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1.  BRAF-V600E mutation in pediatric and adult glioblastoma 
Neuro-Oncology  2013;16(2):318-319.
doi:10.1093/neuonc/not146
PMCID: PMC3895374  PMID: 24311634
brain tumor; children; glioma; microarray
2.  A sensitive and specific histopathologic prognostic marker for H3F3A K27M mutant pediatric glioblastomas 
Acta Neuropathologica  2014;128(5):743-753.
Pediatric glioblastomas (GBM) are highly aggressive and lethal tumors. Recent sequencing studies have shown that ~30 % of pediatric GBM and ~80 % of diffuse intrinsic pontine gliomas show K27M mutations in the H3F3A gene, a variant encoding histone H3.3. H3F3A K27M mutations lead to global reduction in H3K27me3. Our goal was to develop biomarkers for the histopathologic detection of these tumors. Therefore, we evaluated the utility of measuring H3K27me3 global reduction as a histopathologic and prognostic biomarker and tested an antibody directed specifically against the H3.3 K27M mutation in 290 samples. The study cohort included 203 pediatric (including 38 pediatric high-grade astrocytomas) and 38 adult brain tumors of various subtypes and grades and 49 non-neoplastic reactive brain tissues. Detection of H3.3 K27M by immunohistochemistry showed 100 % sensitivity and specificity and was superior to global reduction in H3K27me3 as a biomarker in diagnosing H3F3A K27M mutations. Moreover, cases that stained positive for H3.3 K27M showed a significantly poor prognosis compared to corresponding negative tumors. These results suggest that immunohistochemical detection of H3.3 K27M is a sensitive and specific surrogate for the H3F3A K27M mutation and defines a prognostically poor subset of pediatric GBM.
Electronic supplementary material
The online version of this article (doi:10.1007/s00401-014-1338-3) contains supplementary material, which is available to authorized users.
doi:10.1007/s00401-014-1338-3
PMCID: PMC4201755  PMID: 25200322
Pediatric glioblastoma; H3F3A mutation; K27M mutation; H3K27me3; Diagnostic biomarker; Prognosis; Methylation; Epigenetics
3.  PDGFRA amplification is common in pediatric and adult high-grade astrocytomas and identifies a poor prognostic group in IDH1 mutant glioblastoma 
High-grade astrocytomas (HGAs), corresponding to WHO grades III (AA) and IV (GBM), are biologically aggressive and their molecular classification is increasingly relevant to clinical management. PDGFRA amplification is common in HGAs, although its prognostic significance remains unclear. Using fluorescence in situ hybridization (FISH), the most sensitive technique for detecting PDGFRA copy number gains, we determined PDGFRA amplification status in 123 pediatric and 263 adult HGAs. A range of PDGFRA FISH patterns were identified and cases were scored as non-amplified (normal and polysomy) or amplified (low-level and high-level). PDGFRA amplification was frequent in pediatric (29.3%) and adult (20.9%) tumors. Amplification was not prognostic in pediatric HGAs. In adult tumors diagnosed initially as GBM, the presence of combined PDGFRA amplification and IDH1R132H mutation was a significant independent prognostic factor (p=0.01). In HGAs, PDGFRA amplification is common and can manifest as high-level and focal or low-level amplifications. Our data indicate that the latter is more prevalent than previously reported with copy number averaging techniques. To our knowledge, this is the largest survey of PDGFRA status in adult and pediatric HGAs and suggests PDGFRA amplification increases with grade and is associated with a less favorable prognosis in IDH1 mutant de novo GBMs.
doi:10.1111/bpa.12043
PMCID: PMC3715570  PMID: 23438035
PDGFRA; astrocytoma; FISH; IDH1; isocitrate dehydrogenase 1; prognosis
4.  Pituitary blastoma: a pathognomonic feature of germ-line DICER1 mutations 
Acta neuropathologica  2014;128(1):111-122.
Individuals harboring germ-line DICER1 mutations are predisposed to a rare cancer syndrome, the DICER1 Syndrome or pleuropulmonary blastoma-familial tumor and dysplasia syndrome [online Mendelian inheritance in man (OMIM) #601200]. In addition, specific somatic mutations in the DICER1 RNase III catalytic domain have been identified in several DICER1-associated tumor types. Pituitary blastoma (PitB) was identified as a distinct entity in 2008, and is a very rare, potentially lethal early childhood tumor of the pituitary gland. Since the discovery by our team of an inherited mutation in DICER1 in a child with PitB in 2011, we have identified 12 additional PitB cases. We aimed to determine the contribution of germ-line and somatic DICER1 mutations to PitB. We hypothesized that PitB is a pathognomonic feature of a germ-line DICER1 mutation and that each PitB will harbor a second somatic mutation in DICER1. Lymphocyte or saliva DNA samples ascertained from ten infants with PitB were screened and nine were found to harbor a heterozygous germ-line DICER1 mutation. We identified additional DICER1 mutations in nine of ten tested PitB tumor samples, eight of which were confirmed to be somatic in origin. Seven of these mutations occurred within the RNase IIIb catalytic domain, a domain essential to the generation of 5p miRNAs from the 5′ arm of miRNA-precursors. Germ-line DICER1 mutations are a major contributor to PitB. Second somatic DICER1 “hits” occurring within the RNase IIIb domain also appear to be critical in PitB pathogenesis.
doi:10.1007/s00401-014-1285-z
PMCID: PMC4129448  PMID: 24839956
DICER1; Pituitary blastoma; miRNA; Pediatric tumors
6.  CNS-PNETs with C19MC amplification and/or LIN28 expression comprise a distinct histogenetic diagnostic and therapeutic entity 
Acta Neuropathologica  2014;128(2):291-303.
