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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

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Pathology & Oncology Research

Abstract

Due to the different mechanisms of cell-free DNA production, the single-stranded DNA to double-stranded DNA ratio in blood maybe different between healthy individuals and gastric cancer (GC) patients. We aimed to explore the potential application of this ratio in GC diagnosis. The plasma cell-free DNA extracts from 118 healthy individuals and 106 GC patients were prepared. The levels of single-stranded DNA or double-stranded DNA in plasma, and the single-stranded DNA to double-stranded DNA ratio on the diagnostic efficiency for GC were assessed with ROC curve. The relationships between this ratio and the clinical characteristics of GC patients were analyzed. The ratios in 63 GC patients before and after surgery were compared. In healthy individuals, the single-stranded DNA to double-stranded DNA ratio was not affected by factors including age, gender and BMI, and subjected to normal distribution (P = 0.1090). GC patients had a lower value of this ratio than healthy individuals (P < 0.0001). Considering this ratio as a GC diagnostic indicator, the area under ROC curve (AUC) was 0.923[95% confidence interval (CI):0.880–0.955]. This ratio in unresectable GC was obviously lower than that in resectable GC (P = 0.0045). There was a rank correlation between this ratio and GC TNM staging (rho = −0.266, P = 0.0058), but it had no correlation with tumor size (r = 0.14, P = 0.145). Additionally, this ratio was not affected by hemolysis and repeated freeze-thaw of blood samples, and was significantly elevated after surgery(P < 0.0001). The single-stranded DNA to double-stranded DNA ratio in plasma is a stable non-invasive indicator for GC diagnosis.

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Data Availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

References

  1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A (2018) Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 68(6):394–424. https://doi.org/10.3322/caac.21492

    Article  PubMed  Google Scholar 

  2. Herszenyi L, Tulassay Z (2010) Epidemiology of gastrointestinal and liver tumors. Eur Rev Med Pharmacol Sci 14(4):249–258

    PubMed  Google Scholar 

  3. Kim YI, Kim YW, Choi IJ, Kim CG, Lee JY, Cho SJ, Eom BW, Yoon HM, Ryu KW, Kook MC (2015) Long-term survival after endoscopic resection versus surgery in early gastric cancers. Endoscopy 47(4):293–301. https://doi.org/10.1055/s-0034-1391284

    Article  CAS  PubMed  Google Scholar 

  4. Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, Jemal A, Yu XQ, He J (2016) Cancer statistics in China, 2015. CA Cancer J Clin 66(2):115–132. https://doi.org/10.3322/caac.21338

    Article  PubMed  Google Scholar 

  5. Zeng H, Zheng R, Guo Y, Zhang S, Zou X, Wang N, Zhang L, Tang J, Chen J, Wei K, Huang S, Wang J, Yu L, Zhao D, Song G, Chen J, Shen Y, Yang X, Gu X, Jin F, Li Q, Li Y, Ge H, Zhu F, Dong J, Guo G, Wu M, Du L, Sun X, He Y, Coleman MP, Baade P, Chen W, Yu XQ (2015) Cancer survival in China, 2003-2005: a population-based study. Int J Cancer 136(8):1921–1930. https://doi.org/10.1002/ijc.29227

    Article  CAS  PubMed  Google Scholar 

  6. Ronellenfitsch U, Schwarzbach M, Hofheinz R, Kienle P, Kieser M, Slanger TE, Burmeister B, Kelsen D, Niedzwiecki D, Schuhmacher C, Urba S, van de Velde C, Walsh TN, Ychou M, Jensen K (2013) Preoperative chemo (radio) therapy versus primary surgery for gastroesophageal adenocarcinoma: systematic review with meta-analysis combining individual patient and aggregate data. Eur J Cancer 49(15):3149–3158. https://doi.org/10.1016/j.ejca.2013.05.029

    Article  PubMed  Google Scholar 

  7. Alsina M, Miquel JM, Diez M, Castro S, Tabernero J (2019) How I treat gastric adenocarcinoma. ESMO Open 4(Suppl 2):e000521. https://doi.org/10.1136/esmoopen-2019-000521

    Article  PubMed  PubMed Central  Google Scholar 

  8. Skrypkina I, Tsyba L, Onyshchenko K, Morderer D, Kashparova O, Nikolaienko O, Panasenko G, Vozianov S, Romanenko A, Rynditch A (2016) Concentration and methylation of cell-free DNA from blood plasma as diagnostic markers of renal Cancer. Dis Markers 2016:3693096. https://doi.org/10.1155/2016/3693096

