Blood RNA Expression Research Articles

Multiple Diseases Discriminated by Quantitation of Blood Transcriptome

The steady circulation and physiologically interactive nature of blood ensures that this dynamic system encounters, transmits, and responds to a wide range of biological signals. In this context, we hypothesized that quantitative measurement of the blood transcriptome can enable the identification and validation of RNA transcripts that are specifically associated with the presence of a particular disease or clinical condition. In the present study, we have used 631 blood RNA expression profiling in conjunction with microarray technology to generate highly discriminative panels of 10 pairs of probe sets for each of four separate clinical conditions (gender, colorectal cancer, prostate cancer and osteoarthritis). The robust training set performance for each disease or condition-specific multi-gene panel was corroborated with an independent test set, with areas under the receiver-operating characteristic curves ranging from 0.87 to 0.93 for each of the four conditions in the test set population. This study demonstrates that quantitative measurement of the blood transcriptome, in conjunction with microarray technology, can be used to generate highly discriminative multi-gene panels for many clinical conditions. This approach has great potential to enable the simultaneous monitoring of multiple disease states or clinical conditions from a single blood sample.

Quantitative analysis of Survivin RNA expression in blood as a non‐invasive predictor of response to neoadjuvant radiochemotherapy in esophageal cancer

Analysis of survivin RNA expression in peripheral blood as a non-invasive molecular predictor of response to neoadjuvant radiochemotherapy in patients with locally advanced cancer of the esophagus. Blood samples were drawn from 29 patients with esophageal cancer prior to neoadjuvant radiochemotherapy. After extraction of cellular tumor-RNA from blood samples, quantitative expression analysis of survivin was done by quantitative real-time RT-PCR. Twenty of 29 (69%) of patients showed a minor histopathological response and 9 of 29 (31%) showed a major-response to neadjuvant radiochemotherapy. RNA expression in blood of patients was detectable for survivin in 27.6%, and in 100% for beta-actin. The mean survivin expression was not significantly different between minor- and major-responders. No significant associations were detected between survivin expression levels and patients clinical variables. A high expression level for survivin was significantly associated with a minor-response to neoadjuvant treatment (P = 0.042). Relative survivin expression levels above 0.15 were not associated with major histopathological response (sensitivity: 35%; specificity: 100%). Minor-response to the applied therapy was significantly associated with a high survivin RNA expression level in patient's blood. Survivin appears to be a specific non-invasive predictor of response to neoadjuvant therapy in esophageal cancer.

Functional Analysis of the Chromosome 9p21.3 Coronary Artery Disease Risk Locus

We have investigated the functional significance of conserved sequences within the 9p21.3 risk locus for coronary artery disease (CAD) and determined the relationship of 9p21.3 to expression of ANRIL and to whole genome gene expression. We demonstrate that a conserved sequence within the 9p21.3 locus has enhancer activity and that the risk variant significantly increases reporter gene expression in primary aortic smooth muscle cells. Whole blood RNA expression of the short variants of ANRIL was increased by 2.2-fold whereas expression of the long ANRIL variant was decreased by 1.2-fold in healthy subjects homozygous for the risk allele. Expression levels of the long and short ANRIL variants were positively correlated with that of the cyclin-dependent kinase inhibitor, CDKN2B (p15) and TDGF1 (Cripto), respectively. Relevant to atherosclerosis, genome-wide expression profiling demonstrated upregulation of gene sets modulating cellular proliferation in carriers of the risk allele. These findings are consistent with the hypothesis that the 9p21.3 risk allele contains a functional enhancer, the activity of which is altered in carriers of the risk allele. 9p21.3 may promote atherosclerosis by regulating expression of ANRIL, which in turn is associated with altered expression of genes controlling cellular proliferation pathways.

