in all patients (100%) and T1 contrast-enhanced imaging demonstrated minimal rim enhancement in two patients (25%). In three of eight (38%), a gross total resection (GTR) was achieved, while in four (50%), a near-total resection, and in one (13%), a decompression was performed. Recurrences occurred in two (25%) patients, one with decompression and another with near-total resection, among these 1/2 required repeat surgery after a mean follow-up of 77 months. Conclusion In this series, none from GTR group demonstrated recurrence, while 40% of those receiving less than GTR recurred, underpinning the importance of maximally safe resection in these patients. Overall patients did well without major morbidity from surgery.

BackgroundThe extracellular microenvironment modulates cancer behavior. Although radiographic contrast enhancement is an ominous finding in gliomas, it remains unclear if the associated blood-brain barrier disruption merely reflects or functionally supports tumor aggressiveness.MethodsWe utilized intra-operative microdialysis to sample the extracellular metabolome of radiographically diverse regions during fifteen neurosurgical resections. The global extracellular metabolome of recovered microdialysate was evaluated via ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) and assessed using enrichment and correlation analyses.ResultsAmong 162 named metabolites identified via ultra-performance liquid chromatography tandem mass spectrometry, guanidinoacetate (GAA), was 126.32x higher in enhancing tumor than in adjacent brain. 48 additional metabolites were 2.05-10.18x more abundant in enhancing tumor than brain. With exception of GAA, and 2-HG in IDH-mutant gliomas, differences between non-enhancing tumor and brain microdialysate were comparatively modest and less consistent. The enhancing but not the non-enhancing glioma metabolome was significantly enriched for plasma-associated metabolites largely comprising amino acids and carnitines.ConclusionsOur findings suggest that metabolite diffusion through a disrupted blood-brain barrier may largely define the enhancing extracellular glioma metabolome. Future studies are needed to determine how the altered extracellular metabolome impacts glioma behavior.

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Cerebral amyloidomas, characterized by localized amyloid deposits in the nervous system in the absence of systemic disease, are rare. These typically consist of immunoglobulin light chain (AL)-type, predominantly lambda. Trigeminal nerve involvement is exceptionally rare with only 21 previously reported cases, three with bilateral disease. We report two additional cases of amyloid localized to Meckel’s cave with secondary involvement of the trigeminal nerves bilaterally, with protein characterization by mass spectrometry. The patients, both females, 39 and 49-years-old, respectively, presented with the insidious onset of progressive trigeminal neuropathy, including pain and numbness with sensory loss, refractory to medical therapy. One patient experienced bilateral symptoms. Magnetic resonance imaging demonstrated abnormal thickening and contrast enhancement along Meckel’s cave bilaterally in both cases. The clinical differential diagnosis included benign neoplasms and inflammatory disorders. At the time of biopsy, the trigeminal nerve was noted to be enlarged and multinodular in one case and associated with abnormal soft tan tissue in the other case. Microscopically, the nerve biopsies showed extensive Congo red-positive amyloid deposits. Liquid chromatography tandem mass spectrometry demonstrated that the amyloid was of (AL)-type in both cases (AL [kappa] in one case and AL [lambda] in the other). After extensive evaluation, there was no evidence of systemic involvement. Both patients received localized radiotherapy for their refractory symptoms. One patient has stable symptomatology and imaging. No follow-up is available for the other patient.

Isocitrate dehydrogenase 1/2 (IDH1/2) mutations are common in astrocytic glioma and are frequently coupled with TP53 and ATRX mutations. Collectively, these alterations cause genomic instability leading to high basal DNA double strand breaks (DSBs). Understanding how IDH/TP53/ATRX mutant cells process endogenous DSBs may help exploit inhibitors of DNA damage response (DDR) for the treatment of patients with IDH mutant gliomas. Through systematic effort to uncover the mechanisms involved in repair of endogenous DSBs in IDH1/2 mutant GBMs, we have discovered that high basal phosphorylated DNA-PK (p-DNA-PK) was characteristic of an IDH1/TP53/ATRX mutant GBM164 patient derived xenograft (PDX) but not in another IDH1 mutant GBM196 PDX. Immunofluorescence (IF) studies in patient specimen from which GBM164 was derived showed that p-DNA-PK co-localized with g-H2AX, 53BP1 or H4K20me2 (but not p-RPA) the known surrogates of DSBs. In contrast, p-DNA-PK was absent in the patient specimen from which GBM196 was derived, which otherwise had equally intense g-H2AX immunostaining colocalized with p-RPA. An independent IF study involving 11 IDH1 wild-type (WT) and 11 IDH1 mutant GBM patient samples, the p-DNA-PK was observed in 3 (27%) of 11 IDH1 mutant samples while IDH1 WT tumors were negative for p-DNA-PK. A telomere specific fluorescence in situ hybridization (Tel-FISH) confirmed elevated alternative lengthening of telomere (ALT) activity in GBM196 (but not in GBM164) indicative of HR proficiency. Consistently, HR related genes, including BRCA1 and MRE11A, were found upregulated in ALT-positive GBM196 as compared to those in GBM164. Interestingly, ALT+ GBM196 cells were highly vulnerable to inhibitors of ATM and ATR pathways. In conclusion, IDH1/TP53/ATRX mutant gliomas can be subdivided into HR-mediated ALT-positive group, which repairs the endogenous DSBs by HR (e.g. GBM196) and an ALT-negative/p-DNA-PK group, which repairs DSBs by c-NHEJ (e.g. GBM164) and this subdivision can be developed as a prescient biomarker of sensitivity to DDR inhibitors.

