Clinical validation of multiplex real-time PCR assays for detection of bacterial meningitis pathogens.

Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae are important causes of meningitis and other infections, and rapid, sensitive, and specific laboratory assays are critical for effective public health interventions. Singleplex real-time PCR assays have been developed to detect N. meningitidis ctrA, H. influenzae hpd, and S. pneumoniae lytA and serogroup-specific genes in the cap locus for N. meningitidis serogroups A, B, C, W135, X, and Y. However, the assay sensitivity for serogroups B, W135, and Y is low. We aimed to improve assay sensitivity and develop multiplex assays to reduce time and cost. New singleplex real-time PCR assays for serogroup B synD, W135 synG, and Y synF showed 100% specificity for detecting N. meningitidis species, with high sensitivity (serogroup B synD, 99% [75/76]; W135 synG, 97% [38/39]; and Y synF, 100% [66/66]). The lower limits of detection (LLD) were 9, 43, and 10 copies/reaction for serogroup B synD, W135 synG, and Y synF assays, respectively, a significant improvement compared to results for the previous singleplex assays. We developed three multiplex real-time PCR assays for detection of (i) N. meningitidis ctrA, H. influenzae hpd, and S. pneumoniae lytA (NHS assay); (ii) N. meningitidis serogroups A, W135, and X (AWX assay); and (iii) N. meningitidis serogroups B, C, and Y (BCY assay). Each multiplex assay was 100% specific for detecting its target organisms or serogroups, and the LLD was similar to that for the singleplex assay. Pairwise comparison of real-time PCR between multiplex and singleplex assays showed that cycle threshold values of the multiplex assay were similar to those for the singleplex assay. There were no substantial differences in sensitivity and specificity between these multiplex and singleplex real-time PCR assays.

Tumor-associated methylation of the putative tumor suppressor AJAP1 gene and association between decreased AJAP1 expression and shorter survival in patients with glioma.

Allelic loss of the short arm of chromosome 1 has been observed frequently in a wide spectrum of cancers, most frequently in oligodendroglioma. In our previous studies, we evaluated 177 oligodendroglial tumor samples and identified the AJAP1 gene (formerly Shrew1) in the consensus region of deletion. AJAP1 is a transmembrane protein found in adheren junctions and functions to inhibit glioma cell adhesion and migration. Whereas a putative tumor suppressor gene, we did not detect AJAP1 gene mutations. In subsequent studies, we found that AJAP1 was underexpressed in oligodendrogliomas relative to normal brain tissues. Bioinformatic analysis revealed the presence of CpG islands in the promoter of AJAP1. Methylation analysis of the AJAP1 promoter identified hypermethylation in 21% of oligodendrogliomas (n =27), and the degree of methylation correlated with low levels of AJAP1 expression (P = 0.045). The AJAP1 promoter was also highly methylated in a wide spectrum of cell lines (n = 22), including cell lines of glioblastoma. Analysis of the National Cancer Institute's REMBRANDT dataset, which contains 343 glioma samples, indicated that low AJAP1 gene expression was associated with decreased survival. Thus, both genetic (gene deletion) and epigenetic alterations (promoter methylation) are likely mechanisms that inactivate the putative tumor suppressor AJAP1 in gliomas, which contributes to poor prognosis.

Elevated phospho-S6 expression is associated with metastasis in adenocarcinoma of the lung.

PURPOSE: The primary objective of this study was to determine whether markers of differentiation and activation of the Akt pathway are associated with metastasis in adenocarcinoma of the lung. EXPERIMENTAL DESIGN: Paired primary and metastatic tumor samples were obtained from 41 patients who had undergone resection of both primary lung adenocarcinoma and brain metastatic lesions. Paired samples were compared for relative expression of thyroid transcription factor 1 (TTF-1) and E-cadherin as potential markers of differentiation. Activation of the Akt pathway was assessed by expression of p-Akt and p-S6. Biomarkers that showed relative discordance in expression between the matched pairs were then assessed in a cohort of 77 primary lung adenocarcinomas. Validation was done in an independent cohort of 82 primary lung adenocarcinomas. RESULTS: Among the 41 matched pairs, E-cadherin (23 discordant pairs) and TTF-1 (18 discordant pairs) were overexpressed in primary tumors (20 of 23 and 15 of 18, respectively). In contrast, p-S6 overexpression was significantly associated with metastatic tumors (20 of 21 discordant pairs). The expression of E-cadherin, p-S6, and TTF-1 was evaluated in 77 primary lung adenocarcinomas, in which high p-S6 expression was associated with shorter time to metastasis. The association of p-S6 with metastasis was then validated in an independent set of 82 tumors. In multivariable analysis, p-S6 expression was a negative independent predictor of metastasis-free survival after adjustment for tumor stage. CONCLUSIONS: The biomarker p-S6 is overexpressed in metastatic tumors. In primary tumors, higher p-S6 expression is associated with shorter metastatic-free survival. This biomarker has the potential for risk stratification in future clinical trials.

