Does the Use of the "Proseek® Multiplex Inflammation I Panel" Demonstrate a Difference in Local and Systemic Immune Responses in Endometriosis Patients with or without Deep-Infiltrating Lesions?

Endometriotic lesions are able to infiltrate surrounding tissue. This is made possible partly by an altered local and systemic immune response that helps achieve neoangiogenesis, cell proliferation and immune escape. Deep-infiltrating endometriosis (DIE) differs from other subtypes through the invasion of its lesions over 5 mm into affected tissue. Despite the invasive nature of these lesions and the wider range of symptoms they can trigger, DIE is described as a stable disease. This elicits the need for a better understanding of the underlying pathogenesis. We used the "Proseek® Multiplex Inflammation I Panel" in order to simultaneously detect 92 inflammatory proteins in plasma and peritoneal fluid (PF) of controls and patients with endometriosis, as well as in particular patients with DIE, in order to gain a better insight into the systemically and locally involved immune response. Extracellular newly identified receptor for advanced gycation end-products binding protein (EN-RAGE), C-C motif Chemokine ligand 23 (CCL23), Eukaryotic translation initiation factor 4-binding protein 1 (4E-BP1) and human glial cell-line derived neurotrophic factor (hGDNF) were significantly increased in plasma of endometriosis patients compared to controls, whereas Hepatocyte Growth factor (HGF) and TNF-related apoptosis inducing ligand (TRAIL) were decreased. In PF of endometriosis patients, we found Interleukin 18 (IL-18) to be decreased, yet Interleukin 8 (IL-8) and Interleukin 6 (IL-6) to be increased. TNF-related activation-induced cytokine (TRANCE) and C-C motif Chemokine ligand 11 (CCL11) were significantly decreased in plasma, whereas C-C motif Chemokine ligand 23 (CCL23), Stem Cell Factor (SCF) and C-X-C motif chemokine 5 (CXCL5) were significantly increased in PF of patients with DIE compared to endometriosis patients without DIE. Although DIE lesions are characterized by increased angiogenetic and pro-inflammatory properties, our current study seems to support the theory that the systemic immune system does not play a major role in the pathogenesis of these lesions.

Random rotation for identifying differentially expressed genes with linear models following batch effect correction.

MOTIVATION: Data generated from high-throughput technologies such as sequencing, microarray and bead-chip technologies are unavoidably affected by batch effects (BEs). Large effort has been put into developing methods for correcting these effects. Often, BE correction and hypothesis testing cannot be done with one single model, but are done successively with separate models in data analysis pipelines. This potentially leads to biased P-values or false discovery rates due to the influence of BE correction on the data. RESULTS: We present a novel approach for estimating null distributions of test statistics in data analysis pipelines where BE correction is followed by linear model analysis. The approach is based on generating simulated datasets by random rotation and thereby retains the dependence structure of genes adequately. This allows estimating null distributions of dependent test statistics, and thus the calculation of resampling-based P-values and false-discovery rates following BE correction while maintaining the alpha level. AVAILABILITY: The described methods are implemented as randRotation package on Bioconductor: https://bioconductor.org/packages/randRotation/. CONTACT: p.hettegger@gmail.com. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Oxytocin receptor gene methylation as a molecular marker for severity of depressive symptoms in affective disorder patients.