Introduction: EGFR targeted antibody-drug conjugates (ADCs) show promise as a novel treatment in a subset of glioblastoma (GBM). Two EGFR targeting ADCs include first generation Depatux-M, with an antimitotic toxin monomethyl auristatin F (MMAF), and ABBV-321, with a DNA crosslinking agent pyrrolobenzodiazepine dimer (PBD) toxin. Due to the large molecular weight, poor drug distribution across the blood-brain barrier significantly limits the efficacy in EGFR-amplified GBM. We studied whether convection enhanced delivery (CED) can be used to safely infuse these two EGFR-targeted ADCs in patient-derived xenograft (PDX) models of EGFR-amplified GBM. Methods: The efficacy of Depatux-M and ABBV-321 was evaluated in vitro and in vivo in two EGFRviii-amplified PDXs (GBM6 and GBM108). Immunofluorescence staining was used to evaluate drug distribution along with pharmacodynamics of the ADCs. CED was performed by stereotactic placement of an infusion catheter in the same location as the original tumor implantation. Immunohistochemistry was used to explore mechanisms of normal cell toxicity. Results: Despite potent activity in vitro (high ng/ml IC50), systemic administration of either ADC conferred minimal extension in survival for either GBM6 or GBM108. In contrast, CED significantly enhanced ADC delivery to tumor and peri-tumoral regions and extended survival. In a pilot study in GBM6 with n=3 mice per group, a single CED infusion of 0.002mg ABBV-321 resulted in extended survival beyond 365 days for two mice, while other doses and placebo were associated with shorter median survival (0 mg - 39 days; 0.03 mg - 105 days; 0.3 mg - 49 days). In a subsequent trial evaluating serial infusions performed every 21 days, four infusions at 0.002 mg ABBV-321 resulted in lethal toxicity. In contrast, limiting ABBV-321 to only two serial CED infusions in GBM108 was associated with extended survival of more than 300 days vs. 53 days for AB095 antibody control infusion. In contrast, serial infusion with Depatux-M was much better tolerated. In a single infusion in GBM6, a 0.06 mg dose was well tolerated and associated with a 49-day extension in median survival. Further, four serial infusions at 0.06 mg given every 21 days in GBM6 and GBM108 was associated with 100 day and more than 250-day extension in survival as compared to AB095 antibody control infusion. To investigate the mechanism of toxicity, infusion of non-tumor bearing C57Bl6 mice with ABBV-321 resulted in a marked loss in NeuN staining and elevated CD68 and GFAP staining 7 days later; Depatux-M infusion was only associated with modest elevation in GFAP without loss of NeuN staining or elevated CD68-positive cells. Conclusion: Depatux-M is well tolerated when infused into normal brain and results in extended survival in orthotopic GBM PDXs. In contrast, ABBV-321, with a distinct PBD toxin, had a much narrower therapeutic window when delivered by CED. Citation Format: Kendra A. Porath, Ann Mladek, Rachael A. Vaubel, Michael S. Regan, Sonia Jain, Danielle Burgenske, Katrina Bakken, Brett Carlson, Margaret Connors, Zeng Hu, Lihong He, Paul A. Decker, Gaspar Kitange, Shiv Gupta, Nathalie Y. Agar, Thomas M. Feldsien, Didier R. Lefebvre, Jann N. Sarkaria. Convection enhanced delivery of EGFR-targeting antibody drug conjugates ABBV-321 and Depatux-M [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 334.

Angiomatoid fibrous histiocytoma (AFH) is an uncommon soft-tissue neoplasm that arises mostly in the extremities of young people and generally carries a good prognosis. Intracranial location is unusual and frequently associated with myxoid change. EWSR1 gene fusions with members of the CREB family (CREB1, ATF1, and CREM) are well-established events in AFH. These fusions have also been described in other neoplasms including intracranial myxoid mesenchymal tumor, and it is still uncertain whether the latter is a distinct entity or if it represents a myxoid variant of AFH. Here, we describe a rare falcine AFH presenting in a 50-year-old woman. The most striking feature of this tumor was its diffuse rhabdoid morphology with focal high mitotic activity, raising the consideration of rhabdoid meningioma (WHO grade III). The tumor cells were moderately positive for EMA and negative for progesterone receptor and SSTR2 prompting additional studies. Desmin was strongly positive and CD99 showed membranous immunoreactivity. BAP1, INI-1, and BRG1 expressions were retained. Next-generation sequencing analysis demonstrated an EWSR1-ATF1 gene fusion, supporting the diagnosis of an unusual rhabdoid variant of AFH. After gross total resection of this tumor, the patient remains free of disease 5 months after the surgery without additional treatment.

