Exploiting nanopore sequencing for characterization and grading of IDH-mutant gliomas.

The 2021 WHO Classification of Central Nervous System Tumors recommended evaluation of cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) deletion in addition to codeletion of 1p/19q to characterize IDH-mutant gliomas. Here, we demonstrated the use of a nanopore-based copy-number variation sequencing (nCNV-seq) approach to simultaneously identify deletions of CDKN2A/B and 1p/19q. The nCNV-seq approach was initially evaluated on three distinct glioma cell lines and then applied to 19 IDH-mutant gliomas (8 astrocytomas and 11 oligodendrogliomas) from patients. The whole-arm 1p/19q codeletion was detected in all oligodendrogliomas with high concordance among nCNV-seq, FISH, DNA methylation profiling, and whole-genome sequencing. For the CDKN2A/B deletion, nCNV-seq detected the loss in both astrocytoma and oligodendroglioma, with strong correlation with the CNV profiles derived from whole-genome sequencing (Pearson correlation (r) = 0.95, P < 2.2 × 10-16 to r = 0.99, P < 2.2 × 10-16 ) and methylome profiling. Furthermore, nCNV-seq can differentiate between homozygous and hemizygous deletions of CDKN2A/B. Taken together, nCNV-seq holds promise as a new, alternative approach for a rapid and simultaneous detection of the molecular signatures of IDH-mutant gliomas without capital expenditure for a sequencer.

Lymphomatosis cerebri with coexistent anti-N-methyl-D-aspartate receptor antibody: A case report.

Diagnosis of lymphomatosis cerebri (LC) is usually delayed because of its rarity and the need for pathological confirmation. The association of LC with humoral immunity has scarcely been reported. Herein, we present a woman with a 2-week history of dizziness and gait ataxia, followed by diplopia, altered mental status, and spasticity of all limbs. Magnetic resonance imaging (MRI) of the brain showed multifocal lesions involving bilateral subcortical white matter, deep gray structures, and brainstem. Oligoclonal bands and anti-N-methyl-D-aspartate receptor (NMDAR) antibodies were present in cerebrospinal fluid (CSF) twice. She was initially treated with methylprednisolone but still worsening. A stereotactic brain biopsy confirmed the diagnosis of LC. This is a report on the distinctive coexistence of the rare CNS lymphoma variant and the anti-NMDAR antibody.

Allergic fungal rhinosinusitis presented as a unilateral nasal mass: A first case report from Thailand and literature review.

Background: Allergic fungal rhinosinusitis (AFRS) is a relatively new inflammatory sinonasal disease. Prevalence of the disease is reported to be highly different across Asia. Case presentation: A 23-year-old Thai male came to our hospital with left-sided nasal obstruction. Endoscopic examination found a mass originated from the left sphenoethmoidal recess. Incisional biopsy result of the mass indicated an inflammatory process and high level of serum specific IgE to several aeroallergens was found. Based on the biopsy results and other investigations, the diagnosis of AFRS was made and the patient was treated successfully with endoscopic sinus surgery and postoperative systemic/topical steroids. Discussion: While AFRS is quite common in some regions, the disease is rarely encountered in Thailand and can be presented as a unilateral lesion, mimicking a tumor mass, which could lead to an incorrect diagnosis and inappropriate treatment. Conclusion: Even though AFRS is rarely reported in our country, it still can be found and might be recognized falsely as a neoplastic process. High level of awareness of the disease features could help to minimize inappropriate disease management.

Simplified approach for pathological diagnosis of diffuse gliomas in adult patients.

