Nadejda M. Tsankova

Phenotypic Heterogeneity and Plasticity of Isocortical and Hippocampal Astrocytes in the Human Brain

To examine the diversity of astrocytes in the human brain, we immunostained surgical specimens of temporal cortex and hippocampus and autopsy brains for CD44, a plasma membrane protein and extracellular matrix receptor. CD44 antibodies outline the details of astrocyte morphology to a degree not possible with glial fibrillary acidic protein (GFAP) antibodies. CD44+ astrocytes could be subdivided into two groups. First, CD44+ astrocytes with long processes were consistently found in the subpial area (“interlaminar” astrocytes), the deep isocortical layers, and the hippocampus. Many of these processes ended on blood vessels. Some were also found adjacent to large blood vessels, from which they extended long processes. We observed these CD44+, long-process astrocytes in every brain we examined, from fetal to adult. These astrocytes generally displayed high immunostaining for GFAP, S100β, and CD44, but low immunostaining for glutamine synthetase, excitatory amino-acid transporter 1 (EAAT1), and EAAT2. Aquaporin 4 (AQP4) appeared distributed all over the cell bodies and processes of the CD44+ astrocytes, while, in contrast, AQP4 localized to perivascular end feet in the CD44− protoplasmic astrocytes. Second, there were CD44+ astrocytes without long processes in the cortex. These were not present during gestation or at birth, and in adult brains varied substantially in number, shape, and immunohistochemical phenotype. Many of these displayed a “mixed” morphological and immunocytochemical phenotype between protoplasmic and fibrous astrocytes. We conclude that the diversity of astrocyte populations in the isocortex and archicortex in the human brain reflects both intrinsic and acquired phenotypes, the latter perhaps representing a shift from CD44− “protoplasmic” to CD44+ “fibrous”-like astrocytes.

A qPCR ScoreCard quantifies the differentiation potential of human pluripotent stem cells.

Research on human pluripotent stem cells has been hampered by the lack of a standardized, quantitative, scalable assay of pluripotency. We previously described an assay called ScoreCard that used gene expression signatures to quantify differentiation efficiency. Here we report an improved version of the assay based on qPCR that enables faster, more quantitative assessment of functional pluripotency. We provide an in-depth characterization of the revised signature panel (commercially available as the TaqMan hPSC Scorecard Assay) through embryoid body and directed differentiation experiments as well as a detailed comparison to the teratoma assay. We further show that the improved ScoreCard enables a wider range of applications, such as screening of small molecules, genetic perturbations and assessment of culture conditions. Our approach can be extended beyond stem cell applications to characterize and assess the utility of other cell types and lineages.

Advances in genetic and epigenetic analyses of gliomas: a neuropathological perspective.

Gliomas, the most common malignant primary brain tumors, are universally fatal once they progress from low-grade into high-grade neoplasms. In recent years, we have accumulated unprecedented data about the genetic and epigenetic abnormalities in gliomas; yet, our appreciation of how these deadly tumors arise is still rudimentary. One of the major deterrents in understanding gliomagenesis is the remarkably complex and heterogeneous molecular composition of gliomas, as well as their ability to change phenotypically as they progress and recur. In the past decade, several monumental studies have begun to define better glioma heterogeneity. Four distinct molecular subgroups have emerged: proneural, classical, mesenchymal, and neural; which have unique gene expression signatures and prognostic significance. Of these, gliomas of the proneural subtype, which encompass most grade II/III diffuse gliomas and secondary glioblastomas and often carry isocitrate dehydrogenase (IDH) mutations, have emerged as a distinct tumor subclass with a notably superior prognosis. Important molecular markers with prognostic relevance, such as mutant IDH1/2, have already been incorporated into clinical neuropathological practice. The recent molecular discoveries in gliomas have also emphasized the intimate link between epigenetics and genetics in gliomagenesis. Several of the novel genetic mutations described are responsible for distinct epigenetic remodeling in gliomas, the mechanisms of which are currently being elucidated. Importantly, these epigenetic and genomic alterations represent new and exciting drug targets for future therapeutic interventions in our continuous fight with this fatal malignancy.

Emerging interplay of genetics and epigenetics in gliomas: a new hope for targeted therapy.

