Abstract 2531: Diagnostic evaluation of indeterminate pulmonary nodules via rna-seq of bronchial epithelium
Authors: Shi Xingyi , Billatos Ehab , Zhang Jiarui , Beane Jennifer , Moses Elizabeth , Liu Gang , Stevenson Christopher , Lenburg Marc E. , Spira Avrum 
Published: August 2018

RATIONALE: Diagnosis of the estimated 1.5 million indeterminate pulmonary nodules found annually on chest CT in the US poses a significant clinical challenge. Prior work from our group has identified alterations in the bronchial epithelial cell microarray-derived transcriptome which are associated with lung cancer and which can serve as a clinically useful biomarker for the evaluation of patients undergoing bronchoscopy for suspect lung cancer. Given that RNA sequencing has a broader measurement range and allows for unbiased detection of novel transcripts, we evaluated the ability of bronchial epithelial gene-expression from RNA sequencing to identify patients with lung cancer among individuals with indeterminate pulmonary nodules.

METHODS: Bronchial epithelial brushings were collected from current and former smokers (n=93; age&gt;45, pack-year&gt;20) undergoing diagnostic work up for indeterminate pulmonary nodules (7-30 mm in diameter) at 4 military and 7 VA hospitals within the DECAMP consortium. Patients were followed clinically for up to two years after sample collection until a final diagnosis of lung cancer (n=52) or benign disease (n=41) was made. We performed total RNA sequencing using the Illumina TruSeq Stranded Total RNA Sample Preparation kit. Individual samples were sequenced to generate paired-end reads per sample. Reads were aligned to the human assembly Genome Reference Consortium Human Build 37 (GRCh37) in Ensembl using Spliced Transcripts Alignment to a Reference (STAR) and summarized into count data via RNA-Seq by Expectation Maximization (RSEM).

RESULTS: We found no differences in age, gender, or smoking status between patients with malignant vs. benign nodules. Using a generalized linear model accounting for smoking status, we identified 34 differentially expressed genes (DEGs, FDR q &lt; 0.2) from the bronchial epithelium associated with lung cancer. Of these 34 DEGs, 28 were up-regulated in individuals with lung cancer and enriched for pathways related to the response to hypoxia and p53. The 6 down-regulated DEGs were enriched for immune cell activation, supporting an immunosuppressive field effect of lung cancer. Concordant enrichment of the previous microarray-based bronchial gene-expression classifier was observed using Gene Set Enrichment Analysis (GSEA, FDR q &lt; 0.05), supporting the cross-platform and cross-cohort validity of the microarray-based classifier.

CONCLUSION: Taken together, findings from this pilot study suggest that there are distinct lung-cancer associated airway epithelial gene-expression alterations detected by RNA-sequencing which replicate a previously published microarray-based signature. Additional alterations identified by our RNAseq studies may be leveraged to refine and further develop an RNA-sequencing based airway biomarker for the early detection of lung cancer in high-risk smokers.

Citation Format: Xingyi Shi, Ehab Billatos, Jiarui Zhang, Jennifer Beane, Elizabeth Moses, Gang Liu, Christopher Stevenson, Marc E. Lenburg, Avrum Spira. Diagnostic evaluation of indeterminate pulmonary nodules via rna-seq of bronchial epithelium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2531.