Proteins inform survival-based differences in patients with glioblastoma.

BACKGROUND: Improving the care of patients with glioblastoma (GB) requires accurate and reliable predictors of patient prognosis. Unfortunately, while protein markers are an effective readout of cellular function, proteomics has been underutilized in GB prognostic marker discovery. METHODS: For this study, GB patients were prospectively recruited and proteomics discovery using liquid chromatography-mass spectrometry analysis (LC-MS/MS) was performed for 27 patients including 13 short-term survivors (STS) (≤10 months) and 14 long-term survivors (LTS) (≥18 months). RESULTS: Proteomics discovery identified 11 941 peptides in 2495 unique proteins, with 469 proteins exhibiting significant dysregulation when comparing STS to LTS. We verified the differential abundance of 67 out of these 469 proteins in a small previously published independent dataset. Proteins involved in axon guidance were upregulated in STS compared to LTS, while those involved in p53 signaling were upregulated in LTS. We also assessed the correlation between LS MS/MS data with RNAseq data from the same discovery patients and found a low correlation between protein abundance and mRNA expression. Finally, using LC-MS/MS on a set of 18 samples from 6 patients, we quantified the intratumoral heterogeneity of more than 2256 proteins in the multisample dataset. CONCLUSIONS: These proteomic datasets and noted protein variations present a beneficial resource for better predicting patient outcome and investigating potential therapeutic targets.

Retrospective clinical trial experimentally validates glioblastoma genome-wide pattern of DNA copy-number alterations predictor of survival.

Modeling of genomic profiles from the Cancer Genome Atlas (TCGA) by using recently developed mathematical frameworks has associated a genome-wide pattern of DNA copy-number alterations with a shorter, roughly one-year, median survival time in glioblastoma (GBM) patients. Here, to experimentally test this relationship, we whole-genome sequenced DNA from tumor samples of patients. We show that the patients represent the U.S. adult GBM population in terms of most normal and disease phenotypes. Intratumor heterogeneity affects ≈ 11 % and profiling technology and reference human genome specifics affect <1% of the classifications of the tumors by the pattern, where experimental batch effects normally reduce the reproducibility, i.e., precision, of classifications based upon between one to a few hundred genomic loci by >30%. With a 2.25-year Kaplan-Meier median survival difference, a 3.5 univariate Cox hazard ratio, and a 0.78 concordance index, i.e., accuracy, the pattern predicts survival better than and independent of age at diagnosis, which has been the best indicator since 1950. The prognostic classification by the pattern may, therefore, help to manage GBM pseudoprogression. The diagnostic classification may help drugs progress to regulatory approval. The therapeutic predictions, of previously unrecognized targets that are correlated with survival, may lead to new drugs. Other methods missed this relationship in the roughly 3B-nucleotide genomes of the small, order of magnitude of 100, patient cohorts, e.g., from TCGA. Previous attempts to associate GBM genotypes with patient phenotypes were unsuccessful. This is a proof of principle that the frameworks are uniquely suitable for discovering clinically actionable genotype-phenotype relationships.

Multiscale, multimodal analysis of tumor heterogeneity in IDH1 mutant vs wild-type diffuse gliomas.