Adenovirus-Associated Acute Appendicitis: An Under-Recognized Relationship?

OBJECTIVE: Acute appendicitis (AA) is one of the most common causes of a surgical abdomen worldwide, occurring most frequently in those age 10 to 29 years. Adenovirus (ADV) is a rare but reported cause of AA in children and a well-recognized cause of intussusception in infants and young children. Annually, about 36,000 basic military trainees (BMTs) undergo initial training at Joint Base San Antonio Lackland, Texas. Before reintroduction of the ADV 4/7 vaccine in November 2011, one-third of BMTs developed an adenoviral upper respiratory tract infection (URI) during the 8.5 weeks of training. We hypothesized that ADV may be a common cause of AA in the BMT population given their young age and high incidence of adenoviral URIs. The objective of this study was to determine the frequency with which ADV, cytomegalovirus (CMV), Epstein-Barr virus (EBV), and enterovirus were associated with AA in a population of young adults. MATERIALS AND METHODS: This study was a retrospective review of patient charts and existing pathological tissue specimens of all BMTs who underwent appendectomy at the Wilford Hall Medical Center from January 1, 2003, to August 31, 2011. Pathological tissue samples from 112 BMTs were assayed by quantitative polymerase chain reaction (qPCR) and immunohistochemistry (IHC) for viral targets. RESULTS: ADV DNA was detected in 16 of 112 samples (14%) via qPCR: ADV 4 in 13 cases, ADV B14 in 1 case, and nontypable ADV in 2 cases. IHC was positive in only the ADV B14 case (0.9%). All cases were negative for CMV, EBV, and enterovirus. CONCLUSION: By using qPCR, this study demonstrated an association between ADV and AA higher than has been previously reported: ADV was detected in 14% of AA cases in this series versus in only 0.23% of AA cases in previous studies (p < 0.01). There was no evidence of CMV, EBV, or enterovirus association with AA in this study. Comparison of qPCR to IHC shows that histologic analysis may overlook evidence of ADV in appendiceal tissue: qPCR is significantly more sensitive than light microscopy and IHC for detecting ADV in this setting. Because ADV 4 was detected in 81% of those with positive qPCR, the recently licensed live oral ADV vaccine might be useful for primary prevention against AA. Prospective studies evaluating young adults presenting with AA for evidence of infection with ADV are needed to determine if a causal relationship exists.

The SHREW1 gene, frequently deleted in oligodendrogliomas, functions to inhibit cell adhesion and migration.

Allelic loss of the short arm of chromosome 1 has been observed frequently in oligodendroglioma (60-80%). We evaluated 177 oligodendroglial tumor samples and defined a consensus region of deletion of approximately 630 kb. This region contains a single gene, SHREW1, which encodes a novel transmembrane protein in adherens junctions. Whereas a mutation was not detected in the coding region of the SHREW1 gene in oligodendrogliomas, restoration of SHREW1 expression resulted in suppression of cell adhesion and migration. Thus, SHREW1 inactivation may play a role in the development of oligodendroglial tumors.

Attenuated expression of DFFB is a hallmark of oligodendrogliomas with 1p-allelic loss.

Allelic loss of chromosome 1p is frequently observed in oligodendroglioma. We screened 177 oligodendroglial tumors for 1p deletions and found 6 tumors with localized 1p36 deletions. Several apoptosis regulation genes have been mapped to this region, including Tumor Protein 73 (p73), DNA Fragmentation Factor subunits alpha (DFFA) and beta (DFFB), and Tumor Necrosis Factor Receptor Superfamily Members 9 and 25 (TNFRSF9, TNFRSF25). We compared expression levels of these 5 genes in pairs of 1p-loss and 1p-intact tumors using quantitative reverse-transcriptase PCR (QRTPCR) to test if 1p deletions had an effect on expression. Only the DFFB gene demonstrated decreased expression in all tumor pairs tested. Mutational analysis did not reveal DFFB mutations in 12 tested samples. However, it is possible that DFFB haploinsufficiency from 1p allelic loss is a contributing factor in oligodendroglioma development.

