Hans-Juergen Thiesen

Gene Expression Signatures for Tumor Progression, Tumor Subtype, and Tumor Thickness in Laser-Microdissected Melanoma Tissues

Purpose: To better understand the molecular mechanisms of malignant melanoma progression and metastasis, gene expression profiling was done of primary melanomas and melanoma metastases. Experimental Design: Tumor cell–specific gene expression in 19 primary melanomas and 22 melanoma metastases was analyzed using oligonucleotide microarrays after laser-capture microdissection of melanoma cells. Statistical analysis was done by random permutation analysis and support vector machines. Microarray data were further validated by immunohistochemistry and immunoblotting. Results: Overall, 308 genes were identified that showed significant differential expression between primary melanomas and melanoma metastases (false discovery rate ≤ 0.05). Significantly overrepresented gene ontology categories in the list of 308 genes were cell cycle regulation, mitosis, cell communication, and cell adhesion. Overall, 47 genes showed up-regulation in metastases. These included Cdc6, Cdk1, septin 6, mitosin, kinesin family member 2C, osteopontin, and fibronectin. Down-regulated genes included E-cadherin, fibroblast growth factor binding protein, and desmocollin 1 and desmocollin 3, stratifin/14-3-3σ, and the chemokine CCL27. Using support vector machine analysis of gene expression data, a performance of >85% correct classifications for primary melanomas and metastases was reached. Further analysis showed that subtypes of primary melanomas displayed characteristic gene expression patterns, as do thin tumors (≤1.0 mm Breslow thickness) compared with intermediate and thick tumors (>2.0 mm Breslow thickness). Conclusions: Taken together, this large-scale gene expression study of malignant melanoma identified molecular signatures related to metastasis, melanoma subtypes, and tumor thickness. These findings not only provide deeper insights into the pathogenesis of melanoma progression but may also guide future research on innovative treatments.

Molecular discrimination of responders and nonresponders to anti-TNFalpha therapy in rheumatoid arthritis by etanercept

IntroductionAbout 30% of rheumatoid arthritis patients fail to respond adequately to TNFα-blocking therapy. There is a medical and socioeconomic need to identify molecular markers for an early prediction of responders and nonresponders.MethodsRNA was extracted from peripheral blood mononuclear cells of 19 rheumatoid arthritis patients before the first application of the TNFα blocker etanercept as well as after 72 hours. Clinical response was assessed over 3 months using the 28-joint-count Disease Activity Score and X-ray scans. Supervised learning methods were applied to Affymetrix Human Genome U133 microarray data analysis to determine highly selective discriminatory gene pairs or triplets with prognostic relevance for the clinical outcome evinced by a decline of the 28-joint-count Disease Activity Score by 1.2.ResultsEarly downregulation of expression levels secondary to TNFα neutralization was associated with good clinical responses, as shown by a decline in overall disease activity 3 months after the start of treatment. Informative gene sets include genes (for example, NFKBIA, CCL4, IL8, IL1B, TNFAIP3, PDE4B, PPP1R15A and ADM) involved in different pathways and cellular processes such as TNFα signalling via NFκB, NFκB-independent signalling via cAMP, and the regulation of cellular and oxidative stress response. Pairs and triplets within these genes were found to have a high prognostic value, reflected by prediction accuracies of over 89% for seven selected gene pairs and of 95% for 10 specific gene triplets.ConclusionOur data underline that early gene expression profiling is instrumental in identifying candidate biomarkers to predict therapeutic outcomes of anti-TNFα treatment regimes.

mtDNA nt13708A variant increases the risk of multiple sclerosis.

Background Mitochondrial DNA (mtDNA) polymorphism is a possible factor contributing to the maternal parent-of-origin effect in multiple sclerosis (MS) susceptibility. Methods and Findings In order to investigate the role of mtDNA variations in MS, we investigated six European MS case-control cohorts comprising >5,000 individuals. Three well matched cohorts were genotyped with seven common, potentially functional mtDNA single nucleotide polymorphisms (SNPs). A SNP, nt13708 G/A, was significantly associated with MS susceptibility in all three cohorts. The nt13708A allele was associated with an increased risk of MS (OR = 1.71, 95% CI 1.28–2.26, P = 0.0002). Subsequent sequencing of the mtDNA of 50 individuals revealed that the nt13708 itself, rather than SNPs linked to it, was responsible for the association. However, the association of nt13708 G/A with MS was not significant in MS cohorts which were not well case-control matched, indicating that the significance of association was affected by the population structure of controls. Conclusions Taken together, our finding identified the nt13708A variant as a susceptibility allele to MS, which could contribute to defining the role of the mitochondrial genome in MS pathogenesis.