The most recent WHO classification (2016) for gliomas introduced integrated diagnoses requiring both phenotypic and genotypic data. This approach presents difficulties for countries with limited resources for laboratory testing. The present study describes a series of 118 adult Thai patients with diffuse gliomas, classified by the WHO 2016 classification. The purpose was to demonstrate how a diagnosis can still be achieved using a simplified approach that combines clinical, morphological, immunohistochemical, and fewer molecular assays than typically performed. This algorithm starts with tumor location (midline vs. non-midline) with diffuse midline glioma identified by H3 K27M immunostaining. All other tumors are placed into one of 6 categories, based on morphologic features rather than specific diagnoses. Molecular testing is limited to IDH1/IDH2 mutations, plus co-deletion of 1p/19q for cases with oligodendroglial features and TERT promoter mutation for cases without such features. Additional testing for co-deletion of 1p/19q, TERT promoter mutation and BRAF mutations are only used in selected cases to refine diagnosis and prognosis. With this approach, we were able to reach the integrated diagnosis in 117/118 cases, saving 50 % of the costs of a more inclusive testing panel. The demographic data and tumor subtypes were found to be similar to series from other regions of the world. To the best of our knowledge, this is to the first reported series of diffuse gliomas in South-East Asia categorized by the WHO 2016 classification system.

A Cavernous Venous Malformation of the Orbit Mimicking an Idiopathic Orbital Inflammation.

Orbital cavernous venous malformations (CVMs) are usually slow progressing. Multiple CVMs, bilateral orbital CVMs, and acute presentations are rare. We present a rare, bilateral, orbital CVM with acute painful visual loss in the left eye. The initial clinical presentation mimicked an idiopathic orbital inflammation. Orbital magnetic resonance imaging revealed its rare location at the left orbital apex. Finally, pathology confirmed the presence of an intralesional hemorrhage of a CVM.

Diabetes insipidus and panhypopituitarism as a first presentation of silent adenocarcinoma of lung: a case report and literature review.

BACKGROUND: Pituitary metastasis is a rare condition with a poor prognosis. Very few patients with pituitary metastasis are symptomatic. It is often associated with presence of co-existing metastases to other organs. Isolated pituitary metastasis as the first presentation of primary malignancy is uncommon. CASE PRESENTATION: A 72-year-old woman presented with a 2-month history of polyuria, increasing thirst and unexplained weight loss. Esophagogastroduodenoscopy (EGD) was scheduled as part of the investigation. She was kept nil per os for 10 h prior to EGD, after which she developed alteration of consciousness. Further investigation revealed hypernatremia with sodium level of 161 mmol/L and low urine osmolality of 62 mOsm/kg. Her urine output was 300 mL per hour. Diabetes insipidus (DI) was diagnosed based on evidence of polyuria, hypernatremia, and low urine osmolality. Her urine output decreased and urine osmolality increased to 570 mOsm/kg in response to subcutaneous desmopressin acetate, confirming central DI. Pituitary magnetic resonance imaging showed a heterogeneous gadolinium enhancing lesion at the sellar and suprasellar regions, measuring 2.4 × 2.6 × 3.9 cm compressing both the hypothalamus bilaterally and the inferior aspect of optic chiasm as well as displacing the residual pituitary gland anteriorly. The posterior pituitary bright spot was absent. These MRI findings suggested pituitary macroadenoma. There were also multiple small gadolinium-enhancing lesions up to 0.7 cm in size with adjacent vasogenic brain edema at the subcortical and subpial regions of the left frontal and parietal areas, raising the concern of brain metastases. Pituitary hormonal evaluation was consistent with panhypopituitarism. Histopathological and immunohistochemical studies of the pituitary tissue revealed an adenocarcinoma, originating from the lung. Computed tomography of the chest and abdomen was subsequently performed, showing a 2.2-cm soft tissue mass at the proximal part of right bronchus. There was no evidence of distant metastases elsewhere. The final diagnosis was adenocarcinoma of the lung with pituitary metastasis manifesting as panhypopituitarism and central DI. Palliative care along with hormonal replacement therapy was offered to the patient. She died 4 months after diagnosis. CONCLUSION: Diagnosis of pituitary metastasis is challenging, especially in patients with previously undiagnosed primary cancer. It should be considered in the elderly patients presenting with new-onset central DI with or without anterior pituitary dysfunction.

Efficacy of Thrombus Density on Noninvasive Computed Tomography Neuroimaging for Predicting Thrombus Pathology and Patient Outcome after Mechanical Thrombectomy in Acute Ischemic Stroke.

