Absence of Cytomegalovirus in Glioblastoma and Other High-grade Gliomas by Real-time PCR, Immunohistochemistry, and In Situ Hybridization.

Purpose: Reports of cytomegalovirus (CMV) detection in high-grade gliomas (HGG)/glioblastoma have been conflicting. We undertook a comprehensive approach to determine the presence or absence of CMV in tissue, plasma, and serum of HGG patients.Experimental Design: In a retrospective arm, 25 fresh frozen tissues from glioblastoma patients were tested for CMV by real-time PCR. Tissue microarrays from 70 HGG patients were tested by IHC and 20 formalin-fixed paraffin-embedded (FFPE) glioblastoma tissues by IHC and chromogenic in situ hybridization (CISH), targeting CMV-encoded IE1/2 and pp65. In a prospective arm, 18 patients with newly diagnosed HGG provided tissue and blood samples.Results: All retrospectively collected tissues were negative for CMV by all methods. In the prospective cohort, 18 patients with newly diagnosed HGG provided blood samples at the time of diagnosis and during follow-up. Of 38 plasma specimens, CMV DNA was detected in 3 of 18 samples at baseline and 1 of 20 follow-up samples. Serum CMV IgG was positive in 8 of 15 (53%) of patients. Among the FFPE samples tested in the prospective arm, all were negative for CMV by IHC, CISH, and PCR.Conclusions: Utilizing 6 highly sensitive assays with three orthogonal technologies on multiple specimens and specimen types, no evidence for CMV in glioblastoma tissues was found. Our findings call for multicenter blinded analyses of samples collected from different geographical areas with agreed upon study designs and determination of causality or lack thereof of CMV in HGG/glioblastoma for future guidance on the necessary antiviral and/or CMV-based therapies. Clin Cancer Res; 23(12); 3150-7. ©2016 AACR.

The Accuracy of Clinical Diagnosis of Oral Lesions and Patient-Specific Risk Factors that Affect Diagnosis.

PURPOSE: To examine the rate of discrepancy between clinical impression and histologic diagnosis of oral lesions in patients undergoing biopsy examination and to determine whether there are patient-specific variables associated with a higher rate of discrepancy. MATERIALS AND METHODS: The authors designed and implemented a retrospective cohort study that consisted of patients who underwent biopsy examination of oral lesions from 2005 through 2013 by oral and maxillofacial surgeons at the Massachusetts General Hospital. Accuracy was determined by comparing the clinical impression with the final histologic diagnosis. Clinical and histologic diagnoses were categorized as premalignant or malignant (group 1) or benign (group 2). The primary outcome variable was concordance (yes vs no) between clinical impression and histopathologic diagnosis. The effect of individual predictor variables (age, gender, duration, American Society of Anesthesiology status, cancer history, radiation therapy history, medications, alcohol abuse, and tobacco history) on outcome also was evaluated through univariate and multivariate regression analyses. RESULTS: The study sample was composed of 1,003 oral lesions (74 pathologically confirmed premalignant or malignant and 929 benign) from patients with a mean age of 44.8 years. Of the lesions evaluated, concordance between exact clinical and histologic diagnoses was found in 61% of cases. Overall, the clinical impression, reported as benign versus premalignant or malignant, was 48.6% sensitive and 98.1% specific. Clinicians accurately identified lesions as benign in 95.9% of cases. The most common of these were fibromas (positive predictive value [PPV], 99.2%), mucoceles (PPV, 98.1%), and squamous papillomas (PPV, 96.3%). Several independent risk factors were associated with discrepancy: radiation therapy history (P = .0102), male gender (P = .0381), and patient age (P = .0468). CONCLUSION: The results of this study suggest that the clinical impression, although highly accurate for common benign conditions, is not an acceptable alternative to definitive biopsy findings in other cases, particularly in cases of premalignancy or malignancy. In addition, patients with identified independent risk factors (age, gender, and radiation therapy) should receive timely biopsy examination.

Diagnostic performance of two highly multiplexed respiratory virus assays in a pediatric cohort.

BACKGROUND: Rapid detection of respiratory viruses is important for management and infection control in hospitalized patients. Multiplex nucleic acid tests (NATs) have begun to replace conventional methods as gold standards for respiratory virus detection. OBJECTIVE: To compare the performance of two large multiplex NATS, ResPlex II (RPII) and Respiratory Virus Surveillance kit with electrospray ionization mass spectrometry (RVS/MS) using nasopharyngeal aspirates (NPAs) from hospitalized children who had been tested previously with conventional methods. STUDY DESIGN: Stored residual NPAs (N=306) were tested concomitantly by RPII and RVS/MS. Alternate NATs were used to adjudicate discordant results. RESULTS: More viruses were detected with multiplex NATs (RPII, 110; RVS/MS, 109) than conventional assays (86); diagnostic gain was primarily for fastidious viruses (coronaviruses and enteroviruses [EVs]/human rhinoviruses [HRVs]). Total positive and negative agreement between the multiplex NATs for all viruses detected was quite high (86% positive agreement, 99% negative agreement). Most individual viruses were detected with fairly equivalent accuracy by the multiplex NATs, except for adenoviruses (RPII sensitivity 40%) and human metapneumovirus (RVS/MS sensitivity 42%). RPII had the advantage of detecting EVs and HRVs, however, it demonstrated considerable EV/HRV cross-reactivity (29 HRV-positive specimens by real-time PCR were positive for EV by RPII and 21 specimens positive for HRV only by RT-PCR were dual positive for EV/HRV by RPII). RPII also had reduced sensitivity for HRV detection (in 36 specimens, HRV was detected by RT-PCR but not by RPII). CONCLUSIONS: Both multiplex NATs were promising, but had notable limitations.

Gangliosides block Aggregatibacter Actinomycetemcomitans leukotoxin (LtxA)-mediated hemolysis.

Aggregatibacter actinomycetemcomitans is an oral pathogen and etiologic agent of localized aggressive periodontitis. The bacterium is also a cardiovascular pathogen causing infective endocarditis. A. actinomycetemcomitans produces leukotoxin (LtxA), an important virulence factor that targets white blood cells (WBCs) and plays a role in immune evasion during disease. The functional receptor for LtxA on WBCs is leukocyte function antigen-1 (LFA-1), a ß-2 integrin that is modified with N-linked carbohydrates. Interaction between toxin and receptor leads to cell death. We recently discovered that LtxA can also lyse red blood cells (RBCs) and hemolysis may be important for pathogenesis of A. actinomycetemcomitans. In this study, we further investigated how LtxA might recognize and lyse RBCs. We found that, in contrast to a related toxin, E. coli α-hemolysin, LtxA does not recognize glycophorin on RBCs. However, gangliosides were able to completely block LtxA-mediated hemolysis. Furthermore, LtxA did not show a preference for any individual ganglioside. LtxA also bound to ganglioside-rich C6 rat glioma cells, but did not kill them. Interaction between LtxA and C6 cells could be blocked by gangliosides with no apparent specificity. Gangliosides were only partially effective at preventing LtxA-mediated cytotoxicity of WBCs, and the effect was only observed when a high ratio of ganglioside:LtxA was used over a short incubation period. Based on the results presented here, we suggest that because of the similarity between N-linked sugars on LFA-1 and the structures of gangliosides, LtxA may have acquired the ability to lyse RBCs.