Gene expression in blood of subjects with Duchenne muscular dystrophy

The objective of this study was to examine RNA expression in blood of subjects with Duchenne muscular dystrophy (DMD). Whole blood was collected into PAX gene tubes and RNA was isolated for 3- to 20-year-old males with DMD (n = 34) and for age- and gender-matched normal healthy controls (n = 21). DMD was confirmed by genetic testing in all subjects. RNA expression was measured on Affymetrix whole-genome human U133 Plus 2.0 GeneChips. Using a Benjamini-Hochberg false discovery rate of 0.05 to correct for multiple comparisons, an unpaired t test for DMD versus controls yielded 10,763 regulated probes with no fold change cutoff, 1,467 probes with >|1.5|-fold change, 191 probes with >|2.0|-fold change, and 59 probes with a >|2.5|-fold change. These genes (probes) separated DMD from controls using cluster analyses. Almost all of the genes regulated in peripheral blood were different from the genes reported to be regulated in diseased muscle of subjects with DMD. It is proposed that the genes regulated in blood of subjects with Duchenne muscular dystrophy are indicative, at least in part, of the immune response to the diseased DMD muscle. The regulated genes might be used to monitor therapy or provide novel targets for immune-directed therapy for DMD.

Immunogenicity and Antileukemic Activity of Dendritic Cells Electroporated with Wilms' Tumor WT1 mRNA: A Phase I/II Trial in Acute Myeloid Leukemia

The Wilms' tumor protein WT1 is a target for immunotherapy in malignancies, such as acute myeloid leukemia (AML). Following our demonstration that dendritic cells (DC) can be efficiently transfected by messenger (m)RNA electroporation (Van Tendeloo VF et al. Blood 2001; 98:49–56) and that WT1 mRNA-electroporated DC stimulate WT1-specific T cells in vitro (Van Driessche A et al. Leukemia 2005; 19:1863–1871), we performed a phase I/II dose-escalation trial, in which patients with AML in remission but at high risk of relapse and without a direct sib allo-transplant option (9 patients) or with slowly progressive AML (1 patient) received intradermal injections of WT1 RNA-loaded DC. Following apheresis and CD14 immunomagnetic monocyte separation, DC were generated in 6-day cultures in clinical-grade medium supplemented with serum, granulocytemacrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4, matured with prostaglandin (PG)E2 and tumor necrosis factor (TNF)-alpha, harvested, electroporated with WT1 mRNA and used as vaccines. The patients received four biweekly DC vaccines and a delayed-type hypersensitivity (DTH) test was performed 2 weeks following the last vaccination. Patients were monitored for minimal residual disease (MRD) by analyzing WT1 RNA expression in peripheral blood by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) (Cilloni D et al. Leukemia 2002; 16:2115–2121 & Cilloni D et al. Haematologica 2008; 93:921–924). Before and after vaccination, peripheral blood was collected for immunomonitoring purposes. Feasibility, safety, immunogenicity and effect on MRD were investigated. There was successful DC generation and vaccine production in all 10 patients. No serious adverse events or toxicity were observed and vaccinations were well tolerated. A decrease in WT1 RNA expression was observed during the course of the vaccination in 4/7 patients who had an increased WT1 mRNA level in peripheral blood. Three of those patients are still in complete hematological remission. An in vivo vaccine-specific immune response was demonstrated in 10/10 patients by DTH. Ex vivo immunomonitoring analysis showed a significant increase in circulating activated HLA-DR+ CD4+ T cells and in IL-2 plasma levels following vaccination. Importantly, in vitro restimulation assays of peripheral blood mononuclear cells revealed a significant postvaccination increase in interferon (IFN)-gamma-producing WT1-specific CD8+ T cells (n= 8 evaluable patients), but not in cytokine-producing WT1-specific CD4+ T cells. There was no significant change in WT1-specific antibodies following vaccination. We conclude that vaccination of AML patients with WT1 RNA-loaded DC is feasible and safe. Furthermore, the DC elicit vaccine-specific and WT1-specific CD8+ T-cell responses. The correlation between reduction of circulating WT1 mRNA and the administration of the DC vaccines strongly suggests that this DC vaccine elicits an antileukemic activity.