BACKGROUND AND PURPOSE: The aim of this study was to investigate the efficacy of thrombus density on noninvasive computed tomography (CT) neuroimaging for predicting thrombus pathology and patient outcome after mechanical thrombectomy in acute ischemic stroke. MATERIALS AND METHODS: This retrospective chart and imaging review included patients that were treated by mechanical thrombectomy at Siriraj Hospital according to the American Heart Association/American Stroke Association guidelines for the early management of patients with acute ischemic stroke from March 2010 to February 2015 study period. Preintervention noncontrast CT (NCCT), CT angiography (CTA), and/or contrast-enhanced CT (CECT) images were interpreted using CT densitometry. Pathology results were classified as white, red, or mixed thrombi. The result of treatment was evaluated by the modified Rankin Scale at 90 days after treatment. RESULTS: From 97 included patients - 97 NCCT images, 48 CTA images, 48 CECT images, and 54 pathologic results of cerebral thrombi were included in the final analysis. Mean clot Hounsfield unit values on NCCT, CTA, and CECT were significantly different between red and white thrombus (P = 0.001 on NCCT, P = 0.03 on CTA, and P = 0.001 on CECT), and between red and mixed thrombus (P = 0.043 on NCCT and P = 0.002 on CTA). However, no significant difference was observed between white thrombus and mixed thrombus (P = 0.09 on NCCT, P = 1.00 on CTA, and P = 0.054 on CECT). There was no significant correlation between type of cerebral thrombus or clot density and the result of treatment. CONCLUSION: Thrombus density on CT was found to be a significant predictor of thrombus pathology; however, no significant association was observed between thrombus type or clot density and patient outcome after mechanical thrombectomy.

EpCAM expression in squamous cell carcinoma of the uterine cervix detected by monoclonal antibody to the membrane-proximal part of EpCAM.

BACKGROUND: Epithelial cell adhesion molecule (EpCAM) is a promising biomarker for squamous cell carcinoma (SCC) of the uterine cervix, because it is over-expressed in various cancers of epithelial origin. However, EpCAM expression reported in previous immunohistochemistry (IHC) studies was inconsistent. We hypothesize that the membrane-distal part of EpCAM may be lost during tissue preparation, leaving only the membrane-proximal part of EpCAM available for antibody binding and IHC staining. METHODS: Two new anti-EpCAM MAbs to the membrane-proximal part (WC-2) and the membrane-distal part (WC-1) of EpCAM were generated and characterized. WC-2 was selected for its ability to detect EpCAM in cervical tissues by IHC. One hundred thirty-five archival paraffin-embedded tissues previously diagnosed as cervical SCC (n=44), high-grade (HSIL) (n=43), or low-grade (LSIL) (n=48) squamous intraepithelial lesions were examined. IHC score was collected, recorded, and analyzed for distribution, intensity, and percentage of cancer cells stained for EpCAM. RESULTS: EpCAM expression was consistently detected on cervical tissues by WC-2, but not by WC-1. EpCAM was expressed with high IHC score in the majority of cervical SCC (37/44), but not in normal epithelial area adjacent to SCC. EpCAM was also highly expressed on precancerous lesion of the cervix, particularly in HSIL. More importantly, EpCAM expression could be used to distinguish between HSIL and LSIL, according to staining distribution. HSIL tissues displayed EpCAM expression in two-thirds to full thickness of the epithelium, while in LSIL the staining was limited to the lower one-third of the thickness. The IHC score of EpCAM expression was strongly correlated with cervical cancer and grades of precancerous lesions (r=0.875, p<0.001). CONCLUSION: Only the anti-EpCAM MAb to the membrane-proximal part is able to detect EpCAM on paraffin-embedded cervical cancer tissues. A strong positive correlation between EpCAM expression level and the grades of SILs provides the possibility that EpCAM can be used to predict prognosis and severity in these patients.

High Carbonic Anhydrase-9 Expression Identifies a Subset of 1p/19q Co-Deletion and Favorable Prognosis in Oligodendroglioma.