Elevated type I interferon-like activity in a subset of multiple sclerosis patients: molecular basis and clinical relevance

BackgroundA subset of patients with multiple sclerosis (MS) shows an increased endogenous IFN-like activity before initiation of IFN-beta treatment. The molecular basis of this phenomenon and its relevance to predict individual therapy outcomes are not yet fully understood. We studied the expression patterns of these patients, the prognostic value of an elevated IFN-like activity, and the gene regulatory effects of exogenously administered IFN-beta.MethodsMicroarray gene expression profiling was performed for 61 MS patients using peripheral blood mononuclear cells obtained before and after 1 month of IFN-beta therapy. Expression levels of genes involved in pathways either inducing or being activated by IFN-beta were compared between patients with high (MX1high cohort) and low (MX1low cohort) endogenous IFN-like activity. Patients were followed for 5 years and relapses as well as progression on the expanded disability status scale (EDSS) were documented.ResultsBefore the start of therapy, 11 patients presented elevated mRNA levels of IFN-stimulated genes indicative of a relatively high endogenous IFN-like activity (MX1high). In these patients, pathogen receptors (for example, TLR7, RIG-I and IFIH1) and transcription factors were also expressed more strongly, which could be attributed to an overactivity of IFN-stimulated gene factor 3 (ISGF3, a complex formed by STAT1, STAT2 and IFN regulatory factor 9). After 1 month of IFN-beta therapy, the expression of many pathway genes was significantly induced in MX1low patients, but remained unaltered in MX1high patients. During follow-up, relapse rate and changes in EDSS were comparable between both patient groups, with differences seen between different types of IFN-beta drug application.ConclusionsTherapeutic IFN-beta induces the transcription of several genes involved in IFN-related pathways. In a subgroup of MS patients, the expression of these genes is already increased before therapy initiation, possibly driven by an overexpression of ISGF3. Patients with high and low endogenous IFN-like activity showed similar clinical long-term courses of disease. Different results were obtained for different IFN-beta drug preparations, and this merits further investigation.

Disruption of the Interaction between Transcriptional Intermediary Factor 1β and Heterochromatin Protein 1 Leads to a Switch from DNA Hyper- to Hypomethylation and H3K9 to H3K27 Trimethylation on the MEST Promoter Correlating with Gene Reactivation

Here, we identified the imprinted mesoderm-specific transcript (MEST) gene as an endogenous TIF1beta primary target gene and demonstrated that transcriptional intermediary factor (TIF) 1beta, through its interaction with heterochromatin protein (HP) 1, is essential in establishing and maintaining a local heterochromatin-like structure on MEST promoter region characterized by H3K9 trimethylation and hypoacetylation, H4K20 trimethylation, DNA hypermethylation, and enrichment in HP1 that correlates with preferential association to foci of pericentromeric heterochromatin and transcriptional repression. On disruption of the interaction between TIF1beta and HP1, TIF1beta is released from the promoter region, and there is a switch from DNA hypermethylation and histone H3K9 trimethylation to DNA hypomethylation and histone H3K27 trimethylation correlating with rapid reactivation of MEST expression. Interestingly, we provide evidence that the imprinted MEST allele DNA methylation is insensitive to TIF1beta loss of function, whereas the nonimprinted allele is regulated through a distinct TIF1beta-DNA methylation mechanism.