Amplification of the C19MC oncogenic miRNA cluster and high LIN28 expression has been linked to a distinctly aggressive group of cerebral CNS-PNETs (group 1 CNS-PNETs) arising in young children. In this study, we sought to evaluate the diagnostic specificity of C19MC and LIN28, and the clinical and biological spectra of C19MC amplified and/or LIN28+ CNS-PNETs. We interrogated 450 pediatric brain tumors using FISH and IHC analyses and demonstrate that C19MC alteration is restricted to a sub-group of CNS-PNETs with high LIN28 expression; however, LIN28 immunopositivity was not exclusive to CNS-PNETs but was also detected in a proportion of other malignant pediatric brain tumors including rhabdoid brain tumors and malignant gliomas. C19MC amplified/LIN28+ group 1 CNS-PNETs arose predominantly in children <4 years old; a majority arose in the cerebrum but 24 % (13/54) of tumors had extra-cerebral origins. Notably, group 1 CNS-PNETs encompassed several histologic classes including embryonal tumor with abundant neuropil and true rosettes (ETANTR), medulloepithelioma, ependymoblastoma and CNS-PNETs with variable differentiation. Strikingly, gene expression and methylation profiling analyses revealed a common molecular signature enriched for primitive neural features, high LIN28/LIN28B and DNMT3B expression for all group 1 CNS-PNETs regardless of location or tumor histology. Our collective findings suggest that current known histologic categories of CNS-PNETs which include ETANTRs, medulloepitheliomas, ependymoblastomas in various CNS locations, comprise a common molecular and diagnostic entity and identify inhibitors of the LIN28/let7/PI3K/mTOR axis and DNMT3B as promising therapeutics for this distinct histogenetic entity.
Electronic supplementary material
The online version of this article (doi:10.1007/s00401-014-1291-1) contains supplementary material, which is available to authorized users.
doi:10.1007/s00401-014-1291-1
PMCID: PMC4159569  PMID: 24839957
7.  Histone 3 Lysine 9 Trimethylation Is Differentially Associated with Isocitrate Dehydrogenase Mutations in Oligodendrogliomas and High-Grade Astrocytomas 
Trimethylation of histone 3 lysine 9 (H3K9me3) is a marker of repressed transcription. Cells transfected with mutant isocitrate dehydrogenase (IDH) show increased methylation of histone lysine residues, including H3K9me3, due to inhibition of histone demethylases by 2-hydroxyglutarate. Here, we evaluated H3K9me3 and its association with IDH mutations in 284 gliomas. H3K9me3 was significantly associated with IDH mutations in oligodendrogliomas. Moreover, 72% of World Health Organization grade II and 65% of grade III oligodendrogliomas showed combined H3K9me3 positivity and 1p19q co-deletion. In astrocytic tumors, H3K9me3 positivity was found in all grades of tumors; it showed a significant relationship with IDH mutational status in grade II astrocytomas but not in grade III astrocytomas or glioblastomas. Finally, H3K9me3-positive grade II oligodendrogliomas but not other tumor subtypes showed improved overall survival compared to H3K9me3-negative cases. These results suggest that repressive trimethylation of H3K9 in gliomas may occur in a context-dependent manner and is associated with IDH mutations in oligodendrogliomas, but may be differently regulated in high-grade astrocytic tumors. Further, H3K9me3 may define a subset of grade II oligodendrogliomas with better overall survival. Our results suggest variable roles for IDH mutations in the pathogenesis of oligodendrogliomas vs. astrocytic tumors.
doi:10.1097/NEN.0b013e3182898113
PMCID: PMC3615673  PMID: 23481705
Astrocytoma; Glioma; H3K9me3; Histone methylation; Isocitrate dehydrogenase (IDH); Oligodendroglioma
8.  Embryonal tumor with abundant neuropil and true rosettes (ETANTR), ependymoblastoma, and medulloepithelioma share molecular similarity and comprise a single clinicopathological entity 
Acta Neuropathologica  2013;128(2):279-289.
Three histological variants are known within the family of embryonal rosette-forming neuroepithelial brain tumors. These include embryonal tumor with abundant neuropil and true rosettes (ETANTR), ependymoblastoma (EBL), and medulloepithelioma (MEPL). In this study, we performed a comprehensive clinical, pathological, and molecular analysis of 97 cases of these rare brain neoplasms, including genome-wide DNA methylation and copy number profiling of 41 tumors. We identified uniform molecular signatures in all tumors irrespective of histological patterns, indicating that ETANTR, EBL, and MEPL comprise a single biological entity. As such, future WHO classification schemes should consider lumping these variants into a single diagnostic category, such as embryonal tumor with multilayered rosettes (ETMR). We recommend combined LIN28A immunohistochemistry and FISH analysis of the 19q13.42 locus for molecular diagnosis of this tumor category. Recognition of this distinct pediatric brain tumor entity based on the fact that the three histological variants are molecularly and clinically uniform will help to distinguish ETMR from other embryonal CNS tumors and to better understand the biology of these highly aggressive and therapy-resistant pediatric CNS malignancies, possibly leading to alternate treatment strategies.
doi:10.1007/s00401-013-1228-0
PMCID: PMC4102829  PMID: 24337497
9.  Identification of Gene Markers Associated with Aggressive Meningioma by Filtering across Multiple Sets of Gene Expression Arrays 
Journal of neuropathology and experimental neurology  2011;70(1):10.1097/NEN.0b013e3182018f1c.
