Glioblastoma stem cells generate vascular pericytes to support vessel function and tumor growth.

Glioblastomas (GBMs) are highly vascular and lethal brain tumors that display cellular hierarchies containing self-renewing tumorigenic glioma stem cells (GSCs). Because GSCs often reside in perivascular niches and may undergo mesenchymal differentiation, we interrogated GSC potential to generate vascular pericytes. Here, we show that GSCs give rise to pericytes to support vessel function and tumor growth. In vivo cell lineage tracing with constitutive and lineage-specific fluorescent reporters demonstrated that GSCs generate the majority of vascular pericytes. Selective elimination of GSC-derived pericytes disrupts the neovasculature and potently inhibits tumor growth. Analysis of human GBM specimens showed that most pericytes are derived from neoplastic cells. GSCs are recruited toward endothelial cells via the SDF-1/CXCR4 axis and are induced to become pericytes predominantly by transforming growth factor ß. Thus, GSCs contribute to vascular pericytes that may actively remodel perivascular niches. Therapeutic targeting of GSC-derived pericytes may effectively block tumor progression and improve antiangiogenic therapy.

Glioma stem cell proliferation and tumor growth are promoted by nitric oxide synthase-2.

Malignant gliomas are aggressive brain tumors with limited therapeutic options, and improvements in treatment require a deeper molecular understanding of this disease. As in other cancers, recent studies have identified highly tumorigenic subpopulations within malignant gliomas, known generally as cancer stem cells. Here, we demonstrate that glioma stem cells (GSCs) produce nitric oxide via elevated nitric oxide synthase-2 (NOS2) expression. GSCs depend on NOS2 activity for growth and tumorigenicity, distinguishing them from non-GSCs and normal neural progenitors. Gene expression profiling identified many NOS2-regulated genes, including the cell-cycle inhibitor cell division autoantigen-1 (CDA1). Further, high NOS2 expression correlates with decreased survival in human glioma patients, and NOS2 inhibition slows glioma growth in a murine intracranial model. These data provide insight into how GSCs are mechanistically distinct from their less tumorigenic counterparts and suggest that NOS2 inhibition may be an efficacious approach to treating this devastating disease.

Recurrent Glioblastoma Treated with Recombinant Poliovirus.

BACKGROUND: The prognosis of patients with recurrent World Health Organization (WHO) grade IV malignant glioma is dismal, and there is currently no effective therapy. We conducted a dose-finding and toxicity study in this population of patients, evaluating convection-enhanced, intratumoral delivery of the recombinant nonpathogenic polio-rhinovirus chimera (PVSRIPO). PVSRIPO recognizes the poliovirus receptor CD155, which is widely expressed in neoplastic cells of solid tumors and in major components of the tumor microenvironment. METHODS: We enrolled consecutive adult patients who had recurrent supratentorial WHO grade IV malignant glioma, confirmed on histopathological testing, with measurable disease (contrast-enhancing tumor of ≥1 cm and ≤5.5 cm in the greatest dimension). The study evaluated seven doses, ranging between 107 and 1010 50% tissue-culture infectious doses (TCID50), first in a dose-escalation phase and then in a dose-expansion phase. RESULTS: From May 2012 through May 2017, a total of 61 patients were enrolled and received a dose of PVSRIPO. Dose level -1 (5.0×107 TCID50) was identified as the phase 2 dose. One dose-limiting toxic effect was observed; a patient in whom dose level 5 (1010 TCID50) was administered had a grade 4 intracranial hemorrhage immediately after the catheter was removed. To mitigate locoregional inflammation of the infused tumor with prolonged glucocorticoid use, dose level 5 was deescalated to reach the phase 2 dose. In the dose-expansion phase, 19% of the patients had a PVSRIPO-related adverse event of grade 3 or higher. Overall survival among the patients who received PVSRIPO reached a plateau of 21% (95% confidence interval, 11 to 33) at 24 months that was sustained at 36 months. CONCLUSIONS: Intratumoral infusion of PVSRIPO in patients with recurrent WHO grade IV malignant glioma confirmed the absence of neurovirulent potential. The survival rate among patients who received PVSRIPO immunotherapy was higher at 24 and 36 months than the rate among historical controls. (Funded by the Brain Tumor Research Charity and others; ClinicalTrials.gov number, NCT01491893 .).