ERCC1 RNA Expression in Peripheral Blood Predicts Minor Histopathological Response to Neoadjuvant Radio-chemotherapy in Patients with Locally Advanced Cancer of the Esophagus

ObjectiveThe aim of this study was to determine the gene is spelled excision repair cross-complementing gene 1 (ERCC1) RNA-expression in peripheral blood as a non-invasive molecular predictor of response to neoadjuvant radio-chemotherapy in patients with locally advanced cancer of the esophagus. BackgroundOnly patients with locally advanced cancer of the esophagus with a major histopathological response to neoadjuvant radio-chemotherapy benefit from this treatment. No non-invasive molecular marker exists that can reliably predict response to neoadjuvant therapy in this disease. To improve the treatment of patients with cancer of the esophagus, molecular predictors of response are desperately needed. MethodsBlood samples were drawn from 29 patients with esophageal cancer prior to neoadjuvant radio-chemotherapy. After extraction of cellular tumor-RNA from blood samples, quantitative expression analysis of ERCC1 was done by real-time reverse transcription polymerase chain reaction. ResultsNineteen (65.5%) patients showed a minor and ten (34.5%) a major histopathological response to neoadjuvant therapy. ERCC1 expression in blood of patients was detectable in 82.8%. The median ERCC1 expression was 0.62 (minimum 0.00, maximum 2.48) in minor responders and 0.24 (minimum 0.00, maximum 0.45) in major responders (p = 0.004). No significant associations were detectable between ERCC1 levels and patients’ clinical variables. Relative ERCC1 levels above 0.452 were not associated with major histopathological response (sensitivity, 68.4; specificity, 100%), and 13 of 19 patients with minor response could be unequivocally identified. ConclusionMinor responders to the applied therapy show a significant higher ERCC1 expression level in their blood compared to major responders. ERCC1 appears to be a highly specific non-invasive predictor of response to neoadjuvant therapy in esophageal cancer.

Characterization of novel CAPN3 isoforms in white blood cells: an alternative approach for limb-girdle muscular dystrophy 2A diagnosis

Limb-girdle muscular dystrophy type 2A (LGMD2A) is an autosomal recessive disorder caused by mutations in the CAPN3 gene. Its definitive diagnosis is laborious, since the clinical phenotype is often similar to other types of muscular dystrophy and since the CAPN3 gene encompasses a large genomic region with more than 300 pathogenic mutations described to date. In fact, it is estimated that nearly 25% of the cases with a phenotype suggestive of LGMD2A do not have mutations in the CAPN3 gene and that, in up to 22% of the cases, only one mutation is identified. In the present work, we have characterised CAPN3 messenger RNA (mRNA) expression in peripheral blood, and we have performed a retrospective diagnostic study with 26 LGMD2A patients, sequencing a transcript of CAPN3 present in white blood cells (WBCs). The 25% of the mutations presented in this paper (7/28) act modifying pre-mRNA splicing of the CAPN3 transcript, including the first deep-intronic mutation described to date in the CAPN3 gene. Our results determine that the sequencing of CAPN3 transcripts present in WBCs could be applied as a new approach for LGMD2A diagnosis. This method improves and simplifies diagnosis, since it combines the advantages of mRNA analysis in a more accessible and rapidly regenerated tissue. However, the lack of exon 15 in the CAPN3 isoforms present in blood, and the presence of mRNA degradation make it necessary to combine mRNA and DNA analyses in some specific cases.

948 The Role of ERCC1 RNA Expression in Blood As a Non-Invasive Predictor of Response to Neadjuvant Radio-Chemotherapy in Patients with Locally Advanced Cancer of the Esophagus

