New Brain Tumor Entities Emerge from Molecular Classification of CNS-PNETs.

Primitive neuroectodermal tumors of the central nervous system (CNS-PNETs) are highly aggressive, poorly differentiated embryonal tumors occurring predominantly in young children but also affecting adolescents and adults. Herein, we demonstrate that a significant proportion of institutionally diagnosed CNS-PNETs display molecular profiles indistinguishable from those of various other well-defined CNS tumor entities, facilitating diagnosis and appropriate therapy for patients with these tumors. From the remaining fraction of CNS-PNETs, we identify four new CNS tumor entities, each associated with a recurrent genetic alteration and distinct histopathological and clinical features. These new molecular entities, designated "CNS neuroblastoma with FOXR2 activation (CNS NB-FOXR2)," "CNS Ewing sarcoma family tumor with CIC alteration (CNS EFT-CIC)," "CNS high-grade neuroepithelial tumor with MN1 alteration (CNS HGNET-MN1)," and "CNS high-grade neuroepithelial tumor with BCOR alteration (CNS HGNET-BCOR)," will enable meaningful clinical trials and the development of therapeutic strategies for patients affected by poorly differentiated CNS tumors.

Pediatric bithalamic gliomas have a distinct epigenetic signature and frequent EGFR exon 20 insertions resulting in potential sensitivity to targeted kinase inhibition.

Brain tumors are the most common solid tumors of childhood, and the genetic drivers and optimal therapeutic strategies for many of the different subtypes remain unknown. Here, we identify that bithalamic gliomas harbor frequent mutations in the EGFR oncogene, only rare histone H3 mutation (in contrast to their unilateral counterparts), and a distinct genome-wide DNA methylation profile compared to all other glioma subtypes studied to date. These EGFR mutations are either small in-frame insertions within exon 20 (intracellular tyrosine kinase domain) or missense mutations within exon 7 (extracellular ligand-binding domain) that occur in the absence of accompanying gene amplification. We find these EGFR mutations are oncogenic in primary astrocyte models and confer sensitivity to specific tyrosine kinase inhibitors dependent on location within the kinase domain or extracellular domain. We initiated treatment with targeted kinase inhibitors in four children whose tumors harbor EGFR mutations with encouraging results. This study identifies a promising genomically-tailored therapeutic strategy for bithalamic gliomas, a lethal and genetically distinct brain tumor of childhood.

Recurrent BCOR internal tandem duplication and BCOR or BCL6 expression distinguish primitive myxoid mesenchymal tumor of infancy from congenital infantile fibrosarcoma.

This corrects the article DOI: 10.1038/modpathol.2017.12.

Interrelationship of Primary Virus Replication, Level of Latency, and Time to Reactivation in the Trigeminal Ganglia of Latently Infected Mice.

UNLABELLED: We sought to determine the possibility of an interrelationship between primary virus replication in the eye, the level of viral DNA in the trigeminal ganglia (TG) during latency, and the amount of virus reactivation following ocular herpes simplex virus type 1 (HSV-1) infection. Mice were infected with virulent (McKrae) or avirulent (KOS and RE) strains of HSV-1, and virus titers in the eyes and TG during primary infection, level of viral gB DNA in TG on day 28 postinfection (p.i.), and virus reactivation on day 28 p.i. as measured by explant reactivation were calculated. Our results suggest that the avirulent strains of HSV-1, even after corneal scarification, had lower virus titers in the eye, had less latency in the TG, and took a longer time to reactivate than virulent strains of HSV-1. The time to explant reactivation of avirulent strains of HSV-1 was similar to that of the virulent LAT((-)) McKrae-derived mutant. The viral dose with the McKrae strain of HSV-1 affected the level of viral DNA and time to explant reactivation. Overall, our results suggest that there is no absolute correlation between primary virus titer in the eye and TG and the level of viral DNA in latent TG and time to reactivation. IMPORTANCE: Very little is known regarding the interrelationship between primary virus replication in the eye, the level of latency in TG, and the time to reactivate in the mouse model. This study was designed to answer these questions. Our results point to the absence of any correlation between the level of primary virus replication and the level of viral DNA during latency, and neither was an indicator of how rapidly the virus reactivated following explant TG-induced reactivation.

