A multivariable approach for risk markers from pooled molecular data with only partial overlap.

BACKGROUND: Increasingly, molecular measurements from multiple studies are pooled to identify risk scores, with only partial overlap of measurements available from different studies. Univariate analyses of such markers have routinely been performed in such settings using meta-analysis techniques in genome-wide association studies for identifying genetic risk scores. In contrast, multivariable techniques such as regularized regression, which might potentially be more powerful, are hampered by only partial overlap of available markers even when the pooling of individual level data is feasible for analysis. This cannot easily be addressed at a preprocessing level, as quality criteria in the different studies may result in differential availability of markers - even after imputation. METHODS: Motivated by data from the InterLymph Consortium on risk factors for non-Hodgkin lymphoma, which exhibits these challenges, we adapted a regularized regression approach, componentwise boosting, for dealing with partial overlap in SNPs. This synthesis regression approach is combined with resampling to determine stable sets of single nucleotide polymorphisms, which could feed into a genetic risk score. The proposed approach is contrasted with univariate analyses, an application of the lasso, and with an analysis that discards studies causing the partial overlap. The question of statistical significance is faced with an approach called stability selection. RESULTS: Using an excerpt of the data from the InterLymph Consortium on two specific subtypes of non-Hodgkin lymphoma, it is shown that componentwise boosting can take into account all applicable information from different SNPs, irrespective of whether they are covered by all investigated studies and for all individuals in the single studies. The results indicate increased power, even when studies that would be discarded in a complete case analysis only comprise a small proportion of individuals. CONCLUSIONS: Given the observed gains in power, the proposed approach can be recommended more generally whenever there is only partial overlap of molecular measurements obtained from pooled studies and/or missing data in single studies. A corresponding software implementation is available upon request. TRIAL REGISTRATION: All involved studies have provided signed GWAS data submission certifications to the U.S. National Institute of Health and have been retrospectively registered.

Prognostic impact of CD4-positive T cell subsets in early breast cancer: a study based on the FinHer trial patient population.

BACKGROUND: The clinical importance of tumor-infiltrating cluster of differentiation 4 (CD4) T cells is incompletely understood in early breast cancer. We investigated the clinical significance of CD4, forkhead box P3 (FOXP3), and B cell attracting chemokine leukocyte chemoattractant-ligand (C-X-C motif) 13 (CXCL13) in early breast cancer. METHODS: The study is based on the patient population of the randomized FinHer trial, where 1010 patients with early breast cancer were randomly allocated to adjuvant chemotherapy containing either docetaxel or vinorelbine, and human epidermal growth factor receptor 2 (HER2)-positive patients were also allocated to trastuzumab or no trastuzumab. Breast cancer CD4, FOXP3, and CXCL13 contents were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR), and their influence on distant disease-free survival (DDFS) was examined using univariable and multivariable Cox regression and Kaplan-Meier estimates in the entire cohort and in selected molecular subgroups. Interactions between variables were analyzed using Cox regression. The triple-negative breast cancer (TNBC) subset of the HE10/97 randomized trial was used for confirmation. RESULTS: High CXCL13 was associated with favorable DDFS in univariable analysis, and independently in multivariable analysis (HR 0.44, 95% CI 0.29-0.67, P ≤ 0.001), most strongly in TNBC (HR 0.39, 95% CI 0.19-0.79, P = 0.009). No significant interaction with chemotherapy or trastuzumab administration was detected. Neither tumor CD4 content nor FOXP3 content was associated with DDFS. The favorable prognostic influence of CXCL13 was confirmed in the HE10/97 trial patient population with TNBC (HR 0.30, 95% CI 0.09-0.93; P = 0.038). CONCLUSIONS: The results provide a high level of evidence that humoral immunity influences the survival outcomes of patients with early breast cancer, in particular of those with TNBC. TRIAL REGISTRATION: The study reports retrospective biomarker analyses in the prospective FinHer trial and the prospective HE10/97 trial. ISRCTN76560285 . Registered on 18 March 2005. ACTRN12611000506998 . Registered on 16 May 2011.