Background Antibody drug conjugates (ADCs) targeting the epidermal growth factor receptor (EGFR), such as depatuxizumab mafodotin (Depatux-M), is a promising therapeutic strategy for glioblastoma (GBM) but recent clinical trials did not demonstrate a survival benefit. Understanding the mechanisms of failure for this promising strategy is critically important. Methods PDX models were employed to study efficacy of systemic vs intracranial delivery of Depatux-M. Immunofluorescence and MALDI-MSI were performed to detect drug levels in the brain. EGFR levels and compensatory pathways were studied using quantitative flow cytometry, Western blots, RNAseq, FISH, and phosphoproteomics. Results Systemic delivery of Depatux-M was highly effective in nine of 10 EGFR-amplified heterotopic PDXs with survival extending beyond one year in eight PDXs. Acquired resistance in two PDXs (GBM12 and GBM46) was driven by suppression of EGFR expression or emergence of a novel short-variant of EGFR lacking the epitope for the Depatux-M antibody. In contrast to the profound benefit observed in heterotopic tumors, only two of seven intrinsically sensitive PDXs were responsive to Depatux-M as intracranial tumors. Poor efficacy in orthotopic PDXs was associated with limited and heterogeneous distribution of Depatux-M into tumor tissues, and artificial disruption of the BBB or bypass of the BBB by direct intracranial injection of Depatux-M into orthotopic tumors markedly enhanced the efficacy of drug treatment. Conclusions Despite profound intrinsic sensitivity to Depatux-M, limited drug delivery into brain tumor may have been a key contributor to lack of efficacy in recently failed clinical trials.

Pleomorphic xanthoastrocytoma (PXA) is a rare astrocytoma predominantly affecting children and young adults. We performed comprehensive genomic characterization on a cohort of 67 patients with histologically defined PXA (n=53, 79%) or anaplastic PXA (A-PXA, n=14, 21%), including copy number analysis (ThermoFisher Oncoscan, n=67), methylation profiling (Illumina EPIC array, n=43) and targeted next generation sequencing (n=32). The most frequent alterations were CDKN2A/B deletion (n=63; 94%) and BRAF p.V600E (n=51, 76.1%). In 7 BRAF p.V600 wild-type cases, alternative driver alterations were identified involving BRAF, RAF1 and NF1. Downstream phosphorylation of ERK kinase was uniformly present. Additional pathogenic alterations were rare, with TERT, ATRX and TP53 mutations identified in a small number of tumors, predominantly A-PXA. Methylation-based classification of 46 cases utilizing a comprehensive reference tumor allowed assignment to the PXA methylation class in 40 cases. A minority grouped with the methylation classes of ganglioglioma or pilocytic astrocytoma (n=2), anaplastic pilocytic astrocytoma (n=2) or control tissues (n=2). In 9 cases, tissue was available from matched primary and recurrent tumors, including 8 with anaplastic transformation. At recurrence, two tumors acquired TERT promoter mutations and the majority demonstrated additional non-recurrent copy number alterations. Methylation class was preserved at recurrence. For 62 patients (92.5%), clinical follow-up data were available (median follow-up, 5.4years). Overall survival was significantly different between PXA and A-PXA (5-year OS 80.8% vs. 47.6%; P=0.0009) but not progression-free survival (5-year PFS 59.9% vs. 39.8%; P=0.05). WHO grade remained a strong predictor of overall survival when limited to 38 cases defined as PXA by methylation-based classification. Our data confirm the importance of WHO grading in histologically and epigenetically defined PXA. Methylation-based classification may be helpful in cases with ambiguous morphology, but is largely confirmatory in PXA with well-defined morphology.

BACKGROUND Molecular parameters have been incorporated into the 2016 WHO Classification of CNS Tumors and subsequent cIMPACT-NOW updates to facilitate clinical management of glioma patients. However, there have been few reports of the overall clinical utility of comprehensive genetic testing in adult glioma patients. We report the results of sequencing and chromosomal microarray analysis of adult gliomas seen at the Mayo Clinic and through the Mayo Clinic Laboratories (MCL) reference practice. METHODS A consecutive series of 379 Mayo Clinic adult glioma patients were consented to receive targeted next generation sequencing and chromosomal microarray analysis, regardless of standard clinical ordering practices. Known diagnostic, prognostic, and predictive alterations were annotated for each case. These results were compared to the larger MCL reference practice of 2400 adult glioma samples that received one or both tests clinically. RESULTS Of the consecutive Mayo Clinic cases, 67% had alterations that fell into at least one of the diagnostic, prognostic, and/or predictive categories. Molecular testing generated diagnostic, prognostic, and predictive information in 44%, 34%, and 36% of cases, respectively. Diagnostically, molecular testing mainly aided in arriving at a final integrated diagnosis, and only a small number of cases (n= 5; 1%) had a change in diagnosis. The consecutive cases represent the typical distribution of adult glioma types (47% IDH-wildtype glioblastoma/astrocytoma, 22% IDH-mutant astrocytoma/glioblastoma, 13% IDH-mutant 1p/19q-codeleted oligodendroglioma, and 18% other tumor types). The MCL practice is enriched in tumors with atypical molecular patterns (37% IDH-wildtype glioblastoma/astrocytoma, 17% IDH-mutant astrocytoma/glioblastoma, 18% IDH-mutant 1p/19q-codeleted oligodendroglioma, and 29% other tumor types). CONCLUSIONS Molecular testing provides useful diagnostic, prognostic, and predictive information for the clinical management of adult glioma patients. While the current guidelines for ordering molecular testing are effective for diagnostic purposes, additional prognostic and predictive information can be gleaned from comprehensive molecular testing of adult gliomas.