BACKGROUND: Oxytocin (OXT) is a neuropeptide and hormone involved in emotional functioning and also seems to play a role in moderating the stress response. Both preclinical and clinical studies point to an increased methylation status of the Oxytocin receptor (OXTR) promoter region with concomitant deficits in social, cognitive and emotional functioning. We hypothesize that methylation levels (%) of the oxytocin receptor promoter region correlate with the severity of depression symptoms and/or with the severity of childhood trauma within this present sample of affective disorder patients. METHODOLOGY: Eight hundred forty six (846) affective disorder patients of Central European origin were recruited at the Department of Psychiatry and Psychotherapy of the Medical University Vienna, the Karl Landsteiner University for Health and Science and Zentren für seelische Gesundheit, BBRZ-Med Leopoldau. Psychiatric assessment included a semi-structured diagnostic interview (Schedules for Clinical Assessment in Neuropsychiatry), the Hamilton Depression Scale and the Childhood Trauma Questionnaire. Concomitantly DNA samples of peripheral blood cells were collected for Multiplexed and Sensitive DNA Methylation Testing. RESULTS: Our data suggests a positive but not significant association between OXTR promoter Exons 1-3 methylation levels and severity of depression symptoms as well as severity of emotional neglect in affective disorder patients and no association with childhood trauma. CONCLUSIONS: Our findings contribute to elucidate the role of OXTR in affective disorders, but further longitudinal studies in particular are necessary to broaden the current state of knowledge.

A simple quantitative diagnostic alternative for MGMT DNA-methylation testing on RCL2 fixed paraffin embedded tumors using restriction coupled qPCR.

BACKGROUND: MGMT promoter methylation is associated with favorable prognosis and chemosensitivity in glioblastoma multiforme (GBM), especially in elderly patients. We aimed to develop a simple methylation-sensitive restriction enzyme (MSRE)-based quantitative PCR (qPCR) assay, allowing the quantification of MGMT promoter methylation. METHODS: DNA was extracted from non-neoplastic brain (n = 24) and GBM samples (n = 20) upon 3 different sample conservation conditions (-80 °C, formalin-fixed and paraffin-embedded (FFPE); RCL2-fixed). We evaluated the suitability of each fixation method with respect to the MSRE-coupled qPCR methylation analyses. Methylation data were validated by MALDITOF. RESULTS: qPCR was used for evaluation of alternative tissue conservation procedures. DNA from FFPE tissue failed reliable testing; DNA from both RCL2-fixed and fresh frozen tissues performed equally well and was further used for validation of the quantitative MGMT methylation assay (limit of detection (LOD): 19.58 pg), using individual's undigested sample DNA for calibration. MGMT methylation analysis in non-neoplastic brain identified a background methylation of 0.10 ± 11% which we used for defining a cut-off of 0.32% for patient stratification. Of GBM patients 9 were MGMT methylationpositive (range: 0.56 - 91.95%), and 11 tested negative. MALDI-TOF measurements resulted in a concordant classification of 94% of GBM samples in comparison to qPCR. CONCLUSIONS: The presented methodology allows quantitative MGMT promoter methylation analyses. An amount of 200 ng DNA is sufficient for triplicate analyses including control reactions and individual calibration curves, thus excluding any DNA qualityderived bias. The combination of RCL2-fixation and quantitative methylation analyses improves pathological routine examination when histological and molecular analyses on limited amounts of tumor samples are necessary for patient stratification.

Pelargonium Extract EPs 7630 in the Treatment of Human Corona Virus-Associated Acute Respiratory Tract Infections - A Secondary Subgroup-Analysis of an Open-Label, Uncontrolled Clinical Trial.

