Benjamin H. Hinrichs

Farewell to GBM-O: Genomic and transcriptomic profiling of glioblastoma with oligodendroglioma component reveals distinct molecular subgroups

IntroductionGlioblastoma with oligodendroglioma component (GBM-O) was recognized as a histologic pattern of glioblastoma (GBM) by the World Health Organization (WHO) in 2007 and is distinguished by the presence of oligodendroglioma-like differentiation. To better understand the genetic underpinnings of this morphologic entity, we performed a genome-wide, integrated copy number, mutational and transcriptomic analysis of eight (seven primary, one secondary) cases.ResultsThree GBM-O samples had IDH1 (p.R132H) mutations; two of these also demonstrated 1p/19q co-deletion and had a proneural transcriptional profile, a molecular signature characteristic of oligodendroglioma. The additional IDH1 mutant tumor lacked 1p/19q co-deletion, harbored a TP53 mutation, and overall, demonstrated features most consistent with IDH mutant (secondary) GBM. Finally, five tumors were IDH wild-type (IDHwt) and had chromosome seven gains, chromosome 10 losses, and homozygous 9p deletions (CDKN2A), alterations typical of IDHwt (primary) GBM. IDHwt GBM-Os also demonstrated EGFR and PDGFRA amplifications, which correlated with classical and proneural expression subtypes, respectively.ConclusionsOur findings demonstrate that GBM-O is composed of three discrete molecular subgroups with characteristic mutations, copy number alterations and gene expression patterns. Despite displaying areas that morphologically resemble oligodendroglioma, the current results indicate that morphologically defined GBM-O does not correspond to a particular genetic signature, but rather represents a collection of genetically dissimilar entities. Ancillary testing, especially for IDH and 1p/19q, should be used for determining these molecular subtypes.

Formation of diethylstilbestrol–DNA adducts in human breast epithelial cells and inhibition by resveratrol

Extensive evidence exists that the reaction of estrogen metabolites with DNA produces depurinating adducts that, in turn, induce mutations and cellular transformation. While it is clear that these estrogen metabolites result in a neoplastic phenotype in vitro, further evidence supporting the link between estrogen-DNA adduct formation and its role in neoplasia induction in vivo would strengthen the evidence for a genotoxic mechanism. Diethylstilbestrol (DES), an estrogen analogue known to increase the risk of breast cancer in women exposed in utero, is hypothesized to induce neoplasia through a similar genotoxic mechanism. Cultured MCF-10F human breast epithelial cells were treated with DES at varying concentrations and for various times to determine whether the addition of DES to MCF-10F cells resulted in the formation of depurinating adducts. This is the first demonstration of the formation of DES-DNA adducts in human breast cells. A dose-dependent increase in DES-DNA adducts was observed. Demonstrating that treatment of MCF-10F cells with DES, a known human carcinogen, yields depurinating adducts provides further support for the involvement of these adducts in the induction of breast neoplasia. Previous studies have demonstrated the ability of antioxidants such as resveratrol to prevent the formation of estrogen-DNA adducts, thus preventing a key carcinogenic event. In this study, when MCF-10F cells were treated with a combination of resveratrol and DES, a dose-dependent reduction in the level of DES-DNA adducts was also observed.

Cosmc is an X-linked inflammatory bowel disease risk gene that spatially regulates gut microbiota and contributes to sex-specific risk

