Sheng, John

Abstract Introduction: Long intergenic noncoding RNAs (lincRNAs) and circular (circ) RNAs have been shown to play key roles in carcinogenesis and tumor progression. Urine represents a promising source of tumor-reflective signals shed directly from the urothelial microenvironment in bladder cancer (BC) patients. Here, we evaluate the urine cell-free (cf) RNA transcriptome in a cohort of 50 patients with localized BC. Methods: A total of 50 localized BC patients and 24 healthy adults were enrolled. Pre-operative urine (collected on the day of radical cystectomy) was processed to isolate cfRNA from urine supernatant. Libraries were prepared using an Illumina tagmentation-based RNA enrichment workflow and sequenced to 100M paired-end reads per sample on a NovaSeq X. FASTQ files were concatenated per sample and QC-checked with RNA-SeQC and Samtools. Reads were aligned to GRCh38/hg38 with STAR, and gene counts were generated using Feature Counts. Linear differential expression analysis (DEA) was performed with DESeq2. For discovery we used random halves of TCGA-BLCA and healthy urine cfRNA samples, alongside a second DEA with random halves of urine BC cfRNA vs. controls; overlapping upregulated genes were validated in the remaining samples. For circular (circ) RNAs, annotations from MiOncoCirc were used; junctions with ≥5 supporting reads were retained, and DEA was conducted with edgeR. Results: In this study, the median age was 68 years, and 80% (40/50) were male. Among the cohort, 66% (33/50) had muscle-invasive bladder cancer (MIBC), of whom 54% received neoadjuvant chemotherapy. Differential expression analysis of TCGA-BLCA tumors and BC urine cfRNA, identified 27 genes commonly upregulated, with 23 of the 27 shared genes strikingly being long intergenic noncoding RNAs (lincRNAs). These BC/urine-specific lincRNAs—including LINC00930, LINC01004, DLG5-AS1, APTR, PSORS1C3, and SERPINB9P1—have known roles in proliferation, EMT, metabolism, and immune signaling. Outside of lincRNAs, tumor-reflective coding transcripts in BC/urine were enriched for autophagy, driven by MAP1LC3B2.TCGA-BLCA survival analysis showed that LINC00930 and PSORS1C3 were associated with worse overall survival. Expanding beyond linear RNA sequences, analysis of urine circRNAs from BC patients revealed a strong cancer-specific dysregulation pattern, with 77 circRNAs significantly upregulated compared with healthy controls. Upregulated urine circRNAs arose from host genes involved in BC-relevant pathways, including invasion (DOCK1, ARHGAP5), autophagy/ER stress (VMP1, ERN1), and luminal differentiation (GRHL2, ESRP1). Conclusion: Cell-free RNA profiling reveals a distinct transcriptomic signature including differential lincRNA- and circRNA-driven autophagy, EMT, proliferation, metabolic, differentiation, and immune signaling programs in the urine of bladder cancer patients. Citation Format: Pradeep Chauhan, Li Lin, Irfan Alahi, Jessica Linford, Lilli J. Greiner, Ayesha Hashmi, Anushka Viswanathan, Nathan Colon, Faridi Qaium, Peter K. Harris, John Sheng, Jacob J. Orme, Eric H. Kim, Zachary L. Smith, Woodson Smelser, Christopher A. Maher, Aadel A. Chaudhuri. Urine cell-free RNA captures tumor-reflective long intergenic noncoding and circular RNA programs in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1133.

Abstract Introduction: We previously developed a urine liquid biopsy assay to measure urine tumor DNA (utDNA) levels in bladder cancer (BC) patients. Here, we extended this approach significantly by layering in cell-free DNA fragmentomic and cell-free RNA analysis, and extending the method to the full spectrum of genitourinary (GU) cancers, to enable both GU cancer detection and tumor tissue of origin prediction. Methods: A total of 155 GU cancer patients and 34 healthy adults were enrolled in this prospective study. Preoperative urine was collected from 94 bladder cancer (BC) patients, including 65 with muscle-invasive disease, all undergoing cystectomy. Additionally, urine samples were collected from 18 renal cell carcinoma (RCC) patients undergoing nephrectomy and 42 metastatic prostate cancer (mPC) patients. Urine cfDNA and cfRNA were isolated and sequenced, with cfRNA sequenced to 100 million reads per sample on an Illumina NovaSeq X and cfDNA sequenced at 5x genome-wide coverage on a Novoseq S4 flow cell. To identify malignant tissue of origin, we analyzed somatic copy number alterations (CNAs), genome-wide fragment length ratios, and 4-mer end motifs. Tumor fraction (TFx) was estimated using ichorCNA, while DELFI was employed for fragment length ratios in 5 megabase bins across the genome and chromosome arm-level z-scores. Individual logistic regression models were developed based on each fragmentomic feature, and validated using a training and held-out testing approach. Outputs from these models were combined into a single comprehensive model, also trained and held-out tested using the same approach. Urine cfRNA transcript isoform abundance was quantified using Salmon and normalized to Transcripts Per Million (TPM) to account for sequencing depth variations. Results: Bladder cancer (BC) showed the highest mean tumor fraction (TFx, 10.5%), followed by RCC (3.8%) and mPC (3.4%). Among 94 BC patients, 42% achieved a pCR, while 58% had residual disease and did not. Patients without pCR had significantly higher urine TFx compared to those with pCR (14.7% vs. 4.7%, p=0.0008). A machine learning model based on genome-wide fragment length ratios achieved an ROC AUC of 0.74, while models using genome-wide CNAs and end motifs achieved AUCs of 0.81 and 0.88, respectively. Combining all features into a single model yielded an AUC of 0.92, with classification accuracies of 89% for bladder cancer, 80% for RCC, and 60% for prostate cancer. Additionally, urine cfRNA analysis using the top 1, 000 coding transcripts achieved an AUC of 0.95, with classification accuracies of 100% for mPC, 87% for BC, and 78% for RCC. Conclusion: Fragmentomic features from urine cfDNA and transcriptomic features from urine cfRNA demonstrate strong potential for predicting tumor tissue-of-origin in GU cancers, underscoring the promise of non-invasive diagnosis across the spectrum of GU malignancies through urine liquid biopsy. Citation Format: Pradeep Singh Chauhan, Irfan Alahi, Jessica Linford, Arpit Panda, Nathan Colon, Ryan Mueller, Faridi Qaium, Peter K. Harris, John Sheng, Eric H. Kim, Melissa A. Reimers, Zachary L. Smith, Woodson W. Smelser, Aadel A. Chaudhuri. Predicting genitourinary cancer tissue-of-origin using urine cell-free DNA fragmentomics and cell-free RNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3258.