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Burnham P, Kim MS, Agbor-Enoh S, Luikart H, Valantine HA, Khush KK, De Vlaminck I (2016) Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma. Sci Rep 6:27859. https://doi.org/10.1038/srep27859

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Bettegowda C, Sausen M, Leary RJ, Kinde I, Wang Y, Agrawal N, Bartlett BR, Wang H, Luber B, Alani RM, Antonarakis ES, Azad NS, Bardelli A, Brem H, Cameron JL, Lee CC, Fecher LA, Gallia GL, Gibbs P, Le D, Giuntoli RL, Goggins M, Hogarty MD, Holdhoff M, Hong SM, Jiao Y, Juhl HH, Kim JJ, Siravegna G, Laheru DA, Lauricella C, Lim M, Lipson EJ, Marie SK, Netto GJ, Oliner KS, Olivi A, Olsson L, Riggins GJ, Sartore-Bianchi A, Schmidt K, Shih LM, Oba-Shinjo SM, Siena S, Theodorescu D, Tie J, Harkins TT, Veronese S, Wang TL, Weingart JD, Wolfgang CL, Wood LD, Xing D, Hruban RH, Wu J, Allen PJ, Schmidt CM, Choti MA, Velculescu VE, Kinzler KW, Vogelstein B, Papadopoulos N, Diaz LA Jr (2014) Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med 6(224):224ra224. https://doi.org/10.1126/scitranslmed.3007094

    Article  CAS  Google Scholar 

  11. Siravegna G, Marsoni S, Siena S, Bardelli A (2017) Integrating liquid biopsies into the management of cancer. Nat Rev Clin Oncol 14(9):531–548. https://doi.org/10.1038/nrclinonc.2017.14

    Article  CAS  PubMed  Google Scholar 

  12. Murtaza M, Dawson SJ, Tsui DW, Gale D, Forshew T, Piskorz AM, Parkinson C, Chin SF, Kingsbury Z, Wong AS, Marass F, Humphray S, Hadfield J, Bentley D, Chin TM, Brenton JD, Caldas C, Rosenfeld N (2013) Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA. Nature 497(7447):108–112. https://doi.org/10.1038/nature12065

    Article  CAS  PubMed  Google Scholar 

  13. Wu HC, Yang HI, Wang Q, Chen CJ, Santella RM (2017) Plasma DNA methylation marker and hepatocellular carcinoma risk prediction model for the general population. Carcinogenesis 38(10):1021–1028. https://doi.org/10.1093/carcin/bgx078

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Liao W, Mao Y, Ge P, Yang H, Xu H, Lu X, Sang X, Zhong S (2015) Value of quantitative and qualitative analyses of circulating cell-free DNA as diagnostic tools for hepatocellular carcinoma: a meta-analysis. Medicine (Baltimore) 94(14):e722. https://doi.org/10.1097/MD.0000000000000722

    Article  CAS  Google Scholar 

  15. Volckmar AL, Sultmann H, Riediger A, Fioretos T, Schirmacher P, Endris V, Stenzinger A, Dietz S (2018) A field guide for cancer diagnostics using cell-free DNA: from principles to practice and clinical applications. Genes Chromosom Cancer 57(3):123–139. https://doi.org/10.1002/gcc.22517

    Article  CAS  PubMed  Google Scholar 

  16. Wei L, Wu W, Han L, Yu W, Du Y (2018) A quantitative analysis of the potential biomarkers of non-small cell lung cancer by circulating cell-free DNA. Oncol Lett 16(4):4353–4360. https://doi.org/10.3892/ol.2018.9198

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Ponti G, Maccaferri M, Manfredini M, Kaleci S, Mandrioli M, Pellacani G, Ozben T, Depenni R, Bianchi G, Pirola GM, Tomasi A (2018) The value of fluorimetry (Qubit) and spectrophotometry (NanoDrop) in the quantification of cell-free DNA (cfDNA) in malignant melanoma and prostate cancer patients. Clin Chim Acta 479:14–19. https://doi.org/10.1016/j.cca.2018.01.007

    Article  CAS  PubMed  Google Scholar 

  18. Lee TH, Montalvo L, Chrebtow V, Busch MP (2001) Quantitation of genomic DNA in plasma and serum samples: higher concentrations of genomic DNA found in serum than in plasma. Transfusion 41(2):276–282. https://doi.org/10.1046/j.1537-2995.2001.41020276.x

