Research Article: 3′-End Sequencing for Expression Quantification (3SEQ) from Archival Tumor Samples

Date Published: January 19, 2010

Publisher: Public Library of Science

Author(s): Andrew H. Beck, Ziming Weng, Daniela M. Witten, Shirley Zhu, Joseph W. Foley, Phil Lacroute, Cheryl L. Smith, Robert Tibshirani, Matt van de Rijn, Arend Sidow, Robert B. West, Irene Oi Lin Ng.

Abstract: Gene expression microarrays are the most widely used technique for genome-wide expression profiling. However, microarrays do not perform well on formalin fixed paraffin embedded tissue (FFPET). Consequently, microarrays cannot be effectively utilized to perform gene expression profiling on the vast majority of archival tumor samples. To address this limitation of gene expression microarrays, we designed a novel procedure (3′-end sequencing for expression quantification (3SEQ)) for gene expression profiling from FFPET using next-generation sequencing. We performed gene expression profiling by 3SEQ and microarray on both frozen tissue and FFPET from two soft tissue tumors (desmoid type fibromatosis (DTF) and solitary fibrous tumor (SFT)) (total n = 23 samples, which were each profiled by at least one of the four platform-tissue preparation combinations). Analysis of 3SEQ data revealed many genes differentially expressed between the tumor types (FDR<0.01) on both the frozen tissue (∼9.6K genes) and FFPET (∼8.1K genes). Analysis of microarray data from frozen tissue revealed fewer differentially expressed genes (∼4.64K), and analysis of microarray data on FFPET revealed very few (69) differentially expressed genes. Functional gene set analysis of 3SEQ data from both frozen tissue and FFPET identified biological pathways known to be important in DTF and SFT pathogenesis and suggested several additional candidate oncogenic pathways in these tumors. These findings demonstrate that 3SEQ is an effective technique for gene expression profiling from archival tumor samples and may facilitate significant advances in translational cancer research.

Partial Text: The development of gene expression microarrays in the mid-1990s represented a significant technical achievement that, for the first time, permitted the systematic genome-wide evaluation of gene expression [1], [2]. Since their introduction, these technologies have been widely used for gene expression profiling of cancer samples, leading to the identification of gene expression patterns that predict the biological and clinical features of a wide range of human malignancies [3]–[16].

Since the introduction of gene expression microarrays in the mid-1990s, genome-wide expression profiling has been widely utilized in cancer research [63], [64]. Gene expression profiling experiments have led to significant advances in our understanding of a wide range of human malignancies, but clinical research efforts have been frustrated by lack of specimens. A major hindrance to the translation of gene expression profiling to the clinic is the fact that gene expression microarrays are best performed on fresh frozen tissue, and few samples are stored as fresh frozen. In contrast, essentially all tumor specimens are stored as FFPET [28]. This fixation and storage technique results in extensive RNA fragmentation [29]. Several groups have attempted to use FFPET for gene expression profiling by microarrays [22]–[27] with mixed results. Difficulties of gene expression profiling by microarray on FFPET include the inability to obtain adequate RNA for gene microarray profiling from most archival samples [25] and a lack of sensitivity for identifying genes known to be expressed from frozen tissue in matched FFPET samples [26].



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