The proteomic landscape of glioblastoma recurrence reveals novel and targetable immunoregulatory drivers.

Glioblastoma (GBM) is characterized by extensive cellular and genetic heterogeneity. Its initial presentation as primary disease (pGBM) has been subject to exhaustive molecular and cellular profiling. By contrast, our understanding of how GBM evolves to evade the selective pressure of therapy is starkly limited. The proteomic landscape of recurrent GBM (rGBM), which is refractory to most treatments used for pGBM, are poorly known. We, therefore, quantified the transcriptome and proteome of 134 patient-derived pGBM and rGBM samples, including 40 matched pGBM-rGBM pairs. GBM subtypes transition from pGBM to rGBM towards a preferentially mesenchymal state at recurrence, consistent with the increasingly invasive nature of rGBM. We identified immune regulatory/suppressive genes as important drivers of rGBM and in particular 2-5-oligoadenylate synthase 2 (OAS2) as an essential gene in recurrent disease. Our data identify a new class of therapeutic targets that emerge from the adaptive response of pGBM to therapy, emerging specifically in recurrent disease and may provide new therapeutic opportunities absent at pGBM diagnosis.

Hemorrhagic giant cerebral capillary telangiectasia resulting in death: Case report and literature review.

We report a rare case of death caused by hemorrhage of a giant cerebral central nervous system capillary telangiectasia (CCT). A 49-year-old female presented comatose after suffering a traumatic head injury due to an unwitnessed fall. Computed tomography of the head revealed an acute 8.1 × 5.2 cm right intraparenchymal hematoma. Postmortem pathology found thin-walled dilated capillaries consistent with a giant cerebral CCT. The radiological complexity and rarity of giant CCTs result in these malformations often going undiagnosed. We review other cases of giant intracranial CCTs reported in the English literature and confirm that this is the first case of a hemorrhagic giant cerebral CCT causing death. This report emphasizes the existence and complications of giant CCTs and stresses the importance of their investigation to ensure patients receive optimal treatment and follow-up.

Intraventricular ganglioglioma with extensive hemorrhage.

Gangliogliomas represent a rare form of neuroepithelial tumor (up to 1.3% of brain tumors [1]), which even more rarely present with hemorrhage or localize intraventricularly. To date, only two cases of ganglioglioma with both of these features have been reported. Our patient is a 23-year-old woman who presented with signs and symptoms of increased intracranial pressure (ICPI), with a post-subtotal resection diagnosis of WHO Grade I ganglioglioma localizing bilaterally to the lateral ventricles. One year following the operation, the tumor showed radiologic evidence of interval hemorrhage, which was verified histopathologically following a second subtotal resection. Greater than 95% of the lesion represented a large hematoma with organization and well-defined fibrous pseudo-capsule, with very scanty fragments of adjacent/peripheral low-grade neuroglial tumor. This case represents a very rare presentation of intraventricular ganglioglioma with extensive hemorrhage.
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Radiologic and Histopathologic Features of Calcifying Pseudoneoplasm of the Neural Axis.

AIM: To describe the radiologic and corresponding histopathologic features of calcifying pseudoneoplasms of the neural axis. METHODS: Two cases of calcifying pseudoneoplasm of the neural axis were retrospectively reviewed. The first case was documented in a 64-year-old woman, who presented with lower back pain with radiation to her left leg. The second case was documented in a 70-year-old man, who presented with headaches. Medical records, radiologic and histologic findings, and related literature were reviewed. RESULTS: In the first case, imaging of the lumbar spine revealed a 3.8 × 2.2-cm calcified lesion at the level of vertebrae L5 and S1. A subsequent excision exposed an extradural lesion at L5. Histopathologic examination showed amorphous and granular calcifying material with occasional fibrohistiocytic and giant cell reaction, consistent with calcifying pseudoneoplasm of the neural axis. In the second case, imaging of the head revealed a 2.4 × 2.6-cm well-circumscribed, lobulated, calcified lesion within the basal frontal lobe. Subsequent resection exposed an intradural mass with a nodular arrangement of amorphous and granular calcifying material associated with fibrohistiocytic and giant cell reaction. Both patients had a favorable postoperative course and failed to show any clinical or radiologic sign of recurrence. CONCLUSION: Calcifying pseudoneoplasm of the neural axis is an uncommon condition with an excellent prognosis but is often misdiagnosed due to its nonspecific clinical presentation and varied findings on radiology.

