Andrea Staratschek-Jox

Replication of Lung Cancer Susceptibility Loci at Chromosomes 15q25, 5p15, and 6p21: A Pooled Analysis From the International Lung Cancer Consortium

BACKGROUND Genome-wide association studies have identified three chromosomal regions at 15q25, 5p15, and 6p21 as being associated with the risk of lung cancer. To confirm these associations in independent studies and investigate heterogeneity of these associations within specific subgroups, we conducted a coordinated genotyping study within the International Lung Cancer Consortium based on independent studies that were not included in previous genome-wide association studies. METHODS Genotype data for single-nucleotide polymorphisms at chromosomes 15q25 (rs16969968, rs8034191), 5p15 (rs2736100, rs402710), and 6p21 (rs2256543, rs4324798) from 21 case-control studies for 11 645 lung cancer case patients and 14 954 control subjects, of whom 85% were white and 15% were Asian, were pooled. Associations between the variants and the risk of lung cancer were estimated by logistic regression models. All statistical tests were two-sided. RESULTS Associations between 15q25 and the risk of lung cancer were replicated in white ever-smokers (rs16969968: odds ratio [OR] = 1.26, 95% confidence interval [CI] = 1.21 to 1.32, P(trend) = 2 x 10(-26)), and this association was stronger for those diagnosed at younger ages. There was no association in never-smokers or in Asians between either of the 15q25 variants and the risk of lung cancer. For the chromosome 5p15 region, we confirmed statistically significant associations in whites for both rs2736100 (OR = 1.15, 95% CI = 1.10 to 1.20, P(trend) = 1 x 10(-10)) and rs402710 (OR = 1.14, 95% CI = 1.09 to 1.19, P(trend) = 5 x 10(-8)) and identified similar associations in Asians (rs2736100: OR = 1.23, 95% CI = 1.12 to 1.35, P(trend) = 2 x 10(-5); rs402710: OR = 1.15, 95% CI = 1.04 to 1.27, P(trend) = .007). The associations between the 5p15 variants and lung cancer differed by histology; odds ratios for rs2736100 were highest in adenocarcinoma and for rs402710 were highest in adenocarcinoma and squamous cell carcinomas. This pattern was observed in both ethnic groups. Neither of the two variants on chromosome 6p21 was associated with the risk of lung cancer. CONCLUSIONS In this international genetic association study of lung cancer, previous associations found in white populations were replicated and new associations were identified in Asian populations. Future genetic studies of lung cancer should include detailed stratification by histology.

High-resolution transcriptome of human macrophages.

Macrophages are dynamic cells integrating signals from their microenvironment to develop specific functional responses. Although, microarray-based transcriptional profiling has established transcriptional reprogramming as an important mechanism for signal integration and cell function of macrophages, current knowledge on transcriptional regulation of human macrophages is far from complete. To discover novel marker genes, an area of great need particularly in human macrophage biology but also to generate a much more thorough transcriptome of human M1- and M1-like macrophages, we performed RNA sequencing (RNA-seq) of human macrophages. Using this approach we can now provide a high-resolution transcriptome profile of human macrophages under classical (M1-like) and alternative (M2-like) polarization conditions and demonstrate a dynamic range exceeding observations obtained by previous technologies, resulting in a more comprehensive understanding of the transcriptome of human macrophages. Using this approach, we identify important gene clusters so far not appreciated by standard microarray techniques. In addition, we were able to detect differential promoter usage, alternative transcription start sites, and different coding sequences for 57 gene loci in human macrophages. Moreover, this approach led to the identification of novel M1-associated (CD120b, TLR2, SLAMF7) as well as M2-associated (CD1a, CD1b, CD93, CD226) cell surface markers. Taken together, these data support that high-resolution transcriptome profiling of human macrophages by RNA-seq leads to a better understanding of macrophage function and will form the basis for a better characterization of macrophages in human health and disease.

Unique transcriptome signature of mouse microglia

Microglial cells can be derived directly from the dissociated brain tissue by sorting procedures, from postnatal glial cultures by mechanic isolation or from pluripotent stem cells by differentiation. The detailed molecular phenotype of microglia from different sources is still unclear. Here, we performed a whole transcriptome analysis of flow cytometry‐sorted microglia, primary postnatal cultured microglia, embryonic stem cell derived microglia (ESdM), and other cell types. Microglia and ESdM, both cultured in serum‐free medium, were closely related to sorted microglia and showed a unique transcriptome profile, clearly distinct to other myeloid cell types, T cells, astrocytes, and neurons. ESdM and primary cultured microglia showed strong overlap in their transcriptome. Only 143 genes were differentially expressed between both cell types, mainly derived from immune‐related genes with a higher activation status of proinflammatory and immune defense genes in primary microglia compared to ESdM. Flow cytometry analysis of cell surface markers CD54, CD74, and CD274 selected from the microarray confirmed the close phenotypic relation between ESdM and primary cultured microglia. Thus, assessment of genome‐wide transcriptional regulation demonstrates that microglial cells are unique and clearly distinct from other macrophage cell types.

