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Towards effectiveness of cell free DNA based liquid biopsy in head and neck squamous cell carcinoma.

Author(s): Kowal-Wisniewska E, Jaskiewicz K, Bartochowska A, Kiwerska K, Ustaszewski A, Gorecki T, Giefing M, Paluszczak J, Wierzbicka M, Jarmuz-Szymczak M

Publication: Sci Rep, 2024, Vol. 14, Page 2251

PubMed ID: 38278927 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared cfDNA concentration, size distribution, and integrity between specimens collected from healthy volunteers and patients with head and neck squamous cell carcinoma (HSNCC); among specimens collected from patients with HSNCC at different timepoints relative to diagnosis, surgery, and recurrence; and among specimens collected from HSNCC patients who differed in the anatomical location of the tumor. Blood was collected from 152 patients with HSNCC at multiple timepoints and from 56 healthy volunteers into S-Monovette EDTA tubes. For patients with HSNCC, 137 specimens were obtained at diagnosis, 29 at reoccurrence, 31 specimens 1-14 days post-resection, 20 specimens 15-100 days post-resection, and 135 more than 100 days post-resection.  Plasma was isolated by dual centrifugation at 1200 g for 10 min followed by 2,000 g for 10 min. Hemolysis was assessed visually and by spectrophotometry. Specimens that were not hemolyzed were frozen at -80°C. Specimens were thawed on ice and centrifuged at 16,000 g at 4°C for 10 min before isolation of cfDNA with the QIAamp MinElute ccfDNA Kit. cfDNA was quantified using the Agilent High Sensitivity DNA Kit.

   Summary of Findings:

   The cfDNA concentration was higher in specimens from patients with primary or recurrent HSNCC tumors than from healthy controls (255.63 pg/μl and 261.38 pg/μl, respectively versus 151.92 pg/μl; P<0.0001 and P=0.0006, respectively), but there was no difference in cfDNA concentration between specimens from patients with primary and recurrent tumors. The average median length of cfDNA between 35-250 bp was longer in cancer patients than healthy controls (156 versus 149 bp, P=0.0061) but the median length of cfDNA between 35-1000 bp was shorter in cancer patients than healthy volunteers (270 versus 304 bp, P<0.001). The average median length of cfDNA between 500–10,380 bp and 35–10,380 bp was longer in patients with primary tumors than recurrent tumors (4679 bp versus 4245 bp, P=0.017 and 2224 bp versus 1864 bp, P=0.0435). cfDNA concentrations were higher in specimens collected 1-14 days or 15-100 days post-resection than in specimens from healthy volunteers (458.48 pg/μl and 259.70 pg/μl, respectively, versus 151.92 pg/μl, P<0.0001 and P=0.0168, respectively), but the cfDNA concentration in specimens collected >100 days after resection was comparable to the concentration in specimens from healthy volunteers (138.26 pg/μl). The integrity scores (ratio of 250-1000 bp or 250-10,380 bp fragments to 35-250 bp fragments) were significantly higher in specimens collected >100 days post-resection than those collected at diagnosis (P<0.0001, both) or 1-14 days post-resection (P<0.0001 and P<0.01, respectively). A weak correlation between patient age and cfDNA concentration was observed in both HSNCC patients (r=0.357, P=0.000002) and healthy volunteers (r=0.2849, P=0.04). The authors report no difference in cfDNA concentration overall or broken down by size range among the tumor locations examined or with tumor stage. There was a trend toward a higher concentration of cfDNA in the size range of 35- 250 bp in patients with primary rather than recurrent tumors in the larynx and oral cavity, but the reverse was true for patients with tumors in the nasal cavity/jaw, lip, and oropharynx. Integrity scores (ratio of 250-1000bp or 250-10,380 bp fragments to 35-250 bp fragments) were not significantly different among the tumor locations represented. For patients with tumors in the larynx, there was significantly more cfDNA of 250-500 bp in patients with recurrent tumors than primary tumors (P=0.039).  cfDNA concentrations were comparable among specimens from patients with cancer-free survival (>2 years or < 2 years follow-up), specimens from patients with recurrence at follow-up, and specimens obtained from patients at recurrence, but some differences in the median length of cfDNA between 35–10,380 bp and 500–10,380 bp between the groups were observed.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Patient age	24-86 years
   Preaquisition	Diagnosis/ patient condition	Healthy Head and Neck Squamous Cell Carcinoma Primary tumor Recurrence Tumor in larynx Tumor in oral cavity Tumor in nasal cavity/jaw Tumor in lip Tumor in oropharynx Tumor in hypopharynx
   Preaquisition	Prognostic factor	Stage 1 Stage 2 Stage 3 Stage 4
   Biospecimen Acquisition	Time of biospecimen collection	1-14 days post-resection 15-100 days post-resection >100 days post-resection At diagnosis At recurrence

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