Statin-associated Autoimmune Myopathies: A Pathophysiologic Spectrum.

BACKGROUND: Statins have recently been reported to cause a rare autoimmune inflammatory and/or necrotic myopathy that begins or persists after drug cessation. METHODS: We report on 26 patients seen at a neuromuscular centre between 2005 and 2011 who demonstrated muscle weakness/myalgias and creatine kinase elevations during or after statin treatment with continuation of signs and symptoms despite statin withdrawal. RESULTS: All patients were treated with immunosuppressive therapy with good response; all improved biochemically and 86% improved clinically. Sixty-five percent of patients who attempted to taper off immunosuppressive therapy relapsed. We report on a novel finding whereby five of the seven patients who underwent multiple biopsies throughout their disease demonstrated a transformation of their histological diagnosis, with four progressing from having myofibre necrosis with minimal or no inflammation to a diagnosis of polymyositis. CONCLUSIONS: This study offers preliminary evidence that statin-associated necrotizing myopathy and statin-associated polymyositis may not be separate entities but are part of the same pathophysiological spectrum. Both entities respond well to immunosuppression.

ß-Catenin marks proliferating endothelial cells in glioblastoma.

BACKGROUND: Angiogenesis is a key process in the growth and maintenance of tumors. The Wnt signaling pathway is required for angiogenesis of the central nervous system though development of the blood-brain barrier and subsequent proliferation of endothelial cells during tumor growth. However, the specificity of the Wnt pathway in regulating endothelial cells of different central nervous systems remains to be investigated. MATERIALS & METHODS: Patient-derived tissue samples from 35 paraffin-embedded tumors were used to assess ß-catenin immunoexpression. Tumor samples consisted of the following pathologies: grade II diffuse astrocytoma, glioblastoma, hemangioblastoma, and metastatic adenocarcinoma (lung or breast primary). Average percent reactivity was recorded as a mean observed in ten high-power fields. The following scale was used to grade immunoreactivity: 0 = immunonegative, 1 = 1-25% reactive, 2 = 26-50% reactive, 3 = 51-75% reactive, 4 = 76-100% reactive. RESULTS: While we did not observe nuclear expression of ß-catenin in any samples, there was uniform cytoplasmic expression of ß-catenin within glial tumor cells. There was a clear distinction in tumor endothelial cells whereby diffuse staining was noted in areas of microvascular hyperplasia in GBM and a less immunoreactive profile in low-grade astrocytomas. By contrast, non-glial tumors, contained very minimal cytoplasmic ß-catenin expression in tumor and stromal cells and were devoid of immunoreactivity in endothelial cells. CONCLUSION: ß-catenin is unique marker of proliferating endothelial cells in GBM. Therapies targeting the spatial and structural heterogeneity inherent to GBM may prove to be efficacious and result in an improved survivorship.

Characterization of an RNA binding protein interactome reveals a context-specific post-transcriptional landscape of MYC-amplified medulloblastoma.

Pediatric medulloblastoma (MB) is the most common solid malignant brain neoplasm, with Group 3 (G3) MB representing the most aggressive subgroup. MYC amplification is an independent poor prognostic factor in G3 MB, however, therapeutic targeting of the MYC pathway remains limited and alternative therapies for G3 MB are urgently needed. Here we show that the RNA-binding protein, Musashi-1 (MSI1) is an essential mediator of G3 MB in both MYC-overexpressing mouse models and patient-derived xenografts. MSI1 inhibition abrogates tumor initiation and significantly prolongs survival in both models. We identify binding targets of MSI1 in normal neural and G3 MB stem cells and then cross referenced these data with unbiased large-scale screens at the transcriptomic, translatomic and proteomic levels to systematically dissect its functional role. Comparative integrative multi-omic analyses of these large datasets reveal cancer-selective MSI1-bound targets sharing multiple MYC associated pathways, providing a valuable resource for context-specific therapeutic targeting of G3 MB.