    Article  CAS  PubMed  Google Scholar 

  19. Macheret M, Halazonetis TD (2015) DNA replication stress as a hallmark of cancer. Annu Rev Pathol 10:425–448. https://doi.org/10.1146/annurev-pathol-012414-040424

    Article  CAS  PubMed  Google Scholar 

  20. Branzei D, Foiani M (2010) Maintaining genome stability at the replication fork. Nat Rev Mol Cell Biol 11(3):208–219. https://doi.org/10.1038/nrm2852

    Article  CAS  PubMed  Google Scholar 

  21. Zeman MK, Cimprich KA (2014) Causes and consequences of replication stress. Nat Cell Biol 16(1):2–9. https://doi.org/10.1038/ncb2897

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Gaillard H, Garcia-Muse T, Aguilera A (2015) Replication stress and cancer. Nat Rev Cancer 15(5):276–289. https://doi.org/10.1038/nrc3916

    Article  CAS  PubMed  Google Scholar 

  23. Thierry AR, El Messaoudi S, Gahan PB, Anker P, Stroun M (2016) Origins, structures, and functions of circulating DNA in oncology. Cancer Metastasis Rev 35(3):347–376. https://doi.org/10.1007/s10555-016-9629-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Snyder MW, Kircher M, Hill AJ, Daza RM, Shendure J (2016) Cell-free DNA comprises an in vivo nucleosome footprint that informs its tissues-of-origin. Cell 164(1–2):57–68. https://doi.org/10.1016/j.cell.2015.11.050

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Jahr S, Hentze H, Englisch S, Hardt D, Fackelmayer FO, Hesch RD, Knippers R (2001) DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer Res 61(4):1659–1665

    CAS  PubMed  Google Scholar 

  26. Zhang X, Wu Z, Shen Q, Li R, Jiang X, Wu J, Li D, Wang D, Zou C, Zhong Y, Cheng X (2019) Clinical significance of cell-free DNA concentration and integrity in serum of gastric cancer patients before and after surgery. Cell Mol Biol (Noisy-le-grand) 65(7):111–117

    Article  Google Scholar 

  27. Park JL, Kim HJ, Choi BY, Lee HC, Jang HR, Song KS, Noh SM, Kim SY, Han DS, Kim YS (2012) Quantitative analysis of cell-free DNA in the plasma of gastric cancer patients. Oncol Lett 3(4):921–926. https://doi.org/10.3892/ol.2012.592

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. You Y, Teng W, Wang J, Ma G, Ma A, Wang J, Liu P (2018) Hypertension and physical activity in middle-aged and older adults in China. Sci Rep 8(1):16098. https://doi.org/10.1038/s41598-018-34617-y

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Steevens J, Schouten LJ, Goldbohm RA, van den Brandt PA (2010) Alcohol consumption, cigarette smoking and risk of subtypes of oesophageal and gastric cancer: a prospective cohort study. Gut 59(1):39–48. https://doi.org/10.1136/gut.2009.191080

    Article  CAS  PubMed  Google Scholar 

  30. Sorber L, Zwaenepoel K, Deschoolmeester V, Roeyen G, Lardon F, Rolfo C, Pauwels P (2017) A comparison of cell-free DNA isolation kits: isolation and quantification of cell-free DNA in plasma. J Mol Diagn 19(1):162–168. https://doi.org/10.1016/j.jmoldx.2016.09.009

    Article  CAS  PubMed  Google Scholar 

  31. Sato A, Nakashima C, Abe T, Kato J, Hirai M, Nakamura T, Komiya K, Kimura S, Sueoka E, Sueoka-Aragane N (2018) Investigation of appropriate pre-analytical procedure for circulating free DNA from liquid biopsy. Oncotarget 9(61):31904–31914. https://doi.org/10.18632/oncotarget.25881

    Article  PubMed  PubMed Central  Google Scholar 

  32. Lippi G, Salvagno GL, Montagnana M, Brocco G, Guidi GC (2006) Influence of hemolysis on routine clinical chemistry testing. Clin Chem Lab Med 44(3):311–316. https://doi.org/10.1515/CCLM.2006.054

    Article  CAS  PubMed  Google Scholar 

  33. Chang Y, Tolani B, Nie X, Zhi X, Hu M, He B (2017) Review of the clinical applications and technological advances of circulating tumor DNA in cancer monitoring. Ther Clin Risk Manag 13:1363–1374. https://doi.org/10.2147/TCRM.S141991