Background: Experience in treating human coronavirus (HCoV) infections might help to identify effective compounds against novel coronaviruses. We therefore performed a secondary subgroup-analysis of data from an open-label, uncontrolled clinical trial published in 2015 investigating the proanthocyanidin-rich Pelargonium sidoides extract EPs 7630 in patients with the common cold. Methods: 120 patients with common cold and at least 2 out of 10 common cold symptoms received one film-coated 20 mg tablet EPs 7630 thrice daily for 10 days in an uncontrolled, interventional multicentre trial (ISRCTN65790556). At baseline, viral nucleic acids were detected by polymerase chain reaction. Common cold-associated symptoms and treatment satisfaction were evaluated after 5 days and at treatment end. Based on the data of patients with proof of viral nucleic acids, we compared the course of the disease in patients with or without HCoV infection. Results: In 61 patients, viral nucleic acids were detected. Of these, 23 (37.7%) were tested positive for at least one HCoV (HCoV subset) and 38 (62.3%) for other viruses only (non-HCoV subset). Patients of both subsets showed a significant improvement of common cold symptoms already after 5 days of treatment, although the observed change tended to be more pronounced in the HCoV subset. At treatment end, more than 80% of patients of both groups were completely recovered or majorly improved. In both subsets, less than 22% of patients took concomitant paracetamol for antipyresis. The mean number of patients' days off work or school/college was similar (0.9 ± 2.6 days in HCoV subset vs 1.3 ± 2.8 days in non-HCoV subset). In both groups, most patients were satisfied or very satisfied with EPs 7630 treatment. Conclusion: EPs 7630 treatment outcomes of common cold patients with confirmed HCoV infection were as favourable as in patients with other viral infections. As this trial was conducted before the pandemic, there is currently no evidence from clinical trials for the efficacy of EPs 7630 in patients with SARS-CoV-2 infection. Dedicated non-clinical studies and clinical trials are required to elucidate the potential of EPs 7630 in the early treatment of HCoV infections.

Salivary biomarkers in cardiovascular disease: An insight into the current evidence.

Cardiovascular diseases (CVDs) are the most common cause of mortality worldwide. In acute cardiovascular conditions, time is a crucial player in the outcomes of disease management. Given the ease and noninvasiveness of obtaining saliva, salivary biomarkers may provide a rapid and efficient diagnosis of CVD. Here, we reviewed the published data on the value of salivary molecules for diagnosis of CVD, especially in acute care settings. In this review, we show that some biomarkers such as salivary creatinine kinase myocardial band, C-reactive protein, troponin-1, and myoglobin exhibited promising diagnostic values that were comparable to their serum counterparts. Other molecules were also investigated and showed controversial results, including myeloperoxidase, brain natriuretic peptide, and some oxidative stress markers. Based on our review, we concluded that the clinical use of salivary biomarkers to diagnose CVD is promising; however, it is still in the early stage of development. Further studies are needed to validate these findings, determine cutoff values for diagnosis, and compare them to other established biomarkers currently in clinical use.

Monoamino Oxidase A Gene Single-Nucleotide Polymorphisms and Methylation Status and the Risk of Violent Suicide Attempts in Affective Disorder Patients.

Background: When investigating the neurobiology of suicidal behavior, Monoamino Oxidase A (MAOA) is one of the prime suspects to consider. Interestingly, MAOA dysregulation has also been associated with violent behavior in previous publications. In the present study, we aimed to establish an association between polymorphisms of the MAOA gene and methylation status of the MAOA gene Exon I, and suicide attempts with violent methods in a sample of affective disorder patients. Methods: Eight hundred fourteen Caucasian affective disorder patients were assessed at the Department of Psychiatry and Psychotherapy of the Medical University Vienna, the Karl Landsteiner University for Health and Science and Zentren für seelische Gesundheit, BBRZ-Med Leopoldau. An assemblage of psychiatric interviews was performed (e.g., SCAN, HAMD, SBQ-R, CTQ) and DNA samples of peripheral blood cells were collected for Sequenom MassARRAY® iPLEX Gold genotyping and Multiplexed and Sensitive DNA Methylation Testing. Results: Female affective disorder patients with a history of violent suicide attempt were found to have a significantly increased frequency of the AA genotype in the rs5906957 single nucleotide polymorphism (p = 0.003). Furthermore, the MAOA gene exon I promoter region showed significantly decreased methylation in female violent suicide attempter(s) as opposed to female affective disorder patients who had no history of suicide attempt or no history of suicide attempt with violent method. Limitations: The small sample size hampers to reveal small genetic effects as to be expected in psychiatric disorders. Conclusions: This study offers promising findings about associations between the MAOA gene and violent suicide especially in women.