Significance Inflammatory bowel disease (IBD) is a devastating illness that affects 1.6 million people in the United States and disproportionately affects males in early onset disease. However, IBD genes that contribute to sex-specific risk are unexplored. The gut microbiota interfaces the host with its environment and exhibits alterations in spatial organization in IBD with dysbiosis in the mucosa but a relatively unaffected lumen. Core 1 β3GalT-specific molecular chaperone (Cosmc) was recently identified as an IBD risk gene on the X chromosome by genome-wide association study. We functionally evaluated Cosmc in IBD and discovered that the loss of Cosmc leads to gut inflammation in males but not females and a spatial pattern of dysbiosis resembling IBD. Thus, Cosmc contributes to sex bias in IBD and spatially regulates the gut microbiota. Inflammatory bowel disease (IBD) results from aberrant immune stimulation against a dysbiotic mucosal but relatively preserved luminal microbiota and preferentially affects males in early onset disease. However, factors contributing to sex-specific risk and the pattern of dysbiosis are largely unexplored. Core 1 β3GalT-specific molecular chaperone (Cosmc), which encodes an X-linked chaperone important for glycocalyx formation, was recently identified as an IBD risk factor by genome-wide association study. We deleted Cosmc in mouse intestinal epithelial cells (IECs) and found marked reduction of microbiota diversity in progression from the proximal to the distal gut mucosa, but not in the overlying lumen, as seen in IBD. This loss of diversity coincided with local emergence of a proinflammatory pathobiont and distal gut restricted pathology. Mechanistically, we found that Cosmc regulates host genes, bacterial ligands, and nutrient availability to control microbiota biogeography. Loss of one Cosmc allele in males (IEC-Cosmc-/y) resulted in a compromised mucus layer, spontaneous microbe-dependent inflammation, and enhanced experimental colitis; however, females with loss of one allele and mosaic deletion of Cosmc in 50% of crypts (IEC-Cosmc+/−) were protected from spontaneous inflammation and partially protected from experimental colitis, likely due to lateral migration of normal mucin glycocalyx from WT cells over KO crypts. These studies functionally validate Cosmc as an IBD risk factor and implicate it in regulating the spatial pattern of dysbiosis and sex bias in IBD.

Highlighting nuclear membrane staining in thyroid neoplasms with emerin: Review and diagnostic utility

Immunohistochemical staining (IHC) with emerin, an integral inner nuclear membrane protein, highlights nuclear membrane details in papillary thyroid carcinoma (PTC). We evaluated emerin for highlighting nuclear shape, grooves, inclusions, circumferential nuclear membrane irregularities (“garlands”), deep “stellate” membrane invaginations, and crescents in 34 fine‐needle aspiration (FNA) cell blocks, PTC (n = 24) and follicular neoplasms (FN) (n = 10). Tissue microarrays were also examined for 182 cases, PTC (n = 95) and non‐PTC (n = 87). Emerin IHC of PTC revealed a predominantly oval nuclear shape in the majority of cases, with FN demonstrating round nuclei and FV of PTC showing a roughly equal distribution of round and oval shapes. In addition to oval nuclear shape, the presence of emerin‐positive nuclear grooves, circumferential emerin nuclear “garlands,” nuclear crescent shapes, and chromatin clearing on cell block H&E staining were significant predictors of PTC by regression analysis. Emerin IHC of thyroid FNA and surgical specimens serves as a useful adjunct to conventional H&E staining in the diagnosis of PTC and its distinction from FN by delineating diagnostic nuclear membrane irregularities (“garlands” and crescents), nuclear grooves, and a characteristic oval nuclear shape. In diagnostically challenging cases with limited cellularity, emerin staining can help to provide a more definitive diagnosis of PTC. Diagn. Cytopathol. 2013.

Redox control of Cas phosphorylation requires Abl kinase in regulation of intestinal epithelial cell spreading and migration

Intestinal wounds often occur during inflammatory and ischemic disorders of the gut. To repair damage, intestinal epithelial cells must rapidly spread and migrate to cover exposed lamina propria, events that involve redox signaling. Wounds are subject to extensive redox alterations, particularly resulting from H2O2 produced in the adjacent tissue by both the epithelium and emigrating leukocytes. The mechanisms governing these processes are not fully understood, particularly at the level of protein signaling. Crk-associated substrate, or Cas, is an important signaling protein known to modulate focal adhesion and actin cytoskeletal dynamics, whose association with Crk is regulated by Abl kinase, a ubiquitously expressed tyrosine kinase. We sought to evaluate the role of Abl regulation of Cas at the level of cell spreading and migration during wound closure. As a model, we used intestinal epithelial cells exposed to H2O2 or scratch wounded to assess the Abl-Cas signaling pathway. We characterized the localization of phosphorylated Cas in mouse colonic epithelium under baseline conditions and after biopsy wounding the mucosa. Analysis of actin and focal adhesion dynamics by microscopy or biochemical analysis after manipulating Abl kinase revealed that Abl controls redox-dependent Cas phosphorylation and localization to influence cell spreading and migration. Collectively, our data shed new light on redox-sensitive protein signaling modules controlling intestinal wound healing.