Recurrent BCOR internal tandem duplication and BCOR or BCL6 expression distinguish primitive myxoid mesenchymal tumor of infancy from congenital infantile fibrosarcoma.

Primitive myxoid mesenchymal tumor of infancy is a rare sarcoma that preferentially affects infants. It can be locally aggressive and rarely metastasizes, but the long-term outcome of children with this tumor is mostly unknown. Histologically, it is characterized by primitive cells with abundant myxoid stroma. Internal tandem duplication of B-cell CLL/lymphoma 6 (BCL6)-interacting co-repressor (BCOR) exon 15 has recently been described in clear cell sarcoma of kidney, central nervous system high-grade neuroepithelial tumor with BCOR alteration, and primitive myxoid mesenchymal tumor of infancy. Herein, we report five cases of primitive myxoid mesenchymal tumor of infancy: three girls and two boys with mean age of 6.5 months. The tumors were located in the paraspinal region (n=3), back (n=1), or foot (n=1) and ranged in size from 2.5 to 10.2 cm. BCOR internal tandem duplication was confirmed by PCR and sequencing in all five cases. The minimally duplicated region consisted of nine residues, which is shorter than was previously reported in other BCOR-associated tumors. To assess the clinical value and specificity of the BCOR internal tandem duplication, a group of 11 ETV6-rearranged congenital infantile fibrosarcomas were evaluated and no BCOR internal tandem duplication was identified in any case. Though not detected in congenital infantile fibrosarcomas, BCOR and BCL6 immunoreactivity was present in >90% of the nuclei of tumor cells in each of the five primitive myxoid mesenchymal tumor of infancy. The presence of BCOR internal tandem duplication in all five primitive myxoid mesenchymal tumors of infancy provides evidence that it is a recurrent somatic abnormality and substantiates the concept that this tumor is a unique sarcoma of infancy. Our findings indicate that identification of BCOR internal tandem duplication and/or nuclear immunoreactivity for BCOR or BCL6 can aid in the diagnosis of primitive myxoid mesenchymal tumor of infancy and help to differentiate it from congenital infantile fibrosarcoma.

Coregulatory interactions among CD8α dendritic cells, the latency-associated transcript, and programmed death 1 contribute to higher levels of herpes simplex virus 1 latency.

UNLABELLED: The latency-associated transcript (LAT) of herpes simplex virus 1 (HSV-1), CD8α(+) dendritic cells (DCs), and programmed death 1 (PD-1) have all been implicated in the HSV-1 latency-reactivation cycle. It is not known, however, whether an interaction between LAT and CD8α(+) DCs regulates latency and T-cell exhaustion. To address this question, we used LAT-expressing [LAT(+)] and LAT-negative [LAT(-)] viruses. Depletion of DCs in mice ocularly infected with LAT(+) virus resulted in a reduction in the number of T cells expressing PD-1 in the trigeminal ganglia (TG), whereas depletion of DCs in mice similarly infected with LAT(-) virus did not alter PD-1 expression. CD8α(+) DCs, but not CD4(+) DCs, infected with LAT(+) virus had higher levels of ICP0, ICP4, thymidine kinase (TK), and PD-1 ligand 1 (PD-L1) transcripts than those infected with LAT(-) virus. Coculture of infected bone marrow (BM)-derived DCs from wild-type (WT) mice, but not infected DCs from CD8α(-/-) mice, with WT naive T cells contributed to an increase in PD-1 expression. Transfer of bone marrow from WT mice but not CD8α(-/-) mice to recipient Rag1(-/-) mice increased the number of latent viral genomes in reconstituted mice infected with the LAT(+) virus. Collectively, these data indicated that a reduction in latency correlated with a decline in the levels of CD8α(+) DCs and PD-1 expression. In summary, our results demonstrate an interaction among LAT, PD-1, and CD11c CD8α(+) cells that regulates latency in the TG of HSV-1-infected mice. IMPORTANCE: Very little is known regarding the interrelationship of LAT, PD-1, and CD8α(+) DCs and how such interactions might contribute to relative numbers of latent viral genomes. We show here that (i) in both in vivo and in vitro studies, deficiency of CD8α(+) DCs significantly reduced T-cell exhaustion in the presence of LAT(+) virus but not LAT(-) virus; (ii) HSV-1 infectivity was significantly lower in LAT(-)-infected DCs than in their LAT(+)-infected counterparts; and (iii) adoptive transfer of bone marrow (BM) from WT but not CD8α(-/-) mice to recipient Rag1(-/-) mice restored latency to the level in WT mice following infection with LAT(+) virus. These studies point to a key role for CD8α(+) DCs in T-cell exhaustion in the presence of LAT, which leads to larger numbers of latent viral genomes. Thus, altering this negative function of CD8α(+) DCs can potentially be used to generate a more effective vaccine against HSV infection.