Glioma is recognized to be a highly heterogeneous CNS malignancy, whose diverse cellular composition and cellular interactions have not been well characterized. To gain new clinical- and biological-insights into the genetically-bifurcated IDH1 mutant (mt) vs wildtype (wt) forms of glioma, we integrated data from protein, genomic and MR imaging from 20 treatment-naïve glioma cases and 16 recurrent GBM cases. Multiplexed immunofluorescence (MxIF) was used to generate single cell data for 43 protein markers representing all cancer hallmarks, Genomic sequencing (exome and RNA (normal and tumor) and magnetic resonance imaging (MRI) quantitative features (protocols were T1-post, FLAIR and ADC) from whole tumor, peritumoral edema and enhancing core vs equivalent normal region were also collected from patients. Based on MxIF analysis, 85,767 cells (glioma cases) and 56,304 cells (GBM cases) were used to generate cell-level data for 24 biomarkers. K-means clustering was used to generate 7 distinct groups of cells with divergent biomarker profiles and deconvolution was used to assign RNA data into three classes. Spatial and molecular heterogeneity metrics were generated for the cell data. All features were compared between IDH mt and IDHwt patients and were finally combined to provide a holistic/integrated comparison. Protein expression by hallmark was generally lower in the IDHmt vs wt patients. Molecular and spatial heterogeneity scores for angiogenesis and cell invasion also differed between IDHmt and wt gliomas irrespective of prior treatment and tumor grade; these differences also persisted in the MR imaging features of peritumoral edema and contrast enhancement volumes. A coherent picture of enhanced angiogenesis in IDHwt tumors was derived from multiple platforms (genomic, proteomic and imaging) and scales from individual proteins to cell clusters and heterogeneity, as well as bulk tumor RNA and imaging features. Longer overall survival for IDH1mt glioma patients may reflect mutation-driven alterations in cellular, molecular, and spatial heterogeneity which manifest in discernable radiological manifestations.

Metagenomic next-generation sequencing aids the diagnosis of viral infections in febrile returning travellers.

OBJECTIVES: Travel-associated infections are challenging to diagnose because of the broad spectrum of potential aetiologies. As a proof-of-principle study, we used MNGS to identify viral pathogens in clinical samples from returning travellers in a single center to explore its suitability as a diagnostic tool. METHODS: Plasma samples from 40 returning travellers presenting with a fever of ≥38°C were sequenced using MNGS on the Illumina MiSeq platform and compared with standard-of-care diagnostic assays. RESULTS: In total, 11/40 patients were diagnosed with a viral infection. Standard of care diagnostics revealed 5 viral infections using plasma samples; dengue virus 1 (n = 2), hepatitis E (n = 1), Ebola virus (n = 1) and hepatitis A (n = 1), all of which were detected by MNGS. Three additional patients with Chikungunya virus (n = 2) and mumps virus were diagnosed by MNGS only. Respiratory infections detected by nasal/throat swabs only were not detected by MNGS of plasma. One patient had infection with malaria and mumps virus during the same admission. CONCLUSIONS: MNGS analysis of plasma samples improves the sensitivity of diagnosis of viral infections and has potential as an all-in-one diagnostic test. It can be used to identify infections that have not been considered by the treating physician, co-infections and new or emerging pathogens. SUMMARY: Next generation sequencing (NGS) has potential as an all-in-one diagnostic test. In this study we used NGS to diagnose returning travellers with acute febrile illness in the UK, highlighting cases where the diagnosis was missed using standard methods.

Brain tumor biobanking in the precision medicine era: building a high-quality resource for translational research in neuro-oncology.

The growth of precision medicine has made access to biobanks with high-quality, well-annotated neuro-oncology biospecimens critical. Developing and maintaining neuro-oncology biobanks is best accomplished through multidisciplinary collaboration between clinicians and researchers. Balancing the needs and leveraging the skills of all stakeholders in this multidisciplinary effort is of utmost importance. Collaboration with a multidisciplinary team of clinicians, health care team members, and institutions, as well as patients and their families, is essential for access to participants in order to obtain informed consent, collect samples under strict standard operating procedures, and accurate and relevant clinical annotation. Once a neuro-oncology biobank is established, development and implementation of policies related to governance and distribution of biospecimens (both within and outside the institution) is of critical importance for sustainability. Proper implementation of a governance process helps to ensure that the biospecimens and data can be utilized in research with the largest potential benefit. New NIH and peer-reviewed journal policies related to public sharing of 'omic' data generated from stored biospecimens create new ethical challenges that must be addressed in developing informed consents, protocols, and standard operating procedures. In addition, diversification of sources of funding for the biobanks is needed for long-term sustainability.