Subgroup and subtype-specific outcomes in adult medulloblastoma.

Medulloblastoma, a common pediatric malignant central nervous system tumour, represent a small proportion of brain tumours in adults. Previously it has been shown that in adults, Sonic Hedgehog (SHH)-activated tumours predominate, with Wingless-type (WNT) and Group 4 being less common, but molecular risk stratification remains a challenge. We performed an integrated analysis consisting of genome-wide methylation profiling, copy number profiling, somatic nucleotide variants and correlation of clinical variables across a cohort of 191 adult medulloblastoma cases identified through the Medulloblastoma Advanced Genomics International Consortium. We identified 30 WNT, 112 SHH, 6 Group 3, and 41 Group 4 tumours. Patients with SHH tumours were significantly older at diagnosis compared to other subgroups (p < 0.0001). Five-year progression-free survival (PFS) for WNT, SHH, Group 3, and Group 4 tumours was 64.4 (48.0-86.5), 61.9% (51.6-74.2), 80.0% (95% CI 51.6-100.0), and 44.9% (95% CI 28.6-70.7), respectively (p = 0.06). None of the clinical variables (age, sex, metastatic status, extent of resection, chemotherapy, radiotherapy) were associated with subgroup-specific PFS. Survival among patients with SHH tumours was significantly worse for cases with chromosome 3p loss (HR 2.9, 95% CI 1.1-7.6; p = 0.02), chromosome 10q loss (HR 4.6, 95% CI 2.3-9.4; p < 0.0001), chromosome 17p loss (HR 2.3, 95% CI 1.1-4.8; p = 0.02), and PTCH1 mutations (HR 2.6, 95% CI 1.1-6.2; p = 0.04). The prognostic significance of 3p loss and 10q loss persisted in multivariable regression models. For Group 4 tumours, chromosome 8 loss was strongly associated with improved survival, which was validated in a non-overlapping cohort (combined cohort HR 0.2, 95% CI 0.1-0.7; p = 0.007). Unlike in pediatric medulloblastoma, whole chromosome 11 loss in Group 4 and chromosome 14q loss in SHH was not associated with improved survival, where MYCN, GLI2 and MYC amplification were rare. In sum, we report unique subgroup-specific cytogenetic features of adult medulloblastoma, which are distinct from those in younger patients, and correlate with survival disparities. Our findings suggest that clinical trials that incorporate new strategies tailored to high-risk adult medulloblastoma patients are urgently needed.

Tetanus toxoid and CCL3 improve dendritic cell vaccines in mice and glioblastoma patients.

After stimulation, dendritic cells (DCs) mature and migrate to draining lymph nodes to induce immune responses. As such, autologous DCs generated ex vivo have been pulsed with tumour antigens and injected back into patients as immunotherapy. While DC vaccines have shown limited promise in the treatment of patients with advanced cancers including glioblastoma, the factors dictating DC vaccine efficacy remain poorly understood. Here we show that pre-conditioning the vaccine site with a potent recall antigen such as tetanus/diphtheria (Td) toxoid can significantly improve the lymph node homing and efficacy of tumour-antigen-specific DCs. To assess the effect of vaccine site pre-conditioning in humans, we randomized patients with glioblastoma to pre-conditioning with either mature DCs or Td unilaterally before bilateral vaccination with DCs pulsed with Cytomegalovirus phosphoprotein 65 (pp65) RNA. We and other laboratories have shown that pp65 is expressed in more than 90% of glioblastoma specimens but not in surrounding normal brain, providing an unparalleled opportunity to subvert this viral protein as a tumour-specific target. Patients given Td had enhanced DC migration bilaterally and significantly improved survival. In mice, Td pre-conditioning also enhanced bilateral DC migration and suppressed tumour growth in a manner dependent on the chemokine CCL3. Our clinical studies and corroborating investigations in mice suggest that pre-conditioning with a potent recall antigen may represent a viable strategy to improve anti-tumour immunotherapy.

