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Genome-wide analysis of primary and secondary angiosarcomas using RNA extracted from formalin-fixed parafinembedded samples

ABSTRACT: Angiosarcomas are highly malignant tumors that develop as primary angiosarcomas of unknown cause or as secondary angiosarcomas, most often following radiation therapy for breast cancer. These subsets are morphologically indistinguishable, which motivates our aim to identify genetic classifiers for diagnostic and therapeutic application. We applied the 18k whole-genome c-DNA-mediated annealing, selection, extension, and ligation (WG-DASL) assay for expression profiling of 26 primary and 29 secondary angiosarcomas. Primary and secondary angiosarcomas differed by 103 significantly de-regulated genes with up-regulation of RET, KIT, FLT4, MYC and RASGRP3 and down-regulation of CDKN2C in secondary angiosarcoma. Functional annotation analysis demonstrated involvement of multiple target genes in the receptor protein tyrosine kinase pathway. Up-regulation of RET and down-regulation of CDKN2C characterize secondary angiosarcoma, which implies a mechanistic basis for the evaluation of RET-kinase inhibitors in the treatment of these highly aggressive tumors. Total RNA obtained from primary and secondary angiosarcoma samples from formalin-fixed parafin-embedded samples were compared using WG-DASL. Biological replicates (two samples from the same patient, different parts of the tumor or different operation specimens) Technical replicates (two samples from the same patient and same tumor sample, analyzed in different areas of the bead chip)

ORGANISM(S): Homo sapiens

SUBMITTER: Styring E

PROVIDER: S-ECPF-GEOD-52664 | biostudies-other | 2014 Jul

REPOSITORIES: biostudies-other

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Project description:BackgroundWe have developed a gene expression assay (Whole-Genome DASL), capable of generating whole-genome gene expression profiles from degraded samples such as formalin-fixed, paraffin-embedded (FFPE) specimens.Methodology/principal findingsWe demonstrated a similar level of sensitivity in gene detection between matched fresh-frozen (FF) and FFPE samples, with the number and overlap of probes detected in the FFPE samples being approximately 88% and 95% of that in the corresponding FF samples, respectively; 74% of the differentially expressed probes overlapped between the FF and FFPE pairs. The WG-DASL assay is also able to detect 1.3-1.5 and 1.5-2 -fold changes in intact and FFPE samples, respectively. The dynamic range for the assay is approximately 3 logs. Comparing the WG-DASL assay with an in vitro transcription-based labeling method yielded fold-change correlations of R(2) approximately 0.83, while fold-change comparisons with quantitative RT-PCR assays yielded R(2) approximately 0.86 and R(2) approximately 0.55 for intact and FFPE samples, respectively. Additionally, the WG-DASL assay yielded high self-correlations (R(2)>0.98) with low intact RNA inputs ranging from 1 ng to 100 ng; reproducible expression profiles were also obtained with 250 pg total RNA (R(2) approximately 0.92), with approximately 71% of the probes detected in 100 ng total RNA also detected at the 250 pg level. When FFPE samples were assayed, 1 ng total RNA yielded self-correlations of R(2) approximately 0.80, while still maintaining a correlation of R(2) approximately 0.75 with standard FFPE inputs (200 ng).Conclusions/significanceTaken together, these results show that WG-DASL assay provides a reliable platform for genome-wide expression profiling in archived materials. It also possesses utility within clinical settings where only limited quantities of samples may be available (e.g. microdissected material) or when minimally invasive procedures are performed (e.g. biopsied specimens).

Project description:BACKGROUND: Gene expression profiling of formalin-fixed, paraffin-embedded (FFPE) samples represents a valuable approach for advancing oncology diagnostics and enhancing retrospective clinical studies; however, at present, this methodology still requires optimization and thus has not been extensively used. Here, we utilized thorough quality control methods to assess RNA extracted from FFPE samples and then compared it to RNA extracted from matched fresh-frozen (FF) counterparts. We preformed genome-wide expression profiling of FF and FFPE ovarian serous adenocarcinoma sample pairs and compared their gene signatures to normal ovary samples. METHODS: RNA from FFPE samples was extracted using two different methods, Ambion and Agencourt, and its quality was determined by profiling starting total RNA on Bioanalyzer and by amplifying increasing size fragments of beta actin (ACTB) and claudin 3 (CLDN3) by reverse-transcriptase polymerase chain reaction. Five matched FF and FFPE ovarian serous adenocarcinoma samples, as well as a set of normal ovary samples, were profiled using whole genome Agilent microarrays. Reproducibility of the FF and FFPE replicates was measured using Pearson correlation, whereas comparison between the FF and FFPE samples was done using a Z-score analysis. RESULTS: Data analysis showed high reproducibility of expression within each FF and FFPE method, whereas matched FF and FFPE pairs demonstrated lower similarity, emphasizing an inherent difference between the two sample types. Z-score analysis of matched FF and FFPE samples revealed good concordance of top 100 differentially expressed genes with the highest correlation of 0.84. Genes characteristic of ovarian serous adenocarcinoma, including a well known marker CLDN3, as well as potentially some novel markers, were identified by comparing gene expression profiles of ovarian adenocarcinoma to those of normal ovary. CONCLUSION: Conclusively, we showed that systematic assessment of FFPE samples at the RNA level is essential for obtaining good quality gene expression microarray data. We also demonstrated that profiling of not only FF but also of FFPE samples can be successfully used to identify differentially expressed genes characteristic of ovarian carcinoma.

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Genome-wide analysis of primary and secondary angiosarcomas using RNA extracted from formalin-fixed parafinembedded samples

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