OBJECTIVE: To investigate the relationship between 3 hypoxic markers, carbonic anhydrase-9 (CA-9), hypoxia-inducible factor (HIF)-1α, and HIF-2α and the traditional genetic markers, deletions of chromosomes 1p and 19q and Isocitrate dehydrogenase 1 (IDH1) R132H mutation in oligodendrogliomas. METHODS: Thirty-one oligodendrogliomas (27 World Health Organization Grade [WHO] II and 4 WHO Grade III) were processed into tissue microarray. Fluorescence in situ hybridization was exploited to detect chromosome deletion, whereas immunohistochemistry was performed to assess IDH1R132H mutation, CA-9, HIF-1α, and HIF-2α expression. RESULTS: The frequencies of 1p/19q co-deletion and IDH1 R132H mutation were 68% and 71%, respectively. High expression of CA-9 was observed in 42% and was associated with longer survival (P = 0.04) in WHO Grade II oligodendroglioma. High CA-9 expression also identified 62% of 1p/19q-codeleted oligodendroglioma (P = 0.001). In addition, all tumors with high CA-9 expression displayed 1p/19q-codeletion. HIF-1α and HIF-2α provided no additional prognostic value for survival. CONCLUSIONS: High expression of CA-9, a marker for hypoxia and acidosis, is associated with favorable prognosis in oligodendroglioma. In addition, it may serve as a simple screening test for 1p/19q co-deletion if validated in larger cohorts.

Nine human sparganosis cases in Thailand with molecular identification of causative parasite species.

Human sparganosis is one of the neglected diseases but important food-borne parasitic zoonoses. The disease is caused by larvae (spargana) of diphyllobothriidean tapeworm. Here, we describe nine cases of human sparganosis, caused by Spirometra erinaceieuropaei in a hospital in Thailand during 2001-2012. Clinical characteristics, treatment, and outcome of cases were revealed. Diagnosis and identification of causative parasite species was made by histopathological investigations followed by a polymerase chain reaction-based molecular method using formalin-fixed paraffin embedded tissues. The DNA samples were extracted from tissues and a partial fragment of cytochrome c oxidase subunit 1 (cox1) gene was amplified for the detection of parasitic DNA. Infection could be prevented by increasing activities on health communication by responsible public health agencies.

Sparganosis presenting as cauda equina syndrome with molecular identification of the parasite in tissue sections.

A 52-year-old woman presented with lower back pain, progressive symmetrical paraparesis with sensory impairment, and sphincter disturbance. Magnetic resonance imaging (MRI) of the whole spine revealed multiple intradural extramedullary serpiginous-mass lesions in the subarachnoid space continuously from the prepontine to the anterior part of the medulla oblongata levels, C7, T2-T8, and T12 vertebral levels distally until the end of the theca sac and filling-in the right S1 neural foramen. Sparganosis was diagnosed by demonstration of the sparganum in histopathological sections of surgically resected tissues and also by the presence of serum IgG antibodies by ELISA. DNA was extracted from unstained tissue sections, and a partial fragment of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene was amplified using a primer set specific for Spirometra spp. cox1. After sequencing of the PCR-amplicon and alignment of the nucleotide sequence data, the causative agent was identified as the larva of Spirometra erinaceieuropaei.

Silencing of the imprinted DLK1-MEG3 locus in human clinically nonfunctioning pituitary adenomas.

DLK1-MEG3 is an imprinted locus consisting of multiple maternally expressed noncoding RNA genes and paternally expressed protein-coding genes. The expression of maternally expressed gene 3 (MEG3) is selectively lost in clinically nonfunctioning adenomas (NFAs) of gonadotroph origin; however, expression status of other genes at this locus in human pituitary adenomas has not previously been reported. Using quantitative real-time RT-PCR, we evaluated expression of 24 genes from the DLK1-MEG3 locus in 44 human pituitary adenomas (25 NFAs, 7 ACTH-secreting, 7 GH-secreting, and 5 PRL-secreting adenomas) and 10 normal pituitaries. The effects on cell proliferation of five miRNAs whose expression was lost in NFAs were investigated by flow cytometry analysis. We found that 18 genes, including 13 miRNAs at the DLK1-MEG3 locus, were significantly down-regulated in human NFAs. In ACTH-secreting and PRL-secreting adenomas, 12 and 7 genes were significantly down-regulated, respectively; no genes were significantly down-regulated in GH-secreting tumors. One of the five miRNAs tested induced cell cycle arrest at the G2/M phase in PDFS cells derived from a human NFA. Our data indicate that the DLK1-MEG3 locus is silenced in NFAs. The growth suppression by miRNAs in PDFS cells is consistent with the hypothesis that the DLK1-MEG3 locus plays a tumor suppressor role in human NFAs.