Meningiomasare common intracranial tumors but relatively little is known about the genetic events responsible for their clinical diversity. While recent genomic studies have provided clues, the genes identified often differ among publications. We used microarray expression profiling to identify genes that are differentially expressed, with at least a 4-fold change, between grade I and grade III meningiomas. We filtered this initial set of potential biomarkers through a second cohort of meningiomas and then verified the remaining genes by quantitative polymerase chain reaction followed by examination using a third microarray expression cohort. Using this approach, we identified 9 overexpressed (TPX2, RRM2, TOP2A, PI3, BIRC5, CDC2, NUSAP1, DLG7, SOX11) and 2 underexpressed (TIMP3, KCNMA1) genes in grade III vs. grade I meningiomas. As a further validation step, we analyzed these genes in a fourth cohort and found that patients with grade II meningiomas with high topoisomerase 2-α protein expression (greater than 5% labeling-index) had shorter times to death than patients with low expression. We believe that this multistep, multi-cohort approach provides a robust method for reducing false positives while generating a list of reproducible candidate genes that are associated with clinically aggressive meningioma and are suitable for analysis for their potential prognostic value.
doi:10.1097/NEN.0b013e3182018f1c
PMCID: PMC3839953  PMID: 21157382
Gene expression; Immunohistochemistry; Malignancy; Meningioma; Microarray; Quantitative RT-PCR
10.  A Novel Murine Model for Localized Radiation Necrosis and its Characterization using Advanced Magnetic Resonance Imaging 
International journal of radiation oncology, biology, physics  2009;75(2):10.1016/j.ijrobp.2009.06.007.
Introduction
Magnetic resonance (MR) images following external beam radiotherapy for brain tumors often display signal changes characteristic of either tumor progression and/or radiation injury. No non-invasive diagnostic biomarkers have been identified that clearly distinguish between these two disease processes. This study’s objective was to develop a murine model of radiation necrosis using fractionated, sub-total cranial irradiation and to investigate the imaging signature of radiation-induced tissue damage using advanced MR imaging techniques.
Methods
Twenty four mice each received 60 Gy of hemispheric (left) irradiation in ten equal fractions. MR images at 4.7 T were subsequently collected using T1-, T2- and diffusion-sequences at selected time points following irradiation or implantation. Following imaging, animals were euthanized and their brains were fixed for correlative histology.
Results
Contrast-enhanced T1- and T2-weighted MR images at months 2, 3, and 4 showed changes consistent with progressive radiation necrosis. Quantitatively, mean diffusivity was significantly higher (mean = 0.86, 1.13, and 1.24 μm2/ms at 2, 3, and 4 months, respectively) in radiated brain, compared with contralateral untreated brain tissue (mean = 0.78, 0.82, and 0.83 μm2/ms) (p<0.0001). Histology reflected changes typically seen in radiation necrosis.
Conclusions
This murine model of radiation necrosis will facilitate investigation of imaging biomarkers that distinguish between radiation necrosis and tumor recurrence. In addition, this preclinical study supports clinical data suggesting that DWI may be helpful in answering this diagnostic question in clinical settings.
doi:10.1016/j.ijrobp.2009.06.007
PMCID: PMC3808839  PMID: 19735877
Radiation necrosis; Conformal radiation; Animal models; Magnetic Resonance Imaging; Brain tumor
11.  Gene expression profile identifies tyrosine kinase c-Met as a targetable mediator of anti-angiogenic therapy resistance 
Purpose
To identify mediators of glioblastoma anti-angiogenic therapy resistance and target these mediators in xenografts.
Experimental Design
We performed microarray analysis comparing bevacizumab-resistant glioblastomas (BRGs) to pre-treatment tumors from the same patients. We established novel xenograft models of anti-angiogenic therapy resistance to target candidate resistance mediator(s).
Results
BRG microarray analysis revealed upregulation versus pre-treatment of receptor tyrosine kinase c-Met, which underwent further investigation because of its prior biologic plausibility as a bevacizumab resistance mediator. BRGs exhibited increased hypoxia versus pre-treatment in a manner correlating with their c-Met upregulation, increased c-Met phosphorylation, and increased phosphorylation of c-Met-activated focal adhesion kinase (FAK) and STAT3. We developed two novel xenograft models of anti-angiogenic therapy resistance. In the first model, serial bevacizumab treatment of an initially responsive xenograft generated a xenograft with acquired bevacizumab resistance, which exhibited upregulated c-Met expression versus pre-treatment. In the second model, a BRG-derived xenograft maintained refractoriness to the MRI tumor vasculature alterations and survival-promoting effects of bevacizumab. Growth of this BRG-derived xenograft was inhibited by a c-Met inhibitor. Transducing these xenograft cells with c-Met shRNA inhibited their invasion and survival in hypoxia, disrupted their mesenchymal morphology, and converted them from bevacizumab-resistant to bevacizumab-responsive. Engineering bevacizumab-responsive cells to express constitutively active c-Met caused these cells to form bevacizumab-resistant xenografts.
