Unknown,Transcriptomics,Genomics,Proteomics

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Molecular profiling of cutaneous squamous cell carcinomas and actinic keratoses from organ transplant recipients

ABSTRACT: The risk of developing cutaneous squamous cell carcinoma (SCC) is markedly increased in organ transplant recipients (OTRs) compared to the normal population. Next to sun exposure, the immunosuppressive regimen is an important risk factor for the development of SCC in OTRs. Various gene mutations (e.g. TP53) and genetic alterations (e.g. loss of CDKN2A, amplification of RAS) have been found in SCCs. The aim of this genome-wide study was to identify pathways that are consistently involved in the formation of SCCs and their precursor lesions, actinic keratoses (AKs). To perform the analysis in an isogenic background, RNA and DNA were isolated from normal (unexposed) epidermis, benign AK, and SCC from each of 15 OTRs. Hierarchical cluster analysis of mRNA expression profiles showed SCC, AK and epidermal samples to separate into three distinct groups. Several thousand genes were differentially expressed between epidermis, AK and SCC; most upregulated in SCCs were genes related to hyperproliferation and stress markers, such as keratin 6 (KRT6), KRT16 and KRT17. Matching to oncogenic pathways revealed activation of downstream targets of RAS and cMYC in SCCs and of NFkappaB and TNF already in AKs. Patients were selected from the group of OTRs that are regularly seen at the dermatology clinic of the Leiden University Medical Center. Patients with clinically suspected SCC were informed on the study and after informed consent was obtained, fresh frozen samples were obtained from SCCs (n=15), AKs (n=14) and NS (n=13). RNA was isolated and gene expression profiles were obtained using HumanWG-6 v2 Expression BeadChips (Illumina). The normalized expression data was analyzed for differences in expression between SCC, AK and NS, at single-gene level but also at geneset level.

ORGANISM(S): Homo sapiens

SUBMITTER: Harry Vrieling

PROVIDER: E-GEOD-32628 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:A genome-wide eQTL analysis was performed in whole blood samples collected from 76 Japanese subjects. RNA microarray analysis was performed for 3 independent samples that were genotyped in a genome-wide scan. The correlations between the genotypes of 534,404 autosomal single nucleotide polymorphisms (SNPs) and the expression levels of 30,465 probes were examined for each sample. The SNP-probe pairs with combined correlation coefficients of all 3 samples corresponding to P < 3.10 × 10-12 (i.e., Bonferroni-corrected P < 0.05) were considered significant. SNP-probe pairs with a high likelihood of cross-hybridization and SNP-in-probe effects were excluded to exclude false positive results. We identified 102 cis-acting and 5 trans-acting eQTL regions. The cis-eQTL regions were widely distributed both upstream and downstream of the gene, as well as within the gene. RNA microarray data obtained from 3 independent samples originally recruited for other studies investigating the gene expression levels in psychiatric disorders were used in the present study. For the purpose of the present analyses, genomic DNA was collected from 24 subjects (13 men and 11 women, mean age [SD] = 39.9 [7.6] years) in sample 1, 24 subjects in sample 2 (12 men and 12 women, 34.1 [11.5] years), and 28 subjects (14 men and 14 women, 41.4 [11.8] years) in sample 3. Some of the subjects had depressive symptoms, but all were physically healthy and without clinically significant systemic disease (e.g., malignant disease, diabetes mellitus, hypertension, renal failure, or endocrine disorders). Subjects were recruited from the outpatient clinic of the National Center of Neurology and Psychiatry Hospital, Tokyo, Japan, through advertisements in free local information magazines or through our website announcement. All the subjects were biologically unrelated Japanese individuals who resided in the same geographical area (western Tokyo). The study protocol was approved by the ethics committee at the National Center of Neurology and Psychiatry, Japan. Written informed consent was obtained from every subject after the study was explained to them. Venous blood was collected between 1100 and 1200 h in PAXgene tubes (Qiagen, Valencia) from each subject and was incubated at room temperature for 24 h for RNA stabilization. RNA was extracted from whole blood according to the manufacturer’s guidelines by using the PAXgene Blood RNA System Kit (PreAnalytix GmbH, Hombrechtikon, Switzerland). The RNA was quantified by optical density readings at A260nm by using the NanoDrop ND-1000 (Thermo Scientific, Rockford). Gene expression analysis was performed using Agilent Human Genome 4 × 44 K arrays (Agilent Technologies, Santa Clara). Raw signal data for each of the 3 independent samples were analyzed separately by the GeneSpring GX software (Agilent Technologies). Data were filtered according to the expression level for quality control to eliminate genes that were below the 20th percentile threshold. The expression value of each gene was normalized to the median expression value of all genes in each chip. A total of 30,465 probes were included in the analysis. Genomic DNA was obtained from venous blood samples. Genotyping was performed by Riken Genesis (Yokohama, Japan) using the Illumina HumanOmni1-Quad BeadChip (Illumina, Inc., San Diego). A total of 713,495 autosomal SNPs were assessed for quality using the PLINK v1.07 software. All SNPs with a call rate below 95%, a deviation from Hardy-Weinberg equilibrium at an error level of P < 0.001, or a minor allele frequency of less than 10% were excluded. The remaining 534,404 SNPs were used for further analysis.

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Molecular profiling of cutaneous squamous cell carcinomas and actinic keratoses from organ transplant recipients

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