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Mathurin P, Cadranel JF, Bouraya D, Bronstein JA, Collot G, Devergie B, Poynard T, Opolon P (1996) Marked increase in serum CA 19-9 level in patients with alcoholic cirrhosis: report of four cases. Eur J Gastroenterol Hepatol 8(11):1129–1131. https://doi.org/10.1097/00042737-199611000-00019

    Article  CAS  PubMed  Google Scholar 

  35. Shang X, Song C, Du X, Shao H, Xu D, Wang X (2017) The serum levels of tumor marker CA19-9, CEA, CA72-4, and NSE in type 2 diabetes without malignancy and the relations to the metabolic control. Saudi Med J 38(2):204–208. https://doi.org/10.15537/smj.2017.2.15649

    Article  PubMed  PubMed Central  Google Scholar 

  36. Zali H, Rezaei-Tavirani M, Azodi M (2011) Gastric cancer: prevention, risk factors and treatment. Gastroenterol Hepatol Bed Bench 4(4):175–185

    PubMed  PubMed Central  Google Scholar 

  37. Swets JA (1988) Measuring the accuracy of diagnostic systems. Science 240(4857):1285–1293. https://doi.org/10.1126/science.3287615

    Article  CAS  PubMed  Google Scholar 

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Author notes

  1. Xuewen Huang, Qi Zhao and Xianyuan An contributed equally to this work.

Authors and Affiliations

  1. Department of Clinical Laboratory, the affiliated Wuxi No.2 People’s Hospital of Nanjing Medical University, Wuxi, People’s Republic of China

    Xuewen Huang, Lanfeng Shen, Yiqiu Xu & Dandan Yuan

  2. Department of Clinical Laboratory, the affiliated Wuxi People’s Hospital of Nanjing Medical University, Wuxi, People’s Republic of China

    Qi Zhao

  3. Department of Clinical Laboratory, HuaDong Sanatorium, Wuxi, People’s Republic of China

    Xianyuan An, Jie Pan & Lanjing Zhao

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Contributions

(I) Conception and design: Xuewen Huang.

(II) Administrative support: Xuewen Huang.

(III) Provision of study materials or patients: Qi Zhao, Yiqiu Xu.

(IV) Collection and assembly of data: Xianyuan An, Lanjing Zhao, Dandan Yuan.

(V) Data analysis and interpretation: Xuewen Huang, Jie Pan, Lanfeng Shen, Dandan Yuan.

(VI) Manuscript writing: All authors.

(VII) Final approval of manuscript: All authors.

All authors have read and approved the manuscript.

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Correspondence to Xuewen Huang.

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The study protocol was approved by the Ethics Committee of the affiliated No.2 People’s Hospital of Nanjing Medical University (Wuxi, China) [(2019) Academic Review No. (Y-25)]. The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All participants provided written informed consent.

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Supplementary Fig. 1

Comparison of cfDNA extraction efficiency between two methods. (a-b) A total of 20 randomly selected plasma samples were all subjected to cfDNA extractions with the magnetic bead method and the Qiagen column method, respectively. The concentrations of ssDNA (a) and dsDNA (b) after normalization to total sample volumes were compared. (PNG 213 kb)

High resolution image (TIF 657 kb)

Supplementary Fig. 2

Confirmation of the nature of nucleic acids in cfDNA extracts. (a-d) The cfDNA extracts isolated from the plasma samples of randomly selected 20 GC patients were subjected to RNase A (a-b) and DNase I (c-d) digestion. The concentrations of ssDNA (a and c) and dsDNA (b and d) in the cfDNA extracts before and after RNase A (a-b) or DNase I (c-d) digestions were measured. (e-f) The DNA contents in cfDNA extracts before (e) and after (f) DNase I digestion were examined by the Agilent 2100 bioanalyzer. Representative images of electropherogramwere selected from the data of three GC patients-derived cfDNA extract samples with similar results. (g) Agarose gel images show the electrophoresis results of three GC patients-derived cfDNA extract samples before and after DNase I digestion. (PNG 1297 kb)

High resolution image (TIF 3798 kb)

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Huang, X., Zhao, Q., An, X. et al. The Ratio of ssDNA to dsDNA in Circulating Cell-Free DNA Extract is a Stable Indicator for Diagnosis of Gastric Cancer. Pathol. Oncol. Res. 26, 2621–2632 (2020). https://doi.org/10.1007/s12253-020-00869-1

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