Abstract Introduction: Urine cell-free DNA (cfDNA) is a promising ultra-noninvasive analyte for genitourinary cancers (GU) cancers. In this study, we investigated urine cfDNA fragmentomic features and evaluated their ability to predict the tumor tissue of origin. Methods: A total of 204 GU cancer patients and 34 healthy adults were enrolled in this prospective study. Preoperative urine was collected from 89 bladder cancer (BC) patients (64% muscle-invasive) undergoing cystectomy, 65 renal cell carcinoma (RCC) patients undergoing nephrectomy and 50 metastatic prostate cancer (mPC) patients. Urine cfDNA was isolated and sequenced at 5x genome-wide coverage on a NovaSeq 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. CNAs and tumor fraction (TFx) were quantified using ichorCNA in 1-Mb windows, while chromosome arm-level fragment count z-scores and fragment length ratios were calculated in 5-Mb bins across the genome. Relative frequencies of all 256 possible 4-mer end motifs were calculated for each sample. Individual logistic regression models were developed based on each fragmentomic feature using leave-one-out cross validation (LOOCV). Outputs from these models were combined into multi-feature XGBoost models. Cohort-specific end-motif enrichments were identified using Kruskal-Wallis tests with FDR correction. Motifs were then hierarchically clustered based on their cohort-wise median frequencies to define groups of motifs with shared fragmentation patterns. Results: Across urine cfDNA in the GU cohort, BC showed the highest mean TFx at 10.0%, followed by mPC at 3.6% and RCC at 2.7%. A machine learning model based on 4-mer end motifs achieved a mean ROC AUC of 0.933 with classification accuracies of 63% for healthy, 87% for BC, 72% for mPC, and 83% for RCC. This model outperformed models based on ichorCNA (AUC = 0.767), chromosome arm fragment-count z-scores (AUC = 0.827), and fragment length ratios (AUC = 0.674). Combining end motifs with other fragmentomic features did not improve prediction accuracy. Multi-feature models incorporating end motifs with ichorCNA, chromosome arm-level z-scores, or fragment-length ratios yielded mean AUCs between 0.908-0.924, lower than the end-motif only model. Kruskal-Wallis tests identified 179 of 256 end motifs (79%) as significantly different across healthy, BC, mPC, and RCC. Hierarchical clustering of cohort-level median motifs revealed distinct end motif patterns: Motifs beginning with CT were enriched in RCC; AAAA was strongly enriched in BC; and motifs beginning with CA, AC, and AG were enriched in mPC. Conclusion: Urine cfDNA 4-mer end motifs capture GU cancer-specific biological patterns that enable accurate tissue-of-origin prediction, underscoring their promise as a diagnostic tool. Citation Format: Jessica Linford, Pradeep S. Chauhan, Irfan Alahi, Yohan Kim, Arpit Panda, Nathan Colon, Ryan Mueller, Faridi Qaium, Eric H. Kim, Melissa A. Reimers, Zachary L. Smith, Woodson W. Smelser, Fabrice Lucien-Matteoni, Aadel A. Chaudhuri. Urine cfDNA 4-mer end-motif signatures outperform other fragmentomic features for tissue-of-origin classification in genitourinary cancers 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 2590.
Linford et al. (Fri,) studied this question.