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The Ratio of ssDNA to dsDNA in Circulating Cell-Free DNA Extract is a Stable Indicator for Diagnosis of Gastric Cancer.

Author(s): Huang X, Zhao Q, An X, Pan J, Zhao L, Shen L, Xu Y, Yuan D

Publication: Pathol Oncol Res, 2020, Vol. , Page

PubMed ID: 32632900 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to investigate the effects of extraction method, freeze-thaw cycling and hemolysis on levels of ssDNA and dsDNA and the ssDNA to dsDNA ratio in plasma. The effects gastric cancer diagnosis, tumor size, tumor stage, potential for resection, and surgical excision as well as patient age, gender, BMI, cigarette usage, or alcohol consumption on levels of ssDNA and dsDNA and the ssDNA to dsDNA ratio were also investigated. EDTA blood was collected from 118 healthy patients and 106 with gastric cancer after overnight fast. Post-surgery blood specimens were obtained from 63 of the gastric cancer patients 28 days post-surgery. Plasma was obtained within an hour of blood collection by dual centrifugation at 1900 × g at 4°C for 10 min followed by recentrifugation at 16000 x g at 4°C for 10 min. Plasma was aliquoted and frozen at -80°C and a mixed pool was created by pooling plasma from the 118 healthy patients. cfDNA extraction was performed using Yuan Biotechnology’s magnetic bead-based Mini Cellfree DNA Extraction Kit and stored at -20°C. ssDNA and dsDNA in cfDNA were quantified using the Qubit ssDNA Assay Kit and the Qubit dsDNA HS Assay Kit, respectively. cfDNA fragment size were analyzed by bioanalyzer. Extraction kits were compared by extracting cfDNA from the plasma of 20 patients with gastric cancer using the Mini Cellfree DNA Extraction Kit and the QIAamp Circulating Nucleic Acid Kit. The effect of freeze/thaw cycling was investigated on the pooled plasma by freezing specimen for 4 h at -80°C and thawing at 37°C one to five times. To investigate the effects of hemolysis, blood specimens from 20 subjects were subjected to five freeze/thaw cycles at -80°C for 1 h and thawing at 37°C.

   Summary of Findings:

   The median amount of ssDNA obtained from the plasma was higher using the Mini Cellfree DNA Extraction Kit (12.87 ng/µL) versus the QIAamp Circulating Nucleic Acid Kit (12.87 versus 5.86 ng/µL, P<0.0001), but the median yield of dsDNA was comparable (1.76 versus 1.66 ng/µL). ssDNA and dsDNA levels and the ssDNA to dsDNA ratio were unaffected by freeze/thaw cycling of plasma. While hemolyzed plasma had higher levels of ssDNA and dsDNA than non-hemolyzed plasma (P<0.0001, both), the ratio of ssDNA to dsDNA was unaffected. Plasma ssDNA and dsDNA levels were higher and the ratio of ssDNA to dsDNA was lower in patients with gastric cancer than healthy controls (P<0.0001, all).  The ssDNA to dsDNA ratio had a sensitivity of 86.79% and a specificity of 90.68% for gastric cancer diagnosis and when ssDNA and dsDNA levels were also considered, the sensitivity and specificity were 83.96% and 94.07%, respectively. The ratio of ssDNA to dsDNA was not affected by tumor size but decreased with increasing tumor node metastasis (TNM) stage (P=0.0196) and was higher in resectable than non-resectable tumors (P=0.0045). Plasma ssDNA and dsDNA levels were lower and ratio of ssDNA to dsDNA was higher after surgery (28 days post-surgery) than before (P<0.0001, all). Although healthy males had higher plasma levels of ssDNA and dsDNA than healthy females (P=0.0163 and P=0.0168, respectively), the ratio was comparable between the genders. In contrast, the plasma levels of ssDNA and dsDNA in gastric cancer patients were comparable in males and females, but the ssDNA to dsDNA ratio was higher for males than females (P=0.0430). Alcohol consumption had no effect on ssDNA or dsDNA levels in healthy patients, but the dsDNA levels increased and ssDNA to dsDNA ratio decreased in gastric cancer patients with daily alcohol consumption (P=0.0441 and P=0.0101, respectively). ssDNA and dsDNA levels as well as the ratio of ssDNA to dsDNA were not significantly affected by patient age, BMI, or cigarette usage in either healthy controls or patients with gastric cancer.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Patient gender	Female Male
   Preaquisition	Patient age	31-40 years 41-50 years 51-60 years 61-70 years
   Preaquisition	Patient diet	<5 g alcohol/day 5-15 g alcohol/day >15 g alcohol/day
   Preaquisition	Patient body mass index	18.5-23.9 ≥ 24 <18.5
   Preaquisition	Diagnosis/ patient condition	Never smoker Former smoker Current smoker Gastric cancer Healthy
   Preaquisition	Prognostic factor	Tumor ≤ 20 mm Tumor 20-50 mm Tumor ≥ 50 mm TNM stage I TNM stage II TNM stage III/IV
   Biospecimen Acquisition	Time of biospecimen collection	Pre-surgery 28 days post-surgery
   Biospecimen Aliquots and Components	Hemolysis	Freeze/thaw-induced Not induced
   Storage	Freeze/thaw cycling	0 cycles 1 cycle 2 cycles 3 cycles 4 cycles 5 cycles
   Analyte Extraction and Purification	Analyte isolation method	Mini Cellfree DNA Extraction Kit QIAamp Circulating Nucleic Acid Kit

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