Diffusely infiltrating gliomas are inherently heterogeneous tumors, and there are ongoing efforts to establish a classification scheme that incorporates new molecular and traditional histologic features. In less than a decade, high-throughput sequencing of gliomas has transformed the field, uncovering several pivotal, highly prevalent genetic alterations that stratify patients into different prognostic and treatment-response categories. We highlight the genetic aberrations recently discovered in isocitrate dehydrogenase, alpha thalassemia/mental retardation syndrome X-linked, death-domain-associated protein, histone H3.3, and telomerase reverse transcriptase and discuss how these mutations lead to unexpected changes in the epigenetic landscape in gliomas. We describe the opportunities these discoveries might provide for the development of novel targeted therapy aimed at reversing early epigenetic aberrations in glioma precursor cells. Finally, we discuss the challenges for effective treatment of this fatal disease posed by intratumoral heterogeneity and clonal evolution.

EGFR promoter exhibits dynamic histone modifications and binding of ASH2L and P300 in human germinal matrix and gliomas

Several signaling pathways important for the proliferation and growth of brain cells are pathologically dysregulated in gliomas, including the epidermal growth factor receptor (EGFR). Expression of EGFR is high in neural progenitors during development and in gliomas but decreases significantly in most adult brain regions. Here we show that EGFR expression is maintained in the astrocyte ribbon of the adult human subventricular zone. The transcriptional regulation of EGFR expression is poorly understood. To investigate the role of epigenetics on EGFR regulation in the contexts of neural development and gliomagenesis, we measured levels of DNA methylation and histone H3 modifications at the EGFR promoter in human brain tissues, glioma specimens, and EGFR-expressing neural cells, acutely isolated from their native niche. While DNA was constitutively hypomethylated in non-neoplastic and glioma samples, regardless of their EGFR-expression status, the activating histone modifications H3K27ac and H3K4me3 were enriched only when EGFR is highly expressed (developing germinal matrix and gliomas). Conversely, repressive H3K27me3 marks predominated in adult white matter where EGFR is repressed. Furthermore, the histone methyltransferase core enzyme ASH2L was bound at EGFR in the germinal matrix and in gliomas where levels of H3K4me3 are high, and the histone acetyltransferase P300 was bound in samples with H3K27ac enrichment. Our studies use human cells and tissues undisturbed by cell-culture artifact, and point to an important, locus-specific role for chromatin remodeling in EGFR expression in human neural development that may be dysregulated during gliomagenesis, unraveling potential novel targets for future drug therapy.

Multinodular and vacuolating neuronal tumor of the cerebrum is a clonal neoplasm defined by genetic alterations that activate the MAP kinase signaling pathway

Author(s): Pekmezci, Melike; Stevers, Meredith; Phillips, Joanna J; Van Ziffle, Jessica; Bastian, Boris C; Tsankova, Nadejda M; Kleinschmidt-DeMasters, Bette K; Rosenblum, Marc K; Tihan, Tarik; Perry, Arie; Solomon, David A

Prospective Isolation and Comparison of Human Germinal Matrix and Glioblastoma EGFR+ Populations with Stem Cell Properties

Summary Characterization of non-neoplastic and malignant human stem cell populations in their native state can provide new insights into gliomagenesis. Here we developed a purification strategy to directly isolate EGFR+/− populations from human germinal matrix (GM) and adult subventricular zone autopsy tissues, and from de novo glioblastoma (GBM) resections, enriching for cells capable of binding EGF ligand (LBEGFR+), and uniquely compared their functional and molecular properties. LBEGFR+ populations in both GM and GBM encompassed all sphere-forming cells and displayed proliferative stem cell properties in vitro. In xenografts, LBEGFR+ GBM cells showed robust tumor initiation and progression to high-grade, infiltrative gliomas. Whole-transcriptome sequencing analysis confirmed enrichment of proliferative pathways in both developing and neoplastic freshly isolated EGFR+ populations, and identified both unique and shared sets of genes. The ability to prospectively isolate stem cell populations using native ligand-binding capacity opens new doors onto understanding both normal human development and tumor cell biology.