Conclusion
These findings support the role of c-Met in survival in hypoxia and invasion, features associated with anti-angiogenic therapy resistance; and growth and therapeutic resistance of xenografts resistant to anti-angiogenic therapy. Therapeutically targeting c-Met could prevent or overcome anti-angiogenic therapy resistance.
doi:10.1158/1078-0432.CCR-12-1281
PMCID: PMC3618605  PMID: 23307858
bevacizumab; resistance; glioblastoma; invasion; c-Met
12.  Early recurrence in standard-risk medulloblastoma patients with the common idic(17)(p11.2) rearrangement 
Neuro-Oncology  2012;14(7):831-840.
Medulloblastoma is diagnosed histologically; treatment depends on staging and age of onset. Whereas clinical factors identify a standard- and a high-risk population, these findings cannot differentiate which standard-risk patients will relapse and die. Outcome is thought to be influenced by tumor subtype and molecular alterations. Poor prognosis has been associated with isochromosome (i)17q in some but not all studies. In most instances, molecular investigations document that i17q is not a true isochromosome but rather an isodicentric chromosome, idic(17)(p11.2), with rearrangement breakpoints mapping within the REPA/REPB region on 17p11.2. This study explores the clinical utility of testing for idic(17)(p11.2) rearrangements using an assay based on fluorescent in situ hybridization (FISH). This test was applied to 58 consecutive standard- and high-risk medulloblastomas with a 5-year minimum of clinical follow-up. The presence of i17q (ie, including cases not involving the common breakpoint), idic(17)(p11.2), and histologic subtype was correlated with clinical outcome. Overall survival (OS) and disease-free survival (DFS) were consistent with literature reports. Fourteen patients (25%) had i17q, with 10 (18%) involving the common isodicentric rearrangement. The presence of i17q was associated with a poor prognosis. OS and DFS were poor in all cases with anaplasia (4), unresectable disease (7), and metastases at presentation (10); however, patients with standard-risk tumors fared better. Of these 44 cases, tumors with idic(17)(p11.2) were associated with significantly worse patient outcomes and shorter mean DFS. FISH detection of idic(17)(p11.2) may be useful for risk stratification in standard-risk patients. The presence of this abnormal chromosome is associated with early recurrence of medulloblastoma.
doi:10.1093/neuonc/nos086
PMCID: PMC3379796  PMID: 22573308
FISH; idic(17)(p11.2); i17q; medulloblastoma; pediatric oncology
13.  Isocitrate Dehydrogenase 1 Analysis Differentiates Gangliogliomas from Infiltrative Gliomas 
Recent work has identified novel point mutations in isocitrate dehydrogenase 1 (IDH1) in the majority of the World Health Organization grades II and III infiltrative gliomas and secondary grade IV glioblastomas. Gangliogliomas consist of neoplastic ganglion and glial cells and, in contrast to infiltrative gliomas, are generally indolent. Yet distinguishing between a ganglioglioma and an infiltrative glioma with admixed gray matter can be difficult, perhaps accounting for some “gangliogliomas” that ultimately show aggressive behavior. In this multi-institutional study, 98 cases originally diagnosed as ganglioglioma were analyzed for IDH1 mutations, 86 of which had follow-up data available. Eight cases (8.2%) were positive for R132H IDH1 mutations; six had silent IDH2 mutations and two had nonsense IDH2 mutations. The presence of mutant IDH1 in gangliogliomas correlated with a greater risk of recurrence (P = 0.0007) and malignant transformation and/or death (P < 0.0001) compared with tumors that were IDH1 wild type. Furthermore, the age of patients with IDH1-mutant gangliogliomas was higher than those without mutations (25.5 vs. 46.1 years, P = 0.0033). IDH1/2 testing of tumors suspected of being gangliogliomas may therefore be advisable, particularly in the adult population.
doi:10.1111/j.1750-3639.2011.00480.x
PMCID: PMC3681126  PMID: 21314850
ganglioglioma; glioneuronal; isocitrate dehydrogenase
14.  Distinct Neural Stem Cell Populations Give Rise to Disparate Brain Tumors in Response to N-MYC 
Cancer Cell  2012;21(5):601-613.
SUMMARY
The proto-oncogene MYCN is mis-expressed in various types of human brain tumors. To clarify how developmental and regional differences influence transformation, we transduced wild-type or mutationally-stabilized murine N-mycT58A into neural stem cells (NSCs) from perinatal murine cerebellum, brain stem and forebrain. Transplantation of N-mycWT NSCs was insufficient for tumor formation. N-mycT58A cerebellar and brain stem NSCs generated medulloblastoma/primitive neuroectodermal tumors, whereas forebrain NSCs developed diffuse glioma. Expression analyses distinguished tumors generated from these different regions, with tumors from embryonic versus postnatal cerebellar NSCs demonstrating SHH-dependence and SHH-independence, respectively. These differences were regulated in-part by the transcription factor SOX9, activated in the SHH subclass of human medulloblastoma. Our results demonstrate context-dependent transformation of NSCs in response to a common oncogenic signal.
doi:10.1016/j.ccr.2012.04.012
PMCID: PMC3360885  PMID: 22624711
N-MYC; GFAP; SOX9; medulloblastoma; neural stem cells; glioma
15.  Neuropathology of brain metastases 
Surgical Neurology International  2013;4(Suppl 4):S245-S255.
