BACKGROUND Transrenal cell-free tumor DNA (TR-ctDNA), which transits from the bloodstream into urine, has the potential to enable noninvasive cancer detection for a wide variety of nonurologic cancer types.Methods Using whole-genome sequencing, we discovered that urine TR-ctDNA fragments across multiple cancer types are predominantly ultrashort (<50 bp) and, therefore, likely to be missed by conventional ctDNA assays. We developed an ultrashort droplet digital PCR assay to detect TR-ctDNA originating from HPV-associated oropharyngeal squamous cell carcinoma (HPV+ OPSCC) and confirmed that assaying ultrashort DNA is critical for sensitive cancer detection from urine samples.Results TR-ctDNA was concordant with plasma ctDNA for cancer detection in patients with HPV+ OPSCC. As proof of concept for using urine TR-ctDNA for posttreatment surveillance, in a small longitudinal case series, TR-ctDNA showed promise for noninvasive detection of recurrence of HPV+ OPSCC.Conclusion Our data indicate that focusing on ultrashort fragments of TR-ctDNA will be important for realizing the full potential of urine-based cancer diagnostics. This has implications for urine-based detection of a wide variety of cancer types and for facilitating access to care through at-home specimen collections.Funding NIH grants R33 CA229023, R21 CA225493; NIH/National Cancer Institute grants U01 CA183848, R01 CA184153, and P30CA046592; American Cancer Society RSG-18-062-01-TBG; American Cancer Society Mission Boost grant MBGI-22-056-01-MBG; and the A. Alfred Taubman Medical Research Institute.


Chandan Bhambhani, Qing Kang, Daniel H. Hovelson, Erin Sandford, Mary Olesnavich, Sarah M. Dermody, Jenny Wolfgang, Kirsten L. Tuck, Collin Brummel, Apurva D. Bhangale, Kuang He, Marc G. Gutierrez, Ryan H. Lindstrom, Chia-Jen Liu, Melissa Tuck, Malathi Kandarpa, Michelle Mierzwa, Keith Casper, Mark E. Prince, John C. Krauss, Moshe Talpaz, N. Lynn Henry, Maria D. Giraldez, Nithya Ramnath, Scott A. Tomlins, Paul L. Swiecicki, J. Chad Brenner, Muneesh Tewari


Download this citation for these citation managers:

Or, download this citation in these formats:

If you experience problems using these citation formats, send us feedback.