Interactions between herpesvirus entry mediator (TNFRSF14) and latency-associated transcript during herpes simplex virus 1 latency.

Herpesvirus entry mediator (HVEM) is one of several cell surface proteins herpes simplex virus (HSV) uses for attachment/entry. HVEM regulates cellular immune responses and can also increase cell survival. Interestingly, latency-associated transcript (LAT), the only viral gene consistently expressed during neuronal latency, enhances latency and reactivation by promoting cell survival and by helping the virus evade the host immune response. However, the mechanisms of these LAT activities are not well understood. We show here for the first time that one mechanism by which LAT enhances latency and reactivation appears to be by upregulating HVEM expression. HSV-1 latency/reactivation was significantly reduced in Hvem(-/-) mice, indicating that HVEM plays a significant role in HSV-1 latency/reactivation. Furthermore, LAT upregulated HVEM expression during latency in vivo and also when expressed in vitro in the absence of other viral factors. This study suggests a mechanism whereby LAT upregulates HVEM expression potentially through binding of two LAT small noncoding RNAs to the HVEM promoter and that the increased HVEM then leads to downregulation of immune responses in the latent microenvironment and increased survival of latently infected cells. Thus, one of the mechanisms by which LAT enhances latency/reactivation appears to be through increasing expression of HVEM.

Role of interleukin-2 and herpes simplex virus 1 in central nervous system demyelination in mice.

We have reported previously that ocular infection of different strains of mice with recombinant herpes simplex virus 1 (HSV-1) constitutively expressing interleukin-2 (IL-2) provokes central nervous system (CNS) demyelination and optic neuropathy, as determined by changes in visual evoked cortical potentials and pathological changes in the optic nerve and CNS, whereas recombinant viruses expressing IL-4, gamma interferon, IL-12p35, IL-12p40, or IL-12p70 do not induce this neuropathy. The goal of this study was to dissect the mechanism underlying the interplay between the immune system (elevation of IL-2) and an environmental factor (infection with HSV-1) that elicits this pathology. Similar results were obtained upon delivery of IL-2 into the mouse brain using osmotic minipumps or injection of mice with recombinant IL-2 protein, IL-2 DNA, or IL-2 synthetic peptides prior to infection with wild-type (wt) HSV-1 strains McKrae and KOS. The critical role of IL-2 is further supported by our data, indicating that a single mutation at position T27A in IL-2 completely blocks the HSV-1-induced pathology. This study shows a novel model of autoimmunity in which viral infection and enhanced IL-2 cause CNS demyelination.

Inhibitors of signal peptide peptidase (SPP) affect HSV-1 infectivity in vitro and in vivo.

Recently we have shown that the highly conserved herpes simplex virus glycoprotein K (gK) binds to signal peptide peptidase (SPP), also known as minor histocompatibility antigen H13. In this study we have demonstrated for the first time that inhibitors of SPP, such as L685,458, (Z-LL)2 ketone, aspirin, ibuprofen and DAPT, significantly reduced HSV-1 replication in tissue culture. Inhibition of SPP activity via (Z-LL)2 ketone significantly reduced viral transcripts in the nucleus of infected cells. Finally, when administered during primary infection, (Z-LL)2 ketone inhibitor reduced HSV-1 replication in the eyes of ocularly infected mice. Thus, blocking SPP activity may represent a clinically effective and expedient approach to the reduction of viral replication and the resulting pathology.