Desmoplastic myxoid tumor, SMARCB1-mutant: clinical, histopathological and molecular characterization of a pineal region tumor encountered in adolescents and adults.

Atypical teratoid/rhabdoid tumor (ATRT) is a highly malignant brain tumor predominantly occurring in infants. Mutations of the SMARCB1 gene are the characteristic genetic lesion. SMARCB1-mutant tumors in adolescents and adults are rare and may show uncommon histopathological and clinical features. Here we report seven SMARCB1-deficient intracranial tumors sharing distinct clinical, histopathological and molecular features. Median age of the four females and three males was 40 years (range 15-61 years). All tumors were located in the pineal region. Histopathologically, these tumors displayed spindled and epithelioid cells embedded in a desmoplastic stroma alternating with a variable extent of a loose myxoid matrix. All cases showed loss of nuclear SMARCB1/INI1 protein expression, expression of EMA and CD34 was frequent and the Ki67/MIB1 proliferation index was low in the majority of cases (median 3%). Three cases displayed heterozygous SMARCB1 deletions and two cases a homozygous SMARCB1 deletion. On sequencing, one tumor showed a 2 bp deletion in exon 4 (c.369_370del) and one a short duplication in exon 3 (c.237_276dup) both resulting in frameshift mutations. Most DNA methylation profiles were not classifiable using the Heidelberg Brain Tumor Classifier (version v11b4). By unsupervised t-SNE analysis and hierarchical clustering analysis, however, all tumors grouped closely together and showed similarities with ATRT-MYC. After a median observation period of 48 months, three patients were alive with stable disease, whereas one patient experienced tumor progression and three patients had succumbed to disease. In conclusion, our series represents an entity with distinct clinical, histopathological and molecular features showing epigenetic similarities with ATRT-MYC. We propose the designation desmoplastic myxoid tumor (DMT), SMARCB1-mutant, for these tumors.

Sudden Unexpected Death in a Child From an Anaplastic Ependymoma.

Primary central nervous system tumors are an extremely rare cause of sudden, unexpected death in children as most patients develop symptoms because of increased intracranial pressure and seek medical attention. Rarely, a forensic pathologist may encounter a primary intracranial neoplasm in a pediatric decedent that was not suspected before death. Herein, we present a case of a supratentorial neuroepithelial tumor found at autopsy in a 3-year-old African American boy without any reported significant medical history. The tumor had significant mass effect and caused cerebral edema, which ultimately resulted in transtentorial herniation and death. The gross, histopathological, immunohistochemical, and ultrastructural findings were most consistent with an anaplastic ependymoma.

Heterogeneity within the PF-EPN-B ependymoma subgroup.

Posterior fossa ependymoma comprise three distinct molecular variants, termed PF-EPN-A (PFA), PF-EPN-B (PFB), and PF-EPN-SE (subependymoma). Clinically, they are very disparate and PFB tumors are currently being considered for a trial of radiation avoidance. However, to move forward, unraveling the heterogeneity within PFB would be highly desirable. To discern the molecular heterogeneity within PFB, we performed an integrated analysis consisting of DNA methylation profiling, copy-number profiling, gene expression profiling, and clinical correlation across a cohort of 212 primary posterior fossa PFB tumors. Unsupervised spectral clustering and t-SNE analysis of genome-wide methylation data revealed five distinct subtypes of PFB tumors, termed PFB1-5, with distinct demographics, copy-number alterations, and gene expression profiles. All PFB subtypes were distinct from PFA and posterior fossa subependymomas. Of the five subtypes, PFB4 and PFB5 are more discrete, consisting of younger and older patients, respectively, with a strong female-gender enrichment in PFB5 (age: p = 0.011, gender: p = 0.04). Broad copy-number aberrations were common; however, many events such as chromosome 2 loss, 5 gain, and 17 loss were enriched in specific subtypes and 1q gain was enriched in PFB1. Late relapses were common across all five subtypes, but deaths were uncommon and present in only two subtypes (PFB1 and PFB3). Unlike the case in PFA ependymoma, 1q gain was not a robust marker of poor progression-free survival; however, chromosome 13q loss may represent a novel marker for risk stratification across the spectrum of PFB subtypes. Similar to PFA ependymoma, there exists a significant intertumoral heterogeneity within PFB, with distinct molecular subtypes identified. Even when accounting for this heterogeneity, extent of resection remains the strongest predictor of poor outcome. However, this biological heterogeneity must be accounted for in future preclinical modeling and personalized therapies.