When do myopia genes have their effect? Comparison of genetic risks between children and adults.

Previous studies have identified many genetic loci for refractive error and myopia. We aimed to investigate the effect of these loci on ocular biometry as a function of age in children, adolescents, and adults. The study population consisted of three age groups identified from the international CREAM consortium: 5,490 individuals aged <10 years; 5,000 aged 10-25 years; and 16,274 aged >25 years. All participants had undergone standard ophthalmic examination including measurements of axial length (AL) and corneal radius (CR). We examined the lead SNP at all 39 currently known genetic loci for refractive error identified from genome-wide association studies (GWAS), as well as a combined genetic risk score (GRS). The beta coefficient for association between SNP genotype or GRS versus AL/CR was compared across the three age groups, adjusting for age, sex, and principal components. Analyses were Bonferroni-corrected. In the age group <10 years, three loci (GJD2, CHRNG, ZIC2) were associated with AL/CR. In the age group 10-25 years, four loci (BMP2, KCNQ5, A2BP1, CACNA1D) were associated; and in adults 20 loci were associated. Association with GRS increased with age; ß = 0.0016 per risk allele (P = 2 × 10-8 ) in <10 years, 0.0033 (P = 5 × 10-15 ) in 10- to 25-year-olds, and 0.0048 (P = 1 × 10-72 ) in adults. Genes with strongest effects (LAMA2, GJD2) had an early effect that increased with age. Our results provide insights on the age span during which myopia genes exert their effect. These insights form the basis for understanding the mechanisms underlying high and pathological myopia.

Root coverage with connective tissue graft associated with coronally advanced flap or tunnel technique: a randomized, double-blind, mono-centre clinical trial.

AIM: The aim of this randomized clinical trial was to compare the coronally advanced flap (CAF) with the modified microsurgical tunnel technique (MMTT) for treatment of Miller class I and II recessions. MATERIAL AND METHODS: Forty patients with 71 gingival recessions were recruited and randomly assigned to either CAF or to MMTT. In both groups, a connective tissue graft was applied. Clinical evaluations were performed after 3, 6, and 12 months. Impressions were taken and digitally scanned three-dimensionally to evaluate the quantitative soft tissue changes in the operative region. Patient satisfaction was measured with the root coverage aesthetic score (RES). RESULTS: After a period of 12 months, significant differences were not found between the two groups. Root coverage was 98.3% for CAF and 97.2% for MMTT. The evaluation of the aesthetic outcome using RES showed good results in both groups. The RES score was in accordance with subjective patient satisfaction. There was no significant difference in the amount of volumetric changes. CONCLUSIONS: CAF and MMTT with the additional use of a graft are equally successful in covering gingival recessions of Miller class I and II, with high aesthetic results. All patients indicated their willingness for further periodontal surgery.

A systems level analysis reveals transcriptomic and proteomic complexity in Ixodes ricinus midgut and salivary glands during early attachment and feeding.

Although pathogens are usually transmitted within the first 24-48 h of attachment of the castor bean tick Ixodes ricinus, little is known about the tick's biological responses at these earliest phases of attachment. Tick midgut and salivary glands are the main tissues involved in tick blood feeding and pathogen transmission but the limited genomic information for I. ricinus delays the application of high-throughput methods to study their physiology. We took advantage of the latest advances in the fields of Next Generation RNA-Sequencing and Label-free Quantitative Proteomics to deliver an unprecedented, quantitative description of the gene expression dynamics in the midgut and salivary glands of this disease vector upon attachment to the vertebrate host. A total of 373 of 1510 identified proteins had higher expression in the salivary glands, but only 110 had correspondingly high transcript levels in the same tissue. Furthermore, there was midgut-specific expression of 217 genes at both the transcriptome and proteome level. Tissue-dependent transcript, but not protein, accumulation was revealed for 552 of 885 genes. Moreover, we discovered the enrichment of tick salivary glands in proteins involved in gene transcription and translation, which agrees with the secretory role of this tissue; this finding also agrees with our finding of lower tick t-RNA representation in the salivary glands when compared with the midgut. The midgut, in turn, is enriched in metabolic components and proteins that support its mechanical integrity in order to accommodate and metabolize the ingested blood. Beyond understanding the physiological events that support hematophagy by arthropod ectoparasites, we discovered more than 1500 proteins located at the interface between ticks, the vertebrate host, and the tick-borne pathogens. Thus, our work significantly improves the knowledge of the genetics underlying the transmission lifecycle of this tick species, which is an essential step for developing alternative methods to better control tick-borne diseases.

