Project description: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 SCC development in OTRs. Various gene mutations (e.g. TP53) and genetic alterations (e.g. CDKN2A loss, RAS amplification) have been found in SCCs. The aim of this study was to identify genomic alterations that are consistently involved in the formation of SCCs and their precursor lesions, actinic keratoses (AKs). To perform the analysis in an isogenic background, DNA was isolated from SCC and AK from each of 13 OTRs. Tumor samples and their matching normal control (peripheral blood) were analyzed on the Illumina HumanHap550V3_duo genotyping beadchips. In contrast to previous studies, genome-wide SNP analysis showed very few copy number variations in AKs and SCCs, and these variations had no apparent relationship with observed changes in mRNA expression profiles (GSE32628). Other molecular mechanisms, such as DNA methylation or miRNA alterations, may affect gene expression in SCCs of OTRs.
Project description:RNA-Seq was performed on 8 actinic keratoses (AK) to identify differentially expressed genes in AK vs normal skin and cutaneous squamous cell carcinoma
Project description: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 SCC development in OTRs. Various gene mutations (e.g. TP53) and genetic alterations (e.g. CDKN2A loss, RAS amplification) have been found in SCCs. The aim of this study was to identify genomic alterations that are consistently involved in the formation of SCCs and their precursor lesions, actinic keratoses (AKs). To perform the analysis in an isogenic background, DNA was isolated from SCC and AK from each of 13 OTRs. Tumor samples and their matching normal control (peripheral blood) were analyzed on the Illumina HumanHap550V3_duo genotyping beadchips. In contrast to previous studies, genome-wide SNP analysis showed very few copy number variations in AKs and SCCs, and these variations had no apparent relationship with observed changes in mRNA expression profiles (GSE32628). Other molecular mechanisms, such as DNA methylation or miRNA alterations, may affect gene expression in SCCs of OTRs. 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, including 2 repeats) and AKs (n=11). Peripheral blood was taken as normal control. DNA was isolated and SNP profiles were obtained using HumanHap550V3_duo Genotyping BeadChips (Illumina). Both the log R ratio and the B allele frequency (BAF) were investigated to identify copy number alterations and loss of heterozygosity (LOH) The tumor DNA samples are from the same tumor samples as the RNA samples in GSE32628.
Project description: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.
Project description: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.
Project description:normal skin (no), actinic keratosis (ak), and squamous cell carcinoma (scc) of the skin were examined:; BACKGROUND: Carcinogenesis is a multi-step process indicated by several genes up- or down-regulated during tumor progression. This study examined and identified differentially expressed genes in cutaneous squamous cell carcinoma (SCC). RESULTS: Three different biopsies of 5 immunosuppressed organ-transplanted recipients each normal skin (all were pooled), actinic keratosis (AK) (two were pooled), and invasive SCC and additionally 5 normal skin tissues from immunocompetent patients were analyzed. Thus, total RNA of 15 specimens were used for hybridization with Affymetrix HG-U133A microarray technology containing 22,283 genes. Data analyses were performed by prediction analysis of microarrays using nearest shrunken centroids with the threshold 3.5 and ANOVA analysis was independently performed in order to identify differentially expressed genes (p < 0.05). Verification of 13 up- or down-regulated genes was performed by quantitative real-time reverse transcription (RT)-PCR and genes were additionally confirmed by sequencing. Broad coherent patterns in normal skin vs. AK and SCC were observed for 118 genes. CONCLUSION: The majority of identified differentially expressed genes in cutaneous SCC were previously not described.
Project description:Genome wide DNA methylation profiling in cutaneous squamous cell carcinoma. The Infinium MethylationEPIC BeadChips 850K has been used to interrogate DNA methylation changes. The cohort included 23 patients in total, with precursors of squamous cell carcinoma (actinic keratosis group) and with cSCC at different stages.
Project description:Squamous cell carcinoma is the second most common skin cancer and frequently progress from an intraepithelial actinic keratosis. The role of microRNAs during the progression from actinic keratosis to cutaneous squamous cell carcinoma (cSCC) remains to be elicited. By using an Agilent microRNA expression microarray we found the expression of miR-204 to be markedly downregulated in cSCC when compared to actinic keratoses. DNA methylation of the TRPM3 promoter region upstream of miR-204-5p was identified as one of the repressive mechanisms that accounts for miR-204 silencing in cSCC. Functional studies on HaCaT cells revealed that this microRNA downregulates the Signal Transducer and Activator of Transcription 3 (STAT3) pathway and favours the MAPK signaling pathway, likely acting through PTPN11, a tyrosine phosphatase that is a direct miR-204 target. We found that activated STAT3, as detected by pY705-STAT3 immunofluorescence, is retained in the membrane and cytoplasm compartment in AK, whereas cSCC displayed STAT3 in the nuclei. Taken together, our data indicates that MiR-204 may act as a “rheostat” that controls the signaling towards the MAPK pathway or the STAT3 pathway.