HIV-1 Envelope Mimicry of Host Enzyme Kynureninase Does Not Disrupt Tryptophan Metabolism.

The HIV-1 envelope protein (Env) has evolved to subvert the host immune system, hindering viral control by the host. The tryptophan metabolic enzyme kynureninase (KYNU) is mimicked by a portion of the HIV Env gp41 membrane proximal region (MPER) and is cross-reactive with the HIV broadly neutralizing Ab (bnAb) 2F5. Molecular mimicry of host proteins by pathogens can lead to autoimmune disease. In this article, we demonstrate that neither the 2F5 bnAb nor HIV MPER-KYNU cross-reactive Abs elicited by immunization with an MPER peptide-liposome vaccine in 2F5 bnAb VHDJH and VLJL knock-in mice and rhesus macaques modified KYNU activity or disrupted tissue tryptophan metabolism. Thus, molecular mimicry by HIV-1 Env that promotes the evasion of host anti-HIV-1 Ab responses can be directed toward nonfunctional host protein epitopes that do not impair host protein function. Therefore, the 2F5 HIV Env gp41 region is a key and safe target for HIV-1 vaccine development.

Sulfonylurea receptor 1 expression is variable in adult and pediatric brain tumors.

INTRODUCTION: Edema is a significant cause of neuromorbidity in children and adults with brain tumors. Agents used to control this effect, such as corticosteroids, have their own associated morbidities. Sulfonylurea receptor 1 (SUR1) is a transmembrane protein that regulates the activity of ion channels in neurons, glia, and endothelial cells. SUR1 expression is upregulated in neuroinflammatory conditions. Inhibition of SUR1 with glyburide decreases edema and neuroinflammation by countering cytotoxic edema and apoptosis in rodent models of subarachnoid hemorrhage, stroke, trauma, and cerebral metastases. However, the expression of SUR1 in human brain tumors has not been elucidated. The purpose of this study was to determine SUR1 expression and cellular colocalization in a variety of human brain tumor specimens. MATERIALS AND METHODS: Six glioblastoma, 12 cerebral metastases, 11 medulloblastoma, 9 supratentorial ependymoma, and 8 posterior fossa ependymoma specimens were analyzed using immunofluorescence. SUR1 expression and colocalization with blood vessels, neurons, and glial cells was analyzed and compared using ANOVA. RESULTS: SUR1 expression was found in all specimens examined as a percentage of the total tissue area (mean ± SD): glioblastoma 3.9 ± 4, cerebral metastases 4.1 ± 3.1, medulloblastoma 8.2 ± 7.2, supratentorial ependymoma 9.1 ± 7, and posterior fossa ependymoma 8.1 ± 5.9. SUR1 expression was greater in supratentorial ependymoma compared to glioblastoma and metastases (p < 0.05) and greater in medulloblastoma compared to glioblastoma (p < 0.05). SUR1 colocalized most reliably with the neuronal marker, NeuN, in glioblastoma, metastases, and posterior fossa ependymoma samples (p < 0.05). SUR1 colocalized most reliably with the endothelial cell marker, CD31, in medulloblastoma samples (p < 0.05). CONCLUSION: SUR1 is a putative therapeutic target to reduce neuroinflammation in adult and pediatric brain tumors. Inhibition of SUR1 may result in neuronal stabilization in glioblastoma, cerebral metastases, and posterior fossa ependymoma and reduced edema in medulloblastoma.
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Novel case of recurrent intraventricular atypical central neurocytoma with prominent gangliogliomatous differentiation in a 10-year-old boy with 10 years of follow up.

