Jon D. Wilson

Oct4 Immunohistochemistry Is Superior to Placental Alkaline Phosphatase (plap) in the Diagnosis of Central Nervous System Germinoma

OCT4 is an 18-kDa POU-domain transcription factor encoded by the POU5F1 gene. Also known as OCT3, OTF3, and POU5F1, OCT4 is involved in the initiation, maintenance, and differentiation of pluripotent and germline cells during normal development. It is expressed in mouse and human embryonic stem and germ cells but absent from all differentiated somatic cell types in vitro and in vivo. OCT4 has been detected in primary testicular germ cell tumors with pluripotent potential: seminoma and embryonal carcinoma. We investigated: 1) whether a similar pattern of expression is present in primary intracranial germinomas; and 2) how OCT4 compares with placental alkaline phosphatase (PLAP) in terms of specificity and sensitivity as a potential diagnostic tool. We examined histologic sections from 25 cases of germinoma in which paraffin blocks with sufficient material were available. All cases were reviewed and sections from 32 different blocks were obtained and immunostained for OCT4 and PLAP. Additionally, 49 primary and metastatic brain tumors that may be potentially confused with germinoma, either clinically or histologically, were investigated for OCT4 expression. All but one germinoma were pure (ie, lacking other germ cell components). Intense and often diffuse nuclear staining was detected in 100% of germinomas. PLAP immunoreactivity was detected in 23 of 25 cases and was absent in the remaining 2 cases. The intensity of OCT4 immunostaining was significantly better than that of PLAP. None of the 49 control cases, which included glioblastoma multiforme, pineoblastoma, pituitary adenoma, malignant lymphoma, metastatic melanoma, capillary hemangioblastoma, meningioma, schwannoma, and a variety of metastatic carcinomas showed immunoreactivity for OCT4. Our study demonstrates that OCT4 is a highly specific and sensitive immunohistochemical marker for primary intracranial germinomas. OCT4 should be part of immunoperoxidase staining panels in which germinoma enters the differential diagnosis.

Primary Central Nervous System Lymphoma

Primary central nervous system lymphoma (PCNSL) is an uncommon extranodal non-Hodgkin lymphoma. Its incidence has increased during the last 3 decades and has been reported in both immunocompromised and immunocompetent patients. Immunocompromised patients are affected at a younger age compared with immunocompetent patients. It presents with raised intracranial pressure and focal neurologic and neuropsychiatric symptoms. The lesions are typically solitary. The majority of the lesions are located in the periventricular area, whereas in a few cases they are located in the supratentorial area. Diffuse large B-cell lymphomas constitute most PCNSLs, whereas T-cell, low-grade, anaplastic, and Hodgkin lymphomas are rarely encountered. The morphology of PCNSL shows a characteristic angiocentric pattern and is positive for B-cell markers by immunohistochemistry. The differential diagnosis of PCNSL includes central nervous system gliomas, metastatic tumors, demyelinating disorders, subacute infarcts, and space-occupying lesions due to an infectious etiology. The understanding of the molecular mechanisms involved in the pathogenesis of PCNSL and the identification of molecular biomarkers have lagged behind that of systemic nodal lymphomas. Primary central nervous system lymphomas are treated with combined radiotherapies and chemotherapies. The prognosis for PCNSL is worse than for other extranodal lymphomas.

Heterotopic mesenteric ossification ('intraabdominal myositis ossificans'): report of five cases.

Intraabdominal heterotopic ossification is a very uncommon disorder. We report five new cases, review the previous literature, and discuss the clinical and pathologic features of these lesions. The clinical features of the current cases and of those previously reported are remarkably similar. All patients were middle-aged to elderly men (range, 43-80 years; mean, 61 years) who had small bowel obstruction associated with heterotopic bone formation in the small bowel mesentery, often after one or more abdominal operations. In one case, an initial diagnosis of extraosseous osteosarcoma was considered. This unusual reactive process shares many of the clinical and pathologic features of myositis ossificans, as classically described in somatic soft tissues. We propose to designate this condition heterotopic mesenteric ossification.

Primary bone diffuse large B-cell lymphoma: clinicopathologic study of 21 cases and review of literature.