Organic Solute Transporter α-β Protects Ileal Enterocytes From Bile Acid–Induced Injury

Background & Aims Ileal bile acid absorption is mediated by uptake via the apical sodium-dependent bile acid transporter (ASBT), and export via the basolateral heteromeric organic solute transporter α-β (OSTα-OSTβ). In this study, we investigated the cytotoxic effects of enterocyte bile acid stasis in Ostα-/- mice, including the temporal relationship between intestinal injury and initiation of the enterohepatic circulation of bile acids. Methods Ileal tissue morphometry, histology, markers of cell proliferation, gene, and protein expression were analyzed in male and female wild-type and Ostα-/- mice at postnatal days 5, 10, 15, 20, and 30. Ostα-/-Asbt-/- mice were generated and analyzed. Bile acid activation of intestinal Nrf2-activated pathways was investigated in Drosophila. Results As early as day 5, Ostα-/- mice showed significantly increased ileal weight per length, decreased villus height, and increased epithelial cell proliferation. This correlated with premature expression of the Asbt and induction of bile acid–activated farnesoid X receptor target genes in neonatal Ostα-/- mice. Expression of reduced nicotinamide adenine dinucleotide phosphate oxidase-1 and Nrf2–anti-oxidant responsive genes were increased significantly in neonatal Ostα-/- mice at these postnatal time points. Bile acids also activated Nrf2 in Drosophila enterocytes and enterocyte-specific knockdown of Nrf2 increased sensitivity of flies to bile acid–induced toxicity. Inactivation of the Asbt prevented the changes in ileal morphology and induction of anti-oxidant response genes in Ostα-/- mice. Conclusions Early in postnatal development, loss of Ostα leads to bile acid accumulation, oxidative stress, and a restitution response in ileum. In addition to its essential role in maintaining bile acid homeostasis, Ostα-Ostβ functions to protect the ileal epithelium against bile acid–induced injury. NCBI Gene Expression Omnibus: GSE99579.

Serum Amyloid A1 Is an Epithelial Prorestitutive Factor

Several proteins endogenously produced during the process of intestinal wound healing have demonstrated prorestitutive properties. The presence of serum amyloid A1 (SAA1), an acute-phase reactant, within inflamed tissues, where it exerts chemotaxis of phagocytes, is well recognized; however, a putative role in intestinal wound repair has not been described. Herein, we show that SAA1 induces intestinal epithelial cell migration, spreading, and attachment through a formyl peptide receptor 2-dependent mechanism. Induction of the prorestitutive phenotype is concentration and time dependent and is associated with epithelial reactive oxygen species production and alterations in p130 Crk-associated substrate staining. In addition, using a murine model of wound recovery, we provide evidence that SAA1 is dynamically and temporally regulated, and that the elaboration of SAA1 within the wound microenvironment correlates with the influx of SAA1/CD11b coexpressing immune cells and increases in cytokines known to induce SAA expression. Overall, the present work demonstrates an important role for SAA in epithelial wound recovery and provides evidence for a physiological role in the wound environment.