Pleomorphic xanthoastrocytoma (PXA) is a rare localized glioma characterized by frequent BRAF V600E mutation and CDKN2A/B deletion. We explored the association of copy-number variants (CNVs) with BRAF mutations, tumor grade, and patient survival in a cohort of 41 PXA patients using OncoScan chromosomal microarray. Primary resection specimens were available in 38 cases, including 24 PXA and 14 anaplastic PXA (A-PXA), 23 BRAF V600E mutant tumors (61%). CNVs were identified in all cases and most frequently involved chromosome 9 with homozygous CDKN2A/B deletion (n = 33, 87%), a higher proportion than previously detected by comparative genomic hybridization (50%-60%) (37). CDKN2A/B deletion was present in similar proportion of PXA (83%), A-PXA (93%), BRAF V600E (87%), and wild-type (87%) tumors. Whole chromosome gains/losses were frequent, including gains +7 (n = 15), +2 (n = 11), +5 (n = 10), +21 (n = 10), +20 (n = 9), +12 (n = 8), +15 (n = 8), and losses -22 (n = 11), -14 (n = 7), -13 (n = 5). Losses and copy-neutral loss of heterozygosity were significantly more common in A-PXA, involving chromosomes 22 (P = 0.009) and 14 (P = 0.03). Amplification of 8p and 12q was identified in a single tumor. Histologic grade was a robust predictor of overall survival (P = 0.003), while other copy-number changes, including CDKN2A/B deletion, did not show significant association with survival. Distinct histologic patterns of anaplasia included increased mitotic activity in an otherwise classic PXA or associated with small cell, fibrillary, or epithelioid morphology, with loss of SMARCB1 expression in one case. In 10 cases, matched specimens were compared, including A-PXA with areas of distinct low- and high-grade morphology (n = 2), matched primary/tumor recurrence (n = 7), or both (n = 1). Copy-number changes on recurrence/anaplastic transformation were complex and highly variable, from nearly identical profiles to numerous copy-number changes. Overall, we confirm CDKN2A/B deletion as key a feature of PXA not associated with tumor grade or BRAF mutation, but central to the underlying genetics of PXA.

Pleomorphic xanthoastrocytoma (PXA) is a rare astrocytoma occurring predominantly in children and young adults. It is characterized histologically by large pleomorphic, spindled and lipidized cells with frequent eosinophilic granular bodies and pericellular reticulin deposition. BRAF p.V600E mutation and CKDN2A/B deletion are the most common genetic alterations. We report the integrated genomic characterization of a cohort of 67 patients (37 F, 30 M; median age 20.3 years (interquartile 13.4–32.9) with histologically defined PXA (52, 78%) or anaplastic PXA (A-PXA) (15, 22%), using genome-wide cytogenetic (ThermoFisher Oncoscan, n=67), methylation profiling (Illumina EPIC array, n=43), and targeted next generation sequencing (n=32). BRAF p.V600E mutation (n=51, 76.1%) and CDKN2A/B deletion (n=63; 94%) were the most frequent alterations. Of 16 BRAF p.V600E negative cases, 7 showed an alternative BRAF activating mutation (n=2), NF1 (n=3) mutation or ATG7-RAF1 fusion (n=2). Targeted TERT analysis found promoter mutations in 3 (of 58) cases, but TERT amplification was absent. Supervised and unsupervised methylation profiling against a comprehensive reference cohort demonstrated consensus grouping with the PXA class in 36 of 43 cases; while the minority grouped with a ganglioglioma class (n=3), with reactive brain or had no resolvable subgroup (n=4). Follow-up was available in 61 patients (91.0%) (median 63 months). Overall survival was significantly different between PXA and A-PXA (5-year:80.4% vs. 55.1%; p=0.001), but not progression-free survival (5-year:61.7% vs. 39.8%; p=0.128). Our data confirm the high frequency of MAP-K abnormalities and CDKN2A/B deletion in PXA. WHO grade remains a strong predictor of patient overall survival.