Primary bone diffuse large B-cell lymphomas (PB-DLBCL) are uncommon extranodal lymphomas. Herein, we report the clinical, pathologic, immunohistochemical, and molecular features of 21 cases of PB-DLBCL. The mean age of the patients was 54 years (range: 13 to 85 y). The male and female ratio was 1.6:1. The tumors consisted of diffuse sheets of large atypical cells or a polymorphous mixture of small-to-large cells with large multilobated nuclei, fine chromatin, and inconspicuous to prominent nucleoli. Twelve (57%) cases were non-germinal center B (GCB) and 9 (43%) were GCB subtype based on immunohistochemical classification. B-cell lymphomas (BCL)-2 was positive in 17/21 (81%), TP53 in 11/21 (52%) positive and the mean MIB-1 index was 57%. Polymerase chain reaction showed 10 cases with immunoglobulin heavy-chain (IGH) and 4 cases with IGH/BCL-2 gene rearrangement. The fluorescence in-situ hybridization analyses showed 14% of cases with BCL-6, 19% of cases with BCL-2, and 9% of cases with C-MYC gene rearrangement. Age <60 years and complete response to initial treatment were significant predictors of survival outcome (P≤0.05). Even though no association was observed between the subtype of PB-DLBCL (GCB vs. non-GCB), BCL2, TP53, MIB1 index and overall survival (P>0.05), due to small sample size, and variability in treatment received, this analysis may be interpreted with caution.

Selective Forelimb Impairment in Rats Expressing a Pathological TDP-43 25 kDa C-terminal Fragment to Mimic Amyotrophic Lateral Sclerosis

Pathological inclusions containing transactive response DNA-binding protein 43 kDa (TDP-43) are common in several neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). TDP-43 normally localizes predominantly to the nucleus, but during disease progression, it mislocalizes to the cytoplasm. We expressed TDP-43 in rats by an adeno-associated virus (AAV9) gene transfer method that transduces neurons throughout the central nervous system (CNS). To mimic the aberrant cytoplasmic TDP-43 found in disease, we expressed a form of TDP-43 with mutations in the nuclear localization signal sequence (TDP-NLS). The TDP-NLS was detected in both the cytoplasm and the nucleus of transduced neurons. Unlike wild-type TDP-43, expression of TDP-NLS did not induce mortality. However, the TDP-NLS induced disease-relevant motor impairments over 24 weeks. We compared the TDP-NLS to a 25 kDa C-terminal proaggregatory fragment of TDP-43 (TDP-25). The clinical phenotype of forelimb impairment was pronounced with the TDP-25 form, supporting a role of this C-terminal fragment in pathogenesis. The results advance previous rodent models by inducing cytoplasmic expression of TDP-43 in the spinal cord, and the non-lethal phenotype enabled long-term study. Approaching a more relevant disease state in an animal model that more closely mimics underlying mechanisms in human disease could unlock our ability to develop therapeutics.Pathological inclusions containing transactive response DNA-binding protein 43 kDa (TDP-43) are common in several neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). TDP-43 normally localizes predominantly to the nucleus, but during disease progression, it mislocalizes to the cytoplasm. We expressed TDP-43 in rats by an adeno-associated virus (AAV9) gene transfer method that transduces neurons throughout the central nervous system (CNS). To mimic the aberrant cytoplasmic TDP-43 found in disease, we expressed a form of TDP-43 with mutations in the nuclear localization signal sequence (TDP-NLS). The TDP-NLS was detected in both the cytoplasm and the nucleus of transduced neurons. Unlike wild-type TDP-43, expression of TDP-NLS did not induce mortality. However, the TDP-NLS induced disease-relevant motor impairments over 24 weeks. We compared the TDP-NLS to a 25 kDa C-terminal proaggregatory fragment of TDP-43 (TDP-25). The clinical phenotype of forelimb impairment was pronounced with the TDP-25 form, supporting a role of this C-terminal fragment in pathogenesis. The results advance previous rodent models by inducing cytoplasmic expression of TDP-43 in the spinal cord, and the non-lethal phenotype enabled long-term study. Approaching a more relevant disease state in an animal model that more closely mimics underlying mechanisms in human disease could unlock our ability to develop therapeutics.

Clinical Validation of Targeted Next-Generation Sequencing for Inherited Disorders

CONTEXT Although next-generation sequencing (NGS) can revolutionize molecular diagnostics, several hurdles remain in the implementation of this technology in clinical laboratories. OBJECTIVES To validate and implement an NGS panel for genetic diagnosis of more than 100 inherited diseases, such as neurologic conditions, congenital hearing loss and eye disorders, developmental disorders, nonmalignant diseases treated by hematopoietic cell transplantation, familial cancers, connective tissue disorders, metabolic disorders, disorders of sexual development, and cardiac disorders. The diagnostic gene panels ranged from 1 to 54 genes with most of panels containing 10 genes or fewer. DESIGN We used a liquid hybridization-based, target-enrichment strategy to enrich 10 067 exons in 568 genes, followed by NGS with a HiSeq 2000 sequencing system (Illumina, San Diego, California). RESULTS We successfully sequenced 97.6% (9825 of 10 067) of the targeted exons to obtain a minimum coverage of 20× at all bases. We demonstrated 100% concordance in detecting 19 pathogenic single-nucleotide variations and 11 pathogenic insertion-deletion mutations ranging in size from 1 to 18 base pairs across 18 samples that were previously characterized by Sanger sequencing. Using 4 pairs of blinded, duplicate samples, we demonstrated a high degree of concordance (>99%) among the blinded, duplicate pairs. CONCLUSIONS We have successfully demonstrated the feasibility of using the NGS platform to multiplex genetic tests for several rare diseases and the use of cloud computing for bioinformatics analysis as a relatively low-cost solution for implementing NGS in clinical laboratories.