Blood-Based Gene Expression Signatures in Non–Small Cell Lung Cancer

Purpose: Blood-based surrogate markers would be attractive biomarkers for early detection, diagnosis, prognosis, and prediction of therapeutic outcome in cancer. Disease-associated gene expression signatures in peripheral blood mononuclear cells (PBMC) have been described for several cancer types. However, RNA-stabilized whole blood–based technologies would be clinically more applicable and robust. We evaluated the applicability of whole blood–based gene expression profiling for the detection of non–small cell lung cancer (NSCLC). Experimental Design: Expression profiles were generated from PAXgene-stabilized blood samples from three independent groups consisting of NSCLC cases and controls (n = 77, 54, and 102), using the Illumina WG6-VS2 system. Results: Several genes are consistently differentially expressed in whole blood of NSCLC patients and controls. These expression profiles were used to build a diagnostic classifier for NSCLC, which was validated in an independent validation set of NSCLC patients (stages I–IV) and hospital-based controls. The area under the receiver operator curve was calculated to be 0.824 (P < 0.001). In a further independent dataset of stage I NSCLC patients and healthy controls the AUC was 0.977 (P < 0.001). Specificity of the classifier was validated by permutation analysis in both validation cohorts. Genes within the classifier are enriched in immune-associated genes and show specificity for NSCLC. Conclusions: Our results show that gene expression profiles of whole blood allow for detection of manifest NSCLC. These results prompt further development of gene expression–based biomarker tests in peripheral blood for the diagnosis and early detection of NSCLC. Clin Cancer Res; 17(10); 3360–7. ©2011 AACR.

Liver-Primed Memory T Cells Generated under Noninflammatory Conditions Provide Anti-infectious Immunity

Development of CD8(+) T cell (CTL) immunity or tolerance is linked to the conditions during T cell priming. Dendritic cells (DCs) matured during inflammation generate effector/memory T cells, whereas immature DCs cause T cell deletion/anergy. We identify a third outcome of T cell priming in absence of inflammation enabled by cross-presenting liver sinusoidal endothelial cells. Such priming generated memory T cells that were spared from deletion by immature DCs. Similar to central memory T cells, liver-primed T cells differentiated into effector CTLs upon antigen re-encounter on matured DCs even after prolonged absence of antigen. Their reactivation required combinatorial signaling through the TCR, CD28, and IL-12R and controlled bacterial and viral infections. Gene expression profiling identified liver-primed T cells as a distinct Neuropilin-1(+) memory population. Generation of liver-primed memory T cells may prevent pathogens that avoid DC maturation by innate immune escape from also escaping adaptive immunity through attrition of the T cell repertoire.

Intrahepatic IL-8 producing Foxp3⁺CD4⁺ regulatory T cells and fibrogenesis in chronic hepatitis C.

BACKGROUND & AIMS Regulatory CD4(+) T cells (Tregs) are considered to affect outcomes of HCV infection, because they increase in number during chronic hepatitis C and can suppress T-cell functions. METHODS Using microarray analysis, in situ immunofluorescence, ELISA, and flowcytometry, we characterised functional differentiation and localisation of adaptive Tregs in patients with chronic hepatitis C. RESULTS We found substantial upregulation of IL-8 in Foxp3(+)CD4(+) Tregs from chronic hepatitis C. Activated GARP-positive IL-8(+) Tregs were particularly enriched in livers of patients with chronic hepatitis C in close proximity to areas of fibrosis and their numbers were correlated with the stage of fibrosis. Moreover, Tregs induced upregulation of profibrogenic markers TIMP1, MMP2, TGF-beta1, alpha-SMA, collagen, and CCL2 in primary human hepatic stellate cells (HSC). HSC activation, but not Treg suppressor function, was blocked by adding a neutralizing IL-8 antibody. CONCLUSIONS Our studies identified Foxp3(+)CD4(+) Tregs as an additional intrahepatic source of IL-8 in chronic hepatitis C acting on HSC. Thus, Foxp3(+)CD4(+) Tregs in chronic hepatitis C have acquired differentiation as regulators of fibrogenesis in addition to suppressing local immune responses.