Avelumab first-line maintenance in advanced urothelial carcinoma: Complete screening for prognostic and predictive factors using machine learning in the JAVELIN Bladder 100 phase 3 trial.

BACKGROUND: Avelumab first-line (1 L) maintenance is a standard of care for advanced urothelial carcinoma (aUC) based on the JAVELIN Bladder 100 phase 3 trial, which showed that avelumab 1 L maintenance + best supportive care (BSC) significantly prolonged overall survival (OS) and progression-free survival (PFS) vs BSC alone in patients who were progression free after receiving 1 L platinum-containing chemotherapy. Here, we comprehensively screened JAVELIN Bladder 100 trial datasets to identify prognostic factors that define subpopulations of patients with longer or shorter OS irrespective of treatment, and predictive factors that select patients who could obtain a greater OS benefit from avelumab 1 L maintenance treatment. METHODS: We performed machine learning analyses to screen a large set of baseline covariates, including patient demographics, disease characteristics, laboratory values, molecular biomarkers, and patient-reported outcomes. Covariates were identified from previously reported analyses and established prognostic and predictive markers. Variables selected from random survival forest models were processed further in univariate Cox models with treatment interaction and visually inspected using correlation analysis and Kaplan-Meier curves. Results were summarized in a multivariable Cox model. RESULTS: Prognostic baseline covariates associated with OS included in the final model were assignment to avelumab 1 L maintenance treatment, Eastern Cooperative Oncology Group performance status, site of metastasis, sum of longest target lesion diameters, levels of C-reactive protein and alkaline phosphatase in blood, lymphocyte proportion in intratumoral stroma, tumor mutational burden, and tumor CD8+ T-cell infiltration. Potential predictive factors included site of metastasis, tumor mutation burden, and tumor CD8+ T-cell infiltration. An analysis in patients with PD-L1+ tumors had similar findings to those in the overall population. CONCLUSIONS: Machine learning analyses of data from the JAVELIN Bladder 100 trial identified potential prognostic and predictive factors for avelumab 1 L maintenance treatment in patients with aUC, which warrant further evaluation in other clinical datasets.

Genetic signatures shared in embryonic liver development and liver cancer define prognostically relevant subgroups in HCC.

Multiple activations of individual genes during embryonic liver and HCC development have repeatedly prompted speculations about conserved embryonic signatures driving cancer development. Recently, the emerging discussion on cancer stem cells and the appreciation that generally tumors may develop from progenitor cells of diverse stages of cellular differentiation has shed increasing light on the overlapping genetic signatures between embryonic liver development and HCC. However there is still a lack of systematic studies investigating this area. We therefore performed a comprehensive analysis of differentially regulated genetic signaling pathways in embryonic and liver cancer development and investigated their biological relevance.Genetic signaling pathways were investigated on several publically available genome wide microarray experiments on liver development and HCC. Differentially expressed genes were investigated for pathway enrichment or underrepresentation compared to KEGG annotated pathways by Fisher exact evaluation. The comparative analysis of enrichment and under representation of differentially regulated genes in liver development and HCC demonstrated a significant overlap between multiple pathways. Most strikingly we demonstrated a significant overlap not only in pathways expected to be relevant to both conditions such as cell cycle or apoptosis but also metabolic pathways associated with carbohydrate and lipid metabolism. Furthermore, we demonstrated the clinical significance of these findings as unsupervised clustering of HCC patients on the basis of these metabolic pathways displayed significant differences in survival.These results indicate that liver development and liver cancer share similar alterations in multiple genetic signaling pathways. Several pathways with markedly similar patterns of enrichment or underrepresentation of various regulated genes between liver development and HCC are of prognostic relevance in HCC. In particular, the metabolic pathways were identified as novel prognostically relevant players in HCC development.