Metastatic tumors are the most common neoplasms encountered in the central nervous system (CNS), and continue to be major cause for mortality and morbidity. Macroscopic features and corresponding radiological findings can be diagnostic in majority of the cases, however, microscopic evaluation would be necessary when the differential diagnosis includes a primary CNS tumor, unknown primary tumor site, and when the resection of the tumor is either considered therapeutic or palliative. The first step in the diagnosis of a metastatic brain lesion is to exclude a primary CNS tumor, followed by verification or identification of the primary tumor and the site. Although general approach to a metastatic lesion from an unknown primary tumor is the same everywhere else, there are slight variations for the metastatic lesions in the CNS versus other regions. When morphological features are not enough to establish a definitive diagnosis, additional studies including immunohistochemical stains are applied. With the expending immunohistochemical armamentarium for pathologists, more accurate assessments are possible even in cases of unknown primary tumor. This review summarizes the diagnostic approach to CNS metastases, immunohistochemical assessment of neoplasm of unknown primary, and primary CNS lesions entering in the differential diagnosis of metastases.
doi:10.4103/2152-7806.111302
PMCID: PMC3656562  PMID: 23717796
Brain metastases; central nervous system metastases; metastasis
16.  Pathology of Peripheral Nerve Sheath Tumors: Diagnostic Overview and Update on Selected Diagnostic Problems 
Acta neuropathologica  2012;123(3):295-319.
Peripheral nerve sheath tumors are common neoplasms, with classic identifiable features, but on occasion, they are diagnostically challenging. Although well defined subtypes of peripheral nerve sheath tumors were described early in the history of surgical pathology, controversies regarding the classification and grading of these tumors persist. Advances in molecular biology have provided new insights into the nature of the various peripheral nerve sheath tumors, and have begun to suggest novel targeted therapeutic approaches. In this review we discuss current concepts and problematic areas in the pathology of peripheral nerve sheath tumors. Diagnostic criteria and differential diagnosis for the major categories of nerve sheath tumors are proposed, including neurofibroma, schwannoma, and perineurioma. Diagnostically challenging variants, including plexiform, cellular and melanotic schwannomas are highlighted. A subset of these affects the childhood population, and has historically been interpreted as malignant, although current evidence and outcome data suggests they represent benign entities. The growing current literature and the authors experience with difficult to classify borderline or “hybrid tumors” are discussed and illustrated. Some of these classification gray zones occur with frequency in the gastrointestinal tract, an anatomical compartment that must always be entertained when examining these neoplasms. Other growing recent areas of interest include the heterogeneous group of pseudoneoplastic lesions involving peripheral nerve composed of mature adipose tissue and/or skeletal muscle, such as the enigmatic neuromuscular choristoma. Malignant peripheral nerve sheath tumors (MPNST) represent a diagnostically controversial group; difficulties in grading and guidelines to separate “atypical neurofibroma” from MPNST are provided. There is an increasing literature of MPNST mimics which neuropathologists must be aware of, including synovial sarcoma and ossifying fibromyxoid tumor. Finally, we discuss entities that are lacking from the section on cranial and paraspinal nerves in the current WHO classification, and that may warrant inclusion in future classifications. In summary, although the diagnosis and classification of most conventional peripheral nerve sheath tumors are relatively straightforward for the experienced observer, borderline and difficult to classify neoplasms continue to be problematic. In the current review, we attempt to provide some useful guidelines for the surgical neuropathologist to help navigate these persistent, challenging problems.
doi:10.1007/s00401-012-0954-z
PMCID: PMC3629555  PMID: 22327363
peripheral nerve; neurofibroma; schwannoma; perineurioma; MPNST
17.  Clinical Stratification of Glioblastoma Based on Alterations in Retinoblastoma Tumor Suppressor Protein (RB1) and Association with the Proneural Subtype 
A recent study of CDK4/6-inhibitors in glioblastoma (GBM) xenografts identified retinoblastoma tumor suppressor protein RB1 status as a determinant of tumor therapeutic efficacy. Because of the need for clinically applicable RB1 testing, we assessed the utility of 2 complementary methods for determining RB1 status in GBM. Using fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), we analyzed 34 GBMs that had also undergone molecular characterization as part of The Cancer Genome Atlas (TCGA). By IHC, 4 tumors (11.8%) had complete loss of RB protein expression, including 2 with homozygous deletion of RB1 by FISH and 1 with hemizygous deletion of RB1 by FISH combined with a novel nonsense mutation in RB1. Consistent with these results, in an independent set of 51 GBMs tested by IHC we demonstrated loss of RB1 protein in 5 (9.8%). In GBM molecular subtype analysis of TCGA data, complete loss of RB1 transcript expression was seen in 18 of 170 tumors (10.6%) and these were highly enriched for, but not exclusive to, the proneural subtype (p < 0.01). These data support the use of IHC for determining RB1 status in clinical GBM specimens and suggest that RB1 alterations may be more common in certain GBM subgroups.
doi:10.1097/NEN.0b013e31823fe8f1
PMCID: PMC3246124  PMID: 22157621
Fluorescence in situ hybridization; Glioblastoma; Immunohistochemistry; Patient stratification; RB1; The Cancer Genome Atlas (TCGA)
18.  Medulloblastoma: clinicopathological correlates of SHH, WNT, and non-SHH/WNT molecular subgroups 
Acta neuropathologica  2011;121(3):381-396.