Answer to March 2013 Photo Quiz

Answer: Helically Coiled Clostridium sp. Closely Related to Clostridium ramosum. A manual anaerobic identification panel was performed (RapID ANA II system; Remel, Lenexa, KS), which identified the organism as Clostridium septicum with 99% probability. Although the identification was of high probability, the identification was questioned because the Gram stain and colony morphology were not consistent with C. septicum. Commercial anaerobe kits are not consistently reliable in correctly identifying Clostridium to the species level (1). This is largely due to difficulties in interpreting biochemical reactions and the absence of profile numbers for some species (1). To aid in further identification of the isolate in this case, a sample taken from a colony growing on brucella agar with hemin and vitamin K was interrogated by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (Bruker Daltonics, Billerica, MA). Mass spectrum analysis identified the bacterium as Clostridium ramosum. The isolate was also forwarded to the Centers for Disease Control and Prevention (CDC) via the Georgia Public Health Laboratory, where 16S rRNA sequence analysis identified the bacterium as closely related to C. ramosum. C. ramosum was originally named Bacillus ramosus in 1898 and has a long history of name changes, including Nocardia ramosa (1931), Actinomyces ramosus (1934), Fusiformis ramosus (1936), Bacteroides ramosus (1937), and Ramibacterium ramosum (1938). It was finally placed in the genus Clostridium in 1971, when the bacterium was shown to form spores. Organisms originally named Catenabacterium filamentosum are now also classified as C. ramosum. C. ramosum and the helically coiled clostridia are described as having some features in common (2). All are obligately anaerobic, nonmotile, Gram-positive rods with rounded ends. Spore formation is notoriously difficult to detect and typically requires selection and/or prolonged incubation (e.g., 2 weeks). Unlike most other clostridia, they do not produce butyric acid as a product of glucose fermentation. All are commonly found as normal flora of the intestinal tracts of humans and some animals. They are divided into five groups based on cell morphology, carbohydrate fermentation, and the guanine-plus-cytosine (G+C) content of their DNA (2). Group I is C. ramosum, which consists of straight rods that may develop long filamentous forms under certain growth conditions. Groups II to V are a heterogenous collection of clostridia that are distinguished by their coiled morphology. Group II is Clostridium cocleatum, group III is Clostridium spiroforme, and groups IV and V are not yet named. While C. ramosum strains are usually described as straight rods, coiling morphology has been reported to occur in some strains identified as C. ramosum (2, 3). Historically, some of these C. ramosum strains were renamed based solely on the coiled morphology seen on the Gram stain (3). It may be desirable to find more-accurate phenotypic and genetic characteristics that distinguish the taxonomic genera of C. ramosum and the heterogeneous non-group 1 clostridia (4). Bacteremia caused by Clostridium sp. is rare and is occasionally polymicrobial. Clostridium perfringens, C. septicum, C. tertium, and C. sordelli are the species most commonly recovered from blood cultures. C. ramosum has also been reported, but bacteremia due to the coiled clostridia is very rare (3). The clinical significance of Clostridium species in the bloodstream is frequently unclear. Clostridial bacteremia usually has a gastrointestinal source and occurs most frequently in the setting of underlying medical conditions (e.g., intra-abdominal sepsis, other gastrointestinal disorders, pregnancy, malignancies, and alcoholism with aspiration) and, less commonly, following trauma or surgery (5, 6). Clostridia are often regarded as contaminants or as unimportant causes of transient bacteremia since their presence appears to be unrelated to the clinical condition (5). The clinical significance of Clostridium bacteremia should be interpreted with caution, however, regardless of species, because of the high risk of associated mortality in some patients, particularly those with underlying liver cirrhosis who do not receive appropriate therapy (5). Our patient was covered with broad-spectrum antibiotics (vancomycin and piperacillin-tazobactam) prior to receiving notification of the positive blood culture and Gram stain results. After the Gram stain results were reviewed and it was noted that no other blood cultures were positive, therapy was switched to ceftriaxone for spontaneous bacterial peritonitis prophylaxis since the patient had long-standing ascites but no recent taps indicating infection. (See page 739 in this issue [doi:10.1128/JCM.01268-12] for photo quiz case presentation.)

Photo Quiz: Bacteremia in a Patient with Hepatic Encephalopathy

A 65-year-old male with end-stage liver disease secondary to alcoholic cirrhosis was admitted to Emory University Hospital after experiencing progressive lethargy and displaying combative behavior. The patient was diagnosed with hepatic encephalopathy as well as hepatorenal syndrome. Inpatient work-up indicated that the patient was an appropriate liver transplant candidate, but he developed refractory hypotension within days of being placed on the transplant list. Although the patient was afebrile, the clinical team collected peripheral blood cultures (one set) to rule out bacteremia as a cause of the patients hypotension. Three days after collection, the patients anaerobic blood culture signaled positive (BacT/Alert SN standard anaerobic bottle; bioMerieux, Durham, NC) and the Gram stain revealed aggregates of curled and tightly coiled Gram-positive rods. Aerobic, anaerobic, and thioglycolate broth subcultures were inoculated. Three days later, smooth, circular, convex, semiopaque, gray-white, nonhemolytic colonies were observed growing on brucella agar with hemin and vitamin K (ABAP) and phenyl ethyl alcohol agar (PEA) under anaerobic conditions. A Gram stain of the colonies demonstrated organisms with morphologies similar to those seen in the blood culture, but in smaller aggregates (Fig. 1). Fig 1 Gram stain of colony growing on an anaerobic blood agar plate. Magnification, ×1,000 (oil). Ascites fluid cultures obtained the same day as the initial blood culture showed no aerobic or anaerobic growth. One set of blood cultures obtained 2 days after the initial blood culture also showed no growth. (For answer and discussion, see page 1062 in this issue [doi:10.1128/JCM.01279-12].)