Adaptive and innate transforming growth factor beta signaling impact herpes simplex virus 1 latency and reactivation.

Innate and adaptive immunity play important protective roles by combating herpes simplex virus 1 (HSV-1) infection. Transforming growth factor ß (TGF-ß) is a key negative cytokine regulator of both innate and adaptive immune responses. Yet, it is unknown whether TGF-ß signaling in either immune compartment impacts HSV-1 replication and latency. We undertook genetic approaches to address these issues by infecting two different dominant negative TGF-ß receptor type II transgenic mouse lines. These mice have specific TGF-ß signaling blockades in either T cells or innate cells. Mice were ocularly infected with HSV-1 to evaluate the effects of restricted innate or adaptive TGF-ß signaling during acute and latent infections. Limiting innate cell but not T cell TGF-ß signaling reduced virus replication in the eyes of infected mice. On the other hand, blocking TGF-ß signaling in either innate cells or T cells resulted in decreased latency in the trigeminal ganglia of infected mice. Furthermore, inhibiting TGF-ß signaling in T cells reduced cell lysis and leukocyte infiltration in corneas and trigeminal ganglia during primary HSV-1 infection of mice. These findings strongly suggest that TGF-ß signaling, which generally functions to dampen immune responses, results in increased HSV-1 latency.

CD11c controls herpes simplex virus 1 responses to limit virus replication during primary infection.

CD11c is expressed on the surface of dendritic cells (DCs) and is one of the main markers for identification of DCs. DCs are the effectors of central innate immune responses, but they also affect acquired immune responses to infection. However, how DCs influence the efficacy of adaptive immunity is poorly understood. Here, we show that CD11c(+) DCs negatively orchestrate both adaptive and innate immunity against herpes simplex virus type 1 (HSV-1) ocular infection. The effectiveness and quantity of virus-specific CD8(+) T cell responses are increased in CD11c-deficient animals. In addition, the levels of CD83, CD11b, alpha interferon (IFN-α), and IFN-ß, but not IFN-γ, were significantly increased in CD11c-deficient animals. Higher levels of IFN-α, IFN-ß, and CD8(+) T cells in the CD11c-deficient mice may have contributed to lower virus replication in the eye and trigeminal ganglia (TG) during the early period of infection than in wild-type mice. However, the absence of CD11c did not influence survival, severity of eye disease, or latency. Our studies provide for the first time evidence that CD11c expression may abrogate the ability to reduce primary virus replication in the eye and TG via higher activities of type 1 interferon and CD8(+) T cell responses.

The role of LAT in increased CD8+ T cell exhaustion in trigeminal ganglia of mice latently infected with herpes simplex virus 1.

Herpes simplex virus (HSV) infection is a classic example of latent viral infection in humans and experimental animal models. The HSV-1 latency-associated transcript (LAT) plays a major role in the HSV-1 latency reactivation cycle and thus in recurrent disease. Whether the presence of LAT leads to generation of dysfunctional T cell responses in the trigeminal ganglia (TG) of latently infected mice is not known. To address this issue, we used LAT-positive [LAT(+)] and LAT-deficient [LAT(-)] viruses to evaluate the effect of LAT on CD8 T cell exhaustion in TG of latently infected mice. The amount of latency as determined by quantitative reverse transcription-PCR (qRT-PCR) of viral DNA in total TG extracts was 3-fold higher with LAT(+) than with LAT(-) virus. LAT expression and increased latency correlated with increased mRNA levels of CD8, PD-1, and Tim-3. PD-1 is both a marker for exhaustion and a primary factor leading to exhaustion, and Tim-3 can also contribute to exhaustion. These results suggested that LAT(+) TG contain both more CD8(+) T cells and more CD8(+) T cells expressing the exhaustion markers PD-1 and Tim-3. This was confirmed by flow cytometry analyses of expression of CD3/CD8/PD-1/Tim-3, HSV-1, CD8(+) T cell pentamer (specific for a peptide derived from residues 498 to 505 of glycoprotein B [gB(498-505)]), interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-α). The functional significance of PD-1 and its ligands in HSV-1 latency was demonstrated by the significantly reduced amount of HSV-1 latency in PD-1- and PD-L1-deficient mice. Together, these results may suggest that both PD-1 and Tim-3 are mediators of CD8(+) T cell exhaustion and latency in HSV-1 infection.