Does the Use of the "Proseek® Multiplex Oncology I Panel" on Peritoneal Fluid Allow a Better Insight in the Pathophysiology of Endometriosis, and in Particular Deep-Infiltrating Endometriosis?

Endometriosis appears to share certain cancer-related processes, such as cell attachment, invasion, proliferation and neovascularization, some of which can also be found in other healthy tissues. In order to better understand the altered milieu of the peritoneal cavity, while acknowledging the reported similarities between endometriosis and neoplastic processes, we applied a multiplex oncology panel to search for specific biomarker signatures in the peritoneal fluid of women with endometriosis, women with deep-infiltrating endometriosis (DIE), as well as controls. In total, 84 patients were included in our study, 53 women with endometriosis and 31 controls. Ninety-two proteins were measured in prospectively collected peritoneal fluid (PF) samples, using the "Proseek® Multiplex Oncology I Panel". We first compared patients with endometriosis versus controls, and in a second step, DIE versus endometriosis patients without DIE. Out of the 92 analyzed proteins, few showed significant differences between the groups. In patients with endometriosis, ICOS ligand, Endothelial growth factor, E-selectin, Receptor tyrosine-protein kinase erbB-2, Interleukin-6 receptor alpha, Vascular endothelial growth factor receptor 2, Fms-related tyrosine kinase 3 ligand, C-X-C motif chemokine 10, Epididymal secretory protein E4 and Folate receptor-alpha were decreased, while Interleukin-6 and Interleukin-8 were increased compared to controls. Looking at patients with DIE, we found Chemokine ligand 19, Stem cell factor, Vascular endothelial growth factor D, Interleukin-6 receptor alpha and Melanoma inhibitory activity to be increased compared to endometriosis patients without DIE. We have shown a distinct regulation of the immune response, angiogenesis, cell proliferation, cell adhesion and inhibition of apoptosis in PF of patients with endometriosis compared to controls. The specific protein pattern in the PF of DIE patients provides new evidence that DIE represents a unique entity of extrauterine endometriosis with enhanced angiogenetic and pro-proliferative features.

RGG: a general GUI Framework for R scripts.

BACKGROUND: R is the leading open source statistics software with a vast number of biostatistical and bioinformatical analysis packages. To exploit the advantages of R, extensive scripting/programming skills are required. RESULTS: We have developed a software tool called R GUI Generator (RGG) which enables the easy generation of Graphical User Interfaces (GUIs) for the programming language R by adding a few Extensible Markup Language (XML) - tags. RGG consists of an XML-based GUI definition language and a Java-based GUI engine. GUIs are generated in runtime from defined GUI tags that are embedded into the R script. User-GUI input is returned to the R code and replaces the XML-tags. RGG files can be developed using any text editor. The current version of RGG is available as a stand-alone software (RGGRunner) and as a plug-in for JGR. CONCLUSION: RGG is a general GUI framework for R that has the potential to introduce R statistics (R packages, built-in functions and scripts) to users with limited programming skills and helps to bridge the gap between R developers and GUI-dependent users. RGG aims to abstract the GUI development from individual GUI toolkits by using an XML-based GUI definition language. Thus RGG can be easily integrated in any software. The RGG project further includes the development of a web-based repository for RGG-GUIs. RGG is an open source project licensed under the Lesser General Public License (LGPL) and can be downloaded freely at (http://rgg.r-forge.r-project.org).

PCR-based analysis of differentially methylated regions of GNAS enables convenient diagnostic testing of pseudohypoparathyroidism type Ib.