Double minute amplification of mutant PDGF receptor α in a mouse glioma model

In primary brain tumors, oncogenes are frequently amplified and maintained on extrachromosomal DNA as double minutes (DM), but the underlying mechanisms remain poorly understood. We have generated a mouse model of malignant glioma based on knock-in of a mutant PDGF receptor α (PDGFRα) that is expressed in oligodendrocyte precursor cells (OPCs) after activation by a Cre recombinase. In the tumor suppressor INK4/Arf(-/-) background, mutant animals frequently developed brain tumors resembling anaplastic human gliomas (WHO grade III). Besides brain tumors, most animals also developed aggressive fibrosarcomas, likely triggered by Cre activation of mutant PDGFRα in fibroblastic cell lineages. Importantly, in the brain tumors and cell lines derived from brain tumor tissues, we identified a high prevalence of DM Pdgfra gene amplification, suggesting its occurrence as an early mutational event contributing to the malignant transformation of OPCs. Amplicons extended beyond the Pdgfra locus and included in some cases neighboring genes Kit and Kdr. Our genetically defined mouse brain tumor model therefore supports OPC as a cell of origin for malignant glioma and offers an example of a defined temporal sequence of mutational events, thus providing an entry point for a mechanistic understanding of DM gene amplification and its functionality in gliomagenesis.

BK virus encephalopathy and sclerosing vasculopathy in a patient with hypohidrotic ectodermal dysplasia and immunodeficiency

Human BK polyomavirus (BKV) is reactivated under conditions of immunosuppression leading most commonly to nephropathy or cystitis; its tropism for the brain is rare and poorly understood. We present a unique case of BKV-associated encephalopathy in a man with hypohidrotic ectodermal dysplasia and immunodeficiency (HED-ID) due to IKK-gamma (NEMO) mutation, who developed progressive neurological symptoms. Brain biopsy demonstrated polyomavirus infection of gray and white matter, with predominant involvement of cortex and distinct neuronal tropism, in addition to limited demyelination and oligodendroglial inclusions. Immunohistochemistry demonstrated polyoma T-antigen in neurons and glia, but expression of VP1 capsid protein only in glia. PCR analysis on both brain biopsy tissue and cerebrospinal fluid detected high levels of BKV DNA. Sequencing studies further identified novel BKV variant and disclosed unique rearrangements in the noncoding control region of the viral DNA (BKVN NCCR). Neuropathological analysis also demonstrated an unusual form of obliterative fibrosing vasculopathy in the subcortical white matter with abnormal lysosomal accumulations, possibly related to the patient’s underlying ectodermal dysplasia. Our report provides the first neuropathological description of HED-ID due to NEMO mutation, and expands the diversity of neurological presentations of BKV infection in brain, underscoring the importance of its consideration in immunodeficient patients with unexplained encephalopathy. We also document novel BKVN NCCR rearrangements that may be associated with the unique neuronal tropism in this patient.

Biphasic IDH1 phenotype in a diffusely infiltrating glioma: implications for pathogenesis, treatment and prognosis.

Recent studies suggest isocitrate dehydrogenase 1 (IDH1) mutations are early events in gliomagenesis, given their frequent occurrence in low-grade gliomas, diffuse expression within neoplastic cells, and lack of evidence for preceding TP53 mutations or 1p/19q co-deletion. We present an infiltrating glioma with mixed oligoastroglial morphology and biphasic molecular phenotype. Areas resembling oligodendroglioma by histology expressed mutant IDH1-R132H, and strong ATRX, Olig2, and PDGFR-α by immunohistochemistry. In contrast, astrocytic areas completely lacked the IDH1-R132H mutation, showed loss of nuclear ATRX expression, and only weakly expressed Olig2 and PDGFR-α. Co-deletion of 1p/19q was evident throughout, while p53 expression was largely negative. This case suggests that 1p/19q co-deletion may rarely precede IDH1 mutations or that IDH1 mutations may be secondarily lost, as demonstrated by IDH1-R132H positive and negative cells in a glioma with diffuse 1p/19q co-deletion. The uniquely biphasic molecular phenotype of this tumor supports the rare existence of true mixed oligoastrocytomas that may have significant prognostic and therapeutic implications. The case highlights the variable sequence of key molecular aberrations in gliomagenesis and the difficulty of targeting treatment to genetic profiles in inherently heterogeneous neoplasms.