DNA-methylation profiling distinguishes malignant melanomas from benign nevi.
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ABSTRACT:
SUBMITTER: Conway K
PROVIDER: S-EPMC3073305 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Identification of gene expression changes associated with melanoma development and progression.
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Project description:Despite malignant cutaneous melanoma is relatively rare compared to other skin cancers, it is still responsible for 80% of all skin cancer-related deaths. To identify molecular signatures of melanoma progression, excisional biopsies from 18 common melanocytic nevi (CMN), 8 primary radial growth phase melanomas (RGPM), 15 primary vertical growth phase melanomas (VGPM) and 5 melanoma metastases (MTS) were profiled using whole genome oligo-microarrays. Differentially expressed genes for each progression step were identified, and validation of selected transcripts by qRT-PCR was performed on an independent cohort of fixed samples. The comparison between CMN and RGPM showed an enrichment of Gene Ontology (GO) terms related to inter and intra-cellular junctions, whereas the transition from RGPM to VGPM was characterized by the deregulation of WNT3, MAPK and AKT pathways. In this step, enrichment analysis underlined the alteration of biological processes linked to apoptosis. Upregulation of genes involved into DNA double-strand breaks repair and downregulation of cellular adhesion genes were observed in MTS respect to VGPM. Futhermore, the gene expression profiles of 11 dysplastic nevi (DN) were compared to all the others. Some genes controlling proliferation were found more expressed than in CMN. Overall, DN displayed an heterogeneous behaviour, with relevant oncogenes, such as MYC and BCL6, less expressed than in RGPM, and a modulation pattern similar to VGPM for a subset of gene families, such as mismatch repair. This suggests that DN is not an intermediate step within melanoma progression, but a separate entity with an independent risk of progression to melanoma Experiment Overall Design: In order to identify genes implicated during the course of melanoma development and progression, a total of 57 excisional biopsies from common melanocytic nevi (n = 18), dysplastic nevi (n = 11), primary radial growth phase malignant melanomas (n = 8), primary vertical growth phase malignant melanomas (n = 15) and melanoma metastases (n = 5) were analyzed. Total RNA was isolated by using RNeasy Mini Kit (Qiagen, Dusseldorf, Germany). TotRNA quality was checked by means of RNA 6000 pico chip assays (Agilent Technologies, Palo Alto, CA) run on the Agilent 2100 bioanalyzer. RNA isolated from each fresh tissue and from the human universal reference (BD™ Human Universal Reference Total RNA, Clontech, Palo Alto, CA) was amplified by means of the Amino Allyl MessageAmp II aRNA Kit (Ambion) to obtain amino allyl antisense RNA (aaRNA) following the method developed by Eberwine and coworkers. Two rounds of amplification were performed to obtain the necessary quantity of aaRNA for labeling. Briefly: mRNA was reverse transcribed in cDNA single strand; after the second strand synthesis (in the second round of amplification), cDNA was in vitro transcribed in aaRNA including amino allyl modified nucleotides (aaUTP). Both dsDNA and aaRNA underwent a purification step using columns provided with the kit. Labeling was performed using NHS ester Cy3 or Cy5 dyes (Amersham Biosciences, Buckinghamshire UK) able to react with the modified RNA. mRNA quality was checked by means of RNA 6000 nano chip assays (Agilent Technologies). At least 5 ug of mRNA for each sample were labeled and purified with columns. Equal amounts (0.75 ug) of labeled specimens from sample and reference were put together, fragmented and hybridized to oligonucleotide glass arrays representing 41K human unique genes and transcripts (Human Whole Genome Oligo Microarray Array, Agilent Technologies). All steps were performed using the In Situ Hybridization kit-plus (Agilent Technologies) and following the 60-mer oligo microarray processing protocol (Agilent Technologies). Then, slides were washed with the SSPE wash procedure and scanned with the dual-laser microarray scanner Agilent G2505B. For each sample, a dye-swap replicate was performed. To visually inspect the relationships among samples, unsupervised clustering analysis was applied to the ratios between each DN, RGPM , VGPM or MTS sample and the average profile of all the CMN included into the study, just selecting transcripts with p-value less than 0.01 in at least half of the samples considered. Transcripts modulated in each progression step were then selected by applying a cut-off of 2 for the B value yielded by the limma procedure applied on original ratios (sample over common reference). Initially, DN were included into either the CMN class or the RGPM one, according to the degree of their cyto-architectural atypia However, the presence of such lesions among either CMN or RGPM made the distinction between these two classes and between RGPM and VGPM much less clear. Therefore, to prevent the increasing of heterogeneity, DN were excluded and this allowed the detection of many more differentially expressed transcripts between CMN and RGPM and, especially, between RGPM and VGPM. The great difference found between metastases and the rest of the samples was consistent with unsupervised results. Enrichment of Gene Ontology categories (in particular, biological processes) and gene networks or cellular pathways was performed on each list by using DAVID functional annotation tool and applying a cut-off of 0.01 on the enrichment score obtained. For each progression step, some genes previously described as differentially expressed were found. Furthermore, other novel gene transcripts, never reported as involved in melanoma progression, were identified. The expression deregulation of some of these novel genes was then confirmed on an independent cohort of samples by RT-qPCR.