Purpose: Glioblastoma is the most frequent and lethal primary brain tumor. Development of novel therapies relies on the availability of relevant preclinical models. We have established a panel of 96 glioblastoma patient-derived xenografts (PDX) and undertaken its genomic and phenotypic characterization. Experimental Design: PDXs were established from glioblastoma, IDH-wildtype (n = 93), glioblastoma, IDH-mutant (n = 2), diffuse midline glioma, H3 K27M-mutant (n = 1), and both primary (n = 60) and recurrent (n = 34) tumors. Tumor growth rates, histopathology, and treatment response were characterized. Integrated molecular profiling was performed by whole-exome sequencing (WES, n = 83), RNA-sequencing (n = 68), and genome-wide methylation profiling (n = 76). WES data from 24 patient tumors was compared with derivative models. Results: PDXs recapitulate many key phenotypic and molecular features of patient tumors. Orthotopic PDXs show characteristic tumor morphology and invasion patterns, but largely lack microvascular proliferation and necrosis. PDXs capture common and rare molecular drivers, including alterations of TERT, EGFR, PTEN, TP53, BRAF, and IDH1, most at frequencies comparable with human glioblastoma. However, PDGFRA amplification was absent. RNA-sequencing and genome-wide methylation profiling demonstrated broad representation of glioblastoma molecular subtypes. MGMT promoter methylation correlated with increased survival in response to temozolomide. WES of 24 matched patient tumors showed preservation of most genetic driver alterations, including EGFR amplification. However, in four patient–PDX pairs, driver alterations were gained or lost on engraftment, consistent with clonal selection. Conclusions: Our PDX panel captures the molecular heterogeneity of glioblastoma and recapitulates many salient genetic and phenotypic features. All models and genomic data are openly available to investigators.

Non-genotoxic reactivation of p53 by MDM2 inhibitors represents a promising therapeutic strategy for tumors with wild-type TP53, particularly tumors harboring MDM2 amplification. MDM2 controls p53 levels by targeting it for degradation, while disruption of the MDM2-p53 interaction causes rapid accumulation of p53 and activation of the p53 pathway. We examined the efficacy of the small molecule MDM2 inhibitor KRT-232, alone and in combination with radiation therapy (RT), in MDM2-amplified and/or p53 wildtype patient-derived xenograft (PDX) models of glioblastoma in vitro and in vivo. In vitro, glioblastoma PDX explant cultures showed sensitivity to KRT-232, both tumors with MDM2 amplification (GBM108 and G148) and non-amplified but TP53-wildtype lines (GBM10, GBM14, and GBM39), with IC50s ranging from 300-800 nM in FBS culture conditions. A TP53 p.F270C mutant PDX (GBM43) was inherently resistant, with IC50 >3000 nM. In the MDM2-amplified GBM108 line, KRT-232 led to a robust (5-6 fold) induction of p53-target genes p21, PUMA, and NOXA, with initiation of both apoptosis and senescence. Expression of p21 and PUMA was greater with KRT-232 in combination with RT (25-35 fold induction), while stable knock-down of p53 in GBM108 led to complete resistance to KRT-232. In contrast, GBM10 showed lower induction of p21 and PUMA (2-3 fold) and was more resistant to KRT-232. In an orthotopic GBM108 xenograft model, treatment with KRT-232 +/- RT for one week extended survival from 22 days (placebo) to 46 days (KRT-232 alone); combination KRT-232 + RT further extended survival (77 days) over RT alone (31 days). KRT-232 is an effective treatment in a subset of glioblastoma pre-clinical models alone and in combination with RT. Further studies are underway to understand the mechanisms conferring innate sensitivity or resistance to KRT-232.

BackgroundGlioblastoma (GBM) represents an aggressive cancer type with a median survival of only 14 months. With fewer than 5% of patients surviving 5 years, comprehensive profiling of these rare patients could elucidate prognostic biomarkers that may confer better patient outcomes. We utilized multiple molecular approaches to characterize the largest patient cohort of isocitrate dehydrogenase (IDH)–wildtype GBM long-term survivors (LTS) to date.MethodsRetrospective analysis was performed on 49 archived formalin-fixed paraffin embedded tumor specimens from patients diagnosed with GBM at the Mayo Clinic between December 1995 and September 2013. These patient samples were subdivided into 2 groups based on survival (12 LTS, 37 short-term survivors [STS]) and subsequently examined by mutation sequencing, copy number analysis, methylation profiling, and gene expression.ResultsOf the 49 patients analyzed in this study, LTS were younger at diagnosis (P = 0.016), more likely to be female (P = 0.048), and MGMT promoter methylated (UniD, P = 0.01). IDH-wildtype STS and LTS demonstrated classic GBM mutations and copy number changes. Pathway analysis of differentially expressed genes showed LTS enrichment for sphingomyelin metabolism, which has been linked to decreased GBM growth, invasion, and angiogenesis. STS were enriched for DNA repair and cell cycle control networks.ConclusionsWhile our findings largely report remarkable similarity between these LTS and more typical STS, unique attributes were observed in regard to altered gene expression and pathway enrichment. These attributes may be valuable prognostic markers and are worth further examination. Importantly, this study also underscores the limitations of existing biomarkers and classification methods in predicting patient prognosis.