Convergent evolution and multi-wave clonal invasion in H3 K27-altered diffuse midline gliomas treated with a PDGFR inhibitor.

The majority of diffuse midline gliomas, H3 K27-altered (DMG-H3 K27-a), are infiltrating pediatric brain tumors that arise in the pons with no effective treatment. To understand how clonal evolution contributes to the tumor's invasive spread, we performed exome sequencing and SNP array profiling on 49 multi-region autopsy samples from 11 patients with pontine DMG-H3 K27-a enrolled in a phase I clinical trial of PDGFR inhibitor crenolanib. For each patient, a phylogenetic tree was constructed by testing multiple possible clonal evolution models to select the one consistent with somatic mutations and copy number variations across all tumor regions. The tree was then used to deconvolute subclonal composition and prevalence at each tumor region to study convergent evolution and invasion patterns. Somatic variants in the PI3K pathway, a late event, are enriched in our cohort, affecting 70% of patients. Convergent evolution of PI3K at distinct phylogenetic branches was detected in 40% of the patients. 24 (~ 50%) of tumor regions were occupied by subclones of mixed lineages with varying molecular ages, indicating multiple waves of invasion across the pons and extrapontine. Subclones harboring a PDGFRA amplicon, including one that amplified a PDGRFAY849C mutant allele, were detected in four patients; their presence in extrapontine tumor and normal brain samples imply their involvement in extrapontine invasion. Our study expands the current knowledge on tumor invasion patterns in DMG-H3 K27-a, which may inform the design of future clinical trials.

Immunization with different viral antigens alters the pattern of T cell exhaustion and latency in herpes simplex virus type 1-infected mice.

We have shown previously that immunization with herpes simplex virus type 1 (HSV-1) glycoprotein K (gK) exacerbated corneal scarring (CS) in ocularly infected mice. In this study, we investigated whether higher levels of CS were correlated with higher levels of latency and T cell exhaustion in gK-immunized mice. BALB/c mice were vaccinated with baculovirus-expressed gK or gD or mock immunized. Twenty-one days after the third immunization, mice were ocularly infected with 2 × 10(4) PFU/eye of virulent HSV-1 strain McKrae. On day 5 postinfection, virus replication in the eye was measured, and on day 30 postinfection, infiltration of the trigeminal ganglia (TG) by CD4, CD8, programmed death 1 (PD-1), and T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) was monitored by immunohistochemistry and quantitative real-time PCR (qRT-PCR). This study demonstrated that higher levels of CS were correlated with higher levels of latency, and this was associated with the presence of significantly higher numbers of CD4(+)PD-1(+) and CD8(+)PD-1(+) cells in the TG of the gK-immunized group than in both the gD- and mock-immunized groups. Levels of exhaustion associated with Tim-3 were the same among gK- and mock-vaccinated groups but higher than levels in the gD-vaccinated group. In this study, we have shown for the first time that both PD-1 and Tim-3 contribute to T cell exhaustion and an increase of latency in the TG of latently infected mice.

SMARCB1 deletion in atypical teratoid rhabdoid tumors results in human endogenous retrovirus K (HML-2) expression.

