RNA profiling from ovarian FFPE specimens (Nanostring)
ABSTRACT: To study feasibility of gene expression profiling from FFPE tissues using NanoString nCounter platform, we designed a pilot study utilizing samples from ovarian cancer cohort. We selected samples from large-scale epidemiologic studies and clinical trials representative of a wide variety of fixation times, block ages and block storage conditions. five serous carcinoma and six clear cell carcinoma samples with technical replicates
Project description:To study feasibility of gene expression profiling from FFPE tissues using NanoString nCounter platform, we designed a pilot study utilizing samples from prostate cancer cohort. We selected samples from large-scale epidemiologic studies and clinical trials representative of a wide variety of fixation times, block ages and block storage conditions. five paired tumor and adjacent normal prostate tissue speciemens with technical replicates
Project description:Intrinsic subtyping of breast cancer was performed using an nCounter RUO-PAM50 gene expression assay to determine the ability of instrinsic subtyping to predict what patients may benefit from altered chemotherapy scheduling in the CALGB 9741 clinical trial population. FFPE primary breast tumor samples archived at the CALGB Pathology Coordinating Office (PCO) were used to obtain total RNA for instrinsic subtyping using the nCounter Analysis System. Gene-expression profiles were generated for 1321 of 1471 patient samples (90%) suitable for inclusion in this study.
Project description:The goal of this study is to identify unique miRNA profiles of EVs from MCF7 and MCF10A cells that distinguish their cellular origin. 654 human mature miRNAs were analyzed in NanoString assays to identify miRNA with high abundance in MCF7 EVs and the greatest fold change for MCF7 EVs relative to MCF10A EVs.
Project description:To study feasibility of gene expression profiling from FFPE tissues using NuGen amplified mRNA hybridized on Affymetrix GeneChip Human Gene 1.0 ST arrays, we designed a pilot study utilizing samples from prostate cancer cohort. We selected samples from large-scale epidemiologic studies and clinical trials representative of a wide variety of fixation times, block ages and block storage conditions. Five serous carcinoma and six clear cell carcinoma samples with technical replicates
Project description:The AVAglio and RTOG-0825 randomized, placebo-controlled phase III trials in newly diagnosed glioblastoma reported prolonged progression-free survival (PFS), but not overall survival (OS), with the addition of bevacizumab to radiotherapy/temozolomide. To establish whether certain patient subgroups derived OS benefit from the addition of bevacizumab to first-line standard-of-care therapy, AVAglio patients were retrospectively evaluated for molecular subtype, and bevacizumab efficacy assessed for each patient subgroup. A multivariate analysis accounting for prognostic covariates revealed that bevacizumab conferred a significant OS advantage versus placebo for patients with Proneural IDH1 wild-type tumors (17.1 v 12.8 months, respectively; hazard ratio, 0.43; 95% CI, 0.26 to 0.73; P = .002). This analysis also revealed an interaction between the Proneural subtype biomarker and treatment arm (P = .023). The group of patients with Mesenchymal and Proneural tumors derived a PFS benefit from bevacizumab, compared with placebo; however, this translated to an OS benefit in the Proneural subset only. Retrospective analysis of AVAglio data suggests that patients with IDH1 wild-type Proneural glioblastoma may derive OS benefit from first-line bevacizumab treatment. The predictive value of the Proneural subtype observed in AVAglio should be validated in an independent dataset. A total of 349 (bevacizumab arm, n = 171; placebo arm, n = 178) pretreatment specimens from AVAglio patients (total n = 921) were available for biomarker analysis. Samples were profiled for gene expression and isocitrate dehydrogenase 1 (IDH1) mutation status and classified into previously identified molecular subtypes. PFS and OS were assessed within each subtype.
Project description:Macrophages and dendritic cells are phagocytes present in almost all tissues in mammals and play a pivotal role for tissue homeostasis and during immune responses. Liver phagocytes play a pivotal role in host immune responses and exquisite mechanisms are necessary to govern the density and the location of the different hepatic leukocytes. However in catastrophic events including trauma, infections or toxin ingestion, many of the liver phagocytes can be wiped out leaving large areas devoid of these cells. Here we used a unique combination of mass cytometry (CyTOF), gene expression and liver intravital approaches to precisely determine phagocytic populations within the liver and the functional consequences of their replenishment by myeloid precursors. While Kupffer cells were exclusively located in the sinusoidal lumen, we identified a population of dendritic cells that was mainly located under the liver capsule. After full depletion of dendritic and Kupffer cells, intravascular myeloid precursors replenished location, density and function of both populations. However, these emergency repopulated livers were dysfunctional in their ability to respond to injury and to clear bacteria for at least 30 days. After this “educational period”, new phagocytes returned to normal response to injury and bacterial trapping. Conclusions: Our data shed light on the liver’s ability to locally shape phagocyte precursors into two vastly different immune cells localized to two fundamentally different tissue compartments. 4 different cell types were isolated from mice, RNA was extracted, and gene expression was measured using the Nanostring nCounter Mouse Immunology Panel
Project description:Using outcome after long term follow-up to define risk at disease presentation high risk (n=9 who went on to require liver transplantation) and low risk (n=7 who responded fully to UDCA) patients were identified and their first liver biopsies retrieved. RNA was successfully extracted and analysed using nanostring® transcriptomics. Patients with progressive disease appear to have a distinct molecular signature. high risk (n=9 who went on to require liver transplantation) and low risk (n=7 who responded fully to UDCA) patient material was processed along with non-diseased control liver (n=8)
Project description:To develop a classifier based on microRNAs for primary tumor site identification of liver core biopsies and to explore the influence of surrounding normal liver tissue on classification. Tissue samples from 333 patients, corresponding to one of the following ten assay classes, were obtained from archives of the pathology department, Copenhagen University Hospital, Rigshospitalet, Denmark: Lung cancer, breast cancer, gastric/cardia cancer, colorectal cancer, bladder cancer, pancreatic cancer, hepatocellular carcinoma, cholangiocarcinoma, squamous cell cancers of different origin, and normal liver tissue
Project description:To investigate the cytogenetic and large-scale chromosomal changes in involuted or non-involuted microGISTs using post-whole genome amplification (WGA) FFPE DNA materials Sixteen patients, total 19 FFPE tumor samples (block storage time 4 months to 9 years), including 16 microGISTs and 3 GISTs larger than 1 cm from the same patients harboring microGISTs. All FFPE tumor samples underwent DNA extraction and WGA (modified degenerate oligonucleotide PCR (DOP) method, provided by Sigma). For each tumor sample, a post-WGA DNA extract from the normal tissue in the same block (or block from the same patient with a block storage time differences less than 2 years) was obtained for tumor sample DNA co-hybridization. Tumor and normal areas of interest were marked and collected from 5- to 10-micron unstained or hematoxylin-stained sections by manual or laser (PixCell IITM, Arcturus Bioscience, CA, USA) microdissection. DNAs were then extracted. WGA was performed using GenomePlex® Tissue Whole Genome Amplification WGA5 kit (Sigma, Saint Louis, MO, USA; http://www.sigmaaldrich.com/) in parallel in accordance with the manufacturer's protocols. At least four independent experiments were concurrently performed per template amplification. Four separate WGA reaction products were pooled for each sample.