Medulloblastoma is heterogeneous, being characterized by molecular subgroups that demonstrate distinct gene expression profiles. Activation of the WNT or SHH signaling pathway characterizes two of these molecular subgroups, the former associated with low-risk disease and the latter potentially targeted by novel SHH pathway inhibitors. This manuscript reports the validation of a novel diagnostic immunohistochemical method to distinguish SHH, WNT, and non-SHH/WNT tumors and details their associations with clinical, pathological and cytogenetic variables. A cohort (n = 235) of medulloblastomas from patients aged 0.4–52 years was studied for expression of four immunohistochemical markers: GAB1, β-catenin, filamin A, and YAP1. Immunoreactivity (IR) for GAB1 characterizes only SHH tumors and nuclear IR for β-catenin only WNT tumors. IRs for filamin A and YAP1 identify SHH and WNT tumors. SHH, WNT, and non-SHH/WNT tumors contributed 31, 14, and 55% to the series. All desmoplastic/nodular (D/N) medulloblastomas were SHH tumors, while most WNT tumors (94%) had a classic phenotype. Monosomy 6 was strongly associated with WNT tumors, while PTCH1 loss occurred almost exclusively among SHH tumors. MYC or MYCN amplification and chromosome 17 imbalance occurred predominantly among non-SHH/WNT tumors. Among patients aged 3–16 years and entered onto the SIOP PNET3 trial, outcome was significantly better for children with WNT tumors, when compared to SHH or non-SHH/WNT tumors, which showed similar survival curves. However, high-risk factors (M+ disease, LC/A pathology, MYC amplification) significantly influenced survival in both SHH and non-SHH/WNT groups. We describe a robust method for detecting SHH, WNT, and non-SHH/WNT molecular subgroups in formalin-fixed medulloblastoma samples. In corroborating other studies that indicate the value of combining clinical, pathological, and molecular variables in therapeutic stratification schemes for medulloblastoma, we also provide the first outcome data based on a clinical trial cohort and novel data on how molecular subgroups are distributed across the range of disease.
doi:10.1007/s00401-011-0800-8
PMCID: PMC3519926  PMID: 21267586
19.  LIN28A immunoreactivity is a potent diagnostic marker of embryonal tumor with multilayered rosettes (ETMR) 
Acta Neuropathologica  2012;124(6):875-881.
Embryonal tumor with multilayered rosettes (ETMR, previously known as ETANTR) is a highly aggressive embryonal CNS tumor, which almost exclusively affects infants and is associated with a dismal prognosis. Accurate diagnosis is of critical clinical importance because of its poor response to current treatment protocols and its distinct biology. Amplification of the miRNA cluster at 19q13.42 has been identified previously as a genetic hallmark for ETMR, but an immunohistochemistry-based assay for clinical routine diagnostics [such as INI-1 for atypical teratoid rhabdoid tumor (AT/RT)] is still lacking. In this study, we screened for an ETMR-specific marker using a gene-expression profiling dataset of more than 1,400 brain tumors and identified LIN28A as a highly specific marker for ETMR. The encoded protein binds small RNA and has been implicated in stem cell pluripotency, metabolism and tumorigenesis. Using an LIN28A specific antibody, we carried out immunohistochemical analysis of LIN28A in more than 800 childhood brain-tumor samples and confirmed its high specificity for ETMR. Strong LIN28A immunoexpression was found in all 37 ETMR samples tested, whereas focal reactivity was only present in a small (6/50) proportion of AT/RT samples. All other pediatric brain tumors were completely LIN28A-negative. In summary, we established LIN28A immunohistochemistry as a highly sensitive and specific, rapid, inexpensive diagnostic tool for routine pathological verification of ETMR.
doi:10.1007/s00401-012-1068-3
PMCID: PMC3508282  PMID: 23161096
ETMR; Pediatric brain tumor; LIN28A; Diagnostic marker
20.  Rituximab-Associated Progressive Multifocal Leukoencephalopathy in Rheumatoid Arthritis 
Archives of neurology  2011;68(9):1156-1164.
Objective
To describe the development of progressive multifocal leukoencephalopathy (PML) in patients with rheumatoid arthritis (RA) treated with rituximab.
Design
Case study.
Setting
Clinical care for patients with rheumatologic diseases. Most were referred to academic centers for care after diagnosis (Washington University, St Louis, Missouri; Karolinska Insitute, Stockholm, Sweden; and Royal Melbourne Hospital, Melbourne, Australia) while one was cared for in a neurology practice in Dallas, Texas, with consultation by an academic neurovirologist from the University of Colorado in Denver.
Patients
Four patients developing PML in the setting of rituximab therapy for RA.
