Abstract P3-10-05: Feasibility of detecting long noncoding RNAs in clinical FFPE specimens using clinical grade RNA-sequencing pipeline
Authors: Wei Christina H. , O'Meally Denis , Kirschenbaum Michele , Mao Allen , Kancharla Chetan , Morris Kevin V. , Stewart Daphne 
Published: March 2022

BACKGROUND: RNA-based predictive biomarkers are not assessed in the clinical diagnostic setting. Potential biomarkers include long noncoding RNAs (lncRNAs), a class of molecules emerging as critical regulators of diverse cellular functions, including therapy resistance. Advances in sequencing technologies have made whole transcriptome profiling (RNA-seq) possible in formalin fixed, paraffin embedded (FFPE) tissue. In this study, we assessed the feasibility of detecting lncRNAs in clinical FFPE specimens, using clinical grade RNA-seq technology offered at a CLIA-certified laboratory. Our second aim was to investigate expression of lncRNAs contributing to endocrine resistance in a cohort of metastatic breast cancer patients. METHODS: RNA-seq was performed on clinical FFPE specimens as a physician-ordered diagnostic test in a CLIA-certified laboratory. RNA-seq libraries were constructed using ribosomal RNA depletion method and &gt; 100 M reads were sequenced for each library. We checked for contaminating sequences using fastqScreen against multiple genomes, including human and E. coli among others. To assess the quality of the sequencing libraries, we used the nf-core RNASeq pipeline. Trimmed reads were mapped to the GRCh38 reference and abundance of transcripts from the Gencode comprehensive annotation (v33) was estimated. Each library was assessed for quality control (QC). Mapped reads were merged into a matrix of counts per gene for each sample. Differential expression was assessed between metastatic breast cancers with and without endocrine resistance. RESULTS: We obtained twenty-five cases (23 patients) of ER-positive, HER2 negative metastatic breast carcinoma that had undergone whole transcriptome sequencing at a CLIA-certified molecular laboratory. Of these, 6 (24%) cases had failed QC: 5 patients’ specimens had low RNA content (all core biopsies); 1 patient had total RNA content comprised of less than 75% human reads (lumpectomy). The mean age for specimens that failed sequencing QC was significantly different from those that passed (637.2 days vs 193.7 days, t-test, p = 0.04). After QC, a total of 19 cases were analyzed for differentially expressed lncRNA profiles. Of these, 5 cases were clinically annotated as hormone sensitive; 14 cases had acquired secondary hormone resistance. Thirty-six lncRNAs were identified as differentially expressed (7 antisense; 25 intergenic; 3 intronic; 1 sense-overlapping). Of these, lncRNAs which were found to be over-expressed in endocrine resistant tumors compared to endocrine sensitive tumors are involved in various oncogenic pathways such as cell cycle regulation (Linc0051, MIR205HG, ST8SOA6-AS1), tumor progression (LINC00885, LINC00319), and chemoresistance (SIRLNT). LncRNAs which were downregulated in endocrine resistant tumors compared to endocrine sensitive tumors are involved in stem cell state (CCDC144NL.AS1) and endocrine resistance (MIR2052HG). CONCLUSION: It is feasible to detect lncRNA biomarkers in clinical FFPE tissue specimens, using a laboratory-developed RNA sequencing pipeline in a CLIA-certified genomic laboratory. FFPE-RNA-seq pipeline can potentially expand the molecular information that could be gleamed from clinical FFPE specimens to answer pertinent clinical questions, such as therapy resistance.

Table 1.Summary data of metastatic ER+ breast cancer evaluated in this studyFailed RNA QC Success RNA QCNumber of samples (%)6 (24%)19 (76%)Mean specimen age (range)27 days (11-44)194 days (3-1331)Number of specimens which are biopsies (%)5 (83%)17 (89%)Number of specimens derived from bone source (%)2 (33%)1 (5%)Number of specimens derived from visceral organs (%)2 (33%)12 (63%)Number of specimens derived from soft tissue or lymphoid sources (%)2 (33%)6 (32%)Acquired endocrine resistance5 (83%)14 (74%)Endocrine sensitive1 (17%)5 (26%)

Citation Format: Christina H. Wei, Denis O'Meally, Michele Kirschenbaum, Allen Mao, Chetan Kancharla, Kevin V. Morris, Daphne Stewart. Feasibility of detecting long noncoding RNAs in clinical FFPE specimens using clinical grade RNA-sequencing pipeline [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-10-05.