De novo pathogenic variant in SETX causes a rapidly progressive neurodegenerative disorder of early childhood-onset with severe axonal polyneuropathy.

Pathogenic variants in SETX cause two distinct neurological diseases, a loss-of-function recessive disorder, ataxia with oculomotor apraxia type 2 (AOA2), and a dominant gain-of-function motor neuron disorder, amyotrophic lateral sclerosis type 4 (ALS4). We identified two unrelated patients with the same de novo c.23C > T (p.Thr8Met) variant in SETX presenting with an early-onset, severe polyneuropathy. As rare private gene variation is often difficult to link to genetic neurological disease by DNA sequence alone, we used transcriptional network analysis to functionally validate these patients with severe de novo SETX-related neurodegenerative disorder. Weighted gene co-expression network analysis (WGCNA) was used to identify disease-associated modules from two different ALS4 mouse models and compared to confirmed ALS4 patient data to derive an ALS4-specific transcriptional signature. WGCNA of whole blood RNA-sequencing data from a patient with the p.Thr8Met SETX variant was compared to ALS4 and control patients to determine if this signature could be used to identify affected patients. WGCNA identified overlapping disease-associated modules in ALS4 mouse model data and ALS4 patient data. Mouse ALS4 disease-associated modules were not associated with AOA2 disease modules, confirming distinct disease-specific signatures. The expression profile of a patient carrying the c.23C > T (p.Thr8Met) variant was significantly associated with the human and mouse ALS4 signature, confirming the relationship between this SETX variant and disease. The similar clinical presentations of the two unrelated patients with the same de novo p.Thr8Met variant and the functional data provide strong evidence that the p.Thr8Met variant is pathogenic. The distinct phenotype expands the clinical spectrum of SETX-related disorders.

Hemimegalencephaly: a fetal case with neuropathological confirmation and review of the literature.

Hemimegalencephaly (HME) is a developmental abnormality of the central nervous system, identified by an abnormal increase in the size of one cerebral hemisphere. HME may present as either a syndromic or isolated case. To date the literature on HME has focused primarily on non-fetal pediatric patients, largely related to surgical resection specimens of the HME hemisphere. We present the case of a male fetus at 22 weeks gestation with intracranial abnormalities identified on a follow-up ultrasound. Gross examination of the fetal brain confirmed the increased size of the right cerebral hemisphere. The ipsilateral brain stem and cerebellum were not involved. Light microscopy demonstrated the presence of accelerated cortical differentiation along with several migrational anomalies in the HME hemisphere. Based on the gross and microscopic findings, a diagnosis of fetal hemimegalencephaly was made. The periventricular proliferative zone of the abnormal hemisphere contained a normal population of neuroepithelial precursor cells. An exhaustive immunohistochemical study found immunoreactivity for calretinin and synaptophysin, while the Ki-67 proliferation labeling was not increased in the HME hemisphere. Our case is the first autopsied report on fetal hemimegalencephaly and confirms that the key pathogenic changes may present as early as 20-22 weeks gestation. The major pathological features of our case are in keeping with a disturbance in accelerated neuronal differentiation and migrational abnormalities.

Case report and literature review of Huntington disease with intermediate CAG expansion.

BACKGROUND: Huntington disease (HD) is a genetically inherited neurodegenerative disorder that classically involves a trinucleotide CAG repeat expansion on chromosome 4, with 36 repeats or greater being disease identifying. It generally presents between the age of 30 and 40 years old and is characterised by severe caudate/striatum degeneration with huntingtin protein aggregation. We present here the case of a patient in her early 80s who presented with 5-year history of worsening chorea and family history of HD but an intermediate length CAG expansion. METHODS: Genetic testing of CAG repeats on chromosome 4. Postmortem brain tissue was obtained and stained using immunohistochemistry for amyloid-beta, tau and glial fibrillary acidic protein (GFAP). Sections from the caudate/putamen were also analysed by p62 immunofluorescence. All sections were reviewed by trained neuropathologists. RESULTS: On genetic testing the patient was found to have a 28 CAG repeat on the longest expansion. Microscopic analysis revealed significant neuronal atrophy in the caudate and putamen with gliosis. Immunofluorescent staining demonstrated minimal intranuclear p62 inclusions suggesting little huntingtin aggregation present. Furthermore, there was significant amyloid-beta pathology (Thal-IV stage) and tau involvement in the medial temporal lobe (Braak stage II). CONCLUSION: This case provides clinical and pathological evidence to support an emerging clinical entity involving HD presentation in late age with an intermediate CAG repeat.