Pandemic influenza virus 2009 H1N1 and adenovirus in a high risk population of young adults: epidemiology, comparison of clinical presentations, and coinfection.

BACKGROUND: In 2009, pandemic H1N1 influenza virus (2009 H1N1) emerged worldwide, causing morbidity and mortality that disproportionately affected young adults. Upper respiratory infection (URI), largely due to adenovirus, is an endemic cause of morbidity in military training. Whether clinical presentations differ or excess morbidity results from coinfection is unclear. METHODS: The Center for Advanced Molecular Detection evaluates epidemiology and rapid diagnostics of respiratory pathogens in trainees with URI. From May 1, 2009, to November 30, 2009, demographic, clinical, and PCR data from throat and nasal specimens for adenovirus and 2009 H1N1 were prospectively collected. RESULTS: 375 trainees with URI enrolled and were tested for both adenovirus and 2009 H1N1 by PCR (median age 20; 89% male). Adenovirus PCR was positive in 72% (96% serotype E-4) and 2009 H1N1 in 20%. Males were more likely to have adenovirus and females more likely to have 2009 H1N1 (p  =  0.047). Subjects with 2009 H1N1 presented an average of 1 week earlier in training, had shorter illness duration before enrollment, less sore throat, diarrhea, and fewer abnormal findings on throat exam. Coryza and cough were more common with 2009 H1N1 compared to adenovirus. Subjects with 2009 H1N1 were less likely to have adenovirus than those without, despite persistently high frequencies of adenovirus detections during peak 2009 H1N1 weeks (15% vs. 83%, p < 0.01). Coinfection with adenovirus and 2009 H1N1 was rare (4%). Rates of hospitalization and pneumonia did not differ between the adenovirus, 2009 H1N1, or coinfected groups. CONCLUSION: Military trainees with 2009 H1N1 vs. adenovirus have differing clinical presentations, and males are more likely to have adenovirus. Despite high frequencies of adenovirus infection, coinfection with adenovirus and 2009 H1N1 is rare and apparently does not result in increased morbidity.

A multigene predictor of outcome in glioblastoma.

Only a subset of patients with newly diagnosed glioblastoma (GBM) exhibit a response to standard therapy. To date, a biomarker panel with predictive power to distinguish treatment sensitive from treatment refractory GBM tumors does not exist. An analysis was performed using GBM microarray data from 4 independent data sets. An examination of the genes consistently associated with patient outcome, revealed a consensus 38-gene survival set. Worse outcome was associated with increased expression of genes associated with mesenchymal differentiation and angiogenesis. Application to formalin fixed-paraffin embedded (FFPE) samples using real-time reverse-transcriptase polymerase chain reaction assays resulted in a 9-gene subset which appeared robust in these samples. This 9-gene set was then validated in an additional independent sample set. Multivariate analysis confirmed that the 9-gene set was an independent predictor of outcome after adjusting for clinical factors and methylation of the methyl-guanine methyltransferase promoter. The 9-gene profile was also positively associated with markers of glioma stem-like cells, including CD133 and nestin. In sum, a multigene predictor of outcome in glioblastoma was identified which appears applicable to routinely processed FFPE samples. The profile has potential clinical application both for optimization of therapy in GBM and for the identification of novel therapies targeting tumors refractory to standard therapy.

Epidermal growth factor receptor variant III status defines clinically distinct subtypes of glioblastoma.

PURPOSE: The clinical significance of epidermal growth factor receptor variant III (EGFRvIII) expression in glioblastoma multiforme (GBM) and its relationship with other key molecular markers are not clear. We sought to evaluate the clinical significance of GBM subtypes as defined by EGFRvIII status. PATIENTS AND METHODS: The expression of EGFRvIII was assessed by immunohistochemistry in 649 patients with newly diagnosed GBM. These data were then examined in conjunction with the expression of phospho-intermediates (in a subset of these patients) of downstream AKT and Ras pathways and YKL-40 as well as with known clinical risk factors, including the Radiation Therapy Oncology Group's recursive partitioning analysis (RTOG-RPA) class. RESULTS: The RTOG-RPA class was highly predictive of survival in EGFRvIII-negative patients but much less predictive in EGFRvIII-positive patients. These findings were seen in both an initial test set (n = 268) and a larger validation set (n = 381). Similarly, activation of the AKT/MAPK pathways and YKL-40 positivity were predictive of poor outcome in EGFRvIII-negative patients but not in EGFRvIII-positive patients. Pair-wise combinations of markers identified EGFRvIII and YKL-40 as prognostically important. In particular, outcome in patients with EGFRvIII-negative/YKL-40-negative tumors was significantly better than the outcome in patients with the other three combinations of these two markers. CONCLUSION: Established prognostic factors in GBM were not predictive of outcome in the EGFRvIII-positive subset, although this requires confirmation in independent data sets. GBMs negative for both EGFRvIII and YKL-40 show less aggressive behavior.