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Project description:BackgroundHistopathologic examination is sometimes inadequate for accurate and reproducible diagnosis of certain melanocytic neoplasms. As a result, more sophisticated and objective methods have been sought. The goal of this study was to identify a gene expression signature that reliably differentiated benign and malignant melanocytic lesions and evaluate its potential clinical applicability. Herein, we describe the development of a gene expression signature and its clinical validation using multiple independent cohorts of melanocytic lesions representing a broad spectrum of histopathologic subtypes.MethodsUsing quantitative reverse-transcription polymerase chain reaction (PCR) on a selected set of 23 differentially expressed genes, and by applying a threshold value and weighting algorithm, we developed a gene expression signature that produced a score that differentiated benign nevi from malignant melanomas.ResultsThe gene expression signature classified melanocytic lesions as benign or malignant with a sensitivity of 89% and a specificity of 93% in a training cohort of 464 samples. The signature was validated in an independent clinical cohort of 437 samples, with a sensitivity of 90% and specificity of 91%.ConclusionsThe performance, objectivity, reliability and minimal tissue requirements of this test suggest that it could have clinical application as an adjunct to histopathology in the diagnosis of melanocytic neoplasms.
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Project description:PURPOSE:Recent studies revealed divergent gene expression patterns in Ewing sarcoma (EwS) with canonical EWSR1-ETS gene fusions and undifferentiated round cell sarcomas (URCS) with EWSR1 rearrangements fused to the non-ETS gene NFATc2. Thus, the question arises whether the latter tumors really belong to EwS. METHODS:We collected five cases matching the group of URCS with EWSR1-NFATc2 fusion and performed DNA methylation and copy number profiling. Results were compared to methylation data of 30 EwS with various EWSR1-ETS fusions and one EwS with FUS-ERG fusion, 16 URCS with CIC rearrangement and 10 URCS with BCOR alteration and a total of 81 EWSR1-associated soft tissue sarcomas including 7 angiomatoid fibrous histiocytomas, 7 clear cell sarcomas of the soft tissue, 28 desmoplastic small round cell tumors, 10 extraskeletal myxoid chondrosarcomas and 29 myxoid liposarcomas. RESULTS:Unsupervised hierarchical clustering and t-distributed stochastic neighbor embedding analysis of DNA methylation data revealed a homogeneous methylation cluster for URCS with EWSR1-NFATc2 fusion, which clearly segregated from EwS and the other subtypes. Copy number profiles of EWSR1-NFATc2 cases showed recurrent losses on chromosome 9q and segmental gains on 20q13 and 22q12 involving the EWSR1 and NFATc2 loci, respectively. CONCLUSION:In summary, URCS with EWSR1-NFATc2 fusion share a distinct DNA methylation signature and carry characteristic copy number alterations, which emphasizes that these sarcomas should be considered separately from EwS.
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