Hypertrophic Interstitial Neuropathy of the Trigeminal Nerve: Case Report and Literature Review.

BACKGROUND AND IMPORTANCE: Hypertrophic interstitial neuropathy (HIN) is an uncommon, non-neoplastic lesion typically affecting peripheral nerves. Cranial nerve (CN) involvement is exceedingly rare. We present a case of isolated trigeminal nerve HIN manifesting with V3 distribution neuralgia. CLINICAL PRESENTATION: A 50-yr-old male presented with left sided trigeminal neuralgia refractory to medical management. The patient underwent retromastoid craniectomy for possible microvascular decompression. Intra-operatively, the trigeminal nerve appeared to be focally enlarged with a sausage-like configuration. We selectively resected 1 fascicle which was predominantly involved. Histopathological examination revealed onion bulb formations composed of Schwann cells around centrally placed axons. A diagnosis of HIN was made. Postoperatively, the patient experienced complete resolution of symptoms. CONCLUSION: This is the third case of isolated trigeminal nerve HIN in the literature. We performed a selective resection in a patient presenting with trigeminal neuralgia, resulting in complete resolution of symptoms. It is reported here with intraoperative microscope images, along with a review and analysis of this topic as it related to CN.

A CD133-AKT-Wnt signaling axis drives glioblastoma brain tumor-initiating cells.

Mechanistic insight into signaling pathways downstream of surface receptors has been revolutionized with integrated cancer genomics. This has fostered current treatment modalities, namely immunotherapy, to capitalize on targeting key oncogenic signaling nodes downstream of a limited number of surface markers. Unfortunately, rudimentary mechanistic understanding of most other cell surface proteins has reduced the clinical utility of these markers. CD133 has reproducibly been shown to correlate with disease progression, recurrence, and poor overall survivorship in the malignant adult brain tumor, glioblastoma (GBM). Using several patient-derived CD133high and CD133low GBMs we describe intrinsic differences in determinants of stemness, which we owe to a CD133-AKT-Wnt signaling axis in which CD133 functions as a putative cell surface receptor for AKT-dependent Wnt activation. These findings may have implications for personalized oncology trials targeting PI3K/AKT or Wnt as both pathways may be activated independent of their canonical drivers, leading to treatment resistance and disease relapse.

Wnt activation as a therapeutic strategy in medulloblastoma.

Medulloblastoma (MB) is defined by four molecular subgroups (Wnt, Shh, Group 3, Group 4) with Wnt MB having the most favorable prognosis. Since prior reports have illustrated the antitumorigenic role of Wnt activation in Shh MB, we aimed to assess the effects of activated canonical Wnt signaling in Group 3 and 4 MBs. By using primary patient-derived MB brain tumor-initiating cell (BTIC) lines, we characterize differences in the tumor-initiating capacity of Wnt, Group 3, and Group 4 MB. With single cell RNA-seq technology, we demonstrate the presence of rare Wnt-active cells in non-Wnt MBs, which functionally retain the impaired tumorigenic potential of Wnt MB. In treating MB xenografts with a Wnt agonist, we provide a rational therapeutic option in which the protective effects of Wnt-driven MBs may be augmented in Group 3 and 4 MB and thereby support emerging data for a context-dependent tumor suppressive role for Wnt/ß-catenin signaling.

Central neurocytoma represents a tumor consisting of diverse neuronal phenotypes.