Identification of lung cancer with high sensitivity and specificity by blood testing

BackgroundLung cancer is a very frequent and lethal tumor with an identifiable risk population. Cytological analysis and chest X-ray failed to reduce mortality, and CT screenings are still controversially discussed. Recent studies provided first evidence for the potential usefulness of autoantigens as markers for lung cancer.MethodsWe used extended panels of arrayed antigens and determined autoantibody signatures of sera from patients with different kinds of lung cancer, different common non-tumor lung pathologies, and controls without any lung disease by a newly developed computer aided image analysis procedure. The resulting signatures were classified using linear kernel Support Vector Machines and 10-fold cross-validation.ResultsThe novel approach allowed for discriminating lung cancer patients from controls without any lung disease with a specificity of 97.0%, a sensitivity of 97.9%, and an accuracy of 97.6%. The classification of stage IA/IB tumors and controls yielded a specificity of 97.6%, a sensitivity of 75.9%, and an accuracy of 92.9%. The discrimination of lung cancer patients from patients with non-tumor lung pathologies reached an accuracy of 88.5%.ConclusionWe were able to separate lung cancer patients from subjects without any lung disease with high accuracy. Furthermore, lung cancer patients could be seprated from patients with other non-tumor lung diseases. These results provide clear evidence that blood-based tests open new avenues for the early diagnosis of lung cancer.

Detection of Epstein-Barr Virus in Hodgkin-Reed-Sternberg Cells : No Evidence for the Persistence of Integrated Viral Fragments in Latent Membrane Protein-1 (LMP-1)-Negative Classical Hodgkin’s Disease

Classical Hodgkins disease (HD) is associated with Epstein-Barr virus (EBV) infection. Although in developing countries EBV can be demonstrated in Hodgkin-Reed-Sternberg (H-RS) cells in up to 95% of HD cases, in industrialized countries only about 50% of HD cases are associated with EBV. An open question remains whether EBV in the EBV-negative cases has escaped detection by standard screening procedures due to deletions in the viral genome associated with integration of viral fragments into the host cell genome. We, among others, recently described this phenomenon in Burkitts lymphoma cells. To investigate whether H-RS cells in latent membrane protein-1 (LMP-1)-negative HD cases harbor fragments of the EBV genome, we combined fluorescence in situ hybridization (FISH) using a set of six overlapping DNA probes spanning the whole EBV genome with immunophenotyping of fresh frozen lymphoma sections. Results in the eight cases analyzed were as follows: in three LMP-1-positive cases, FISH analysis yielded specific signals for each EBV DNA probe in H-RS cells, which had been identified by morphology and CD30 staining. In contrast, none of the EBV DNA probes hybridized to the H-RS cells in the five LMP-1-negative cases. Thus, there is no evidence for the presence of fragments of the viral genome integrated into the host cell genome in the LMP-1-negative cases. Furthermore, in the LMP-1-positive cases analyzed, no large deletions in the viral genome were detected. These results show that, in classical HD, LMP-1-negative cases do not harbor EBV DNA within the H-RS cells. Whether, in these cases, a still unknown virus contributes to the transformation and maintenance of the malignant phenotype remains to be established.

Non-Hodgkin lymphoma expressing high levels of the danger-signalling protein HMGB1

HMGB1 is a high mobility group protein that can act either as a DNA binding protein or extracellularly as a cytokine-like danger signal. Extracellular HMGB1, either actively secreted or passively released by necrotic cells, is linked to inflammation and cancer. Herein, the results of a study to quantify the expression of HMGB1 in lymphomas by quantitative real-time RT-PCR are presented. HMGB1 expression was analysed in 18 non-Hodgkin lymphomas and two lymphoma cell lines. 11/18 primary lymphomas expressed HMGB1 mRNA at a level exceeding the average of normal lymph nodes. Immunohistochemistry showed that HMGB1 positivity is confined to the lymphoma cells. No correlation between HMGB1 expression and grading was found. However, a high percentage of lymphomas is overexpressing a danger-signalling protein. This protein can support the growth and angiogenesis of lymphoma cells in a paracrine way when released e.g. due to necrosis. Thus it constitutes an interesting therapeutic target as well.

RNA-Stabilized Whole Blood Samples but Not Peripheral Blood Mononuclear Cells Can Be Stored for Prolonged Time Periods Prior to Transcriptome Analysis

Microarray-based transcriptome analysis of peripheral blood as surrogate tissue has become an important approach in clinical implementations. However, application of gene expression profiling in routine clinical settings requires careful consideration of the influence of sample handling and RNA isolation methods on gene expression profile outcome. We evaluated the effect of different sample preservation strategies (eg, cryopreservation of peripheral blood mononuclear cells or freezing of PAXgene-stabilized whole blood samples) on gene expression profiles. Expression profiles obtained from cryopreserved peripheral blood mononuclear cells differed substantially from those of their nonfrozen counterpart samples. Furthermore, expression profiles in cryopreserved peripheral blood mononuclear cell samples were found to undergo significant alterations with increasing storage period, whereas long-term freezing of PAXgene RNA stabilized whole blood samples did not significantly affect stability of gene expression profiles. This report describes important technical aspects contributing toward the establishment of robust and reliable guidance for gene expression studies using peripheral blood and provides a promising strategy for reliable implementation in routine handling for diagnostic purposes.