Atypical Teratoid Rhabdoid Tumor (AT/RT) is a rare pediatric central nervous system cancer often characterized by deletion or mutation of SMARCB1, a tumor suppressor gene. In this study, we found that SMARCB1 regulates Human Endogenous Retrovirus K (HERV-K, subtype HML-2) expression. HML-2 is a repetitive element scattered throughout the human genome, encoding several intact viral proteins that have been associated with stem cell maintenance and tumorigenesis. We found HML-2 env expression in both the intracellular and extracellular compartments in all AT/RT cell lines (n = 4) and in 95% of AT/RT patient tissues (n = 37) evaluated. SMARCB1 knock-down in neural stem cells (NSCs) led to an upregulation of HML-2 transcription. We found that SMARCB1 binds adjacent to the HML-2 promoter, repressing its transcription via chromatin immunoprecipitation; restoration of SMARCB1 expression in AT/RT cell lines significantly downregulated HML-2 expression. Further, targeted downregulation of HML-2 transcription via CRISPR-dCas9 coupled with suppressor proteins led to cellular dispersion, decreased proliferation, and cell death in vitro. HML-2 knock-down with shRNA, siRNA, and CRISPR-dCas9 significantly decreased Ras expression as measured by qRT-PCR, suggesting that HML-2 modulates MAPK/ERK signaling in AT/RT cells. Overexpression of NRAS was sufficient to restore cellular proliferation, and MYC, a transcription factor downstream of NRAS, was bound to the HERV-K LTR significantly more in the absence of SMARCB1 expression in AT/RT cells. We show a mechanism by which these undifferentiated tumors remain pluripotent, and we demonstrate that their formation is aided by aberrant HML-2 activation, which is dependent on SMARCB1 and its interaction with MYC.

Level of herpes simplex virus type 1 latency correlates with severity of corneal scarring and exhaustion of CD8+ T cells in trigeminal ganglia of latently infected mice.

A hallmark of infection with herpes simplex virus type 1 (HSV-1) is the establishment of latency in ganglia of the infected individual. During the life of the latently infected individual, the virus can occasionally reactivate, travel back to the eye, and cause recurrent disease. Indeed, a major cause of corneal scarring (CS) is the scarring induced by HSV-1 following reactivation from latency. In this study, we evaluated the relationship between the amount of CS and the level of the HSV-1 latency-associated transcript (LAT) in trigeminal ganglia (TG) of latently infected mice. Our results suggested that the amount of CS was not related to the amount of virus replication following primary ocular HSV-1 infection, since replication in the eyes was similar in mice that did not develop CS, mice that developed CS in just one eye, and mice that developed CS in both eyes. In contrast, mice with no CS had significantly less LAT, and thus presumably less latency, in their TG than mice that had CS in both eyes. Higher CS also correlated with higher levels of mRNAs for PD-1, CD4, CD8, F4/80, interleukin-4, gamma interferon, granzyme A, and granzyme B in both cornea and TG. These results suggest that (i) the immunopathology induced by HSV-1 infection does not correlate with primary virus replication in the eye; (ii) increased CS appears to correlate with increased latency in the TG, although the possible cause-and-effect relationship is not known; and (iii) increased latency in mouse TG correlates with higher levels of PD-1 mRNA, suggesting exhaustion of CD8+ T cells.

Involvement of STAT4 in IgG subtype switching and ocular HSV-1 replication in mice.

PURPOSE: To assess the relative impact of elevated T-helper 2 (T(H)2)- and reduced T-Helper 1 (T(H)1)-dependent immune responses on ocular herpes simplex virus type 1 (HSV-1) infection. METHODS: Signal transducer and activator of transcription protein 4 knockout mice (BALB/c-STAT4(-/-)) and wild-type BALB/c control mice were immunized with avirulent HSV-1 strain KOS or were mock-immunized. Three weeks after the third immunization, neutralizing antibody titers were determined by plaque reduction assays. Following ocular infection with virulent HSV-1 strain McKrae, viral replication in the eye, blepharitis, corneal scarring (CS), survival, and immunoglobulin (Ig) isotypes in sera were determined. RESULTS: Vaccinated STAT4(-/-) and BALB/c mice contained significant and similar neutralizing antibody titers and were completely protected against HSV-1-induced death and CS. In contrast to vaccinated STAT4(-/-) mice, mock-vaccinated STAT4(-/-) mice had higher ocular HSV-1 titers than mock-vaccinated BALB/c mice on days 2-3 post-ocular infection. There were also significant differences in the levels of IgG2a, IgG2b, and IgG3 in the sera of STAT4(-/-) mice when compared to the control BALB/c mice. CONCLUSIONS: These results suggest that the absence of T(H)1 cytokine responses did alter protection against viral replication and IgG isotypes but not eye disease or survival.