Patient derived xenograft (PDX) models have shown great utility for pre-clinical and translational studies for a range of malignancies. We have established a comprehensive, publically available collection of 95 high-grade glioma flank PDX models. Viable PDX were derived from Glioblastoma, IDH-wildtype (n=91), Glioblastoma, IDH-mutant (n=2), Diffuse Midline Glioma, H3 K27M-mutant (n=1), and Anaplastic Oligodendroglioma (n=1) and include both primary (n=60) and recurrent (n=35) tumors. Comprehensive molecular characterization of PDX is ongoing and, to date, has included whole exome sequencing (WES, n=82), RNA-sequencing (n=40), and genome-wide methylation profiling (n=78) that included MGMT promoter, with data available in cBioPortal. PDX reflected the genetic characteristics of glioblastoma, with the majority harboring TERT promoter mutations, chromosomal gain +7, loss -10 and homozygous deletion of CKDN2A/B. EGFR alterations were frequent (~40%) and included amplification, point mutation, EGFRvIII, and other splice variants. Other common alterations, including amplifications of MET, CDK4, CDK6, MDM2, MDM4 and mutation of TP53, PTEN, NF1, RB1, PIK3CA, PIK3R1 were present at similar frequencies reported by TCGA, with the exception of PDGFRA alterations that were underrepresented in PDX. RNA-sequencing showed representation of each of the glioblastoma gene expression subtypes. To assess preservation of genetic features during xeongrafting, we performed WES on 20 matched patient tumors. The vast majority of genetic driver alterations were shared between patient and PDX, including EGFR, EGFRvIII, CDK4, MDM2, and MET amplifications. In 3 PDX, subclonal selection events were observed, including amplifications of MYCN, CDK6 and an EGFR splice variant, as well as selection against PDGFRA amplification. Overall, our large panel of PDX models reflects the genetic heterogeneity of glioblastoma and largely preserves the genetic features of the primary patient tumors. The PDX National Resource is a powerful tool for neuro-oncology research, with all PDX models and genomic data openly available (http://www.mayo.edu/research/labs/translational-neuro-oncology/mayo-clinic-brain-tumor-patient-derived-xenograft-national-resource).

SOX2 is a transcription factor that is essential for maintenance of pluripotency and has several conserved roles in early embryonic development. Heterozygous loss-of-function variants in SOX2 are identified in approximately 40% of all cases of bilateral anophthalmia/micropthalmia (A/M). Increasingly SOX2 mutation-positive patients without major eye findings, but with a range of other developmental disorders including autism, mild to moderate intellectual disability with or without structural brain changes, esophageal atresia, urogenital anomalies, and endocrinopathy are being reported, suggesting that the clinical phenotype associated with SOX2 loss is much broader than previously appreciated. In this report we describe six new cases, four of which carry novel pathogenic SOX2 variants. Four cases presented with bilateral anophthalmia in addition to extraocular involvement. Another individual presented with only unilateral anophthalmia. One individual did not have any eye findings but presented with a suprasellar teratoma in infancy and was found to have the recurrent c.70del20 mutation in SOX2 (c.70_89del, p.Asn24Argfs*65). This is this first time this tumor type has been reported in the context of a de novo SOX2 mutation. Notably, individuals with hypothalamic hamartomas and slow-growing hypothalamo-pituitary tumors have been reported previously, but it is still unclear how SOX2 loss contributes to their formation.

Background. Patients with meningiomas have widely divergent clinical courses. Some entirely recover following surgery alone, while others have relentless tumor recurrences. This clinical conundrum is exemplified by rhabdoid meningiomas, which are designated in the World Health Organization Classification of Tumours as high grade, despite only a subset following an aggressive clinical course. Patient management decisions are further exacerbated by high rates of interobserver variability, biased against missing possibly aggressive tumors. Objective molecular determinants are needed to guide classification and clinical decision making. Methods. To define genomic aberrations of rhabdoid meningiomas, we performed sequencing of cancer-related genes in 27 meningiomas from 18 patients with rhabdoid features and evaluated breast cancer [BRCA]1-associated protein 1 (BAP1) expression by immunohistochemistry in 336 meningiomas. We assessed outcomes, germline status, and family history in patients with BAP1-negative rhabdoid meningiomas. Results. The tumor suppressor gene BAP1, a ubiquitin carboxy-terminal hydrolase, is inactivated in a subset of high-grade rhabdoid meningiomas. Patients with BAP1-negative rhabdoid meningiomas had reduced time to recurrence compared with patients with BAP1-retained rhabdoid meningiomas (Kaplan-Meier analysis, 26 mo vs 116 mo, P < .001; hazard ratio 12.89). A subset of patients with BAP1-deficient rhabdoid meningiomas harbored germline BAP1 mutations, indicating that rhabdoid meningiomas can be a harbinger of the BAP1 cancer predisposition syndrome. Conclusion. We define a subset of aggressive rhabdoid meningiomas that can be recognized using routine laboratory tests. We implicate ubiquitin deregulation in the pathogenesis of these high-grade malignancies. In addition, we show that familial and sporadic BAP1-mutated rhabdoid meningiomas are clinically aggressive, requiring intensive clinical management.