Central neurocytoma is a rare neuronal tumor that typically occurs in young adults. Infrequently, these tumors exhibit advanced neuronal maturation and glial differentiation, giving rise to a histologically diverse tumor, in contrast to a typical central neurocytoma. We present a novel case of intraventricular central neurocytoma with prominent gangliogliomatous differentiation that developed atypical features upon recurrence after 10 years of follow up in a 10-year-old boy. Our case provides insight into the divergent differentiation capability of a neurocytic tumor and illustrates the diverse histological features of this rare entity.

Epilepsy in neurofibromatosis type 1.

OBJECTIVES: To describe the characteristics of epilepsy in patients with Neurofibromatosis type 1 (NF1). METHODS: Analysis of a cohort of consecutive NF1 patients seen in our NF1 clinic during a three-year period. RESULTS: Of the 184 NF1 patients seen during that period, 26 had epilepsy and three had febrile seizures. Of the 26, 17 (65%) had localization-related epilepsy, seven of whom (41%) were drug resistant. Six (23%) had apparently primary generalized epilepsy (0/6 drug resistant), two (8%) Lennox-Gastaut syndrome, and one (4%) West syndrome (all three were drug-resistant). As compared to the patients with no epilepsy, those with epilepsy were more likely to have MRI findings of mesial temporal sclerosis (MTS) (23% vs. 5%, p=0.0064), and cerebral hemisphere tumors (31% vs. 10%, p=0.0079), but not of the other MRI findings including neurofibromatosis bright objects, or optic gliomas. Three of the six patients with MTS underwent temporal lobectomy with subsequent control of their seizures with confirmation of MTS on pathology in 3/3 and presence of coexisting focal cortical dysplasia (FCD) in 2/3. We also have observed three additional patients outside the above study with the association of NF1, MTS, and intractable epilepsy. SIGNIFICANCE: Epilepsy is relatively common in NF1, often occurs in patients with brain tumors or with MTS which can coexist with FCD, can be associated with multiple types of epilepsy syndromes, and when localization-related is often drug-resistant. Patients with NF1 and MTS can respond to medial temporal lobectomy and may have coexisting medial temporal lobe cortical dysplasia.

Prognostic value of medulloblastoma extent of resection after accounting for molecular subgroup: a retrospective integrated clinical and molecular analysis.

