ctDNA transiting into urine is ultrashort and facilitates noninvasive liquid biopsy of HPV+ oropharyngeal cancer
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
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
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Clinical Research and Public Health Oncology

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

Abstract

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.

Authors

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

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Figure 3

Proof of concept for earlier detection of cancer recurrence via serial urine HPV16 TR-ctDNA measurements in 4 patients with HPV+ OPSCC.

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Proof of concept for earlier detection of cancer recurrence via serial u...
Shown are ddPCR results from testing of longitudinal urine collections from 4 patients, using the stem-loop 42 bp urine TR-ctDNA assay. Number of HPV16 copies detected (y axis) was plotted on a log10 scale (patient 1 and patient 3) or linear scale (patient 2 and patient 4) as HPV16 ctDNA values (cumulative of ddPCR triplicates) from urine cfDNA samples collected at different time points over several months (x axis). Samples were analyzed from 7 time points for patient 1, 4 time points for patient 2, 4 time points for patient 3, and 3 time points for patient 4. Closed symbols represent HPV16 molecules detected above LoD (magenta dotted line), and open symbols represent values that were below the LoD, with serially collected samples at baseline prior to treatment in black, samples collected during treatment in green, and samples collected during posttreatment surveillance for recurrence in red. The day of treatment is marked as 0 on the x axis. Patients 1–3 underwent chemoradiation (CRT) treatment (7 weeks) and showed detectable HPV16 TR-ctDNA prior to initiation of treatment. Patient 4 underwent surgical resection but, notably, had baseline urine DNA of poor quality and, therefore, that time point could not be accurately analyzed for HPV16 TR-ctDNA. In 3 patients, HPV16 TR-ctDNA was detected in urine during the surveillance period prior to clinically detected recurrence.