BACKGROUND: Pseudohypoparathyroidism type Ib (PHPIb) is characterized by parathyroid hormone (PTH) resistance, which can lead to hypocalcemia, hyperphosphatemia, and increased serum PTH. The disorder is caused by mutations in regulatory regions of the GNAS gene (GNAS complex locus) that lead to interferences in the methylation status of alternative GNAS promoters, such as exon A/B, NESP55, and XL alpha-s. PHPIb comprises disorders that show distinctive changes in methylation status but share the same clinical phenotype: (a) loss of methylation only at exon A/B of the GNAS gene and involving no other obvious epigenetic abnormalities [e.g., those caused by heterozygous microdeletions in the STX16 (syntaxin 16) region and found in many patients with autosomal dominant (AD) PHPIb]; (b) methylation abnormalities at several differentially methylated regions (DMRs), which are observed in most patients with sporadic PHPIb and some families with AD PHPIb. METHODS: To permit early and reliable diagnosis of suspected PHPIb, we designed methylation-sensitive restriction enzyme-based and bisulfite deamination-based PCR tests for exon A/B and NESP55 DMRs. RESULTS: Both PCR strategies permit proper methylation testing of GNAS and NESP55 DMRs and elucidate different disease subtypes. We have identified a novel microsatellite repeat polymorphism within GNAS exon A/B, and pedigree analyses have shown its presence to be conclusive evidence for familial disease. CONCLUSIONS: We provide a simple diagnostic test for PHPIb, an imprinting disorder caused by different molecular changes within the GNAS complex locus. PHPIb, a complex and diagnostically challenging clinical phenotype, can be treated successfully by taking steps before the manifestation of symptoms to avoid clinical complications in affected patients or asymptomatic members of affected families who show positive results in genetic tests.

Multiplexed and Sensitive DNA Methylation Testing Using Methylation-Sensitive Restriction Enzymes "MSRE-qPCR".

DNA methylation is a chemically stable key-player in epigenetics. In the vertebrate genome the 5-methyl cytosine (5mC) has been found almost exclusively in the CpG dinucleotide context. CpG dinucleotides are enriched in CpG islands very frequently located within or close to gene promoters. Analyses of DNA methylation changes in human diagnostics have been conducted classically using methylation-sensitive restriction enzymes (MSRE). Since the discovery of bisulfite conversion-based sequencing and PCR assays, MSRE-based PCR assays have been less frequently used, although especially in the field of cancer epigenetics MSRE-based genome-wide discovery and targeted screening applications have been and are still performed successfully. Even though epigenome-wide discovery of altered DNA methylation patterns has found its way into various fields of human disease and molecular genetics research, the validation of findings upon discovery is still a bottleneck. Usually several multiples of 10 up to 100 candidate biomarkers from discovery have to be confirmed or are of interest for further work. In particular, bisulfite PCR assays are often limited in the number of candidates which can be analyzed, due to their low multiplexing capability, especially, if only small amounts of DNA are available from for example clinical specimens. In clinical research and diagnostics a similar situation arises for the analyses of cell-free DNA (cfDNA) in body fluids or circulating tumor cells (CTCs). Although tissue- or disease- (e.g., cancer) specific DNA methylation patterns can be deduced very efficiently in a genome-wide manner if around 100 ng of DNA are available, confirming these candidates and selecting target-sequences for studying methylation changes in liquid biopsies using cfDNA or CTCs remains a big challenge. Along these lines we have developed MSRE-qPCR and introduce here method details, which have been found very suitable for the efficient confirmation and testing of DNA methylation in a quantitative multiplexed manner (e.g., 48-96 plex) from ng amounts of DNA. The method is applicable in a standard qPCR setting as well for nanoliter scaled high-throughput qPCR, enabling detection of <10 copies of targets, thus suitable to pick up 0.1-1% of specific methylated DNA in an unmethylated background.

DNA Methylation Signatures Predicting Bevacizumab Efficacy in Metastatic Breast Cancer.