Ep-CAM RNA expression predicts metastasis-free survival in three cohorts of untreated node-negative breast cancer.

Epithelial cell adhesion molecule (Ep-CAM) recently received increased attention as a prognostic factor in breast cancer. We aimed to validate the influence of Ep-CAM RNA expression in untreated node-negative breast cancer. Ep-CAM RNA expression was evaluated utilizing microarray-based gene-expression profiling in 194 consecutive node-negative breast cancer patients with long-term follow-up not treated in the adjuvant setting. The prognostic significance of Ep-CAM RNA expression for disease-free survival (DFS), metastasis-free survival (MFS), and breast cancer-specific overall survival (OS) was evaluated in univariate and multivariate analysis adjusted for age, grading, pTstage, ER as well as PR receptor and HER-2 status. Additionally, Ep-CAM RNA expression was compared with immunohistochemistry (IHC) for Ep-CAM in 194 patients. The prognostic impact of Ep-CAM gene expression was validated in further 588 node-negative breast cancer patients. Levels of Ep-CAM RNA expression showed a significant correlation with IHC (P = 0.001) and predicted in univariate analysis DFS (P = 0.001, HR = 2.4), MFS (P = 0.003, HR = 2.5), and OS (P = 0.002, HR = 3.1) accurately. The prognostic influence of Ep-CAM RNA was significant also in multivariate analysis for DFS (P = 0.017, HR = 2.0), MFS (P = 0.049, HR = 1.9), and OS (P = 0.042, HR = 2.3), respectively. The association with MFS was confirmed in an independent validation cohort in univariate (P = 0.006, HR = 1.9) and multivariate (P = 0.035, HR = 1.7) analysis. Ep-CAM RNA correlated with the proliferation metagene (P < 0.001, R=0.425) Nevertheless, in multivariate analysis, Ep-CAM was associated with MFS independent from the proliferation metagene (P = 0.030, HR = 1.8). In conclusion, Ep-CAM RNA expression is associated with poor MFS in three cohorts of untreated node-negative breast cancer.

Fertility After Ovarian Cystectomy: How Does Surgery Affect IVF/ICSI Outcomes?

Introduction For patients considering undergoing assisted reproductive techniques (ART), many concerns arise when persistent ovarian cysts are found. This large study aimed to determine how ovarian cyst removal affects success rates of IVF/ICSI therapies. Methods 550 patients who underwent an IVF/ICSI treatment between 2002 and 2011 with a persistent ovarian cyst ≤ 5 cm before treatment were analyzed retrospectively. 328 patients' preference was to undergo a laparoscopic cystectomy and 222 patients opted for a conservative management. Control subjects included 13 552 patients undergoing IVF/ICSI at the same period of time without an ovarian cyst. Results After adjusting for age, patients with ovarian cysts without surgery needed a significant higher stimulation dose than the control group (2576.4 vs. 2207.5 IU, p < 0.001). However, on average, they had 1.13 (- 0.25 - 2.01) higher oocyte number retrieved compared to the operated patients (9.0 ± 5.5 vs. 8.2 ± 5.0) (p = 0.012). Patients after surgical cyst removal had a significant lower number of oocytes retrieved (MNOR) in comparison to the control group (8.2 ± 5.0 vs. 9.5 ± 5.4) (p = 0.00). Compared to controls, operated patients had similar clinical pregnancy rate (CPR) (34.2 vs. 33.5%) OR 1.031 (95% CI 0.817 - 1.302) (p = 0.815). Compared to controls, patients without surgery showed significant lower pregnancy rate (34.2 vs. 25,7%) OR 1.428 (95% CI 1.054 - 1.936) (p = 0.002) and lower live birth rate (LBR) (21.9 vs. 13.5%) OR 1.685 (95% CI 1.143 - 2.485) (p = 0.008). Conclusions Ovarian cystectomy did not negatively impact the pregnancy rate or the live birth rate compared to controls.