Expression of transcription factor E2F1 and telomerase in glioblastomas: mechanistic linkage and prognostic significance.

BACKGROUND: Several tumor suppressor pathways have been identified as modulators of telomerase function. We examined the functional role of the retinoblastoma-E2F1 pathway in regulating telomerase activity in malignant gliomas. METHODS: Adenovirus vectors were used to transfer cDNAs into human glioblastoma and sarcoma cells. Telomerase activity was assessed with a telomere repeat amplification protocol. Promoter activity in cancer cells was assessed with promoter-luciferase reporter constructs. Promoter binding was assessed with the chromatin immunoprecipitation (ChIP) assay. We isolated astrocytes from E2F1 transgenic mice and normal mice for in vivo studies. We evaluated the expression of E2F1 and hTERT (the catalytic subunit of human telomerase) mRNAs by reverse transcriptase-polymerase chain reaction and proteins in human glioblastoma samples by immunoblot analysis. Associations between survival among 61 glioblastoma multiforme patients and expression of E2F1 and hTERT mRNA and protein were examined with Kaplan-Meier analysis, the log-rank test, and Cox proportional hazards regression models. All statistical tests were two-sided. RESULTS: Ectopic E2F1 expression increased hTERT promoter activity in cancer cells. We detected an interaction between E2F1 protein and the hTERT promoter. Transgenic E2F1 astrocytes contained functional telomerase protein. E2F1 mRNA expression and hTERT mRNA expression were statistically significantly correlated in human glioblastoma specimens (R = .8; P < .001). Longer median survival was statistically significantly associated with lower E2F1 mRNA expression in tumors (103.6 weeks) rather than with higher expression (46.1 weeks) (difference = 57.5 weeks; 95% confidence interval [CI] = 14.7 to 159.7; log-rank P = .002). E2F1 mRNA was the only factor that was statistically significantly associated with overall survival in a multivariable model (P = .04). Among 27 patients with glioblastoma multiforme samples, the expression of E2F1 protein was statistically significantly associated with survival (log-rank P < .001). CONCLUSIONS: E2F1 may participate in telomerase activity regulation in malignant glioma cells. Its expression appears to be strongly associated with the survival of patients with malignant brain tumors.

The prognostic impact of histology and 1p/19q status in anaplastic oligodendroglial tumors.

BACKGROUND: It has been reported previously that the combined loss of chromosomal arms 1p and 19q is a significant predictor of outcome for patients with anaplastic oligodendroglial (AO) tumors and that such chromosomal loss correlates with classic histology in AO. The authors sought to determine whether histology was an equivalent or superior predictor of outcome compared with 1p/19q status in 131 patients with AO tumors. METHODS: The status of 1p and 19q was determined using real-time, quantitative polymerase chain reaction analysis and/or fluorescence in situ hybridization. Clinical features (response to adjuvant therapy and tumor location) and molecular genetic abnormalities (9p and 10q deletions, overexpression of p53 and epidermal growth factor receptor) were determined on available specimens. Histologic assessments for classic oligodendroglial features were performed by five neuropathologists. RESULTS: Classic histology was associated closely with 1p/19q loss, as reported previously. Patients who had tumors that were considered classic by at least four of the five neuropathologists showed significantly increased progression-free and overall survival compared with the patients who had less classic tumors. The authors also tested the correlation between 1p/19q status and outcome in subsets of patients stratified according to classic tumor features. The association of 1p/19q status with survival was related closely to the presence of classic histology. Loss of 1p/19q was predictive of improved outcome only among patients who had tumors with classic histologic features. CONCLUSIONS: The current results suggested that, in addition to 1p/19q status, histologic features contribute information to the prediction of outcome in patients with AO. Loss of 1p and 19q appeared to be a prognostic marker only in the subset of patients who had AO tumors with classic histologic features.