Long-term genome-wide blood RNA expression profiles yield novel molecular response candidates for IFN-β-1b treatment in relapsing remitting MS

AIMS In multiple sclerosis patients, treatment with recombinant IFN-beta (rIFN-beta) is partially efficient in reducing clinical exacerbations. However, its molecular mechanism of action is still under scrutiny. MATERIALS & METHODS We used DNA microarrays (Affymetrix, CA, USA) and peripheral mononuclear blood cells from 25 relapsing remitting multiple sclerosis patients to analyze the longitudinal transcriptional profile within 2 years of rIFN-beta administration. Sets of differentially expressed genes were attained by applying a combination of independent criteria, thereby providing efficient data curation and gene filtering that accounted for technical and biological noise. Gene ontology term-association analysis and scientific literature text mining were used to explore evidence of gene interaction. RESULTS Post-therapy initiation, we identified 42 (day 2), 175 (month 1), 103 (month 12) and 108 (month 24) differentially expressed genes. Increased expression of established IFN-beta marker genes, as well as differential expression of circulating IFN-beta-responsive candidate genes, were observed. MS4A1 (CD20), a known target of B-cell depletion therapy, was significantly downregulated after one month. CMPK2, FCER1A, and FFAR2 appeared as hitherto unrecognized multiple sclerosis treatment-related differentially expressed genes that were consistently modulated over time. Overall, 84 interactions between 54 genes were attained, of which two major gene networks were identified at an earlier stage of therapy: the first (n = 15 genes) consisted of mostly known IFN-beta-activated genes, whereas the second (n = 12) mainly contained downregulated genes that to date have not been associated with IFN-beta effects in multiple sclerosis array research. CONCLUSION We achieved both a broadening of the knowledge of IFN-beta mechanism-of-action-related constituents and the identification of time-dependent interactions between IFN-beta regulated genes.

Immunomodulatory effects of holothurian triterpene glycosides on mammalian splenocytes determined by mass spectrometric proteome analysis

Spleen is a prime organ in which immuno-stimulation takes place in mammalians. Proteome analysis was used to investigate the elicited effects on mouse splenocytes upon exposure to holothurian triterpene glycosides. Cucumarioside A(2)-2, and Frondoside A, respectively, have been used to in-vitro stimulate primary splenocyte cultures. Differential protein expression was monitored by 2D gel analysis and proteins in spots of interest were identified by MALDI ToF MS and nano LC-ESI Q-ToF MS/MS, respectively. Differential image analysis of gels from control vs. gels from stimulated primary splenocyte cultures showed that approximately thirty protein spots were differentially expressed. Prime examples of differentially expressed proteins are NSFL1 cofactor p47 and hnRNP K (down-regulated), as well as Septin-2, NADH dehydrogenase [ubiquinone] iron-sulfur protein 3, and GRB2-related adaptor protein 2 (up-regulated). Immuno-analytical assays confirmed differential protein expression. Together with results from proliferation and cell adhesion assays, our results show that cellular proliferation is stimulated by holothurian triterpene glycosides. In conclusion, holothurian triterpene glycosides are thought to express their immuno-stimulatory effects by enhancing the natural cellular defense barrier that is necessary to fight pathogens and for which lymphocytes and splenocytes have to be recruited constantly due to limited lifetimes of B-cells and T-cells in the body.

Reassessment of Blood Gene Expression Markers for the Prognosis of Relapsing-Remitting Multiple Sclerosis

Despite considerable advances in the treatment of multiple sclerosis, current drugs are only partially effective. Most patients show reduced disease activity with therapy, but still experience relapses, increasing disability, and new brain lesions. Since there are no reliable clinical or biological markers of disease progression, long-term prognosis is difficult to predict for individual patients. We identified 18 studies that suggested genes expressed in blood as predictive biomarkers. We validated the prognostic value of those genes with three different microarray data sets comprising 148 patients in total. Using these data, we tested whether the genes were significantly differentially expressed between patients with good and poor courses of the disease. Poor progression was defined by relapses and/or increase of disability during a two-year follow-up, independent of the administered therapy. Of 110 genes that have been proposed as predictive biomarkers, most could not be confirmed in our analysis. However, the G protein-coupled membrane receptor GPR3 was expressed at significantly lower levels in patients with poor disease progression in all data sets. GPR3 has therefore a high potential to be a biomarker for predicting future disease activity. In addition, we examined the IL17 cytokines and receptors in more detail and propose IL17RC as a new, promising, transcript-based biomarker candidate. Further studies are needed to better understand the roles of these receptors in multiple sclerosis and its treatment and to clarify the utility of GPR3 and IL17RC expression levels in the blood as markers of long-term prognosis.