Sex-specific differences in pulmonary embolism.

INTRODUCTION: Sex-specific differences regarding risk factors, symptoms and prognosis have been reported for several cardiovascular diseases. For patients with pulmonary embolism (PE), sex-specific data are limited and inconsistent. We aimed to investigate sex-specific differences in PE. MATERIALS AND METHODS: Over a 10-year period (01/2003-09/2013), patients with confirmed PE were enrolled in a prospective single-centre cohort study. RESULTS: We prospectively examined 569 PE patients (55.9% women). Men more often had cancer (20.7% vs. 13.5%, p = 0.024) and unprovoked PE (61.0% vs. 47.5%, p = 0.001) while women more frequently presented with risk factors for venous thromboembolism such as older age (median, 71 [IQR, 55-79] vs. 67 [53-75] years, p = 0.008), surgery/trauma/immobilisation (38.4% vs. 29.5%, p = 0.026) and sex-hormone therapy (14.8% vs. 0.8%, p < 0.001). Overall, 84 patients (14.8%) had an adverse 30-day outcome and 43 (7.6%) died within 30 days; outcomes did not differ between males and females and were not influenced by the patients' sex. Risk stratification markers and models such as right ventricular dysfunction on TTE/CT, cardiac troponin, sPESI, Bova score and 2014 ESC guidelines algorithm predicted adverse outcome in normotensive female patients only, while tachycardia, hypoxia, NT-proBNP and modified FAST score were able to predict an adverse outcome in both sexes. Using sex-specific biomarker cut-off values, the 2014 ESC guidelines algorithm was able to predict adverse outcome in both sexes. CONCLUSIONS: The 30-day adverse outcomes did not differ between male and female PE patients and were not influenced by the patients' sex despite sex-specific differences in the prognostic performance of risk stratification markers/models.

Quality of life and functional limitations after pulmonary embolism and its prognostic relevance.

BACKGROUND: While the importance of patients' quality of life (QoL) in chronic cardiac or pulmonary disease is uncontroversial, the burden of an acute pulmonary embolism (PE) on QoL has received little attention thus far. OBJECTIVES: We aimed to validate the German PEmb-QoL questionnaire, identify associations between QoL and clinical/functional parameters, and investigate the prognostic relevance of QoL for long-term survival in survivors of an acute PE episode. PATIENTS/METHODS: Patients were invited for a clinical follow-up visit including assessment of QoL using the German PEmb-QoL questionnaire 6 months after an objectively confirmed PE at a single center. Internal consistency reliability, construct-related validity, and regressions between PEmb-QoL and clinical patient-characteristics were assessed using standard scale construction techniques. RESULTS: Overall, 101 patients [median age, 69 ([interquartile range] IQR 57-75) years; women, 48.5%] were examined 208 (IQR 185-242) days after PE. Internal consistency reliability and construct-related validity of the PEmb-QoL questionnaire were acceptable. As many as 47.0% of patients reported dyspnea, 27.5% had right ventricular (RV) dysfunction on transthoracic echocardiography (TTE), and 25.3% were diagnosed with post-PE impairment (PPEI) at 6-month follow-up. Furthermore, 15.9% of patients were diagnosed with depression 6 months after an acute PE. The QoL was affected by dyspnea, preexisting pulmonary disease, and PPEI, and a reduced QoL was associated with an increased risk for long-term mortality after an observation period of 3.6 years. CONCLUSIONS: The German PEmb-QoL questionnaire is a reliable instrument for assessing QoL 6 months after PE. The QoL was affected by dyspnea, preexisting pulmonary disease, and PPEI and was associated with long-term mortality.

Multi-trait genome-wide association study identifies new loci associated with optic disc parameters.