Complete and sustained response of adult medulloblastoma to first-line sonic hedgehog inhibition with vismodegib

ABSTRACT Medulloblastoma is an aggressive primitive neuroectodermal tumor of the cerebellum that is rare in adults. Medulloblastomas fall into 4 prognostically significant molecular subgroups that are best defined by experimental gene expression profiles: the WNT pathway, sonic hedgehog (SHH) pathway, and subgroups 3 and 4 (non-SHH/WNT). Medulloblastoma of adults belong primarily to the SHH category. Vismodegib, an SHH-pathway inhibitor FDA-approved in 2012 for treatment of basal cell carcinoma, has been used successfully in the setting of chemorefractory medulloblastoma, but not as a first-line therapy. In this report, we describe a sustained response of an unresectable multifocal form of adult medulloblastoma to vismodegib. Molecular analysis in this case revealed mutations in TP53 and a cytogenetic abnormality, i17q, that is prevalent and most often associated with subgroup 4 rather than the SHH-activated form of medulloblastoma. Our findings indicate that vismodegib may also block alternate, non-canonical forms of downstream SHH pathway activation. These findings provide strong impetus for further investigation of vismodegib in clinical trials in the first-line setting for pediatric and adult forms of medulloblastoma.

Detection of PAX2 Deletions and Duplications Using Multiplex Ligation-Dependent Probe Amplification

BACKGROUND Renal coloboma syndrome (RCS) is a rare inherited disorder caused by mutations in the PAX2 gene. Clinical testing is currently performed by bidirectional Sanger sequencing of all 12 coding exons of the PAX2 gene, which detects point mutations or small insertion/deletion mutations. Large genomic deletions of PAX2 have been identified in 3/90 known RCS families, accounting for approximately (3%) of RCS cases. In these cases, the deletion was detected by cytogenetic techniques such as G-banding or array comparative genomic hybridization. While these methods would be sufficient to identify whole gene deletions, they may not be able to identify smaller rearrangements affecting single exons. Similarly, such deletions would not be detected by Sanger sequencing. AIM The aim of this study was to determine whether mutation-negative RCS probands harbor a genomic deletion or duplication involving one or more exons of the PAX2 gene. We evaluated this hypothesis in 46 patients with a clinical suspicion of RCS in whom no mutations were identified. RESULTS We developed a multiplex ligation-dependent probe amplification assay to detect gene deletion/duplication in all 12 exons of the PAX2 gene. Of the 46 PAX2 mutation-negative samples tested, none demonstrated deletions or duplications in the PAX2 gene. This suggests that deletions or duplications in PAX2 are unlikely to significantly contribute to the pathogenesis of RCS, beyond the known 3% of cases that have been attributed to whole gene deletions. Given these results, we hypothesize that other genes and/or locus control regions regulating PAX2 may be involved in the pathogenesis of PAX2 mutation-negative cases of RCS.

Association between gene expression of peripheral blood oxidative stress markers and Alzheimer's disease: The nun study

Background: A definite diagnosis of Alzheimer’s disease (AD) is determined following brain autopsy by detection of hallmark pathologies of the disease: an extracellular aggregates of amyloid-b (Ab) peptides, termed as Ab plaques, and an intracellular neurofibrillary tangles. Presently, noninvasive monitoring of Ab plaques in the brains of living patients is, however, inadequate due to restricted resolution and specificity. We, thus, hypothesized that the eyes can serve as a window to the brain for direct and noninvasive imaging of AD. Methods: To this end, we used APPswe/PS1dE9 double-transgenic AD mice, which faithfully represent some of the key pathologies found in the human disease. We investigated presence and formation of amyloid plaques in retinas from these AD mice, as well as estimated the correlation between retinal amyloid plaques and the plaque pathology in the brain. Lastly, we evaluated responsiveness of retinal plaques to an immune-based therapy and the ability to image them in live mice. Results: Here, we detected Ab plaques in retinal samples of AD mice, and found that Ab plaques appear in the retina significantly earlier than in the brain. Retinal plaques accumulated during disease progression, and in good correlation with brain pathology. Moreover, retinal Ab plaque number and size were significantly reduced following an immune-based therapy, which we found to be effective in reducing plaques in the brain. Systemic administration of a natural compound that binds and labels Ab plaques, allowed in vivo noninvasive visualization of retinal Ab plaques in live AD mice, with high resolution and specificity. Conclusions: These studies establish the potential of direct retinal Ab plaque imaging in live subjects as a promising tool for early AD diagnosis, prognosis and assessment of therapies.