Background: Biomarkers predicting response to bevacizumab in breast cancer are still missing. Since epigenetic modifications can contribute to an aberrant regulation of angiogenesis and treatment resistance, we investigated the influence of DNA methylation patterns on bevacizumab efficacy. Methods: Genome-wide methylation profiling using the Illumina Infinium HumanMethylation450 BeadChip was performed in archival FFPE specimens of 36 patients with HER2-negative metastatic breast cancer treated with chemotherapy in combination with bevacizumab as first-line therapy (learning set). Based on objective response and progression-free survival (PFS) and considering ER expression, patients were divided in responders (R) and non-responders (NR). Significantly differentially methylated gene loci (CpGs) with a strong change in methylation levels (Δß>0.15 or Δß<-0.15) between R and NR were identified and further investigated in 80 bevacizumab-treated breast cancer patients (optimization set) and in 15 patients treated with chemotherapy alone (control set) using targeted deep amplicon bisulfite sequencing. Methylated gene loci were considered predictive if there was a significant association with outcome (PFS) in the optimization set but not in the control set using Spearman rank correlation, Cox regression, and logrank test. Results: Differentially methylated loci in 48 genes were identified, allowing a good separation between R and NR (odds ratio (OR) 101, p<0.0001). Methylation of at least one cytosine in 26 gene-regions was significantly associated with progression-free survival (PFS) in the optimization set, but not in the control set. Using information from the optimization set, the panel was reduced to a 9-gene signature, which could divide patients from the learning set into 2 clusters, thereby predicting response with an OR of 40 (p<0.001) and an AUC of 0.91 (LOOCV). A further restricted 3-gene methylation model showed a significant association of predicted responders with longer PFS in the learning and optimization set even in multivariate analysis with an excellent and good separation of R and NR with AUC=0.94 and AUC=0.86, respectively. Conclusion: Both a 9-gene and 3-gene methylation signature can discriminate between R and NR to a bevacizumab-based therapy in MBC and could help identify patients deriving greater benefit from bevacizumab.

Comparison of RNA amplification techniques meeting the demands for the expression profiling of clinical cancer samples.

Available ribonucleic acid (RNA) amplification methods are extensively tested for reproducibility, but only a few studies additionally deal with potential amplification bias. On targeted arrays, we evaluated three amplification protocols, which are less time consuming than the commonly used T7-RNA polymerase based in vitro transcription protocols and therefore may be more suitable for clinical use: Template-switching polymerase chain reaction (PCR), Ribo-single primer isothermal amplification and a random primer-based PCR. Additionally, a more sensitive labelling method, Dendrimer labelling, was evaluated. All methods were compared to unamplified RNA labelled at reverse transcription. From our results, we conclude that RNA amplification with template-switching PCR is highly reproducible and results in a reliable representation of the starting RNA population. We then assessed whether RNA amplification of clinical breast and thyroid cancer samples with template-switching PCR showed robust performance when altered cycle numbers or partially degraded RNA were used. Template-switching PCR proved to be a very reliable method for global RNA amplification, even when starting from partially degraded RNA down to a RNA Integrity Number of 4.3. In conclusion, template-switching PCR amplification promises to help micro-array expression profiling of limited amounts of human samples on its way to a clinical routine.

Inhibitors of DNA methylation support TGF-ß1-induced IL11 expression in gingival fibroblasts.