Intervention
Rituximab therapy.
Main Outcome Measures
Clinical and pathological observations.
Results
Four patients from an estimated population of 129 000 exposed to rituximab therapy for RA are reported in whom PML developed after administration of this drug. All were women older than 50 years, commonly with Sjögren syndrome and a history of treatment for joint disease ranging from 3 to 14 years. One case had no prior biologic and minimal immunosuppressive therapy. Progressive multifocal leukoencephalopathy presented as a progressive neurological disorder, with diagnosis confirmed by detection of JC virus DNA in the cerebrospinal fluid or brain biopsy specimen. Two patients died in less than 1 year from PML diagnosis, while 2 remain alive after treatment withdrawal. Magnetic resonance scans and tissue evaluation confirmed the frequent development of inflammatory PML during the course of the disease.
Conclusion
These cases suggest an increased risk, about 1 case per 25 000 individuals, of PML in patients with RA being treated with rituximab. Inflammatory PML may occur in this setting even while CD20 counts remain low.
doi:10.1001/archneurol.2011.103
PMCID: PMC3428054  PMID: 21555606
21.  Gliomas in Neurofibromatosis Type 1: A Clinicopathologic Study of 100 Patients 
There are few pathologic studies of gliomas in patients with neurofibromatosis type 1. We analyzed clinical and pathologic features of gliomas from 100 neurofibromatosis type 1 patients (57 men; 43 women). The median age at tumor diagnosis was 13 years (range, 4 months to 68 years). Most tumors were typical pilocytic astrocytoma (PA) (49%) or diffusely infiltrating astrocytoma (DA) (27%) that included World Health Organization Grades II (5%), III (15%), and IV (7%); others were designated as low-grade astrocytoma, subtype indeterminate (LGSI; 17%). Two pilomyxoid astrocytomas, 1 desmoplastic infantile ganglioglioma and 1 conventional ganglioglioma, were also identified. The tumors in 24 cases arose in the optic pathways and included PA (n = 14), LGSI (n = 4), DA (n = 4), pilomyxoid astrocytoma (n = 1), and ganglioglioma (n = 1). The prognoses of the PA and LGSI gliomas overall were generally favorable; there were no survival differences between PA and LGSI groups based on site, tumor size, mitotic activity, or MIB-1 labeling index. In the combined PA and LGSI group, age younger than 10 years and gross total resection were associated with an increased overall survival rate (p = 0.047 and 0.002, respectively). Compared with the combined group (PA + LGSI), patients with DA at all sites had decreased overall and recurrence-free survival times (p < 0.001 and p = 0.003, respectively). This study emphasizes the wide histologic spectrum of gliomas that occur in patients with neurofibromatosis type 1. Classic PA and LGSI are the most common, and most have favorable prognoses. By contrast, DAs are more aggressive, similar to those that arise sporadically.
doi:10.1097/NEN.0b013e318165eb75
PMCID: PMC3417064  PMID: 18344915
Astrocytoma; Brain tumor; Central nervous system; Glioma; Neurofibromatosis; Pilocytic astrocytoma
22.  Identification of transcriptional regulatory networks specific to pilocytic astrocytoma 
BMC Medical Genomics  2011;4:57.
Background
Pilocytic Astrocytomas (PAs) are common low-grade central nervous system malignancies for which few recurrent and specific genetic alterations have been identified. In an effort to better understand the molecular biology underlying the pathogenesis of these pediatric brain tumors, we performed higher-order transcriptional network analysis of a large gene expression dataset to identify gene regulatory pathways that are specific to this tumor type, relative to other, more aggressive glial or histologically distinct brain tumours.
Methods
RNA derived from frozen human PA tumours was subjected to microarray-based gene expression profiling, using Affymetrix U133Plus2 GeneChip microarrays. This data set was compared to similar data sets previously generated from non-malignant human brain tissue and other brain tumour types, after appropriate normalization.
Results
In this study, we examined gene expression in 66 PA tumors compared to 15 non-malignant cortical brain tissues, and identified 792 genes that demonstrated consistent differential expression between independent sets of PA and non-malignant specimens. From this entire 792 gene set, we used the previously described PAP tool to assemble a core transcriptional regulatory network composed of 6 transcription factor genes (TFs) and 24 target genes, for a total of 55 interactions. A similar analysis of oligodendroglioma and glioblastoma multiforme (GBM) gene expression data sets identified distinct, but overlapping, networks. Most importantly, comparison of each of the brain tumor type-specific networks revealed a network unique to PA that included repressed expression of ONECUT2, a gene frequently methylated in other tumor types, and 13 other uniquely predicted TF-gene interactions.
Conclusions
These results suggest specific transcriptional pathways that may operate to create the unique molecular phenotype of PA and thus opportunities for corresponding targeted therapeutic intervention. Moreover, this study also demonstrates how integration of gene expression data with TF-gene and TF-TF interaction data is a powerful approach to generating testable hypotheses to better understand cell-type specific genetic programs relevant to cancer.
doi:10.1186/1755-8794-4-57
PMCID: PMC3200988  PMID: 21745356
23.  Assessment of Epidermal Growth Factor Receptor (EGFR) expression in human meningioma 
Purpose
This study explores whether meningioma expresses epidermal growth factor receptor (EGFR) and determines if there is a correlation between the WHO grade of this tumor and the degree of EGFR expression.