Review: Pituitary adenomas in children and adolescents.

Clinical manifestations and outcomes of pituitary adenomas in children are not clearly defined. We retrospectively reviewed cases of pituitary adenomas in children 0-18 years treated at MassGeneral Hospital for Children over 15 years. Thirty-five patients were identified. Age at presentation was 7-18 years. Seventeen had prolactinomas, 3 had somatotropinomas, and 15 had Cushing disease. Thirteen prolactinoma patients were female and most commonly presented with oligomenorrhea (10/13) and galactorrhea (7/13). Nine were successfully treated medically. Two somatotropinoma patients presented with visual disturbances; the third was an incidental finding. Two were cured by trans-sphenoidal surgery (TSS). Thirteen Cushing disease patients were initially cured by TSS; six recurred after 3-6 years. Patients with or without recurrence did not differ for age, tumor-size and hormone levels. The high recurrence rate of Cushing disease in our series (46%) compared with adults treated surgically at this institution (7%) emphasizes the need for long-term follow-up.

Activation of paternally expressed genes and perinatal death caused by deletion of the Gtl2 gene.

The Dlk1-Gtl2 imprinting locus is located on mouse distal chromosome 12 and consists of multiple maternally expressed non-coding RNAs and several paternally expressed protein-coding genes. The imprinting of this locus plays a crucial role in embryonic development and postnatal growth. At least one cis-element, the intergenic differentially methylated region (IG-DMR) is required for expression of maternally expressed genes and repression of silenced paternally expressed genes. The mechanism by which the IG-DMR functions is largely unknown. However, it has been suggested that the unmethylated IG-DMR acts as a positive regulator activating expression of non-coding RNAs. Gtl2 is the first non-coding RNA gene downstream of the IG-DMR. Although its in vivo function in the mouse is largely unknown, its human ortholog MEG3 has been linked to tumor suppression in human tumor-derived cell lines. We generated a knockout mouse model, in which the first five exons and adjacent promoter region of the Gtl2 gene were deleted. Maternal deletion of Gtl2 resulted in perinatal death and skeletal muscle defects, indicating that Gtl2 plays an important role in embryonic development. The maternal deletion also completely abolished expression of downstream maternally expressed genes, activated expression of silenced paternally expressed genes and resulted in methylation of the IG-DMR. By contrast, the paternal inherited deletion did not have this effect. These data strongly indicate that activation of Gtl2 and its downstream maternal genes play an essential role in regulating Dlk1-Gtl2 imprinting, possibly by maintaining active status of the IG-DMR.

Increased expression of angiogenic genes in the brains of mouse meg3-null embryos.

Maternally expressed gene 3 (MEG3) is a noncoding RNA highly expressed in the normal human brain and pituitary. Expression of MEG3 is lost in gonadotroph-derived clinically nonfunctioning pituitary adenomas. Meg3 knockout mice were generated to identify targets and potential functions of this gene in embryonic development and tumorigenesis. Gene expression profiles were compared in the brains of Meg3-null embryos and wild-type littermate controls using microarray analysis. Microarray data were analyzed with GeneSifter, which uses Kyoto Encyclopedia of Genes and Genomes pathways and Gene Ontology classifications to identify signaling cascades and functional categories of interest within the dataset. Differences were found in signaling pathways and ontologies related to angiogenesis between wild-type and knockout embryos. Quantitative RT-PCR and immunohistological staining showed increased expression of some Vascular Endothelial Growth Factor pathway genes and increased cortical microvessel density in the Meg3-null embryos. In conclusion, Meg3 may play an important role in control of vascularization in the brain and may function as a tumor suppressor in part by inhibiting angiogenesis.

Maternally expressed gene 3, an imprinted noncoding RNA gene, is associated with meningioma pathogenesis and progression.