Unbiased Metabolic Profiling Predicts Sensitivity of High MYC-Expressing Atypical Teratoid/Rhabdoid Tumors to Glutamine Inhibition with 6-Diazo-5-Oxo-L-Norleucine.

PURPOSE: Atypical teratoid/rhabdoid tumors (AT/RT) are aggressive infantile brain tumors with poor survival. Recent advancements have highlighted significant molecular heterogeneity in AT/RT with an aggressive subgroup featuring overexpression of the MYC proto-oncogene. We perform the first comprehensive metabolic profiling of patient-derived AT/RT cell lines to identify therapeutic susceptibilities in high MYC-expressing AT/RT. EXPERIMENTAL DESIGN: Metabolites were extracted from AT/RT cell lines and separated in ultra-high performance liquid chromatography mass spectrometry. Glutamine metabolic inhibition with 6-diazo-5-oxo-L-norleucine (DON) was tested with growth and cell death assays and survival studies in orthotopic mouse models of AT/RT. Metabolic flux analysis was completed to identify combination therapies to act synergistically to improve survival in high MYC AT/RT. RESULTS: Unbiased metabolic profiling of AT/RT cell models identified a unique dependence of high MYC AT/RT on glutamine for survival. The glutamine analogue, DON, selectively targeted high MYC cell lines, slowing cell growth, inducing apoptosis, and extending survival in orthotopic mouse models of AT/RT. Metabolic flux experiments with isotopically labeled glutamine revealed DON inhibition of glutathione (GSH) synthesis. DON combined with carboplatin further slowed cell growth, induced apoptosis, and extended survival in orthotopic mouse models of high MYC AT/RT. CONCLUSIONS: Unbiased metabolic profiling of AT/RT identified susceptibility of high MYC AT/RT to glutamine metabolic inhibition with DON therapy. DON inhibited glutamine-dependent synthesis of GSH and synergized with carboplatin to extend survival in high MYC AT/RT. These findings can rapidly translate into new clinical trials to improve survival in high MYC AT/RT.

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.

Genomic analysis demonstrates that histologically-defined astroblastomas are molecularly heterogeneous and that tumors with MN1 rearrangement exhibit the most favorable prognosis.

Astroblastoma (AB) is a rare CNS tumor demonstrating abundant astroblastomatous pseudorosettes. Its molecular features have not been comprehensively studied and its status as a tumor entity is controversial. We analyzed a cohort of 27 histologically-defined ABs using DNA methylation profiling, copy number analysis, FISH and site-directed sequencing. Most cases demonstrated mutually exclusive MN1 rearrangements (n = 10) or BRAFV600E mutations (n = 7). Two additional cases harbored RELA rearrangements. Other cases lacked these specific genetic alterations (n = 8). By DNA methylation profiling, tumors with MN1 or RELA rearrangement clustered with high-grade neuroepithelial tumor with MN1 alteration (HGNET-MN1) and RELA-fusion ependymoma, respectively. In contrast, BRAFV600E-mutant tumors grouped with pleomorphic xanthoastrocytoma (PXA). Six additional tumors clustered with either supratentorial pilocytic astrocytoma and ganglioglioma (LGG-PA/GG-ST), normal or reactive cerebrum, or with no defined DNA methylation class. While certain histologic features favored one genetic group over another, no group could be reliably distinguished by histopathology alone. Survival analysis between genetic AB subtypes was limited by sample size, but showed that MN1-rearranged AB tumors were characterized by better overall survival compared to other genetic subtypes, in fact, significantly better than BRAFV600E-mutant tumors (P = 0.013). Our data confirm that histologically-defined ABs are molecularly heterogeneous and do not represent a single entity. They rather encompass several low- to higher-grade glial tumors including neuroepithelial tumors with MN1 rearrangement, PXA-like tumors, RELA ependymomas, and possibly yet uncharacterized lesions. Genetic subtyping of tumors exhibiting AB histology, particularly determination of MN1 and BRAFV600E status, is necessary for important prognostic and possible treatment implications.