A new avenue of mining published genome-wide association studies includes the joint analysis of related traits. The power of this approach depends on the genetic correlation of traits, which reflects the number of pleiotropic loci, i.e. genetic loci influencing multiple traits. Here, we applied new meta-analyses of optic nerve head (ONH) related traits implicated in primary open-angle glaucoma (POAG); intraocular pressure and central corneal thickness using Haplotype reference consortium imputations. We performed a multi-trait analysis of ONH parameters cup area, disc area and vertical cup-disc ratio. We uncover new variants; rs11158547 in PPP1R36-PLEKHG3 and rs1028727 near SERPINE3 at genome-wide significance that replicate in independent Asian cohorts imputed to 1000 Genomes. At this point, validation of these variants in POAG cohorts is hampered by the high degree of heterogeneity. Our results show that multi-trait analysis is a valid approach to identify novel pleiotropic variants for ONH.

Genotypic resistance testing in HIV by arrayed primer extension.

The analysis of mutations that are associated with the occurrence of drug resistance is important for monitoring the antiretroviral therapy of patients infected with human immunodeficiency virus (HIV). Here, we describe the establishment and successful application of Arrayed Primer Extension (APEX) for genotypic resistance testing in HIV as a rapid and economical alternative to standard sequencing. The assay is based on an array of oligonucleotide primers that are immobilised via their 5'-ends. Upon hybridisation of template DNA, a primer extension reaction is performed in the presence of the four dideoxynucleotides, each labelled with a distinct fluorophore. The inserted label immediately indicates the sequence at the respective position. Any mutation changes the colour pattern. We designed a microarray for the analysis of 26 and 33 codons in the HIV protease and reverse transcriptase, respectively, which are of special interest with respect to drug resistance. The enormous genome variability of HIV represents a big challenge for genotypic resistance tests, which include a hybridisation step, both in terms of specificity and probe numbers. The use of degenerated oligonucleotides resulted in a significant reduction in the number of primers needed. For validation, DNA of 94 and 48 patients that exhibited resistance to inhibitors of HIV protease and reverse transcriptase, respectively, were analysed. The validation included HIV subtype B, prevalent in industrialised countries, as well as non-subtype B samples that are more common elsewhere.

The immune contexture of hepatocellular carcinoma predicts clinical outcome.

The general relevance of the immune system for cancer development and therapy is increasingly recognized. However and although the immune contexture of most human cancer types has been determined, a global characterisation of the immune tumour microenvironment in hepatocellular carcinoma (HCC) is lacking. Equally, differences in the immune contexture of HCC between different patient subgroups and its effect on survival remain to be established. Here we report an in silico analysis of the immune contexture of human HCC. Using large deep sequencing HCC tumour, adjacent non-tumour and healthy liver high-dimensional data sets, we were able to reveal previously unrecognized differences in the immune contexture of HCC. Strikingly, we found that different etiologies and HCC stages were not associated with major changes in the immune contexture. In contrast, the presence of T cells and cytotoxic cells as well as the absence of macrophages and Th2 cells positively correlated with patient survival. Based on these novel findings, we developed a prognostic score that accurately distinguishes between patients with good and poor survival. Our study provides the first global characterisation of the immune contexture of HCC and will have direct implications for future HCC therapies.

Expressional analysis of disease-relevant signalling-pathways in primary tumours and metastasis of head and neck cancers.

Head and neck squamous cell carcinoma (HNSCC) often metastasize to lymph nodes resulting in poor prognosis for patients. Unfortunately, the underlying molecular mechanisms contributing to tumour aggressiveness, recurrences, and metastasis are still not fully understood. However, such knowledge is key to identify biomarkers and drug targets to improve prognosis and treatments. Consequently, we performed genome-wide expression profiling of 15 primary HNSSCs compared to corresponding lymph node metastases and non-malignant tissue of the same patient. Differentially expressed genes were bioinformatically exploited applying stringent filter criteria, allowing the discrimination between normal mucosa, primary tumours, and metastases. Signalling networks involved in invasion contain remodelling of the extracellular matrix, hypoxia-induced transcriptional modulation, and the recruitment of cancer associated fibroblasts, ultimately converging into a broad activation of PI3K/AKT-signalling pathway in lymph node metastasis. Notably, when we compared the diagnostic and prognostic value of sequencing data with our expression analysis significant differences were uncovered concerning the expression of the receptor tyrosine kinases EGFR and ERBB2, as well as other oncogenic regulators. Particularly, upregulated receptor tyrosine kinase combinations for individual patients varied, implying potential compensatory and resistance mechanisms against specific targeted therapies. Collectively, we here provide unique transcriptional profiles for disease predictions and comprehensively analyse involved signalling pathways in advanced HNSCC.