Epilepsy is commonly encountered in forensic pathology and is ultimately determined to be the cause of death in 1–2% of medicolegal death investigations. Epilepsy is a risk factor for death from external causes, including accidents and drowning. More commonly, deaths result from the underlying epilepsy pathology, including intracranial neoplasms, cerebrovascular disease, status epilepticus, and sudden unexpected death in epilepsy (SUDEP). SUDEP refers to the sudden death in an epilepsy patient that lacks an alternative anatomic or toxicological cause of death. At autopsy, intracranial pathology is present in the majority of epilepsy-related deaths and is more likely to be identified following brain fixation. Common findings include brain tumors, mesial temporal sclerosis, and malformations of cortical development. Death investigators should pay particular attention to clinical history to establish a clear history of epilepsy and to determine seizure type, frequency, underlying etiology, and prior medical and surgical treatments as well as other comorbid medical conditions. A complete autopsy with toxicology is necessary to identify other causes of death, particularly in cases of suspected SUDEP. While toxicology may be helpful in some cases, caution must be taken in interpreting postmortem antiepileptic drug concentrations as levels decrease postmortem.

Lisavanbulin (LIS; BAL101553) is the prodrug of BAL27862, a microtubule-binding, tumor checkpoint controller and potential radiosensitizer. These studies evaluated optimal integration of LIS with standard of care radiation therapy (RT) and/or temozolomide (TMZ) using GBM PDX models. Distribution across the blood brain barrier was evaluated after a single 30 mg/kg oral LIS dose, and concentrations of the active metabolite BAL27862 were measured by liquid chromatography-tandem mass spectrometry. Similar BAL27862 concentrations were detected in the brain (B) and plasma (P) at both two (B:P ratio 1.29) and six hours (B:P ratio 1.64) post-dose. An in vivo screen of LIS monotherapy across 14 orthotopic GBM PDX models showed significant survival benefit (p Citation Format: Danielle M Burgenske, Ann C Mladek, Jenny L Pokorny, Heidi A Lane, Felix Bachmann, Rachael A Vaubel, Mark A Schroeder, Katrina K Bakken, Lihong He, Zeng Hu, Brett L Carlson, Surabhi Talele, Gautham Gampa, Matthew L Kosel, Paul A Decker, Jeanette E Eckel-Passow, William F Elmquist, Jann Sarkaria. Modeling the clinical paradigm of lisavanbulin (BAL101553) deployment in patient-derived xenografts (PDX) of glioblastoma (GBM) [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C096. doi:10.1158/1535-7163.TARG-19-C096

Lettera - non ha abstract

Dihydrolipoamide dehydrogenase (DLD) is a multifunctional protein well characterized as the E3 component of the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase complexes. Previously, conditions predicted to destabilize the DLD dimer revealed that DLD could also function as a diaphorase and serine protease. However, the relevance of these cryptic activities remained undefined. We analyzed human DLD mutations linked to strikingly different clinical phenotypes, including E340K, D444V, R447G, and R460G in the dimer interface domain that are responsible for severe multisystem disorders of infancy and G194C in the NAD(+)-binding domain that is typically associated with milder presentations. In vitro, all of these mutations decreased to various degrees dihydrolipoamide dehydrogenase activity, whereas dimer interface mutations also enhanced proteolytic and/or diaphorase activity. Human DLD proteins carrying each individual mutation complemented fully the respiratory-deficient phenotype of yeast cells lacking endogenous DLD even when residual dihydrolipoamide dehydrogenase activity was as low as 21% of controls. However, under elevated oxidative stress, expression of DLD proteins with dimer interface mutations greatly accelerated the loss of respiratory function, resulting from enhanced oxidative damage to the lipoic acid cofactor of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase and other mitochondrial targets. This effect was not observed with the G194C mutation or a mutation that disrupts the proteolytic active site of DLD. As in yeast, lipoic acid cofactor was damaged in human D444V-homozygous fibroblasts after exposure to oxidative stress. We conclude that the cryptic activities of DLD promote oxidative damage to neighboring molecules and thus contribute to the clinical severity of DLD mutations.

The behavior of rhabdoid meningiomas otherwise lacking malignant features remains unknown as most of the originally reported aggressive cases showed anaplastic histologic features independently of rhabdoid phenotype. We studied 44 patients with rhabdoid meningiomas lacking anaplastic features. Median age at diagnosis was 48.6 years (range 10-79). Location was supratentorial in 28 (63.6%), skull base in 15 (34.1%), and spinal in 1 (2.3%). Tumor grade was otherwise World Health Organization grade I (n = 22, 50%) or II (n = 22, 50%). Rhabdoid cells represented 50% in 14 (31.8%). Median clinical follow-up, available for 38 patients, was 5.0 years (range 0.17-14.2). Recurrence occurred in 9 patients (5-year recurrence-free survival, 73.7%) with a significantly higher risk in subtotally resected tumors (p = 0.043). Rhabdoid cell percentage was not associated with recurrence. Six patients died (4 of disease, 2 of unclear causes); 5-year overall survival was 86.7%, a mortality in excess of that expected in grade I-II meningiomas but much lower than originally reported. Review of 50 similar previously reported cases confirmed our findings. We suggest that rhabdoid meningiomas be graded analogously to nonrhabdoid tumors, with caution that some may still behave aggressively and close follow-up is recommended.