Central neurocytoma (CN) has long been regarded as a neuronal tumor based on the immunohistochemical expression of synaptophysin and the ultrastructural observation of neurosecretory granules, neurites, and synapses. Having diagnosed 11 CNs at our institution over the past thirty years, we set out to conduct an immunohistochemical study to assess the expression profile of neuronal markers across our cases. Markers of interest included synaptophysin, alpha-synuclein, chromogranin, neurofilament, and calretinin. Intriguingly, we observed a dichotomous expression profile between neurofilament and calretinin, suggesting the presence of histologic variants of CN based on the degree of neuronal maturation. We have further provided an overview of the clinico-pathologic heterogeneity within our series with respect to age of onset, overall outcome, and presence of anaplastic features. In highlighting the case of an infant with an incidental CN diagnosed at autopsy, we have discussed the role of a primitive neural cell of origin for driving tumor formation and accounting for our proposed differences in neuronal maturation within CN.

Multiple recurrences require long-term follow-up in patients diagnosed with spindle cell oncocytoma of the sella turcica.

Spindle cell oncocytoma (SCO) of the sella turcica is characterized as a WHO grade I non-endocrine neoplasm of the sella turcica. Histologically, these tumors contain spindled and variably oncocytic cellular processes. Although SCOs lack immunoreactivity for neuroendocrine markers and pituitary hormones, they are clinically indistinguishable from non-functioning pituitary adenomas. In contrast to the initially described benign clinical course, several reports have subsequently illustrated cases with multiple recurrences with or without histological features of anaplasia in the form of nuclear pleomorphism, frequent mitoses, high Ki-67 index, and/or necrosis. With a follow-up of 14years, we report a case of SCO with multiple recurrences along with an exhaustive clinico-pathological review of all 41 cases of SCO reported in the literature, of which recurrence has been described in 11 cases. Collectively, this report highlights the importance of long-term follow-up and the possible need for adjuvant radiotherapy in patients diagnosed with a sellar SCO and provides a comprehensive review of this rare nonadenomatous sellar tumor.

The identification of human pituitary adenoma-initiating cells.

Classified as benign central nervous system (CNS) tumors, pituitary adenomas account for 10% of diagnosed intracranial neoplasms. Although surgery is often curative, patients with invasive macroadenomas continue to experience significant morbidity and are prone to tumor recurrence. Given the identification of human brain tumor-initiating cells (TICs) that initiate and maintain tumor growth while promoting disease progression and relapse in multiple CNS tumors, we investigated whether TICs also drive the growth of human pituitary adenomas. Using a nanoString-based 80-gene custom codeset specific for developmental pathways, we identified a differential stem cell gene expression profile within human pituitary adenomas. Prospective functional characterization of stem cell properties in patient-derived adenomas representing all hormonal subtypes yielded a subtype-dependent self-renewal profile, which was enriched within the CD15+ cell fraction. The tumor-initiating capacity of CD15high adenoma cells was assayed in comparison to CD15low adenomas using in vivo limiting dilutions, which maintained the rare frequency of TICs. Repeated analyses using sorted cell populations for CD15+ TICs compared to CD15- adenoma cells provided further evidence of xenograft tumor formation to support CD15+ cells as putative pituitary adenoma-initiating cells (PAICs). The clinical utility of our findings was established through in silico analyses and comparative gene expression profiling of primary and recurrent pituitary adenomas. CD15 was enriched in recurrent adenomas, which was validated using routine clinical immunohistochemistry in a limited number of samples. Our work reports the first prospective identification of human PAICs using CD15. Patients with CD15high adenomas may therefore benefit from more aggressive surgical interventions and chemo/radiotherapy.

Pyrvinium Targets CD133 in Human Glioblastoma Brain Tumor-Initiating Cells.