Methods
Following institutional review board approval, 113 meningioma specimens from 89 patients were chosen. Of these, 85 were used for final analysis. After a blinded review, immunohistochemical stains for EGFR were performed. Staining intensity (SI) was scored on a scale 0-3 (from no staining to strong staining). Staining percentage of immunoreactive cells (SP) was scored 1-5 (from the least to the maximum percent of the specimen staining). Immunohistochemical score (IHS) was calculated as the product of SI and SP.
Results
Eighty-five samples of meningioma were classified in accordance with World Health Organization (WHO) criteria: benign 57/85 (67%), atypical 23/85 (27%), and malignant 5/85 (6%). The majority of samples demonstrated a moderate SI for EGFR. IHS for EGFR demonstrated a significant association between SI and histopathologic subtype. Also, there was a correlation between the SP and histopathologic subtype (p = 0.029). A significant association was determined when the benign and the atypical samples were compared to the malignant with respect to the SP (p = 0.009). While there was a range of the IHS for the benign and the atypical histologic subtypes, malignant tumors exhibited the lowest score and were statistically different from the benign and the atypical specimens (p < 0.001).
Conclusions
To our knowledge, this represents the largest series of meningioma samples analyzed for EGFR expression reported in the literature. EGFR expression is greatest in benign meningiomas and may serve a potential target for therapeutic intervention with selective EGFR inhibitors.
doi:10.1186/1748-717X-5-46
PMCID: PMC2890616  PMID: 20509969
24.  Oligodendroglial neoplasms with ganglioglioma-like maturation: a diagnostic pitfall 
Acta Neuropathologica  2010;120(2):237-252.
Although oligodendroglial neoplasms are traditionally considered purely glial, increasing evidence suggests that they are capable of neuronal or neurocytic differentiation. Nevertheless, ganglioglioma-like foci (GGLF) have not been previously described. Herein, we report seven examples where the primary differential diagnosis was a ganglioglioma with an oligodendroglial component. These five male and two female patients ranged in age from 29 to 63 (median 44) years at initial presentation and neuroimaging features were those of diffuse gliomas in general. At presentation, the glial component was oligodendroglioma in six and oligoastrocytoma in one; one was low-grade and six were anaplastic. A sharp demarcation from adjacent GGLF was common, although some intermingling was always present. The GGLF included enlarged dysmorphic and occasionally binucleate ganglion cells, Nissl substance, expression of neuronal antigens, GFAP-positive astrocytic elements, and low Ki-67 labeling indices. In contrast to classic ganglioglioma, however, cases lacked eosinophilic granular bodies and CD34-positive tumor cells. Scattered bizarre astrocytes were also common and one case had focal neurocytic differentiation. By FISH analysis, five cases showed 1p/19q codeletion. In the four cases with deletions and ample dysmorphic ganglion cells for analysis, the deletions were found in both components. At last follow-up, two patients suffered recurrences, one developed radiation necrosis mimicking recurrence, and one died of disease 7.5 years after initial surgery. We conclude that GGLF represents yet another form of neuronal differentiation in oligodendroglial neoplasms. Recognition of this pattern will prevent a misdiagnosis of ganglioglioma with its potential for under-treatment.
doi:10.1007/s00401-010-0695-9
PMCID: PMC2892612  PMID: 20464403
Anaplasia; FISH; Ganglioglioma; Immunohistochemistry; Oligodendroglioma; Neuronal
25.  Absence of PIttsburgh Compound B Detection of CerebralAmyloid Beta in a Patient With Clinical, Cognitive, and Cerebrospinal FluidMarkers of Alzheimer Disease 
Archives of neurology  2009;66(12):1557-1562.
Objective
To determine the temporal relationships of clinical, cognitive, Pittsburgh Compound-B (PiB) amyloid imaging, and cerebrospinal fluid (CSF) markers of Alzheimer’s disease (AD).
Design
A case report of a longitudinally assessed participant in a memory and aging study who had serial clinical and psychometric assessments over 6 years, in addition to PiB imaging and CSF biomarker assays, prior to coming to autopsy.
Setting
Alzheimer’s Disease Research Center
Findings
An 85-year old individual was cognitively normal at his initial and next 3 annual assessments. Decline in measures of episodic memory and, to a lesser degree, working memory began at about age 88 years. PiB-PET amyloid imaging was negative at age 88.5 years, but at age 89.5 years there was reduced amyloid-beta 42 (Aβ42) and elevated levels of tau in the CSF. At his 6th assessment, when he was 90 years old, he was diagnosed with very mild dementia of the Alzheimer type. After death at age 91 years, the autopsy revealed foci of frequent neocortical diffuse Aβ plaques, sufficient to fulfill Khachaturian neuropathologic criteria for AD, but neuritic plaques and neurofibrillary tangles were sparse. Postmortem biochemical analysis of the cerebral tissue confirmed that PiB-PET-binding was below the level needed for in vivo detection.
Conclusion
Clinical, cognitive, and CSF markers consistent with AD may precede detection of cerebral Aβ with amyloid imaging agents such as PiB, which primarily label fibrillar Aβ plaques.
doi:10.1001/archneurol.2009.279
PMCID: PMC2796200  PMID: 20008664

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