Methylation markers of malignant potential in meningiomas.

OBJECT: Although most meningiomas are benign, about 20% are atypical (Grade II or III) and have increased mortality and morbidity. Identifying tumors with greater malignant potential can have significant clinical value. This validated genome-wide methylation study comparing Grade I with Grade II and III meningiomas aims to discover genes that are aberrantly methylated in atypical meningiomas. METHODS: Patients with newly diagnosed meningioma were identified as part of the Ohio Brain Tumor Study. The Infinium HumanMethylation27 BeadChip (Illumina, Inc.) was used to interrogate 27,578 CpG sites in 14,000 genes per sample for a discovery set of 33 samples (3 atypical). To verify the results, the Infinium HumanMethylation450 BeadChip (Illumina, Inc.) was used to interrogate 450,000 cytosines at CpG loci throughout the genome for a verification set containing 7 replicates (3 atypical), as well as 12 independent samples (6 atypical). A nonparametric Wilcoxon exact test was used to test for difference in methylation between benign and atypical meningiomas in both sets. Heat maps were generated for each set. Methylation results were validated for the 2 probes with the largest difference in methylation intensity by performing Western blot analysis on a set of 20 (10 atypical) samples, including 11 replicates. RESULTS: The discovery array identified 95 probes with differential methylation between benign and atypical meningiomas, creating 2 distinguishable groups corresponding to tumor grade when visually examined on a heat map. The validation array evaluated 87 different probes and showed that 9 probes were differentially methylated. On heat map examination the results of this array also suggested the existence of 2 major groups that corresponded to histological grade. IGF2BP1 and PDCD1, 2 proteins that can increase the malignant potential of tumors, were the 2 probes with the largest difference in intensity, and for both of these the atypical meningiomas had a decreased median production of protein, though this was not statistically significant (p = 0.970 for IGF2BP1 and p = 1 for PDCD1). CONCLUSIONS: A genome-wide methylation analysis of benign and atypical meningiomas identified 9 genes that were reliably differentially methylated, with the strongest difference in IGF2BP1 and PDCD1. The mechanism why increased methylation of these sites is associated with an aggressive phenotype is not evident. Future research may investigate this mechanism, as well as the utility of IGF2BP1 as a marker for pathogenicity in otherwise benign-appearing meningiomas.

Family history of cancer in benign brain tumor subtypes versus gliomas.

PURPOSE: Family history is associated with gliomas, but this association has not been established for benign brain tumors. Using information from newly diagnosed primary brain tumor patients, we describe patterns of family cancer histories in patients with benign brain tumors and compare those to patients with gliomas. METHODS: Newly diagnosed primary brain tumor patients were identified as part of the Ohio Brain Tumor Study. Each patient was asked to participate in a telephone interview about personal medical history, family history of cancer, and other exposures. Information was available from 33 acoustic neuroma (65%), 78 meningioma (65%), 49 pituitary adenoma (73.1%), and 152 glioma patients (58.2%). The association between family history of cancer and each subtype was compared with gliomas using unconditional logistic regression models generating odds ratios (ORs) and 95% confidence intervals. RESULTS: There was no significant difference in family history of cancer between patients with glioma and benign subtypes. CONCLUSION: The results suggest that benign brain tumor may have an association with family history of cancer. More studies are warranted to disentangle the potential genetic and/or environmental causes for these diseases.

Tobacco use in youth with mental illnesses.

Despite an abundance of literature documenting the prevalence and dangers of youth tobacco use, there is a relative dearth of literature in the area of effective cessation treatments for youth (Fiore et al. in Clinical tobacco guideline: treating tobacco use and dependence, 2008). Additionally, although it has been widely accepted that mental illness is highly correlated with tobacco use and dependence, little research has been done to support prevention and cessation efforts for youth with mental illnesses. This paper summarizes the literature on tobacco use and cessation in youth, with a focus on describing the existing knowledge base for youth with mental illnesses.

