Comment[ArrayExpressAccession] E-GEOD-28042 MAGE-TAB Version 1.1 Public Release Date 2013-10-15 Investigation Title Peripheral Blood Mononuclear Cell Gene Expression Profiles May Predict Poor Outcome in Idiopathic Pulmonary Fibrosis [Agilent] Comment[Submitted Name] Peripheral Blood Mononuclear Cell Gene Expression Profiles May Predict Poor Outcome in Idiopathic Pulmonary Fibrosis [Agilent] Experiment Description Background: In this study we aimed to identify peripheral blood mononuclear cell (PBMC) gene expression profiles predictive of poor outcomes in idiopathic pulmonary fibrosis (IPF) Methods: Microarray analyses of PBMC were performed in 120 patients from discovery (n=45) and replication cohorts (n=75). Genes and pathways associated with transplant-free survival (TFS) were identified and confirmed by qRT-PCR. Findings: 52 genes were predictive of TFS in a discovery cohort (FDR<5%, Cox score above 2.5 or below -2.5). Clustering the replication cohort samples using these genes distinguished two patient groups with significantly different TFS (hazard ratio 1.96, 95%CI 1.01-3.8, P=0.018). Decreased expression of M-bM-^@M-^\The co-stimulatory signaling during T cell activationM-bM-^@M-^] Biocarta pathway and in particular CD28, ICOS, LCK and ITK was associated with shorter TFS times in each cohort (FDR<5%). qRT-PCR expression of CD28, ICOS, LCK and ITK correlated with the microarray results in the discovery cohort (P<0.05) and their decreased expression was predictive of shorter TFS in the replication cohort (P<0.05). A genomic and clinical model demonstrated an area under the ROC curve of 78.5% at 2.4 months for death and lung transplant prediction. Interpretation: Our results suggest that CD28, ICOS, LCK and ITK are outcome biomarkers in IPF. PBMC from 75 patients with the diagnosis of IPF were obtained within 30 minutes from blood draw. Total RNA was extracted, labeled and hybridized to Agilent Whole Human Genome Oligo Microarray, 4 x 44K. Patients were followed from blood draw until death, transplant or last follow up. Hierarchical clustering and gene-set analysis with censored outcome data were used to study the association of gene expression and outcome in this cohort (replication cohort) Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name HERAZO MAYA Herazo Maya Person First Name JOSE Jose Person Mid Initials DAVID Person Email geo@ncbi.nlm.nih.gov Person Affiliation Yale University Person Address Medicine, Yale University, 300 Cedar Street, New Haven, CT, USA Person Roles submitter Protocol Name P-GSE28042-1 P-GSE28042-2 P-GSE28042-4 P-GSE28042-7 P-GSE28042-5 P-GSE28042-3 P-GSE28042-6 Protocol Description The scanned images were analyzed with Feature Extraction Software 10.7.1.1 (Agilent) using default parameters (GE1_107_Sep09 and Grid: 014850_D_20070820) to obtain background subtracted and spatially detrended Processed Signal intensities. Gene expression data was normalized to the geometric mean of all the IPF samples and controls using cyclic loess, for the normalization and statistical analysis we used the average gene expression values of the replicated probes, the number of probes after the average and normalization process was 29807. ID_REF = VALUE = Normalized signal intensity The scanned images were analyzed with Feature Extraction Software 10.7.1.1 (Agilent) using default parameters (GE1_107_Sep09 and Grid: 014850_D_20070820) to obtain background subtracted and spatially detrended Processed Signal intensities. Gene expression data was normalized to the geometric mean of all the IPF samples and controls using cyclic loess, for the normalization and statistical analysis we used the average gene expression values of the replicated probes, the number of probes after the average and normalization process was 29807. ID_REF = VALUE = Normalized signal intensity Labeling reactions were performed for each sample using Agilent Low RNA Input Linear Amplification Kit PLUS, One-Color (5184-3523, Agilent Technologies, Santa Clara, CA). Briefly, with a starting concentration of 400 nanograms of total RNA, an initial cDNA strand was synthesized using a oligo(dT)24 primer containing a T7 RNA polymerase, this cDNA was then used as a template to generate Cy3 labeled cRNA by a reverse transcriptase enzyme. After the cRNA was obtained a purification step was performed using RNeasy Mini Kit (74104, Qiagen, Valencia, CA) followed by measurement of the yield and specific activity of each sample 1.65 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60M-0C for 30 minutes in a reaction volume of 55 ml containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ml of 2x GEx Agilent hybridization buffer HI-RPM was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112F) for 17 hours at 65M-CM-^BM-0C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37M-CM-^BM-0C GE Wash buffer 2 (Agilent), then dried immediately. 1.65 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60M-0C for 30 minutes in a reaction volume of 55 ml containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ml of 2x GEx Agilent hybridization buffer HI-RPM was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112F) for 17 hours at 65M-0C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37M-0C GE Wash buffer 2 (Agilent), then dried immediately. 10 ml of blood was drawn by venipuncture in each participant and collected in a cell preparation tube (CPT) followed by centrifugation at 1500 g for 30 minutes at room temperature. After plasma removal, the cell layer was transferred into a 15 ml conical tube and 5 ml of RNase free PBS was added, the contents were mixed by turning upside down followed by centrifugation at 300 g for 15 minutes. Once the supernatant was removed, the PBS washing step previously described was repeated using 10 ml of PBS, with centrifugation for 10 minutes. After the PBMC isolation was completed, 1 ml of QIAzol (79306, Qiagen, Valencia, CA) was added to each sample and they were snap frozen in -80 celsius. Total RNA was extracted and purified using the miRNeasy Mini Kit (217004, Qiagen, Valencia, CA) under fully automated sample preparation by the assistance of the QIAcube device (9001292, Qiagen, Valencia CA) following the manufacturerM-bM-^@M-^Ys protocol.After extraction, total RNA yield and quality were evaluated using NanoDrop at 260 nm and the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) Slides were scanned immediately after washing on the Agilent DNA High Resolution Microarray Scanner (G2505C US45102918) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5 M-NM-