Background: Only patients with locally advanced cancer of the esophagus with a major histopathological response to neadjuvant radio-chemotherapy benefit from this treatment. Unfortunately, no non-invasive molecular marker exists that can reliably predict response or non-response to neadjuvant therapy in this disease. To further improve the treatment of patients with locally advanced cancer of the esophagus, molecular predictors of response are desperately needed. The aim of this study was to determine the value of ERCC1 RNA-Expression in sera of patients with locally advanced cancer of the esophagus as a non-invasive molecular predictor of response to neoadjuvant radio-chemotherapy. Material and methods: A total of 29 patients with locally advanced cancer (cT3-T4, Nx, M0) of the esophagus were included in this study. Blood samples from each patient were drawn prior to neoadjuvant radio-chemotherapy (cis-Platin, 5-FU, 36Gy). Transthoracic en-bloc esophagectomy was performed in all patients following completion of neadjuvant therapy. After extraction of cellular tumorRNA from sera samples, quantitative expression analysis of ERCC1 and the internal reference gene beta-Actin was done by real-time RT-PCR (Taqman©). Histomorphological regression was defined as major response when resected specimen contained 10 % of vital residual tumor cells. Results: Nineteen of 29 (65.5 %) of patients showed a minor histopathological response and 10 of 29 (34.5 %) showed a major-response to neadjuvant radio-chemotherapy. ERCC1 RNA expression in sera of patients was detectable for ERCC1 in 82.8 % (24 of 29) and 100 % (29 of 29) for beta-Actin. The median ERCC1 expression was 0.62 (min.: 0.00, max.: 2.48) in minor-responders and 0.24 (min.: 0.00, max.: 0.45) in major-responders and was significantly different (p = 0.004 Mann-Whitney test). No significant associations were detected between ERCC1 expression levels and patients clinical variables (histology, tumor stage, gender etc.). Relative ERCC1 expression levels above 0.452 were not associated with major histopathological response (sensitivity: 68.4; specificity: 100 %) and 13 of 19 patients with minor histopathological response to the delivered neadjuvant therapy could be unequivocally identified. This association of dichotomized relative ERCC1 expression and histopathological response was statistically significant (P < 0.001). Conclusion: The applied method is technically feasible for the analysis of cellular ERCC1 RNA expression in sera of patients with locally advanced cancer of the esophagus. Minor-responders to the applied radiochemotherapy show a significant higher ERCC1 expression level in their sera compared to major-responders prior to therapy. ERCC1 expression levels in sera appear to be highly specific to predict minor-response to neoadjuvant radiochemotherapy in patients with locally advanced esophageal cancer and could be applied to prevent expensive, noneffective, and potentially harmful therapies in a substantial number (45 %) of patients. 92 VI. Molekulare Onkologie: Pradiktion und Therapie

SLC2A9 influences uric acid concentrations with pronounced sex-specific effects

Serum uric acid concentrations are correlated with gout and clinical entities such as cardiovascular disease and diabetes. In the genome-wide association study KORA (Kooperative Gesundheitsforschung in der Region Augsburg) F3 500K (n = 1,644), the most significant SNPs associated with uric acid concentrations mapped within introns 4 and 6 of SLC2A9, a gene encoding a putative hexose transporter (effects: -0.23 to -0.36 mg/dl per copy of the minor allele). We replicated these findings in three independent samples from Germany (KORA S4 and SHIP (Study of Health in Pomerania)) and Austria (SAPHIR; Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk), with P values ranging from 1.2 x 10(-8) to 1.0 x 10(-32). Analysis of whole blood RNA expression profiles from a KORA F3 500K subgroup (n = 117) showed a significant association between the SLC2A9 isoform 2 and urate concentrations. The SLC2A9 genotypes also showed significant association with self-reported gout. The proportion of the variance of serum uric acid concentrations explained by genotypes was about 1.2% in men and 6% in women, and the percentage accounted for by expression levels was 3.5% in men and 15% in women.

Vaccination of Acute Myeloid Leukemia Patients with Dendritic Cells Electroporated with mRNA Encoding the Wilms' Tumor Protein WT1: A Phase I/II Trial.

To date, Wilms' tumor protein (WT1) is acknowledged as a valuable target for active specific immunotherapy in several solid and hematological malignancies, such as leukemia. Preclinical data from our laboratory and that of Hans Stauss have already shown that WT1 RNA-electroporated dendritic cells (DC) stimulate WT1-specific T cells in vitro (Van Driessche A et al. Leukemia 2005; 19:1863–1871). Therefore, we started a phase I/II dose-escalation trial in which patients with acute myeloid leukemia (AML) in remission received intradermal injections with WT1 RNA-loaded DC. Feasibility, safety and immunogenicity of the vaccine were investigated. Seven patients received four biweekly DC vaccines. A delayed-type hypersensitivity (DTH) test was performed 2 weeks following the last vaccination. Patients underwent an apheresis and monocytes were isolated using CD14-labeled magnetic beads by CliniMACS. DC were generated in 6-day cultures in clinical-grade medium supplemented with serum, GM-CSF and IL-4 and maturated with PGE2 and TNF-a. Keyhole limpet hemocyanin (KLH) was added during maturation as a CD4+ helper antigen. Mature DC were harvested, electroporated with WT1 mRNA and used as vaccines. Patients were monitored for minimal residual disease (MRD) by analyzing WT1 RNA expression in peripheral blood by qRT-PCR. When the patient was HLA-A2+, tetramer staining was performed to detect WT1-specific CD8+ T cells. Before and after the vaccination cycle, peripheral blood was collected for immunomonitoring purposes. There was successful DC generation and vaccine production in all patients selected. No serious adverse events or toxicity was seen and all vaccinations were well tolerated. A decrease in WT1 RNA expression was observed during the course of the vaccination in 3/5 patients who had an increased WT1 mRNA level in peripheral blood at the start of DC vaccination. A vaccine-specific immune response was demonstrated in 7/7 patients by an in vivo DTH reaction both to KLH as well as to WT1. By tetramer analysis, detectable levels of WT1-specific CD8+ T cells could be demonstrated during the course of the vaccination both in the peripheral blood as well as in the expanded DTH-infiltrating T cells from the skin biopsies. Preliminary data from immunomonitoring in pre- and post-vaccination T cell samples from 3 patients show a mixed T helper (Th)1/Th2 response towards the KLH and the WT1 protein following vaccination. We conclude that vaccination of AML remission patients with WT1 RNA-loaded DC is feasible and safe. Furthermore, the vaccine elicits anti-vaccine T-cell responses in vivo and a decrease in WT1 RNA expression levels was observed during MRD monitoring in some vaccinated patients.