Background Tenosynovial giant cell tumors are a group of slowly growing benign neoplasms of synovial membrane of joints, tendons, and bursae. The localized type or giant cell tumor of tendon sheath (GCTTS) is the extra-articular form of tenosynovial giant cell tumors. We describe two patients with a GCTTS, confirmed histologically at the time of surgical resection, that was adherent to peripheral nerves. Rare GCTTS can cause extrinsic compression of major nerves. Case Descriptions The first patient was a 36-year-old man with a left wrist mass associated with pain and paresthesia in the radial three digits. On ultrasound and magnetic resonance imaging (MRI), the mass appeared arising from the left median nerve with a picture suggestive of an atypical neurogenic tumor; however, the possibility of GCTTS could not be excluded. Intraoperatively, the tumor was adherent to the median nerve without a connection to nearby intercarpal joints. The second patient was a 25-year-old woman with a history of malignant melanoma and an incidentally discovered mass on routine follow-up. MRI of the pelvis showed an ovoid mass related to the right sciatic nerve. The MRI picture was suggestive of a GCTTS, although a benign neurogenic tumor was favored given the anatomic relation to the sciatic nerve. Intraoperatively, the tumor appeared as a nodule implanted on the nerve, and it was easily peeled off. Conclusion We present a new, rare presentation of GCTTS adherent to peripheral nerves with extrinsic compression. We suggest either an implantation mechanism or an unrecognized extrasynovial origin for such tumors.

Friedreich ataxia (FRDA) is an autosomal recessive, multi-systemic degenerative disease that results from reduced synthesis of the mitochondrial protein frataxin. Frataxin has been intensely studied since its deficiency was linked to FRDA in 1996. The defining properties of frataxin - (i) the ability to bind iron, (ii) the ability to interact with, and donate iron to, other iron-binding proteins, and (iii) the ability to oligomerize, store iron and control iron redox chemistry - have been extensively characterized with different frataxin orthologs and their interacting protein partners. This very large body of biochemical and structural data [reviewed in (Bencze et al., 2006)] supports equally extensive biological evidence that frataxin is critical for mitochondrial iron metabolism and overall cellular iron homeostasis and antioxidant protection [reviewed in (Wilson, 2006)]. However, the precise biological role of frataxin remains a matter of debate. Here, we review seminal and recent data that strongly link frataxin to the synthesis of iron-sulfur cluster cofactors (ISC), as well as controversial data that nevertheless link frataxin to additional iron-related processes. Finally, we discuss how defects in ISC synthesis could be a major (although likely not unique) contributor to the pathophysiology of FRDA via (i) loss of ISC-dependent enzymes, (ii) mitochondrial and cellular iron dysregulation, and (iii) enhanced iron-mediated oxidative stress. This article is part of a Special Issue entitled 'Mitochondrial function and dysfunction in neurodegeneration'.

OBJECTIVE To determine the influence of a positive genetic test for hypertrophic cardiomyopathy (HCM) on clinical outcome. PATIENTS AND METHODS A cohort of 203 unrelated patients with HCM (mean ± SD age, 50±18 years) was enrolled from January 1, 2002, through December 31, 2003. They were followed up for a mean ± SD time of 4.0±1.7 years after genetic testing of the 8 HCM-susceptibility genes that encode key sarcomeric/myofilament proteins. The clinical phenotype of those with a positive genetic test (myofilament-positive HCM) was compared with those with a negative genetic test (myofilament-negative HCM). RESULTS In this cohort of 203 patients, 87 mutations were identified in 126 patients (myofilament-positive HCM, 62%); the remaining 77 patients (38%) were myofilament-negative. Despite similar baseline features, patients with myofilament-positive HCM showed increased risk of the combined end points of cardiovascular death, nonfatal stroke, or progression to New York Heart Association class III or IV compared with the patients with myofilament-negative HCM (25% vs 7%, respectively; independent hazard ratio, 4.27; P=.008). These end points occurred at any age among patients with myofilament-positive HCM (range, 14-86 years), but only in those aged 65 years and older among patients with myofilament-negative HCM. Moreover, patients with myofilament-positive HCM showed greater probability of severe left ventricular systolic and diastolic dysfunction, defined as an ejection fraction of less than 50% and a restrictive filling pattern (P=.02 and P CONCLUSION Screening for sarcomere protein gene mutations in HCM identifies a broad subgroup of patients with increased propensity toward long-term impairment of left ventricular function and adverse outcome, irrespective of the myofilament (thick, intermediate, or thin) involved.