ctDNA transiting into urine is ultrashort and facilitates noninvasive liquid biopsy of HPV⁺ oropharyngeal cancer

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 TRctDNA 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.