Multiple perspectives on tobacco use among youth with mental health disorders and addictions.

PURPOSE: Youth and young adults with mental health disorders and addictions are at a high risk of becoming nicotine dependent, and at least half will die of tobacco-related diseases. In comparison to the general population, this population also faces neurobiological and psychosocial vulnerabilities. There is a critical need for community services and research targeting tobacco interventions for these individuals. METHODS: A concurrent mixed methods study was conducted by collecting data from in-depth key informant interviews, focus groups, and a survey. Qualitative key informant interviews with healthcare professionals (n = 11) and youth focus groups (n = 32) were conducted by using semi-structured questioning regarding barriers and facilitators to tobacco interventions. Content analysis was used to code transcripts and categorize themes. Survey data were also collected from 230 smokers ages 13 to 17 years (n = 62) and young adults ages 18 to 25 years (n = 40) at three community mental health centers. The survey inquired about tobacco use, motivation to quit, history of quit attempts, and treatment preferences. RESULTS: Five thematic categories were identified in both the adult key informant interviews and the focus groups with youth: (1) motivation to quit, (2) cessation treatment needs, (3) social influence, (4) barriers to treatment, and (5) tobacco-free policy. Among those surveyed, 44% currently smoked. Youth and young adult survey respondents who smoked were often motivated to quit, few had used proven tobacco cessation aids, but there was interest in access to nicotine replacement therapy. CONCLUSION: Merged qualitative and quantitative findings support past literature regarding youth in the general population but also expand upon our knowledge of issues specific to youth and young adults with mental health disorders and addictions. Findings suggest interventions warranting further attention in community treatment settings.

A method comparison study to assess the reliability of urine collection pads as a means of obtaining urine specimens from non-toilet-trained children for microbiological examination.

BACKGROUND: In young, non-toilet-trained children, the collection of a urine sample for microbiology can be challenging, with the application of a urine bag being the main method of collection. However, recent research has shown that absorbent pads can be used yielding similar results to bag specimens. However, weaknesses in study design erode confidence in research findings. Therefore, improvements in research design are required to fully evaluate the reliability of pad collection. AIMS OF THE STUDY: This pilot study sought to test the feasibility of a technique for the collection of concurrent bag/pad urine samples from non-toilet-trained children, and to assess the reliability of urine pads over bags as a collection method for urine specimens for microbiological evaluation. DESIGN: A pilot, method comparison study. METHODS: Twenty concurrent bag and pad specimens were collected from non-toilet-trained children, following parental consent. Urine specimens were analysed for presence or absence of white cell count (WBC), and bacterial growth, using standard laboratory methods. DATA ANALYSIS: The Kappa (kappa) statistics and confidence interval (CI) estimation were used to assess agreement between the two collection methods. RESULTS: Despite concurrent samples there was a lack of agreement between bag and pad specimens on both main outcome measures. Agreement between bag and pad specimens for the presence of WBC yielded a kappa=0.10 (95% CI: 0.19, 0.39), indicating poor agreement, while a kappa of 0.5 (95% CI: 0.12, 0.88) was calculated for the degree of agreement in bacterial growth reflecting moderate agreement. Differences in proportions of the presence of WBC between bag and pad did not quite reach significance at the 5% level 0.2 (95% CI: 0.00, 0.42, P=0.062). For cultures the difference was calculated as 0.15 (95% CI: 0.05, 0.35, P=0.125). CONCLUSION: The pilot study demonstrates that concurrent urine samples can be obtained without difficulty. Despite poor to moderate agreement on outcome measures the level of agreement is greater than reported in those other studies, that use non-current methods of urine collection, suggesting an advantage of the concurrent technique. It is recommended that larger scale studies be undertaken using the concurrent collection technique to assess reliability of these findings.