PURPOSE: Clonal evolution of cancer may be regulated by determinants of stemness, specifically self-renewal, and current therapies have not considered how genetic perturbations or properties of stemness affect such functional processes. Glioblastoma-initiating cells (GICs), identified by expression of the cell surface marker CD133, are shown to be chemoradioresistant. In the current study, we sought to elucidate the functional role of CD133 in self-renewal and identify compounds that can specifically target this CD133(+) treatment-refractory population. EXPERIMENTAL DESIGN: Using gain/loss-of-function studies for CD133 we assessed the in vitro self-renewal and in vivo tumor formation capabilities of patient-derived glioblastoma cells. We generated a CD133 signature combined with an in silico screen to find compounds that target GICs. Self-renewal and proliferation assays on CD133-sorted samples were performed to identify the preferential action of hit compounds. In vivo efficacy of the lead compound pyrvinium was assessed in intracranial GIC xenografts and survival studies. Lastly, microarray analysis was performed on pyrvinium-treated GICs to discover core signaling events involved. RESULTS: We discovered pyrvinium, a small-molecule inhibitor of GIC self-renewal in vitro and in vivo, in part through inhibition of Wnt/ß-catenin signaling and other essential stem cell regulatory pathways. We provide a therapeutically tractable strategy to target self-renewing, chemoradioresistant, and functionally important CD133(+) stem cells that drive glioblastoma relapse and mortality. CONCLUSIONS: Our study provides an integrated approach for the eradication of clonal populations responsible for cancer progression, and may apply to other aggressive and heterogeneous cancers.

A cancer stem cell model for studying brain metastases from primary lung cancer.

BACKGROUND: Brain metastases are most common in adults with lung cancer, predicting uniformly poor patient outcome, with a median survival of only months. Despite their frequency and severity, very little is known about tumorigenesis in brain metastases. METHODS: We applied previously developed primary solid tumor-initiating cell models to the study of brain metastases from the lung to evaluate the presence of a cancer stem cell population. Patient-derived brain metastases (n = 20) and the NCI-H1915 cell line were cultured as stem-enriching tumorspheres. We used in vitro limiting-dilution and sphere-forming assays, as well as intracranial human-mouse xenograft models. To determine genes overexpressed in brain metastasis tumorspheres, we performed comparative transcriptome analysis. All statistical analyses were two-sided. RESULTS: Patient-derived brain metastasis tumorspheres had a mean sphere-forming capacity of 33 spheres/2000 cells (SD = 33.40) and median stem-cell frequency of 1/60 (range = 0-1/141), comparable to that of primary brain tumorspheres (P = .53 and P = .20, respectively). Brain metastases also expressed CD15 and CD133, markers suggestive of a stemlike population. Through intracranial xenotransplantation, brain metastasis tumorspheres were found to recapitulate the original patient tumor heterogeneity. We also identified several genes overexpressed in brain metastasis tumorspheres as statistically significant predictors of poor survival in primary lung cancer. CONCLUSIONS: For the first time, we demonstrate the presence of a stemlike population in brain metastases from the lung. We also show that NCI-H1915 tumorspheres could be useful in studying self-renewal and tumor initiation in brain metastases. Our candidate genes may be essential to metastatic stem cell populations, where pathway interference may be able to transform a uniformly fatal disease into a more localized and treatable one.

Bmi1 marks intermediate precursors during differentiation of human brain tumor initiating cells.

The master regulatory gene Bmi1 modulates key stem cell properties in neural precursor cells (NPCs), and has been implicated in brain tumorigenesis. We previously identified a population of CD133+ brain tumor cells possessing stem cell properties, known as brain tumor initiating cells (BTICs). Here, we characterize the expression and role of Bmi1 in primary minimally cultured human glioblastoma (GBM) patient isolates in CD133+ and CD133- sorted populations. We find that Bmi1 expression is increased in CD133- cells, and Bmi1 protein and transcript expression are highest during intermediate stages of differentiation as CD133+ BTICs lose their CD133 expression. Furthermore, in vitro stem cell assays and Bmi1 knockdown show that Bmi1 contributes to self-renewal in CD133+ populations, but regulates proliferation and cell fate determination in CD133- populations. Finally, we test if our in vitro stem cell assays and Bmi1 expression in BTIC patient isolates are predictive of clinical outcome for GBM patients. Bmi1 expression profiles show a marked elevation in the proneural GBM subtype, and stem cell frequency as assessed by tumor sphere assays correlates with patient outcome.