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 2

Ultrashort amplicon HPV16 stem-loop ddPCR assay detects TR-ctDNA fragments in urine from patients with HPV+ OPSCC and shows concordance with results from plasma ctDNA analysis.

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Ultrashort amplicon HPV16 stem-loop ddPCR assay detects TR-ctDNA fragmen...
(A) Schematic of the short-amplicon (42 bp) stem-loop 2-stage PCR approach (17) used to detect and quantify ultrashort HPV16 TR-ctDNA present in urine (see Supplemental Figure 3 for details). (B) Analytical validation of the stem-loop assay using a 2-fold dilution series to define the detectable range (top plots). Expected copies of synthetic spiked-in HPV16 target DNA (left) or the GEs of HPV16+ cancer cell line UM-SCC-104 tested (right) were plotted against the measured copies (cumulative of triplicates). LoD was determined to be 4.2 copies (see Methods). Bottom plots show observed coefficient of variation (%CV) corresponding to measurement at each dilution of synthetic HPV16 DNA or UM-SCC-104 gDNA. (C) Stem-loop assay was used to detect HPV16 ctDNA in matched urine and plasma samples collected before treatment, from 32 HPV+ OPSCC patients representing both locally advanced (LA) and metastatic (M) cases, and 12 negative controls (1 HPV18+ OPSCC patient, 6 HPV– head and neck squamous cell carcinoma [HNSCC] patients, and 5 healthy individuals without cancer). The assay detected HPV16 ctDNA in both urine and plasma in 27 of the 32 cases of HPV+ OPSCC. HPV16 ctDNA was not detected in any of the negative controls (12 of 12 cases), showing high concordance between plasma-based and urine-based ctDNA detection. (D) Comparison of log10 HPV16 ctDNA values in urine and matched plasma from 31 patients with HPV+ OPSCC (see Methods). Significant correlation was found between HPV16 copies detected in urine and plasma based on Pearson’s correlation test; *P (2-tailed) = 0.0014. HPV16 TR-ctDNA values (cumulative of ddPCR triplicates) correspond to the mean value of 30 mL urine samples from 2 bottles; and ~0.9 ml for plasma samples..