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
View: Text | PDF
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

×

Figure 1

Fragment size analysis of urine cfDNA WGS from patients with nonurologic cancers, showing TR-ctDNA enrichment in ultrashort fragments.

Options: View larger image (or click on image) Download as PowerPoint
Fragment size analysis of urine cfDNA WGS from patients with nonurologic...
(A) The plots show log2(copy number ratio) (Log2CNRatio) calls on the y axis for plasma cfDNA or urine cfDNA for 3 patients, with the x axis showing the genomic position of the mapped DNA fragments across the indicated chromosomes. Low-pass coverage urine cfDNA WGS data, unfiltered for fragment length, showing CNA patterns qualitatively concordant with those from matched plasma cfDNA WGS (also unfiltered for length) in some but not all patients; cancer-associated CNAs are visible in patients AML14 and AML13 but not in patient ST5. After stratification of analysis by fragment length into 20 bp wide bins, CNA plots showed that restriction to ultrashort bins (<50 bp; i.e., 30–50 bp and 20–40 bp) revealed tumor-associated CNA more robustly than unfiltered data. (B) Heatmap of estimated percentage of tumor DNA content from WGS data. We observed increased enrichment for tumor DNA with ultrashort fragments (i.e., <50 bp) relative to larger fragment size bins, consistent with the qualitative differences evident in A. (C) Composite curves of urine cfDNA WGS data from 3 patients with solid tumors and 3 patients with AML. The curves labeled as focally amplified, gain, and loss are implicitly derived from tumor-enriched DNA (fragment length of mapped reads emanating from regions with known tumor-associated CNA), while the unaltered curves reflect inferred fragment lengths for reads mapping to the remainder of the genome. As shown, the majority of DNA fragments and, importantly, those mapping to genomic regions of tumor-associated CNA are ultrashort (<50 bp) in length.