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Expression profiling of 275 lung cancer specimens

ABSTRACT: From ~1,700 non-small cell lung cancer specimens collected at the MD Anderson Cancer Center over the years 1997 to 2005, we selected 914 tumors with detailed clinical and pathological information. We extracted RNA and DNA from frozen tissues using histology quality control from 700 cases. RNA was examined for quality using Agilent Bioanalyzer and RNA integrity number (RIN) was obtained for all specimens. A final selection of 275 tumor specimens was based on the following criteria: 1) Select mainly adenocarcinomas (n = 183) and squamous carcinomas (n = 80); 2) Tissue material contains at least 70% tumor by section examination; 3) within the tumor section, there should be at least 30% tumor cells (as opposed to stromal cells); 4) RIN should be at least 4.0 (range 0 - 10). Various profiling experiments were then performed including mRNA expression (this study), miRNA profiling, aCGH (Agilent), Reverse-phase protein array (RPPA), mutation analysis of 20 genes (Sequenom) and MS-MALDI-TOFF analysis.

ORGANISM(S): Homo sapiens

PROVIDER: GSE41271 | GEO | 2013/07/01

SECONDARY ACCESSION(S): PRJNA176417

REPOSITORIES: GEO

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Project description:We searched the head and neck pathology database at The University of Texas MD Anderson Cancer Center (Houston, Texas) to identify all patients with SGCs who had been treated surgically from 1995 to 2008. Twenty cases from each tumor type, ACCs, MECs, and SDCs were selected based on the availability of at least 1.0 gm of fresh frozen tumor tissues. Seventeen salivary gland tissues (10 from matching tumor cases and 7 from neck resections from patients with other diseases) were used as control (Supplemental Table 1). The 20 ACCs had previously been included in our earlier comparative genomic hybridization analysis. 32 The tissues had been harvested immediately after resection by experienced head and neck pathologists, placed in liquid nitrogen, and stored at -80°C until use. Frozen sections of each tissue specimen were evaluated for tumor content and quality. Specimens with more than 20% of host non-neoplastic elements in any given sample underwent macrodissection to enrich the tumor percentage to more than 80%. One ACC tumor was disqualified because of excessive stroma; of the 59 tumor specimens, 14 had undergone mass dissection to remove non-tumor elements. All tissues were obtained according to an institutional review board-approved protocol for non-mucosal head and neck cancer. DNA extraction: Fresh-frozen tissue was processed using the Gentra Puregene tissue kit (QIAGEN, Valencia, CA). In brief, normal and tumor tissues were carefully dissected, and proteinase K was immediately added for cell digestion. After protein precipitation, DNA was precipitated using isopropanol, followed by a 70% ethanol washing procedure. Purified DNA was dissolved and eluted in DNA hydration solution and stored at -80°C. The quality of DNA was assessed using electrophoresis and nano-drop measurement. A 260/280 wavelength ratio of at least 1.8 was used to qualify specimens for analysis. Microarray analysis: We used the Affymetrix 250k Nsp SNP array (Affymetrix, Inc., Santa Clara, CA) to survey the genotype and DNA CNA copy number data for 59 tumor specimens and 17 healthy salivary gland specimens. Each array contains 261,563 SNP sites, which are approximately uniformly distributed throughout the genome. The SNP site mapping information was provided by Affymetrix, Inc., using human genome sequence version NCBI36/hg18. Two samples were repeatedly sampled to assess data reproducibility.

Project description:MicroRNAs (miRNAs) are a novel class of short RNAs which have shown to be dysregulated in a variety of cancers including squamous cell carcinoma (SCC) of the head&neck. Microarray based miRNA expression profiles of cutaneous SCC (cSCC) however have not been investigated so far. Seven patients with cutaneous SCC were enrolled in the study. Tumor biopsies (n=7) were taken from the center of the tumor. Adjacent healthy skin (n=7) was biopsied as a control (intraindividual control). miRNA expression profiles of all specimen were detected by mircroarray miRNA expression profiling based on miRBAse 16 and compared. In 7 patients biopsy specimens of cutaneous SCC and control specimens from an adjacent healthy skin site near the tumor border (intraindividual control) were obtained during surgical removal of the tumor. Specimes were immediately stored in RNAlater (Qiagen, HIlden, Germany) at -80 M-BM-0C. Total RNA including miRNAs were isolated with miRNeasy Mini Kit (Quiagen, Hilden, Germany) according to the manufacturerM-bM-^@M-^Ys protocol. For RNA quality control purposes we determined RNA concentration, purity and RNA integrity number (RIN) with Agilent 2100 Bioanalyzer, RNA 6000 NanoLabCHip Kits (both Agilent Technologies, Santa Clara, USA) and the NanoDrop ND-1000 spectral photometer (Peqlab, Erlangen, Germany). All of the following steps described were carried out according to the manufacturerM-BM-4s protocol. In order to enable assessment of labeling and hybridization efficiencies total RNA samples were spiked with MicroRNA Spike-In Kit (Agilent Technologies, Santa Clara, USA). After treatment with calf intestine phosphatase (CIP), a labeling reaction was started with 100 ng total-RNA per sample. For labeling dephosphorylated RNA, T4 RNA ligase, incorporating Cyanine 3-Cytidine biphosphate (miRNA Complete Labeling and Hyb Kit, Agilent Technologies, Santa Clara, USA) was used. The Cyanine-3-labeled miRNA samples were then prepared for One-Color based hybridization (Complete miRNA Labeling and Hyb Kit, Agilent Technologies, Santa Clara, USA). Hybridization was performed at 55M-BM-0C for 20 hrs with Human miRNA Microarrays Release 16.0, 8x60K format (Agilent Technologies, Santa Clara, USA). Microarray slides were washed (Gene Expression Wash Buffers, Agilent Technologies, Santa Clara) and dried with acetonitrile (Sigma-Aldich, St.Louis, USA). Fluorescent signal intensities were detected with Agilent DNA Microarray Scanner (Agilent Technologies, Santa Clara, USA) and Scan Control A.8.4.1 Software (Agilent Technologies, Santa Clara, USA). Extraction of data was done by using the Feature Extraction 10.7.3.1 Software (Agilent Technologies, Santa Clara, USA).

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Expression profiling of 275 lung cancer specimens

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