PURPOSE: Oral wound healing requires gingival fibroblasts to respond to local growth factors. Epigenetic silencing through DNA methylation can potentially decrease the responsiveness of gingival fibroblasts to local growth factors. In this study, our aim was to determine whether the inhibition of DNA methylation sensitized gingival fibroblasts to transforming growth factor-ß1 (TGF-ß1). METHODS: Gingival fibroblasts were exposed to 5-aza-2'-deoxycytidine (5-aza), a clinically approved demethylating agent, before stimulation with TGF-ß1. Gene expression changes were evaluated using quantitative polymerase chain reaction (PCR) analysis. DNA methylation was detected by methylation-sensitive restriction enzymes and PCR amplification. RESULTS: We found that 5-aza enhanced TGF-ß1-induced interleukin-11 (IL11) expression in gingival fibroblasts 2.37-fold (P=0.008). 5-aza had no significant effects on the expression of proteoglycan 4 (PRG4) and NADPH oxidase 4 (NOX4). Consistent with this, 5-aza caused demethylation of the IL11 gene commonly next to a guanosine (CpG) island in gingival fibroblasts. The TGF-ß type I receptor kinase inhibitor SB431542 impeded the changes in IL11 expression, indicating that the effects of 5-aza require TGF-ß signaling. 5-aza moderately increased the expression of TGF-ß type II receptor (1.40-fold; P=0.009), possibly enhancing the responsiveness of fibroblasts to TGF-ß1. As part of the feedback response, 5-aza increased the expression of the DNA methyltransferases 1 (DNMT1) (P=0.005) and DNMT3B (P=0.002), which are enzymes responsible for gene methylation. CONCLUSIONS: These in vitro data suggest that the inhibition of DNA methylation by 5-aza supports TGF-ß-induced IL11 expression in gingival fibroblasts.

MethylMeter(®): bisulfite-free quantitative and sensitive DNA methylation profiling and mutation detection in FFPE samples.

AIM: Development of a sensitive method for DNA methylation profiling and associated mutation detection in clinical samples. MATERIALS & METHODS: Formalin-fixed and paraffin-embedded tumors received by clinical laboratories often contain insufficient DNA for analysis with bisulfite or methylation sensitive restriction enzymes-based methods. To increase sensitivity, methyl-CpG DNA capture and Coupled Abscription PCR Signaling detection were combined in a new assay, MethylMeter(®). Gliomas were analyzed for MGMT methylation, glioma CpG island methylator phenotype and IDH1 R132H. RESULTS: MethylMeter had 100% assay success rate measuring all five biomarkers in formalin-fixed and paraffin-embedded tissue. MGMT methylation results were supported by survival and mRNA expression data. CONCLUSION: MethylMeter is a sensitive and quantitative method for multitarget DNA methylation profiling and associated mutation detection. The MethylMeter-based GliomaSTRAT assay measures methylation of four targets and one mutation to simultaneously grade gliomas and predict their response to temozolomide. This information is clinically valuable in management of gliomas.

Analysis and Visualization Tool for Targeted Amplicon Bisulfite Sequencing on Ion Torrent Sequencers.

Targeted sequencing of PCR amplicons generated from bisulfite deaminated DNA is a flexible, cost-effective way to study methylation of a sample at single CpG resolution and perform subsequent multi-target, multi-sample comparisons. Currently, no platform specific protocol, support, or analysis solution is provided to perform targeted bisulfite sequencing on a Personal Genome Machine (PGM). Here, we present a novel tool, called TABSAT, for analyzing targeted bisulfite sequencing data generated on Ion Torrent sequencers. The workflow starts with raw sequencing data, performs quality assessment, and uses a tailored version of Bismark to map the reads to a reference genome. The pipeline visualizes results as lollipop plots and is able to deduce specific methylation-patterns present in a sample. The obtained profiles are then summarized and compared between samples. In order to assess the performance of the targeted bisulfite sequencing workflow, 48 samples were used to generate 53 different Bisulfite-Sequencing PCR amplicons from each sample, resulting in 2,544 amplicon targets. We obtained a mean coverage of 282X using 1,196,822 aligned reads. Next, we compared the sequencing results of these targets to the methylation level of the corresponding sites on an Illumina 450k methylation chip. The calculated average Pearson correlation coefficient of 0.91 confirms the sequencing results with one of the industry-leading CpG methylation platforms and shows that targeted amplicon bisulfite sequencing provides an accurate and cost-efficient method for DNA methylation studies, e.g., to provide platform-independent confirmation of Illumina Infinium 450k methylation data. TABSAT offers a novel way to analyze data generated by Ion Torrent instruments and can also be used with data from the Illumina MiSeq platform. It can be easily accessed via the Platomics platform, which offers a web-based graphical user interface along with sample and parameter storage. TABSAT is freely available under a GNU General Public License version 3.0 (GPLv3) at https://github.com/tadkeys/tabsat/ and http://demo.platomics.com/.