Standardized weaning of infants <32 weeks of gestation from continuous positive airway pressure - a feasibility study.

The practice of weaning premature infants from continuous positive airway pressure (CPAP) varies considerably and is usually performed without written standards. In this study, the feasibility of a standardized weaning approach was evaluated. In a quasi-experimental design, data from a prospective, post-intervention cohort (n=41) were compared to data from a pre-intervention cohort (n=36). Standardized weaning was feasible but no significant differences in short-term respiratory outcomes were observed. Weaning from CPAP was achieved at 32.1 ± 1.6 (post-intervention) versus 32.5 ± 2.3 weeks (pre-intervention) postmenstrual age. More rigorous, large-scale clinical trials are necessary before firm recommendations on distinct weaning regimens can be made.

Kinome Profiling of Regulatory T Cells: A Closer Look into a Complex Intracellular Network.

Regulatory T cells (Treg) are essential for T cell homeostasis and maintenance of peripheral tolerance. They prevent activation of auto-reactive T effector cells (Teff) in the context of autoimmunity and allergy. Otherwise, Treg also inhibit effective immune responses against tumors. Besides a number of Treg-associated molecules such as Foxp3, CTLA-4 or GARP, known to play critical roles in Treg differentiation, activation and function, the involvement of additional regulatory elements is suggested. Herein, kinase activities seem to play an important role in Treg fine tuning. Nevertheless, our knowledge regarding the complex intracellular signaling pathways controlling phenotype and function of Treg is still limited and based on single kinase cascades so far. To gain a more comprehensive insight into the pathways determining Treg function we performed kinome profiling using a phosphorylation-based kinome array in human Treg at different activation stages compared to Teff. Here we have determined intriguing quantitative differences in both populations. Resting and activated Treg showed an altered pattern of CD28-dependent kinases as well as of those involved in cell cycle progression. Additionally, significant up-regulation of distinct kinases such as EGFR or CK2 in activated Treg but not in Teff not only resemble data we obtained in previous studies in the murine system but also suggest that those specific molecular activation patterns can be used for definition of the activation and functional state of human Treg. Taken together, detailed investigation of kinome profiles opens the possibility to identify novel molecular mechanisms for a better understanding of Treg biology but also for development of effective immunotherapies against unwanted T cell responses in allergy, autoimmunity and cancer.

Activation of the basal cell carcinoma pathway in a patient with CNS HGNET-BCOR diagnosis: consequences for personalized targeted therapy.

High grade neuroepithelial tumor of the central nervous system with BCOR alteration (CNS HGNET-BCOR) is a recently described new tumor entity with a dismal prognosis. The objective of this study was to identify and validate pathways deregulated in CNS HGNET-BCOR as basis for targeted therapy approaches.We characterized the BCOR alteration in a pediatric patient with CNS HGNET-BCOR diagnosis by Sanger sequencing and demonstrated an elevated BCOR expression by qRT-PCR and western blot. By whole transcriptome sequencing and Ingenuity Pathway Analysis, we identified the activation of the Sonic Hedgehog (SHH) and of the WNT signaling pathway in two different regions of the primary tumor and of one inoculation metastasis compared to normal brain. We validated the activation of the SHH and of the WNT pathway by qRT-PCR analysis of GLI1 and AXIN2 respectively. GLI1 and AXIN2 were upregulated in the primary tumor and in two inoculation metastases compared to normal brain. Mutational analysis of SMO, PTCH1 and SUFU, three key components of the SHH pathway, revealed a Single Nucleotide Polymorphism (SNP) in PTCH1 (rs357564). We tested the effect of the GLI-inhibitor arsenic trioxide (ATO) on a short-term cell culture isolated from the metastasis. ATO was able to reduce the viability of the cells with an IC50 of 1.3 µM.In summary, these results provide functional evidence of altered BCOR expression and homogeneous coactivation of both the SHH and WNT signaling pathways, building the basis for potential novel therapeutic approaches for patients with a CNS HGNET-BCOR diagnosis.