BACKGROUND: Patients with incomplete surgical resection of medulloblastoma are controversially regarded as having a marker of high-risk disease, which leads to patients undergoing aggressive surgical resections, so-called second-look surgeries, and intensified chemoradiotherapy. All previous studies assessing the clinical importance of extent of resection have not accounted for molecular subgroup. We analysed the prognostic value of extent of resection in a subgroup-specific manner. METHODS: We retrospectively identified patients who had a histological diagnosis of medulloblastoma and complete data about extent of resection and survival from centres participating in the Medulloblastoma Advanced Genomics International Consortium. We collected from resections done between April, 1997, and February, 2013, at 35 international institutions. We established medulloblastoma subgroup affiliation by gene expression profiling on frozen or formalin-fixed paraffin-embedded tissues. We classified extent of resection on the basis of postoperative imaging as gross total resection (no residual tumour), near-total resection (<1·5 cm(2) tumour remaining), or sub-total resection (≥1·5 cm(2) tumour remaining). We did multivariable analyses of overall survival and progression-free survival using the variables molecular subgroup (WNT, SHH, group 4, and group 3), age (<3 vs ≥3 years old), metastatic status (metastases vs no metastases), geographical location of therapy (North America/Australia vs rest of the world), receipt of chemotherapy (yes vs no) and receipt of craniospinal irradiation (<30 Gy or >30 Gy vs no craniospinal irradiation). The primary analysis outcome was the effect of extent of resection by molecular subgroup and the effects of other clinical variables on overall and progression-free survival. FINDINGS: We included 787 patients with medulloblastoma (86 with WNT tumours, 242 with SHH tumours, 163 with group 3 tumours, and 296 with group 4 tumours) in our multivariable Cox models of progression-free and overall survival. We found that the prognostic benefit of increased extent of resection for patients with medulloblastoma is attenuated after molecular subgroup affiliation is taken into account. We identified a progression-free survival benefit for gross total resection over sub-total resection (hazard ratio [HR] 1·45, 95% CI 1·07-1·96, p=0·16) but no overall survival benefit (HR 1·23, 0·87-1·72, p=0·24). We saw no progression-free survival or overall survival benefit for gross total resection compared with near-total resection (HR 1·05, 0·71-1·53, p=0·8158 for progression-free survival and HR 1·14, 0·75-1·72, p=0·55 for overall survival). No significant survival benefit existed for greater extent of resection for patients with WNT, SHH, or group 3 tumours (HR 1·03, 0·67-1·58, p=0·89 for sub-total resection vs gross total resection). For patients with group 4 tumours, gross total resection conferred a benefit to progression-free survival compared with sub-total resection (HR 1·97, 1·22-3·17, p=0·0056), especially for those with metastatic disease (HR 2·22, 1·00-4·93, p=0·050). However, gross total resection had no effect on overall survival compared with sub-total resection in patients with group 4 tumours (HR 1·67, 0·93-2·99, p=0·084). INTERPRETATION: The prognostic benefit of increased extent of resection for patients with medulloblastoma is attenuated after molecular subgroup affiliation is taken into account. Although maximum safe surgical resection should remain the standard of care, surgical removal of small residual portions of medulloblastoma is not recommended when the likelihood of neurological morbidity is high because there is no definitive benefit to gross total resection compared with near-total resection. FUNDING: Canadian Cancer Society Research Institute, Terry Fox Research Institute, Canadian Institutes of Health Research, National Institutes of Health, Pediatric Brain Tumor Foundation, and the Garron Family Chair in Childhood Cancer Research.

Preclinical toxicity evaluation of a novel immunotoxin, D2C7-(scdsFv)-PE38KDEL, administered via intracerebral convection-enhanced delivery in rats.

D2C7-(scdsFv)-PE38KDEL (D2C7-IT) is a novel immunotoxin that reacts with wild-type epidermal growth factor receptor (EGFRwt) and mutant EGFRvIII proteins overexpressed in glioblastomas. This study assessed the toxicity of intracerebral administration of D2C7-IT to support an initial Food and Drug Administration Investigational New Drug application. After the optimization of the formulation and administration, two cohorts (an acute and chronic cohort necropsied on study days 5 and 34) of Sprague-Dawley (SD) rats (four groups of 5 males and 5 females) were infused with the D2C7-IT formulation at total doses of 0, 0.05, 0.1, 0.4 µg (the acute cohort) and 0, 0.05, 0.1, 0.35 µg (the chronic cohort) for approximately 72 h by intracerebral convection-enhanced delivery using osmotic pumps. Mortality was observed in the 0.40 µg (5/10 rats) and 0.35 µg (4/10 rats) high-dose groups of each cohort. Body weight loss and abnormal behavior were only revealed in the rats treated with high doses of D2C7-IT. No dose-related effects were observed in clinical laboratory tests in either cohort. A gross pathologic examination of systemic tissues from the high-dose and control groups in both cohorts exhibited no dose-related or drug-related pathologic findings. Brain histopathology revealed the frequent occurrence of dose-related encephalomalacia, edema, and demyelination in the high-dose groups of both cohorts. In this study, the maximum tolerated dose of D2C7-IT was determined to be between 0.10 and 0.35 µg, and the no-observed-adverse-effect-level was 0.05 µg in SD rats. Both parameters were utilized to design the Phase I/II D2C7-IT clinical trial.

