Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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

Author(s): Bhambhani C, Kang Q, Hovelson DH, Sandford E, Olesnavich M, Dermody SM, Wolfgang J, Tuck KL, Brummel C, Bhangale AD, He K, Gutierrez MG, Lindstrom RH, Liu CJ, Tuck M, Kandarpa M, Mierzwa M, Casper K, Prince ME, Krauss JC, Talpaz M, Henry NL, Giraldez MD, Ramnath N, Tomlins SA, Swiecicki PL, Brenner JC, Tewari M

Publication: JCI Insight, 2024, Vol. 9, Page

PubMed ID: 38516891 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the stability of a low-range DNA ladder in non-stabilized urine stored at room temperature for up to 24 h,  urine stabilized with EDTA (10 mM, or 40 mM) and/or Tris (10 mM, or 30 mM), and in urine containing 100 mM EDTA that was stored for up to 7 days at room temperature. Additionally, the authors evaluated the effect of urine cell-free DNA (cfDNA) fragment size on the detection of circulating tumor DNA (ctDNA) and copy number variations (CNV) using next-generation sequencing (NGS). The study also compared the detection of Human Papillomavirus (HPV) DNA in matched plasma and urine specimens using a stem-loop 42 bp ddPCR assay and a conventional 77 bp ddPCR assay and investigated the ability of the stem-loop assay to detect HPV DNA prior to clinical recurrence. Matched blood in either K₂EDTA or Streck BCT tubes and urine were collected from 20 patients with locally advanced HPV+ OPSCC, 12 patients with metastatic HPV+ OPSCC, 1 patient with HPV18+ OPSCC, and six patients with HPV- HNSCC; urine was also collected from 5 volunteers who were free of cancer.  Plasma was separated from blood by centrifugation at 1,600 g for 10 min, followed by 16,000 g for 10 min, and stored at –80°C until DNA isolation with the QIAamp Circulating Nucleic Acid Kit. Unless otherwise specified, urine was preserved with 100 mM EDTA (pH 8.0), and then either double centrifuged (1,600 g for 10 min, followed by 3,000 g for 10 min) or centrifuged at 3,000 g for 10 minutes, followed by filtration through a 0.45 µm polyethersulfone filter. Urine was stored at –80°C until cfDNA isolation using a Q Sepharose resin-based binding method.  CPV was analyzed using low-pass ssDNA whole genome sequencing (details not provided). HPV 16 DNA was detected using a stem-loop 42 bp ddPCR assay and a conventional 77 bp PCR assay. To investigate the effect of EDTA and Tris on urine stability, matched urine aliquots from two donors were spiked with an Ultra Low Range DNA ladder and were preserved with water (pH 6.), 10 mM EDTA (pH 6.0), 40 mM EDTA (pH 7.0), 30 mM Tris (pH 7.5), 10 mM EDTA and 50 mM Tris (pH 7.5), or 40 mM EDTA and 50 mM Tris (pH 7.5). The aliquots were then stored at room temperature for 0, 1, 7 or 24 h and analyzed by electrophoresis. Additionally, matched urine aliquots from two donors were spiked with an Ultra Low Range DNA ladder, stabilized with 40 mM EDTA (pH 6.0) or 100 mM EDTA (pH 7.0), and stored at room temperature for 0, 3, 5 or 7 days before storage at –20° C until DNA extraction and bioanalyzer analysis.

   Summary of Findings:

   A spiked DNA ladder in urine degraded within 1 h when water with a pH of 6 was added (control) or when in urine contained 30 mM Tris (pH 7.5), but this degradation was partially attenuated in urine specimens that contained 10 mM EDTA (pH 6.0) and was prevented for 24 h when urine contained 40 mM EDTA (pH 7.0). Greater stability of the spiked DNA ladder was observed in urine specimens that contained 10 mM EDTA and 50 mM Tris (pH 8) compared to those that contained 10 mM EDTA alone. All DNA ladder bands (20-200 bp) were stable for at least 7 days of room temperature storage when urine contained 100 mM EDTA.

   CNVs found in plasma were also detected in urine by low-pass coverage NGS, but their detection improved when analysis was limited to short fragments (20-40 or 30-50 bp). Short DNA (<50 bp) present in urine was found to have a higher percentage of tumor DNA than longer DNA fragments.  Using a stem-loop 42 bp ddPCR assay, HPV 16 DNA was detected in matched plasma and urine specimens from all 20 patients with locally advanced HPV+ oropharyngeal squamous cell carcinoma (OPSCC). In specimens from patients with metastatic HPV+ OPSCC, HPV 16 DNA was detected in the plasma of 9 of 12 patients and the urine of 8 of 12 patients, with concordant results for 9 of 12 patients (7 positives and 2 negatives). Importantly, HPV 16 was not detected in either urine or plasma of the one patient with HPV18+ OPSCC, the six patients with HPV- head and neck squamous cell carcinoma (HNSCC) or the urine (plasma not tested) of 5 non-cancer patients. The number of copies of HPV 16 DNA were modestly correlated between matched plasma and urine from patients with HPV+ OPSCC (r=0.5483, P=0.0014). In contrast to the stem-loop 42 bp ddPCR assay, a conventional 77 bp ddPCR assay detected very low (0-4 copies in 5 of 11 specimens) or no copies (6 of 11 specimens) of HPV in urine specimens that were positive (31-4,628 copies) using the stem-loop assay. HPV16 DNA was detected in urine using the stem loop assay prior to clinical recurrence in 3 of 4 patients.

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Urine
   • Bodily Fluid - Plasma

   Preservative Types

   • Other Preservative
   • Frozen

   Diagnoses:

   • Not specified
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Automated electrophoresis/Bioanalyzer
   DNA	Electrophoresis
   DNA	Digital PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Locally advanced HPV+ OPSCC Metastatic HPV+ OPSCC HPV18+ OPSCC HPV- HNSCC Non-cancer patients
   Biospecimen Acquisition	Biospecimen location	Plasma Urine
   Biospecimen Acquisition	Time of biospecimen collection	Prior to treatment After treatment/surgery Prior to recurrence At clinical recurrence
   Storage	Time at room temperature	0 h 1 h 7 h 24 h 0 days 3 days 5 days 7 days
   Digital PCR Specific	Length of gene fragment	Stem-loop 42 bp amplicon Conventional 77 bp amplicon
   Storage	Short-term storage solution	Water (pH 6) added to urine Urine containing 10 mM EDTA (pH 6.0) Urine containing 40 mM EDTA (pH 7.0) Urine containing 30 mM Tris (pH 7.5) Urine containing 10 mM EDTA and 50 mM Tris (pH 7.5) Urine containing 40 mM EDTA and 50 mM Tris (pH 7.5 Urine containing 100 mM EDTA (pH 7.0)

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