Insights into the Pathogenesis of Anaplastic Large-Cell Lymphoma through Genome-wide DNA Methylation Profiling.

Aberrant DNA methylation patterns in malignant cells allow insight into tumor evolution and development and can be used for disease classification. Here, we describe the genome-wide DNA methylation signatures of NPM-ALK-positive (ALK+) and NPM-ALK-negative (ALK-) anaplastic large-cell lymphoma (ALCL). We find that ALK+ and ALK- ALCL share common DNA methylation changes for genes involved in T cell differentiation and immune response, including TCR and CTLA-4, without an ALK-specific impact on tumor DNA methylation in gene promoters. Furthermore, we uncover a close relationship between global ALCL DNA methylation patterns and those in distinct thymic developmental stages and observe tumor-specific DNA hypomethylation in regulatory regions that are enriched for conserved transcription factor binding motifs such as AP1. Our results indicate similarity between ALCL tumor cells and thymic T cell subsets and a direct relationship between ALCL oncogenic signaling and DNA methylation through transcription factor induction and occupancy.

Evidence of a polyclonal nature of myositis ossificans.

Myositis ossificans is a localized, self-limiting, reparative lesion that is composed of reactive hypercellular fibrous tissue and bone. Although it is clearly a benign lesion, its clinical, radiological, and histological appearance may sometimes mimic a malignant tumor. Whether myositis ossificans represents a monoclonal or polyclonal hyperplastic proliferation is not yet known. To address this question, we therefore extracted DNA from the respective paraffin-embedded tumor tissues of nine women with a median age of 50 years at diagnosis (range: 20-84 years) and studied the X inactivation pattern by means of methylation-sensitive polymerase chain reaction and primers that target the polymorphic CGG trinucleotide repeat of the FMR1 gene. The fact that we did not detect any skewing of the X inactivation pattern in the five successfully analyzed cases corroborates the notion that myositis ossificans results from a polyclonal proliferation and confirms that it is a reactive, reparative process. Analysis of the X inactivation pattern may, thus, supplement the differential diagnostic work-up of cases with an uncertain histology, at least in the informative proportion of female patients.

Immune-Signatures for Lung Cancer Diagnostics: Evaluation of Protein Microarray Data Normalization Strategies.

New minimal invasive diagnostic methods for early detection of lung cancer are urgently needed. It is known that the immune system responds to tumors with production of tumor-autoantibodies. Protein microarrays are a suitable highly multiplexed platform for identification of autoantibody signatures against tumor-associated antigens (TAA). These microarrays can be probed using 0.1 mg immunoglobulin G (IgG), purified from 10 µL of plasma. We used a microarray comprising recombinant proteins derived from 15,417 cDNA clones for the screening of 100 lung cancer samples, including 25 samples of each main histological entity of lung cancer, and 100 controls. Since this number of samples cannot be processed at once, the resulting data showed non-biological variances due to "batch effects". Our aim was to evaluate quantile normalization, "distance-weighted discrimination" (DWD), and "ComBat" for their effectiveness in data pre-processing for elucidating diagnostic immune­signatures. "ComBat" data adjustment outperformed the other methods and allowed us to identify classifiers for all lung cancer cases versus controls and small-cell, squamous cell, large-cell, and adenocarcinoma of the lung with an accuracy of 85%, 94%, 96%, 92%, and 83% (sensitivity of 0.85, 0.92, 0.96, 0.88, 0.83; specificity of 0.85, 0.96, 0.96, 0.96, 0.83), respectively. These promising data would be the basis for further validation using targeted autoantibody tests.