Histologically benign, clinically aggressive: Progressive non-optic pathway pilocytic astrocytomas in adults with NF1.

Although optic pathway gliomas are the most common brain tumors associated with neurofibromatosis type 1 (NF1), extra-optic gliomas occur and may behave more aggressively with outcomes that differ by age. A retrospective case-control study was designed to describe the clinical course of adult NF1 patients with progressive extra-optic pilocytic astrocytomas (PAs) and compare to a pediatric cohort. Data for patients treated at the Johns Hopkins Comprehensive Neurofibromatosis Center from 2003 to 2013 were reviewed to identify cases (adults, age >18) and controls (pediatric, age <18) with clinically or radiographically progressive extra-optic PAs. Demographic, clinical, histologic, and radiographic data were collected. Three adult NF1 cases and four pediatric NF1 controls were identified. Mean age was 32.3 ± 9.5 years, 66% male (cases); 12.8 ± 4.2 years, 100% male (controls). Symptomatic progression occurred in two-of-three adults (67%) while the majority of pediatric patients presented with isolated radiographic progression (n = 3, 75%). Onset tended to be more rapid in adults (4 ± 1 vs. 14 ± 8.3 months, P = 0.10). Subtotal resection was the treatment for all pediatric patients. Radiotherapy (n = 2), chemotherapy (n = 2), and targeted, biologic agents (n = 2) were administered in adults. Although all pediatric patients are living, outcomes were universally poor in adults with progression to death in all (median survival 17.1 months, range 6.6-30.3). In conclusion, despite grade I histology, all three adult NF1 patients with progressive extra-optic PAs suffered an aggressive clinical course which was not seen in pediatric patients. Clinicians should be aware of this clinico-histologic discrepancy when counseling and managing adult NF1 patients with progressive extra-optic PAs. © 2016 Wiley Periodicals, Inc.

TERT promoter mutations occur frequently in gliomas and a subset of tumors derived from cells with low rates of self-renewal.

Malignant cells, like all actively growing cells, must maintain their telomeres, but genetic mechanisms responsible for telomere maintenance in tumors have only recently been discovered. In particular, mutations of the telomere binding proteins alpha thalassemia/mental retardation syndrome X-linked (ATRX) or death-domain associated protein (DAXX) have been shown to underlie a telomere maintenance mechanism not involving telomerase (alternative lengthening of telomeres), and point mutations in the promoter of the telomerase reverse transcriptase (TERT) gene increase telomerase expression and have been shown to occur in melanomas and a small number of other tumors. To further define the tumor types in which this latter mechanism plays a role, we surveyed 1,230 tumors of 60 different types. We found that tumors could be divided into types with low (<15%) and high (≥15%) frequencies of TERT promoter mutations. The nine TERT-high tumor types almost always originated in tissues with relatively low rates of self renewal, including melanomas, liposarcomas, hepatocellular carcinomas, urothelial carcinomas, squamous cell carcinomas of the tongue, medulloblastomas, and subtypes of gliomas (including 83% of primary glioblastoma, the most common brain tumor type). TERT and ATRX mutations were mutually exclusive, suggesting that these two genetic mechanisms confer equivalent selective growth advantages. In addition to their implications for understanding the relationship between telomeres and tumorigenesis, TERT mutations provide a biomarker that may be useful for the early detection of urinary tract and liver tumors and aid in the classification and prognostication of brain tumors.

Proteomic profiling of patient-derived glioblastoma xenografts identifies a subset with activated EGFR: implications for drug development.