Meningiomas are common tumors, representing 15% to 25% of all central nervous system tumors. NF2 gene inactivation on chromosome 22 has been shown as an early event in tumorigenesis; however, few factors underlying tumor growth and progression have been identified. The chromosomal abnormalities of 14q32 are often associated with meningioma pathogenesis and progression; therefore, it has been proposed that an as yet unidentified tumor suppressor is present at this locus. Maternally expressed gene 3 (MEG3) is an imprinted gene located at 14q32 which encodes a noncoding RNA with an antiproliferative function. We found that MEG3 mRNA is highly expressed in normal arachnoidal cells. However, MEG3 is not expressed in the majority of human meningiomas or the human meningioma cell lines IOMM-Lee and CH157-MN. There is a strong association between loss of MEG3 expression and tumor grade. Allelic loss at the MEG3 locus is also observed in meningiomas, with increasing prevalence in higher grade tumors. In addition, there is an increase in CpG methylation within the promoter and the imprinting control region of MEG3 gene in meningiomas. Functionally, MEG3 suppresses DNA synthesis in both IOMM-Lee and CH157-MN cells by approximately 60% in bromodeoxyuridine incorporation assays. Colony-forming efficiency assays show that MEG3 inhibits colony formation in CH157-MN cells by approximately 80%. Furthermore, MEG3 stimulates p53-mediated transactivation in these cell lines. Therefore, these data are consistent with the hypothesis that MEG3, which encodes a noncoding RNA, may be a tumor suppressor gene at chromosome 14q32 involved in meningioma progression via a novel mechanism.

Rathke's cleft cysts in children and adolescents: association with female puberty.

There are few pediatric data regarding manifestations and outcomes of Rathke's cleft cysts (RCC). We retrospectively reviewed 13 cases treated at Massachusetts General Hospital over 10 years. Age at presentation was 12-17 years, except for one 7-year-old who presented with sexual precocity. There was a female preponderance [11 females, 2 males, p = 0.01], and all were pubertal at diagnosis. Common features at presentation were headaches (11/13), endocrine abnormalities (5/13) and visual disturbances (2/13). Four patients underwent transsphenoidal surgery. Symptoms improved in all but one, in whom headaches persisted. Recurrent growth in one patient was treated successfully by excision. For conservatively treated patients, cyst size was unchanged over follow-up (6 months-5 years). Female preponderance and pubertal presentation suggest a possible link between sex hormones and RCC pathogenesis. Although estrogen and progesterone receptor immunostaining was negative in the cyst lining, estrogen receptor immunostaining was positive in adjacent pituitary cells. Further investigations regarding this issue are warranted.

Surgical pathology and etiology of 278 surgically removed mitral valves with pure regurgitation in Thailand.

INTRODUCTION: There are multiple causes of mitral regurgitation. Its etiology includes floppy valve, postinflammatory disease, infective endocarditis, and other disorders. Recently, there has been an increased tendency to remove only portions of the mitral valve, causing difficulty in the determination of etiology. Our objective was to study the pathology and etiology of mitral regurgitation from surgically removed specimens. METHODS: Native mitral valve specimens surgically excised due to mitral insufficiency were examined. Etiology was determined according to macroscopic, microscopic, clinical, and operative findings. RESULTS: Among 278 mitral valve specimens, 43% were classified as floppy valve, 31% as postinflammatory disease (presumably associated with rheumatic fever), 12% as infective endocarditis, and 14% as miscellaneous group. In floppy valves, diffuse myxoid change and chordal rupture were the main findings. In postinflammatory disease, moderate neovascularization and chronic inflammatory cell infiltration were most commonly found. Aschoff bodies were found in two cases. In infective endocarditis, gram-positive cocci were found in 70% of cases. In the miscellaneous group, three cases were related to Marfan syndrome and one case was related to papillary muscle necrosis. In comparison with postinflammatory disease, the posterior leaflet in the floppy valve had a significantly longer basal free-edge length, a more frequent chordal rupture, and an higher mean age of patients. Among completely and partially excised specimens with postinflammatory disease, there were no significant differences in microscopic findings. CONCLUSION: The three most common etiologies in mitral regurgitation were floppy valve, postinflammatory disease, and infective endocarditis. Macroscopic, microscopic, clinical, and operative findings are important in the evaluation of etiology, especially in partially excised specimens.