Abstract 1396: A novel approach of target enriched next generation sequencing reveals differences in expression of lncRNAs in stage II colorectal cancer formalin fixed paraffin embedded (FFPE) tumors
Authors: Brunet-Vega Anna , Pegueroles Cinta , Iraola-Guzmán Susana , Vilà Laia , Casalots Alex , Ribera Paula , Macias Ismael , Hovhannisyan Hrant , Saus Ester , Pericay Carles , Gabaldón Toni 
Published: September 2020

Abstract
The aim of this study was to identify and quantify lncRNAs in formalin-fixed paraffin-embedded (FFPE) samples from stage II colorectal cancer (CRC) patients.
Colorectal cancer (CRC) is the third most common cancer worldwide, and has high metastasis and recurrence rates. Use of adjuvant chemotherapy in stage II CRC patients is challenging, and new biomarkers are required to identify patients with high probability of relapse. Long noncoding RNAs (lncRNA) play an important role in cancer, awakening especial interest as potential novel cancer biomarkers and therapeutic targets.
Resected tissues and biopsy preserved as formalin-fixed, paraffin-embedded (FFPE) samples are the most abundant supply for translational research. Unfortunately, long-chain RNA molecules, such as lncRNAs, may suffer degradation in those conditions. This fact coupled with their low expression levels challenges their detection with current techniques.
Thus, in this study we developed a new approach which couples target enrichment and RNAseq to enhance detection of lncRNAs. Our three main goals are: I) evaluate the efficiency of this methodology on FFPE samples, II) investigate the lncRNAs involved in stage II CRC, and III) check for the presence of lncRNA differentially expressed in tumors of individuals who relapsed after surgery and not.
We designed a custom-made target enrichment based on Nimblegen (Roche) technology to sequence a total of 8,048 lncRNAs (258 of interest for CRC). We tested 36 paired stage II CRC tumor FFPE tissues and their adjacent normal tissue, 12 from patients who relapsed after surgery. We quantified the expression of the targeted lncRNA and performed a differential expression analysis. Our approach detected expression of numerous lncRNA from FFPE samples. Importantly, we validated 5 target lncRNAs expression values by real time qPCR using two independent housekeeping genes, and found significant correlation between RNAseq (log2 (TPM)) to the qPCR (deltaCT) values.
In addition, we identified a total of 83 lncRNAs which are differentially expressed between normal and tumoral samples. This list is enriched in genes previously reported as being involved in CRC, but includes many novel candidates of unknown function.
Finally, the expression profile of tumors from patients with and without relapse seems quite similar, although we identified 5 lncRNAs that are significantly differentially expressed. Further validation in a larger cohort is needed to determine whether they could have prognostic value.
In summary, our study shows that SeqCap Enrichment System is a good strategy to determine lncRNAs expression in FFPE tissue. Of note, we identified a list of new lncRNAs that are deregulated in stage II CRC tumor. Further research on these lncRNAs may lead to novel clinical applications in cancer biogenesis and prognosis.
Citation Format: Anna Brunet-Vega, Cinta Pegueroles, Susana Iraola-Guzmán, Laia Vilà, Alex Casalots, Paula Ribera, Ismael Macias, Hrant Hovhannisyan, Ester Saus, Carles Pericay, Toni Gabaldón. A novel approach of target enriched next generation sequencing reveals differences in expression of lncRNAs in stage II colorectal cancer formalin fixed paraffin embedded (FFPE) tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1396.