Interleukin-8 is associated with circulating CD133+ progenitor cells in acute myocardial infarction

Release of progenitor cells is observed during inflammatory conditions and contributes to neovascularization. We, therefore, sought to investigate the relationship of circulating progenitor cells and interleukin (IL)-8 in acute myocardial infarction (AMI). From patients with stable angina and AMI, serial venous blood samples were obtained. The number of circulating CD133+CD45- progenitor cells, endothelial progenitor cells (EPCs), and circulating endothelial P1H12+CD45- cells was analyzed by flow cytometry. After stenting in patients with AMI, an increase in plasma IL-8 and vascular endothelial growth factor (VEGF) concentrations was observed, which was only minimal in patients with stable angina. Only in patients with AMI, this was followed by an increase in circulating CD133+CD45- progenitor cells. In contrast, circulating endothelial P1H12+CD45- cells and E-selectin RNA expression in peripheral blood were only elevated early in AMI, indicating shedding of activated endothelial cells. Multivariable analysis revealed an association of IL-8 and circulating CD133+CD45- progenitor cells in AMI, in addition to statin therapy and risk factor profile. In AMI, IL-8 is associated with circulating progenitor cells. In addition to the pro-angiogenic functions of IL-8 and VEGF, this mechanism may contribute to new vessel generation and, thereby, improve myocardial function.

Reduced CTLA-4 protein and messenger RNA expression in umbilical cord blood T lymphocytes

A favorable incidence and severity of graft-vs-host disease is observed in patients transplanted with banked, unrelated, HLA-mismatched umbilical cord blood (UCB) grafts, while the incidence of malignant relapse remains low. CTLA-4 mediates negative T-cell signaling and may contribute to the development of allogeneic tolerance. In this study, we compared protein and mRNA expression of CTLA-4 in stimulated UCB and adult peripheral blood T cells. T cells were isolated from UCB and adult peripheral blood and stimulated with anti-CD3 and anti-CD28 monoclonal antibodies. Cells were immunostained and analyzed by flow cytometry for both surface and intracellular expression of CTLA-4 in the presence and absence of cyclosporin A, and kinetics of CTLA-4 expression compared. CTLA-4 mRNA expression was measured using quantitative real-time polymerase chain reaction. NFAT1 protein levels were measured by Western blot analysis. These studies demonstrate reduced surface and intracellular expression of CTLA-4 in stimulated UCB T cells compared to adult controls. Furthermore, reduced CTLA-4 protein expression in UCB T cells was noted to be in part transcriptionally regulated, as CTLA-4 mRNA levels also were significantly lower. Reduced CLTA-4 expression by UCB T cells followed the kinetics of delayed and reduced expression of the transcription factor NFAT1 by UCB T lymphocytes during primary stimulation. Moreover, cyclosporin A, which is known to modulate NFAT activation, reduced CTLA-4 protein expression in adult and UCB T cells. Reduced expression of the key regulatory proteins CTLA-4 and NFAT-1 may contribute to favorable UCB T lymphocyte allogeneic responses.