The γH2AX assay for genotoxic and nongenotoxic agents: comparison of H2AX phosphorylation with cell death response.

DNA double-strand breaks (DSBs) and blocked replication forks resulting from bulky adducts and inhibitors of replication activate the DNA damage response (DDR), a signaling pathway marked by phosphorylation of histone 2AX (H2AX). The phosphorylated form, γH2AX, accumulates at the site of the damage and can be visualized as foci by immunocytochemistry. The objective of this study was to assess if γH2AX is a reliable biomarker for genotoxic exposures. To this end, we selected 14 well-known genotoxic compounds and compared them with 10 nongenotoxic chemicals, using CHO-9 cells because they are well characterized as to DNA repair and DDR. We quantified γH2AX foci manually and automatically. In addition, total γH2AX activation was determined by flow cytometry. For all chemicals the cytotoxic dose response was assayed by a metabolic cytotoxicity assay. We show that (1) all genotoxic agents induced γH2AX dose-dependently whereas nongenotoxic agents do not; (2) γH2AX was observed for genotoxicants in the cytotoxic dose range, revealing a correlation between cytotoxicity and γH2AX for genotoxic agents; for nongenotoxic agents cytotoxicity was not related to γH2AX; (3) manual scoring of γH2AX and automated scoring provided comparable results, the automated scoring was faster and investigator independent; (4) data obtained by foci counting and flow cytometry showed a high correlation, suggesting that γH2AX scoring by flow cytometry has the potential for high-throughput analysis. However, the microscopic evaluation can provide additional information as to foci size, distribution, colocalization and background staining; (5) γH2AX foci were colocalized with 53BP1 and Rad51, supporting the notion that they represent true DSBs. Collectively, the automated analysis of γH2AX foci allows for rapid determination of genetic damage in mammalian cells. The data revealed that the induction of γH2AX by genotoxicants is related to loss of viability and support γH2AX as a reliable bio-indicator for pretoxic DNA damage.

Immunoglobulin kappa C predicts overall survival in node-negative breast cancer.

BACKGROUND: Biomarkers of the immune system are currently not used as prognostic factors in breast cancer. We analyzed the association of the B cell/plasma cell marker immunoglobulin kappa C (IGKC) and survival of untreated node-negative breast cancer patients. MATERIAL AND METHODS: IGKC expression was evaluated by immunostaining in a cohort of 335 node-negative breast cancer patients with a median follow-up of 152 months. The prognostic significance of IGKC for disease-free survival (DFS) and breast cancer-specific overall survival (OS) was evaluated with Kaplan-Meier survival analysis as well as univariate and multivariate Cox analysis adjusted for age at diagnosis, pT stage, histological grade, estrogen receptor (ER) status, progesterone receptor (PR) status, Ki-67 and human epidermal growth factor receptor 2 (HER-2) status. RESULTS: 160 patients (47.7%) showed strong expression of IGKC. Univariate analysis showed that IGKC was significantly associated with DFS (P = 0.017, hazard ratio [HR] = 0.570, 95% confidence interval [CI] = 0.360-0.903) and OS (P = 0.011, HR = 0.438, 95% CI = 0.233-0.822) in the entire cohort. The significance of IGKC was especially strong in ER negative and in luminal B carcinomas. In multivariate analysis IGKC retained its significance independent of established clinical factors for DFS (P = 0.004, HR = 0.504, 95% CI = 0.315-0.804) as well as for OS (P = 0.002, HR = 0.371, 95% CI = 0.196-0.705). CONCLUSION: Expression of IGKC has an independent protective impact on DFS and OS in node-negative breast cancer.