The development of drugs to inhibit glioblastoma (GBM) growth requires reliable pre-clinical models. To date, proteomic level validation of widely used patient-derived glioblastoma xenografts (PDGX) has not been performed. In the present study, we characterized 20 PDGX models according to subtype classification based on The Cancer Genome Atlas criteria, TP53, PTEN, IDH 1/2, and TERT promoter genetic analysis, EGFR amplification status, and examined their proteomic profiles against those of their parent tumors. The 20 PDGXs belonged to three of four The Cancer Genome Atlas subtypes: eight classical, eight mesenchymal, and four proneural; none neural. Amplification of EGFR gene was observed in 9 of 20 xenografts, and of these, 3 harbored the EGFRvIII mutation. We then performed proteomic profiling of PDGX, analyzing expression/activity of several proteins including EGFR. Levels of EGFR phosphorylated at Y1068 vary considerably between PDGX samples, and this pattern was also seen in primary GBM. Partitioning of 20 PDGX into high (n = 5) and low (n = 15) groups identified a panel of proteins associated with high EGFR activity. Thus, PDGX with high EGFR activity represent an excellent pre-clinical model to develop therapies for a subset of GBM patients whose tumors are characterized by high EGFR activity. Further, the proteins found to be associated with high EGFR activity can be monitored to assess the effectiveness of targeting EGFR. The development of drugs to inhibit glioblastoma (GBM) growth requires reliable pre-clinical models. We validated proteomic profiles using patient-derived glioblastoma xenografts (PDGX), characterizing 20 PDGX models according to subtype classification based on The Cancer Genome Atlas (TCGA) criteria, TP53, PTEN, IDH 1/2, and TERT promoter genetic analysis, EGFR amplification status, and examined their proteomic profiles against those of their parent tumors. Proteins found to be associated with high EGFR activity represent potential biomarkers for GBM monitoring.

Increased proportion of FoxP3+ regulatory T cells in tumor infiltrating lymphocytes is associated with tumor recurrence and reduced survival in patients with glioblastoma.

Glioblastoma multiforme (GBM) is an aggressive malignancy associated with profound host immunosuppression mediated in part by FoxP3 expressing regulatory CD4+ T lymphocytes (Tregs) that down-regulate anti-tumor immunity. In order to assess whether FoxP3 was an independent driver differentially expressed in primary versus recurrent GBMs, we stained resected primary and recurrent GBM tumors for CD3, CD4, CD8 and FoxP3 expression using standard immunohistochemistry. Slides were scanned with a high-resolution scanner (ScanScope CS; Aperio), and image analysis software (Aperio ScanScope) was used to enumerate lymphocyte subpopulations allowing for high-throughput analysis and bypassing manual selection bias. As shown in previous studies, enumeration of individual lymphocyte populations did not correlate with clinical outcomes in patients with GBM. However, the CD4+ to regulatory FoxP3+ T cell ratio was diminished in recurrent disease, and increased CD3 and CD8+ to regulatory T cell ratios showed a positive correlation with survival outcomes in primary GBM. These results suggest that while absolute numbers of tumor infiltrating lymphocytes may not be informative for predicting clinical outcomes in patients with GBM, the effective balance of CD3, CD4 and CD8+ T cells to immunosuppressive FoxP3+ regulatory cells may influence clinical outcomes in this patient population.

The zinc finger transcription factor ZFX is required for maintaining the tumorigenic potential of glioblastoma stem cells.

Glioblastomas are highly lethal brain tumors containing tumor-propagating glioma stem cells (GSCs). The molecular mechanisms underlying the maintenance of the GSC phenotype are not fully defined. Here we demonstrate that the zinc finger and X-linked transcription factor (ZFX) maintains GSC self-renewal and tumorigenic potential by upregulating c-Myc expression. ZFX is differentially expressed in GSCs relative to non-stem glioma cells and neural progenitor cells. Disrupting ZFX by shRNA reduced c-Myc expression and potently inhibited GSC self-renewal and tumor growth. Ectopic expression of c-Myc to its endogenous level rescued the effects caused by ZFX disruption, supporting that ZFX controls GSC properties through c-Myc. Furthermore, ZFX binds to a specific sequence (GGGCCCCG) on the human c-Myc promoter to upregulate c-Myc expression. These data demonstrate that ZFX functions as a critical upstream regulator of c-Myc and plays essential roles in the maintenance of the GSC phenotype. This study also supports that c-Myc is a dominant driver linking self-renewal to malignancy.