The ancient mammalian KRAB zinc finger gene cluster on human chromosome 8q24.3 illustrates principles of C2H2 zinc finger evolution associated with unique expression profiles in human tissues

BackgroundExpansion of multi-C2H2 domain zinc finger (ZNF) genes, including the Krüppel-associated box (KRAB) subfamily, paralleled the evolution of tetrapodes, particularly in mammalian lineages. Advances in their cataloging and characterization suggest that the functions of the KRAB-ZNF gene family contributed to mammalian speciation.ResultsHere, we characterized the human 8q24.3 ZNF cluster on the genomic, the phylogenetic, the structural and the transcriptome level. Six (ZNF7, ZNF34, ZNF250, ZNF251, ZNF252, ZNF517) of the seven locus members contain exons encoding KRAB domains, one (ZNF16) does not. They form a paralog group in which the encoded KRAB and ZNF protein domains generally share more similarities with each other than with other members of the human ZNF superfamily. The closest relatives with respect to their DNA-binding domain were ZNF7 and ZNF251. The analysis of orthologs in therian mammalian species revealed strong conservation and purifying selection of the KRAB-A and zinc finger domains. These findings underscore structural/functional constraints during evolution. Gene losses in the murine lineage (ZNF16, ZNF34, ZNF252, ZNF517) and potential protein truncations in primates (ZNF252) illustrate ongoing speciation processes. Tissue expression profiling by quantitative real-time PCR showed similar but distinct patterns for all tested ZNF genes with the most prominent expression in fetal brain. Based on accompanying expression signatures in twenty-six other human tissues ZNF34 and ZNF250 revealed the closest expression profiles. Together, the 8q24.3 ZNF genes can be assigned to a cerebellum, a testis or a prostate/thyroid subgroup. These results are consistent with potential functions of the ZNF genes in morphogenesis and differentiation. Promoter regions of the seven 8q24.3 ZNF genes display common characteristics like missing TATA-box, CpG island-association and transcription factor binding site (TFBS) modules. Common TFBS modules partly explain the observed expression pattern similarities.ConclusionsThe ZNF genes at human 8q24.3 form a relatively old mammalian paralog group conserved in eutherian mammals for at least 130 million years. The members persisted after initial duplications by undergoing subfunctionalizations in their expression patterns and target site recognition. KRAB-ZNF mediated repression of transcription might have shaped organogenesis in mammalian ontogeny.

MS characterization of apheresis samples from rheumatoid arthritis patients for the improvement of immunoadsorption therapy – a pilot study

Identification of proteins from apheresis samples was performed by both SDS‐PAGE and 2‐D gel separation of eluted proteins from staphylococcal protein A‐based immunoadsorption columns (Prosorba®) followed by MS peptide mass fingerprinting and MS/MS peptide sequencing on a MALDI QIT TOF mass spectrometer. MS/MS peptide sequencing was performed in conjunction with a micro reversed phase HPLC configured with an online MALDI plate‐spotting device. Apheresis treatment had been performed in three patients with longstanding therapy refractory rheumatoid arthritis. 2‐D gels displayed ca. 500 spots representing proteins that were eluted from the Prosorba® columns. From 54 gels, a total of 1256 protein spots had been picked and yielded in the identification of 56 non‐redundant proteins without counting isoforms. Proteins from the eluates belong to five major groups comprising (i) immunoglobulins (IgG, IgA, IgM heavy and light chains; about 40% of the spots), (ii) proteins involved in coagulation, (iii) HDL/LDL‐associated proteins, (iv) proteins from the complement system, and (v) acute phase proteins. MS analysis showed that the full‐length C3 complement protein had been cleaved upon complement activation, presumably on the column, such that the anaphylatoxin C3a was produced and released during therapy. Our results are consistent with clinical observations on both patient responses to therapy and reported adverse events. For the first time, direct molecular information has become available to support mechanistic reasoning for the principle of function of staphylococcal protein A‐based immunoadsorption therapy and for the explanation of adverse events. According to our results, removal and/or modulation of immune complexes together with complement activation can be regarded as the major events that are taking place during Prosorba® therapy. In order to avoid complement activation and induction of an inflammatory cascade, we suggest the prevention of C3a anaphylatoxin‐related reactions during immunoadsorption therapy.